1st FEMS Congress / Posters 103^505
P1^1
IDENTIFICATION OF A NOVEL LACTOCOCCAL
BACTERIOCIN FROM A L. LACTIS SUBSP. LACTIS
STRAIN
M. Akcelik(1), C
 . Tu«kel(2)
(1) Ankara University, Faculty of Science, Department of
Biology, Tandogflan, 06100, Ankara, Turkey ; (2) Ankara
University, Faculty of Agriculture, Department of Food En-
gineering, D|skap|, 06110, Ankara, Turkey
40 di¡erent L. lactis strains were isolated from traditional
fermented milk products and screened for their antimicro-
bial activities. Eight of the isolates showed inhibitory e¡ect
against di¡erent indicator bacteria and identi¢ed as L.
lactis subsp. lactis. Nisin was characterized in three and
lacticin 481 was characterized in four of eight bacteriocin-
producing isolates by PCR-techniques. A broad inhibitory
spectrum bacteriocin produced by one of the L. lactis
subsp. lactis strains didn’t show any similarity between
any known lactococcal bacteriocins. The production was
associated with a 50 kb plasmid.
P1^2
BIOGENIC AMINES PRODUCTION BY LACTOBA-
CILLI AND STAPHYLOCOCCI ISOLATED FROM
FERMENTED SAUSAGES
A. Storti, C. Tutta, C. Andrighetto, G. Marcazzan, A.
Lombardi
Veneto Agricoltura Istituto per la Qualita' e le Tecnologie
Agroalimentari, via San Gaetano 74, 36016 Thiene, Vice-
nza, Italy
In several ripened or fermented foods biogenic amines,
such as hystamine, tyramine, phenyletilamine and tript-
amine may be present; according to their concentration,
to the particular product composition and to the individ-
ual health response may cause tossic e¡ect of di¡erent
intensity to the human body. Biogenic amines production
in fermented sausages is often related to the decarboxylat-
ing activity of lactic acid bacteria that play an important
role in meat fermentation and ripening processes. To con-
trol biogenic amines in fermented meat products, it is very
important to have information about the microorganisms
that during may contribute to the synthesis or degradation
0378-1097 ? 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.
FEMSLE Congress 2-6-03
of biogenic amines. The aim of this work was to study
biogenic amines production by lactic acid bacteria and
coagulase negative staphylococci isolated from fermented
sausages. These strains were evaluated for their ability to
produce tyramine, hystamine, cadaverine and putrescine in
a di¡erential plating medium containing the corresponding
aminoacid. A total of 73 lactobacilli and 23 coagulase-
negative staphylococci isolated from traditional fermented
meat products were screened for biogenic amine produc-
tion. Among lactobacilli 8 strains identi¢ed as Lactobacil-
lus curvatus displayed decarboxylase activity (4 strains
produced putrescine and 4 putrescine and tyramine); re-
garding staphylococci, 2 strains identi¢ed as Staphylococ-
cus xylosus were able to produce hystamine and putrescine
and 1 strain belonging to the species Staphylococcus car-
nosus produced hystamine, putrescine and cadaverine.
Work is now in progress in order to evaluate the ability
of strains of Staphylococcus xylosus to degrade biogenic
amines. Strains exhibiting such activity might have a po-
tential to prevent an accumulation of biogenic amines dur-
ing sausage fermentation.
P1^3
GROWTH OF YEAST STRAINS DURING BATCH
PRODUCTION OF SINGLE-CELL PROTEIN FROM
CHEESE WHEY
B. AŁ sva¤nyi(1), G. Bugyi(2), L. Daro¤czi(3), R. Kova¤cs(1),
J. Szigeti(1) and L. Varga(1)
(1) University of West Hungary, Faculty of Agricultural
and Food Sciences, Institute of Food Science, 15-17 Luc-
sony Street, 9200 Mosonmagyaro¤va¤r, Hungary ; (2) Mik-
roauto Inc., Cegle¤d, Hungary; (3) Y-Food Inc., Berettyo¤u¤j-
falu, Hungary
The objective of this research was to evaluate the suitabil-
ity of various Kluyveromyces species for use in single-cell
protein (SCP) production. The maximum viable cell
counts reached by selected strains of K. fragilis, K. lactis,
and K. marxianus during batch production of SCP were
determined. The Kluyveromyces strains were grown in un-
fractionated, heat-treated cheese whey, which had a lac-
tose content of 4.5%, thereby providing optimum growth
conditions for yeasts. Fermentations were run batchwise in
an automated BIOFLO III0 batch/continuous fermenter
under identical conditions with respect to pH, aeration,
and agitation rate. The parameters set were computer-con-
trolled using Advanced Fermentation Software version
104 1st FEMS Congress / Posters 103^505
3.42. Samples were taken at regular preset intervals, their
viable cell counts were determined by the pour-plate tech-
nique, and the strains were then ranked in order of growth
rate. By reaching the highest maximum cell counts of all
the Kluyveromyces strains tested within 24 h of fermenta-
tion, K. marxianus NCAIM Y.00933 proved to be the
strain most suitable for SCP production. An economical
technology for the large-scale production of SCP is still to
be developed. The use of SCP for animal nutrition might
be an alternative to the traditional uses of cheese whey.
P1^4
ISOLATION OF MICROORGANISMS FROM LOOG-
PAENG FOR RICE KOJI PREPARATION
S. Attrapadung(1), S. Bovonsombut(2), A. Plikomol(1)
and S. Bovonsombut(1)
(1) Department of Biology, Faculty of Science, Chiang Mai
University, Chiang Mai 50200, Thailand; (2) Department
of Food Technology, Faculty of Engineering and Agro-In-
dustry, Maejo University, Chiang Mai 50290, Thailand
For improving the quality of Thai rice wine, A number of
loog-paeng (Thai traditional rice wine starter) was col-
lected from 8 di¡erent areas in the northern part of Thai-
land for isolation of useful microorganisms. These isolates
were 67 fungi , 55 yeasts and 37 bacteria. Rhizopus spp.,
Bacillus spp. and Saccharomyces spp. were dominant spe-
cies of fungi, bacteria and yeast respectively.The types of
microorganism were not the same pattern from one loca-
tion to another according to the formulation of loog-
paeng. Whilst the amount of these ones decrease along
with the age of loog-paeng. The isolates were tested for
amylolytic activity by using starch agar medium. The re-
sult showed 83 isolates were able to produce clear zone.
Among them, 30 isolates which made a large clear zone
were selected for determining amylase activity and some
quality attributes on rice koji.
P1^5
ANTIMUTAGENICITY OF PROBIOTIC BACTERIUM
ENTEROCOCCUS FAECIUM M-74
A. Belicova¤, L. Kriz›kova¤, J. Dobias, L. Ebringer, J. Kraj-
c›ovic›
Institute of Cell Biology, Comenius University, Odbora¤rske
na¤m. 5, 811 07 Bratislava, Slovak Republic
Live and killed cells of probiotic bacterium Enterococcus
faecium M-74 grown in the presence and absence of so-
dium selenite pentahydrate (Se) as well as the MRS media
with selenium (+Se) and MRS media without selenium (-
FEMSLE Congress 2-6-03
Se) extracts and the diethyl ether extracts from unfer-
mented milk and milk fermented by E. faecium M-74
were prepared and their possible protective e¡ects on the
mutagenicity of selected mutagens in Salmonella and Eu-
glena gracilis assays were studied. MRS media extract
(+Se) after cultivation of E. faecium M-74 showed a sig-
ni¢cant higher antimutagenic activity in reducing genotox-
icity of o£oxacin, N-methyl-N’-nitro-N-nitrosoguanidine
(MNNG) and 5-nitro-2-furyl acrylic acid (NFA) than
MRS (-Se) extract in Salmonella Typhimurium TA98,
TA100 and TA102. The live cells of the probiotic strain
M-74 showed higher antimutagenic activity in inhibiting
the mutagens [CM1]than the killed bacterial cells. How-
ever, the live bacterial cells grown in the presence of Se
showed signi¢cantly higher antimutagenic activity. The di-
ethyl ether extracts isolated from unfermented milk and
milk fermented by E. faecium M-74 exhibited a signi¢cant
dose-dependent protective e¡ect against MNNG-, nitro-
vin- (NIT), NFA- and ultraviolet (UV) irradiation-induced
mutagenicity on the S. Typhimurium TA97, TA100 and E.
gracilis. Overall, the fermented milk extract was the most
active against UV irradiation, less active against NIT and
MNNG, and the least active against NFA on bacteria.
The highest antibleaching e¡ects were observed against
MNNG on E. gracilis. The di¡erences between antimuta-
genic e¡ects from fermented and unfermented milk ex-
tracts were statistically signi¢cant.
P1^6
IMPORTANCE OF NUTRITION CONTROL OF SAC-
CHAROMYCES CEREVISIAE IN WINEMAKING
S. Belviso(1), L. Bardi(2), A. B. Bartolini(3), M. Marzo-
na(1)
(1) Dept. Applied General and Organic Chemistry, Univer-
sity of Turin, Via Pietro Giuria 7, 10125 Turin, Italy ; (2)
Plant Nutrition Experimental Institute, Via Pianezza 115,
10151 Turin, Italy ; (3) INTEC S.r.l., Via Monti Berici 4,
S.Giovanni Lupatoto (Verone), Italy
It is well known that sluggish and stuck fermentations are
very important problems in winemaking. To prevent them
correct yeast nutrition gestion and careful biological pro-
cesses control during fermentation are required. Among
biological processes linked to alcoholic fermentation, lipid
metabolism is little studied. Sometimes happens that grape
must is a low lipid medium and, as a consequence, during
fermentation lipid biosynthesis is activated. Since anaero-
biosis condition is present, UFA and sterol biosynthesis
which are considered among lipids ‘‘surviving factors’’ for
Saccharomyces cerevisiae is not possible. The lack of these
components can cause the arrest of cell multiplication and
then of sugar consumption resulting in stuck fermentation.
In our laboratory we carried out fermentations without
 1st FEMS Congress / Posters 103^505
any nutritional apport, fermentations where a lipid source
in form of inactivated yeast was added as nutritional ap-
port (Saccharomyces cerevisiae can include fatty acids and
sterol from the growth medium in cell membranes), and
fermentations where de¢ned volumes of oxygen were
added (UFA and sterol biosynthesis need molecular oxy-
gen). In the two last series of fermentations more additions
were carried out at established values of sugar concentra-
tion in growth medium. We checked: growth kinetics,
sugar consumption, yeast cell composition, acetic acid
production as symptom of lipid synthesis stress and nitro-
gen consumption to assure that it was never a limiting
growth factor. Results con¢rmed that it was the lack of
lipid compounds to cause fermentation arrest. Additions
of inactivated yeast and oxygen, in fact, improved fermen-
tation performances. These e¡ects can be very important
for winemakers in order to prevent sluggish and stuck
fermentations.
P1^7
LACTOBACILLUS GASSERI LF221 AND K7 PRE-
VENT LATE BLOWING OF PROBIOTIC CHEESE
B. Perko, B. Bogovic› Matijas›ic¤, R. Marins›ek-Logar and I.
Rogelj
University of Ljubljana, Biotechnical Faculty, Chair of
Dairy Science, Groblje 3, 1230 Domz›ale, Slovenia
Among di¡erent media which have been tested as carriers
for probiotic bacteria, cheese has appeared as an attractive
one. Human isolates Lactobacillus gasseri K7 and LF221
produce bacteriocins which inhibit several Clostridium ty-
robutyricum strains, therefore we tested their ability to
prevent late blowing of cheese used as their carrier. A
mixture of C. tyrobutyricum 1551 and 1559 spores
(2.3
103/ml) or a silage juice (10 spores/ml) and derivatives
of L. gasseri LF221 and K7 strains resistant to rifampicin
(107 cfu/ml) were added to cheese milk in di¡erent combi-
nations. Cheese samples were aseptically collected every
week, and analysed for n-butyric acid, cfu/g of lactobacilli
(Rogosa), clostridia spores (RCM), LF221 and K7 strains
(Rogosa with 250 Wg/ml rifampicin). During ripening the
level of added probiotics in general remained the same or
was slightly increased (¢nal cfu/g was 3.5
108 ^ 1,1
109).
While in cheeses without probiotics, the number of non-
starter lactobacilli increased up to 6.5
106 ^ 2.5
107 cfu/g,
LF221 and K7 strains prevailed in cheeses where added.
The identity of the colonies grown on agar with rifampicin
was con¢rmed by RAPD analysis. The visual inspection
and the values of n-butyric acid content in cheese with
added clostridia spores (849 mg/kg in cheese with silage
juice and 2185 mg/kg in cheese with spores from pure
cultures) clearly showed the late-blowing of cheeses with-
out probiotics. On the other hand, the n-butyric acid con-
FEMSLE Congress 2-6-03
105
tent in the cheeses with added probiotic strains, was ob-
viously lower from those of cheeses without probiotics.
P1^8
STUDY OF ARGININE DEGRADATION BY LACTIC
ACID BACTERIA IN RELATION WITH POSSIBLE
APPEARANCE OF ETHYL CARBAMATE IN WINES
A. Bordons, J. Gil, I. Araque, S. Romero, M. C. Masque¤,
C. Reguant and R. Carrete¤
Departament de Bioqu|¤mica i Biotecnologia, CeRTA, Uni-
versitat Rovira i Virgili, Tarragona, Catalonia, Spain
Ethyl carbamate (EC) is a potential carcinogenic com-
pound sometimes found in wines. EC can appear by re-
action of ethanol, at the acid pH of wine, with urea pro-
duced by yeasts, or with citrulline or carbamyl phosphate,
both produced by lactic acid bacteria (LAB) from argi-
nine. On the other hand, wine LAB such as Oenococcus
oeni are well known by the malolactic fermentation (MLF)
they carry out. The main positive e¡ects of MLF are the
reduction of wine acidity (conversion of l-malic to l-lactic
acid) and the modi¢cation of £avor properties. Complete
degradation of arginine by LAB occurs via the ADI path-
way, leading to ammonia, ornithine, ATP and CO2, but
several strains do not degrade it completely and can pro-
duce citrulline or carbamyl phosphate. In order to mini-
mize the appearance of EC in wines, we have studied
¢rstly the ability for degrading arginine in a lot of strains
of di¡erent species of LAB wines. We have found di¡erent
responses in Oenococcus depending on the strain, and that
some homofermentative Pediococcus could degrade argi-
nine. Activities of enzymes of the ADI pathway (arginine
deiminase [ADI], ornithine transcarbamylase [OTC] and
carbamate kinase [CK]) have been measured in cells ex-
tracts of di¡erent strains and species. On the other hand,
in order to detect the genes of ADI pathway in strains of
O. oeni, speci¢c PCR primers have been designed for each
gene (ADI, OTC and CK) and have been tested in several
strains.
106 1st FEMS Congress / Posters 103^505
P1^9
RAPID ASSESSMENT OF PHYSIOLOGICAL STATE
OF OENOCOCCUS OENI BY FLOW CYTOMETRY
DURING MALOLACTIC FERMENTATION
M. Bouix, S. Ghorbal, J. L. Tholozan, J. Y. Leveau
Dept of Industrial Microbiology, Ecole Nationale Supe¤r-
ieure des Industries Agricoles et Alimentaires, Massy,
France
We proposed a simple £ow cytometric method to evaluate
the intracellular pH (pHi) and the membrane potential of
the cells of Oenococcus oeni during the malolactic fermen-
tation.
At the begining of the malolactic fermentation, the pHi of
cells was low and equal to the external pH of the medium,
and the membrane potential was weak. During the
growth, the pHi raised to a maximum value of 6 whatever
the external pH (3 or 5), and the membrane potential also
raised. When the malic acid was exhausted of the medium,
the pHi dropped down to the external pH value, and the
cells appeared depolarized. The assessment of these pa-
rameters let to understand the cellular physiology of Oe-
nococcus oeni and then to better get under control the
malolactic fermentation in the wines.
P1^10
THE TECHNOLOGICAL ACCEPTABILITY OF EN-
TEROCOCCI ISOLATED FROM CHEESES
S. Bulajic¤, Z. Mijac›evic¤
Faculty of Veterinary Medicine, University of Belgrade, 11
000 Belgrade, Serbia
Enterococci are part of the commensal microbiota of ani-
mals and humans. In the area of food microbiology they
have been considered as indicators of fecal contamination.
Also, they have been regarded as index microorganisms
for unhygienic food processing. Additionaly, studies on
the micro£ora of traditional cheeses in Mediterranean
contries have clearly indicated that enterococci strains dis-
play diversity enzymatic activities that may have desirable
role in the process of ripening and contribute to typical
taste and £avour. However, their presence in food system
is still a matter of controversy owing to their pathogenic
potential and also potent ability to exchange genetic ma-
terial comprimases the gene pool of antibiotic resistance.
In this study, enterococci strains isolated from cheeses
originated from our local market were subjected to tech-
nological characterisation regarding their proteolytic activ-
ity. Proteolytic pro¢les of investigated strains were exam-
ined by electrophoresis. On the basis of our results we
FEMSLE Congress 2-6-03
were conducted the selection of the most appropriate cul-
tures to be used for the enchancment of the organoleptic
properties of cheese.
P1^11
INFLUENCE OF YEAST ON POLYPHENOL COM-
POSITION OF WINE
A. Caridi(1), A. Cufari(1), R. Lovino(2), R. Palumbo(3)
and I. Tedesco(3)
(1) Dipartimento di Scienze e Tecnologie Agro-Forestali e
Ambientali, Reggio Calabria University, Piazza San Fran-
cesco 7, I-89061 Gallina (RC), Italy; (2) Istituto Speri-
mentale per l’Enologia, Sezione Operativa di Barletta, Via
Vittorio Veneto 26, I-70051 Barletta (BA), Italy ; (3) Is-
tituto di Scienze dell’Alimentazione, Consiglio Nazionale
delle Ricerche, Via Roma 52A/C, I-83100 Avellino (AV),
Italy
In red wine production, the type and quantity of polyphe-
nols play a major role in wine quality. Anthocyanins, £a-
vonols, catechins and other £avonoids contribute to the
di¡erent characteristics of wine, particularly color and as-
tringency; in addition, recently it has been shown that
they possess a wide range of antioxidant and pharmaco-
logical e¡ects. The objective of this research was to exam-
ine the in£uence of yeast used for winemaking on the type
and quantity of phenolic compounds, elucidating possible
relationships with the antioxidant capacity of wine. Two
strains of Saccharomyces, previously selected for enology
and for their di¡erent interaction with grape polyphenols,
were employed. A sample of Gaglioppo must from Cala-
brian black grapes was utilized. This variety was employed
because it has limited anthocyan content so it requires
careful handling to protect the phenolic compounds.
Winemakings were carried out in triplicate using stainless
steel vessels of 600 l, containing about 400 l of must with
grape skins and seeds. Twenty-seven physicochemical and
polyphenolic parameters were determined in the red wines
produced. Among the polyphenolic parameters, very sig-
ni¢cant (p 6 0.01) di¡erences were observed for color in-
tensity, total polyphenols, and non-anthocyanic £avo-
noids. Moreover, signi¢cant (p 6 0.05) di¡erences were
observed for OD520 and monomeric anthocyans. This re-
search elucidates for the ¢rst time how yeasts interact with
speci¢c polyphenols, so varying the wine concentration of
these compounds. This behavior modi¢es signi¢cantly the
physicochemical and antioxidant characteristics of wines
and, probably, also the sensorial and volatile constituents.
 1st FEMS Congress / Posters 103^505
P1^12
COMPARATIVE EFFECTS OF FEEDING CONTAIN-
ING FLAVOMYCIN, BIOTEKSIN-L AND DRY
YEAST (SACCHAROMYCES CEREVISIAE ) ON
BROILER PERFORMANCE
M. Denli(1), K. C
 elik(2), F. Okan(1)
(1) C ukurova University Animal Sci. Dept., 01330 Adana,
Turkey ; (2) C  anakkale Onsekiz Mart University, Animal
Sci. Dept., C
 anakkale 17100, Turkey
A 6-week study was conducted to determine the e¡ects of
feeding diets containing commercial probiotic (Biyoteksin-
L), antibiotic (Flavomycin) and dry yeast (Saccharomyces
cerevisiae) upon would a¡ect performance ,abdominal fat
weight, carcass weight and carcass yield of broiler chicks.
Four dietary treatments were randomly assigned to four
groups of chicks. A control or containing 0,15% commer-
cial probiotic (bioteksin-L), 0,15% antibiotic,(£avomycin)
and 0,3 % dry yeast. A signi¢cant increase in body weight
gain, feed conversion rate and carcass weight of birds was
observed in birds fed £avomycin group and dry yeast
group in end of 6-wk period compaired to the control
(P 6 0.05).This increase was partly accounted for by in-
creased feed intake. The results obtained in the experiment
showed that Biyoteksin-L and Saccharomyces cerevisiae
plus supplementation to diets tended to decrease, abdomi-
nal fat weight and abdominal fat percentage
(P 6 0.5).while having no signi¢cant e¡ects on carcass
yield.
P1^13
PURIFICATION AND PARTIAL CHARACTERIZA-
TION OF A NOVEL BACTERIOCIN PRODUCED
BY A THERMOPHILIC ENDOSPORE-FORMING
STRAIN GEOBACILLUS STEAROTHERMOPHILUS
32A
K. Pokusajeva, M. Stuknyte, N. Kuisiene, D. Chitavichius
Department of Microbiology and Plant Physiology, Vilnius
University, Chiurlionio 21/27, Vilnius, LT-2009, Lithuania
Aerobic, endospore-forming thermophilic strain Geobacil-
lus stearothermophilus 32A was identi¢ed as a two bacte-
riocins producer with a bactericidal activity against Geo-
bacillus subterraneus DSM 13552, G. uzenensis DSM
13551, G. thermocatenulatus DSM 730, and G. thermoleo-
vorans DSM 5366. These bacteriocins are produced during
log-phase growth and are inhibitory to active growing
cells. The antimicrobial activities of these substances di¡er.
Twofold dillution tests showed that the activity of one of
the bacteriocins appeared later after another one disap-
FEMSLE Congress 2-6-03
107
peared. The antimicrobial activity of the bacteriocins on
the sensitive indicatory cells disappeared completely by
treatment with proteinase K, which indicates its proteina-
ceous nature. Bactericidal activity was kept during storage
at 4‡C and was remarkably stable in the wide range of pH.
In SDS-PAGE analysis, only two peptide bands displayed
antimicrobial activity against thermophilic indicatory
strain 35C. The inhibitory peptides had a molecular weight
of approximately 12 and 6.8 kDa. Results of this study
suggest that these antimicrobial substances produced by
the wild type strain of Geobacillus stearothermophilus
32A may be the new bacteriocins.
P1^14
PHENOTYPIC, GENOTYPIC AND TECHNOLOGI-
CAL CHARACTERISTICS OF LACTOCOCCI ISO-
LATED FROM TRADITIONAL FIORE SARDO
CHEESE
S. Cosentino, M. B. Pisano, C. Piras and A. Corda
Department of Experimental Biology, Section of Hygiene,
University of Cagliari, S.S. 554, Km. 4,500, 09042 Monser-
rato (CA), Italy
The evolution and composition of dairy micro£ora is of
particular interest for PDO cheeses such as Fiore Sardo,
that is manufactured with raw ewe’s milk without the
addition of any natural or starter cultures. In this case,
the ripening process relies entirely on the indigenous £ora
present in the milk and in the dairy environment. Studies
carried out on the micro£ora of Fiore Sardo have shown
lactococci and enterococci to be the dominant bacterial
population. This paper reports the phenotypic, genotypic
and technological characterization of lactococcal strains
isolated from 14 batches of artisanal Fiore Sardo cheese,
in order to assess the biodiversity within this wild micro-
bial population. Lactococci were isolated from M17 agar
plates incubated at 30‡C and were initially identi¢ed by
morphological, physiological and biochemical tests. This
identi¢cation was con¢rmed by a polymerase chain reac-
tion analysis with Lactococcus genus specii¢c primers LC1
and LC2. Randomly ampli¢ed polymorphic (RAPD)
DNA technique was used for the genetic typing of the
isolates. A total of 80 isolates were identi¢ed as Lactococ-
cus lactis. Most strains were able to hydrolyse casein, none
produced lipolytic reactions on tributyrin agar and several
were good acid-producers. The RAPD patterns from 50
representative strains were analysed by UPGMA dendo-
grams. At a similarity level of 50% two main clusters with
several subclusters were distinguished, showing a high het-
erogeneity in the biotypes. The results of our survey con-
¢rm that wild bacterial population should be preserved to
protect the traditional raw milk cheeses and to select new
starter strains for the dairy industry.
108 1st FEMS Congress / Posters 103^505
This work was supported by a grant from MURST, Plan
‘‘Agroalimentary products: dairy products’’, Cluster 08B,
Project n.7.
P1^15
INFLUENCE OF THREE BOTRYTICIDES ON WINE
FERMENTATION DYNAMICS AND YEAST POPU-
LATION BIODIVERSITY
N us›(1), A. Amalietti(2), N. C
F. C N adez›(2), A. Gregorc›ic›(3)
and P. Raspor(2)
(1) University of Ljubljana, Biotechnical faculty, Agronomy
Department, Chair of Viticulture, Jamnikarjeva 101, 1000
Ljubljana, Slovenia; (2) University of Ljubljana, Biotech-
nical faculty, Food Science and Technology Department,
Chair of Biotechnology, Jamnikarjeva 101, 1000 Ljubljana,
Slovenia; (3) Agriculture Institute of Slovenia, Hacquetova
17, 1000 Ljubljana, Slovenia
Wine fermentation depends on many viticulture and eno-
logical practices. One of them, not yet clearly understood,
is a usage of botrtryticides in vineyard and in£uence of
their residues on fermentation dynamics and yeast popu-
lation biodiversity. In our research, three botryticides of
di¡erent chemical groups were used: iprodione, pyrime-
thanil and cyprodinil plus £udioxonil. The grape of the
cultivar Rebula (Vitis vinifera L.) was sprayed at the clo-
sure of the berries and at the beginning of the grape ripen-
ing. At the harvest, the botryticides residues on the grape
were below prescribed tolerance measured with a combi-
nation of gas chromatography and mass spectroscopy. To
preserve indigenous microbial populations grape process-
ing was performed aseptically in three parallels for each
treatment. No sul¢te and wine yeasts were added. The
must composition during the fermentations was analyzed
by HPLC. The yeast population dynamics was followed
by colony counting, colony morphotyping and by molec-
ular methods, electrophoretic karyotyping and PCR-
RFLP of the rDNA. There was a signi¢cant di¡erence
in fermentation duration between the treatments: 36
days for the control and iprodione fermentations, 50
days for the cyprodinil plus £udioxonil fermentations
and 68 days for pyrimethanil fermentations. The non-Sac-
charomyces yeasts persisted during the fermentations, with
Candida stellata and, to lesser extent, Hanseniaspora uva-
rum remaining long after the fermentations were either
dominated or not by Saccharomyces cerevisiae.
This research was supported by the Ministry of Education
Science and Sport and by the Ministry of Agriculture
Forestry and Nutrition (Project no. V4-0591-01).
FEMSLE Congress 2-6-03
P1^16
CULTIVATION OF KOMBUCHA ON SWEETENED
ECHINACEA TEA
D. Cvetkovic, J. Canadanovic-Brunet, S. Markov
Faculty of Technology, University of Novi Sad, Boulevar
Cara Lazara 1, 21 000 Novi Sad, Yugoslavia
Kombucha is a beverage with special therapeutic proper-
ties produced by metabolic activity of yeasts (Schizosac-
charomyces pombe, Saccharomyces ludwigii, Saccharomy-
ces cerevisiae...) and acetic acid bacteria (Acetobacter
xylinum, Acetobacter aceti, Gluconobacter oxydans...) in
cultivation medium (sweetened black tea). The ability of
black tea, as a traditional source of nitrogen compounds
in cultivation medium, to be replaced with echinacea tea
(radix and herba) was investigated. It is well known that
Echinacea spp. have proved pharmacological properties.
The use of echinacea tea in preparation of kombucha
would produce beverage with increased therapeutic prop-
erties in comparing to traditional beverage produced with
black tea. Fermentation process of sweetened echinacea
tea was observed during ¢ve days of incubation by deter-
mination of total account of yeast cells and acetic acid
bacteria, as well as chemical parameters. The investiga-
tions showed that fermentation of sweetened echinacea
tea was very e⁄cient. Also, higher acidity was gained in
comparing with traditional kombucha for the same incu-
bation period. Antioxidative capacity of black and echina-
cea tea and their kombucha beverages was investigated by
electron spin resonance (ESR) spectroscopy. Results
showed that echinacea tea (especially tea prepared from
herba of the plant) have signi¢cantlly higher antioxiadtive
properties in model system then black tea. On the other
hand, kombucha beverages, produced by fermentation of
sweetened black and echinacea tea, showed an aditionally
higher antioxidative capacity then the black and echinacea
tea. This e¡ect was caused by compounds produced during
kombucha culture fermentation and/or their synergestic
e¡ect with extracted compounds from teas.
 1st FEMS Congress / Posters 103^505
P1^17
SOME ASPECTS ON THE ACTION AND APPLICA-
TION OF BACTERIOCINS PRODUCED BY LACTO-
BACILLUS STRAINS ISOLATED FROM FERMENT-
ING TABLE-OLIVES
A. Delgado(1), D. Brito(2), M. Caeiro(3) and C.
Peres(2)
(1) ITQB, Oeiras, Portugal; (2) INIAP/EAN, Oeiras,
Portugal; (3) Instituto Piaget, Almada, Portugal
Two bacteriocin producers (Lactobacillus plantarum
LB17.2b and Lactobacillus pentosus LBB96) were previ-
ously isolated from fermenting olive brines, of distinct
technological procedures. A brief study on their bacterio-
cins revealed that the bacteriocin from L. pentosus LBB96
is apparently activated by heat (65 ‡C to 100 ‡C). It also
seems to have a broad activity spectrum, including Entero-
coccus spp. strains that are multiresistant to antibiotics.
The in£uence of technologically relevant factors from
brine, such as oleuropein and di¡erent NaCl concentra-
tions, was evaluated on growth, on bacteriocin production
and on bacteriocin bioavailability. Oleuropein (0.4%)
seemed to have a limited in£uence on growth or on bac-
teriocin production. NaCl enhanced the activity of both
bacteriocins but a fall in bacteriocin production, by L.
plantarum LB17.2b, was registered for 2% NaCl whereas
the growth was not a¡ected. Bacteriocin production by L.
pentosus LBB96 seemed to be stimulated by 2 to 4% NaCl.
This last strain showed to be more salt-tolerant being able
to grow and to produce bacteriocin in MRS broth with
8% NaCl and 0.4% oleuropein. This study supports the
hypothesis that LAB bacteriocins can be active in complex
food systems and some of them may also have health-care
applications.
P1^18
ISOLATION, IDENTIFICATION AND CHARACTER-
ISATION OF GLYCEROL-DEGRADING LACTIC
ACID BACTERIA FROM SOUTH AFRICAN RED
WINES
S. J. Krieling, I. S. Pretorius and M. du Toit
Institute for Wine Biotechnology, Department of Viticulture
and Oenology, Stellenbosch University, Stellenbosch ZA-
7600, South Africa
Two-hundred-and-forty lactic acid bacteria (LAB) were
isolated from Pinotage, Merlot and Cabernet Sauvignon
grapes and wine samples obtained from ¢ve wineries. In
2001, the LAB population on Cabernet Sauvignon grapes
ranged from 102 to 104 cfu/ml. In both 2001 and 2002, the
FEMSLE Congress 2-6-03
109
LAB population on Pinotage and Merlot grapes ranged
from 102 to 103 cfu/ml. The Cabernet Sauvignon grapes
carried a LAB population of between 103 and 104 cfu/ml
in 2002. The number of LAB in Cabernet Sauvignon wine
after alcoholic fermentation (AF) ranged from 103 to 105
cfu/ml in 2001 and from 102 to 105 cfu/ml in 2002. After
AF, Pinotage and Merlot wines carried a LAB population
ranging from 102 to 104 cfu/ml in 2001. In 2002, the num-
ber of LAB in Merlot and Pinotage ranged from 102 to
105 cfu/ml. Twenty-eight strains were identi¢ed as Oeno-
coccus oeni, four as Leuconostoc mesenteroides, ¢fteen
strains were identi¢ed as Lactobacillus brevis, another ¢f-
teen strains as Lactobacillus hilgardii by means of species-
speci¢c primers. Ninety-eight strains were identi¢ed as
Lactobacillus plantarum, three as Lactobacillus paraplanta-
rum and 12 as Lactobacillus pentosus by means of a multi-
plex PCR assay using species-speci¢c primers. Primers spe-
ci¢c for Lactobacillus paracasei were used to identify 28
strains. Two strains of the obligately homofermentative
group were identi¢ed as Pediococcus acidilactici and 35
strains were taken as Pediococcus spp., based on cell mor-
phology. Twenty-six of the isolated strains were found to
contain the glycerol dehydratase gene sequence. Prelimi-
nary results suggest that the GD-possessing strains exhibit
an inhibitory activity against Gram-positive and Gram-
negative bacteria, and that this antimicrobial activity is
similar to that of reuterin, which is produced by Lb. reu-
teri.
P1^19
SCREENING, ISOLATION AND CHARACTERISA-
TION OF ANTIMICROBIAL / ANTIFUNGAL PEP-
TIDES PRODUCED BY LACTIC ACID BACTERIA
ISOLATED FROM WINE
J. Morgan, M. A. Vivier, I. S. Pretorius and M. du Toit
Institute for Wine Biotechnology, Department of Viticulture
and Oenology, Stellenbosch University, Stellenbosch ZA-
7600, South Africa
A total of 170 lactic acid bacteria (LAB) were isolated
from Pinotage, Merlot and Cabernet Sauvignon cultivars
in the Western Cape region. Samples were collected from
grapes and during di¡erent stages of the winemaking pro-
cess and screened for antimicrobial activity. Of the total
isolates, 25 showed activity predominantly towards Lacto-
bacillus- and Pediococcus-sensitive strains. Two isolates
were selected for further characterisation due to their
stability in bacteriocin production and constant high ac-
tivity against the indicator organism, Lactobacillus planta-
rum LMG 13556. The two bacteriocin-producing strains
were identi¢ed as Lactobacillus paracasei #77 and Lacto-
bacillus brevis #81.1. The bacteriocins were inactivated by
proteinase K, K-chymotrypsin and lysozyme, but not by
110 1st FEMS Congress / Posters 103^505
catalase. The bacteriocins were heat stable and displayed
the highest activity from pH 3 to pH 7. The selected iso-
lates were found both to be bacteriostatic in their mode of
action. The highest production of bacteriocin occurred
after approximately 16 h of growth at 30‡C. The molec-
ular weight of the bacteriocins, as determined by tricine
SDS-PAGE, was between 6.5 and 14.0 kDa. The LAB
isolates were also tested for antifungal activity against Bo-
trytis cinerea. The most predominant activity was seen
after 24 h of incubation with germinating spores. This
study shows that LAB found in South African vineyards
and in the winemaking process produce antimicrobial sub-
stances that have the potential to inhibit or out-compete
other non-producing or sensitive LAB naturally present in
the wine and also to in£uence the proliferation of fungal
spores.
P1^20
CHARACTERISATION OF WINE-ISOLATED ACE-
TIC ACID BACTERIA FROM SOUTH AFRICAN
RED WINES
A. Oelofse, M. G. Lambrechts, I. S. Pretorius and M. du
Toit
Institute for Wine Biotechnology, Department of Viticulture
and Oenology, Stellenbosch University, Stellenbosch ZA-
7600, South Africa
Acetic acid bacteria (AAB) isolated from South African
wine were screened for the production of antimicrobial
peptides, production of extracellular enzymes and charac-
terised by their ability to cause volatile acidity (VA). The
supernatant of two isolates exhibited bioactivity against
other strains of AAB and other Gram-negative microor-
ganisms at a pH of 6.5 with the agar di¡usion method. By
means of biochemical tests and PCR-RFLP analysis of the
16S rDNA, one of the producer strains was identi¢ed as
Gluconobacter frateurii. This revealed the occurrence of an
AAB species that has not yet been found in the winemak-
ing environment. The antibacterial compound was deter-
mined to be a proteinaceous substance, since it lost its
activity after proteolytic enzyme treatment. Preliminary
characterisation indicated that the antimicrobial substance
was stable in a pH range from 3.0 to 8.0 and that it was
temperature sensitive, with the activity diminishing slightly
with the temperature increase from 4‡C to 65‡C and there-
after, all activity was lost. The screening of AAB for extra-
cellular enzymes revealed the production of pectinases,
proteases, xylanases, L-glycosisdases, cellulases and amy-
lases. The range of enzymes produced varied with di¡erent
isolates of the same species. This study also revealed the
ability of some of these aerobic microorganisms to pro-
duce large quantities of acetic acid ( s 3.5 g/l) under an-
aerobic conditions. It was evident that AAB could easily
FEMSLE Congress 2-6-03
survive and grow during the strenuous conditions of fer-
mentation and consequently contribute to the wine’s vol-
atile acidity. Undesirable levels of volatile acidity a¡ect
wine quality negatively and serve as indication of micro-
bial spoilage, mainly by AAB.
P1^21
FISH PROTEIN HYDROLYSATES AS NITROGEN
SOURCE FOR MICROBIAL GROWTH, PROTEASE
AND LIPASE PRODUCTION
N. Souissi(1), L. Dufosse¤(2), R. Ghorbel(1), Y. Triki-El-
louz(1), F. Guerard(2) and M. Nasri(1)
(1) Unite¤ de Technologie Enzymatique et de Microbiologie,
Ecole Nationale d’Inge¤nieurs de Sfax, B.P. ‘‘W’’ 3038 Sfax,
Tunisie ; (2) Laboratoire de Microbiologie Applique¤e, LU-
MAQ, E.A. 2651, I.U.P. Innovation en Industries Alimen-
taires, Creac’h Gwen F-29000 Quimper, France
Fish protein hydrolysates from low cost ¢sh species (Sar-
dinella aurita) were prepared and tested as nitrogen source
for microbial growth and lipase production by Rhizopus
oryzae and Staphylococcus simulans. The strains tested ex-
hibited a greater lipase production than that obtained with
casein peptone. Higher lipase production was achieved
only when cells were grown in media containing defatted
meat ¢sh protein hydrolysates, indicating the presence in
lipid fraction of some constituents, which may repress li-
pase synthesis. Protease production by Bacillus subtilis and
Bacillus licheniformis was assayed in media containing
only ¢sh substrates at a concentration of 10 g/L and com-
pared with control media. The best results were obtained
with combined heads and viscera £ours indicating that it
was not necessary to add ingredients (such as salts, glucose
and yeast extract) to ¢sh medium. Furthermore, it seems
that heads and viscera £ours contain bioactive molecules
that stimulated enzyme synthesis. The high lipase and pro-
tease activities obtained with substrates from cheap ¢sh
species clearly indicated that these substrates could be
used in industrial fermentation processes.
 1st FEMS Congress / Posters 103^505
P1^22
SEMI-INDUSTRIAL AND INDUSTRIAL BAKER’S
YEAST STRAINS IMPROVED FOR USE IN FROZEN
DOUGHS
F. Dumortier(1), A. Teunissen(1a), M-F. Gorwa(3b), J.
Bauer(3c), A. Tanghe(1), A. Lo|«ez(3), P. Smet(4), P.
Van Dijck(1,2) and J. M. Thevelein(1)
(1) Laboratorium voor Moleculaire Celbiologie and (2)
Vlaams Interuniversitair Instituut voor Biotechnologie-
VIB, Institute of Botany and Microbiology, Katholieke Uni-
versiteit Leuven, B-3001 Leuven-Heverlee, Flanders, Bel-
gium; (3) Lesa¡re De¤veloppement, F-59706 Marcq-en-
BarRul, Cedex, France; (4) Algist Bruggeman N.V., B-
9000 Gent, Belgium. Present addresses : aDepartment of
Pharmacology, ErasmusMC,3000 DR Rotterdam, The
Netherlands; bDepartment of Applied Microbiology, Lund
University/Lund Institute of Technology, S-22100 Lund,
Sweden; cBASF-LYNX Bioscience AG, D-69120 Heidel-
berg, Germany.
The possibility to store doughs in the freezer permits the
separation of the processes of dough production and bak-
ing. However, routine production and storage of frozen
doughs are still problematic. Although commercial baker’s
yeast is highly resistant to environmental stress conditions,
it rapidly looses stress resistance during dough preparation
due to initiation of fermentation. As a result, the yeast
signi¢cantly looses gassing power during storage of frozen
doughs. We obtained freeze-tolerant mutants of semi-in-
dustrial (i.e. strains used in crossing schemes resulting in
improved industrial strains) and industrial strains by se-
lection of UV-mutagenized strains for increased stress re-
sistance. In the ¢rst instance, strains were tested for freeze
tolerance by suspending the cells in water and freezing
them at -20‡C for 12 days. Several improved mutant
strains were obtained, but when tested in pilot scale, no
improved freeze tolerance was observed. Therefore it was
decided to carry out the selection in conditions that were
more close to reality: selection with frozen doughs. In this
way the industrial strain S47 gave rise to two mutant
strains (AT25 and AT28) which maintained a better
dough-rising capacity during frozen storage of the dough.
Further investigation of AT25, the most promising of
both, showed that other industrially important properties
such as yield, growth rate, nitrogen assimilation and phos-
phorus content were very similar to those in the original
S47 strain.
Teunissen et al. (2002). Isolation and characterization of a
freeze-tolerant diploid derivative of an industrial baker’s
yeast strain and its use in frozen doughs. Appl. Environ.
Microbiol. 68: 4780-4787.
FEMSLE Congress 2-6-03
111
P1^23
POLYMORPHISM OF LACZ AND LACS GENES OF
STREPTOCOCCUS THERMOPHILUS FROM DAIRY
ENVIRONMENT AS REVEALED BY SEQUENCE
ANALYSIS
D. Ercolini, V. Fusco, G. Blaiotta and S. Coppola
Dipartimento di Scienza degli Alimenti, Universita' Federico
II di Napoli, via Universita' 100, 80055 Portici (NA), Italy
Streptococcus thermophilus is widely used in food fermen-
tation. Lactose, the principal energy source used by Strep-
tococcus thermophilus for growth in milk, is transported
into the cell by a permease (LacS) and then hydrolysed
within the cell into glucose and galactose by L-galactosi-
dase. The aim of this study was to investigate the sequence
heterogeneity of lacZ and lacS genes of Streptococcus ther-
mophilus in order to di¡erentiate strains arising from dif-
ferent dairy environments. Strains of Streptococcus ther-
mophilus isolated from a variety of dairy products were
initially characterised at strain level by RAPD-PCR anal-
ysis. Primers were designed from the published sequences
of the lacZ and lacS genes and used in PCR experiments
in order to obtain fragments of the genes. The primers
were shown to work for all the Streptococcus thermophilus
strains used. The amplicons from lacZ and lacS genes
were digested with restriction enzymes and di¡erences
were detected between the strains analysed suggesting the
nucleotide sequence of the genes to be di¡erent. Moreover,
direct sequencing of the PCR ampli¢ed products was per-
formed. Comparison of the sequences obtained from the
wild as well as reference strains showed several di¡erences
in nucleotides distributed throughout the genes. Several
base changes were also found in lacZ genes of Streptococ-
cus thermophilus by other authors. The corresponding pro-
tein sequence was also virtually determined and the di¡er-
ences in aminoacid composition of the enzymes evaluated.
Furthermore, the possibility to design strain-speci¢c mo-
lecular markers from the polymorphic sequences of the lac
genes is currently under evaluation.
P1^24
APPLICATION OF MIXED YEAST CULTURES IN
BREWING
G. Farkas, J. Rezessy-Szabo, A. Hoschke
Department of Brewing and Distilling, Szent Istvan Univer-
sity, 1118 Budapest, Menesi ut 45, Hungary
Production of low-alcoholic or non-alcoholic beers is
based on two methods : by removal of alcohol, or by re-
striction of alcohol formation. In the latter method the
112 1st FEMS Congress / Posters 103^505
application of non-brewer’s yeast is one of the possibil-
ities. With use of a yeast strain that does not utilize mal-
tose, the ethanol content of beer will be low, but will also
taste sweet. In our work four non-brewer’s yeast strains
were tested. Saccharomycodes ludwigii, Saccharomyces ex-
iguus, Saccharomyces delbrueckii and Torulaspora del-
brueckii are yeast strains which do not ferment maltose.
Beside them a well characterized brewer’s yeast, Saccharo-
myces cerevisiae WS34/70 was used. Mono- and mixed-
culture fermentations in wort were carried out. In the lat-
ter ones ratio of brewer’s and non-brewer’s yeast were 1:1,
1:2, 1:5 and 1:10. Products were analyzed by beer ana-
lyzer and gas chromatography. Cell concentrations were
determined by Buerker chamber. In mixed-culture fermen-
tations Saccharomyces cerevisiae dominated, regardless of
initial cell ratios. Beer analysis showed that degree of at-
tenuation and ethanol concentration was low. Some
mixed-culture samples gave favorably reduced alcohol
content values. Non-brewer’s yeast mono-culture samples
provided values that do not match the £avor pro¢le of a
beer: low concentration of esters was experienced. Results
of mixed-culture fermentation gave values that are within
acceptable concentration limits of the £avor active com-
pounds. Although the results of the analysis suggests that
the use of non-brewer’s yeast in mono-culture fermenta-
tion would not provide a fully acceptable product, appli-
cation of mixed-cultures that includes a brewer’s yeast
strains with good fermentation characteristics can com-
pensate for insu⁄ciencies.
P1^25
ANTIFUNGAL ACTIVITY OF MONOTERPENES
AGAINST TROPICAL FRUITS FUNGI
M. Pupo(1), E. S. S. Alves(1), R. B. Santos(2), J. A.
Ventura(3) and P. M. B. Fernandes(1)
(1) Dept. de Cie“ncias Fisiolo¤gicas and (2) Dept. de Qu|¤-
mica, Universidade Federal do Espirito Santo ^ UFES ; (3)
Instituto Capixaba de Pesquisa, Assiste“ncia Te¤cnica e Ex-
tensa‹o Rural ^ INCAPER
Papaya, Banana and Pineapple are the most consumed
tropical fruits in the world, being Brazil one of the main
worldwide producers. Diseases caused by fungus that in-
fects plants lead to losses in the production and demand
the use of chemical fungicides. This work aimed to eval-
uate the e⁄ciency of ¢ve monoterpenes isolated from
plant essential oils in the inhibition of the mycelial growth
and conidia germination of the three pathogens Colleto-
trichum gloeosporioides, Colletotrichum musae, Fusarium
subglutinans fsp ananas in vitro. The inhibitory activity
of the monoterpenes compounds in the concentration
ranging from 20 to 100 % was tested against the fungi.
The essential oils constituents’ citral, citronellal and L-
FEMSLE Congress 2-6-03
carvone showed a potent fungicidal activity, but little or
no activity was observed from the treatments with K-
pinene and isopulegol. Based in the results with the mono-
terpenes, plants that present greater amounts of citral and
citronellal had been selected and their oily extracts have
been tested. The oil extracts tested were from the plants:
Cymbopogon citratus, Cymbopogon nardus, Eucalyptus cit-
rodora, Eucalyptus globosus and Lippia alba. Those results
may be an indication to the use of those essential oils as
natural pesticides in the control of fruit diseases.
Financial support: finep, cnpq, funcitec
P1^26
EVOLUTION OF NATURAL MYCOBIOTA GROW-
ING ON THE SURFACE OF THE SPANISH CURED
MEAT PRODUCT CECINA DURING ITS RIPENING
PROCESS
F. Fierro, F. Laich and J. F. Martin
Institute of Biotechnology of Leon (INBIOTEC), Avenida
Real 1, 24006-Leon, Spain
Cecina is a cured meat product typical of Leo¤n (North-
West of Spain) that is elaborated from meat pieces of
bovine animals. Throughout the ripening process, which
takes approximately one year and involves a smoking
stage, di¡erent microorganisms grow on the surface of
the piece. The presence of yeasts, bacteria from the family
Micrococcaceae and ¢lamentous fungi is considered as a
signal of a good curing process. The fungi participate in
the ripening process and contribute to the £avour and
taste of the ¢nal product. We have carried out a quanti-
tative study of the fungal species growing on the surface of
cecina pieces during the ripening process, and the main
conclusions are reported here. Isolates were identi¢ed by
morphological characters and RAPD analysis. Species of
the genus Penicillium are preponderant throughout the
process, especially in the ¢rst stages, being the most fre-
quently isolated : P. solitum, P. verrucosum, P. implicatum,
P. nalgiovense and P. chrysogenum and P. commune. After
the smoking stage, species more tolerant to high salt con-
centrations and lower water activity appear, among them
some species of the genus Eurotium. Together with species
considered bene¢cial for the product, as P. nalgiovense,
commonly used as surface starter for fermented meats
(salamis), other species were isolated which are highly my-
cotoxigenic, like P. verrucosum. The convenience to use
methods of control of the natural mycobiota during the
curing process is discussed.
 1st FEMS Congress / Posters 103^505
P1^27
POPULATION DYNAMICS OF ACETIC ACID BAC-
TERIA DURING RED WINE FERMENTATIONS
A. Gonza¤lez, N. Hierro, M. Poblet, N. Roze¤s, A. Mas, J.
M. Guillamo¤n
Departament de Bioqu|¤mica i Biotecnolog|¤a, Unitat d’Eno-
logia del CeRTA, Facultat d’Enologia, Universitat Rovira i
Virgili, Tarragona, Spain
In oenology, acetic acid bacteria (AAB) have received little
attention so far. However, the increase in acetic acid in
wines as a consequence of the AAB growth is a common
problem for wineries. In order to study the AAB popula-
tion dynamics during the wine-making process we have
used two rapid and reliable molecular techniques such as
Enterobacterial Repetitive Intergenic Consensus-PCR
(ERIC-PCR) and Repetitive Extragenic Palindromic-
PCR (REP-PCR), which proved useful for characterising
AAB strains. A total amount of 440 strains originated
from 2001 and 2002 vintages and di¡erent fermentation
conditions (yeast inoculation, SO2) of red grenache grape-
musts were ¢ngerprinted. We detected a high degree of
strain diversity in the ¢rst stage of fermentation that de-
creased throughout the process. However, several strains
and species were dominant in the alcoholic fermentation
phases. Gluconobacter oxydans dominated the fresh must,
while Acetobacter aceti was the only isolated species at the
end of the process. Gluconoacetobacter hansenii and Glu-
conoacetobacter liquefaciens were also isolated in signi¢-
cant numbers at the beginning of fermentation.
P1^28
ANTIMUTAGENICITY OF GREEN TEA ANALYZED
BY USING MICROORGANISMSa¤ TESTS
R. Hlad|¤kova¤(1), I. Ma¤rova¤(1), P. Pta¤c›ek(1), A. Mikul-
cova¤(1), M. Ne›mec(2)
(1) Faculty of Chemistry, Brno University of Technology,
Purkyn›ova 118, 612 00 Brno; (2) Faculty of Science, Ma-
saryk University, Tvrde¤ho 14, 602 00 Brno
Green tea extract contains various antioxidant substances,
such as tea polyphenols and ascorbic acid. Numbers of
these antioxidants inhibit the formation of mutagenic ac-
tivity because oxygen free radicals and lipid peroxidation
processes are the most important factors in the induction
of mutagenesis and carcinogenesis. Aim of this work is a
study of antimutagenic and antioxidant e¡ects of tea ex-
tracts. Quanti¢cation of active substances in tested ex-
tracts was performed using HPLC. Biological e¡ects of
extracts were analyzed using four independent tests of
FEMSLE Congress 2-6-03
113
genotoxicity: 1) The Ames test with Salmonella typhimu-
rium TA98, 2) Cytogenetic analysis of peripheral blood
lymphocytes (CAPL), 3) test with Saccharomyces cerevisie
D7 and 4) test with Euglena gracilis. Extracts were al-
lowed to be positive antimutagens based on their ability
to inhibit mutagenic e¡ects of standard mutagens. Anti-
oxidant capacity of extracts was analyzed using ‘‘Randox
Total Antioxidant Status’’ kit. No toxic e¡ects of green
tea extract were found. Green tea extracts exhibited high
antioxidant as well as high antimutagenic e¡ects (more
than 60 percent of mutagenicity inhibition). Antioxidant
and antimutagenic e¡ects (more than 40 percent of muta-
genicity inhibition) were proved with standard tea cate-
chins : (-)catechin and (-)catechin-gallate too.
P1^29
HYDROPHOBICITY AND ADHESIVE PROPERTIES
OF LACTOBACILLI USED IN PROBIOTIC YO-
GHURTS
C. Guigas, U. Schillinger and W. H. Holzapfel
Bundesforschungsanstalt fu«r Erna«hrung, Institut fu«r Hy-
giene und Toxikologie, Haid-und-Neu-Str. 9, D-76131
Karlsruhe
Cell surface hydrophobicity is one of the factors that may
contribute to the adhesion of bacterial cells to host tissues.
It may play an important role in the interaction of pro-
biotic lactobacilli and other lactic acid bacteria with the
epithelial cells of the gastrointestinal tract. The adherence
of these organisms to mucosal structures is generally be-
lieved to facilitate colonisation and persistence of probi-
otic strains in the normal intestinal population. 19 Lacto-
bacillus strains isolated from probiotic yoghurts were
examined for adhesion to the mucus-secreting human en-
terocyte-like cell line HT29 MTX and to several extracel-
lular matrices such as collagen, ¢brinogen and ¢bronectin
as well as for their cell surface hydrophobicity. The inves-
tigations included well-studied probiotic strains such Lac-
tobacillus johnsonii LC-1 and Lactobacillus rhamnosus GG
as controls. Strain-speci¢c di¡erences in the adhesive
properties were found. Hydrophobicity, however, did not
necessarily correlate with the adhesion properties, indicat-
ing that adhesion to the host cell is a very complex process
mediated by many di¡erent mechanisms.
114 1st FEMS Congress / Posters 103^505
P1^30
EFFECT OF BACTERIOCIN-PRODUCING ENTERO-
COCCUS FAECALIS BFE 1071 ON THE GUT FLORA
OF MALE SPRAGUE-DAWLEY RATS
A. Wijaya, Ch. Neudecker, C. M. A. P. Franz and W. H.
Holzapfel
Bundesforschungsanstalt fu«r Erna«hrung, Institut fu«r Hy-
giene und Toxikologie, Haid-und-Neu-Str. 9, D-76131
Karlsruhe
Bacteriocin production has been described as a functional
trait of probiotic bacteria, and probiotic strains that pro-
duce bacteriocins have found application in foods. To
date, information on the in£uence of bacteriocin produc-
tion by probiotic strains on the autochthonous £ora of the
host is rare. In this investigation, the in£uence of the bac-
teriocin-producing and potentially probiotic strain Entero-
coccus faecalis BFE 1071 on the gut £ora of healthy male
Sprague-Dawley rats, was studied. Rats (weight, 125 to
150 g; n = 36,), were separated into 4 groups and fed
25g of each of the following diets per day: group A
(n=6) received basal diet (Altromin C1000); group B
(n=6) basal diet containing 1% of heat-inactivated bacter-
iocin-producing E. faecalis BFE 1071 biomass; group C
(n=12) basal diet containing 1% bacteriocin-producing E.
faecalis BFE 1071 biomass (3.6 x 1011 CFU/g); and group
D (n=12) basal diet containing 1% of the bacteriocin-neg-
ative mutant E. faecalis BFE 1071/79(-) (1.9 x 1011 CFU/
g). The faeces of all groups was collected daily and ana-
lysed. Lactobacilli numbers in faeces ranged from 107 to
108 CFU/g with no noticeable di¡erence in numbers be-
tween the diet groups. However, enterococcal numbers in
groups C and D were ca. 1 log higher at 109 CFU/g when
compared to groups A and B. From the predominant
Lactobacillus and Enterococcus representatives, respec-
tively 101 and 139 strains were isolated in total from the
four diet groups, and characterised by phenotypic features
and RAPD ¢ngerprinting. The predominant lactobacilli
from the rat GI tract comprised L. gasseri, L. johnsonii
and presumptive L. murinus isolates. Interestingly, the ad-
ministration of bacteriocin-producing enterococci ap-
peared to promote L. johnsonii and L. gasseri numbers.
RAPD ¢ngerprinting proved suitable to recognise the bac-
teriocin-producing Enterococcus strain or its mutant.
Thereby, the predomination of the test strain in the faeces
and its survival during passage of the rat intestinal tract
could be con¢rmed.
FEMSLE Congress 2-6-03
P1^31
TOTAL NUMBER OF MICROORGANISMS IN
SHEEP’S MILK AND WHITE ^ SOFT CHEESE IN
DIFFERENT PHASES OF TECHNOLOGICAL PRO-
CESS IN MACEDONIA
N. D. Hristovski(1), E. Krsteva(2), N. Kozarovski(1), M.
K. Arapceska(1) and J. N. Hristovska(1)
(1) Faculty of Biotechnical Sciences, 7000 Bitola, Macedo-
nia; (2) Institute for Health protection 7000 Bitola, Mace-
donia
In Pelister’s region of Macedonia exists, traditional pro-
duction of sheep’s white ^ soft cheese in home condition.
The aim of research was to standardize process of produc-
tion of this kind of cheese, from raw sheep’s milk to ¢nal
product with high quality, which can been sold at a mar-
ket in vacuum form with weight of 0.3 ^ 0.6 kg. During
research was conducted monitoring of bacteriological sta-
tus of raw milk and cheese in di¡erent phases of techno-
logical process. The following bacteriological examination
were exerted: total number of microorganisms in broth
agar in fresh milk: 1000 ^ 400 microorganisms /ml, total
number of microorganisms in broth agar in pasteurized
milk before curdling: 200 microorganisms /ml, total num-
ber of microorganisms in broth agar in fresh cheese: 1000
microorganisms /ml, total number of microorganisms in
broth agar in fermented cheese: 20 microorganisms /ml.
In technological process of cheese’s production were used
low pasteurization at 65‡C with duration of 30 minutes,
3% starter culture, 0.015% CaCl2, cheese ferment and the
milk was at the temperature of 30‡C. The ripening of the
cheese was exerted in metal cans in duration of 30 days.
After that the cheese was vacuumed and sold at a market
as high quality sheep’s white ^ soft cheese from Pelister’s
region of Macedonia. With the holding to prescribed tech-
nology was gotten a cheese with high quality according to
chemical composition, aroma, £avor and acceptable price,
which gives opportunity for pro¢table production of this
kind of cheese.
 1st FEMS Congress / Posters 103^505
P1^32
T-RFLP AS A METHOD FOR MONITORING ANTI-
BIOTIC INDUCED DISTURBANCES IN HUMAN
NORMAL MICROFLORA
C. Jernberg(1,2), A. Sullivan(2), C. Edlund(2) and J. K.
Jansson(3)
(1) Sodertorn University College, Department of Natural
Sciences, S-141 89 Huddinge, Sweden ; (2) Karolinska In-
stitute, Department Laboratory Medicine, Division of Clin-
ical Bacteriology, S-141 86 Huddinge, Sweden; (3) Depart-
ment of Microbiology, Swedish University of Agricultural
Sciences, Box 7025, S-750 07 Uppsala, Sweden
Clindamycin is an antibiotic used to treat infections
caused by obligate anaerobes such as Bacteroides species.
During antibiotic treatment, the natural intestinal micro-
£ora can be disturbed allowing pathogens, such as Clos-
tridium di⁄cile, to overgrow and produce toxins. These
negative impacts may be counterbalanced by use of pro-
biotics during antibiotic treatment. In this study, changes
in the composition of the human faecal bacterial micro-
£ora of healthy volunteers due to the use of clindamycin
and a probiotic was analysed qualitatively and quantita-
tively, primarily using a conventional culturing method.
Since only a small fraction of the intestinal micro£ora
can be cultivated, the faecal samples were also analyzed
using a culture independent molecular ¢ngerprinting tech-
nique, terminal restriction fragment length polymorphism
(T-RFLP). It was shown that antibiotic treatment had a
signi¢cant impact on several bacterial populations in the
intestinal community. Di¡erent populations either in-
creased or decreased in relative abundance in both the
placebo and the probiotic treated groups. Furthermore,
some populations did not seem to be a¡ected at all. Se-
lected pure isolates were also characterized by T-RFLP.
Attempts were made to match terminal restriction frag-
ments (TRFs) of the isolates with speci¢c TRFs in the
bacterial community ¢ngerprint pattern. The data result-
ing from culturing methods and T-RFLP analyses were
compared. For example, the culture-based results for the
Bacteroides group were in accordance with the T-RFLP
results.
FEMSLE Congress 2-6-03
115
P1^33
BOTRYTICIDES INFLUENCE THE YEAST MICRO-
FLORA OF GRAPE BERRIES
N adez›(1), F. C
K. Jug(1), N. C N us›(2), P. Raspor(1)
(1) University of Ljubljana, Biotechnical faculty, Food Sci-
ence and Technology Department, Chair of Biotechnology,
Jamnikarjeva 101, 1000 Ljubljana, Slovenia; (2) University
of Ljubljana, Biotechnical faculty, Agronomy Department,
Chair of Viticulture, Jamnikarjeva 101, 1000 Ljubljana,
Slovenia
Grapes are a primary source of indigenous yeast £ora,
which plays an important role in wine fermentation with
either their impact on distinctive wine style, or causing
stuck or sluggish fermentation. The composition of the
yeast population of grape berries is in£uenced by several
factors such as ripening stage of the grape, weather con-
ditions, geographic location, grape cultivar and viticulture
practice, in particular the time and intensity of disease and
pest management. The aim of this study was to determine
the in£uence of three commonly used botryticides such as
iprodione, pyrimethanil and cyprodinil + £udioxonil on
yeast £ora composition of the grape berries of cultivar
Rebula (Vitis vinifera L.) in Primorska vine growing re-
gion. The agar plate counting results of yeast populations
varied among the treatments from 9x102 to 4x103 CFU
per ml. Further, colony morphotypes were described and
enumerated. Using the combination of molecular tech-
nique (PCR-RFLP of 18S+ITS ribosomal DNA) and
standard yeast identi¢cation tests, 18 species among 308
yeasts isolates were identi¢ed. The basidiomyceteous
yeasts, such as Rhodotorula glutinis, R. minuta, Cryptococ-
cus luteolus and Sporobolomyces sp. predominated in all
samples. Hanseniaspora species, which are mostly predom-
inant species on grapes and at the beginning of wine fer-
mentation, were only detected on grape berries of un-
treated control.
This work was ¢nanced by the Slovenia Ministry of Edu-
cation, Science and Sport and Ministry of Agriculture,
Forestry and Food project No. V4-0591 (CRP).
116 1st FEMS Congress / Posters 103^505
P1^34
COMPARISON OF GROWTH AND GLYCEROL
PRODUCTION KINETICS OF TWO ENDOGENIC
WINE YEAST STRAINS IN DIFFERENT SUBSTRATE
MEDIA
º zbas
S. Karasu Yalc|n, Z. Y. O
Food Engineering Department, Hacettepe University, Bey-
tepe 06532, Ankara, Turkey
Glycerol has been known as an important by-product of
wine fermentations improving sensory quality of wine.
This study was carried out with two endogenic wine yeast
strains, Saccharomyces cerevisiae Kalecik 1 and Saccharo-
myces cerevisiae Narince 3. E¡ects of di¡erent sustrates on
growth and glycerol production characteristics of the
yeasts were investigated in batch system. The kinetics of
growth and glycerol biosynthesis were analysed at various
initial concentrations of glucose, fructose, and sucrose
which take place in the sugar composition of must. Glu-
cose was found as the substrate which S. cerevisiae Kale-
cik 1 showed most tendency to use for growth, while it
was sucrose for S. cerevisiae Narince 3. For both of the
strains, speci¢c glycerol production rate was highest in
fructose medium and reached maximum level at 350 g/L
of initial concentration. Anyway, S. cerevisiae Narince 3
could not produce glycerol below 100 g/L initial concen-
tration of fructose. No glycerol production was observed
for the strain Kalecik 1 below 200 g/L initial sucrose con-
centration, and very low speci¢c glycerol production rates
and glycerol yields were obtained below this concentra-
tion. When natural white grape juice was used as fermen-
tation medium, S. cerevisiae Kalecik 1 and S. cerevisiae
Narince 3 reached a maximum dry weight of 9.3 g/L and
8.0 g/L, respectively. The strain Kalecik 1 produced glyc-
erol at a maximum concentration of 11.8 g/L while Nar-
ince 3 produced 14.1 g/L in grape juice.
P1^35
ACID RESISTANCE OF BACTERIOCIN-PRO-
DUCERS ISOLATED FROM BOUZA: A TRADITION-
AL EGYPTIAN FERMENTED BEVERAGE
A. A. M. Khalil
Department of Protein Research, Genetic Engineering and
Biotechnology Institute, Mubarak City for Science and Ap-
plied Technology, Research Zone, Borg Al-Arab, Alexan-
dria, Egypt. On leave from Department of Biotechnology,
Lund University, Lund, Sweden
Bouza [Written also bosa, bozah, bouza] as a lactic acid
fermented food is produced in many mid-Asian, Middle
FEMSLE Congress 2-6-03
East, the Balkans, and African countries. Bouza is a tradi-
tional Egyptian beverage made by yeast and lactic acid
bacteria fermentation of barely, wheat or millet seed and
has high alcohol content (up to 7% by volume). These
products have many advantages such as destroying unde-
sirable factors in the raw products, and providing a safer
product with high acidity. Ability of any bacterial isolate
to grow at low pH values is a precondition for them to be
selected for a consequent study for proving their probiotic
characteristics. The aim of this study is to examine in vitro
the ability bacteriocin-producers isolated from Bouza to
tolerate low pH values. Viable counts were determined
and resistance to pH (R%-pH) was determined as the per-
centage of surviving cells after incubation for 1 h at pH of
2.5, 3.0, and 3.5. Lactobacillus fermentum was not signi¢-
cantly in£uenced by pH values of 3.5 and 3.0 and this
strain showed the highest survival percentage within the
pH range tested. The R%-pH values obtained were 83.5,
64.9, and 51.3% at pH values of 3.5, 3.0 and 2.5 respec-
tively. Lactobacillus casei showed a signi¢cant decrease in
viable cells at all tested pHs. However, Lactobacillus plan-
tarum cells were completely killed at pH values of 2.0 and
2.5. The obtained results proofed that Lactobacillus fer-
mentum is a promising probiotic culture.
P1^36
INFLUENCE OF SODIUM CHLORIDE AND SO-
DIUM NITRITE ON ANTIBACTERIAL PROTEIN
PRODUCTION BY LACTOBACILLUS SAKEI LB 706
A. A. M. Khalil
Department of Protein Research, Genetic Engineering and
Biotechnology Institute, Mubarak City for Science and Ap-
plied Technology, Research Zone, Borg Al-Arab, Alexan-
dria, Egypt. Present address : Department of Biotechnology,
Lund University, Lund, Sweden
Fermentation of meats is an ancient and excellent preser-
vation technique which results in stable and safe end prod-
ucts. However, the possibility of using bacteriocin-produc-
ing starter cultures is still being questioned since the
amount of available bacteriocin in the meat environment
appears to be lower than expected. In this study, Lacto-
bacillus sakei Lb 706 was used as a producer of sakacin A
with Weissella paramesenteroides DSM 20288 as an indi-
cator organism. A series of in vitro fermentation experi-
ments was performed with MRS broth containing di¡erent
concentrations of sodium chloride and sodium nitrite in
order to examine the e¡ects of these compounds on both
cell growth and the production of sakacin A by L. sakei
Lb 706. It was found that sodium chloride and sodium
nitrite interfere with cell growth and bacteriocin activity.
The growth of L. sakei Lb 706 is sometimes enhanced in
the presence of low concentrations of sodium chloride but
 1st FEMS Congress / Posters 103^505
was clearly inhibited in the presence of high NaCl concen-
trations. Increases in salt concentration decrease the
growth rate of L. sakei Lb 706 linearly. Moreover, sodium
chloride negatively a¡ects the production of sakacin A by
L. sakei Lb 706. On the other hand, sodium nitrite had an
insigni¢cant e¡ect on bacteriocin activity. At higher so-
dium nitrite concentration the sakacin A ceased accompa-
nied with lower cell masses. Production of sakacin A de-
creases because the amount of biomass formed decreases.
It has been suggested that the decrease in bacteriocin pro-
duction in the presence of salt is due to interference of
sodium chloride molecules with binding of the induction
factor, which is essential for bacteriocin production, to its
receptor.
P1^37
CRYORESISTANT LACTATE LEAVEN IN BREAD-
MAKING
S. V. Kitaevskaya, O. A. Reshetnik
Department of Food Technology, Kazan State Technologi-
cal University, K. Marks str., 68, Kazan, Russia
Recently, the technologies of manufacture of £acky small-
piece products from wheat £our on the base of the frozen
semi-products are widely used, however, there are no any
processing lines making bread from frozen rye or wheat-
rye semi-products. Special attention should be devoted to
the correct selection of compounding components, lactic
acid bacteria and yeast strains, freezing-defrosting regimes
and the test semi-products storage conditions. A restora-
tive e¡ect of freezing on the blended rye and wheat £our
dough of various moistures has been discovered. The ex-
pediency of application of the technology of bread manu-
facture from such a £our blend on the base of frozen semi-
products was shown. The technological parameters of
dough batch, freezing and defrosting of wheat-rye bread
semi-products were optimized, aiming the conservation of
test micro£ora cells. Research work for selection of cryor-
esistant lactic acid bacteria strains was carried out. The
bringing in of lactic acid bacteria Lactobacillus casei
TMB-D on the stage of dough batch was shown as stabi-
lizing the qualities of wheat-rye semi-products during
freezing. The positive e¡ect of researched strain metabo-
lites on rheological characteristics of dough and the qual-
ity of ¢nished articles was established.
FEMSLE Congress 2-6-03
117
P1^38
MICROMANIPULATION AND FLUORESCENCE IN
SITU HYBRIDISATION FOR THE ISOLATION AND
IDENTIFICATION OF OENOCOCCUS OENI
STRAINS FROM WINE
H. Ko«nig, J. Fro«hlich, S. Hirschha«user
Institute of Microbiology and Wine Research, Johannes Gu-
tenberg-University, 55099 Mainz, Germany
The Gram positive bacterium Oenococcus oeni plays an
important role in winemaking and it is bene¢cial to wine
quality. Oenococci possess a greater tolerance of acid and
ethanol than many other lactobacilli from wine belonging
to the genera Lactobacillus, Leuconostoc and Pediococcus.
Oenococcus strains are involved in deacidi¢cation, £avor
modi¢cation, and increasing the microbial stability of
wine. The malolactic fermentation usually occurs after al-
coholic fermentation. The acidity of wine is decreased due
to the conversion of malate to lactate and carbon dioxide.
Since Oenoccocus strains vary in malolactic activity, etha-
nol, acid and temperature tolerance and fermentation pat-
tern the identi¢cation of new strains with novel feature for
special applications in wine-making is desirable. We have
developed speci¢c molecular probes for the rapid identi¢-
cation of oenococci. In combination with micromanipula-
tion Oenococcus strains were rapidly isolated from di¡er-
ent wines.
P1^39
MANUFACTURE OF HONEY BEER WITH SACCHA-
ROMYCES CEREVISIAE AND SACCHAROMYCES
PASTORIANUS
R. Kova¤cs(1), B. Vecseri-Hegyes(2), B. A Ł sva¤nyi(1), L.
Varga(1), J. Szigeti(1), L. Daro¤czi(3) and G. Bugyi(4)
(1) University of West Hungary, Faculty of Agricultural
and Food Sciences, Institute of Food Science, Mosonma-
gyaro¤va¤r, Hungary ; (2) Szent Istva¤n University, Faculty
of Food Science, Department of Brewery and Distillery,
Budapest, Hungary; (3) Y-Food Inc., Berettyo¤u¤jfalu, Hun-
gary; (4) Mikroauto Inc., Cegle¤d, Hungary
The aim of this research was to produce two di¡erent
kinds of honey beer, a high-alcohol and a low-alcohol
product with unique sensory properties. First, the ethanol
production of two yeast species (Saccharomyces cerevisiae
and S. pastorianus) was determined by lab-scale experi-
ments. Fermentations were conducted at 15 and 30‡C
for 14 days using glucose and fructose as substrates in
fermentation equipment speci¢cally designed for this pur-
118 1st FEMS Congress / Posters 103^505
pose. Glucose, fructose, and ethanol levels were measured
with the use of a high-performance liquid chromatography
(HPLC) system. Both yeast species proved to be capable
of producing enough alcohol to make honey beer from
substrates containing 25 to 30% honey. Honey beer was
manufactured then under industrial pilot plant conditions
using the same two Saccharomyces species. Mixtures of
malt and honey were used as raw material. The total car-
bohydrate content was set at 10%. Honey made up 10 to
50% of total carbohydrates. At the end of the ripening
process, the chemical composition, i.e., volatile com-
pounds, diacetyl, ethanol, and residual carbohydrate con-
tents, of beers was determined and their sensory properties
were also evaluated. In conclusion, a delicious beer rich in
aroma compounds was produced with S. cerevisiae. This
product was similar in alcohol content to the traditional
beer varieties. In contrast, S. pastorianus was hardly capa-
ble of fermenting maltose, thus producing a low-alcohol
beer whose aroma pro¢le also fell short of expectations.
Therefore, the sensory properties of this latter product
should be improved by addition of £avouring substances.
P1^40
ANTIBACTERIAL AND ANTIMUTAGENIC ACTIV-
ITY OF PROBIOTIC BACTERIUM ENTEROCOCCUS
FAECIUM M-74 AND THEIR MODULATION BY SE-
LENIUM
L. Kriz›kova¤, A. Belicova¤, J. Dobias, J. Krajc›ovic› and L.
Ebringer
Comenius University, Faculty of Natural Sciences, Institute
of Cell Biology, Odbora¤rske na¤m. 5, 811 07 Bratislava,
Slovak Republic
The antibacterial activity of the probiotic bacterium En-
terococcus faecium M-74 was assessed on De Man, Rogo-
sa, Sharpe (MRS), Todd Hewitt (T-H), M17 (M-17) and
Brain Heart Infusion (BHI) media with sodium selenite
pentahydrate (+Se) and without sodium selenite pentahy-
drate (-Se) under aerobic or anaerobic conditions against
nine bacterial pathogens. The highest antibacterial activity
was found to be in the MRS medium under anaerobic
conditions. There were no di¡erences in the antibacterial
activity between MRS(+Se) and MRS(-Se) media. The
antimutagenic activity of MRS(+Se) and MRS(-Se) ex-
tracts after cultured with E. faecium M-74 as well as of
live and killed cells of E. faecium M-74 grown in the pres-
ence or absence of Se against genotoxicity of o£oxacin
(OFL) and acridine orange (AO) was determined in Eu-
glena gracilis assay. The MRS(+Se) extracts showed a sig-
ni¢cant higher activity in reducing genotoxicity of OFL
and AO than MRS(-Se) extracts. The live cells of the pro-
biotic strain M-74 exhibited higher antimutagenic activity
than the killed bacterial cells, but di¡erent depending on
FEMSLE Congress 2-6-03
the mutagen used. However, the live bacterial cells grown
in the presence of Se showed signi¢cantly higher antimu-
tagenic activity. These results suggest a potential bene¢t
for the future development of new Se-enriched probiotics
exhibiting higher antimutagenic properties.
P1^41
THE MYOELECTRICAL MIGRATING COMPLEX
(MMC) OF PIG SMALL INTESTINE EXCITE AU-
TOLYSIS OF PEDIOCOCCUS PENTOSACEUS
D. Kruszewska, AY . Ljungh, P. Podgurniak, M. Da
 bek, and
S. G. Pierzynowski
Department of Medical Microbiology, Dermatology and In-
fection, Lund University, So«lvegatan 23, 223-62 Lund, Swe-
den
It is known that some mammalian organs and tissues gen-
erate extremely low electromagnetic ¢elds (ELEF). The
small intestine MMC generates such ELEF and natural
micro£ora is exposed to ELEF. We test if the ELEF hav-
ing characteristics similar to those produced by the MMC
of the pig small intestine a¡ect the production of the pep-
tidoglycan hydrolases of Pediococcus pentosaceus in in vi-
tro culture. The autolytic phenotype of P. pentosaceus
strain was evaluated under starvation, in PBS at 300C
with or without the action of the MMC. For stimulations,
the GGP-3 MMC Simulator (Flow Instruments, Poland)
was used. The electric current emitted by the device was
transmitted to the tubes using platinum plate electrodes.
Released peptidoglycan hydrolases were evaluated by re-
naturing two-dimensional gel electrophoresis (2-DE). The
¢rst dimension was performed in a pH gradient in the
range from 3 to 10; in the second dimension SDS-
PAGE with P. pentosaceus extracted peptidoglycan was
supplemented. The antibacterial activity of P. pentosaceus
peptidoglycan hydrolases against lactic acid bacteria
(LAB) : Lactobacillus plantarum, L. paracasei, Leuconostoc
mesenteroides was evaluated. As a result of autolytic ac-
tivity the 30% of the P. pentosaceus cells were destroyed in
the suspension after 24h. Twenty-¢ve extracellulary pro-
teins were separated. The major activity band was ob-
served to migrate at about 45 kDa. The MMC induced
the appearance of a diverse group of hydrolases with low-
er Mr and di¡erent pI. The antibacterial activity of the
MMC stimulated peptidogylycans hydrolases of P. pento-
saceus was more variable than those from the untreated
cultures.
 1st FEMS Congress / Posters 103^505
P1^42
APPLICATION OF TEMPORAL TEMPERATURE
GRADIENT GEL ELECTROPHORESIS TO THE
BACTERIAL DIVERSITY OF RAW MILKS
V. Lafarge(1), J.-C. Ogier(2), V. Girard(1), V. Malad-
en(1), O. Son(2), C. Bach(2), A. Delacroix-Buchet(2), J.-
Y. Leveau(3)
(1) Agence Francaise de Se¤curite¤ des Aliments, Maisons
Alfort, France; (2) Institut National de la Recherche
Agronomique, Jouy en Josas, France; (3) Ecole Nationale
Supe¤rieure des Industries Agricoles et Alimentaires, Massy,
France
Until now, the bacterial community of raw milk was de-
scribed by classical microbiological methods. In practice,
these methods are long and tedious, and allow only a par-
tial inventory of the bacterial micro£ora. In this study, we
applied molecular biological techniques based on the di-
rect analysis of DNA to identify the micro-organisms
present in raw milks. The Temporal Temperature Gra-
dient gel Electrophoresis (TTGE) method was adapted
to identify the diversity and the evolution of the bacterial
£ora in refrigerated and non-refrigerated raw milk sam-
ples. TTGE was run under conditions that optimise sepa-
ration of ribosomal DNA fragments from species bacteria
whose genomes have either low or high GC content. Using
TTGE, we examined raw milk samples before and after
their incubation at 4‡C for 24 hours. The raw milk sam-
ples gave rise to TTGE pro¢les that varied in complexity.
A given sample generated a TTGE pro¢le of between 3
and 10 bands. Most samples displayed a dominant band
of ‘‘Lactococcus lactis’’. After incubation of raw milk sam-
ples at 4‡C for 24 hours, TTGE pro¢les were modi¢ed,
with an increase of psychrotroph £ora (Listeria, Pseudo-
monas £uorescens, Hafnia alvei).
P1^43
THE STRUCTURES OF EXOPOLYSACCHARIDES
PRODUCED BY STRAINS OF LACTOBACILLUS
FERMENTUM 133 AND 108
T. Lipinski, A. Korzeniowska-Kowal, M. Gawlik, M. Strus,
P. B. Heczko, C. Jones, A. Gamian
Institute of Immunology and Experimental Therapy, Polish
Academy of Sciences, 53-114 Wroclaw, Poland
Bacteria of the genus Lactobacillus are Gram-positive mi-
croorganisms, known as natural inhabitants of mammali-
an gastrointestinal tract. These bacteria can bene¢cially
a¡ect the health of the host by improving the indigenous
microbial balance. Colonisation of the intestinal mucosa
FEMSLE Congress 2-6-03
119
by probiotic microorganisms is considered to be important
for antagonistic activity against enteropathogens, modula-
tion the immune system, and for increased healing of dam-
aged gastric mucosa. Essential role in the adhesion phe-
nomenon play the surface antigens of bacterial cell e.g.
polysaccharides and proteins. Exopolysaccharides (EPS)
produced by lactic acid bacteria are excreted to the growth
medium or remain attached to the bacterial cell wall. Such
polysaccharides have growing interest also due to the suit-
able rheological properties for the dairy industry and the
structures of EPS from several Lactobacillus strains have
been established. Our research are focused on probiotic
properties of Lactocbacillus polysaccharides. Strains 108
and 133 have been isolated from gastrointestinal tract of
healthy mice and from mice with experimentally induced
IBD (in£ammatory bowel disease) respectively. Compara-
tive studies of exopolysaccharides produced by Lactobacil-
lus found in healthy mice and mice with IBD may help to
improve our knowledge on the IBD pathogenesis. Here we
report the structures of the polysaccharides isolated from
Lactobacillus fermentum strain 133 and 108.
P1^44
IDENTIFICATION AND CHARACTERIZATION OF
LACTIC ACID BACTERIA AND YEASTS ISOLATED
FROM SOURDOUGHS
F. Zilio(1), L. Zampese(2), C. Andrighetto(1), A. Lom-
bardi(1)
(1) Veneto Agricoltura, Istituto per la Qualita' e le Tecno-
logie Agroalimentari, Via San Gaetano 74, 36016, Thiene
(VI), Italy ; (2) Bioagro srl, Via San Gaetano 76, 36016,
Thiene (VI), Italy
Sourdough is a complex fermentation of cereals in which
lactic acid bacteria and yeasts are involved. In the present
study 78 lactic acid bacteria and 31 yeasts were isolated
from sourdoughs used for the production of traditional
bread and cakes in Veneto region, Northern Italy. The
sourdoughs were produced from Triticum aestivum wheat.
Isolates were identi¢ed by conventional physiological and
biochemical tests and by molecular methods (RAPD-
PCR). The results showed di¡erences between sourdoughs
regarding their microbial pro¢le and revealed an high di-
versity at species and strain level. Among lactic acid bac-
teria Lactobacillus sanfranciscensis was the most frequently
found species (60%). Inside this species, RAPD-PCR anal-
ysis showed the presence of di¡erent groups of strains
within the same sourdough. Other lactic acid bacteria
were identi¢ed as Leuconostoc citreum, Lactococcus lactis,
Lactobacillus plantarum. Technological relevant properties
such as gas production, acetate/lactate production and
acidifying activity were evaluated. A good acidifying abil-
ity, with pH ranging from 3.60 to 4.10 after 18 hours of
120 1st FEMS Congress / Posters 103^505
incubation in a wheat £our-based substrate, was found in
all the tested strains. Regarding yeasts, all the sourdoughs
contained Saccharomyces barnettii as dominant species;
Saccharomyces cerevisiae and S. pastorianus were also iso-
lated but with a lower frequency.
P1^45
BIOCHEMICAL CHARACTERIZATION OF LACTIC
ACID BACTERIA ISOLATED FROM FRENCH DRY
FERMENTED SAUSAGES
M. C. Lopez-Mendoza(1), R. Huerta(2), C. M. Mata(1)
and R. Jordano(2)
(1) Department of Animal Production and Food Science
and Technology, Cardenal Herrera-CEU University, Edi¢-
cio Seminario sn, E-46113 Moncada (Valencia), Spain;
(2) Department of Food Science and Technology, Univer-
sity of Cordoba, Campus de Rabanales, Edi¢cio Darwin,
Anexo. E-14071, Cordoba, Spain
The use of starter cultures to control and run the fermen-
tative process is a usual way of manufacturing sausages in
meat industries. Food technologists have developed sev-
eral types of starter cultures to be used for sausages pro-
duction, but sometimes these microorganisms are di¡erent
than those found in natural fermented sausages. The aim
of this study was to check if the strains of lactic acid
bacteria present in French dry-sausages were according
to the starter added initially. A total of 180 strains of
lactic acid bacteria isolated from four batches of two dif-
ferent French dry-sausages types (type A and type B),
elaborated in pilot plant, were characterised. The main
test used to identify the isolated bacteria were: microscop-
ic-morphologic characteristics, catalase activity, produc-
tion of gas, growth at 4, 15 and 45 ‡C, fermentation of
¢fteen carbohydrates, growth at pH 3.9, growth in the
presence of 7% and 10% (w/v) NaCl, hydrolysis of argi-
nine, gas CO2 production from glucose, production of
hydrogen sulphide, production of hydrogen peroxide, pro-
duction of dextrane and production of acetoin. The iso-
lated species of lactic acid bacteria corresponded, in higher
percentages, to those added as starters: Lactobacillus sake
(74 % of strains) for dry-sausage type A and Pediococcus
pentosaceus (47.5% of strains) for type B. But other species
such Lactobacillus curvatus and Pediococcus acidilactici
(24.5% and 1.5% of strains respectively) have been also
isolated in product A and L. sake, L. curvatus, Pediococcus
urinaeequi and P. acidilactici (25%, 20%, 5% and 2.5% of
strains respectively) in product B.
FEMSLE Congress 2-6-03
P1^46
CHARACTERISATION OF BACTERIOCINS PRO-
DUCED BY NATURAL ISOLATE LACTOBACILLUS
PARACASEI SUBSP. PARACASEI BGBUK2-16 AND
ITS ANTIMICROBIAL ACTIVITY AGAINST SOME
PATHOGENIC STRAINS
J. Lozo(1), M. Vukasinovic(2) and Lj. Topisirovic(1)
(1) Institute of Molecular Genetics and Genetic Engineer-
ing, Vojvode Stepe 444A, P.O.Box 794, 11000 Belgrade,
Yugoslavia; (2) Faculty of Technology and Metallurgy,
Karnegieva 4, 11000 Belgrade, Yugoslavia
The strain Lactobacillus paracasei subsp. paracasei
BGBUK2-16 produces two bacteriocins, designated
Bac216 and Bac217. Bacteriocin Bac217 is inactivated by
the heat treatment at 100‡C for 60 minutes, whereas
Bac216 retained activity after heating at 100‡C for 90 min-
utes. Both bacteriocins also retain their activities after
storage at -20‡C for 9 months and at 4‡C for 4 months.
These bacteriocins showed activity within pH range from 3
to 12. After treatment with the various proteolitic enzymes
they lost their activity. These results strongly suggested
that Bac216 and Bac217 should be proteins. Antimicrobial
activity of bacteriocins against some pathogenic strain was
tested by using well di¡usion assay. These analyses showed
that these bacteriocins exhibited a wide range of antimi-
crobial activity on many di¡erent pathogenic strains in-
cluding some spoilage and food-born pathogenic bacteria
such as Staphylococcus aureus ATCC25923, Listeria mono-
cytogenes and Bacillus cereus ATCC 11778. The e¡ect of
BGBUK2-16 on the growth of S. aureus in mixed culture
was observed. Treatment of S. aureus with 1600 AUml-1 of
each bacteriocins led to considerable decrease in CFUml-1
and after 7 hours of treatment all S. aureus cells were
killed. In the case when the initial inoculum of S. aureus
was lower all viable cells were killed after 4 hours. These
results showed that Lactobacillus paracasei subsp. paraca-
sei BGBUK2-16 could be used as a co-protective culture
in food fermentation.
 1st FEMS Congress / Posters 103^505
P1^47
BROAD AND COMPLEX ANTIFUNGAL ACTIVITY
OF LACTIC ACID BACTERIA
J. Magnusson(1), K. Stro«m(1), J. Sjo«gren(2) and J.
Schnu«rer(1)
(1) Department of Microbiology, Swedish University of
Agricultural Sciences, P.O.Box 7025, S-750 07 Uppsala,
Sweden; (2) Department of Chemistry, Swedish University
of Agricultural Sciences, P.O. box 7015, S-750 07 Uppsala,
Sweden
More than 1200 isolates of lactic acid bacteria isolated
from di¡erent environments were screened for antifungal
activity in a dual-culture agar plate assay. Approximately
10% of the isolates showed inhibitory activity and 4%
showed strong activity against the indicator mould Asper-
gillus fumigatus. A smaller number of isolates was studied
further to elucidate the nature of the antifungal e¡ect. By
di¡erent separation protocols, it was possible to purify
and characterize several di¡erent antifungal compounds
of both proteinaceous, and low molecular weight nature
from these isolates. Examples are antifungal cyclic dipep-
tides, phenyllactic acid and various fatty acids. Addition
of glycerol to the growth medium strongly enhanced the
antifungal e¡ect of Lactobacillus coryniformis. The current
study shows that lactic acid bacteria from di¡erent envi-
ronments and from di¡erent genera and species can exhib-
it antifungal activity against a number of common spoil-
age moulds and yeasts. The inhibitory activity is caused by
several di¡erent compounds with varying inhibitory spec-
tra and degree of e¡ect. Complex synergistic interactions
occur between the primary fermentation products lactate
and acetate, and the more speci¢c antifungal compounds.
P1^48
METABOLIC PATHWAYS INVOLVED IN THE PRO-
DUCTION OF CHEESE AROMA
L. Marilley, H. Sollberger, J. O. Bosset and M. G. Casey
Swiss Federal Research Station, Schwarzenburgstr. 161,
CH-3003 Bern, Switzerland
Cheese manufacture begins with the adjunction of starter
bacteria, which initiate the fermentation process. During
the ripening stage of Swiss semi-hard and hard cheeses,
which are produced from raw milk, a second bacterial
population, composed mainly of mesophilic facultatively
heterofermentative lactobacilli reachs cell densities of 107-
109 cfu /g cheese. This second micro£ora also in£uences
the quality of cheeses by producing £avour compounds.
The objective of this study was to determine the aroma-
FEMSLE Congress 2-6-03
121
relevant end-products of the lactic acid bacteria metabo-
lism, and thus to determine the metabolic pathways in-
volved in the production of cheese aroma. The production
of aromatic volatile compounds was measured by gas
chromatography followed by mass spectrometry (GC-
MS) using a purge and trap enrichment system. The alde-
hydes 3-methylbutanal, 2-methylbutanal, 2-methylpropa-
nal, the secondary alcohols 3-methylbutanol, 2-methylbu-
tanol, 2-methylpropanol (catabolism of branched-chain
amino acids), and the methyl ketones 2-pentanone, 2-hep-
tanone and 2-nonanone (putative incomplete L-oxidation)
ranked among the main compounds. The production of
these volatiles di¡ered between the strains analysed, and
thus some strains may have a stronger ability to in£uence
the aroma of cheeses. Swiss Emmentaler cheeses were
manufactured with adjunctions of di¡erent facultatively
heterofermentative lactobacilli. In those manufactured
with Lactobacillus casei adjunct cultures, chemical analysis
showed an increased concentration of the above-men-
tioned aldehydes and secondary alcohols. A better under-
standing of these metabolic routes is a prerequisite to the
biotechnological development of new high quality cheese
products.
P1^49
CONTROL OF FOOBORNE PATHOGENS AND
SPOILAGE BACTERIA BY STARTER CULTURES
AND BIOPRESERVATIVES IN SPANISH DRY FER-
MENTED SAUSAGE
C. M. Mata-Anguiano and M. C. Lo¤pez
Department of Animal Production and Food Science and
Technology, Cardenal Herrera-CEU Universitiy, Edi¢cio
Seminari s/n, E-46113, Moncada (Valencia), Spain.
‘‘Salchicho¤n’’ is a Spanish dry fermented sausage pro-
duced by the hurdles technology being implicated the
¢ve factors following: nitrite and salt as preservatives,
decreased of Eh, competitive lactic £ora, and lowered
pH and aw. Lactic acid bacteria (LAB) produces the re-
duction of pH and gives place to the appearance of some
metabolites with antimicrobial action (lactic acid, hydro-
gen peroxide and bacteriocines). Also the addition of start-
er cultures or biopreservatives to these meat products try
to inhibit the growth of foodborne pathogens and spoilage
bacteria. The aim of this study was to observate the e¡ects
of di¡erents starter cultures and biopreservatives on the
growth of foodborne pathogens (coliforms, enterococci,
Escherichia coli, Staphylococcus aureus, Salmonella spp
and Listeria) in sausage during ripening. Five di¡erents
batch of sausage were prepared each one with a di¡erent
starter culture (batch D and E) and biopreservatives
(batch B and C). Control is batch A. The samples with
starter cultures added showed a signi¢cantly smaller
122 1st FEMS Congress / Posters 103^505
counts of coliforms and enterococci than the other batch
(p 6 0,001), and the sample with preservative 1 (batch B)
presented signi¢cantly smaller coliforms and enterococci
counts than the control and batch C (p 6 0,001). In the
same way, the sausage added with biopreservatives pre-
sented a signi¢cantly higher S. aureus counts than those
added with starter cultures (p 6 0,001). E. coli, Salmonella
spp and Listeria was not found in any sample.
P1^50
BIFIDOBACTERIUM BIFIDUM B7.1 ISOLATED
FROM HUMAN ORIGIN IS A PROMISING PROBI-
OTIC STRAIN
A. Mayer, J. Rezessy-Szabo, B. Kondas, A. Hoschke
Szent Istvan University, Faculty of Food Sciences, Depart-
ment of Brewing and Distilling, Menesi ut. 45, Budapest,
Hungary
Probiotic dairy products have a market share of around
70% on the functional food market. Several probiotic
foods containing bi¢dobacteria are commercially avail-
able. One of the most important criteria of a probiotic
strain is human origination. Therefore, our aim was to
isolate bi¢dobacteria from human sources, which have
good probiotic (produce SCFA, adherence capability, an-
tagonistic e¡ect to enteropathogens) and moreover tech-
nological (acid and oxygen tolerance) features. The strains
isolated from healthy adults and infants were identi¢ed
with physiological, biochemical and molecular methods.
From infants mainly B. bi¢dum and B. breve, from adults
B. longum were obtained. Adherence capability to human
tissue culture and antagonistic e¡ects to enteropathogens
of the isolated strains were tested. The resistance of the
most promising strains was examined against gastric and
bile acid and digestive enzymes in test tube methods. Fur-
thermore they were tested in a simulator of the human
gastrointestinal tract. The strain B. bi¢dum B7.1 proved
to be the best among the tested strains, it was able to
survive the stomach-small intestine model in 80% and
had good e¡ects on the microbiota and the production
of SCFA in the colon model. Elaboration of reliable
mass propagation of this strain is one of the guarantees
for its application as functional food ingredient and/or
supplement. To achieve this it was cultivated in TPY broth
and soymilk in two-litre laboratory fermentor. After the
24-hours incubation time we got approx. 109 cfu/ml con-
centration. After separation of the ferment liquor we got a
product with 1010 cfu/ml concentration.
FEMSLE Congress 2-6-03
P1^51
THE CAPABILITY OF SPIRULINA PLATENSIS
CELLS TO ACCUMULATE COPPER AND MANGA-
NESE
A. A. Nalimova, V. V. Popova, N. A. Pronina
Institute of Plant Physiology, Russian Academy of Sciences,
127276, Botanicheskaya, 33, Moscow, Russia
Growth and accumulation of Cu and Mn in Spirulina
platensis cells, cultivated on media enriched with these
metals have been investigated. It was shown that growth
of cyanobacteria was stimulated by low (up to 2.55 mg/l
Cu, 138.9 mg/l Mn) and inhibited by high concentrations
of the elements in cultivation media. 5 mg/l Cu, 280 mg/l
Mn were lethal for cells. Maximal value of intracellular Cu
concentration was observed in S. platensis cells during the
¢rst hours after addition of CuSO4 following by copper
decrease in the course of cell cultivation. It was the result
of intracellular reduction Cu2+ to Cu1+ with the secretion
of latter into the medium. On the contrary, continuously
increase in the intracellular level of Mn was detected dur-
ing cultivation of S.platensis in the presence of MnCl2. S.
platensis incorporated Cu and Mn preferably in the frac-
tion of soluble proteins (above 55% for Cu, above 90% for
Mn). The accumulation of Cu is light dependent and was
not detected in the dark. On the contrary, Mn was accu-
mulated in higher concentration on the dark as compared
to light conditions.
P1^52
MICROBIAL ECOLOGY OF DAIRY PRODUCTS BY
TEMPORAL TEMPERATURE GEL ELECTROPHO-
RESIS (TTGE) AND DENATURING GRADIENT GEL
ELECTROPHORESIS (DGGE)
J.-C. Ogier(1), V. Lafarge(2), A. Rault(2), V. Malad-
en(2), O. Son(1), C. Bach(1), J.-Y. Leveau(3) and A.
Delacroix-Buchet(1)
(1) Institut National de la Recherche Agronomique, Jouy en
Josas, France; (2) Agence Francaise de Se¤curite¤ des Ali-
ments, Maisons Alfort, France; (3) Ecole Nationale Supe¤r-
ieure des Industries Agricoles et Alimentaires, Massy, France
Numerous micro-organisms, including bacteria, yeasts,
and moulds, are present in dairy products, forming a com-
plex ecosystem. Our goal is to characterise the complex
bacterial ecosystem of the raw milk micro£ora and to
follow its evolution during dairy processing. We applied
Temporal Temperature Gradient Electrophoresis (TTGE)
and Denaturing Gradient Gel electrophoresis (DGGE) to
identify the bacterial species present in dairy products.
 1st FEMS Congress / Posters 103^505
Total DNA was extracted from dairy products, and a var-
iable region (V3) of the 16S rDNA gene was ampli¢ed by
PCR with universal bacterial primers, resulting in a mix-
ture of amplicons separable via TTGE or DGGE. We
developed an original approach to rapidly identify the
bacteria present in an unknown dairy ecosystem; each
band was assigned to a bacterial identity by comparing
against an exhaustive bacterial reference database (V150
species) that includes useful dairy micro-organisms (lactic
acid bacteria), spoilage bacteria (e.g., Pseudomonas and
Enterobacteria) and a few pathogenic bacteria (e.g., Liste-
ria monocytogenes and Staphylococcus aureus). We used
TTGE to distinguish between bacteria species with a low
GC V3 sequence ( 6 55%), whereas DGGE was more ef-
fective in separating bacterial species with a high GC V3
sequence ( s 55%). Some related species could not be dif-
ferentiated using the V3 region. We therefore compared
di¡erent regions, either within the 16S rDNA region
(V6, V8), or within other functional genes (i.e., tmRNA
and rpoB) to improve resolution. We also developed spe-
ci¢c primers to detect pathogens (e.g., Listeria). TTGE
and DDGE have proven to be powerful and simple meth-
ods to rapidly identify the main bacterial species within
dairy products by using a combination of universal prim-
ers, or to detect contaminants by using speci¢c primers.
P1^53
INFLUENCE OF ETHANOL QUANTITY IN A NU-
TRITIVE MEDIA ON SACCHAROMYCES CEREVISI-
AE
D. J. Pejin, J. D. Pejin, O. S. Grujic and S. D. Kocic
Faculty of Technology, University of Novi Sad, Boulevar
Cara Lazara 1, 21 000 Novi Sad, Yugoslavia
The aim of this study was to determine in£uence of etha-
nol on metabolic activities and content of cytochromes,
alcohol dehydrogenase, isocitrate lyase and malate dehy-
drogenase in yeast cells. From the obtained results it can
be concluded that the increase of ethanol concentration in
a nutritive media in£uences cytochromes concentration in
yeast. Most signi¢cant decrease is observed in content of
cytochromes aa3 though the content of cytochromes b and
cytochromes c also decreased. This decrease can be ex-
plained by the ethanol concentration increase that disturbs
cytohromes formation and thus the intensity of yeast cell
breathing. From the results obtained the conclusion can be
drawn that ethanol concentration exceeding 7.94 vol %
decreases the metabolic activity as the ability of yeast to
respire glucose is reduced about three times at the ethanol
concentration of 11.45 vol %. The ability of yeast to utilize
oxygen at 11.45 vol % when ethanol is the media is also
lower. The activity of malate dehydrogenase is found to be
depressed by augmentation of ethanol concentration in the
FEMSLE Congress 2-6-03
123
initial media. For example, malate dehydrogenase activ-
ities are twice as low in the medium containing 11.45 vol
% compared to the value obtained with the ethanol con-
centration of 7.94 vol %. Alcohol dehydrogenase activity
remains high in the cells even at the ethanol concentration
of 11.45 vol %, i.e. it does not essentially change. The
activity of isocitrate lyase is also slightly lower when the
ethanol concentration of 11.45 vol % is used in compar-
ison to that of 7.94 vol %. It is necessary to perform more
investigations of the mentioned metabolic and enzymatic
activities in the media with the initial amount ranging
from 7.94 vol % to 11.45 vol %. Besides, dependence of
the activity of malate synthetase upon ethanol concentra-
tion in the initial media is to be established.
P1^54
PRELIMINARY CHARACTERISATION OF LACTO-
BACILLUS PLANTARUM STRAINS ISOLATED
FROM SOURDOUGHS
O. Pepe, G. Blaiotta, M. Anastasio, D. Ercolini and F.
Villani
Dipartimento di Scienza degli Alimenti, Universita' degli
Studi di Napoli Federico II, 80055 Portici, Italy
Twenty-nine Gram-positive rods, non-producing gas from
glucose, were isolated from sourdoughs and recognised as
Lactobacillus spp. Twenty-eight of them were identi¢ed as
belonging to the species Lactobacillus plantarum by both
phenotypic (API System) and genotypic (PCR speci¢c am-
pli¢cation) methods. Moreover, a high degree of discrim-
ination was obtained by pulsed-¢eld electrophoresis
(PFGE). In fact Lactobacillus plantarum strains were gath-
ered in 12 di¡erent genomic groups indicating a remark-
able genetic polymorphism within the species. This micro-
bial diversity was con¢rmed by the technological
performances of fourteen Lactobacillus plantarum strains,
associated with maltose positive or maltose negative Sac-
charomyces cerevisiae strains, tested in dough making ex-
periments. The microbial contents and fermentation prop-
erties of the starter cultures were greatly in£uenced by the
interaction between LAB and the type of associated
yeasts. The time of leavening of doughs prepared with
Lactobacillus plantarum and yeast strains appeared shorter
than those prepared with the yeast alone, in spite of the
detrimental e¡ect on the yeast growths. However, requir-
ing a shorter production time from 7 to around 5 hours, it
is worth noting that the association with the maltose pos-
itive Saccharomyces cerevisiae can be regarded as more
e¡ective. Moreover, the presence of Lactobacillus planta-
rum strains in the starter enhanced the acidi¢cation activ-
ities. The results showed as some technological properties
are dependent on the type of the LAB strains, besides the
type of species, included in the starter.
124 1st FEMS Congress / Posters 103^505
P1^55
THE OLIVE MILL WASTEWATER: A CULTURE
MEDIUM FOR THE GROWTH OF LACTIC ACID
BACTERIA
N. Roze'z(1), Ma M. Oliveira(2), L. Catulo(2) and C.
Peres(2)
(1) ITQB, Oeiras, Portugal; (2) INIAP/EAN, Oeiras,
Portugal
Highly pollutant olive mill wastewaters (OMW) are still
produced by a large number of Portuguese units that use
traditional olive oil extraction processes. OMW is rich in
organic compounds such as polyphenols, fats and sugars
but poor in nitrogen. Several dilutions of OMW supple-
mented with di¡erent nitrogen levels were inoculated with
lactic acid bacteria isolated from brined olives. Liquid and
liquid/solid systems were tested in order to maximise deg-
radation/growth. Performance was also evaluated by com-
paring the results of trials that were carried out under
aseptic and non-aseptic conditions. The presence of a ni-
trogen source such as tryptone and/or yeast extract was
indispensable for growth. Degradation of polyphenols was
observed to be dependent on nitrogen source and its con-
centration. Also, OMW compounds were used as carbon
and energy source. Lactate production increased with the
augmentation of tryptone and OMW levels. The cells in-
corporated oleic and linoleic acids present in OMW and
synthesised dihydrosterculic acid from oleic acid and so
increase the degree of unsaturation of the plasmatic mem-
brane. Unfortunately OMW could not be fermented with-
out any dilution.
P1^56
GROWTH AND ADAPTATION OF LACTOBACIL-
LUS PLANTARUM TO OLIVE FERMENTATION
CONDITIONS
N. Roze's(2), T. Baptista da Silva(2), Ma M. Oliveira(1)
and C. Peres(1)
(1) INIAP/EAN, Oeiras, Portugal; (2) ITQB, Oeiras,
Portugal
The lactic acid fermentation of plant materials is presented
from an ecological perspective emphasising microbial in-
teractions and their in£uence on production of fermented
plant foods. In olive fermentation lactobacilli and yeasts
are the most important genera involved in the processes.
Among the lactic acid bacteria (LAB) found on olives
there appears to be a predominance of homolactic LAB,
with Lactobacillus plantarum being the most frequently
occurring species with an important role on lactic olive
FEMSLE Congress 2-6-03
fermentation. The e¡ect of some parameters of olive brine
ecosystem on the growth, physiology and cell composition
of L. plantarum isolated from traditional homemade olive
brines were determined. Oleuropein is the most important
phenolic compound of olives. Sodium chloride is already
present in olive brines. Results indicated that the speci¢c
growth rate was not modi¢ed by the glucose concentra-
tions, but in the presence of fructose it was lower. Con-
centrations of sodium chloride had a strong e¡ect on the
speci¢c growth rate and it decreased with increasing NaCl
amounts. When oleuropein was the only source of carbon
L. plantarum exhibited growth and synthetised lactic acid.
Nevertheless, both compounds oleuropein and NaCl dis-
played greater inhibition and showed a bactericidal e¡ect
whatever the growth temperatures used. Growth was de-
layed at 15‡C and rapid at 37‡C. However the antibacte-
rial e¡ect of oleuropein was more pronounced. The fatty
acid pro¢les and phospholipid composition were weakly
a¡ected by growth conditions. This study allowed some
new aspects of olive fermentation to be shown.
P1^57
THIAMINE REQUIREMENTS IN RELATION TO
PYRUVIC ACID PRODUCTION FOR YARROWIA
LIPOLYTICA
O. A. Perevoznikova, S. V. Kamzolova, I. G. Morgunov
G.K. Skryabin Institute of Biochemistry and Physiology of
Microorganisms, Russian Academy of Sciences, pr-t Nauki
5, Pushchino, Moscow region, Russia 142290
In this study, to a pyruvate producing yeast strain Yarro-
wia lipolytica YKM-Y 2407 and variations in their thi-
amine requirements with regard to the productivity of
pyruvic acid under growth on di¡erent substrates (glucose
or glycerol) are presented. With various carbon sources,
the limitation of yeast growth by thiamine was shown to
be the main condition of the production of pyruvic acid by
Y. lipolytica which are enable to synthesize the pyridine
part of thiamine molecule and requires addition of this
vitamin to the medium. Excretion of pyruvic acids starts
simultaneously with a decrease in the growth rate, when
the thiamine content in proliferating cells, and, hence, the
activity of thiamine-dependent enzymes involved in metab-
olism is drastically decreased. The decrease in the activity
of pyruvate dehydrogenase stimulates the production of
pyruvic acid. The threshold concentration of thiamine at
which Y. lipolytica ceased to synthesize pyruvic acid was
2.0 and 4.5 Wg/l on glucose and glycerol, respectively.
Based on the results obtained under yeast cultivation in
£asks, the optimal condition for pyruvic production by
batch culture of Y. lipolytyca were proposed; under glyc-
erol as substrate of carbon and energy and the limiting
thiamine (2.0 mg/l) pyruvic acid production was as high
 1st FEMS Congress / Posters 103^505
as 67.0 g/l ; speci¢c rate of pyruvic acid synthesis reached
85mg pyruvic acid (g cells h) -1, mass yield coe⁄cient was
0.70
P1^58
EFFECT OF THE ADDITION OF AUTOCHTHO-
NOUS STARTER CULTURES ON THE MANUFAC-
TURING OF FIORE SARDO CHEESE
M. B. Pisano, M. Deplano, M. E. Fadda, A. Senis and S.
Cosentino
Department of Experimental Biology, section of Hygiene,
Univ. of Cagliari, SS 554, Km 4.500, 09042 Monserrato
(CA), Italy
In order to improve the quality of Fiore Sardo cheese
productions, still maintaining its traditional organoleptic
characteristics, 8 batches were elaborated, in a pilot dairy
plant, using raw milk with and without the addition of
autochthonous starter cultures. The lactic acid bacteria
used as starters were previously isolated from traditionally
made Fiore Sardo cheese. The evolution of chemical and
microbial characteristics as well as the sensory parameters
were determined for each batch of cheese at di¡erent
stages of ripening. No signi¢cant di¡erences in total solid
or in the mean content of NaCl, fat, protein and total
nitrogen were found in cheeses made with both manufac-
turing processes. Nevertheless lower values of standard
deviations were observed in cheeses manufactured with
the addition of starters. As for the microbiological param-
eters, at 3 months of ripening the mean count of Gram-
negative bacteria increased at 48h of ripening, then de-
creased to levels of 3 log units lower in cheeses manufac-
tured with starters. A similar trend was observed for
Staphylococci. Lactic acid bacteria reached a maximum
at 48h of ripening and then remained at high levels during
ripening with mean counts at least 2 log units higher in
cheeses made with starters. As for sensory analysis,
cheeses made with starters received higher scores, partic-
ularly for openings, texture and taste. These preliminary
results seem to con¢rm the potential use of autochthonous
£ora as adjunct cultures in the manufacturing of Fiore
Sardo cheese in a attempt to standardize the production
of this artisanal ewe’s product.
This work was supported by a grant from MURST, Plan
‘‘Agroalimentary products: dairy products’’, Cluster 08B,
Project n. 7.
FEMSLE Congress 2-6-03
125
P1^59
INITIAL LOADING OF SACCHAROMYCES CEREVI-
SIAE DICTATES BIOPROCESS KINETICS IN SIN-
GLE AND COMPOSED CULTURES WITH NON-
SACCHAROMYCES YEAST
K. Povhe Jemec and P. Raspor
Department of Food Science and Technology, Biotechnical
Faculty, University of Ljubljana, Jamnikarjeva 101, 1000
Ljubljana, Slovenia
The dynamics of three non-Saccharomyces strains (Hanse-
niaspora uvarum, Candida stellata and Metschnikowia pul-
cherrima) in the combination of Saccharomyces cerevisiae
at di¡erent starting concentrations in the inoculated Mal-
vasia grape must have been studied. The yeast population
of each strain has been evaluated by morphological exami-
nation. Stability of S. cerevisiae karyotype was followed
during the fermentation process. The quantity of S. cere-
visiae population in the ¢rst ¢ve days of fermentation has
been evaluated on the selective and non-selective media.
After one day of fermentation in all fermenters H. uvarum
took over 50% of whole yeast population, even in ferment-
ers with highest concentration of S. cerevisiae cells. As S.
cerevisiae population was in the progress, H. uvarum pop-
ulation was in the regression. Population of M. pulcherri-
ma yeasts was even more inhibited by S. cerevisiae and
rising concentration of the ethanol, whereas C. stellata
population showed to be more ethanol tolerant. At the
thirty-¢rst day in all fermenters, no matter on the S. cere-
visiae cell starting concentration, S. cerevisiae was, as the
only one species, detected in the must. The highest density
of yeast population counted 3x107 cfu/ml in the must in-
oculated only with S. cerevisiae while in the fermenters
inoculated with combined inoculum has not exceeded
2x107cfu/ml. Conversion of the sugars to the ethanol was
more e⁄cient in the fermentation processes conducted by
higher concentration of S. cerevisiae cells. Glucose was
almost completely exhausted in all fermenters, nondepend-
ent on the type of the inoculum used, while in fermenters
with lower S. cerevisiae starting concentration, more of
residual fructose was detected.
This study was supported in part by the Ministry of Sci-
ence and Technology of the Republic of Slovenia (Project
no. L4-8849-490). K. Povhe Jemec is grateful to the Min-
istry of Science and Technology of the Republic of Slov-
enia for grant (no.: S 38-490-007/18126/97). We are grate-
ful to Vinakoper wine industry to kindly supplying grape
must, and to M. SVergan for some chemical analyses and
K. Matastik for her technical assistance.
126 1st FEMS Congress / Posters 103^505
P1^60
IDENTIFICATION OF LACTOCOCCUS LACTIS
SUBSPECIES USING REP-PCR FINGERPRINTING
J. Prodelalova, A. Spanova, B. Rittich
Department of Microbiology, Faculty of Science, Masaryk
University, Tvrdeho 14,
602 00 Brno, Czech Republic
Bacteria of the species Lactococcus lactis are important
members of lactic acid bacteria. Two Lactococcus lactis
subspecies, lactis and cremoris, are widely used in the man-
ufacture of cheese and other fermented dairy products. L.
lactis subsp. hordniae is not used in dairy technology. Dif-
ferent procedures of DNA isolation for DNA ampli¢ca-
tion were tested. Repetitive element sequence-based PCR
(rep-PCR) was used to identify three subspecies of Lacto-
coccus lactis mentioned above. Single primer LL-REP1 [1]
was used in ampli¢cation reaction. Ampli¢ed fragments
were resolved by 1,5% agarose gel and stained with ethid-
ium bromide. The use of LL-Rep1 primer resulted in a
banding pattern containing approximatelly 6-10 visualised
PCR products. The size of the DNA fragments obtained
after ampli¢cation using LL-Rep1 primer ranged between
300-2000 bp. Obtained data indicate that used method is
suitable for distinguish between subspecies of bacteria
Lactococcus lactis. The ¢ngerprinting rep-PCR was used
for subspecies identi¢cation of di¡erent Lactococcus lactis
strains collected in Czech Republic.
[1] E. Urbach et al (1998). FEMS Microbiol. Lett. 162:
111-115.
P1^61
THE EFFECT OF FRUCTOOLIGOSACCHARIDES
AND POLYFRUCTANS ON THE DEVELOPMENT
OF BIFIDOBACTERIA
M. Bekers, M. Marauska, P. Semjonovs, M. Grube, A.
Rapoport, R. Ionina
Institute of Microbiology and Biotechnology, University of
Latvia, Kronvald Blvd., 4, LV-1586 Riga, Latvia
The production of functional food products increases rap-
idly. Probiotics and prebiotics as well as dietary ¢bers are
signi¢cant components of functional foods. Methods of
biotechnology can be used for production of the above-
mentioned functional food components. Polyfructan ^ le-
van was produced by Zymomonas mobilis in a laboratory
scale experiments. Levan ^ levansucrase complex was ob-
tained from the cell-free culture liquid with ethanol and
further used as biocatalyst for fructooligosaccharides
(FOS) production in sucrose syrup. Obtained fructan syr-
FEMSLE Congress 2-6-03
up (FS) containing FOS and levan was investigated and
compared with glucose, commercial FOS and inulin prep-
arations as carbon sources for cultivation of Bi¢dobacte-
rium lacticum 12. It was established that Bi¢dobacterium at
physiological conditions could not directly utilize inulin
and levan. However, levan was hydrolyzed to FOS by
lactic acid. FOS was a good carbon source for Bi¢dobac-
terium. Inulin was more inert component as compared
with levan if yeasts (invertase) as hydrolyzing agent were
used. FS was an excellent medium component for Bi¢do-
bacterium. FS contais 65% of total carbohydrates includ-
ing 20-25% FOS and 6-7% dietary ¢ber ^ levan. Tests on
animals (mice) proved that FS is a non-toxic product with
cholesterol lowering properties. FS has a pleasant honey-
like taste and reduced (30%) energetic value taking into
account the total carbohydrate content. A good quality
yogurt was produced from fat-free milk with 5-10% FS
additive.
P1^62
MICROBIAL ECOLOGY OF PORTUGUESE EWE’S
CHEESE
H. I. Santos(1,2), J. J. Figueiredo Marques(1,3), L. Ra-
poso(1), R. Tenreiro(4), M. T. Barreto Crespo(1) #
(1) IBET/ITQB, Apartado 12, 2781-901 Oeiras, Portugal;
(2) Universidade Agostinho Neto, Luanda, Angola; (3)
Estaca‹o Agrono¤mica Nacional, INIA, Oeiras, Portugal;
(4) FCUL, Campo Grande, Edif|¤cio C2, Piso 4, 1700 Lis-
boa, Portugal
Traditional cheeses constitute a group of fermented food
products that are highly appreciated due to their nutri-
tional and organoleptic properties. Portuguese ewe’s
cheese, produced from unpasteurized milk, depends on
the environmental microbial innocula to develop its char-
acteristic texture and £avour, as no starter cultures can be
added to these traditional products. The maturation peri-
od, when well succeeded, leads to an exquisite ¢nal prod-
uct. The knowledge on the microbial succession in such a
process is therefore very important to preserve the same
quality standards. Many of the published works on the
microbiology of traditional cheese mainly focus on the
culturable microorganisms in usual laboratory media.
Part of the information on the microbial evolution can
than be lost if non-culturable strains are present, as well
as, information on the sources of environmental innocula.
This work will present results of the microbial ¢ngerprints
of the manufacturing areas, utensils and raw material and
¢ngerprints on the microbial succession along the matura-
tion process. Axial distribution of the microbial commu-
nity along the cheese will also be accessed. The evaluation
of those populations will be performed using total DNA
extracts from samples, ampli¢cation of 16SrDNA and
 1st FEMS Congress / Posters 103^505
subsequent temperature gradient gel electrophoresis
(TGGE). Culturable microorganisms of the same samples
will be obtained by traditional plating methods, and will
be used for comparison and identi¢cation of the members
of the community.
P1^63
THE INFLUENCE OF NEW GROWTH REGULATOR
OF MICROORGANISMS ‘‘HYDROPTERINE’’ ON
THE LACTIC ACID FERMENTATION PROCESSES
M. Shamtsyan(1), O. Pantyuk(1), A. Bochkova(2)
(1) St. Petersburg State Institute of Technology (Technical
University), St. Petersburg, Russia ; (2) All-Russian Insti-
tute of Food Acidulates, Aroma and Colors, St. Petersburg,
Russia
The in£uence of hydropterine, a novel, chemically synthe-
sized growth regulator on the lactic acid fermentation pro-
cesses of Lactobacillus delbrueckii and Streptococcus bovis
was studied. It was determined, that this substance can be
easily dissolved in water, is not toxic for human and ani-
mals use and is ecologically safe.The experiments prove
high e⁄ciency of this regulator. Its concentrations of 10-
6
, 10-5 g/l were signi¢cantly stimulating the processes of
biomass accumulation and lactic acid production of both
producers. Addition of hydropterine to the fermentation
media at the beginning of the process was causing the
reduction of fermentation period of L. delbrueckii for 11-
15% and for 18-20% for S. bovis. Daily addition of hydro-
pterine to the fermentation media was valuably more af-
fective. The in£uence of this substance on several key en-
zymes of lactic acid fermentation was also, studied.
P1^64
DEHYDRATION OF PROBIOTIC STRAINS LACTO-
BACILLUS GASSERI K7 AND LF221
S. Stojkovic¤, B. Bogovic› Matijas›ic¤, I. Rogelj
University of Ljubljana, Biotechnical Faculty, Zootechnical
Department, Chair of Dairying, Groblje 3, 1230 Domz›ale,
Slovenia
Freeze-drying and spray drying were used to dehydrate
Lactobacillus gasseri K7 and LF221 strains. In freeze-dry-
ing process various protective media (1: MRS + 20 %
glycerol; 2: 10 % NFDMS (CaCO3)-non-fat dry milk sol-
ids (cultivated in MRS broth supplemented with 1 g/l
CaCO3); 3: 10 % NFDMS + 5 % glycerol; 4: 10 %
NFDMS + 10 % sucrose + 0,5 % ascorbic acid + 0,5 %
NH4Cl) were applied. The best survival rates determined
after freeze-drying and storage, especially at room temper-
FEMSLE Congress 2-6-03
127
ature were obtained in the protective medium 4. The ad-
dition of Ca2+ increased the survival rate of both strains
during 1 month storage at room temperature. In general,
survival at room-temperature was the worst, while there
were no di¡erences between results of the storage at 4‡C
and at ^ 20‡C. Bacteriocin production ability of K7 strain
was not altered in any of protective media during freezing,
but later (after drying) it decreased substantially in me-
dium 1, while remained unchanged in other media. Spray
drying was conducted in a laboratory-scale spray dryer at
constant inlet (170‡C) and outlet (95‡C) air temperature.
Heat pre-adaptation (52‡C for 15 min) improved the sur-
vival of K7 cells and their bacteriocin production ability
during 14-days-storage at 4‡C, while it had negative e¡ect
on the survival of LF221 cells.
P1^65
VARIETY OF SOYBEAN ON THE QUALITY OF
THAI FERMENTED SOYBEAN ‘‘THUA-NAO’’ INOC-
ULATE WITH BACILLUS SUBTILIS
L. Sutthirangkun and M. Sukchotiratana
Department of Biology, Faculty of Science, Chiang Mai
University, Chiang Mai 50200, Thailand
Thai fermented soybean from the northern provinces, lo-
cally called ‘‘Thua-Nao’’ is comparable to ‘‘natto’’ but the
inoculum is natural. The product is subsequently non
sticky. The varieties of soybean might have some e¡ect
on PGA production. Therefore, four stains of Bacillus
subtilis i.e. B. subtilis IFO16449 and B. subtilis natto
from Japan, B. subtilis SS9 and B. subtilis SS11 isolated
from thua-nao which were found to produce Q-polygluta-
mic acid (PGA) were used as inoculum for 5 varieties of
soybean i.e. Chiang Mai 3(CM 3), Chaing Mai 60(CM
60), SJ.2 , SJ.4 and SJ.5 and incubated at 45‡C for 48
hours. The amount of PGA produced was then detected
by colorimetry method at 520 nm. B. subtilis SS9 was
found to give maximum PGA from all the varieties of
soybean. The amount of PGA produced on the soybean
substrate was CM 3, 7.30 mg/ml ; CM 60, 7.64 mg/ml;
SJ.2, 7.10 mg/ml; SJ.4,7.20 mg/ml; SJ.5, 7.15 mg/ml re-
spectively. B. subtilis SS9 also produce highest PGA in
PGA producing medium.
128 1st FEMS Congress / Posters 103^505
P1^66
Q-POLYGLUTAMIC ACID PRODUCTION BY BA-
CILLUS SUBTILIS FROM THUA-NAO
M. Sukchotiratana and P. Noisuwan
Microbiology Section, Department of Biology, Faculty of
Science, Chiang Mai University, Chiang Mai 50200, Thai-
land
Forty isolates of bacteria were obtained from Thua-nao,
fermented soybean of Northern Thailand. Twenty-six iso-
lates were found to produce Q- polyglutamic acid (PGA)
detected by Colorimetry method at 520 nm when they
were grown aerobically in PGA producing medium con-
taining 2 % glucose and 1% ammonium sulfate as carbon
and nitrogen sources respectively. The isolate SS11 from
Thua-nao in Sansai district, Chiang Mai which was iden-
ti¢ed to be Bacillus subtilis produced the maximum PGA
of 9.63 mg/ml. The spore suspension was inoculated in
PGA producing medium for optimizing the PGA produc-
tivity condition. The medium containing 2% glucose and
3% ammonium sulfate incubated at 45 OC pH 8.0 for 48
hr with shaking at 200 rpm gave the maximum PGA of
10.44 mg/ml. Two other isolates from Thua-nao i.e. isolate
SS2 and MC18 which were Lactobacillus sp. and Micro-
coccus sp. were found to produce PGA hydrolase enzyme,
endo-type speci¢city, in PGAH medium. This enzyme
caused the unstickiness of Thua-nao in contrast to the
sticky Japanese natto.
P1^67
SYNBIOTIC EFFECT OF LACTOBACILLUS ACIDO-
PHILUS M92
J. SNus›kovic¤, B. Kos, S. Beluhan, J. Frece and S. Matos›ic¤
Department of Biochemical Engineering, Faculty of Food
Technology and Biotechnology, University of Zagreb, Pier-
ottijeva 6, 10 000 Zagreb, Croatia
The scienti¢c basis for the development of probiotics, pre-
biotics or their combination (synbiotics) stems from the
knowledge that the gastrointestinal micro£ora is involved
in protecting the host (man or animals) against coloniza-
tion of the intestinal tract by non-indigenous microorgan-
isms. The use of probiotics (live bene¢cial microorgan-
isms) and prebiotics (non-digestible oligosaccharides) as
food/feed supplements bene¢cially a¡ect the host by im-
proving its intestinal microbial balance. The probiotic
strain Lactobacillus acidophilus M92 was examined for its
ability to assimilate the various kinds of prebiotic sub-
strates (sorbitol, manitol, lactulose, ra⁄nose, oligofructose
and inulin). Bacterial cell yields in the exponential growth
FEMSLE Congress 2-6-03
phases was higher on prebiotic substrates than on glucose.
Calculated parameters of metabolic activity of Lactobacil-
lus acidophilus M92 have shown slightly better assimilation
of polyols and lactulose than the other tested prebiotics.
Increase of the cell number and stimulation of metabolic
activity of examined probiotic strain can lead to enhanced
e¡ect, termed the synbiotic e¡ect.
P1^68
CHARACTERIZATION OF LACTOBACILLUS SPP.
STRAINS ISOLATED FROM DAIRY PRODUCTS
P. Svec(1), V. Drab(2), E. Durnova(3), P. Roubal(4) and
I. Sedlacek(1)
(1) Masaryk University, Faculty of Science, Czech Collec-
tion of Microorganisms, Tvrdeho 14, 602 00 Brno, Czech
Republic; (2) Dairy Research Institute, Sobeslavska 841,
390 02 Tabor, Czech Republic; (3) Institute of Hygiene,
Partyzanske nam. 7, 728 92 Ostrava, Czech Republic; (4)
Dairy Research Institute, Ke Dvoru 12, 160 00 Praha 6,
Czech Republic
A series of twenty biotechnologically usable Lactobacillus
strains with unclear taxonomical position were analysed
and characterized by biochemical tests as well as by ribo-
typing. All strains were characterized by using API 50CH
kit and by conventional tests key for this bacterial group.
Biochemical test results assigned 17 strains to the species
L. acidophilus ; two strains were identi¢ed as L. rhamnosus
and one strain were identi¢ed only as Lactobacillus sp.
Ribotyping with EcoRI and a probe complementary to
16S and 23S rRNA divided all strains analysed into a
few groups. Both L. rhamnosus strains as well as one Lac-
tobacillus sp. strain were di¡erentiated in the full agree-
ment with biochemical test results. Ribotyping revealed
intraspecies diversity among seventeen L. acidophilus
strains and divided this group into four clusters. The
most numerous cluster including eleven strains and the
type strain L. acidophilus CCM 4833T. Obtained results
suggested that ribotyping as applied in this study could
be useful for di¡erentiation of L. acidophilus complex.
This study was supported by Ministry of Agriculture proj-
ect QE 1382.
 1st FEMS Congress / Posters 103^505
P1^69
USE OF REP-PCR TECHNIQUE TO CLASSIFY AND
IDENTIFY A WIDE RANGE OF LACTIC ACID BAC-
TERIA
F. Feutry, M. Oneca, P. Torre
Laboratorio de lactologia, Area de Nutricio¤n y Bromatolo-
g|¤a, Departamento Ciencias del Medio Natural, Universidad
Pu'blica de Navarra, 31006 Pamplona, Spain
This study was performed to evaluate the suitability of the
REP-PCR (Repetitive Extragenic Palindromic-Polymerase
Chaine Reaction) for classifying and identifying a wide
range of lactic acid bacteria. The primer pair REP1R-Dt
and REP2-D was employed. First, the possibility of work-
ing directly with colonia without extraction of DNA was
successfully attempted. In a second step, the REP-PCR
was applied to some reference strains belonging to di¡er-
ent species of the genera Lactococcus, Leuconostoc, En-
terococcus, Lactobacillus, Streptococcus and Pediococcus.
The dendogram generated after cluster analysis showed
that the REP-PCR technique was enable to discriminate
lactic acid bacteria at the genera, at the species and at the
subspecies level. The method was then extended at identi-
¢ed strains isolated from food. It resulted that each one of
the strains studied was located in a distinct cluster includ-
ing the corresponding reference strain and was even enable
to distinguish them at the strain level. In conclusion, REP-
PCR was proven to be a useful gentotypic tool for rapid
and reliable screenning, speciation and strain detection of
a wide range of lactic acid bacteria from food.
P1^70
THE ROLE OF YEAST AND LACTIC ACID BACTE-
RIA ON THE ACCUMULATION OF BIOGENIC
AMINES IN WINE
F. Gardini(1), S. Torriani(2) and G. Suzzi(3)
(1) Dipartimento di Protezione e Valorizzazione Agroali-
mentare, Universita' di Bologna, Via Fanin 46, 40127 Bolo-
gna, Italy ; (2) Dipartimento Scienti¢co Tecnologico, Uni-
versita' di Verona, Strada Le Grazie 15, 37134 Verona,
Italy ; (3) Dipartimento di Scienze degli Alimenti, Univer-
sita' di Teramo, Mosciano Sant’Angelo, Teramo, Italy
Biogenic amines (BA) are basic compounds whose pres-
ence in foods can be a cause of disease for consumers.
They are usually produced by microbial decarboxylation
of amino acids and their presence can be relevant in fer-
mented foods in which the desired growth of microorgan-
isms can be associate with their accumulation. The pres-
ence of such compounds in wine is particularly dangerous
FEMSLE Congress 2-6-03
129
because ethanol inhibits the amine oxidases, which are the
enzymes responsible for their detoxi¢cation. Recently, a
particular attention has been posed on the role of malo-
lactic bacteria on the accumulation of BA. Nevertheless,
also yeasts can contribute to the production of these
amines, either directly or in£uencing the metabolism of
lactic acid bacteria. This work was aimed to the study of
the e¡ects of the yeast strains and some wine-making pro-
cedures on the accumulation of BA in wines subjected to
malolactic fermentation by using a strain of Oenococcus
oeni. The results underlined the potential role of yeasts in
the production of some amine (putrescine and cadaverine),
while bacteria were responsible for the accumulation of 2-
phenylethylamine. In addition, a key role of the type of
must and of the length of alcoholic fermentation was evi-
denced, as well as the presence of precursors in must and
wine.
P1^71
APPLICATION OF DGGE IN PROFILING MICRO-
BIAL COMMUNITIES OF TRADITIONAL FER-
MENTED FOODS
S. Torriani, S. Fasoli, V. Gatto, M. Marzotto, L. Rizzotti
and F. Rossi
Dipartimento Scienti¢co e Tecnologico, Universita' degli
Studi di Verona, Strada le Grazie 15, 37134 Verona, Italy
In this research, we applied the Polymerase Chain Reac-
tion Denaturing Gradient Gel Electrophoresis (PCR-
DGGE) technique to study the microbial communities of
several traditional fermented foods, i.e. fermented milks,
cheeses, sourdoughs, sausages and wines. For each prod-
uct, a speci¢c DNA extraction protocol was developed to
recover ampli¢able DNA from the main microbial groups
associated with these complex matrices. Several sets of
universal and taxon-speci¢c primers, targeting rDNA
genes, were selected on the bases of their ability to di¡er-
entiate the species of interest. A large number of type
strains belonging to the bacterial and yeast species com-
monly found in such foods were used to optimise the
assays and to generate reference ladders. Using the se-
lected sets of primers, di¡erent composite DGGE pro¢les
were obtained for each food. The majority of bands in
DGGE pro¢les were correlated to a de¢ned species either
by using the reference ladders or by sequencing. The dy-
namics of the microbial population were monitored during
manufacturing processes of some foods. Di¡erences in the
composition of the microbial communities were observed,
as indicated by the presence and intensity of the obtained
bands. Generally, no discrepancies were found when the
results of the PCR-DGGE were compared with conven-
tional plate counts integrated by molecular identi¢cation
of single isolates. The use of such culture-independent ap-
130 1st FEMS Congress / Posters 103^505
proach should enable microbiologists to gain, in a short
time, a reliable overview of the microbial components that
more likely determine overall quality of the food products.
P1^72
EXOPOLYSACCHARIDES PRODUCTION BY LAC-
TOBACILLUS PLANTARUM EP56: PHYSIOLOGI-
CAL AND BIOCHEMICAL ASPECTS
R. Tallon(1), P. Bressollier(1,2), and M. C. Urdaci(1)
(1) Ecole Nationale d’Inge¤nieurs des Techniques Agricoles
de Bordeaux (ENITAB), 1 cours du Ge¤ne¤ral de Gaulle,
33175 Gradignan cedex, France; (2) IUT de Limoges, De¤-
partement Ge¤nie Biologique, Alle¤e Andre¤ Maurois, 87000
Limoges, France
Exopolysaccharides (EPS) produced by lactic acid bacteria
present a growing interest because of their rheological
properties and health bene¢ts. To ¢nd new EPS producers,
a total of 32 Lactobacillus plantarum strains were screened
for mucoid phenotypes on MRS-agar plates. One strain,
EP56, was isolated and was grown in a chemically de¢ned
medium (CDM). Two EPS fractions were isolated: 1) a
cell-bound EPS fraction (EPS-b) composed of a single
high molecular weight polymer containing glucose, galac-
tose, N-acetyl galactosamine and traces of glycerol; 2) a
released EPS fraction (EPS-r) composed of the EPS-b
polysaccharide and a second low molecular weight poly-
mer containing glucose, galactose, rhamnose and traces of
glycerol. Presence of phosphate (1.6% w/w) in both EPS-b
and EPS-r, contributes to give a negative net charge to the
polymers. Studies on polysaccharides production kinetics
and location showed that both polysaccharides are synthe-
sized during the exponential growth phase and that cap-
sular material i.e. high molecular mass polysaccharide is
progressively released into the culture medium during sta-
tionary growth phase. EPS production by L. plantarum
EP56 is in£uenced by environmental parameters. When
incubation temperature was reduced from the optimal val-
ue of 37‡C to 18‡C, the amount of polysaccharide is four-
fold increased. EPS-producing properties of L. plantarum
EP56 may present interests for potential applications in
fermented food and probiotics.
FEMSLE Congress 2-6-03
P1^73
TRACE ELEMENT ACCUMULATION BY SPIRULI-
NA PLATENSIS (CYANOBACTERIA) AND ITS DIE-
TARY IMPLICATIONS
L. Varga, J. Szigeti and N. Molna¤r
Institute of Food Science, Faculty of Agricultural and Food
Sciences, University of West Hungary, 15-17 Lucsony
Street, 9200 Mosonmagyaro¤va¤r, Hungary
The purpose of this study was to investigate the accumu-
lation of trace elements by the cyanobacterium Spirulina
platensis, which was cultivated for 8 days in arti¢cial cul-
ture media containing KI, ZnCl2, and Na2SeO3
5H2O at
concentrations ranging from 0.03 to 30 mg/L. Iodine, zinc,
and selenium levels in the Spirulina dry matter (DM) were
then determined. The maximum iodine intake of S. platen-
sis was found to be 450 mg/kgDM. This was observed
when the initial KI level of the culture medium was 30
mg/L. A concentration of 0.03 mg/L KI in the culture
medium resulted in a 370-fold accumulation of iodine in
the cyanobacterial DM. As for zinc, its level was slightly
less than 100 mg/kgDM when 10 mg/L ZnCl2 was present
in the culture medium. The lowest ZnCl2 concentration
tested (0.3 mg/L) caused the highest accumulation of
zinc (47-fold) in the Spirulina DM. Selenium content of
the cyanobacterial biomass was 330 mg/kg when S. platen-
sis was cultivated in a medium containing 30 mg/L Na-
selenite. Similarly to what was experienced with the other
two minerals, the lowest level (0.03 mg/L) of Na2-
SeO3
5H2O in the culture medium resulted in the highest
accumulation of selenium (58-fold) in the Spirulina bio-
mass. The cyanobacteria accumulating trace elements in
their cells are highly suitable for human consumption be-
cause minerals are present in the Spirulina cells in organic
or complex bonds, thus trace elements have an increased
absorption rate and their bene¢cial e¡ects are further im-
proved by the proteins, vitamins and other bioactive sub-
stances of cyanobacteria.
P1^74
THE TECHNOLOGY OF PRODUCTION OF DRY
DAIRY STARTER ON THE BASIS LACTOBACILLUS
ACIDOPHILUS OL4 STRAIN FROM NON-COM-
MERCIAL DAIRY PRODUCT
G. V. Yamborko, L. V. Kaprelyants, V. O. Ivanytsya
I. I. Mechnikov Odessa National University, Odessa, Uk-
raine
From the result of screening biological and technical char-
acteristics of 40 Lactobacillus strains isolated from non-
 1st FEMS Congress / Posters 103^505
commercial fermented products manufactured in the
Odessa region, the strain L. acidophilus OL4, perspective
for obtaining starter has been selected. This strain has
more intensive antagonistic activity than some other in-
dustrial Lactobacillus strains, polyresistance to antibiotic
drugs used in medical practice, and saves viability in mod-
el conditions of a gastrointestinal tract. The technology of
obtaining of a starter contains the following operations:
the growth of cells of L. acidophilus OL4 in the optimized
nutrient medium ; the concentrating bacterial mass with
using ultra¢ltrational membrane and desiccating of a bac-
terial concentrate in a £uidized layer on the inert stu¡. The
usage of the given technology allows to lower energy out-
put in the process of obtaining of a dry bacterial concen-
trate with the high quality and activity in 2 times. The dry
bacterial concentrate L. acidophilus OL4 is powder acquir-
ing the following characteristics : the total quantity of lac-
tobacteria in 1 gram ^ 2,0 X 109 CFU, the level of humid-
ity ^ 3,0 %, the index of dissolubility ^ 0,5 Y 0,2 ml of raw
sediment, the speed of acidoformation ^ 12,6 Y 1,4 oT/h,
the speed of milk fermentation ^ 3,8 Y 0,5 h, the acidity of
a clot ^ 100 Y 4 oT. The dry bacterial concentrate L.
acidophilus OL4 has the capacity to correct normal biota
of intestine of experimental animals caused by antibiotic
therapy, and it colonizes vaginal ecosystem, supporting
constant population of lactobacilli. The bacterial concen-
trate may be used for manufacturing dairy products of
preventive assigning, and it can be applied as a separate
treatment for correction of disbacteriosis.
P1^75
USE OF RIBOTYPING AND PROTEIN PROFILE
ANALYSIS IN THE CHARACTERIZATION OF
BEER CONTAMINANTS
A. Yansanjav(1), I. Hollerova(2), P. SNvec(3) and M.
Neflmec(1)
(1) Department of Microbiology, Faculty of Science, Ma-
saryk University, Tvrdeho 14, 60200 Brno, Czech Republic;
(2) Research Institute of Brewing and Malting, Plc., Lipova
15, 120 44 Praha, Czech Republic; (3) Czech Collection of
Microorganisms, Faculty of Science,Masaryk University,
Tvrdeho 14, 60200 Brno, Czech Republic
Mankind began brewing beer in Mesopotamia around
2000 BC without any knowledge of microorganisms or
enzymes. Hop compounds were presumably introduced
into beer as a strong preservative of quality. Nevertheless,
unwanted microorganisms still grow in beer, including a
few lactic acid and Gram negative bacteria and wild yeasts
which are able to grow even in ¢nished beer with a low
concentration of nutrients and oxygen. These contami-
nants bother consumers and cause economic losses to
breweries. The major contaminants of Czech beer are lac-
FEMSLE Congress 2-6-03
131
tic acid bacteria. An API 50 CH kit was used to identify
lactic acid bacteria but the method gave doubtful results.
Therefore, this work at ¢rst was focused to try to di¡er-
entiate beer spoilage strains from non-spoilage strains us-
ing ribotyping and protein pro¢le analysis using SDS-
PAGE. Secondly, to make clear the classi¢cation of
some closely related strains such as Lactobacillus colli-
noides and Lactobacillus brevis. Identi¢cation of isolates
was made on the basis of locations of the type strains
on the clusters. Ribotyping with EcoRI restriction enzyme
and protein pattern analysis was shown to be a good tool
to di¡erentiate closely related taxons such as L. brevis and
L. collinoides. No correlation was observed between the
ability to spoil beer and their ribotypes or respective pro-
tein pro¢les of tested strains.
P1^76
BIOLOGICAL PROPERTIES OF LACTOBACILLI
ISOLATED FROM HEALTHY CHILDREN
N. O. Yelinska, I. V. Fabiyanska, V. O. Ivanitsa
Odessa National University, Microbiology and Virology De-
partment, Dvoryanskaya St., 2, Odessa, 65 026, Ukraine
From contents of gastric-intestinal tract of healthy chil-
dren at the age of 1-7 years which are habitants of Odessa
and were prophylactic examined has been isolated 67
strains of lactobacilli bacteria. The isolated strains have
been classi¢ed as genus Lactobacillus on the grounds of
studying morphological, cultural and physiological-bio-
chemical properties. The species identi¢cation of the iso-
lated strains has shown that the majority (15,1%) are L.
delbrueckii subsp. bulgaricus. It has been determined a
nature of antagonistic activity of the isolated strains
against opportunistic bacteria and yeast-like fungi. It has
been discovered that lactic acid decreasing medium pH,
play predominant role in depression of the test-culture
growth. Insigni¢cant role peroxide of hydrogen and bac-
tericidal substances in depression pro- and eukaryotic mi-
croorganisms has been shown. It has been demonstrated
that the investigated strains of lactobacilli were character-
ised by strong resistance to the most drugs ^ to polyenilike
antibiotics, phusidine, imidasoles, chinolines, polymixine
and phtorchinolines. More low resistance to macrolides,
tetracyclines, cephalosporines has been noted. The isolated
strains were sensitive to ryphampicines, levomycitines,
penicilines and other L- lactamic antibiotics. The strains
of lactobacilli have strong antagonistic activity to oppor-
tunistic microorganisms and high resistance to antibiotics
and may be recommended as the basis of probiotic prep-
arations for intestinal microbiocenosis have been selected.
132 1st FEMS Congress / Posters 103^505
P1^77
COMPARATIVE STUDY OF BACTERIOCIN PRO-
DUCTION OF TWO LACTOBACILLUS GASSERI
PROBIOTIC STRAINS
N anz›ek Majhenic›, B.
M. Zoric›, H. Holo, I. F. Nes, A. C
› ¤
Bogovic Matijasic and I. Rogelj
Chair of Dairying, Zootechnical Department, Biotechnical
Faculty, University of Ljubljana, Groblje 3, 1230 Domz›ale,
Slovenija
The production of similar or even identical bacteriocins by
di¡erent lactic acid bacteria is not rare, especially if the
strains colonise the same ecological niche. Lactobacillus
gasseri LF221 and K7 are isolates from faeces of two
babies and are producing similar Class II bacteriocins
with a wide antimicrobial activity.The structural genes of
K7 bacteriocins that have been sequenced are shown to be
identical with corresponding parts of LF221 A and LF221
B acidocin genes of LF221 strain which suggests that the
bacteriocins from the two strains are identical. The genes
are in both strains chromosomally located. In the present
study, the missing region of orf* sequence, located up-
stream of orf69 (acidocin LF221 A gene) was obtained.
The complete sequence of orf* was revealed to encode a
79 amino acids long peptide (orf79), the putative comple-
mentary peptide, required for the acidocin LF221 A activ-
ity. The upstream region of acidocin LF221 B genes (the
structural bacteriocin and immunity genes) revealed an orf
with strong homology with ABC-transporter of other
LAB bacteriocin encoding gene clusters. At the same
time, the corresponding DNA regions in K7 were se-
quenced as well and the same gene organisation was found
in that bacteriocin system, too. However, both strains dif-
fer in some phenotypical characteristics, such as the level
and optimal conditions for growth and bacteriocins pro-
duction, RAPD and plasmid pro¢le and adhesion to
Caco-2 cells. Additional gene and expression studies might
explain the di¡erences observed in the bacteriocin produc-
tion between K7 and LF221 strains.
P2^1
ANTIBACTERIAL ACTIVITY OF LACTOBACILLUS
SAKEI ISOLATED FROM TRADITIONAL DRY
SAUSAGE
S. Ammor, I. Chevallier and E. Dufour
Unite¤ de Recherche Typicite¤ des Produits Alimentaires,
ENITA-CF, Site de Marmilhat, 63370, Lempdes, France
Traditional dry sausages rely on natural contamination by
environmental £ora. Therefore, an important role of the
FEMSLE Congress 2-6-03
lactic acid bacteria is to inhibit the competing natural £ora
which includes spoilage bacteria and, occasionally, patho-
gens such as Staphylococcus aureus and Listeria monocy-
togenes. In most cases, the formation of lactic and acetic
acids from carbohydrates and the resulting pH decrease,
are responsible for the antagonistic e¡ect. However, a
pathogenic bacterium such as Listeria monocytogenes is
able to survive both the fermentation and drying stages
of sausage-manufacturing process because of its ability to
survive acidic conditions and its tolerance to considerable
amounts of sodium and nitrite. The use of bacteriocino-
genic starter cultures and/or co-cultures may o¡er promis-
ing solution which minimizes the risks associated with the
growth of undesirable bacteria. A total of 20 Lactobacillus
sakei isolated from traditional dry sausages have been
screened for bacteriocins production towards some indica-
tor organisms belonging to pathogenic (Listeria innocua,
Staphylococcus aureus and Escherichia coli), spoilage
(Pseudomonas £uorescens, Pseudomonas putida, Hafnia al-
vei and Enterococcus faecium) and aromatic (Staphylococ-
cus xylosus and Staphylococcus carnosus) bacteria. Ob-
tained results showed the capacity of one strain to
inhibit the growth of Listeria innocua, Staphylococcus aur-
eus, Pseudomonas £uorescens and Enterococcus faecium,
without inhibition of the growth of the aromatic staph-
ylococci.
P2^2
ENTEROCOCCI IN FOOD PRODUCTS CONSTI-
TUTE A RESERVOIR FOR TRANSFERABLE TETRA-
CYCLINE RESISTANCE GENES
S. R. Andersen, A. Wilcks and T. R. Licht
Institute of Food Safety and Nutrition, Danish Veterinary
and Food Administration, M\rkh\j Bygade 19, DK ^ 2860
S\borg, DK
A signi¢cant amount of tetracycline resistant faecal enter-
ococci can be isolated from Danish food product samples
collected at retail level. Among randomly collected isolates
of E. faecalis from various uncooked food sources, ap-
proximately 30% were resistant to tetracycline with MIC
values exceeding 8 Wg/ml of this drug. We studied the
transferability in vitro on agar surfaces and in broth of
tetracycline resistance from food-borne E. faecalis isolates
to a known E. faecalis recipient strain. Our study shows
that transfer does indeed take place under laboratory con-
ditions. The recipient E. faecalis strain received the tetra-
cycline resistance phenotype in approximately 75% of the
mating experiments (app. 70% on agar surfaces and app.
43% in broth). Further analysis by PCR revealed that
tet(M) was present in approximately 73% of the donor
isolates and that Tn916 was present in several, but not
all of these investigated donor isolates. Results will be
 1st FEMS Congress / Posters 103^505
presented further revealing the genetic basis for the trans-
ferability of the tetracycline resistance phenotype. Our
data provide a measure for potential human exposure to
transferable tetracycline resistance genes deriving from in-
dicator faecal enterococci in raw food shortly before it
enters the consumer kitchen.
P2^3
MICROBIAL CONTAMINATION OF TROUT AND
POND ENVIRONMENTS
C. Armbrust and H.-D. Werlein
Universita«t Hannover, Institute of Food Science, Wunstorfer
Str. 14, 30453 Hannover, Germany
In recent years the proceeds of ¢shery decline. Intensive
aquacultures became important to satisfy the increasing
demand for ¢sh and ¢sh products. Aquaculture is cur-
rently one of the fastest growing food production sectors
in the world. Fish is generally regarded as safe, but prod-
ucts from aquaculture have sometimes been associated
with certain food safety issues. Increasing stocking rates
e.g. can lead to higher microbial contaminations of the
¢sh. Own studies were undertaken to investigate three
trout farms of greater Hannover, Germany for a period
of three month. The experimental approach consisted of
enumerating aerobic psychrotrophic and mesophilic bac-
teria and anaerobic bacteria in pond water, sediment sam-
ples and on the skin and in ¢sh £esh of trout. In addition
all samples were assayed for Pseudomonas, Enterobacter-
iaceae, Salmonella, Aeromonas, yeasts and molds, Clostri-
dium and Clostridium spores. The objective was to eluci-
date the relationship between the obtained results and the
pond environment, temperature of water, stocking rate
and supply of water. It could be detected that the micro-
bial contamination of water was nearly changeless in all
ponds over the whole period although the total viable
counts of sediment and ¢sh samples varied. Stocking
rate and supply of water predominantly in£uenced the
incidence of Enterobacteriaceae in sediment and ¢sh sam-
ples. A decreasing water temperature and stocking rate
tend to result in decreasing microbial contamination of
¢sh samples. Salmonella could not be detected in any of
the samples. Clostridium was always present in the sedi-
ment samples. If Clostridium could be detected in water
samples it mostly occurred in the ¢sh samples too.
FEMSLE Congress 2-6-03
133
P2^4
VIT GENE PROBE METHOD FOR THE RAPID AND
SPECIFIC DETECTION OF LISTERIA MONOCYTO-
GENES IN FOOD SAMPLES
B. Becker(1), A. Sabrowski(2), M. Lohneis(2), W. H.
Holzapfel(1)
(1) Institut fu«r Hygiene und Toxikologie der Bundesfor-
schungsanstalt fu«r Erna«hrung, Haid-und-Neu-Str. 9, D-
76131 Karlsruhe ; (2) Chemisches und Veterina«r-Untersu-
chungsamt, D-76131 Karlsruhe
On account of the high morbidity ( s 30%) experienced
with listeriosis in humans, and the wide distribution of
Listeria species in the food environment, the exact and
rapid identi¢cation of food-borne Listeria strains is of ut-
most importance. Among the recently developed acceler-
ated detection and identi¢cation methods for Listeria, and
especially for L. monocytogenes, those involving gene
probes promise special advantages. Bene¢ts may be ex-
pected in their application in routine control procedures
and in support of quality and safety assurance operations
in the food industry. The objective of this model study was
to assess the applicability of the VIT (‘‘Vermicon Identi-
¢cation Technology’’) System (Vermicon, Mu«nchen, Ger-
many) for the detection of L. monocytogenes in 30 com-
mercial samples of vacuum packaged smoked salmon. The
o⁄cial method according to ‰ 35 LMBG served as refer-
ence procedure. The VIT-system was developed to enable
the application of FISH on a routine basis. Fluorescence
labelled gene probes are applied directly in the sample.
After reaction with target bacteria, these are not removed
by subsequent washing. Upon stimulation with high en-
ergy light source, Listeria spp. show a green shine and L.
monocytogenes both green and red. Using the o⁄cial refer-
ence method, 15 (50%) of the 30 samples studied were
identi¢ed as Listeria positive, and were also con¢rmed
positive by the VIT system. Moreover, 13 samples could
be identi¢ed as L. monocytogenes positive. For 12 samples,
the Listeria numbers were 6 102 cfu/g, and ranged for 3
samples between 150 and 1.9 x 103 cfu/g. The direct ap-
plication of VIT to food samples appears feasible, pro-
vided the cell numbers exceed 103 cfu/g. As Listeria num-
bers are generally 6 102 cfu/g, a pre-enrichment step
seems necessary. Application of the VIT procedure re-
quires 3-4 h. A particular advantage is the speci¢c and
simultaneous detection of bacteria of the genus Listeria
and of L. monocytogenes in a single test.
134 1st FEMS Congress / Posters 103^505
P2^5
OCCURRENCE OF MICROBES OF FOOD SAFETY
IMPORTANCE IN SOILS TREATED WITH SEWAGE
SLUDGE
J. Beczner(1), B. Biro¤(2), Sz. Janko¤(3)
(1) Central Food Research Institute, P.O. Box 393, 1537
Budapest, Hungary ; (2) Research Institute for Soil Science
and Agricultural Chemistry HAS, Herman Otto u. 15, 1022
Budapest, Hungary ; (3) Budapest University of Technology
and Economy, Gellert ter 4, 1111 Budapest, Hungary
A number of food-borne human pathogens are naturally
present in soils. Stabilised, composted and pollutant-free
sewage sludge can be utilised in the agriculture as nitro-
gen-, phosphor-, microelement- and organic matter sup-
plements. Biological treatments of the sludge generally re-
duce the abundance of human pathogens, nevertheless
absolutely pathogen-free status can rarely be reached.
Four representative Hungarian soil-types (calcareous cher-
nozem-, acidic forest-, calcareous sandy- and acidic sandy-
soils) were loaded with two di¡erent sewage-sludge forms
at di¡erent concentrations for three consecutive years.
Soil-samples were quantitatively investigated (in the last
two years) for the presence of the total count, moulds,
coliforms, E. coli, sulphite-reducing clostridia, Salmonella,
Listeria spp., Bacillus cereus by standard food microbio-
logical methods. No essential di¡erence was found be-
tween the e¡ect of sludge-types on the microbiological
status of the soils in general. The number of most mi-
crobes investigated increased as a function of increasing
sludge doses. Salmonellae were eliminated during sludge
treatment, however microbes indicating faecal contamina-
tion survived (coliforms, Enterobacteriaceae). Moulds pre-
ferred acidic soils. The abundance of sulphite-reducing
clostridia increased as a function of sludge dose, more in
the sandy than in the loamy soils. Spores of Bacillus cereus
occurred in all soil types. In spite of Listeria spp. occur-
ring in every soils and treatments, neither the presence nor
the absence of L. monocytogenes could be unanimously
proved. The sludge-doses increased the microbial abun-
dance of the soils, however the cell counts indicated rela-
tively fast elimination of the introduced microbes probably
due to the activity of indigenous competitive micro-£ora,
or some other environmental factors.
The work was supported by Ministry of Education:
NKFP-4/0028/2002, Ministry of Agriculture and Regional
Development: KF-78/7/2001, and COST 838.
FEMSLE Congress 2-6-03
P2^6
ENDOTOXINS ON GRASSES INFLUENCED BY DIF-
FERENT INTENSITY LEVELS OF MANAGEMENT
U. Behrendt(1) and A. Ulrich(2)
Centre for Agricultural Landscape and Land Use Research
Mu«ncheberg (ZALF e.V.), Institute of Primary Production
and Microbial Ecology (1) Gutshof 7, D-14641 Paulinenaue
and (2) Eberswalder Str. 84, D-15374 Mu«ncheberg, Ger-
many
Measures of extensi¢cation for sustainable pasture man-
agement are accompanied by a reduced frequency of uti-
lization and a lower intensity of fertilization. Especially
the delay of the ¢rst use from May until mid July, which
is required within the framework of bird protection pro-
grammes, goes along with microclimatic changes in the
¢eld as well as with changes in the physiological status
of the host plant. A consequence can be a structural and
quantitative alteration of microbial communities that in-
£uence the formation and enrichment of microbial toxins
on over-matured herbages. The endotoxins (lipopolysac-
charides) of Gram-negative bacteria are of special interest
since increased concentrations in feed-stu¡s of ruminants
can trigger certain endotoxin associated diseases. Thus, the
content of bioactive endotoxin in herbages that di¡er in
senescence, plant species composition and history of man-
agement was determined by LAL (Limulus amebocyte ly-
sate) testing. Furthermore, the bacterial community struc-
ture of the phyllosphere was characterized by cultural and
culture-independent analyses (selective media; terminal re-
striction fragment length polymorphism of 16S rDNA) in
order to ¢nd relations considering the enrichment of en-
dotoxins. Results of this study revealed the senescence of
plant material to be the main cause for increased endotox-
in concentrations on grasses. In over-matured plants of
mid July, contents were found that are probable to a¡ect
animal performance. Additionally, seasonal changes in the
microbial community were a further factor in£uencing en-
dotoxins on herbages.
P2^7
SCREENING FOR PLANT ANTIMUTAGENS WITH
MICROBIAL TESTS
T. Beric-Bjedov, J. Knezevic-Vukcevic, B. Vukovic-Gacic,
D. Mitic, B. Nikolic, J. Stanojevic, S. Stankovic, D. Simic
Chair of Microbiology, Faculty of Biology, University of
Belgrade, Belgrade, Yugoslavia
The naturally occurring dietary constituents might modu-
late the mutagenesis and possibly carcinogenesis process.
 1st FEMS Congress / Posters 103^505
In the study of plant antimutagens, microorganisms pro-
vide a powerful experimental tool. For screening of plant
antimutagens and for detection of mechanisms of antimu-
tagenesis, we used E. coli K12 assay (Simic at al., 1998).
The test consist of: Test A ^ repair pro¢cient strain and its
uvrA counterpart for detection of induced mutations; Test
B -isogenic mutator strains (i) de¢cient in methyl ^ di-
rected mismatch repair for detection of spontaneous mu-
tations due to replication errors and (ii) de¢cient in remov-
ing 8-oxo-G for detection of spontaneous mutations due
to oxidative damage; Test C ^ isogenic repair pro¢cient
strain constitutive for alkaline phosphatase carrying
s¢A: :lacZ fusion for measuring the level of SOS induction
(induction of mutagenic SOS repair); Test D ^ strains
carrying di¡erent recA alleles and two non-overlapping
deletions in duplicated lac operon for measuring intra-
chromosomal recombination. The results obtained in E.
coli are compared with standard S. typhimurium (Ames)
and S. cerevisiae D7 tests. We screened more than 25
di¡erently prepared extracts of wild and cultivated sage
(Salvia o⁄cinalis L.) and basil (Ocimum basilicum L.), as
well as their pure constituents. Monoterpenes from culti-
vated sage and basil inhibit UV mutagenesis in bioantimu-
tagenic manner. Protective e¡ect of essential oils and pure
monoterpenes is due to modulation of DNA repair, i.e. by
enhancement of the error-free (excision, recombination)
and by inhibition of the error-prone (SOS mutagenic) re-
pair pathways.
P2^8
MULTIPLEX-PCR AS A CONVENIENT TOOL IN
FOOD SAFETY TO DETECT SIMULTANEOUSLY
DIFFERENT FUSARIUM SPECIES IN WHEAT
B. Birzele, J. Bu«nker, J. Kra«mer and A. Prange
Dept. of Agricultural and Food Microbiology, University of
Bonn, Meckenheimer Allee 168, 53115 Bonn, Germany
Mycotoxin producing and phytopathogenic fungi of the
genus Fusarium infect wheat during the vegetation period.
Their mycotoxins, e.g. deoxynivalenol (DON), are pre-
dominantly produced in the ¢eld, but also after harvest
when storage conditions are impropriate or when grains
have to be harvested at too high moisture contents and
immediate drying of grains is impossible. When storing
wheat under suboptimal conditions further mycotoxin in-
crease is possible lowering wheat quality and food safety.
In order to investigate the in£uence of suboptimal storage
conditions on the ability of Fusarium spp. to survive and
to produce DON, di¡erent storage trials were conducted
with moisture contents of 17% and 20% and temperatures
of 15‡C and 20‡C. DON contents were determined by
ELISA. A multiplex-PCR assay was designed to detect
the four Fusarium species, which are most frequently iso-
FEMSLE Congress 2-6-03
135
lated from grains (grown in the Rhineland [1]) by cultural
methods. By this PCR assay a precise and simultaneous
detection of F. graminearum, F. culmorum, F. poae and F.
avenaceum on the basis of established PCR reactions [2,3]
was achieved thus o¡ering a time e⁄cient and convenient
means of quality control to detect Fusarium species in
food. To validate the PCR results, Fusarium species were
also isolated by incubating 200 grains per sample on se-
lective media and were subsequently di¡entiated micro-
scopically.
[1] B. Birzele et al. (2002) Eur. J. Plant Pathol. 108: 667-
673. [2] A.G. Schilling et al. (1996) Phytopathol. 86: 515-
522. [3] D.W. Parry and P. Nicholson (1996) Plant Pathol.
45: 383-391.
P2^9
TOXOPLASMOSIS IN SERBIA : A DOMINANTLY
MEAT-BORNE INFECTION
B. Bobic, A. Nikolic and O. Djurkovic-Djakovic
Toxoplasmosis Research Laboratory, Institute for Medical
Research, P.O. Box 102, 11129 Belgrade, Yugoslavia
No program of prevention of congenital toxoplasmosis
has ever been implemented in Serbia. To de¢ne the sub-
groups of the women of generative age at particular risk of
Toxoplasma gondii infection during pregnancy, a study to
identify the risk factors for infection was conducted. The
series involved 2936 women 15-45 years of age from
throughout Serbia serologically tested for anti-Toxoplasma
antibodies (Sabin-Feldman dye test) during the 1988-1997
period. Analysis of the data collected by means of an
epidemiological questionnaire showed undercooked meat
consumption to be the single predictor of infection (of
those usually considered) in the whole series (P=0.003).
Undercooked meat consumption signi¢cantly (P=0.000)
contributed to infection in the Belgrade suburban area,
as opposed to both urban and rural settings. Its in£uence
continuously decreased over the study period and paral-
leled the decrease in the prevalence of infection from
85.9% in 1988 to 39.1% in 1997. Analysis of food meats
by animal species showed an association between infection
and beef consumption (P=0.002), which triggered prelimi-
nary studies of the infection rate in various food animals
in Serbia currently underway. Consumption of meat and
meat products out of home also contributed to infection
(P=0.001 and P=0.000, respectively). The same analysis
performed in women grouped according to obstetric his-
tory showed a higher infection rate in women with a his-
tory of pathological pregnancies who aknowledged under-
cooked meat consumption than in those who denied it,
while there was no such di¡erence among women with
no pathological pregnancies regardless of eating habits
(P=0.420).
136 1st FEMS Congress / Posters 103^505
P2^10
THERMAL INACTIVATION OF LISTERIA INNO-
CUA IN A RANGE OF SOUS VIDE MODEL PROD-
UCTS : SOME EFFECTS OF PRIOR HEAT STRESS
AND ACID ADAPTATION
P. Bourke, D. O’ Beirne
Food Science Research Centre, University of Limerick, Cas-
tletroy, Limerick, Ireland
Thermal inactivation D- and z- values of Listeria innocua
were determined in a range of sous vide processed model
products: sliced potato, broccoli £orets, and beef and sal-
mon pieces. The thermal inactivation of populations sub-
jected to prior heat stress or acid adaptation was also
examined in broccoli and potato model products. Two
commonly used commercial processes for sous vide foods:
70‡C for 2 minutes and 85‡C for 11 minutes were also
challenged with acid adapted populations. The e¡ects of
some post processing storage conditions were also inves-
tigated. D-values were a¡ected by product type, with sig-
ni¢cantly higher D-values in salmon and beef products
than in potato and broccoli samples. Prior heat stress or
acid adaptation of the inoculum resulted in greater heat
resistance in potato samples (p 6 0.05). Similar e¡ects
were observed for the broccoli model product but were
not always signi¢cant. The commercial pasteurisation pro-
cess of 70‡C for 2 minutes resulted in 6 log reductions in
both acid adapted and non-adapted populations, but Lis-
teria innocua was still detected using conventional plate
count methods. Following a process of 11 minutes at
85‡C, the organism was detected by enrichment proce-
dures and was found in all model products. Subjecting
samples to freezing (liquid nitrogen) followed by frozen
storage at -40‡C reduced populations compared with sam-
ples refrigerated (8‡C) post processing. Commercial pro-
cesses need to address e¡ects of product and stresses on
survival of pathogens in products subjected to sous vide
processing.
FEMSLE Congress 2-6-03
P2^11
MICROBIAL INSPECTION OF ICE-CREAM PLANT
FOR MINIMIZING THE RISK OF CONTAMINA-
TIONS
S. Bovonsombut(1), K. Kongsoonthorn(1), S. Buabarn(1)
and S. Bovonsombut(2)
(1) Department of Biology, Faculty of Science, Chiang Mai
University, Chiang Mai 50202, Thailand; (2) Department
of Food Technology, Faculty of Engineering and Agro-In-
dustry, Maejo University, Chiang Mai, 50290, Thailand
This article describes a microbial inspection carried out on
ice-cream plant in Chiang Mai province (north of Thai-
land). The samples obtained were the intermediate prod-
ucts of ice cream from suspected equipments in the process
line, the ¢nal products and water. And the swab test con-
ducted on the surface of the equipments as such and the
hands of some employees. Using classical methods, aero-
bic plate count, coliform, faecal coliform, Escherichia coli
and Staphylococcus aureus were enumerated. S. aureus
were the main species found throughout the experiment
due to the cross contamination by the human. Water sup-
ply system still gives a good quality of drinking water ac-
cording to the law.
P2^12
CHARACTERISATION OF LISTERIA MONOCYTO-
GENES, SEROTYPE 1/2b, USING IN VIVO PATHO-
GENICITY TESTS AND AMPLIFICATION PRO-
FILES ANALYSIS OF THE VIRULENCE GENES iap
AND inl
P. Cabrita(1), S. Ferreira-Dias(2) and L. Brito(1)
(1) Laborato¤rio de Microbiologia, DBEB and (2) Centro
de Microbiologia e Indu¤strias Agr|¤colas DAIAT, Instituto
Superior de Agronomia, Tapada da Ajuda 1349-017 Lisboa,
Portugal
Listeria monocytogenes, a foodborne pathogen, is capable
of causing serious invasive disease in both humans and
animals. The iap and inl genes of L. monocytogenes, which
code for important virulence proteins present variable re-
gions among strains belonging to the same serovar. In this
work, a total of 39 L. monocytogenes isolates from milk,
smoked meat and chicken carcass, belonging to serotypes
1/2a, 1/2b, 3b, 4a and 4c, were analysed by a combination
of PCR and restriction enzyme analysis (REA) of the gene
inl and by PCR ampli¢cation with speci¢c primers for the
gene iap. The same isolates were typed by RAPD ampli-
¢cation with three di¡erent primers. Serial dilutions of cell
suspensions, from isolates belonging to serotype 1/2b, were
 1st FEMS Congress / Posters 103^505
also tested for pathogenicity by intraperitoneal injection
into immunocompetent mice. The population of L. mono-
cytogenes necessary to kill about 50% of the mice (LD50)
in each test, set within 7 days, ranged from 104 to 106 cfu.
The association among pathogenicity, ampli¢cation pro-
¢les cleavage patterns and origin of the strains was eval-
uated using multivariate data analysis (Principal Compo-
nent Analysis, Cluster Analysis and Discriminant
Analysis). According to cleavage patterns and ampli¢ca-
tion pro¢les, strains belonging to the same serovar may be
divided into di¡erent groups, which may di¡er in virulence
potential.
We thank M.Guerra for providing the isolates of L. mono-
cytogenes.
P2^13
IMMUNOCHEMICAL DETECTION OF NISIN FROM
LACTOCOCCUS LACTIS IN BIOLOGICAL SAM-
PLES
C.-H. Brogren, I. Badiola, A. Johansen. B. Jelle and F. K.
Vogensen
Department of Dairy and Food Chemistry, Centre of Ad-
vance Food Sciences, The Royal Veterinary and Agricultur-
al University, Frederiksberg, Denmark
The bactericin nisin A is used as a natural antibiotic ad-
ditive in dairy products to inhibit growth of Gram-positive
pathogens, e.g. Listeria monocytogenes. It is not known
whether nisin impacts on the natural intestinal £ora in
vivo. As it is produced by the non-probiotic Lactococus
lactis, no in situ intestinal colonisation is expected, and
any nicin in the intestine must derive directly from oral
intake. Proteolitic enzymes such as alpha-chymotrypsin
can cleave nisin and abolish its activity. It is not known
if the digestion fragments have any in£uence on the nor-
mal intestinal £ora. This study concerns immunochemical
detection of nisin and its degradation product. Polyclonal
antiserum was raised in rabbit against the intact puri¢ed
nisin conjugated to KLH. The no-nonsense antibodies spe-
ci¢c to nisin were puri¢ed by a⁄nity chromatography on
nisin-ECH-Sepharose columns. Pure antibody or antigen
was used to coat ELISA plates for competitive and non-
competitive immunoassays, respectively. A detection limit
of 5 ng/ml was obtained, compared to about 1 ug/ml in the
bioassay. This is, to our knowledge, the most sensitive
immunoassay so far described. Although biological activ-
ity is abolished after alpha-chymotrypsin digestion or in
the presence of faecal matrix, fragments of nisin could still
be detected by the immunochemical methods. Proteolytic
degration was studied further by gel electrophoreses and
immunoblotting. The sensitive immunoassays will now be
applied to faecal samples obtained from in vivo experi-
ments in rats given oral challenge with pure nisin or Lac-
FEMSLE Congress 2-6-03
137
tococcus lactis producing or non-producing mutants. We
are attempting to develop suspension array beads immu-
noassay applicable to fast £ow cytometric analysis. It is
unlikely that degraded nisin has any in£uence on the nat-
ural intestinal £ora, since the biological activity is lost in
the gastro-intestinal tract. The level of nisin and its deg-
radation fragments in the intestine will be signi¢cantly
lower than observed in pure culture supernatant, and is
expected to be below the bioassay detection limit but
above the immunochemical detection limit.
P2^14
HYGIENIZATION OF CHICKEN EGGS BY IONIS-
ING RADIATION LEADS TO SAFETY EGGS
S. Cabo Verde(1), R. Tenreiro(2) and M. L. Botelho(1)
(1) Instituto Tecnolo¤gico e Nuclear, Estrada Nacional 10,
Apartado 21, 2686-953 Sacave¤m, Portugal; (2) Departa-
mento de Biologia Vegetal e Centro de Gene¤tica e Biologia
Molecular, Faculdade de Cie“ncias da Universidade de Lis-
boa, 1749-016 Lisboa, Portugal
The purpose of this study is to develop the application of
irradiation technology to chicken eggs in order to get a
product free of pathogenic microorganisms. Bioburden
values of eggs from chicken with di¡erent ages (n=150)
were found to be no signi¢cantly di¡erent (p 6 0.05).
An average value of (2.0 Y 0.3).105 cfu/egg was obtained
for the shell. Two major morphological types were char-
acterized in eggs natural microbiota, but no Salmonella
and Campylobacter were detected. HACCP studies indi-
cated the feed as a critical point. Dosimetry studies were
carried out in gamma and electron beam facilities to ¢nd
the best geometries and dose rate for irradiation. Whole
eggs were arti¢cially contaminated with control strains of
Salmonella typhimurium and Campylobacter coli and irra-
diated in the Q facility at sub-lethal doses (0.2 to 1 kGy)
with a dose rate of 1.0 kGy/h. Dvalue varied between 0.31
kGy and 0.26 kGy in S. typhimurium and between 0.21
kGy and 0.18 kGy in C. coli, for shell and yolk+white
respectively. Using sub-lethal doses up to 5 kGy, the Dval-
ue of natural microbiota in whole eggs was 1.29 kGy for
gamma radiation and 1.42 kGy using the electron beam.
Surviving microbiota after egg irradiation includes the
same two major morphological types, although a selection
of original pigmented strains seems to occur. As no Sal-
monella and Campylobacter were also detected and biobur-
den was e¡ectively reduced, results point out that low
irradiation doses could guarantee egg safety.
138 1st FEMS Congress / Posters 103^505
P2^15
DETECTION OF MICROBIAL SEQUENCES IN-
SERTED INTO GENETICALLY MODIFIED PLANTS
N el
K. Cankar, T. Dreo, K. Gruden, M. Ravnikar and J. Z
National Institute of Biology, Dept. of Plant Physiology and
Biotechnology, Vec›na pot 111, 1000 Ljubljana
Products from GM soybeans, corn and oilseed rape are on
the European market for some years. The products that
contain genetically modi¢ed organisms (GMO) in concen-
tration above 1% must be labelled in order to guarantee
the consumer’s choice between GM and non-GM prod-
ucts. Methods for GMO detection are predominantly
based on detection of DNA sequences inserted into genet-
ically modi¢ed plants. At the moment four transgenic lines
of corn, one transgenic line of soybean and 4 lines of
rapeseed are approved for use in food and feed. Promoters
and terminators used in genetically modi¢ed plants origi-
nate from plant associated viruses and bacteria of which
35S promoter from cauli£ower mosaic virus (CaMV) and
NOS terminator from Agrobacterium tumefaciens are most
frequently used and enable screening of above described
maize and soybean lines. In our laboratory qualitative
screening methods have been adapted for Real-time
PCR, thus minimising the possibility of contamination
with PCR products and increase of reliability of the meth-
od. The major disadvantage of 35S and NOS screening is
the possibility of false positive results in case of presence
of CaMV virus or bacterium Agrobacterium tumefaciens.
Positive results must be therefore con¢rmed and GMO
content quanti¢ed by construct speci¢c ampli¢cation
methods. To di¡erentiate between virus infected plants
and genetically modi¢ed plants a system for Real-time
PCR was designed that allows recognition of virus coat
protein gene. This assay is especially important for intro-
duction of detection methods for GM rapeseed, since ra-
peseed is susceptible to CaMV virus infections.
FEMSLE Congress 2-6-03
P2^16
INACTIVATION OF SUSPENDED AND ATTACHED
TO FOOD CONTACT MATERIALS SALMONELLA
AND LISTERIA MONOCYTOGENES CELLS BY
HEAT AND COMMERCIAL DISINFECTANTS
J. Carballo, R. Marra and A. B. Arau¤jo
Ł rea de Microbiolog|¤a, Departamento de Biolog|¤a Funcio-
A
nal y Ciencias de la Salud, Universidad de Vigo, Facultad de
Ciencias, Campus de Ourense, As Lagoas s/n, 32004 Our-
ense, Spain
Bacterial adherence and colonization of surfaces may
cause problems in the food industry. Food can be contam-
inated with spoilage and/or pathogenic bacteria because
attached cells become more resistant to cleaning and dis-
infection procedures. Salmonella and Listeria monocyto-
genes are two pathogenic bacteria which are able to adhere
to surfaces.The objective of this study was to compare the
e¡ect of di¡erent concentrations of two commercial sani-
tizers, heat (85 ‡C, 15 minutes) and their combinations on
Salmonella spp. and L. monocytogenes cells in suspension
and attached to food contact materials (stainless steel,
polytetra£uorethylene and rubber). Only attached L.
monocytogenes cells survived heat treatment, although
the percentage of survival was very low ( 6 1%). Attached
bacteria survived the treatment with the concentrations
recommended by the manufacturers of two disinfectants
with di¡erent composition. Concentrations double and
quadruple than that recommended by the manufacturer
of the sanitizer based on alquyldiethylenediamineglicyne
and di-alquyldiamineethylglicyne reduced in 1 or 2 log
cycles, respectively, the surviving population to the recom-
mended one. Sanitizer containing quaternary ammonium
compounds used at double concentration reduced attached
bacteria by 2-3 log cycles and no attached bacteria sur-
vived the treatment with the concentration quadruple than
the recommended, irrespective to the surface to which
bacteria were adhered. Bacteria were eradicated from the
surfaces with the combination of heat treatment with each
of the sanitizers at recommended concentrations. Thus,
combining heat treatment with chemical treatment would
be useful to improve the desinfection of food industry
surfaces without increasing the in use concentrations of
sanitizers.
 1st FEMS Congress / Posters 103^505
P2^17
INHIBITORY ACTION OF NISIN AGAINST LISTE-
RIA MONOCYTOGENES ON SURFACES
J. Carballo, P. Morais and A. B. Arau¤jo
Ł rea de Microbiolog|¤a, Departamento de Biolog|¤a Funcio-
A eight expert laboratories in the United Kingdom. Samples
nal y Ciencias de la Salud, Universidad de Vigo, Facultad de
Ciencias, Campus de Ourense, As Lagoas s/n, 32004 Our-
ense, Spain
Listeria monocytogenes are pathogenic bacteria that can
contaminate food and cause listeriosis (a food-borne dis-
ease with high mortality rate). These bacteria were isolated
from walls, £oors and equipment of the food industry.
Their ability to attach to di¡erent surfaces and form bio-
¢lms increases their resistance to cleaning and desinfection
operations. It was demonstrated that nisin adsorbed on
surfaces reduced L. monocytogenes adherence to them
and inhibited the growth of this pathogen in food when
adsorbed on food packaging ¢lms. The objective of this
study was to investigate the e¡ect of nisin suspensions
with di¡erent concentrations against planktonic and at-
tached (to stainless steel, polyethyleneterephthalate and
rubber) L. monocytogenes cells. Attached cells were more
resistant than their planktonic counterparts. Planktonic
cells in populations of 105 and 108 CFU were eradicated
with concentrations of nisin of 5 and 50 Wg/mL (depending
on the strain) and 1000 Wg/mL, respectively. On the con-
trary, amounts of attached cells between 6x106 CFU (case
of strain ES25 attached to rubber) and 9x107 CFU (strain
ES24 adhered to stainless steel) survived the treatment
with 10.000 Wg/mL of nisin, although the degree of surviv-
al was very low. Reductions in 3-4 log cycles were
achieved, independently of the material to which bacteria
were attached. The treatment of surfaces with nisin could
contribute to control L. monocytogenes in surfaces of the
food industry.
P2^18
DEVELOPMENT AND VALIDATION OF A METH-
OD TO DETECT CRYPTOSPORIDIUM PARVUM
ON RASPBERRIES: TOWARDS A STANDARD
METHOD
N. Cook(1), N. Wilkinson(1), K. Paton(2), K. Barker(1),
R. Nicholls(2) and H. V. Smith(2)
(1) Central Science Laboratory, Sand Hutton, York YO41
1LZ, UK; (2) Scottish Parasite Diagnostic Laboratory,
Stobhill Hospital, Balornock Road, Glasgow G21 3UW, UK
A method was developed based on elution of C. parvum
oocysts from raspberries by rolling in 1 M glycine pH 5.5,
FEMSLE Congress 2-6-03
139
concentration of oocysts by centrifugation, IMS separa-
tion, labelling of oocysts with FITC-MAb / DAPI, and
microscopic identi¢cation and enumeration. In the origi-
nating laboratories the recovery e⁄ciency of the method
was 41.0 Y 13.0 % (n = 30). The method was subjected to
interlaboratory collaborative trial, involving eight expert
laboratories in the United Kingdom. The trial involved
comprised 60 g raspberries. They were inoculated at three
levels : low (8.5 ^ 26.8 oocysts), medium (29.7 ^ 65.7 oo-
cysts), and high (53.9 ^ 131.3 oocysts). Blank, or uninocu-
lated, samples were also tested. The method had a overall
sensitivity (correct identi¢cation of all inoculated samples)
of 90.9 %, and a speci¢city (correct identi¢cation of unin-
oculated samples) of 83.3 %. The total mean percentage
recovery (from all inoculated samples) produced by the
method was 49.2 Y 28.3 %. Testing all samples whether
inoculated or blank, the overall accordance, or repeatabil-
ity, was 80.4 %. The overall concordance, or reproducibil-
ity, was 80.2 %. The qualitative performance aspects of the
method were just as reproducible between laboratories, as
repeatable within a laboratory. The results of the collab-
orative trial indicate that the assay may be con¢dently
applied in analytical microbiological laboratories.
The work was supported by the United Kingdom Food
Standards Agency.
P2^19
DEVELOPMENT AND VALIDATION OF A METH-
OD TO DETECT CRYPTOSPORIDIUM PARVUM
ON LETTUCE: TOWARDS A STANDARD METHOD
N. Cook(1), N. Wilkinson(1), K. Paton(2), K. Barker(1),
R. Nicholls(2) and H. V. Smith(2)
(1) Central Science Laboratory, Sand Hutton, York YO41
1LZ, UK; (2) Scottish Parasite Diagnostic Laboratory,
Stobhill Hospital, Balornock Road, Glasgow G21 3UW, UK
A method was developed based on elution of C. parvum
oocysts from lettuce by stomaching with 1 M glycine pH
5.5, concentration of oocysts by centrifugation, IMS sep-
aration, labelling of oocysts with FITC-MAb / DAPI, and
microscopic identi¢cation and enumeration. In the origi-
nating laboratories the recovery e⁄ciency of the method
was 59 Y 12 % (n = 30). The method was subjected to
interlaboratory collaborative trial, involving eight expert
laboratories in the United Kingdom. Samples comprised
30 g lettuce. They were inoculated at three levels : low (8.5
^ 14.2 oocysts), medium (53.5 ^ 62.6 oocysts), and high
(111.3 ^ 135.0 oocysts). Blank, or uninoculated, samples
were also tested. The method had an overall sensitivity
(correct identi¢cation of all inoculated samples) of 89.6
%, and a speci¢city (correct identi¢cation of uninoculated
samples) of 85.4 %. The total mean percentage recovery
140 1st FEMS Congress / Posters 103^505
(from all inoculated samples) produced by the method was
40.0 Y 26.3 %. Testing all samples whether inoculated or
blank, the overall accordance, or repeatability, was 80.1
%. The overall concordance, or reproducibility, was 79.5
%. The qualitative performance aspects of the method
were just as reproducible between laboratories, as repeat-
able within a laboratory. The results of the collaborative
trial indicate that the assay may be con¢dently applied in
analytical microbiological laboratories.
The work was supported by the United Kingdom Food
Standards Agency.
P2^20
A VALIDATED PCR ASSAY FOR DETECTION OF
LISTERIA MONOCYTOGENES : TOWARDS AN IN-
TERNATIONAL STANDARD
M. D’Agostino(1), M. Wagner(2), J. A. Vazquez-Bo-
land(3), J. Hoorfar(4), R. Karpiskova(5), S. Novella(3),
J. Ellison(1), A. Murray(1) and N. Cook(1)
(1) DEFRA Central Science Laboratory (CSL), Sand
Hutton, York, YO41 1LZ, UK; (2) Institute for Milk Hy-
giene, Milk Technology and Food Science, Veterinaerplatz
1, Vienna 1210, Austria ; (3) University Complutense de
Madrid, 28040 Madrid, Spain; (4) Department of Bacteri-
ology, Danish Veterinary Institute, 27 Bulowsvej, Copenha-
gen 1790, Denmark; (5) National Institute of Public
Health, Palackeho 1-3, Brno 61242, Czech Republic
Successful validation of any method should be necessary
for its adoption as a standard. We report a PCR assay for
Listeria monocytogenes. It has a detection limit of less than
10 bacterial cells per reaction. When tested against 38 L.
monocytogenes strains and 52 non-target strains (29 non-
monocytogenes Listeria spp. and 23 non-Listeria spp.), the
assay had a high diagnostic accuracy, being 100 % inclu-
sive and exclusive. It was evaluated in an international
ring trial involving 12 participating European laboratories
(from Austria, Czech Republic, Denmark, Germany (3
participating laboratories), Greece, Ireland, Poland, Spain
(2 participating laboratories), and Sweden). In this trial,
the method was tested against an additional 14 L. mono-
cytogenes strains and 12 non-monocytogenes Listeria
strains. Here, the inclusivity was 100 % and the exclusivity
was 99.4 %. The PCR assay was just as reproducible be-
tween laboratories, as repeatable within a laboratory, and
thus may be con¢dently applied in analytical microbiolog-
ical laboratories.
FEMSLE Congress 2-6-03
P2^21
A VALIDATED PCR-BASED METHOD FOR DETEC-
TION OF LISTERIA MONOCYTOGENES IN RAW
MILK : TOWARDS AN INTERNATIONAL STAN-
DARD
M. D’Agostino(1), J. Hoorfar(2), and N. Cook(1)
(1) DEFRA Central Science Laboratory (CSL), Sand
Hutton, York, YO41 1LZ, UK; (2) Department of Bacte-
riology, Danish Veterinary Institute, 27 Bulowsvej, Copen-
hagen 1790, Denmark
An enrichment / PCR method for the detection of Listeria
monocytogenes in raw milk was tested in an international
collaborative trial, involving thirteen European laborato-
ries (in Austria, Czech Republic (2), Denmark, France,
Germany, Greece, Ireland, The Netherlands, Poland, Por-
tugal, Slovakia, and Spain) in December 2002 and January
2003. Samples of raw milk inoculated at approximately 2,
20 and 200 cells per 25 ml were enriched in half-Frasers’s
broth, and sent to each participant. The participants incu-
bated the samples in the secondary enrichment broth
LEM2 and then applied the PCR. The accuracy parame-
ters ^ sensitivity and speci¢city ^ of the method were de-
termined. Sensitivity is the percentage of inoculated sam-
ples that gave a positive result. Speci¢city is the percentage
of uninoculated samples that gave a negative result. Re-
peatability and reproducibility were determined by calcu-
lating accordance and concordance values. Accordance is
de¢ned as the percentage chance of ¢nding the same result
(i.e. both positive or negative whether correctly or not)
from two identical inoculated samples analysed in the
same laboratory under standard repeatability conditions.
Concordance is de¢ned as the percentage chance of ¢nding
the same result (i.e. both positive or negative whether cor-
rectly or not) from two identical inoculated samples ana-
lysed in two di¡erent laboratory under standard repeat-
ability conditions. The concordance odds ratio was
calculated in order to assess the degree of between-labo-
ratory variation in results. The completed results of the
trial and evaluation of the method will be available at
the congress.
 1st FEMS Congress / Posters 103^505
P2^22
EFFECT OF THE ADDED STARTER CULTURE ON
THE SURVIVAL OF LISTERIA MONOCYTOGENES
IN DRY SAUSAGES
L. De Zutter, K. Houf
Department of Veterinary Food Inspection, Faculty of Vet-
erinary Medicine, Ghent University, Merelbeke, Belgium
Traditional dry sausages are frequently contaminated with
L. monocytogenes. The present paper reported the behav-
iour of L. monocytogenes during the production and stor-
age of such sausages inoculated with 2 di¡erent starter
cultures (SM272 and TSC150). For the production of all
sausages one batch of meat was used. After comminuting
the meat and addition of the adjectives, one part was in-
oculated with SM272 and the other with TSC150. Each
portion was further divided in 3 parts : 1/ no addition of L.
monocytogenes (controls), 2/ addition of 102/g and 3/ ad-
dition of 105 L. monocytogenes/g. All sausages were pro-
duced and stored in the same conditions as the commercial
ones. From each type of sausages a portion was individu-
ally packaged in MAP after 7 days.The control sausages
were initially L. monocytogenes positive. During produc-
tion and storage less sausages were found positive contain-
ing the starter culture SM272. For inoculated sausages a
larger reduction was observed with the starter culture
SM272 than with TSC150: in low level inoculated sau-
sages (102/g) reduction levels of 103 and 101 and in high
level inoculated sausages (105/g) reduction levels of 105
and 102 were stated respectively. In MAP packed sausages
L. monocytogenes levels stabilized during the second half
of the storage period. In conclusion these challenge tests
showed that the reduction of L. monocytogenes depended
on the starter culture used and the number of L. mono-
cytogenes initially presented. At the moment that such
sausages are commercialized (7 days after production),
absence of L. monocytogenes cannot be guaranteed.
P2^23
SURVEY OF ANTIBIOTIC SUSCEPTIBILITY IN
LACTIC ACID BACTERIA ISOLATED FROM CAB-
RALES, A TRADITIONAL BLUE-VEINED SPANISH
CHEESE
A. B. Flo¤rez, S. Delgado and B. Mayo
Instituto de Productos La¤cteos de Asturias (CSIC), 33300-
Villaviciosa, Asturias, Spain
At present, there is a great concern about the spread of
antibiotic resistance determinants by bacteria from the
food chain [1]. Thus, strains intended to be used on
FEMSLE Congress 2-6-03
141
food systems have to be systematically examined for anti-
biotic susceptibility. Species of lactic acid bacteria isolated
from Cabrales cheese were investigated for their antibiotic
susceptibility. It seems clear that the use as starters of
antibiotic resistant lactic acid bacteria should be avoid
[1]. Ninety-¢ve lactic acid bacteria strains isolated from
di¡erent batches of artisanal starter-free Cabrales cheese
were tested against 15 antibiotics with the ‘‘Sensitrite
Anaero3’’ kit (Treck Diagnostic System). MICs to some
antibiotics have to be completed on Mueller-Hinton plates
by the NCCLS procedure. Cefoxitin and metronidazole
were not e¡ective against this group of bacteria. Lactococ-
cus and Enterococcus strains displayed antibiotic resistance
pro¢les di¡erent from those of Lactobacillus and Leuco-
nostoc ones. Penicillins MICs were always higher for Lac-
tobacillus and Leuconostoc species. However, in clinical
terms, none of them was resistant. MICs to tetracycline
were also higher for the Lactobacillus and Leuconostoc
isolates. All Leuconostoc isolates and all but four Lacto-
bacillus were resistant to vancomycin, a resistance that was
only found in two related Enterococcus isolates, whereas it
was absent in Lactococcus. Enterococci were more resis-
tant and/or presented higher MICs to most antibiotics
than lactococci. Moreover, three Enterococcus strains pre-
sented a kind of multiresistance. In spite of this, a few
Lactococcus isolates were resistant to tetracycline and
chloramphenicol, a fact that justify this analysis.
[1] Teuber, M., L. Meile, and F. Schwarz. 1999. Acquired
antibiotic resistance in lactic acid bacteria. Antonie van
Leeuwenhoek 76:115-137.
P2^24
ANTIBIOTIC SUSCEPTIBILITY OF LACTOBACIL-
LUS AND BIFIDOBACTERIUM SPECIES FROM
THE HUMAN GASTROINTESTINAL TRACT
S. Delgado(1), L. Otero(2) and B. Mayo(1)
(1) Instituto de Productos La¤cteos de Asturias (CSIC),
33300-Villaviciosa; (2) Servicio de Microbiolog|¤a del Hos-
pital de Cabue•es, 33394-Gijo¤n, (Asturias), Spain
At present, there is a great concern about the spread of
antibiotic resistance determinants by bacteria from the
food chain [1]. Thus, strains intended to be used on
food systems have to be systematically examined for anti-
biotic susceptibility. We are characterizing several Lacto-
bacillus and Bi¢dobacterium species from the human gas-
trointestinal tract that could eventually be used as
probiotics. The absence of antibiotic resistance is one of
the criterium to select presumptive useful strains. Seventy
strains of Bi¢dobacterium and Lactobacillus species have
been tested against 15 antibiotics with the ‘‘Sensitrite
Anaero3’’ kit (Treck Diagnostic System). MICs to some
antibiotics were completed on Mueller-Hinton plates by
142 1st FEMS Congress / Posters 103^505
the NCCLS procedure. All strains were sensitive to chlor-
amphenicol, imipenem and the group of penicillins. On the
other hand, most of the strains were resistant to metroni-
dazole. All bi¢dobacteria isolates were susceptible to ce-
foxitin, whereas about half of the lactobacilli were resis-
tant. Single strains of Bi¢dobacterium infantis/longum, B.
pseudocatenulatum, Lb. acidophilus and Lb. rhamnosus
were resistant to erythromycin and/or clyndamicin. Ap-
proximately, ¢fty per cent of the Bi¢dobacterium isolates
were resistant to tetracycline, as well as two Lb. acidophi-
lus strains and one Lb. plantarum. None of the tested
Bi¢dobacterium infantis/longum isolates was resistant to
vancomycin, while around ¢fty per cent of the lactobacilli
were found to be resistant. Most of the observed resistan-
ces seemed to be intrinsic and were similar to those re-
viewed on the literature [1, 2].
[1] Teuber, M., L. Meile, and F. Schwarz. 1999. Acquired
antibiotic resistance in lactic acid bacteria. Antonie van
Leeuwenhoek 76:115-137. [2] Gasser, F. 1994. Safety of
lactic acid bacteria and their occurrence in human clinical
infections. Bull. Inst. Pasteur 92:45-67.
P2^25
PROBIOTIC ENTEROCOCCI IN ANIMAL FEEDS:
PROPOSED METHODS FOR THE OFFICIAL QUAL-
ITY ASSESSMENT
K. J. Domig(1), A. Weiss(1), H. K. Mayer(1), R. G. K.
Leuschner(2) and W. Kneifel(1)
(1) Department of Dairy Science and Microbiology, BOKU
^ University of Natural Resources and Applied Life Scien-
ces, Vienna, Austria ; (2) Central Science Laboratory, De-
partment for Environment, Food and Rural A¡airs, York,
UK
The enumeration and identi¢cation of probiotic enterococ-
ci strains has become an important item within quality
control of probiotic animal feeds. As an important part
of the EU project ‘‘Methods for the o⁄cial control of
probiotics (microorganisms) used as feed additives
(SMT4-CT98-2235), protocols for the selective enumera-
tion of enterococci and molecular biological procedures
for the identi¢cation on strain level were developed and
validated. Aiming at the selective enumeration of enter-
ococci either present as a single component or in combi-
nation with other microorganisms (bacilli, bi¢dobacteria,
lactobacilli, pediococci, yeasts), varieties of culture meth-
ods applying selective and non-selective agar media were
screened. Bile Esculin Azide Agar was shown to be the
most suitable medium. The enumeration method was val-
idated within a collaborative study involving 20 laborato-
ries from 12 European countries. For identi¢cation of pro-
biotic strains, the performance of molecular biological
typing methods based on Polymerase Chain Reaction
FEMSLE Congress 2-6-03
(PCR) and Pulsed Field Gel Electrophoresis (PFGE)
were investigated using 74 enterococcal test strains. In
contrast to the other typing methods, which vary regard-
ing their reproducibility and discriminatory properties,
PFGE is currently considered to be the ‘‘golden standard’’
for sub-typing enterococci. A proven PFGE protocol was
slightly modi¢ed and examined with nine restriction endo-
nucleases. It could be demonstrated that this method
shows a pronounced e⁄cacy. The PFGE protocol was
proposed as a standard method that is capable of checking
the identity of enterococcal strains used as probiotic feed
additives.
P2^26
COMPROMISED PLANT HEALTH INCREASES THE
RISK OF OPPORTUNISTIC COLONISATION BY
HUMAN PATHOGENIC BACTERIA
B. Du¡y
Swiss Federal Research Center for Fruit Production, Viti-
culture and Horticulture (FAW), CH-8820 Wa«denswil,
Switzerland
Human disease outbreaks traced to foodborne Salmonella
and E. coli O157:H7 are increasing worldwide. These bac-
teria opportunistically colonize a variety of plants, with
some degree of host speci¢city. Here we demonstrate
that host health in£uences colonization. S. enteriditis, S.
typhimurium and E. coli O157:H7 populations established
at low levels on healthy wheat roots (103 cfu/g root), to-
mato leaves (103 /g leaf) and strawberry (102 cfu/fruit).
Human pathogen colonization was signi¢cantly increased
on hosts infected with a fungal pathogen. Populations on
wheat roots infected by Rhizoctonia solani or Gauemanno-
myces graminis, on tomato leaves infected by Botrytis cin-
erea, and on strawberry infected by B. cinerea averaged
104-105 cfu/g host tissue depending on the bacterium-dis-
ease combination. None of the human pathogens a¡ected
fungal growth or plant disease development. Human
pathogenic bacteria were not detected on conidia of B.
cinerea sporulating on tomato leaves. These results indi-
cate that increased risk of contamination of diseased crops
should be considered in designing intervention strategies
to reduce produce-linked outbreaks.
 1st FEMS Congress / Posters 103^505
P2^27
ANTIMUTAGENIC EFFECT OF HIGH PRESSURE-
TREATED AND UNTREATED BRASSICA OLERA-
CEA EXTRACTS ON HETEROCYCLIC AMINES-IN-
DUCED SALMONELLA TYPHIMURIUM MUTANTS
E. Edalucci(1), M. Benincasa(2), P. Rovere(3) and M.
Tamaro(1)
(1) Department of Biomedical Sciences, Microbiology Sec-
tion, University of Trieste, via Fleming 22, I-34127 Trieste,
Italy ; (2) Department of Biochemistry, Biophysics and
Macromolecular Chemistry, University of Trieste, via Gior-
geri 1, 34127 Trieste, Italy ; (3) SSICA, viale Tanara 31/A,
I-43100 Parma, Italy
The protective e¡ect exerted by vegetables in the diet
against various toxic substances is well known. Several
epidemiological studies indicate that the consumption of
vegetables might deter the occurrence of cancer. The Ames
test which has been extensively carried out to identify mu-
tagens potential carcinogens is increasingly being used to
evaluate the antimutagenic activity of potential anticarci-
nogens. In this study we have assayed the in£uence of
di¡erent methods of food stabilisation, high pressure
(HP) vs. pasteurisation (P), on the antimutagenic response
to mutagenicity induced by heterocyclic aromatic amines
(HAAs). HP-treated Brassica oleracea extracts displayed
high inhibitory activity against mutagenicity induced by
heterocyclic aromatic amines, as well as untreated sam-
ples; whereas conventional pasteurisation process reduced
the antimutagenic properties of B. oleracea extracts. Pro-
tective e¡ects of the consumption of Brassica vegetables
are related to the presence of a variety of molecules and/or
enzymatic proteins, i.e. peroxidase. In this study has been
showed that the peroxidase activity was present in both
HP-treated and untreated samples. High pressure treat-
ment of food as a means of preservation can guarantee
high quality, elevated microbiological safety and the pres-
ervation of the organoleptic properties of the product.
When the consumption of fresh vegetables is not achiev-
able, the use of vegetable products treated with non-con-
ventional methods of preservation, such as HP, should not
be dismissed as a viable alternative, since in our study this
method has been shown to maintain the biological proper-
ties of the fresh product.
FEMSLE Congress 2-6-03
143
P2^28
INHIBITORY EFFECT OF ENTEROCIN EJ97
AGAINST BACILLUS MACROIDES/B. MAROCCA-
NUS ISOLATED FROM SPOILED VEGETABLE
PUREE
A. Ga¤lvez, Ma T. Garc|¤a, R. Lucas, N. Ben Omar, R. Pe¤r-
ez-Pulido, and M. Mart|¤nez-Ca•amero
Ł rea de Microbiolog|¤a, Facultad Ciencias Experimentales,
A
Universidad de Jae¤n, 23071-Jae¤n, Spain
Enterocin EJ97 is an antimicrobial peptide isolated from
Enterocococus faecalis EJ97. This bacteriocin is active
against food-borne pathogenic and/or spoilage bacteria
(Listeria monocytogenes, Bacillus coagulans, B. macroides/
B. maroccanus, Staphylococcus aureus), a reason why it
shows great interest for biopreservation. The peptide se-
quence and the genetic determinants of EJ97 have been
elucidated. The puri¢ed bacteriocin showed bactericidal
activity against strains of B. macroides/B. maroccanus iso-
lated from spoiled zucchini puree. At low concentrations,
enterocin EJ97 killed all viable cells from strain INRA
P53-2 within 4 h of incubation at 37‡C, 24 h at 15‡C or
48 h at 4‡C. The bactericidal e¡ect was more pronounced
at pH 7.0 compared to pH 9.0 or pH 5.0, and was poten-
tiated by sodium nitrite. Inhibition of B. macroides/B. mar-
occanus INRA P53-2 in a commercial vegetable puree re-
quired a ten-fold higher bacteriocin concentration. The
rate of bacterial killing by EJ97 in a vegetable puree in-
creased as the incubation temperature was higher in the
range of 4‡C to 37‡C. These results suggest that enterocin
EJ97 may have a potential for use in the prevention of
food spoilage caused by endospore-forming bacteria.
P2^29
PATHOGENS BEHAVIOUR IN LOW-ACID FER-
MENTED SAUSAGES TREATED BY HIGH PRES-
SURE
M. Garriga, T. Aymerich, B. Mart|¤n, B. Marcos and M.
Hugas
Institute for Food Research and Technology (IRTA), Meat
Technology Centre, 17121 Monells, Spain
Low-acid fermented sausages form a group of traditional
Mediterranean products with a great variety within the
di¡erent regions. Because of its physico-chemical charac-
teristics, mainly a high pH, several pathogens of concern
like Salmonella, Listeria monocytogenes and Staphylococ-
cus aureus, could survive during the ripening process and
even multiply. High hydrostatic pressure is an emergent
technology with potential application for the extension
144 1st FEMS Congress / Posters 103^505
of shelf life of several meat products and is already being
applied in sliced cooked meat products. However, few in-
vestigations have been done with fermented meat prod-
ucts. The purpose of this study was to assess the applica-
tion of high pressure processing for the safety of low-acid
fermented sausages ripened at 12‡C. Two di¡erent prod-
ucts ‘‘fuet’’ and ‘‘chorizo’’ were manufactured and inocu-
lated (103 cfu/g) with a cocktail of Salmonella, L. mono-
cytogenes and S. aureus. Four batches were designed, 2 of
them with additional inoculation of a mixture of selected
lactic acid bacteria and staphylococci. The level of pres-
sure (200 MPa, 10 min., 16‡C) applied before ripening
gave the correct appearance for colour and texture of
both products but was not e¡ective in diminishing the
pathogens inoculated, compared with the non treated
products. The inoculation of a mixture of selected strains
gave better results than high pressure processing, specially
regarding L. monocytogenes. No di¡erences between treat-
ments were recorded for Salmonella, which diminished in
treated and not treated products below the detection limit
( 6 10 cfu/g). When the non treated ripened products were
treated at higher pressure (400 MPa), absence of salmo-
nella in 25 g was achieved.
P2^30
BIFIDOBACTERIA AS FAECAL INDICATORS: DE-
TECTION AND IDENTIFICATION OF STRAINS ISO-
LATED FROM RAW MILK AND IN ENVIRONMENT
OF FARMS
F. Gavini, C. Grare, H. Ramboainiaina and M. Ca¡e¤
Institut National de la Recherche Agronomique, BP 39,
59650 Villeneuve d’Ascq, France
Within the framework of an European project (QLK1-CT-
2000-00805) the bi¢dobacteria are studied as new standard
for ensuring hygienic conditions through the food chain
(raw milk and meat products). Their use as indicators of
faecal contamination in food is particularly interesting be-
cause : (1) they are an important part of the micro£ora in
human and animal faeces; (2) the dominant Bi¢dobacte-
rium species are di¡erent in human and in animal £ora;
(3) they are anaerobes and thus cannot multiply in most
foods. Bi¢dobacteria were isolated from 83% of 300 sam-
ples of raw milk at the teat from farms in North of France
and in Belgium. Bi¢dobacteria were detected in all samples
of manure tested and in air of 3 farms.The bi¢dobacteria
were detected in 97% of cow dung (30 samples) and in
48% of faeces from other animal species (52samples) on
farm. The same major species , B. pseudolongum (subspe-
cies not identi¢ed yet), was isolated in all animal faeces
that present bi¢dobacteria, as in cow dung, except for
pigeons. The second isolated species was B. thermophilum
in 20% of the faecal samples containing bi¢dobacteria in
FEMSLE Congress 2-6-03
animal species other than cow. The species B. pseudolon-
gum was also isolated from 96% of raw milk at the teat
samples that contained bi¢dobacteria, followed by B. ther-
mophilum (in 4% of the raw milk at the teat samples, 4%
of tanks). These results show that the main faecal contam-
ination origin of raw milk is animal and likely cow dung.
P2^31
LACTIC ACID BACTERIA ISOLATED FROM SOUR-
DOUGH EXHIBIT ANTIFUNGAL PROPERTIES
M. Giesova(1), L. B. Bullerman(2), J. Chumchalova(1)
and M. Plockova(1)
(1) Department of Dairy and Fat Technology, Institute of
Chemical Technology, Technicka 5, 166 28 Prague 6, Czech
Republic; (2) Department of Food Science and Technology,
University of Nebraska, Lincoln, Nebraska, 68583-0919,
USA
In this study 60 Lactobacillus strains were isolated from 2
sourdough cultures of di¡erent origin. These strains were
screened for their antifungal activity using Penicillium ver-
rucosum as an indicator strain. Based on these preliminary
results, 8 sourdough Lactobacillus strains together with
Lactobacillus casei 154 and Lactobacillus rhamnosus VT1
were used for further studies. These strains were screened
for their antifungal activity against 14 mold strains from
the genera Aspergillus, Penicillium, Rhizopus, Cladospo-
rium, Geotrichum and Fusarium. Each of Lactobacillus
strains was inoculated into MRS or modi¢ed MRS
(mMRS) agar, after solidi¢cation MRS (or mMRS) agar
was overlaid with soft potato dextrose agar (PDA) (0.75
%w/w of agar). Suspension of mold spores (103) was spot-
ted onto the PDA surface. Plates were incubated 21 days
at 30‡C, colony diameter was measured every day. Asper-
gillus niger, Penicillium roqueforti, Penicillium digitatum
and Geotrichum candidum were found to be the least in-
hibited mold strains and their growth and sporulation was
delayed for 1-4 days. Aspergillus ochraceus, Penicillium
commune, Cladosporium cladosporoides and Fusarium pro-
liferatum were found to be the most inhibited mold strains.
The highest inhibitory e¡ect was caused by Lactobacillus
strains originating from ‘‘original’’ sourdough culture,
while growing on MRS agar. Lactobacillus casei 154 ex-
hibited the highest inhibitory e¡ect while growing on
mMRS agar.
 1st FEMS Congress / Posters 103^505
P2^32
MICROBIOLOGICAL QUALITY OF SLOVENIAN
CHEESE MADE FROM RAW COW’S MILK
K. Godic› Torkar, M. Smolej, S. Golc Teger
Laboratory for Dairying, zootechnical Dept., Biotechnical
Faculty, University of Ljubljana, Groblje 3, 1230 Domz›ale,
Slovenia
The presence of pathogen and indicator microorganisms in
14 semi-hard cheeses after one month ripening made from
raw cow’s milk by individual Slovenian producers was
studied. Salmonella spp., Listeria spp., Proteus, sulphite-
reducing clostridia and Campylobacter spp. were not de-
tected in cheese samples. E. coli was found in 4 (30 %) of
samples on levels from 10 to 3400 CFU/g of cheese. Co-
agulase positive staphylococci were present in 9 (64 %) of
samples ranged from 100 to 50 000 CFU/g of cheese. High
number of enterococci (from 3.103 to 15.107 CFU/g) and
coliforms (from 10 to 19.105 CFU/g) were detected. The
same microorganisms were established in total of 98 sam-
ples taken during milking and processing of cheeses men-
tioned before: swabs from udders and milking machine,
fresh raw and mixed milk from vat, whey and salt water.
Listeria spp. was isolated from four cow’s udders, one
swab from milking machine, two milk and one whey sam-
ples, while none of examined samples were positive to
presence of Salmonella spp. and Campylobacter spp. Pro-
teus was present in 7 (7 %) cases of milk and whey. Clos-
tridia were detected in 10 (10 %) samples (swabs, raw milk,
whey). E. coli was isolated from 12 (12 %) samples of
swabs, raw and mixed milk, whey and salt water. Because
of improper milking and processing hygiene conditions,
which are expressed also with high number of enterococci
and coliforms, three (21 %) of tested cheese samples did
not correspond to the microbiological criteria according
valid regulations.
P2^33
ASSESSMENT OF UNCERTAINTY OF MEASURE-
MENT IN PLATE COUNT METHOD ON 21‡C
WITH PREINCUBATION
M. Gos›njak
Institute of Public Health, 3000 Celje, Slovenia
No measurement is perfect because it is always a¡ected by
many factors. Uncertainty of measurement (u.m.) quanti-
¢es the range within the true value is likely to fall. Labo-
ratories need to produce the estimated uncertainties where
it is required by client or by the test speci¢cation or where
the estimated uncertainty is necessary for the validity or
FEMSLE Congress 2-6-03
145
application of the result. The most important factors that
may contribute to the uncertainty of measurement in mi-
crobiology are uncertainty of the inoculum volume, uncer-
tainty due to the random scatter of the particles and un-
certainty caused by human factor. We assessed the
uncertainty of the plate count method at 21‡C with pre-
incubation. The factors that could a¡ect the quality of the
result were determined, their in£uence on the ¢nal result
was calculated or assessed. The results of calibrations of
pipettes were the base for the calculation of their uncer-
tainty. Their in£uence was considered in the process of the
preparation of test sample (0.24% of combined u.m.) and
in the preparation of dilutions (0.81% of combined u.m.).
We considered also the uncertainty of measurement aris-
ing from the non uniform distribution of micro-organisms
in the sample (Poisson distribution) which represents
22.7% of combined u.m.. Technologist to technologist var-
iation during the sample preparation accounted for 66.4%
and 8.4% of combined u.m. during the reading of results.
We estimated also the personal error of the analyst at the
reading of results (1.45% of combined u.m.).
P2^34
COMPARISON OF PCR-BASED TECHNIQUES TO
DISTINGUISH FUNGI ON HAZEL NUTS
M. Grube(1), K. Pelant(1) and M. Stelzl(2)
(1) Institute of Botany, Holteigasse 6, 8010 Graz, Austria;
(2) Hygienicum, Andritzer Reichsstrasse 44, A-8045 Graz/
Austria
Food spoilage fungi produce a range of toxic compounds
and thus represent a risk to human health. The detection
of toxins usually involves chromatographic and other
techniques for direct risk assessment associated with a
food item. However, fungi often remain undetermined in
such assays, which ignores the biological diversity of toxin
production in di¡erent fungal strains. Classic determina-
tion of food fungi involves phenotypic or physiological
characters, such as morphology expressed on various cul-
ture media. Beside this, numerous molecular typing tech-
niques based on PCR have been developed for recognition
of fungal strains. Some of the genetic markers amplify
variable portions of speci¢c genes, which are then se-
quenced for strain comparison. Other approaches distin-
guish strains by the presence of PCR-fragments after am-
pli¢cation with toxin speci¢c genes or with primers for
anonymous loci which may be present in the entire ge-
nome. We applied various PCR-techniques to distinguish
strains of fungi isolated from hazel nuts and present a
comparison of these approaches.
146 1st FEMS Congress / Posters 103^505
P2^35
HEAT, ACID AND OSMOTIC SHOCK AND NISIN
RESISTANCE IN LISTERIA MONOCYTOGENES
M. M. Guerra(1), F. A. Bernardo(2), and M. Adams(3)
(1) Escola Superior de Hotelaria e Turismo do Estoril,
Portugal; (2) CIISA/Laborato¤rio de Inspecca‹o Sanita¤ria,
Faculdade de Medicina Veterina¤ria, Universidade Te¤cnica
de Lisboa, Portugal; (3) School of Biomedical and Life
Sciences, University of Surrey, UK
Food-borne pathogens are commonly stressed during food
processing. This may reduce the e¡ectiveness of food pres-
ervation hurdles and food safety may be compromised if
pathogens develop stress resistance as a response. The ef-
fect of the application of several preliminary stress shocks
(heat, acid and osmotic) on the resistance of Listeria
monocytogenes Scott A to nisin action was investigated.
A sublethal dose of acid (HCl, pH 5.5) or NaCl (7%,
wt/vol) was added to Listeria monocytogenes broth cul-
tures in both the exponential and stationary phases of
growth, and the cells were allowed to grow for 1h. Cells
in both phases were also heat shocked at 45‡C for 1h. The
stress-adapted cells were then exposed to 100 IU/ml of
nisin for 90 minutes. Viable counts of the pathogen were
determined at time intervals throughout the experiment.
The preliminary stresses increased resistance to the anti-
listerial activity of the bacteriocin only in stationary phase
cells ( s 1 log di¡erence in the inactivation of non-
shocked and shocked cells). This di¡erence decreased
over time for acid and osmotic shocked cells. Acid shock
provided the highest initial protection to stationary phase
cells, but heat shock was the one that provided a more
prolonged resistance. These results clearly show that the
sensitivity of Listeria monocytogenes to nisin will depend
on the cell’s previous history. This could have important
pratical implications on how best to use bacteriocons in
multifactorial food preservation systems. (age of culture
phase growth, the type of stress, time of exposure to nisin).
P2^36
THE MICROBIAL QUALITIES OF MILK AND ITS
BY-PRODUCTS IN TIRANA CITY
Z. Haxhiaj and Y. Xhumari
Institute of Public Health, Rruga Alexander Moisiu, No.
80, Tirana, Albania
The transition from a centralized state controlled system
towards a free market based one comported drastic
changes in the food production and distribution system
of Albania. The lack of incentives in the former system,
FEMSLE Congress 2-6-03
gradually installed a chronicle insu⁄ciency of availability
for food products. The adopted initial policy for ¢lling this
gap was an unconditional support of all free initiatives
within the food system. This policy, created within a short
time the expected abundance of food products on the
market, but it was associated to the creation of consider-
able problems on food safety and the consequent impair-
ment of public health. The aim of the present study is to
perform an assessment of the microbial qualities of milk
and its by-product and to identify dangerous products on
public health. Samples related products bought randomly
on the Tirana market. The samples (about 350) were an-
alytically examined for the following parameters as well :
Aerobe Meso¢le microorganisms, Coliforms, Escherichia
coli, Salmonella, Staphylococcus aureus, Molds. The micro-
biological quality of milk and its products is not in needed
level. The results over the norms are: Aerobe Meso¢le 53.4
%, Coliforms 54 %, E.coli 45 % , Molds 43.1%. All the
kind of analysed products presented high values for pre-
vious parameters, The faecal contamination is primary in
general contamination. Staphylococcus aureus is founded
in one sample of butter and two samples of curd. Salmo-
nella and Shigella are not present in examined samples. At
the end of our study we have given some recommenda-
tions for improvement of the situation.
P2^37
GROWTH OF SALMONELLA SEROVARS IN HENS’
EGG ALBUMEN AS AFFECTED BY STORAGE
W. Messens, L. Duboccage, K. Grijspeerdt, M. Heyndrickx
and L. Herman
Ministry of the Flemish Community, Agricultural Research
Centre-Ghent, Department of Animal Product Quality and
Transformation Technology, Brusselsesteenweg 370, B-9090
Melle, Belgium
Salmonella enterica serovar Enteritidis is a common food-
borne pathogen, the transmission of which is primarily
associated with the consumption of contaminated shell
eggs. There is no agreement about the growth of Salmo-
nella when deposited in egg albumen near room temper-
ature. The objective of our study was to aid solving the
problem of con£icting results of growth/no growth of Sal-
monella in fresh and stored albumen at room temperature.
Di¡erent S. enterica serovars, including serovar Enteriti-
dis, were tested for growth at 20‡C in separated albumen,
both fresh and up to 3 weeks stored, upon inoculation
with approx. 31 cells per ml. Shell eggs were stored as
such or the albumen was stored separately. The serovar
Enteritidis did not behave di¡erently compared to the oth-
er serovars. A pronounced growth occurred more fre-
quently and up to a one-log unit higher level in fresh
than in stored albumen. Since growth in the albumen
 1st FEMS Congress / Posters 103^505
was una¡ected by the storage condition, we have no in-
dication that nutrients or some factors negating the inhib-
itory properties of the albumen leak out from the yolk
during the 3 weeks storage at 20‡C. A better growth in
fresh albumen was also observed when inoculating approx.
8 cells in the albumen of whole shell eggs. In this case,
growth occurred up to higher levels than when separated
albumen was inoculated possibly due to migration of Sal-
monella towards the yolk. It was proven that the enhanced
growth in fresh albumen compared to the stored albumen
was at least partly caused by a pH e¡ect.
P2^38
ANTIMICROBIAL, ANTIOXIDANT AND ANTIMU-
TAGENIC ACTIVITY OF PLANT EXTRACTS CON-
TAINING PHENOLIC COMPOUNDS
M. Hnilova¤, R. Hlad|¤kova¤ and M. Drda¤k
Department of Food Technology and Biotechnology, Fac-
ulty of Chemistry, Brno University of Technology, Purky-
n›ova 118, 61200 Brno, Czech Republic
Plant phenolics, especially £avonoids, are currently of
growing interest owing to their supported functional prop-
erties in promoting human health. The plant extracts pre-
pared from Daisy (Bellis perennis), Shepera¤s purse (Cap-
sella Bursa-pastoris), Colta¤s tail (Equisetum articum), Sage
(Salvia o⁄calis), Chamomile (Matricaria chamomilla),
Plantain (Plantago lanceolata), Green tea (Camellia sinen-
sis) and standards of £avonoids were used to evaluate
their antimicrobial, antioxidant and antimutagenic activ-
ities. Antimicrobial screening of plant extracts and stan-
dards of £avonoids against selected foodborne microbes
(Bacillus subtilis, Micrococcus luteus, Escherichia coli, Sac-
charomyces cerevisiae, Zygosaccharomyces rouxii, Hanse-
nula anomala, Pichia farinosa, Candida vini) was conducted
in this study. The tests were carried out using di¡usion
methods ^ Hole Di¡usion Assay (HDA) and Minimal In-
hibition Concentration (MIC). The plant extracts showed
an entire antimicrobial spectrum, especially extract pre-
pared from Daisy (Bellis perennis) had excellent antimi-
crobial activity against common foodborne microorgan-
isms and on growth on Bacillus subtilis, Micrococcus
luteus, Saccharomyces cerevisiae and Zygosaccharomyces
rouxii exhibited comparable inhibition as a chemical pres-
ervative, potassium sorbate. The antioxidant activity of
plant extracts and standards of £avonoids was determi-
nated by a radical (DPPH) decoloration assay and by a
Randox test. Observed antioxidant activity was compared
with a total content of £avonoids in plant extracts. The
antimutagenic activity of plant extracts and standards of
£avonoids was determinated by Ames test on auxotri¢c
mutant Salmonella typhimurium TA98. All studied plant
extracts showed slight antimutagenic activity.
FEMSLE Congress 2-6-03
147
P2^39
THE INDICATION OF CHROMIUM (TRIS) PICOLI-
NATE AS DNA DOUBLE STRAND BREAKER
S. Jenko-Brinovec(1), A. Plaper(2), M. Golob(2), J.
Kos(2,3), and P. Raspor(1)
(1) University of Ljubljana, Biotechnical Faculty, Ljubljana
Slovenia; (2) KRKA d.d., Research Dept. of New Entities,
Novo mesto, Slovenia; (3) Jozef Stefan Institute, Dept. of
Biochemistry and Molecular Biology, Ljubljana, Slovenia
Trivalent chromium is an essential mineral important in
the metabolism of fats and carbohydrates. Its dietary in-
take is suboptimal and therefore chromium supplements
are used frequently. Among them chromium picolinate
(Cr(pic)3) is the most popular. Recently, questions have
been raised about the safety of Cr(pic)3, especially with
regards to its mutagenic and clastrogenic e¡ects. Our
work demonstrates that Cr(pic)3, in the presence of cellu-
lar reductant ascorbate and H2O2, cleaves DNA. Both
single-strand and double-strand DNA breaks are formed.
The cleavage is probably the consequence of hydroxyl
radicals formed during the reoxidation of reduced form
of Cr(pic)3 with H2O2, since the addition of DMSO, an
e¡ective ]OH scavenger, completely prevented the forma-
tion of nicked or linearized plasmids. Interestingly, chro-
mium chloride in the same system produced only single
strand DNA breaks. It seems that the same picolinate li-
gands which enable trivalent chromium to pass the cell
membranes more easily also make the chromium more
dangerous once it gets inside the cell. Besides the forma-
tion of ROS and consequently oxidative DNA damage,
Cr(pic)3 also inhibited the gyrase relaxation activity in
vitro. As Cr(pic)3 does not bind to the DNA the inhibition
is most likely due to the binding of Cr(pic)3 to the enzyme,
and in this way preventing its enzymatic activity or its
binding to the substrate. Chromium picolinate also had
citotoxic e¡ect on mammalian cells in the cell culture.
Obviously, the risk-bene¢t ratio of Cr(pic)3 has not yet
been adequately characterized and further studies should
be employed before taking chromium supplements on dai-
ly basis.
We thank the Ministry of Science and Technology of the
Republic of Slovenia (Project no. : J4-7454-490) for ¢nan-
cial support and S.J.B. would like to thank to the Ministry
of Education, Science and Sport of the Republic of Slov-
enia for grant (S4-490-007/21462/2000).
148 1st FEMS Congress / Posters 103^505
P2^40
CYTOTOXICITY ASSESSMENT OF AEROMONAS
HYDROPHILA ON VERO CELL CULTURES
N. R. Karabasil
Department for Food Hygiene and Technology of Animal
Origin, Faculty of Veterinary Medicine, University of Bel-
grade, 11000 Belgrade, Bulevar JNA 18. Serbia and Mon-
tenegro
Aeromonas hydrophila, introduced as a ‘‘new’’ foodborne
pathogen, are environmental bacteria, ubiquitous in water,
soli, and in many foods. The purpos of this paper is to
provide data for cytotoxic acitvity of Aeromonas hydro-
phila. Six Aeromonas hydrophila strains were used, ¢ve
food-derived strains previosly isolated from freshwater
¢sh obtained on local retail market place and one type
strain (CCM 4528). Cytotoxicity were estimated on Vero
cells monolayers, 45 minutes to 24 h at 37‡C after ¢ltrates
of A. hydrophila added. In e¡orts to obtain is it heat-labile
or heat-stable cytotoxin, ¢ltrates of A. hydrophila were
heated during 30 minutes at 56‡C and 5 minutes at
100‡C. Treated ¢ltrats were added to Vero cell monoleyers
and cytotoxicity were estimated after 24 h at 37‡C. Tested
¢ltates of Aeromonas hydrophila caused cytotoxic e¡ect at
the Vero cells, but the intensity of changes di¡ered in
dependance of the ¢ltrate. In all tested strains, cytotoxic
e¡ect was induced with heat-labile cytotoxin.
P2^41
OLIGODYNAMIC WATER DISINFECTION METH-
OD
R. R. Khaydarov and R. A. Khaidarov
Scienti¢c Devices Designing Department, Institute of Nu-
clear Physics, 702132, Ulugbek, Tashkent, Uzbekistan
The purpose of this work was to improve existing oligo-
dynamic water disinfecting method: destroying impacting
low concentrations of metal ions on bacteria in the water.
Experiments have shown that the maximum disinfecting
e¡ect is obtained by using the composition of 4 various
metal ions with Ag as the main component. It was inves-
tigated dependence of bacteria killing time against e¡ective
concentration of metal ions in the water, initial bacteria
concentration (from 103 to 1012 CFU/L) and their types,
in£uence of di¡erent cations and anions (Cl-, SO42-, S2-,
Fe2+, Fe3+, etc.) in the water on disinfecting process. It
was shown that when the concentration of metal ions in
treated water does not exceed American drinking water
regulation limit, the killing time of Legionella-pneumophi-
la and typhoid-paratyphoid is 60 minutes, salmonellas and
FEMSLE Congress 2-6-03
cholera is 30 minutes when their concentration is 1000 1/
liter. Besides during 20 days there was not bacteria growth
in treated water samples. Increasing the initial bacteria
concentration causes increasing the bacteria killing time
in accordance with given above formula. Spores are
more resistant to destruction than vegetative bacteria.
Combination of described method with ultraviolet and
electric ¢eld was used for killing Bacillus subtilis as the
acceptable analogue spores. Bacteria destruction time
was 2 hours at an initial concentration of 1000 CFU/litre.
P2^42
INFLUENCE OF METHODOLOGICAL CRITERIA
ON THE RESULTS OF ANTIBIOTIC SUSCEPTIBIL-
ITY TESTING OF LACTIC ACID BACTERIA
B. Ko«gler(1), K. Domig(1), H. P. Lettner(2), W.
Kramer(2) and W. Kneifel(1)
(1) Department of Dairy Research & Bacteriology, Agri-
cultural University, Gregor Mendel-Str. 33, A-1180 Vienna,
Austria ; (2) Lactosan Starterkulturen GMBH & Co KG,
Industriestr. West 5, Kapfenberg, Austria
Known as safe starter cultures lactic acid bacteria (LAB)
have not received much attention in regard to antimicro-
bial resistance. Recently, the susceptibility of LAB to
transferable antibiotic resistance has become a subject of
concern. Therefore, antibiotic susceptibility testing pro-
grammes gain in signi¢cance. The present study demon-
strates that there are many variables a¡ecting the results
of antimicrobial susceptibility tests. Di¡erences in media,
inoculum density, incubation conditions and testing meth-
ods in£uence the interpretation of antibiotic breakpoints.
In the current study several lactobacilli strains were tested
for susceptibility and resistance against thirteen antibiotics
by the agar dilution method and the broth microdilution
method as suggested by the National Committee for Clin-
ical Laboratory Standards (NCCLS). Modi¢cations of the
standardized NCCLS methods concerned the culture me-
dium, the inoculation density and the incubation condi-
tions. An obvious increase of the MIC value was noted
when an unde¢ned medium (e.g., MRS or BHI) and an
inoculum density (e.g., 106 CFU/spot) was used. The same
tendencies in MIC di¡erences were seen upon aerobic and
anaerobic conditions as well as agar and broth method.
There is still a lack of an international agreement on a
standardized antimicrobial susceptibility testing procedure
and on breakpoints. Consequently, the establishment of a
standardized antimicrobial resistance surveillance system
of LAB, at least within Europe, would facilitate the com-
parison of resistance data among di¡erent laboratories.
 1st FEMS Congress / Posters 103^505
P2^43
EXTREMOPHYLIC MICROSCOPIC FUNGI FROM
THE SOUTHERN SLOPES OF THE CAUCASUS AS
A PRODUCERS OF STABLE ENZYMES
G. I. Kvesitadze, L. I Kutateladze, T. I. Alexidze, T. A.
Sadunishvili, E. G. Kvesitadze
Durmishidze Institute of Biochemistry and Biotechnology,
Academy of Sciences of Georgia, 380059 Tbilisi, Georgia
Extremophilic microorganisms attract special attention be-
cause of their ability to produce enzymes stable to critical
conditions. Such strains are also suitable for the cloning of
genes of stable enzymes. With this aim, 3996 micromycete
cultures were isolated from di¡erent soil-climatic zones of
the Caucasus: 15% were toxic, 6% thermophilic, 1.5% al-
kaliphilic, 0.8% acidophilic and above 2% halophylic. The
collection was screened on the ability to produce enzymes
stable to extreme conditions. Among the cultures of ex-
tremophiles, over 30 strains producing stable enzymes
widely used in food processing, such as cellulases and xy-
lanases, heat and acid-stable glucoamylases, acid-stable
protease and some other enzymes has been revealed. The
majority of these enzymes were puri¢ed and their basic
physical-chemical properties determined. Stability, kinetic
and thermodinamic characteristics of these enzymes allows
their wide application in food processing. For instance, the
increase of the cultivation temperature of Allesheria ter-
restris from 40 to 48-490 C resulted in the formation of a
new endoglucanase which was inactivated only by 20% at
650C for 6h in the absence of substrate. The molecular
weight of the heat-stable enzyme is 69 kD; the isoelectric
point (pI) is 6.4 and the Michaelis constant (Km) is 6.6 g/
liter. The thermostable endoglucanase of Allescheria ter-
restris reacts with polyclonal antibodies against the unsta-
ble endoglucanase of Trichoderma reesie. The physiology
of the strain-producers have been investigated. Using mu-
tagens (UV irradiation, ethylenimine, and nitrosomethy-
lurea), 12 mutant strains synthesizing considerable
amounts of extracellular cellulases have been created. Pro-
toplasts fussion of obligately thermophilic (Allesheria ter-
restris) and mesophilic (Aspergillus niger) strains resulted
in a fusant displaying a higher thermal stability of endol-
gucanase compared with the parents. Some strains (Asper-
gillus, Trichoderma, Allesheria, etc) are being screened on
their growth inhibition by plant preparation which based
on current investigation (INTAS-Food 00-0727) could be
successfully applied for food spoilage prevention to im-
prove its quality and extend shel£ife.
FEMSLE Congress 2-6-03
149
P2^44
CHARACTERISATION OF STAPHYLOCOCCUS EPI-
DERMIDIS ENTEROTOXIN TYPE C FROM A FOOD
OUTBREAK
S. Loncarevic, T. Mathisen, R. Kristensen, L. M. Rorvik
Section for Feed and Food Microbiology, National Veteri-
nary Institute, P.B. 8256 Dep., 0033 Oslo, Norway
Although Staphylococcus aureus intoxication is a common
food-borne illness, no case of S. epidermidis food-borne
intoxication has been reported. However, S. epidermidis
producing staphylococcal enterotoxins (SET) has been re-
covered from sheep and goat milk in some investigations.
In 2001, a series of suspected staphylococcal intoxications
occurred in a hotel in Norway. Foodstu¡s obtained at the
three di¡erent occasions showed the presence of di¡erent
staphylococcal species. SET was recovered only from few
bacterial cultures and not directly from the implicated
food samples. In the ¢rst outbreak only one isolate of S.
aureus produced SET A, in the second two isolates of S.
epidermidis isolates produced SET C and in the third no
SET or staphylococcal bacterial isolates were detected
from food samples. Two isolates from meat pudding and
cheesecake, con¢rmed by API and additional biochemical
tests as S. epidermidis, were shown to produce SET C.
Reversed passive latex agglutination (SET-RPLA) was
used for detecting the SET A-D production, while multi-
plex PCR-technique was used to reveal genes coding for
SET types A-E and G-J. Furthermore, molecular typing of
the S. epidermidis isolates by Pulsed Field Gel Electropho-
resis (PFGE) con¢rmed that they shared identical pattern.
In order to compare these S. epidermidis SET C genes with
published S. aureus SET C genes, sequencing will be per-
formed. The results will give more information about SET
produced by S. epidermidis and possible involvement of S.
epidermidis in food poisoning.
150 1st FEMS Congress / Posters 103^505
P2^45
MOLECULAR IDENTIFICATION OF TRICHOSPOR-
ON CASEORUM SP. NOV. AND TRICHOSPORON
LACTIS SP. NOV. ISOLATED FROM CHEESES
K. Lopandic(1), T. Sugita(2), W. J. Middelhoven(3), H.
Prillinger(1), M. Herzberg(4), J. W. Fell(5)
(1) Institute of Applied Microbiology, University of Agri-
culture, Muthgasse 18, A-1190 Vienna, Austria ; (2) De-
partment of Microbiology, Meiji Pharmaceutical University,
2-522-1 Noshio, Kiyose, Tokyo, 204-8588 Japan; (3) Lab-
oratory of Microbiology, Wageningen University, P.O. Box
8033, 6700 EJ, Wageningen, The Netherlands; (4) Philips-
University Marburg, Karl von Frisch Str., 35043 Marburg,
Germany ; (5) University of Miami, Rosenstiel School of
Marine and Atmospheric Sciences, 4600 Rickenbacker Cau-
seway, Key Biscayne, FL 33149, USA
During the studies of yeast contaminants associated with
milk products two strains from unripened soft and fresh
cheese (Salzburg region Austria) were isolated showing
phylogenetic relatedness to the genus Trichosporon. The
extensive analysis included biochemical and physiological
characterisation, partial sequencing of 26S rDNA and
complete sequences of 18S rDNA, ITS1/ ITS2 and IGS1
regions as well as RAPD-PCR ¢ngerprinting. The results
demonstrated that the strains represent two new species
and in reference to the source of isolation the strains are
named T. caseorum and T. lactis. The new species are
closely related to the human pathogens T. ovoides and
T. inkin on the phylogenetic trees based on 26S and 18S
rRNA encoding genes and the internal transcribed spacer
(ITS) regions. The length and sequences of the IGS1 re-
gions as well as the RAPD-PCR patterns are remarkable
di¡erent in comparison to those of the closest phylogenetic
relatives. Both species could be distinguished phenotypi-
cally and show strong ability to decompose mutagenic and
toxic compounds such as phenol, hydroquinone, cresole
and cinnamate. The two new Trichosporon species did
not grow well in liquid media below pH 5.5 that made a
deviation from standard physiological tests necessary. The
failure to assimilate inositol and growth on D-glucosamine
can be used to distinguish both species from T. inkin and
T. ovoides.
FEMSLE Congress 2-6-03
P2^46
ANTIBIOTIC RESISTANCE IN DAIRY ENTEROCOC-
CI: WHAT SHOULD WE FEAR?
F. S. Lopes(1), T. C. Ribeiro(1), A. Euge¤nio(1), M.
Abrantes(1), R. Tenreiro(2), M. T. Barreto Crespo(1)
(1) IBET/ITQB, Apartado 12, 2781-901 Oeiras, Portugal;
(2) FCUL, Campo Grande, Edif|¤cio C2, Piso 4, 1700 Lis-
boa, Portugal
Enterococci are part of the comensal microbiota of hu-
mans and animals, and are present in the environment,
namely in some food products, like milk and cheese. In
recent years they have become a subject of increasing con-
cern due to their multiple antibiotic resistance to the ma-
jority of antibiotics used in the health care system. The
emergence of multiple resistant strains is, in part, a con-
sequence of their capability to exchange genetic material,
namely through plasmids and transposons. Studies con-
ducted in the last decades have been concerned mostly
with clinical isolates and the environmental contribution
to this serious antibiotic problem has been neglected.
Therefore, some antibiotics, for which resistance is usually
associated with mobile genetic elements, namely gentamy-
cin, erythromycin, vancomycin and tetracycline, have been
a subject of study in 200 enterococci isolated from milk
and cheese in our laboratory. High-level resistance to gen-
tamycin has not been detected in these isolates, and MIC
values for this antibiotic are still low. Nevertheless, eryth-
romycin resistance is widely disseminated in these dairy
isolates, but not transposon Tn917. However, this resis-
tance is easily lost, together with resistance to chloranphe-
nicol, cipro£oxacin, spiramycin, nor£oxacin, o£oxacin and
ceftriazone, in the presence of the curing agent acridine
orange, indicating a mobile element mediated transfer.
Therefore, studies of transfer of resistance of these resis-
tances are also being performed.
P2^47
RAPID DETECTION OF BACILLUS CEREUS FROM
FOODS BY PCR ANALYSIS
M. Manzano, C. Giusto, L. Iacumin, C. Cantoni and G.
Comi
Department of Food Science, University of Udine, via Mar-
angoni 97, 33100 Udine, Italy
B. cereus, a well known food poisoning organism, has
been identi¢ed as the causative agent in a number of
food poisoning outbreaks many of which unreported be-
cause confused with those of other pathogens. Improving
microbial safety and extending the shelf-life of industrial
 1st FEMS Congress / Posters 103^505
food products has always been an important concern to
the food industry. There is still confusion regarding how
many di¡erent enterotoxins are produced by B. cereus. In
fact it produces a high variety of toxins. Furthermore
enterotoxins could be produced also by non-B. cereus spe-
cies. Isolates of B. circulans, B. lentus, B. mycoides and B.
thuringiensis, which are strictly related to B. cereus, dem-
onstrated positive results using RPLA assay. Some au-
thors proved di⁄culties to distinguish between B. cereus,
B. mycoides and B. thuringiensis because they referred as
B. cereus also the B. mycoides and B. thuringiensis. In this
work we developed a couple of speci¢c primers for B.
cereus and speci¢c DNA extraction techiques to obtain
PCR products from food without any previous enrich-
ment. Salad, meat, rice, co¡ee and milk samples were
used to verify the procedure. The gyrB gene used as target
for the primer design was useful to obtain ampli¢ed prod-
ucts only for B. cereus also in the presence of a low num-
ber of B. cereus cells and a high number of other natural
contaminant microrganisms. Moreover positive results
were obtained using the PCR detection of B. cereus di-
rectly in food samples, and the data are con¢rmed by
classical microbiological analyses.
P2^48
CHARACTERIZATION OF S. JORGE CHEESE, A
PORTUGUESE RAW COW’S MILK CHEESE: EVO-
LUTION OF MICROBIAL AND PHYSICOCHEMI-
CAL PROFILES
J. Marcelino Kongo(1,2) and F. X. Malcata(2)
(1) CIRN ^ Universidade dos Acores, Rua Ma‹e de Deus,
9500 Ponta Delgada, Acores-Portugal; (2) Universidade
Cato¤lica Portuguesa ^ Escola Superior de Biotecnologia,
Rua Dr. Antonio Bernardino de Almeida, 4200-072 Porto-
Portugal
S. Jorge cheese is a Portuguese dairy product, which has
been manufactured for more than 350 years in the Azores
island with the same name from cow’s raw milk, in the
absence of any commercial starters; this cheese was
granted an AOC status in 1984. Emerging market rules
have enforced stricter quality and safety requirements, so
a full characterization of the Portuguese artisanal cheese
sold in largest numbers is in order, especially because such
a cheese is the main economic support of S. Jorge is-
landers. The aim of this work was thus to produce exper-
imental data that would characterize S. Jorge cheese. To-
ward such goal, the artisanal technology used in the
production of the cheese was monitored for six months.
Milk and cheese samples were collected every three weeks
at seven di¡erent factories, and standard plating on such
media as KAA, Rogosa, MSE, VRBGA, M17 and
OGYEA was done so as to ascertain the evolution of
FEMSLE Congress 2-6-03
151
the microbial pro¢le, followed by tentative identi¢cation
via API galleries. In addition, plating on XLDN, PAL-
CAM, OXFORD, MSA and VRBGA media was done
so as to grasp the safety of actual consumption of said
cheese, namely in terms of Listeria, Enterobacteriaceae,
Staphylococcus and Salmonella spp. The physicochemical
and microbiological data produced were subject to multi-
variate statistical analysis, in attempts to correlate them.
P2^49
DETECTION OF HISTIDINE DECARBOXYLASE
GENE IN LACTIC ACID BACTERIA ISOLATED
FROM PORTUGUESE WINES
A. P. Marques(1), M. C. Leita‹o(1), V. Basto(1), R. Ten-
reiro(3), M. V. San Roma‹o(1,2)
(1) Instituto de Biologia Experimental e Tecnolo¤gica/Insti-
tuto de Tecnologia Qu|¤mica e Biolo¤gica ^ Universidade
Nova de Lisboa. Apt, 12, 2781-901 Oeiras, Portugal; (2)
Estaca‹o Vitivin|¤cola Nacional, 2565-191 Dois Portos, Por-
tugal; (3) Departamento de Biologia Vegetal/Centro de
Gene¤tica e Biologia Molecular, Faculdade de Cie“ncias, Uni-
versidade de Lisboa, Campo Grande, 1740-016 Lisboa, Por-
tugal
Biogenic amines (BA) are basic nitrogenous compounds
found in wine and in other fermented food products. His-
tamine is one of the major BA in wine and one of the
causative agent of physiological distress such as hypoten-
sion and digestive disturbances. Lactic acid bacteria are
considered the main responsible for the BA formation.
Some of these bacteria contain enzymatic systems that
allow them to metabolise the peptides accumulated after
alcoholic fermentation of wine, during yeast autolysis,
with the subsequent formation of amino acids that can,
after decarboxilation, originate BA. Some Portuguese
wines, special red wines, present histamine contents rang-
ing the 8 mg/L. It is therefore important to ¢nd the micro-
organisms responsible for histamine formation in Portu-
guese wines. The aim of this work was the detection of the
histidine decarboxylase (HDC) gene in strains of lactic
acid bacteria (LAB) isolated from Portuguese wines. The
detection of HDC gene was done by PCR and colony
hybridization with non-isotopic DNA probe. DNA hy-
bridization is a convenient way to detect undesirable his-
tamine-forming strains. Speci¢c primers designed from
hdcA gene of Lactobacillus buchneri (DSMZ 5987) were
used to amplify the internal part of the HDC gene. Am-
pli¢ed DNA was puri¢ed and sequenced. The sequence
showed highest degree of homology with sequences of
HDC gene in the database of Genbank. DNA probe
was generated by PCR and labelled with digoxigenin
(DIG High Prime DNA Labelling and Detection Starter
152 1st FEMS Congress / Posters 103^505
Kit II ^ Roche). The HDC gene was detected in some of
the tested strains.
This work was partially supported by Agro Medida 8.1
Program, Project n.‡ 33 ^ Formac^a‹o de aminas bioge¤nicas
no vinho ^ caracterizac^a‹o de produtos enolo¤gicos existen-
tes no mercado.
P2^50
º RC
BRUCELLOSIS IN MUNICIPAL OF KE 6 OVEº DUR-
ING THE PERIOD 1983-90
I. Z. Mehmedi and M. R. Mehmedi
The Department of Infective Diseases, Medical Center Ki-
chevo, Kichevo 6250, M.Tito bb, Republic of Macedonia
Objective: to present clinical features and serological data
of brucellosis in the municipal of Kercove, Republic of
Macedonia. Methods : patients with brucellosis followed
between January 1983 ^ December 1990 were retrospec-
tively evaluated. Initial evaluation included complete
blood count urinalysis and serological tests (standard
tube Brucella agglutination test-STA). Diagnosis of brucel-
losis was made by following criteria : a) history of expo-
sure, b) compatible clinical picture of disease, c) brucella
serum agglutination titer more then 1/160. Results: In the
above mentioned period were registered 66 cases with bru-
cellosis, 14 of which are female and 52 male. The average
age was 41Y30 years. The most frequent features in the
patients disease of acute Brucellosis are: fever 46(69,69%);
joints pain( mostly in the spine and in the hips) 53
(80,30%); sweat 45(68,18%); loss of appetite 30(45,45%);
headache 34(51,51%); hepatosplenomegaly 7 (10,60%). In
the diagnosis of human brucellosis the best results were
shown with the Rose Bengal test, which was positive
100% of patients with acute brucellosis, then with the
Coombs test positive in 96,96%. The test by Wright with
93,93%. Conclusion : The most frequent features in the
patients disease of acute Brucellosis are: joint
pain(80,30%), fever (69,69%), sweat (68,18%). In the diag-
nosis of human brucellosis the best results were shown
with the Rose Bengal test, which was positive 100% of
patients with acute brucellosis.
FEMSLE Congress 2-6-03
P2^51
EFFECT OF TEMPERATURE AND VISCOSITY ON
BACTERIAL INACTIVATION BY HIGH PRESSURE
HOMOGENIZATION
A. Diels, E. Y. Wuytack and C. W. Michiels
Department of Food and Microbial Technology, Katholieke
Universiteit Leuven, Leuven, Belgium
High pressure homogenization is a unit process used in the
food and pharmaceutical industry primarily to make or
stabilize emulsions or suspensions. The development of
applications at increasingly higher pressure has created
an interest in taking bene¢t from the microbial inactiva-
tion that occurs during this process. We have studied the
inactivation of E. coli by high pressure homogenization
(100 ^ 300 MPa) in bu¡ered suspensions adjusted to di¡er-
ent viscosities with polyethylene glycol, and at di¡erent
initial temperatures of the cell suspensions (5 ^ 50‡C).
The results indicate that (i) bacterial inactivation increases
with initial temperature and decreases with viscosity of the
suspension; (ii) at least up to 35‡C, the increased inacti-
vation due to temperature can be entirely explained by the
reduced viscosity of the suspensions at elevated temper-
ature; (iii) above a certain temperature, bacterial inactiva-
tion exceeds the inactivation that would be predicted by
the reduced viscosity e¡ect, suggesting that thermal e¡ects
start to contribute to cellular inactivation in addition to
mechanical e¡ects. These experiments should contribute to
a better understanding of the bactericidal mechanisms of
high pressure homogenization, and to the development of
novel applications.
P2^52
IMPROVING THE MICROBIOLOGICAL SAFETY OF
SEEDS AND SPROUTS BY LOW-DOSE GAMMA IR-
RADIATION
Cs. Moha¤csi-Farkas, EŁ. Andra¤ssy, A. Bru«ckner, V. Gergely
Department of Microbiology and Biotechnology, Szent Ist-
va¤n University, Somlo¤i u¤t 14-16, 1118 Budapest, Hungary
Consumption of raw seed sprouts has been linked to sev-
eral outbreaks during the last decade in countries around
the world. Most of these foodborne outbreaks were due to
contamination with Salmonella spp. or Escherichia coli
O157:H7. In order to avoid these outbreaks, treatments
to reduce the pathogenic microorganisms on sprouts or on
seeds are needed. In the frame of an IAEA Co-ordinated
Research Project our aim was to investigate the e¡ect of
low dose gamma-radiation on natural micro£ora and ger-
mination of selected seeds and sprouts. Furthermore, an
 1st FEMS Congress / Posters 103^505
optimal dose that provides their microbiological safety
was also to be determined. Alfalfa and radish seeds were
irradiated using 60Co gamma-radiation source and were
treated with doses of 1, 2 and 3 kGy. After the treatment,
seeds germination or yield ratio and changes of microbial
load were analysed. To investigate the e¡ect of irradiation
on pathogenic microorganisms, seeds were inoculated with
a suspension of avirulent strains of E. coli O157 and Lis-
teria monocytogenes. Results showed that irradiation with
2 kGy dose decreased the total aerobic plate count by 2-3
log cycles on seeds. The yield ratio decreased by 30%.
Total aerobic plate count of sprouts germinated from ir-
radiated seeds achieved the same level on all samples. 1
kGy dose decreased the number of L. monocytognes by 1-
1.5 log cycles on seeds, and 3 log cycles on sprouts. E. coli
O157 proved to be much more radiation -sensitive than L.
monocytogenes. On the seeds 1 kGy dose reduced the num-
ber of E. coli O157 by 3 log cycles, and on sprouts a
100,000-fold reduction was achieved.
P2^53
FREQUENCY OF LISTERIA MONOCYTOGENES IN
DAIRY AND MEAT PRODUCTS
J. Nowroozi(1), A. J. Negad(2), S. Shahidi(2)
(1) Iran University of Medical Sciences and (2) Islamic
Azad University, Tehran, Iran
Listeria minocytogenes is a Gram positive, non-sporeform-
ing, rod shaped bacterium and has often been associated
with raw and pasteurized milk, dairy products, such as
soft cheese, meat products and vegetables. The aim of
this study was to ¢nd the frequency of L. monocytogenes
in dairy and meat products distributed in Tehran city and
detection of plasmids by electrophoresis. Samples of dairy
products (such as cheese, butter, cream), milk; sausage
and frankfurt was added to TSYEB and was refregrated
(cold enrichment method). Then, two selective medium
(Palcam agar and Listeria Selective agar) was used for
growth and biochemical tests for identi¢cation. Bacterial
plasmids were isolated by alkali lysis and has been elec-
trophoresis through agarose gel, then, observed and pho-
tographed. From 61 dairy products, 18 samples (29.5%)
and from 52 sausage and frankfurt, 8 samples (15.38%)
were contaminated with L. monocytogenes. From 26 L.
monocytogenes isolates, 5 (19.23%) of them contained plas-
mid DNA with 2.3 kbp molecular weight. Contamination
of dairy products and meat products (sausage and frank-
furt) by L. monocytogenes can occur during preparation,
transport and distribution. So, because of the high con-
sumption of these products in our country, it is necessary
to surveillance the production and distribution to prevent
of incidence of listeriosis in susceptible people.
FEMSLE Congress 2-6-03
153
P2^54
COMBINED ACTIVITY OF BACTERIOCINS AND
HIGH PRESSURE TREATMENTS ON THE SURVIV-
AL OF STAPHYLOCOCCUS AUREUS IN RAW MILK
CHEESE
J. L. Arque¤s(1), E. Rodr|¤guez(1), P. Gaya(1), M. Medi-
na(1), B. Guamis(2) and M. Nu•ez(1)
(1) Tecnolog|¤a de Alimentos, INIA, Carretera La Coru•a
km 7, Madrid, 28040 Spain; (2) CeRTA, Universitat
Auto'noma de Barcelona, Bellaterra,08193 Spain.
Staphylococcus aureus is one of the most common causes
of food-borne illness. The combined activity of bacterio-
cin-producing lactic acid bacteria used as protective cul-
tures and high pressure treatments on the survival of S.
aureus during ripening of raw milk cheese was investi-
gated. S. aureus was inoculated at approximately 105
cfu/ml into raw milk. Milk cultures of ¢ve strains of lactic
acid bacteria producing di¡erent bacteriocins were added
at 0.1%. Semi-hard cheese was manufactured according to
usual procedures. High pressure treatments of 300 MPa
(P300) during 10 min at 10‡C or 500 MPa (P500) during 5
min at 10‡C were applied to 2-day-old cheeses. On day 3,
S. aureus reached 6.46 log cfu/g in control cheese. Counts
were 0.16-0.46 log units lower in cheeses with bacteriocin
producers, and 0.45 and 2.43 log units lower respectively
in P300 and P500 cheeses without bacteriocin producers.
Synergistic inhibitory activity was detected in treatments
with P500 and bacteriocin producers, the highest inhibi-
tory activity being recorded for the combined treatment
P500 and nisin A. S. aureus was not detected on day 20 in
any P500 cheese. On day 60, counts of S. aureus were 5.30
log cfu/g in control cheese, and 0.02-0.81 log units lower in
cheeses with bacteriocin producers. Counts in P300
cheeses without bacteriocin producers were 2.84 log cfu/
ml, and 0.05-0.58 log units lower with bacteriocin pro-
ducers.
154 1st FEMS Congress / Posters 103^505
P2^55
MYCOLOGICAL AND MYCOTOXICOLOGICAL IN-
VESTIGATIONS DURING MICROMALTING PRO-
CESS OF BARLEY
M. Skrinjar(1), O. Grujic(1), I. Peric(1), J. Pejin(1), V.
Krstanovic(2) and S. Kocic(1)
(1) Faculty of Technology, University of Novi Sad, Bulevar
Cara Lazara 1, 21000 Novi Sad, Yugoslavia; (2) Faculty
of Food Technology, University of J. J. Strossmayer in
Osijek, F. Kuhaca 18, 31001 Osijek, Croatia
Mycological and mycotoxicological analyses were under-
taken of raw materials (unsorted barley, ¢rst class barley),
semi-products (green malt, dried malt) and ¢nal products
(malt), for brewing industry. Water samples used for bar-
ley steeping were also analyzed. The total count of moulds
in 1g/ml of sample was determined, followed by isolation
and identi¢cation of the mould. Mycotoxicological analy-
sis by TLC enabled qualitative and quantitative determi-
nation of a£atoxins B1 and G1, ochratoxin A and zeara-
lenone. Determination of the total count of moulds per 1g
of barley before and after germination, as well as malt was
carried out by grains disposal (10) directly to Saubouraud
maltose agar containing chloramphenicol and oxytetracy-
clin, while in 1ml of steeping water it was determined by
the application of the standard Koch’s method. Average
count of moulds per 1g of unsorted barley before malting
process was 2.5X102, and after ¢rst, second and third day
of steeping this was 3.7X102, 5.6X102 and 2.1X103, respec-
tively. First class barley contained 2.6X102 moulds per g,
and after the ¢rst, second and third day of steeping
3.5X102, 4.1X102 and 1.4X103, respectively. 1 ml of water
taken from steeping vessel before process of malting con-
tained 81.5 moulds, and after the ¢rst, second and third
day of barley steeping 1.0X102, 2.8X102 and 1.2X104, re-
pectively. After germination, the total count ranged from
3.6X105 (unsorted barley) to 5.6X105 (¢rst class barley)
moulds per g. In 1g of malt with roots, the total count
ranged from 8.7X104 (unsorted barley) to 2.5X104 (¢rst
class barley). Similarly, in malt this ranged from 3.4X104
(unsorted barley) to 2.4X104 (¢rst class barley) moulds per
g. Between 65 and 80% of moulds isolated from barley
were from the group Dematiaceous Hyphomycetes. After
steeping, their share in mycopopulations increased to 90-
95%. During the third day of steeping and during the
period of germination, signi¢cant presence of Penicillium
species was registered. In the course of malt drying, Pen-
icillium spp. and Zygomycetes were registered again at
high levels. None of the samples investigated was contam-
inated by toxins.
FEMSLE Congress 2-6-03
P2^56
A BIOSENSOR SYSTEM WITH PARAMECIUM CAU-
DATUM FOR THE ASSESSMENT OF POTEN-
TIALLY TOXIC FUNGAL METABOLITES
B. Pernfuss, J. Stemer, R. Poeder and H. Strasser
Institute of Microbiology, Leopold-Franzens University of
Innsbruck,Technikerstrasse 25, 6020 Innsbruck, Austria
In the course of the EU-funded project QLK1-2001-01391
‘‘Risk Assessment of Fungal Biological Control Agents
(BCA’s)’’ it is investigated by a multidisciplinary Euro-
pean consortium if fungal compounds enter the food chain
and if they pose a health risk. Besides the extraction, iden-
ti¢cation and quanti¢cation of fungal metabolites from
BCA’s information on intra-species and inter-species var-
iability in the production of selected metabolites, as well as
data on their spatial-temporal distribution and their rate
of decomposition will be provided. Fast and accurate
screening and testing protocols should be developed taking
into account existing regulatory testing programmes and
the need to integrate endpoints. One of the key issues of
the project is the development of in vitro toxicity assays
such as sensitive cell lines (i.e. biosensors) to detect se-
lected metabolites. Plant and animal cell cultures should
act as a substitute for animal testing, and must facilitate
high throughput screening for detecting toxicity and in
vitro toxicity. In this context we have optimised conditions
for the assessment of the ciliate Paramecium caudatum as a
‘‘biosensor’’. This sensitive test organism can detect fungal
secondary metabolites in an inexpensive testing system and
give an indication of their toxic potential. E¡ective dose
data (ED50) of P. caudatum with oosporein, one of the
secondary metabolites produced and excreted by the en-
tomopathogenous anamorphic fungus Beauveria brong-
niartii, indicate that this biosensor system is even more
sensitive than hamster tumour cells, as well as selected
human and insect cell lines.
P2^57
THE INFLUENCE OF COOKING ON THE GENO-
TOXIC POTENTIAL OF MEAT AND MEATSTUFFS
CONTAINING FOOD ADDITIVES AND MEGATERIN
V. Y. Ponomarev, O. B. Ivanchenko, O. A. Reshetnik
Dep. of Food Technology, Kazan State Technological Uni-
versity, K. Marks str., 68, Kazan, Russia
Traditionally, microbial enzymes are widely used as a re-
search tools in genetic engineering, medicine and molecu-
lar biology. Last time, microbial protease was used in food
industry for improving the quality of meat. The animal
 1st FEMS Congress / Posters 103^505
meat and model meat products containing food additives
and microbial enzyme of Bacillus megaterium (commer-
cially known as ‘‘megaterin’’) were investigated for geno-
toxicity before and after cooking under high temperature.
The liquid procedure was used for assessing the potency of
meat extracts to cause DNA damages in DNA-repair test.
The repair-pro¢cient and repair-de¢cient (recA-, polA-,
uvrA-) Escherichia coli trp auxotrophs were used. Water
extracts from meat and meat products were tested with
and without microsomal activation mixture. The animal
meat and the multicomponent salting mixture, containing
sodium nitrite, phosphates, chlorides, showed no geno-
toxic properties. Enzyme of B. megaterium appears to pos-
sess DNA damaging activity in uvr A- and pol A- mutants
of E. coli strains. Perhaps, the growth inhibition was re-
lated with proteolytic activity of enzyme. The genotoxicity
of meat with food additives increased after addition of
megaterin. The considerable DNA-damaging e¡ect on E.
coli test strains was shown. Cooking at the high temper-
atures led to the loss the DNA-damage activity of model
systems. However, the extracts from roasted meat and
model meat products induced DNA-damage after meta-
bolic activation in vitro with enzymes of rat liver micro-
somal fraction.
P2^58
PREVALENCE OF LISTERIA MONOCYTOGENES IN
BROILER MEAT AT RETAIL LEVEL IN ESTONIA
K. Praakle, J. Lunden, M. Lindstro«m, M.-L. Ha«nninen and
H. Korkeala
Department of Food and Environmental Hygiene, Faculty of
Veterinary Medicine, P. O. Box 57, FIN-00014 University
of Helsinki, Finland
Listeria monocytogenes is a ubiquitous pathogen that has
been implicated within the past decade as the causative
organism in several outbreaks of foodborne disease. It is
well established that any fresh food product of animal or
plant origin may harbour varying numbers of L. monocy-
togenes. However, there are no studies on the occurrence
of L. monocytogenes done in Estonia. Therefore we have
studied the prevalence of L. monocytogenes in broiler meat
at retail level in Estonia. A total of 92 samples of broiler
thigh meat produced in Estonia and 87 samples imported
to Estonia (56 from Denmark, 21 from Hungary, and 10
from other countries) were examined. For the detection of
L. monocytogenes the ISO 11290-1:1996 method was used.
The method includes pre-enrichment in half-Fraser broth
and selective enrichment in Fraser broth. Both enrichment
media were plated on selective palcam agar and LMBA
(Listeria monocytogenes Blood Agar). Typical colonies
were further streaked on blood agar plates and veri¢ed
as L. monocytogenes by Gram-reaction, catalase test, and
FEMSLE Congress 2-6-03
155
API LISTERIA. The contamination rates for L. monocy-
togenes were 87% (80) for the broiler meat produced in
Estonia, 38% (21) for the meat imported from Denmark,
and 76% (16) for the broiler meat imported from Hun-
gary. The characterization of the isolates by genotypical
method is in progress.
P2^59
TRANSFER OF ANTIBIOTIC RESISTANCE GENES
IN DAIRY ENTEROCOCCI
T. C. Ribeiro(1), F. Lopes(1), A. Euge¤nio(1), M.
Abrantes(1), R. Tenreiro(2), T. Crespo(1)
(1) IBET/ITQB, Apartado 12, 2781-901 Oeiras, Portugal;
(2) FCUL, Campo Grande, Edif|¤cio C2, Piso 4, 1700 Lis-
boa, Portugal
Enterococcus are ubiquitous bacteria frequently found in
the environment and they are part of the commensal mi-
crobiota of humans and animals. They can occur in sev-
eral traditional cheeses made with raw milk. Enterococcus
are considered as emerging human pathogens and they are
often associated with hospital-acquired infections. A rea-
son for the rise of nosocomial infections related to enter-
ococci is their ability to develop resistance against most
antibiotics currently used. The rapid spread of antibiotic
resistances in a wide variety of bacteria is mainly due to
the location of antibiotic resistance genes on mobile genet-
ic elements such as plasmids and transposons. Food-borne
enterococci, like those from milk and cheese, are able to
rapidly horizontally transfer their antibiotic resistance
genes to other members of the genus and potentially to
other bacteria of clinical importance. To evaluate this risk,
conjugation phenomena are being studied for antibiotics
for which resistance is probably associated with plasmids,
such as kanamycin, tetracycline, erythromycin, ampicillin
and vancomycin. In order to identify the plasmid(s) carry-
ing these resistance determinants, PFGE and southern blot
hybridisation are being used. These ¢lter mating assays are
being performed using Enterococcus faecalis FA2-2, 13
food isolates previously cured of erythromycin resistance
and 1 cured of tetracycline resistance (acridine orange, 45
Wg/mL) as recipient strains. Enterococci with vancomycin
resistance genotype (vanA, vanB and vanC), already de-
termined, are being used as donor strains.
156 1st FEMS Congress / Posters 103^505
P2^60
SEROLOGICAL IDENTIFICATION OF LISTERIA
MONOCYTOGENES ISOLATED FROM FOOD US-
ING SLIDE AGGLUTINATION TEST
T. Rupel, T. Majstorovic¤, M. SNedlbauer, N. Trunk
Institute of Public Health of the Republic of Slovenia,
Centre for Environmental Health, Department of Sanitary
Microbiology, Grablovic›eva 44, 1000 Ljubljana, Slovenia
Listeria monocytogenes (LM) is subdivided into serotypes
on the basis of somatic (O) and £agellar (H) antigens.
Thirteen serotypes of LM have been identi¢ed. Because
the vast majority of cases of human disease are caused
by only three serotypes (1/2a, 1/2b and 4b), serotyping is
only minimally helpful in epidemiologic investigations.
Strains of LM isolated from food have been collected at
Department of Sanitary Microbiology, since 1994. Pure
cultures of Listeria isolated from foods were con¢rmed
with the api-Listeria test (bioMerieux). Serological slide
agglutination tests were done on all isolates of LM, using
commercially prepared antisera Bacto- Listeria O Antisera
Types 1,4 and Poly (Difco). The 56 strains were subdi-
vided into 2 serovars. The 46 strains of LM were serotype
1, six strains were serotype 4 and the test for 4 strains was
negative. The goal of subtyping LM isolates was to deter-
mine which serotypes were the most frequently isolated
from food samples.
P2^61
A MODIFIED NESTED PCR METHOD TO DETECT
NON-CULTURABLE E. COLI CELLS IN DRINKING
WATER
A. Rust, A. J. B. Zehnder and W. Ko«ster
Swiss Federal Institute For Environmental Science and
Technology, Ueberlandstr. 133, CH-8600 Duebendorf, Swit-
zerland
In order to guarantee safe drinking water, the microbial
quality is controlled routinely by analysing water samples
for bacterial parameters. A simple, inexpensive but time
consuming culturing method for the detection of E. coli
has been used for many decades. We developed a faster
polymerase-chain-reaction (PCR)-method with two subse-
quent runs to detect E. coli in drinking water. A DNAse
digestion step was introduced in the course of the sample
preparation to avoid false positive results by free DNA of
dead bacteria. The PCR method was evaluated as alter-
native to the classical culture method. We spiked drinking
water samples with E. coli cells at low concentrations and
stored them at cold temperature intending to simulate en-
FEMSLE Congress 2-6-03
vironmental conditions. Tests with both methods were
performed at intervals ranging from days up to weeks.
At early time points, the two methods delivered compara-
ble results. However, at later time-points, the E. coli cells
became non-culturable, whereas the results obtained by
PCR showed no signi¢cant decrease of ampli¢able
DNA. The PCR gives positive results for all E. coli cells
containing chromosomal DNA inside an intact cell enve-
lope, also for those cells which are in a non-culturable
state. At the moment we do not recommend the applica-
tion of this PCR technique for routine analysis but see its
bene¢ts in environmental water research.
P2^62
QUANTITATIVE RISK ASSESSMENT FOR SALMO-
NELLA IN FEED FOR FATTENING PIGS IN SWIT-
ZERLAND
I. Sauli(1), J. Danuser(1), A. H. Geeraerd(3), K. Bo«gli-
Stuber(1), B. Bissig-Choisat(1), K. D. C. Sta«rk(1) and C.
Wenk(2)
(1) Swiss Federal Veterinary O⁄ce, Schwarzenburgstrasse
161, 3003 Bern, Switzerland; (2) Swiss Federal Institute of
Technology, Institute for Animal Sciences, Universita«t-
strasse 2, 8092 Zu«rich, Switzerland ; (3) BioTeC ^ Biopro-
cess Technology and Control, Department of Chemical En-
gineering, Katholieke Universiteit Leuven, W. de Croylaan
46, B-3001 Leuven, Belgium
In pig production, the carrier pig, contaminated feed and
environment are currently considered the most signi¢cant
sources of infection. Pigs rarely show clinical signs, thus
undetected Salmonella carriers can enter the food produc-
tion chain. In a ‘‘Farm to Fork’’ food safety concept, the
production of safe feed is the ¢rst step for ensuring safe
foods and is therefore crucial. The aims of this study were
(1) to investigate the prevalence of Salmonella throughout
the production of feed for fattening pigs in Switzerland,
and (2) to perform a quantitative risk assessment for esti-
mating the risk that Salmonella-contaminated batches of
feed are produced in Swiss mills. Samples of major ingre-
dients were collected at delivery, and samples of ¢nished
feed were collected in the storage bins. Dust samples were
moreover collected at critical points in one feedmill. A
model for simulating the behaviour (growth, survival or
inactivation) of the pathogen throughout the production
steps was build using predictive microbiology tools. Vari-
ous scenarios were created considering the production pro-
cesses commonly implemented in Switzerland. Using the
results of the prevalence survey as input data in the sim-
ulation model, the risk that Salmonella-contaminated
batches of feed are produced in Swiss mills was assessed.
Preliminary results of the survey revealed a low prevalence
of Salmonella in both ingredients and ¢nished feed. The
 1st FEMS Congress / Posters 103^505
risk that Salmonella-contaminated batches of feed are pro-
duced depended on the production process, but was low
for all scenarios. Final results of the prevalence survey and
of the quantitative risk assessment will be presented.
P2^63
OCCURENCE, FACTORS INFLUENCING THE RE-
SISTANCE AND SAFETY IMPLICATIONS OF
HIGHLY HEAT RESISTANT SPORES
P. Scheldeman(1,2), K. Goossens(1), A. Pil(1), L. Her-
man(1), P. De Vos(2), O. Guillaume-Gentil(3), J. Han-
sen(4), S. Foster(4) and M. Heyndrickx(1)
(1) DVK-CLO, Dept. Animal Product Quality, Brusselses-
teenweg 370, 9090 Melle, Belgium ; (2) Universiteit Gent,
Laboratory for Microbiology (WE10V), K. Ledeganck-
straat 35, 9000 Gent, Belgium ; (3) Nestle¤ Research Center,
P.O. Box 44, CH-1000 Lausanne 26, Switzerland ; (4) Uni-
versity of She⁄eld, Dept. Molecular Biology and Biotech-
nology, Firth Court, Western Bank, She⁄eld S10 2TN, UK
Highly heat resistant spores (HRS) of Bacillus sporother-
modurans may survive sterilizing and ultra high temper-
ature (UHT) treatment of milk. Spores originating from
the dairy farm display variable but lower heat resistances
than spores freshly isolated from UHT-treated dairy prod-
ucts. REP-PCR and ribotyping indicated that the majority
of these B. sporothermodurans isolates from UHT- prod-
ucts belong to a clonal lineage (HRS-clone), whereas
clearly more heterogeneity was found among dairy farm
isolates. Also, HRS-clone spores grown under lab condi-
tions seem to gradually lose their heat resistance upon
successive culturing. It was investigated if these features
of clonal origin but di¡erent heat resistances were corre-
lated with the in£uence of sublethal stress conditions. In-
deed, a sublethal H2O2 treatment resulted in a signi¢cant
heat resistance increase of HRS-clone spores. To evaluate
the importance of this experience, the intrinsic heat resis-
tance of spores from a variety of Bacillus species present at
the dairy farm was studied next to the question whether an
increased heat resistance following peroxide stress is re-
stricted to B. sporothermodurans or if other spore forming
bacteria such as Bacillus cereus can also show this. Under-
standing the mechanism of this heat resistance induction is
of great importance to accommodate industrial heat- and/
or other treatments in the light of food safety and quality.
Therefore, a fundamental study of endospore properties
important for heat resistance was initiated, aiming to com-
pare the chemical structure (spore peptidoglycan compo-
sition and mineralization) of B. sporothermodurans, Paeni-
bacillus sp. and B. cereus spores displaying di¡erent heat
resistances under di¡erent stress conditions, and to deter-
mine any correlation between these parameters and the
spore resistance properties.
FEMSLE Congress 2-6-03
157
P2^64
EFFECTS OF GROWTH CONDITIONS ON BACIL-
LUS CEREUS GROWTH AND TOXIN PRODUCTION
P. Schmitt, T. Clavel, M. Jobin and C. Duport
UMR A408 INRA/Universite¤ d’Avignon ‘‘Se¤curite¤ et Qual-
ite¤ des Produits d’Origine Ve¤ge¤tale’’ INRA Domaine St
Paul 84914 Avignon cedex 9, France
The objective of this presentation is to report and dis-
cussed about our results and those cited in literature on
the e¡ects of growth rate, physico-chemical and nutrition-
al parameters in£uencing Bacillus cereus growth and tox-
ins production. B. cereus occurs widely in the environment
and is frequently isolated from cooked chilled food and
vegetable. B. cereus is responsible for two types of food
associated illness: the emetic syndrome and the diarrheal
syndrome. The former is due to a small molecular weight
cyclic toxin, cereulide. The diarrheal syndrome results
from enterotoxins production by B. cereus growing in
the small intestine of host following ingestion of viable
cells and/or spores, making this condition a true toxicoin-
fection. Some contradictories results had been published
resulting from (i) the high diversity among B. cereus strain
(ii) use of rich or poor media, with or without various
glucides (iii) di¡erent physico-chemical conditions such.
In addition, majority of experiments were performed on
uncontrolled batch cultures. Conversely to batch culture,
chemostat permits cells to grow inde¢nitely at a de¢ned
growth rate and biomass concentration, in an environment
of stable substrates and products. For this reason, B. ce-
reus was cultivated in regulated conditions and in chemo-
stat. Our results shown that in aerobiosis, the maximum
HBL amount was detected during the middle of exponen-
tial growth phase, whereas it took place at the early sta-
tionary growth phase for anaerobic growth cultures. The
greatest HBL production occurred under anaerobiosis In
this condition, experiments carried out in chemostat
shown that the production of toxin was regulated by the
growth rate and glucose: HBL production decreased as
growth rate increased and was inhibited by high glucose
concentrations (300 mM, 5.4 % w/v).
158 1st FEMS Congress / Posters 103^505
P2^65
RISK ASSESMENT OF FUNGAL BIOLOGICAL CON-
TROL AGENTS ^ BEAUVERIA BRONGNIARTII AND
ITS METABOLITE OOSPOREIN: NEEDS AND
DEEDS
C. Seger(1), D. Erlebach(2), U. Griesser(1), S. Sturm(1),
H. Stuppner(1), and H. Strasser(2)
(1) Institute of Pharmacy, Department of Pharmacognosy,
Leopold-Franzens University of Innsbruck, Innrain 52, A-
6020 Innsbruck; (2) Institute of Microbiology, Leopold-
Franzens University of Innsbruck, Technikerstra_e 25, A-
6020 Innsbruck
Much has been investigated in the ¢eld of mycotoxins
produced on feed and fodder however ecological studies
on the formation, signi¢cance and fate of secondary me-
tabolites produced by fungi in the environment are scarce.
In order to perform a responsible risk-assessment of fun-
gal biological pest control agents investigations on the en-
vironmental enrichment and the signi¢cance of secondary
metabolites released by entomopathogenic fungi are
needed. It is the major aim of the EU-RTD-project
RAFBCA (QLK1-CT-2001-01391) to obtain information
on the temporal-spatial distribution of metabolites in the
biocontrol agents as well as in the applicated environment.
Beauveria brongniartii (Ascomycota, anamorph of Clavici-
pitales) is known to be a very selective and highly virulent
entomopathogenic fungus used in the control of Melolon-
tha melolontha (chockchafer; coleoptera, scarabaeidae).
Although there have been numerous experiences in the
use of this fungus in infested areas since the seventies
only few data have been published on the ecotoxicological
impact and relevance of metabolites synthesised and se-
creted by B. brongniartii. The major metabolite ^ the
bis-dihydroxybenzoquinone derivative oosporein is poorly
characterized and its fate in biological matrices is un-
known (i.e. soil and crop). Data on the physico-chemical
characterization of oosporein will be presented and their
impact on the risk assessment process will be discussed.
P2^66
ENTRY OF ESCHERICHIA COLI INTO CORN
PLANT VIA THE ROOT SYSTEM
S. Sela(1), N. Bernstein(2), and R. Pinto(1)
(1) Department of Food Science ; (2) Institute of Soil
Water and Environmental Sciences, Agricultural Research
Center, The Volcani Center, Beth-Dagan, Israel
The incidence of human bacterial pathogens on fresh fruits
and vegetables has been a growing concern in industrial-
FEMSLE Congress 2-6-03
ized countries. Contamination of crops might occur at any
stage of the food chain. In several cases, manure or con-
taminated irrigation water has been suspected as the pri-
mary source of contamination. Recent studies have dem-
onstrated that E. coli and Salmonella enterica can enter the
plants via the root system, translocate to the shoot, and
survive in the internal tissues of lettuce and tomato, re-
spectively. Since conventional washing treatments are in-
e¡ective in removal of bacteria from inner tissues, such
contaminated plants might pose a health hazard for con-
sumers. To verify that other plants are susceptible to this
route of contamination we have examined the uptake of
antibiotic-resistant E. coli by corn plants. Young corn
seedlings were grown hydroponically in nutrient solution
supplemented with E. coli. Plants were divided into three
groups, one with intact roots, another with root-tip-re-
moved (damaged) and the third group with the entire
root system removed. The upper plant parts were tested
for bacterial internalization at 48 hrs after inoculation. We
found that E. coli was indeed capable of internalization
via the root system of both intact and root-damaged corn
seedling. The uptake of bacteria was signi¢cantly higher in
plants with damaged or without roots compared to intact
plants. These results support the notion that human
pathogens might contaminate fresh vegetables pre-harvest
by internalization via the root system.
P2^67
ISOLATION AND IDENTIFICATION OF XEROTO-
LERANT YEASTS FROM FRUIT YOGHURT AND
QUARK SAMPLES
º zbas
S. Senses, Z. Y. O
Food Engineering Department, Hacettepe University, Bey-
tepe 06532, Ankara, Turkey
Xerotolerant yeasts have been known to be the major
spoilage £ora in high-sugar foods. In order to prevent
high ¢nancial losses caused by xerotolerant yeasts, isola-
tion and identi¢cation of this group of yeasts should be
performed, properly. Additionally, recent developments in
the medical ¢eld have resulted in a number of emerging
pathogens, also including some foodborne strains. In this
study, swollen packages of 50 fruit yoghurt and 4 quark
samples were collected from retail markets and investi-
gated for the presence of xerotolerant yeasts. During the
study, Dichloran 18% Glycerol (DG18) agar, Tryptone
Glucose Yeast Extract (TGY) agar, Malt Extract Yeast
Extract 50% Glucose (MY 50G) agar and Malt Extract
Yeast Extract 40% Sucrose (MY 40S) agar which has been
modi¢ed by us, were used as isolation media. In total, 13
yeast strains were isolated from the samples. For the iden-
ti¢cation of these isolates, ID 32C strips and some other
identi¢cation tests were performed. 13 strains of asporog-
 1st FEMS Congress / Posters 103^505
enous yeasts were identi¢ed as eight species belonging to
two genera. The major species identi¢ed were; Candida
famata, C. guilliermondii, C. glucosophila, C. kefyr, C. kru-
sei, C. lambica, C. parapsilosis, Rhodotorula glutinis. Using
the ID 32C, 31 di¡erent carbon assimilations were inves-
tigated for the isolates. All isolates were capable of glucose
assimilation, whereas only 77% of them assimilated su-
crose, 69% maltose, 69% galactose, 54% ra¢nose and
15% lactose. Approximately 69% of the isolates were
found as xerotolerant yeasts. Also approximately 84% of
the isolates were found to ferment glucose.
P2^68
THE INHIBITION OF GROWTH OF VARIOUS HU-
MAN AND PHYTHOPATHOGENIC FUNGI BY TEA
EXTRACTS IN VITRO
M. Shamtsyan, A. Komolov, N. Zolnikova
St. Petersburg State Institute of Technology (Technical
University), St. Petersburg, Russia
Microscopic fungi are the main pathogens of agricultural
plants and are causing valuable losses of the harvest. Also
they can produce various mycotoxins, which are the rea-
son of heavy disfunctions of animal and human organ-
isms. Our studies were focused on the e¡ect of green
and black tea extracts on the growth of microscopic fungi,
belonging to 20 di¡erent genera. Micromycetes were culti-
vated on the agar media with or without addition of tea
extracts. It appears, that the addition of de¢nite amounts
(0.05 ^ 0.5 %) of tea extracts to the growth media causes a
signi¢cant slowing in the growth of all of the species of
tested micromycetes. It was also estimated, that the fungi-
static e¡ect of green tea extracts was higher than of the
extracts obtained from black tea. Green tea extracts
caused the most pronounced e¡ect in growth reduction
on the tested species from the genera of Fusarium (growth
was reduced for 55 ^ 75 % for di¡erent species), Alternaria
(60 ^ 70 % reduction), Aspergillus (60 ^ 65 % reduction)
and Botritis (55 ^ 60% reduction). Investigations on the
in£uence of green tea extract on the morphology of fungi
were carried out. The obtained results indicate the possi-
bility for the utilization of green tea extracts as a natural
fungistatic product. One that can protect plants, animal
feed and food from the pathogenic fungi and act as a
natural antifungal preservative, and also in research prac-
tices for the isolation of micromycetes and their puri¢ca-
tion.
FEMSLE Congress 2-6-03
159
P2^69
EFFECTS OF LACTATE AND DIACETATE ON VIA-
BILITY OF LISTERIA MONOCYTOGENES AND
CHANGES IN ITS CYTOPLASMIC PROTEINS
E. Mbandi and L. A. Shelef
School of Science, Wayne State University, Detroit, Mich-
igan, U.S.A.
The antilisterial e¡ects of sodium lactate, diacetate, and
their combination in ready-to-eat meats, pH 6.3, were
studied using Listeria monocytogenes Scott A (serotype
4b). Each of the salts alone (2.5% lactate and 0.2% diac-
etate) delayed growth of the pathogen during refrigerated
(5‡C) and abuse (10‡C) storage for up to 45 days. Combi-
nation of the salts was most inhibitory, resulting in lister-
icidal e¡ects at 5‡C and listeriostatic e¡ects at 10‡C. A
proteomic approach was used to study changes in the
cytoplasmic proteins of the organism. Cells were treated
for 6 h with each salt and their combination as above in a
chemically de¢ned medium, pH 6.3.SDS-PAGE gels
showed similar band patterns in untreated and treated
samples. Data from two-dimensional electrophoresis
showed that diacetate caused the highest changes in total
number of proteins (198 vs. 131 in control, and 150 in
lactate treatment), highest unmatched proteins (124 vs.
53 in lactate), highest increase in expression (20 vs. 5 in
lactate), and highest number of novel protein (90 vs. 45 in
lactate). The highest number of repressed proteins was
observed in the combination treatment (41 vs. V30 in
the single salt treatment). No di¡erence showed in the
expression level of prfA protein, the regulator of virulence
genes of the pathogen, using Western blot analysis. Re-
sults suggest that survival of L. monocytogenes in the pres-
ence of lactate and diacetate is similar to survival mecha-
nisms during NaCl or acid stress conditions, and that
expression of virulence proteins may not be a¡ected by
treatment with these salts.
160 1st FEMS Congress / Posters 103^505
P2^70
HIGH DOSES OF PROBIOTICS (DP16, DP28, DP38,
DP66, AND DP88) HAVE NO ADVERSE EFFECT ON
THE HEALTH OF MICE
A. Liu(1), Y. Gao(1,3) and Q. Shu(2)
(1) Mt Albert Health Research Laboratory and (2) Micro-
bial Science Research Group of the New Zealand Institute
for Crop and Food Research Limited, Private Bag 92 169,
Auckland, New Zealand ; (3) Instititute of Food, Nutrition
and Human Health, Massey University, Auckland, New
Zealand
The safety of probiotic strains DP16, DP28, DP38, DP66,
DP88, and LA1 (a commercial probiotic strain used as a
positive control), was studied in mice (BALB/c) by feeding
at a high dose. Mice (6V8 week-old) were acclimatized on
a skim milk powder based diet for 14 days prior to being
randomly allocated into 7 groups (n=8). Following accli-
matization, mice were orally administered DP88 [Group
(G) 7], DP66 (G6), DP38 (G5), DP28 (G4), DP16 (G3),
and LA1 (G2) (5 x 1010 CFU/mouse/day) from day 0 to
day 7. The negative control mice (G1) did not receive any
probiotic organisms. The e¡ect of the probiotics on the
health of mice was assessed by monitoring the health ap-
pearance, behavior, presence of diarrhoea, feed intake,
water intake, and liveweight gain of the animals. On day
14, mice were euthanased by an iso£uorance overdose and
bacterial translocation to mesentery lymphatic node
(MLN), spleen, liver, kidney, and blood were also mea-
sured. No abnormal clinical signs were observed in any of
the groups during the period of the experiment. Also,
there was no signi¢cant di¡erence in feed intake, water
intake, and liveweight gain between the control and other
groups (P s 0.05). No bacteria were detected in blood,
kidney, liver, and spleen of animals from any group. There
were no bacteria in the MLNs of the negative control
mice. However, small numbers of bacteria were found in
the MLNs of some animals from G2 (2/8), G3 (1/8), G4
(1/8), G5 (2/8), G6 (1/8) and G7 (1/8); The numbers of
bacteria in the MLNs were positively correlated with the
faecal numbers of lactic acid bacteria (P 6 0.01, r=0.6).
Interestingly, DNA ¢nger printing analysis showed that
the bacteria isolated from the MLNs were di¡erent from
the strains used in this study. These results demonstrated
that probiotic strains DP16, DP28, DP38, DP66, and
DP88 did not translocate to systemic tissues and had no
adverse e¡ect on the health of mice.
FEMSLE Congress 2-6-03
P2^71
MYCOLOGICAL AND MYCOTOXICOLOGICAL
QUALITY OF SOME AGRICULTURAL PRODUCTS
TAKEN FROM ‘‘HEALTHY FOOD’’ STORES
M. Skrinjar, V. Injac, I. Sedej and S. Kocic
University of Novi Sad, Faculty of Technology, Boulevard
Cara Lazara 1, 21000 Novi Sad, Yugoslavia
Mycological and mycotoxicological investigations of some
agricultural products (corn £akes, integral rice, dried
grapes, wheat £akes, oats £akes) were performed. The
products were taken from ‘‘healthy food’’ shops. Five
samples of every product were tested. In all samples the
total number of moulds in 1g was examined, as well as
identi¢cation of isolated species. Investigation of the pres-
ence of a£atoxin B1 (AB1), G1 (AG1), ochratoxin A (OA)
and zearalenone (ZEA) have been involved in mycotoxi-
cological analysis. Two media were used for isolation of
moulds: Sabouraud maltose agar (SMA) and MY50G me-
dium for xerophiles. Qualitative and quantitative determi-
nation of mycotoxin was done applying the TLC method
according to A.O.A.C. The greatest number of investi-
gated samples were contaminated with moulds. In 1g of
corn £akes from 3 to 9,6X102 , integral rice from 10 to 40,
dried grapes from 2 to 7 and in wheat £akes from 2 to
2,8X102 moulds were found. The most frequent species
belonged to genera Cladosporium, Alternaria, Eurotium,
Aspergillus and Penicillium. OA was established in 1 sam-
ple of corn £akes (40.0 Wg
kg -1) and integral rice (80.0 Wg

kg -1), in 2 samples of dried grapes (40.0 and 56.0 Wg
kg
-1
) and wheat £akes (80.0 and 160.0 Wg
kg -1) and in 3
samples of oats £akes in traces up to 160.0 Wg
kg -1. ZEA
was found in 1 sample of corn £akes and wheat £akes at
concentration of 192.0 Wg
kg -1 and in 2 samples of oats
£akes (384.0 and 480.0 Wg
kg -1). No sample was con-
taminated with AB1 and AG1.
P2^72
VANCOMYCIN RESISTANT ENTEROCOCCI FROM
NORWEGIAN POULTRY FARMS ESTABLISHED
AFTER THE BAN OF AVOPARCIN
M. S\rum(1), G. Holstad(1), A. Lillehaug(1) and H.
Kruse(2)
(1) National Veterinary Institute, Oslo, Norway; (2) Nor-
wegian zoonotic centre, Oslo, Norway
An association has been documented between the use of
avoparcin as a growth promoter and the occurrence of
vancomycin resistant enterococci (VRE) in poultry pro-
duction. Studies have shown that VRE have persisted up
 1st FEMS Congress / Posters 103^505
to 3-4 years after avoparcin was banned in Norway in
1995. VRE have also been shown to survive on the farms.
The aim of this study was to examine the prevalence of
VRE in poultry at Norwegian poultry farms established
after the ban of avoparcin was implemented. Faecal sam-
ples from 39 farms established after 1995 (sample farms)
and from 25 farms previously exposed to avoparcin (con-
trol farms) were examined. The concentration of VRE and
generic enterococci were determined. One colony from
each positive sample was selected, identi¢ed, and tested
for presence of the vanA gene by PCR. The susceptibility
to narasin was also examined. VanA-type VRE were de-
tected in samples from 25 out of 39 sample farms (64%)
compared to 23 out of 25 of the control farms (92%).
There seemed to be higher concentration of VRE in the
control samples. No resistance towards narasin was de-
tected. This study shows that VRE are present at poultry
farms previously not exposed to avoparcin. The propor-
tion of VRE positive farms was lower for recently estab-
lished farms than for farms previously exposed to avopar-
cin. The concentration of VRE at the recently established
farms seems to be lower than at farms previously exposed
to avoparcin.
P2^73
MYCOLOGICAL AND MYCOTOXICOLOGICAL
PICTURE OF SOME CATEGORIES OF WHEAT
KERNELS
T. Stojanovic(1), M. Skrinjar(2), M. Saric(2)
(1) High School of Food Technology, 18400 Prokuplje,
Yugoslavia; (2) Faculty of Technology, 21000 Novi Sad,
Yugoslavia
According to the type and degree of infection, all samples
of wheat were classi¢ed into four groups: healthy, dark
germed, little fusarious and very fusarious kernels. They
all derived from the two experimental localities, 1 and 2,
and they were of the following sorts : Kg56S and Evropa
90 from the experimental locality 1 and Kg56S, and Nora
from the experimental locality 2. Sixteen samples from
each group were analysed. Mycotoxicological examination
(a£atoxin B1 and G1, ochratoxin A and zearalenone) were
carried out by using mycotoxin method which was de-
scribed by Balzer et al. (1978). A total number of moulds
per kernel was as folowing: in healthy kernels from 0.75 to
1.21, dark germed from 1.52 to 2.51, little fusarious from
2.12 to 2.94, and in very fusaruous kernels between 2.20
and 3.21. Mould isolated from all wheat samples were
classi¢ed into 13 genera and 34 species. The following
genera of mould were isolated: Acremonium, Alternaria,
Aspergillus, Botrytis, Chaetomium, Cladosporium, Fusa-
rium, Gilmaniella, Helminthosporium, Monilia, Penicillium,
Verticillium and Ulocladium. A£atoxin B1 isolated from
FEMSLE Congress 2-6-03
161
very fusarious wheat kernels deriving from the experimen-
tal locality 1 at the concentration to 2.3 mkg/kg. Contam-
ination by ochratoxin A was more frequent, and the con-
centrations ranged from 11 mkg/kg at the healthy kernels
to 48 mkg/kg at the very fusarious ones. The most severe
infections were caused by zearalenone which was found in
as many as 75% of the analysed samples. Its concentra-
tions ranged from 160 mkg/kg to 500 mkg/kg.
P2^74
MEASUREMENT OF STAGES DURING GERMINA-
TION AND OUTGROWTH OF INDIVIDUAL SPORES
OF CLOSTRIDIUM BOTULINUM
S. C. Stringer, M. D. Webb and M. W. Peck
Institute of Food Research, Norwich Research Park, Col-
ney, Norwich NR4 7UA, UK
Clostridium botulinum is a group of spore forming anaer-
obic bacteria that produce the extremely potent botulinum
neurotoxin. It is vital to prevent growth of this organism
in foods as consumption of as little as 30 ng of the toxin
can result in severe illness or death. Clostridium botulinum
is a particular concern in heat-treated products as compet-
ing vegetative £ora is eliminated. In such products growth
is likely to initiate from just a few spores so the distribu-
tion of time to growth in packs will re£ect the heteroge-
neity of times to growth from individuals. Knowing the
times to germination and growth from individual spores
rather than just time to growth from a population will
allow more accurate calculation of the risk of toxin pro-
duction in a pack. Additionally, although the time to
growth of an individual cannot be predicted from knowl-
edge of the behaviour of the entire population, the time to
growth of any size of population can be predicted if the
underlying distribution of times to growth of individuals is
known. An image analysis system and phase contrast mi-
croscopy has been used to follow individual spores of non-
proteolytic Clostridium botulinum strain Eklund 17B from
dormancy, through germination and emergence, to cell
division. Frequency distributions ¢tted to the data show
there is considerable variability within the spore popula-
tion and there is poor correlation between germination
and subsequent growth events such as emergence and
cell division.
162 1st FEMS Congress / Posters 103^505
P2^75
SUBLETHAL INJURY AND DETECTION OF GRAM-
NEGATIVE BACTERIA AND YEASTS
E. Svira¤kova¤, L. Horn|¤kova¤ and M. Plockova¤
Institute of Chemical Technology Prague, Department of
Dairy and Fat Technology, 166 28 Prague 6, Czech Repub-
lic.
This work was directed to study the e¡ect of physical
(heating, freezing) and chemical (EDTA, mixture of or-
ganic acids, NaCl, nisin) stress factors to growth and via-
bility of Gram-negative bacteria (Escherichia coli DMF
7502, Pseudomonas sp. DMF 9010) and yeasts (Candida
sp. DMF 1021, Kluyveromyces marxianus var. marxianus
DMF 1005), and to possibilities of detection of sublethally
injured bacteria and yeasts. The maximal inhibitive e¡ects
to growth of bacteria and yeasts were reached with the
EDTA e¡ect (20 mmol l-1) (99% reduction of both types
of microorganisms relative to their original cells popula-
tions). Both bacterial strains showed to direct inhibitive
e¡ect of nisin (0,1; 1; 10; 100 IU ml-1) lower sensitivity
than the sublethally (physically or chemically) damaged
bacteria. Both yeast strains showed to direct inhibitive
e¡ect of nisin (0,1; 1; 10; 100 IU ml-1) only slight sensi-
tivity ; the growth of sublethally injured yeasts was stimu-
lated by nisin (100 IU ml-1). A thorough determination of
the behaviour of sublethally injured bacteria and yeasts
can be achieved using a combination of detection meth-
ods, for example, for bacteria, spectrophotometry together
with selective and non-selective media, and for yeasts, £ow
cytometry together with some selective and non-selective
media.
P2^76
INFLUENCE OF LACTIC ACID BACTERIA ON
SOME FOOD-BORNE PATHOGEN MICRO-ORGAN-
ISMS
K. Szeker, J. Beczner
Dept of Microbiology, Central Food Research Institute,
Herman Otto ut 15, H-1022 Budapest, Hungary
Modern consumers nowadays demand natural food prod-
ucts. The traditional food preservation processes (for ex-
ample thermal processing at high temperature, sugaring,
pickling, drying) although kill pathogen bacteria, often
decrease the nutritional value and taste of food products
as well. Instead of drastic preservation processes more
attentions are being given to physical and chemical meth-
ods alone or in combinations in low doses in order to keep
the freshness of food products (minimal processing). These
FEMSLE Congress 2-6-03
novel, mild technics are for example the microwave treat-
ment, intensive light impulse, high pressure treatment,
electrical and magnetic pulses, bacteriocins and bacteriocin
producing cultures (mainly lactic acid bacteria). The in£u-
ence of these novel treatments on pathogen micro-organ-
isms are not completely known yet, so every new combi-
nation must be examined previously, whether the new
methods ensure the microbiological safety of the food
product. The bacteriocin production of 23 lactic acid bac-
terium strains against vegetative cells of Bacillus cereus T
and Listeria monocytogenes 4ab was tested. The in£uence
of lactic acid bacteria on the growth and survival of
pathogen bacteria in co-culture was examined. The
changes of the CFU were followed also by impedance
measurement (Malthus System V). Selective media were
needed to detect the lactic acid bacteria and the test mi-
crobes derived from the co-culture selectively quantita-
tively. MRS was found as suitably selective medium for
lactic acid bacteria and polymyxin B-containing PCA (PB-
PCA) for Bacillus cereus. Lactococus lactis subsp. lactis
(106 cm-3) in co-culture did not inhibit the growth of B.
cereus (104 cm-3) in milk at 30 0C.
The work was supported by the Hungarian National Sci-
enti¢c Research Foundation: OTKA T038215, Ministry of
Education: NKFP-4/0028/2002, and Ministry of Agricul-
ture and Regional Development: KF-79/7/2001.
P2^77
A STUDY OF MICROBIAL CHARACTERS OF SAF-
FRON IN IRAN
F. Tabatabaee
Department of Food Science and Technology, Faculty of
Agriculture, Ferdowsi University, Mashad-Iran
Sa¡ron (Crocus sativus) is an important traditional plant
from the Iridaceae Family that is produced a lot in the
eastern area of Iran. It is a bene¢cial plant that has red
color, suitable £avour and fragrance, that is used in food,
cosmetic and drug industries and a lot of this product is
exported every year. In this research, microbial characters
of sa¡ron were tested by standard microbiological meth-
ods and determined coliform bacteria as the most impor-
tant £or in Iranian sa¡ron, especially when the sa¡ron has
high wet. Also were determined the total count factor in
sa¡ron of Salmonella typhi, Bacillus reus, and Clostridium
perferingens.
 1st FEMS Congress / Posters 103^505
P2^78
STUDY OF ANTIMICROBIAL EFFICACY OF SOME
PLANT INHIBITORS
M. Tediashvili, T. Koberidze, K. Porchkidze, N. Janelidze,
G. Tsertsvadze, K. Akhvlediani
G. Eliava Institute of Bacteriophage, Microbiology and Vi-
rology, 3 Gotua str., Tbilisi 380060, Georgia
Screening of potential antibacterial plant compounds in 64
strains of di¡erent species of food pathogenic bacteria and
4 phages was carried out. Evaluation of MIC of antibac-
terial substances for each group of test bacteria was per-
formed by dilution and disc di¡usion method. Bacterio-
phage susceptibility towards Inhibitor T3 was assessed by
Phage Plaque assay. Transmission Electron Microscopy
was used to register morphological changes under in£u-
ence of tested inhibitors. The highest inhibitory activity of
T3, proportional to the dose of inoculated substances, was
observed for S. aureus, Sh. sonnei, Sh. £exneri, P. vulgaris
and P. mirabilis. T1, T2, T3, preparations as well as Green
Tea extract exhibited a higher activity against gram- pos-
itive bacteria, S. aureus, in particular. Obvious e⁄cacy was
revealed against meticillin-resistant S. aureus (MRSA)
strains. EM studies demonstrated cell damage and aggre-
gation in S. aureus cell culture after 24 hours incubation
with T3 preparation. For suppression of growth of some
gram-negative bacteria such as S typhimurium and enter-
ohemoragic E. coli, as well as Pseudomonas sp. inoculation
of more then 1000 mkg/ml of T3 was necessary. On the
contrary, these concentrations of plant compounds do not
a¡ect food bene¢cial and probiotic bacteria like B. bi¢-
dum, Lb. plantarum, Lb. delbruki etc. Anti -viral potential
of plant antibacterial substances was demonstrated by in-
hibitory e⁄cacy of T3 preparation towards Un phage
leading to 3 log decrease of phage titer after 24 hours
exposition with 500 mkg/ml of the inhibitor.
P2^79
ASSESSMENT OF MUTAGENICITY AND CARCINO-
GENICITY EFFECTS OF PLASTICS BAGS AND DIS-
POSABLE FOOD CONTAINERS IN THE SALMO-
NELLA/MICROSOME TEST
M. Tohidpour, S. Mehrabian, M. Emtyazjoo, H. Assem-
pour
Dept. of Microbiology, Faculty of Basic Sciences, Univer-
sity of Azad Islamic, Branch of North of Tehran, Iran
Nowadays, plastics bags and disposable food containers
made of high and low-density polyethylene have found
increasing use in Iran. As there is a direct relation between
FEMSLE Congress 2-6-03
163
the safety of such a type a product with the society’s
health, in this research the mutagenicity and carcinogenic-
ity of these products have been investigated using Ames
test and Salmonella typhimurium (TA100, TA104). As the
result of the e¡ects of mutation, these bacteria strains have
no potential to produce histidine on culturing in a minimal
glucose agar medium. In general, carcinogen materials will
cause a reverse mutation of these bacteria and results in
production of histidine by the bacteria in the MGA me-
dium for the Ames test. To investigate the mutagenicity of
the PE bags and containers, we faced the pieces of these
products on a test plate S. typhimurium (TA100, TA104)
and the results of the experiments were these compared
with MGA medium containing sodium azide as the pos-
itive control and distilled water as the negative control.
The comparison of the colonies in the negative control,
which are produced spontaneously, with those of the in-
vestigating PE samples a way to prove the mutagenicity of
the experimental samples. The result showed that the
HDPE grades as the raw material and their products
made in form of disposable glasses and containers, drink-
ing bottles and milk bags don’t possess the mutagenicity
property. The thin ¢lm as plastics bags caused reverse
mutation of S. typhimurium of these products. If these
products coated by liquid or solid oils were found to
have much more a¡ects on mutagenicity of the ¢lms. Fol-
lowing the above tests, rat liver tissue microsomes it was
later obtained under sterile conditions and was added to a
MGA medium including the suspected PE samples. The
results obtained from the latter experiments con¢rmed
that the PE thin bags used for packaging of the food
products in Iran su¡er from carcinogenicity.
P2^80
OCCURRENCE OF ANTIBIOTIC-RESISTANCE IN
THE PRODUCTION CHAINS OF ITALIAN DAIRY
AND MEAT COMMODITIES
S. Torriani(1), L. Rizzotti(1), F. Clementi(2), L. Aquilan-
ti(2), F. Biavasco(3), C. Vignaroli(3), P. S. Cocconcel-
li(4), S. Gazzola(4), B. Cenci-Goga(5) and F. Della-
glio(1)
(1) Dipartimento Scienti¢co e Tecnologico, Universita' di
Verona, Italy ; (2) Dipartimento di Biotecnologie Agrarie
e Ambientali, Universita' di Ancona, Italy ; (3) Istituto di
Microbiologia, Facolta' di Scienze MM. FF. NN. e di Me-
dicina, Universita' di Ancona, Italy ; (4) Istituto di Micro-
biologia, Facolta' di Agraria, Universita' Cattolica del Sacro
Cuore di Piacenza, Italy ; (5) Dipartimento di Scienze degli
Alimenti, Universita' di Perugia, Italy
Antibiotic resistance (AR) in bacteria is a concrete threat
to human health, emerging in the last decades as conse-
quence of the large-scale use of antibiotics as growth pro-
164 1st FEMS Congress / Posters 103^505
moters and in human and veterinarian medicine. The phe-
nomenon of acquired resistance becomes more worrisome
as these genes normally are on mobile genetic elements
and this makes their intra-, inter-speci¢c and inter-generic
transfer possible. AR bacteria, belonging to the genera
Enterococcus and Staphylococcus and the lactic acid bac-
teria group, are increasingly isolated from foods with a
large consumption. To deepen the knowledge of the role
that foods play as AR vectors, seems therefore of great
interest. The present research surveys the di¡usion and
distribution of AR genes and microorganisms in some
production chains of food of animal origin (breeding-
milk-cheese ; breeding-fresh meat/processed meat) in di¡er-
ent Italian geographic areas. The presence of genes codify-
ing for the most important resistances (e.g. vancomycin,
methicillin, erythromycin, gentamycin) was investigated
using speci¢c PCR assays performed directly on DNA
from food matrices. We found that several samples of
animal faeces, feedstu¡s, dairy and meat products gave
positive ampli¢cation reactions. Bacteria, resistant to anti-
biotics and belonging to the above-mentioned groups,
were isolated from these positive samples. Their character-
isation by means of molecular techniques allowed the
monitoring of single strains along the production chain.
The ¢ndings of this study indicate a frequent occurrence
of AR in food-associated bacteria at all levels of the food
production chains. The usefulness of molecular ¢nger-
printing to individuate points in which contamination
might occur was emphasised.
P2^81
PHARMACEUTICAL MICROBIOLOGY ON GUARD
DRUGS QUALITY
S. Tyski
National Institute of Public Health, Warsaw, Poland
Microbiology is very important in assessing the quality of
pharmaceutical preparations. There are several steps in
pharmaceutical industry, where control of microbial con-
tamination is crucial. Monitoring of production environ-
mental in clean areas during aseptic manufacturing, in-
volve measurement of viable microorganisms content in
the air, on the surfaces and on personal gloves. The usage
of disinfecting products with wide antimicrobial spectrum,
dedicated to the appropriate surfaces is recommended to
limit the bioburden level. Microbial status (bacteria and
fungi contamination) of starting materials and water used
for production are essential for microbiological quality
and safety of ¢nal product. It is di⁄cult to understand
that microbiological requirements for drinking water are
higher than for puri¢ed pharmacopoeial water. Microbial
quality of pharmaceutical preparations, which are on the
market, is described in Pharmacopoeias. There is a strict
FEMSLE Congress 2-6-03
borderline between sterile products and those, which can
contain permitted level of microorganisms. Preparations
for topical use, for oral and rectal administration as well
as herbal medicinal products have limited quantity and
quality of contaminating microorganisms described in
Pharmacopoeias. Sometimes antimicrobial preservatives
may be added, to prevent proliferation or to limit micro-
bial contamination, which during normal conditions of
storage and use, particularly multidose containers could
occur in the product. It may present a hazard to the pa-
tient from infection and spoilage of the preparation. The
e⁄cacy of an antimicrobial preservative may be demon-
strated by the pharmacopoeial test with bacterial and fun-
gal test strains. Proper test microorganisms are used also
for determination of antibiotics and antiseptics activity.
P2^82
THE EVALUATION OF MICROBIAL POPULATION
OF PIZZA CHEESE IN REFRIGERATOR AND
FREEZER CONDITIONS
A. Valaei, J. Fooladi and S. Mehrabian
Azzahra University, Vanak St. Tehran, Iran
Cheese is produced by the lactic fermentation of milk.
Mozzarella is an Italian soft cheese made of cow’s milk.
One type of this cheese which has more fat and less water
is pizza cheese ; the amount of fat and water are 24 % and
48 %, respectively (compared to normal mozzarella at 18
% and 52 %, respectively). In this research, 280 samples of
pizza cheese at +4‡C and -20‡C at these times of exami-
nation 0, 1, 3, 5, 7, 10, 14, 30, 60 days and 10-1 10-2
dilutions, were studied. Microbial contamination of pizza
cheese was examined for 5 bacteria: E. coli, coliform, S.
aureus, Enterococcus spp. and Cl. perfringenes. Pizza
cheese was not found to be contaminated by E. coli, in-
dicating that the milk was pasteurized. The result of 0 time
showed that there was no E. coli, but coliforms (Citrobater
freundii, Aerobacter aerogenes ), Cl. perfringenes, Entero-
coccus and S. aureus were observed above the standard
level. The result for +4‡C in survival of bacteria showed
that coliforms, Cl. perferingens and Enterococcus increased
in temperature of refrigerator compared to 0 time, but S.
aureus decreased. During the ¢rst days, the amount of
Enterococcus and Coliforms increased in the temperature
of -20‡C. From ¢fth day, bacteria decreased gradually, but
from fourteenth day bacteria decreased strongly, but S.
aureus at the ¢rst days decreased. According to the results
obtained, the following suggestions are o¡ered : 1) Pizza
cheese should be kept 15 days of storage after production
in -20‡C freezer then be contributed to the market; 2)
Quality control producing factories should be sampled
after 15 days of production storage in fridge -20‡C; 3)
For home usage, pizza cheese should be packed in small
 1st FEMS Congress / Posters 103^505
sizes, so that the whole package would be consumed after
freezing; 4) Pizza cheese should be used cooked to kill
micro-organisms in this cheese.
P2^83
COMPARISON OF FOOD AND CLINICAL ISO-
LATES OF ENTEROCOCCUS FAECIUM AND EN-
TEROCOCCUS FAECALIS
L. Vannini(1), G. Suzzi(2), P. S. Cocconcelli(3) and M.
E. Guerzoni(1)
(1) Dipartimento di Protezione e Valorizzazione Agroali-
mentare, Facolta' di Agraria, Universita' degli Studi di Bo-
logna Via Fanin 46, 40127 Bologna, Italy ; (2) Dipartimen-
to di Scienze degli Alimenti, Facolta' di Agraria, Universita'
degli Studi di Teramo, Via Spagna 1, Mosciano S. Angelo,
Teramo, Italy ; (3) Istituto di Microbiologia, Universita'
Cattolica del Sacro Cuore, Via Emilia Parmense 84,
29100 Piacenza, Italy
Enterococci are ubiquitous microorganisms that can colo-
nise di¡erent niches and may serve as indicators of the
sanitary quality of foods. Enterococci commonly occur
in vegetables and fermented foods of animal origin. In
fermented meats, enterococci can be detrimental because
they can cause spoilage. On the contrary, they have im-
portant implications in the dairy industry as they contrib-
ute to the development of organoleptic characteristics dur-
ing cheese ripening. However, enterococci have recently
assumed major importance in clinical microbiology due
to their enteric habitat, transmission through the food
chain, antibiotic resistance and their possible involvement
in food-borne illness or environmental contamination in
hospitals due to the presence of virulence factors. The aim
of this work was to study isolates of Enterococcus spp. of
food and clinical origin for their physiological and molec-
ular characteristics. In particular they were isolated from
fermented foods and cheeses, from blood, catheters, endo-
carditis, meningitis and sepsis. All the isolates were sub-
typed with the Automated RiboPrinter System, screened
for resistance to di¡erent antibiotics, investigated for plas-
mid analysis, the presence of virulence factors and growth
ability at di¡erent Aw and pH values. The comparison of
the ribotype patterns evidenced the existence of di¡erent
pro¢les between the isolates of the two main species con-
sidered (E. faecium and E. faecalis). However, no ribo-
types speci¢cally associated with strains isolated from
foods or from clinical sources were observed. This could
indicate a possible transmission of such isolates along the
food chain to humans. Moreover, the ability of the isolates
of clinical origin to grow in environmental stress condi-
tions suggests that some of the isolates from nosocomial
infections may have food origin.
FEMSLE Congress 2-6-03
165
P2^84
SEARCH FOR NEW PROBIOTIC CANDIDATES :
SAFETY AND QUALITY CRITERIA
A. Weiss(1), H. K. Mayer(1), H. P. Lettner(2), W.
Kramer(2) and W. Kneifel(1)
(1) Department of Dairy Research and Bacteriology, Uni-
versity of Agricultural Sciences, Gregor-Mendel-Strasse 33,
A-1180 Vienna, Austria ; (2) Lactosan Starterkulturen
GmbH & Co KG, Industriestrasse West 5, A-8605 Kapfen-
berg, Austria
Over the past decades probiotic micro-organisms have
gained a great market potential for both human and ani-
mal nutrition. In parallel, the demand for new, more suit-
ably performing probiotic strains has grown. Before a giv-
en strain is incorporated into a product, several safety and
quality (functional and technological) aspects have to be
considered. At ¢rst, strains should be isolated prevailingly
from the site of the intended application and should have
a history of being non-pathogenic. The safety properties of
strains as intrinsic properties, pharmacokinetics and inter-
actions between probiotic strain and host have to be as-
sessed. It is of utmost importance that strains do not carry
transmissible antibiotic resistance genes in order to fore-
stall the spread of antibiotic resistance. In addition to the
proper identi¢cation, in the present study the probiotic
strains were tested concerning several quality criteria to
allow the prediction of the tolerance of the manufacturing
process, the shelf life and the passage through the intesti-
nal tract: tolerance of acid, bile, sodium chloride and en-
zymes of the gastrointestinal tract, tolerance of oxygen,
autoaggregation, fermentation of carbohydrates, growth
stimulation by prebiotic carbohydrates, enzyme pro¢le,
antagonistic potential against potentially pathogenic mi-
cro-organisms, fermentation pro¢les and -products as
well as incubation temperature. Exemplary results of these
tests will be presented.
P2^85
THERMAL INACTIVATION OF SALMONELLA
SENFTENBERG W775 ON MUNGBEAN SEEDS
USED FOR SPROUT PRODUCTION
A. Weiss and W. P. Hammes
Institute of Food Technology, University of Hohenheim,
70593 Stuttgart, Germany
Seed sprouts have frequently been implicated as vehicle in
food borne disease outbreaks. The sprouting process pro-
vides optimal conditions for bacterial growth and there-
fore, the absence of pathogens on seeds can be de¢ned as
166 1st FEMS Congress / Posters 103^505
the critical control point in sprout production. The Food
and Drug Administration recommended a 5 log units re-
duction of pathogens to minimise the risk of food poison-
ing, and for this purpose a treatment with 20.000 ppm
calcium hypochlorite is in use. For the production of or-
ganic food in general, the use of disinfectants is commonly
not accepted. To design an easy and economic alternative
we studied the applicability of thermal processing. Mung-
bean seeds were inoculated with 1 x 108 cfu/g Salmonella
Senftenberg W775 by immersion. This strain is well known
as extremely heat resistant organism. The seeds were dried
and stored at 2 ‡C. The Salmonella counts on the dried
seeds remained unchanged for 6 weeks at a level of 1-5 x
108 cfu/g. The contaminated seeds were treated in a water
bath at 55, 58 and 60 ‡C for 0,5-16 minutes. The experi-
ments were repeated 3 times. The D-values were deter-
mined for 55, 58 and 60‡C and the z-value of 6,2 (r2=
0,99) was calculated for the inactivation of S. Senftenberg
W775 on the mungbean seeds. The thermal treatment at
time/ temperature regimes of 55‡C/20 min, 60‡C/10 min,
70‡C/5 min and 80‡C/2 min reduced the pathogens for
more than 5 log units without a¡ecting sprouting.
P2^86
COMPARISON OF DRYCULT0 TPC, PETRIFILM
AND STANDARD METHOD FOR THE DETERMINA-
TION OF MICROBIAL QUALITY IN WATER, JUI-
CES AND SOFT DRINKS
H.-D. Werlein and C. Armbrust
University of Hannover, Institute of Food Science, Wunstor-
fer Strasse 14, D-30453 Hannover, Germany
DryCult0 TPC (Orion Diagnostica, Espoo Finland) is a
new simple system to investigate the microbial quality of
water and other liquid samples on location. Dry Cult0
consists of a dehydrated medium which absorbs 1 ml of
the liquid sample and should show comparable results to
the standard methods. Samples of water, mineral water,
beer, milk, orange-, apple- and grape juice were examined.
Some of the samples were arti¢cially inoculated with dif-
ferent microorganisms. DryCult0 was compared with the
standard and the petri¢lm method. DryCult0 and Petri-
¢lm were treated as described in the protocols of the sup-
pliers, respectively the standard method. Di¡erent investi-
gations carried out with E. coli, S. aureus, Saccharomyces
cerevisiae and naturally contaminated samples showed,
that DryCult0 TPC is able to detect di¡erent kinds of
microorganisms. Water, mineral water, beer and soft
drinks are suitable products. Milk and some juices are
only limited suitable. The results are comparable to the
standard methods and to the petri¢lm method. The Dry-
Cult0 TPC system is very easy to handle. The DryCult0
method shows a tendency to lower results compared to the
FEMSLE Congress 2-6-03
standard methods. It was remarkable if the sample is
highly contaminated no growth is visible.
P2^87
DETECTION OF AFLATOXINOGENIC FUNGI US-
ING PCR METHOD
I. Zachova¤(1), J. Vytr›asova¤(1), M. Pejchalova¤(1), G.
Tavc›ar-Kalcher(2)
(1) Department of Biological and Biochemical Sciences,
Faculty of Chemical Technology, University of Pardubice,
Pardubice, Czech Republic, SNtrossova 239, CZ-530 03; (2)
University of Ljubljana, Veterinary Faculty, Institute for
Hygiene and Pathology of Animal Nutrition, Gerbic›eva
60, 1000 Ljubljana, Slovenia
This work covers the possibility of using PCR method for
acceleration and more accurate identi¢cation of the a£a-
toxinogenic fungi isolated from feed and foodstu¡s. The
method was optimalized on pure cultures (Aspergillus £a-
vus CCM F-108 and Aspergillus parasiticus CCM F/550).
The speci¢city of the optimalized PCR method was veri-
¢ed using various fungal strains. 50 samples of feed were
examined, of which 18 were positive for the presence of
the a£atoxinogenic fungi on AFPA medium. Isolated As-
pergillus strains were examined using PCR method. The
results obtained almost always agreed with the results
gained on the AFPA medium. This method is a possible
starting point for accelerating the detection of the a£atox-
inogenic fungi, but it will be necessary to solve certain
non-speci¢c reactions, which are caused by a complex
sample matrix. The results obtained are an initial step
for further research. The PCR technique has proved itself
in the detection of a£atoxinogenic fungi isolated from
feed.
This study was supported by Research Project msmt No.
253100002 and Project kontakt me 241.
P2^88
COAGULASE-POSITIVE STAPHYLOCOCCI : EN-
TEROTOXIN PRODUCTION AND ANTIBIOTIC RE-
SISTANCE
P. Zangerl(1), M. Gonano(2), A. Rammelmayr(3), M.
Wagner(2)
(1) Federal Institute of Alpine Dairying, A-6200 Rotholz;
(2) University of Veterinary Medicine, A-1210 Vienna; (3)
Austrian Agency for Health and Food Safety, A-3261 Stei-
nakirchen am Forst, Austria
To study the enterotoxigenicity of coagulase-positive
staphylococci, 247 dairy isolates (121 and 126 isolates
 1st FEMS Congress / Posters 103^505
from raw milk and raw-milk cheeses, respectively), 91 iso-
lates from mastitis milk and 48 clinical human isolates
were analysed for the production of the enterotoxins
(SEs) A, B, C, D, and E by a commercially available
ELISA test kit (Tecra). Additionally PCR-assays for the
detection of the enterotoxin genes sea, seb, sec, sed, see,
seg, seh, sei, and sej were carried out for the mastitis and
human strains, together with 91 strains selected from the
dairy isolates. These and further 43 human strains (91
human strains in total) were analysed for the resistance
against 14 antibiotics by an agar di¡usion test. The ability
to produce SEA-SEE was strongly dependent on the ori-
gin of strains. While 41.7% of the human isolates were
found to produce SEA-SED, dairy and mastitis isolates
showed much lower frequency of enterotoxin production
(13.0% and 10.8%, respectively). Irrespective of origin,
SEC was found to be the predominant enterotoxin and
none of the strains harboured the see gen. The genes en-
coding the recently characterised enterotoxins G-J were
detected much more frequently. The most frequent combi-
nation of enterotoxin genes was seg/sei. Antibiotic resis-
tance testing revealed that 44.6% of the dairy isolates,
67.3% of the mastitic isolates and only 2.7% of the human
isolates were susceptible to all antibiotics tested. Penicillin
resistant strains exhibited utmost importance since 10.9%
of the mastitic isolates, 35.5% of the dairy isolates and
60.1% of the human isolates were resistant to penicillin G.
P2^89
HUMAN CALICIVIRUS OUTBREAKS IN SLOVENIA
IN 2000-2002
J. Zims›ek(1), M. Poljs›ak-Prijatelj(1), D. Barlic›-Magan-
ja(2), A. Hoc›evar-Grom(3)
(1) Institute of Microbiology and Immunology, Medical
Faculty, University of Ljubljana, Slovenia; (2) Veterinary
Faculty, University of Ljubljana, Slovenia; (3) Institute of
Public Health of the Republic of Slovenia, Ljubljana
Noroviruses, members of the family Caliciviridae, are small
non-enveloped viruses with single-stranded polyadenilated
genome of positive polarity. The way of transmission for
these viruses is via faecal-oral route; viruses can spread
between people by direct contact or through contaminated
food and water, causing sporadic and epidemic gastroen-
teritis in children and adults. We have investigated the
incidence of noroviruses associated with outbreaks of
acute nonbacterial gastroenteritis in Slovenia, since 2000.
Stool specimens from outbreaks were tested during this
period. After ultracentrifugation stool samples were exam-
ined by direct electron microscopy. RNA extracted from
stool specimens with SV Total RNA Isolation system
(Promega) was used as the template for reverse transcrip-
tion, followed by ampli¢cation in a single tube, two-en-
FEMSLE Congress 2-6-03
167
zyme system. Gene fragments were ampli¢ed using generic
primers targeting the polymerase region of the genome.
The identity and the speci¢city of amplicons belonging
to genogroup II were con¢rmed by hybridisation with bio-
tin labelled capture probes. Genogroup II dominated
among the specimens of infants, children and adults. Dur-
ing 2000 most frequent genotype were Lordsdale and Ha-
waii, during 2001 genotype Lordsdale and Melksham; in
2002 genotype Lordsdale and multiple genetic type were
detected. During 2002 the strains, which could not be de-
tected before increased and were further preceded for se-
quence analysis. Most outbreaks occurred in canteens,
homes for the elderly, holiday lodges and schools.
P3^1
HALOBACILLUS KARAJENSIS SP. NOV., A NEW
MODERATELY HALOPHILIC SPECIES ISOLATED
FROM SALINE SOIL
M. A. Amoozegar(1), F. Malekzadeh(1), K. A. Malik(2),
P. Schumann(2) and C. Spro«er(2)
(1) Department of Biology (Microbiology unit), Faculty of
Science, University of Tehran, Tehran, Iran; (2) DSMZ ^
Deutsche Sammlung von Mikroorganismen und Zellkulturen
GmbH, Mascheroder Weg 1 B. 38124 Braunschweig, Ger-
many
A moderately halophilic, Gram-positive, spore-forming
bacterium was isolated from surface saline soil of the re-
gion of Karaj, Iran. The strain, designated MA-2 was
strictly aerobic, rod-shaped, occurring singly, in pairs or
short chains, contained L-orn-D-asp type peptidoglycan,
and the major respiratory lipoquinone was MK-7. It was
non-motile, having ellipsoidal endospore located centrally
or sub-terminally. Growth occurred at 10-49 ‡C and the
pH range was 6-9.6. Strain MA-2T grew at salinities 1-24%
(w/v) NaCl, showing optimal growth at 10% (w/v). The
guanine-plus-cytosine content of the DNA was 41.3
mol%. Phylogenetic analysis based on 16S rRNA sequen-
ces showed that strain MA-2T was associated with rRNA
group 1 Bacillus. The microorganisms showing the closest
phylogenetic relationship to strain MA-2T were members
of the genus Halobacillus: Halobacillus litoralis and Hal-
obacillus trueperi. On the basis of phenotypic and chemo-
taxonomic characteristics, 16S rRNA sequence analysis
and DNA-DNA similarity data, it is proposed that strain
MA-2T should be placed in the genus Halobacillus as a
new species, Halobacillus karajensis sp. nov.
168 1st FEMS Congress / Posters 103^505
P3^2
GENETIC POPULATION ANALYSIS OF PSEUDO-
MONAS AERUGINOSA BY MULTILOCUS SE-
QUENCE TYPING
I. Vernez, P.M. Hauser, D. S. Blanc
University hospital of Lausanne, Lausanne, Switzerland
In order to study the population genetics structure of
Pseudomonas aeruginosa, we developed a multilocus se-
quence typing (MLST) scheme. The sequences of internal
fragments of seven housekeeping genes were obtained for
34 P. aeruginosa isolates from patients hospitalized in 5
di¡erent European cities. Twenty six di¡erent allelic pro-
¢les were identi¢ed. The mean allelic diversity was 0.854
(range: 0.606 to 0.978), which was about 6X greater than
the results obtained with the multilocus enzyme electro-
phoresis (MLEE) method. Linkage disequilibrium was
measured with the index of association. An index of
1.95+0.24 was calculated when all the strains were taken
into consideration. This index was 1.76+0.27, when only
one strain per sequence type was taken into consideration.
Both results are di¡erent from 0, indicating that there is an
association within loci, which means that the population
structure of P. aeruginosa is clonal. These results are in
contradiction with a previous study based on MLEE data,
which showed that this structure was non-clonal.
P3^3
THE FUNCTIONAL EVOUTION OF STREPTOKI-
NASE DOMAINS IN PLASMINOGEN ACTIVATION
D. M. Kim, J. H. Chung and S. M. Byun
Department of Biological Sciences, Korea Advanced Insti-
tute of Science and Technology, 305-701 Daejon, South
Korea
Streptokinase is a human plasminogen activator, secreted
from pathogenic bacteria, Streptococcus species. Random
mutagenesis of streptokinase was carried out to determine
functional amino acid residues in plasminogen activation.
Fourteen streptokinase mutants without plasminogen acti-
vation activity on skim milk-plasminogen overlay plate
were selected and sequenced. Six mutants (D41C, S44K,
S44P, R45P, H48T and D220G) showed substantial ami-
dolytic activities as compared with that of wild type.
Moreover, ¢ve point mutations in Asp41-His48 region of
a streptokinase plays an important role in binding to a
substrate plasminogen. Because of the structural and se-
quence similarity of SKK domain to those of staphyloki-
nase, the chimeric proteins substituted SKK domain with
staphylokinase were examined for their activity of plas-
FEMSLE Congress 2-6-03
minogen activation. The kinetic data showed that these
fusion proteins bound to human plasmin for plasminogen
activation. Additionally the N-terminus of staphylokinase-
streptokinase fusion protein was substituted by the N-ter-
minus of streptokinase. The serial substitution showed
that the non-hydrophilic residues at the N-terminus of
the fusion proteins without SKQ domain did not a¡ect
the formation of the active plasmin-activator complex.
However, the fusion mutants with Q domain shortened
the lag times in kinetic data. These results propose that
streptokinase requires the N-terminal non-hydrophilic res-
idues and Q domain for formation of the active streptoki-
nase-plasminogen complex. Recombinant bovine plasmin-
ogen activator also activated human plasminogen by
staphylokinase-like activation mode. Because the activator
has homology with streptokinase, the bovine plasminogen
activation activities of streptokinase fragments were exam-
ined. Streptokinase fragment without Q domain among
them activated bovine plasminogen. These data suggest
that the Q domain of streptokinase determines plasmino-
gen species necessary for activation, and converses ability
of substrate recognition to human species.
P3^4
CLONAL DIFFUSION AND HORIZONTAL TRANS-
MISSION OF MEC DETERMINANT IN METHICIL-
LIN-RESISTANT STAPHYLOCOCCUS AUREUS ISO-
LATED IN ITALY
F. Campanile(1), M. Santagati(1), D. Bongiorno(1), S.
Borbone(1), M. Cassone(2) and S. Stefani(1)
(1) Department of Microbiological and Gynaecological Sci-
ence, University of Catania; (2) Department of Clinical
Medicine, Rome, Italy
The key genetic component of methicillin-resistance ^ the
mecA determinant ^ is not native of Staphylococcus aur-
eus. The origins of the major MRSA clones are still poorly
understood; previous reports have suggested a common
origin for all MRSA from a single ancestral S. aureus
strain that acquired the mec complex. Recent studies
have shown that some MRSA strains are very divergent,
implying that mecA has been transferred among S. aureus
lineages. Multilocus-sequence typing (MLST) of S. aureus
has been used to probe population biology of bacterial
pathogens and to predict ancestral genotypes and evolu-
tionary descendent within groups of related genotypes.
Studies carried out in our laboratory revealed the presence
of ¢ve MRSA clones in Italy: the archaic clone, the multi-
resistant Iberian and Brazilian clones and two new ones,
classi¢ed as Italian and Rome clones. The purposes of this
study were to explore the evolution of early and contem-
porary MRSA isolates in a selected sample of strains; to
compare their clonal type and their genetic backgrounds
 1st FEMS Congress / Posters 103^505
with main Italian MRSA clones, re-examined for their
genetic relatedness by MLST. Our results show that the
Rome clone have undergone an evolution due to the ac-
quisition of two copies of Tn554 and their modi¢cation of
mecA polymorphism II to I. A closed correlation among
four previously classi¢ed clones (Archaic, Iberian, Brazil-
ian and Rome) has been showed by the same allelic pro¢le
(3-3-1-12-4-4-16). Surprisingly, the Italian clone belongs to
a totally di¡erent genetic background (1-4-1-4-12-24-29),
suggesting horizontal mecA transfer among di¡erent S.
aureus ancestral lineages.
P3^5
TRANSPOS-ID : A PECULIAR STRUCTURE OF
Tn231LM
D. Xu and J.-C. Co“te¤
Agriculture and Agri-Food Canada, 430 Gouin Blvd., Saint-
Jean-sur-Richelieu, QC, J3B 3E6, Canada
Composite transposons contain a central region £anked
by 2 Insertion sequences (IS). The central region, uncon-
nected to transposition, usually carries drug resistance.
Both IS can be found in same or in inverted orientations.
We report here the peculiar structure of a novel composite
element, which we tentatively named Tn231LM. It was
isolated from Bacillus thuringiensis strain M15. This com-
posite transposon is delimited by two IS elements, IS231L
and IS231M. Both IS are found in the same orientation.
Each IS contains 18-bp inverted terminal repeats (IR) and
generates 9-bp direct repeats (DR) at each end. The se-
quence from the right IR through the right DR of IS231M
complements that of the left IR and left DR of IS231L.
This complementary sequence extends an additional 9-bp,
named inner extended IR (ieIR). The sequence of the right
IR and right DR of IS231L perfectly complements that of
the left IR and left DR of IS231M. This complementary
sequence extends an additional 27-bp, named outer ex-
tended IR (oeIR). The oeIR ends in an 8-bp DR. Based
on the structure, two composite transposons are possible.
One, in which the central region is 448-bp between both
oeIR ends. Alternatively, should the transposon be con-
sidered ending at these 8-bp DR, the central region of the
transposon would be an autonomously replicated mega-
plasmid. In the latter case, Tn231LM can be considered
having the capacity of autonomous replication. We thus
propose a new name for this special apparatus: ‘‘transpos-
id’’. In the strain M15, there are about 20 copies of IS231
sequences. But the Tn231LM remains unique based on a
result of Southern hybridization with a 5’ end £uorescein-
labeled probe with the sequence of 27-nt oeIR.
FEMSLE Congress 2-6-03
169
P3^6
DISTRIBUTION OF IS231-LIKE SEQUENCES
AMONG BACILLUS THURINGIENSIS SEROVARS
K.-B. Joung and J.-C. Co“te¤
Agriculture and Agri-Food Canada, 430 Gouin Blvd., Saint-
Jean-sur-Richelieu, QC, J3B 3E6, Canada
Insertion sequences (IS) are the simplest transposable ele-
ments, and usually contain only the genes responsible for
their transposition. Some IS (IS231, IS232, IS233, IS240,
ISBT1 and ISBT2) and two class II transposons (Tn4430
and Tn5401) have been isolated from a limited number of
Bacillus thuringiensis strains. IS231 are found closely asso-
ciated with cry genes and are believed to play a role in
their multiple locations and genetic mobility, and in the
generation of novel speci¢city. We set to analyze the dis-
tribution of IS231-like sequences among Bacillus thurin-
giensis serovars. A total of 95 bacterial strains were tested
in the present study. These include the 82 known B. thur-
ingiensis serovars type strains, ¢ve more B. thuringiensis
intra-serovar strains, kurstaki HD-1, subtoxicus, dendroli-
mus, tenebrionis and sandiego, a non-motile hence non-se-
rotypeable strain, B. thuringiensis var wuhanensis, six B.
cereus strains, and the B. mycoides type strain. A 723-bp
HaeII conserved fragment from IS231M has been used as
a probe against EcoRI-digested B. thuringiensis total DNA
to yield serovar-speci¢c hybridization pro¢les. The ap-
proach was useful at revealing the extent of distribution
of IS231-like sequences between and within strains. Of the
88 B. thuringiensis strains tested, 70 showed hybridization
banding patterns that comprised between one and 20 dis-
tinct bands. These 70 B. thuringiensis strains were grouped
based on banding patterns similarities. Interestingly, intra-
serovar strains did not necessarily cluster together.
P3^7
PATTERNS OF 16S-23S INTERGENIC TRAN-
SCRIBED SPACER (ITS) AND DISTRIBUTION OF
A 16-BP MOTIF, BOX X, PRESENTS IN THE 5’ CON-
SERVED ITS IN BACTERIA.
D. Xu and J.-C. Co“te¤
Agriculture and Agri-Food Canada, 430 Gouin Blvd., Saint-
Jean-sur-Richelieu, QC, J3B 3E6, Canada
Nucleotide sequence comparisons done in our lab, be-
tween and within bacterial species, show that the bacterial
16S-23S intergenic transcribed spacer (ITS) can be divided
into four regions: I, II, III and IV. Region I, which cor-
responds to the 5’ end of 16S-23S ITS, is the foremost
inter-allelically conserved region within species ; Region
170 1st FEMS Congress / Posters 103^505
II is variable; Region III is inter-allelically conserved; and
Region IV, at the 3’ end of 16S-23S ITS, is variable. Some
ITS contain all 4 regions, others contain regions I, II and
III, and others contain only regions I and III. An analysis
of 197 16-23S ITS sequences from 115 ¢rmicute and mol-
licute species revealed that a 16-nt conserved motif located
near the 3’ end of Region I is present in all ITS. We called
this motif ‘‘box X’’. Part of the 3’ end sequence of box X
could form a hairpin structure with the downstream se-
quence. An analysis of the RNA secondary structure
shows that box X and its £anking sequences can form a
double-stranded structure with part of the leader region of
the 16S rRNA gene. Presumably, this structure could serve
as an RNA double-stranded processing site (DsPS) for
RNase III. The variable Region II may contain tRNA
genes in some of the alleles. This Region can be absent
in others. Region III contains, near its 5’ end, the con-
served box A ^ antitermination sequence. Region IV is
variable and is not present in all species. Interestingly, in
Bacillus halodurans, as many as three copies of box X can
be found in tandem in Region I. This suggests that box X
may play an important role, in addition to the formation
of DsPS, possibly during the transcription of the rrn op-
erons.
P3^8
METHICILLIN RESISTANCE IN STAPHYLOCOC-
CUS SCIURI
I. Couto(1,2,3), S. Wei Wu(2), A. Tomasz(2) and H. de
Lencastre(1,2)
(1) Laborato¤rio Gene¤tica Molecular, Instituto de Tecnolo-
gia Qu|¤mica e Biolo¤gica (ITQB/UNL), Rua da Quinta
Grande, 6, Apartado 127, 2780-156 Oeiras, Portugal; (2)
Laboratory of Microbiology, The Rockefeller University,
New York, NY 10021, USA; (3) Centro de Recursos Mi-
crobiolo¤gicos, Faculdade de Cie“ncias e Tecnologia (FCT/
UNL), Quinta da Torre, 2829-516 Monte da Caparica,
Portugal.
Resistance to beta-lactam antibiotics in staphylococci is
associated with the presence of the mecA gene that en-
codes PBP2A, a penicillin-binding protein with greatly re-
duced a⁄nity to these antibiotics. While studying the ori-
gin of mecA, which is exogenous to S. aureus and to other
clinically relevant staphylococci, we identi¢ed a genetic
element closely related to it in the animal commensal spe-
cies S. sciuri. The S. sciuri mecA homologue is native to
this species, being found in all S. sciuri isolates tested to
date ( s 160). Despite the similarities between the S. sciuri
mecA and the methicillin-resistant S. aureus (MRSA)
mecA, the great majority of S. sciuri isolates showed no
appreciable resistance to beta-lactam antibiotics. We now
describe two unusual S. sciuri strains isolated from hu-
FEMSLE Congress 2-6-03
mans that showed resistance to methicillin associated
with high rates of transcription of the S. sciuri mecA ho-
mologue and production of a protein resembling PBP2A.
In one of these strains increased transcription of the mecA
homologue was related to the insertion of IS256 upstream
mecA while the other strain had single nucleotide altera-
tions in the promoter region of the gene. A third methi-
cillin-resistant human isolate of S. sciuri that carries both
the native mecA homologue and a MRSA type mecA was
shown to be unstable in the absence of drug selection,
segregating antibiotic susceptible cells, which had lost the
MRSA-type mecA. These observations illustrate the re-
markable variety of strategies available to bacteria to ac-
quire mechanisms of drug resistance in the in vivo environ-
ment.
P3^9
ACINETOBACTER PARVUS SP. NOV., A SMALL
COLONY FORMING SPECIES ISOLATED FROM
HUMAN SPECIMENS
A. Nemec(1), L. Dijkshoorn(2), I. Cleenwerck(3), T. De
Baere(4), D. Janssens(3), T. J. K. van der Reijden(2), P.
Jez›ek(5) and M. Vaneechoutte(4)
(1) National Institute of Public Health, Prague, Czech Re-
public ; (2) Department of Infectious Diseases, Leiden Uni-
versity Medical Center, The Netherlands; (3) BCCM/
LMG Bacteria Collection, University of Ghent, Gent, Bel-
gium; (4) Department of Clinical Chemistry, Microbiology
and Immunology, Gent, Belgium ; (5) Department of Clin-
ical Microbiology, Pr›¤|bram, Czech Republic
The taxonomic status of seven glucose non-acidifying,
non-proteolytic Acinetobacter strains characterized by
forming small colonies (from 0.1 to 0.7 mm in diameter
after 24 h of incubation at 30 ‡C on di¡erent kinds of agar
media). With one exception, all strains were from human
specimens. They could be distinguished from all described
Acinetobacter (genomic) species by their ability to grow on
ethanol and acetate as sole sources of carbon but not on
22 other substrates tested including D,L-lactate or D,L-4-
aminobutyrate. DNA-DNA hybridization studies, 16S
rRNA gene sequence analysis, ampli¢ed ribosomal DNA
restriction analysis, and DNA polymorphism analysis by
AFLPTM showed that these strains represent a hitherto
unknown species for which the name Acinetobacter parvus
sp. nov. is proposed. The type strain is LMG 21765T (=
CCM 7030T) isolated from an ear of an outpatient. The
EMBL accession number for its 16S rDNA sequence is
AJ293691 and its DNA G+C content is 41,8 %.
 1st FEMS Congress / Posters 103^505
P3^10
APPLICATION OF AMPLIFIED RIBOSOMAL DNA
RESTRICTION ANALYSIS TO THE IDENTIFICA-
TION OF ENVIRONMENTAL ACINETOBACTER
ISOLATES REVEALS A NEW RESTRICTION PAT-
TERN AND SEVERAL UNDESCRIBED PROFILES
L. Dolzani, F. Gombac, M. Bellino, C. Lagatolla, R. Bres-
san, E. Edalucci, E. Tonin, and C. Monti-Bragadin
Dipartimento di Scienze Biomediche, Universita' di Trieste,
via Fleming 22, 34127 Trieste, Italia
The genus Acinetobacter, including both environmental
and clinically relevant organisms, comprises more than
twenty DNA genomic groups or genomospecies, de-
lineated by DNA/DNA hybridization. Genomic species
identi¢cation, however, may still be problematic. Among
the available methods, Ampli¢ed Ribosomal DNA Re-
striction Analysis (ARDRA) showed a good correlation
with DNA/DNA hybridization (System. Appl. Micro-
biol.21,33-39, 1998), but, although developed as a general
method, its usefulness was mainly tested on clinical iso-
lates. In this study, we evaluated the usefulness of AR-
DRA for the identi¢cation of 46 environmental Acineto-
bacter isolates, collected during the year 2000 from
samples of soil and water. Isolation strategy included en-
richment in Baumann medium followed by plating on
LAM selective medium. Identi¢cation at the genus level
was con¢rmed by the transformation assay of Juni. AR-
DRA results showed the following: only 26 (57%) isolates
gave already described restriction patterns and patterns
combinations, and could therefore be identi¢ed to the ge-
nomospecies level. Six isolates showed a new RsaI pattern.
All of them had identical restriction pro¢les with the other
endonucleases and performed homogeneously in a series
of biochemical tests, so they are likely to belong to the
same genomospecies. The remaining isolates showed un-
described combinations of already described patterns. Phe-
notypic characterization of the isolates that could be iden-
ti¢ed by ARDRA gave a concordant identi¢cation in most
cases, while it was unconclusive for the other strains.
Overall results indicated that the diversity of environmen-
tal Acinetobacter strains is higher than previously de-
scribed and that ARDRA is at present of limited useful-
ness for the identi¢cation of such strains.
FEMSLE Congress 2-6-03
171
P3^11
PHYLOGENY OF LACTIC ACID BACTERIA BASED
ON recA AND 16S rDNAS SEQUENCE ANALYSIS
G. E. Felis, F. Dellaglio, E. Knij¡ and S. Torriani
Dipartimento Scienti¢co e Tecnologico, Universita' degli
Studi di Verona, Strada le Grazie 15, 37134 Verona Italy
Current delineation of the evolutionary relationships
among bacteria and, in particular, among Lactic Acid
Bacteria (LAB) is largely stated on 16S rRNA sequence
analysis. Based on branching patterns in the 16S rRNA
trees, LAB belong to the Gram-positive low GC division
(Firmicutes) and are separated in several genera, phyloge-
netically intermixed (Lactobacillus and Pediococcus) and/
or internally sub-divided (Lactobacillus). In addition, they
exhibit di¡erent morphology, GC content, metabolic and
phenotypic traits, that only partially correlate with their
phylogenetic placement. Such premises support the oppor-
tunity of a deeper investigation on genealogical inter-rela-
tionships among LAB, to depict a more robust taxonomic
scheme for these bacteria of relevant ecological and food
interest. The present study addressed a comprehensive
phylogenetic analysis of several LAB genera and species
by integrating the information deriving from di¡erent mo-
lecular markers, i.e. recA gene and 16S rDNAs. Partial
sequences of RecA protein and its coding gene have suc-
cessfully been used to perform phylogenetic analysis of
many bacterial genera, but rarely on LAB. Complete
recA primary structures, available from databases or di-
rectly sequenced, were analysed for a major number of
species belonging to the genera Lactobacillus, Pediococcus,
Enterococcus, Leuconostoc, Oenococcus, Lactococcus, Car-
nobacterium and Weissella. A comparison of 16S rDNAs
and recA phylogenetic trees obtained by the same bioin-
formatic methods was carried out. Statistical robustness of
the deduced trees, in£uence of genome GC content on tree
reconstruction and correlation among phylogenetic, phe-
notypic and genotypic classi¢cation schemes of LAB were
discussed in a polyphasic scenario.
172 1st FEMS Congress / Posters 103^505
P3^12
DEVELOPMENT OF AN INSECT MODEL SYSTEM
FOR THE EVOLUTION OF VIRULENCE AND THE
ROLE OF VIRULENCE FACTORS IN THE IMPOR-
TANT HUMAN PATHOGEN STAPHYLOCOCCUS
AUREUS
V. M. Fleming and E. J. Feil
Department of Biology and Biochemistry, University of
Bath, Claverton Down, Bath, BA2 5AB, England, UK.
Staphylococcus aureus represents an increasing public
health burden and is a major cause of community-ac-
quired and nosocomial sepsis. Although commonly re-
garded as an opportunistic pathogen, recent evidence has
suggested that virulence may be a cumulative e¡ect related
to the total number of bacterial virulence determinants
present in the genome of a given strain. In order to exam-
ine this hypothesis, we have developed an in vivo model of
virulence based on the insect larvae Manduca sexta (the
Tobacco Horn Worm). Preliminary research on this model
suggests an LD50 of approximately 104 cells of S. aureus at
37‡C, with viable S. aureus cells readily recovered from the
cadavre, suggesting that in vivo colonisation has occurred.
Through the use of a well-characterised bacterial strain
collection, we have also demonstrated consistent di¡eren-
ces in virulence (as measured by weight change in the
larvae) between bacterial strains, and noted correlations
with the strain-speci¢c virulence gene-pro¢le. Further-
more, those strains which have most commonly been re-
covered from cases of serious disease in humans also tend
to show the greatest virulence in the M. sexta model. This
model is being further developed in order to investigate
the conditions under which S. aureus is able to adapt to
the in vivo caterpillar environment, assay the relative ¢t-
ness of strains through competition experiments, and in-
dex the relative gain in virulence potential through mea-
surements of Manduca weight loss or death. Once strains
of di¡ering pathogenic potential have been identi¢ed, it
may be possible to track changes in putative virulence
loci, or metabolic genes that may be linked to such loci,
by the generation of nucleotide sequence data.
FEMSLE Congress 2-6-03
P3^13
CRYOCOLA ANTIQUUS GEN. NOV., SP. NOV., ISO-
LATED FROM SIBERIAN PERMAFROST
E. Yu. Gavrish(1,2), S. G. Karasev(3), N. E. Suzina(2),
D. A. Gilichinsky(4) and L. I. Evtushenko(1,2)
(1) Pushchino State University, Pushchino, Moscow Re-
gion, 142290, Russia ; (2) All-Russian Collection of Micro-
organisms (VKM), G. K. Skryabin Institute of Biochemis-
try and Physiology of Microorganisms RAS, Pushchino,
Moscow region, 142290 Russia; (3) Kuban State Univer-
sity, Krasnodar, 350040, Russia ; (4) Institute of Physico-
chemical and Biological Problems of Soil Science RAS,
Pushchino, Moscow region, 142290, Russia
Two yellow-pigmented actinomycetes (strains VKM Ac-
2070T and VKM Ac-2278) were isolated in the course of
studying the microorganisms from frozen Late Pliocene
samples 1.8-3.0 million years old (Kolyma lowland, Rus-
sia). The bacteria were characterized by non-spore-form-
ing, straight or curved long cells (0.3 ^ 0.4 x 3.0 ^ 4.5 Wm)
fragmenting into shorter motile rods (0.3 ^ 0.5 x 0.7 ^ 1.2
Wm) with perithrichous £agella. The growth temperature
range was 4 ^ 26‡C; thermal optimum 18 ^ 20 ‡C. The
strains contained 2,4-diaminobutyric acid in their peptido-
glycan (B2Q type), and rhamnose as the predominant cell
wall sugar. The major menaquinones were MK-11 and
MK-12. The principal phospholipids were phosphatidyl-
glycerol and diphosphatidylglycerol, predominant fatty
acids were anteiso-15:0, anteiso-17:0 and iso-16:0. The
G+C content of DNA was approximately 65 mol%. The
phylogenetic analysis based on 16S rDNA sequences re-
vealed that the permafrost isolates formed a separate
branch within the Microbacteriaceae phylogenetic cluster,
which is closely linked to the Cryobacterium psychrophi-
lum and exhibited 96.3-96.6% 16S rDNA binary sequence
similarity to this species. However, the isolates clearly dif-
fered from Cryobacterium psychrophilum in the cell mor-
phology and pigmentation, growth temperature range and
optimum, and the composition of menaquinones and fatty
acids. On the basis of both phenotypic and molecular ge-
netic ¢ndings, we propose a new genus and species, Cry-
ocola antiquus gen. nov., sp. nov. in the family Micro-
bacteriaceae.
 1st FEMS Congress / Posters 103^505
P3^14
PHYLOGENY OF PHOTOTROPHIC GREEN SUL-
FUR BACTERIA AND DIVERSITY IN MARINE HAB-
ITATS
B. Alexander, J. F. Imho¡
Institut fu«r Meereskunde, Du«sternbrooker Weg 20, D-24105
Kiel, Germany
Phototrophic green sulfur bacteria represent a separate
phylogentic line of the eubacteria. They are common with-
in the light £ooded anoxic marine environment, in partic-
ular under conditions of low light intensities and high
sul¢de concentrations. Little is known about the diversity
of green sulfur bacteria in marine habitats and a limited
number of green sulfur bacteria have been isolated from
the marine environment. Therefore, their phylogeny was
studied on the basis of gene sequences of the 16S rRNA
and of the Fenna-Matthews-Olson (FMO) protein. Prim-
ers were designed that allowed speci¢c ampli¢cation of
green sulfur bacterial 16S rDNA and of the major part
of the fmoA gene. The largely congruent phylogenetic re-
lationship of gene sequences of the fmoA gene and of 16S
rDNA obtained by pure culture studies facilitated the
comparison of results with both sets of primers with nat-
ural samples. Marine strains were found to form clusters
separate from freshwater strains (Alexander et al., 2002).
The analysis of samples from coastal habitats from geo-
graphical distant areas showed that so far known green
sulfur bacteria occur around the world at habitats that are
suitable and typical for green sulfur bacteria. Their natural
diversity in marine habitats is relatively low and the major
phylogenetic lines were known from the analysis of avail-
able cultures of green sulfur bacteria.
P3^15
COMPARISON OF THE PHYLOGENETIC PLACE-
MENT OF OCHROBACTRUM AND BRUCELLA
C. Teyssier(1), M. Sime¤on de Buochberg(1), H. March-
andin(2), J. L. Jeannot(1) and E. Jumas-Bilak(1)
(1) Laboratoire de Bacte¤riologie, Faculte¤ de Pharamacie,
15, av. C. Flahault, BP 14491, 34093 Montpellier cedex 5,
France; (2) Laboratoire de Bacte¤riologie, Hopital Arnaud
de Villeneuve, Montpellier, France
The bacteria belonging to the genus Ochrobactrum are
likely members of the microbiota of soil more and more
frequently isolated from immunocompromised patients.
The genus Ochrobactrum include 4 species: Ochrobactrum
anthropi, Ochrobactrum intermedium, Ochrobactrum tritici
and Ochrobactrum grignonense and is the closest known
FEMSLE Congress 2-6-03
173
relative of Brucellae. Oddly enough, 16S rRNA based phy-
logeny placed the species O. intermedium in an intermedi-
ate position between O. anthropi and Brucella spp. We
constructed a phylogenetic tree based on 16S rDNA se-
quences of 11 clinical isolates of O. anthropi (N=5) and O.
intermedium (N=6) and 7 selected type strains representa-
tive of the genera Ochrobactrum and Brucella. The tree
con¢rmed that O. intermedium isolates are closer relative
from Brucella spp. than from O. anthropi. This paradox-
ical branching was also found when we performed a phy-
logenetic analysis based on 23S rDNA sequences. Finally,
we ampli¢ed and sequenced the rpoB gene of Ochrobac-
trum isolates and type strains. In the rpoB-based tree con-
structed, the genus Brucella was clearly branched (boot-
strap value of 91%) out of the genus Ochrobactrum. Thus,
the paradoxical grouping of Ochrobactrum and Brucella in
the rRNA-based trees was not con¢rmed by rpoB phylog-
eny. These results highlighted the need of di¡erent
markers to assess the phylogenetic placement of an organ-
ism, especially in groups where the rRNA polymorphism
is too low to clearly resolve the relationships between spe-
cies.
P3^16
GENOME SIZING AND RRN COPY NUMBERING IN
SOME REPRESENTATIVE MEMBERS OF THE
SPOROMUSA SUB-BRANCH
H. Marchandin(1), C. Teyssier(2), M. Sime¤on de Buoch-
berg(2), H. Jean-Pierre(1) and E. Jumas-Bilak(2)
(1) Laboratoire de Bacte¤riologie, Ho“pital Arnaud de Ville-
neuve, 371 Avenue du Doyen Gaston Giraud, F-34292
Montpellier Cedex 5, France; (2) Laboratoire de Bacte¤r-
iologie, Faculte¤ de Pharmacie, 15 Avenue Charles Flahault,
BP 14491,F-34093 Montpellier Cedex 5, France
Chromosome number, genome size and rRNA copy num-
ber are parameters that displayed great variability among
bacteria. Although several members of the Sporomusa sub-
branch are considered as putative human pathogens, the
genome organization and structure of the bacteria belong-
ing to this sub-branch of the Gram positive bacteria phy-
lum has yet never been investigated. We performed the
¢rst genomic investigations for 31 representative strains
of ¢ve genera belonging to this phyletic group: Acidami-
nococcus, Anaeroglobus, Dialister, Megasphaera, Selenomo-
nas, and Veillonella. Number, size, and topology of bacte-
rial chromosome, as well as 16S rRNA gene copy number
were determined using a combination of Pulsed-Field Gel
Electrophoresis of full-length or I-CeuI restricted chromo-
somal DNA and hybridization approaches. All strains
were shown to possess one circular chromosome ranging
in size from approximately 1380 kb for Dialister pneumo-
sintes to 2580 kb for Selenomonas sp. Copy number of the
174 1st FEMS Congress / Posters 103^505
rrn loci varied from 3 in the genus Acidaminococcus to 7
for Megasphaera elsdenii. Mapping analysis revealed that
one of the rrn operon was inverted in orientation with
respect to the others and low-resolution maps could be
established for 3 Veillonella type strains. These data are
the ¢rst available for members of the Sporomusa sub-
branch and although no correlation seemed to exist be-
tween chromosome size and rRNA genes number in bac-
teria, we observed that all the strains we have studied
displayed the smallest genome sizes described according
to their rrs copy number.
P3^17
REVEALING OF COLLAGEN-LIKE SEQUENCES IN
THE MICROORGANISMS OF DIFFERENT TAXO-
NOMIC GROUPS
E. V. Karpova, O. V. Alekseeva , T. S. Kalebina, I. S.
Kulaev
Moscow State University, Moscow, Russia
Collagens and collagen-like sequences are widely repre-
sented in higher eukaryotes. While much is know about
animal collagens, these proteins have not been found in
plants yet. The distribution of collagen-like proteins
among microorganisms is poorly investigated. Collagens
are the proteins characterized by a speci¢c primary struc-
ture, and the presence of the unique triple helical domain
formed by three polypeptide chains. Earlier we have de-
tected the genomic DNA fragments in prokaryotes (Mi-
crococcus luteus and Nocardia sp.) and yeasts (Candida
utilis and Candida maltosa) homologous to the chicken
A1(1) collagen-encoding cDNA. In present work we
used the highly puri¢ed speci¢c enzyme ^ collagenase
from Clostridium histolyticum for the detection of colla-
gen-like sequences in the proteins of di¡erent microorgan-
isms. Using the selective biotinilation by NHS-LC-Biotin,
electrophoresis and electron microscopy we have identi¢ed
the proteins hydrolyzed by this enzyme in Halobacterium
salinarium, Candida utilis and Hansenula polymorpha cells.
We have shown that predominantly collagenase-sensitive
proteins are revealed the cell wall localization and, evi-
dently, serve as structural proteins. The data obtained al-
low us to suggest that collagen-like sequences are widely
spread among microorganisms of di¡erent taxonomic
groups and collagens, apparently, are evolutionary ancient
proteins.
This work has been supported by grants 00-04-48356, 01-
04-06571 and 00-15-97851 of Russian Foundation for Ba-
sic Research and 01-0583 INTAS.
FEMSLE Congress 2-6-03
P3^18
gyrB AS A PHYLOGENETIC MARKER FOR SPECIES
IDENTIFICATION
H. Kasai, K. Watanabe, and S. Harayama
Marine Biotechnology Institute, 3-75-1 Heita, Kamaishi,
Japan
The current progress in bacterial systematics can be traced
back to the introduction of 16S rDNA-based techniques.
By using the techniques for 16S rDNA cataloguing and
sequencing, prokaryotic taxa can be ordered hierarchically
among the ranks of genera and kingdoms. Determination
of inter- and intraspecies relatedness has been facilitated
by the techniques for protein-coding gene sequencing,
DNA pro¢ling and DNA microarrays. On 1995, Yama-
moto and Harayama developed the universal primers to
amplify the gyrB genes of various bacteria. Because the
evolutionary rate is faster than the gene for 16S rDNA, it
can be applied for identi¢cation and classi¢cation of
closely related bacteria. From the results of comparative
studies between the gyrB-based phylogenetic classi¢cation
and DNA-DNA hybridization, it is likely that the gyrB is
useful to classify the bacteria at the genomic species level.
Then, we have established the gyrB database for identi¢-
cation and classi¢cation of bacteria since 1998. The data-
base contain the pages for the gyrB sequence data, for the
methods for ampli¢cation of the gyrB, for the multiple
alignment of the gyrB, for the blast search tools, for recent
topics on the gyrB, and so on. Recently, we have released
nearly 2,000 gyrB sequence data from the type strains of
various taxa. From the accumulated data, we would like
to introduce the primer sets for amplify the gyrB fragment
e⁄ciently. In parallel, we would show the results of com-
parative studies between the gyrB phylogeny and DNA-
DNA hybridization on several.
P3^19
APPLICABILITY OF BOX-PCR FINGERPRINTING
TO DIFFERENTIATE STREPTOMYCES SPECIES
B. Lanoot(1), M. Vancanneyt(1), P. Dawyndt(2), M.
Cnockaert(1), H. Ying(3), Z. Liu(3) and J. Swings(1)
(1) BCCMTM / LMG Bacteria Collection, K.L. Lede-
ganckstraat 35, 9000 Gent, Belgium ; (2) Laboratorium
voor Microbiologie, Universiteit Gent, K.L. Ledeganck-
straat 35, 9000 Gent, Belgium ; (3) Institute of Microbiol-
ogy, Chinese Academy of Sciences, P.O. Box 2714, Beijing
100080, P.R. China
The type strains of 476 validly described Streptomyces
species were screened using the rep-PCR ¢ngerprint tech-
 1st FEMS Congress / Posters 103^505
nique. The aim of the study was to evaluate the taxonomic
relevance of the rep-PCR technique as a tool for species
delineation within Streptomyces systematics. Of the prim-
ers tested BOX, (GTG)5 and primer pair REP, the BOX
primer yielded the less complex and more robust patterns
if applied under highly standardised conditions. A good
correlation was observed between rep-PCR ¢ngerprinting
and DNA-DNA pairing studies. Species sharing nearly
identical rep patterns were correlated with DNA-DNA
homology values above 70%. These data clearly demon-
strate that the genus is largely overspeciated and that at
least 38 species should be reclassi¢ed as junior synonyms.
Emended descriptions are proposed for eight species.
However BOX-PCR does not allow one to reveal all syn-
onymous taxa. Within a species the dispersion of the re-
petitive BOX element over the genome can however be
very diverse as strains sharing a DNA-DNA homology
value at or above 70% can have di¡erent rep patterns.
The taxonomic level of discrimination is quite high namely
between intra-species and species level depending on the
species analysed. We concluded that rep-PCR is a repro-
ducible and easy-to-perform technique valuable for speci-
ation and typing of streptomycetes which will help in the
establishment of a genomically based classi¢cation of all
validly described Streptomyces species.
P3^20
DESCRIPTION OF A NOVEL VIBRIO SPECIES ISO-
LATED FROM SEA BREAM, BIVALVES AND SEA
WATER
M. C. Macian(1,2), M. J. Pujalte(1,2), P. A. D. Gri-
mont(3) and E. Garay(1,2)
(1) Department of Microbiology and Ecology, University of
Valencia; (2) Cavanilles Institute of Biodiversity and Evol-
utive Biology (ICBBE), Burjassot E-46100, Valencia,
Spain; (3) Unity of Emergent Pathogen Bacteria Biodiver-
sity, Institut Pasteur, Paris, France
A group of 18 strains that behaved as typical Vibrio spe-
cies has been subjected to a polyphasic taxonomic study,
including numerical taxonomy, oxidation pro¢le (Biolog),
ribotyping (RiboPrinter0), DNA-DNA hybridization (S1
nuclease/trichloroacetic acid method) and phylogenetic
analysis of the 16S rRNA gene sequences. Three strains
were isolated during an annual survey of Vibrio species
from bivalves and marine water on the Spanish Mediter-
ranean coast in the 80’s. More recently, a group of 15
strains were isolated from kidney of diseased Sparus aur-
ata (sea bream) cultured at Spanish ¢sh farms at the Med-
iterranean coast. The isolates were ¢rst phenotypically
characterized, and the results revealed a very high degree
of phenotypic similarity between them. Thereafter, other
tests were performed. Oxidation pro¢le obtained with the
FEMSLE Congress 2-6-03
175
Biolog system was characteristic and di¡erent from those
pro¢les included in the system. Ribotypes obtained with
two restriction endonucleases showed a moderate variabil-
ity within the strains. The almost complete 16S rDNA
sequences of ¢ve isolates were determined, and the com-
parative analysis con¢rmed their a⁄liation to the genus
Vibrio, with levels of sequence similarity between strains
higher than 98 %, and of 95.5-96.5 % with other Vibrio
species. Phylogenetic analysis performed by applying three
alternative treeing methods situated our strains in the vi-
cinity of the V. £uvialis-V. furnissi cluster. DNA-DNA
hybridization results con¢rmed the independence of the
isolates in a new genospecies. All data support a very close
relationship between the 18 strains and suggest that they
could constitute a new species within genus Vibrio.
P3^21
NOVEL HALOARCHAEAL ISOLATES FROM SLO-
VENIAN AND CROATIAN SALTERNS
N rnigoj, B. Herzog-Velikonja, N. Poklar and L. Pas›ic¤
M. C
Biotechnical Faculty, University of Ljubljana, Vec›na pot
111, 1000 Ljubljana, Slovenia
Until recently, the presence of halophilic archaea in hyper-
saline habitats in eastern Adriatic coast has not been in-
vestigated. Among numerous habitats, the Slovenian salt-
erns in city of Sec›ovlje and Ston salterns, located at
Croatian Peljes›ac peninsula, were chosen. The salterns dif-
fer in ground foundation. The ground foundation of Se-
c›ovlje ponds is made out of arti¢cial gypsum crust rich in
green algae known as ‘‘petola’’, while the ground forma-
tion of Ston salterns is cemented. Both Sec›ovlje salterns
and Ston salterns were repeatedly sampled from august
2000 to august 2002. Inoculation of ¢ltered hypersaline
samples into standard halophilic media led to isolation
of more then 50 strains in pure culture. Several phenotypic
characteristics, which include antibiotic susceptibility, ge-
latine, casein, and starch hydrolysis, ability to degrade
DNA, production of acids from sugars, catalase and oxi-
dase activity, were tested. A number of strains were found
to produce proteinaceous compounds which, like halobac-
terial halocins, inhibited the growth of Halobacterium hal-
obium. Based on phenotypic characteristics tested we pre-
sumptively identi¢ed these isolates as the members of the
Halobacteriaceae family. Based on partial sequence of 16S
rDNA, isolates were classi¢ed as Haloarcula, Haloterrige-
na, Halobacterium, Haloferax and Natrialba, respectively.
176 1st FEMS Congress / Posters 103^505
P3^22
NUCLEOTIDE SEQUENCE ANALYSIS AND MO-
LECULAR COMPARISON OF THE NOVEL INCJ
CONJUGATIVE TRANSPOSON-LIKE CTNR391
AND THE SXT ELEMENTS: MOBILE DNA REPAIR
CASSETTES ?
J. T. Pembroke(1), J. O’Halloran(1), D. Boeltner(2), A.
M. Osborn(2), P. Strike(3), C. MacMahon(1), and B.
McGrath(1)
(1) Molecular Biochemistry, University of Limerick, Ire-
land; (2) Department of Biological Sciences,University of
Essex, UK; (3) Donnan Laboratories, University of Liver-
pool, UK
The prototype IncJ group mobile genetic element
CTnR391, originally classi¢ed as a plasmid, has been
shown to integrate speci¢cally in E. coli into the prfC
gene and behave as a conjugative transposon. This ele-
ment is related to a small group of other IncJ elements
pMERPH, R997 and R748 with apparently a very re-
stricted global distribution. Nucleotide sequence analysis
of the 89-kb element has revealed some 96 ORFs with 30
ORFs that could not be assigned function. Much of the
sequence showed homology to the mobile SXT element of
Vibrio. Comparative analysis has revealed a high level of
evolutionary conservation in respect to nucleotide se-
quence and gene order. We have identi¢ed a number of
putatitive insertions into the core of each element di¡ering
in length and sequence that appear in identical loci in both
elements, which in the main encode unknown ORFs.
Functional analysis of both CTnR391 and SXT reveals a
mosaic structure consisting of bacteriophage, plasmid and
transposable elements. The CTnR391 integrase resembles
phi-80 like integrases while there are a series of transfer
modules which resemble the transfer system of the Salmo-
nella plasmid R27. Most interestingly analysis has revealed
that both SXT and CTnR391 possess a large number of
DNA repair like genes involved in induced mutagenesis
(rumAB), UV sensitisation (ORF13,14,15), repair ATP-
ases (ORF’s 25,26) recombination (ORF’s 68,75), DNA
binding (ORF’s 4,67), repair induction (lon ORF31).
These functions are conserved in both elements and sug-
gest that the element may have evolved as a mobile repair
cassette.
FEMSLE Congress 2-6-03
P3^23
DEVELOPMENT OF INTERACTIVE IDENTIFICA-
TION SYSTEM OF UNIQUE AND EXTREMOPHILIC
MICROORGANISMS ‘‘SINIDEM’’ IN INTERNET
I. Sorokin, N. Galchenko and V. F. Galchenko
Institute of Microbiology of RAS, Prospekt 60-let Oktjabr-
ja, 7/2, 117312 Moscow, Russia
The full-function microbiological database program si-
nidem and the setup version used for installation of this
program to a computer with Microsoft Windows 95, 98,
2000, XP operating systems have been created. The soft-
ware/hardware platform for sinidem database was as-
sembled and tested. The ¢rst version of sinidem that al-
lows the viewing of properties, values, and descriptions of
microorganisms using a web-browser and provides the
base possibilities of The interactive identification was
started up and published in Internet (http://uniqem.ru).
Thus, the microbiological database program sinidem per-
mits the fast search of information on microorganisms
described in the database. sinidem will provide probabil-
istic attribution by speci¢ed properties of microorganism
that is not described in the database to speci¢c microor-
ganism or the group of similar ones described in the data-
base; that is, it gives opportunity of The interactive
probabilistic identification of microorganisims. The
vocabulary of main properties used for description in si-
nidem was created and prepared for publication in Inter-
net. The complex of sinidem software tools makes it pos-
sible to add new properties and their values after the
reviewing by specialists.
P3^24
MOSAICISM OF THE LARGE NATURAL ESCHERI-
CHIA COLI PLASMID PRK100
M. Starcic Erjavec(1,2), W. Gaastra(2), J. van Putten(2)
N gur-Bertok(1)
and D. Z
(1) Department of Biology, Biotechnical Faculty, Univer-
sity of Ljubljana, Vec›na pot 111, 1000 Ljubljana, Slovenia;
(2) Department of Infectious Diseases and Immunology,
Utrecht University, P.O. Box 80.165, 3508 TD Utrecht,
The Netherlands
In search for the evolutionary origin of the conjugative F-
like plasmid pRK100, we determined the plasmid’s func-
tional replication region(s) and performed targeted genetic
analysis (hybridization, PCR, DNA sequencing) of the
plasmid. Construction of minireplicons via ligation of
Tn1725 with plasmid fragments and targeted cloning of
putative replication regions, followed by sequence analysis
 1st FEMS Congress / Posters 103^505
indicated two functional replication regions, a plasmid F-
like RepFIB and a plasmid R1-like RepFIIA replication
region. Partial nucleotide sequencing of regions of the
plasmid revealed genes that encode a putative enterochelin
iron uptake system previously associated with an Escheri-
chia coli pathogenicity island including genes with similar-
ity to PAI III536-like enterochelin and the pColV-like aero-
bactin genes. In addition, a homologue of the plasmid
R100-like RmoA gene was found that exhibits strong sim-
ilarity to the Hha/YmoA class of modulators of gene ex-
pression. PCR and hybridization experiments further dem-
onstrated that pRK100 harbors multiple IS2 and IS3
insertion sequences that may have facilitated in the acqui-
sition of elements from other plasmids. These data togeth-
er with the previous identi¢cation of a F-like tra region
and a pColIa-like colicin Ia, indicate that pRK100 has a
highly mosaic structure with elements derived from many
di¡erent known large natural plasmids and the chromo-
some.
P3^25
CONSERVATION OF THE MOSAIC STRUCTURE
OF THE FOUR INTERNAL TRANSCRIBED
SPACERS AND LOCALIZATION OF THE RRN OP-
ERONS ON THE STREPTOCOCCUS PNEUMONIAE
GENOME
V. Giannino', M. Santagati, G. Guardo, C. Cascone and S.
Stefani
Department of Microbiological and Gynaecological Scien-
ces, University of Catania, Italy
The detection of heterogeneity of the 16S-23S ribosomal
intergenic transcribed spacer (ITS) region has become
rather common for identi¢cation and typing purposes of
bacteria. ITS not only varies in sequence and length, but
also in number of alleles per genome and in their position
on the chromosome, allowing discrimination of several
species within a genus. Also variation in ITS sequences
between the multiple rrn operons present within a genome
may be as high or greater than between strains of the same
species or subspecies. The aim of this study was: i) to test
the polymorphism of the spacer by PCR-ribotyping for
typing and identi¢cation purposes in Streptococcus pneu-
moniae; ii) to characterise the molecular structure of the
ITS sequence; iii) to determine the number and localiza-
tion of rrn operons in S. pneumoniae by PFGE of I-Ceu I
restriction fragments of entire genome. Our results show
that the genome of S. pneumoniae contains 4 ribosomal
operons, localised in the same place on the chromosome,
each containing a single ITS allele of 241bp: thus, the
PCR-ribotyping in S. pneumoniae is not useful as a mo-
lecular typing method, but can be use only for identi¢ca-
tion purposes. The ITS sequence presents a mosaic organ-
FEMSLE Congress 2-6-03
177
isation of blocks highly conserved intra and inter-species,
giving no possibility to mutations to arise. The observa-
tion that the sequence blocks of the ITS mosaic of S.
pneumoniae are sharing with oral streptococci localised
in a di¡erent branch in the phylogenetic tree, leads to
consider that the causative recombination events are an-
cient.
P3^26
WHAT IS THE VALUE OF CHEMICAL DATA IN
THE TAXONOMY OF THE FAMILY HALOMONA-
DACEAE?
C. Belloch and B. J. Tindall
DSMZ-Deutsche Sammlung von Mikroorganismen und
Zellkulturen GmbH., Mascheroder Weg 1b, D-38124
Braunschweig, Germany
Members of the family Halomonadaceae have been chosen
as a model system for study, in order to appreciate the
signi¢cance of genetic, chemical and biochemical/physio-
logical data in the current taxonomy. Previous work
showed that the genera Halomonas and Deleya did not
form clear coherent groups and led to the proposal to
combine these two genera. Subsequent work has relied
heavily on 16S rDNA squence data as the basis for the
current taxonomy. At present the family Halomonadaceae
comprises the genera Halomonas, Cobetia, Chromohalo-
bacter, Zymomonas, and Carnimonas. Although there are
currently some clear groupings within the family, as more
species are added it is becoming more di⁄clut to assign
new taxa to existing groups or to clear distinguish the
current groupings based on the 16S rDNA data alone.
In the present study we have returned to the chemical
data, which initially showed that the genera Halomonas
and Deleya were not coherent groups, with a view to ex-
amining the relationship between the 16S rDNA and
chemical data as a means of resolving some of the present
problems in the taxonomy of this family.
P3^27
ANCIENT PROKARYOTES ^ WHAT IS THEIR SIG-
NIFICANCE IN PROKARYOTIC SYSTEMATICS?
B. J. Tindall
DSMZ-Deutsche Sammlung von Mikroorganismen und
Zellkulturen GmbH., Mascheroder Weg 1b, D-38124
Braunschweig, Germany
In recent years there have been a number of reports of
organisms associated with geologically ancient material
(rock salt or amber). Although there are some doubts
178 1st FEMS Congress / Posters 103^505
about the source of isolated strains or 16S rDNA sequen-
ces from such ancient material, these reports have a num-
ber of consequences in the interpretation of the evolution-
ary history of the organisms concerned. One of the major
problems is that some of the logical consequences of the
hypothesis that the organisms or the sequences associated
with them are ancient have not been fully explored. An
interesting paradox is that if we accept that the biological
material examined really is ancient then some of our cur-
rent theories and hypotheses on which our understanding
of prokaryotic evolutionary history and systematics is
based may not be supported.
P3^28
RECONSTRUCTING THE PAST ^ TAKING A CLOS-
ER LOOK AT THE COMPLEXITY
B. J. Tindall
DSMZ-Deutsche Sammlung von Mikroorganismen und
Zellkulturen GmbH., Mascheroder Weg 1b, D-38124
Braunschweig, Germany
The goal of modern prokaryotic systematics is to group
organisms according to their evolutionary relationships.
One of the major di⁄culties is that there is no reliable
fossil record for prokaryotes which allows us to trace their
evolution over geological time. This applies to morphol-
ogy and molecular (protein and DNA sequence) data.
Thus we tend to extrapolate backwards using a set of
assumptions, some of which may not re£ect the true pic-
ture. When two data sets are compared one of the most
important aspects is that they meet certain criteria, in par-
ticular that homologous parameters are being analysed.
Over the past 30 years concepts, such as orthology, parol-
ogy, xenology, paraxenology, and plerology have been
formulated. Unfortunately, such concepts are usually ne-
glected in modern prokaryotic systematics. Taking a closer
look at the way these concepts a¡ect our interpretation of
data provides an interesting insight into the di⁄culties
associated with unravelling the evolutionary history of
prokaryotes.
FEMSLE Congress 2-6-03
P3^29
HOW IMPORTANT IS THE PHENOTYPE IN EVAL-
UATING 16S rDNA DATA?
C. Belloch and B. J. Tindall
DSMZ-Deutsche Sammlung von Mikroorganismen und
Zellkulturen GmbH., Mascheroder Weg 1b, D-38124
Braunschweig, Germany
The analysis of the small subunit rRNA molecule (or its
gene sequence) has become a routine method in microbi-
ology. The way in which we interpret the signi¢cance of
the sequence data is strongly in£uenced by concepts devel-
oped in the mid-1980’s. However, recent advances in the
study of the ribosome, including the study of RNA and
protein interactions in intact ribosome particles, has led to
the availability of important information of signi¢cance to
the way in which the rRNA sequence data may be inter-
preted. Using a model set of organism and taking the
structure and function of the 16S rRNA molecule into
consideration (including both rRNA primary and second-
ary structure, together with protein interactions) it is pos-
sible to show a number of novel aspects in the way in
which this data may be interpreted. This approach is not
only important in understanding the relevance of this mol-
ecule in determining taxonomic groupings in prokaryotes,
but it also helps to appreciate the evolutionary constraints
on this molecule.
P3^30
CHARACTERIZATION OF PSEUDOALTEROMO-
NAS SP. STRAIN CP76: AN EXTRACELLULAR PRO-
TEASE PRODUCER
C. Sa¤nchez-Porro, E. Mellado, S. Mart|¤n, D. R. Arahal, M.
C. Ma¤rquez and A. Ventosa
Department of Microbiology and Parasitology, University
of Sevilla, 41012 Sevilla, Spain
During a large screening for the isolation of moderately
halophilic bacteria (able to grow optimally in media con-
taining between 3 and 15 % NaCl) showing hydrolytic
activities, we isolated a total of 26 proteolytic moderately
halophilic bacteria from several hypersaline environments
(salterns and saline soils) in South Spain. One of the iso-
lates, designated as strain CP76, was selected as the best
producer and most appropriate strain to carry out further
studies. The extracellular protease was puri¢ed by a two
steps process and characterized biochemically. This en-
zyme showed optimal activity over a wide range of salt
concentrations, from 0 to 20 % NaCl, and at 55‡C and pH
8.5; The molecular mass was estimated to be of 38 Kda.
 1st FEMS Congress / Posters 103^505
We sequenced its N-terminal amino acid sequence and it
showed high similarity to several metalloproteases. The
genetic characterization of the gene involved in the pro-
duction of this enzyme is under way. Besides, we have
characterized taxonomically strain CP76. Phenotypic
data as well as the comparison of 16S rRNA complete
sequences showed that this bacterium belongs to the genus
Pseudoalteromonas. The highest 16S rRNA similarity with
most Pseudoalteromonas species is 95.6% or lower, except
for the recently described species Pseudoalteromonas ruth-
enica, to which it was closely related (99% similarity). P.
ruthenica has been described very recently (2002) and is a
slight halophile (optimal growth at 1-3 %NaCl) isolated
from marine invertebrates. Althoght there are several phe-
notypic di¡erences between this species and strain CP76,
DNA hybridization studies will determine if our isolate
constitutes a separate species of the genus Pseudoalteromo-
nas.
P4^1
CHARACTERIZATION OF PSYCHROTROPHIC
BACTERIA ISOLATED FROM FISH AND SEA
WATER OFF THE ICELANDIC COAST, BASED ON
16S GENE ANALYSIS AND BIOCHEMICAL REAC-
TIONS
E. Benediktsdo¤ttir(1) and V. Th. Marteinsson(2)
(1) Institute of Biology, Microbiology Laboratory, Univer-
sity of Iceland, Armuli 1A, IS-108 Reykjavik, Iceland; (2)
Prokaria, Gylfa£ot 12, IS-112 Reykjavik, Iceland
Thirty-six sea samples and samples taken from skin, gills
and intestines of 19 individual ¢sh of ¢ve species, were
cultivated at 15‡C on Marine agar, TCBS agar and in
alkaline peptone water supplemented with minerals. All
samples were taken from pelagic ¢sh and water o¡ the
Icelandic coast, with water temperatures at or below
11‡C. One hundred thirty-three strains isolated from ¢sh
and 193 strains isolated from sea water were presump-
tively identi¢ed as Vibrio and Photobacteria based on
Gram reaction, oxidative/fermentative ability, oxidase re-
actions and inability to grow on CLED agar. These strains
and selected reference strains were subjected to biochemi-
cal tests and numerical taxonomy. Forty-six of the strains
were subjected to partial or complete 16S ribosomal anal-
ysis. According to the 16S gene sequencing, the isolates
belonged to 6 genera, most strains belonged to the Vibrio
genus, but Photobacterium, Enterovibrio, Psychromonas,
Moritella and Shewanella were also represented. Nine
strains belonged to unknown species, with 97 % or less
identity to strains of known species: Three of these be-
longed to the Vibrio genus, 2 to the Psychromonas genus,
2 to the Shewanella and 2 to a possible new genus. The
results of the biochemical tests clustered strains into four
FEMSLE Congress 2-6-03
179
main clusters, V. splendidus-like cluster, V. logei-like clus-
ter, Photobacterium-like cluster, and an unreactive group
including the Moritella and Shewanella strains. The rele-
vance of the di¡erent tests for psychrotrophic bacteria will
be discussed.
P4^2
PRIMARY STRUCTURE OF S-LAYER PROTEINS
FROM MESOPHILIC, THERMOPHILIC AND HY-
PERTHERMOPHILIC METHANOCOCCI
H. Ko«nig(1), E. Akca(1), H. Claus(1), B. Schlott(2), T.
Debaerdemaeker(3) and J.-P. Declercq(4)
(1) Institut fu«r Mikrobiologie und Weinforschung, Johannes
Gutenberg-Universita«t, 55099 Mainz, Germany ; (2) Insti-
tut fu«r Molekulare Biotechnologie (IMB), 07745 Jena, Ger-
many; (3) Sektion Ro«ntgen- und Elektronenbeugung, Uni-
versita«t, 89069 Ulm, Germany; (4) Laboratoire de Chimie
et de Cristallographie, Universite¤ Catholique de Louvain,
1348 Louvain-la-Neuve, Belgium
Cells of methanococci are covered by a single layer of
protein subunits (S-layer) in hexagonal arrangement,
which are directly exposed to the environment and cannot
be stabilized by cellular components. We have isolated S-
layer proteins from cells of Methanococcus vannielii (Topt.=
37 ‡C), Methanococcus thermolithotrophicus (Topt.= 65 ‡C)
and Methanococcus jannaschii (Topt.= 85 ‡C). The primary
structure of the S-layer proteins was determined by se-
quencing the corresponding genes. According to the pre-
dicted amino acid sequence the molecular masses of the S-
layer proteins of the di¡erent methanococci are in a small
range between 59064 Da and 60547 Da. Compared to its
mesophilic counterparts the observation is noteworthy
that in the S-layer protein of the extreme thermophile
Mc. jannaschi the acidic amino acid Asp is predominant,
the basic amino acid Lys occurs in higher amounts and
Cys and His are only present in this organism. Despite the
di¡erences in the growth optima and the predominance of
some amino acids, the comparative total primary structure
revealed a relatively high degree of identity (38 ^ 45 %)
between the investigated methanococci. This observation
indicates that the amino acid sequence of the S-layer pro-
teins is signi¢cantly conserved from the mesophilic to ex-
tremely thermophilic methanococci.
180 1st FEMS Congress / Posters 103^505
P4^3
MODERATELY ACIDOPHILIC, THERMOPHILIC
ANAEROBIC BACTERIA FROM KAMCHATKA HOT
SPRINGS
I. V. Kublanov, M. I. Prokofeva, I. V. Subbotina, T. P.
Tourova and E. A. Bonch-Osmolovskaya
Institute of Microbiology, Russian Academy of Sciences,
Prospekt 60 Let Oktyabrya 7 bldg 2, 117312, Moscow,
Russia
Acidic hot springs are quite common in volcanic environ-
ments, but most known thermoacidophilic prokaryotes are
either aerobes, or organisms with a di¡erent type of an-
aerobic respiration. Thermoacidophiles with tentatively
fermentative type of metabolism are represented by Ar-
chaea of genera Thermoplasma and Acidilobus and by
the only representative of Bacteria ^ Thermoanaerobacte-
rium aotearoense, which is a moderate thermophile and
moderate acidophile. 15 samples of water and sediment
from Kamchatka hot springs from 35 to 80‡C and from
pH 2.0 to 4.0 were used for inoculation of anaerobic me-
dium with di¡erent sugars as growth substrates. pH of the
medium and incubation temperature were the same as in
sampling sites. Four very active enrichment cultures grow-
ing on sucrose or starch at 60‡C at pH 3.5 were obtained,
and from two of them dominating organisms were isolated
in pure culture and characterized. Strain 761 had rod-
shaped spore-forming cells, motile with one £agellum. It
grew optimally at 55‡C and pH 5.7; lower pH limit of
growth was 3.2. Isolate 761 was able to ferment a wide
range of mono-, di- and polysaccharides; when thiosulfate
was added, the formation of elemental sulfur was ob-
served. G+C content of DNA was 33.8 mol.%. Analyses
of 16S rDNA sequence placed the new isolate in genus
Thermoanaerobacterium. Cells of strain 711 were also
spore-forming motile rods. It grew optimally at 60‡C
and pH 5.0 and was able to ferment many sugars. Thio-
sulfate, when added, stimulated the growth on fermentable
substrates, being reduced to hydrogen sul¢de. Hybridiza-
tion with a molecular probe speci¢c for genus Thermoa-
naerobacter revealed strain 711 as a member. Thus, both
new isolates were organisms with fermentative metabo-
lism, moderate thermophiles and moderate acidophiles,
belonging to bacterial genera that included (with the ex-
ception of T. aotearoense) only neutrophilic species.
FEMSLE Congress 2-6-03
P4^4
NOVEL THERMOPHILIC BACTERIA FROM DEEP-
SEA HYDROTHERMAL VENTS
M. L. Miroshnichenko(1), S. L’Haridon(2), O. Nerses-
sian(2), S. Spring(3), P. Schumann(3), E. Stacke-
brandt(3), E. Bonch-Osmolovskaya(1), C. Jeanthon(1)
(1) Institute of Microbiology, Russian Academy of Scien-
ces, 117312 Moscow, Russia; (2) UMR 6539, Institute
Universitaire Europeen de la Mer, 29280, Plouzane,
France ; (3) German Collection of Microorganisms and
Cell Cultures, Mascheroder Weg 1b, 38124 Braunschweig,
Germany
Five new genera of moderately thermophilic bacteria were
isolated from the deep-sea hydrothermal habitats. New
isolates were found to be diverse both phylogenetically
and phenotypically, possessing chemolithoautotrophic,
chemoorganoheterotrophic, or mixotrophic types of me-
tabolism. Epsilon-Proteobacteria isolated from the East
Paci¢c Rise and Mid-Atlantic Ridge were represented by
anaerobic thermophiles Nautilia lithotrophica gen. nov.,
sp. nov. and Caminibacter profundus sp. nov., growing
chemolithoautotrophically with molecular hydrogen in
the process of sulfur reduction. The ability of chemolitho-
trophic growth was also shown for the new representatives
of the family Thermaceae. These are Oceanothermus pro-
fundus gen. nov., sp. nov. and Vulcanithermus mediatlanti-
cus gen. nov., sp. nov. Both microorganisms were able to
microaerophilic growth with a wide range of substrates,
including molecular hydrogen. They could also grow an-
aerobically in the presence of nitrate. From the hot vents
of Mid Atlantic Ridge Caldithrix abyssi gen. nov., sp. nov.
was isolated. This strictly anaerobic moderate thermo-
phile, capable of mixotrophic growth, represented a new
phylum. It was able to grow by means of fermentation of
proteinaceous substrates, or chemolithoheterotrophically
by oxidizing molecular hydrogen in the course of reduc-
tion nitrate to ammonium. Marinobacillus modestocaldus
gen. nov., sp. nov. was found to be able to oxidize organic
substrates in the presence of nitrate or grew chemolitho-
heterotrophically with molecular hydrogen as electron do-
nor and nitrate as electron acceptor.
 1st FEMS Congress / Posters 103^505
P4^5
DETECTION OF HYPERTHERMOPHILIC CREN-
ARCHAEOTA OF THE GENUS DESULFUROCOC-
CUS BY HYBRIDIZATION WITH OLIGONUCLEO-
TIDE PROBES
A. A. Perevalova, A. V. Lebedinskii, N. A. Chernyh, and E.
A. Bonch-Osmolovskaya
Institute of Microbiology, Russian Academy of Sciences,
117312 Moscow, Russia
The goal of this study was to design oligonucleotide prim-
ers and probes that would allow chemio£uorescent detec-
tion and identi¢cation of representatives of the genus De-
sulfurococcus in pure cultures, enrichments, and natural
samples. The Crenarchaeota-speci¢c primer pair that we
designed, Cren 7F (5’-TTCCGGTTGATCCYGCCG-
GACC-3’) and Cren 518R (5’- GCTGGTWTTACCGC-
GGCGGCTGA-3’), was used in order to obtain PCR
products on the DNA of pure cultures, enrichments, and
natural samples. The PCR products were hybridized with
oligonucleotide probes targeting the genus Desulfurococcus
(Dco. 198, 5’-CGTTAACYCCYGCCACACC-3’) and its
species Dco. mobilis (Dco_mob 198, 5’-CGTTAACCCC-
TGCCACACC-3’) and Dco. amylolyticus (Dco_amy 198,
5’-CGTTAACCCCCGCCACACC-3’). With the use of
these primers and probes, four new strains of hyperther-
mophilic archaea were identi¢ed as members of the species
Desulfurococcus amylolyticus. We also managed to detect
representatives of the genus Desulfurococcus in marine nat-
ural samples, where they had not earlier been found.
P4^6
DETECTION OF MEMBERS OF THE GENUS THER-
MOANAEROBACTER USING OLIGONUCLEOTIDE
PROBES
I. V. Subbotina, N. A. Chernyh, A. V. Lebedinskii, E. A.
Bonch-Osmolovskaya
Institute of Microbiology, Russian Academy of Sciences,
117312 Moscow, Russia
The genus Thermoanaerobacter comprises Gram-positive
thermophilic anaerobic bacteria which can chemoorgano-
trophically reduce thiosulfate, sulfur and iron as electron
acceptors. These organisms have been isolated from a wide
variety of environments including hot springs, oil reser-
voirs and anaerobic digestors, but their distribution has
not been thoroughly investigated. We designed oligonu-
cleotide probes that target the genus Thermoanaerobacter
and applied these for detection and identi¢cation of these
organisms in pure cultures, enrichments and natural sam-
FEMSLE Congress 2-6-03
181
ples. The genus-level probe Tab 827 (5’-GCTTCC-
GCDYCCCACACCTA-3’) was suggested, as well as
probes for three phylogenetic groups of genus Thermoa-
naerobacter: TabI 844 (5’-TTAACTACGGCACGR-
AATGCTTC-3’) speci¢c for T. wiegelii, T. siderophilus,
T. ulfurophilus, T. brockii, T. kivui, T. ¢nii, T. ethanolicus,
T. acetoethylycus and T. thermohydrosulfuricus ; TabII 424
(5’-CACTAMYGGGGTTTACAACC-3’) speci¢c for T.
thermocopriae, T. mathranii and T. italicus ; TabIII 184
(5’-TCCTCCATCAGGATGCCCTA-3’) speci¢c for T.
tencongensis, T. yonseiensis and Carboxydobrachium pacif-
icum (the latter organism is related to the genus Thermoa-
naerobacter according to its 16S rRNA sequence). Speci-
¢city and sensitivity of all probes were evaluated by
hybridization of DIG-11-dUTP labelled probes to 16S
rDNA PCR products of bacterial strains on nylon mem-
brane. Optimum conditions for the hybridization were de-
termined. The probes were speci¢c for the target Thermoa-
naerobacter spp. and did not hydridize to the rDNA of
non-target bacteria. Eight pure unidenti¢ed cultures and
¢ve enrichments were analyzed. All cultures were pheno-
typically similar to members of the genus Thermoanaero-
bacter. On the basis of the hybridization results it was
concluded that seven of the 13 samples contained repre-
sentatives of Thermoanaerobacter. One of the strains was
isolated from a deep-sea hydrothermal habitat, where this
genus was not previously found. Thus, our method en-
abled identi¢cation of representatives of the genus Ther-
moanaerobacter without traditional culture-based and mi-
croscopic techniques.
P4^7
ALKALIPHILIC ANAEROBES FROM SODA LAKES
T. N. Zhilina and G. A. Zavarzin
Institute of Microbiology, Russian Academy of Sciences,
Moscow, Russia
Soda lakes represent an extreme example of terrestrial
athalassophilic environment. Due to the evaporation these
lakes accumulate carbonate salts and have high mineral
content. Microbial community in this habitat is subjected
to extreme alkalinity and osmotic stress. High productivity
of soda lakes is caused mainly by cyanobacteria with an-
oxigenic phototrophic bacteria as the main secondary pro-
ducers, closing pathway of decomposition. Anaerobic de-
composers perform metabolic pathways between these two
groups. Alkaliphilic microbial community of soda lakes is
enormously rich in representatives. Isolated alkaliphilic
anaerobes belong to new genera of most diverse phyloge-
netic a⁄liation and comprise complete trophic network. In
total we have described 9 new genera and 14 species from
di¡erent functional groups making preliminary overview
of trophic interrelations in this complicated community.
182 1st FEMS Congress / Posters 103^505
Among alkaliphilic anaerobes are described: sulfate reduc-
ers Desulfonatronovibrio and Desulfonatronum; haloanae-
robes: Natroniella, Halonatronum; acetogens and ammo-
niefers Tindallia, Natronincola ; saccharolytic
Anoxynatronum, Alkalibacterium, species of spirochaetes;
aerotolerant formiate producing bacilli Amphibacillus si-
biricum and A. fermentum, fumarate producing gliding Al-
kali£exus.
P4^8
A STUDY OF MICROBIAL POPULATIONS AND
THEIR CONTROL IN LIBYAN OILFIELD
M. Gaja, H. Ben Hussein and N. Sharaa
Production Technologies and processing research Depart-
ment, Petroleum Research Centre, P.O. Box 6431, Tripoli,
Libya
This study aim to assess and enumerate various trouble
microbial groups; such as general aerobic bacteria, sul-
phur-oxidizing bacteria, sulphate-reducing bacteria and
iron related bacteria in injection water and produced water
at Libyan oil ¢eld. Results showed that bacterial levels
were high however e¡ective biocides were used. In addi-
tion, bacterial populations were also evaluated at reservoir
and upstream temperatures in these commingled waters in
presence and absence of biocides. This study also discusses
failure reasons of currently used biocides in these studied
conditions.
P4^9
LIGNOCELLULOLYTIC MICROORGANISMS IN A
COMPOSTING ENVIRONMENT: UBIQUITY OF
LIGNOCELLULOSE-DEGRADING ENZYMES AND
POTENTIAL USE
G. Guisado, M. J. Lo¤pez, M. C. Vargas-Garcia, F. Sua¤rez-
Estrella and J. Moreno
Area de Microbiolog|¤a, Departamento de Biolog|¤a Aplicada,
CITE II-B, Universidad de Almer|¤a, La Ca•ada de San
Urbano, 04120 Almer|¤a, Spain
The capacity of microorganisms to assimilate organic mat-
ter during composting depends on their ability to produce
the enzymes needed for degradation of the substrate. Plant
wastes are the most usual materials for composting. Those
materials are mainly composed by cellulose, hemicellulose,
and lignin. Thus, e⁄cient degradation during composting
means that biodegradation of lignin is also needed togeth-
er with cellulose and hemicellulose. Lignocellulolytic mi-
croorganisms may increase substrate bioavailability and
improve humi¢cation processes. Compost is a naturally
FEMSLE Congress 2-6-03
enriched environment in microorganisms showing these
activities and may be viewed as a natural source of these
interesting microorganisms. In this study, lignocellulolytic
activities were analysed in 211 microorganisms isolated
from composting piles, including bacteria (173 strains)
and fungi (38 strains). These microorganisms were selected
using a screening procedure according to their abilities to
grow from cellulose, lignin and hemicellulose. Several en-
zymatic activities related to lignocellulose degradation
were evaluated. Hemicellulolytic activity was more often
found than cellulolytic or ligninolytic activities. Most of
the isolates showed peroxidase and oxidase activities. The
number of strains with laccase activity ranged from 10 to
30% depending on the detection method used. Tyrosinase
and polyphenoloxidase were the less usual activities with a
5% of the microorganisms analysed being positives. Some
activities related to lignin degradation typically found in
white rot fungi were also detected in several bacteria. Ac-
cording to the results obtained in this work, lignocellulosic
enzymatic activities are more widespread than usually as-
sumed. The analyses carried out allowed the selection of
several microorganisms according to their enzymatic fea-
tures. These microorganisms may serve as inoculants for
composting improvement, biomass treatment operations
or e¥uents depuration.
P4^10
DIVERSITY AND EXPRESSION OF CATABOLIC
GENES INVOLVED IN CATABOLISM OF PHENOL
AND p-CRESOL IN PSEUDOMONADS ISOLATED
FROM THE POLLUTED AREA
A. Heinaru, M. Merimaa, M. Lehiste, J. Truu and E. Hei-
naru
Institute of Molecular and Cell Biology, Tartu University,
23 Riia Street, Tartu 51010, Estonia
Three di¡erent catabolic types were revealed for the bio-
degradation of phenol and p-cresol in bacteria isolated
from the river water continuously polluted with phenolic
compounds of oil shale leachate. Phenol and p-cresol were
degraded via catechol meta (type I strains like P. mendo-
cina PC1), p-cresol via protocatechuate ortho, and phenol
either via catechol ortho (type II strains like P. £uorescens
PC24) or catechol meta pathway (type III strains like P.
£uorescens PC18). Catabolic potential of the 39 strains
were studied on the basis of genetic diversity of phenol
hydroxylase, p-cresol methylhydroxylase and catechol
2,3-dioxygenase genes. It was found that 11 strains pos-
sessed almost identical single-component phenol monoox-
ygenase gene pheA and the remaining 28 strains had multi-
component phenol hydroxylases. The phylogenetic
analysis of the partial amino acid sequences of the largest
subunit of this enzyme among these strains showed strong
 1st FEMS Congress / Posters 103^505
similarity to the MopN and PoxD. The partial nucleotide
sequences of catechol 2,3-dioxygenases of the 26 strains
clustered predominantly with xylE DK1 and nahH genes.
Phenol hydroxylase and catechol 2,3-dioxygenase genes
did not cluster with dmp, phl and phh genes which were
expected to be most characteristic for pseudomonads. De-
spite the fact that the activity of p-cresol methylhydroxy-
lase was detected in both strains PC18 and PC24, only
phenol in strain PC18 was an inducer of p-cresol methyl-
hydroxylase. The nucleotide sequences of the correspond-
ing gene clusters pchC-pchX-pchF revealed a high similar-
ity rate (97, 94 and 86% of corresponding genes), being
much closer to each other than the corresponding genes in
the strain NCIMB9866 (sharing only 61-76% similarity
rate).
P4^11
IN SITU IDENTIFICATION OF OIL-DEGRADING
RHODOCOCCI USING IMMUNOFLUORESCENT
TECHNIQUE
I. B. Ivshina(1), M. S. Kuyukina(1), C. J. Cunningham(2)
and J. C. Philp(3)
(1) Institute of Ecology and Genetics of Microorganisms,
Russian Academy of Sciences, 13 Golev str., Perm 614081,
Russia; (2) Contaminated Land Assessment and Remedia-
tion Research Centre (CLARRC), The King’s Buildings,
Edinburgh EH9 3JN, Scotland, UK; (3) School of Life
Sciences, Napier University, 10 Colinton Road, Edinburgh
EH10 5DT, Scotland, UK
Indirect immuno£uorescence (iIF) appeared to be e⁄cient
for studying in situ diversity of the genus Rhodococcus
bacteria belonging to mycolic acid-containing actinobacte-
ria. Polyclonal immune sera against most valid Rhodococ-
cus species were used. High sensitivity of the iIF method,
short sample processing time (1.5-2.0 hrs) and species
speci¢city of anti-rhodococcal sera used allowed study of
a great number of soil and subsurface samples from oil-
¢elds and oil-contaminated sites. Individual Rhodococcus
species were shown to be associated with hydrocarbon
accumulations. R. ruber dominated in subsurface samples
from oil¢elds. The predominant components of biocenoses
of oil-contaminated soil were represented by R. erythrop-
olis, R. opacus and R.‘‘longus’’. Rhodococci were detected
in numbers 103-104 cells g-1 in the presence of 106-108 cells
g-1 of attendant microorganisms. The iIF speci¢city was
tested by immuno£uorescent staining of Kocuria rosea,
Micrococcus luteus and Pseudomonas spp., isolated and
identi¢ed as predominant components of alkanotrophic
rhodococci-associated micro£ora. Rapid detection of rho-
dococci in natural samples allowed us to develop a ¢eld
method for microbiological prospecting of oil deposits.
This method is currently used in ¢eld-scale bioremediation
FEMSLE Congress 2-6-03
183
of oil-contaminated soil for monitoring indigenous and
introduced oil-degrading bacteria.
This work was supported by INTAS grant 2001-2151.
P4^12
CLASSIFICATION OF NAPHTHALENE DEGRADA-
TION IncP-9 PLASMIDS OF FLUORESCENT PSEU-
DOMONAS STRAINS
T. Yu. Izmalkova, S. L. Sokolov, I. A. Kosheleva, A. M.
Boronin
Institute of Biochemistry and Physiology of Microorganisms
RAS, Pushchino, Russia
The collection of naphthalene degrading £uorescent Pseu-
domonas strains isolated from di¡erent coal tar- and oil-
contaminated sites was tested on the presence of IncP-9
plasmids. Seventeen strains of twenty-¢ve IncP-9 plasmid-
bearing pseudomonads were selected for the further study.
Fifteen strains are P. putida, one (37NF) ^ P. £uorescens
and one (8909N) ^ P. aeruginosa. Most of plasmids were
placed into IncP-9 L-subgroup, while four plasmids
(pBS216, pSN11, pNF142 and pBS1145) ^ into N-sub-
group. Plasmid pBS2 contains fused replicons IncP-9
and IncP-7. According to the RFLP patterns generated
by EcoRI digestion, the plasmids were divided into three
groups (A, B, C). IncP-9N subgroup plasmids and pBS2
belong to the group C. Thus, plasmid replicon organiza-
tion correlates with the whole plasmid structure. PCR am-
pli¢cation of nahAc DNA fragment of these plasmids and
subsequently digestion of amplicons with HaeIII has dem-
onstrated that nahAc sequences are highly conserved.
Comparison of nahG restriction patterns revealed that
nahG from P. £uorescens 37NF is similar to nahG from
archetypal plasmid NAH7, while the other studied strains
contain nahG alike one from pDTG1. Ampli¢cation of the
regulatory gene nahR was observed only in the case of B
and C plasmid groups. Group A plasmids did not give
ampli¢cation of nahR fragment. Earlier we have demon-
strated the coexistence of two di¡erent salicylate hydrox-
ylase genes in P. putida strains BS202, BS3701 and
BS3790 ; one of these has no homology with classic
nahG. All representatives of group A plasmids are sug-
gested to have clustered genetic structure.
This work was supported by INTAS, grants NN 99-1481
and 01-2383.
184 1st FEMS Congress / Posters 103^505
P4^13
DIRECT AND SPECIFIC DETECTION OF AIR-
BORNE THERMOPHILIC ACTINOMYCETES
FROM COMPOSTING FACILITIES
P. Ka«mpfer(1), R. Scha«fer(1), C. Beimfohr(3), A.
Neef(2)
(1) Institut fu«r Angewandte Mikrobiologie, Justus-Liebig-
Universita«t Giessen, Heinrich-Bu¡-Ring 26-32, D-35392
Giessen, FRG; (2) GBF ^ Gesellschaft fu«r Biotechnologi-
sche Forschung, Bereich Mikrobiologie, Mascheroder Weg
1, D-38124 Braunschweig, FRG; (3) vermicon AG, Emmy-
Noether-Str. 2, D-80992 Mu«nchen, FRG
The high number of biowaste treatment facilities like com-
posting plants has led to an increased attention for the
detection of potentially pathogenic microorganisms from
the airborne state. Certain groups of compost inhabiting
thermophilic actinomycetes (TPAs) which can be emitted
from treated biomaterials into the atmosphere are of hy-
gienical importance. For detection and identi¢cation of
di¡erent TPAs, nowadays the time consuming cultiva-
tion-based approach is most often used. Application of
FISH (Fluorescence in situ hybridisation) o¡ers a more
rapid and speci¢c identi¢cation of TPAs. Speci¢c
16S rRNA-targeted oligonucleotide probes were devel-
oped and evaluated in a FISH format with agar-grown
whole cells for the detection of the species Saccharopoly-
spora rectivirgula, the genera Thermoactinomyces and Sac-
charomonospora as well as for the groups of Nocardiopsis /
Thermobi¢da and certain thermophilic Streptomyces. Con-
ditions for cell wall permeabilisation and hybridisation
stringency were optimised independently for all groups
of organisms. The probes proved to be speci¢c for the
target organisms and enabled the detection of ¢laments
and spores. E⁄zient permeabilization of the cells was
achieved via combined acetic acid/lysozyme or hydrochlor-
ic acid treatments prior to the hybridization step.
FEMSLE Congress 2-6-03
P4^14
FUNCTIONAL AND SPECIES DIVERSITY OF SOIL
OIL-OXIDIZING BACTERIA IN CONTRASTING CLI-
MATIC ZONES
E. V. Karaseva(1), S. G. Karasev(1), I. E. Girich(1), V.
V. Koksina(1), I. B. Ivshina(2), M. S. Kuyukina(2) and J.
C. Philp(3)
(1) Biology Faculty, Kuban State University, 7 Stavropol-
skaya str., Krasnodar 350040, Russia; (2) Institute of Ecol-
ogy and Genetics of Microorganisms, Russian Academy of
Sciences, 13 Golev str., Perm 614081, Russia ; (3) School of
Life Sciences, Napier University, 10 Colinton Road, Edin-
burgh EH10 5DT, Scotland, UK
Crude oil-contaminated soils of di¡erent types and geo-
graphical locations, particularly ‘‘podzol’’ from Middle
Urals and ‘‘chernozem’’ from Kuban region were studied.
A common characteristic of the oil-degrading bacterioce-
noses was that representatives of several actinobacteria
genera, e.g. Rhodococcus and Gordonia predominated in
heavily (above 10 % w/w) oil-polluted soils, with some
isolated strains according to the morphological, cultural
and chemotaxonomical properties being accommodated
to the genera Arthrobacter, Nocardia, Nocardioides, Strep-
tomyces. However, as natural oil degradation proceeded,
the microbial community of soils studied shifted towards
an increased proportion of proteobacteria, represented by
various Pseudomonas species, and to relatively high num-
ber of micrococcii among soil actinobacteria. Seasonal dy-
namics in oil-oxidising bacteriocenoses of Kuban soils dis-
played an increase in spore-forming bacteria at
temperatures above 35‡C and prolonged periods of desic-
cation. A speci¢c feature of oil-degrading bacteriocenoses
of Urals soils was found to be a lack of sharp seasonal
£uctuations in number of Rhodococcus spp. The average
level of these actinobacteria, determined in situ by indirect
immuno£uorescent microscopy, was 103-104 cells g-1 soil.
Physiological properties and ecological behaviour of acti-
nobacterial species (G. rubropertincta, G. terrae, R. eryth-
ropolis, R. opacus, R. ruber), dominant in oil-oxidising
bacteriocenoses, were examined. Isolated bacterial strains
^ active biodegraders of petroleum (aliphatic, aromatic,
polycyclic) hydrocarbons were deposited in the Regional
Specialized Alkanotrophic Microorganism Collection
IEGM (www.ecology.psu.ru/iegmcol/). The information
on these strains as potential bioaugmentation agents for
oil-contaminated soil remediation is included into the
IEGM Collection database.
This work was supported by INTAS grant 2001-2151.1.
 1st FEMS Congress / Posters 103^505
P4^15
CHANGING OF MICROBIOLOGICAL VARIETY IN
OIL-CONTAMINATED SOILS
N. A. Kireeva, E. M. Tarasenko, M. D. Bakaeva
Biology Department, Bashkir State University, Ufa, Russia
Getting in soils, the oil ambiguously in£uences all complex
of the microorganisms describing the given ecosystem.
During laboratory and ¢eld experiments it was obtained,
that the number of geterotrophic microorganisms in oil-
contaminated wood gray soils at high pollutant concen-
trations is much lower than in uncontaminated control
soil. At low concentrations of contamination the number
of microorganisms is increased. Species variety however is
considerably creased. As the result of oil a¡ecting the
atypical strains are widely presented. Thus the number
of phytotoxic forms is increasing and it leads to soil phy-
totoxic to plants. By the increasing of time of oil staying in
the soil the number of hydrocarbon-oxidizing microorgan-
isms is proportionally increased. The increasing of hydro-
carbon-oxidizing microorganisms number in oil contami-
nation conditions promotes an intrinsic self-cleaning of
soil. On the other hand, population ‘‘£ash’’ leads to a
degradation of environment, where the nutritious substan-
ces are exhausted. In ¢eld conditions the highest values of
number are found out for chronic oil contaminated soil. It
is connected to increasing of hydrocarbon-oxidizing fun-
guses number, which one are Fusarium sp., Aspergillus
fumigatus, Aspergillus niger. Screening of hydrocarbon-ox-
idizing bacteria’s has shown their accessory to genus’s Ar-
throbacter, Pseudomonas, Rhodococcus. Including hydro-
carbon-oxidizing micromycetes and bacteria, the
biological preparation for bioremediation of oil-contami-
nated soils is created.
P4^16
BACTERIAL GUT MICROFLORA OF WOODLICE
PORCELLIO SCABER AS HEAVY METAL IMOBILI-
SATION AGENTS
A. Lapanje, D. Drobne, M. Rupnik
Biotechnical Faculty, Department of Biology, Vec›na pot
111, 1000 Ljubljana, Slovenia
Metal pollution is a serious environmental problem. The
toxicity of metals is primarily dependant on its bioavail-
ability. Metal bioavailability is changed by chemical and
biotic transformations. Bacterial activity is responsible for
metal bioavailability in a great deal. Sulfate-reducing bac-
teria (SRB) are well known to precipitate metals. The SRB
can produce H2S, which precipitate the metal and reduce
FEMSLE Congress 2-6-03
185
its bioavailability. The other type of bacterial activity re-
lated to metal transformations is production of more bio-
available and toxic metal forms. Our research on mercury
uptake, elimination and transformation in a terrestrial iso-
pod Porcellio scaber provide evidence on transformation
of inorganic mercury into methyl-mercury in the isopod
gut (Jereb et al., 2003). This fact triggered our research to
explore presence of SRB in the emptied gut of P. scaber.
Namely, SRB have a distinctive capability of transforma-
tion of mercury into methyl-mercury. We used culture
dependant as well as molecular methods for identi¢cation
of SRB in the emptied gut. We found at least three di¡er-
ent morphotypes of colonies of SRB and we isolated one
species. In the gut of P. scaber we found also some other
bacterial species, which could in£uence metal bioaviability.
They are from the genus Shewanella. Some of them be-
longed to the genus Paracoccus which members can de-
grade organic contaminants. These bacterial species were
detected with primers which were basically designed for
detection of Desulfoccocus, Desulfonema and Desulfosarci-
na phylogeneticaly distinct group. The speci¢ty of primer
pairs designed by Daly et al. (2000) is discussed. We also
discuss the presence of strictly anaerobic bacteria in the
gut of P. scaber, which was unexpected because of unfa-
vourable physico-chemical properties of the gut lumen.
P4^17
MOLECULAR MONITORING OF MICROBIAL DI-
VERSITY IN ANAEROBIC WASTEWATER TREAT-
MENT SYSTEMS WITH AN IDENTIFICATION DNA
ARRAY
K. Roest, H. G. H. J. Heilig, H. Smidt, A. D. L. Akker-
mans, A. J. M. Stams and W. M. de Vos
Laboratory of Microbiology, Wageningen University, Hes-
selink van Suchtelenweg 4, NL-6703 CT Wageningen, The
Netherlands
Wastewater treatment in Up£ow Anaerobic Sludge Blan-
ket (UASB) bioreactors is a commonly applied technol-
ogy, especially for industrial wastewater streams with
high organic matter content. Although these anaerobic
systems are working highly e⁄cient, only limited knowl-
edge is available on the microbial processes that take place
inside the reactors. Microbial diversity of sludge from an-
aerobic wastewater treatment systems was studied using
both cultivation techniques and 16S rRNA/DNA analysis
methods. Besides Denaturant Gradient Gel Electrophore-
sis ¢ngerprinting, cloning and sequencing, and Fluorescent
In Situ Hybridisation, a macro array for detection of mo-
lecular diversity in UASB sludge was developed. Approx-
imately 80-bp fragments of the V6 variable region of
cloned and identi¢ed 16S rDNA sequences from sludge
were ampli¢ed, ¢xed on nylon membranes, and subjected
186 1st FEMS Congress / Posters 103^505
to reverse hybridisation with 32P-labelled 16S rDNA from
a selection of the initial clones. To circumvent cross-hy-
bridisation, the highly conserved primer sites were re-
moved by enzymatic cleavage of introduced phosphoro-
thioate-bonds, or blocked with anti-sense probes before
hybridisation. Furthermore, di¡erent target/probe ratios
were used. The microbial diversity of sludge samples
from di¡erent anaerobic wastewater treatment systems
was screened with the macro array and compared to other
available molecular techniques. This showed the capacity
and strength of the array. To further improve reliability
and taxonomic resolution of the array, additional variable
regions of sequenced clones will be used for the develop-
ment of a multiple probe array. The developed macro
array will be miniaturised to an identi¢cation DNA micro
array and used for rapid molecular detection of microbial
diversity in sludge of wastewater treatment samples.
P4^18
COMETABOLISM OF ANILINE AND ITS CHLORI-
NATED DERIVATIVES IN AZOTOBECTER SP. CUL-
TURE MEDIUM
S. Russel
Department of Soil Environmental Sciences, Division of
Agricultural Microbiology, Warsaw Agricultural University,
02-528 Warsaw, ul. Rakowiecka 26/30, Poland
Aromatic amines are established intermediary products
during the decomposition of numerous pesticides. Aniline
and its derivatives are used as a substrate for chemical
synthesis of dye, explosives, synthetic polymers etc. All
these compounds are degraded during microbial metabo-
lism to anilines, but the fate of intermediates in nature is
only partially clari¢ed. Several di¡erent transformation
products of the halogenated anilines have been found,
but a pathway of complete degradation, i.e., decomposi-
tion into mineral products, has not yet been elaborated. In
this presentation, the activity of an Azotobacter sp. iso-
lated from soil, capable of transforming various anilines
under aerobic condition is described. Approximately 30
bacterial strains were isolated from soil. The bacterium
which was most active in metabolism of 4-chloroaniline
was an Azotobacter sp. and this strain was selected for
further study. The bacterium was grown in a nitrogen-
free medium at 280C under aerobic conditions. The ¢nal
concentration of various anilines in the growth medium
was 20 ppm. After 2 days of incubation the Azotobacter
isolate decomposed 100% unsubstituted aniline and 73 to
85% dichlorinated its derivatives. Clear evidence of the
ability of the Azotobacter sp. to metabolize anilines was
obtained with GC and TLC analysis. Using radioactive 4-
chloroaniline it was possible to ¢nd in bacterial culture
medium 3 metabolites which were named M-1, M-2 and
FEMSLE Congress 2-6-03
M-3. Gas chromatography indicated production by Azo-
tobacter sp. four metabolites. Based on its chromato-
graphic characteristics, melting point, UV- and mass-spec-
tra, metabolite M-1 was identi¢ed as 4-chloroacetanilide
and M-2 as 4-chloropropionanilide. The infrared spectra
of chemically synthesized 4-chloroacetanilide and 4-chlor-
opropionanilide and isolated metabolites were identical.
P4^19
BASIC REPLICONS OF BIODEGRADATION PLAS-
MIDS OF PSEUDOMONAS STRAINS
S. L. Sokolov(1), I. A. Kosheleva(1), N. P. Kovalenko(1),
A. M. Boronin(1), K. Smalla(2) and C. M. Thomas(3)
(1) Institute of Biochemistry and Physiology of Microor-
ganisms RAS, Pushchino, Russia ; (2) Federal Research
Centre for Agriculture and Forestry, Braunschweig, Ger-
many; (3) University of Birmingham, School of Bioscien-
ces, Birmingham, UK
Since biodegradative plasmids are of environmental signif-
icance, the studying of their replicons is important in cat-
aloguing the diversity and phylogeny of these plasmids.
The pathway for the catabolism of polycyclic aromatic
hydrocarbons is often found in £uorescent Pseudomonas
on large conjugative catabolic plasmids. Screening of lab-
oratory collection revealed that most studied plasmids for
biodegradation of naphthalene belong to Pseudomonas P-9
and P-7 incompatibility groups. Using two pairs of prim-
ers speci¢c for IncP-9 replicons: korA3Fa-rep3Rc and
mpfA1Fa-korA2Ra, it was shown that HaeIII digested
amplicons of catabolic/resistance plasmids vary and fall
into three subgroups. Resistance plasmids such as pM3
and pMG18 belong to K-subgroup, when caprolactam
degradation plasmids pBS265, pBS267 and pBS268 ^ to
Q-subgroup. Majority of tested naphthalene biodegrada-
tion plasmids and toluene degradation plasmid pWWO
di¡er in the structure of their backbone segments from
plasmids determining the antibiotic resistance and degra-
dation of caprolactam and were placed in L-subgroup of
IncP-9 plasmids. However, it has been shown that in the
case of some naphthalene degradation plasmids belonging
to IncP-9 group the PCR product with RepA-RepB prim-
ers was not obtained, and these plasmids can be referred
to the fourth, N-subgroup. A mini-replicon of N-plasmid
containing all necessary genes for plasmid replication
and maintenance was constructed by ligation of 6 kb plas-
mid fragment with Tcr-cassette. In order to develop mo-
lecular tools for classi¢cation of the IncP-7 plasmids and
to determine the sequence diversity within IncP-7 family
the mini-replicon of pBS213 was constructed.
This work was supported by INTAS, grants NN 99-1481
and 01-2383.
 1st FEMS Congress / Posters 103^505
P4^20
INOCULATION OF COMPOSTING WINDROWS
WITH SELECTED MICROORGANISMS
F. Sua¤rez-Estrella, M. C. Vargas-Garc|¤a, M. J. Lo¤pez, G.
Guisado and J. Moreno
Area de Microbiolog|¤a, Departamento de Biolog|¤a Aplicada,
CITE II-B, Universidad de Almer|¤a, La Ca•ada de San
Urbano, 04120 Almer|¤a, Spain
In the past decades, the role of intensive agriculture in the
economy of the South of Spain has become determinant.
This important expansion involves negative aspects, such
as the production of enormous amounts of horticultural
wastes. Di¡erent solutions have been proposed in relation
to this concern, being composting one of the most inter-
esting. This wastes treatment alternative allows the elimi-
nation of potentially dangerous wastes at the same time as
an interesting product is obtained, compost, which is used
as an organic amendment in agriculture. The aim of this
work was to study the e¡ect of inoculation with micro-
organisms on the ¢nal product quality and the duration of
a composting process of melon plant wastes. Microorgan-
isms used for inoculation were selected by their aptitude to
grow on solid wastes at 40 and 60‡C, as well as by their
ability to produce great quantities of biomass on ordinary
liquid media in a short time. A total of 23 isolates from
horticultural wastes were screened. This experiment al-
lowed the selection of six bacterial strains that showed
the adequate characteristics. To test the ability of these
microorganisms to persist in the windrows, as well as their
in£uence on the properties of the ¢nal product, a compost-
ing assay was achieved. Melon plants were arranged in
seven 1,56 m3 piles (Dimensions: length 2,5 m; height
0,8 m; base 1,10 m). Each windrow was inoculated with
a bacterial suspension to reach a concentration between
107-108 cfu/g of waste, being the last windrow used as
uninoculated control. Samples were extracted and tested
in relation to strain persistence. Physical and chemical
characteristics of compost were also evaluated.
FEMSLE Congress 2-6-03
187
P4^21
DIVERSITY OF THE EPIPHYTIC BACTERIAL COM-
MUNITY ASSOCIATED WITH THE GREEN ALGA
ULVA LACTUCA
N. A. Tujula(1,2), T. L. Skovhus(2,3), C. Holm-
strom(1,2), J. S. Webb(1,2), P. Steinberg(2) and S. Kjel-
leberg(1,2)
(1) School of Microbiology and Immunology, School of
Biotechnology and Biomolecular Sciences, University of
New South Wales, Sydney 2052, Australia ; (2) Centre
for Marine Biofouling and Bioinnovation, University of
New South Wales, Sydney 2052, Australia; (3) Depart-
ment of Microbial Ecology, Aarhus University, DK-8000
Aarhus, Denmark
Pseudoalteromonas tunicata is a dark green-pigmented bac-
terium that produces anti-fouling compounds against in-
vertebrate larvae, algal spores, bacteria, fungi, protozoa
and diatoms. It was originally isolated from the surface
of the tunicate Ciona intestinalis and has since been re-
ported in marine environments across the world. In Aus-
tralia it has been isolated from the marine alga Ulva lac-
tuca. Both C. intestinalis and U. lactuca remain relatively
free from fouling organisms in the ¢eld. Recent qualitative
analysis of the distribution of Pseudoalteromonas spp. on
living surfaces in the marine environment support the no-
tion of a host speci¢c association. It has been proposed
that some living surfaces may be colonised by antifoulant-
producing bacteria (like P. tunicata) which can help in
protecting the host organisms from biofouling. The aim
of our research is to examine the ecological role of the
inhibitory bacterium P. tunicata within the epiphytic bac-
terial community on U. lactuca. In this study we report the
¢rst molecular bacterial community analysis of U. lactuca.
A Eubacterial 16S rDNA clone library was constructed
from U. lactuca samples collected from Shark Point, Syd-
ney, Australia. Two hundred clones were initially screened
by using the DNA ¢ngerprinting method of restriction
fragment length polymorhisms (RFLP). The result demon-
strated 70 di¡erent RFLP patterns, indicating a high level
of diversity on the plant surface. A representative from
each class of the di¡erent RFLP patterns was sequenced.
The majority of the clones belong to the Proteobacteria.
Futhermore, denaturing gradient gel electrophoresis has
been used to assess spatial variablity of the epiphytic com-
munity of U. lactuca.
188 1st FEMS Congress / Posters 103^505
P4^22
MATHEMATIC MODEL OF MICROBIOLOGICAL
PROCESSES IN THE OIL-CONTAMINATED SOIL
V. V. Vodopyanov, N. A. Kireeva, E. M. Tarasenko
Biology Department, Bashkir State University, Ufa, Russia
In resent years the study of microbiological processes,
which are taking place in oil-contaminated soils was car-
ried out by methods of mathematical models. Oil in£uenc-
ing on kinetics of loss and growth of microorganisms,
growth and progressing of microscopical funguses, kinetics
of enzymatic reacting was studied. The special attention
was given on a problem: how the microbiological process-
es, which are taking place in oil-contaminated soils, in£u-
ence speed of destruction of oil in soil. By methods of
mathematical models was found out, that per the ¢rst
year after contamination the number of microorganisms
in soil practically irrelevant with dynamics of oil destruc-
tion. It was pointed, that in soil per the ¢rst year after
pollution, there is destruction of oil by two factors: abo-
rigine microbiota, existing in soil before its contamination,
physicochemical factors. It was found out, that under op-
erating of the physicochemical factors in ground is decom-
posed about 20 % of oil. In view of variation of fractional
composition of oil during degradation and di¡erent in£u-
encing of the imported biological components in soil (fer-
tilizers, hydrocarbon-oxidizing microorganisms associa-
tion, etc.) on di¡erent fractions of oil the
multicomponent model of oil degradation was studied.
The obtained model describes the dynamics of oil degra-
dation in di¡erent situations well.
P4^23
INFLUENCE OF VARIOUS CONCENTRATION OF
2,4,6-TRINITROTOLUENE ON STRATEGY OF ITS
TRANSFORMATION BY BACILLUS SUBTILIS SK1
G. Yu. Yakovleva and B. M. Kurinenko
Department of Microbiology, Laboratory of Engineering
Enzymology, Kazan State University, Kazan, 420008, Rus-
sia
The in£uence of various 2,4,6-trinitrotoluene (TNT) con-
centrations (from 20 to 200 mg/l) on the viability of the
Bacillus subtilis SK1 and the strategy of transformation of
the xenobiotic has been studied. It is shown that all the
TNT concentrations under consideration are found toxic
for this strain. The toxic action of the xenobiotic results in
suppression of culture growth, reduction of the glucose
utilization, amount of reduced piridine and oxidated £a-
vine cofactors. We have found that the quantity of toxic
FEMSLE Congress 2-6-03
e¡ect depends on the TNT concentration. At low concen-
trations of xenobiotic (20 mg/l), B. subtilis SK1 saves its
active metabolism and viability ; after the TNT elimination
it begins the active growth. The high concentration of
TNT suppresses the growth of the strain and reduces the
number of viable cells. Although the toxic e¡ect of the
TNT concentration from 20 to 200 mg/l is essentially dif-
ferent, it does not in£uence on the speed of the TNT
transformation during the ¢rst two hours of the experi-
ment. But the strategy of the TNT transformation at low
and high concentration is found to be di¡erent for this
strain. At low concentrations of xenobiotic (20 mg/l), B.
subtilis SK1 reduced nitro groups of TNT producing the
products of nitroreduce, at high concentrations (100-200
mg/l) ^ eliminated nitro groups from the ring with accu-
mulation nitrites in the medium. To our knowledge, two
di¡erent mechanisms of TNT transformation for a single
strain have been observed for the ¢rst time.
P4^24
RHIZOSPHERE-ASSOCIATED MICROORGAN-
ISMS: DIVERSITY, ANTAGONISTIC POTENTIAL
AND APPLICATIONS IN BIOCONTROL
G. Berg(1), J. Lottmann(1), S. Bru«ckner(2) and K.
Smalla(3)
(1) University of Rostock, Institute for Molecular Physiol-
ogy and Biotechnology, Albert-Einstein-Str. 3, D-18051 Ro-
stock, Germany; (2) Prophyta Biologischer P£anzenschutz
GmbH, D-23999 Malchow/Poel, Germany ; (3) Federal
Biological Research Centre for Agriculture and Forestry,
Messeweg 11/12, D-38104 Braunschweig, Germany
The study of rhizosphere-associated microorganisms and
their antagonistic potential is important not only for
understanding their ecological role and the interaction
with plants and plant pathogens but also for any biotech-
nological application. The interface between soil and plant
roots ^ the rhizosphere ^ is a microbial hot spot where
root-colonizing antagonistic bacteria play an important
role in plant health and growth. The fungus Verticillium
dahliae Kleb. responsible for high yield losses in many
crops worldwide was chosen as model organism for antag-
onism studies. In comparison to other microenvironments
of plants, the abundance, diversity and antagonistic activ-
ity in the rhizosphere of ¢eld grown potato plants was
enhanced. In another study analyzing three di¡erent po-
tential Verticillium host plants (potato, oilseed rape and
strawberry) the abundance, the phenotypic and genotypic
diversity of rhizobacteria, which showed antagonistic ac-
tivity towards Verticillium was strongly plant species de-
pendent. Altogether more than 7,000 isolates belonging to
81 di¡erent bacterial species identi¢ed by their fatty acid
pro¢les (FAME) and/or sequencing the 16S rRNA were
 1st FEMS Congress / Posters 103^505
analyzed. To allow a cultivation-independent analysis, 16S
rDNA fragments ampli¢ed by PCR from soil or rhizo-
sphere bacterial DNA and analyzed by denaturing gra-
dient gel electrophoresis (DGGE) con¢rmed the plant-
speci¢city of rhizosphere-associated microorganisms. To
assess the potential of bacterial strains in biocontrol
(from this and other studies), a screening strategy using
di¡erent in vitro and ad planta steps from laboratory to
¢eld trials was used. Altogether, ¢ve biocontrol agents
were found. Based on them two biocontrol products (Rhi-
zoStar0, RhizoVit0) were developed.
P4^25
IDENTIFICATION AND CHARACTERISATION OF
PSEUDOMONAS SPECIES ISOLATED FROM SEMI
ARID REGION OF UZBEKISTAN
D. Egamberdiyeva, J. Dilafruz and D. Kakhramon
Institute of Microbiology, A.Qadiriy str.7 B, 700128 Tash-
kent, Uzbekistan
The purpose of this study was to investigate the Pseudo-
monas species isolated from the Rhizosphere of di¡erent
agricultural crops grown in ¢eld location in semi arid re-
gion Uzbekistan and to characterize their functions and
physiological activity. Pseudomonas species were isolated
from the Rhizosphere of cotton, wheat, alfalfa, melon,
tomato and maize grown at the ¢eld location in Surchan-
darya region Uzbekistan. Isolated strains identi¢ed using
fundamental selection methods, the standard battery of
biochemical and physiological characterization tests. It
was found that the most commonly genera were Pseudo-
monas denitri¢cans, P. mendocina, P.rathonis, P. alcali-
genes, P. aurantiaca, P. £uoro-violaceus. and P.stutzeri.
These strains were well distributed among the isolates
from the wheat, maize, alfalfa, cotton and tomato. Bacte-
rial strains hydrolyzed Tween 20, Tween 60, and lecithin,
produced Auxin. The isolated strains produced B group
vitamins: thiamine, pantothenic acid, pyridoxal, nicotinic
acid and biotin, di¡erent amino acids: lysine, asparagine,
histidine, leucine, valine, glutamine and enzymes : lipase,
nitratreductase, amylase, and arginine dihydrolase. P. al-
caligenes PsA15, P. denitri¢cans PsD6 reacted antagonis-
tically to soil- borne plant pathogens (e.g. Fusarium cul-
morum, Verticillum loteritum). Several strains had
distinctive patterns of antibiotic resistance that are poten-
tially useful as genetic markers. All of the strains oligoni-
trophil, and they have ability to survive N-de¢cient soils.
Most of Pseudomonas species were salt tolerant, being able
to grow in media containing 7% NaCl. P. alcaligenes
PsA15, P. denitri¢cans PsD35, P.rathonis PsR47 survived
under high temperature (50‡C) conditions. These abilities
contributed the isolated bacteria to survive in the environ-
mentally stressed conditions.
FEMSLE Congress 2-6-03
189
P4^26
THE INFLUENCE OF SOME HUMAN ACTIVITIES
ON THE OCCURRENCE OF THE ECTOMYCORRHI-
ZAL FUNGI AND ECTOMYCORRHIZAE
T. Grebenc, H. Kraigher
Slovenian Forestry Institute, Vecna pot 2, SI-1000 Ljublja-
na, Slovenia
The community of ectomycorrhizal fungi can re£ect the
natural or human-in£uenced changes in the forest ecosys-
tems. In our study the in£uence of the new felled gap (30m
diameter, no natural regeneration) in the mature beech-
silver ¢r forest stand was compared with the naturally
occurring gap of about the same size and with abundant
regeneration. Sporocarps of ectomycorrhizal fungal spe-
cies were mapped and types of ectomycorrhizae were col-
lected in a transect line through the gap and identi¢ed
after anatomical characteristics and with PCR-ITS-
RFLP and sequence analysis of as yet undescribed types.
In two years samplings the occurrence of the sporocarps in
the natural regenerating gap was not signi¢cantly di¡erent
from the undamaged part of the research plot while the
number of types of ectomycorrhizae only decreased in
samples collected on a footpath. The analysis of ectomy-
corrhizae revealed some not yet described types of ecto-
mycorrhizae, predominantly on silver ¢r, which were par-
tially determined with sequence analysis. In the centre of
the newly felled gap the abundance of ectomycorrhizal
fungi (sporocarps and ectomycorrhizae) decreased to al-
most zero due to the complete removal of the symbiotic
partner indicating unsuitable in£uence to the forest eco-
system.
P4^27
EFFECT OF NODULATION AND MYCORRHIZA-
TION ON MICROBIAL COMMUNITIES IN MEDICA-
GO TRUNCATULA RHIZOSPHERE
C. Mougel(1), L. Ranjard(1), T. Corberand(1), D. Mer-
dinoglu(2) and P. Lemanceau(1)
(1) INRA ^ CMSE, UMR ‘Microbiologie et Ge¤ochimie des
Sols’, 17 rue de Sully, B.P. 86510, 21065 Dijon Cedex,
France ; (2) INRA ^ Laboratoire de Ge¤ne¤tique et d’Ame¤li-
oration des Plantes, 28 rue de Herrlisheim, B.P. 507, 68021
COLMAR Cedex, France
Plant root provide suitable habitats for the growth of mi-
croorganisms, as indicated by the high number of di¡erent
microbial populations associated with the roots. Among
the plants ^ microbes interactions occurring in the rhizo-
sphere, symbiotic associations are known to improve plant
190 1st FEMS Congress / Posters 103^505
growth in low fertility conditions. The model plant Med-
icago truncatula has the ability to develop symbiotic asso-
ciation with rhizobia and glomalean fungi. Mutants of M.
truncatula a¡ected in their ability to establish symbiotic
association with one or both symbionts are available.
The e¡ect of M. truncatula wild-type and mutants on bac-
terial and fungal communities was analyzed on DNA di-
rectly extracted from rhizospheric soil and root tissue. The
community structures were assessed by RISA (ribosomal
intergenic spacer analysis) ¢ngerprinting obtained on au-
tomatic sequencer. Variations among the structures of
bacterial (B-ARISA) and fungal (F-ARISA) communities
were processed using principal component analysis. These
analyses indicated that the communities had di¡erent ge-
netic structure according to the compartments sampled
(bulk soil, rhizospheric soil and root tissue). Furthermore,
in each rhizospheric compartment (rhizospheric soils, root
tissues), the genetic structure of the microbial communities
varied between the genotypes of M. truncatula (wild-type
and mutants). The biological meaning of the shifts in the
structure of the microbial communities associated with
roots nodulated and/or mycorrhized compared to roots
impaired in their ability to develop symbiotic relations
are discussed.
P4^28
EFFECTS OF MYCORRHIZATION AND NODULA-
TION ON THE STRUCTURE AND DIVERSITY OF
FLUORESCENT PSEUDOMONADS ASSOCIATED
WITH MEDICAGO TRUNCATULA ROOTS
A. Robin(1), T. Corberand(1), C. Robin(2), E. Beniz-
ri(2), Ph. Lemanceau(1) and C. Mougel(1)
(1) UMR INRA/Universite¤ de Bourgogne ‘Microbiologie et
Ge¤ochimie des Sols’, INRA CMSE, BP 86510 21065 Dijon
Cedex, France; (2) UMR ‘Agronomie et Environnement’,
INPL (ENSAIA) ^ INRA, BP 172, F54505 Vandoeuvre-
le's-Nancy, France
Despite the importance of mycorrhizae and Rhizobia on
plant growth and health of leguminous plants, little is
know on their interaction with other rhizospheric micro-
organisms. The aim of our study was to assess the e¡ect of
mycorrhization and nodulation on the structure and diver-
sity of indigenous populations of £uorescent pseudomo-
nads. The study strategy consisted in comparing the pop-
ulations of £uorescent pseudomonads associated with the
roots of M. truncatula wild-type J5, mycorrhized and
nodulated (Nod+Myc+), to that of mutants impaired in
their ability to establish symbiotic relations: mutant
TRV48 (Nod-Myc+) and mutant TRV25 (Nod-Myc-).
The phenotypes of the wild-type and mutants of M. trun-
catula were characterized (dry weight matter, mycorrhiza-
tion rate, presence/absence of nodules). Fluorescent pseu-
FEMSLE Congress 2-6-03
domonads were numerated and isolated from three
compartments (rhizospheric soil, root tissues and unculti-
vated soil). These populations were characterized by
AFLP (Ampli¢ed Fragments Length Polymorphism).
Comparison of densities of pseudomonads con¢rmed the
rhizosphere e¡ect and indicated that the carrying capaci-
ties for £uorescent pseudomonads of the rhizosphere of
the three plant phenotypes were similar. The root tissues
of the mutants Nod- harbored lower £uorescent pseudo-
monads populations than those of the wild-type (Nod+)
suggesting an analogy of recognition process between
plant/Rhizobia and plant/pseudomonads. Statistic analyses
of the data showed (i) that the frequency distributions of
populations associated with roots and soil signi¢cantly
di¡ered and (ii) that the diversity of the rhizospheric pop-
ulations was higher than that of the soil populations.
However, the data yielded did not allow the identi¢cation
of populations of £uorescent pseudomonads preferentially
associated with mycorrhized and/or nodulated roots.
P4^29
STRUCTURE OF MICROBIAL COMMUNITY IN
RHIZOSPHERE OF DIFFERENT WHEAT SPECIES
A. I. Melentiev, L. Yu. Kuzmina, N. F. Galimzianova and
T. F. Boyko
Institute of Biology, Ufa Research Center RAS, prospekt
Oktiabria, 69, Ufa, 450054, Russia
The growth and development of micro£ora in the plant
rhizosphere promotes the nutrient uptake, inhibits the de-
velopment of plant pathogens and in any cases meets the
requirements in phytogormones. The microbial communi-
ty of plant rhizosphere is formed from aboriginal micro-
£ora of a soil. According some data the structure of rhizo-
sphere microbial community is speci¢c for each genera of
plant. The in£uence of genome composition of wheat (Tri-
ticum L.) on the structure of forming rhizosphere microbe
community in typical chernozem soil was studied. The
experiments were carried out in ¢eld conditions with the
next wheat: T. monococcum (Ab), T. timopheevii (AbG), T.
dicoccum (AuB), T. durum (AuB) and T. aestivum (AuBD).
As a result it was ascertained that the main di¡erences of
microbial community in rhizosphere are located in the
population density of some groups of microorganisms.
The number of heterotrophs and number of nitrogen
transforming bacteria in rhizosphere of wheat is increased
in the order T. timopheevii, T. dicoccum, T. aestivum, T.
monococcum, T. durum. The population density of these
bacterial groups di¡ered in 1000 and 100 times accord-
ingly between the extremes. At the same time, the di¡er-
ences in number of spore-forming bacteria, fungi and ac-
tynomycetes were not revealed in rhizosphere of
investigated wheat.
 1st FEMS Congress / Posters 103^505
P4^30
NITROGEN-FIXING ACTIVITY OF RHIZOBIUM GA-
LEGAE STRAINS SPECIFIC FOR GALEGAE ORIEN-
TALIS LAM. AND GALEGAE OFFICINALIS L.
B. Milicic, Dj. Kuzmanovic, D. Josic and A. T. Vidojevic
Institute for Soil Science, Belgrade, Yugoslavia
In two parallel experiments conducted in test tubes on
agreded Jansen’s substrate, the nitrogen-¢xing activity of
three strains of Rhizobium galegae (speci¢c for G. orientalis
Lam.) was tested on G. orientalis and two indigenous
strains (speci¢c for G. o⁄cinalis L.) on G. o⁄cinalis. In-
oculation of G. orientalis with its speci¢c strains 801, 802
and 803 in the form of a ‘single strain inoculum’ caused a
2.3 ^ 2.9 fold increase in the yield of dry shoot mass and a
6.0 ^ 8.6 fold increase of N content in it as compared with
the control. The percentage of ¢xed N in these strains was
83.5 ^ 88.4%. Autochthonous strains 821 and 822 of G.
o⁄cinalis prepared as a single strain inoculum caused a 1.7
fold increase yield of dry shoot mass and 4.6 ^ 5.0 fold
increase of N content in it compared with the control. The
percentage of ¢xed N in these strains was 78.4 and 79.8%.
An inoculum composed of a mixture of strains 801 + 802
(‘double strain inoculum’) speci¢c for G. orientalis
prompted the roots of G. o⁄cinalis to from inactive nod-
ules ( Nod + Fix-) that do not ¢x N from the atmosphere.
In the same way, an inoculum composed of a mixture of
strains 821+822 (double strain inoculum) speci¢c for G.
o⁄cinalis elicited formation of likewise inactive nodules (
Nod + Fix-) on roots of G. orientalis. These results ob-
tained in our investigation clearly indicate that strains of
R. galegae are strictly speci¢c for plant species of the ge-
nus Galega (G. orientalis and G. o⁄cinalis).
P4^31
DIVERSITY OF phlD GENE POOLS OF THE WHEAT
RHIZOSPHERE IN A RICE-WHEAT CROPPING
SYSTEM (MIDDLE GANGA PLAIN, INDIA)
G. Imfeld(1), D. Roesti(1) N. Shani(1), S. Sharma(2), R.
Gaur(2), K. Jeet(2), M. Aragno(1), B. N. Johri(2) and P.
Rossi(1)
(1) Laboratory of Microbiology (LAMUN), University of
Neucha“tel, 11 rue Emile-Argand, 2007 Neucha“tel Switzer-
land; (2) G.B. Pantangar University of Agriculture and
Technology, Department of Microbiology, Pantnagar, India
Among the antifungal compounds synthesized by biocon-
trol strains, the 2,4-diacetylphloroglucinol (DAPG) is a
key metabolite associated with disease suppression in sev-
eral pathosystems. The phlD gene is involved in the bio-
FEMSLE Congress 2-6-03
191
synthesis of a DAPG precursor and is used as a genetic
marker to detect putative DAPG producing strains. The
goal of this study was to assess the impact of fertilization
level and yields of three wheat ¢elds on the diversity of
phlD gene pools of the wheat rhizospheric soil and rhizo-
plane/endorhizosphere fractions. Diversity of putative
DAPG producers was assessed after combining RFLP
analysis of the phlD gene from DNA of isolated strains
and environmental samples. In parallel, RFLP analysis
was also performed on the 16S-23S rDNA Intergenic
Spacer Region of the isolated strains in order to improve
the strain characterization. Operating Taxonomic Units
obtained by the restriction patterns lead to the distinction
of several dominant groups among the phlD positive bac-
teria that were correlated with the speci¢c ¢eld character-
istics. The combined approach allowed a better under-
standing of the phlD population structure of the studied
agricultural system. Moreover, this study constitutes a pre-
liminary basis for time and spatial follow-up of multiple
samples as well as for gene expression analysis in order to
enhance potential information concerning phlD population
dynamics and their role in agricultural systems.
P4^32
INFLUENCE OF DIFFERENT PLANT SPECIES ON
THE DIVERSITY OF BURKHOLDERIA STRAINS
AND SELECTION OF ANTAGONISTIC ISOLATES
J. F. Salles(1), P. Garbeva(1), J. A. van Veen(2), J. D.
van Elsas(1)
(1) Plant Research International, P.O. Box 16, 6700 AA
Wageningen, NL; (2) NIOO-CTO, P.O. Box 40, 6666 ZG
Heteren, NL
The genus Burkholderia has great potential for agricultural
use such as in biocontrol, due to the production of anti-
biotic compounds and ability to colonise the rhizosphere
of many plants in high population density. The aim of this
work was to evaluate the diversity of Burkholderia species
in the rhizosphere of di¡erent crops, as well as the ability
of these to select Burkholderia strains with antagonistic
activity against the soilborne pathogen Rhizoctonia solani.
Therefore, various crops were planted in pots containing
soil obtained from areas with di¡erent history. Burkholde-
ria isolates were obtained by plating in selective medium
and further checked for antagonistic activity in dual-cul-
ture assay. The diversity was evaluated by PCR-DGGE
with speci¢c primers, allowing the assessment of Burkhol-
deria populations. Furthermore, in order to determine the
part of the Burkholderia community responding to crop
plants, root exudates were collected and added to soil in
the presence of bromodeoxyuridine (BrdU). DNA from
the active population (BrdU-labelled) was extracted by
immunocapture and used for PCR-DGGE. The results
192 1st FEMS Congress / Posters 103^505
showed that although crop history had a major impact on
Burkholderia diversity, plant species played an important
role as well, since the communities associated with maize
and grass, or oat and barley were similar. The analysis of
DGGE patterns obtained from total and culturable pop-
ulations showed that the presence of speci¢c bands on
DGGE could be correlated to those crops. Although no
di¡erences in the selection of antagonistic isolates were
observed between treatments, the e¡ect of root exudates
on the organisms will be shown.
P4^33
DIVERSITY AND FUNCTIONS OF THE BACTERIA
ASSOCIATED WITH THE CLUSTER ROOTS OF
WHITE LUPIN. EFFECTS OF ROOT EXUDATES
ON THE SURROUNDING MICROFLORA
L. Weisskopf(1,2), N. Fromin(1), E. Abou-Mansour(3),
E. Martinoia(2), N. Tomasi(2), R. Tabacchi(3) and M.
Aragno(1)
(1) Laboratory of Microbiology, University of Neucha“tel,
CH-2007 Neucha“tel, Switzerland ; (2) Laboratory of Plant
Physiology, University of Zurich, CH-8008 Zurich, Switzer-
land; (3) Laboratory of Analytical Organic Chemistry,
University of Neucha“tel, CH-2007 Neucha“tel, Switzerland
White lupin (Lupinus albus) is a non-mycorrhized legumi-
nous plant which is able to grow on soils with sparingly
available phosphate by producing cluster roots (so-called
proteoid roots). These cluster roots exude large amounts
of organic acids, mainly citrate and malate, and secondary
compounds such as phenolics, causing fast and consider-
able changes in their proximity. These compounds, in ad-
dition to their role in phosphate acquisition, are very likely
to in£uence the surrounding micro£ora. In contrast to the
physiology of cluster roots, which has been intensively
studied in Lupinus albus, little attention has been paid so
far to the associated micro£ora, with respect to its diver-
sity as well as to its potential promoting or deleterious
e¡ect on root growth and nutrition. Here we report the
diversity and functions of bacteria associated to the cluster
roots of L. albus. We investigated the diversity of bacterial
communities by ¢ngerprinting methods based on PCR-
DGGE analysis of the V3 region of 16S rDNA and 16S
rRNA. We also assessed the functions of isolated strains
by testing some PGPR traits in vitro, like the ability to
produce auxins or siderophores, to solubilize inorganic
phosphate or to inhibit the growth of pathogenic fungi.
Since our ¢rst results showed a strong impact of the plant
on the bacterial micro£ora, we then analyzed the e¡ects of
di¡erent root exudates components on the isolated strains
in order to elucidate chemical components of the plant
bacteria communication.
FEMSLE Congress 2-6-03
P4^34
INTERSPECIES AND SUBSPECIES DIVERSITY
AMONG LEPTOSPIRES IN TERMS OF ECOLOGY
Yu. V. Ananyina, A. P. Samsonova
Gamaleya Instititute for Epidemiology and Microbiology of
the Russian Academy of Medical Sciences, Gamaleya str.18,
123098, Moscow, Russia
Pathogenic leptospires (Leptospira interrogans sensu lato)
are known worldwide to play an important role in human
and animal infectious pathology. Some Leptospira agents
(for example, of copenhageni, icterohaemorrhagiae and
canicola serovars) can cause severe diseases with high
case fatality rates or resulting in late clinical sequelae in
humans a¡ected. Meanwhile others, though less virulent
(grippotyphosa, pomona serovars, etc.), are able to induce
cluster or outbreak, mainly water-borne, morbidity involv-
ing dozens and even hundreds of human beings. At
present pathogenic leptospires are classi¢ed as belonging
to at least seven genomospecies and more than 230 sero-
vars have been so far recognized. Experimental evidence
testi¢es that leptospires of di¡erent species and subspecies
taxons display not only substantial diversity in pathogenic
potential, but also ecology. These spirochaetes are highly
variable in terms of host speci¢city (speci¢c mammalian
host range), selective tropism to renal and nervous tissues
as well as ability to compete and persist in vivo as well as
under environmental conditions. Experimental data on in-
terspecies and subspecies biodiversity among leptospires
will be presented and analyzed in relevance to leptospirosis
epidemiological and clinical patterns.
P4^35
IDENTIFICATION, BASIC GENETIC CHARACTER-
ISATION AND REGIONAL DISTRIBUTION OF VIR-
ULENT LACTOCOCCAL BACTERIOPHAGES DE-
TECTED IN POLISH DAIRIES
A. Szczepan¤ska, W. Krysa and J. Bardowski
Department of Microbial Biochemistry, IBB PAS, Pawin-
skiego 5a, 02-106 Warsaw, Poland
Here presented are the results of our research on virulent
bacteriophages infecting dairy lactococcal starter cultures.
The type, morphology and geographical distribution of
lactococcal bacteriophages, which are a serious problem
in dairy plants, were investigated. After detection and iso-
lation of phages from industrial whey samples DNA was
extracted from each isolated phage and characterised with
the use of restriction endonucleases. Detected phages were
assigned into several types, regarding the restriction pat-
 1st FEMS Congress / Posters 103^505
tern and the genome size. The phage host range was de-
termined against industrial lactococcal strains as well as
against two model strains of Lactococcus lactis ^ IL1403
and MG1363. Subsequently, the identi¢ed phages were
classi¢ed into three major genetic groups ^ P335, 936
and C2 ^ by multiplex PCR analysis. Several phages
showed speci¢city to more than one genetic group which
could indicate either presence of a mosaic phage or co-
isolation of a prophage. This result needs further exami-
nation. Additionally, from distribution studies we could
conclude that one type of phage was equally disseminated
throughout the regional network of dairies. The remaining
phages exhibited speci¢city to certain geographical re-
gions. Overall, we have isolated and characterised over
150 individual phages from 14 whey samples, which
form di¡erent restriction groups. Majority of them belong
to the c2 and 936 genetic groups. These studies allowed us
to assess the type of phages a¡ecting the fermentation
processes. Furthermore, we were able to determine that
not one but several phages dominate in the analysed in-
dustrial dairy samples.
This work was supported by the KBN grant No 3 P06T
051 23.
P4^36
POPULATION GENETICS OF IXODES RICINUS
TICKS AND BORRELIA BURGDORFERI SENSU
LATO: PRELIMINARY ASPECTS
S. Casati(1), M. V. Bernasconi(1), L. Gern(2), and J.-C.
Pi¡aretti(1)
(1) Istituto Cantonale di Microbiologia, Via Mirasole 22A,
6500 Bellinzona, Switzerland ; (2) Institut de Zoologie,
Universite¤ de Neucha“tel, Rue Emile-Argand, 2000 Neucha“-
tel, Switzerland
The present work is inserted into a major project in prog-
ress aimed at analysing at the genetic level both the vector
(the tick) and the micro-organisms, they may carry. We
intend to identify a possible correlation between the vector
and the pathogen. The study will consider a collection of
about 1500 Ixodes ricinus ticks and the pathogens Borrelia
burgdorferi s.l., Babesia sp. and TBE virus. A ¢rst step
involves 600 ticks collected from vegetation and animals
in Neucha“tel and in Ticino. By means of PCR and direct
sequencing (using 162 bp of the recA gene) we tested the
presence and the identity of Borrelia burgdorferi s.l. 32%
of the ticks of Neucha“tel and 15% of the ticks of Ticino
were positives The following genospecies have been iden-
ti¢ed: B. afzelii, B. garinii, B. valaisiana, B. lusitaniae and
B. burgdorferi s.s.. The intra-speci¢c variation ranged from
1.8 to 9.2%. Thus, we found a considerable amount of
inter- and intra-speci¢c variation : di¡erent lineages of
Borrelia have been met in limited area. A second step
FEMSLE Congress 2-6-03
193
investigates the genetic variability within I. ricinus (from
Switzerland and Scandinavia) using 12S and 16S rDNA
genes. Both genes show a low variability (2-3%) precluding
the discrimination of unique geographic populations and
hence also a correlation between the vector and the patho-
gens. In conclusion, the inter- and intra-speci¢c variability
found in the Borrelia strains re£ects the variability found
in Europe. Whereas, the I. ricinus ticks from Europe seem
to be extremely homogeneous from a population genetics
point of view.
P4^37
CLONAL ANALYSIS OF SOME MULTIPLE ANTIBI-
OTIC RESISTANT AND HEAVY-METAL TOLERANT
E. COLI STRAINS ISOLATED FROM POLLUTED
WATERS
R. Cernat, V. Lazar, C. Balotescu, E. Coipan, C. Cojocaru
and C. Bucur
Dep. Microbiology-Immunology, Faculty of Biology, Uni-
versity of Bucharest, Aleea Portocalelor 1-3, Sect.5, Bu-
charest 77206, Romania
Self-transmissible plasmids conferring multiple antibiotic
resistance are wide-spread in coliforms populations. In
soil and water, multiple antibiotic resistance is clearly as-
sociated with resistance/tolerance to heavy-metals (Hg2+,
Cu2+, Pb2+, Zn2+, Cd2+). For di¡erent genera the genes
for heavy-metals resistance are often plasmid encoded.
Since these genes are clustered on the same plasmids,
heavy-metals and drugs can serve as selective pressure
factors for the populations of these plasmid-harboring
bacteria. The aims of this preliminary study were (1) to
¢nd possible correlation between resistance genotype de-
termined by genetic analysis and antibiotic and heavy-met-
al resistance patterns and (2) to assess the genetic diversity
of several E. coli strains isolated from chronically polluted
waters. Antibiotic susceptibility testing was performing for
aminoglycosides (amikacin, gentamycin, and kanamycin),
L-lactams (ampicillin, imipenem), amoxicillin/clavulanate,
cephalosporins (ceftazidime, cefotaxime and cephalotin),
quinolones (cipro£oxacin, nor£oxacin and nalidixic acid),
tetracycline and chloramphenicol, as described by Kirby-
Bauer disk di¡usion method following NCCLS recom-
mendations. MICs values of antimicrobials and heavy-
metal salts (CuSO4, CdCl2, Co(NO3)2, Cr(NO3)3, HgCl2,
NiCl2 and ZnSO4) were determined by dilution method.
For the data analysis NCCLS breakpoints for resistance
and sensitivity were used. Plasmid DNA was isolated from
E.coli strains by an alkaline lysis method and digested to
completion with EcoRI enzyme. Genetic similarity and
clustering were calculated using NTSIS program. The phe-
notypic data shows the direct association between multiple
antibiotic and heavy-metal resistance for E.coli strains in
194 1st FEMS Congress / Posters 103^505
polluted waters. DNA ¢ngerprinting with plasmid DNA
RFLP suggested that, depending of source of isolation, E.
coli strains could be grouped in distinct populations with a
di¡erent plasmid diversity.
P4^38
GENETIC DIVERSITY OF BORRELIA BURGDOR-
FERI SENSU LATO IN IXODES RICINUS TICKS
COLLECTED FROM TWO AREAS IN SLOVAKIA
AND CZECH REPUBLIC
M. Derdakova(1), L. Beati(3), B. Petko(1), M. Stan-
ko(2) and D. Fish(3)
(1) Parasitological Institute, Slovak Academy of Scien-
ce,Hlinkova 3, Kosice, Slovak Republic ; (2) Institute of
Zoology, Slovak Academy of Science, Kosice, Slovak Re-
public; (3) School of Epidemiology and Public Health,
Yale University, New Haven, USA
The causative agent of Lyme borreliosis belongs to the
Borrelia burgdorferi sensu lato complex. At least 5 distinct
genospecies has been detected in Ixodes ricinus ticks from
Europe: B. afzelii, B. garinii, B. burgdorferi sensu stricto,
B. valaisiana, B. lusitaniae and B. bissetti. Geographic dis-
tribution of distinct genospecies in Europe varies. The het-
erogeneity of B. burgdorferi s.l. in I. ricinus populations
from two geographically distinct areas was compared in
this study. The genetic variability of B. burgdorferi s.l. was
established by PCR-SSCP analysis of the rrfA-rrlB inter-
genic spacer. By this method we were able to detect also
di¡erences within single genospecies and 16 di¡erent ge-
notypes were described. The overall prevalence of infec-
tion was similar in both areas. 20.3% and 25.7% of I.
ricinus ticks were found to be infected with B. burgdorferi
s.l. in Ceske Budejovice, Czech republic and Kosice, Slo-
vakia respectively. B. afzelii, B. garinii, B. valaisiana, B.
burgdorferi s.s. were present in both sites. In addition, the
presence of genomic group A14 S was detected in Ceske
Budejovice. The predominant genospecies in Ceske Bude-
jovice was B. afzelii (56.5%) in contrast to Kosice where B.
burgdorferi s.s. was detected in 39.6% of positive ticks.
Higher occurence of mixed infection of two and in one
case three di¡erent Borrelia genospecies was detected in
ticks from Kosice. Di¡erences were also detected among
Borrelia population according to the developmental stage
and sex of I. ricinus from both study sites.
FEMSLE Congress 2-6-03
P4^39
BACTERIAL COLONIZATION OF THE DOLPHIN
FOREGUT
M. G. Dom|¤nguez-Bello(1,2), G. Godette(3), P. Gue-
neau(1), C. Bonaventura(3), A. Read(3), D. Gannon(3),
W. McLellan(4)
(1) Laboratorio de Fisiologia Gastrointestinal, IVIC, Ca-
racas, 1020A, Venezuela; (2) University of Puerto Rico,
San Juan, Puerto Rico; (3) Duke Marine/Freshwater Bio-
medical Center, Duke University, Beaufort, NC 28516-
9721, USA; (4) University of North Carolina Willmington,
USA
The mammalian digestive tube, even the hostile acid stom-
ach, has been colonized by microorganisms. Forestomach
compartmentalization allows ruminants to nourish on fer-
mentation products and microbial biomass. Bacterial pro-
tein can be digested thanks to a gastric acid resistant ly-
sozyme ruminants have recruited as a digestive enzyme. In
herbivores, evolution of forestomachs appear to have been
driven by the selective pressure of dietary cellulose. The
only known case of gastric compartmentalization not as-
sociated with herbivorous diet is that in cetaceans. In this
paper we have performed a preliminary characterization of
bacteria colonizing gastric chambers of bottlenose dol-
phins and determined expression of gastric lysozyme in
the acidic pouch. Bacteria were cultured anaerobically
from frozen forestomachs of dolphins, and identi¢ed ac-
cording to culture characteristics and membrane fatty
acids analysis. Detection of Archaea in forestomach con-
tents and Helicobacter spp in the acidic sac was performed
by speci¢c PCR ampli¢cations. Bacteriolytic activity of
gastric protein extracts and proteins puri¢ed with cationic
exchange chromatography, was assayed in lysoplates with
M luteus. Proteins were electrophoresed on 8-25% SDS
gradient gels. We found anaerobic cultivable bacteria,
most closely related to the proteolytic Clostridium, Eubac-
terium, and also to Enterobacter and Streptococcus strains.
There was no ampli¢cation of Archaebacteria, in forest-
omach contents. Most (14/17) dolphins were colonized by
gastric Helicobacter spp. We report for the ¢rst time, the
expression of gastric lysozyme in Cetaceans. We propose
that the major function of the cetacean foregut is the di-
gestion of dietary compounds that would otherwise be
poorly digested, such as chitin and wax ethers. As cellulose
for ruminants, chitin may have exerted a selective pressure
for the evolution of a complex foregut in crustacean feeder
Cetaceans.
 1st FEMS Congress / Posters 103^505
P4^40
MOLECULAR IDENTIFICATION OF BIFIDOBACTE-
RIA FROM SMALL INTESTINE OF RATS FED WITH
RAW KIDNEY BEAN (PHASEOLUS VULGARIS L.)
L. Fanedl, F. V. Nekrep and G. Avgus›tin
University of Ljubljana, Biotechnical Faculty, Zootechnical
Dept., Groblje 3, SI-1230 Domz›ale, Slovenia
During a raw kidney been feeding trial, which was set in
order to study the e¡ect of antinutrients i.e. phytohemag-
glutinin (PHA) on rat gut microbial community, several
Gram-positive bacteria, phenotypically identi¢ed as bi¢do-
bacteria were isolated from rat small intestines. The mo-
lecular investigation of their relatedness to each other and
to type strains of 20 bi¢dobacterial species by RAPD tech-
nique revealed, that they fall into two main groups and
that none is related to any of the analysed type strains.
Representative strains from the larger group of isolates
were phylogenetically analysed by comparative sequence
analysis of the PCR ampli¢ed 16S rRNA genes. All se-
quences clustered together and formed a coherent group
within the phylogenetic subgroup Bi¢dobacterium lactis as
de¢ned by the Ribosomal Database Project. Their closest
relatives were found to be the bacteria from B. pseudolon-
gum subsp. pseudolongum and B. pseudolongum subsp. glo-
bosum, generally with less than 97% sequence homology.
The similarity levels among 16S rRNA sequences of iso-
lated strains also showed considerable genetic variability,
ranging from 91.4 to 95.8%. In conjunction with other
molecular, phenotypic and ecological data, the 16S
rRNA sequence analysis supports the proposal for novel
bi¢dobacterial species inhabiting the rat small intestines.
Their role during the antinutrient i.e. lectin administration
to the rat gut, still remains hidden.
P4^41
PLASMID pC PRESENT IN SALMONELLA ENTER-
ICA SEROVAR ENTERITIDIS INFLUENCE PHAGE
RESISTANCE
D. Gregorova(1), M. Pravcova(1), A. Sebkova(1), R.
Karpiskova(2) and I. Rychlik(1)
(1) Veterinary Research Institute, Hudcova 70, 621 32
Brno, Czech Republic; (2) National Institute of Public
Health, Center for Food Chain Hygiene, Palackeho 1-3,
612 42 Brno, Czech Republic
Salmonella enterica serovar Enteritidis (S. Enteritidis) pos-
sesses plasmids of di¡erent sizes and roles. Besides the
serovar-speci¢c virulence plasmid present in most ¢eld
strains, S. Enteritidis can harbour plasmids of low molec-
FEMSLE Congress 2-6-03
195
ular mass whose biological role is poorly understood. We
therefore identi¢ed and sequenced the most frequent low
mass plasmids in S. Enteritidis. Using DNA hybridisation
we found that there are at least three distinct groups of
plasmids, which had negative cross hybridisation. We
therefore sequenced 5 representative plasmids from each
group and we concluded that they belong to ColE1, ColE2
and rolling circle replicating plasmids. From ColE1 group,
plasmid pC present in S. Enteritidis strains belonging to
phage type PT14b, was sequenced. The size of plasmid
was determined to be 5 269 bp and it was predicted to
encode 4 ORFs. The ¢rst two ORFs were found (initial
3230 bp) to be highly homologous to ORFs on ColE1
plasmid of E. coli. Proteins encoded by other ORFs
were 99% homologous to a restriction methylase and re-
striction endonuclease encoded by plasmid pECO29 of a
¢eld strain of E. coli. Using insertional mutagenesis we
con¢rmed experimentally the function of the plasmid pC
encoded restriction modi¢cation system. The presence of
the plasmid encoded restriction modi¢cation system ex-
plains the high resistance to phage infection of S. Enter-
itidis PT14b strains.
P4^42
DIVERSITY OF MESOPHILIC SHEWANELLA ISO-
LATED FROM THE NORTH-WESTERN PART OF
THE PACIFIC OCEAN
E. P. Ivanova(1), T. Sawabe(2), N. V. Zhukova(3), N. M.
Gorshkova(4), O. I. Nedashkovskaya(4), G. M. Frolo-
va(4), A. F. Sergeev(5), V. V. Mikhailov(4), R. Chris-
ten(6), D. V. Nicolau(1)
(1) Industrial Research Institute Swinburne University of
Technology PO Box 218, Hawthorn, Vic 3122, Australia;
(2) Graduate School of Fisheries Sciences, Faculty of Fish-
eries, Hokkaido University, 3-1-1 Minato-cho, Hakodate
041-8611, Japan; (3) Institute of Marine Biology of the
Far-Eastern Branch of the Russian Academy of Sciences,
690032, Vladivostok, Palchevskogo St. 17, Russia ; (4) Pa-
ci¢c Institute of Bioorganic Chemistry of the Far-Eastern
Branch of the Russian Academy of Sciences, 690022 Vladi-
vostok, Pr. 100 Let Vladivostoku 159, Russia ; (5) Paci¢c
Oceanological Institute of the Far-Eastern Branch of the
Russian Academy of Sciences, Baltiiskaya Str., 43,
690017, Vladivostok, Russia; (6) UMR 6078 CNRS &
Universite¤ Nice Sophia-Antipolis, Bat. J. Maetz, F06238
Villefranche sur mer cedex, France
Although bacteria of the genus Shewanella belong to one
of the readily cultivable group of Q-Proteobacteria, little is
known about the occurrence and abundance of these mi-
croorganisms in marine ecosystem. Studies revealed that
of 654 isolates obtained from marine invertebrates (ophiu-
roid Amphiopholis kochii, sipuncula Phascolosoma japoni-
196 1st FEMS Congress / Posters 103^505
cum, and holothurian Apostichopus japonicus, Cucumaria
japonica), seawater and sediments of the North-Western
part of the Paci¢c Ocean (i.e. the Sea of Japan and i.
Iturup, Kurile Islands), 10.7% belonged to genus Shewa-
nella. The proportion of viable Shewanella species varied
from 4% to 20% depending on the source of isolation.
From the isolation study, representative strains of di¡erent
phenotypes (out of seventy presumptive Shewanella
strains) were selected for detailed characterization using
phenotypic, chemotaxonomic, and phylogenetic testing.
16S rDNA sequence-based phylogenetic analysis con-
¢rmed the results of tentative identi¢cation and placed
the majority of these strains within only a few species of
the genus Shewanella, namely S. japonica, S. colwelliana,
and two novel species S. ¢delis and S. waksmanii. Numeri-
cally dominant strains of S. japonica were metabolically
active and produced proteinases (gelatinases, caseinases),
lipases, amylases, agarases, and alginases. Shewanella
strains studied demonstrated weak antimicrobial and anti-
fungal activities that might be an indication of their pas-
sive role in colonization of living and non-living surfaces.
P4^43
CHARACTERIZATION OF RHIZOBIUM GALEGAE
STRAINS BY PLASMID PROFILES, INTRINSIC
ANTIBIOTIC RESISTANCE AND RAPD
D. Josic, Dj. Kuzmanovic, B. Milicic, B. Misic
Institute for Soil Science, Belgrade, Yugoslavia
Five Rhizobium galegae strains ^ three strains speci¢c for
Galega orientalis Lam. (801, 802 and 803) and two indig-
enous strains speci¢c for Galega o⁄cinalis L. (821 and
822) were characterized by intrinsic antibiotic resistance
(IAR), plasmid pro¢le and RAPD. Characterization of
strains by plasmid pro¢le analysis and IAR showed high
similarity of strains. Plasmid pro¢les of strains 821 and
822 were identical, as well as plasmid pro¢les of strains
801 and 803. All strrains were found to carry one plasmid
band (size 200-330kb). IAR tests for eight antibiotics (Am-
picilline, Tetracycline, Penicilline, Neomycine, Streptomy-
cine, Erythromycine, Rifampycine and Chloramphenycol)
and RAPD pro¢les showed di¡erences between three
strains speci¢c for G. orientalis. However, strains 821
and 822 speci¢c for G. o⁄cinalis had identical IAR pat-
terns as well as plasmid pro¢les, since RAPD analysis
showed the di¡erences between them.
FEMSLE Congress 2-6-03
P4^44
IDENTIFICATION OF DOMINANT RHIZOBIUM LE-
GUMINOSARUM BV. TRIFOLII ISOLATED FROM
DIFFERENT TYPES OF SOIL IN SERBIA
D. Josic, B. Milicic, A. Terzic-Vidojevic
Institute for Soil Science, Belgrade, Yugoslavia
Rhizobium leguminosarum bv. trifolii is microsymbiont Tri-
folium pratense and Trifolium repens, very important le-
gumes in Serbia. Since nitrogen is often the limiting nu-
trient in soil, the use of appropriate strains of Rhizobium
to supply nitrogen to the plant is economically and eco-
logically justi¢ed. That is why the natural nodulating pop-
ulation of those bacteria was collected during 2002 year.
We planed to estimate biodiversity distribution by moni-
toring dominant genotypes of these bacteria. The popula-
tion of Rhizobium leguminosarum bv. trifolii were colected
from 50 marked locations of 11 types of soil in Serbia. 437
natural isolates, rescued from nodules of Trifolium repens
or Trifolium pratense, were analysed by phenotypic ap-
proache. We obtained 162 di¡erent isolates on the basis
of di¡erences in their IAR ^ intrinsic antibiotic resistance
(¢ve antibiotics) and HMT- heavy methal tolerance (¢ve
heavy methal). We investigated 32 dominant isolates with
more than three di¡erences in IAR-HMT patterns by plas-
mid pro¢les analysis and RAPD ¢ngerprinting. The results
showed vide diversity of dominant Rhizobium leguminosa-
rum bv. trifolii ¢eld isolates and o¡ered the possibility to
assess their changes on marked locations during time and
under di¡erent environmental conditions.
P4^45
THE RELATIONSHIP BETWEEN HLYA AND SHEA
GENES OF EXTRAINTESTINAL ESCHERICHIA
COLI
M. Kere¤nyi, I. Ba¤tai, G. Mestya¤n, L. Emòdy and T. Pa¤l
Department of Medical Microbiology, Pe¤cs University,
Pe¤cs, Szigeti u. 12. H-7643, Hungary
Extraintestinal Escherichia coli most frequently causes uri-
nary tract infection. It can also be isolated from blood
culture and abdominal wound. The alpha-hemolysin,
which is one of the most important virulence factors, is
present in about 60 % of isolated strains from urinary
tract infection. The exact role of silent hemolysin in viru-
lence has not been determinated. In this study hemolytic
phenotype and occurrence of hlyA and sheA genes were
determined and compared in 520 clinical isolates of E. coli.
The hlyA gene carrier strains do not cause hemolysis, if
their secretory mechanism is defected. 13 strains of clinical
 1st FEMS Congress / Posters 103^505
isolates carried structure hemolysin gene, but hemolysis
zone were not developed around their colonies in de¢-
ciency of secretion. In spite of lack of hemolysin structure
gene seven strains were hemolytic on blood agar. These
strains were pozitive for sheA gene. This suggests that
increased expression of silent hemolysin or its releasing
is responsible for hemolysis. These hemolysis zones
around colonies are similar to the ones caused by alpha-
hemolysin. The alpha-hemolysin is coded on chromosome
(most often on PAI) of E. coli in human. SheA gene was
also determined on chromosome in lab strains, in animal,
and human isolates. GC % content in hlyA and sheA genes
is similar, but it is di¡erent from E. coli genom. A further
question if there is incompatibility between these genes on
the chromosome and what is responsible for it. We con-
clude that hemolysis around colonies of E. coli was caused
by other than alpha-hemolysin.
P4^46
PHYLOGENETIC AFFILIATION OF BACTERIA AT-
TACHED TO THE HINDGUT CUTICLE OF TERRES-
TRIAL ISOPOD PORCELLIO SCABER
R. Kostanjs›ek(1), J. SNtrus(1), G. Avgus›tin(2)
(1) University of Ljubljana, Biotechnical Faculty, Biologi-
cal department, Vec›na pot 111, 1000 Ljubljana, Slovenia;
(2) University of Ljubljana, Biotechnical Faculty, Zooteh-
nical department, Groblje 3, 1234 Domz›ale, Slovenia
Porcellio scaber is a terrestrial isopod crustacean involved
in decomposition of decayed plant material. Observations
of P. scaber tube-like hindgut revealed the presence of
¢lamentous bacteria attached to spine-shaped protuberan-
ces formed by cuticular lining of the inner gut surface. In
order to determine the phylogenetic position of the at-
tached bacteria the 16S rRNA gene sequences were re-
trieved directly and analysed. The phylogenetic analysis
of attached bacteria revealed that they form a single clus-
ter within Mycoplasmales group, clearly separated from
other mycoplasmas. The nearest sequences clustered in
so called group ‘A’ consisting of bacterial 16S rRNA se-
quences retrieved directly from bovine, pig and human
intestine. The phylogenetic analysis further showed that
the 16S rRNA sequences of attached bacteria from P.
scaber gut and of group ‘A’ are monophyletic and deeply
branched groups positioned between Spiroplasma and
Acholeplasma-Anaeroplasma group. A⁄liation of attached
bacteria from P. scaber to the mollicutes was further con-
¢rmed with electron microscopy observations, which re-
vealed the absence of cell wall. The microscopy also re-
vealed the presence of spherical shaped attachment
structure at the end of bacterial cells. In situ hybridisation
with speci¢c oligonucleotide probe con¢rmed the presence
of the targeted sequences to attached bacteria in P. scaber
FEMSLE Congress 2-6-03
197
hindgut. Given the speci¢c host, unique attachment struc-
ture and phylogenetic distance we propose that the ¢la-
mentous bacteria attached to the cuticular spines of P.
scaber hindgut represent a novel bacterial taxon within
mollicutes.
P4^47
COMPARISON BETWEEN RAPD-PCR AND RAP-
PCR IN OENOCOCCUS OENI STRAINS
T. Lechiancole, D. Messina and G. Salzano
Dept. of Biology, Univerity of Basilicata, Campus Macchia
Romana, 85100 Potenza, Italy
Oenococcus oeni, formely Leuconostoc oenos is the species
of lactic acid bacteria most frequently associated with the
malolactic fermentation in wine. O. oeni strains are mem-
bers of a genomically homogeneous species, in spite of
remarkable divergences in their genomic organization. as
supported by chromosomal DNA homology studies, 16S
and 23S rRNA sequencing, and more recently, sequence
analysis of the 16S-23S rDNA intergenic spacer region.
Results of several studies on genotypic diversity among
strains of O. oeni, carried out using di¡erent molecular
techniques (DNA ¢ngerprinting, PFGE, RAPD-PCR)
suggest that this species is also genomically homogeneous.
PCR-based methods, such as RNA arbitrarily primed
PCR (RAP-PCR), have recently been developed to iden-
tify di¡erentially expressed genes in eukaryotic organisms
However, to our knowledge, there is only one report on
successful identi¢cation of di¡erentially expressed genes in
bacteria using RAP-PCR. In this work two molecular
tools were applied to discriminate among O. oeni strains
isolated from Aglianico wines coming from Basilicata :
RAPD-PCR (Random Ampli¢ed Polymorphic DNA-
Polymerase Chain Reaction) and RAP-PCR (RNA Arbi-
trarily Primed-Polymerase Chain Reaction). Our results
demonstrate that RAP-PCR gives the best results. In
this work we demonstrated that the analysis on expressed
genes is useful tool to evaluated biodiversity among iso-
lates belonging to the same species, in particular O. oeni
strains isolated from wines coming from the same region.
198 1st FEMS Congress / Posters 103^505
P4^48
NOVEL SOURCES OF ACTINOMYCETE DIVER-
SITY FOR DETECTION OF ANTIMICROBIAL
AGENTS WITH PHARMACEUTICAL APPLICA-
TIONS
E. M. H. Wellington(1), F. Marinelli(2), H.-P. Fiedler(3),
L. Dijkhuizen(4), J. Vater(5), M. Goodfellow(6), S. A.
Fotinos(7), and A. D. Karagouni(8)
(1) Department of Biological Sciences, University of War-
wick ; (2) Biosearch Italia SpA; (3) Mikrobiologisches In-
stitut, Universita«t Tu«bingen; (4) Microbiology, University
of Groningen; (5) Max-Volmer-Institut fu«r Biophysikali-
sche Chemie und Biochemie, Technische Universita«t Berlin;
(6) Department of Agricultural and Environmental Science,
University of Newcastle upon Tyne; (7) Lavipharm S A;
(8)Department of Biology, University of Athens
Actinobacteria represent a signi¢cant proportion of the
terrestrial and aquatic bacteria in oligotrophic environ-
ments. The biosynthetic potential of actinobacteria is far
from being fully realized due to problems of selective iso-
lation and cultivation. The key aspect of this project (ac-
tapharm qlrt-2000-01783 2001-2004) is the combination
of expertise for detection and recovery of highly diverse
actinobacteria populations through isolation and cultiva-
tion approaches and avoiding culture by provision of en-
vironmental gene libraries, with advanced detection tech-
nology for identi¢cation of bioactive metabolites. In order
to provide an improved strategy for the detection and
exploitation of microbial metabolic diversity, the project
was divided into work packages that include the develop-
ment of molecular tools to detect genetic diversity of acti-
nobacteria and the application of novel isolation tech-
niques for recovery of actinobacteria from natural
habitats. As a further step, the development of hosts and
vectors will allow the construction and expression of BAC
environmental gene libraries, so that genetic diversity and
biosynthetic potential of uncultured actinobacteria will be
captured. Finally, characterization of the chemical diver-
sity within metabolite pro¢les of isolates and clone libra-
ries and development of a data-base to combine chemical
and biological activity data with genetic diversity data is
held to enable discovery and exploitation of novel bioac-
tive compounds.
FEMSLE Congress 2-6-03
P4^49
MYXOBACTERIA AS POTENTIAL SOURCE OF
NEW ANTI-INFECTIVES
F. Gaspari(1), Y. Paitan(2), D. Losi(1), E. Z. Ron(2)
and F. Marinelli(1)
(1) Biosearch Italia S.p.A., Via R. Lepetit 34, Gerenzano
(VA), Italy ; (2) Faculty Of Life Sciences, Tel-Aviv Uni-
versity, Ramat Aviv, Tel-Aviv, Israel
Pharmaceutical industry is constantly searching for new
biologically active molecules. One strategy is to seek
them among microbial metabolites, and to set out the
e¡orts for new potential producer microorganisms. Myx-
obacteria are known to be proli¢c producers of a variety
of bioactive secondary metabolites including antibacterial
and antifungal compounds. About 80 basic structures and
450 structural variants have been described up to now,
most of them being exclusively produced by this microbial
group. Even though these microorganisms are attractive
sources of new compounds, they have not been extensively
exploited in pharmaceutical screening because their isola-
tion is time consuming and they are quite di⁄cult to han-
dle and cultivate. During the collaboration between Bio-
search Italia and Tel Aviv University, 100 myxobacteria
belonging to 5 of the 12 described genera, were isolated
from 36 soil and 14 tree bark samples collected in di¡erent
areas inside the State of Israel. Four isolation methods,
previously reported in the literature and based on the pe-
culiar metabolic and cell cycle aspects of myxobacteria,
were combined with puri¢cation procedures and optimisa-
tion of cultivation conditions and medium composition. 97
out of 100 were fermented and screened against a panel of
clinically relevant pathogens including fungi and bacteria.
High frequency of antibacterial and antifungal activities
produced by these isolates con¢rms the attractiveness of
mixobacteria for screening projects. Typical mixobacterial
antibiotics such as Mixovirescin have been identi¢ed as
well as other anti-infective molecules usually produced
by actinomycetes as in the case of Althiomycin.
 1st FEMS Congress / Posters 103^505
P4^50
MOLECULAR TYPING ANALYSIS OF LACTIC ACID
BACTERIA FROM BEER BY USING PCR-BASED
METHODS AND PULSED-FIELD GEL ELECTRO-
PHORESIS
M. Marino, M. Maifreni and G. Rondinini
Dipartimento di Scienze degli Alimenti, Universita' degli
Studi di Udine, via Marangoni 97, 33100 Udine, Italy
Beer production carries a risk of microbial contamination
at all stages of the process. Only a few species of the lactic
acid bacteria, i.e., Lactobacillus and Pediococcus, are su⁄-
ciently resistant to the antibacterial properties of beer to
grow at any stage of the process, causing turbidity and on
o¡ £avour, often attributable to diacetyl. Superattenua-
tion by starch-fermenting lactobacilli and slime formation
by pediococci are other possible faults. The growth of
bacteria is possible in beers because of a normal pH range
of 4-5 and a good content of utilisable nutrients. Identi-
¢cation of brewery bacterial isolates has traditionally been
accomplished biochemically by determining the assimila-
tion and fermentation patterns of a number of carbohy-
drates and nitrogen sources. However, biochemical identi-
¢cation is not accurate for determining the genotypic
di¡erences of microorganisms. Alternatively, there are
many recently developed approaches for the molecular
¢ngerprinting of bacteria. Ribotyping is the only molecu-
lar technique which has been successfully used to charac-
terize di¡erent brewery contaminants. Pulsed-¢eld gel elec-
trophoresis also allows an overall clear identi¢cation
potential at the strain level. Alternative simple and faster
genotypical approaches involve the use of PCR for molec-
ular ¢ngerprinting. In this work a preliminary character-
ization of lactic acid bacteria isolated from artisanal beers
was performed using RAPD analysis, REP-PCR tech-
niques and pulsed-¢eld gel electrophoresis.
P4^51
GENETIC CHARACTERIZATION OF THE NITRATE-
REDUCING COMMUNITY IN FRENCH SOILS
L. Philippot, E. Mounier, D. Che¤neby, S. Piutti, S. Hallet,
F. Martin-Laurent, J. C. Germon
UMRA 111, INRA ^ CMSE, Microbiologie des Sols- Ge¤o-
sols, 17 rue Sully, B.V. 86510, 21065 Dijon Cedex, France
Microbial respiratory nitrate reduction is the ¢rst step of
the denitri¢cation and dissimilatory reduction of nitrate to
ammonium pathway, which are considered as important
processes since they contribute to the global cycling of
nitrogen. This ability of facultative anaerobes to respire
FEMSLE Congress 2-6-03
199
nitrate has been ascribed mainly to the activity of a mem-
brane bound nitrate reductase encoded by the narGHJI
operon. In this study, we employed direct PCR and clon-
ing of narG gene fragments to determine the phylogenetic
a⁄liation of nitrate-reducing bacteria occurring in di¡er-
ent soils. The clones from each soil speci¢c library were
grouped by restriction fragment length polymorphism, and
representatives from each group were sequenced and ana-
lyzed. Phylogenetic analysis of narG sequences separated
the clone families into two main groups that represent the
Gram-positive and Gram-negative nitrate-reducing bacte-
ria. Novel narG lineages that branch distinctly from all
currently known membrane bound nitrate-reductase en-
coding genes were detected within the Gram-negative
branch for most of the studied soils. In addition each
soil exhibited some speci¢c narG gene clusters. Altogether,
our results revealed a more complex nitrate-reducing com-
munity than did previous culture-based studies.
P4^52
DIVERSITY OF STREPTOMYCETES ISOLATED
FROM INDOOR DUST
H. Rintala, A. Hyva«rinen, A. Nevalainen
National Public Health Institute, Department of Environ-
mental Health, P.O. Box 95, 70701 Kuopio, Finland
Streptomycetes are Gram-positive, ¢lamentous bacteria of
the order Actinomycetales. They are common in soil, but
also present in aquatic habitats, composts and fodder.
During their vegetative growth, they form extensive
branching mycelium, but as soon as the environmental
conditions worsen, spores are formed. Streptomycetes
are able to produce thousands of di¡erent secondary me-
tabolites having a broad spectrum of biological activities.
In buildings they can colonise moist building materials,
grow and proliferate there, and thus can produce volatile
secondary metabolites, such as geosmin, and spores into
the indoor air. Spores of streptomycetes isolated from
moisture-damaged buildings have been shown to induce
in£ammatory responses in cell cultures and laboratory an-
imals. In mice, also systemic e¡ects similar to those in-
duced by anthracyclines have been observed. Indoor dust
consists of biological and non-biological material originat-
ing from both indoor and outdoor sources. However, it
re£ects the microbial condition of a building. We isolated
14 Streptomyces -strains from dust collected from regular
vacuum cleaner dust bags of the occupants of 12 moisture-
damaged residences that were located in eastern Finland.
The 16S rRNA gene was partially sequenced and a phy-
logenetic tree was constructed. The strains clustered in
four groups, the largest group consisting of eight strains
a⁄liated with Streptomyces anulatus and Streptomyces hal-
stedii, which are members of the largest species cluster of
200 1st FEMS Congress / Posters 103^505
streptomycetes, the Streptomyces albido£avus cluster. That
cluster also contains many producers of secondary metab-
olites. We also screened the strains for genes coding for
the biosynthesis of secondary metabolites.
P4^53
DIVERSITY OF THE NITRATE REDUCTASE GENES
IN THE PSEUDOMONAS GENUS
L. Roussel-Delif(1), S. Tarnawski(1), J. Hamelin(1), L.
Philippot(2), M. Aragno(1) and N. Fromin(1)
(1) Laboratoire de Microbiologie, Universite¤ de Neucha“tel,
CP 2, CH-2007 Neucha“tel, Switzerland; (2) INRA CMSE,
Laboratoire de Microbiologie des sols, 17 rue Sully, F-
21034 Dijon cedex, France
Denitri¢cation is the reductive respiration of nitrate to
gaseous N compounds. This function is present in di¡erent
phyla including Q-Proteobacteria. The nitrate reductase en-
zymes catalyse the ¢rst step of denitri¢cation pathway.
Two isoenzymes were described for nitrate reductase: the
periplasmic form encoded by nap genes and the mem-
brane-bound form encoded by nar genes. We investigated
nitrate reduction and denitri¢cation in Pseudomonas pop-
ulations in the rhizosphere of two perennial grasses di¡er-
ing in trophic requirements: Lolium perenne and Molinia
coerulea under ambient or elevated atmospheric CO2 con-
tent, at ¢ve sampling dates. 1246 Pseudomonas strains
were checked for nitrate reduction and denitri¢cation ac-
tivities. 40% and 20% of Pseudomonas strains isolated
from L. perenne and M. coerulea respectively, were able
to reduce nitrate only to nitrite and 13% were complete
denitri¢ers for both plants. The proportions of both ni-
trate reducing and denitrifying Pseudomonas were gener-
ally higher in soil fraction and under elevated CO2. At the
gene level, the occurrence and diversity of napA and narG
genes among nitrate reducing Pseudomonas were assessed
by using PCR-RFLP analysis. We detected a large diver-
sity of narG and napA genes. The proportion of the mem-
brane-bound form was higher in root fractions and under
elevated CO2. These results are discussed in relationship
with the structuration of Pseudomonas populations in rhi-
zosphere environments.
FEMSLE Congress 2-6-03
P4^54
PCR DETECTION OF MEMBERS OF GEODERMA-
TOPHILUS AND NOCARDIACEAE IN DNA OB-
TAINED FROM STONE SURFACES
O. Salazar, A. Valverde, A. Villalba and O. Genilloud
Centro de Investigacio¤n Ba¤sica de Espa•a (CIBE), Merck
Research Laboratories, Merck Sharp & Dohme de Espa•a,
S. A. Josefa Valcarcel 38, 28027 Madrid, Spain
Actinomycetes of the genus Geodermatophilus and the
family Nocardiaceae have been recovered from stone sur-
faces of rocks and altered surfaces of monuments [1-3]
Nevertheless, they only represent a minor group of the
actinomycetes that can be obtained from diverse rock en-
vironments using standard isolation methods [4]. Given
that their isolation frecuency can be biased with these
procedures, we have focused on the development of mo-
lecular tools that could provide us with a deeper insight on
their natural occurrence in the di¡erent habitat studied.
Frequently, the wide range of morphologies and pheno-
types of actinomycetes, and the frequent absence of spor-
ulation in laboratory conditions di⁄cult the current iden-
ti¢cation of many wild type isolates and make necessary
the use of alternative molecular tools for identi¢cation.
We present here the design of two pairs of speci¢c oligo-
nucleotides for the rapid detection of members of Geoder-
matophilus and Nocardiaceae by speci¢c ampli¢cation of
16S rDNA region. The speci¢city of these primers was
validated with type strains and these primers were used
to detect the presence of representatives of these taxa di-
rectly from rock surfaces DNA collected in di¡erent Span-
ish locations. This study presents the usefulness of the
application of these identi¢cation tools to assess the oc-
currence of members of these taxa directly from this alter-
native isolation source as well as from other environments
considered of high interest for microbial isolation pro-
grams.
[1] Eppard et al. (1996) Arch. Microbiol 166, 12-22. [2]
Urzi et al. (2001) Environmental Microbiology 3, 471-
479. [3] Groth et al. (1999) J. Microbiol. Met. 36, 115-
122. [4] Gonza¤lez et al (2003). Accompaning poster.
 1st FEMS Congress / Posters 103^505
P4^55
DIVERSITY OF AUTOTROPHIC BACTERIA HAR-
BOURING cbbL GENES IN DIFFERENT AGRICUL-
TURAL SOILS
D. Selesi, I. Pattis, M. Schmid and A. Hartmann
GSF National Research Centre for Environment and
Health, Institute of Soil Ecology, Ingolsta«dter Landstrasse
1, 85764 Mu«nchen-Neuherberg, Germany
Autotrophic bacteria harbouring the Calvin cycle enzyme
ribulose-1,5-bisphosphate carboylase/oxygenase (Rubis-
CO) may play a signi¢cant part in the conversion of car-
bon dioxide into organic matter and microbial biomass
and may thus contribute to the global carbon cycling.
To gain an insight into the genetic diversity of CO2-¢xing
bacteria in soil habitats we developed PCR-based assays
targeting the large subunit gene cbbL of the form I Ru-
bisCO. Based on the calculation of phylogenetic relation-
ships we designed di¡erent primer sets having strong spec-
i¢city for ‘red-like’ and ‘green-like’ cbbL sequences of
selected terrestrial autotrophs. Bulk genomic DNA was
isolated from agricultural soils with rye crop at di¡erent
long-term fertilization as well as from a soil under clover/
gras cover. The RFLP and phylogenetic analysis of the
ampli¢ed cbbL sequences indicated a high diversity of
‘red-like’ and a low diversity of ‘green-like’ cbbL sequences
in these soils. Additionally variations in community com-
position on the basis of cbbL genes could be observed in
the di¡erently managed soils.
P4^56
AN UPDATE OF VIBRIONACEAE TAXONOMY
F. L. Thompson and J. Swings
Laboratory for Microbiology and BCCMTM/LMG Culture
Collection, Ghent University, K.L. Ledeganckstraat 35,
Ghent 9000, Belgium
The family Vibrionaceae (Gammaproteobacteria) is prob-
ably one of the best-documented bacterial groups from
the aquatic environment. Six main groups of vibrios are
delineated on the basis of their 16S rDNA sequences i.e.
the Vibrio core group, the psychrophilic Vibrio species, V.
hollisae, Enterovibrio and Salinivibrio. Vibrios are ubiqui-
tous and abundant in the aquatic environment. These mi-
crobes were initially supposed to represent the dominant
marine micro£ora as deduced from plate counts. Recent
studies based on cultivation independent approaches have
shown vibrios compose about 4 % (V108 cells liter-1) of
the Bacteria in aquatic environments. High abundance of
vibrios is also detected in tissues and/or organs of certain
FEMSLE Congress 2-6-03
201
marine animals e.g. ¢sh, squids, abalones, bivalves,
shrimps, copepods and corals. The Vibrionaceae harbours
a wealth of diverse genomes as revealed by AFLP genomic
analysis of 506 strains. It was found that 236 isolates dis-
tributed in 31 clusters have genomes unrelated to any type
strain and are thus potential new taxa. Among these un-
known isolates, a novel genus i.e. Enterovibrio norvegicus
and ¢fteen novel species i.e. V. barceloniensis, V. brasilien-
sis, V. chagasii, V. coralliilyticus, V. fortis, V. gallicus, V.
kanaloae, V. neptunius, V. pomeroyi, V. pacinii, V. probio-
ticus, V. rotiferianus, V. superstes, V. tasmaniensis and V.
xuii have been described. Several of these new species have
ubiquitous occurrence, but their exact ecological role is
unknown at present.
Acknowledgement: F.L.T. has a Ph.D. Scholarship from
CNPq, Brazil.
P4^57
CULTURE COLLECTION IBSO AND ELECTRONIC
COLLECTION ‘‘BIOLUMBASE’’: STUDY AND
MAINTAINANCE OF LUMINOUS BACTERIA DI-
VERSITY
G. A. Vydryakova, S. E. Medvedeva, A. M. Kuznetsov, D.
A. Kotov, J. V. Chugaeva, and E. K. Rodicheva
Institute of Biophysics SB RAS, 660036, Krasnoyarsk, Rus-
sian Federation
There are di¡erent bacterial species able to emit light. The
Culture Collection of the Institute of Biophysics of the
Siberian Branch of the Russian Academy of Sciences
(CC IBSO) deposits about 700 strains of marine luminous
bacteria and genetically modi¢ed E. coli strains with
marker lux-gene. Luminous bacteria are producers of
many enzymes: L-ornithine-, L-arginine-, L-lysine-decar-
boxylases, neuraminidase, chitinase, cellulase, alginate
lyase, endonucleases of restriction. The most interesting
are luciferase, oxidoreductase and dehydrogenases. While
relatively little is known about the biological signi¢cance
of bacterial light emission, much has been published about
the biochemistry and regulation of bioluminescence in lu-
minous bacteria. But real structure of emitter is unknown.
Luminescence spectra of luminous bacteria (Vibrio ¢scheri,
Vibrio harveyi, Photobacterium phosphoreum, Photobacte-
rium leiognathi) and recombinant E. coli (with lux-genes
from P. leiognathi) have been studied in our research. It
was shown that V. harveyi, P. phosphoreum, P. leiognathi
wavelength spectrum peaked at about 475 nm, where as E.
coli and V. ¢scheri wavelength spectrum peaked at about
490 nm. So, it is interesting to note di¡erences at wave-
length spectrum peaks of E. coli (with lux-genes from P.
leiognathi) and P. leiognathi. As we can assume such dif-
ferences possible are dependent on reaction microenviron-
ment. There is the work version of electronic collection
202 1st FEMS Congress / Posters 103^505
HBiolumBaseg of natural and transgenic luminous mi-
croorganisms that are maintained in microbial collections
of IBP SB RAS (http://lux.ibp.ru). It contains information
about their main morphological, physiological-biochemi-
cal and genetic features and bioluminescent systems of
di¡erent bacteria.
This work was supported by RFBR (grant 00-07-9011).
P4^58
RFLP rDNA CHARACTERISATION OF THE GENUS
CLADOSPORIUM ISOLATED FROM BATHROOMS
M. Butala, P. Zalar and N. Gunde-Cimerman
University of Ljubljana, Biotechnical Faculty, Biology De-
partment, Vec›na pot 111, 1000 Ljubljana, Slovenia
Saprotrophic fungi from the genus Cladosporium are usu-
ally the dominant microoorganisms in outdoor as well as
in indoor air. They are not known as agents of the ‘‘sick
building syndrom’’, which was assigned mainly to the
presence of airborne Penicillium and Stachybotrys spp,
but are sometimes described as agents of pulmonary and
cutaneous infections. Inside of every building, not neces-
sary sick building, there are places where occasionally
warm and moist atmosphere develops. Bathrooms and
kitchens represent such special habitats, which are quickly
occupied by moulds. In our study we sampled 26 bath-
rooms all over Slovenia with the aim to study the presence
and a species composition of the genus Cladosporium. Dif-
ferent species from this genus were present in 80 % of the
sampled bathrooms. Isolates were compared morphologi-
cally and with the molecular characterisation ^ RFLP of
rDNA (partially SSU, whole ITS and partially LSU).
Reference strains for common species, type and neotype
material where described, and strains isolated from plant
material were included in the study. According to the spe-
ci¢c restriction pattern, obtained by selection of speci¢c
restriction enzymes, fungi were identi¢ed to the species
level. By this method, a quick identi¢cation tool for the
members of the most common saprotrophic species from
the genus Cladosporium was established. The prevailing
species was Cladosporium sphaerospermum, which was
present in 70 % of sampled bathrooms, followed by C.
cladosporioides and C. herbarum, present in 22 and 8 %
of sampled bathrooms, respectively.
FEMSLE Congress 2-6-03
P4^59
MOLECULAR IDENTIFICATION OF HANSENIAS-
PORA AND KLOECKERA SPECIES
N adez›(1), M. Th. Smith(2), P. Raspor(1)
N. C
(1) University of Ljubljana, Biotechnical Faculty, Depart-
ment of Food Science and Technology, Jamnikarjeva 101,
1000 Ljubljana, Slovenia; (2) Centraalbureau voor Schim-
melcultures, Yeast Division, P.O. Box 85167, 3508 AD
Utrecht, The Netherlands
The yeast genus Hanseniaspora and its anamorph Kloeck-
era are morphologically characterized as apiculate yeasts
with bipolar budding. The Hanseniaspora / Kloeckera
yeasts are frequently isolated from various natural sources
such as soil, fruits and insects as well as from fermented
foods and beverages. As predominant inhabitants on the
surface of grape berries and in starting wine fermentations,
these genera have been intensely studied to determine their
role on the quality of the ¢nal fermentation product. For
monitoring the presence apiculate yeasts in wine fermen-
tations the rapid and accurate identi¢cation method is
required. The identi¢cation of Hanseniaspora and Kloeck-
era species using traditional, physiological testing is ham-
pered by the low number of positive growth characters
and moreover, inconsistencies in identi¢cation results.
The recent development of molecular techniques for yeast
identi¢cation has substantially reduced time needed for
identi¢cation and has improved the reliability through
the direct analysis of DNA, thus identi¢cation does not
vary with the physiological state of organism. In our study
three molecular methods, PCR ¢ngerprinting, electropho-
retic karyotyping and RFLP of the PCR-ampli¢ed ITS
regions (ITS1, ITS2 and the intervening 5,8S rDNA),
were studied for accurate identi¢cation of 92 strains of
Hanseniaspora and Kloeckera isolated from di¡erent sour-
ces as well as from geographically distinct regions. Of
these three methods PCR-RFLP analysis of ITS regions
with three restriction enzymes is proposed as rapid and
accurate identi¢cation technique in a form of a two-step
molecular identi¢cation key to Hanseniaspora and Kloeck-
era species.
This study was partly supported by a FEMS fellowship.
 1st FEMS Congress / Posters 103^505
P4^60
INJURIES OF GRAPE BERRIES HAS IMPACT ON
BIODIVERSITY OF YEAST POPULATION ISO-
LATED FROM GRAPE BERRY SURFACES
N adez›, S. Smole Moz›ina, P. Raspor
D. Miklic› Milek, N. C
University of Ljubljana ; Biotechnical faculty, Chair of Bio-
technology, Jamnikarjeva 101, 1000 Ljubljana, Slovenija
Yeasts are widespread in nature and are found in soils, on
surfaces of vegetables and in the digestive tract of animals.
Yeasts of di¡erent genera, species and strains found on the
surface of grapes and associated with surfaces of winery
equipment are the main microorganisms responsible for
wine fermentations. The aim of this study was to deter-
mine the in£uence of geographic locations and health con-
dition on the indigenous yeast £ora of grape berries. Sam-
ples of grape berries were collected from grape cultivar
Modra frankinja, which is one of the main vine cultivars
for production of red vine Cvicek in Slovenia. The healthy
and slightly injured grapes were collected during the rip-
ening in September 1999 on ¢ve di¡erent locations of Do-
lenjska vine growing region. The grape berries were ho-
mogenized and plated at serial dilutions on media. The
microbial population that consisted mostly of yeasts and
¢lamentous fungi was enumerated. In order to identify the
yeast colonies on surfaces of di¡erent grapes a combina-
tion of restriction analysis of PCR ampli¢ed rDNA-ITS
region and morphological and physiological tests were
used. Yeast population on healthy grapes was numerically
lower than the population of yeasts isolated from dam-
aged grape berries. Further, sampling location had great
impact on yeast number and on diversity of yeast species.
In most samples the following yeast species predominated:
Hanseniaspora uvarum, Rhodotorula glutinis, Aureobasidi-
um pullulans, Metschnikowia pulcherrima and Cryptococcus
spp.. Saccharomyces cerevisiae was not found.
This work was ¢nanced by the Slovene Ministry of Edu-
cation, Science and Sport (project No. L4-8849-98).
P4^61
TYPING OF DEBARYOMYCES HANSENII AND
KLUYVEROMYCES LACTIS STRAINS FROM FIORE
SARDO CHEESE
M. E. Fadda, V. Mossa, M. B. Pisano and S. Cosentino
Department of Experimental Biology, section of Hygiene,
University of Cagliari, S.S 554, Km 4,500, 09042 Monser-
rato (CA), Italy
Over the last decade various methods based on molecular
biological techniques to characterize yeasts isolated from
FEMSLE Congress 2-6-03
203
food products have been developed. An extensive study
carried out on the natural £ora of Fiore Sardo showed
the di¡use presence, throughout ripening, of several yeast
species. Since Debaryomyces hansenii and Kluyveromyces
lactis were found to be predominant, in this study we
investigated the genetic heterogeneity among the isolates
of these species. 20 D. hansenii and 15 K. lactis strains
isolated from Fiore Sardo cheese were typed by RAPD-
PCR with the M13 primer and by the restriction pattern
of 18S rDNA digested with two restriction enzymes
(HaeIII and HpaII). DNA banding patterns were analysed
using the Gel Compar software package, version 2.5 (Ap-
plied Maths, Kortrijk, Belgium). Similarities among iso-
lates were estimated using the Pearson coe⁄cient and clus-
tering was based on the UPGMA method. The species D.
hansenii and K. lactis originated distinct RAPD patterns
and therefore were clearly separated in the dendogram. At
a 70% level of similarity 2 clusters were identi¢ed for both
D. hansenii and K. lactis. Restriction enzyme analysis with
HaeIII and HpaII showed the presence of one pro¢le with-
in K. lactis and D. hansenii strains. Both techniques were
found useful for the characterization of yeasts. PCR am-
pli¢cation with M13 gave good results for the identi¢ca-
tion at species level and also presented a good discrimina-
tion power at subspecies level. In comparison, rDNA
restriction analysis was useful for discrimination at species
level but it was not satisfactory for identi¢cation at sub-
species level.
This work was supported by a grant from MURST, Plan
‘‘Agroalimentary products : dairy products’’, Cluster 08B,
Project n.7.
P4^62
EXTRACHROMOSOMAL DNAs IN YEASTS ISO-
LATED FROM SPRING SAPWOOD FLUXES
A. Fejfa¤r(1), J. Kucsera(1), W. I. Golubev(2)
(1) Department of Microbiology, University of Szeged,
P.O.Box 533, Szeged, Hungary, H-6701; (2) Institute of
Biochemistry and Physiology of Microorganisms, Russian
Academy of Sciences, Pushchino, Russia
Exudate of deciduous trees in the spring provides a carbo-
hydrate-rich media for yeast species. The three dominant
species of this habitat are Nadsonia fulvescens var. elonga-
ta, Trichosporon pullulans and Xanthophyllomyces dendro-
rhous. Their extrachromosomal DNA elements were inves-
tigated during this study. The existence of DNA plasmids
was revealed in all of the examined species. Two strains of
N. fulvescens var. elongata contained three or four types of
DNA plasmids (strain VKM Y-268: 4.70, 1.09 and 0.97
kb; strain VKM Y-1653 : 5.20, 4.34, 0.94 and 0.83 kb,
respectively), Trichosporon pullulans VKM Y-273 har-
boured one species of it (4.1 kb), while a number of
204 1st FEMS Congress / Posters 103^505
were identi¢ed in ¢ve strains of X. dendrorhous with sizes
6.5-2.0 kb. All the plasmids were sensitive to exonuclease
treatment what indicates their linear structure. The plas-
mids of N. fulvescens var. elongata and X. dendrorhous
strains had buoyant density similar to that of the nuclear
DNA. Contrary plasmid from T. pullulans copuri¢ed with
the mitochondrial DNA on CsCl gradient, showing lower
G+C content than the nuclear DNA. Plasmids from Nad-
sonia and Trichosporon strains were absent from puri¢ed
organelles suggesting their possible cytoplasmic location;
X. dendrorhous plasmids were located in the mitochondrial
fraction. Plasmids of Nadsonia and Trichosporon strains
are cryptic while DNA plasmids of X. dendrorhous could
take part in the mitochondrial genome organisation.
This work was supported by FKFP 0091/2001.
P4^63
MOLECULAR ASSESSMENT OF YEAST DIVERSITY
AT THE SA= O DOMINGOS MINE (PORTUGAL), A
METAL POLLUTED ACIDIC ENVIRONMENT IN
THE IBERIAN PYRITE BELT
M. Gadanho and J. P. Sampaio
Centro de Recursos Microbiolo¤gios (CREM), Secca‹o
Auto¤noma de Biotecnologia, Faculdade de Cie“ncias e Tec-
nologia, Universidade Nova de Lisboa, 2829-516 Caparica,
Portugal
The Iberian Pyrite Belt (IPB) is a vast mining area in the
South of Portugal and Spain and one of the most impor-
tant pyrite regions in the world. At several sites in the IPB,
like the S. Domingos abandoned mines, Acid Rock Drain-
age is responsible for the contamination of soils and
groundwater, giving rise to acidic reddish-brown waters
with high concentrations of metals. Since yeasts are well
adapted to grow in acidic conditions, this study attempted
to address their occurrence, diversity and potential in bio-
remediation. At S. Domingos mine, four sites were inves-
tigated. The pH of the water samples ranged from 1.7 to
2.7 and the concentration of Fe2+ was 0.069-25.9 g/l.
Water samples (5 liters) were collected in di¡erent seasons
of the year and yeasts were investigated using pure cul-
ture-independent approaches. Total DNA was extracted
and PCR-TGGE (Temperature Gradient Gel Electropho-
resis) was employed. Since yeasts appeared to be present in
low densities, the same approach was used in enrichment
studies. Nutrients were added to the water samples and
yeasts were monitored between the ¢rst and 15th day of
incubation. DNA from various species was detected and
identi¢cation was achieved by sequence analysis. Pure cul-
ture studies were performed in parallel using di¡erent
growth conditions and culture media employing water
from the sites under investigation. The most frequent
taxa belonged to new species of the genera Rhodosporidi-
FEMSLE Congress 2-6-03
um and Cryptococcus. The temporal and spatial distribu-
tion of yeast populations was characterized and the results
obtained in the molecular and traditional approaches were
compared.
M.Gadanho was supported by a grant N‡ SFRH/BD/
1170/2000.
P4^64
INTERPRETATION OF mtDNA POLYMORPHISMS
OF ASPERGILLUS TUBINGENSIS STRAINS
Ł . Juha¤sz, H. Engi, Zs. Hamari, F. Kevei
A
Department of Microbiology, Faculty of Sciences, Univer-
sity of Szeged, P.O. Box 533, H-6701 Szeged, Hungary
In previous studies considerable inter- and intraspeci¢c
mtDNA polymorphisms were detected among black As-
pergilli. Aspergillus tubingensis strains proved to be highly
variable they could be divided into six groups (labeled 2a-
2f) based on their mtDNA pro¢les. To study the reason of
mtDNA variability physical and partial functional maps
were developed. Physical map were constructed by four
restriction enzyme (EcoRI, EcoRV, BglII, HindIII) apply-
ing reciprocal digestion technique (each fragment was iso-
lated and re-digested by another enzyme). Functional
maps were developed by sequence analysis of certain re-
striction fragment clones and size determination of PCR
products. The precise size of mitochondrial genes was es-
tablished by the size of PCR products ampli¢ed using
forward primers designed to match the start point of A.
nidulans and/or A. niger mitochondrial genes and reverse
primers designed to match the end of these genes. Gene
order and size of the intergenic regions between two neigh-
boring genes were also determined by PCR using various
combinations involving forward primers designed to
match the end of the mitochondrial genes and reverse
primers designed to match the start of those same genes.
The basic organisation of mtDNAs was highly similar but
intergenic sequences and optional intron content showed
some alterations. The results will be discussed in detail.
This work was supported by grants (T 037584 F032704
and) of Hungarian Scienti¢c Research Foundation
(OTKA).
 1st FEMS Congress / Posters 103^505
P4^65
STUDIES ON THE HETEROGENEITY OF THE CAR-
OTENOGENIC YEAST RHODOTORULA MUCILAGI-
NOSA FROM PATAGONIA, ARGENTINA
D. Libkind(1), M. Gadanho(2), M. van Broock(1) and J.
P. Sampaio(2)
(1) Laboratorio de Microbiolog|¤a Aplicada y Biotecnolog|¤a,
Universidad Nacional del Comahue, Centro Regional Uni-
versitario Bariloche (CRUB) ^ CONICET (Consejo Na-
cional de Investigaciones Cient|¤¢cas y Tecnolo¤gicas), Bar-
iloche, R|¤o Negro, Argentina; (2) Centro de Recursos
Microbiolo¤gicos, Secca‹o Auto¤noma de Biotecnologia, Fac-
uldade de Cie“ncias e Tecnologia, Universidade Nova de Lis-
boa, 2829-516 Caparica, Portugal
The basidiomycetous red yeast Rhodotorula mucilaginosa
is a ubiquitous species and can be found both in natural
and in human-related environments. Due mostly to similar
physiological pro¢les, several species are presently re-
garded as synonyms of Rh. mucilaginosa, although very
few have been investigated using molecular methods. In
this study, forty-¢ve Rh. mucilaginosa isolates obtained
from diverse natural and arti¢cial environments (lakes,
soil, nectar, wild fruits, grape surfaces) from Patagonia,
Argentina, were studied. The methods employed encom-
passed morphological and physiological characterisations,
DNA ¢ngerprinting using MSP-PCR (mini/micro-satellite
primed-PCR), single nucleotide sequencing (SNS) and
DNA sequence analysis of the D1/D2 and ITS regions.
Preliminary MSP-PCR experiments using primer (GTG)5
revealed considerable heterogeneity in Rh. mucilaginosa.
Subsequent studies with primer M13 allowed the forma-
tion of three groups, one of them encompassing the type
strain and 84% of the studied strains. Physiological and
morphological results correlated with the presence of the
three groups. No sexual compatibility reactions were ob-
served between or within these groups. Selected isolates
were investigated by sequence analysis. In contrast with
the D1/D2 region, the less conserved ITS region revealed
di¡erences between the tested strains of the di¡erent
groups. However, no correlations were found between
the three groups and the geographic origin of the isolates,
or their isolation source. The observed heterogeneity is
presently interpreted as intraspeci¢c variability. Future
work will expand the set of studied isolates, will address
the molecular relationships of the current synonyms of Rh.
mucilaginosa and will conceivably allow a better under-
standing of the heterogeneity presently reported.
FEMSLE Congress 2-6-03
205
P4^66
COMPLEX COMPOSITION OF THE YEAST ZYGO-
FABOSPORA / KLUYVEROMYCES LACTIS: GENET-
IC AND MOLECULAR DIFFERENTIATION OF SUB-
POPULATIONS
G. I. Naumov, N. N. Sukhotina and E. S. Naumova
State Institute for Genetics and Selection of Industrial Mi-
croorganisms, I-Dorozhnyi, 1, Moscow 117545, Russia
Our new experimental and literature data (Naumov, 1986;
Sidenberg and Lachance, 1986; Fuson et al., 1987 ; Nau-
mov and Naumova, 2002) are discussed in the light of
heterogeneity of the Z. lactis species. It includes both
wild lactose-negative geographic populations, having par-
tial genetic isolations, and domesticated lactose-positive
dairy strains. Using di¡erent molecular markers we di¡er-
entiated eight wild populations of Z. lactis: ¢ve North-
American (including the known varieties Z. lactis var.
drosophilarum and Z. lactis var. phaseolospora), one Euro-
pean (Z. lactis var. krassilnikovii), one South-African (Z.
lactis var. vanudenii), and one Far East Asian. The results
of genetic hybridisation, PCR analysis and sequencing
rDNA will be presented.
P4^67
GENETIC VARIABILITY IN THE SPECIES RHIZO-
PUS STOLONIFER ASSESSED BY RAPD ANALYSIS
Ł cs(2) and Cs. Va¤gvo«l-
T. Papp(1), H. Heinrich(2), K. A
gyi(2)
(1) HAS-USZ Microbiological Research Group, University
of Szeged, Faculty of Sciences, Department of Microbiol-
ogy, P.O. Box 355, H-6701, Szeged, Hungary; (2) Univer-
sity of Szeged, Faculty of Sciences, Department of Micro-
biology, P.O. Box 355, H-6701, Szeged, Hungary
Rhizopus stolonifer is one of the most important members
of the class Zygomycetes. Recent taxonomy based on mor-
phological and growth characteristics reduces the number
of species di¡erentiating only two varieties (var. stolonifer
and re£exus) within this species. The objective of this
study was to examine this situation with molecular
markers. Twenty-nine R. stolonifer strains isolated from
various locations and substrates were characterized by
random ampli¢ed polymorphic DNA (RAPD) analysis.
The numerical analysis of the RAPD data revealed four
main clusters with extremely high dissimilarity values, at
the same time, low or moderate variabilities were observed
within these groups. R. stolonifer var. stolonifer, R. stolo-
nifer var. re£exus and R. niveus showed species level ge-
netic distances with this method. These results suggest
206 1st FEMS Congress / Posters 103^505
higher genetic heterogeneity in the case of R. stolonifer,
than as could be expected on the basis of the recent species
concept.
P4^68
DNA POLYMORPHISM FOUND IN THE INTERNAL
TRANSCRIBED SPACER (ITS) REGION OF UDE-
NIOMYCES PYRICOLA
M. Takashima(1), T. Nakase(2), J. Tornai-Lehoczki(3),
T. Deak(4) and T. Kudo(1)
(1) Japan Collection of Microorganisms, Bioscience Tech-
nology Center, RIKEN (The Institute of Physical and
Chemical Research), Wako, Saitama, Japan; (2) National
Center for Genetic Engineering and Biotechnology (BIO-
TEC), National Science and Technology Development
Agency (NSTDA), Bangkok, Thailand; (3) National Col-
lection of Agricultural and Industrial Microorganisms,
Szent Istva¤n University; (4) Department of Microbiology,
Szent Istva¤n University, Budapest, Hungary
Udeniomyces pyricola, an anamorphic basidiomycetous
yeast, was isolated from Switzerland, Canada, Japan,
New Zealand and Tasmania, Australia, but has not been
isolated from the tropical or subtropical area of southeast-
ern Asia (Nakase, T. 2000. J. Gen. Appl. Microbiol. 46:
189-216). Of a total of 100 ballistoconidium-forming
yeasts isolated from plants in Hungary, 10 strains from
10 plants were identi¢ed as U. pyricola by their morpho-
logical, physiological and biochemical characteristics. In
addition, 9 strain maintained at the Japan Collection of
Micoorganisms (JCM), from Switzerland (JCM 2958, type
strain), Canada (neotype strain of Bullera grandispora
JCM 8249 plus two isolates), Japan (3), New Zealand
(1) and Tasmania (1), were used in this study. The se-
quence of the D1/D2 region of 26S rDNA of 16 strains
including the type strain, JCM 2958, were identical, and
one base di¡erence was found in the remaining three
strains. Based on the sequences of the internal transcribed
spacer (ITS) region, they were divided into three clusters.
JCM 2958 and strains isolated from Hungary (7 out of
10), Canada, New Zealand and Tasmania composed clus-
ter 1, and three Japanese and three Hungarian isolates
each made a distinct cluster. In cluster 1, four or one
base insertions/deletions were detected at two positions,
respectively, near the 3’ end of ITS2 region. Based on
these insertions/deletions, the Hungarian strains were di-
vided into three groups, and the Canadian strains be-
longed one of these ITS2 type group.
FEMSLE Congress 2-6-03
P4^69
POLYKETIDE SYNTHASE GENE SEQUENCES IN
OCHRATOXIN-PRODUCING ASPERGILLUS SPE-
CIES
J. Varga, K. Rigo¤, S. Kocsube¤ and B. Farkas
Department of Microbiology, University of Szeged, Faculty
of Sciences, P.O.Box 533, H-6701 Szeged, Hungary
Ochratoxin A (OA) is a pentaketide dihydroisocoumarin
derivative linked to an L-phenylalanine moiety. OA is fre-
quently encountered in various foods including cereals,
co¡ee, spices and wine, and exhibits nephrotoxic, immu-
nosuppressive, teratogenic and carcinogenic properties.
Although several e¡orts have been made, none of the en-
zymes or genes responsible for OA production has been
isolated or characterized up to date. Our aim was to iden-
tify polyketide synthase (PKS) gene sequences in ochratox-
in producing Aspergillus species (A. ochraceus, A. niger, A.
muricatus, A. albertensis) using primer pairs developed for
the ketosynthase (KS) domain of fungal PKSs. Ketosyn-
thase domain probes ampli¢ed a single DNA fragment of
about 700 bp in each examined isolate. Sequences of these
domains were aligned and analyzed by phylogenetic meth-
ods. The KS domain sequences were highly diverse indi-
cating that they most probably represent PKSs responsible
for di¡erent functions. A. albertensis and A. niger KS do-
main sequences clustered together with sequences of genes
required for pigment biosynthesis (wA) in A. nidulans and
P. patulum, the KS sequence of N. muricatus was most
closely related to an A. parasiticus wA type domain se-
quence, while those of the A. ochraceus isolates were
highly similar to an A. terreus naphthopyrone synthase
gene. Since some A. fumigatus isolates are also able to
produce OA, KS domain sequences were also used to
search the TIGR A. fumigatus genomic database for
PKS related sequences. At least 12 putative PKS genes
were identi¢ed, two of which also carry a C-methylation
domain which was suggested earlier to be part of a hypo-
thetical ‘‘ochratoxin synthase’’ gene. Further studies are in
progress to see whether the identi¢ed KS sequences are
part of the PKS gene responsible for the make-up of the
isocoumarin skeleton of OA.
 1st FEMS Congress / Posters 103^505
P4^70
EVOLUTIONARY RELATIONSHIPS AMONG AS-
PERGILLUS TERREUS AND ITS RELATIVES
J. Varga(1), B. To¤th(2), K. Rigo¤(1), S. Kocsube¤(1), B.
Farkas(1) and J. Te¤ren(3)
(1) Department of Microbiology, University of Szeged,
Faculty of Sciences, P.O.Box 533, H-6701 Szeged, Hun-
gary ; (2) Cereal Research non-Pro¢t Company, P.O.
Box 391, H-6701 Szeged, Hungary ; (3) Animal Health
and Food Control Station, P.O. Box 446, H-6701 Szeged,
Hungary
Aspergillus terreus is a ubiquitous fungus in our environ-
ment. This fungus is an opportunistic human pathogen,
and economically important as the main producer of lov-
astatin, a cholesterol lowering drug. Lovastatin has re-
cently also been suggested to have potent anti-cancer ef-
fects. Our aim was to examine the genetic variability of A.
terreus and closely related species (22 isolates representing
12 species) using molecular and analytical techniques.
Lovastatin production was examined by HPLC. Lovasta-
tin was produced by some isolates belonging to the species
A. terreus, A. niveus and A. carneus. RAPD analyses were
carried out using 20 di¡erent random decamers. Neighbor-
joining analysis of RAPD data (245 characters) let us
cluster the isolates into distinct groups. Since previous
studies indicated that isolates of A. terreus and closely
related species have identical 28 S rRNA gene sequences,
we sequenced the intergenic spacer region and the 5.8 S
rRNA gene of the isolates. Phylogenetic analysis of se-
quence data let us classify the isolates into di¡erent clades.
A. terreus and its subspecies and A. allahabadii formed one
clade, another clade included A. £avipes, A. iizukae and an
A. niveus isolate, a distinct clade included A. carneus iso-
lates and A. obscurus, while the other examined species (A.
janus, A. anthodesmis, A. ambiguus) formed distinct
branches on the tree. The related species A. £avipes was
more heterogeneous than A. terreus isolates, indicating
that isolates currently assigned to the species A. £avipes
possibly represent a number of undescribed species.
FEMSLE Congress 2-6-03
207
P4^71
WALLEMIA : THE GENUS OF OSMOPHILIC FUNGI
INHABITING SALTERNS
P. Zalar(1), G. S. de Hoog(2) and N. Gunde Cimer-
man(1)
(1) University of Ljubljana, Biotechnical Faculty, Biology
Department, Vec›na pot 111, 1000 Ljubljana, Slovenia; (2)
Centraalbureau voor Schimmelcultures, P.O.Box 85167,
3508 AD Utrecht, The Netherlands
Wallemia is an important genus of xerophilic fungi in-
volved in the spoilage of low water activity food commod-
ities. It is only occasionally described from natural low
water activity environments. Up to now only one species,
W. sebi was recognised in this genus and a number of
synonyms proposed. For several strains of the species
W. sebi the production of considerably toxic metabolites
walleminol and walleminon were reported, which were
shown in bio-experiments to be of comparable toxicity
with citrinin and pennicillic acid and it thus represents a
potential health hazard. In the past also medical cases
were reported which indicated its possible pathogenic
role to humans. Therefore it is very important to under-
stand the natural ecology of this fungus. In our study of
halophilic fungi living in the hypersaline water of salterns
worldwide, fungi from the genus Wallemia were repeatedly
isolated. On the basis of ITS rDNA variability of a set of
strains including reference strains, and comparing their
morphological and physiological characters, we propose
division of the genus Wallemia into three species. In order
to determine their phylogenetic position, SSU rDNA do-
mains of the proposed species representatives were se-
quenced and compared with the other representatives of
the fungal kingdom. According to this it is placed among
Basidiomycetes.
P4^72
CHARACTERIZATION OF THE BACTERIOPHAGES
OF THE PT-SERIES RELATED TO PS. AERU-
GINOSA
N. Chanishvili(1), M. Merabishvili(1), T. Glonti(1), M.
Vaneehautte(2)
(1) George Eliava Institute of Bacteriophage, Microbiology
and Virology, Tbilisi, Georgia; (2) Department of Clinical
Chemistry, Microbiology and Immunology, University Hos-
pital, University of Gent, 185, De Pintelaan, 9000, Gent,
Belgium
Spread of bacterial drug-resistance in modern hospital set-
tings put into discussion a potential application of bacter-
208 1st FEMS Congress / Posters 103^505
iophages as alternatives of antibiotics and/or disinfectants.
Phage cocktails have been successfully used in medical
practice in the FSU for longer than 70 years. Nevertheless,
the genetic characteristics of the therapeutic bacterio-
phages is poorly known. The goal of this study was to
¢ll in some gaps regarding the phages related to Ps. aeru-
ginosa. Five phages have been chosen for these studies:
PT-1, PT-2, PT-4, PT-5 and PT-8. The AFLP has been
applied as a tool for gene typing of these phages. Obtained
results showed that the phage clones : PT-1, PT-4 & PT-8
have the same genetic patterns. The electron microscopic
studies and serological studies proved this similarity as
well. These facts are especially interesting, since these
phages have been isolated in various times and in di¡erent
ecological niches. The phage clone PT-1 is one of the
components of the traditional commercial preparation ‘‘
Intesti- bacteriophage’’, the clone PT-4 has been isolated
from the river Mtkvari and the clone PT-8 from the Lake
Lisi in Tbilisi, Georgia. Despite of the morphological sim-
ilarity, the genomic patterns of the phages PT-2 and PT-5,
signi¢cantly di¡er from each other and the rest of the
chosen phages. The phages of PT series have been
screened against 200 strains. They showed high e⁄ciency
towards the strains of Ps. aeruginosa, including imipineme-
resistant and mucoid bacteria. These results prove genetic
diversity of the therapeutic bacteriophages applicable for
combating drug-resistant bacteria.
P4^73
GENETIC CHARACTERIZATION OF STREPTOCOC-
CUS THERMOPHILUS BACTERIOPHAGES ISO-
LATED FROM RAW MILK SAMPLES
Zh. P. Dimitrov
ELBY Bulgaricum, Research Department, 12-a Malashev-
ska Str. 1202 So¢a, Bulgaria
The bacteriophage attacks against starters for milk prod-
ucts can lead to serious problems in dairy industry. Strep-
tococcus thermophilus is widely used as a component of
dairy starters in the manufacture of yoghurt and several
kinds of cheeses. Two bacteriophages active against Strep-
tococcus thermophilus strains were isolated from 50 raw
milk samples with di¡erent geographic origins in Bulgaria.
Considering very weak phage absorption onto the cells in
conventional broth medias the protocol for propagation of
these phages was optimized. Restriction analysis with dif-
ferent restriction endonucleases was carried out in order to
estimate the molecular weight and to compare the restric-
tion pro¢les with published ones. Applying partially diges-
tion approach the physical maps of the both phage DNA-s
was created. Both phages had cohesive ends. Using several
known primer pairs for PCR, di¡erent phage DNA frag-
ments were ampli¢ed. After labeling these fragments were
FEMSLE Congress 2-6-03
tested as hybridization probes with purpose to detect ly-
sogeny among Streptococcus thermophilus strains. Some of
primers were used for fast PCR detection of the phage
content in milk samples.
P4^74
UZBEK COLLECTION OF AGRICULTURAL MI-
CROORGANISMS: ITS DEVELOPMENT
D. Egamberdiyeva and K. Davranov
Institute of Microbiology, A.Qadiriy str. 7 B, 700128 Tash-
kent, Uzbekistan
Uzbek National Collection of Agricultural microorgan-
isms has been established in order to improve the native
microbial resources of Uzbekistan, to study the diversity
of native microbial population and to preserve a valuable
microorganisms. The main objectives of our Collection are
deposit of agriculturally useful microbial strains from Sci-
enti¢c community, their preservation and distribution for
education, research, training courses, isolation, identi¢ca-
tion of strains. At present we have salt tolerant, heat re-
sistance, plant growth promoting bacterial strains, nitro-
gen ¢xers, fungi, plant pathogenic fungi, some plant
viruses. Because of limited ¢nancial resources there were
lack of chemicals and equipment’s for preservation of mi-
croorganisms. We are grateful for Society of General Mi-
croorganisms (SGM, UK) for supporting the development
of Uzbek Culture Collection. With this support we will use
also freeze dried ampoules for preservation, develop strain
data management and publish ¢rst catalogue Uzbek Cul-
ture Collection.
P4^75
CYANOBACTERIAL DIVERSITY ON HISTORIC
BUILDINGS
C. A. Crispim(1), P. M. Gaylarde(1), C. C. Gaylarde(1),
B. A. Neilan(2)
(1) Federal University of Rio Grande do Sul, Porto Alegre,
Brazil ; (2) University of New South Wales, Sydney, Aus-
tralia
Algae and cyanobacteria, together with fungi, are largely
responsible for biodeterioration of external surfaces of
buildings. Traditional and molecular techniques were
used to analyse cyanobacterial populations in bio¢lms
on outer walls of historic buildings in Brazil. In mature
bio¢lms, members of cyanobacterial sub-sections 1 and 2
were generally the major biomass; occasionally ¢lamen-
tous genera from the botanical families Scytonemataceae,
Microchaetaceae and Rivularaceae were dominant. Tradi-
 1st FEMS Congress / Posters 103^505
tional culture techniques, however, resulted in the isola-
tion of mainly ¢lamentous organisms of sub-sections 3
and 4. Sub-section 1 organisms were found growing endo-
lithically. PCR products were obtained from morphologi-
cally identi¢ed organisms using 16S rDNA primers, 271F
(universal) and 408R (cyanobacteria speci¢c). The sequen-
ces of these DNA fragments were compared with known
deposited sequences using the BLAST facility. Phyloge-
netic analysis indicates that all the cyanobacteria se-
quenced in this study corresponded to their appropriate
group. However, sequence homologies with deposited se-
quences were, on the whole, low and the positions of the
isolates in the dendrogram showed that they were deeply-
rooted. For our analysis, we used only deposited sequen-
ces for which morphological identi¢cations were available.
These were mainly aquatic cyanobacteria from environ-
ments very di¡erent from our dry, sun-exposed walls,
where cells often had thick pigmented sheaths. One of
our isolates was very closely related phylogenetically to a
specimen obtained from a high, hot dessert (Arches Na-
tional Park, Utah), where survival strategies are similar to
those in the extreme environments of external walls. None
of our isolates showed close similarity to extremophiles
from very cold or hypothermal regions.
FEMSLE Congress 2-6-03
209
P4^76
CONSTRUCTION OF METAGENOMIC LIBRARIES
OF UNCULTURED ELECTROCHEMICALLY AC-
TIVE MICROORGANIMS
J. H. Back(1), H. Cho(1), I. S. Chang(2), B. H. Kim(2)
and Y. S. Han(1)
(1) Biomedical Research Center, Korea Institute of Science
and Technology, P. O. Box 131, Cheongryang, Seoul, Ko-
rea; (2) Water Environment Research Center, Korea Insti-
tute of Science and Technology, P. O. Box 131, Cheon-
gryang, Seoul, Korea
We constructed metagenomic libraries that would be the
basis to identify the genes related electron transfer. Meta-
genomic libraries are a powerful tool for exploring uncul-
tured microorganisms. Traditional methods for culturing
microorganism limit analysis to those that grow under
laboratory condition. Given the profound utility and im-
portance of uncultured electrochemically active microor-
ganisms in water industries, methods are needed to explore
their unique characteristics using the metagenome. This
study is to develop a strategy for accessing the genetic
diversity and identify the electron transfer mechanism of
electrochemically active microorganisms. Also it is aiming
to characterize electrochemically active microbes in a me-
diator-less microbial fuel cell. Metagenomic libraries will
be a good resource for discovering new and useful genes
among the uncultivated electrochemically active microor-
ganisms.
P4^77
A NEW CRYOPRESERVATION METHOD FOR FUN-
GI USING PERLITE
L. Homolka, L. Lisa, I. Eichlerova and M. Tomsovsky
Institute of Microbiology AS CR, Videnska 1083, 142 20
Prague 4, Czech Republic
E⁄cient microbiological work requires a reliable source of
cultures, which is ensured by its safe storage. Routine
subculturing does not prevent genetic and physiological
changes. Various storage methods have been developed
in order to eliminate these disadvantages. The storage in
liquid nitrogen is a safe and perspective method of a long-
term maintenance of most fungal species, especially those
not amenable to freeze-drying. A new alternative method
using perlite as a particulate solid carrier in the growth
medium with a cryoprotectant was tested for cryopreser-
vation of several basidiomycete species from di¡erent gen-
era (Armillaria, Pleurotus, Pluteus, Polyporus) which failed
to survive or retain their properties in routinely used cry-
210 1st FEMS Congress / Posters 103^505
opreservation procedures. Frozen basidiomycete strains
were kept in cryovials submerged in liquid nitrogen and
were after a 12-month storage thawed and checked for
viability, purity and changes in growth, morphology and
biochemical characteristics. All cultures survived the cry-
opreservation procedure and no negative e¡ects of cryo-
preservation by this method have been observed. As the
method turned out to be successful, it was used for cry-
opreservation of further several hundreds di¡erent basi-
diomycete strains and also several micromycete strains.
A more detailed study was performed with selected strains
from the genus Trametes. This method, useful for both
sporulating and non-sporulating fungal cultures, has sev-
eral other important advantages: protection of cultures
from contamination (reduced number of transfers) and
the saving of time, work and room.
This work was supported by grant no. 526/02/1216 from
the Grant Agency of the Czech Republic and by Institu-
tional Research Concept no. AV0Z5020903.
P4^78
COMPLETE GENOMIC SEQUENCE OF THE LYTIC
BACTERIOPHAGE P1203 OF CORYNEBACTERIUM
GLUTAMICUM CCRC 18238
W. H. Hsu(1), T. Y. Pan(1), C. L. Chen(1), C. S.
Sheu(2) and H. Y. Hu(3)
(1) Institute of Molecular Biology, National Chung Hsing
University, Taichung 402, Taiwan ; (2) Vedan Enterprise
Co., Taichung 433, Taiwan; (3) Department of Food Sci-
ence and Nutrition, Hung Kuang Institute of Technology,
Taichung 433, Taiwan
Coryneform bacteria are widely used in the industrial pro-
duction of amino acid and various other biotechnological
application such as bioconversion. Bacteriophage infection
leads to the lysis of cells and thereby interrupts the pro-
duction of amino acid. Despite their economic importance,
genomic sequences of corynebacteriophage are not avail-
able to provide valuable insight regarding the phage-host
interaction and bacteriophage evolution which will un-
doubtedly be necessary for developing long-term phage-
resistant strains. Lytic corynebacteriophage P1201 was iso-
lated from Corynebacterium glutamicum CCRC18238, a
glutamic acid hyperproducing strain, that had become
contaminated during an industrial fermentation. P1203
belongs to the Styloviridae family (B1 morphotype) with
an isometric head of 52 nm wide and a long non-contrac-
tile and striated tail of 348 nm. It had a narrow host range
and adsorption to its host was enhanced in the presence of
Mg2+ ion. The nucleotide sequence of the 70-kb double
stranded DNA genome was determined. A total of 44
open reading frames were predicted from the nucleotide
sequence, and analysis of the corresponding proteins was
FEMSLE Congress 2-6-03
used to construct a functional map. The Sau3A1 digested
genomic DNA was cloned into a promoter probed vector
with lac Z as a reporter gene and expressed in Escherichia
coli. Several clones with high speci¢c activity of L-galac-
tosidase were found, indicating that these promoters were
controlled by the host RNA polymerase. This is the ¢rst
corynebacteriophage genome sequence to be reported.
P4^79
MOLECULAR TOOLS APPLIED FOR DIVERSITY
STUDIES OF CULTURABLE BACTERIAL POPULA-
TIONS IN OIL-CONTAMINATED RHIZOSPHERE
M. M. Jussila, L. Suominen and K. Lindstro«m
Department of Applied Chemistry and Microbiology, Viikki
Biocenter, P.O. Box 56,
FIN-00014 University of Helsinki, Helsinki, Finland
A culture collection of 52 indigenous meta-toluate tolerat-
ing bacteria isolated from oil-contaminated rhizosphere of
Galega orientalis was characterized and identi¢ed by clas-
sical and molecular biological methods. The phylogenetic
diversity was indicated by the presence of ¢ve major line-
ages of the Bacteria domain, while gram-positive bacteria
was the most dominating group. A TOL plasmid-speci¢c
xylE-PCR was developed in order to detect both active
and potential degraders of m-toluate. The ability to de-
grade m-toluate in the presence of the gene xylE was de-
tected only within the genus Pseudomonas. The genetic
diversity was indicated by cluster analysis of the data,
revealing 13 16S rDNA ribotypes and 23 (GTG)5-geno-
types among various bacterial isolates ranging from sim-
ilar strains to di¡erent genera. Generally, the 16S-ribotype
and the (GTG)5-genotype corresponded to each other very
well and grouped the strains at the species level. 16S
rDNA PCR-RFLP ribotyping and (GTG)5-PCR genomic
¢ngerprinting methods combined with partial sequencing
of 16S rRNA genes of representatives of the main clusters
was used to construct a reference dendrogram in order
later to rapidly group and search for new and interesting
bacterial species from oil-contaminated rhizosphere.
 1st FEMS Congress / Posters 103^505
P4^80
THE INFLUENCE OF NATIVE, HYDROPHOBIZED
AND DIMERIC FORM OF RIBONUCLEASE BACIL-
LUS INTERMEDIUS ON THE LYSOSOME-PHAGO-
SOME FUSION IN PERITONEAL MACROPHAGES
OF RAT
N. V. Kalacheva, B. M. Kurinenko
Department of Microbiology, Laboratory of Engineering
Enzymology, Kazan State University, Kazan, 420008, Rus-
sia
The e¡ect of native RNAse Bac. intermedius depends on
the concentration: comparatively low concentrations (0.5
^ 50 Wg ml-1 ) stimulate the phagosome-lysosome fusion
whereas high concentrations (above 70 Wg ml-1 ) inhibit
this process. RNAse modi¢ed by surfactant oxanol-KD6
and dimeric form RNAse possesses only the inhibitory
e¡ect that appears at concentrations considerably lower
than those of native enzyme. The stimulatory e¡ect of
native RNAse and the inhibitory e¡ect of hydrophobized
derivative do not depend on the catalytic activity. The
results obtained allow us to conclude that the stimulatory
e¡ect of RNAse is caused by weak irritation of the cyto-
plasm tic membrane leading to trigging o¡ cell activation
mechanism. But the inhibitory e¡ect is connected with a
stronger action of compounds on cellular membranes and
it may be caused by the modi¢cation of properties both of
phagosome and lysosome membranes. Thus, the common
peculiarity of the e¡ect of native, hydrophobized and di-
meric form of RNAse on phagocytic cells is the ability to
inhibit the fusion between lysosomes and phagosomes.
This property of RNAse and its modi¢ed derivatives
may be used in anti-in£ammatory therapy and for de-
crease of cytotoxic reactions dependent on phagocytosis.
P4^81
THE ROLE OF MICROBIAL AUTOREGULATORS
FROM ALKYL HYDROXYBENZEN GROUP IN THE
FORMATION OF HYPOMETABOLIC FORMS OF
BACTERIA
A. B. Margulis, O. N. Ilinskaya, A. I. Kolpakov
Microbiology Department, Kazan State University, Krem-
levskaya 18, Kazan, 420008, Russia
It is well-known fact, that in response to unfavourable
conditions the microorganisms can stop their division
and begin to form dormancy (anabiotic) or hypometabolic
forms with high resistance to environmental in£uences.
The autoregulatory d1 factors of microorganisms may in-
duce the transition of vegetative microbial cells into the
FEMSLE Congress 2-6-03
211
anabiotic state, when they acquire the higher resistance to
unfavourable conditions. The present investigation showed
that long incubation of gram-positive and gram-negative
bacteria in distilled water maintained the viability of bac-
teria (Bacillus subtilis and Escherichia coli) and their col-
ony formation ability during 12 weeks. Character of these
processes under starvation conditions was similar both for
gram-positive and gram-negative bacteria. However, after
4th week of incubation a part of bacteria in variant con-
taining 50 Wg/ml of hexylresorcinol (the microbial growth
regulator from alkyl hydroxybenzen group) transited into
hypometabolic state. Hexylresorcinol induced the forma-
tion of hypometabolic forms of gram-negative bacteria
after 8 weeks of starvation. It was registered by the de-
crease of number of colony forming units accompanied
with some increase of bacterial suspension density. In
Gram-positive bacteria hexylresorcinol induced the forma-
tion of new morphotype (small untypical colonies mostly
consisted of the cells without endospores). It was found,
that about 47% of Bacillus cells after 8 weeks of starvation
with hexylresorcinol had no response on addition of exo-
genic nutrient substrate, i.e. were in hypometabolic state.
P4^82
STUDIES ON STABILITY OF BREWERS’ STRAINS
OF SACCHAROMYCES CEREVIASIAE YEASTS
DURING LONG-TERM PRESERVATION IN IAFB
CULTURE COLLECTION
A. Misiewicz, J. Czuba, I. Sikorska, B. Sieliwanowicz, K.
Baranowski
Institute of Agricultural and Food Biotechnology, Warsaw,
Poland
Brewer’s strains of Saccharomyces cerevisiae yeasts, top
and bottom fermentation are one of the group of micro-
organisms preserving in Culture Collection of Industrial
Microorganisms. Stability of morphological, physiological
and biotechnological properties after 50-years preservation
were studied. Morphological properties were documented
using digital image analysis (images from light and elec-
tron microscopy). Physiological features were checked by
traditional tests as well as API test with computer soft-
ware. Biotechnological properties of strains were checked
using HPLC and GC techniques. All conducted tests con-
¢rm that preserving strains conserve their quality after 50
years.
212 1st FEMS Congress / Posters 103^505
P4^83
SEROLOGICAL DIAGNOSIS OF LEISHMANIA DO-
NOVANI WITH IIF METHOD
L. Puzderliska, M. Dimitrova, L. Karakerezova
Department of Preventative Health, Stip, Macedonia
Leishmania donovani is a provocator of a very serious
transmissible parasitic disease also called visceral leishma-
niosis. This parasite has a container (dog), a transmitter
Phlebotomus papatassi and ¢nal consument sick person. In
our examination we would like to present the presence and
attendance of local visceral leishmaniosis on the territory
of Republic of Macedonia. The total number of examined
sera was 74, 67 of which were statistically elaborated, be-
cause up to 74 with repeated positive controls. From 67
examined sera with IIF, 19 sera were with new infection of
Leishmania donovani or 28.3% and 71.7 were negative.
Conclusion : In the presence of prolonged febrile condi-
tions that don’t react on antibiotic therapy to take into
consideration for possible parasitic with Leishmania dono-
vani.
P4^84
RUSSIAN NATIONAL COLLECTION OF INDUSTRI-
AL MICROORGANISMS ^ MICROBIAL GENETIC
RESOURCES FOR BIOTECHNOLOGY
S. P. Sineoky
Russian National Collection of Industrial Microorganisms,
FGUP GosNII genetika, 1-st Dorozhny proezd 1, 113545,
Moscow, Russian Federation
Russian National Collection of Industrial Microorganisms
(VKPM) is a largest Russian service collection for non-
pathogenic microbial strains. Over 15 000 strain are main-
tained for application in fundamental and applied inves-
tigations. At the present time, VKPM is functioned as
National Bioresource center and is involved in the decision
of various problems essential for successful development
of Biotechnology. Among such problems ^ biosafety for
natural and genetically modi¢ed strains, developing nor-
mative base for strains deposition and distribution, pro-
tection of intellectual rights in biotechnology and method-
ology of the strains identi¢cation using PCR technique.
VKPM activity in these areas will be presented in the
communication.
FEMSLE Congress 2-6-03
P4^85
EXPANDING THE BIODIVERSITY OF MICROOR-
GANISMS IN JAPAN COLLECTION OF MICROOR-
GANISMS
M. Takashima, Y. Benno, and T. Kudo
Japan Collection of Microorganisms, Bioscience Technology
Center, RIKEN (The Institute of Physical and Chemical
Research), Wako, Saitama, Japan
To keep pace with the progress in research on microbial
diversity, the sphere of microorganisms that must be man-
aged in culture collection is expanding. The Japan Collec-
tion of Microorganisms, which was established in 1980,
works to contribute to domestic, regional, and global im-
provements in the conservation of microbiological resour-
ces in cooperation with other culture collections and in-
stitutions, and to supply authentic microorganisms to
researchers in the ¢elds of life sciences and biotechnology.
In answer to user requests, JCM has developed an e⁄cient
anaerobic chamber to cultivate the extremely oxygen-sen-
sitive (EOS) anaerobes, and is maintaining these micro-
organisms including the genera Treponema, Prevotella
and Tannerella. So-called extremophiles such as methano-
gens, hyperthermophiles, and subterraneous and piezo-
philic bacteria are also introduced. As of the end of March
2002, a total of 10,354 strains (6,589 bacteria, 205 archaea,
3,539 ¢lamentous fungi and yeasts, and 21 others) were
listed in JCM. Study is continuing on the unknown, that is
to say, the undescribed, not yet isolated, or yet-to-be cul-
tured microorganisms using molecular phylogenetic tech-
niques in addition to the usually applied morphological,
physiological, biochemical and chemotaxonomic methods.
Also, identi¢cation of microorganisms that have been in
the collection for some time, of which the taxonomic po-
sition at the species level has not been determined, is being
made and newly collected strains examined.
 1st FEMS Congress / Posters 103^505
P4^86
A NOVEL APPROACH FOR MOLECULAR CHAR-
ACTERIZATION OF BACTERIAL POPULATIONS
FROM NATURAL STARTER CULTURES
E. Soldati(1), V. Corich(1), G. Poletto(1), C. Andrighet-
to(2), A. Lombardi(2) and A. Giacomini(1)
(1) Dipartimento di Biotecnologie Agrarie, Universita' di
Padova, viale dell’Universita', I-35020 Legnaro (PD), Italy ;
(2) Veneto Agricoltura ^ Istituto per la Qualita' e le Tec-
nologie Agroalimentari, via San Gaetano 74, I-36016 Thiene
(VI), Italy
Natural starter cultures used in cheese manufacturing are
complex bacterial communities with unknown composi-
tion in terms of species and strains, whose identi¢cation,
possibly directly within the matrix, has great technological
importance. A natural whey starter culture from Grana
Padano cheese production was used to develop such an
identi¢cation system. A preliminary characterization at
species level by ARDRA on the culturable fraction and
by TGGE on 16SrDNA target sequence from DNA ex-
tracted from whey, revealed a population composed by
Lactobacillus helveticus (90%) and Lactobacillus delbrueckii
subsp. lactis (10%). A characterization at strain level was
carried out by PCR-based techniques, one exploiting the
presence of particular DNA sequences to originate genetic
polymorphism (RAPD-PCR with primers M13 and
D8635) and a second newly developed approach (Sau-
PCR) which uses a primer based on the restriction enzyme
Sau3AI sequence to amplify genomic DNA digested with
the same enzyme. Analysis of electrophoretic pro¢les evi-
denced two main groups of isolates including 12 minor
subgroups. Ten representatives from di¡erent subgroups
were screened for the presence of useful polymorphic
DNA sequences. Published sequences for aminopeptidases
and proteases highly speci¢c for L. helveticus were exam-
ined, along with highly strain-conserved fragments from
RAPD analyses. The aim of the research was to ¢nd out
sequences either highly (for identi¢cation at species level)
or poorly (identi¢cation at strain level) conserved among
the isolates. Data presented report the results of such anal-
ysis.
FEMSLE Congress 2-6-03
213
P4^87
INVESTIGATION OF THE YEAST MICROFLORA
OF ‘‘TOKAJ ESSENCE’’
H. Csoma, M. Sipiczki
Department of Genetics, University of Debrecen, Debrecen,
Hungary
Tokaj is one of the major European wine regions, where
botrytized wines can be produced. Due to the unique cli-
matic conditions of the region, the grapes infected by Bo-
trytis cinerea develop what is called noble rot. The noble-
rotted berries can be harvested selectively from the in-
fected bunches and stored in containers where the weight
of the grapes presses out some of the juice. This juice that
drains away freely from the stored botrytized grapes is
called ‘‘essence’’. The collected essence is placed in wooden
barrels for fermentation and maturation (usually at tem-
peratures below 15‡C). Because of the low temperature
and the very high sugar content of the juice, occasionally
over 50%, fermentation occurs very slowly and often pro-
duces little more than 5 to 7 % alcohol before termination.
To investigate the micro£ora, we took samples from fer-
menting essence in four wineries and isolated yeasts from
the samples. The molecular analysis of the isolates (elec-
trophoretic karyotyping, PCR-RFLP and sequencing of
the ITS1-5S-ITS2 region of rDNA) revealed a high degree
of heterogeneity in the fermenting populations, but most
isolates proved to be strains of Zygosaccharomyces, Can-
dida stellata, Torulaspora and Saccharomyces sensu stric-
to species. The composition of the populations changed
with time, usually in favour of Saccharomyces. Di¡erences
were also detected between samples taken from the bottom
and the surface of the fermenting essence.
P4^88
Withdrawn.
214 1st FEMS Congress / Posters 103^505
P4^89
IDENTIFICATION OF CULTIVABLE BACTERIA
FROM ACTIVATED CARBON FILTERS USED FOR
WATER TREATMENT
A. Magic¤-Knez›ev, B. Wullings and D. van der Kooij
Kiwa Research and Consultancy, P.O. Box 1072, 3430 BB
Nieuwegein, The Netherlands
Biological activated carbon ¢ltration (BACF) is frequently
used in water treatment for the removal of undesirable
organic compounds. Optimisation of the biological activ-
ity during BACF may reduce both the high costs and en-
vironmental burden associated with thermal regeneration
of activated carbon. Bacteria were isolated from BACF
¢lters at several Dutch water treatment plants as part of
a study aiming at acquiring information about the nature
of the bacterial activity in BACF. Microorganisms were
removed from the surface of activated carbon by de¢ned
ultrasonic treatment and cultured at solid and liquid me-
dia. Although the cultivability was higher in the liquid
media, less than 1% of bacteria was cultivated. A total
of 260 obtained isolates were grouped by the analysis of
repetitive extragenic palindromic DNA (REP) patterns in
12 major clusters. From 60 representative isolates, for
which the sequence of 16S rDNA has been determined,
28 were identi¢ed as representatives of the family of Co-
mamondacaea. Within this group 19 isolates were closely
related (99%) with uncultured bacterial clones from pol-
luted groundwater or soil. Smaller numbers of isolates
were either related to the genus Hydrogenophaga or Sphin-
gomonas, or matched with the sequence of MTBE degrad-
ing bacterium. Other isolates were related to unidenti¢ed
bacterial clones. These data show that even with molecular
techniques it is di⁄cult to identify cultivable bacteria,
which in turn represent less than 1% of the total number
of bacteria in BAC ¢lters. Further studies with selected
pure cultures will be conducted to elucidate essential phys-
iological properties of these bacteria.
P4^90
MOLECULAR CHARACTERIZATION OF LACTIC
ACID BACTERIA INVOLVED IN NATURAL FER-
MENTATION OF TURNIP
M. Maifreni, M. Marino and G. Rondinini
Dipartimento di Scienze degli Alimenti, Universita' degli
Studi di Udine, via Marangoni 97, 33100 Udine, Italy
Lactic acid fermentation, an ancient preservation method,
is nowadays especially favoured as a ‘‘natural’’ process to
increase the shelf-life of various products (dairy, meat,
FEMSLE Congress 2-6-03
vegetable). Most vegetables can be lactic acid-fermented,
so far cucumber, cabbage and olives are the only vegeta-
bles that are fermented in large volumes for human con-
sumption. However, the are agricultural, nutritional, sen-
sory and preservation reasons for evaluating lactic acid
fermentation as a potential process for making new prod-
ucts from other vegetables. Lactic acid bacteria are the
main responsible for the fermentation of vegetable such
as cabbage, carrots, beets, but the indigenous LAB £ora
varies as a function of the quality of the raw material,
tempertaure and harvesting conditions. Spontaneus fer-
mentation thus leads to variations in the sensory proper-
ties of the products. Brovada is an ancient traditional
product from the North-East Italian region, included in
the list of Italian typical products, which is obtained mak-
ing the turnips (Brassica rapa) ferment naturally. At
present, there is few informations on the spectrum of mi-
crorganisms associated with the fermentation of Brassica
rapa and the development of £avour compounds during
the process. These knowledge are essential for the develop-
ment of the product with improved quality. The present
study reports on the characterization of lactic acid bacteria
associated with the natural fermentation of Brovada using
PCR-based molecular techniques. Application of RAPD
and REP-PCR allowed the grouping of lactic acid bacteria
population in Lactobacillus spp. (L. hilgardii, L. planta-
rum, L. coryniformis) and Pediococcus parvulus.
P4^91
BIODIVERSITY AND TECHNOLOGICAL PROPER-
TIES OF LEUCONOSTOC SP. ISOLATED FROM
TRADITIONAL DAIRY PRODUCTS
B. Martinez, J. I. Sanchez, T. Delgado and A. Rodriguez
Instituto de Productos Lacteos de Asturias (IPLA ^
CSIC), Crta. In¢esto s/n, 33300 Villaviciosa (Asturias),
Spain
Leuconostoc sp. are commonly used in mesophilic starters
for the production of fresh or soft cheeses and fermented
milks due to their ability to produce £avours and to mod-
ify the appearance and texture of the product. Since these
microorganisms are relatively inert in milk they are always
cultured together with an acid producing Lactococcus lac-
tis strain. The aim of this work was to determine the
technological ability of eleven wild strains of Leuconostoc
isolated from traditional dairy products and to select those
which present the best properties in order to include them
in mixed starter cultures. Molecular characterization of
the strains was initially carried out by Pulse Field Gel
Electrophoresis (PFGE) and RAPD-PCR techniques. Pro-
duction of dextran, organic acids and volatile compounds
were studied in order to check the texturing and £avouring
properties of the strains. The resistance to di¡erent param-
 1st FEMS Congress / Posters 103^505
eters such as nisin, acid and salt concentration as well as
the viability after freezing was also determined. Generally,
a large variability was found among the isolated strains,
mainly regarding the above mentioned resistances. Finally,
analyses of the metabolite pro¢le and biomass production
in batch co-cultures of Leuconostoc and L. lactis are in
progress to de¢ne the right ratio for large-scale mixed
starter production. The obtained results clearly showed
that natural environment provides a good source of strains
with appropriate technological properties.
P4^92
INFLUENCE OF BACTERIA IN RIPENING ENVI-
RONMENTALS OF PECORINO FILIANO CHEESE
AND STUDY OF BACTERIAL EVOLUTION DURING
RIPENING
D. Messina, T. Lechiancole and G. Salzano
Dept. of Biology, Univerity of Basilicata, Campus Macchia
Romana, 85100 Potenza, Italy
It is well know that the Pecorino is a hard cheese manu-
factured in di¡erent areas of Italy. Pecorino Filiano is a
traditional hard cheese produced in Basilicata (Southern
Italy) without addition of selected or natural starter cul-
ture. The fermentation and ripening processes of this
cheese are entirely performed by the indigenous micro£ora
present in the milk. Ripening of this cheese occurs in the
cave, this is special environment at controlled temperature
and humidity. Air microrganisms can represent surface
cheeses micro£ora and they have a strong impact on the
appearance, £avour and texture development of the
cheeses, and usually lead to shorter ripening periods. It
is well known that bacteria play an important role during
the ripening of cheeses. In this work two Pecorino Filiano
cheeses, one is manufactured by using raw whole ewe’s
milk with addition of 10% raw whole goat’s milk (Pecor-
ino A); the other is manufactured by using only raw whole
ewe’s milk (Pecorino B), ripened into a cold store (refer-
ence frame) and in a cave were studied. RAPD-PCR is an
important tool for a rapid and reliable screening of a lot
of strains. This molecular technique was applied on all
isolated bacteria to evaluate interaction between air bac-
teria and surface cheese bacteria and also to study bacte-
rial evolution of these two cheeses. In Pecorino Filiano
cheese interaction between air bacteria and surface cheeses
bacteria occur, especially during ripening in the cave.
Moreover during ripening of two cheeses there is a very
strong heterogeneity of bacterial micro£ora.
FEMSLE Congress 2-6-03
215
P4^93
DIFFERENTIATION OF YEASTS ISOLATED FROM
ARTISANAL EWE’S CHEESES USING RAPD-PCR
ANALYSIS
V. Mossa, M. E. Fadda, M. Deplano and S. Cosentino
Department of Experimental Biology, section of Hygiene,
University of Cagliari, S.S 554, Km 4,500, 09042 Monser-
rato (CA), Italy
The yeasts, with their metabolic properties, are increas-
ingly considered as important agents in the maturation
process of several cheeses. In a previous study we have
shown that yeast population from artisanal Sardinian
ewe’s cheese, was mainly represented by Debaryomyces
hansenii, Kluyveromyces lactis, Candida lambica, Candida
zeylanoides and Geotrichum candidum species. In the
present work RAPD-PCR with the core sequence of the
phage M13 as primer was used for typing yeast strains
belonging to this predominant species. DNA banding pat-
terns were analysed using the Gel Compar software pack-
age, version 2.5 (Applied Maths, Kortrijk, Belgium). Sim-
ilarities among isolates were estimated using the Pearson
coe⁄cient and clustering was based on the UPGMA meth-
od. At a similarity level of 50%, the ¢ve prevalent species
were well separated in six clusters, one of which was rep-
resented by a single strain. RAPD-PCR analysis showed
good capacity to produce species-speci¢c patterns, in fact
all the isolates were grouped with the type strains of the
species considered, thus con¢rming the results of previous
classi¢cation based on phenotypic traits. This analysis
have also showed a certain degree of genetic polymor-
phism among the strains: in fact a similarity of about
55% for the strains of the species C. zeylanoides, K. lactis
and C. lambica, and 65% for D. hansenii and G. candidum
was observed. Our results indicate the usefulness of
RAPD-PCR analysis for di¡erentiation of yeast species.
This work was in part supported by a grant from
MURST, Plan ‘‘Agroalimentary products: dairy prod-
ucts’’, Cluster 08B, Project n.7.
216 1st FEMS Congress / Posters 103^505
P4^94
DIVERSITY IN THE TEHNOLOGICAL PROPERTIES
OF YOGHURT BACTERIA ISOLATED FROM HOME
MADE YOGHURT
K. Pashova-Baltova(1), M. Michailova(1), M. Fukui(2)
(1) ELBY Bulgaricum, Research Department, 12A Mala-
shevska Str., 1202 So¢a, Bulgaria ; (2) Japan Internetional
Cooperaton Agency, JICA
The characterization of monocultures is the ¢rst step in
the development of new successful starter culture for fer-
mented products. Bulgaria is famous with its traditional
product ^ Bulgarian yoghurt. The purpose of this study
was to isolate lactic acid bacteria from home made yo-
ghurt from ecological areas and to perform analysis of
important technological properties of the collected mono-
cultures. 73 strains L. bulgaricus and 114 strains S. ther-
mophilus were isolated from home made yoghurt. After 10
times of reinoculation strains from both species were
tested for: fermentative activity after incubation at 16hr
and 40hr (at 370C); osmotic tolerance in 15% sugar syrup;
antibiotic tolerance against traces of penicillin, viability of
strains during preservation for 20 days at 50C; kinematic
and visual viscosity. The results showed that the techno-
logical properties of the collected strains of yoghurt bac-
teria are quite variable. The created database permitted
classi¢cation of the tested strains into groups with high,
medium and low values of the analyzed technological pa-
rameters. The large variations in the properties of the
tested strains presents a good potential for the develop-
ment of new starters for fermented milk products with
desired characteristics.
P4^95
PHENOTYPIC AND GENETIC VARIABILITY OF
KLOECKERA APICULATA STRAINS OF WINE ORI-
GIN
P. Romano, C. Fiore, A. Capece, M. Caruso and M. Para-
ggio
Dipartimento di Biologia, Difesa e Biotecnologie Agro-
Forestali, Universita' degli Studi della Basilicata, Campus
Macchia Romana, 85100 Potenza, Italy
The species of the genera Hanseniaspora (and its ana-
morph form Kloeckera) are frequently isolated from vari-
ous source as soil, fruits and insects, as well as from fer-
mented foods and beverages. As predominant inhabitants
on the surface of grape berries and in early must fermen-
tation, apiculate yeasts perform biochemical activities,
producing some important reactions, thus in£uencing pos-
FEMSLE Congress 2-6-03
itively and/or negatively the ¢nal wine quality. During the
years, we have collected a great number of apiculate yeasts
and in this paper we report a study on about two hun-
dreds K. apiculata wine strains. The strains were tested for
enzymatic activities of interest in winemaking, such as L-
glucosidase, L-xylosidase and protease, and for the pro-
duction of fermentation metabolites, a¡ecting wine £a-
vour, such as higher alcohols, carbonyl compounds and
acetic acid. A signi¢cant variability was found, with the
individuation of di¡erent phenotypes. Fifty strains, as rep-
resentatives of the di¡erent phenotypes, were further ana-
lysed for genetic polymorphism, by using RAPD analysis
and ARDRA technique. The RAPD analysis showed a
low degree of similarity between the strains, whereas no
di¡erences were recorded by ARDRA technique, con¢rm-
ing that strains belonged to the species K. apiculata. Our
results emphasized the existence of a considerable biodi-
versity among wine apiculate strains and this variability is
of technological interest as these yeasts, contrary to a gen-
eral opinion that they are wine spoilage micro-organisms,
constitute £avour potential producers. In this context, it
becomes advantageous to select, for each wine, also suit-
able strains of K. apiculata to use in mixed or sequential
cultures with Saccharomyces cerevisiae.
P4^96
HIGH THROUGHPUT SCREENING ON FLAVOUR
FORMATION BY BACTERIAL CULTURES TO MAP
BIO-DIVERSITY AMONG LACTIC ACID BACTERIA
B. A. Smit, W. J. M. Engels, J. T. M. Wouters and G. Smit
NIZO Food Research, Department of Flavour, Nutrition
and Ingredients, PO-box 20, 6710 BA Ede, The Netherlands
Flavour is a very important characteristic of foods and
drinks. In fermented products like cheeses, sausages and
alcoholic beverages formation of odour and taste active
compounds by micro-organisms contributes considerably
to this £avour. The process of £avour formation is inves-
tigated intensively to improve production processes, devel-
op new varieties and accelerate £avour formation while
maintaining balanced product characteristics. Flavour for-
mation can be enhanced in several ways, e.g. by the use of
alternative (strains of) starter micro-organisms, by the use
of adjunct cultures (working additional, or synergetic), by
changing of process parameters, or by modi¢cation of
strains. In practice, genetically modi¢ed strains are not
used in food applications, thus exploration of the natural
bio-diversity among bacteria is a powerful alternative. To
explore this biodiversity new, fast and £exible screening
methods on functional characteristics like production of
£avour compounds, relevant enzyme activities, and so on
are necessary. In or lab, automated 96-well enzyme and
bioassays, in combination with direct mass- spectrometric
 1st FEMS Congress / Posters 103^505
methods for £avour analysis have been developed. These
techniques enable fast screening on various enzyme activ-
ities in relation to overall £avour production by (a mixture
of) strains. An example is the screening on branched chain
aldehyde production by lactic acid bacteria. This screening
revealed large di¡erences in the actual production and the
levels enzyme activities involved. With this knowledge we
can select lactic acid bacteria for production of high aro-
ma formation levels.
P4^97
CORK STOPPERS INDUSTRY: PENICILLIUM DI-
VERSITY COLONIZING CORK SLABS
G. A. M. Soares(1), A. C. Oliveira(1), M. C. Basilio(1),
R. Tenreiro(2), M. V. San Roma‹o(1,3)
(1) Instituto de Biologia Experimental e Tecnolo¤gica/Insti-
tuto de Tecnologia Qu|¤mica e Biolo¤gica-Universidade Nova
de Lisboa, Apt. 12, 2781-901 Oeiras, Portugal; (2) Depar-
tamento de Biologia Vegetal-Faculdade de Cie“ncia da Uni-
versidade de Lisboa, Lisboa, Portugal; (3) Estaca‹o Viti-
vin|¤cola Nacional, 2565-191 Dois Portos, Portugal
Cork stoppers are made from the bark of the cork oak
tree, Quercus suber L., widely found in Portugal, Spain
and in other Mediterranean countries. Nevertheless Portu-
gal is the world’s largest producer of cork stoppers for the
wine industry. The manufacturing process of cork stoppers
includes a stabilization period during which moulds grow
on the cork slabs. This process, in which cork slabs are
traditionally considered to be of good quality for stopper
manufacturing when they are completely covered with
moulds, has been used for several decades. A study intend-
ing to obtain information on the mycobiota associated
with the Portuguese cork throughout the manufacturing
process of stoppers was carried out. Chrysonilia sitophila
showed to be the most dominant fungus, followed by a
signi¢cant occurrence of the genus Penicillium. This work
concerns the identi¢cation at species level of the isolates
belonging to the genus Penicillium. The strains were grown
for 7 days in CYA at 5, 25 and 37‡C, in MEA at 25‡C and
in 25% glycerol nitrate agar at 25‡C. Fungal DNA was
also isolated and molecular methods were used to con¢rm
the identities of some strains. The ribosomal DNA region
containing spacers ITS1, 5.8S and ITS2 was PCR-ampli-
¢ed and the products analysed. The restriction patterns of
the PCR products were con¢rmed by using reference
strains. The results show the diversity of mould species
colonizing the cork slabs and the use of restriction length
polymorphism of the rDNA as a potential tool to con¢rm
the identity of Penicillium species.
This work was partially supported by amorim & irma‹os.
Sta. Maria de Lamas. Portugal.
FEMSLE Congress 2-6-03
217
P4^98
RAPID IDENTIFICATION AND TYPING OF LACTO-
BACILLUS PLANTARUM STRAINS IN DAIRY
PRODUCTS BY USING POLYMERASE CHAIN RE-
ACTION AND RANDOMLY AMPLIFIED POLY-
MORPHIC DNA
M. Oneca, F. Feutry, M. Ortigosa, A. Irigoyen and P.
Torre
Laboratorio de Lactolog|¤a, Area de Nutricio¤n y Bromato-
log|¤a, Dpto. C.C. del Medio Natural, Universidad Pu¤blica
de Navarra, Campus Arrosad|¤a s/n, 31006 Pamplona, Nav-
arra, Spain
Roncal Cheese is a ripened uncooked cheese made from
raw ewea¤s milk in the Autonomous Community of Nav-
arra (Spain) and it has the Guarantee of Protected Origin.
The dairies that manufactured this cheese take the milk
from the three main cattle raising areas of Navarra. Re-
cent research carried out in this cheese show that di¡erent
species of Lactobacilli are present in high number (108
CFU/g), although they are not added to the cheese in
the starters. One of the main species found is Lb. planta-
rum. This species included in the group of Non Starter
Lactic Acid Bacteria (NSLAB) contribute to the ¢nal
properties of this cheese. The aim of this study is the
identi¢cation and typing of Lb. plantarum strains in milk
and cheese with the Guarantee of Protected Origin and to
search if the strains present in cheeses come from the orig-
inal milk. The PCR speci¢c reaction to Lb. plantarum was
performed using the speci¢c primers Lb Pl1/Lb Pl2 and
the semi-universal primers Lb1/Lb2. The RAPD reactions
used the primers OPA-3 and P1. Six clusters at the sim-
ilarity level of 50% were obtained. Comparing the strains,
it was observed that the main part of the strains found in
cheese didn’t come from the original milk.
P4^99
EXPLORING MICROBIAL DIVERSITY IN BALTIC
SEA SEDIMENTS
A. Edlund(1), T. Soule(2) and J. K. Jansson(3)
(1) So«derto«rn University College, Department of Natural
Sciences, 141 89 Huddinge, Sweden; (2) University of Ida-
ho, Department of Computer Science, Janssen Engineering
Building, Moscow, ID 83844-1010, USA; (3) Swedish Uni-
versity of Agricultural sciences, Department of Microbiol-
ogy, Box 7025, 750 07 Uppsala, Sweden
The aim of this study was to conduct a survey of the
dominant prokaryotic species in Baltic Sea sediment, a
previously unexplored environment. Sampling took place
218 1st FEMS Congress / Posters 103^505
during September 2002 and started in the inner parts of
the southern Stockholm archipelago. Triplicate sediment
cores were collected from areas which were highly polluted
with heavy metals and PCBs. Samples were also collected
from eutrophied areas which are strongly a¡ected by the
release of sewage treatment e¥uents. References were col-
lected from an area with relatively low levels of pollutants.
Total DNA was extracted from the sediment cores at dif-
ferent depths throughout the pro¢les. Terminal Restriction
Fragment Length Polymorphism (T-RFLP) analysis was
performed using primers speci¢c for eubacterial or archael
16S rRNA genes and the products were digested with
three restriction enzymes. To visualise the changes in com-
munity structures between the sampling areas and between
di¡erent depths, relative abundance values and species-
speci¢c base pair values were correlated using non-para-
metric multivariate analyses. Principal component analy-
ses were performed to test correlations between environ-
mental variables such as pollutant levels, depths and
community structures. Our results suggest that species
abundance changes according to depths and according to
environmental parameters. For bacteria, the species abun-
dance decreased in the areas with heavy metal pollutants
when compared to the reference site. The T-RFLP results
were compared to plate counting on di¡erent media. Pu-
tative identities of individual terminal restriction frag-
ments (TRFs) were estimated using the Ribosomal Data
Base Project II and by comparison to TRFs of isolates.
P4^100
MICROBIAL ECOLOGY OF TNT-CONTAMINATED
SOILS AND ANAEROBIC TNT BIODEGRADATION
PROCESSES
L. Eyers, B. Stenuit, S. El Fantroussi and S. N. Agathos
Unit of Bioengineering (GEBI), Catholic University of Lou-
vain, Place Croix du Sud 2/19, B-1348 Louvain-la-Neuve,
Belgium
2,4,6-trinitrotoluene (TNT) is one of the most common
explosives. In spite of its known toxicity and mutagenicity
for many organisms, soils and groundwater are still fre-
quently contaminated at manufacturing, disposal and
TNT-destruction sites. Fifteen soil samples collected
from two TNT-destruction ¢elds were investigated for
their concentrations of TNT and its derivatives. They con-
tained TNT at various concentrations, ranging from 4 to
114 g TNT/kg soil. Various metabolites were also detected,
including 2,4,6-trinitrobenzaldehyde, 4-amino-2,6-dinitro-
toluene, 2-amino-4,6-dinitrotoluene and 2,4-dinitroto-
luene. Microbial communities of these soil samples were
characterized by means of a 16S rRNA PCR-DGGE tech-
nique using bacterial universal primers. The DGGE pat-
terns of these soil communities were compared with non-
FEMSLE Congress 2-6-03
contaminated soils found at the two TNT-destruction
sites. Non-polluted soils revealed complex ¢ngerprints of
microorganisms while the contaminated samples showed
the presence of dominant bands, indicating that a strong
selection had occurred. Interestingly, some of these con-
taminated soils contained a dominant band matching the
one of the previously isolated TNT-degrading strain Pseu-
domonas sp. JLR 11 (Esteve-Nunez A. and J. L. Ramos,
Env. Sci. Technol. 1998, 32, 3802-3808). The biodegrada-
tion capacities of these polluted soils were evaluated by
enrichment cultures with TNT as sole N-source under an-
oxic conditions. Nitrite release together with growth of the
consortia suggest that anoxic denitration activities oc-
curred. TNT biodegradation activities under Fe-reducing,
sulphate reducing and methanogenic conditions are cur-
rently under investigation.
P4^101
DIVERSITY OF BENZENE-DEGRADING BACTERIA
IN A CONTAMINATED SANDSTONE AQUIFER
A. Fahy(1), A. S. Ball(1), T. J. McGenity(1), A. J.
Hart(2), K. N. Timmis(1)
(1) Department of Biological Sciences, University of Essex,
Colchester CO4 3SQ, UK; (2) Environment Agency, Sol-
ihull B92 7HX, UK
The diversity of aerobic benzene-degrading bacteria from
a contaminated sandstone aquifer was evaluated in a mi-
crocosm experiment. The aquifer, situated below a large
chemical plant, is the focus of research on natural attenu-
ation. Contaminants include BTEX (benzene, toluene, eth-
ylbenzene, xylene), polyaromatic hydrocarbons and chlori-
nated aliphatic hydrocarbons. Benzene is the most
abundant and persistent pollutant, and groundwater sam-
ples were collected from the four most contaminated mon-
itoring wells. Degradation occurred under di¡erent condi-
tions in three groundwater samples: it was stimulated by
the addition of nitrate in one sample, both phosphate and
nitrate in another, but appeared to be inhibited by nitrate
in a third sample. No degradation occurred in a fourth
sample.Microbial community dynamics of the ground-
water in which benzene degradation occurred were moni-
tored using T-RFLP (terminal restriction fragment length
polymorphism), and compared to the communities found
in situ. The inhibition or stimulation of benzene degrada-
tion was re£ected in the community structures. The main
bacterial populations were identi¢ed by cloning and par-
tial sequencing of 16S rDNA. One community was dom-
inated by beta Proteobacteria (90% of the clone library),
another by gamma Proteobacteria (54%); the third com-
prised 47% beta Proteobacteria and 43% Firmicutes. Four
of the dominant benzene-degrading bacteria were isolated
on minimal medium, and were most closely related to the
 1st FEMS Congress / Posters 103^505
following organisms : Hydrogenophaga £ava (98.5% iden-
tity), Pseudomonas anguilliseptica (97.9%), and two di¡er-
ent Rhodococcus erythropolis strains (100 and 99.8%).
P4^102
DIVERSITY OF METHANOGEN ARCHAEA IN
DRAINED FINNISH PEATLAND
P. E. Galand(1), H. Juottonen(1), H. Fritze(2) and K.
Yrjala(1)
(1) Department of Biosciences, Division of general micro-
biology, P.O. Box 56, FIN-00014 University of Helsinki,
Finland; (2) Finnish Forest Research Institute, Vantaa Re-
search Center, P.O. BOX 18, FIN-01301 Vantaa, Finland.
Methane (CH4) is an e¡ective greenhouse gas produced
during anaerobic microbial processes by methanogen
Archaeae. Wetlands (bogs, fens, etc) are the main source
of natural CH4 emission. Possible changes in CH4 produc-
tion in these habitats will have an impact on the global
climate change. Peatlands originally constituted a third of
the land area in Finland, before large areas were drained
for forestry plantations in the 1960’s-70’s. Ash fertilization
in peat lands has been found to promote tree growth and
has been used in Finland to enhance reforestation. Fertil-
isation of peatland is been thought to decrease CH4 emis-
sions but its e¡ects on the community of methane pro-
ducers have never been studied. We report results from a
study on the impact of ash fertilization on the methanogen
Archaea community in Finnish peatland soil. Possible
changes in the community structure were examined in re-
lation to the potential CH4 production of the soil. The
methanogen diversity was studied by using molecular
methods (PCR-DGGE, cloning and sequencing) targeting
the functional methyl coenzyme M reductase gene.
P4^103
DIVERSITY OF ACTINOMYCETES ISOLATED
FROM STONE SURFACES
I. Gonza¤lez, M. I. Cercenado, M. J. Gregorio, A. Villalba,
and O. Genilloud
Centro de Investigacio¤n Ba¤sica de Espa•a (CIBE), Merck
Research Laboratories, Merck Sharp and Dohme de Es-
pa•a, S. A. Josefa Valca¤rcel 38, 28027 Madrid, Spain
It is well known that actinomycetes are one of the major
communities of the microbial population present in soil
but they can also be isolated from a variety of sources
such as microbial mats, marine sediments, seaweeds, li-
chens or stone surfaces among others. The isolation of
actinomycetes of the genus Geodermatophilus and the fam-
FEMSLE Congress 2-6-03
219
ily Nocardiaceae has previously been described from de-
serts and rock surfaces highly exposed to UV radiation [1-
5]. We have attempted the use of di¡erent stones as an
alternative source for the isolation of novel microorgan-
isms to be tested in natural products screening programs.
We have studied the diversity of the actinomycetes popu-
lation isolated from 5 di¡erent samples of stones, includ-
ing limestone, slate and granite rocks collected in Mallorca
island and in the Madrid mountains (Spain). Samples were
treated according to two general isolation methods
coupled to eight selective isolation media and including
the use of di¡erent bacterial growth inhibitors. To assess
the diversity of the microbial population obtained follow-
ing the di¡erent procedures, the 360 isolates were initially
identi¢ed to the genus level on the basis of their micro-
morphology. Among these strains were identi¢ed represen-
tatives of the genus Streptomyces, as well as members of
the families Nocardiaceae, Geodermatophilaceae, Pseudono-
cardiaceae and Micromonosporaceae. As many as 270
Streptomyces isolates were further characterized chemo-
taxonomically on their fatty acid composition. In this
study we evaluate the diversity of the actinomycete isolates
obtained from stones according to the di¡erent nature of
the rock and their taxonomic position.
[1] Eppard et al. (1996) Arch. Microbiol 166, 12-22. [2]
Urzi et al. (2001) Environmental Microbiology 3, 471-
479. [3] Groth et al. (1999) J. Microbiol. Met. 36, 115-
122. [4] Schumann et al. (1997) Int J Syst Bacteriol 47,
278-83. [5] Salazar et al (2003). Accompanying poster at
1st Congress of European Microbiologists.
P4^104
OCCURRENCE OF OLIGONITROPHILIC YEASTS
IN ACID FOREST SOILS
G. GorzaTa and S. Russel
Department of Soil Environmental Sciences, Division of
Agricultural Microbiology, Warsaw Agricultural University,
02-528 Warsaw, ul. Rakowiecka 26/30, Poland
It is estimated that soil microorganisms constitute 25% of
the total biomass on earth. Despite their wide occurrence
and extensive role in soils, only about 40,000 species of
soil microorganisms have been cultured and identi¢ed.
Yeasts are one of the most important groups of soil micro-
organisms, although their ecological role is still insu⁄-
ciently recognized. The term "oligonitrophilic yeasts’’ des-
ignates a physiological group of yeasts able to grow in an
environment containing trace amounts of nitrogen. The
yeasts of genera Lipomyces are the best-known represen-
tatives of oligonitrophilic organisms. This work studied
the occurrence and distribution of oligonitrophilic yeasts
in selected types of acid, forest soils collected in White
Forest about 70 km north-east Warsaw. Sample were tak-
220 1st FEMS Congress / Posters 103^505
en ¢ve-times within vegetation season from six soil types:
pseudogley soil, rusty soil, muck soil, gley-podzol soil, gley
soil and black earth. The total number of oligonitrophilic
yeasts was counted by plate method using nitrogen-free
medium amended with cycloheximide and streptomy-
cin.The plate cultures were incubated for 7 days at 280C,
then 7 days at room temperature. Yeast colonies were
observed under the microscope, counted and isolated for
further characterization. Oligonitrophilic yeasts are con-
centrated near the surface of the pro¢le. The highest num-
ber of yeasts (respectively 312 and 251 CFU per g od d.m.
of soil) was observed in AM horizon of muck soil and in
A horizon of pseudogley soil. In all soils, the highest
amounts of yeasts were found in the upper layers, with
the exception of litter layers where the content of yeasts
ranged from 0 CFU (genetic horizon Ofh of gley soil) to
just 84 CFU (genetic horizon Ofh of gley-podzol soil) per
g of d.m. of litter. The concentration of oligonitrophilic
yeasts in upper horizons of soil pro¢les is to a large extent
the result of the greater abundance of readily available
organic matter. Below 50 cm, yeasts occur sporadically.
20 pure strains of nitrophilic yeasts were isolated and par-
tially characterized. All strains probably belong to Lipo-
myces, with the exception of 1 strain that intensively pro-
duces pseudomycelium.
P4^105
DIVERSITY OF BACTERIA AND ARCHAEA IN BAL-
TIC SEA SEDIMENT
F. Hafirdeman and S. Sjo«ling
Section for Natural Sciences, So«derto«rns ho«gskola, 141 89
Huddinge, Sweden
Marine prokaryotes play a signi¢cant role in remineraliza-
tion of organic matter within the aquatic ecosystem and
are vitally important for the biogeochemical cycling and
eventual degradation of pollutants in the Baltic Sea sedi-
ments. However, the role of bacterial and archaeal diver-
sity in the Baltic Sea benthic ecosystems is still a ‘‘black
box’’ as no empirical data are available. Therefore, 16S
rDNA analysis was used to assess the prokaryotic diver-
sity and community structure of Baltic Sea sediment from
the Asko« archipelgo. Ampli¢ed rDNA restriction analysis
(ARDRA) was performed on 16S rDNA clone libraries
( s 103 unique clones/library) of di¡erent sediment depth.
Results indicate a high bacterial diversity of 16S rDNA
sequences. Approximately 80 % of the screened bacterial
clones showed unique OTUs (restriction patterns). The
diversity of Archaea was lower. Seasonal and spatial dis-
tribution is discussed.
FEMSLE Congress 2-6-03
P4^106
METHANOTROPHIC BACTERIA IN BOREAL FOR-
EST SOIL : LONG-TERM EFFECTS OF PRESCRIBED
BURNING AND ASH FERTILIZATION
K. Jaatinen(1), C. Knief(2), P. Dun¢eld(2), K. Yrja«la«(3)
and H. Fritze(1)
(1) Finnish Forest Research Institute, Vantaa Research
Centre, P.O. Box 18, FIN-01301 Vantaa, Finland; (2)
Max-Planck-Institut fu«r terrestrische Mikrobiologie, Karl-
von-Frisch-Strasse, D-35043 Marburg, Germany; (3) De-
partment of Biosciences, Division of General Microbiology,
P.O. Box 56, University of Helsinki, FIN-00014 Helsinki,
Finland
Methane (CH4) is an e¡ective greenhouse gas and its con-
centration in the atmosphere has been increasing at a rate
of 1% per year. Forest soils are a major terrestrial sink for
atmospheric methane. Methane oxidizing bacteria (MOB)
are responsible for the atmospheric methane consumption.
MOB can use methane as a sole source of carbon and
energy. Little is known, however, about the high-a⁄nity
MOB inhabiting soil, and factors that in£uence their ac-
tivity and diversity in boreal forests. Prescribed burning
and wood ash fertilization are common forestry practises
used for improving the acid neutralization capacity of nat-
urally acidic soils. These practises have been shown to
a¡ect soil microbial community structure and activity.
Ash fertilization (amounts of 1000, 2500 and 5000 kg ha-
1
) and prescribed burning experiments were established in
1990, in a 100 yr old Scots pine (Pinus sylvestris) stand in
Central Finland. Mineral soil samples were taken in 2002
and potential atmospheric CH4 oxidation rates were mea-
sured. MOB were characterized by PCR and DGGE with
primer sets targeting the pmoA gene, coding for the a-sub-
unit of the particulate methane monooxygenase. Even
though the treatments increased soil pH, there was no
linear correlation with methane oxidation rates. The com-
munity structures of MOB were very similar compared to
the control plots. A majority of the pmoA sequences ob-
tained from DGGE bands were only distantly related to
known methanotrophs.
 1st FEMS Congress / Posters 103^505
P4^107
CHARACTERISTICS OF CELLULOLYTIC ACTINO-
MYCETES ISOLATED FROM SELECTED FOREST
SOILS
D. JabTon¤ska-GorzaTa and S. Russel
Department of Soil Environmental Sciences, Laboratory of
Agricultural Microbiology, Warsaw Agricultural University,
Rakowiecka Str. 26/30, 02-526 Warsaw, Poland
Cellulose is a major structural component of plant cell
walls and the most abundant polysaccharide in the bio-
sphere. It is a linear polymer built from 100 to 14,000
glucose molecules linked by -1,4-glycosidic bonds, and is
a highly recalcitrant substrate for enzymatic action. In
soil, its degradation by di¡erent systematic groups of cel-
lulolytic microorganisms, e.g. bacteria, actinomycetes and
fungi, represents a signi¢cant part of the carbon cycle.
Although soil actinomycetes, including members of Strep-
tomyces, Micromonospora, Streptosporangium and Ther-
momonospora, are one of the most active groups of cellu-
lolytic microorganisms, they remain poorly characterised.
The aim of this study was to evaluate the quantitative and
qualitative occurrence of cellulolytic actinomycetes in mor-
phologically di¡erent soil types in White Forest located 70
km north-east of Warsaw. The soil samples were collected
¢ve times within vegetation season from six soil types:
pseudogley soil, rusty soil, muck soil, gley-podzol soil,
gley soil and black earth. The total number of actinomy-
cetes in the soil samples was analyzed by plate method:
using mineral medium containing ¢lter paper as a sole
carbon source. Isolated strains were classi¢ed by morpho-
logical, physiological and biochemical characteristics. The
activity of enzymes degrading ¢lter paper (FP-ase) and
carboxymethyl cellulose (CMC-ase) was assayed by Man-
dels method. For electron microscopy of cellulosomes, the
cytochemical technique with cationionized ferritine CF
was used. The highest amounts of actinomycetes were
found in muck soil. The greatest concentration of actino-
mycetes population was observed in the organic, upper
horizons of soils. 25 strains of cellulolytic actinomycetes
were isolated and characterized as belonging to genera
Streptomyces, Micromonospora and Streptosporangium.
Electron microscopy showed the presence of cellulosome
resembling structures which appear as spherical units on
the surface of bacterial cells. It was possible to observe
penetration of actinomycete mycelium through the cellu-
lose ¢bres using the laser confocal scanning microscope.
FEMSLE Congress 2-6-03
221
P4^108
CHARACTERIZATION OF METHANE-OXIDIZING
BACTERIAL COMMUNITIES IN UPLAND SOILS
WITH MOLECULAR METHODS
C. Knief and P. F. Dun¢eld
Max-Planck-Institute for Terrestrial Microbiology, Karl-
von-Frisch Str., 35043 Marburg, Germany
We characterized the community of methane-oxidizing
bacteria (MOB) in di¡erent upland soils that displayed
atmospheric methane uptake. For molecular characteriza-
tion, soil DNA was extracted and the gene of the partic-
ulate methane monooxygenase subunit A (pmoA), a useful
phylogenetic marker for MOB, was ampli¢ed by PCR.
Mixed PCR-products were separated by denaturing gra-
dient gel electrophoresis and individual bands were se-
quenced. Comparative sequence analysis to a pmoA-data-
base revealed that some detected sequences were closely
related to sequences of the genera Methylocaldum, Meth-
ylosinus and Methylocystis. Further sequences belonged to
two di¡erent phylogenetic clusters for which there are no
known cultured representatives. The ¢rst cluster was pre-
viously detected in acidic upland soils and is related to
pmoA-sequences of K-Proteobacteria. In several pH-neu-
tral upland soils we found a second cluster of sequences
distantly related to pmoA-sequences of the Q-Proteobacte-
ria (upland soil cluster Q). Evidence that not only K-Pro-
teobacteria but also Q-Proteobacteria are involved in the
process of atmospheric methane uptake in soils was given
by incubating selected soils with 13C-labeled methane at a
mixing ratio below 50 ppmv. All selected soils contained
pmoA-sequences of the upland soil cluster Q but no other
pmoA-sequences related to Q-Proteobacteria. In some soils
pmoA-sequences related to K-Proteobacteria were also
present. In all soils phospholipid fatty acids 14:0,
16:1g7c and 16:0, characteristic for methane-oxidizing Q-
Proteobacteria, were labeled with 13C. This suggests that Q-
Proteobacteria contribute to the process of atmospheric
methane oxidation and that the organisms containing
pmoA sequences of the upland soil cluster Q are indeed
methanotrophic.
P4^109
DYNAMICS OF ARCHAEAL COMMUNITIES IN
ARABLE SOILS
A. Gattinger(1), M. Schloter(1), M. G. Ho«£e(2) and M.
Labrenz(2)
(1) GSF ^ Forschungszentrum fu«r Umwelt und Gesundheit
GmbH, Institut fu«r Bodeno«kologie, 85764 Neuherberg, Ger-
222 1st FEMS Congress / Posters 103^505
many; (2) GBF ^ German National Research Centre for
Biotechnology, 38124 Braunschweig, Germany
In an interdisciplinary research project interactions be-
tween organic matter and microorganisms in arable soils
from three di¡erent research sites are investigated. The soil
in Scheyern, a Gleyic Cambisol is integratedly managed,
whereas the Orthic Luvisol in Merzenhausen is managed
conventionally at a normal intensity level. In Bad Lauch-
sta«dt three di¡erent fertilisation levels of a Haplic Phae-
zoem (HP) are investigated (no fertilisation, mineral and
mineral+organic fertilisation). Phospholipid fatty acid
(PLFA) pro¢ling as well as single strand conformation
polymorphism (SSCP) of samplings taken in spring,
summer and autumn revealed that microbial community
composition were di¡erent in the three soil types accord-
ing to soil type, fertilisation level and soil depth. More-
over, Archaea-speci¢c phospholipid etherlipids (PLEL)
with distinctive pro¢les were predominantly detected in
samples from organically fertilized HP. Molecular analyses
of archaeal 16S rDNA extracted from SSCP-gels revealed
that these sequences belonged to the Euryarchaeota (Ther-
moplasma and methanogens) as well as to uncultured
Crenarchaeota. However, sequences clustering with Ther-
moplasma were only distantly related to cultivated strains
or environmental clones (82 ^ 84 % similarity). In samples
without organic enrichment signi¢cantly lower PLEL con-
centrations were measured indicating that archaeal abun-
dance is rather in£uenced by fertilisation than by soil type.
P4^110
ESTIMATION OF PSEUDOMONAS POPULATIONS
IN SOIL
G. Lloyd-Jones(1), A. Tizzard(2), A. D. Laurie(1)
(1) Landcare Research, PO Box 69, Lincoln 8152, New
Zealand; (2) Lincoln Technology, Lincoln Ventures Lim-
ited, PO Box 133, Lincoln, Christchurch, New Zealand
Pseudomonas are fast growing and nutritionally versatile
bacteria that are able to utilise a wide variety of carbon
sources. The abundance of the genus has been highlighted
by conventional microbiology and the genus is well repre-
sented in collections of cultured bacteria. We have eval-
uated the culturability of the Pseudomonas population by
comparing culture-dependent with culture-independent ap-
proaches for estimating populations in two New Zealand
soils. Four di¡erent incubations, a beech-forest soil and a
permanent pasture soil incubated at room temperature
under constant moisture conditions and also by exposure
to toluene vapour, were used to corroborate our observa-
tions. Total Pseudomonas populations were enumerated
using quantitative £uorogenic PCR (Taqman) to target a
63-bp amplicon within the 16S gene that is highly speci¢c
FEMSLE Congress 2-6-03
for the £uorescent Pseudomonas (sensu stricto) group of
bacteria, and the culturable £uorescent Pseudomonas pop-
ulation by plating dilutions of soil extracts onto Pseudo-
monas-speci¢c agars (King’s B and Gould’s S1) and count-
ing £uorescent colonies. Incubation of these soils led to
population changes manifested as a signi¢cant increase in
the total Pseudomonas population as estimated using the
culture-independent method, while the culturable popula-
tion showed at least a 10-fold decrease. The culturable
Pseudomonas population (a genus thought highly cultura-
ble) represented only a small fraction (V0.1%) of the total
Pseudomonas population present in these soils. New iso-
lation approaches to target previously uncultured mem-
bers of the genus should provide a rich source of material
with potentially useful properties for biotechnological ap-
plications.
P4^111
EFFECTS OF TETRACYCLINE ANTIBIOTICS ON
SOIL MICROBIAL COMMUNITY PROFILES
L. Macovei, G. Jandl, S. Thiele-Bruhn
Institute of Soil Sciences and Plant Nutrition, University of
Rostock, Justus-von-Liebig Weg 6, 18059 Rostock, DE
Antibiotic pharmaceuticals administered to livestock are
for the most part excreted. With the contaminated manure
used as fertiliser, they are spread onto agricultural land.
Consequently residual concentrations of antibiotics in soils
were reported. Since antibiotics are highly e¡ective sub-
stances even at low concentrations, they a¡ect soil micro-
organisms. This was determined for various activity pa-
rameters, total microbial biomass and the concentration
of ergosterol. The aim of this study was to identify in
more detail the e¡ects of tetracycline antibiotics on the
community pro¢le of soil microorganisms. For this pur-
pose phospholipid fatty acid (PLFA) patterns of two dif-
ferent soils were analysed, following incubation after soil
amendment with di¡erent tetracycline compounds of var-
ied.
P4^112
SPATIAL LINKAGE BETWEEN NirK DIVERSITY
AND DENITRIFICATION ACTIVITY IN THREE
ARABLE FIELDS
S. M. Mitchell, R. E. Wheatley, J. Squires and T. J. Dan-
iell
Plant Soil Interactions, Scottish Crop Research Institute,
Invergowrie, Dundee, DD2 5DA, Scotland, UK
Denitri¢cation is an anaerobic process in which nitrogen
 1st FEMS Congress / Posters 103^505
compounds are utilised as alternative electron acceptors
for respiration. Incomplete denitri¢cation can lead to the
release of nitrogen oxide gases which are potent green-
house gases. Biological denitri¢cation is widespread in
prokaryotic systems with a wide range of bacterial and
archaeal species capable of the process. Nitrite reductase
is a key enzyme in denitri¢cation catalysing the conversion
of nitrite (NO2-) to nitric oxide, a form which is no longer
available to most biological systems. A high throughput
sequence approach was utilized to analyse the sequence
diversity of a nitrite reductase gene in soil sampled from
three Scottish arable ¢elds. It has been previously ob-
served that the gene encoding the copper containing nitrite
reductase is dominant in terrestrial systems. A fragment of
the NirK gene was ampli¢ed by PCR, cloned and approx-
imately 1000 clones have been sequenced. Rarefaction
analysis suggests that this sampling has exhausted all dom-
inant types from this system. Sequence information gen-
erated has been used to design a T-RFLP strategy allow-
ing for high throughput analysis of NirK relative
abundance. Potential and actual nitri¢cation and denitri-
¢cation rates have been measured in one of the three ¢elds
and shifts have been observed both temporally and spa-
tially at a range of scales. Work is in progress to link
activity measures for denitri¢cation and the diversity of
the NirK gene in order to examine if £uctuations in activ-
ity re£ect shifts in the structure of the denitrifying com-
munity.
P4^113
A METAGENOMIC FOSMID LIBRARY OF WADDEN
SEA SEDIMENTS
M. Mu_mann, J. Ku«ver, B. Meyer, A. Ellrott, R. I. Amann
Max Planck Institute for Marine Microbiology, Celsiusstr.
1, 28359 Bremen, Germany
The metabolic properties of environmentally abundant but
uncultivated bacteria are mostly unknown. Recent inves-
tigations have demonstrated the power of the metagenom-
ic approach in elucidating potential activities of these bac-
teria (Beja¤ et al. 2000). Therefore we established a
metagenomic fosmid library (V 34,000 clones) from wad-
den sea sediment DNA with average insert sizes of 38 kb.
The library was screened for adenosine 5’-phosphosulfate
reductase (APS-reductase), a functional and phylogenetic
marker gene of putative sul¢de oxidizing and sulfate re-
ducing bacteria. Flanking regions of the APS-reductase
gene were sequenced and annotated to look for encoding
functional genes. The diversity of the APS gene in PCR-
and non-PCR-based libraries was compared with the
16S rDNA diversity of PCR based clone libraries. The
environmental relevance of putative sul¢de oxidizing and
FEMSLE Congress 2-6-03
223
sulfate reducing bacteria was shown by £uorescence in situ
hybridization (FISH).
Reference : Beja¤ et al., Nature 2000
P4^114
MYXOBACTERIA OF THE SOUTH-WEST OF UK-
RAINE
O. L. Rakhimova, V. O. Ivanitsa
Odessa National University, Microbiology and Virology De-
partment, Dvoryanskaya St. 2, Odessa, 65 026, Ukraine
24 myxobacteria strains were isolated from the soils of
Ukraine south-west and Bleak sea contact zones. Isolated
strains were identi¢ed as species Myxococcus fulvus, Myx-
ococcus xanthus, Myxococcus stipitatus, Archangium ge-
phyra, Cystobacter fuscus, Polyangium vitellinum, Nanno-
cystis exedens. The isolated strains have wild spectrum of
lytic enzymes and can produce antagonistic substances.
Certain myxobacteria strains were included to Ukrainian
Collection of Microorganisms (UCM). Long term preser-
vation possibility of these strains was studied. It was
shown that addition of antioxidants: cysteamine, ionol,
K-tokopherol to protective medium (sucrose-gelatin agar)
doesn’t in£uence on the viability of myxobacteria lyophi-
lizated cells. It was found the possibility for using of the
accelerated storage test in the case of lyophilizated M.
xanthus UCM 10041 and P. cellulosum UCM 10043 via-
bility predicting. The biological rhythms in vital activity of
myxobacteria strains M.xanthus UCM 10041, P.cellulosum
UCM 10043 was found. Part of viable cells and ability to
form fruiting bodies are £uctuating cyclic. The e¡ect of
heavy metals on physiological activity, gliding motility,
fruiting body formation was estimated. Ability of M. xan-
thus strains to accumulate heavy metals was estimated too.
It was shown that M. xanthus strains are able to accumu-
late zinc, cadmium, nickel, chromium, copper, lead in the
quantity close to their dry weight. Each from the men-
tioned heavy metals at certain concentration is toxic for
the studied M. xanthus strains. Toxic e¡ect was manifested
as depression of growth, worsening of motility, breaking
of fruiting body formation, reducing of respiration and
biomass synthesis. However, active adsorption of heavy
metals occurs even under such conditions when heavy met-
als have concentrations, which are toxic for these strains.
224 1st FEMS Congress / Posters 103^505
P4^115
MICROBIAL POPULATIONS IN DEEP MARINE
SEDIMENTS
H. Sass, B. Engelen and H. Cypionka
Institute for Chemistry and Biology of the Marine Environ-
ment, University of Oldenburg, P.O. Box 2503, D-26111
Oldenburg, Germany
Only a small percentage of the prokaryotes present in ma-
rine sediments can be cultured in traditional media. Most
sediment bacteria are classi¢ed as ‘unculturable’, although
they have thrived in their natural environment. Aim of
study is to enhance the cultivation success of marine sedi-
ment bacteria. Our approaches involve some new aspects
designed to increase the cultivation success. These include
the use of non-selective but de¢ned media with di¡erent
carbon sources, like polymers and monomers, all at low
concentrations. Other variations are the use of substrate
gradients, the variation of the incubation temperature, and
the addition of particles (e.g. FeS precipitates). Promotion
of syntrophic interactions is achieved by addition of back-
ground bacteria to MPN series. In our media the bacteria
cannot grow to high densities. Therefore growth analysis
has to make use of sensitive detection techniques, like
counting of grown bacteria after staining with £uorescent
DNA stains, FISH or other molecular biological tech-
niques like PCR and DGGE. These di¡erent cultivation
techniques were applied to sediment samples from the
North Sea taken from depths down to 6 m and to sedi-
ments from the Eastern Mediterranean Sea with ages of
up to 200 000 years. The di¡erent cultivation approaches
were compared with respect to the cultivation success and
the dominating phylogenetic and physiological types ob-
tained.
P4^116
BIODIVERSITY OF THE MICROORGANISMS NEAR
AN OUTCROP OF GAS HYDRATES OF LAKE BAI-
KAL
O. V. Shubenkova(1), T. I. Zemskaya(1), O. M. Khlys-
tov(1), S. M. Chernitsina(1), B. B. Namsaraev(2), O. P.
Dagurova(2)
(1) Limnological Institute SB RAS, Box 4199, 664033,
Irkutsk, Russia ; (2) Institute of general and Experimental
Biology of Siberian Branch of RAS, Ulan-Ude, Russia
Bacterial biodiversity of the sediments sampled in the
Southern Basin of Lake Baikal (at the water depth of
1400 m) near the gas hydrates outcrop was studied. Ear-
lier, existence of gas hydrates here was predicted by nu-
FEMSLE Congress 2-6-03
merous studying (Rensbergen et al., 2001). Area near the
outcrop was determined by geophysics data (M. De Batist
et al., 1998, 1999, 2000; Klerx et al., 2000). Sediments
were analyzed with using complex methods of geochemis-
try and microbiology. The crystals are situated in silty
gray clays. Microbiological studying was realized with us-
ing classic and molecular biology methods. Bacteria grow-
ing were noted on media with adding methane in anaero-
bic and aerobic conditions. Total bacterial DNA was
extracted from sediments and its concentrations were mea-
sured. It was 1.17 Wg/g sediment for surface sediment. In
deeper sediment horizons the concentration was lower.
Possibilities of spectrophotometric method were limited
by equipment sensitiveness. However polymerase chain re-
action was successful that suggests of DNA presence.
Methanotrophic bacteria were found in the surface sedi-
ment. Archebacreria and sulfatereducing bacteria were
found in dipper horizons. This biodiversity is compounded
on the data of 16S RNA. Analysis of lipids con¢rms it.
Studying of bacterial biodiversity of this area is continu-
ous.
P4^117
IDENTIFICATION OF INTRONS IN BACILLUS
STRAINS ISOLATED FROM SOIL
S. Stankovic(1), V. Lazarevic(2), T. Beric-Bjedov(1), J.
Knezevic-Vukcevic(1), D. Simic(1)
(1) Faculty of Biology, University of Belgrade, Yugoslavia;
(2) Institut de Microbiologie Fundamentale, Batiment de
Biologie, Universite de Lausanne, Switzerland
Collection of Bacillus sp. strains isolated from soil was
screened by low stringency PCR, using oligonucleotides
corresponding to the insertion sites of the three interven-
ing sequences in the B. subtilis 168 SPL prophage ribonu-
cleotide reductase genes bnrdE and bnrdF. In 3 (SS6, SS72
and SS114) out of 212 strains tested strong bands were
generated. The sequencing of the PCR products revealed
one (SS114), two (SS72) and three (SS6) group I introns in
the bnrdE-bnrdF tandem. The introns that are inserted at
the analogous positions have the same length and exhibit
over 97% identity. SS6 bnrdE-I1, SS72 bnrdE-I1 and their
168 homologue are 252 nt long and contain no ORF. SS6
bnrdE-I2, SS72 bnrdE-I2 and SS114 bnrdE-I span 280 nt
and also contain no ORF. bnrdF-I from strain SS6 con-
tains a putative HNH endonuclease ORF that is 98%
identical to its counterpart. In B. subtilis 168 the nrdE
and nrdF genes are separated by 17 nt. The bnrdE-bnrdF
intergenic spacers in the three strains are much longer,
ranging from 230 to 602 nt. The bnrdE-bnrdF intergenic
region in the strain SS6 failed to disclose any protein cod-
ing sequences. In contrast, bnrdE-bnrdF intergenic regions
in strains SS72 and SS114 contain highly similar ORFs
 1st FEMS Congress / Posters 103^505
(orf435) whose deduced products have no homologues in
databases. The in vivo intron splicing was demonstrated by
reverse transcription, followed by PCR ampli¢cation of
synthesized cDNA. The exact exon-intron junctions were
con¢rmed by cloning and sequencing of the RT-PCR
products.
P4^118
DIVERSITY OF SULFATE-REDUCING BACTERIA
FROM THE ROMANIAN BLACK SEA BOTTOM
SEDIMENTS
E. Stoica(1), J. Kuever(2), M. Dragan-Bularda(3)
(1) National Institute for Marine Research and Develop-
ment ‘‘Grigore Antipa’’, Bd. Mamaia 300, Constanta RO-
8700, Romania; (2) University of Bremen, Center for En-
vironmental Research and Technology, Institute for Soil
Science, Leobener Strasse, D-28359 Bremen, Germany ;
(3) Babes-Bolyai University, Department of Plant Biology,
3400 Cluj-Napoca, Romania
Seven strains (Et1, But1, But2, But3, Lac1, Isob1, Benz1) of
Gram negative, mesophilic, nonsporing sulfate-reducing
bacteria were isolated from bottom sediments of the Ro-
manian Black Sea coast. Analysis of partial 16S rDNA
sequences obtained from pure cultures of isolated by
PCR, revealed that all strains belonged to the N ^ subclass
of Proteobacteria. Three strains (But1, But3, Lac1) were
morphologically and nutritionally similar. According to
their 16S rDNA sequences, the isolates were a⁄liated
with the following species: Desulfofrigus fragile (But1,
But3, Lac1, 97.9 ^ 98% similarity), Desulfovibrio acrylicus
(Et1, 98% similarity), Desulfobacterium autotrophicum
(But2, 99% similarity), Desulfobacterium niacini (Isob1,
99% similarity). This is a ¢rst description of sulfate-reduc-
ing bacteria diversity from the Romanian Black Sea coast,
after the recent changes of environmental conditions from
the NW shelf of the Black Sea as a consequence of eutro-
phication.
FEMSLE Congress 2-6-03
225
P4^119
DIVERSITY OF nosZ GENE FRAGMENTS IN NA-
TIVE AND CULTIVATED SOIL
B. Stres(1,3), I. Mahne(1), G. Avgustin(2), J. M.
Tiedje(3)
(1) Dept. of Food Science and Technology, Biotechnical
Faculty, University of Ljubljana, Vecna pot 111, 1000
Ljubljana, Slovenia ; (2) Zootechnical Dept., Biotechnical
Faculty, University of Ljubljana, Groblje 3, 1234 Domzale,
Slovenia; (3) Michigan State University, Center for Micro-
bial Ecology, PSSB 540, East Lansing, MI -48824, USA
Denitri¢cation in conjunction with nitri¢cation leads to
loss of plant available nitrogen compounds in soil habi-
tats. Agricultural soil at Kellogg Biological Station, MSU,
USA, has been subjected to de¢ned long-term ¢eld experi-
ments to evaluate the e¡ects of land use on various soil
properties (http://lter.kbs.msu.edu). In the frame of this
experiment the two most divergent plots, i.e. cultivated
(standard chemical input, crop rotation, conventional till-
age) and native (unspoiled reference site, early successional
community, never tilled), were selected to analyze indige-
nous nosZ gene diversity. The gene nosZ codes for nitrous
oxide reductase, the last enzyme in denitri¢cation chain
and is believed to be present in majority of denitrifying
bacteria. Rarefaction analysis of more than 550 nosZ
clones revealed major di¡erences between the two com-
munities and T-RFLP analysis of ampli¢ed indigenous
nosZ fragments gave similar results. However, further phy-
logenetic analysis of 48 selected clones representing major
groups of clones and some randomly selected clones re-
vealed less drastic di¡erences in the two communities,
pointing to rather minor changes caused by the two treat-
ments and raising an interesting question of suitability of
restriction pro¢ling to describe complex microbial com-
munities.
P4^120
HETEROTROPHIC BACTERIAL DYNAMICS AND
DIVERSITY IN ANNECY, BOURGET AND GENEVA
LAKES
U. Dorigo, S. Jacquet and J. F. Humbert
UMR CARRTEL, INRA, BP 511, 74203 Thonon-les-Bains
cedex, France
A comparative study of the dynamics and diversity of
heterotrophic bacterioplankton of three Alpine lakes in
France was undertaken. These lakes are characterised by
di¡erent nutrient loads and in particularly by the presence
(lake Geneva and lake Bourget) or absence (lake of Ann-
226 1st FEMS Congress / Posters 103^505
ecy) of toxic proliferations of the cyanobacterium Plank-
tothrix rubescens. Each lake was sampled at least once a
month and at di¡erent water depths in order to establish
relationships between toxic proliferations and changes in
the bacterioplankton community, which may be responsi-
ble of the lysis of the cyanobacterial cells and toxins deg-
radation. PCR-Denaturing Gradient Gel Electrophoresis
(DGGE) and sequencing of 16S rDNA were performed
to examine the bacterioplankton community composition
and to observe changes in the numerically dominating
populations. The DGGE-patterns were analysed in rela-
tion to lake characteristics, season and dynamics of Plank-
tothrix rubescens. The phylogenetic a⁄liation of the pre-
dominant members of the microbial communities
(Proteobacteria, Cytophage-Flavobacterium-bacteroides)
was also inferred by £uorescence in situ hybridisation
(FISH). Flow cytometry was used to establish the total
number of heterotrophic bacteria and to discriminate ma-
jor populations. First results indicate that the diversity and
dynamics of the bacterioplankton vary in relation to the
lake and the period of study. Temporal di¡erences in com-
munity composition seem to be greater than the spatial
di¡erences during either season.
P4^121
MICROBIAL DIVERSITY OF MID-ATLANTIC
RIDGE HYDROTHERMAL VENTS : FROM CUL-
TURE TO COMMUNITY MOLECULAR FINGER-
PRINTS
M. Gadanho(1), S. Chaves(2), T. Tenreiro(2), J. P. Sam-
paio(1) and R. Tenreiro(2)
(1) Centro de Recursos Microbiolo¤gios (CREM), Secca‹o
Auto¤noma de Biotecnologia, Faculdade de Cie“ncias e Tec-
nologia, Universidade Nova de Lisboa, 2829-516 Caparica,
Portugal; (2) Centro de Gene¤tica e Biologia Molecular,
Departamento de Biologia Vegetal, Faculdade de Cie“ncias
de Lisboa, Rua Ernesto Vasconcelos, Ed. C2, Piso 4, Cam-
po Grande, 1749-016 Lisboa, Portugal
On August 2002, ¢ve hydrothermal sites (Menez Gwen,
Rainbow, Lucky Strike, Saldanha Mount and Menez
Hom) located on the Mid-Atlantic ridge were visited dur-
ing the Portuguese mission SEAHMA-1. Deep-sea sam-
pling was performed using the submersible VICTOR
6000 on board of the French research vessel L’Atalante.
Samples obtained from animals, sediment, chimneys and
water were immediately processed on-board for enrich-
ment, in several culture media, under aerobic and anaer-
obic conditions, and at incubation temperatures ranging
from 10 ‡C to 85 ‡C. Crude samples were also frozen at ^
70‡C for further analysis. A total of nearly 500 isolates
belonging to the Domains Bacteria and Archae and to the
Kingdom Fungi (yeasts) were isolated in pure culture from
FEMSLE Congress 2-6-03
the on-board enrichments. DNA was extracted from all
the isolates and M13-PCR ¢ngerprinting was performed
for similarity grouping. From each M13-cluster one isolate
was chosen and 16S or 26S rDNA sequencing was per-
formed for phylogenetic allocation. Preliminary results in-
dicated that anaerobic isolates obtained at the highest
temperatures were mainly members of the Thermococ-
cales, Thermotogales and Clostridiales. Yeast isolates
were obtained at 20-25‡C. In parallel, total DNA was
extracted from the crude samples and from all the enrich-
ments. After ampli¢cation with primers for 16S and 18S
rDNA, the amplicons were separated by Temperature
Gradient Gel Electrophoresis (TGGE) generating molecu-
lar community ¢ngerprints. Cluster analysis of these pro-
¢les and identi¢cation of common and speci¢c organisms
by direct sequencing of TGGE bands, followed by com-
parison with the culture-based approach, allowed the as-
sessment of microbial diversity of sampled hydrothermal
vents.
This work was supported by FCT, Project SEAHMA
PDCTM/C/MAR/15281/99.
P4^122
GENUS EUROTIUM ^ HALOPHILIC FUNGAL IN-
HABITANTS OF HYPERSALINE WATERS IN THE
SALTERNS
N. Gunde-Cimerman(1), J. C. Frisvad(2), L. Butinar(3)
and P. Zalar(1)
(1) Biology Department, Biotechnical Faculty, University
of Ljubljana, Vec›na pot 111, 1000 Ljubljana, Slovenia;
(2) BioCentrum-DTU, Technical University of Denmark,
2800 Kgs. Lyngby, Denmark; (3) National institute of
Chemistry, Department of Biotechnology, Hajdrihova 19,
1000 Ljubljana, Slovenia
In the course of the mycodiversity study of hypersaline
environments, di¡erent species of the known food-borne
xerophilic genera, such as Wallemia, Penicillium, Aspergil-
lus, and its teleomorphic stage, Eurotium as well as halo-
philic black yeasts were isolated from the man made salt-
erns. Genus Eurotium was represented by ¢ve species: E.
amstelodami, E. herbariorum, E. repens, E. rubrum and E.
chevalieri. Strains of E. amstelodami were most consis-
tently isolated from the Slovenian salterns and later as
well in other salterns (Spain, Israel, Dominican Republic,
Namibia), while E. herbariorum, E. repens and E. rubrum
were isolated frequently. Therefore these species probably
contribute to the indigenous fungal community in hyper-
saline environments. To determine their halophilic adap-
tation to long-term survival in hypersaline environments,
spores of E. amstelodami, E. herbariorum, E. repens and E.
rubrum were in vitro exposed to prolonged suspensions in
water with di¡erent salt concentrations. They survived
 1st FEMS Congress / Posters 103^505
from 0-30% NaCl for three months and more. Only E.
chevalieri spores did not survive the long-term exposure
to NaCl concentrations from 20-30% and were recovered
as well from the hypersaline waters just once. Therefore E.
chevalieri is probably a temporal inhabitant of brine at
lower salinities.
P4^123
BLACK HOLES OF SOUTH ANDROS, THE BAHA-
MAS : WHAT THEY ARE AND WHY THEY ARE
BLACK
R. A. Herbert(1) and S. Schwabe(2)
(1) Division of Environmental and Applied Biology, Biolog-
ical sciences Institute, University of Dundee, Dundee DD1
4HN, Scotland; (2) International Blue Holes Foundation,
7/121 Sir Fred Schonell Drive, St. Lucia, Brisbane, Queens-
land, Australia 4067
Black holes are vertical limestone cave systems which have
no known lateral passages. One particularily spectacular
black hole in the Bahamas is the ‘‘Black Hole of South
Andros which has an opening of 300m diameter with a
water depth of 47m. The water column is strati¢ed with an
upper oxic low salinity water mass overlying a deeper sa-
line layer with a salinity of 33-35psu. Temperature pro¢les
show an almost uniform 290 C in the upper water layer
(17.8m) with a very sharp increase at the pycnocline to
360C.Co-incident with the pycnocline is a 1m thick layer
of phototrophic purple sulfur bacteria with a cell density
of V107/ml. Assuming a uniform cell density this equates
to a dry weight biomass of 5 tonnes. Classical taxonomy
and 16S rDNA sequencing has identi¢ed the dominant
phototrophic bacteria as belonging to the genera Allochro-
matium and Thiocapsa. Both bacteria are characterised by
the presence of bacteriochlorophyll a and carotenoids of
the normal spirilloxanthin series. Spirilloxanthin absorbs
maximally at 550nm which prevents light scattering which
would explain why from the air the water surface appears
black. Both bacteria have a low e⁄ciency (20-30% ) in
channelling captured light energy to the photosynthetic
reaction centres. We postulate that the excess captured
light is dissipated as heat which would account for the
sharp increase in temperature recorded at the pycnocline.
These data will be discussed with respect to the sulfur
cycle operating in these unique ecosystems.
FEMSLE Congress 2-6-03
227
P4^124
LACK OF GENETIC DIFFERENTIATION IN
BENTHIC AND PELAGIC SUB-POPULATIONS OF
MICROCYSTIS AERUGINOSA IN A FRENCH STOR-
AGE RESERVOIR
J. F. Humbert(1), D. Duris-Latour(2), B. Le Berre(1) and
M. J. Salencon(3)
(1) UMR CARRTEL, INRA, BP 511, 74203 Thonon-les-
Bains Cedex, France; (2) Universite¤ J. Monnet, Lab. de
Biologie Animale et Applique¤e, 42023 St Etienne, France;
(3) EDF, Laboratoire National d’Hydraulique et Environ-
nement, Site de Chatou, 6, Quai Watier, 78401 Chatou
Cedex
We compared the genetic diversity of the ITS1 of the
rRNA gene in benthic and pelagic sub-populations of Mi-
crocystis aeruginosa isolated at two di¡erent sampling sta-
tions and at di¡erent sampling time (winter and summer)
in the French storage reservoir of Grangent. For each
sampling point, a gene clone library was constructed after
PCR ampli¢cation of the large ITS1 fragment. Genetic
diversity was estimated by random sequencing of several
clones per library. 66 ITS1 sequences could be obtained.
Nucleotide diversity of all the sampling sub-populations
was in the same range (average number = 0.022) whatever
their origin revealing that di¡erent clones are involved
during the summer bloom event and contribute to the
high biomass production. By phylogenetic study and by
analysis of molecular variance (AMOVA), we also found
no genetic di¡erentiation between benthic and pelagic sub-
populations. Our result con¢rm data on population dy-
namics for these two sub-populations and show that life
cycle of M. aeruginosa in temperate area is characterized
by a benthic phase in winter and spring that allows the
survey of this organism in unfavorable environmental con-
ditions, followed by a pelagic phase in summer and au-
tumn when environmental conditions permits their
growth.
228 1st FEMS Congress / Posters 103^505
P4^125
FEMTO-, PICO- AND ULTRAPLANKTON DYNAM-
ICS AND DIVERSITY IN THE 3 LARGEST NATURAL
FRENCH ALPINE FRESHWATER ECOSYSTEMS:
ANNECY, BOURGET AND GENEVA LAKES
S. Jacquet(1), U. Dorigo(1), J.-F. Humbert(1) and I. Bie-
gala(2)
(1) UMR CARRTEL, Station INRA d’Hydrobiologie La-
custre, 75 Avenue de Corzent, 74203 Thonon, France; (2)
Station Biologique de Rosco¡, Equipe Phytoplancton Oce¤-
anique, Place Georges Tessier, 29680 Rosco¡, France
To date, almost nothing is known about microbial com-
munities inhabiting the three largest and deepest French
alpine lakes, i.e. Annecy, Bourget and Geneva lakes. Us-
ing a combination of techniques and methods such as
analytical £ow cytometry and sorting, epi£uorescence mi-
croscopy, PCR-DGGE, culture enrichment, TSA-FISH, a
¢rst attempt was made to observe and compare the com-
position and dynamics of viral, heterotrophic bacterial and
picophytoplanktonic communities within these 3 ecosys-
tems. The poster will present our ¢rst results and research
perspectives.
P4^126
DESIGN AND EVALUATION OF 16S rRNA OLIGO-
NUCLEOTIDE PROBES TO ASSESS THE STRUC-
TURAL COMPOSITION OF THE ARCHAEAL COM-
MUNITIES THRIVING IN HOT BIOTOPES
O. G. Nercessian(1), M. I. Prokofeva(2), A. V. Lebedin-
skii(2) and C. Jeanthon(1)
(1) UMR 6539, Centre National de la Recherche Scienti-
¢que and Universite¤ de Bretagne Occidentale, Institut Uni-
versitaire Europe¤en de la Mer, 29280 Plouzane¤, France; (2)
Institute of Microbiology, Russian Academy of Sciences,
Prospect 60 Let Oktyabrya 7/2, 117811 Moscow, Russia
Hyperthermophilic cultured Archaea and several new un-
cultivated lineages are usually encountered in hot bio-
topes. However, because little e¡ort has been made to
study the structural composition of these microbial com-
munities, molecular tools such as 16S rRNA-based oligo-
nucleotide probes remain poorly available. We developed
and validated by dot-blot hybridizations sixteen archaeal
oligonucleotide probes targeting thermophilic microorgan-
isms and environmental clades frequently detected in high-
temperature ecosystems. These probes were designed to
target the sulfur-reducing heterotrophic Thermococcales,
the sulfate-reducers Archaeoglobus, the lithoautotrophic
methanogens Methanococcales and Methanopyrus, the De-
FEMSLE Congress 2-6-03
sulfurococcales, the Pyrodictiaceae and Ignicoccus, the
thermoacidophiles Sulfolobales and Thermoproteales. We
also designed probes targeting uncultivated archaeal
groups so far detected in hydrothermal ecosystems: these
included the Korarchaeota and the groups 2 and 8 of
Deep-sea Hydrothermal Vent Euryarchaeotes. The probes
allowed us to determine the structure of archaeal commu-
nity associated with colonization devices deployed at 13‡N
(East Paci¢c Rise) and chimney and sediment samples
originating from geographically distant deep-sea hydro-
thermal ecosystems (9‡N on the East Paci¢c Rise and
36‡N on the Mid-Atlantic Ridge). The Pyrodictiaceae, Sul-
folobales, Thermoproteales and Korarchaeota were not de-
tected but the remaining probes gave positive signals in
most samples. The richness of hyperthermophilic Archaea
in colonization devices and chimney fragments was higher
than in sediments. Interestingly, uncultivated groups
DHVE2 and DHVE8 were mostly detected associated
with colonization devices and chimney fragments suggest-
ing a thermophilic way of life. This extensive set of archae-
al 16S rRNA-based oligonucleotide probes proved to be
useful to compare the structural composition of archaeal
communities in numbers of environmental samples.
P4^127
ANTARCTIC LAKES ^ ‘HOT-SPOTS’ FOR MICRO-
BIAL DIVERSITY AND BIOTECHNOLOGICAL
SCREENING
R. De Wit(1), P. Dyer(2), O. Genilloud(3), E. Go«t-
tlich(4), D. Hodgson(5), S. de Hoog(6), B. Jones(7), J.
Laybourn-Parry(2), F. Marinelli(8), E. Stackebrandt(9),
J. Swings(10), B. J. Tindall(9), W. Vyverman(10), A.
Wilmotte(11)
(1) Biological Oceanography, University of Bordeaux 1,
33120 Arcachon, France; (2) Biological Sciences, Univer-
sity of Nottingham, Loughborough LE12 5RD, UK; (3)
Merck-Sharp-Dohme Espana, 28027 Madrid; (4) IWW,
45476 Mu«lheim, Germany ; (5) British Antarctic Survey,
CB3 OET Cambridge, UK; (6) Centraalbureau voor
Schimmelcultures, 3740 AG Baarn, Netherlands; (7) Gen-
encor, PO Box 218, 2300 AE Leiden, Netherlands; (8)
Biosearch Italia SPA, 21040 Gerenzano (VA), Italy ; (9)
DSMZ, 38124 Braunschweig, Germany; (10) Microbiol-
ogy/Protistology, University of Gent, 9000 Gent, Belgium ;
(11) Botany B22, University of Lie'ge, 4000 Lie'ge, Belgium
The EC project MICROMAT (BIO4-98-0040) has been
the ¢rst multi-disciplinary study on the diversity of a
broad spectrum of microorganisms in benthic microbial
mats of Antarctic lakes using conventional and molecular
methods. It has arisen from a cooperation between Euro-
pean bacteriologists, protistologists, mycologists, micro-
bial ecologists, paleolimnologists and industrial microbiol-
 1st FEMS Congress / Posters 103^505
ogists from 3 companies involved in biotechnological and
pharmaceutical research. We have characterised the diver-
sity of bacteria, cyanobacteria, algae, protozoans and fun-
gi in microbial mat samples of several Antarctic lakes by
conventional (microscopy, cultivation) and genotypic
(clone libraries and DGGE based on the SSU rDNA
from the samples) methods. Isolation of strains yielded
1500 bacterial, 60 cyanobacterial, 230 fungal, 91 algal
and 50 protozoan isolates from 3 to 24 lakes. The geno-
typic data comprised 470 prokaryotic and 300 eukaryotic
SSU rDNA sequences and showed the usual discrepancy
with cultivated diversity for all organisms, except for the
cyanobacteria. A low eukaryotic diversity, dominated by a
few highly specialised and often endemic taxa was ob-
served. In contrast, the prokaryotic diversity was ex-
tremely high and numerous novel phylotypes were discov-
ered. These newly discovered ‘hot-spots’ of microbial
biodiversity have stimulated the interest of biotechnolog-
ical companies who have screened 1700 strains for new
cold enzymes and antimicrobial compounds.
P4^128
DESCRIPTION OF BIODIVERSITY AND ENZYME
ACTIVITY OF MICROBIAL COMMUNITY OF
LAKE BAIKAL WATER MASS
V. V. Parfenova, N. L. Bel’kova, J. R. Zakharova, S. J.
Maksimenko
Limnological Institute SB RAS P.O. Box 4199, 664033,
Irkutsk, Russia
Lake Baikal is the most ancient freshwater lake of the
world, which is characterized by its unique ecological con-
ditions. The peculiarities of hydrological and hydrochem-
ical factors such as big depths, low water temperature,
high content of oxygen and low concentration of organic
matters, stipulate the speci¢c conditions of vital functions
of microorganisms. We isolated 1000 strains of psychro-
phylic and oligotrophyc bacteria from the water mass and
determined their lipase, phosphatase and protease activity.
These organisms possessed high enzyme activity. The re-
sults of research of biodiversity of the studied lake micro-
organisms were treated with the help of methods both of
classical and molecular biology. The taxonomic composi-
tion of cultivated and non-cultivated strains was analyzed.
The diversity of cultivated strains is presented mainly by
the species, which were observed also in a number of other
reservoirs. The microorganisms of known and described
species were mainly cultivated. They are Acinetobacter,
Artrobacter, Alcaligenes, Bacillus, Flavobacterium, Caulo-
bacter, Micrococcus, Pseudomonas, Sarcina. The majority
of bacteria are known not to be cultivated on certain nu-
trient mediums. The analysis of nucleotide sequences of
fragments of the gene 16S of mtDNA revealed a wider
FEMSLE Congress 2-6-03
229
diversity of non-cultivated microorganisms, which be-
longed to di¡erent phylogenetic groups. However, the
overwhelming majority of sequences possess a low homol-
ogy with the known data bank and form clusters at con-
structing phylogenetic trees in which there are sequences
revealed by the similar methods from other natural eco-
systems. The data obtained allow to make a conclusion
that the microbial community of the lake is unique con-
sisting both of known species observed in other reservoirs
and new ones not studied yet and probably distributed
only in Lake Baikal. Moreover, taking into account the
low concentration of organic matter in the lake, the bac-
teria possess high degree of enzyme activity.
P4^129
BACTERIA OF GENUS PSEUDOMONAS IN MICRO-
BIAL COMMUNITY OF LAKE BAIKAL
O. N. Pavlova, V. V. Drucker
Limnological Institute of SB RAS, P. O. Box 4199, 664
033, Irkutsk, Russia
The distribution and species variability of bacteria of ge-
nus Pseudomonas in microbial community of Lake Baikal
known as an oligotrophic and deep water lake was
studied. 382 water samples and bottom sediments, col-
lected in di¡erent basins of Lake Baikal, were analyzed
during 1998 ^ 2002. 277 pure cultures of genus Pseudomo-
nas was isolated and identi¢ed. Species P. aeruginosa, P.
alcaligenes, P. cepacia (Burkholderia cepacia), P. caryo-
phylli (Burkholderia caryophylli), P. diminuta (Brevundi-
monas diminuta), P. £uorescens, P. mendocina, P. putida,
P. stutzeri were found more often. Is established that bac-
teria of genus Pseudomonas dominate in heterotrophic
community of microorganisms in Lake Baikal. While
studying seasonal dynamics of quantitative development
of bacteria in the shallow zone of Southern Lake Baikal
it was stated that pseudomonades are prevailed in March-
May and in October-November periods. It was studied
protease, lipase and phosphatase enzyme activities of iso-
lated cultures of Pseudomonas genus. The most active
strains are isolated from shallow water zone and from
bottom sediments of Lake Baikal.
P4^130
MICRO-MAR: A MARINE PROKARYOTES DATA-
BASE TO CORRELATE HABITAT WITH TAXONO-
MY
F. Rodr|¤guez-Valera and J. C. Alba
Divisio¤n de Microbiolog|¤a, Campus de San Juan, Universi-
dad Miguel Herna¤ndez, Alicante, Spain
230 1st FEMS Congress / Posters 103^505
The last 15 years a large amount of 16S rDNA sequences
from microbes has become available. Many if not most
correspond to PCR products directly ampli¢ed from nat-
ural samples and then cloned and sequenced. The number
of sequences retrieved from marine environments only ex-
ceeds 5000. On the other hand, oceanographic and ecolog-
ical databases provide a wealth of information about en-
vironmental features at di¡erent locations. Here we
describe a public data base where both types of informa-
tion sources have been combined with sequence based
taxonomy to provide a grid of geographic and habitat
characteristics for every taxon of marine prokaryotes in-
cluding non-cultured types known only by sequence.
P4^131
ACTINOMYCETES IN MICROBIAL COMMUNITY
OF LAKE BAIKAL
I. A. Terkina, V. V. Drucker
Limnological Institute of SB RAS, P. O. Box 4199, 664033,
Irkutsk, Russia
The distribution, numbers and species diversity of actino-
mycetes dominant genera in Lake Baikal are inverstigated
for the ¢rst time. It was revealed that actinomycetes in
water, in bottom sediments, and in sponges are presented
by Streptomyces, Micromonospora, Nocardia and Arthro-
bacter. There are heterotrophic, olygotrophic and psycro-
phillic strains among the genera. Actinomycetes can be
found in the waterbody of Southern, Middle and North-
ern Baikal, beginning from the depth of 400 m. Nocardia
and Arthrobacter in the deep water regions can be found
only up to the depth of 400 m, Streptomyces and Micro-
monospora inhabit also in the near bottom layer like in
lithoral zone but at the greater depths. Nocardia can be
met occasionally in the near bottom layers, bottom sedi-
ments and in sponges of shallow water zone of Lake Bai-
kal. From 65 strains identi¢ed up to species belonging to
Streptomyces and Micromonospora the most often avail-
able in bottom sediments, waterbody and sponges in Lake
Baikal are Streptomyces globisporus and Micromonospora
purpurea. Laboratory studies showed that a lot of repre-
sentatives Streptomyces and Micromonospora are able to
produce various hydrolytic enzymes, such as proteases,
lipases, phosphatases and cellulases at low temperature.
Besides, these actiomycetes exhibit remarkable biological
activity against another bacterial strains. Thus, we can
suggest, actinomycetes can be participants of a number
of important degradation processes and in£uence on struc-
ture and functioning of microbial community in lake Bai-
kal.
FEMSLE Congress 2-6-03
P4^132
BACTERIOPLANKTON COMMUNITY COMPOSI-
TION OF NEIGHBOURING EUTROPHIC SIBERIAN
RESERVOIRS
M. Yu. Trusova, M. I. Gladyshev
Institute of Biophysics of Siberian Branch of Russian Acad-
emy of Sciences, 660036, Krasnoyarsk, Russia
Bacterioplankton are among the most abundant and im-
portant components of aquatic ecosystems. The taxonomic
composition of bacterial assemblages and their spatiotem-
poral dynamics in freshwater lakes and reservoirs are
likely to be of major importance in determining the role
of bacteria in aquatic food web and biogeochemistry. The
study of bacterial diversity in water habitats has strongly
advanced with the recent introduction of molecular tech-
niques. Due to cultivation-independent methods for iden-
ti¢cation of microorganisms a large number of previously
unknown taxa were detected. The existence of globally
distributed taxa has been discovered. The rRNA approach
has provided a powerful instrument to study interactions
of bacteria with other components of aquatic ecosystems,
and to relate community composition data to environmen-
tal data and trophic status of the ecosystem. Using 16S
rRNA partial gene sequence analyses we have investigated
the bacterial diversity of winter bacterioplankton of two
neighbouring eutrophic Siberian reservoirs. The reservoirs
are similar in phytoplankton community composition in
spring and autumn but tend to di¡er in summer in exhib-
iting cyanobacterial bloom. Forty-eight unique partial 16S
rRNA gene sequences retrieved from two libraries were
mostly a⁄liated with the class Actinobacteria, beta subdi-
vision of the class Proteobacteria, and the phylum Cyto-
phaga-Flavobacterium-Bacteroides. The clone library of the
reservoir exhibiting summer cyanobacterial bloom showed
more diversity in sequence composition. A signi¢cant
number of bacterial 16S rRNA gene clones were closely
related to freshwater bacteria previously found in di¡erent
aquatic ecosystems. This ¢nding con¢rms the assumption
that some bacterial clades are globally distributed.
P4^133
GENOMIC DIVERSITY OF HETEROTROPHIC BAC-
TERIA ISOLATED FROM ANTARCTIC MICROBIAL
MATS
S. Van Trappen(1), J. Mergaert(1) and J. Swings(1,2)
(1) Laboratorium voor Microbiologie, Vakgroep Bioche-
mie, Fysiologie en Microbiologie, Universiteit Gent, Bel-
gium; (2) BCCM/LMG Bacteria Collection, Universiteit
Gent, Belgium
 1st FEMS Congress / Posters 103^505
The genomic diversity of heterotrophic bacteria, isolated
from microbial mats in 10 lakes in 3 di¡erent Antarctic
regions (Vestfold Hills, McMurdo Dry Valleys and Larse-
mann Hills) was further investigated. In a previous study
[1], 746 strains have been assigned to 41 clusters by nu-
merical analysis of their fatty acid pro¢les, and 16S rDNA
sequence analysis of representative strains showed that
they belong to the alpha-, beta- and gamma-subclasses
of the Proteobacteria, the high and low percent G+C
Gram-positives and to the Cytophaga-Flavobacterium-Bac-
teroides branch. The aim of this study was to investigate in
more detail the genomic diversity of 451 strains belonging
to 20 fatty acid clusters, of which representative strains
were phylogenetically related to the Proteobacteria. Repet-
itive extragenic palindromic DNA (rep)-PCR ¢ngerprint-
ing was performed on these clusters and the wealth of
di¡erent rep-¢ngerprints obtained, illustrates that the ge-
nomic diversity of heterotrophic bacteria in Antarctic mi-
crobial mats is extremely high. To investigate the genomic
relatedness between the di¡erent rep-PCR groups, DNA-
DNA hybridisations between representative strains are
being carried out. Several fatty acid clusters contain or
represent di¡erent hybridisation groups and 16S rDNA
sequence analysis shows that these groups often represent
new species related to recently described species from cold
environments.
[1] S. Van Trappen et al. Diversity of 746 heterotrophic
bacteria isolated from microbial mats from ten Antarctic
lakes, Systematic and Applied Microbiology. In press
P4^134
NEW PROKARYOTIC GENE DIVERSITY IN ANT-
ARCTIC DRY VALLEYS
S. J. Whiting(1), J. M. Ward(1), K. D. Bruce(2) and D.
C. Cowan(3)
(1) Dept. of Biochemistry and Molecular Biology, Darwin
Building, University College London, Gower St., London
WC1E 6BT, UK; (2) Department of Life Sciences, Frank-
lin-Wilkins Building, King’s College London, 150 Stamford
Street, London SE1 9NN, UK; (3) Department of Biotech-
nology, University of the Western Cape, Bellville 7535,
Cape Town, South Africa
The Dry Valleys of South Victoria Land, Antarctica con-
tain some of the most extreme biotopes on Earth and have
long been considered as valid Martian analogues. Life
within the Antarctic desert soils, where water contents
range from 0.2-2.0% w/w and mean annual temperatures
fall below 6 -20‡C , must additionally contend with des-
iccating winds, diurnal freeze-thaw cycles, and high sea-
sonal UVA/UVB radiation. Molecular phylogenetic anal-
ysis of Antarctic desert mineral soils have revealed this
biotope to support a wide diversity of bacterial species,
FEMSLE Congress 2-6-03
231
the majority of which have no close cultured representa-
tives. Divisions represented in 16S rDNA clone libraries
include the Proteobacteria, Actinobacteria, Acidobacteria
and Verrucomicrobia along with candidate division TM7.
Analysis of Archaeal 16S rDNA sequences has identi¢ed
the presence of non-thermophilic Crenarchaeota in the
Dry Valley environment that cluster within Group I.b of
the uncultured Crenarchaeota, a group represented by
clones recovered from forest and agricultural soils. Our
investigation of prokaryote diversity is currently being ex-
tended through the incorporation of cultivation techniques
and additionally, in the generation of a soil metagenomic
DNA library from which to probe for 16S rDNA sequen-
ces. Further to the characterization of microbial diversity,
we are also examining functional gene diversity focussing
on the recovery of integrons and their associated gene
cassettes. Integrons are genetic elements that permit gene
acquisition and expression through the capture of mobile
gene cassettes and they are well recognised for their role in
the dissemination of antibiotic resistance. Using a PCR-
based strategy, we have evidence of integrons and their
associated genes in pristine Antarctic environments.
P4^135
ANTIFUNGAL SUBSTANCES OF ANTAGONISTIC
BACTERIA BACILLUS SP. 739
G. E. Aktuganov, A. I. Melentiev, A. V. Shirokov
Institute of Biology, Ufa Research Center RAS, Prospekt
Oktiabria, 69, Ufa, 450054, Russia
Three groups of extracellular antifungal substances were
detected in cultural broth of Bacillus sp. 739 ^ the strain,
known as antagonist toward several phytopathogenic fun-
gi. The ¢rst group contains hydrolytic enzymes including
chitinase (EC 3.2.1.14), chitosanase (EC 3.2.1.132) and L-
1,3-glucanase (EC 3.2.1.39). Other two groups of the anti-
fungal substances are low-molecular proteins (MwV10-20
kD) and peptide or lipo-peptide antibiotics, respectively.
Chitinase and L-1,3-glucanase from Bacillus sp. 739 caused
degradation of intact and dead mycelium a lot species of
soil fungi. Besides, these enzymes were respond for myco-
lytic e¡ect of Bacillus sp. 739 toward Helminthosporium
sativum and Fusarium culmorum in mixed culture. How-
ever, partially puri¢ed enzyme preparations did not in£u-
ence on the mycelium growth or fungal spore germination
in vitro. Three low-molecular protein and peptide fungi-
cides were found in cultural broth of Bacillus sp. 739 by
thin-layer and paper chromatography. Preparative isola-
tion of the components was performed by sulfate ammo-
nium saturation, acetone extraction and gel-chromatogra-
phy on Toyopearl HW-40. Amino acid analysis of total
antifungal fraction indicated high content of alanine, va-
line, leucine and phenylalanine among sixteen amino acids
232 1st FEMS Congress / Posters 103^505
found in acid hydrolysis products. Two antibiotic compo-
nents were identi¢ed as polypeptides (MwV1-2 kD), while
third one was a protein (MwV14 kD). Protein inhibited
spore germination only, whereas low-molecular antibiotics
caused mass formation of spheroplasts in hyphae of grow-
ing H. sativum mycelium and its lysis. Minimal inhibiting
concentration of protein 14 kD was about 100-150 Wg/ml.
Presence of di¡erent mechanism of fungal growth inhibi-
tion provided by Bacillus sp. 739 indicates a complex na-
ture of interactions between antagonistic bacteria and soil
fungi.
P4^136
PHYLOGENETIC ANALYSIS OF CHITIN SYN-
THASE GENES FROM PHAEOMONIELLA CHLA-
MYDOSPORA
A. Alves(1), A. J. L. Phillips(2) and A. Correia(1)
(1) Centro de Biologia Celular, Campus Universita¤rio de
Santiago, Departamento de Biologia, Universidade de
Aveiro, 3810-193 Aveiro, Portugal; (2) Centro de Recursos
Microbiolo¤gicos, Faculdade de Cie“ncias e Tecnologia, Uni-
versidade Nova de Lisboa, Quinta da Torre, 2829-516, Ca-
parica, Portugal
Esca syndrome is one of the most destructive diseases of
grapevine. It is widespread in most countries where vines
are grown, and has been already identi¢ed in several Por-
tuguese regions. Apparently it results from the action of
several fungi acting in combination or in succession. The
primary pathogens in this succession are mitosporic fungi
of the genera Phaeoacremonium (Pm) and Phaeomoniella
(Pa). The taxonomy of these genera is still subject to dis-
cussion and is not fully de¢ned. In this work, 14 fungal
strains isolated from grapevines a¡ected by esca, and be-
longing to the species Phaeomoniella chlamydospora (=
Phaeoacremonium chlamydosporum), Phaeoacremonium
aleophilum, Pm. angustius and Pm. viticola were studied.
Primers speci¢c for class I and class II chitin synthase
genes were used. A chitin synthase DNA fragment was
ampli¢ed, cloned and sequenced for all Pa. chlamydospora
strains. In all other species it was not possible to detect
homologues of this fragment. The nucleotide and deduced
amino acid sequence analysis revealed that all the frag-
ments belonged to a class I chitin synthase. Class II chitin
synthase genes were not detected. The phylogenetic anal-
ysis based on the deduced a.a. sequences revealed that Pa.
chlamydospora forms a group distinct from the other spe-
cies analysed. Nevertheless, among these Pa. chlamydo-
spora seems more closely related to A. niger and A. nidu-
lans, ascomycetes of the genus Emericella (Eurotiales), B.
dermatitidis and H. capsulatum ascomycetes of the genus
Ajellomyces (Onygenales). These results con£ict with phy-
FEMSLE Congress 2-6-03
logenetic analysis based on ITS sequences which places
Pa. chlamydospora in the Chaetothyriales.
P4^137
IS PICHIA ANOMALA THE ONLY YEAST SPECIES
INHIBITING MOULD GROWTH DURING AIR-
TIGHT STORAGE OF MOIST CEREAL GRAIN?
º . Druvefors, J. Schnu«rer
U. A
Department of Microbiology, Swedish University of Agri-
cultural Sciences, Box 7025, 750 07 Uppsala, Sweden
Penicillium roqueforti is the most important spoilage fungi
in airtight stored cereal grain. We have previously shown
that the biocontrol yeast Pichia (Hansenula) anomala
strain J121 can reduce growth of P. roqueforti both in vitro
and in high-moisture cereal grain in a test-tube version of
a malfunctioning storage system. The ability of P. anomala
J121 to prevent growth of inoculated P. roqueforti and
other moulds has been validated using 0.21 m3 pilot scale
silos for outdoor airtight storage of 160 kg batches of
moist grain during 14 months. We have now investigated
whether the inhibiting e¡ect on mould growth in airtight
storage is a unique ability of P. anomala J121. More than
85 strains from 34 species from di¡erent yeast genera were
evaluated. All strains of P. anomala had high biocontrol
activity, including all tested haploid strains. The absolute
majority of other yeast species, including several strains of
Saccharomyces cerevisiae, Debaromyces hansenii, and
Cryptococcus albidus had no or very limited biocontrol
activity. Hypopichia burtonii and Pichia guillermondii in-
hibited P. roqueforti, but not to the same extent as P.
anomala. Where several strains of the same species were
evaluated no di¡erences between strains were detected.
P4^138
BIOLOGICAL CONTROL OF FUNGAL STRAW-
BERRY DISEASES BY THE CHITINOLYTIC SERRA-
TIA PLYMUTHICA STRAIN HRO-C48
G. Berg(1), S. Kurze(1), J. Frankowski(1), A. Wolf(1),
R. Dahl(2) and H. Bahl(1)
(1) University of Rostock, Microbiology, Albert-Einstein-
Str. 3, D-18051 Rostock, Germany ; (2) Strawberry Farm
Ro«vershagen, D-18182 Purkshof, Germany
The rhizobacterium Serratia plymuthica C48 was previ-
ously selected as an antagonist to phytopathogenic fungi.
The antifungal activity of the strain mainly based on an
e⁄cient chitinolytic system. One chitinase (E.C. 3.2.1.14)
CHIT60 and one N-acetyl-_-1,4-D-hexosaminidase
(E.C. 3.2.1.52) CHIT100 were puri¢ed and characterized.
 1st FEMS Congress / Posters 103^505
The nucleotide sequence of the gene encoding the chitinase
CHIT60 was highly similar to that of chitinase A from
Serratia liquefaciens. The puri¢ed CHIT100 (100 Wg ml-1)
inhibited the spore germination and germ tube elongation
of the phytopathogenic fungus Botrytis cinerea by 28 %
and 31.6 % respectively. With CHIT60 (100 Wg ml-1) the
e¡ect was more pronounced: 78 % inhibition of germina-
tion and 63.9 % inhibition of germ tube elongation. In ¢ve
consecutive vegetation periods, ¢eld trials were carried out
in soils naturally infested by the soilborne pathogens. The
root application of S. plymuthica prior planting reduced
Verticillium wilt compared with the nontreated control by
0 to 37.7%, with an average of 24.2%, whereas the increase
of yield ranged from 156 to 394%, with an average of
296%. Additionally, in£uence of the biocontrol agent on
the bacterial communities (non-target microorganisms) of
the strawberry rhizosphere was monitored by analysis of
PCR-ampli¢ed fragments of the 16S rRNA genes of the
bacterial community after separation by denaturing gra-
dient gel electrophoresis (DGGE) and by analysis of the
in£uence on fungal and bacterial diversity. Under ¢eld
conditions, the strain survived at approximately log10 3-7
CFU g-1 root in the strawberry rhizosphere at 14 months
after root application.
P4^139
EVALUATION OF OXILITE AS A BIOCIDE
AGAINST OIL FIELD BACTERIA
M. Gaja, H. Ben Hussein and K. El-Barouni
Production Technologies and processing research Depart-
ment, Petroleum Research Centre, P.O. Box 6431, Tripoli,
Libya
Laboratory experiments were carried out to determine the
biocidal activities of Oxilite for treatment applications in
water systems of oil producing and drinking water. Oxilite
(oxidizing agent) is cost e¡ective product that is known to
be non-toxic mixture of all individually oxidants in ionic
and radical form. The biocidal e¡ects of Oxilite on harm-
ful micro-organisms such as sulphate-reducing bacteria
(SRB) in 103D and Augila oil¢eld injection waters and
against Escherichia coli (E. coli) in local drinking water
have been studied by measurement of their cell numbers
at 37‡C versus time. It is apparent that such product
showed bactericidal or killing e¡ect (100%) against SRB
in 103D injection water at all tested dilutions. However
Oxilite in Augila injection water generally showed bacter-
iostatic activity with disappearance of ferrous sulphide
precipitation by SRB. In drinking water, Oxilite was found
(100%) e¡ective to kill E. coli cells. The laboratory results
showed that Oxilite can be considered as e¡ective biocides
in water systems.
FEMSLE Congress 2-6-03
233
P4^140
MOLECULAR TYPING OF MYCOBACTERIUM
AVIUM SUBSP. PARATUBERCULOSIS ISOLATED
FROM GOATS AND CATTLE IN NORWAY
B. Dj\nne(1), P. Ahrens(2), I. Pavlik(3), P. Svastova(3),
and G. Holstad(1)
(1) National Veterinary Institute, P.O. Box 8156 Dep., N-
0033 Oslo, Norway; (2) Danish Veterinary Laboratory,
Bulowsvei 27 DK-1790, Copenhagen, Denmark; (3) Veteri-
nary Research Institute, Hudcova 70, Brno 621 32, Czech
republic
In Norway, clinical paratuberculosis has been frequently
diagnosed in goats, while cattle have been almost free of
the disease. This di¡erence in disease prevalence between
goat and cattle, has led to speculations about the existence
of a Mycobacterium avium subsp. paratuberculosis (M. a.
paratuberculosis) isolate that is not pathogenic for cattle.
There is little available information about genotypic var-
iations of M. a. paratuberculosis isolated from animals in
Norway. The aims of the present study were to obtain
genotypic information of 58 isolates from goats and four
isolates from cattle in Norway by use of ampli¢ed frag-
ment length polymorphism (AFLP) and restriction frag-
ment length polymorphism (RFLP) analysis. All M. a.
paratuberculosis isolates from cattle and 91% of the iso-
lates from goats belonged to the same AFLP type (type
G). The other ¢ve isolates belonged to four di¡erent
AFLP types. It was also demonstrated that the isolates
from cattle and 76% of the isolates from goats had the
same RFLP pattern, B-C1, but some RFLP patterns not
previously detected were also found. No genotypic varia-
tion that could explain a di¡erence in virulence was found
between the isolates from cattle and the majority of the
Norwegian goat isolates. This indicates that the most com-
mon M. a. paratuberculosis isolates in Norway are able to
infect both cattle and goats.
234 1st FEMS Congress / Posters 103^505
P4^141
ISOLATION OF CLOSTRIDIUM DIFFICILE FROM
THE PATIENTS, OBJECTS OF HOSPITAL ENVI-
RONMENT AND ANIMALS (REPUBLIC OF BELA-
RUS)
N. V. Lebedkova(1), L. P. Titov(1), A. Yu. Finogenov(2),
N. N. Androsik(2), E. P. Schesljenok(1), I. Grigoro-
vich(1), V. F. Korotkina(3), J. Silva(4)
(1) Research Institute for Epidemiology and Microbiology,
Minsk, Belarus ; (2) S. N. Vyshelesski Institute of Exper-
imental Veterinary Medicine, Minsk, Belarus ; (3) Hospital
of Infectious Diseases, Minsk, Belarus; (4) Division of In-
fectious and Immunologic Diseases, University of Califor-
nia, Davis Medical Center, Sacramento, California 958174
The infections caused C. di⁄cile are registered in many
countries of the world. This microorganism is isolated
from the patients and carriers, objects of hospital environ-
ment and animals. The purpose : to establish distribution
C. di⁄cile at the patients, on objects of hospital environ-
ment and at animals. Materials and methods. Samples
faeces of the patients, animals and wash-out from objects
of hospital environment. Specimens were inoculated onto
cycloserine-cefoxitin-fructose agar. The samples were incu-
bated anaerobically for 48-72 h at 37 0C. Identi¢cation C.
di⁄cile carried out by cultural and biochemical properties.
C. di⁄cile isolates were tested for the presence of genes
toxins A and B by polymerase chain reaction (PCR). Re-
sults. From the patients we obtained 22 isolates C. di⁄cile.
The frequency of isolation C. di⁄cile were from dogs 4,0%
(n=151), foals 2,8% (n=106), cats 1,6% (n=63). C. di⁄cile
are isolated from objects of hospital environment in 2,5%
(n=1700) of cases. Basically, this pathogen was found out
on objects of hospital environment of intensive care units
(ICU)-5,7% (n=700). Most contaminated were bed-pans
22,1% (n=77), £oors 3,1% (n=160), wash-stands 2,2%
(n=225), night-tables 2,0% (n=151), walls 1,7 % (n=239),
tidying up instruments 1,7% (n=237). All isolates C. di⁄-
cile expressed fragments tox genes A and B. Conclusion.
Thus, toxigenic isolates C. di⁄cile are isolated from the
patients, objects of hospital environment and animals.
Most contaminated were objects of hospital environment
of ICU, which can be the factors of transmission, this
pathogen.
FEMSLE Congress 2-6-03
P4^142
LACTIC UNLIKE OTHER BACTERIA, PROLIFER-
ATE UNDER ATMOSPHERE CONTAINING CAR-
BON-MONOXIDE ONLY
U. Maor(1), N. Shaklai(1), N. Gollop(2) and V. A. Tse-
makhovich(1)
(1) Department of Human Genetics and Molecular Medi-
cine, Faculty of Medicine, Tel-Aviv University, P.O. Box
39040, Tel-Aviv 69978 ; (2) Dept. of Food Science, The
Vulcani Center ARO, P.O. Box 6, Bet-Dagan, 50250, Israel
It has been long known that carbon-dioxide inhibits of
anaerobic bacterial growth. The role of carbon-monoxide,
an inert molecule, in anaerobic bacterial proliferation is
less clear. The literature contains some information sug-
gesting that carbon monoxide can inhibit anaerobic bac-
terial growth as well but the picture is not clear. We tested
the involvement of, carbon-monoxide, in growth of facul-
tative/anaerobic bacteria. We compared the growth of E.
coli (386) on liquid and solid nutrient-rich media under
three atmospheres containing; air, nitrogen or carbon-
monoxide. We found as expected slower growth under
nitrogen in compared to air. However, growth under car-
bon-monoxide was much inhibited as compared to nitro-
gen. Exchange of carbon-monoxide by nitrogen resulted in
accelerated growth reaching the original level of growth
under nitrogen. This data suggested that binding of car-
bon-monoxide to an iron containing protein site essential
for the metabolism of this bacteria is involved. We next
tested growth of several lactic bacteria (Lactobacillus plan-
tarum Lactobacillus leichmanii, Lactobacillus casaei, Lacto-
bacillus delbrukii, Leuconostock mesenteroides) under the
above atmospheres. We found practically no di¡erence
in growth of all above lactic bacteria under the three at-
mospheres. These data can be explained by lack of iron
containing, carbon-monoxide binding sites on key proteins
/ enzymes in the lactic bacteria. In correlation, lactic bac-
teria have long been shown as the sole bacteria with no
need of iron as an essential element.
P4^143
STUDY ON BIASES IN TEMPLATE-TO-PRODUCT
RATIOS OF MULTI -TEMPLATE PCR
M. Palatinszky, M. Nikolausz, R. Sipos, K. Ma¤rialigeti
Department of Microbiology, Eo«tvo«s Lora¤nd University,
Budapest 1117, Pa¤zma¤ny P. se¤ta¤ny 1/C, Hungary
Molecular ¢ngerprinting methods are widely used in mi-
crobial diversity examinations of environmental samples.
However any conclusions drawn about the relative abun-
 1st FEMS Congress / Posters 103^505
dance of certain taxons within a community should always
take the limitations of the applied techniques into consid-
eration. Among molecular ¢ngerprinting methods T-
RFLP (Terminal Restriction Fragment Length Polymor-
phism) is a semi-quantitative technique, based on the sep-
aration of multitemplate PCR amplicons according to
their size by high-throughput capillary electrophoresis.
The preceding steps (DNA isolation, PCR) carry a poten-
tial bias distorting template ratios, therefore the T-RFLP
quantitation of a given community only applies to the
relative abundance of the multi-template PCR products
and not to the original composition of the environmental
sample. Our aim was to explore how multitemplate PCR
alters DNA concentration ratios within the community
DNA sample. We have constructed a model community
of ¢ve collection strains of bacteria (Aeromonas hydrophi-
la, Bacillus cereus, Bacillus subtilis, Pseudomonas aeru-
ginosa, Pseudomonas £uorescens) with di¡erent genome
sizes, G+C content and rrn copy number. Following
DNA isolations the amount of DNA from each strain
was determined by spectrophotometric quantitation so
that the ratio of the 16S rDNA copy number could be
set at prede¢ned values. The distortion of the relative
abundance of the di¡erent amplicons, acquired from the
PCR performed on the di¡erent template mixtures, was
measured by T-RFLP. Our results show that both the
genomic properties of the model bacteria and the condi-
tions of the PCR itself (cycle number, annealing temper-
ature, touch down protocol) in£uence the predicted ampli-
con ratios.
P4^144
DEVELOPMENT OF A HIGH THROUGHPUT DI-
VERSITY DNA ARRAY FOR THE HUMAN INTESTI-
NAL MICROBIOTA
M. Rajilic, H. G. H. J. Heilig, L. Rigottier-Gois, W. M. de
Vos, E. E. Vaughan
Laboratory of Microbiology, Wageningen University, 6703
CT Wageningen, The Netherlands
The human gastrointestinal tract is inhabited by a diverse
microbial community termed microbiota, which contains
up to 1014 total bacteria that have a profound in£uence on
human health. The composition and activity of microbiota
is host dependent, but still a¡ected by environmental fac-
tors such as diet, age, lifestyle, and may also be altered due
to intestinal or other diseases. For following changes in
microbiota on a large-scale, high throughput methods
such as DNA arrays are required. We have developed a
diversity macroarray for the detection of the numerically
dominant human intestinal bacteria. The array is made by
spotting speci¢c 16S ribosomal DNA based probes from
over 100 bacterial species on a nylon membrane. Valida-
FEMSLE Congress 2-6-03
235
tion was performed by hybridizing the array with the 16S
rDNA from various bacterial species present on the array,
labelled by incorporation of radioactive [K-32P] dATP. The
results indicated speci¢c hybridisation suggesting the
method has a potential for the development of a micro-
biota microarray. Furthermore, DNA and RNA isolated
from faecal or mucosal samples will be tested to investi-
gate the diversity and activity of the microbiota within the
di¡erent niches in the human intestinal ecosystem.
P4^145
CURRENT STATE OF ANTIMICROBIAL RESIS-
TANCE OF S. PYOGENES (GAS) IN RUSSIA : RE-
SULTS OF PROSPECTIVE MULTICENTER STUDY
(PEHASus-I, PHASE’’B’’)
O. V. Sivaja(1), R. S. Kozlov(2), L. S. Stratchounski(2)
and PEHASus Project Group
(1) Department of Clinical Pharmacology of Smolensk
State Medical Academy, Smolensk, Russia ; (2) Institute
of Antimicrobial Chemotherapy, Smolensk, Russia
The study was conducted in 16 cities (Chelyabinsk, Eka-
terinburg, Irkutsk, Jakutsk, Jaroslavl, Kazan, Krasnodar,
Moscow, Novokuznetsk, Saint-Petersburg, Smolensk,
Stavropol, Tjumen, Tomsk, Rjazan, Voronezh) in Russia
in 2001-2002. The total of 683 non-duplicate clinical iso-
lates of Streptococcus pyogenes (GAS) were included in
this study. Identi¢cation of the strains was done on the
basis of colony morphology, Gram stain, bacitracin (0.02
IU) susceptibility and latex agglutination tests. Suscepti-
bility testing to penicillin G (PEN), erythromycin (ERY),
azithromycin (AZI), clarithromycin (CLA), midecamycin
(MID), clindamycin (CLI), telithromycin (TEL), levo£ox-
acin (LEV), tetracycline (TET), chloramphenicol (CHL),
vancomycin (VAN) and linezolid (LIN) was performed
centrally by broth microdilution method. Breakpoints
were those of NCCLS (2002), except for TEL (9 0.5; 1;
v 2 mg/L), SPI (9 1; s 4 mg/L) and MID (9 1; s 4
mg/L). There were no resistance detected to PEN, TEL,
LEV, VAN and LIN. Percentage of non-susceptible (in-
termediate and highly resistant) to macrolides and linco-
samides isolates was as follows : ERY ^ 8% (MIC-0.06 mg/
L), AZI ^ 9% (MIC-0.25 mg/L), CLA ^ 7% (MIC-0.125
mg/L), MID 1% (MIC-0.5 mg/L), and CLI ^ 1% (MIC-
0.03 mg/L). The highest non-susceptibility was observed to
CHL (51%) and TET (47%). PEN remains 100% active
against all GAS isolates. High resistance to TET and
CHL compromises their usage in streptococcal infections.
16-membered midecamycin possessed the highest activity
again all strains. Obtained date can be helpful for choice
of appropriate antimicrobial for streptococcal infection
therapy.
236 1st FEMS Congress / Posters 103^505
P4^146
DIVERSITY OF NANOBACTERIA AND PROVOKED
MINERAL DEPOSITS IN CALCIFIED PLACENTA
T. N. Abashina(1) and R. M. Agababov(2)
(1) Pushchino State University, Pushchino, 142290 Russia ;
(2) Central Municipal Hospital No3, Donetsk, 83014 Uk-
raine
Calci¢cation of tissues is a common case of disorders in
placenta. Current knowledge of the calci¢cation is still
limited and controversial. Potential relations between ap-
pearance of the mineral deposits and bacterial factors are
unknown. Up to present, calci¢cation of placental tissues
has never been shown in relation with some bacterial dis-
semination. Our electron microscopy of placental calci¢ed
sites discovered: micro-cavities in the tissues; nanobacte-
ria; mineral micro-deposits of the calci¢cation. The micro-
cavities were of 1 x 6 Wm size, they served as a basic place
of localisation of the nanobacteria and the mineral micro-
deposits. The revealed new and unstudied nanobacteria
were presented with small cells (0.15-0.20 Wm in diameter)
containing RNA and DNA and coated with cellular mem-
branes, the cell wall was absent. Low electron density of
the micro-cavity in comparing with the surrounding tissues
testi¢es to the fact that the cavities were ful¢lled with so-
lution and were favourable for accumulation of dissolved
inorganic salts, i.e. for deposition of calcium as mineral
storage. Redox potential on membrane surface of the
nanobacteria could stimulate the process. Thus, our ¢nd-
ing con¢rms the early proposed mechanism of placenta
calci¢cation : rapid formation of apatite mineral in placen-
ta means existence of supersaturated environment and cat-
alysts of the process (i.e. nanobacteria). Thus, the present
study is the ¢rst demonstration of a relation between the
placenta calci¢cation and nanobacteria.in micro-cavities in
tissues.
The authors are grateful to Dr.P.Schwartsburd and
Dr.N.Suzina (Pushchino Research Centre RAS) for their
support during the investigations.
FEMSLE Congress 2-6-03
P4^147
TWO GAMMA-PROTEOBACTERIA GROW IN PRO-
TOZOA AND REQUIRE CHARCOAL FOR GROWTH
IN LABORATORY MEDIA. DESCRIPTION OF AQUI-
CELLA LUSITANA GEN. NOV., SP. NOV. AND
AQUICELLA SYPHONIS, SP. NOV.
P. Santos(1), I. Pinhal(1), F. A. Rainey(2), J. Costa(3),
N. Empadinhas(3), B. Fields(4), R. Benson(4), A. Ver|¤s-
simo(1) and M. S. da Costa(3)
(1) Departamento de Zoologia and Centro de Neurocie“n-
cias, Universidade de Coimbra, 3004-517 Coimbra, Portu-
gal; (2) Department of Biological Sciences, Louisiana
State University, Baton Rouge, LA 70803, USA; (3) De-
partamento de Bioqu|¤mica, Universidade de Coimbra, 3001-
401 Coimbra, Portugal; (4) Respiratory Diseases Branch,
Centers for Disease Control and Prevention, Mailstop G03,
1600 Clifton Road, Atlanta, GA 30333, USA
Several isolates, belonging to two new species of the same
novel genus of Gamma-Proteobacteria, were recovered
from borehole and spa water at Sa‹o Gemil in Central
Portugal. These organisms are allied to the strictly intra-
cellular species of the genus Ricketsiella, which cause de-
sease in arthropods, and to the facultatively intracellular
species of the genus Legionella, some of which cause Le-
gionnaires’ desease and Pontiac fever. These organisms
only grew on bu¡ered charcoal yeast extract (BCYE) me-
dium because, like the species of the genus Legionella, they
require activated charcoal for growth. Unlike, the vast
majority of the strains of Legionella, the new isolates do
not require L-cysteine or pyrophosphate for growth.
Strains SGT-39T and SGT-56 grew consistently between
30 and 43‡C, while strains SGT-108T and SGT-109 grew
between 30 and 40‡C. The pH range for growth of these
organisms was surprisingly narrow; strain SGT-39T and
SGT-56 grew between pH 6.3 and 7.3, while strain SGT-
108T and strain SGT-109 grew between pH 6.3 and 7.0.
Both organisms infected and proliferated in the amoeba
Hartmannella vermiformis but did not grow in U937 hu-
man cells. On the basis of 16S rRNA sequence analysis,
physiological, biochemical and chemical analysis we are of
the opinion that strain SGT-39T represents a new species
of a novel genus for which we propose the name Aquicella
lusitana, while strain SGT-108T represents a second species
of the same novel genus for which we propose the name
Aquicella syphonis.
 1st FEMS Congress / Posters 103^505
P4^148
TEPIDIMONAS AQUATICA SP. NOV., A NEW
SLIGHTLY THERMOPHILIC BETA-PROTEOBAC-
TERIUM ISOLATED FROM A HOT WATER TANK
M. Freitas(1), F. A. Rainey(2), M. F. Nobre(1), A. J. D.
Silvestre(3) and M. S. da Costa(4)
(1) Departamento de Zoologia and Centro de Neurocie“n-
cias, Universidade de Coimbra, 3004-517 Coimbra, Portu-
gal; (2) Department of Biological Sciences, Louisiana
State University, Baton Rouge, La 70803, USA; (3) De-
partamento de Qu|¤mica, Universidade de Aveiro, 3810-193,
Aveiro, Portugal; (4) Departamento de Zoologia, Univer-
sidade de Coimbra, 3004-517 Coimbra, Portugal
A bacterial isolate, with an optimum growth temperature
of about 50‡C, was recovered from a domestic hot water
tank in Coimbra. Phylogenetic analysis using 16S rRNA
gene sequence indicated that strain CLN-1T is a member
of the beta-Proteobacteria and represents a new species of
the genus Tepidimonas. The major fatty acids of strain
CLN-1T are C16:0 and 17:0 cyclo. Ubiquinone 8 is the
major respiratory quinone, the major polar lipids are
phosphatidylethanolamine, and phosphatidylglycerol. The
new isolate is aerobic and facultatively chemolithohetero-
trophic. Thiosulfate and tetrathionate are oxidized to sul-
fate in the presence of a metabolizable carbon source.
Heterotrophic growth of strain CLN-1T occurs on amino
acids and organic acids, but this organism does not assim-
ilate carbohydrates. Glycerol is the only polyol assimi-
lated. Another slightly thermophilic organism, designated
DhA-73 that, based on 16S rRNA gene sequence analysis,
belongs to an undescribed species of the genus Tepidimo-
nas, is also unable to assimilate carbohydrates and polyols.
This organism was isolated years ago was capable of de-
grading tricyclic diterpenes, such as abietic acid, dehydroa-
bietic acid and palustric acid derived from conifer resin.
Resinic acids, namely abietic acid, dehydroabietic acid and
isopimaric acid are not degraded. On the basis of the
phylogenetic analyses, physiological and biochemical char-
acteristics, we propose that strain CLN-1T represents a
new species for which we o¡er the name Tepidimonas
aquatica.
FEMSLE Congress 2-6-03
237
P4^149
AKKERMANSIA MUCINIPHILA, GEN. NOV., SP.
NOV., A NOVEL INTESTINAL MUCIN-DEGRADING
BACTERIUM
M. Derrien(1), E. E. Vaughan(1,2), C. M. Plugge(1) and
W. M. de Vos(1,2)
(1) Laboratory of Microbiology, Wageningen University,
Wageningen, The Netherlands; (2) Wageningen Centre
for Food Sciences, Wageningen, The Netherlands
The gastro-intestinal tract (GI-tract) harbours a diverse
and abundant microbiota. Recent studies based on 16S
rDNA (16S ribosomal DNA) revealed that up to 60 to
80% of the intestinal microbiota has not yet been culti-
vated. Intestinal mucus, mainly composed of glycoproteins
named mucins, forms a crucial intermediate layer between
host and microbe since it provides protection against
pathogens on one hand and constitutes an important en-
ergy source for both commensal and potentially pathogen-
ic micro-organisms on the other. The aim of this study was
to monitor the diversity of mucin-degrading bacteria from
feces by both a 16SrDNA based approach, and to quanti-
fy them by enrichment and dilution. In the present study
we describe the isolation and characterisation of a novel
intestinal mucin-degrading organism. It was isolated by
combining enrichment in liquid and soft agar basal media
containing mucin as sole carbon source. It is an oval-
shaped, Gram-negative organism, strictly anaerobic, non-
motile and non-spore forming. A striking characteristic
was the presence of ¢laments arising from the cells, which
appeared to connect cells together leading to aggregates in
the mucin-containing medium. The16S rDNA gene se-
quence analysis revealed that the strain is distantly related
to the previously described genera Verrucomicrobium and
Prosthecobacter. On the basis of physiological and molec-
ular properties the isolate is considered to represent a new
genus and a new species, for which the name Akkermansia
muciniphila gen. nov., sp. nov. is proposed in honour of a
renouned dutch microbiologist Antoon D.L Akkermans.
238 1st FEMS Congress / Posters 103^505
P4^150
ALKALIBACTERIUM SACCHAROVORANS GEN.
NOV., SP. NOV. ^ A NEW ALKALIPHILIC SACCHA-
ROLYTIC ANAEROBIC BACTERIUM FROM CEL-
LULOLYTIC COMMUNITY OF LAKE NIZHNEE BE-
LOE (SOUTH-EASTERN SIBERIA, RUSSIA)
E. S. Garnova, T. N. Zhilina, T. P. Tourova
Laboratory of Relict Microbial Communities, Institute of
Microbiology RAS, Prospect 60-letiy Octyabrya, 7/2,
117312 Moscow, Russia
Alkaliphilic anaerobic microbial community of epiconti-
nental soda lakes represents an example of natural system
where organic matter is completely decomposed (Zavarzin
et al., 1999). Saccharolytic pathway, which connects hy-
drolytic and secondary anaerobic bacteria, is one of the
main metabolic routes in the community. Saccharolytic
anaerobic alkaliphilic microorganisms have been isolated
from geographically di¡erently-located soda lakes and in-
volve a variety of phylogenetic groups: spirochetes (Zhili-
na et al., 1996), haloanaerobes (Zhilina et al., 2001a), ba-
cilli (Zhilina et al., 2001b) and clostridia strains (Jones et
al., 1998).
Central Asian soda lakes represent an extreme type of
prairie lakes with considerable input of cellulose material.
During the study of anaerobic cellulose decomposition in
these lakes (Kevbrin et al., 1999) several strains of anaer-
obic alkaliphilic microorganisms that grew on glucose
were isolated (Tourova et al., 1999). One of them was a
member of clostridia cluster XI where it represented a new
genus named Anoxynatronum sibiricum gen.nov., sp.nov.
(Garnova et al., in press). The other three strains had the
high level of DNA-homology (96-100%). Strain Z-79820
was studied in further detail. It was Gram positive aspor-
ogenous non-motile short rod. The new isolate was true
alkaliphile : growth occurred from pH 7.2 to 10.2 with an
optimal pH of 9.0. Strain Z-79820 obligately depended on
Na+ but needed neither HCO3-/CO3- nor Cl-. It was hal-
otolerant and mesophile, which ¢tted the geological and
climatic conditions of its habitat. The organism fermented
mono- and disaccharides with production of acetate, etha-
nol, formate, H2 and CO2. The new bacterium fell into the
clostridium cluster XV where it formed an individual
branch. Among the representatives of this cluster, it was
the ¢rst alkaliphilic organism. According to growth char-
acteristics and genotypic traits of strain Z-79820 we pro-
posed the new genus and species with the name Alkalibac-
terium saccharovorans.
FEMSLE Congress 2-6-03
P4^151
NOVEL GROUPS OF CULTURABLE BACTERIA ARE
NUMERICALLY DOMINANT IN THE PORCINE
GASTROINTESTINAL TRACT
O. Hojberg, L. L. Mikelsen and B. B. Jensen
Department of Animal Nutrition and Physiology, Danish
Institute of Agricultural Sciences, PO Box 50, DK-8830
Tjele, Denmark
Fast elucidation of bacterial diversity by 16S rDNA clon-
ing and sequencing reveal the gaps in our culture collec-
tions and expedite the quest for important, yet uncultured
groups of bacteria. We analysed V2000 porcine gastro-
intestinal tract isolates and classi¢ed them into so far
V120 operational taxonomic units (otus) on the basis of
morphology, physiology and 16S rDNA sequences. De-
spite high diversity, 75% of the isolates were covered by
eighteen otus (each constituted s 1%) representing twelve
genera. This could be due to selectivity of culturing, but is
actually in accordance with a comprehensive 16S rDNA
clone library (Appl. Environ. Microbiol., 2002, 68:673-
690), where 23 of 375 de¢ned otus ( s 1% each) made up
52% of 4270 clones. Seven of our eighteen dominant otus
showed less than 97% similarity to identi¢ed species in the
16S rDNA databases, however most isolates matched por-
cine bacterial clone sequences. Clearly, some dominant
clone library otus were not among our isolates. On the
other hand, one of our dominant species (4% of colon
isolates) belonging to the Sporomusa subgroup showed
less than 97% similarity to any database sequence. The
latter as well as other unknown species were major pro-
ducers of butyrate and lactate. These metabolites are con-
sidered crucial for preventing pathogen invasion and de-
velopment of colon cancer. To our knowledge, the present
work is one of the most comprehensive studies on the
culturable microbiota of monogastrics and indicates that
major groups of so far unidenti¢ed bacteria potentially
involved in stabilising a healthy gut can actually be cul-
tured and characterised.
 1st FEMS Congress / Posters 103^505
P4^152
ORANGE-PIGMENTED YEASTS FROM THE PHYL-
LOPLANE: TAXONOMIC CHARACTERISATION
AND DIRECT MOLECULAR DETECTION ON
LEAVES
Ł . Fonseca and I. Spencer-Martins
J. Ina¤cio, A
Centro de Recursos Microbiolo¤gicos (CREM), Biotechnol-
ogy Unit, Faculty of Sciences and Technology, New Univer-
sity of Lisbon, 2829-516 Caparica, Portugal
An investigation of the mycobiota on leaves from selected
plant species (Acer monspessulanum, Quercus faginea, Cis-
tus albidus, Pistacia lentiscus and Osyris quadripartite) col-
lected at the ‘‘Arra¤bida Natural Park’’, an ecosystem of
Mediterranean characteristics in Portugal, yielded about
830 yeast isolates. Five percent of these isolates (42
strains) produced orange-pigmented colonies. They were
assigned to the Tremellales lineage of the Hymenomycetes
using a combination of phenotypic and molecular meth-
ods, which included PCR ¢ngerprinting and rDNA se-
quence analysis. According to the molecular data, part
of the phylloplane isolates was related to species of Dio-
szegia, whereas others clustered in di¡erent clades within
the Tremellales. Among those representing undescribed
species, Cryptococcus cistialbidi sp. nov. was notable for
its exclusive occurrence on Cistus albidus at high frequen-
cies. A speci¢c oligonucleotide probe, based on a region of
the 26S rRNA gene, was designed for this yeast, and £uo-
rescently labelled for direct detection and quanti¢cation of
Cr. cistialbidi sp. nov. on the phylloplane by in situ hybrid-
isation.
J.I. receives a PhD grant (Praxis XXI/BD/19833/99) from
‘‘Fundac^a‹o para a Cie“ncia e a Tecnologia’’, Portugal.
P4^153
ISOLATION AND CHARACTERIZATION OF A
HIGHLY XYLANOLYTIC RUMEN BACTERIAL
STRAIN Mz5, TYPE STRAIN OF A NEW SPECIES
PSEUDOBUTYRIVIBRIO XYLANIVORANS
M. Zorec(1), J. Kopecny(2), Y. Kobayashi(3), F. V. Nek-
rep(1) and R. Marinsek-Logar(1)
(1) University of Ljubljana, Biotechnical Faculty, Zootech-
nical Department, Domzale, Slovenia; (2) Institute of Ani-
mal Physiology and Genetics, Czech Academy of Sciences,
Prague, Czech Republic; (3) Hokkaido University, Gradu-
ate School of Agriculture, Sapporo, Japan
Several bacterial isolates were obtained from the rumen
£uid of a cow using the medium with oat spelts xylan.
Strain Mz5 showed the highest cell-associated xylanolytic
FEMSLE Congress 2-6-03
239
activity, it was curved rod with one subpolar £agellum,
stained gram-negative, was anaerobic, non-spore-forming
and produced butyrate as main fermentation product on
di¡erent carbohydrates. Other fermentation products were
lactate and hydrogen gas and during growth acetate was
utilized. High activities of xylanase, proteinase, pectin hy-
drolase and DNase were determined. The complete 16S
rDNA sequence was obtained and phylogenetic relation-
ships were determined. On the basis of other comparative
phenotypic and genotypic analyses (FAMES, RFLP, 16S
rDNA sequences, mol% G+C) for the group of 17 closely
related strains assignment to a new species was proposed ^
Pseudobutyrivibrio xylanivorans with Mz5 as a type strain.
Xylanolytic activity of Mz5T was mainly cell-associated
probably due to the production of extracellular polysac-
charides that hinder di¡usion of the enzymes. Mz5T pro-
duces 11 electrophoretically multiple xylanases (MW rang-
ing from 27 to 146 kDa). Xylanase activity was strongly
inducible with oat spelts xylan (130-times) and 146 kDa
xylanase was proved to be contitutive. Strain Mz5 could
be used as a probiotic in animal feed because of its high
xylanolytic activities and production of butyrate that has
bene¢cial e¡ects on colonocytes. Competitive ¢tness is fur-
thermore assured by the ability of fermentation of di¡er-
ent substrates and fast adaptability.
P5^1
CLONING AND STABLE EXPRESSION OF THE a-
ACETOLACTATE DECARBOXYLASE GENE FROM
BACILLUS LICHENIFORMIS IN YEAST
X. Bao, Y. Qin, H. Zheng, A. Hou, G. Yang, and D. Gao
State Key Laboratory of Microbial Technology, Depart-
ment of Microbiology,
Shandong University, Jinan, P.R. China 250100
The genomic library of Bacillus licheniformis was con-
structed using plasmid pUC19 in Escherichia coli. The
gene encodinga-acetolactate decarboxylase(a-ALDC) was
isolated from the library by directly detecting enzyme ac-
tivity on the agar plate. The nucleotide sequence of a 1.6-
kilobase DNA fragment containing the a-acetolactate de-
carboxylase gene was determined. An open reading frame
(GenBank accession number AF428095) that may encode
a protein composed of about 265 amino acids was found.
The a-acetolactate decarboxylase from B. licheniformis was
puri¢ed and characters of the enzyme were studied, the
results indicate that its pI was 4.5 and the enzyme showed
optimal activity at pH 6 and 4. The DNA fragment coding
for a-acetolactate decarboxylase was placed under the con-
trol of the phosphoglycerate kinase (PGK) promoter and
terminator of the Saccharomyces cerevisiae. Then the a-
ALDC gene expression cassette was ligated to the multiple
integration plasmid pMIRY2 which has the rDNA region
240 1st FEMS Congress / Posters 103^505
as the integrating site. The new recombinant plasmid
pMIRY2-ALDC, which was linearized at the SmaI site
in the rDNA region, was co-transformed into the beer
yeast industrial strain together which a plasmid containing
G418 gene. The multiple integration of the plasmid in the
transformants was proved by Southern blot. Yeast cells
containing pMIRY2-ALDC plasmids showed a-acetolac-
tate decarboxylase activity. The result of laboratory -scale
fermentation reveals that the diacetyl concentration in
wort was signi¢cantly lower by recombinant strain than
by the host strain.
P5^2
ARCHICTECTURAL PROPERTIES OF ARAR, THE
KEY REGULATOR OF ARABINOSE UTILIZATION
IN BACILLUS SUBTILIS
I. Franco(1), R. G. Saraiva(1) and I. de Sa¤-Nogueira(1,2)
(1) Instituto de Tecnologia Qu|¤mica e Biolo¤gica, Universi-
dade Nova de Lisboa. Avenida de Repu¤blica, Apartado 127,
2781-901 Oeiras, Portugal; (2) Faculdade de Cie“ncias e
Tecnologia, Universidade Nova de Lisboa, Quinta da Torre,
2825 Monte de Caparica, Portugal
The transcriptional factor AraR plays an important role in
carbohydrates catabolism in Bacillus subtilis. AraR is the
key regulator of arabinose utilization controlling tran-
scription from the araABDLMNPQ-abfA metabolic oper-
on and the araE/araR divergent unit by di¡erent mecha-
nisms, which allow a tight and £exible control of the
system. Additionally, AraR regulates the utilization of xy-
lose and galactose since the main transporter of arabinose
AraE is a non-speci¢c permease also responsible for the
transport of those carbohydrates into the cell. The amino
acid sequence of AraR suggests a chimeric organization
for the protein. A small N-terminal domain homologous
to the GntR family of bacterial repressors is involved in
DNA-binding, whereas a larger C-terminal region typical
of the LacI/GalR family of bacterial regulators binds the
inducer and is also responsible for oligomerization. Re-
cently, a subdivision of the GntR family according to
the structural, phylogenetic and functional characteristics
of the C-terminal domain failed to include AraR suggest-
ing the emergence of a new sub-family typi¢ed by AraR.
In this work, a collection of AraR mutants displaying
single amino acid substitutions was obtained by random
mutagenesis of the araR allele and in vivo screening for
defects, either associated with a constitutive phenotype or
ability to respond to the inducer arabinose. These mutant
proteins were characterized by in vivo regulation studies.
De¢nition of the AraR functional domains and its impli-
cations in the mechanistic mode of action of the repressor
will be discussed.
FEMSLE Congress 2-6-03
P5^3
REGULATORY MECHANISMS OF THE ARABINAN-
DEGRADING COMPLEX IN BACILLUS SUBTILIS
J. M. Ina¤cio(1), M. P. Raposo(1), L. J. Mota(1a) and I.
de Sa¤-Nogueira(1,2)
(1) Instituto de Tecnologia Qu|¤mica e Biolo¤gica, Universi-
dade Nova de Lisboa. Avenida de Repu¤blica, Apartado 127,
2781-901 Oeiras, Portugal; (2) Faculdade de Cie“ncias e
Tecnologia, Universidade Nova de Lisboa, Quinta da Torre,
2825 Monte de Caparica, Portugal. aCurrent Address: Bio-
zentrum der Universita«t Basel, Klingelbergstr. 50-70 CH-
Basel, Switzerland.
Hemicellulose is composed of xylans, arabinans, galactans
and mannans, which contribute to the texture of plant
tissues as structural components of the cell wall. Bacillus
subtilis produces three di¡erent types of enzymes involved
in degradation of the homoglycane arabinan, an endo-ara-
banase and two arabinofuranosidases, capable of releasing
arabinosyl oligomers and arabinose from plant cell walls.
These arabinan-degrading activities are able to disintegrate
potato tissue and to solubilize sugar beet protopectina.
The two arabinofuranosidases are most probably encoded
by the last gene of the arabinose metabolic operon ara-
ABDLMNPQ-abfA, and the xsa gene, located 23-kb
downstream from the operon. The abnA gene, positioned
immediately upstream from the metabolic operon, most
probably encodes an endo-arabanase. In vivo RNA anal-
ysis indicates that abnA and xsa are monocistronic, and
transcribed from sigma-A like promoters. Furthermore,
transcriptional studies showed that expression of the ara-
binases is induced by arabinose and arabinan, and re-
pressed by glucose. The induction mechanism is mediated
by the key regulator of arabinose metabolism, AraR.
However, the regulatory mechanisms of these three genes
appear to be di¡erent and its physiological implications
will be discussed.
This work was supported by grant POCTI/AGR/36212/
from FCT to I. S-N. J. I. was the recipient of fellowship
034/BIC/2001.
 1st FEMS Congress / Posters 103^505
P5^4
GENE CLONING AND CHARACTERIZATION OF
CITROBACTER FREUNDII L-METHIONINE -Q-
LYASE
I. V. Manukhov(1), S. M. Rastorguev(1), G. B. Zavilgel-
sky(1), D. V. Mamaeva(2), T. V. Demidkina(1)
(1) State Scienti¢c Center (GosNIIgenetika), 1st Dorozh-
nii pr. 1, 117545, Moscow, Russia ; (2) Engelhardt Institute
of Molecular Biology, Vavilov str. 32, Moscow 119991,
Russia
L- methionine-Q-lyase (MGL) catalyzes Q-elimination of L-
methionine. The enzyme was puri¢ed from Citrobacter
freundii cells. Its N-terminal sequence was determined. Us-
ing oligonucleotide primers constructed on the basis of N-
terminal sequence and sequences of active center of MGL
from di¡erent bacteria we obtained 500 kb PCR-product.
It was cloned in pUC19. Sequences of some clones were
determined. One clone contained a fragment (500 bp) with
a sequence homologous to that of Pseudomonas putida
MGL. EcoR1 genomic library of Citrobacter freundii was
constructed in pUC18 and screened by 500 bp fragment.
Two 3.0 kb identical fragments showed MGL activity
while expressed in E. coli. The sequence of the 3.0 kb
fragment contained two ORF. Region of 1,194 nucleotides
has 56% and 61 % sequence identity with MGLs from
Pseudomona putida and Fusobacterium nucleatom and
thus encodes the MGL gene. It was subcloned and homo-
geneous MGL was obtained. Kinetic parameters of Q-elim-
ination reactions for the wild type and recombinant en-
zymes were determined and proved to be identical.
Comparison of Citrobacter freundii MGL kinetic parame-
ters with those of MGLs from di¡erent bacteria revealed
that enzymes do not di¡er in their a⁄nity to substrates
but di¡er in rates of Q-elimination reactions.
P5^5
REDOX BALANCING IN RECOMBINANT XYLOSE
UTILISING SACCHAROMYCES CEREVISIAE
E. Rintala, L. Salusjarvi, L. Ruohonen and M. Penttila
VTT Biotechnology, Technical Research Centre of Finland,
P.O. Box 1500, FIN-02044 VTT, Finland
Lignocellulosic biomass is an attractive substrate to be
used in the production of ethanol as a renewable energy
source to replace fossil fuels. Hexose sugars of lignocellu-
losics are readily fermented by many microorganisms,
whereas pentoses, the major constituent hemicellulose are
a challenge in the process to make it economically feasible.
S. cerevisiae, a well-known fermentation process organism,
FEMSLE Congress 2-6-03
241
cannot naturally utilise xylose although it is able to fer-
ment xylulose, an isomer of xylose. Introduction of Pichia
stipitis genes encoding xylose reductase (XR) and xylitol
dehydrogenase (XDH) gives S. cerevisiae the ability to
utilise xylose. Over-expression of its own xylulokinase-en-
coding gene (XKS1) further enhances xylose consumption
[1]. The XR and XDH reactions generate a cofactor im-
balance into the cell because XR has a preference for
NADPH, whereas XDH is speci¢c for NAD+. One at-
tempt to solve this imbalance has been the introduction
of a transhydrogenase cycle into the cells, to convert
NADP+ and NADH, the products of XR and XDH, to
NADPH and NAD+, the substrates for XR and XDH [2].
We have studied a of transhydrogenase cycle, which relies
on overexpression of malic enzyme. We have also used
genome wide approaches to study the physiology of xylose
utilisation in recombinant S. cerevisiae. We have made
studies both on proteomic and genomic level to reveal
novel and unpredictable changes in the metabolism of xy-
lose fermenting yeast [3,4].
[1] Toivari et al. (2001). Metab.Eng.3, 236-249. [2] Aristi-
dou et al.(1999). Patent Application. PCT/FI99/00185. [3]
Salusjarvi et al.(2002). Yeast 20,295-314. [4] Salusjarvi et
al. Manuscript in preparation.
P5^6
PEROXISOME FORMATION AND REGULATION
OF PEROXISOME HOMEOSTASIS IN CANDIDA
PSEUDOTROPICALIS DURING VARIOUS CARBON
SOURCE UTILIZATION
I. Rusyn(1), O. Kulachkovsky(2), O. Moroz(2), S.
Gudz(1)
(1) Department of Microbiology and (2) Interdepartment
Laboratory of Electron Microscopy, Biological Faculty,
Ivan Franko National University of Lviv, Hrushevskoho
St. 4, UA-79005 Lviv, Ukraine
Microbodies (peroxisomes, glyoxysomes) play important
role in yeast cell metabolism. Peroxisomes contain many
of enzyme systems, through which cells are capable to
metabolize various carbon compounds. Quantity of per-
oxisomes, their fermentative maintenance changes accord-
ing to change of cultivation medium. Regulation of per-
oxisome homeostasis realize by means of induction of
peroxisome biogenesis and catabolite regulation: repres-
sion of microbody formation and also peroxisome degra-
dation. Peroxisome formation and regulatory mechanisms
of their homeostasis in C. pseudotropicalis were insu⁄-
ciently investigated. Aime of this work was investigation
of microbody formation in presence of various carbon
compounds and regulatory mechanisms of their homeo-
stasis in C. pseudotropicalis. Induction of microbody bio-
genesis during ethanol, ethylamine, lactose, oleate and hy-
242 1st FEMS Congress / Posters 103^505
drocarbons utilization was showed. Microbodies were re-
vealed on each growth phase during lactose utilization in
aerobic conditions. Results demonstrated noncomplete
glucose catabolite repression of this organelles biogenesis.
De¢cient provide of culture with oxygen was the cause of
limited utilization of derived from hexoses ethanol. In
these conditions of nutrient deprivation despite on pres-
ence in cultivation medium of ethanol (as inductor) micro-
bodies biogenesis slow down and their autophagic degra-
dation take place. Ethylamine utilization only as nitrogen
source but not carbon source, which is result repressive
‘‘ammonium e¡ect’’, was showed. Cells needed addition
of carbone source ^ glucose to cultivation medium for
ethylamine utilization. Large peroxisomes were revealed
in yeast cells during oleate and ethylamine (in presence
of glucose) utilization. C. pseudotropicalis are capable to
utilize hydrocarbons. Small peroxisomes were revealed in
cells during hydrocarbon utilization.
P5^7
STRUCTURAL AND FUNCTIONAL ANALYSIS OF
NITRILE DEGRADATION CLUSTER FROM RHO-
DOCOCCUS RHODOCHROUS M8
L. Ryabchenko, E. Kotlova, G. Larikova, A. Novikov, D.
Podchernjaev, G. Chestukhina, A. Yanenko, V. Debabov
Institute of Genetics and Selection of Industrial Microor-
ganisms, 1-st Dorozhny proezd, 1, 113545, Moscow, Russia
R. rhodococcus M8 is used as a biocatalyst for industrial
production of acrylamide. E⁄ciency of the catalyst is de-
pendent upon two enzymes ^ cobalt-dependent nitrile hy-
dratase (NH), catalysing transformation of acrylonitrile to
acrylamide, and amidase (AM), hydrolysing amide to
acrylic acid. NH and AM were isolated from strain M8
and its mutants producing enzymes in great amounts. NH
is a heteromer of two types of subunit (26 and 23 kD),
while AM comprises 4 identical subunits (Mb 42 kDa).
The best substrates for NH and AM are short-chained
aliphatic nitriles and amides. NH and AM are completely
inhibited by Hg and Ag ions, which are typical reagents on
sulfohydrylic group. Activity of NH, in contrast to AM,
was decreased in presence of PMSF ^ an inhibitor of ser-
ine proteinases. Maximum activity of NH and AM was
observed at 45‡C and 55-60‡C, respectively. We cloned
and sequenced the chromosomal loci that control synthesis
of both enzymes in R. rhodochrous M8. One of them,
designated NH locus, contains genes coding subunits of
NH and regulatory genes controlling induction of NH
and AM. The amidase gene was found in the other locus
which was not linked to NH genes. The AM gene from
M8 had a high level of homology with aliphatic amidases
found in Pseudomonas aeruginosa. Thus, the absence of
operon structure for NH and AM genes is the key feature
FEMSLE Congress 2-6-03
of the strain M8. Expression of NH genes was shown in
Rhodococcus erythropolis, and expression of AM gene in
E. coli. We introduced amino acid changes into the Co-
binding site of NH. Although these mutations did not
a¡ect positions which directly interact with Co, all mu-
tants were inactive. The mutant strain M33, which pos-
sesses high constitutive synthesis of NH and is defective in
AM synthesis, has all regulatory genes in the NH locus
deleted. M33 now will be utilized as an industrial bioca-
talyst to attain 50 % solutions of acrylamide, which are
applicable for polymerization.
without additional concentration.
P5^8
CLONING AND EXPRESSION OF MYO-INOSITOL
OXYGENASE FROM THE YEAST CRYPTOCOCCUS
NEOFORMANS
W. A. Schroeder, S. C. McFarlan and P. M. Hicks
Biotechnology Development Center, Cargill Inc., P.O. Box
5702, Minneapolis, MN, 55440-5702, USA
The oxidation of myo-inositol to D-glucuronate, the ¢rst
dedicated step in myo-inositol catabolism is catalysed by
the enzyme myo-inositol oxygenase (MIO). This enzyme
could be used to develop novel biosynthetic routes to vi-
tamin C, D-glucaric acid, and D-glucurono-Q-lactone di-
rectly from D-glucose or myo-inositol by fermentation in
yeast or bacteria. The 35 kDa enzyme was puri¢ed from
the yeast Cryptococcus terreus by a combination of anion
exchange chromatography, a⁄nity chromatography, and
2-D gel puri¢cation. A peptide ¢ngerprint of the puri¢ed
protein was used to identify mio homologs and ESTs in
tissues of fungi, higher plants, and in kidney tissues of
mammals. Homologs of mio were cloned from human,
rat, Arabidopsis thaliana and Cryptococcus neoformans
cDNA libraries. Recombinant MIO was produced in
yeast, bacterial and insect cells. Several groups have iden-
ti¢ed mammalian homologs of these genes that were ex-
pressed in the cortex of kidneys and that repression of mio
was associated with acute renal failure, diabetic nephrop-
athy and incorrect embryonic nephrogenesis. In plants,
mio expression has been found in response to environmen-
tal stresses and during seedling development. Thus, in ad-
dition to its value in providing novel routes to fermenta-
tion products from D-glucose, myo-inositol oxygenase
may be useful in the treatment of kidney disease and in
the development of crops with improved agronomic traits.
 1st FEMS Congress / Posters 103^505
P5^9
DISRUPTION OF THE GENE ENCODING TRYPSIN-
LIKE ENZYME FROM STREPTOMYCES RIMOSUS
M. SNtaudohar Kozjan(1), H. Petkovic¤(2), M. Legis›a(3)
(1) Krka d.d., Novo mesto, Slovenia; (2) Biotica Technol-
ogy Limited, Cambridge, United Kingdom; (3) National
Institute of Chemistry, Ljubljana, Slovenia
Bacterium Streptomyces rimosus is known to produce at
least ¢ve extracellular proteases. A 0.4 kb DNA fragment
of a trypsin-like gene was ampli¢ed using primers designed
on the basis of the sal gene from Streptomyces lividans.
Genomic DNA from S. rimosus was used as a template in
the polymerase chain reaction. The sequence of the frag-
ment showed 72 % similarity to trypsin-like gene from
Streptomyces griseus. The 0.4 kb fragment was used to
construct a vector for gene disruption experiment. Four
S. rimosus thiostrepton resistant transformants, SRT2,
SRT3, SRT5 and SRT6, displaying wild type morphology
were selected. Southern hybridization analysis revealed
that transformants SRT2 and SRT3 shared the same re-
striction/integrational pattern, but subsequently lost thio-
strepton resistance.The plazmid had integrated by multiple
copies into 19 kb SstI fragment of the recipient strain. In
the case of SRT5 transformant, the plasmid or at least its
part apparently integrated into 3,2 kb SstI fragment of the
recipient strain. No large-scale DNA ampli¢cation/rear-
angement could be detected on the gel electrophoresis
due to genetic instability that was well documented for a
number of other gene disruptions in streptomycetes. The
total proteolytic activity of SRT5 was lower than in the
parent strain and SRT2, SRT3, SRT6.
P5^10
OPTIMISATION OF THE TRANSCRIPTION TUN-
ING CONCEPT FOR LACTOSE INDUCTION
G. Striedner, V. Scherrer, A. Landberg, K. Du«rrschmid, M.
Cserjan-Puschmann, F. Po«tschacher, J. Kern and K. Bayer
Institute of Applied Microbiology, University of Natural
Resources and Applied Life Sciences, Nussdorfer La«nde
11, 1190 Vienna, Austria
Optimal exploitation of the metabolic potential of the cell
factory provides an e¡ective approach to obtain high yield
of recombinant protein in bacteria. Since the thereto used
strong expression vectors overburden host cell metabo-
lism, adaptation of recombinant gene expression to the
capabilities of the synthesis machinery is essential. There-
fore a novel concept of transcription rate control based on
continuous supply of limiting amounts of inducer in a
FEMSLE Congress 2-6-03
243
constant ratio to biomass was developed and implemented
in a fed batch process with carbon limited exponential feed
regime. As a whole, this approach proved to be a very
e⁄cient strategy for the optimisation of recombinant pro-
tein production processes due to increased yield, enhanced
process reproducibility and controllability. Under these
conditions the substitution of IPGT by lactose is enabled,
thereby complying with regulatory issues of the produc-
tion of recombinant proteins on industrial scale. However,
the concept of tuning transcription rate, which was devel-
oped for induction with IPTG has to be adapted to the use
of lactose, whereby the main issue is maintaining the ap-
propriate induced state versus lactose consumption. To
gain optimal yields the behaviour of the lac based T7
expression system during lactose induction and the inter-
action of L-galactosidase, which is controlled by the lac
promotor as well must be investigated. Therefore mRNAs
of T7-RNA polymerase, L-galactosidase and of the target
protein were quanti¢ed by real time PCR, L-galactosidase
was quanti¢ed by ELISA and an activity assay. These
data provide signi¢cant information for optimised process
design using lactose as inducer.
P5^11
HYDROGENASES OF THE MARINE FILAMEN-
TOUS, NON-HETEROCYSTOUS, CYANOBACTE-
RIUM LYNGBYA MAJUSCULA CCAP 1446/4 ^ BIO-
TECHNOLOGICAL APPLICATIONS
E. Leita‹o(1), F. Oxelfelt(1), P. Oliveira(2), A. M. Bur-
ja(3), P. C. Wright(4) and P. Tamagnini(1,2)
(1) IBMC ^ Cellular and Applied Microbiology Unit, Uni-
versity of Porto, Rua do Campo Alegre 823, 4150-180 Por-
to, Portugal; (2) Dept. Botany, Fac. Sciences, Univ. Porto,
Rua do Campo Alegre 1191, 4150-181 Porto, Portugal; (3)
Dept of Chemical and Process Engineering, Heriot-Watt
University, Edinburgh EH14 4AS, UK; (4) Biological
and Environmental Systems Group, Dept. Chemical and
Process Engineering, University of She⁄eld, She⁄eld S10
2TN, UK
Molecular hydrogen has been purported as a potential
energy source and carrier for the 21st century. Hydrogen
can be produced using both chemical and biological
means. The use of various groups of microorganisms,
such as cyanobacteria, for H2 production has received in-
creased attention within the past decade. The majority of
research within this ¢eld has focussed on heterocystous
cyanobacteria, to the detriment of both unicellular and
¢lamentous non-heterocystous strains. Here we report pre-
liminary research focussed on hydrogenases structural and
accessory genes within the marine, ¢lamentous, non-heter-
ocystous cyanobacterium, Lyngbya majuscula CCAP 1446/
4. L. majuscula was found to contain the structural genes
244 1st FEMS Congress / Posters 103^505

(hupSL) encoding an uptake hydrogenase, the gene (hoxY)
encoding the small subunit of the hydrogenase moiety of
the bi-directional enzyme and the accessory genes hypD
and hypF. In silico analysis highlighted two sets of long
repetitive sequences within the hupSL intergenic region.
5’RACE experiments revealed the presence of a transcrip-
tion start site, putatively for the hupSL operon, 59 bp
upstream of the hupS start-codon. Hydrogen uptake activ-
ity was con¢rmed via H2 electrode measurements. In par-
allel, large scale production of L. majuscula biomass was
performed, as a future lead to integrated growth and pro-
duction of hydrogen and other natural products.
cyclases of higher plants and a cyanobacterium but also
from lycopene L-cyclases of other eubacteria.
P5^13
HYDROGEN PHOTOPRODUCTION BY PURPLE
BACTERIA. HAS IT A PRACTICAL POTENTIAL?
A. A. Tsygankov
Institute of Basic Biological Problems RAS, Pushchino,
Moscow Region, 142290, Russia

P5^12 Hydrogen can be produced by purple bacteria under the

light via nitrogenase system. Optimal conditions for hy-
STRUCTURE AND FUNCTIONAL ANALYSIS OF A drogen photoproduction (temperature, pH, light intensity,
LYCOPENE L-MONOCYCLASE GENE ISOLATED electron donor excess, nitrogen de¢ciency, and anaerobic
FROM A UNIQUE MARINE BACTERIUM PRODUC- conditions) are already de¢ned. The rates of the reaction
ING MYXOL as high as 3.6 l per l of matrix in one hour by immobilized
cells were measured. However, before the practical consid-
M. Teramoto(1), S. Takaichi(2), Y. Inomata(1), H. Ike- eration of photobiological hydrogen production many sci-
naga(1) and N. Misawa(1) enti¢c and technological problems should be solved. Some
of them are: low e⁄ciency of light energy bioconversion;
(1) Marine Biotechnology Institute, Kamaishi-shi, Iwate sensitivity of key enzymes (nitrogenase and hydrogenase)
026-0001, Japan ; (2) Nippon Medical School, Biological to oxygen; low saturating light intensity comparing with
laboratory, Kosugi-cho, Nakahara, Kawasaki-shi 211-0063, sun light ; low speci¢c rates of hydrogen photoproduction;
Japan decrease of speci¢c rates during scale-up procedure. A
numerous reviews and research articles were published
Cyclization of the linear, pink-colored carotenoid lycopene last decades. Simultaneously, the development of cultiva-
(i,i-carotene) is a pivotal branching point in the diver- tion regimes, immobilization procedures, and photobior-
gent carotenoid biosynthetic (crt) pathways. In the present eactors brings some methods from the state-of-the art to
study, we found for the ¢rst time a gene coding for lyco- the technology. In this presentation the application of
pene L-monocyclase, which is responsible for the synthesis modern technologies for solution of problems shown
of Q-carotene (L,i-carotene) from lycopene. The crt gene above is discussed.
cluster including this lycopene L-monocyclase gene (desig-
nated crtYm) was isolated from a unique marine bacte-
rium, strain P99-3, that produces myxol. The sequence
analysis revealed that this cluster contained the genes ho-
mologous to the known crtI, crtB, crtZ, crtY, and crtA
genes in addition to two unknown open reading frames,
and that the crtA homolog was divergently organized rel-
ative to the others in a tail-to-tail orientation. CrtYm, a
CrtY homolog, metabolized lycopene to Q-carotene in ad-
dition to a small amount of L-carotene (L,L-carotene),
which was con¢rmed by deletion/expression analysis of
the crtYm and by subsequent analysis of the metabolites
from lycopene based on the retention times on high-per-
formance liquid chromatography, spectral data through
UV-visible absorption, and mass spectrometry. CrtYm ac-
tivity was markedly inhibited by coexpression of orf1,
which was localized upstream of crtYm, suggesting that
the myxol synthetic pathway is controlled by an unknown
Orf1-mediated mechanism in strain P99-3. A phylogenetic
analysis showed the lycopene L-monocyclase from strain
P99-3, CrtYm, to be distant not only from the lycopene L-

FEMSLE Congress 2-6-03

 1st FEMS Congress / Posters 103^505
P6^1
ANTIMICROBIAL RESISTANCE PHENOTYPES IN
MRSA STRAINS ISOLATED FROM INVASIVE IN-
FECTIONS IN ROMANIA ^ 1998 ^ 2001
I. Codita(1), V. Dumitrescu(1), L. Grigore(1), E. Moldo-
veanu(1), O. Dorobat(2), I. Nistor(3), R. Papa-
gheorghe(4), N. Popescu(4), M. Ghita(5), E. Mitache(6)
and T. Turcu(7)
(1) Cantacuzino Institute, Splaiul Independentei 103, Bu-
charest, R-70.100 Romania; (2) Infectious and Tropical
Diseases Clinical Hospital ‘‘ Victor Babes’’, Bucuresti;
(3) Emergency Pediatric Clinical Hospital ‘‘ Gr. Alexan-
drescu’’, Bucuresti; (4) Clinical Hospital ‘‘Coltea’’, Bucur-
esti; (5) Clinical Institute Fundeni, Bucuresti; (6) ‘‘Ma-
tei Bals’’ Institute for Infectious Diseases, Bucuresti; (7)
Infectious and Tropical Diseases Clinical Hospital, Iasi
40 strains were isolated from bloodcultures and identi¢ed
as methicillin resistant Staphylococcus aureus (MRSA), of
which 20 strains were isolated and characterised in 1998
and 20 in 2001.The study of antimicrobial resistance phe-
notypes using the main antimicrobials active on staphylo-
cocci revealed the following: (1) 12 from the 20 strains
isolated in 1998 and 8 from those isolated in 2001 showed
high resistance to methicilline (Thomasz 1991); (2) of the
strains isolated in 1998, 2 were classi¢ed in the ‘‘reduced
susceptibility’’ or ‘‘GISA candidate’’ MRSA (CMI = 4
mg/L) category. All strains isolated in 2001 had CMI val-
ues 9 2 mg/L ; (3) 8 of the strains isolated in 1998 and 6 of
those isolated in 2001 showed inductible resistance to
erythromycin ( ER My S phenotype); one strain isolated
in 2001 showed intermediate phenotype to erythromycine
(E) and clyndamycine (My) ; (4) one strain in each year
showed an KR TS GS / APH (3’’) III amynoglicoside phe-
notype, while the rest of the strains harboured the KR TR
GR / AAC (6’) ^ APH (2’’) phenotype, which is respon-
sible for resistance to all aminoglicosides ; (5) 18 strains in
each of the two groups were resistant to £uoroquinolones.
This study which examined a greater number of strains
isolated from invasive infections yielded information on
the susceptibility level and on the prevalence of the ac-
quired resistance to the main classes of antimicrobial sub-
stances that are active on MRSA strains. These data can
be used both for de¢ning local S/I/R critical values and for
de¢ning the activity spectra and the indications for the use
of these classes of antimicrobials in invasive infections, in
Romania. Identifying the rare resistance phenotypes can
help orient the research in order to clarify the resistance
mechanisms before resistance spread and for adopting a
more appropriate regementation looking to the politics of
antimicrobial substances utilisation.
FEMSLE Congress 2-6-03
245
P6^2
ANTIMICROBIAL RESISTANCE AMONG DIAR-
RHEAGENIC ESCHERICHIA COLI IN SERBIA
N. Galic(1) and M. Cobeljic(2)
(1) Institute of Public Health of Serbia, Belgrade, Yugo-
slavia ; (2) Military Medical Academy, Belgrade, Yugosla-
via
The objective of this study was to determine the frequency
of antibiotic resistance among diarrheagenic E. coli iso-
lated from feces of patients with diarrhea and healthy
persons in Serbia from 1982 to 1999. In total, 884 E.
coli strains were identi¢ed and all of them were tested
for the presence of all known virulence factors of diarrhea-
genic E. coli. Among them, 235 (26.6%) strains of enter-
otoxigenic (ETEC), 26 (2.9%) strains of enteropathogenic
(EPEC) and 48 (5.4%) strains of di¡usely adherent
(DAEC) E. coli were detected. Enteroaggregative, shiga-
toxin producing and enteroinvasive E.coli were not found.
The remaining E. coli strains did not express virulence
factors. All the strains were tested for antimicrobial resis-
tance by disc-di¡usion method. Resistance to one or more
antibiotics was found in 113 (48%) ETEC, 26 (100%)
EPEC, 38 (79%) DAEC and 575 (60%) strains nonpatho-
genic E. coli, what was signi¢cantly di¡erent when com-
paring these groups of strains. Resistance was not detected
to quinolones and third generation cephalosporins in diar-
rheagenic E. coli. ETEC, EPEC and DAEC strains were
resistant to two or more antibiotics at a similar rate (84%,
96% and 92%, respectively). The most frequent resistance
patterns were ApRClR and StRApRClR in ETEC,
StRApRClR and StRTcR in EPEC, StRApRTcRT/SR
and ApRClR in DAEC. Common ¢nding of multiple anti-
biotic resistance among diarrheagenic E. coli in this study
may be due to antimicrobial selective pressure and stabil-
ity of antibiotic genes in the environment.
P6^3
ANTIMICROBIAL SUSCEPTIBILITIES OF HAEMO-
PHILUS INFLUENZAE AND HAEMOPHILUS PARA-
INFLUENZAE RESPIRATORY TRACT ISOLATES IN
A LARGE GREEK HOSPITAL
S. Kanavaki, E. Faviou, S. Karambela, M. Makarona, E.
Eustathiadou, E. Alexandraki, P. Koumantakis, A. Pefanis
Sotiria General Hospital, Athens, Greece
A total of 247 isolates of Haemophilus spp [119 H. in£u-
enzae (HI), and 128 H. parain£uenzae (HP)] were recov-
ered in our hospital laboratory from sputum cultures,
from September 2001 to September 2002, and were char-
246 1st FEMS Congress / Posters 103^505
acterized with respect to beta-lactamase production, and
the in vitro activities of ten antimicrobial agents, by apply-
ing the Kirby-Bauer method. The overall percentages of
HI isolates found to be resistant to amoxicillin, amoxiclav,
cephalothin, cefuroxime, cefotaxime, tetracycline, erythro-
mycin, cotrimoxazole, chloramphenicol, and cipro£oxacin
were as follows : 11, 0, 2.5, 0, 0, 4, 50, 27, 0, and 0, re-
spectively. Regarding HP the respective percentages were:
7, 0, 3, 0, 0, 1, 58, 9, 0, and 0. For HI the prevalence of
beta-lactamase production was 11%, while for HP was 7%.
All beta-lactamase positive isolates were resistant to ampi-
cillin. None of the beta-lactamase negative isolates were
resistant to ampicillin (BLNAR). This is in contrast with
reports from Spain and USA, where BLNAR strains rep-
resent 9.3% and 2.5% of the isolated HI strains. Our ¢nd-
ings demonstrate that antimicrobial resistance pro¢les of
HI and HP di¡er between Greece and other EU countries.
Most of the EU countries (with the exception of Germany
and Italy) report higher percentages of resistance to amox-
icillin, amoxiclav, and cotrimoxazole. In contrary, the fre-
quency of erythromycin resistance in Greece, is very high.
P6^4
ANTIMICROBIAL SUSCEPTIBILITY OF VIBRIO
PARAHAEMOLYTICUS IN KOREA, 1997 TO 2002
J. Kim, S. Kim, H. Jang, H. Nam, Y. Kang, B. Lee
Lab. of Enteric Infections, Dept. of Microbiology, National
Institute of Health, Nokneon-Dong, Eunpyeong-Gu, Seoul,
Korea 122-701
Vibro parahaemolyticus is a major agent of diarrheic dis-
ease in Korea. In this study we reviwed the antimicrobial
susceptiblility patterns of V. parahaemolyticus to L-lac-
tams, and quinolones. A total of 3,319 isolates of V. para-
haemolyticus were received by the national public health
network of Korea from 1997 to 2002. Allowing for epide-
miological properties, 250 strains among these isolates
were selected. Their susceptibility to nalidixic acid (NA),
nor£oxacin (NFX), or£oxacin (OFX), spar£oxacin (SPX),
and cipro£oxacin (CIP) was tested by agar dilution meth-
od. The disk di¡usion method was adopted for the sus-
ceptibility to ampicillin (AM), ampicillin/sulbactam
(SAM), cefoxitin (FOX), cephalothin (CF), amoxicillin/
clavulanic acid (AMC), azteonam (ATM), ceftriaxone
(CRO), ticarcilli (TIC), ceftazidime (CAZ), cefotxime
(CTX) and trimethoprim/sulfamethoxazole (SXT). The
susceptibility of tested V. parahaemolyticus was 42.9% to
AM, 98.2% to SAM, 98.6% to FOX, 95.2% to CEP, 98.0%
to AMC, 66.7% to ATM, 98.0% to CRO, 14.3% to TIC,
98.6% to CAZ , and 98.6% to CTX. DNA sequence anal-
ysis of the quinolone resistance determining regions
(QRDR) of gyrA and parC genes was carried out for the
strains which is weakly susceptible to £uoroquinolones.
FEMSLE Congress 2-6-03
The V. parahaemolyticus isolates showed a marked de-
crease in susceptibility to SPX measured by geometric-
mean MICs, reduced from 57.9% in 1997 to 0% in 2002,
and other £uoloquinolones showed similar tendency.
Many tested strains have a silent mutation in gyrA and
parC gene, but no amino acid substitution was observed.
And no isolates showed decrease in susceptibility to SXT.
In this study we have not observed an important resistance
pattern to all the tested antibiotics, but we may predict the
emergence from the decreasing pro¢le of susceptibility,
emergence of resistant strain is predicted.
P6^5
MUTATION IN gyrA, gyrB, parC AND parE GENES OF
QUINOLONE RESISTANT SALMONELLA SPP. ISO-
LATED IN KOREA
J. Kim, S. Kim, H. Jang, Y. Kang, B. Lee
Lab. of Enteric Infections, Dept. of Microbiology, National
Institute of Health, Nokneon-Dong, Eunpyeong-Gu, Seoul,
Korea 122-701
Salmonella spp. strains obtained during 1997 to 2001 were
tested for their susceptibilities to quinolones. Among the
tested strains, 6 quinolone-resistant strains were con¢rmed
(Salmonella Typhi; 1 strain, Salmonella Enteritidis; 5
strains). They were highly resistant to nalidixic acid
(MIC, 320 to 1280Wg/ml) and cipro£oxacin (MIC, 0.4 to
0.8Wg/ml). In order to characterize the quinolone-resistant
isolates, sequencing of the quinolone resistance determin-
ing regions (QRDR) of gyrA, gyrB, parC and parE genes
was done. gyrA gene in six quinolone-resistance strains
had three mutations in di¡erent amino acid substitution
codon serine-83, aspartate acid-87 and glutamate-133. The
amino acid substitution in serine-83 was TCC (Ser) !
TTC (Phe). The amino acid substitutions in aspartate-87
were GAC (Asp) ! AAC (Asn) and GAC (Asp) ! GCC
(Gly). The amino acid substitution in glutamate-133 was
GAA (Glu) ! GGA (Gly). No mutations were found in
the QRDR regions of gyrB, parC and parE genes. These
¢ndings indicated this mutation in gyrA plays an impor-
tant role in acquisition of quinolone-resistance in clinical
isolates of Salmonella spp
 1st FEMS Congress / Posters 103^505
P6^6
CHARACTERIZATION OF MULTIDRUG RESIS-
TANCE SHIGELLA SONNEI IN KOREA 1998 TO 2002
S. Kim, J. Kim, H. Jang, Y. Kang, B. Lee
Lab. of Enteric Infections, Dept. of Microbiology, National
Institute of Health, Nokneon-Dong, Eunpyeong-Gu, Seoul,
Korea 122-701
During the last few years, there has been a continuous
increase in the incidence of Shigella infection in Korea.
Although the antibiotic therapy may reduce the duration
of symptoms and excretion of Shigella, it may increase the
risk of developing antibiotic resistance. This study was
conducted in order to monitor resistant rates of Shigella
sonnei isolates and explore multi-drug resistance pattern.
A total of 5,542 S. sonnei strains obtained from 1998 to
2002 were tested by disk di¡usion method for their anti-
biotic susceptibility to ampicillin (AM), amikacin (AN),
ampicillin / sulbactam (SAM), cephalothin (CF), chloram-
phenicol (C), cipro£oxacin (CIP), ceftriaxone (CRO), ce-
foxitin (FOX), gentamicin (GM), kanamycin (K), nalidixic
acid (NA), streptomycin (S), trimethoprim / sulfamethox-
azole (SXT), ticarcillin (TIC), tetracycline (TE), and
amoxicillin / clavulanate (AMC). The type speci¢c PCR
was used for the classi¢cation of ESBL encoding genes,
and genes related to tetracycline and streptomycin resis-
tance. Strains tested showed high resistance to S, TE, SXT
(876%, 94.6%, 94.7%), and were susceptible to CIP, C,
FOX, AN. 86.9% of tested strains were resistant to 4 or
more drugs. 35.2% of strains had resistance to NA, TE, S
and SXT. 1,030 strains showed resistance to AM, SAM,
K, S, SXT, TIC, TE and AMC. 24 ESBL (TEM, CTX-M,
and CMY types) producing strains were con¢rmed. The
tetA and strA were common genes related to tetracycline
and streptomycin resistance, respectively. Some of the
tested NA resistant isolates have a mutation, which causes
substitution of Ser-83 in gyrA. This suggests that the de-
creased quinolone susceptibility was due to the mutation
of gyrA gene. Even though the increase of antibiotic resis-
tance over time was not remarkable during this period,
decrease in susceptibility to cephalosporins and quinolones
was noticeable. These results suggest the limitation of the
commonly used antibiotics, and it may emerge as a serious
threat to the public health in Korea.
FEMSLE Congress 2-6-03
247
P6^7
REVIEW OF ANTIMICROBIAL RESISTANCE OF
SHIGELLA FLEXNERI ISOLATED IN KOREA DUR-
ING 1998 TO 2002
S. Kim, J. Kim, H. Jang, Y. Kang, B. Lee
Lab. of Enteric Infections, Dept. of Microbiology, National
Institute of Health, Nokneon-Dong, Eunpyeong-Gu, Seoul,
Korea 122-701
Recently, shigellosis has reemerged as a major infectious
disease in Korea. The large scale outbreaks have been in
1998 through 2002. Shigella £exneri is the second major
cause of shigellosis in Korea. In this study, the patterns of
antimicrobial susceptibility of S. £exneri were reviewed. A
total of 396 S. £exneri strains obtained from 1998 to 2002
were tested for their antibiotic susceptibility to ampicillin
(AM), amikacin (AN), ampicillin/sulbactam (SAM), ceph-
alothin (CF), chloramphenicol (C), cipro£oxacin (CIP),
ceftriaxone (CRO), cefoxitin (FOX) gentamicin (GM), ka-
namycin (K), nalidixic acid (NA), streptomycin (S), trime-
thoprim/sulfamethoxazole (SXT), ticarcillin (TIC), tetracy-
cline (TE), and amoxicillin/clavulanate (AMC) by disk
di¡usion method. Tested S. £exneri isolates showed high
resistance to penicillins ; AM 96.6%, TIC 90.5%, but in
combination with beta-lactamase inhibitor, showed low
resistance, especially to AMC, but the resistance to
AMC is increasing over time (1998, 0% to 2001, 47.1%).
No ESBL producing strain was found from the tested
strains. Similar resistance pattern was observed to quino-
lones. Although resistance rate was low ^ NA (8.6%) and
CIP (0.6%), the resistance is increasing year by year. Two
CIP resistant strains were isolated in 2002. And the resis-
tance to TE and S was high (89.6% and 96.9%), but on the
other hand it was low to AN (0.3%), GM (0.9%), K
(2.4%), and CRO (0%). The multi-resistance patterns of
S. £exneri are quite di¡erent from S. sonnei. A 96.2% of
tested strains were resistant to 4 or more antibiotics, 16.9%
of strains have resistance to AM, C, S, SXT, TIC and TE,
and 10.1% have resistance to AM, SAM, C, S, SXT, TIC
and TE. These observations of emergence of the resistant
strains to commonly used antibiotics can be a serious
threat to the public health in Korea.
248 1st FEMS Congress / Posters 103^505
P6^8
SUSCEPTIBILITY OF TOXIGENIC AND NON-TOXI-
GENIC CORYNEBACTERIUM DIPHTHERIAE ISO-
LATED IN 1997-2002 TO COMMONLY PRESCRIBED
ANTIMICROBIALS: A CAUSE FOR CONCERN?
R. S. Kozlov(1), M. Warner(2), C. Kelly(3), M. Sukhor-
ukova(1) and A. Efstratiou(3)
(1) Institute of Antimicrobial Chemotherapy, Smolensk
State Medical Academy, P.O. Box 57, 28 Krupskaya
Street, Smolensk, 214019, Russian Federation; (2) Antibi-
otic Resistance Monitoring & Reference Laboratory, Cen-
tral Public Health Laboratory, 61 Colindale Avenue, Lon-
don NW9 5HT, UK; (3) PHLS Respiratory & Systemic
Infection Laboratory, Central Public Health Laboratory, 61
Colindale Avenue, London NW9 5HT, UK
Diphtheria re-emerged in countries of the former USSR in
the 1990s with over 170,000 cases and 4,000 deaths re-
ported to WHO during 1990-1998. The disease still re-
mains endemic in many countries of South-East Asia,
Africa and South America. The epidemiology of C. diph-
theriae is changing as there have been many documenta-
tions of signi¢cant increases in the number of both inva-
sive and non-invasive diseases caused by non-toxigenic
strains. WHO currently recommends penicillin as the
drug of choice and erythromycin as an alternative for
patients with diphtheria and their close contacts. Macro-
lide resistance in corynebacteria has been previously docu-
mented. The susceptibility to penicillin G, amoxicillin /
clavulanate, erythromycin, clindamycin, telithromycin
and levo£oxacin was determined by agar dilution method
on 89 toxigenic and non-toxigenic C. diphtheriae isolated
from patients during the period 1997-2002. C. diphtheriae
biotype mitis NCTC 11397, Staphylococcus aureus ATCC
29213, ATCC 25923 and Enterococcus faecalis ATCC
29212 were used as controls. In comparison by MIC90,
telithromycin was the most active agent (MIC90 = 0.004
mg/l), followed by erythromycin (MIC90 = 0.015 mg/l),
clindamycin (MIC90 = 0.125 mg/l), levo£oxacin (MIC90
= 0.125 mg/l), penicillin G (MIC90 = 0. 25 mg/l) and
amoxicillin / clavulanate (MIC90 = 0.5 mg/l). Penicillin
G and erythromycin retain their activity against C. diph-
theriae and should be considered as a ¢rst choice in pa-
tients with diphtheria and close contacts. Telithromycin,
clindamycin, levo£oxacin and amoxicillin/clavulanate
demonstrated favourable in vitro activity and may be con-
sidered as the alternatives based upon relevant data from
clinical trials.
FEMSLE Congress 2-6-03
P6^9
SEROTYPE DISTRIBUTION AND ANTIMICROBIAL
RESISTANCE IN STREPTOCOCCUS PNEUMONIAE
CAUSING INVASIVE AND OTHER INFECTIONS IN
THE FAR EAST OF RUSSIA (VLADIVOSTOK)
A. V. Martynova, V. B. Turcutiyucov, I. V. Strizhak, N. M.
Kondrashova
Epidemiology Department, the Medical University of Vla-
divostok, Russia
Streptococcus pneumoniae continues to be a major cause
of morbidity and mortality throughout the world and the
Far East of Russia is not an exception. The emerging re-
sistance to some common antibiotics compounds this
problem. There arises a need to monitor the resistance
pattern and map serotype distribution in di¡erent geo-
graphic locations. The present study was undertaken to
determine the serotype prevalence and antibiotic suscepti-
bility of clinically signi¢cant S. pneumoniae isolated from
the patients of young age (18-20 years) with pneumonia in
the State Navy Hospital (Vladivostok, Russia). A total of
120 clinical isolates from invasive and other clinically sig-
ni¢cant pneumococcal infections were serotyped and
screened for susceptibility to commonly used antibiotics
by the NCCLS standards and modi¢ed laboratory proce-
dures. The majority (59.3%) of the isolates belonged to
one or other of the serotypes 1, 6, 19, 5, 23 and 7. Sero-
type 1 was the commonest isolate from patients of men-
ingitis and empyema followed by pneumonia. Nineteen
isolates (12.6%) were non-vaccine type. Eleven (7.3%) iso-
lates were relatively resistant to penicillin (minimum inhib-
itory concentration was between 0.1 and 1 microgram/ml)
and 16,4% were resistant to one or more antibiotics. Re-
sistance was distributed equally among the predominant
serotypes. In conclusion : the common serotypes responsi-
ble for signi¢cant infections were similar to those reported
in some other studies from Russia, with minor variations.
Resistance to cotrimoxazole and tetracycline was predom-
inant followed by chloramphenicol. Low level resistance to
penicillin was observed but no isolate had absolute resis-
tance. This calls for monitoring of resistance and mapping
of serotype distribution from various parts of Russia.
 1st FEMS Congress / Posters 103^505
P6^10
INTEGRON PATTERNS AND SELECTED ANTIBI-
OTIC RESISTANCE GENES OF DT104 AND NON-
DT104 STRAINS OF SALMONELLA TYPHIMURIUM
IN HUNGARY
N. No¤gra¤dy(1), I. Gado¤(2), J. Pa¤szti(2) and M. Kira¤ly(2)
(1) Veterinary Medical Research Institute of the Hungarian
Academy of Sciences, Budapest; (2) National Center of
Epidemiology, Budapest
The integron pattern (IP) and resistance genes of 44
DT104 and 35 non-DT104 Salmonella Typhimurium
strains isolated in Hungary in 1997-1999 were studied.
Di¡erent IPs were determined by 5’CS/3’CS PCR in case
of the two groups. An IP system was set up which par-
tially di¡ered from the published systems, di¡ering mainly
in respect of the non-DT104 strains. The genetic back-
ground of Amp and Chl resistances of both groups was
established by PCR. Characteristically : pse-1 and £o at
DT104 strains, oxa-1 and cat at non-DT104 strains were
found with a few exceptions. Tetracyline resistance was
coded by TetB in case of non-DT104 strains which gene
was not found in DT104 strains. Str/Spc resistance was
coded by genes aadA in both groups.The resistance cas-
sette content of the main integrons and their positions
were determined by sequencing in case of some represen-
tative strains. The sequences of the amplicons covering the
aadA genes were blast analysed at http://www.ncbi.nlm.-
nih.gov. In case of DT104 strains 96-100% identity was
found with the aadA2 gene (accession No. AF071555)
and the non-DT104 showed 96-100% identity with the
aadA1 gene (accession No. AJ009819). PCR applying
RH50/RH51 primers resulted in amplicons only in the
presence of aadA2 gene, because the binding site of
RH51 in the gene aadA1 was inactive. This fact gave the
opportunity of a quick discrimination between aadA1 and
aadA2.
P6^11
MICROBIAL-MONITORING IN POULTRY SLAUGH-
TERHOUSES WITH SPECIAL CONSIDERATION OF
ENTEROCOCCI
S. Schmidt, W. Sixl, A. Eisner, G. Wu«st, G. Feierl, J.
Posch, E. Marth
Hygiene Institut University of Graz, Universita«tsplatz 4,
8010 Graz, Austria
Fighting infections the medicine nowadays is confronted
with the problem of resistance against antibiotics more
and more. Enterococci belonging to the physiological £ora
FEMSLE Congress 2-6-03
249
are only facultative pathogenous, however, if anti-infective
treatment is required there are only few options due to
natural resistance. Therefore resistance against vancomy-
cin is of special relevance, which was described for the ¢rst
time in 1988 and is seen in Austria since 1991. Especially
because of possible transfer of the van-genes into isolates
of Staphylococcus aureus making them resistant to vanco-
mycin this problem is of great importance. In this connec-
tion the in£uence of resistant Enterococci in animals is
discussed controversially. During HACCP investigation
in a slaughterhouse for poultry the spectrum of microor-
ganisms was examinated during half a year. In all samples
Enterococci were found, nine of which were resistant to
vancomycin and teicoplanin, another nine isolates of E.
faecium demonstrated lack of sensitivity to Quinupristin
/ Dalfopristin. Furthermore we could ¢nd campylobacter
sp. in 70% and salmonella sp in 25% of the samples. This
decrease of salmonella in poultry is in contrast to a high
level of reported cases in humans. Our investigations dem-
onstrates the need of microbiological monitoring of food
animals in connection with development and spread of
antimicrobial resistance.
P6^12
STREPTOCOCCAL SEROGROUPS AND SERO-
TYPES CIRCULATING IN ROMANIA (1999-2002).
MLS RESISTANCE PHENOTYPES
V. Ungureanu, I. Ciucafl, M. Gheorghe
Cantacuzino Institute, Splaiul Independentei 103, Bucharest,
R-70.100 Romania
The objective was to survey the circulation and the macro-
lide resistance of L-hemolytic streptococci in our country.
The streptococcal strains were collected by the National
Reference Laboratory for Streptococci between 1999 ^
2002. The identi¢cation of streptococcal strains was per-
formed by slide agglutination, using the co-agglutination
reagents for A, B, C, G serogrouping and antisera for T
serotyping ^ prepared by the Cantacuzino Institute. The
susceptibility to macrolides and other antibiotics was
tested by the agar di¡usion method and MIC determina-
tion by agar dilution method. Results: 55.14% of strains
were group A streptococci; 23.62% group G; 5.79% group
C; 5.17% group B and 10.28% were non-groupable.
46.41% of group A were isolated from respiratory infec-
tions (acute tonsilitis and scarlet fever), 7.87% from skin
infections, 41.71% from healthy carriers. The origin of
non-group A and non-groupable streptococci was: respi-
ratory tract (47.20%), carriers (46.69%), other infections
(3.40%). The incidence of pattern T 5/11/12/27 was pre-
dominant (56.77%), followed by the pattern 2/4/6/28
(10.50%). The resistance to erythromycin, clindamycin,
tetracycline and chloramphenicol was found. 12.32% of
250 1st FEMS Congress / Posters 103^505
group A streptococci were erythromycin resistant. The
MLS resistance phenotypes (M, iMLS, cMLS) were deter-
mined by erythromycin-clindamycin double disc test. Con-
clusions: The group A streptococci infection still presents
a health public problem in our country as all over the
world. The pattern T 5/11/12/27 is predominant. The
rate of erythromycin resistance is increasing.
P6^13
PHENOTYPIC AND GENOTYPIC DETERMINATION
OF ANTIBIOTIC RESISTANCE OF CAMPYLO-
BACTER JEJUNI AND C. COLI FROM HUMANS
AND POULTRY
T. Zorman(1), L. Herman(2), I. Berce(3), S. Uzunovic¤-
Kamberovic¤(4), A. Klanc›nik(1), S. Smole Moz›ina(1)
(1) University of Ljubljana, Biotechnical Faculty, Depart-
ment for Food Science and Technology, Ljubljana, Sloven-
ia; (2) Center for Agricultural Research ^ Ghent, Depart-
ment for Animal Product Quality and Transformation
Technology, Melle, Belgium ; (3) Institute of Public Health
Nova Gorica, Nova Gorica, Slovenia; (4) Cantonal Center
for Public Health in Zenica, Zenica, Bosnia and Herzego-
vina
Macrolides and £uoroquinolones are regarded as the
drugs of choice for the treatment of human Campylobacter
infections. The use of antimicrobials for the treatment of
infections and as growth promoters has induced antimi-
crobial resistance of Campylobacter spp. Since poultry is a
potential source of human infections the use of antibiotics
in animal production can a¡ect the lifetime of therapeutics
for human use. In Slovenia, antibiotic resistance of Cam-
pylobacter spp. is still not monitored at the national level.
There are some data available for clinical but not for
poultry isolates. More than 150 poultry and human cam-
pylobacters were isolated in the years 2000-03, some of
them were tested for susceptibility to eight antimicrobials.
The resistance was studied by disc di¡usion method and
MICs for cipro£oxacin and erythromycin were determined
by E-tests. Higher portion of isolates was resistant to ci-
pro£oxacin than to erythromycin. The occurrence of resis-
tance was higher at C. coli than at C. jejuni as well as at
poultry compared to human isolates. This is noteworthy
as the extent of C. coli contamination of poultry in Balkan
region is signi¢cant, so transfer of resistance genes to hu-
man is relieved. Susceptibility tests are not internationally
standardised. To con¢rm the accuracy of our test results
we used a mismatch ampli¢cation mutation assay
(MAMA) PCR to detect the gyrA mutation in 30 quino-
lone resistant C. jejuni isolates from clinical and food en-
vironment. There was 100% correlation between results of
both techniques. The PCR procedure to detect the resis-
tance genes in C. coli is still under investigation.
FEMSLE Congress 2-6-03
P6^14
CORRELATION BETWEEN BACTERIAL VAGINO-
SIS AND pH VALUE OF VAGINAL SECRETION
V. Vuksanovic¤ and M. Bujko
Institute of Health, Faculty of Medicine, University of
Montenegro, Podgorica, Montenegro
Bacterial vaginosis (BV) is disbalance of vaginal ecosystem
which is caused by changing of dominant lactobacillus
with association of bacteria Gardnerella vaginalis, anaer-
obic bacteria and Mycoplasma hominis. Normal pH value
of vaginal secretion is 3.8-4.2 Pathogenic bacteria grow
rapidly when pH value is pH s 5. The aim is to examine
whether there is correlation between present BV and pH
value of vaginal secretion and to determinate whether pH
value of vagina has the practical signi¢cance for estima-
tion of the status of vaginal ecosystem. The presence of
BV in vaginal secretion was examined in 642 women by
using Nugent method. The pH value of vaginal secretion
was paralelly tested by using pH indicator strips. Diagno-
sis of Trichomonas vaginitis was made by native prepara-
tion, and Candida vaginitis was diagnosed by stained
preparation methylene blue. Diagnosis of other bacterial
infections was made by using appropriate base. BV was
diagnosed in 120 (18.7%) women, and intermediate £ora
in 17 (2.6%) women. Analysis of pH value of vaginal se-
cretion in women with BV showed that patients with BV
had increased pH values pH s 4,5 of vaginal medium
and this condition was found in 118 (98.3%) patients.
This correlation is statistically signi¢cant for level
p 6 0.001. Trichomonas vaginitis was found in 51 (7.9%)
patients, and 45 (88.2%) of them had pH s 4.5. The num-
ber of women whose microbiological test was normal was
152 (23.7%) and 123 (80.9%) of them had normal pH
value ^ pH 6 4.5. Statistical signi¢cance was not deter-
mined for Candida vaginitis (pH 6 4.5 was found in 76 ^
58.9% women). In conclusion : There is signi¢cant corre-
lation between BV/intermediate condition and increased
pH value of vaginal secretion. Since the method of deter-
mination of disbalance of vaginal ecosystem is simple,
rapid, economic and e¡ective, the routine following of
pH value of vaginal secretion as screening method that
can suggest need for further microbiological and biochem-
ical analysing is recomanded.
 1st FEMS Congress / Posters 103^505
P6^15
MULTIPLE BACTERIAL SPECIES COLONIZE
CHRONIC WOUNDS
K. Gjodsbol(1), T. Karlsmark(2), B. Jorgensen(2) and K.
A. Krogfelt(1)
(1) Statens Serum Institut, Artillerivej 5, DK-2300 Copen-
hagen S, Denmark; (2) Bispebjerg University Hospital,
Bispebjerg Bakke 23, DK-2400 Copenhagen NV, Denmark
The aim of the study was to investigate the bacterial pro-
¢le of chronic venous leg ulcers. Forty-six patients were
included in the study. All had persisting ulcers for more
than three months. Of the patients 29 were female and 17
were male, age 29-91 years (median 78 years). The healing
of the ulcers was followed for 8 weeks or until the ulcer
healed. Every second week swaps; biopsies and ¢lter paper
pads were collected. Specimens were plated on 5 % blood
agar plates and anaerobic agar plates and incubated at
37‡C both aerobic and anaerobic. Primary isolation plates
were examined after 24 hours and then incubated for at
least 4 days. Bacteria were isolated and identi¢ed at spe-
cies level by standard methods. More than one bacteria
species were detected in close to all the samples (94,4 %).
The most common species found in the ulcers were Staph-
ylococcus aureus (isolated at least once during the study
period in 97,8 % of the patients), E. faecalis (71,7 %),
Pseudomonas species (58,7 %, mainly P. aeruginosa), coag-
ulase-negative staphylococci (45,7 %), Proteus species (41,3
%), anaerobic bacteria (39,1 %), E. cloacae (37,0 %), E.
coli (32,6 %) and corynebacteria (30,4 %). Identi¢cation of
the bacteria species is essential for supplying the proper
treatment, since colonisation may be a key parameter in
sustaining chronicity in venous leg ulcers. Further inves-
tigation of the importance of the bacterial pro¢le of the
chronic wounds will be performed, by correlating the bac-
terial pro¢le to the course of the ulcer healing.
P6^16
INFECTIVE AGENTS IN EARLY CHILDHOOD
PNEUMONIA
M. Maneva, Z. Sarovska and L. Stojanovska
Institute for Respiratory Diseases in Children, Skopje, Mac-
edonia
Object: Retrograde study involved evaluation of clinic his-
tories of patients that were treated at the Department for
patients under age of three years. Material: In the period
of three years 1.204 children were hospitalized for di¡erent
respiratory diseases. Pneumonia was diagnosed and
treated in 321 patients (26.66%). From them, 161 patients
FEMSLE Congress 2-6-03
251
were selected in order to clear clinical, radiological and
laboratory characteristics. Results: It has been proved
that infective agents as causes of pneumonia were ¢nd
out in 59 children (36,6%). Viruses were the most frequent
cause (37 / 23.0%). Bacteria was determined in 16 (9.9%)
patients and mixed (viral-bacterial) infection in 6(3.7%). In
the group of the patients with viral infection there was
absolute domination of adenoviruses with 70.3%, than
combination of AD and In£uenzae A (16.2%) and In£u-
enzae A with 13.5%. Among the patients with bacterial
cause of pneumonia, Staphylococcus was with the greatest
percentage (87.5%), while E. coli and Staphylococcus + E.
coli were represented with 6.2%. It was evident that mixed
infection (combination of AD + Staphylococcus / 50.0%)
were presented in the great number of children. The other
agents were more rare (16.7%). Conclusion : The small
percentage of con¢rmed agents was based on delayed ad-
mission in hospital, previous antibiotic treatment, lack of
fast, exact and sophisticated methods of discharge. Con-
¢rmation of the exact cause is the base of successful treat-
ment and determinated prognosis.
P6^17
ETIOLOGY OF BACTERIAL MENINGITIS IN CHIL-
DREN DURING 5 YEARS (1997-2002) IN MINSK
O. M. Morozova, L. P. Titov, A. A. Astapov, N. L. Klyuy-
ko, L. A. Grak
Byelorussian Research Institute Epidemiology and Microbi-
ology (BelRIEM), Minsk, Belarus.
The aim of the study was to assess the etiology of bacterial
meningitis in children during 5 years (1997-2002). Material
for research (CSF, blood and nasopharyngeal swabs) was
immediately inoculated on medium and incubated at 35-
370C for 24 h under CO2 (5-10%) atmosphere. Typing of
strains was carried out by api BioMerieux and latex ag-
glutination (KIT5 BioMerieux). 142 bacterial cultures
from children with bacterial meningitis were isolated. 85
^ N. meningitides (group B 69 strains, group C -12 and
group A -4), 16 ^ Haemophilus in£uenzae type b, 15 ^
Streptococcus pneumoniae, 9 ^ Streprococcus spp, 7 ^ Lis-
teria spp, 3- Staph.aureus, 3 ^ Candida, 2 ^ Enterobacter, 1
^ Pseudomonas aeruginoza, 1 ^ Salmonella enteritidis. 22
strains of N. meningitides (25,9 %) was isolated from CSF
and blood, 28 (32,9 %) ^ from CSF, 20 (23,5 %) ^ only
from blood, 16 (17,7 %) ^ only from nasopharynx. 6
strains of H. in£uenzae type b (37,5 %) was isolated
from CSF and blood, from CSF ^ 9 (56,25 %) and only
from blood ^ 1 strain. (6,25%). 5 strains of Streptococcus
pneumoniae (33,3 %) was isolated from CSF and blood, 8
(53,3 %) from CSF and 2 (13,4 %) ^ from blood. In con-
clusion : In etiological structure of bacterial meningitis in
children in Minsk dominate N. meningitides (59,9 %) with
252 1st FEMS Congress / Posters 103^505
prevalence N. meningitides group B (81,2 %). Second of
the importance agents was H. in£uenzae type b (11,3 %),
third ^ Streptococcus pneumoniae (10,6 %).
P6^18
EVALUATION OF TWO CASES OF MALARIA IN
NISB REGION-SERBIA IN THE LAST FIVE YEARS
S. Tasic¤(1), N. Miladinovic¤(1), A. Tasic¤(2), A. Ni-
kolic¤(2), Z. Velic›kovic¤(1) and S. Pes›ic¤(1)
(1) Medical Faculty, Nis›, Serbia; (2) Institute of Public
Health, Nis›, Serbia
In the ¢ve last years in laboratory of parasitology in In-
stitute of Public health in Nis› only two cases of malaria
were diagnosed. The aim of this study was to evaluate
these two cases. In both of cases malaria was diagnosed
by parasitical investigation of blood smears stained by
Giemsa. First case was the man who returned from Zaire.
Patient was hospitalized in Clinic of In¡ective disease with
typical symptoms of malaria (fever, anemia, weakness),
and he did not take the chemioprophylactic drugs for
malaria even before or after his stay in Zaire. Malaria
vivax was diagnosed based on morphologically character-
istics of evolution forms of plasmodium. Second case was
the man from group of our workers in Zambia. Also, in
this case patient was hospitalized with typical symptoms of
malaria. In this case mixed infection with Plasmodium vi-
vax and Plasmodium falciparum was found by parasitically
investigation of blood. Patient did not receive drugs for
chemioprophylaxis before stay, but he started with Arte-
misinin immediately after the ¢rst sings of malaria appear-
ance. The singes of illness were less pronounced than in
the ¢rst case. Other workers from the same group received
drug during the stay and no one obtained disease. In con-
clusions, only two cases of malaria were diagnosed in the
¢ve last year in region of Nis›-Serbia. In both of cases
patients did not receive drugs before stay in endemic
area, but one of them started Artemisinin after appearance
of singes and illness was less pronounced.
FEMSLE Congress 2-6-03
P6^19
FREQUENCY OF UREAPLASMA UREALITYCUM
AND MYCOPLASMA HOMINIS IN FEMALE’S GEN-
ITAL TRACT AND THEIR SUSCEPTIBILITY TO
ANTIMICROBIAL AGENTS
S. Stojanova, M. Panovska, Z. Bozinova and S. Ivic-Kolev-
ska
Republic Institute for Health Protection, 50 Divizija br.6
1000 Skopje, Macedonia
There are 6 mycoplasmas in the female genital tract, out of
which the most common are Ureaplasma urealyticum (U.
urealyticum) and Mycoplasma hominis (M. hominis). The
role of U. urealyticum and M. hominis in pathogenesis of
certain genital infections has been proved. The aim of this
investigation was to determine the incidence of the genital
U. urealyticum and M. hominis alone and in combination
with other microorganisms and to assess their susceptibil-
ity towards antimicrobial agents. During our work 180
patients were examined. Three cervical swabs of each pa-
tient were used: the ¢rst for examination of the quantita-
tive frequency of U. urealyticum and M. hominis detected
by Mycoplasma IST kit; the second was used for isolation
of cultivable pathogenic and opportunistic pathogenic mi-
croorganisms by standard microbiological techniques; the
third for detection of antigens of Chlamydia trachomatis
using the ELFA-technique on the VIDAS-analyzer. From
180 patients, in 63 patients(34.99%) U. urealyticum and/or
M. hominis were isolated. In 13 patients U. urealyticum
and M. hominis were both isolated, from which 4 patients
associated with Candida albicans. In 6 patients only M.
hominis was isolated from which 2 patients associated
with E. coli. In 44 patients U. urealyticum was isolated,
from which 7 patients associated with Candida albicans.
During our work was examined the susceptibility of U.
urealyticum and M. hominis to 8 antimicrobial agents.
The highest susceptibility was found towards doxycycline,
josamycin and tetracycline. The resistance was found to-
wards erythromycin and cipro£oxacin. There is a high pres-
ence of U. urealyticum and M. hominis alone or in associ-
ation with other microorganisms in patients with genital
infections, which is showing their role in these infections.
 1st FEMS Congress / Posters 103^505
P6^20
SEROSITIS AS A COMPLICATION OF SEPTICAE-
MIA, WITHOUT MENINGITIS, CAUSED BY N.
MENINGITIDIS: A CASE REPORT
E. Trikka-Graphakos, Z. Salem, N. Charalambakis, M.
Nepka, Ch. Pappas, F. Ioannidou
Department of Clinical Microbiology, ‘‘ Thriassio’’ General
Hospital, Athens, Greece
Complications after meningococcal septicaemia have be-
come extremely rare since antibiotics have been used. Se-
rositis caused by Neisseria menigitidis, especially group C,
have been described mainly in patients with underlying
disease (diabetes mellitus, complement component de¢-
ciency). We describe a case of a diabetic patient with peri-
carditis and pleuritis caused by N. meningitidis group C. A
68-year-old diabetic patient was admitted to the emer-
gency department with high fever, chills and sore throat.
During physical examination there were not meningeal
signs or other abnormal ¢ndings. On admission, white
blood cell count was increased with a shift to the left while
CRP was also elevated. The laboratory and immunologi-
cal ¢ndings were negative. The chest X- ray revealed opac-
ity in the lower lobe of the right lung. N. meningitidis
group C (2a:P1.2, 5) was isolated from 2 sets of blood
cultures. According to the MIC (E- test), the microorgan-
ism was sensitive to penicillin, 2nd and 3rd generation
cephalosporins sulfonamides, tetracycline, erythromycin,
rifampicin, chloramphenicol, and quinolones. The patient
was treated with a 3rd generation cephalosporin, 2 g i.v.,
every 12 h, and his clinical condition improved rapidly. On
the 12th day of hospitalization, the patient’s clinical con-
dition was deteriorated with fever, dyspnoea and chest
pain while during physical examination paroxysmal pulse
rate and hypotention were present. From the cardiac U.S
pericardial £uid collection with ¢ndings of pericardial
tamponate and from the chest X- ray, pleural e¡usion
were present. The pleural £uid was exudate while the cul-
ture was negative. Dramatic improvement of patient’s clin-
ical condition followed after immediate periocardiocentesis
and administration of prednisolone (1 mg/kg). In conclu-
sion, Neisseria meningitidis group C, can cause serositis,
with negative culture, in patients with underlying disease.
FEMSLE Congress 2-6-03
253
P6^21
EMERGENCE OF METALLO-L-LACTAMASE PRO-
DUCING PSEUDOMONAS AERUGINOSA AS NOSO-
COMIAL PATHOGEN
E. Edalucci, C. Lagatolla, E. Medessi, L. Dolzani, F. Gio-
nechetti, F. Gombac, C. Monti-Bragadin and E. A. Tonin
Department of Biomedical Sciences, Microbiology Section,
University of Trieste, via Fleming 22, I-34127 Trieste, Italy
In the last three years, 165 imipenem resistant (MIC =16
Wg/ml) Pseudomonas aeruginosa clinical isolates were col-
lected at the Laboratory for Clinical Microbiology of the
‘‘Cattinara’’ Hospital (Trieste-Italy). Total DNA was ex-
tracted from all strains and the presence of the blaVIM
determinant was investigated by dot blot. Among the
165 strains, 122 (about 74%) were blaVIM-positive. 110 of
them carried the allelic form blaVIM1 (90%) and 12 (10%)
the blaVIM2. This is the ¢rst report of high-level endemicity
of metallo-L-lactamase producing P. aeruginosa. Typing of
the isolates, performed by two di¡erent methods (RAPD
and AFLP analysis), revealed that most of the isolates
(78%) might be considered related. In fact, both methods
identi¢ed two clusters (indicated as cluster A and B) which
included strains with a Dice similarity coe⁄cient higher
than 88%. 4 blaVIM-positive isolates were unrelated both
with either clusters and among each other, so they were
considered sporadic. Cluster A included 119 isolates : 109
of them (92%) carried the blaVIM1 determinant, 10 were
blaVIM-negative. They were widely distributed in the hos-
pital and were also found in 11 outpatients. Cluster B
included 10 isolates, 9 of them carried the blaVIM2 deter-
minant, 1 was blaVIM-negative. They were collected from 4
wards. 28 of the 122 blaVIM-positive isolates have been
typed by macrorestriction analysis (PFGE) after digestion
of total DNA with SpeI. This technique con¢rmed sub-
stantially the previous results but, being more discrimi-
nant, allowed the identi¢cation of di¡erent subtypes inside
the previously identi¢ed clusters.
P6^22
STENOTROPHOMONAS MALTOPHILIA. AN
EMERGING PATHOGEN IN A LARGE GREEK GEN-
ERAL HOSPITAL
S. Kanavaki, E. Galani, S. Karambela, M. Makarona, E.
Alexandraki, A. Pefanis, H. Giamarellou
Sotiria General Hospital, Athens, Greece
During the ¢ve year period 1998-2002 we observed a sig-
ni¢cant increase in the incidence of Stenotrophomonas mal-
tophilia (SM) isolation from clinical specimens from hos-
254 1st FEMS Congress / Posters 103^505
pitalized patients. From a total of 31298 isolated microbial
strains, 238 strains were characterised as SM. The inci-
dence was increased by sixfold from the beginning to the
end of the ¢ve year period (from 0,2 to 1,8%, respectively).
One hundred and seventy nine (75%) strains were isolated
between 1/1/2001 and 30/9/2002. Eighty six (48%) of the
strains were isolated from sputum, 48 (26,8%) from blood,
22 (12,3%) from bronchial secretions, 12 (6,7%) from pus,
5 (2,8%) from urine, 5 from pleural £uid and one from the
tip of a central vein catheter. Some of these isolations
probably represent colonisation rather than true infection.
However, the fact that a signi¢cant percent of SM strains
were isolated from the blood underscores the signi¢cance
of SM as an emerging nosocomial pathogen. Regarding
the antibiotic susceptibility, 97,2% of the strains were sus-
ceptible to cotrimoxazole, 90% were susceptible to cipro-
£oxacin, and 82,7% were susceptible to ticarcillin/clavu-
lanic acid, while 98,4% of the strains were resistant to
imipenem/cilastatin. A variety of antibiotic susceptibility
phenotypes were observed, suggesting that there were no
SM epidemics in the hospital. These ¢ndings were con-
¢rmed in part by applying molecular techniques (PFGE)
in a small (n=30) number of strains isolated from blood
cultures. In conclusion, Stenotrophomonas maltophilia rep-
resents a signi¢cant emerging nosocomial pathogen in our
hospital.
P6^23
INVESTIGATION OF AN OUTBREAK OF VANCO-
MYCIN RESISTANT ENTEROCOCCI
J. Papaparaskevas(1), D. Katsarelias(2), D. Voros(2), P.
T. Tassios(1), E. Kouskouni(1,3), N. J. Legakis(1) and L.
Zerva(1,3)
(1) Department of Microbiology; (2) Department of Sur-
gery; (3) Laboratory of Microbiology, Areteion Hospital,
Medical School, University of Athens, Athens, Greece
The objective of this study was to investigate an outbreak
of vancomycin resistant enterococci (VRE) in a Greek
University Hospital. During the period 05/2001-04/2002,
13 VRE isolates were studied. Species identi¢cation and
susceptibility to ampicillin, erythromycin, cipro£oxacin,
rifampicin, tetracycline, gentamicin, streptomycin, vanco-
mycin and teicoplanin were performed by the VITEK and
the ATB systems (bioMerieux), while susceptibility to line-
zolide and quinupristine-dalfopristine by disk di¡usion.
Genes vanA, vanB, vanC, ddlE. faecalis and ddlE. faecium
were detected by multiplex PCR. Pulsed Field Gel Elec-
trophoresis (PFGE) was used for DNA ¢ngerprinting.
Five VRE strains were isolated from patients’ clinical sam-
ples in ICU or a clinical ward, 5 from ICU environmental
cultures and 3 from patients’ surveillance cultures in two
clinical wards, all harbouring the vanA gene. Twelve iso-
FEMSLE Congress 2-6-03
lates were identi¢ed as Enterococcus faecium and 1 as En-
terococcus faecalis. All E. faecium isolates were resistant to
ampicillin, erythromycin and cipro£oxacin and susceptible
to linezolid and dalfopristine+quinupristine, whilst the E.
faecalis strain was susceptible only to ampicillin and line-
zolide. PFGE allocated 8 isolates (all environmental, 1
clinical and 2 colonizing strains) into 1 cluster (A), 4 clin-
ical strains into 2 clusters (B and D) and 1 strain into
cluster C. A thorough cleaning in ICU resulted in out-
break eradication. This constitutes the second report of
a VRE nosocomial outbreak in Greece. The majority of
VRE were multiresistant E. faecium. A link was estab-
lished between environmental, colonizing and clinical
strains. The outbreak started in ICU and involved two
clinical wards. Infection control measures e¡ectively eradi-
cated the outbreak.
P6^24
BACTERIA CARRIERS IN SOME PROFESSIONAL
GROUPS IN THE MUNICIPALITY OF OHRID
WITHIN THE PERIOD FROM 2000 TO 2002
J. Sturlakova-Korovesovska and S. Dimovska
Republic Sanitary and Health Inspectorate (RSHI), Re-
gional Unit Ohrid, 16 Lazo Trposki, 6000 Ohrid, Republic
of Macedonia
Objective: To show the most frequent bacteria carriers
found in some professional groups during periodical
health examinations. Material and Methods : The data
are taken from the records of the RSHI, Regional Unit
^ Ohrid, and are obtained during the microbiological ex-
aminations of throat and nose smear, feces, and cello-
phane smear for parasite identi¢cation of some professio-
nal groups within the period 2000-2002. Standard
statistical data processing method was used. Results :
Within the given period, a total number of 5240 individ-
uals were examined, of which 902 education employees;
1338 health-care employees ; 62 pharmaceutical employees
and 2938 employees involved in food processing. The per-
centage of positive nose smear ¢ndings ranged from 1.9%
in health-care and pharmaceutical employees, 2.7% in ed-
ucation employees and 5.4% in employees engaged in food
production and tra⁄c. The most frequent cause of bacte-
ria carriers in all cases was Staphylococcus aureus. The
presence of positive throat smear ¢ndings was the follow-
ing: 0.2% in pharmaceutical employees, 1.1% in education
employees, 0.8% in health-care employees and 0.3% in
employees engaged in food processing. The most fre-
quently isolated bacteria was Streptococcus pyogenes. No
bacteria carrier was detected in the copraculture examina-
tions. Conclusion: In evaluating the results obtained, it
has been stated that Staphylococcus aureus dominates as
a bacteria carrier. Nose was found as the most frequent
 1st FEMS Congress / Posters 103^505
location, while the frequency of incidence was the greatest
in employees engaged in food processing and tra⁄c. Due
to the increased consumption of creams and ice creams in
summer, systematic examinations should be more fre-
quent.
P6^25
E. COLI O6 NOSOCOMIAL OUTBREAK IN NEONA-
TAL INTENSIVE CARE UNIT
I. Tomova(1), E. Georgieva(1), T. Kantardjiev(1), S. Sta-
neva(2), M. Christova(1), P. Vajarova(2)
(1) National Center of Infectious and Parasitic Diseases-
So¢a, Bulgaria ; (2) Hygienic Epidemiological Inspector-
ate-Varna, Bulgaria
The nosocomial outbreak concerns a neonatal infection of
the type endotoxic shock (septicemia) in the background
of immaturity and preterm birth. A thorough laboratory
control has been initiated establishing contamination of
some solutions and equipment for infusion therapy. All
isolates from clinical materials and parenteral solutions
perfained to Escherichia coli and were identi¢ed as O6
serotype of the enterotoxigenic group (ETEC). The strains
were tested by complex of bacteriological and genetic
methods, demonstrating their identity. Dissemination of
infection was realized by the contact route, the hands of
personnel and special apparatuses being proved to be of
primary importance among factors for distribution of in-
fection.
P6^26
USE OF PFGE AND FLAA-RFLP FOR TYPING OF
CAMPYLOBACTER JEJUNI AND C. COLI FROM
POULTRY AND CLINICAL ENVIRONMENTS
T. Zorman(1), M. Heyndrickx(2), S. Uzunovic¤-Kamber-
ovic¤(3), S. Smole Moz›ina(1)
(1) University of Ljubljana, Biotechnical Faculty, Depart-
ment for Food Science and Technology, Ljubljana, Sloven-
ia; (2) Center for Agricultural Research Ghent, Depart-
ment for Animal Product Quality and Transformation
Technology, Melle, Belgium; (3) Cantonal Center for Pub-
lic Health in Zenica, Zenica, Bosnia and Herzegovina.
Thermotolerant Campylobacter species C. jejuni and C.
coli are recognised as one of the major causes of acute
bacterial enteritis in many countries as well as in Slovenia
and Bosnia. Our previous study indicated that high degree
of poultry meat on Slovenian and Bosnian retail market is
contaminated with campylobacters. In order to indicate
the animals and meat as reservoirs of campylobacters for
FEMSLE Congress 2-6-03
255
causing the disease in the community two high resolution
genotyping techniques were applied. Pulsed-¢eld gel elec-
trophoresis (PFGE) and £aA typing were used to inves-
tigate the genetic diversity among isolates from chickens in
farm environment, chicken meat from retail market and
sporadic cases of campylobacteriosis in the same geo-
graphical area. Macrorestriction pro¢les were analysed us-
ing the GelCompar software. Among 124 analysed isolates
we observed 78 di¡erent SmaI macrorestriction pro¢les.
FlaA typing by using CfoI distributed the strains into 10
£aA types, so PFGE technique was much more discrimi-
native. Di¡erent C. jejuni and C. coli clones were isolated
from the same meat product. On the other side, the same
macrorestriction pro¢les of human isolates in the same
geographical area show on their clonal origin. Very similar
or identical genotypes indicate the transmission of these
pathogens from chickens in farm environment to poultry
meat on retail market, as well as from meat to humans.
There is an evidence for subsequent contamination of
poultry meat with Campylobacter strains present in the
market environment. The results of this study suggest a
link between campylobacters in farm environment with
those causing disease in the community.
P6^27
PLASMID AND DIGESTION OF CHROMOSOMAL
DNA OF L. MONOCYTOGENES
J. Nowroozi, M. Hakemi
Iran University of Medical Science, Tehran, Iran
L. monocytogenes is a Gram positive, facultative anaerobic
and intracellular bacteria. Epidemiological studies sug-
gested that contaminated foods such as pasteurized milk,
soft cheese, raw vegetables and etc, are probable vehicle
of infection to human. The purpose of this study was to
compare plasmid DNA pro¢les and chromosomal DNA
after digestion with Hpa-1 in L. monocytogenes isolated
from dairy and clinical specimens. Plasmid DNA was pre-
pared by alkali lysis and chromosomal DNA was obtained
by using lysozyme, pronase and extracted by phenol /
chloroform. Concentration of DNA was measured by us-
ing a spectophotometer. Cleavage reaction with Hpa-1 re-
striction enzyme was done for three hours. In clinical
specimens, 2 bands of plasmid DNA, and in dairy speci-
mens, one band of plasmid DNA were detected. After
digestion, 4 bands of chromosomal DNA in clinical speci-
mens and 2 bands in dairy specimens were found. All L.
monocytogenes isolated from di¡erent sources had one
band of similar of plasmid DNA and also, after digestion,
2 bands of similar chromosomal DNA in clinical and
dairy specimens, but another one band of plasmid DNA
and two bands of chromosomal DNA were detected in
256 1st FEMS Congress / Posters 103^505
clinical specimens. This probably indicated di¡erent viru-
lent strains of Listeria monocytogenes.
P6^28
TOXOPLASMA INFECTION AT EYE LEVEL
D. Steriu(1), M. Pop(2), C. Cedru(1)
(1) Cantacuzino Institute, Splaiul Independentei 103, Bu-
charest, R-70100, Romania; (2) Hospital of Ophthalmol-
ogy, Bucharest, Romania
595 serum samples from patients with di¡erent eye dis-
eases, with a possible toxoplasmic etiology, were investi-
gated by ELISA. 341 positive sera were detected. Starting
from the idea that toxoplasmosis is a systemic infection
frequently encountered in the healthy population, only
those cases in which the titers pointed to an exacerbation
of the infection were taken into consideration. Thus, 131
such cases were reported. Most of them presented initial
or recurrent chorioretinitis (77 cases) and uveitis (34
cases), most of them showing a posterior localization. It
is worth mentioning that no positive IgM samples were
identi¢ed, therefore no possibly toxoplasmic primary in-
fection could be detected. At the same time, the lesions
that occurred, even the recurrent ones, do not have a his-
tory that can be traced back to the early childhood. Of
particular interest was the 0 ^ 5 years age group that
included 27 children, of whom 12 had positive titers for
toxoplasmosis. In ten of these children, the positive titres
were indicative of an exacerbation of infection. The obser-
vation that in all the cases recurrent chorioretinitis was
incriminated enables us to presume that even in the ab-
sence of investigations performed at birth, these can be
considered to be congenital toxoplasmic infection.
P6^29
ENTEROBACTER SAKAZAKII IN MILK POWDER
A. E. Heuvelink, M. Ahmed, F. D. Kodde, J. T. M. Zwartk-
ruis-Nahuis, H. van der Zee and E. de Boer
Inspectorate for Health Protection and Veterinary Public
Health, P.O. Box 202, 7200 AE Zutphen, The Netherlands
Enterobacter sakazakii is a rare but important cause of
life-threatening neonatal sepsis and meningitis. We exam-
ined the occurrence of this bacterium in di¡erent milk
powders. A portion of 25 g of milk powder was carefully
added to 225 ml bu¡ered pepton water (BPW), dissolved
by slowly swinging and incubated for 18 to 24 h at
37‡Cfollowed by inoculating1 Wl onto violet red bile lac-
tose (VRBL) agar (18 to 24 h at 37‡C ) and 10 ml was
transferred to 90 ml of Enterobacteriaceae enrichment
FEMSLE Congress 2-6-03
(EE) broth (18 to 24 h at 37‡C). Then, also 1 Wl of EE
culture was inoculated onto VRBL agar (18 to 24 h at
37‡C). Typical colonies (Enterobacteriaceae) on VRBL
were tested for the formation of a yellow pigment and a-
glucosidase activity on tryptone soy agar (TSA) and nu-
trient agar supplemented with 4-methylumbelliferyl-a-D-
glucopyranoside (NA-MUG), respectively. Yellow-pig-
mented colonies (2 d at 25‡C) which produced £uorescent
colonies on NA-MUG agar (366 nm) were also tested for
a-glucosidase activity with the 4-paranitrofenol (PNP) test.
Colonies from TSA were suspended in a tube with 2 ml
0.85% NaCl. After the addition of 2 ml PNP-a-glucosidase
solution (5 g/l, 1.15 M phosphate bu¡er, pH 7.0) the tubes
were incubated at 37‡C for 4 h. The formation of yellow-
coloured PNP was examined by measuring the absorbance
at 405 nm. Isolates that showed a-glucosidase activity (94
uur) were streaked onto sorbitol-MacConkey agar (18 to
24 h at 37‡C) and identi¢ed as E. sakazakii by using an
API 20E biochemical test strip. Additionally, isolates were
tested with a tDNA-PCR and subtyped with PFGE. A
total of 211 samples of milk powder were examined, in-
cluding 40 samples of powdered-infant formula. Strains of
E. sakazakii were isolated from 8 samples : 1 sample of
infant formula and 7 samples collected from cargos and
bulk goods of milk powder. Among the 8 isolates, 7 dis-
tinct restriction patterns could be discriminated. During a
follow-up study, 102 samples of powdered-infant formula
were examined. E. sakazakii strains were isolated from 2
of these samples.
P6^30
SURFACE BIOPOLYMERS OF THE MELIOIDOSIS
AGENT AS POSSIBLE COMPONENTS OF CHEMI-
CAL VACCINE
S. I. Zhukova, V. S. Rybkin, N. N. Piven, I. V. Avrorova,
N. G. Plekhanova, D. V. Viktorov and O. B. Proshina
Antiplague Research Institute, Volgograd, Russia
The problem of creating good specimens of prophylaxis
and treatment of melioidosis infection has been actual and
important recently. In the present work there have been
studied characteristics of some antigen complexes of the
melioidosis agent including exoprotease, biopolymers of
exterior and interior membranes of a microbe cell and
surfactant antigen complexes contained the antigen 8,
one of the pathogenic factors. Experiments were made
on white mice and white rats. Animals were immunized
twice with antigen specimens in the mixture (1:1) with the
Freind full adjuvant in doses of 30 mkg(for mice) and 100
mkg(for rats). Animals were infected with the virulent cul-
ture of the melioidosis agent C-141(for mice) and 100 (for
rats) 14 days after the second immunization. There have
been shown exoprotease increased the quantity of alive
 1st FEMS Congress / Posters 103^505
mice in 2,5-4 times more, compared with the control
group, but the median duration of life of dead animals
increased on 36-66 %. The specimen of cytoplasmatic
membranes protected 30% of mice from 30 LD50 melioi-
dosis agent. Mice were also protected with surfactant anti-
gen fractions C, C1 and B. Common using of these frac-
tions with immunomodulators(IM) (bromantan and
glutaxim) increases the survival rate of mice on 30-40%.
The specimen of exterior membranes without immunomo-
dulators didn’t have protective characteristics. Surfactant
antigen fractions C, C1, D,H were tested in experiments
with rats. C and D fractions were mostly protective de-
fending 50-58% of rats from 18-180 LD50 strain 100 of the
melioidosis agent. So, surfactant fractions of the melioido-
sis agent used in the coplex with IM should be the most
perspective for creating chemical vaccine. We have tested a
lot of IM and chosed adaptogens of adamantanic group(-
bromantan) and specimens from tiopoetins type(glutoxim)
as the most e¡ective.
P6^31
MULTIPLEX PCR FOR IDENTIFICATION OF ES-
CHERICHIA COLI O157 :H7, AS APPLIED TO MON-
ITORING OF HEMORRHGIC COLITIS CAUSATIVE
AGENT IN RUSSIA
V. A. Bannov , N. V. Volozhantsev , E. A. Svetoch, V. V.
Verevkin
State Research Center for Applied Microbiology, 142279,
Obolensk, Moscow region, Russia
A multiplex polymerase chain reaction (PCR) assay was
designed to simplify detection of Escherichia coli O157:H7
and to identify the H serogroup and the type of Shiga
toxin produced by this bacterium. The system contains
four pairs of primers complementary to nucleotide se-
quence of cytotoxin genes (stx1 and stx2), and intimin
protein responsible for the microbe adhesion (eae A),
and gene rfb responsible for antigen 0157 synthesis. Be-
sides, primers for a plasmid-encoded hemolysin gene
(hly933), or chromosomal £agellar (£iC h7; £agellar struc-
tural gene of H7 serogroup) gene were used in a multiplex
PCR for coampli¢cation of the corresponding DNA se-
quences from enterohemorrhagic E. coli (EHEC)
O157:H7. The PCR system was tested by using 17 refer-
ence strains of E. coli O157:H7 isolated from hemorrhagic
colitis patients in Japan and USA, 12 strains of E.coli of
other serotypes and 26 strains of other species of bacteria.
Samples of clinical materials isolated in 1999-2002 from
patients su¡ered from diarrhea, including patients with
HUS ; from samples of calf and pigs fecal mass have
been analysed by the developed PCR system. PCR gene
typing revealed the availability of rfb, eae, stx, ehx and
£iC genes. Samples of epizootic material from poultry
FEMSLE Congress 2-6-03
257
farms of 8 regions of Russia were analysed. PCR with
chicken samples produced only rfb amplicons, and was
not produce eae speci¢c amplicon. In samples from dairy
plant ¢lters were isolated strains bearing eae, rfb, stxII and
£iC(h7) genes.
P6^32
EVOLUTION OF ANALYTICAL METHODS FOR
CRYPTOSPORIDIUM PARVUM AND GIARDIA SPP
IN SURFACE WATER: RESULTS OF A TIME EX-
TENDED MONITORING IN THE NORTH WEST OF
ITALY
E. Carraro(1), S. Bonetta(1), S. Bonetta(1), P. Secco(2),
E. Fea(3), C. Pignata(3), G. Gilli(3)
(1) Dept. of Science and Advanced Technology, University
of Piemonte Orientale A. Avogadro, Alessandria, Italy; (2)
Dept. of Medical Sciences, University of Piemonte Orien-
tale A. Avogadro, Novara, Italy ; (3) Dept. of Public
Health and Microbiology, University of Turin, Torino, Italy
Our research group started a study in 1992 to assess mi-
crobiological risk related to the presence of Cryptospori-
dium parvum and Giardia spp. in a surface source for
drinking water in the North West of Italy. Sampling, con-
centration and determination of the protozoa were per-
formed according to standard methods (APHA-AWWA-
WEF, 1995; US-EPA, 1999). The overall analysis of the
results underlines the occurrence of the protozoa in the
raw water examined (Giardia cysts 0,002 ^ 67/L , and
Cryptosporidium oocysts 0 ^ 36,86/L) pointing out an as-
sociation between protozoa contamination levels and run-
o¡. Nevertheless, cysts and oocysts were never found in
drinking water, showing the ability of the water multistage
treatment to remove the contamination. Since standard
methods for a routine detection of Cryptosporidium and
Giardia in water samples only provide presumptive infor-
mation about cyst/oocysts species identity and viability,
RT-PCR and PCR-based assays have been developed to
increase detection sensitivity and speci¢city. The recovery
e⁄ciency of IMS-RT-PCR assay for detection of viable
Cryptosporidium parvum and Giardia spp. in spiked water
samples was evaluated. The IMS showed a recovery of 90-
99% for Cryptosporidium and 88-100% for Giardia and a
sensitivity of 10 cysts/oocysts was obtained with IMS/RT-
PCR method. The occurrence of Cryptosporidium oocysts
and Giardia cysts in surface raw water points out the im-
portance of routinary control of water for human con-
sumption. Considering how complex the analytical meth-
ods are, the setting up references laboratories for Giardia
and Cryptosporidium control in the waters is of paramount
importance.
258 1st FEMS Congress / Posters 103^505
P6^33
PREVALENCE AND PROPERTIES OF VEROCYTO-
TOXIN-PRODUCING ESCHERICHIA COLI ISO-
LATED FROM SHEEP IN SERBIA
N obeljic¤, B. Dimic¤
D. Opac›ic¤, M. C
Military Medical Academy, Veterinary Centre ‘Belgrade’,
Belgrade, Serbia
The aim of this study was to elucidate the importance of
sheep as reservoir of verocytotoxin-producing E. coli
(VTEC) in Serbia. In the surroundings of Belgrade, from
March through June 2002, fecal samples were collected
from lambs and mutton sheep and cultured on Mac Con-
key agar plates. Isolated E. coli strains were tested for
verocytotoxin production on Vero tissue culture cells. De-
tected VTEC were examined for phenotypic traits that are
frequently exhibited by VTEC associated with illness in
humans (lack of sorbitol fermentation, enterohemolysin
production, expression of characteristic O antigen, local-
ized adherence to epithelial tissue culture cells). Of 202
examined lambs and mutton sheep, VTEC were isolated
from feces of 135 (66.8%).Of them, 16 (11.8%) were sor-
bitol-negative and 6 were hemolytic (all produced alfa he-
molysin). Agglutination with antisera for typical human
VTEC serogroups (O26, O55, O111, O128, and O157)
revealed that only 7 strains belonged to O128, and 1 to
O55 serogroup. By using HEp-2 cells adherence test it was
found that 17 (12.6%) strains adhered to these cells (14
displayed aggregative, 2 di¡use, and 1 localized pattern of
adherence). 29 (21.5%) VTEC strains showed resistance to
antimicrobial drugs: 28 were resistant to cephalexin only,
and 1 strain displayed multiple resistance to 4 of 11 tested
antibiotics. Although sheep are frequent carrier of VTEC,
their importance as a reservoir of these pathogenic agents
should be further investigated since most of the isolated
strains in this study lacked properties that are commonly
encountered among human VTEC.
P6^34
COLD, HEAT SHOCK AND STARVATION INFLU-
ENCE VIABILITY, CULTURABILITY AND MOR-
PHOLOGY OF CAMPYLOBACTER JEJUNI CELLS
A. Klanc›nik, P. Skoc›ir, T. Zorman, S. Smole Moz›ina
University of Ljubljana, Biotechnical Faculty, Department
for Food Science and Technology, Ljubljana, Slovenia
Campylobacter jejuni is a leading cause of acute bacterial
foodborne gastroenteritis worldwide. Its physiology is in
many senses unusual. It seems to lack many of adaptive
responses to environmental stresses known in other food-
FEMSLE Congress 2-6-03
borne pathogens. However, under stress conditions cam-
pylobacters transit from a spiral-shaped to the more resis-
tant coccoid form, usually accompanied by a
transformance into a viable but nonculturable state
(VBNC). More research is needed to elucidate the impor-
tance of this phenomenon for survival of pathogenic cam-
pylobacters during food production chain. This was the
reason for our investigation of transition of C. jejuni ac-
tively growing and stationary phase cells into a VBNC and
coccoid form following their viability, culturability and
cellular morphology after cold shock at 25‡C and heat
shock at 55‡C. Acridine orange direct count (AODC),
growth on Karmali agar as well as £uorescent and elec-
tron microscopy were methods used, respectively. In addi-
tion, the e¡ect of starvation before cold as well as before
heat shock and cold shock before heat shock was inves-
tigated. In some experiments, chloramphenicol was added
as protein synthesis inhibitor after 1, 2, 3, 4, 5 and 24 h of
starvation before heat shock. We con¢rmed that low tem-
peratures enhanced survival of C. jejuni cells for cca 20%,
viability and culturability were preserved at least 30 days
in rich medium (Preston) and more than 40 days in
starved cells. High temperature provoked quick transfor-
mation from culturable spiral-shaped to nonculturable
coccoid cells. It is remarkable that starved C. jejuni cells
increased cold and heat resistance. However, the latter was
more pronounced, especially in exponentially growing cells
after 5 hours of starvation before heat treatment (25%
increase of survival). The e¡ect of chloramphenicol treat-
ment was more obvious, when added as earlier after start
of starvation. The results indicate the absence of pheno-
typic stationary phase response of Campylobacter cells.
P6^35
PCR DETECTION OF CRYPTOSPORIDIUM SP. IN
HUMAN AND ANIMAL FAECES IN SLOVENIA
B. Soba(1), M. Petrovec(1), A. Vergles Rataj(2), J. Log-
ar(1)
(1) Institute of Microbiology and Immunology, Medical
Faculty, University of Ljubljana, Ljubljana, Slovenia; (2)
Institute of Microbiology and Parasitology, Veterinary Fac-
ulty, University of Ljubljana, Ljubljana, Slovenia
Cryptosporidium is a pathogenic protozoon of humans and
animals. It can cause acute or chronic diarrhoea or even
life-threatening disease in immunocompromised host. An
infection, cryptosporidiosis, can be transmitted by con-
taminated food or water. In recent years, molecular biol-
ogy-based methods have provided insight into the trans-
mission dynamics of cryptosporidiosis. The aim of this
study was to develop a method based on polymerase chain
reaction ^ PCR for detection of Cryptosporidium in faecal
samples. We extracted DNA from four human and ¢ve
 1st FEMS Congress / Posters 103^505
calf faecal samples in which Cryptosporidium oocysts were
previously detected by modi¢ed Ziehl-Neelsen stain. A
fragment of Cryptosporidium 18S rRNA gene encompass-
ing the hypervariable region was ampli¢ed by polymerase
chain reaction. In all nine faecal samples a fragment of
this gene was sucessfully ampli¢ed. In the future it is sug-
gested that detection of Cryptosporidium sp. by polymerase
chain reaction and by its genotyping could provide in-
sights into the transmission dynamics, epidemiology and
possible zoonotic origin of this parasite in Slovenia.
P6^36
IDENTIFICATION OF HELICOBACTER SPECIES IN
DIFFERENT MOUSE STRAINS BY PCR-DGGE AND
DNA SEQUENCING
Y . Ljungh, T. Wadstro«m
W. A. Al-Soud, H.-O. Nilsson, A
Department of Medical Microbiology, Dermatology and In-
fection, Lund University, Lund, Sweden
The genus Helicobacter now comprise at least 24 formally
named species, which are widely distributed in the gastro-
intestinal tract of mammals, birds and other animals. The
mouse models are widely used in research. Recently, the
complete mouse genome has been published, which will
have a huge impact on the use of mouse models. The
aim of this study was to address the problem of Helico-
bacter gut colonisation and infections of di¡erent mouse
strains. A PCR-denaturing gradient gel electrophoresis
(PCR-DGGE) technique was optimised for the detection
and identi¢cation of mice Helicobacter species. Eight mice
strains were included in the study. They were obtained
from four di¡erent laboratory animal facilities C57Bl/6
(n=12), SPF-SCID (n=5), conventional SCID (n=2),
B6sJl (n=3), Balb/cA (n=5), C3H/HEJ (n=4), and Apo-E
(n=5), and IL-10 de¢cient C57Bl/6 (n=6). Mice were sac-
ri¢ced and blood, liver, stomach, the small intestine, and
faecal samples were collected. DNA from faeces was ex-
tracted by the Qiagen DNA Stool Kit (Qiagen), and from
all other samples using the Qiagen DNA Kit. Helicobacter
DNA was detected by genus-speci¢c semi-nested PCR as-
say, in all mice strains tested except the IL-10 de¢cient
C57Bl/6 mice. Using this assay 85 of 204 (42%) samples
were positive. Approximately 6% of the mice were found
co-infected by v 2 Helicobacter species. The identi¢cation
results of PCR-DGGE were con¢rmed by DNA sequenc-
ing and H. ganmani speci¢c PCR designed in our labora-
tory, which allowed identi¢cation of H. typhlonius, H. ro-
dentium, H. bilis, H. ganmani, H. hepaticus, and H.
trogontum. We showed that a number of Helicobacter spe-
cies are colonising the gastrointestinal tract of di¡erent
mouse strains. In addition, di¡erences in colonisation
among di¡erent mouse strains were found, which may
be related to the level of immune de¢ciency and to di¡er-
FEMSLE Congress 2-6-03
259
ences among breeding houses. The PCR-DGGE tech-
nique, which was optimised in this study, was found e⁄-
cient and simple for determination of the Helicobacter
status in laboratory mice.
P6^37
EPIDEMIOLOGY OF HELICOBACTER SP. INFEC-
TION OF DOGS IN SLOVENIA
N erne, M. Pogac›nik, I. Gruntar, J. Mehle,
M. Gombac›, M. C
B. Krt, M. Ocepek
Institute of Pathology, Administrative and Forensic Veteri-
nary Medicine, Veterinary Faculty, Gerbic›eva 60, Ljubljana,
Slovenia
Our research included 213 randomly chosen dogs of 44
di¡erent breeds from all parts of Slovenia (6 regions),
which died or had been euthanised. Both genders were
included in the age from 9 days to 15 years. They lived
either indoor or outdoor and were fed either with com-
mercially prepared food or home made food or were still
suckling. Using toluidine-blue staining method on nec-
ropsy taken stomach tissue we detected Helicobacter in
92,4 % of dogs in all examined parts of stomach i.e. fun-
dus, corpus and antrum. We determined a mild infection
in 17,3% of dogs, medium in 48,1 % and a very strong
infection in 27 % of dogs. Helicobacters were located in
gastric mucus, gastric pits and in gastric glands’ lumen.
For the determination of e¡ects of epidemiological param-
eters (gender, age, breed, nutrition, region and housing
conditions) on infection and the infection rate with heli-
cobacters we calculated the prevalence of infection and
statistically evaluated results.We determined that age and
feeding regimen a¡ect the infection and the degree of in-
fection, whereas gender, breed, location and indoor/out-
door living conditions do not. The rate of infection was
33,3 % in dogs up to 2 months of age, still living with the
bitch and in dogs older than 2 months 94,4 %. The infec-
tion rate of dogs fed with mixed (home prepared) food
was 95,9 % and for dogs fed with commercially prepared
dog food 91,5 %. All suckling puppies were uninfected.
Di¡erences in the prevalence of infection between the re-
gions were small, with the highest infection rate detected
in Primorje region.
260 1st FEMS Congress / Posters 103^505
P6^38
NEW APPROACHES IN ISOLATION OF HELICO-
BACTER SPP. FROM GASTRIC MUCOSA IN DOGS
I. Gruntar, J. Mehle, B. Krt, M. Ocepek, M. Gombac, M.
Cerne, M. Pogacnik
Veterinary Faculty, Ljubljana University, Gerbiceva 60,
1000 Ljubljana, Slovenia
Helicobacters are spiral shaped Gram-negative bacteria,
frequently present in stomach and intestine of humans
and several animal species. Clinical importance of these
bacteria is unclear, with an exception of H. pylori, a caus-
ative agent of chronic gastritis in humans. One of the
reasons for such situation arises from the fact that re-
search on Helicobacter sp. is seriously hampered by con-
siderable di⁄culties in isolation of these bacteria. In our
study we tried to increase the isolation e⁄ciency of Heli-
cobacter spp. by introducing some new approaches in cul-
turing these bacteria from gastric mucosa from freshly
euthanised dogs.
Suitability of di¡erent isolation media was evaluated, as
well as an e¡ectiveness of a variety of antibiotics added to
these media. Diverse physical and chemical methods of
sample preparation and sample decontamination were
evaluated in order to get rid of contaminants that fre-
quently obstructed successful isolation of helicobacters de-
spite great numbers of spiral bacteria present in gastric
mucosa. Brain Heart Infusion (BHI) agar supplemented
with 10% de¢brinated horse blood, 7% horse serum, Van-
comycin, Polymyxin B, Trimetoprim and Amphotericin B
was found to be the best medium for the recovery of
Helicobacter spp. Among the standard media, chocolate
agar gave the best results. We noticed that isolation rate
was considerably better when gastric mucus was lique¢ed
using cysteine solution before plating the samples. The
initial growth of helicobacters was enhanced by use of
few drops of BHI broth, supplemented with foetal calf
serum and freshly prepared yeast extract. A combination
of these methods resulted in successful isolation of Heli-
cobacter spp. from virtually every sample with a positive
preliminary urease test. Moreover, using our techniques,
viable helicobacters could be recovered even from cor-
rectly frozen gastric mucosa samples.
FEMSLE Congress 2-6-03
P6^39
PECULIARITIES OF PATHOGENESIS AND ADAP-
TATION MECHANISMS OF ANTIGENIC VARIANTS
OF BURKHOLDERIA PSEUDOMALLEI BY EXPERI-
MENTAL MELIOIDOSIS
V. V Alekseev, T. N. Vjazmina, N. G. Plekhanova, S. V.
Zamaraeva, V. I. Kapliev and T. Y. Kivokurtseva
Antiplague Research Institute, Volgograd, Russia
The present study examines the pathogenesis of melioido-
sis, induced by typical and defected in the synthesis of
capsule antigen and protease strains, and the enzyme ac-
tivity of the microbe on the di¡erent stages of the disease.
Tests with subcutaneous infection of the animals with
strains and their antigenic variants showed that Ag8 is
the main factor, indicating pathogenicity of the microbe,
and proteases played a minor role. Some di¡erences in
pathogenic action of the defected strains were determined
by the level of the resistance of macroorganism to the
infection. By aerogenic infection the antigen 8 of the mi-
crobe had a relative meaning, as the strain displayed a
pathogenic action, inducing death of 50% of animals in
comparisom with the subcutaneous infection. The respira-
tory melioidosis developed atypical course including mac-
ro- and micromorphological changes, dissemination of the
microbe and its antigens, interaction with phagocytes, clin-
ical manifestations. Long-duration bacterium carrying by
the infection with Australian serovar of the agent was
noted. The strains, not producing hydrolase, were aviru-
lent for the animals. This fact con¢rms that hydrolyse is
considered to be the main factor of pathogenicity by aero-
genic infection. It was determined that realization of the
pathogenic properties of strains B.pseudomallei free of a
capsule occurred by means of signi¢cant activation of hy-
drolytic enzymes. It is considered as the manifestation of
the adaptation mechanism of the agent. The necessity of
the correction of the tactics of laboratory diagnostics by
respiratory melioidosis, induced by atypical strains, and
means of production of diagnostic preparations have
been shown.
P6^40
THE IMMUNE RESPONCE IN MICE AND RATS BY
B. PSEUDOMALLEI ANTIGENS VACCINATION
I. V. Avrorova, V. S. Rybkin, N. N. Piven, S. I. Zhukova,
N. P. Khrapova, D. V. Viktorov and N. M. Drefs
Antiplague Research Institute, Volgograd, Russia.
Improving the methods of prophylaxis and treatment of
melioidosis infection is impossible without studying its
 1st FEMS Congress / Posters 103^505
pathogenesis and immunogenesis. Therefore the dynamics
of making of the immune responce in animals with di¡er-
ent susceptibility to melioidosis has been interesting. The
purpose of this work is studying peculiarities of the vacci-
native process caused with protective antigens of the me-
lioidosis agent in mice and rats. Surfactant antigen com-
plexes C, C1, D and H of the meliodosis agent were used
for the immunization of animals. Mice and rats were de-
¢ned the titer of speci¢c ILISA antibodies, the delayed-
type hypersensitivity level and phagocytic activity of peri-
toneal macrophages using the chemiluminescentic method.
The protective characteristics of melioidosis antigens were
assessed on the mortality rates: the percentage of dead
animals and the median life duration were calculated.
There have been shown similar antigens of melioidosis
agent in host of mice and rats have di¡erent immune ac-
tivity. Most studied specimens inoculated to mice didn’t
in£uence cellular or humoral immunity or highly de-
pressed it. The immunization of mice with these antigens
didn’t prevent animals from infecting with the virulent
strain of the melioidosis agent.However inoculating these
specimens to rats su⁄ciently stimulated the cellular im-
munity which increased the protective activity by the
highly virulent strain B.pseudomallei 100. So, the inocula-
tion of studied antigens to rats (not to mice) made the
adequate cellular immune responce which was the most
su⁄cient part in the protection of melioidosis infection.
All experienced fractions decreased mortality rates in
rats. Antigens C and D had mostly expressed protective
activity in rats by melioidosis.
P6^41
ADAPTATION MECHANISMS OF YERSINIA PSEU-
DOTUBERCULOSIS AND LISTERIA MONOCYTO-
GENES TO ABIOTIC FACTORS OF ENVIRONMENT
L. S. Buzolyova(1,2), G. P. Somov(1)
(1) Institute of Epidemiology and Microbiology of Siberian
Branch Russian Academy of Medical Sciences; (2) The Far
East National University, Russia
Yersinia pseudotuberculosis and Listeria monocytogenes
can be adapted to di¡erent conditions of ecological niches,
in animals as well as in environmental objects with regu-
larly changing abiotic factors. Adaption of biochemical
mechanisms explain the wide ecological plasticity of Yer-
sinia and Listeria to changing environmental conditions.
Our experiments showed that during long habitation in
soil and water, these bacteria change their biological prop-
erties. But these changes have an adverse e¡ect. We ar-
ranged that Y. pseudotuberculosis and L. monocytogenes
can pass to gasotrophy in the conditions of limited carbon
and nitrogen. In starvation period, they can use the autol-
ysis products of dead bacteria. Y. pseudotuberculosis and
FEMSLE Congress 2-6-03
261
L. monocytogenes can store nourishing substances. L.
monocytogenes changed their morphological properties.
We also arranged that Yersinia in low temperature con-
ditions supports the necessarily vital level of metabolism,
using conformational and enzymolysis strategy. We found
that Yersinia have a cholinesterase enzyme (HE) with high
catalytic activity. A fall in temperature to 4 0C accelerates
hydrolysis of acetylcholinesterase (ATH) and propionil-
cholinesterase (PTH) by 20-50 % (for butyrylcholinesterase
(BTH) the speed didn’t change). When Yersinia was at 4
0
C, Km of HE increased (with hydrolysis ATH and BTH)
2-fold; PTH 6-fold. So, the temperature e¡ect was para-
doxical: reducing the cultivation temperature resulted in
increasing enzymolysis of substrates. To reveal changes in
enzymolysis structure with changing temperature, we used
phosphororganic combinations which speci¢cally block
the hydroxyl group of serine of HE active center. We
found a distinction in the quantity of inhibition of enzy-
molysis activity between low and high temperature, show-
ing that the conformation of active surface structure of
Yersinia HE depends on temperature of habitation. Evi-
dently, the adaptation temperature of Yersinia is accom-
panied by conformational changes in the proteins of mo-
lecular enzymolysis.
P6^42
EFFECTS OF SUBINHIBITORY CONCENTRATIONS
OF ANTIBIOTICS ON ADHESION TO AND INVA-
SION OF HEP-2 CELLS BY SOME BACTERIAL
STRAINS WITH DIFFERENT SOURCES OF ISOLA-
TION
V. Lazar(1), C. Balotescu(1), R. Cernat(1), A. M. Is-
rail(2), L. Petrache(1), C. Dinu(2)
(1) Dep. Microbiology-Immunology, Faculty of Biology,
University of Bucharest, Aleea Portocalelor 1-3, Sect.5, Bu-
charest 77206, Romania ; (2) Cantacuzino Intitute for Re-
search and Development in Microbiology and Immunology,
Spaiul Independentei 23-26, Sect.5, Bucharest, Romania
Adherence of microorganisms to epithelial cells is a pivo-
tal event in the pathogenesis of infectious diseases. The
anti-adhesion e¡ects of antibiotics were intensively studied
and con¢rmed for abiotic surfaces (polymers, metals and
cerams), but studies on the e¡ect of antibiotics on bacte-
rial adhesion to cellular substrate are very scant and con-
troversial. The aim of this study was to evaluate the e¡ects
of subinhibitory concentrations of PEN, AMP, AMC,
CAZ, VAN, CHL, KAN, ERY, STR, NOR on the adhe-
sion and invasion capacity of some Aeromonas hydrophila,
Citrobacter freundii, Enterobacter cloacae, Escherichia coli
and Listeria monocytogenes strains isolated from water,
food, acute diarrhea, urine and stool cultures. The pro/
anti adhesion and invasion e¡ects were evaluated quanti-
262 1st FEMS Congress / Posters 103^505
tatively by Cravioto’s modi¢ed method (gentamycin pro-
tection assay). As concerning the Gram-negative bacterial
strains, all antibiotics exhibited overall inhibitory e¡ect on
the adherence/invasion capacity, with no respect of species
or source of isolation. As concerning the Gram-positive
strains, it is to be noticed that AMP, AMX and VAN at
subtherapeutic concentrations promoted the adherence
and invasion capacity of Listeria monocytogenes strains.
These antibiotics have also changed the adherence pattern
of these strains, shifting from di¡use to localized adher-
ence, with the occurrence of speci¢c bacterial clusters,
probably by changing the external electric charge of bac-
terial wall. Our results demonstrated that some antibiotics
could speci¢cally stimulate the synthesis of adhesion mol-
ecules in certain bacterial strains. The pro-adhesion e¡ects
demonstrated by the cell wall acting antibiotics on Gram-
positive strains might have important implications in the
selection of the appropriate antibiotic treatment of pa-
tients with systemic infections.
P6^43
THE ROLE OF TOXINS IN CLOSTRIDIUM DIFFI-
CILE-ASSOCIATED DISEASE
B. Geric, M. Grabnar, and M. Rupnik
Department of Biology, Biotechnical Faculty, University of
Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia
Clostridium di⁄cile is a Gram positive rod causing anti-
biotic-associated diarrhea and potentially lethal pseudo-
membranous colitis and is the most common infectious
cause of nosocomial diarrhea. Two toxins were up to
now recognized as main virulence factors responsible for
development of disease. Recently, some C. di⁄cile strains
have been reported to produce an additional toxin, binary
toxin CDT. Among C. di⁄cile strains we can therefore
di¡erentiate six toxin production types according to the
combination of one, two or all three toxins produced.
Toxin A (TcdA), primarily an enterotoxin, and toxin B
(TcdB), a cytotoxin, are GTP- ribosyltransferases which
cause depolymerisation of actin micro¢laments and de-
stroy the cytoskeleton of cells. This, as well as some other
systemic e¡ects of both toxins, cause extensive tissue dam-
age and £uid response in the gut. Binary toxin is an ADP-
ribosyltransferase which most probably works synergisti-
caly with TcdA and TcdB in the destruction of actin mi-
cro¢laments of the cytoskeleton. The production of binary
toxin is less than 1% of extracelulary proteins, but the
concentrated binary toxin was shown to be cytotoxic in
cell cultures. The prevalence of binary toxin producing
strains is estimated to be from 1.6 to 6 %. Most of the
binary toxin producing strains also produce TcdA and/or
TcdB, but we have also found a few strains that produce
the binary toxin alone. Since the role of binary toxin in the
FEMSLE Congress 2-6-03
development of disease is not understood, we used these
latter strains to study the role of binary toxin in the devel-
opment of disease in di¡erent animal models.
P6^44
A NEW TWO-COMPONENT SIGNAL TRANSDUC-
TION SYSTEM IN BARTONELLA HENSELAE
K. Hercik and P. Branny
Institute of Microbiology, Czech Academy of Sciences, Vi-
denska 1083, Prague 4, 142 20, Czech Republic
A major mechanism of signal transduction, widespread in
bacteria, is the so-called two-component system. These
systems are structured around two conserved proteins: a
histidine kinase and a response regulator protein that are
phosphorylated at His and Asp residues, respectively.
Pathogenic bacteria often use two-component system to
regulate expression of the virulence factors. We have suc-
ceeded to found both members of a new two-component
signal transduction system in Bartonella henselae, a major
causative agent of ‘‘cat scratch disease’’. Based on their
sequence similarities, the genes belong to the family of
NtrY/NtrX systems. Deduced amino acid sequences of
both genes contain all conserved domains typical for
two-component systems. The corresponding genes were
cloned and overexpressed in E. coli. Autophosphorylation
activity of isolated histidine kinase, on its conserved His
residue, was veri¢ed by kinase reaction in vitro. The func-
tion of identi¢ed system is not known.
P6^45
ADAPTIVE RESISTANCE TO BIOCIDES AND
CROSS-RESISTANCE TO ANTIMICROBIAL
AGENTS IN SALMONELLA ENTERICA AND ES-
CHERICHIA COLI
M. Braoudaki and A. C. Hilton
Aston University, Birmingham, UK
The inappropriate use of domestic disinfectants has raised
concern about promoting microbial resistance and poten-
tial cross-resistance to therapeutic antibiotics. The aim of
this study was to investigate the potential for adaptive
resistance in Salmonella and Escherichia coli O157 to com-
monly used biocides, to identify mechanisms underlying
resistance and any cross-resistance to antibiotics. Salmo-
nella and E. coli were serially exposed in sub-inhibitory
concentrations to erythromycin, benzalkonium chloride,
chlorohexidine and triclosan. Following each passage
any adaptive resistance, or cross-resistance in a panel of
antibacterials was recorded. Adaptive resistance was ob-
 1st FEMS Congress / Posters 103^505
served in all strains investigated. Erythromycin-resistant S.
enteritidis expressed cross-resistance to chloramphenicol,
whereas in erythromycin- resistant S. typhimurium cross-
resistance to chlorohexidine was detected. Benzalkonium
chloride resistant S. virchow showed an elevated resistance
to chlorohexidine, however chlorohexidine resistant S.
virchow did not demonstrate cross-resistance to benzalkon-
ium chloride suggesting speci¢c rather than general mech-
anisms. Triclosan-resistant E. coli O157 strains repeatedly
exerted decreased susceptibility to various antimicrobials,
including chloramphenicol, erythromycin, imipenem, tet-
racycline and trimethoprim, as well as to a number of
biocides. Conversely, E. coli O55 when adapted to triclo-
san did not show any cross-resistance to any of the anti-
microbial agents tested. The adaptive mechanisms under-
lying resistance were stable for up to 30 days when strains
were passaged in antibiotic/biocide free media. It appears
possible that the domestic kitchen may provide a selective
environment for adaptive resistance to biocides. This may
eventually lead to the undesirable situation of resident
strains becoming resistant to disinfection and cross-resis-
tant to other antimicrobials.
P6^46
VACCINE POTENTIAL OF S. PNEUMONIAE (PNC)
SURFACE PROTEINS
Y. Mizrachi-Nebenzahl(1), G. Feldman(1), M. Portnoi(1),
R. Dagan(1), K. Overweg(2), J. Wells(2), E. Ling(1)
(1) Ben Gurion University, Beer Sheva, Israel; (2) Insti-
tute of Food Research, Norwich, UK
Pnc is an important respiratory pathogen that induces a
wide range of diseases, varying from asymptomatic car-
riage to lethal meningitis and sepsis. Children are partic-
ularly susceptible to Pnc. The current polysaccharide-
based vaccines are only partially e¡ective in children.
Pnc surface proteins are highly immunogenic and the anti-
body response to these proteins is enhanced with the age.
We hypothesize that immunization with Pnc surface pro-
teins that are common to virulent and non virulent strains
and are immunogenic later in life can elicit protective im-
mune response. Pnc cell wall (CW) proteins were isolated
and subjected to 2-D PAGE. Proteins selected according
to the above mentioned criteria were sequenced and
cloned. Mice were immunized with CW, recombinant Al-
dolase and GAPDH proteins or with aldolase cDNA and
challenged intranasally with 108 cfu of Pnc serotype 3
(WU2). Immunization with CW and aldolase cDNA in-
duced antibody response, determined by Western blot.
79.1% of CW, 43% of rAldolase, 33% with pVAC Aldo-
lase, 36% of rGAPDH and 40% with pVAC GAPDH
immunized mice and 0% of control mice survived chal-
lenge. The non-survivals remained alive for 48 hours lon-
FEMSLE Congress 2-6-03
263
ger then controls. The ability of these proteins to circum-
vent the immune evasion of Pnc WU2, previously
observed in our laboratory, is currently studied.
P6^47
OBSERVATION OF LISTERIA MONOCYTOGENES
IN HELA CELL CULTURE
J. Nowroozi(1), J. V. Youse¢(2), R. K. Moakhar(2), M.
A. Jebelli(1)
(1) Iran University of Medical Sciences and (2) Institute of
Vaccines and Serum Hesarak Razi, Tehran, Iran
Listeria monocytogenes can grow inter and intracellular.
Immunosuppressed individuals, elderly people and preg-
nant woman are at speci¢c risk with this bacteria. So,
the aim of this study was to determine the incidence of
this bacteria in the local cheese, observation them in Hela
cell culture by light microscopy. In this experimental
study, selective enrichment medium with yeast extract,
Palkam and listeria selective agar with cold enrichment
was used. Then, after isolation of bacteria, di¡erent con-
centration of L. monocytogenes obtained from local cheese
in qum city were inoculated in Hela cell culture. Then,
samples were taken intervally (12, 24, 36, 48 hrs), stained
with Gimsa, detected by light microscopy and photo-
graphed. It was found that 3.5% of local cheese in qum
city was contaminated by L. monocytogenes. Pictures were
taken by microscope, showed that this bacteria in concen-
tration more than 5x10 5 after 24 hours can enter into cells
and after 36 hours lysed the host cells.The results showed
that L. monocytogenes exist in contaminated local cheese.
Contamination of dairy products by this bacteria can oc-
cur during preparation, transport and distribution. So,
because of the high consumption of these products in
our country, it is necessary to surveillance the production
and distribution to prevent of incidence of listeriosis in
susceptible people.
P6^48
INVESTIGATION OF CTPA AMONG LISTERIA
MONOCYTOGENES AND ITS TRANSFER INTO E.
COLI
J. Nowroozi
Iran University of Medical Sciences, Tehran, Iran
Listeria monocytogenes is a Gram positive bacteria, fre-
quently found in the environment and is responsible for
serious food-borne diseases such as perinatal infections,
septicaemia and meningoencephalitis in humans and ani-
mals. For this reason, distribution of the ctpA (copper
264 1st FEMS Congress / Posters 103^505
transport protein A) among L. monocytogenes isolated
from clinical, environment, dairy, and poultry samples
were investigated. Then, ctpA gene was transferred into
E. coli DH5-a. This investigation was carried out in two
steps (ctpA was found in L. monocytogenes isolated from
di¡erent sources, then ctpA gene was transferred into E.
coli DH5-a). CtpA DNA from L. monocytogenes was am-
pli¢ed by PCR, identi¢ed on agarose gel, puri¢ed by phe-
nol, and ligated into pGEM-T vector. Then transferred on
X-gal plate containing ampicillin. The sequencing of ctpA
DNA was determined by using dye terminator kit and
sequencing machine. Using PCR to identify the homolo-
gous DNA in 69 isolates, 38% of isolates tested contained
the ctpA deteminant. Our results showed that 90% of clin-
ical and dairy isolates, 85% of environmental isolates and
7% of poultry isolates of L. monocytogenes contained ctpA
in chromosome DNA. Fortunately, transformation of
ctpA from L. monocytogenes into E. coli DH5-a was suc-
cessful. Since, the existance of ctpA in clinical, dairy and
environmental samples was 90% and in poultry was 7%,
so, the virulence of all strains of this bacteria are not the
same. By introducing of such clone (ctpA gene) into suit-
able carrier strains, could be expected to produce a good
oral immunogen against L. monocytogenes.
P6^49
INFLUENCE OF CAPSULE FORMATION IN ME-
LIOIDOSIS AGENT ON ITS PERSISTENCE IN
HOST ORGANISM
S. F. Popov, V. V. Alekseev and V. Y. Kurilov
Antiplague Research Institute, Volgograd, Russia
The melioidosis agent of Burkholderia pseudomallei is
highly pathogenic for humans and animals. Mechanisms
of its survival in vivo haven’t been studied thoroughly.
The present study evaluates the in£uence of capsule for-
mation in melioidosis agent on its interaction with host
cells. The typical strains of B. pseudomallei were used:
C-141, 57576, 60806. To reproduce a subacute form of
the disease per 6 guinea-pigs were infected subcutaneously
with 24-hours cultures with the doses of 103, 104, 105 mi-
crobe cells per each animal respectively. The animals were
killed with ether at the 22-26 day post inoculation. By
autopsy the infection process had a generalized character
with multiple necrosis in lungs, liver and spleen. Electron
microscopic examination of ultrathin sections of the given
organs showed absence of direct damage of parenchyma-
tosous cells by bacteria. Nevertheless, distrophic changes
and common intoxication were observed. The other pecu-
liarity of the melioidosis infection was the presence of the
agents predominantly in cells of mononuclear phagocytes.
One can see bacteria, parasitizing intracellular and having
normal ultrastructure, to obtain a capsule. The capsule
FEMSLE Congress 2-6-03
was presented by a wide electron transparent zones, evenly
surrounding a procaryote body. These zones moderately
¢lled electron solid granulous capsule substance. The part
of intact bacteria was in phagolysosomes of macrophages.
In these cases the capsule prevented contact of lysosomal
contents and the cell wall of microbes. The other part of
undamaged incupsulated bacteria was free arranged in cy-
toplasma. The bacteria, obtaining properties of destruc-
tion were revealed, as a rule, in phagolysosomes and
hadn’t a capsule. Thus, the ability of capsule formation
in B. pseudomallei may be considered as a factor of path-
ogenicity, in£uencing persistance of an agent in a macro-
organism.
P6^50
CLINICAL AND ENVIRONMENTAL STENOTRO-
PHOMONAS STRAINS : DIVERSITY AND DIFFER-
ENTIATION
K. Ribbeck, A. Roder, M. Hagemann and G. Berg
University of Rostock, Institute for Molecular Physiology
and Biotechnology, Albert-Einstein-Str. 3, D-18051 Ro-
stock, Germany
In recent years, the importance of the Gram-negative bac-
terium Stenotrophomonas maltophilia in biotechnology and
as a nosocomial pathogen has increased, giving rise to a
need for new information about its diversity and interac-
tions with other organisms. Stenotrophomonas isolates
from clinical and environmental sources including strains
of biotechnological interest were characterized regarding
the production of antifungal metabolites and enzymes, os-
moprotective substances and plant growth promoting sub-
stances as well as by in vitro interactions with fungi and
plants. The 16S rDNA sequence analysis supported the
high intraspeci¢c diversity of the isolates found for all
parameters and, together with phenotypic properties led
to the proposal of a new plant-associated species: Steno-
trophomonas rhizophila. The production of osmoprotective
substances was di¡erent in clinical and environmental
strains while the former ones produced only trehalose ad-
ditionally glucosylglycerol was found in the latter one. The
genes encoding for the biosynthetic enzymes, trehalose-
phosphate synthase (Tps) or glucosylglycerol-phosphate
synthase (GgpS), were partly cloned and sequenced from
S. rhizophila strain DSM 14405. Using Southern-blot ex-
periments and PCR applying universal primers designed to
amplify speci¢cally partial tps and ggpS fragments from
many isolates a clear correlation between the exclusive
accumulation of trehalose and the only detection of tps
in S. maltophilia strains was found, while in the glucosyl-
glycerol and trehalose accumulating S. rhizophila strain
both genes were detectable. The absence or presence of
glucosylglycerol and the ggpS gene could be used as a
 1st FEMS Congress / Posters 103^505
speci¢c indicator to distinguish between potential human-
pathogenic S. maltophilia strains and environmental
strains of the species S. rhizophila.
P6^51
COMPARISON OF THE FATTY ACID PROFILES
AND WHOLE-CELL PROTEINS OF BORRELIA
BURGDORFERI SENSU LATO
L. Cechova(1), M. Nemec(1), E. Durnova(2), J. Halouz-
ka(3)
(1) Department of Microbiology, Faculty of Science, Ma-
saryk University Brno, Tvrdeho 14, 602 00 Brno, Czech
Republic; (2) Regional Institute of Hygiene Ostrava, Par-
tyzanske nam. 7, 728 92 Ostrava, Czech Republic ; (3) In-
stitute of Vertebrate Biology AVCR Brno, Department of
Medical Zoology, Klasterni 2, 691 42 Valtice, Czech Re-
public
Ten strains of Borrelia burgdorferi sensu lato were ana-
lysed in this study. There are represented strains that are
undoubtedly associated with Lyme disease (B. burgdorferi
sensu stricto, B. garinii, B. afzelii), next B. valaisiana,
strains (nearer unappointed) isolated from arthropods
and rodents and one non-Borrelia strain. Analysis of the
fatty acid methyl esters (FAMEs) of bacteria is a com-
monly used chemotaxonomic technique. Application of
this method to spirochaetes associated with Lyme borre-
liosis is important for more detailed identi¢cation of bac-
teria Borrelia burgdorferi sensu lato. Fatty acids are of
interest because of their role in pathogenesis of several
bacterial diseases. Fatty acid metabolism is not under plas-
mid control and the presence of certain fatty acids has
been shown to correlate with taxonomic conventions.
Analysis was performed using a gas liquid chromatogra-
phy column in conjunction with Microbial Identi¢cation
System software (MIS). In B. burgdorferi s. l. were recog-
nized fatty acids with number of carbon atoms from 12 to
18. The major fatty acid components of the Borrelia spe-
cies used in this study are hexadecanoate (C16:0), cis-oc-
tadec-9-enoate [C18:1(9c)] and octadecanoate (C18:0).The
cellular debris obtained after lysis of Borrelia were solubi-
lized by sodium dodecylsulfate and studied by polyacryl-
amide electrophoresis, comparing the molecular weight
and relative concentration of the protein bands. Cluster
analysis of the PAGE protein pro¢le grouped Borrelia
strains into a single cluster at r=0.7. A higher number of
all bands in the area between 18 and 60 kDa were ob-
served in the pro¢le of Borrelia strains. In conclusion,
the FAME analysis and analysis of whole-cell protein pro-
¢les by SDS-PAGE proved to be useful to contribute to
more detailed study of bacteria Borrelia burgdorferi sensu
lato.
FEMSLE Congress 2-6-03
265
P6^52
SEROLOGICAL DIFFICULTIES IN NEUROBORRE-
LIOSIS DIAGNOSTICS
O. Dakovic¤ Rode, T. Maretic¤
University Hospital for Infectious Diseases HDr. Fran Mi-
haljevic¤g, Mirogojska 8, 10000 Zagreb, Croatia
Neuroborreliosis includes a variety of neurological disor-
ders caused by Borrelia burgdorferi sensu lato. Clinical
manifestation of neuroborreliosis is not pathognomonic.
The etiological diagnosis is based mainly on serological
tests and determination of speci¢c anti-B. burgdorferi anti-
bodies in CSF. Speci¢c antibodies response is in£uenced
by phenotypic di¡erences among B. burgdorferi species,
di¡erent antigenic structure, their di¡erent geographic
spreading, and patient’s possibility to react to infection.
In some patients with neuroborreliosis antibodies could
be produced only intrathecally. Therefore, it is always
necessary to test serum and CSF simultaneously and de-
termine the CSF/serum index. In our study serological
results of 28 patients with clinical diagnosis of neurobor-
reliosis were analyzed. Serum and CSF anti-B. burgdorferi
IgM and IgG antibodies were determined by recombinant
indirect ELISA (Biomedica, Wien, Austria) and capture
ELISA (IDEIA Lyme Neuroborreliosis, Dako, Denmark).
All sera positive results were con¢rmed by Western blot
(Mikrogen, Germany or DPC Biermann GmbH, Ger-
many). The speci¢c antibody index in capture ELISA
was calculated as de¢ned by manufacturer. 11/28 (39,3%)
patients had proved intrathecal antibodies to B .burgdor-
feri by capture ELISA antibody index. 21/28 (75,0%) pa-
tients had signi¢cant CSF antibody titre by indirect ELI-
SA. 19/28 (67,9%) patients had con¢rmed EIA results by
IgM and/or IgG WB. One patient with capture positive
ELISA antibodies had no con¢rmation by WB. While
serologic assays for anti-B. burgdorferi antibodies have
not been standardized yet, all serologic results should be
interpreted according to epidemiological and clinical data
and used to con¢rm the clinical diagnosis.
266 1st FEMS Congress / Posters 103^505
P6^53
THE MOLECULAR EVIDENCE OF BABESIA MI-
CROTI INFECTION IN SMALL MAMMALS COL-
LECTED IN SLOVENIA
D. Duh(1), M. Petrovec(1), T. Trilar(2), K. Prosenc(1)
and T. Avsic-Zupanc(1)
(1) Institute of Microbiology and Immunology, Medical
Faculty, Ljubljana, Slovenia; (2) Slovenian Museum of
Natural History, Ljubljana, Slovenia
Human babesiosis is an important emerging tick-borne
disease. Life cycle of Babesia microti, the main causative
agent of this zoonosis in the United States of America, has
been well described. In Europe, the zoonotic cycle of B.
microti awaits to be determined. Eventhough, B. microti
was found in a variety of small mammals in di¡erent
countries across Europe, it was only recently detected in
Ixodes ricinus ticks in Slovenia by using molecular meth-
ods. In order to investigate the mammalian hosts of B.
microti in Slovenia we collected 261 small mammals rep-
resenting 11 species. They were tested for the presence of
babesial parasites with a PCR assay based on the nuclear
small subunit rRNA gene (nss-rDNA). Only two species,
the bank vole (Clethrionomys glareolus) and yellow-necked
mouse (Apodemus £avicollis), were infected with B. micro-
ti. The prevalence rate was 15.9 % for C. glareolus and
11.8 % for A. £avicollis. Nucleotide sequences of ampli¢ed
portions of B. microti nss-rDNA from C. glareolus and A.
£avicollis were indistinguishable from each other and iden-
tical with those previously described in I. ricinus ticks col-
lected in Slovenia. The results of this study represent the
¢rst molecular evidence of B. microti in small mammals in
Europe. Furtheron, the molecular characterisation of B.
microti in small mammals as well as in ticks in Slovenia
clearly indicates the wide distribution of this parasite in
nature.
P6^54
IN VITRO EFFECT OF SALIVARY GLAND AND
MIDGUT EXTRACTS FROM IXODID TICKS ON
THE GROWTH OF BORRELIA GARINII
I. Rudolf and Z. Hubalek
Medical Zoology Laboratory, Institute of Vertebrate Biol-
ogy, Academy of Sciences of the Czech Republic, Klasterni
2, CZ-69142, Valtice, Czech Republic
The e¡ect of salivary gland extract (SGE) and midgut
extract (MGE) from Ixodes ricinus and Dermacentor retic-
ulatus was tested in vitro on the growth, motility and
morphology of Borrelia garinii. Concentration of motile
FEMSLE Congress 2-6-03
spirochetes (the number of motile cells/ml) was determined
at speci¢c intervals using dark¢eld microscopy. In I. rici-
nus, the concentration of motile spirochetes increased sig-
ni¢cantly between days 2 and 11 post inoculation (p.i.)
with both SGE and MGE compared to control (C). In
D. reticulatus, a signi¢cant increase in concentration of
motile spirochetes was only detected with SGE (compared
to control) on day 5 p.i., while a marked decrease in con-
centration of motile spirochetes was observed on day 9 p.i.
due to MGE e¡ect, and on day 12 p.i. due to the e¡ect of
both organ extracts compared to control. The e¡ect of
SGE and MGE on the growth of B. garinii in vitro dif-
fered between the two tick species tested : while the ex-
tracts derived from I. ricinus (a competent vector for
Lyme borreliosis) stimulated signi¢cantly the growth,
those from D. reticulatus (a non-competent species) did
not a¡ect the growth of borreliae markedly or even inhib-
ited it on days 9 (MGE) and 12 p.i. (MGE and SGE). The
results indicate that the tick organ extracts surprisingly
need not be inhibitory for pathogen survival in the body
of even a non-competent tick species like D. reticulatus in
a short-term exposure.
P6^55
IMMUNITY IN RABBITS IMMUNIZED WITH
CHALMYDOPHILA PSITTACI
W. DeptuTa and M. Pawlikowska
Chair of Microbiology and Immunology, Faculty of Natural
Sciences, University of Szczecin, ul. Felczaka 3a, 71-412
Szczecin, Poland
Present study aimed at determining parameters of non-
speci¢c cell-mediated and humoral immunities in rabbits
experimentally immunized with a killed antigen of Chla-
mydophila (Chl.) psittaci strain 6BC (the earlier avian
strain of Chlamydia psittaci). The studies were performed
on 20 rabbits, 10 received Chl. psittaci strain 6BC antigen,
and 10 rabbits served as a control. Blood for the tests was
sampled from the marginal ear vein on day 1, 7, 14, 21, 28,
35, 42, 49 and 56 of the experiment. In the blood, the
capacity of neutrophilic granulocytes to adhere (ZA) and
to ingest standard bacterial strains (expressed by index of
phagocytosis, IP, and by the percentage of phagocytes,
%kp), reduce nitrotetrazolium blue (NBT) in the spectro-
photometric, spontaneous and stimulated NBT test, coef-
¢cient of granulocyte metabolic activity (WAMG), estab-
lished in spontaneous and stimulated NBT tests,
stimulation index (IS), activity of myeloperoxidase
(MPO), concentration and activity of lysozyme (LZM)
were estimated in serum. Titre of speci¢c serum antibodies
anti-Chlamydia was estimated by complement-¢xation
(CF) test. Analysis of the results demonstrated that four
out of 12 examined parameters demonstrated augmented
 1st FEMS Congress / Posters 103^505
values (concentration and activity of LZM, activity of
MPO, IP), 6 parameters manifested augmented values
(ZA, spectrophotometric NBT reduction test, spontrane-
ous and stimulated, spontaneous and stimulated WAMG)
while two parameters showed no alterations (%kp, IS).
The alterations began on day 7-14 and persisted to days
49-56 of the experiment. Speci¢c anti-Chlamydia antibod-
ies could not be detected by CF test until the day 42 of the
experiment.
P6^56
VIRULENCE CHARACTERS OF RABBIT ENTERO-
PATHOGENIC ESCHERICHIA COLI (REPEC) IN
HUNGARY AND CZECH REPUBLIC
M. A. Dow(1), I. To¤th(1), P. Zs. Fekete(1), P. Alexa(2),
E. Oswald(3) and B. Nagy(1)
(1) Veterinary Medical Research Institute of the Hungarian
Academy of Sciences, Budapest, Hungary ; (2) Veterinary
Research Institute, Brno, Czech Republic; (3) UMR960
INRA de Microbiologie Moleculaire, Ecole Nationale Vet-
erinaire de Toulouse, France
In a collection 70 E. coli strains, isolated from cases of
post weaning diarrhoea of rabbits in Hungary and Czech
Republic, PCR investigations were performed for viru-
lence genes. As many as 47 E. coli strains proved to
have the eae gene (encoding for intimin) and all of them
proved to be of f- type of intimin. All eae+ strains had the
LEE (locus of enterocyte e¡acement) indicated by the
presence of espD gene. Neither the enteropathogen E.
coli speci¢c EAF (EPEC Adhesion factor) plasmid nor
bfp (bundle forming pili) gene (characteristic for human
EPEC) were detected. None of the strains had genes of stx
(Shiga-like toxins: stx1 or stx2), lt (thermolable enterotox-
in), or sta or stb (thermostable enterotoxin) genes. How-
ever, the sequence of porcine attachment associated (paa)
gene was detected in 20 strains. The eae+ strains belonged
mainly to serogroups O15, O55, O49, O103, O132, O145,
O153, and O157. By PCR, the ¢rst described rabbit adhe-
sive factor (AF/R1) sequence was detected in two strains
(both of O153 serogroup). Adhesive factor/rabbit2 (AF/
R2) sequence was detected in eleven strains (all of O103
serogroup), and the REPEC adherence locus (ralG) gene
was detected in eighteen strains representing: O145, O153,
O49, O15, O55, O157 and O132 serogroups. The ral spec-
i¢city was veri¢ed with colony hybridization. The tested
eae+ strains strongly adhered to HeLa cells in culture as-
says in a di¡use way. The type or intensity of HeLa ad-
hesion did not seem to relate to the ¢mbrial type carried
by the strains investigated.
FEMSLE Congress 2-6-03
267
P6^57
INFECTIONS OF EXOTIC FISH IN SLOVENIA
V. Jencic, M. Ocepek, I. Zdovc, P. Hostnik
University of Ljubljana, Veterinary Faculty, Gerbiceva 60,
Ljubljana, Slovenia
Aquarium and other exotic ¢sh breeding is a popular and
economically important amateurish activity. Although ¢sh
kept in the closed systems causative agents of diseases can
endanger populations in open waters and in the intensive
¢sh farming. Some ¢sh Mycobacteria can be harmful for
¢sh breeders as well. Developed and organised diagnostics
of exotic ¢sh diseases can help to reduce the current ¢sh
health problems. From epizootic, environmental and eth-
ical point of view, education the owners of exotic ¢sh is
extremely important, as well. Diverse ¢sh health problems
could be established through systematic clinical examina-
tions. High incidence of bacterial infection caused by My-
cobacterium (TBC) was found and control methods were
applied. The acidoresistent bacteria have been identi¢ed in
clinically changed ¢sh using direct Ziel Nielson staining
method. The majority of clinically examined suspicious
aquarium ¢sh were positive on Mycobacteriae using spe-
cial media for bacteriological examinations. Isolated bac-
teria were M. fortuitum, M. chelonae and M. gordonae.
Stained and native bacteriological samples we examined
and super¢cial bacteria from the family Myxobacteriae
were established. When bacterial nutrient media were em-
ployed most often bacteria from the family Aeromonas
and species hydrophila/caviae and A. sobria were deter-
mined. Carp pox ^ herpes virus was established using
pathohistological method in clinically suspicious coi carp
(Cyprinus carpio) All clinically suspicious ¢sh were found
negative when virologicaly examined, using di¡erent cell
cultures.
P6^58
MOLECULAR EVIDENCE OF BACTERIA FROM
THE GENUS BARTONELLA SP. IN SMALL MAM-
MALS COLLECTED IN SLOVENIA
N. Knap, M. Petrovec, D. Duh and T. Avsic-Zupanc
Institute of Microbiology and Immunology, Medical Fac-
ulty, Ljubljana, Slovenia
Bacteria of the genus Bartonella are emerging human
pathogens causing an increasing variety of diseases, most
of which are thought to be zoonoses. Therefore, it is im-
portant to determine di¡erent vectors and mammalian res-
ervoir hosts of bartonellae. The aim of our study was to
investigate the prevalence and the genetic diversity of bar-
268 1st FEMS Congress / Posters 103^505
tonellae in small mammals in Slovenia. We collected 146
animals representing 4 species, yellow-necked mouse (Apo-
demus £avicollis), wood mouse (Apodemus sylvaticus),
striped ¢eld mouse (Apodemus agrarius) and bank vole
(Clethrionomys glareolus), in 3 di¡erent zoogeographical
regions of Slovenia. Animals were tested for the presence
of bartonellae with PCR assay based on the ITS regions of
Bartonella genome. On overall, 48.6 % (71 of the 146) of
small mammals were infected with bartonellae. The prev-
alence rate di¡ered among 4 species of small mammals
tested: 71.8 % (23 of the 32) for A. sylvaticus, 62.7 %
(27 of the 43) for A. £avicollis, 31.7 % (13 of the 41) for
C. glareolus and 26.6 % (8 of the 30) for A. agrarius. In
addition, the evident di¡erence in the prevalence rate of
infection was noticed regarding the zoogeographical re-
gions in which the animals were trapped. The PCR ampli-
¢cation yielded products of di¡erent lengths implying ge-
netic diversity of bartonellae detected in small mammals.
To characterise and identify the species of bartonellae the
representative amplicons were sequenced.
P6^59
IDENTIFICATION AND ANALYSIS OF VIBRIO VUL-
NIFICUS BIOTYPE 2-SPECIFIC DNA SEQUENCES
C.-T. Li(1), E. Sanjuan(2), C. Amaro(2) and L.-I.
Hor(1)
(1) Department of Microbiology and Immunology, National
Cheng Kung University, Tainan, Taiwan, R.O.C ; (2) De-
partment of Microbiology and Ecology, University of Va-
lencia, Spain
Vibrio vulni¢cus is a bacterial species from warm brackish
water with pathogenic potential for humans and several
species of aquatic animals. In humans, this pathogen
causes wound infections and, occasionally, septicemia. In
eels and other aquatic animals, it causes an emergent dis-
ease whose symptoms are similar to the vibriosis due to V.
anguillarum. This disease was ¢rst registered in Europe in
1989, and since then is the main cause of economic losses
in anguilliculture. The eel-pathogenic strains are classi¢ed
in the biotype 2 of the species. These strains share high-
Mw plasmids and belong to di¡erent serovars, being the
serovar E the most virulent one. The main objective of our
work is to determine the genetic basis of eel virulence in V.
vulni¢cus. This objective will be developed in di¡erent
phases, and we present here the results obtained in the
¢rst one. Suppression subtractive hybridization was used
to select biotype 2-speci¢c DNA fragments. The obtained
fragments were cloned and analyzed by Southern hybrid-
ization, DNA sequence determination and searching of
homologous sequences. The selected sequences and the
primer pairs derived from them were tested for speci¢city
with more than eighty strains, including V. vulni¢cus and
FEMSLE Congress 2-6-03
non-V. vulni¢cus isolates. The results obtained in the
screening showed that three sequences were speci¢c for
serogroup E, and two sequences for eel-virulence. The
last sequences were located on one of the common plas-
mids. The entire nucleotide sequence of this plasmid is
now under determination.
P6^60
ATTACHING EFFACING ESCHERICHIA COLI
(AEEC) BACTERIA IN WEANED PIGS
A. Malik(1), I. To¤th(1), L. Beutin(2), B. Nagy(1)
(1) Veterinary Medical Research Institute of the Hungarian
Academy of Sciences, Budapest, Hungary ; (2) Robert
Koch Institute, Berlin, Germany
The aim of these studies was to examine the occurrence
and the characteristics of the AEEC in postweaning diar-
rhoea of piglets. Therefore we collected mucosal mem-
branes from the ileum and colon of weaned pigs that
died as a result of postweaning diarrhoea. Besides, we
have collected rectal swabs from healthy and diarrhoeal
weaned piglets. The eae+ isolates were identi¢ed by PCR
and further investigated for additional virulence genes (tir,
EAF, bfp, stx1, stx2, espD, paa). From 133 piglets that
died as a result of postweaning diarrhoea 37 eae+ of
1702 strains were detected (representing 9 % of the pigs).
Further 45 eae+ strains were identi¢ed among 535 strains,
(from rectal swabs of 57 healthy and 31 diarrhoeal live
pigs). The eae-positivity in non-diarrhoeal animals was
14 % and in diarrhoeal animals was 29 %. The main O
types of eae+ strains were O45, O49, O28 and O108. The
dominant group was O4/O123 carrying both antigens.
None of the strains had stx1, stx2, EAF or bfp genes,
but 91% was espD+ indicating the presence of the LEE
pathogenecity island in almost all strains. The eae genes
were typed by PCR: 72 % of the intestinal strains were of
L-type, while only 31% of the rectal swab strains were L-
type, the rest having untypable eae gene, suggesting the
existence of new variant(s) of eae gene in this collection.
Our results indicate that the AEEC bacteria in weaned
pigs are relatively frequent and they may have some new
types of EPEC virulence traits.
 1st FEMS Congress / Posters 103^505
P6^61
EVALUATION OF IMMUNOPROFILACTIC EFFEC-
TIVENESS OF LEUKOTOXIN INACTIVATED WITH
VARIOUS METHODS
P. Mikucki, A. Wernicki, A. Puchalski, R. Urban-Chmiel
and M. FaTkowska-Podstawka
Department of General Prophylaxis and Avian Diseases,
Faculty of Veterinary Medicine, Agricultural University Lu-
blin, ul. Akademicka 12, 20-033 Lublin, Poland
Mannheimia haemolytica serotype 2 and especially leuko-
toxin (Lkt) produced by it, is known to be as one of the
essential virulence agents participating in etiopathognesis
of pulmonary diseases in sheep. Subunit vaccines used so
far contain chromatographically puri¢ed or biologically
inactivated Lkt. The purpose of the study was to compare
protection e¡ectiveness of antibodies induced by Lkt ei-
ther native or inactivated with formaldehyde or glutaral-
dehyde. The experiment was carried out on sheep, immu-
nized twice at a two-week interval with various Lkt forms
and with commercial vaccine including Lkt. The control
animals received only an adjuvant. The immunogenic
properties of antigens used, was estimated based on absor-
bance values of sera antibodies (ELISA). Antibodies pro-
tective e¡ect, after intratracheal challenge (1x108 cfu/ml
M. haemolytica serotype 2) was estimated on the basis of
the extent of lung lesions. Immunized sheep lungs, in com-
parison with the control group, showed lower extent of
in£ammatory process. However, no signi¢cant di¡erences
in lung lesions were observed in all experimental groups.
Statistically signi¢cant higher absorbance values were ob-
served in all immunized sheep. The relationship between
intensity of lung lesion extent, and absorbance values in
experimental sera, suggest correlation between antibodies
level and their protective e¡ect. The results obtained in
our study suggest no signi¢cant in£uence of aldehydes
used to Lkt inactivation on immunogenicity and thus on
protective e¡ect of antibodies produced. This asssumption,
however, needs con¢rmation in further experiments.
P6^62
DEVELOPMENT OF A PROTOCOL FOR THE SPE-
CIFIC ISOLATION OF THE PATHOGEN VIBRIO
VULNIFICUS BIOTYPE 2
E. Sanjua¤n and C. Amaro
Dpto. Microbiolog|¤a y Ecolog|¤a, Universidad de Valencia,
Valencia, Spain
The haemorrhagic septicemia due to Vibrio vulni¢cus bio-
type 2 is the main cause of economic loss in eel culture.
FEMSLE Congress 2-6-03
269
This disease emerged in Japan in 1975, arrived to Spain in
1989 and spread to Northern Europe in 1995. Since then,
the number of human infections associated with ¢sh ma-
nipulation has increased. Some of these cases have been
caused by V. vulni¢cus biotype 2, which even though it has
pathogenic potential for humans, has been epidemiologi-
cally underestimated. This biotype, or more precisely path-
ovar, comprises strains of at least three di¡erent serovars,
and serovar E is the most virulent of them. The recom-
mended protocols for the isolation of V. vulni¢cus from
water and seafood samples involve an enrichment step in
alkaline peptone water, supplemented or not with poly-
myxin B, followed by seeding on selective media contain-
ing colistin and polymyxin B as selective compounds, and
cellobiose as the di¡erential compound. In this work we
tested the e⁄cacy of the recovery of biotype 2 strains from
environmental samples of these classical procedures and of
a new protocol based on the exclusive capacity of biotype
2 to grow in eel blood. The results obtained in the labo-
ratory and the ¢eld demonstrate that this new protocol is
more e¡ective, suggesting that its use for the isolation of
the biotype 2 of this species from environmental samples
could clarify its epidemiological importance in human and
eel health.
P6^63
THE CHICKEN HUMORAL IMMUNE RESPONSE IS
INVOLVED IN PROTECTION AGAINST ORNITHO-
BACTERIUM RHINOTRACHEALE INFECTION
D. F. Schuij¡el(1), P. J. M. Nuijten(1), A. M. M. A.
Pennings(1), J. van Putten(2), P. C. M. van Empel(1)
(1) Intervet International BV, Bacteriology R&D, Wim de
Ko«rverstraat 35, 5830 AA Boxmeer, The Netherlands; (2)
Utrecht University, Faculty of Animal Health, Department
of Bacteriology, Yalelaan 1, 3584 CL Utrecht, The Nether-
lands
Over the last decade, Ornithobacterium rhinotracheale has
emerged as an important pathogen associated with respi-
ratory tract infections in poultry. To study the chicken
immune response after O. rhinotracheale infection, the
role of antibody-mediated immunity was investigated by
using two di¡erent strategies. First, young broiler chickens
were treated with immunosuppressive cyclophosphamide,
thereby depleting antibody-producing B-lymphocytes, fol-
lowed by a challenge with O. rhinotracheale serotype A. At
post-mortem, organ lesion-scores were compared to those
of challenged birds with a normal immune system. Im-
mune-suppressed birds su¡ered from signi¢cant higher le-
sion-scores than the control animals. In the second strat-
egy, B-lymphocyte depleted broiler chickens received sera
from protected animals prior to a homologous challenge
with O. rhinotracheale serotype A. Lesion-scores from im-
270 1st FEMS Congress / Posters 103^505
munized and non-immunized birds were compared. The
immune-suppressed birds that received a dose of anti-se-
rotype A antibodies showed lower lesion scores after ho-
mologous challenge. Passive immunization with non-spe-
ci¢c antiserum prior to challenge did not reduce organ
lesions. The results of both studies point out the impor-
tance of speci¢c O. rhinotracheale circulating antibodies in
protection against infection. Furthermore, administration
of serotype A antiserum prior to a heterologous challenge
with O. rhinotracheale serotype G signi¢cantly reduced
organ lesions, indicating an important role for the humor-
al immune response in cross-protection against infection
with other O. rhinotracheale serotypes. The next step is to
identify cross-protective antigens of O. rhinotracheale that
induce humoral immunity in order to develop a suitable
cross-protective vaccine.
P6^64
SOME LESS FREQUENT POTENTIAL ZOONOTIC
ANIMAL PARASITES IN SLOVENIA
A. Vergles Rataj, A. Dovc›, J. Posedi, J. Rac›nik
Veterinary Faculty, Gerbic›eva 60, Ljubljana
Some less frequent potential zoonotic animal parasites
were found in Slovenia in the past few years. Pentastomids
were found in 8.3% of examined geckos, 12.2 % of exam-
ined monitor lizards and in 16.1% of examined snakes.
Acarine ectoparasites ^ Ophionyssus natricis was found
on boa skin. Dermanyssus gallinae were often detected
on poultry and other birds. It can infestate humans, caus-
ing serious problems. Among free-living birds, Argas sp.
was noted. Potential zoonotic parasites were detected and
determined in pet animals : Tryxacarus caviae in guinea
pigs, Cheyletiella sp. in rabbits, Notoedres cati and Oto-
dectes cynotis in cats, Sarcoptes scabiei in dogs. Hymeno-
lepis nana was found in one mouse. Strongyloides sp. was
found in monkey. In this article, adult parasites and some
of their developing forms and eggs are described and pre-
sented.
P6^65
FOOT-AND-MOUTH DISEASE EMERGENCE IN U.P.
(NORTH INDIA)
S. K. Yadav
Dept. of Epidemiology and Preventive Medicine, College Of
Veterinary Science & Animal Husbandry Mathura U.P.,
Veterinary University & Cow Research Institute, India
The continents like North America, Australia and Antarc-
tica are currently reported free from FMD. But the virus is
FEMSLE Congress 2-6-03
still endemic in Asia, Africa, the Middle East and Japan.
The other regions supposed to be free are sporadically
a¡ected as reported by World organization for Animal
Health. It has also been observed that FMD occurred in
Italy in 1993, in the eastern part of Greece close to the
Turkish border in 1994, 1996 and 2000 and the UK, Ire-
land, France and the Netherlands in 2001, with the cessa-
tion of vaccination against FMD. A systematic epidemio-
logical study on Foot-and-mouth disease was conducted
to Asses geographical distribution and seasonal occurrence
of di¡erent types and subtypes of the virus associated with
this disease and the factors associated with the mainte-
nance and spread of infection in selected areas of North
India. For FMD virus isolation and typing work, a total
of 21 specimens of vesicular epithelium were collected dur-
ing this year from di¡erent outbreaks of FMD. All of
these were collected from bu¡aloes, the main species in-
volved during the various outbreaks. While 11 of the
strains were typed O FMD virus. A bu¡alo trembled
and died instantly; autopsy revealed presence of a single
small vesicular lesion on the dorsum linguae and pinpoint
haemorrhages on the myocardium.The piece of myocardi-
um yielded type O virus. Reports are also available from
Mexico and Brazil recording occurrence of malignant
form of FMD. The zoonotic impotence of the disease
was also studied.
P6^66
THE POST FLOOD LEPTOSPIROSIS IN CZECH RE-
PUBLIC
K. Zitek and C. Benes
Institute for Public Health, Centre for Epidemiology and
Microbiology, Prague, Czech Republic
Leptospirosis is a typical zoonosis with natural nidality
and its occurrence in the climatic conditions of the Czech
Republic is sporadic and the incidence is normally about
0,3 cases per 100.000 inhabitants. In 1997 and 2002, how-
ever, the incidence of leptospirosis has been in£uenced by
the actual natural phenomenon ^ catastrophic £oods,
which increased the numbers of serologically diagnosed
and registered cases three times, i.c. to 0,9/100.000 in com-
parison with previous years. In 1997 there have been ex-
amined for a leptospirosis a total of 7,156 subjects in the
Czech Republic, and the disease was diagnosed and regis-
tered in 94 of them (and in 2002 in 92 patients respec-
tively). Two-third of these cases came from inundate areas,
half of them in direct relation with the £oods. Four of
registered cases (1997) of Weil disease have died in the
Czech Republic. The di¡erence between the actual and
reported morbidity is critically discussed. The poster con-
tains graphs, maps and tables which described over men-
tioned.
 1st FEMS Congress / Posters 103^505
P7^1
IDENTIFICATION OF HELICOBACTER PYLORI:
COMPARISON OF STAINING METHODS
T. Babic(1), H. Basic(2), M. Otasevic(1)
(1) Institute for Public Health, Dept. of Microbiology,
Brace Taskovic 50, 18000 Nis, Yugoslavia; (2) Clinical
Centre Nis, Institute of Pathology, Brace Taskovic 48,
18000 Nis, Yugoslavia
Aim : To compare the sensitivity of detecting H. pylori in
gastric biopsy and resection specimens using modi¢ed
Giemsa stain and immunohistochemistry using a commer-
cially available anti-H. pylori antibody (Dako, Denmark).
Methods: Gastric antral biopsy specimens showing
chronic gastritis (27 cases) together with tissue blocks
from gastrectomy specimens for duodenal ulcer were his-
tology reviewed. The para⁄n sections were stained with
modi¢ed Giemsa and immunoenzymatic by alcaline phos-
phatase anti-alkaline phosphatase (APAAP) method. Re-
sults : The modi¢ed Giemsa and immunoenzymatic treated
sections were carefully examined for the presence of H.
pylori. Using a modi¢ed Giemsa stain, the spiral shaped
bacteria of H. pylori stained blue, were attached to the
brush border of the gastric foveolar epithelial cells and
inside gastric pits. In some cases masked bacteria hidden
within mucous were obvious only in immunostained prep-
arations (red deposits). Similarly, in modi¢ed Giemsa
treated sections, coccoid forms, which were particularly
seen in sections from resection specimens, caused some
uncertainty. These coccoid H. pylori were obvious in im-
munostained preparations. Immunoenzymatic staining can
be performed on cryostat and para⁄n tissue sections, but
reaction was more intense and di¡use in a cryostatic sec-
tions. H. pylori was identi¢ed in 75.9% sections stained
with modi¢ed Giemsa, but it could be identi¢ed with
greater frequency in sections stained with APAAP
(93.1%). In all cases the bacteria were more prominent
and easier to detect in the immunostained sections than
in sections stained tinctorially. Conclusion : immunohisto-
chemical identi¢cation of H. pylori by the APAAP proce-
dure is a highly sensitive and easy to use method for de-
tecting this organism.
FEMSLE Congress 2-6-03
271
P7^2
TOXOPLASMA GONDII ANTIBODIES IN WOMEN
DURING REPRODUCTIVE PERIOD ^ OUR EXPERI-
ENCES
D. Cvetkovic, T. Grdanoska, N. Panovski, M. Petrovska
Institute of Microbiology and Parasitology, Medical Fac-
ulty, Vodnjanska 17, 1000 Skopje, Macedonia
The aim of this study was to analyse the seroprevalence of
anti T. gondii antibodies in women during their reproduc-
tive period in our country. A total of 400 women were
examined: 200 women with pathologic pregnancy (103
with spontaneous abortion, 19 habitual abortion, 39 ster-
ility and 39 with still-born) and 200 who have never been
pregnant or had normal pregnancy, as a control group.
The sera from women with pathologic pregnancy were
collected at the Institute of Microbiology, and the sera
from the control group were received from the Republic
Center for Blood Transfusion. Following tests were used
for detection of speci¢c IgG antibodies : Indirect immuno-
£uorescent assay (IIF) ( Toxo-Spot IF bioMerieux),
France; direct agglutination test (DA) (Toxo-Screen DA
bioMerieux), France; enzyme immunosorbent assay (EIA)
EIA DIALAB, Wien. The tests for detection of speci¢c
IgM antibodies: EIA EIA DIALAB, Wien and immuno-
sorbent agglutination test (ISAGA) (toxo Isaga bioMer-
ieux), France. Obtained results, according to IgG antibod-
ies, are the following: 74.5% of the female patients in
reproductive period had no and 25.5% had contact with
Toxoplasma gondii. Acute and chronic infection was more
frequent in the women with pathology during the preg-
nancy versus control group. Acute infection was serodiag-
nosed with presence of IgM antibodies in 10 women from
the examined group and one from the control group
(p 6 0.01). Acute and chronic infection was more fre-
quently detected in women with diagnosis of habitual
abortion.
P7^3
AN ENZYME LINKED IMMUNOSORBENT ASSAY
(ELISA) FOR THE DETECTION OF THE FISH
PATHOGEN MORITELLA VISCOSA
K. J. Heidarsdottir and E. Benediktsdottir
Institute of Biology, Microbiology Labaratory, University
of Iceland, AŁ rmu¤li 1A, 108 Reykjav|¤k, Iceland
Moritella viscosa is a psychrotrophic bacteria causing win-
ter ulcers in salmonid ¢sh reared in saline water at temper-
atures below 10‡C. The clinical signs of the disease are
skin lesions and sometimes haemorrhages in internal or-
272 1st FEMS Congress / Posters 103^505
gans. The bacteria has been isolated from winter ulcers in
Iceland, Norway and Scotland. The occurrence of the dis-
ease might have been underestimated, because M. viscosa
is slow growing and possible overgrown by other marine
bacteria. Identi¢cation of M. viscosa is also impeded by
rather fastidious growth and weak reactivity in most bio-
chemical tests. Therefore an immunosorbent assay (ELI-
SA) with horse radish-peroxidase system was developed.
Research have shown that the main antigens of M.viscosa
are lipooligosaccharides and a 17-19 kDa antigen in the
wall. For coating, two di¡erent antibodies were tested,
polyclonal antibodies raised against the cell wall produced
in mice, and puri¢ed IgG from rabbits that were raised
against whole cell. Puri¢ed and cross-adsorbed polyclonal
antibodies from sheep (Microtek) were used for catching.
Di¡erent treatment of kidney samples spiked with bacte-
rial cells were conducted to ¢nd the optimal treatment. No
cross reaction was observed against non pathogenic strains
of M. viscosa or other marine bacterial species tested.
P7^4
INCIDENCE OF HEMOLYSIN AND GELATINASE
AMONG UROISOLATED ENTEROCOCCI
G. Jankoska, M. Petrovska, G. Mirchevska, B. Trajkovska-
Curchic, T. Spasenovski
Institute of Microbiology and Parasitology, Medical Fac-
ulty, Vodnjanska 17, 1000 Skopje, R. Macedonia
Hemolisyn, aggregation substance and enterococcal pro-
tease, commonly called gelatinase, are some phenotypic
markers that have been proposed as possible virulence
factors of enterococci. The aim of this study was to deter-
mine the production of hemolysin and gelatinase among
di¡erent species of enterococci isolated from urine sam-
ples. Total of 45 strains of Enterococcus spp. isolated
from urine samples were examined. CPS ID2 agar (bio-
Me¤rieux) was used for isolation and identi¢cation of the
strains as Enterococcus spp. VITEK automated system
(GPI card) was included in identi¢cation of di¡erent spe-
cies. Hemolisyn production was detected on Columbia
CNA agar as clear zone of L-hemolysis around the streak.
Production of gelatinase was determined by using of tripti-
case soy agar supplemented with 1.5% skim milk. A clear
halo around the colonies after overnight incubation at
37‡C was considered positive for gelatinase production.
The strains were identi¢ed as: Enterococcus faecalis (39),
Enterococcus faecium (6 isolates). Only 3 strains (6.67% of
all) were negative for both hemolysin and gelatinase pro-
duction. In 14 (31.12% isolates of all) there were present
both factors. The rest of isolates (22-48.89% in total) were
positive for only one factor, hemolysin or gelatinase (he-
molysin in 10, gelatinase in 12 strains). There weren’t
found any signi¢cant di¡erence in the presence of hemo-
FEMSLE Congress 2-6-03
lysin and gelatinase within the di¡erent species. These two
markers of virulence, either together or separated, were
present in 80% of uroisolated enterococci.
P7^5
CARBON SOURCE UTILIZATION FOR DIFFEREN-
TIATION OF THE TEMPE FUNGUS RHIZOPUS
OLIGOSPORUS AND RELATED SPECIES
J. Jennessen(1), A. R. B. Eriksson(1), J. Olsson(2) and J.
Schnu«rer(1)
(1) Department of Microbiology, Swedish University of
Agricultural Sciences, P.O. Box 7025, SE-750 07 Uppsala,
Sweden ; (2) Centre for Clinical Trials of Foodstu¡s, Upp-
sala University, P. O. Box 609, SE-751 25 Uppsala, Sweden
The zygomycete Rhizopus oligosporus is used in the pro-
duction of tempe, a fermented food traditionally made
from soybeans, but which also can be made from cereal
grains. Di¡erent strains of R. oligosporus di¡er in proper-
ties such as mycelium growth, metabolite production,
spore production, and optimal growth conditions. The
substrate also in£uences the growth and metabolism of
R. oligosporus. This makes knowledge from soybean pro-
duction not directly transferable to production of cereal
grain tempe. Increased understanding of basic features of
R. oligosporus, e.g. growth on di¡erent substrates, is
needed in order to produce tempe of good quality. R.
oligosporus and R. microsporus are morphologically very
similar. Some strains of R. microsporus have, as opposed
to R. oligosporus, been found to produce toxic metabolites.
There is thus a need for new methods able to distinguish
R. oligosporus from closely related species, as well as to
di¡erentiate between R. oligosporus strains. A range of
factors in£uences the biology of the tempe fungus. One
important factor is the composition of carbon sources of
the substrate. Rhizopus species and R. oligosporus strains
have been characterised according to their carbon source
utilisation patterns during growth in microtiter plates.
This presentation will discuss the importance of this new
tool for elucidating Rhizopus taxonomy, as well as its use
in strain di¡erentiation.
 1st FEMS Congress / Posters 103^505
P7^6
IMPROVED SERODIAGNOSIS OF LYME BORRE-
LIOSIS BY BORRELIA AFZELII IMMUNOBLOT
V. L. J. Jovicic(1), E. M. Grego(2), B. L. Lako(3), B. M.
Ristovic(3), Z. A. Lepsanovic(3), N. T. Stajkovic(3)
(1) Institute of Public Health of Serbia ‘‘Dr Milan Jova-
novic, Department for Microbiology; (2) Institute of Mo-
lecular Genetics and Genetic Engineering; (3) Institute for
Microbiology Military Medical Academy, Belgrade, Yugo-
slavia
To improve serodiagnosis of Lyme borreliosis (LB) the
performances of two tests were evalueted.An indirect im-
muno£uorescent assay (IFA) based on Borrelia burgdorferi
sensu stricto, and immunoblot (IB) based on local isolate
of Borrelia afzelii were prepared. The serum panels con-
tained 199 serum samples: control group (n=70), and pa-
tients at di¡erent stages of LB (n=129). The speci¢city of
IB was 96%, and 89% of IFA. In early LB the sensitivity
of the IFA was 55% and 92% of IB. In late stage of LB
sensitivity was: 70% of IFA and 93% of IB. IgM and IgG
antibodies from sera of patients with early and late LB,
the most frequently demonstrated reactivity to the OspC.
The other signi¢cant proteins in early LB were: p39, p41
in IgM IB, and p83/100, p39, Osp17 in IgG IB; in late LB:
p39, p41 in IgM IB, and p83/100, Osp17, p43, p30, p14,
and p21. In our study OspC was the most important pro-
tein in early and late disease and we postulate that these
results are the outcome of performance of IB based on
local Borrelia afzelii strain abundantly expressing OspC.
The using IB as a con¢rmatory test improves serodiagno-
sis in our geographical region.
P7^7
SUSCEPTIBILITY OF EXTENDED SPECTRUM L-
LACTAMASES (ESBL) PRODUCING STRAINS OF
KLEBSIELLA SPP. AND E. COLI TO ANTIMICRO-
BIAL AGENTS
A. Kaftandzieva, V. Kotevska, K. Popovska-Jovanovska, N.
Panovski
Institute of Microbiology and Parasitology, Medical Fac-
ulty, ul. Vodnjanska br. 17, 1000 Skopje, Macedonia
ESBL enzymes confer resistance to oxyimino-cephalospor-
ins and monobactams. These enzymes occur predomi-
nantly in Klebsiella spp. and E. coli. ESBL producing or-
ganisms contain multi-resistant plasmids. As a result, they
are often resistant to other classes of antibiotics. We aimed
to detect ESBL producing strains of Klebsiella spp. and E.
coli and to determine their susceptibility to di¡erent
FEMSLE Congress 2-6-03
273
classes of antimicrobial agents. All strains of Klebsiella
spp. and E. coli, isolated from 8 di¡erent clinics from
Clinical center in Skopje in a 10-month period, were tested
by the disc di¡usion method for antibiotic susceptibility
(NCCLS). Intermediate susceptible or resistant to ceftriax-
one (CRO) were 144 strains. They were tested for ESBL
production by the disc di¡usion test (DDT) and combina-
tion disc (CD) method. Identical results were obtained
using DDT and CD. In the group of 144 strains, ESBL
positive were 86 (59.7%): Klebsiella spp. 56/110 and E. coli
30/34. The DDT method is useful and necessary since
ESBLs producers often have a low level resistance to third
generation cephalosporins. Moreover, imipenem, merope-
nem, amikacin and cipro£oxacin showed good in vitro
activity against ESBL producing strains of Klebsiella
spp. and E. coli.
P7^8
THE POTENTIAL OF MABS FOR DETECTION OF
BURKHOLDERIA PSEUDOMALLEI
N. P. Khrapova, S. N. Tikhonov, E. V. Prokhvatilova and
M. Y. Kulakov
Antiplague Research Institute, Volgograd, Russia
Burkholderia pseudomallei is widely distributed in tropical
and subtropical areas. The removal of infection is possible
o¡ the endemic areas with persons who temporarily stayed
on these territories. Intensive tourism, business trips, mi-
gratory movement promote original export of B. pseudo-
mallei. Risk of spreading of melioidosis exists constantly.
The actuality of rapid and reliable detection of B. pseudo-
mallei in di¡erent samples increases. That is why national
programms of preventing and control the spread of dan-
gerous diseases include the section providing improve-
memnt of e¡ectiveness of immunodiagnostic means and
methods for express detection and identi¢cation of B.
pseudomallei. We have created the collection of di¡erent
Mabs against conserved termostable epitopes of exopoly-
saccharide and LPS of B. pseudomallei. On the basis of
some of evaluated Mabs preparations for immuno£uores-
cent test, indirect hemagglutination test and ELISA were
prepared. These preparations allow to detect the B. pseu-
domallei and/or B. mallei and di¡erentiate these two mem-
bers of the genus. They don’t bind to the epitopes located
on the antigens of microorganisms of other genera. The
qualitive parameters of these preparations make the detec-
tion of 80% Burkholderia strains available. The e¡ective-
ness of using of these preparations on the basis of eval-
uated Mabs prevails the e¡ectiveness of using of
monoclonal analogues, makes the researches of various
specimens of the environment and clinical material more
reliable.
274 1st FEMS Congress / Posters 103^505
P7^9
MICROBIOLOGICAL MONITORING IN PROPHY-
LAXIS OF POSTOPERATIVE COMPLICATIONS AT
RECONSTRUCTION OPERATIONS OF CHILD-
REN’S MAXILLOFACIAL SURGEON
R. Kipshakbayev
Kazakh National Medical University, Department of Child-
ren’s Stomatology, Tole bi st. 88, Almaty, Kazakhstan,
480090
Studying the micro£ora of an oral cavity both in the pre-
operative and postoperative periods is of great importance
in prophylaxis of post-operative complications following
maxillofacial surgery. This research studied the micro£ora
of the oral cavity, its sensitivity dynamics in children with
congenital clefts of a lip and palate, the development of an
optimum algorithm of sampling in an oral cavity and the
upper respiratory ways, an optimum set of nutrient media
for these micro£ora, and an algorithm of the circuit for
e¡ective antibacterial prophylaxis of postoperative compli-
cations. We surveyed 240 children from 6 months to 6
years of age with congenital clefts of a lip (118) and palate
(122). Material was taken from: 1) border of skin and a
mucous upper lip in the ¢eld of a cleft, 2) area of nose on
the defect side, 3) edge of palate cleft, 4) back wall of
larynx, 5) pharynx. Identi¢cation of speci¢c structure
was made by the developed original technique (scienti¢c
know-how No 537, 20.12.1999). The research designed an
optimum algorithm of 5-days sampling, an optimum set of
nutrient media, and tests for identi¢cation of microorgan-
isms in conditions of limited resources. Microbiological
monitoring for contamination of postoperative wounds
under in£uence of antiseptics and antibiotics was made.
We o¡ered a method of unitary introduction of the anti-
biotic ceftriaxone before operation and processing of a
postoperative wound by a 0,01% miramistine (scienti¢c
know-how No 518, 2.06.1999). Thus, on the basis of com-
plex qualitative and quantitative comparison of micro£ora
of an oral cavity and para-oral areas of healthy children
and children with congenital clefts of a lip and the palate,
the development of dysbiotic changes for the ¢rst time is
revealed at congenital clefts of a lip and the palate which
are the important risk factor of development of postoper-
ative complications. Introduction in clinic of the new cir-
cuit of postoperative microbiological monitoring have
lowered postoperative complications of in£ammatory
character and have raised results of surgical treatment
by 20% and 12.5% accordingly.
FEMSLE Congress 2-6-03
P7^10
ALTERNATIVE METHOD FOR EFFICIENT CON-
CENTRATION OF PLANT VIRUSES USING MONO-
LITHIC CHROMATOGRAPHIC SUPPORTS
P. Kramberger(1), N. Petrovic›(2), A. SNtrancar(1) and M.
Ravnikar(2)
(1) BIA Separations, d.o.o., Teslova 30, SI-1000 Ljubljana,
Slovenia; (2) National Institute of Biology, Vec›na pot 111,
POB141, SI-1001 Ljubljana, Slovenia
It was shown that plants can acquire a number of plant
viruses through the roots. In these cases irrigation water
can play a vector role. To detect plant viruses, which are
highly diluted in water, a concentration step prior to the
detection procedure is essential. Conventional procedures
for virus concentration are di⁄cult and time-consuming.
We have applied new chromatographic media, named
CIM Convective Interaction Media0 disk monolithic col-
umns for virus concentration. We have tested the useful-
ness of these columns on two model single stranded RNA
plant viruses: rod-shaped Tomato mosaic virus (ToMV)
and isometric Cucumber mosaic virus (CMV). We success-
fully concentrated ToMV on a strong anion exchanger
(QA disk monolithic column) and CMV on a weak anion
exchanger (DEAE disk monolithic column). The elution of
concentrated virus from the monolith was performed by
high salt concentration. As shown by ELISA, a few orders
of magnitude higher virus titter has occurred in the eluent.
Described procedure enables more e⁄cient and, compared
to other methods, much faster concentration of plant vi-
ruses. The procedure could be conveniently used in mon-
itoring plant viruses in irrigation waters, and applied to
other laboratory manipulations of plant viruses.
P7^11
EVALUATION OF FOUR DIFFERENT MEDIA FOR
ISOLATION AND IDENTIFICATION METHICILLIN
RESISTANT STAPHYLOCOCCUS AUREUS FROM
REGIONAL HOSPITAL DERIVED SAMPLES
P. Kuralt, T. Z N retnik, A. SNtorman
N ohar-C
Institute of Public Health Celje, Department of Medical
Microbiology, Gregorc›ic›eva 5, 3000 Celje, Slovenia
Rapid isolation and identi¢cation of Methicillin resistant
Staphylococcus aureus from samples, especially surveil-
lance cultures is critical step in preventing spread of bac-
teria inside the hospital environment. 1089 swabs from
patient in the regional hospital were collected from Octo-
ber to December 2002 and 117 were MRSA positive. The
purpose of our work was to compare three di¡erent media
 1st FEMS Congress / Posters 103^505
recommended by NCCLS and Phenyl Red Mannitol
Broth with aztreonam and ceftizoxime (PRMB) recently
described in the literature. All the clinical samples were
submerged in Todd Hewitt Broth with supplement
(THBS), and PRMB and then streaked onto Manitol
Salt Agar with oxacillin plates (MSA) and 5% bovine
blood agar plates (KA). The frequency and speed of iso-
lation of MRSA were recorded for each medium sepa-
rately. The limit of detection was determined with ¢ve
control ATCC MRSA strains. In 47% of positive samples
MRSA was isolated from all four media, in 9.4% from
both broths, in 9.4% only from THBS and in 2.6 % only
from PRMB. As the PPV for PRMB after 24 hours was
only 19.7%, the combination of solid and liquid media for
rapid detection of MRSA is still needed. Interestingly
growth of ¢ve control MRSA strains was supported to a
very di¡erent extent by each medium.
FEMSLE Congress 2-6-03
275
P7^12
INCIDENCE OF ACTIVE TOXOPLASMOSIS IN RE-
GION OF NISH, SERBIA
N. Miladinovic-Tasic(1), S. Tasic(1), A. Tasic(2), B. Pet-
rovic(1), D. Zdravkovic(2)
(1) Medical Faculty, Nis; (2) Institute of Public Health,
Nis, Serbia
Toxoplasmosis, world-spread antroposoonosis, is great
problem in many countries today. The aim of the study:
The occurrences of active toxoplasmosis on Nis region, in
the period from 1998 to 2002, have been surveyed. Sam-
ples and methods : the study used the data obtained from
the Parasitology Department of the Institute of Public
Health, Nis and has examined patients who were perma-
nent citizens of Nis region in the period from 1998 to
2002. Active toxoplasmosis incidence rates in this region
have been determined of the sample of 100,000 citizens
according to the census from 1991 (396.043). The diagno-
sis was made on the bases of clinical report and positive
immune test by ¢nding speci¢c IgM antibodies. Speci¢c
IgM and IgG antibodies have beed detected by indirect
immunoenzyme assay. Results : In last ¢ve years in Para-
sitology Department 1467 patients were examined for spe-
ci¢c antibodies to toxoplasmosis. Prevalence of chronic
toxoplasmosis was 17,85%, and active toxoplasmosis was
diagnosed in 2,93% of patients. The cumulative rate of
active toxoplasmosis in ¢ve year period is 10,85, while
the average cumulative incidence per a year is 2,17. During
the studied period the values of incidence varied from 1,51
in 1998 to 3,02 in 2002. The highest incidence was regis-
tered in 2002 (3,02). The incidence of active toxoplasmosis
in other years was 2,01 (1999, 2001) and 2,27(2000). Con-
clusion : In the city of Nis region the incidence of active
toxoplasmosis was not high in the last ¢ve years and there-
fore does not represent a great clinical or epidemiological
problem.
P7^13
CHLAMYFAST AND MYCOFAST TEST IN DIAGNO-
SIS OF GENITAL TRACT INFECTIONS IN MEN
A. Vasic(1), S. Tasic(2) and N. Miladinovic-Tasic(2)
(1) Institute of Public Health, Nis; (2) Medical Faculty,
Nis 18000, Yugoslavia
Infections with Chlamydia trachomatis (Ch. trachomatis),
Mycoplasma hominis (M. hominis) and Ureaplasma urealy-
ticum (U. urealyticim) have been recognised as being com-
mon sexually transmitted disiase in industrial countries,
and all there suspected to have a pathogenic role in human
276 1st FEMS Congress / Posters 103^505
reproductive failure.The prevalence of Ch. trachomatis, M.
hominis and U. urealyticum infections in male patients
were evaluated. In this study 400 men with symptoms of
urethritis were analyzed in the Bacteriology Section of In-
stitute of Public Health in Nis, Serbia. Infection with Ch.
trachomatis was ditected with Chlamyfast (International,
Mycoplasma ^ France). Mycofast (International, Myco-
plasma, France) was used for identi¢cation of M. hominis
and U. urealyticum. Ch. trachomatis infection was detected
in 35,55% of male patients.In signi¢cant lower percents of
patients M. hominis (2,32%) and U. urealyticum (6,20%)
infections were found. Mixed infections were detected in
only 8 cases.In one case mixed infection was with Ch.
trachomatis and M. hominis, and in second case mixed
infetion with Ch. trachomatis and U. urealytica was found.
M. hominis and U. urealyticum were identi¢ed in material
from 6 patients.
P7^14
A SCANNER READING ASSISTED MIC (COLORI-
METRIC) METHOD FOR SUSCEPTIBILITY TEST-
ING OF ENVIRONMENTAL GRAM NEGATIVE FER-
MENTATIVE BACTERIA
M. Rahman(1), I. Ku«hn(1), M. Rahman(2) and R. Mo«ll-
by(1)
(1) Microbiology and Tumor Biology Center (MTC), Kar-
olinska Institutet, SE-171 77 Stockholm, Sweden; (2) In-
ternational Center for Diarrhoeal Disease Research, GPO
Box 128, Dhaka 1000, Bangladesh
The ScanMIC method is a scanner reading assisted colori-
metric MIC determination method, designed for suscepti-
bility testing of environmental gram-negative fermentative
bacteria. The method is a slight modi¢cation of the
NCCLS recommended broth micro dilution method, using
a tetrazolium salt (TTC) as indicator to estimate the end-
point of growth in a microtiter plate (i.e. growth inhibi-
tion) and a £atbed scanner to capture the image of the
microtiter plate. The choice of TTC as indicator was based
upon toxicity values, ease to read by scanner and relation
to bacterial growth. A simple in-house software was de-
veloped to transform the microtiter plate scan image into
numerical values of the pellet sizes and automatically to
generate the MIC values. We compared the ScanMIC
method for 119 separate coliform strains against seven
antimicrobial agents to the NCCLS recommended agar
dilution method and another 98 separate coliform strains
to the NCCLS recommended broth microdilution method.
‘‘Absolute categorical agreement’’ was obtained in 91 %.
Agreement for MIC di¡erences (within Y 1 log2 dilution)
was obtained in 94 % for ScanMIC versus agar dilution
and 98.5 % for ScanMIC versus broth micro dilution. This
ScanMIC method was found to be labour saving and
FEMSLE Congress 2-6-03
needed only a cheap scanner device for reading, and it
may thus be useful for epidemiological surveys of suscep-
tibility pattern in large numbers of environmental isolates.
P7^15
X-RAY MICROANALYSIS ^ A POWERFUL METH-
OD FOR DETECTION OF MICROBIAL CELLS IN
THE ENVIRONMENT
V. Sorokin, A. Mulyukin, G. El’-Registan and V. Galchenko
Institute of Microbiology of RAS, Prospekt 60-let Oktjabr-
ja, 7/2, 117312 Moscow, Russia
Standard microbiological culture methods make it possible
to reveal only a small fraction of the microorganisms ac-
tually present in the environment and gives limited infor-
mation on the physiological slate of microbial cells in situ.
It is often di⁄cult to distinguish microbial cells and abio-
tic particles in environmental samples by using only the
electron microscopy. The content of S, P, Ca, and K and
the Ca/K and P/S ratios considerably di¡er in cells with
di¡erent physiological state (vegetative cells, resting cells,
endospores, dead cells) and abiotic particles. X-ray micro-
analysis allowed us to reveal the remarkable and statisti-
cally signi¢cant di¡erences of these parameters in various
bacterial cells. Also, we used these parameters as markers
for the direct detection of microbial cells in natural hab-
itats, such as permafrost. X-ray microanalysis was success-
fully used for the detection of microbial cells among cell-
like particles immediately in the samples of ancient Ant-
arctic permafrost deposits (170 thousand years old). The
X-ray spectra of cell-like particles were compared with the
data obtained for microbial cells of various physiological
state. Thus, the absence of P and S peaks in X-ray spectra
in the most of cell-like particles allowed us to regard them
as non-living objects. Among some other particles that
were preliminary recognized as most probable microbial
cells by a set of characteristics of the element composition,
we were able to ¢nd the resting forms of microorganisms.
It had the increased intracellular level of Ca, high Ca/K
ratio and low P/S ratio. The distinguishing of cells with
di¡erent physiological statuses is important for ecological
monitoring of the environment. X-ray microanalysis can
be a regarded as a promising tool for a primary detection
of microbial cells in situ and to predict their physiological
state.
 1st FEMS Congress / Posters 103^505
P7^16
COMPARISON OF TWO METHODS FOR DETERMI-
NATION OF GIARDIA LAMBLIA CYSTS IN FECES
N. Svent-Kucina, I. Grmek-Kosnik, M. Ravnik, B. Virnik,
M. Petrevcic, M. Erzar
Institute of Public Health Kranj, Gosposvetska ulica 12,
4001 Kranj, Slovenia
We made comparison of two methods for determination
Giardia lamblia cysts in feces in laboratory for gastrointes-
tinal diseases in department of medical microbiology of
Institute of public health in Kranj. First method was ex-
amination of native samples with Lugol under light micro-
scope, made by two independent examinators. Second
method was ELISA test for determination Giardia lamblia
cysts from feces. We examined 322 samples from patients
and from people who prepare food (preventive examina-
tion). 318 samples were negative by both methods. Two
samples were positive by both methods. Two samples were
negative by ¢st Lugol and positive by ELISA method.
However, by second Lugol examination, those two sam-
ples turned out to be positive. We conclude that ELISA
method is more sensitive for determination of Giardia lam-
blia cysts from feces.
P7^17
EVALUATION OF FLUORESCENT POLARIZATION
TEST IN DIAGNOSIS OF HUMAN BRUCELLOSIS
V. Taleski
Institute of Preventive Medicine, Military Hospital, Depart-
ment of Microbiology,
Skopje, Macedonia
Human brucellosis is a zoonosis that remains a worldwide
veterinary, medical, and economical problem. Last 8
years, approximately 500-600 new cases of human brucel-
losis are registered yearly in Macedonia. Tests ranging
from culture to serodiagnostic tests to the recent molecular
techniques are available. Mostly used are conventional
serologic techniques such as RBT (Rose Bengal, Slide Ag-
glutination Test), Wright (Tube Agglutination Test) and
Coombs (Antihuman Globulin Test), than ELISA and
competitive-ELISA. Fluorescence Polarization Assay
(FPA) has been validated for number of species including
cattle, swine, bison (Nielsen and Gall). Objectives: Evalu-
ation of Fluorescence Polarization Assay (FPA) in com-
parison with ELISA and the Conventional serologic tech-
niques in diagnosis of human brucellosis. Patient samples
were collected at 5 regional hospitals in Macedonia. Many
of the patients were on treatment when blood samples
FEMSLE Congress 2-6-03
277
were collected. Samples were held frozen at -200C until
they were processed. A total of 217 sera samples were
tested. Initial diagnoses were made by serology: RBT,
Wright and Coombs tests. To detect IgM and IgG anti-
brucella antibodies NOVUM Diagnostica micro plates,
coated with Brucella LPS antigen and reader ELISA TE-
CAN-Classic, were used. In FPA, O-polysaccharide pre-
pared from B. abortus strain 1119-3 conjugated with FITC
(Fluorescein isothiocyanate) and Fluorescence Polariza-
tion Analyzer FPM-1, were used. Results and conclusions:
Sensitivity of Wright, Coombs, ELISA and FPA were
82%, 89%, 98% and 86% respectively. Speci¢city of
Wright, Coombs, ELISA and FPA were 98%, 100%,
100% and 92% respectively. FPA is a very promising
tool in diagnosis of human brucellosis beside diagnosis
in animals, further studies concerning sensitivity and spec-
i¢city are needed. ELISA remains a reference method in
serologic diagnosis of human brucellosis.
P7^18
MORPHOLOGICAL AND MORPHOMETRIC CHAR-
ACTERISTICS OF DIROFILARIA SPECIES
A. Tasic(1), S. Tasic(2), N. Miladinovic(2), D. Zdrav-
kovic(1) and N. Bakrae'(3)
(1) Institute of Public Health Nis; (2) Medical Faculty
Nis; (3) Veterinary Faculty Beograd, Yugoslavia
A large number of publications and scienti¢c studies from
Europe and other parts of the world show that ¢lariasis of
dogs have an important in£uence on the health of dogs
and on human health. The aim of this study is the di¡er-
entiation of ¢lariosis caused by Diro¢laria immitis and
Diro¢laria repens in Zrenjanin, the territory known as a
district of mosquitoes. In this study there were included
peripherical blood smears of 32 dogs. Nostandardized
Knott’s test with modi¢cation and DIFIL test (evsco,
buena, nj, usa) were used for the detection of micro¢lar-
iae. Micro¢laria identi¢cation and determination were
based on their morphologic and morfometrical character-
istics. Softver Lucia 32G, version 4.11 was used for the
determination of morphological and morphometric char-
acteristics of diro¢laria species. Micro¢lariae of Diro¢laria
repens were found in all investigated dogs (32), but in two
dogs mixed infection with Diro¢laria immitis end Diro¢-
laria repens was found.
278 1st FEMS Congress / Posters 103^505
P7^19
IMMUNOLOGICAL INVESTIGATION IN VAGINAL
MYCOSES
S. Tasic(1), N. Miladinovic(1), A. Tasic(2) and D. Zdrav-
kovic(2)
(1) Medical Faculty and (2) Institute of Public Health,
Nis, Yugoslavia
Despite numerous researches about primary recurrent gen-
ital candidiasis which a¥icts 10% of female population in
the world, the cause of it is still unknown. The aim of this
study was to investigate whether the disregulation of sys-
tem immune response, that is to say, the domination TH-2
immune response, could be the cause of recurrent vaginal
candidiasis in women. The examination of the parameters
of cellular immunity included 20 women with RGC (group
A), who were in the remission phase at the moment of
examination and 20 women of the control group (group
B), who had never had a diagnosis of genital candidiasis.
The production of IFN-gamma and IL-4 by mononuclear
cells of peripheral blood was stimulated by joined activity
of standard mitogen Concavalin-A and speci¢c antigen
HKB (heat killed blastospores of Candida albicans). The
quantity of produced interferon gamma and IL-4 was de-
tected by immunoenzyme test Quantikine (R/D system
USA). The production of cytokine by a joint stimulation
of the mononuclear cells of the women’s peripheral blood
did not di¡erentiate in the examined groups (A-IFN-gam-
ma-average value (AV)1115pg/ml; IL-4-AV-59 pg/ml i B-
IFN-gamma-AV-1371 pg/ml; IL-4-AV-48pg/ml). The rela-
tionship between two ‘‘key’’ cytokines was important for
the detection of the relationship between Th-2 i Th-1 im-
mune response. In the relation expressed as a quotient of
IL-4 cytokine values and IFN-gamma values there was not
any statistically signi¢cant di¡erence between the exam-
ined groups, or in other words, in all the women there
was proved the domination of TH-1 immune response.
A- IL-4/IFN-gamma= 55x10-3 ; B- IL-4/IFN-gamma =
51x10-3, p 6 0.05.
P7^20
DETECTION OF SERUM SPECIFIC IgG AND IgA
ANTIBODIES TO CANDIDA ALBICANS IN WOMEN
WITH RECURRENT GENITAL CANDIDIASIS
S. Tasic(1), N. Miladinovic(1), A. Tasic(2), P. Vla-
hovic(3), B. Petrovic(1) and
D. Zdravkovic(2)
(1) Medical Faculty, (2) Institute of Public Health and (3)
Clinical Centre, Nis, Yugoslavia
FEMSLE Congress 2-6-03
There have been only a few studies so far which attempted
to examine the role of speci¢c serum and secretolytic im-
munoglobulin in women with recurrent genital candidiasis
(RGC). The aim of this study was to prove a possible
cause-e¡ect relation between speci¢c immunoglobulin ¢nd-
ings and the ¢ndings of Candida sorts in women’s genital
tract by the detection of anti-Candida IgG and IgA anti-
bodies in the blood of examined women. The examined
test group for serological analysis included 60 women with
RGC who had positive ¢ndings of Candida albicans in
vaginal mucosa. The control group included 60 women
without Candida sp. infection/colonization of genital tract.
The speci¢c anti-Candida IgG and IgA antibodies in the
blood of the examined women were found by a non-stan-
dardized indirect imuno£uorescent test. The speci¢c anti-
Candida IgG antibodies were proved in 90% of the women
with RGC and in 45% of the women in the control group.
IgA speci¢c immunoglobulin were found in 12 women of
the test group and in only one women in the control
group. Lower titer of IgG antibodies was proved in almost
the same number of the women in both examined groups.
Higher titer of IgG antibodies was proved in a signi¢-
cantly greater number of the women with a chronic fungal
genital infection (34) in comparison to the women in the
control group (9).
P7^21
DETECTION OF SPECIFIC ANTIBODIES FOR ACTI-
NOBACILLUS PLEUROPNEUMONIAE IN SWINE
BLOOD SERA
B. Vidic, Z. Grgic, S. Savic-Jevdjenic
Scienti¢c Veterinary Institute ‘‘Novi Sad’’, Rumenacki put
6, 21 000 Novi Sad, Yugoslavia
Actinobacillus pleuropneumoniae, also known as Haemo-
philus pleuropneumoniae, causes pleuropneumonia in
swine, a disease that presents a signi¢cant health and eco-
nomical problem in intensive swine production. The dis-
ease mostly appears in fattened swine, at the age of 2-6
months, but spreads to older and younger animals in the
herd. In Europe, mostly serotype 2, 8 and 9 of A. pleuro-
pneumoniae are present. Serologic methods are very impor-
tant for diagnostics of pleuropneumonia in swine, but also
for the determination of infection prevalence in swine-
herds. Few methods are yet described for diagnosis : ag-
glutination, indirect haemagglutination, complement ¢xa-
tion, ELISA, hemolisin-neutralisation test and cytotoxin-
neutralisation test. The goal of our research was to apply
several serologic methods to analyze for the ¢rst time the
presence of A. pleuropneumoniae in swineherds in this
country. Analysis of 764 swine sera was done, from boars,
sows and piglets of 4 swineherds. The samples were taken
by free choice, from those that came for regular yearly
 1st FEMS Congress / Posters 103^505 279

examination for brucellosis and leptospirosis. For detec-
tion of speci¢c antibodies to A. pleuropneumoniae, the fol-
lowing methods were used: microagglutination, 2-ME-ag-
glutination and complement ¢xation (CF). Antigens were
prepared from a reference strain of A. pleuropneumoniae
serotype 2. Antibodies speci¢c for A. pleuropneumoniae
were found in all analyzed swineherds. By microagglutina-
tion, 27,5-66,6% of animals were positive, with the anti-
body titre from 1:8 to 1:128. 2-ME-agglutination detected
a lower percentage of positive swine and also a lower anti-
body titre (GMT 16). By modi¢ed CF reaction, 21,1-
47,2% of positive swine were found with the titre of com-
plement ¢xation antibodies from 1:4 to 1:64. Our results
con¢rm the presence of A. pleuropneumoniae infection in
swineherds and show that there is a need for wider epi-
sootiologic research in our region.
for expanding the tools available to the public health sci-
entist and molecular epidemiologist.
P7^23
PCR EVALUATION OF ESCHERICHIA COLI
STRAINS PATHOGENICITY
M. Damian(1), C. R. Usein(1), C. Capusa(2), R. Faga-
ras(2), M. Cosman(1), D. Tatu-Chitoiu(1), M. Nica(3),
M. Florea(1) and G. Mircescu(2)
(1) Cantacuzino Institute, Splaiul Independentei 103, Bu-
charest, R-70.100, Romania; (2) ‘‘Dr. C. Davila’’ Clinical
Hospital of Nephrology, Bucharest, Romania; (3)’’Victor
Babes’’ Infectious Diseases Hospital, Bucharest, Romania

P7^22 Escherichia coli is a heterogeneous species consisting of

both enteric commensal and pathogenic strains. Di¡erent
A PCR MICROARRAY BASED APPROACH FOR types of E. coli cause various diseases in a range of hosts
IDENTIFICATION OF ROTAVIRUS STRAINS including intestinal and extra-intestinal infections. Classi-
cal bacteriological methods are not able to discriminate
C. Booth(1), N. Boonham(1), R. Stones(1), M. Iturriza- among the pathogenic and non-pathogenic E. coli strains.
Go¤mara(2), J. Gray(2) and N. Cook(1) PCR technique was used to detect virulence encoding
genes in a collection of isolates from patients with di¡erent
(1) Central Science Laboratory, Sand Hutton, York YO41 types of urinary tract infections. In order to establish the
1LZ, UK; (2) Enteric Virus Unit, Enteric, Respiratory and pathogenic power of the strains, the genes coding for ad-
Neurological Virus Laboratory, Central Public Health Lab- herence (pap, sfa/foc, afa) and for toxins (hly, cnf) was
oratory, London NW5 9HT targeted. Urinary and fecal isolates from patients were
compared with fecal isolates from controls.
Microarrays, containing immobilised gene-speci¢c DNA
or oligonucleotide probes, allow monitoring and analysis
of a large number of genetic features in a single hybrid-
isation test. This technology has been used for gene ex-
pression analysis, bacterial phylogenetic classi¢cation,
gene mapping, and polymorphism analysis. It can be
used to identify variants of speci¢c genes, and thus to
determine the strain type of a microorganism. In this
study, the potential of a PCR microarray-based approach
for the detection and identi¢cation of rotavirus strains was
demonstrated. The array was designed to identify ¢ve ro-
tavirus genotypes found in the UK. Seven oligonucleotide
probes, speci¢c for the VP7 and VP4 genotypes, were
spotted onto the glass slides. Rotavirus particles were ex-
tracted from faeces and infected cell cultures, using a silica
microcolumn. Nucleic acid was extracted and single tube
RTPCR was performed using conserved primers, allowing
the incorporation of amino-allyl dNTPs. Amplicons were
then labelled with £uorescent dyes (Cy3 and Cy5), and
hybridised to the microarray. The array was scanned
and the identity of the isolate determined by the hybrid-
isation pattern. All rotavirus isolates were correctly iden-
ti¢ed by the microarray. The method described is capable
of performing all the steps in the analysis from sample
treatment to strain identi¢cation, and holds the potential

FEMSLE Congress 2-6-03

280 1st FEMS Congress / Posters 103^505
P7^24
MOLECULAR IDENTIFICATION BY ITS-PCR OF
STAPHYLOCOCCAL ISOLATES FROM OVINE
SUB-CLINICAL MASTITIS
S. F. F. Pereira(1), I. Couto(1,2), C. Queiroga(3), A.
Marinho(3), C. L. Vilela(4), I. Santos-Sanches(1,5) and
H. de Lencastre(1,6)
(1) Laborato¤rio Gene¤tica Molecular, Instituto de Tecnolo-
gia Qu|¤mica e Biolo¤gica (ITQB/UNL), Rua da Quinta
Grande, 6, Apartado 127, 2780-156 Oeiras, Portugal; (2)
Centro de Recursos Microbiolo¤gicos, Faculdade de Cie“ncias
e Tecnologia (FCT/UNL), Quinta da Torre, 2829-516
Monte da Caparica, Portugal; (3) Departamento de Sani-
dade Animal e Vegetal, Universidade de EŁvora, Apartado
94, 7002-554, EŁvora, Portugal; (4) Centro Interdisciplinar
de Investigaca‹o em Sanidade Animal, Faculdade de Medic-
ina Veterina¤ria, Polo Universita¤rio Alto da Ajuda, 1300-477
Lisboa, Portugal; (5) Faculdade de Cie“ncias e Tecnologia
da Universidade Nova de Lisboa (FCT/UNL), Quinta da
Torre, 2829-516 Monte da Caparica, Portugal; (6) Labo-
ratory of Microbiology, The Rockefeller University, New
York, NY 10021, USA
Infectious ovine mastitis is responsible for serious econom-
ic losses in sheep farms, as a result of the decreased milk
production, decline in milk quality and increased demand
for use of drugs and veterinary care. Coagulase-negative
Staphylococci species, in particular S. epidermidis, are the
most frequent causative agents of ovine intramammary
infections. The use of molecular methods for the precise
identi¢cation of the staphylococcal species is of enormous
importance for the recognition of the etiology of infection.
In this study, Internally Transcribed Spacer PCR (ITS-
PCR) was used to identify a collection of 97 staphylococ-
cal isolates collected from milk samples of 88 ewes with
sub-clinical mastitis in 19 herds from EŁvora, Portugal.
Eighty-three isolates were identi¢ed : the majority (47) as
S. epidermidis and the other isolates as S. xylosus (12), S.
warneri (8), S. chromogenes (7), S. simulans (4), S. haemo-
lyticus (3), S. lentus (3) and S. hyicus (1). The remaining 14
isolates could not be identi¢ed by ITS-PCR because their
ampli¢cation patterns di¡ered from those of the staphylo-
coccal control strains used. The ITS-PCR results were
compared with the ones previously obtained by the com-
mercial kit API STAPH (BioMe¤rieux) and 11 isolates were
found to be misidenti¢ed by the kit. ITS-PCR proved to
be a simple method that met our needs for the rapid and
more reliable identi¢cation of large numbers of isolates.
Further characterisation of the S. epidermidis isolates by
molecular typing methods should identify di¡erences in
strain populations between sheep herds.
FEMSLE Congress 2-6-03
P7^25
EFFECTIVENESS OF DIFFERENT DNA EXTRAC-
TION METHOD FOR SALMONELLA ENTERITIDIS
DETECTION IN MEAT BY SYBR-GREEN REAL
TIME PCR
E. Delibato, S. Di Pasquale, D. De Medici, L. Croci and L.
Toti
Food department, Italian National Institute of Health, Viale
Regina Elena 299, 00161 Rome, Italy
The objective of this study was to develop a rapid, repro-
ducible and robust method for detecting Salmonella Enter-
itidis in poultry samples, ¢rst comparing four methods of
DNA extraction and puri¢cation from the pre-enrichment
culture (i.e., boiling, alkaline lysis, nucleospin and Dyna-
beads DNA Direct System I) and then combining the most
e¡ective method with a Real-Time PCR method based on
double-stranded DNA binding dye SYBR Green I using
the ABI Prism1 7700 system. The speci¢city of the reac-
tion was determined by the melting temperature (Tm) of
the amplicon obtained. The experiments were conducted
both on samples of chicken experimentally contaminated
with S. Enteritidis and on commercially available poultry
samples, which were also used for comparisons with the
standard cultural method (i.e., ISO 6579/2001). The com-
parison between the four DNA extraction methods
showed signi¢cant di¡erences for all comparisons, except
when comparing boiling and nucleospin (i.e., the two
methods that produced the lowest threshold cycle). Boiling
was chosen because of its simplicity and rapidity and was
combined with SYBR Green I Real-Time PCR, using
primers SEFA-1 and SEFA-2. The speci¢city of the reac-
tion was con¢rmed by the Tm, which was consistently
speci¢c for the amplicon obtained; the mean peak Tm
obtained with curves speci¢c for S. Enteritidis was
82.56Y0.22. The standard curve constructed using the
mean threshold cycle and various concentrations of S.
Enteritidis (ranging from 103 to 108 cfu/ml) showed a
good linearity (R2=0.9767) and a sensitivity limit of less
than 103 cfu/ml. The results of this study demonstrate that
this SYBR Green I Real-Time PCR constitutes an e¡ec-
tive and easy-to-perform method for detecting S. Enter-
itidis in poultry samples.
 1st FEMS Congress / Posters 103^505
P7^26
UREAPLASMA SPP. DETERMINATION IN WOMEN
ATTENDING A FAMILY PLANNING CLINIC IN
GUINEŁ-BISSAU, USING POLYMERASE CHAIN RE-
ACTION OF THE MULTIPLE-BANDED ANTIGEN
GENE
D. Domingues(1), L. Ta¤vora Tavira(1), A. Duarte(2), A.
Sanca(3), E. Prieto(1), F. Exposto(1)
(1) Unidade de DST/IHMT, Universidade Nova de Lisboa,
Lisboa, Portugal; (2) Departamento de Microbiologia/Fac-
uldade de Farma¤cia/Universidade de Lisboa, Lisboa, Portu-
gal; (3) Centro de Medicina Tropical de Bissau/IHMT,
Bissau, Guine¤-Bissau
Although commonly found in the genital tract of assymp-
tomatic women, it has been suggested that only certain
subgroups of Ureaplasma spp. are disease associated. Vag-
inal specimens were collected for identi¢cation of Urea-
plasma spp. and to estimate a possible association of this
microorganisms with age, absence of lactobacilli, and tet-
racycline resistance. From the 94 women studied, 40 (43%)
carried Ureaplasmas spp. and therefore were biotyped by
PCR. Twenty-nine (73%) strains belonged to Ureaplasma
parvum, 10 (25%) were Ureaplasma urealyticum and one
patient (2.5%) carried both species. Ureaplasma parvum
was the most frequently found in all age groups and seems
to be more frequent in young women in the 20 to 25 years
age group (41%), while Ureaplasma urealyticum was com-
mon in women who had between 30-35 years of age (22%).
In this study, Ureaplasma spp. was not associated with
changes in the vaginal £ora, although the inverse seemed
to be true for Mycoplasma hominis. However, Ureaplasma
urealyticum was more associated with the loss of lactoba-
cilli than Ureaplasma parvum. The number of Ureaplasma
urealyticum strains that presented tetracycline (40%) and
multiple resistance (100%) was higher than that presented
by Ureaplasma parvum strains (27% and 69%, respec-
tively).
P7^27
THE IMPORTANCE OF MOLECULAR TESTING OF
PARVOVIRUS B19 INFECTION IN IMMUNOCOM-
PETENT INDIVIDUALS
D. Duh, M. Petrovec and T. Avsic-Zupanc
Institute of Microbiology and Immunology, Medical Fac-
ulty, Ljubljana, Slovenia
Human parvovirus B19 is associated with a growing spec-
trum of disease manifestations. Infection with parvovirus
B19 is usually a mild self-limiting disease. However, im-
FEMSLE Congress 2-6-03
281
munosuppressed and immunocompromised individuals as
well as pregnant women and their fetuses are at great risk
for serious consequences from parvovirus B19 infection.
Thus, the diagnostic method for detection of parvovirus
B19 must be chosen carefully considering the type of path-
ology and the type of patient. While molecular testing
should be the test of choice when diagnosing parvovirus
B19 infections in patients at great risk, serological testing
should be performed in immunocompetent individuals. In
some cases, viral DNA may persist at low level in the
blood despite the presence of circulating IgG antibody
and therefore virus can not be detected by serological
methods. In our study, 11 immunocompetent patients
with an acute infection were tested by serological and mo-
lecular methods. In consecutive serum samples speci¢c
IgM antibodies were detected up to 3 months. The results
of serologic testing agreed with the results of molecular
methods used, nested and real-time PCR. No viral DNA
was detected after 3 months, when IgM antibodies de-
creased to the undetectable levels. The sensitivity of both
molecular methods used was the same. Real-time PCR
allowed us to detect minimum of 3 copies of viral genome.
Among 11 patients there was 1 individual in which viral
DNA persisted for more than a year yet IgM antibodies
were no longer detectable.
P7^28
CHARACTERISATION OF ANTIBIOTIC RESISTANT
SALMONELLA TYPHIMURIUM STRAINS ISO-
LATED IN THE CZECH REPUBLIC BETWEEN 1984
AND 2002
M. Faldynova(1), M. Pravcova(1), F. Sisak(1), H. Hav-
lickova(1), I. Kolackova(2), A. Cizek(3), R. Karpisko-
va(2) and I. Rychlik(1)
(1) Veterinary Research Institute, Hudcova 70, 621 32
Brno, Czech Republic; (2) National Institute of Public
Health, Center for Food Chain Hygiene, Palackeho 1-3,
612 42 Brno, Czech Republic; (3) University of Veterinary
and Pharmaceutical Sciences, Palackeho 1-3, 612 42 Brno,
Czech Republic
In 1985, a new clone of pentadrug resistant Salmonella
enterica serovar Typhimurium appeared. Since that time
it has spread throughout the world. In this study we fo-
cused on the introduction of this clone in the Czech Re-
public and in the evolution of the resistance to antibiotics
in general. In a collection of 66 strains isolated between
1984 and 2002, genes coding for the antibiotic resistance
were determined using speci¢c PCRs and DNA sequenc-
ing. We found that the pentadrug resistant clone ¢rst ap-
peared in the Czech Republic in 1990. The genetic basis of
antibiotic resistance in strains isolated before this date was
considerably di¡erent from that observed in current
282 1st FEMS Congress / Posters 103^505
strains. Strains isolated in 1984-85 were typical by the
presence of strA, tetB, tetC, cat, TEM and sul2 genes.
Most of these strains were free of integrons. Rarely iso-
lated integron-positive strains from this period were al-
ways of di¡erent genetic composition from recent isolates
i.e. they never coded for aadA2, £oR, tetG and PSE1.
Such genes are on the other hand found in 80% of present
antibiotic resistant strains. In a single strain isolated in
1991 we found a new variant of the aadA gene designated
aadA21, the 5’ end of which was identical with aadA2 and
the 3’ end of which was identical with aadA1. This ¢nding
further con¢rms introduction and mixing of a new bacte-
rial population in the beginning of the 1990’s in the Czech
Republic.
P7^29
THE IDENTIFICATION AND DISCRIMINATION BE-
TWEEN EUROPEAN AND AMERICAN PATHOGEN-
IC STRAINS OF YERSINIA ENTEROCOLITICA BY
PCR-MSSCP TECHNIQUE
R. Gierczynski, M. Jagielski, W. Rastawicki
National Institute of Hygiene, 00-791 Warsaw, Chocimska
24, Poland
The species Yersinia enterocolitica includes human-patho-
genes as well as strains considered non-pathogenic. Hence,
strains of Y. enerocolitica are divided to two phylogenetic
groups called European and American according to re-
gions of their predomination. The European strains are
considered less virulent than American but they often
show the higher resistance to L-lactams. Pathogenic strains
of both groups possessing gene ail, encoding an adhesine
Ail. The nucleotide sequences of ail from European and
American isolates show some stable di¡erences. In this
report we show the simple, sensitive and highly reliable
method for identi¢cation of pathogenic strains of Y. enter-
ocolitica and determination of its phylogenetic group by
the multitemperature single strand conformation polymor-
phism (MSSCP) technique. We compared the single strand
conformation pro¢les of 425 bp fragment of ail gene from
6 strains of Y. entrocolitica O :8 (American) and 32 Euro-
pean strains representing serogroups O:3 (28 strains), O:9
(3), O:5,27 (1). All tested DNA fragments of European
strains revealed identical pattern that was easily distin-
guishable from pattern observed for American strains.
The comparison of the nucleotide sequences of analysed
amplicons of O:8 and O :3 strains indicated the presence
of 15 point mutations. All the test procedure including
PCR, MSSCP and silver staining was ¢nished within 6
hours. If comparing with classic RFLP, the PCR-MSSCP
appears to be more reliable and sensitive but less time and
funds consuming when the large numbers of samples are
analysed.
FEMSLE Congress 2-6-03
P7^30
EPIDEMIOLOGICAL STUDIES OF A SALMONELLA
ENTERICA SEROVAR TYPHIMURIUM OUTBREAK
IN HUMANS AND ANIMALS IN ICELAND
E. Gunnarsson(1), S. Gudmundsdottir(2), H. Hardardot-
tir(3), S. Bjarnadottir(1)
(1) Institute for Experimental Pathology, University of Ice-
land, Keldur, 112 Reykjavik ; (2) Icelandic Fisheries Labo-
ratories, Sku¤lagata 4, 101 Reykjav|¤k; (3) Department of
Microbiology, Landspitali University Hospital, 101 Reykja-
vik, Iceland
The present study deals with the use of Pulsed-Field Gel
Electrophoresis (PFGE) and phage typing (PT) as an epi-
demiological tool in a study of an outbreak of Salmonella
enterica Serovar Typhimurium (S. Typhimurium) in Ice-
land. S. Typhimurium was diagnosed as a cause of death
of two foals on one farm late in September 1999. Two
months later S. Typhimurium was isolated from a dead
cow on a big dairy farm in the same district. In a survey of
all cattle on the farm S.Typhimurium was found in 40 %
of the samples examined. During the next weeks and
months this serovar was isolated in connection with dis-
ease or deaths of horses on three additional farms. S.
Typhimurium was also traced back to one more farm after
isolation in a slaughterhouse. In a survey on those farms
involved S. Typhimurium was found in cattle, horses,
sheep and dogs. On two of the farms some people in the
household got sick. During the winter S. Typhimurium
was also isolated from a poultry farm, horses and a dog
in the Reykjavik area and from horses in the south-west-
ern part of the country. Many isolates of Salmonella from
humans at this time were identi¢ed as S. Typhimurium but
no strains of this serovar were isolated from food in Ice-
land. It was suspected that the transmission was from
animals to humans. The typing revealed that the majority
(84%) of the strains belonged to the same PFGE and
phage type. This outbreak strain might be endemic in Ice-
land.
 1st FEMS Congress / Posters 103^505
P7^31
DYNAMIC MODIFICATION OF MICROORGAN-
ISMS BY PYRENE-BUTANOATE FOR FLUORO-
METRIC DETECTION IN CAPILLARY ELECTROKI-
NETIC TECHNIQUES
M. Horka¤(1), F. Rufiz›ic›ka(2), K. SNlais(1)
(1) Institute of Analytical Chemistry, Academy of Sciences
of the Czech Republic, Vever›¤| 97, 611 42 Brno, Czech Re-
public; (2) Department of Microbiology, Faculty of Med-
icine, Masaryk University Brno, Czech Republic
The capillary electrokinetic techniques such as capillary
zone electrophoresis and capillary isoelectric focusing can
be used to on-line rapidly separate, identify, quantify or
study the mixed bacterial cultures and fungi. The capillary
isoelectric focusing of the low-molecular-mass pI markers
and mixed cultures of microbial populations of Escherichia
coli, Candida albicans, Staphylococcus epidermidis and En-
terococcus faecalis with UV detection were recently shown.
It was possible to quantify bacterial cells according their
peak areas; the minimum detectable number of microbial
cells was 5 x 102 ^ 1 x 103. The infectious doses of patho-
genic bacteria can be very low in order of tens to hundreds
of cells. Therefore, the sensitive and selective £uorescence
detection is needed. Pyrenebutanoate and non-ionogenic
tenside on the basis of pyrenebutanoate as the £uorescent
compounds were used as a bu¡er additives in capillary
zone electrophoresis or capillary isoelectric focusing, re-
spectively, for a dynamic modi¢cation of the microbial
samples. Using the deuterium lamp UV excitation for
the on column £uorometric detection, the minimum de-
tectable amounts of the microorganisms in order of ones
to tens of cells sampled on the separation capillary was
achieved.
P7^32
FTIR MICROSPECTROSCOPY AS A NOVEL METH-
OD FOR DETECTION OF MALIGNANT CELLS IN-
FECTED WITH RETROVIRUSES AND CELLS IN-
FECTED WITH HERPES VIRUSES
M. Huleihel, M. Talyshinsky, Y. Souprun, I. Mukmanov
and V. Erukhimovitch.
The Institute for Applied Biosciences, Ben-Gurion Univer-
sity of the Negev, Beer-Sheva, Israel
In the present study we used microscopic Fourier-Trans-
form Infrared spectroscopy (FTIR) to investigate and to
detect malignant cells which were transformed in culture
by murine sarcoma virus (MuSV) and cells infected with
herpes viruses (herpes simplex 1,2 and varicella viruses).
FEMSLE Congress 2-6-03
283
For this study various primary cells from di¡erent animals
were used for transformation with MuSV and Vero cells
(cell line) were used for infection with herpes viruses. The
advantage of microscopic FTIR spectroscopy over conven-
tional FTIR spectroscopy is that it facilitates inspection of
restricted regions of cell culture or the tissue. Our results
showed signi¢cant and consistent di¡erences between the
various tested normal cells and malignant cells trans-
formed by MuSV and cells infected with herpes viruses.
A considerable decrease in carbohydrates and phosphates
levels was seen in malignant cells compared to the normal
cells. Whereas, a signi¢cant increase in phosphates levels
and a decrease in carbohydrates levels were seen in in-
fected cells with herpes viruses compared to uninfected
cells. In addition, the peak attributed to the PO2- symmet-
ric stretching mode at 1082 cm-1 in normal cells was
shifted signi¢cantly to 1087 in malignant cells, whereas,
in herpes infected Vero cells there was no shift in this
peak. These results in addition to further di¡erences in
the shapes of various bands throughout the spectrum
strongly support the possibility of developing the FTIR
microscopy as diagnostic method for the detection of ma-
lignant and virus infected cells.
P7^33
STUDY OF SPECIFIC BINDING OF BACTERIA AND
ENTEROVIRUSES ONTO THE AFFINE SUB-
SRTRATES BY ATOMIC FORCE MICROSCOPY
T. E. Ignatyuk(1), I. A. Golutvin(1), S. N. Virjasov(2), S.
G. Ignatov(2), N. S. Nasikan(1), I. A. Kabanov(1), A. L.
Suvorov(1)
(1) State Science Center Institute for Theoretical and Ex-
perimental Physics, 25 B. Cheremushkinskaya, Moscow,
117259 Russia ; (2) Science Research Center of Applied
Microbiology, Obolensk, Moscow Region 142279, Russia
Onrush of nanotechnologies has resulted in a large scale
production of scanning probe microscopes (SPM), operat-
ing with nanometer resolution, which in its own turn gave
rise to the tendency for creation of biosensors that can
detect single biological interactions. We used an atomic
force microscope (AFM) to study morphological and im-
mune properties of bacteria (Legionella micdadei, Escheri-
chia coli, Micobacterium tuberculosis) and enteroviruses
(poliovirus, adenovirus, rotavirus), adsorbed on the sur-
face of gold, silicon nitride, glass, mica and graphite.
Both speci¢c and non speci¢c binding to the substrates
was studied. In case of bacteria we used Protein A/ anti-
body complexes to produce a⁄ne substrates. In case of
enteroviruses, amphiphilic polyelectrolites were used to
form Langmuir ¢lms of antibodies which were transferred
onto the solid substrates. Special image processing soft-
ware which a¡ords to extensively automatize biological
284 1st FEMS Congress / Posters 103^505
objects identi¢cation procedure according to their AFM
images was developed. Atomic force microscopy based
method can be used for rapid detection and identi¢cation
enteroviruses and microbiological objects in water quality
assay.
P7^34
MOLECULAR TYPING N. GONORRHOEAE
STRAINS IN RUSSIA
E. N. Ilina, M. V. Malahova, V. A. Vereschagin, V. M.
Govorun, M. M. Zubcov and A. A. Kubanova
Research Institute of Physico-Chemical Medicine and Cen-
tral Research Institute of Dermatology and Venerology,
Moscow, Russia
The ¢rst typing method for N. gonorrhoeae was developed
with monoclonal antibodies in 1984 year. This typing sys-
tem based on the study of structure of gonococcal major
outer membrane protein I (Por I, PI). Peptide mapping
analysis divided N. gonorrhoeae strains into two distinct
groups designated serovar A (PIA) and serovar B (PIB). It
was shown that PIA strains especially associated with dis-
seminating gonococcal infection and PIB strains ^ with
antibiotic resistance to wild specter of drugs. Now the
molecular biology methods using to typing N. gonorrhoeae
strains. During last years gonococcal serovars distribu-
tions were studied in several countries. There are no
data about gonococcal serovars distribution in Russia.
The aim of our study was to use the genetics methods to
typing Russian N. gonorrhoeae clinical strains. The N. gon-
orrhoeae strains were obtained from patients in Moscow
and its region. The resistance to quinolones, macrolides,
tetracycline and cefatoxime were analyzed by serial agar
dilution method using chocolate agar with the selective
additions + antibiotics in various concentrations. We re-
port the typing of 41 clinical strains by protein I gene
sequencing and Opa gene PCR-RFLP analysis. Genotyp-
ing showed there were 11 various groups according Opa-
typing. Among 41 N. gonorrhoeae clinical strains 34 (83%)
believed to PIB serovar and 7 (17%) ^ to PIA serovar. All
resistant strains to £uoroquinolones were found to have
the PIB type, which is associated to multidrug resistant.
FEMSLE Congress 2-6-03
P7^35
SIMULTANEOUS GROWTH AND DETECTION OF
SALMONELLA AND LISTERIA MONOCYTOGENES
B. Jers›ek
Department of Food Science and Technology, Biotechnical
Faculty, University of Ljubljana, Jamnikarjeva 101, SI-1000
Ljubljana, Slovenia
Salmonella spp. belong to important food contaminating
pathogenic bacteria causing a high number of human in-
fection. Listeria spp. are widely distributed in nature and
are frequently isolated from foods. Among Listeria spp. L.
monocytogenes has been recognised as the most threaten-
ing human pathogen. Traditional culture detection meth-
ods take 4 ^ 6 days to obtain results, are time consuming
and laborious. The objective of the present study was to
detect Salmonella and L. monocytogenes simultaneously.
As the best broth for growth of both bacteria universal
enrichment broth (UPB) was chosen. It was incubated at
least 24 h at 35‡ C. There was no di¡erence in growth of
Salmonella strain when incubated alone or with L. mono-
cytogenes strain, but L. monocytogenes strain grew at slow-
er rate in the presence or absence of Salmonella strain. As
simultaneous detection method for Salmonella and L.
monocytogenes multiplex PCR was used. Two sets of pre-
viously published speci¢c primers were combined in one
reaction. DNAs of Salmonella and L. monocytogenes were
prepared together. Crude cell bacterial lysate were pre-
pared by mixing bacterial cultures after growth in UPB
and lysis bu¡er containing NaOH and SDS and subse-
quent heating of samples at 94‡ C for 15 min. The lowest
detection level for both bacteria was 104 cfu/ml in the
original culture media or 500 bacterial cells in the PCR
reaction. The combination of simultaneous growth of Sal-
monella and L. monocytogenes and simultaneous detection
of these bacteria with PCR prove to be sensitive and time-
e⁄cient and therefore promising for further development
of detection method.
P7^36
INFECTION WITH CHLAMYDIA TRACHOMATIS
AMONG FEMALES IN GREECE
K. Avdeliodi, M. Simou, E. Glynou, and H. Kada
Laboratory of Microbiology, Elena Venizelou Hospital, 2 E.
Venizelou Sq., Athens 115 21, Greece
The objective of this study was to determine the frequency
of infection with Chlamydia trachomatis among female
outpatients attended at a Greek Hospital during a 6-year
period. Materials and methods: During a 6-year period (1/
 1st FEMS Congress / Posters 103^505
1/1996 ^ 31/12/2001) specimens from 7.366 consenting out-
patients examined at the Department of Obstetrics and
Gynaecology were submitted for testing for C. trachoma-
tis. All consecutive specimens were included in the study.
Diagnosis of infection was established by a molecular
commercial method, the Ligase Chain Reaction (LCR,
Abbott) according to the instructions of the manufacturer.
A questionnaire was developed for the collection of pa-
tient demographical data. Results : Among the total group
of patients, there were 6.876 women of Greek nationality
(93%) and 488 foreigners (7%). There were 3.315 sympto-
matic (45%) and 4.051 asymptomatic patients (55%), who
were referred for some other reason. Overall, 266 out of
7.366 women were positive by LCR (infection incidence:
3.6%). Among the 266 C. trachomatis infected women 202
were Greek (infection incidence among Greek patients
2.9%) and 64 foreigners (infection incidence among for-
eigners patients 13.1%) (p 6 0,00001). Only 122 women
out of 266 C. trachomatis infected patients were sympto-
matic (46%). Conclusions : This is the ¢rst report on the
incidence of infection with C. trachomatis among patients
in Greece as detected by a molecular method. An overall
incidence of 3.6% was detected, however infection was
more prevalent among foreign patients (13.1%)
(p 6 0,00001).
P7^37
BACTERIAL IDENTIFICATION AND FINGERPRINT-
ING BASED ON SIMPLE SEQUENCE REPEATS
(SSR)
Y. Kashi, L. Somer, E. Diamant, R. Gur-Arie, Y. Danin-
Poleg and Y. Palti.
Department of Food Engineering and Biotechnology and
Grand Water Research Institute, Technion, Haifa 32000,
Israel
Analysis of the complete genomic DNA sequences of pro-
karyotes showed the existence of tens of thousands well
distributed Simple Sequence Repeat (SSR) tracts, with
core motifs ranging from 1-6 nucleotides. DNA sequences
at arbitrarily chosen SSR tracts were compared among
strains of E. coli and Listeria. Polymorphisms of SSR
copy number were observed at mononucleotide SSR tracts
screened, with all polymorphisms occurring in non-coding
regions. The identi¢ed SSR polymorphism could prove
important as a genome-wide source of variation, both
for practical applications (including rapid detection, strain
identi¢cation) and for evolutionary adaptation of mi-
crobes. SSRs loci in the non-coding portion of microbial
genomes are located in areas near gene regulatory ele-
ments, supporting hypothesis that SSR polymorphisms
may be a part of a ¢ne-tuning mechanism in molecular
processes that e¡ect gene regulation. The ¢nding that SSR
FEMSLE Congress 2-6-03
285
tracts are hyper-variable in bacterial genomes, but stable
at the strain level, enables the use of SSR for bacterial
strains identi¢cation, including discrimination of patho-
genic and non-pathogenic strains. The SSR based ¢nger-
printing method identi¢es bacterial strains, assigns an
identity number to bacterial strain, and can be developed
to a rapid high-throughput typing technology.
P7^38
RETROSPECTIVE PCR ANALYSIS OF VIBRIO
CHOLERAE EL TOR STRAINS ISOLATED DURING
DIFFERENT EPIDEMIC SITUATIONS IN TURKME-
NISTAN
E. A. Kostromitina, N. B. Cheldyshova, N. I. Smirnova and
V. V. Kutyrev
Russian Anti-Plague Research Institute ‘‘Microbe’’, Univer-
sitetskaya Str. 46, 410005, Saratov, Russia
PCR is important in the diagnosis of dangerous infections
including cholera. PCR makes it possible not only to iden-
tify the infectious agent early, but also to retrospectively
analyse the data enabling the epidemiologic situation to be
prognosticated. The aim of this work was to identify genes
associated with virulence (ctxA, tcpA, toxR, zot, ace, rtxA,
attRS1) in the genome of 123 V. cholerae E l Tor strains
isolated in Turkmenistan in the 70-80’s from people and
the environment using PCR. The complete set of the main
virulence genes (ctxA+ tcpA+ toxR+ ) was present only in
strains isolated during cholera epidemics from clinical
cases or carriers as well as from the environment. 77%
of the interepidemic environmental isolates lacked the
genes for both the cholera toxin and toxin-coregulated
piles. About 43% of vibrio carriers in Turkmenistan were
also shown to be infected with non-toxigenic variants that
contained the tcpA genes whose product provides for e⁄-
cient small-intestinal colonization. Such strains inhabited
the surrounding medium, too (18%). Some V. cholerae El
Tor strains whose genotype was ctxA- ace+ zot+ were
found to carry CTX phi bacteriophage with an incomplete
set of virulence genes. Gene rtxA encoding the main sub-
unit of the RTX toxin capable of causing gastroenteritis,
was present in the chromosome of 92% of isolates ana-
lysed. In fact, all ctxA- tcpA+ strains were also shown to
carry a genomic site, attRS1, indicative of their possible
infection with CTX phi bacteriophage. Thus, our data
may suggest that under the environmental conditions in
Turkmenistan in the 70-80’s, toxigenic strains could orig-
inate from non-toxinogenic ones, because a large portion
of the El Tor cholera vibrio population living in vibrio
carriers and in open-water reservoirs during the interepi-
demiologic period contained not only the tcpA genes en-
coding the receptor for CTX phi bacteriophage, but also a
genomic attRS1-site necessary for bacteriophage invasion
286 1st FEMS Congress / Posters 103^505
into the chromosome resulting in the formation of stable
lysogens.
P7^39
A STUDY OF THE POPULATION STRUCTURE OF
NONTOXIGENIC VIBRIO CHOLERAE O1 ISO-
LATED FROM THE IMMUNOCOMPROMISED PA-
TIENT
E. A. Kostromitina, N. I. Smirnova
Russian Anti-Plague Research Institute ‘‘Microbe’’, Univer-
sitetskaya Str. 46, 410005, Saratov, Russia
A single case of cholera is described in a 69-year old-man,
su¡ering from hormonal bronchial asthma and chronic
hypoacidic gastritis for a long time. A Vibrio cholerae
O1 El Tor Inaba serotype strain with typical morpholog-
ical, cultural and biochemical properties with low viru-
lence for suckling-rabbits and epidemically safe according
to epidemiological observations was isolated from the pa-
tient. Despite the typical clinical symptoms (more than 30
liters liquid loss), the chromosome of the isolate was neg-
ative by PCR for cholera toxin gene (ctxA), responsible
for severe watery diarrhea, and for toxin-coregulated pilus
gene (tcpA), encoding critical colonization factor. It was
positive for essential regulatory gene toxR and gene of
soluble hemagglutinin/protease (hapA). To understand
the nature of virulence of this strain we investigated the
structure of its population. The study of 2000 clones of V.
cholerae El Tor strain revealed the uniformity of its pop-
ulation in production of some pathogenic enzymes : all the
clones expressed hemolysin, phospholipase and hemagglu-
tinin / protease activity. PCR assay of 30 random clones
demonstrated the absence of ctxA (that was con¢rmed by
DNA-hybridization with CT-probe) and of tcpA, as well
as of zot and ace, encoding additional toxins and situated
on CTX-genetic element along with genes ctxAB, and ad-
ditional regulatory gene toxT. All clones were positive for
rtxA, forming a part of a RTX-genetic element which en-
codes biosynthesis of some enterotoxin able to cause weak
diarrhea, and were negative for the NAG-speci¢c st gene,
encoding heart-stable toxin. We came to the following
conclusions : (i) the examined population of V. cholerae
El Tor is homogeneous in structure and does not contain
ctxA and tcpA genes con¢rming its epidemiological safety;
(ii) the severe gastroenteritis in the patient was probably
caused by other toxins (RTX-toxin, hemolysin, as well as
Sanyal toxin) on the background of the obvious decrease
of host antibacterial resistance resulting from hypoacidity
and immunosuppression, caused by steroid hormones giv-
en to the patient over a long period.
FEMSLE Congress 2-6-03
P7^40
A THREE-DAYS PCR-BASED METHOD FOR THE
DETECTION OF LISTERIA MONOCYTOGENES IN
FOOD
E. Kacl|¤kova¤(1), D. Pangallo(1), H. Drahovska¤(2), K.
Oravcova¤(1), T. Kuchta(1)
(1) Department of Microbiology and Chemistry, Food Re-
search Institute, Priemyselna¤ 4, P.O. Box 25, SK-82475
Bratislava 26, Slovakia ; (2) Department of Molecular Bi-
ology, Faculty of Natural Sciences, Comenius University,
Mlynska¤ dolina B-2, SK-84215 Bratislava, Slovakia
A three-days PCR-based method is presented for the de-
tection of Listeria monocytogenes in food. The method is
equivalent to EN ISO 11290-1 or ISO 10560 in terms of
the same detection limit of 100 cfu per 25 g or 10 g and
100 % relative accuracy. The version alternative to EN
ISO 11290-1 begins with a primary enrichment in half
Fraser broth (24 h), secondary enrichment in Fraser broth
(24 h) and post-enrichment in Brain heart infusion broth
(5 h). The version alternative to ISO 10560 begins with
enrichment in Listeria enrichment broth (48 h) and post-
enrichment in Tryptone soya broth (5 h). These steps are
followed by bacterial cell lysis by boiling, PCR oriented to
inlB gene using a mimic internal control, and agarose gel
electrophoresis. At the evaluation of the method on model
food samples arti¢cially contaminated with decimal dilu-
tions of a L. monocytogenes culture (cheese, smoked ¢sh,
ready-to-eat meat products; 21 samples altogether), a de-
tection limit of 100 cfu per 25 g or 10 g was determined.
Dead L. monocytogenes cells did not cause false positivity,
as determined using model food samples arti¢cially con-
taminated with decimal dilutions of dead L. monocyto-
genes cells. At the evaluation of the method on naturally
contaminated food samples (same as above, 140 samples
altogether) identical results (8 positives) as with the refer-
ence method were obtained.
P7^41
MULTIPLEX PCR ANALYSIS FOR SIMULTANEOUS
DETECTION OF GENERA CARNOBACTERIUM
AND LEUCONOSTOC
M. C. Macian(1,2), E. Chenoll(2), R. Aznar(1,2)
(1) Department of Microbiology and Ecology, University of
Valencia ; (2) Institute of Agrochemistry and Food Tech-
nology (IATA), Spanish Council for Scienti¢c Research
(CSIC), P. O. Box 73, Burjassot E-46100, Valencia, Spain
Members of genera Carnobacterium and Leuconostoc have
been reported during last years as potential food spoilers,
 1st FEMS Congress / Posters 103^505
especially of vacuum-packed meat products. Several au-
thors focused their interest on developing rapid and reli-
able methods for detection of both LAB genera, and sub-
sequently several genus-speci¢c primers for PCR detection
are available in the literature. However, as a result of
taxonomic rearrangements new species have been de-
scribed in both genera. In the present work we have eval-
uated the speci¢city of the reported PCR primers and have
designed new genus- , group- and species-speci¢c primers
in order to accommodate speci¢city to the new taxonomic
situation. As a result a Multiplex-PCR assay has been
developed that allow simultaneous detection of members
of both genera as well as members of the non-motile Car-
nobacterium species: C. divergens, C. maltaromicum and C.
gallinarum, that are the most frequently isolated as poten-
tial food spoilers. Species-speci¢c identi¢cation is also pos-
sible by applying another Multiplex-PCR reaction. Both
PCR procedures have been tested using reference strains
as template and have proved useful for colony identi¢ca-
tion of isolates (grown on MRS or Blood-Agar plates). In
addition, they could be successfully applied for PCR de-
tection of genus Carnobacterium and Leuconostoc, and
non-motile Carnobacterium species, in arti¢cially inoculat-
ed as well as naturally contaminated vacuum-packed meat
products.
P7^42
CORRELATION BETWEEN THE PLASMID PRES-
ENCE AND THE RESISTANCE TOWARDS BETA-
LACTAMS IN KLINICAL ISOLATES OF KLEBSIEL-
LA SPECIES
I. H.-P. Meloska, G. Jankoska, G. Mircevska, M. Petrov-
ska
Institute for Microbiology and Parasitology, Medical Fac-
ulty, Vodnjanska 17, Skopje, Macedonia
The increasing use of extended spectrum beta-lactams in
the last decade has been associated with the emergence of
plasmid spread of resistant bacterial strains. The aim of
the present study was to determine, if the resistance of our
hospital isolates of Klebsiella species towards beta lactams
is correlated with their plasmid content. This study in-
cluded a total of 60 (45 K pneumoniae and 15 K oxytoca)
strains, originating from in-patients in Surgical Clinics in
Skopje and isolated from respiratory samples, wound
swabs and drains. The susceptibility testing was preformed
with disc di¡usion method and VITEK (bioMerieux,
France) automated technique. The plasmid content was
determined by thermal-alkal lyses method, as described
by Kado and Liu followed by agarose gel electrophoresis.
The resistance of Klebsiella spp was the following: amox-
icillin clavulanic acid (68.3%), piperacillin (100.0%), piper-
acillin tazobactam (78.3 %), ceftazidime (83.3%), ceftriax-
FEMSLE Congress 2-6-03
287
one (90.0%), cefotaxime (85.0%) and ce¢pime (41,7%).
Plasmids sized : 55kb, 65kb, 75kb, (50 & 80kb) and (55
& 85kb), were isolated from 53.3% of examined Klebsiella
spp strains. The presence of plasmids was signi¢cantly
higher (84.4% and 96.9%) in strains resistant to penicillins
and third generation cefalosporins (p 6 0.0000). However,
out of 25 strains resistant to ce¢pime, 18 (72.0%) had
plasmids. The plasmids analyses showed that all Klebsiella
spp strains with one 65kb plasmid were resistant to pen-
icillins and to cephalosporins. Only two strains without
plasmids were resistant to all four examined cephalospor-
ines. The predominant isolation of plasmids from strains
resistant to beta-lactam antibiotics suggests the plasmid
origin of this resistance.
P7^43
ARE THE ONCOGENIC HUMAN PAPILLOMA VI-
RUS TYPES INCLUDED IN CERVICAL HPV INFEC-
TION OF WOMEN IN MONTENEGRO?
G. Mijovic(1,2), N. Jokmanovic(3), Z. Zekovic(1), M.
Bujko(1,2)
(1) Institute of Public Health, (2) School of Medicine,
University of Montenegro, and (3) Clinical center of Mon-
tenegro, Podgorica, Montenegro
Human papillomaviruses (HPV) are small DNA viruses
associated with epithelial lesions ranging from certain be-
nign proliferative lesions to invasive carcinomas. More
than 100 HPV types were identi¢ed, but some of them
have certain oncogenic potential, and are associated with
cervical neoplasia. The aim of this study was to investigate
if the oncogenic HPV types are included in cervical HPV
infection of women in Montenegro. HPV infection was
diagnosed by using the method of hybridization in situ,
and 115 cervical swabs of women (aged 17- 62 years)
who visited Gynecologic Ambulance of Clinical Center
in Podgorica from April 2000 to November 2001 were
examined. Cervical samples of 85 women with clinical
signs of cervicitis and 30 cervical swabs of women without
any clinical signs of cervical disorder were tested. Screen-
ing test was positive in 51 cases (44,3 %). HPV typing was
done in 41 samples and the results were positive for: HPV
type 6/11 in 18 (35,3%), type 16/18 in 21 (51,2%), types 31/
33/35/51 in 23 (45,1%). 30% of women without clinical
signs of cervical disorder were HPV positive. The results
of this investigation showed that among women with cer-
vical HPV infection, oncogenic HPV 16/18, and HPV 31/
33/51 types were present in 51% and 45% cases, respec-
tively. These results indicated that HPV infection, espe-
cially caused by oncogenic types, represent a signi¢cant
public health concern in Montenegro.
288 1st FEMS Congress / Posters 103^505
P7^44
COMPARISON OF THREE PCR-BASED METHODS
FOR THE DETECTION OF LISTERIA MONOCYTO-
GENES IN FOOD
E. Kacl|¤kova¤, K. Oravcova¤, T. Kuchta
Department of Microbiology and Chemistry, Food Research
Institute, Priemyselna¤ 4, P.O. Box 25, SK-82475 Bratislava
26, Slovakia
Following PCR-based methods for the detection of Liste-
ria monocytogenes in food were compared: 1. BAX for
Screening L. monocytogenes (Cat. No. 1771 0605 ; Quali-
con, Wilmington, DE, USA), 2. Probelia L. monocyto-
genes (Ampli¢cation Kit Cat. No. 357 8004 and Detection
Kit Cat. No. 357 8005; Bio-Rad, Marnes-la-Coquette,
France), 3. three-days PCR-based method of Kacl|¤kova¤
et al. presented elsewhere in this Congress. Method 1 fea-
tures all PCR reagents supplied in a tablet, the ampli¢ca-
tion product is detected by gel electrophoresis and a 2-
days enrichment is required prior to PCR. Method 2 fea-
tures PCR reagents in a stabilized everything-containing
mastermix, the ampli¢cation product is detected by hy-
bridization and subsequent ELISA and a 2-days (1-day
for non-meat samples) enrichment is required prior to
PCR. Method 3 is a cheap protocol requiring pipetting
all reagents, the ampli¢cation product is detected by gel
electrophoresis and a 2-days enrichment is required prior
to PCR. All the three methods employ an internal ampli-
¢cation control. The methods were evaluated at the anal-
ysis of model food samples arti¢cially contaminated with
L. monocytogenes (4 types of cheese, 3 types of meat prod-
ucts, 1 type of smoked ¢sh and 1 type of frozen vegetable
salad) along with the standard method for the detection of
L. monocytogenes in food, EN ISO 11290-1. All the three
PCR-based methods had the same detection limit as the
standard method, 100 cfu / sample. However, methods 1
and 2 produced false positive results for samples contam-
inated with dead L. monocytogenes cells.
FEMSLE Congress 2-6-03
P7^45
DIAGNOSTICS OF DIARRHEAGENIC ESCHERI-
CHIA COLI BY USE OF MULTIPLEX-PCR DI-
RECTED TOWARDS CHARACTERISTIC VIRU-
LENCE GENES
S. Persson, K. E. Olsen, F. Scheutz, J. Sonne-Hansen, K. A.
Krogfelt, V. Fussing and P. Gerner-Smidt
Dept. of Gastrointestinal Infections, Statens Serum Institut,
Artillerivej 5, 2300S Copenhagen, Denmark
Diagnostics of diarrheagenic E. coli groups have tradition-
ally been based on culturing faecal samples, followed by
biochemical characterization, DNA hybridisation, serotyp-
ing and/or detection of toxins. Many reports have all
ready shown the potential of PCR as a diagnostic tool,
by directing primer towards the virulence genes that char-
acterise these pathogens. However, most of these methods,
are not taking the routine diagnostic problems of good
sensitivity limits and proper positive controls into account.
The present method relies on multiplex-PCR on a simple
DNA preparation from plate grown cells, followed by
PCR product detection by gel electrophoresis. This was
done by designing one multiplex-PCR containing 6 genes
necessary for classifying the E. coli groups: ETEC, VTEC,
EPEC and EIEC, where each amplicon had a distinguish-
able size. The assay was also designed to contain a primer-
set directed towards a universal 16S DNA sequence, serv-
ing as a positive control for the PCR. By this assay we
were able to detect as little as 1 pathogenic colony in a
bacterial mixture of 104 commensal colonies on the plate.
Compared to a probe-hybridisation method previously
used in our laboratory, the PCR assay showed superior
sensitivity, when both methods were applied on the same
425 clinical samples. Compared to culturing the faecal
samples, PCR on template DNA puri¢ed directly from
faeces is an attractive strategy. This would result in a
faster analysis, which is not a¡ected by the selectivity of
in vitro growth, and should be able to detect dead cells.
The performance of a number of commercial DNA puri-
¢cation kits will be compared.
 1st FEMS Congress / Posters 103^505
P7^46
KINETIC PARAMETERS OF IMMOBILIZED DEOX-
YRIBONUCLEASE I ON CIM0 MONOLITHIC SUP-
PORT
N. Berginc(1), K. Benc›ina(2), M. Benc›ina(1), A. SNtran-
car(2), A. Podgornik(2)
(1) Laboratory of Biotechnology, National Institute of
Chemistry, Hajdrihova 19, P.O.B. 600, SI-1001 Ljubljana,
Slovenia; (2) Bia separations d.o.o., Teslova 30, SI-1001
Ljubljana, Slovenia
Traces of DNA in RNA samples present impurities that
could a¡ect results of mRNA quanti¢cation and cDNA
synthesis. In most cases, the DNA impurities in RNA
samples are removed using enzyme deoxyribonuclease
(DNase), which speci¢cally breaks down DNA impurities.
In order to avoid the addition of DNase into the analyzing
sample, the use of immobilized DNase on solid support is
recommended. Because of the size of DNA however, very
few supports available to the matrix enable e⁄cient inter-
action between immobilized enzyme and DNA. Further-
more, since DNA mobility inside the closed pores of the
supports is extremely slow the entire removal of DNA is
extremely long and consequently very ine⁄cient. In recent
years a new group of support named monoliths was intro-
duced. They consist of a single piece of highly porous
material. Pores are interconnected forming a network of
channels through which the liquid is pumped. Because of
enhanced exchange between mobile and stationary phase
separation and bioconversion processes are signi¢cantly
accelerated. Therefore also the e⁄ciency of DNA removal
might be competitive to the degradation with free enzyme.
In this work we immobilized DNase on CIM monoliths.
The kinetic parameters of immobilized DNase were deter-
mined compared with kinetic parameters of free DNase.
The use of immobilized DNase in real samples was tested.
FEMSLE Congress 2-6-03
289
P7^47
NUCLEIC ACID SEQUENCE-BASED AMPLIFICA-
TION (NASBA) DETECTS DNA IN MYCOBACTE-
RIUM AVIUM SUBSP. PARATUBERCULOSIS
D. Rodriguez-Lazaro(1), J. Lloyd(2), M. Pla(1), I. Iko-
nomopoulos(3) and N. Cook(2)
(1) Institute of Food and Agricultural Technology (IN-
TEA). University of Girona, Campus Montilivi s/n. E-
17071 Girona, Spain; (2) Central Science Laboratory
(CSL), Sand Hutton, York, YO41 1LZ, UK; (3) Depart-
ment of Histology-Embryology, School of Medicine, Uni-
versity of Athens, Athens, Greece
Mycobacterium avium subsp. paratuberculosis (MAP) is the
etiological agent of Johne’s disease in ruminants, and has
been implicated as the agent of Crohn’s in humans. Three
nucleic acid sequence-based ampli¢cation (NASBA) assays
were developed for MAP, based on di¡erent target se-
quences. NASBA has been previously shown to selectively
mediate the detection of RNA in microbial cells. Nucleic
acids were extracted from MAP and Salmonella enterica
serotype Typhimurium, and subjected to four enzymatic
treatments prior to ampli¢cation, to determine the nature
of the template nucleic acid which was ampli¢ed. These
enzymatic treatments were DNase, RNase, S1 nuclease,
and RNase / S1 nuclease. With each assay, the latter three
treatments had no e¡ect on the MAP NASBA signal,
whereas DNase treatment abolished it. As expected, the
converse was observed with the S. Typhimurium signal.
Thus, whereas NASBA selectively ampli¢ed RNA in S.
Typhimurium, it ampli¢ed DNA in Mycobacterium avium
subsp. paratuberculosis.
P7^48
DETECTION OF ENDOTOXIN USING GAS-LIQUID
CHROMATOGRAPHY CONNECTED WITH MASS
SPECTROMETRY (GLC/MS)
J. Rybka, A. Gamian
Institute of Immunology and Experimental Therapy, Weigla
12, 53-114 Wroclaw, Poland
LPS (endotoxin) is a major component of the outer mem-
brane of Gram-negative bacterial cell. The LPS molecule
consists of three structurally di¡erent regions: lipid A,
core oligosaccharide and O-antigenic polysaccharide.
LPS possess immunostimulatory and toxic properties to
human organism even in trace amounts. Detection and
quantitation of endotoxin in biological £uids, environment
or biotechnological products is a matter of great impor-
tance for medicine as well as for the industry. Nowadays
290 1st FEMS Congress / Posters 103^505
used endotoxin quantitation methods: biological rabbit
pyrogen test and Limulus Amebocyte Lysate (LAL) test
are indirect and expensive methods with several limita-
tions. There is no valid diagnostic method for direct de-
tection of endotoxin in body £uids for example in blood or
blood serum so far. Sepsis ^ development of bacterial in-
fection in blood ^ and septic (endotoxic) shock are ones of
the most important medical problems leading frequently
to the death of the patient. Despite intensive investigations
there is no reliable procedure of diagnosis and monitoring
of septic shock. Elaboration of a simple and reliable meth-
od of chemical endotoxin detection and its quantitation
would be very helpful in sepsis and septic shock treatment.
In the present work we propose a detection of endotoxin
by one of its integral component : 2-keto-3-deoxyoctulo-
sonic acid (Kdo). Kdo is one of the components, inherent
of the inner part of the LPS core region; up to three Kdo
residues form the Kdo region of the lipopolisaccharide
molecule. Kdo, after chemical derivatization, is detected
as a chemical marker of endotoxin using gas-liquid chro-
matography/mass spectrometry analysis (GLC/MS). The
detected Kdo derivative is acetylated methyl ester of
Kdo methyl glycoside.
P7^49
APPLICATION OF MOLECULAR METHODS TO
THE EPIDEMIOLOGY OF DRUG-RESISTANT TU-
BERCULOSIS IN POLAND
A. Sajduda(1), A. Dela(2), J. Dziadek(2), E. Augustyno-
wicz-Kopec¤(3), and F. Portaels(4)
(1) University of Jo¤dz¤, Jo¤dz¤, Poland ; (2) Centre for Mi-
crobiology and Virology, Polish Academy of Sciences, Jo¤dz¤,
Poland; (3) National Research Institute of Tuberculosis
and Lung Diseases, Warsaw, Poland ; (4) Institute of Trop-
ical Medicine, Antwerp, Belgium
The aim of this study was to investigate drug-resistant M.
tuberculosis isolates from tuberculosis patients in Poland
by molecular epidemiological methods. A total of 268
drug-resistant M. tuberculosis isolates from 251 patients
were analyzed by IS6110 restriction fragment length poly-
morphism (RFLP) and spoligotyping. The generated
IS6110 ¢ngerprint patterns were highly variable. The num-
ber of IS6110 copies per isolate varied from 5 to 20. 203 of
the 251 strains (81%) contained 6 to 11 IS6110 copies, with
a mean of 10 bands. 203 distinct IS6110 ¢ngerprint pat-
terns were observed in the 251 isolates analysed, of which
179 patterns were observed only once each, whereas 24
were shared by two or more isolates. Some strains with
identical IS6110 patterns were identi¢ed in patients of the
same family or patients living in the same area, indicating
active transmission. 2 clusters, consisting of 2 and 3 iden-
tical strains, respectively, were isolated both from Polish
FEMSLE Congress 2-6-03
and foreign-born patients. The 3 members of this latter
cluster exhibited an IS6110 RFLP pattern characteristic
for Beijing genotype family. Altogether, 7 strains (2.8%)
of Beijing genotype were found. All the isolates were sub-
ject to spoligotyping analysis and the results compared
with those obtained by IS6110-associated RFLP. A total
of 96 spoligotypes were identi¢ed in the 251 isolates. There
were 20 spoligotypes identi¢ed among 72 isolates assigned
to 24 IS6110 clusters. In addition, among 179 isolates with
unique IS6110 pro¢les, 88 spoligotypes were identi¢ed.
Twelve spoligotypes were shared by clustered and unique
isolates. By spoligotype alone, 183 isolates were divided
into 33 spoligotypes, whereas 63 isolates showed unique
spoligopatterns. The results obtained in this study indicate
that transmission of drug-resistant M. tuberculosis strains
contributes to the emergence of drug-resistant tuberculosis
in Poland, including members of the Beijing genotype fam-
ily.
This work was supported by grant from the State Com-
mittee for Scienti¢c Research (KBN, contract no. PBZ030-
15). A. S. was supported by FEMS Fellowship in the In-
stitute of Tropical Medicine, Antwerp, Belgium
P7^50
CLONALITY OF GROUP A STREPTOCOCCI FROM
CARRIAGE
I. Santos-Sanches(1,2), G. Ribeiro(1,2), and D. Rolo(1,2)
(1) Centro de Recursos Microbiolo¤gicos, Faculdade de
Cie“ncias e Tecnologia (FCT/UNL), Quinta da Torre,
2829-516 Monte da Caparica, Portugal; (2) Laborato¤rio
de Patoge¤nese Microbiana, Instituto de Tecnologia Qu|¤mica
e Biolo¤gica Rua da Quinta Grande, 6, Apartado 127, 2780-
156 Oeiras, Portugal
Group A streptococci (GAS) are responsible for a multi-
plicity of human diseases. In many countries, macrolides
are extensively used in the empirical treatment of pharyng-
itis and other upper respiratory track infections, contrib-
uting to the decreased susceptibility against these antimi-
crobials. There is no evidence of the role of asymptomatic
carriers in the dissemination of drug-resistant GAS in Por-
tugal. The aim of this study was the recognition of drug-
resistant GAS clones capable of colonizing unrelated pop-
ulations from di¡erent settings in the district of Lisbon.
Antimicrobial resistance was determined by disc di¡usion.
Macrolides resistance phenotypes: M and MLSB types
were de¢ned by a triple-disc test with erythromycin, clin-
damycin and josamycin. Clonality of drug-resistant strains
was assessed by pulsed-¢eld gel electrophoresis (PFGE)
using SmaI and S¢I restriction enzymes. In 2000-2001,
2,066 oropharyngeal swabs were taken from children at
day care centers, elementary schools students, personnel
and family members. A total of 218 GAS were isolated
 1st FEMS Congress / Posters 103^505
and 32 were resistant to antimicrobials (15%). Resistance
to macrolides was 66% (21/32) and M-type was prevalent
(16/21). Resistance to tetracycline (Te) was 34% (11/32).
The M-type isolates were uncut by SmaI but clustered into
four S¢-I PFGE patterns with one comprising 13 isolates.
MLSB-type strains were genotipically unrelated. Strains
resistant to Te were grouped in ¢ve patterns with SmaI
and six with S¢I. Two major disseminated clones were
recognized, one resistant to macrolides (M-type) and the
other resistant to Te. Further comparison with disease-
causing isolates will contribute to a better understanding
of the molecular epidemiology of GAS.
P7^51
RESTRICTION ANALYSIS OF CONSERVATIVE RE-
GIONS IN THE GENOME OF ACETOBACTER SPP.
M. Kretova(1), P. Siekel(2), J. Grones(1)
(1) Department of Molecular Biology, Faculty of Natural
Sciences, Comenius University, Mlynska¤ dolina B-2, SK-
84215 Bratislava, Slovakia; (2) Food Research Institute,
Priemyselna¤ 4, P.O. Box 25, SK-82475 Bratislava 26, Slo-
vakia
Acetic bacteria are ubiquitous microorganisms which have
found use in food and pharmaceutical technologies be-
cause they can be cultured at di⁄cult conditions, at low
pH, and are able to produce organic acids from ethanol,
mannose, xylose and other saccharides. Phenotypic identi-
¢cation of acetic bacteria is di⁄cult, unprecise and time-
consuming. One of the reasons is a high frequency of
spontaneous mutations and the presence of insertion ele-
ments plays a role as well. An improvement in identi¢ca-
tion of Acetobacter spp. is achieved by the use of DNA-
based methods. In this study, regions of 23S rRNA which
are conserved within individual genera as well as 16S-23S
rRNA spacer regions were ampli¢ed by PCR. Di¡erences
in the primary structure of 23S rRNA were observed for
Acetobacter pasteurianus 3614, A. pasteurianus 3612,
A. pasteurianus 2374 a A. aceti 3620, which cannot be
distinguished by microbiological identi¢cation methods.
For a more detailed identi¢cation of individual species,
a fragment of the 16S-23S rRNA spacer region was cloned
in a pBSSK+ plasmid and analysed by restriction using
endonucleases HaeII and HpaII. By this method, di¡erent
restriction pro¢les were obtained for the tested strains.
Primary structures of the 16S-23S rRNA spacer region
of selected Acetobacter species were compared with previ-
ously published data in DNA databases.
FEMSLE Congress 2-6-03
291
P7^52
PEPTIDE NUCLEIC ACID-MEDIATED PCR CLAMP-
ING AS A MOLECULAR METHOD FOR DIFFEREN-
TIATION OF tox-GENE OF TOXIGENIC FROM THE
‘tox-GENE’ OF NON-TOXIGENIC CORYNEBACTE-
RIUM DIPHTHERIAE
N. V. Volozhantsev, V. A. Bannov and K. I. Volkovoi
State Research Center for Applied Microbiology, 142279,
Obolensk, Moscow Region, Russia
In nature, both toxigenic and non-toxigenic strains of Cor-
ynebacterium diphtheriae exist. Toxigenic strains bear pro-
page containing tox-gene. This gene expresses diphtheria
toxin, which contributes essentially to pathogenesis of
diphtheria. Non-toxigenic strains mostly do not contain
tox-gene and do not cause clinical diphtheria. During epi-
demic rise in the 1990s in Russia, non-toxigenic tox-gene
bearing (NTTB) C. diphtheriae strains were often isolated.
In most of NTTB strains, two mutations in the tox-gene
were detected. One of them (one nucleotide deletion in
region 52-55) results in the shift of the DNA open reading
frame due to which toxin formation is not observed.
NTTB strains can hardly play an epidemiologically signi¢-
cant role in diphtheria. Nevertheless, the probable phage
conversion along with DNA recombination suggests the
possibility of ‘‘silent’’ tox-gene recovery in these strains.
Therefore, monitoring of such strains is important. To
provide rapid identi¢cation of the NTTB strains and to
di¡erentiate them from toxigenic strains, peptide-nucleic
acid (PNA)-mediated PCR clamping was used. Oligonu-
cleotide primers for tox-gene fragment ampli¢cation and
PNA molecule complementary to the nucleotide sequences
located between the PCR primers were constructed. PNA
was bound to complementary sequences of native tox-
gene, thus blocking PCR, and was not bound to the nu-
cleotide sequences of mutant gene. In the latter case, PCR
was not blocked, and ampli¢cation products appeared.
Simultaneous performance of PCR in the presence and
absence of PNA allows for clear di¡erentiation of toxigen-
ic C. diphtheriae strains containing native tox-gene, non-
toxigenic strains not containing tox-gene, and non-toxi-
genic strains bearing ‘‘silent’’ tox-gene.
292 1st FEMS Congress / Posters 103^505
P7^53
BACTERIAL VIRULENCE AND ACQUISITION OF
ERYTHROMYCIN RESISTANCE IN ITALIAN ISO-
LATES OF STREPTOCOCCUS PYOGENES
C. Zampaloni, P. Cappelletti, M. S. Pasquantonio, D. Pet-
relli, M. Prenna, S. Ripa, L. A. Vitali
Department of MCA Biology, University of Camerino, via
Camerini 2, 62032 Camerino (MC), Italy
The in£uence of macrolides on the expression of bacterial
virulence mechanisms has been extensively studied. How-
ever, the relationship between bacterial genetic back-
ground and acquisition of speci¢c antibacterial resistance
has not been yet addressed. The aim of this work was to
determine the level of genetic correlation between macro-
lide resistance and M protein virulence factor distribution
in S. pyogenes. 167 erythromycin (ER) resistant S. pyo-
genes were analyzed for the macrolide resistance pheno-
type and subsequently screened for the genetic determi-
nants of resistance (erm(A) and mef(A) genes). This set
of isolates plus 48 ER sensitive isolates were then analyzed
to determine the emm gene type. The following distribu-
tion of resistance phenotypes was obtained: 18% cMLSB,
31% iMLSB, 51% M phenotype. All emm2 strains showed
the M phenotype, while all strains harboring emm22 gene
were cMLSB. More than 85% of the emm89 positive
strains was iMLSB and the same proportion of emm4
strains showed the M phenotype. emm3 is recorded only
among sensible strains. The distribution of frequencies of
the emm gene type was signi¢cantly di¡erent both when
compared to the type of resistance and when the resistant
versus the sensible population of S. pyogenes under study
were considered. Therefore, resistance genes and the cor-
responding phenotypes are not randomly distributed
among the ER resistant S. pyogenes strains, indicating
that the virulence potential and its related expression
may a¡ect the acquisition of speci¢c resistance traits. Pre-
sented data indicate that particular genetic backgrounds
render this bacterium less prone to acquire resistance to
macrolides (i.e. emm3 strains).
FEMSLE Congress 2-6-03
P7^54
CIPROFLOXACIN RESISTANT STRAINS 0F NEIS-
SERIA GONORRHOEAE IN SLOVENIA
A. Zore(1), M. Petrovec(1), D. Kes›e(1), E. Ruz›ic¤-
Sabljic¤(1), M. Potoc›nik(2), M. Gubina(1)
(1) Institute of Microbiology and Immunology, Medical
Faculty, Zalos›ka 4, Ljubljana, Slovenia; (2) Dept. of Der-
matology, Univ. Medical Centre Ljubljana, Zalos›ka 4,
Ljubljana, Slovenia
Sensitivity of Neisseria gonorrhoeae to antibiotics is chang-
ing. Oral penicillins and tetracyclines are no longer recom-
mended for therapy. Fluoroquinolones are recommended
for therapy of uncomplicated infections (MMWR, 1998).
The ¢rst £uoroquinolone resistant strains of N. gonor-
rhoeae have been diagnosed in Slovenia in 2002. The
aim of the study was to determine the susceptibility of
N. gonorrhoeae isolates from patients in Slovenia to anti-
biotics and to determine underlying genetic mechanisms of
£uoroquinolone resistance. In a two-year period (2001-
2002), we isolated N. gonorrhoeae from 40 patients (35
male, 5 female) treated at the outpatient clinic of Dept.
of Dermatology. Swabs of urethra and cervix were trans-
ported to laboratory in transport culture system. GC-Agar
plates with 1% de¢ned growth supplement were used for
isolation of N. gonorrhoeae and determination of minimal
inhibitory concentrations by E-test (AB Biodisc) to peni-
cillin, tetracycline, azithromycin, cefotaxime, ceftriaxone
and cipro£oxacin. GC-Agar plates, supplemented by Vitox
(Oxoid) were cultured at 5% CO2 , and 35‡C for 1-2 days.
L-lactamase was determined by nitrocephine test. For
quality control, we used N. gonorrhoeae strain ATCC
49226 (NCCLS standards: M 100-S12, M7-A5, 2002). Ge-
netic heterogeneity of £uoroquinolone resistant isolates
was determined for gyrA and parC genes by sequencing
PCR products. Nine of 40 (22.5%) N. gonorrhoeae strains
were resistant to cipro£oxacin. No strain was resistant to
other antibiotics or produced (-lactamase. All cipro£oxa-
cin resistant strains had point mutations in gyrA (on co-
don Ser-91 and Asp-95) or parC genes (on codon Asp-86
and Leu-131). Because all resistant strains were found in
2002, the actual £uoroqinolone resistance of N. gonor-
rhoeae in Slovenia might be even higher than the 22.5%
we found. Double alterations at Ser-91 and Asp-95 in
GyrA plus a single or double ParC alteration probably
play an important role in the development of high-level
£uoroquinolone resistance in N. gonorrhoeae.
 1st FEMS Congress / Posters 103^505
P8^1
GENES INVOLVED IN BIOSYNTHESIS OF LINCO-
MYCIN AND RELATED ANTIBIOTICS ^ REGULA-
TION OF EXPRESSION
N erma¤k, J.
M. Albrechtova¤, M. Jel|¤nkova¤, J. Kopecky¤, L. C
¤ ›
Janata and J. Sp|zek
Institute of Microbiology, Academy of Sciences of the
Czech Republic, V|¤den›ska¤ 1083, 142 20 Prague-4, Czech
Republic
Lincomycin, produced by Streptomyces lincolnensis, and
its derivatives (e.g. clindamycin) are important, clinically
used antibiotics. Twenty-seven putative open reading
frames with biosynthetic or regulatory functions (lmb
genes) and three resistance genes (lmrA, lmrB, lmrC)
have been identi¢ed. A distinct order of gene transcription
initiation was observed. The aim of this experiment was to
¢nd possible regulatory genes. S. lincolnensis was culti-
vated in a semi-synthetic production media and samples
of culture were collected from 18h of cultivation. Total
RNA was isolated and cDNA from random primers was
prepared. By using RT PCR the presence of particular
transcripts was demonstrated during cultivation. It was
found that transcripts of lmbU, lmbR, lmbQ, and lmbV
genes were present in 18h of cultivation. All genes of the
lincomycin gene cluster were transcribed starting from 20h
of cultivation. cDNA from speci¢c primers was further
prepared from 28h total RNA. An arrangement of partic-
ular genes in transcriptional units was ascertained using
RT PCR. A set of prepared probes was also used to search
for homologous genes in Streptomyces caelestis producing
a structurally related antibiotic celesticetin. Homologues
of lmbQ and lmbF were detected. Chromosomal DNA of
producer of anthramycin, an antibiotic structurally di¡er-
ent, but biosynthetically related to lincomycin is screened
for implied analogues of lmb genes coding for the shared
biosynthetic pathway. In order to test the regulatory rela-
tionships several constructs for a directed inactivation
were prepared (e.g. construct for lmbJ inactivation with
respect to the expected interconnection between lmbJ
and lmbIH expression).
FEMSLE Congress 2-6-03
293
P8^2
THE INFLUENCE OF URINE ON THE IN VITRO
ANTIMICROBIAL ACTIVITY OF VARIOUS ANTIBI-
OTICS AGAINST DIFFERENT ESCHERICHIA COLI
PHENOTYPES
M. Ang-Kucuker(1), N. Gonullu(2), O. Buyukbaba-Bo-
ral(1), O. Kucukbasmaci(2), O. Ang(1)
(1) Department of Microbiology and Clinical Microbiol-
ogy, Istanbul Faculty of Medicine, University of Istanbul,
34390 Capa, Istanbul, Turkey; (2) Institute for Experimen-
tal Medical Research, University of Istanbul, Istanbul, Tur-
key
The purpose of this study was to assess the in£uence of
urine on the in vitro activities of various antibiotics used
in the therapy of this clinical infections. Thirty Escherichia
coli strains isolated from patients with urinary infections
were used in this study : the ¢rst group was susceptible to
ampicillin. The second group was resistant to ampicillin
and ampicillin + sulbactam with an inhibitor resistant
beta-lactamases (IRT) producer phenotype. The third
group was an extended spectrum beta-lactamases (ESBL)
producing E. coli group detected by the double disk syn-
ergia between ceftazidime and amoxicillin/clavulanic acid.
MICs were determined by the microbroth dilution meth-
od. The media were Mueller-Hinton broth (Oxoid, Istan-
bul, Turkey) and human, pooled antibiotic-free urine with
a pH of 6.4 and sterilized by passage through a 0.2 Wm
¢lter. MICs of approximately all antibiotics were higher in
the urine media. We obtained an equal MIC90 for cepha-
lothin in broth and urine media in ampicillin susceptible
and IRT producer groups. The most evident di¡erence of
MIC90 (128 fold higher in urine) was obtained for amika-
cin and cipro£oxacin in the E. coli susceptible group. The
lowest di¡erence resulted for ampicillin + sulbactam (two
fold higher in urine) in IRT and ESBL producer groups.
In conclusion, we assessed the activity of di¡erent anti-
biotics in broth and urine media and we found a lower
activity of these antibiotics in urine media with the excep-
tion of cephalothin.
294 1st FEMS Congress / Posters 103^505
P8^3
THE EVALUATION OF MUMMY’S INHIBITORY EF-
FECT ON SOME BACTERIAL AGENTS OF WOUND
INFECTION
Sh. Assar, M. Khaksari, M. Allahtavakoli and A. R. Rezaee
Rafsanjan School of Medicine, Rafsanjan University of
Medical Sciences, Rafsanjan 77179, Iran
One of the most important goals of therapeutic medicine is
to amend wounds in a short time and safe position as
possible. Since natural drugs have these characters and a
variety of e¡ective compounds, medical communities and
World Health Organization paid more attention to them.
The aim of this study was to evaluate the inhibitory e¡ect
of mummy on some agents of wound infection. We col-
lected Mummy, a natural, semisolid, black or brown sub-
stance, from ¢ssures of some mountain caves. Then, the
inhibitory e¡ect of the mummy in concentration of 30, 50,
80 and 100 percent was measured on growth of Pseudo-
monas aeroginosa, Staphylococcus aureus, Escherichia coli
by kirby-bauer as an antibiogram method. The mummy in
above concentrations did not show any inhibitory e¡ect
on Staphylococcus aureus or E-coli’s growth, but Pseudo-
monas aeroginosa was a¡ected in a upgrading manner.
This natural drug had an inhibitory e¡ect on the most
important agent of wound infection, Psedomonas aerogi-
nosa. Because of its routine application among some peo-
ple, we suggest it for pharmacological investigations and
con¢rmation of usage in concentration about 18 gm/dl.
P8^4
THE ACTIVITY OF L-LACTAMASES AGAINST NEW
CEPHALOSPORIN ANTIBIOTICS IN KLEBSIELLA
PNEUMONIAE ISOLATES
J. Blahova, K. Kralikova, V. Krcmery
Institute of Preventive and Clinical Medicine, Limbova 14,
83301 Bratislava, Slovakia
Cefoperazone is one of the most promising third-genera-
tion cephalosporins, active also against strains of Pseudo-
monas aeruginosa and/or Stenotrophomonas maltophilia.
Favorable e¡ects of cefoperazone or cefoperazone/sulbac-
tam against nosocomial bacteria resistant to a palette of
other antibiotics leads the clinicians to use it extensively
mainly in hospitals and their intensive care units (ICU).
However, this habit might produce an unwanted and in-
creased selection pressure in direction of emergence of
cefoperazone-resistant strains. Among 22 isolates of Kleb-
siella pneumoniae, two strains phenotypicaly expressed the
production of extended-spectrum L-lactamases (ESBL) hy-
FEMSLE Congress 2-6-03
drolyzing cefoperazone, ceftazidime, cefotaxime and cefe-
pime and transferred the genes of the resistance to these
antibiotics to susceptible recipient strains indicating that
the gene(s) for ESBL production are localized on a plas-
mid. Hydrolytic activity of lactamases in cell lysate was
determined also by macroidometric method and con¢rmed
active hydrolysis of cefoperazone, cefotaxime and ceftazi-
dime in original strains and also in transconjugant clone.
Clavulanate added to the cell lysate together with antibi-
otics, actively inhibited hydrolytic activity of lactamases
(decreasing almost to 25 %) in both, original strain and
transconjugant. Similar activity was shown also when sul-
bactam was used as a L-lactamase inhibitor. ESBL pro-
ducing strains have a strongly negative epidemiological
signi¢cance being coded by transmissible plasmids. The
appearance of transmissible genes coding for resistance
to these drugs might further deteriorate the outlook of
successful therapy of a series of infectious diseases caused
by these problematic bacteria. It seems reasonable to per-
form at least phenotypic tests to detect strains producing
ESBLs in all clinical relevant Gram negative bacteria.
P8^5
A SEARCH FOR SUBUNITS OF N-DEMETHYLLIN-
COMYCIN SYNTHETASE
D. Byrtusova¤, J. Novotna¤, S. Kadlc›¤|k, J. Janata, J. Ko-
pecky¤, J. Sp|¤z›ek
Institute of Microbiology, Academy of Science of the Czech
Republic, V|¤den›ska¤ 1083, Prague, Czech Republic
N-demethyllincomycin (NDL)-synthetase is one of the en-
zymes involved in biosynthesis of the antibiotic lincomycin
in Streptomyces lincolnensis. This enzyme, consisting of
several nonidentical subunits, catalyzes formation of the
amide bond between propylproline and methylthiolincosa-
mide. To identify its still unknown subunits we used two
approaches. First, we searched for proteins with similar
function in databases and selected proteins LmbC,
LmbD, LmbE and LmbF. Protein LmbC has a conserved
domain, which is responsible for amino acid activation.
An analog of LmbE catalyzes cleavage of the amide
bond in the detoxi¢cation pathway in Mycobacterium. Se-
lection of proteins LmbD and LmbF was based on the
localization of their coding sequences in the neighborhood
of the gene lmbE. Second, we looked for interactions
among proteins involved in lincomycin biosynthesis. We
assume that the detection of these interactions should help
us to reveal subunits of di¡erent enzyme complexes and of
some regulatory proteins. At present, we test in vivo inter-
actions using the yeast two-hybrid system. We prepared
soluble proteins LmbC, LmbD, LmbE, LmbF by heterol-
ogous production in E. coli BL21(DE3) and developed the
protocol for the puri¢cation and storage of these proteins.
 1st FEMS Congress / Posters 103^505
The ability of the puri¢ed proteins to catalyze the forma-
tion of NDL from supplied substrates is now tested. Re-
combinant proteins can also be used to detect the inter-
actions in vitro.
P8^6
GLYCOPEPTIDE RESISTANCE IN THE TEICOPLA-
NIN PRODUCER STRAIN ACTINOPLANES TEI-
CHOMYCETICUS ATCC 31121 AND IN THE A40926
PRODUCER STRAIN NONOMURAEA SP.
ATCC39727
A. Consolandi, B. Fabrizio, F. Bagatin, R. Rossi, F. Ma-
rinelli
Biosearch Italia S.p.A., Via R. Lepetit, 34, 21040 Gerenza-
no (VA), Italy
Glycopeptide antibiotics are active against several gram-
positive bacteria due to their ability to bind the D-alanyl-
D-alanine terminal dipeptide of cell wall intermediates.
Two di¡erent mechanisms of resistance are reported for
pathogens. In enterococci the van genes encode for a re-
sistance apparatus that determines the synthesis of altered
peptidoglycan by replacing terminal D-alanyl-D-alanine
dipeptide with D-alanyl-D-lactate. In staphylococci, resis-
tance originates from the accumulation of several di¡erent
mutations determining the increase in cell wall thickness.
The presence of van-like genes has been reported in the
glycopetide producer strains Streptomyces toyocaensis,
Amycolatopsis orientalis and Actinoplanes teichomyceticus
ATCC 31121. However, genetic data have not been so far
supported by microbiological and physiological studies. In
order to characterize resistance mechanisms in the pro-
ducer strains A. teichomyceticus, and Nonomuraea sp. we
developed systems for the determination of Minimal In-
hibitory and Bactericidal concentrations in these myceliar
microorganism. A. teichomyceticus was resistant to high
levels of vancomycin and teicoplanin showing a typical
VanA phenotype of resistance. On the other side, Nono-
muraea sp. showed the typical response to glycopeptides
observed for staphylococci. This was also consistent with
the genetic studies already reported for A. teichomyceticus.
So far, typical van glycopetide resistance genes in Nono-
muraea sp. have not been evidenced.
FEMSLE Congress 2-6-03
295
P8^7
SEROTYPES AND ANTIMICROBIAL SUSCEPTIBIL-
ITY OF SALMONELLA SPP. ISOLATED FROM PA-
TIENTS WITH ACUTE DIARRHEA IN BELGRADE,
SERBIA
I. Dakic(1), I. Cirkovic(1), M. Vivoda(1), S. Stepa-
novic(1), B. Stosovic(2), M. Svabic Vlahovic(1)
(1) Institute of Microbiology and Immunology, School of
Medicine, Belgrade, Serbia; (2) Institute of Infectious and
Tropical Diseases ‘‘Dr Kosta Todorovic’’, Belgrade, Serbia
The inappropriate use of antibiotics in the clinical practice
as well as in the agriculture, resulted in increased antibi-
otic resistance in Salmonella spp. The objective of the
present study was to determine the serotype distribution
and antimicrobial susceptibility of Salmonella spp. isolated
from patients with acute diarrhea in Belgrade. A total of
150 Salmonella strains were isolated from patients with
acute diarrhea at the Institute of Infectious and Tropical
Diseases in Belgrade, between August 2001 and July 2002.
The strains were identi¢ed and serotyped by conventional
methods. Antimicrobial susceptibility tests for 11 antibiot-
ics were performed by disk-di¡usion method according to
NCCLS recommendations. Extended-spectrum beta-lacta-
mases (ESBL) were screened by double disk synergy meth-
od using amoxicillin-clavulanate, ceftazidime and cefotax-
ime. S. enteritidis (127 isolates; 84.7%) was the
predominant serotype, followed by S. typhimurium (13;
8.7%) and S. infantis (4; 2.6%), while the remaining 6
isolates (4%) were identi¢ed as members of other se-
rogroups. Susceptibility to all antibiotics was established
in 78 (52%) Salmonella isolates. All the isolates were sen-
sitive or intermediary susceptible to cefuroxime and cefo-
taxime, and fully susceptible to gentamicin and cipro£ox-
acin. However, 35 (23.3%) isolates were resistant to
nalidixic acid, 18 (12%) to tetracycline, 14 (9.3%) to am-
picillin and amoxicillin-clavulanate, 8 (5.3%) to trimetho-
prim-sulfamethoxazole, 5 (3.3%) to chloramphenicol and 5
(3.3%) to kanamycin. Nine (6%) isolates showed multiple-
resistance, while ESBL production was observed in 3 (2%)
isolates. The presence of 48% strains resistant to one or
more antibiotics suggests the need for continuous surveil-
lance of changes in Salmonella spp. antimicrobial suscep-
tibility patterns.
296 1st FEMS Congress / Posters 103^505
P8^8
ISOLATION AND CHARACTERIZATION OF A NEW
MICROCIN PRODUCED BY ESCHERICHIA COLI:
MICROCIN F
S. Dandrieux, S. Sable, C. Barthelemy, G. Cottenceau, A.
M. Pons
Laboratoire de Ge¤nie Prote¤ique et Cellulaire, Ba“timent
Marie Curie, Universite¤ de La Rochelle, Avenue Michel
Cre¤peau, 17042 La Rochelle CEDEX 01, France
Numerous members of the Enterobacteriaceae produce
substances that inhibit phylogenetically related species.
Microcins (Mcc) form a family of inhibitory substances
that can be distinguished from the colicins by their much
smaller size ( 6 10 kDa), and the fact that their synthesis
non lethal for the producing strains, is not inducible by
DNA-damaging SOS agents. The production of microcins
depends predominantly on plasmids. Microcins exhibit
di¡erent mechanisms of action: inhibition of DNA repli-
cation, inhibition of protein synthesis or abolition of mem-
brane potential. Usually, their synthesis is optimal in min-
imal medium, early in the stationary phase. Up to date,
nine microcins have been identi¢ed according to the cross
resistance of the mono-producers strains, as well as the
biochemical or genetic properties: MccB17, C7, D93,
E492, H47, J25, L, N24 and V. Here, we report the iso-
lation and the characterization of a new microcin pro-
duced by an Escherichia coli strain: MccF. The wild
type strain also produces MccV that has been previously
characterized. MccF is an original compound produced
both in minimal and rich media, at the beginning of the
stationary phase. This molecule presents a molecular
weight ranging from 12 to 20 kDa, as revealed by ultra-
¢ltration and tricine-SDS-Page electrophoresis. Microcin
activity is not modi¢ed by pH variation from 4 to 12
values. MccF loses activity upon heat treatment (70‡C,
30min). The genetic determinants for MccF production
are located on plasmid.
P8^9
Withdrawn.
FEMSLE Congress 2-6-03
P8^10
RESISTANCE OF GENITAL MYCOPLASMAS
M. Dimitrova, L. Puzderliska, and L. Karakerezova
Department Of Preventive Health, Stip, Macedonia
In our laboratory the susceptibility of genital mycoplas-
mas was followed in the period of three years i.e. 2000’02.
For this purpose Mycoplasma JST were used. We exam-
ined 471 endocervical swabs, from which 256 (54%) were
positive to mycoplasma. Ureaplasma urealyticum were 209
(82%), Mycoplasma hominis were 13 (5%) and 34 (13%)
were combined isolates of both species. U. urealyticum
shows intermediary susceptibility to O£oxacin in 49%, al-
most identical moderate resistance is shown to Erythro-
mycin 7%, Tetracyclin 11%, O£oxacin 6% and to Doxy-
cyclin 3%. M. hominis is resistant to Erythromycin 100%,
to Tetracyclin 46% and it shows intermediary susceptibil-
ity to O£oxacin in 15%. Both combined isolates of U.
urealyticum and M. hominis were resistant to Erythromy-
cin in 82%, Tetracyclin 20%, intermediary susceptibility to
O£oxacin in 70%. In the current year, there is increasing
isolation of M. hominis (in relation) compared with the
same one in the last few years. There are also more often
combined isolates of both species. Because of resistance
occurrence of great importance is the examination of my-
coplasmas susceptibility in the most frequently used anti-
biotics. All examined mycoplasmas do not have resistance
to Josamycin and Pristinamycin.
 1st FEMS Congress / Posters 103^505
P8^11
EFFECT OF ESSENTIAL OIL OF SAGE-BRUSH ON
RESISTANCE OF STAPHYLOCOCCUS TO ANTIBI-
OTICS
T. S. Ermakova, L. P. Titov, E. F. Panshina
Research Institute for Epidemiology and Microbiology,
Minsk, Republic of Belarus
Proceeding increase of microbial resistance to antibiotics
becomes very acute problem. The purpose of present re-
search was study of in£uence of essential oil of sage-brush
bolchan (Artemisia balchanorum Krasch) (EOS) in subbac-
tericidal doses on resistance of Staphylococcus spp. to anti-
biotics. The kinetics of microbial growth in liquid medium
at presence of various concentration of oil was recorded
by spectrophotometer. Sensitivity of cultures to antibiotics
was investigated after 2, 4 and 8 passages by disc-di¡usion
method. At 0,05% of the oil solution in medium the lag-
phase of bacterial growth was extended up to 5h (in the
control 3h), and in a log-phase bacterial growth does not
reach a control level. The oil concentration in thin agar
layer 0,05, 0,02 and 0,01% brakes the formation of micro-
colonies. The EOS inhibiting e¡ect ampli¢es with increas-
ing of their contents in medium. At study 99 cultures of
staphylococcus isolated from carriers, changes of their sen-
sitivity to antibiotics under in£uence EOS were marked. In
result of passages with bacteriostatic concentration of
EOS (0,01%) increase quantity of cultures sensitive to
tested preparations (more often Neomycin, Tetracycline
and Erythromycin). After 8 passages 80-90% of investi-
gated cultures became sensitive to one and more antibiot-
ics. Spectrum and level of resistance in control (passages
without oil) did not change. Thus, e¡ect of sage-brush
essential oil components there was a loss of staphylococ-
cus resistance to many antibiotics. It is interesting to in-
vestigate biochemical and genetic basis of this phenome-
non, equal as possibility of practical application of this
property of sage-brush essential oil.
P8^12
PHENOTYPIC CHARACTERIZATION OF MACRO-
LIDE RESISTANCE IN PHARYNGEAL ISOLATES
OF GROUP A STREPTOCOCCUS
Sp. Fokas, St. Fokas, F. Markatou and M. Dionysopoulou
Clinical Microbiology Department, General Hospital of
Sparta, Sparta, Lakonia 23100, Greece
Resistance to macrolides within Group A Streptococcus
(S. pyogenes) is an increasing problem worldwide making
necessary the prudent use of this class of antibiotics. The
FEMSLE Congress 2-6-03
297
aim of our study was to record the current trend regarding
macrolide resistance in S. pyogenes in our region (Lako-
nia, Southern Greece) and determine the phenotypes of
resistance. We investigated 231 non duplicated S. pyogenes
strains isolated in 2 years period (2000-2001) from patients
with the clinical diagnosis of pharyngitis. The identi¢ca-
tion to the species level was achieved by colony morphol-
ogy, beta hemolysis on blood agar, and agglutination tech-
nique. The pattern of susceptibility to erythromycin,
clindamycin and penicillin was examined for all the strains
performing the disk di¡usion method according to the
criteria of NCCLS (1999). All the isolates of S. pyogenes
tested were susceptible to penicillin while the resistance to
macrolides was signi¢cant (20,78%, 48 strains). The ex-
pression (%) of the macrolide resistance phenotypes
among the resistant strains as they were evaluated by the
erythromycin-clindamycin double disk test were : M-phe-
notype 69%, inducible iMLS(B) phenotype 29% and the
constitutive cMLS(B) phenotype 2%. The overall resis-
tance rate to clindamycin was 6,5%. Macrolide resistance
within S. pyogenes is relatively frequent and responsible
for many cases of treatment failure in patients treated
with macrolides. Continued epidemiological surveillance
of the resistance is required. However, in our region treat-
ment of S. pyogenes infections with clindamycin in pa-
tients allergic to penicillin is considered to be adequate
alternative option due to the high prevalence of the M-
phenotype.
P8^13
PURIFICATION, CLONING AND CHARACTERIZA-
TION OF BACTERIOCIN PRODUCED BY PROPIO-
NIBACTERIA THEONII P-127
N. Gollop, V. Zakin and G. Ben-Shushan
Dept of Food Science, ARO, P.O. Box 6, Bet-Degan, 50250
Israel
The bacteriocin GZB-1 with molecular weight of 3000
Dalton, was puri¢ed from the growth media of Propioni-
bacteria theonii P-127, a strain which is know to produce,
under speci¢c growth conditions, the bcateriocin PLG-1
with molecular weight of 9,500. The N-terminal of GZB-
1 was microsequenced, the gene was cloned and the DNA
sequence was determined. GZB-1 is highly homologous to
PAMP (Faye et al, J. Bacteriol. (2002) 184, 3649-3656),
but in contrast to PAMP it was puri¢ed in its active form
and no protease digestion was needed. The survival curve
of indicator bacteria Lactobacillus delbrukii with 100 units
per ml of GZB-1 showed two phases. The fast phase of 20
minutes was followed by a slow phase. During the fast
phase bacterial survival was reduced by two logs and dur-
ing the slow phase bacterial survivals was reduced by 3
additional logs in 200 minutes. GZB-1 activity is a¡ected
298 1st FEMS Congress / Posters 103^505
by magnesium and is activity is completely abolished at 50
mM magnesium chloride. Other divalent cations had no
e¡ect on GZB-1 activity. This is the ¢rst report, to our
knowledge that bacteria may produce two di¡erent bacte-
riocins under di¡erent growth condition.
P8^14
RESISTANCE TO ANTIBIOTICS STRAINS OF S.
AUREUS ISOLATED AT DIFFERENT INFECTIONS
IN REPUBLIC OF BELARUS
V. A. Gorbunov, L. P. Titov, T. S. Ermakova, K. H. Blyha
Research Institute for Epidemiology and Microbiology,
Minsk, Belarus
S. aureus is an important causative agent of nosocomial
infections. Monitoring antimicrobial resistance of S. aur-
eus forms the basis of measures to prevent the spread of
multiresistant strains, the development of e¡ective prophy-
laxis and treatment of infections caused by them. Identi-
¢cation and de¢nition of susceptibility to antibiotics was
carried out by ATB expression (BioMerieux, France). The
frequency of isolation S. aureus from urological infections
was 2,99% (n=1440), intestinal 15,35% (n=1590), respira-
tory 5,88% (n=204), infections of skin and soft tissues
10,0% (n=110), sepsis 13,97% (n=136). Percentage of re-
sistant strains S. aureus in 1996-1999 was: urinary infec-
tions to penicillin 92,3%, oxacillin 38,5%, cefazolin 2,6%,
gentamicin 20,7%, tetracycline 75,0%, erythromycin
48,7%, lincomycin 84,6%, cipro£oxacin 15,4%, co-trimox-
azole 17,1%, vancomycin 10,3%; from respiratory infec-
tions to penicillin 92,3%, oxacillin 53,8%, cefazolin 0,0%,
gentamicin 12,5%, tetracycline 53,8%, erythromycin
30,8%, lincomycin 53,8%, cipro£oxacin 25,0%, co-trimox-
azole 0,0%, vancomycin 0,0%; from intestinal infections to
penicillin 100,0%, oxacillin 100,0%, cefazolin 9,7%, genta-
micin 9,7%, tetracycline 19,4%, erythromycin 33,3%, lin-
comycin 22,6%, cipro£oxacin 3,2%, co-trimoxazole 12,9%,
vancomycin 9,7%; from skin infections to penicillin 92,5%,
oxacillin 25,5%, cefazolin 3,0%, gentamicin 12,1%, tetracy-
cline 89,0%, erythromycin 53,5%, lincomycin 82,0%, cipro-
£oxacin 2,1%, co-trimoxazole 13,0%, vancomycin 9,0%;
from blood infections to penicillin 100,0%, oxacillin
62,5%, cefazolin 12,5%, gentamicin 50,0%, tetracycline
56,3%, erythromycin 56,3%, lincomycin 87,5%, cipro£ox-
acin 16,7%, co-trimoxazole 0,0%, vancomycin 0,0%. In
conclusion, the frequency of isolation of resistant strains
of S. aureus varies from 25,5 to 100% and depends on the
form of infection and such as a drug. Methicillin-resistant
staphylococci are more often isolated from intestinal in-
fections and sepsis (62,5-100%). They are found much less
often at urological and skin infections. The high frequency
of cipro£oxacin-resistant staphylococci isolated from res-
piratory tract and blood (16,7 and 25,0%, repectively), and
FEMSLE Congress 2-6-03
also the increase in resistance to vancomycin (up to 10%)
demands attention. Monitoring MRSA is crucial in Bela-
rus.
P8^15
RESISTANCE TO ANTIBIOTICS OF STRAINS E.
COLI ISOLATED FROM PATIENTS WITH URO-
LOGICAL AND INTESTINAL INFECTIONS IN RE-
PUBLIC OF BELARUS
V. A. Gorbunov, L. P. Titov, T. S. Ermakova, K. H. Blyha
Research Institute for Epidemiology and Microbiology,
Minsk, Belarus
Monitoring of resistance clinically signi¢cant microorgan-
isms to antibiotics is a part of strategy of containment
their spread in clinical terms. Increasing resistance of E.
coli emphasizes the necessity to monitor antibacterial sus-
ceptibility of this pathogen. Identi¢cation and de¢nition of
susceptibility to antibiotics was carried out on the ATB
expression (BioMerieux, France) Results: E. coli is one of
the main causative agent of urological and intestinal in-
fections. The frequency of these isolation at urological
infections was 39,0% (n=1440), intestinal-12,1%
(n=1590). Strains E. coli isolated from patients with uri-
nary infections in 1996-1999 were resistant to amoxicillin
in 59,2-60,4%, cefotaxime-13,1-16,6%, aztreonam-21,3-
27,3%, imipenem-1,1-4,4%, gentamicin-15,3-26,2%, amika-
cin-29,2-42,3%, chloramphenicol ^ 66,7-68,7%, tetracy-
cline-86,5-92,3%, o£oxacin-15,2-24,4%, cipro£oxacin-
10,2-16,3%. Strains E. coli isolated from patients with in-
testinal infections in 1996-1999 were resistant to amoxicil-
lin in 58,21-70,2%, cefotaxime-4,1-18,2%, aztreonam ^ 0,0-
54,5%, imipenem ^ 0,0-54,5%, gentamicin ^ 17,2-52,6%,
amikacin ^ 22,1-42,1%, chloramphenicol ^ 47,3-63,6%, tet-
racycline ^ 61,4-54,5%, o£oxacin ^ 0,0%, cipro£oxacin ^
0,0-54,5%. In conclusion: Strains E. coli are highly resis-
tent to amoxicillin, chloramphenicol, tetracycline. The re-
liable increase of resistance E.coli to aminoglycozides is
observed at urinary infections, to aztreonam, imipenem
and cipro£oxacin at intestinal infections. Probably, it is
connected to high frequency of using these drugs in the
investigated period of time.
 1st FEMS Congress / Posters 103^505
P8^16
NaCl IN THE SELECTIVE SALT ENRICHMENT
BROTH SUPRESS GROWTH OF MRSA
I. Grmek-Kosnik, U. Dermota, B. Krivic, Z. Jokovic, A.
Kodran, J. Smrekar, N. Meglic and T. Blazic
Institute of Public Health Kranj, Gosposvetska ulica 12,
4001 Kranj, Slovenia
Study of the growth kinetics in the selective salt (NaCl)
enrichment broth showed that high concentration of salt
in the selective enrichment broth inhibits the growth of
MRSA (2). It is thus essential to test the salt tolerance
of an epidemic MRSA isolate before undertaking wide-
spread and costly screening programmes. We compared
the growth of 10 isolates (2 ATCC strains and 8 domestic
strains) MRSA after 24 hours incubation in the di¡erent
concentrations of NaCl salt enrichment broth. Growth
kinetics demonstrated a logarithmic inhibition of growth
of MRSA with increase of NaCl concentration. Growth of
MRSA in salt enrichment broth was signi¢cantly sup-
pressed even with NaCl concentration of 0.5% (p 6 0.05,
Student’s t-test) but growth was detected in all tubes even
at NaCl concentration of 10%. We con¢rmed, that con-
centration of NaCl salt enrichment broth suppresses the
growth of MRSA. Our ¢ndings and ¢ndings in literature
(2) suggest that the sensitivity of the screening method
might be diminished for at least some isolates of MRSA
at the recommended concentration of NaCl (7%). Thus, a
policy of screening to control an outbreak could be seri-
ously undermined by a high level of false negative results.
P8^17
EFFECT OF pH VARIATIONS ON MEASUREMENTS
OF INHIBITION ZONE AROUND ANTIBIOTIC-IM-
PREGNATED DISKS
Z. Jokovic, U. Dermota, I. Grmek Kosnik, M. Ceh, C.
Stropnik
Institute of Public Health Kranj, Gosposvetska ulica 12,
4001 Kranj, Slovenia
We studied the pH variations in Mueller-Hinton medium
on reference strain susceptible to antibiotics. NCCLS
standardizes antibiotic susceptibility testing by the disk-
di¡usion (Kirby-Bauer) method with agar medium pH
7.2 to 7.4. We compared zones of inhibition around pen-
icillin, vancomycin, gentamycin and erythromycin disks
with reference strain Staphylococcus aureus ATCC 25923
on Mueller-Hinton medium contained di¡erent pH value
(from 6.2 to 8.4 with intervals of 0.2). Accuracy pH mea-
surements were obtained with a pH meter. We measured
FEMSLE Congress 2-6-03
299
inhibition zone diameter. Using a standard table of anti-
biotics susceptibilities, we determined if the strain was re-
sistant, intermediate or susceptible. We also looked, if the
control limits for reference strain were appropriate. Zones
of inhibition around penicillin deviated at pH 8.2 and 8.4,
around vancomycin at pH 8.0, 8.2 and 8.4, around genta-
mycin at pH 7.6, 7.8, 8.0, 8.2 and 8.4, around erythromy-
cin at pH 8.0, 8.2 and 8.4. All measures the diameter of
the zones of growth inhibition around penicillin, vanco-
mycin and gentamycin were within the limits of suscepti-
bility, for erythromycin the zones were out of the expected
ranges. Inhibition zones around erythromycin at pH 6.2
and 6.4 were within the limits of intermediate, at pH from
6.6 to 8.4 was within the limits of susceptibility. Our ¢nd-
ings are, if the pH is not between 7.2 and 7.4, the rate of
the di¡erent antimicrobial agents or the activity of the
drugs may be a¡ected. Zones of inhibition with reference
strain are out the limit, if the pH is from 7.6 to 8.4.
P8^18
DETECTION OF EXTENDED-SPECTRUM L-LACTA-
MASES : COMPARISON OF THE MAST DD TEST,
THE DOUBLE DISK, E-TEST ESBL AND VITEK 2
B. Jutersek, T. Zohar Cretnik, A. Storman, V. Bozanic
Institute of Public Health Celje, Department of Microbiol-
ogy, Gregorciceva 5, 3000 Celje
Extended-spectrum L-lactamase (ESBL) type of resistance
is an important problem in the treatment of nosocomial
infections. Detection of ESBL expression has proven to be
di⁄cult with routine susceptibility test, because in-vitro
tests may not determine resistance to cephalosporins at
the NCCLS interpretative breakpoint for susceptibility.
In our laboratory, double disk di¡usion test is used rou-
tinely for ESBL detection for all K. pneumoniae isolates.
298 isolates of ESBL producing K. pneumoniae were col-
lected from patients in the regional hospital in last 5 years
and 40 of them were randomly selected for the study. The
aim of the study was to evaluate and compare the ability
of four di¡erent methods to detect ESBL production :
double disk di¡usion test (DD), ESBL E-test method,
MAST disk test and VITEK 2. Detection of genes coding
for SHV family L-lactamases was carried out by PCR with
SHV speci¢c primers. The sensitivity of di¡erent methods
were as follows : DD 100%, Mast disks 90%, E-tests 100%
and VITEK 2 70%. DD is reliable, easy to and relatively
cheap. E-tests and Mast disks are also practically useful
for detection of ESBL. Problem of VITEK 2 is a limited
database for recognition of ESBL producers. Co-opera-
tion with manufacturer and upgrading of their data is
necessary.
300 1st FEMS Congress / Posters 103^505
P8^19
MYCOPLASMA IDENTIFICATION AND SENSITIV-
ITY TEST IN FEMALE GENITAL TRACT : A PRO-
SPECTIVE STUDY
M. Simou, S. Tzortzatou, T. Lazaraki, E. Thomou, E.
Kada
Laboratory of Microbiology, Elena Venizelou Hospital,
Athens, Greece
This is a prospective study to assess the prevalence of
Mycoplasma hominis and Ureaplasma urealyticum in cer-
vical smears and their susceptibility to antimicrobial
agents. Methods: 430 cervical smears of women in repro-
ductive age who attended the Outpatient Department of
our Hospital were tested for the presence of Mycoplasma
hominis and Ureaplasma urealyticum. The identi¢cation of
mycoplasma and the sensitivity test were performed using
the Mycofast Screening Evolution 2, kit (International
Microbio, France). Results : Out of 430 specimens 145
(33.7%) were positive for mycoplasma. 134 specimens
(31.16%) were positive for Ureaplasma urealyticum. From
134 U. urealyticum positive specimens 20 gave positive
results in 1/1000 dilution, 35 in 1/10.000 dilution and 79
in 1/100.000 dilution. 11 specimens (2.5%) were positive
for M.hominis in s 1/10.000 dilution. In 9 cases (2.0 %)
both Ureaplasma urealyticum and Mycoplasma hominis
were identi¢ed. Resistance to antimicrobial agents was
9.6 % to Roxithromycine (14 strains were resistant : 8 M.
homiiniss and U. urealyticum , 4 U. urealyticum and 2 M.
hominis), 6.2 % to O£oxacine (9 strains : 4 M. hominis and
U. urealyticum and 5 U. urealyticum) and 0.6 % to Dox-
ycycline (1 strain of U. urealyticum). In the total 24 (16.55
%) strains of mycoplasma were resistant to antibiotics.
Conclusions : The incidence (33.7 %) of mycoplasma in
genital tract of women in reproductive age is relatively
high. The percentage 16.5 % of resistance to antimicrobials
could be a therapeutic problem in£uencing conception and
leading to infertility.
FEMSLE Congress 2-6-03
P8^20
IN VITRO ACTIVITY OF TELITHROMYCIN COM-
PARED TO MACROLIDES AND FLUOROQUINO-
LONES AGAINST STREPTOCOCCUS PNEUMO-
NIAE, HAEMOPHILUS INFLUENZAE AND
MORAXELLA CATARRHALIS
º . Ku«cu«kbasmaci(1), N. Go«nu«llu«(1), Z. Aktas(2), D.
O
Gu«rol(2), R. Berkiten(2)
(1) Istanbul University, Institute of Experimental Medical
Research, 34390 Capa, Istanbul, Turkey ; (2) Istanbul Uni-
versity, Istanbul Faculty of Medicine, Department of Micro-
biology, 34390 Capa, Istanbul, Turkey
This study has the objective of comparing the in vitro
activity of the ketolide telithromycin with those of macro-
lides and £uoroquinolones against major respiratory
pathogens recently isolated in a Turkish university hospi-
tal. A total of 336 strains including 83 S. pneumoniae, 168
H. in£uenzae and 85 M. catarrhalis isolated from outpa-
tients with CA-RTIs were examined. MICs were deter-
mined by the agar dilution method. Of the 83 S. pneumo-
niae isolates, 25.3% (21/83) were intermediately resistant
and 10.8% (9/83) were highly resistant to penicillin G.
Telithromycin demonstrated 4-8 fold higher potent activity
against S.pneumoniae than all the macrolide antibiotics. In
fact, telithromycin (MIC90, 0.008 Wg/ml) was the most ac-
tive compound against S. pneumoniae among all the anti-
biotics tested. Both penicillin G resistant and penicillin G
susceptible S. pneumoniae strains were highly susceptible
to telithromycin, moxi£oxacin and gemi£oxacin. MIC90 of
gemi£oxacin was two fold lower than that of moxi£oxacin
and 32 fold lower than those of both cipro£oxacin and
levo£oxacin. Of the 168 H. in£uenzae isolates, seven (4%)
were L-lactamase positive. Telithromycin (MIC90, 4 Wg/ml)
was more active than erythromycin (MIC90, 8 Wg/ml) or
clarythromycin (MIC90, 16 Wg/ml) and was as active as
azithromycin (MIC90, 4 Wg/ml). L-lactamase status did
not seem to a¡ect the activity of telithromycin, macrolides
or £uoroquinolones. All four £uoroquinolones were highly
active against H. in£uenzae and the most active compound
was gemi£oxacin with a MIC90 of 0.015 Wg/ml. The MIC90
values for M. catarrhalis of macrolides and telithromycin
ranged from 0.015 to 0.06 Wg/ml. Against M. atarrhalis,
the activities of the four £uoroquinolones were similar and
their MIC90 were within two dilution steps of each other
while gemi£oxacin was superior to others with a mode
MIC of 0.015 Wg/ml.
 1st FEMS Congress / Posters 103^505
P8^21
COMPARISON OF INTRAVENOUS AMINOGLYCO-
SIDE THERAPY WITH SWITCH THERAPY TO CE-
FIXIME IN URINARY TRACT INFECTIONS
A. R. Lari(1), S. Noorbakhsh(2), F. Masjediyan(1), M.
Habibi(1), R. Alaghehbandan(1) and H. Mostafavi(3)
(1) Department of Microbiology, Iran University of Medi-
cal Sciences, P.O. Box 14515-717, Tehran, Iran; (2) De-
partment of Pediatric, Iran University of Medical Sciences,
P.O. Box 14515-717, Tehran, Iran; (3) Exir Pharmaceut-
ical Company, Tehran, Iran
Urinary tract infections (UTIs) are one of the most im-
portant causes of hospitalization among children in Iran.
As intravenous antibiotic therapy is associated with side
e¡ects, toxicity, high cost, and long hospitalization period
in treatment of UTIs, ‘‘switch’’ therapy (intravenous-to-
oral antibiotic) has been considered. The aim of this study
was to compare the e⁄cacy of intravenous aminoglycoside
(amikacin or gentamicin) therapy with intravenous cef-
triaxone and switch therapy to ce¢xime in children with
UTIs. In this single-blind randomized clinical trial, 54 chil-
dren aged 9 10 years with complicated urinary tract in-
fections, who were determined by positive urine analysis/
culture, were enrolled and divided in two groups A and B.
Children in group A (n = 30) treated with intravenous
amikacin (15 mg/kg daily) or gentamicin (3 mg/kg daily)
with ampicillin (100 mg/kg daily) for 7-10 days. Patients in
group B (n = 24) treated with intravenous ceftriaxone (50
mg/kg daily) for the ¢rst 2 days and then switch to ce¢x-
ime (8 mg/kg daily) orally for 8 days. One week after
treatment, patients were evaluated clinically/microbiologi-
cally. Rate of response (clinically/microbiologically) to in-
travenous aminoglycoside therapy in patients of group A
was 80% (24/30). Children of group B, who received cef-
triaxone with switch to ce¢xime, had 88% (21/24) response
rate. However, there was not statistical signi¢cant di¡er-
ence between rate of response in two groups (P (2) = 0.82).
Switch therapy with ce¢xime in children with UTIs in-
creases e¡ectiveness and convenience. Switch therapy
shortens duration of hospitalization, and of course, de-
creases costs and risk of nosocomial infections. Also, ce-
¢xime could be considered as switch therapy in children
with UTIs.
FEMSLE Congress 2-6-03
301
P8^22
ANTIMICROBIAL ACTIVITY OF ESSENTIAL OIL
OF SAGE (SALVIA OFFICINALIS L.) AND DIFFER-
ENT TERPENOID FRACTIONS
I. Bratic, A. Bratic, D. Mitic, J. Knezevic-Vukcevic, B.
Vukovic-Gacic, D. Simic
Chair of Microbiology, Faculty of Biology, University of
Belgrade, Serbia
Herbs, spices and fruits have been used through the ages
as a source of £avour in food and parfume preparation.
Many essential oils and their ingredients have been shown
to exhibit a range of biological activities, including anti-
bacterial and antifungal activity. This study was per-
formed to investigate the antimicrobial activity of essential
oil of Salvia o⁄cinalis L., and its fractions distinguished
by di¡erent content of mono- and sesqui- terpenoids. For
this purpose we used:disk di¡usion test for pre-screening
of antimicrobial potential of test agents; minimum inhib-
itory concetration (MIC) tests to identifying the amount
of antimicrobial agent required for visible antimicrobial
e¡ect and time-kill assay to measure the time required
for antimicrobial e¡ect.The essential oil and their fractions
demonstrated a signi¢cant antimicrobial e¡ect on Staph-
ylococcus aureus ATCC 25923 and Bacillus subtilis ATCC
10707 in disk di¡usion test. Moreover, B. subtilis was
more sensitive than S. aureus. The MIC concentration
for S. aureus was 1.25Wl/ml for essential oil and all tested
fractions except F2 fraction (2.5 Wl/ml). B. subtilis was
more sensitive than S. aureus and the MIC concetration
was in the range 0.1-2.5 Wl/ml. The time-kill test showed
that essential oil and its fractions have bactericidal e¡ect
on S. aureus, while the e¡ect on B. subtilis is bacteriostatic.
P8^23
MECHANISMS OF RESISTANCE TO MACROLIDES
AND LINCOSAMIDES IN METHICILLIN RESIS-
TANT STAPHYLOCOCCI
G. Novotna¤, K. L|¤c›kova¤, J. Janata, and J. Sp|¤z›ek
Institute of Microbiology, Academy of Science of the Czech
Republic, V|¤den›ska¤ 1083, Prague 14220, Czech Republic
We identi¢ed di¡erent types of resistance mechanisms to
macrolides and lincosamides in a group of methicillin re-
sistant coagulase negative staphylococci. One-hundred-¢ve
clinical isolates in vitro resistant to one of erythromycin
(macrolide), lincomycin or clindamycin (lincosamide) were
tested by southern blot analysis for the presence of the
genes ermA, ermC (resistance by target site alteration)
msrA (e¥ux resistance) and linA (resistance by antibiotic
302 1st FEMS Congress / Posters 103^505
inactivation). The resistance was mainly due to the pres-
ence of msrA gene, which was detected in 57 strains [54%].
Genes ermC and ermA were detected in 48 strains [46%]
and the linA was detected in 22 strains [21%]. The dissem-
ination of resistance types di¡ers strongly from the pub-
lished data. While in other countries cross-resistance to
macrolides and lincosamides conferred by ermA and
ermC predominates, in the Czech Republic the gene
msrA is the most frequent genetic determinant conferring
resistance only to macrolides, while sensitivity to lincosa-
mides is preserved. We observed a relationship between
the type of resistance and the level of resistance to eryth-
romycin: whereas MICERY for the strains bearing msrA
only was 16 to 128 mg/l, for the strains with erm it was
MICERY 512 to 1024 mg/l. In 14 strains we detected no
resistance genes although the strains were resistant to lin-
comycin and clindamycin. The fact that lincosamides are
not inactivated indicates that a new type of resistance
di¡erent from inactivation is involved.
P8^24
EFFECTIVENESS OF TREATMENT OF RESPIRA-
TORY FORM OF ACUTE MELIOIDOSIS USING
LIPOSOMAL CEFAZOLIN IN EXPERIMENT
K. A. Rotov, V. V. Alekseev, V. I. Kapliev and S. N. Ti-
khonov
Antiplague Research Institute, Volgograd, Russia
The choice of antibiotics is very important due to a wide
spread of Pseudomonas pseudomalei. To solve this prob-
lem, one may use liposomal forms of antibacterial agents.
These forms improve the penetration into the cells and
provide an increased time of drug elimination with mini-
mized toxicity, immunologic reaction. In this study we
have determined the e¡ectivity in treatment of respiratory
form of melioidosis using cefazolin in free and liposomal
forms in mice. Liposomal cefazolin was prepared by re-
verse phase evaporation and was sterilized by Q-radiation
in doses of 500 krad. Experimentaly respiratory form of
acute melioidosis was induced by aspiration of P. pseudo-
malei 100. E¡ectivity of treatment was determined by per-
cent of defence, ED50 of drugs, morphology during autop-
sy, bacteriology of organs. Usage of cefazolin made it
possible to raise the percent of defence from 50% for
free antibiotic to 75% for immobilized form. It was found
out that ED50 of the drug was 3.59 mg in liposomal form
and 9.77 mg in free antibiotic. Thus, inclusion of cefazolin
in liposomes helped to raise e¡ectiveness in treatment of
respiratory form of acute melioidosis in mice.
FEMSLE Congress 2-6-03
P8^25
DISTRIBUTION OF NIM AND CFI A GENES IN BAC-
TEROIDES SPP. IN BELARUS
V. Slizen(1), L. P. Titov(1), J. S. Brazier(2), M. Gal(2)
(1) Belarussian State Medical University, Department of
Microbiology, Immunology, Virology, Minsk, Belarus;
(2) PHLS Anaerobe Reference Unit, Cardii¡, UK
Despite the widespread use of metronidazole and imipe-
nem, bacteroides resistance to these antibiotics remains
low (about 1%). A rise in resistance to 3-5% was recorded.
Nim (A,B,C,D,E) and c¢A genes confer resistance to these
drugs, respectively, though these genes frequently are not
expressed. Low incidence of c¢A and nim genes is indica-
tive of recent acquisition of these resistance determinants.
We investigated the distribution of c¢A and nim genes
among Bacteroides spp. to evaluate the risk of emergence
of phenotypic resistance via gene activation. 40 isolates of
Bacteroides spp from patients with perirectal abscesses
were screened for the presence of c¢A and nim (A, B, C,
D, E) genes by PCR technique with the universal primers.
RFLP analysis (HpaII and Taq I restriction) of ampli¢ca-
tion products from nim gene PCR produced pro¢les that
enabled identi¢cation of nim (A,B,C,D,E) genes. Agar disk
di¡usion method was used to test phenotypic resistance to
metronidazole (5 Wg) and imipenem (10 Wg). All isolates
were susceptible to metronidazole and did not give posi-
tive reaction in detection of nim (A,B,C,D) by PCR. All
tested bacteroides were susceptible to imipenem in routine
disk testing and lacked c¢A gene (absence of PCR prod-
ucts after ampli¢cation with the speci¢c primers). The data
support the concept of low distribution of nim and c¢A
genes in bacteroides. Imipenem and metronidazole remain
to be highly potent drugs against bacteroides in Belarus.
PCR-RFLP technique will permit improved identi¢cation
of Bacteroides spp. and facilitate the recognition of nitro-
imidazole resistance determinants.
 1st FEMS Congress / Posters 103^505
P8^26
MONITORING OF ANTIMICROBIAL RESISTANCE
IN REPUBLIC OF BELARUS
L. P. Titov(1), V. A. Gorbunov(1), T. S. Ermakova(1), K.
H. Blyha(1), E. F. Panshina(1), N. I. Tochko(2), E. A.
Selesneva(3), N. A. Matveeva(4)
(1) Research Institute for Epidemiology and Microbiology,
Minsk, Belarus; (2) City Center of Hygiene and Epidemi-
ology, Minsk, Belarus ; (3) Region Center of Hygiene and
Epidemiology, Grodno, Belarus ; (4) Region Center of Hy-
giene and Epidemiology, Gomel, Belarus
Microbiological monitoring allows increased e⁄ciency of
microbiological diagnostics, rational antimicrobial chemo-
therapy and improvement in their control. Creation of a
really functioning national surveillance system for contain-
ment of antimicrobial resistance ^ an important task of
public health services. Material for research : sputum,
pleural liquid, tracheal aspirations, blood, urine, faeces
etc. The monitoring was carried out using ATB-expression
(BioMerieux) in three Belarus regions (Minsk, Gomel,
Grodno) in 1996-1999. Results I. 6584 strains of micro-
organisms were isolated. 218 species of bacteria and fungi
are identi¢ed. Most frequent microorganisms were: Staph-
ylococcus spp.- 28,8%; Escherichia spp. ^ 17,7%; Strepto-
coccus spp. ^ 6,3%; Candida spp. ^ 5,8%; Enterobacter
spp. ^ 4,0%; Pseudomonas spp. ^ 3,9%; Citrobacter spp.
^ 3,5%; Klebsiella spp. ^ 3,3%. II. Increasing resistance of
strains S. aureus to ampicillin (39%), oxacillin (73%), ce-
furoxime (14%) was observed. III. Signi¢cant increase of
resistance E. coli in 1996-1999 was observed to moxalac-
tam (19%), aztreonam (16%), cipro£oxacin (16%), ticarcil-
lin/clav.acid (15%), amikacin (14%), gentamicin (13%),
imipenem (11%), netilmicin (10%). IV. The share of resis-
tant strains Candida spp. in 1996-97 were 2-5% depending
on a drug, whereas in 1998-99 these indexes have increased
in 2 and more time (9-14%). V. Analysis of dynamics
antimicrobial resistance P. aeruginosa for the period with
1996-97 on 1998-99 has revealed increase of resistant
forms to ticarcillin / clav.acid (55%), imipenem (30%), az-
treonam (68%), gentamicin (77%), netilmicin (86%), cipro-
£oxacin (51%). Conclusion: Increase of antimicrobial re-
sistance of microorganisms from 1996 to 1999 bears
witness to the necessity of carrying out constant monitor-
ing of multidrug resistance, including introduction of hos-
pital formularies for the practical doctors, control of anti-
biotics usage in hospitals and other measures, whose
ultimate goal is containment of antimicrobial resistance.
FEMSLE Congress 2-6-03
303
P8^27
ANTIBIOTIC RESISTANCE IN BANK VOLES
(CLETHRIONOMYS GLAREOLUS) AND WOOD
MICE (APODEMUS SYLVATICUS)
N. J. Williams(1), T. R. Jones(1), D. Hunt(1), N. P.
French(1), M. Begon(2), M. Bennett(1) and C. A.
Hart(3)
(1) University of Liverpool Veterinary School, Leahurst,
Chester High Road, Neston, South Wirral, CH64 7TE;
(2) School of Biological Sciences, Nicholson Building, Uni-
versity of Liverpool, L69 3GP; (3) Department of Medical
Microbiology, Duncan Building, University of Liverpool,
L69 3GA
A previous survey of wild bank voles and wood mice in a
woodland site, demonstrated antibiotic resistant organisms
in their faecal £ora, even though they had no obvious
contact with antibiotics. These results raised a number
of questions: such as the source of the antibiotic resistance
genes; their transferability; and whether a selective pres-
sure was required for their maintenance. This report de-
scribes attempts to address some of these questions,
through a longitudinal survey of the antibiotic susceptibil-
ity of E. coli and carriage of vancomycin-resistant enter-
ococci (VRE) in wild rodents. Rodents have been live-
trapped monthly since June 2001, using 200 traps placed
in pairs at 10m intervals over a permanently marked grid
of 100m2 of woodland. Faecal samples have been collected
from traps and associated with individual animals identi-
¢ed by transponder chips. E. coli is isolated from faeces
using media with and without antibiotics. Isolates are
screened for resistance against six commonly used antibi-
otics. The prevalence of antibiotic resistance in faecal E.
coli and faecal carriage of VRE have both been found to
vary with season. Furthermore, bank voles appear to be
half as likely to excrete an ampicillin (OR 0.49, P=0.03) or
trimethoprim (OR 0.44, P=0.07) resistant E. coli than
wood mice and one-third less likely to excrete a chloram-
phenicol (OR 0.28, P=0.005) or tetracycline (OR 0.33,
P= 6 0.001) resistant E. coli. Overall a higher prevalence
of VRE was also found in wood mice (7%) than in bank
voles (0.5%). The genes responsible for resistance are cur-
rently being investigated.
304 1st FEMS Congress / Posters 103^505
P9^1
ANALYSIS OF THE BILE STRESS RESPONSE IN
LISTERIA MONOCYTOGENES LO28
M. Begley, C. G. M. Gahan and C. Hill
Department of Microbiology, National University of Ire-
land, Cork, Ireland
Bile is one of the many barriers that pathogens must over-
come in the human gastrointestinal tract in order to infect
and cause disease. We have demonstrated that Listeria
monocytogenes LO28 can tolerate the average concentra-
tion of bile/bile acids encountered in vivo. Data indicates
that prior adaptation of the bacterium to sublethal levels
of bile or other stresses encountered during food process-
ing and sanitizing treatments (acid, heat, salt, SDS) may
alter cellular physiology to enhance bile resistance. Present
studies are focusing on the molecular mechanisms under-
lying bile tolerance. Three genes possibly involved in the
degradation / transformation of bile salts were identi¢ed
following analysis of the recently published L. monocyto-
genes EGDe genome. These genes are absent from the
genome of the non-pathogenic strain L. innocua. Initial
physiological analyses of deletion mutants suggest that
all three genes contribute to listerial bile tolerance. In or-
der to identify other genetic loci involved in bile stress,
transposon and plasmid integration banks were screened
for bile sensitive mutants. This revealed a role for a num-
ber of genes including btlA, gadE and zurR in bile resis-
tance. Interestingly, all loci identi¢ed so far play putative
roles in the maintenance of the cell envelope or in stress
responses.
P9^2
ACTIVITY OF ASCORBIC ACID AGAINST HELICO-
BACTER PYLORI
E. T. Dokic, M. Petrovska, N. Panovski
Institute of Microbiology, Medical faculty, Skopje, Republic
of Macedonia
Introduction : The most successful treatment for the erad-
ication of H. pylori includes combination of proton pump
inhibitors with antibiotics such as amoxicillin, clarithro-
mycin, tetracycline and metronidazole. Toxic e¡ects of
ascorbic acid in the presence of metal ions and oxygen
were reported for a number of viruses and bacteria. The
role of vitamin C, both in vitro and in vivo, in H. pylori
infection hasn’t been studied enough so far. The aim of
this study is to determine the in vitro activity of vitamin C
against 30 H. pylori clinical isolates. Materials and meth-
ods 30 H. pylori clinical isolates cultured from gastric bi-
FEMSLE Congress 2-6-03
opsies taken at routine endoscopy were studied. Ascorbic
acid was diluted and 2-fold dilution prepared to obtain a
¢nal concentration range of 4000 to 62,5 mg/L. An agar
dilution method using Columbia agar supplemented with
yeast extract and sheep blood was used. A high inoculum
(approximately 106 CFU/ml) was applied using a replica-
tor and plates were incubated in a microaerophilic atmo-
sphere at temperature of 370 for 5 days. Minimum inhib-
itory concentration (MIC) was recorded as the lowest
concentration that inhibited visible growth. Results The
number of strains that were inhibited at each concentra-
tion of ascorbic acid were as follows : 3 strains with
MIC=125 mg/L, 8 with MIC= 250 mg/L, 11 with
MIC=500 mg/L, 6 with MIC=1000 mg/L and 2 with
MIC=2000 mg/L. Conclusions Ascorbic acid shows good
in vitro activity against H. pylori clinical isolates and could
explain some of the bene¢cial properties of vitamin C.
P9^3
MEASUREMENT OF GLUTATHIONE AS STRESS
INDICATOR IN ACTIVATED SLUDGES
M.-A. Dziurla(1), P. Leroy(2), G. Stru«nkmann(3), M.
Salhi(4), P. Camacho(5), V. Heinz(6), J. Mu«ller(3), E.
Paul(4), Ph. Ginestet(5) and J. C. Block(1)
(1) Laboratoire de Chimie, Physique et Microbiologie pour
l’Environnement ^ LCPME, Unite¤ Mixte de Recherche ^
UMR 7564, CNRS ^ Universite¤ Henri Poincare¤, Nancy 1,
Faculte¤ de Pharmacie ^ Po“le de l’eau, 15, avenue du Char-
mois ^ 54500 Vandoeuvre-les-Nancy, France; (2) Equipe
Thiols et Fonctions Cellulaires, Faculte¤ de Pharmacie, Uni-
versite¤ Henri Poincare¤ Nancy 1, 30 rue Lionnois, 54000
Nancy, France; (3) Institute of Mechanical Process Engi-
neering, Technical University of Braunschweig, Volkmar-
oder Str. 4/5, 38104 Braunschweig, Germany ; (4) Institut
National des Sciences Applique¤es de Toulouse (INSA-Tou-
louse), Complexe Scienti¢que de Rangueil, 31077 Toulouse
Cedex 04, France ; (5) CIRSEE-ONDEO Services, 38, rue
du Pre¤sident Wilson, 78230 Le Pecq, France ; (6) Institut
fu«r Lebensmitteltechnologie und Prozesstechnik, Technische
Universita«t Berlin, Ko«nigin Luise Strasse 22, 14195 Berlin,
Germany
Excess biomass produced during the biodegradation of
waste water by activated sludges requires costly disposal.
Production of excess sludge can be reduced by submitting
sludges to mechanical, electrical, thermal, or oxidative
stress. These treatments induce biological stress responses
allowing the restoration of cellular structures and meta-
bolic pathways. The adaptation and the resistance of the
sludge microbial ecosystem to stress conditions is a major
question as it may de¢nitively limit the e¡ect of stress
treatments. Defense mechanisms developed, in particular
in response to oxidative stress involve various antioxidant
 1st FEMS Congress / Posters 103^505
activities and compounds such as glutathione. In order to
determine the redox status of activated sludges before and
after stress, an HPLC method was developed for measur-
ing reduced and total glutathione (GSH and GSHt) in
perchloric acid sludge extracts. The method was sensitive,
highly speci¢c and validated for linearity, precision and
recovery. To our knowledge, this is the ¢rst report on
GSH measurement in activated sludges. Extraction yield
for GSH was estimated to be 84 %. Considering an oxi-
dation of 30 % of GSH during extracts storage at -80‡C,
the concentration of GSH measured was estimated to rep-
resent 60 % of the e¡ective GSH content in activated
sludges. Total glutathione ranged from 0.32 to 3.34
Wmol per g volatile solids. In ‘‘normal’’ activated sludges,
GSH and GSHt concentrations varied without knowing
the reason for the variations. In sludges submitted to ther-
mal and mechanical stress, decrease in glutathione and
oxygen uptake rate occured indicating that glutathione is
a stress indicator.
P9^4
GENERATION OF REACTIVE OXYGEN SPECIES
(ROS) IN YEAST CANDIDA INTERMEDIA AS RE-
SULT OF COPPER AND ZINC EXPOSURE
SN. Fujs(1), Z. Gazdag(2), M. Pesti(2), J. Bela¤gyi(3), P.
Raspor(1) and M. Batic›(1)
(1) Department of Food Science and Technology, Biotech-
nical Faculty, University of Ljubljana, Jamnikarjeva 101,
1000 Ljubljana, Slovenia ; (2) Department of General and
Environmental Microbiology, Faculty of Sciences, Univer-
sity of Pecs, P.O. Box 266, H-7601 Pecs, Hungary ; (3)
Central Research Laboratory, University of Pecs, P. O.
Box 266, H-7601 Pecs, Hungary
Reactive oxygen species (ROS) are generated during nor-
mal cellular metabolism and can damage cellular compo-
nents by oxidizing lipids, proteins and nucleic acids. The
redox active metal copper can be toxic at high concentra-
tion, due to generation of reactive hydroxyl radical (OH])
via a Fenton-type reaction and other reactive oxygen spe-
cies, including H2O2 and superoxide radical (O2]). The less
redox active zinc has important role in antioxidant pro-
tection of DNA. Zinc displaces bound iron from macro-
molecules thereby reduces iron catalysed Fenton reaction
of OH] generation. The objective of this study was estima-
tion of ROS generation in yeast Candida intermedia to 5
mg/l of copper and 50 mg/l of zinc exposure. To estimate
intracellular peroxide and superoxide levels, £uorescence
indicators dihydrorhodamine 123 (DHR) and dihydroethi-
dium were used, respectively. The formation of rhodamine
and ethidium (ET) was quanti¢ed spectro£uorimetrically.
Hydroxyl radical formation induced by Cu and Zn was
measured by electron spin resonance spectroscopy in cell
FEMSLE Congress 2-6-03
305
extract of C. intermedia by using the spin trap N-tert-bu-
tyl-a-phenyl nitrone (PBN). The cellular copper and zinc
concentrations in biomass were determined by £ame
atomic absorption spectrometry (FAAS). O2]-dependent
ET £uorescence in yeast C. intermedia to copper and
zinc exposure was increased by 111% and 18%, respec-
tively. Opposite, H2O2-dependent DHR oxidation was de-
creased compared to control sample. Hydroxyl radical for-
mation in yeast extract exposed to 2 mM copper and zinc
was not detected by ESR. High concentrations of copper
and zinc stimulated superoxide radical generation in yeast
C. intermedia. Moreover, copper exposure showed more
deleterious ROS generation compared to zinc exposure.
We would like to acknowledge the Ministry of Agricul-
ture, Forestry and Food and Ministry of Education, Sci-
ence and Sport of the Republic Slovenia for support proj-
ect No. V4-0402-00.
P9^5
CELL CYCLOPROPANE FATTY ACID CHANGES AS
A KEY FACTOR IN STRESS TOLERANCE OF SAL-
MONELLA
M. E. Guerzoni and L. Vannini
Dipartimento di Protezione e Valorizzazione Agroalimen-
tare, Facolta' di Agraria, Universita' degli Studi di Bologna
Via Fanin 46, 40127 Bologna, Italy
To date most of the work on stress response of pathogenic
species as well as the relationship between stress exposure
and virulence has been focused on de novo protein syn-
thesis while the potential role of phospholipid or fatty acid
modi¢cation has been virtually ignored. In this work fatty
acid adaptation, and particularly cyclopropane fatty acid
(CFA) changes, following oxidative and thermal stresses in
12 strains of Salmonella was analysed. The 12 strains, that
were characterised by di¡erent antibiotic resistances, plas-
mid pro¢les and ribotype patterns, showed di¡erent via-
bility responses and CFA changes when exposed to vari-
ous H2O2 concentrations and cold and heat stresses. For
some of the strains a relevant increase in CFA percentage
with a consequent decrease in unsaturated fatty acid con-
tent was observed following the stress exposure; on the
other hand negligible changes in fatty acid composition
was evidenced with other ones. The relationship between
stress tolerance, virulence factors and fatty acid modula-
tion patterns will be discussed.
306 1st FEMS Congress / Posters 103^505
P9^6
CILIATE Cd-METALLOTHIONEIN GENES: A
HIGHLY CONSERVED MICROBIAL RESPONSE
TO HEAVY METAL STRESS
J. C. Gutie¤rrez, S. D|¤az, L. Ben|¤tez and A. Mart|¤n-Gonza¤lez
Dpto. Microbiolog|¤a-III, Facultad de Biolog|¤a, C/. Jose¤ An-
tonio Novais 2, Universidad Complutense (UCM), 28040
Madrid, Spain
Metallothioneins (MTs) are cysteine (Cys)-rich heavy met-
al-binding proteins, which are present in animals, plants as
well as in cyanobacteria and eukaryotic microorganisms.
MTs are induced by heavy metals (Cd, Zn and Cu), and
they are responsible for the bioaccumulation and inactiva-
tion of these metals in the cell. We report three di¡erent
macronuclear gene isoforms (MTA, MTB and MTC) en-
coding Cd-MTs isolated from very di¡erent ciliates. All
these MTs show a high average identity among them;
the identity among MTAs is about 74%, for MTB iso-
forms is 97-100% and for MTCs is about 89%. A lower
average identity is showed among the three isoforms
(MTA ^ MTB : 32%, MTA-MTC : 35%, MTB ^ MTC
: 55%). Ciliate MTs present unique features with regard
the rest of known MTs: a)- They show Cys motifs com-
posed by three Cys residues (-CCC-) very conserved
among ciliates MTs, but not reported in other MTs. b)-
The total number of Cys residues is higher than that in
other MTs [ MTA (22 ^ 31), MTB (33 ^ 34), MTC (48) ].
c)- MTB and MTC isoforms have aromatic amino acids
(Tyr, Phe) and His, which are not present in any MT. d)-
Their molecular weights are higher (11-16 KDa) than
those ( 6 7 ^ 10 KDa) for typical MTs. e)- They are
over-expressed after heavy metal exposure. The singular
features of ciliate MTs make possible that they can be
used as biological tools to design molecular biosensors
to detect environmental pollution by heavy metals.
P9^7
CHAOTROPICITY: A NEW CONCEPT IN WATER
STRESS
J. E. Hallsworth(1), S. Heim(2) and K. N. Timmis(1,2)
(1) Department of Biological Sciences, University of Essex,
Colchester CO4 3SQ, UK; (2) Division of Microbiology,
GBF ^ National Research Centre for Biotechnology,
Braunschweig, D-38124, Germany
Low water availability is the most ubiquitous cause of
stress for terrestrial plants, animals and microorganisms,
and has a major impact on ecosystem function and agri-
cultural productivity. Studies of water stress have largely
FEMSLE Congress 2-6-03
focused on conditions that a¡ect cell turgor, ie osmotic
stress. We show that chaotropic chemicals that do not
a¡ect turgor reduce water activity, perturb macromole-
cule-water interactions and thereby destabilize cellular
macromolecules, inhibit growth, and are powerful media-
tors of water stress in a typical soil bacterium, Pseudomo-
nas putida. Such bacteria mineralise organic wastes, in-
cluding chaotropic xenobiotics, play a critical role in the
carbon cycle, and detoxify polluted habitats by natural
attenuation or bioremediation. Chaotropic solute-induced
water stress resulted mostly in the up-regulation of pro-
teins involved in stabilization of biological macromole-
cules and membrane structure. We suggest that some
chemicals currently considered to be toxic are primarily
chaotropic agents of water stress, and that associated
changes in entropy represent a common factor in di¡erent
sources of stress and the corresponding cellular stress re-
sponses.
P9^8
MEASURING VITALITY AND GENETIC STRESS RE-
SPONSE OF BACILLUS LICHENIFORMIS FOR OP-
TIMIZATION OF PROPAGATION METHODS
T. HornbAk(1,2), A. Nielsen(1), M. Jakobsen(2) and J.
Dynesen(1)
(1) Novozymes A/S, Sm\rmosevej 25, DK-2880 BagsvArd,
Denmark; (2) KVL, Dairy and Food Science, Food Micro-
biology, Rolighedsvej 30 4th, DK-1958 Frederiksberg, Den-
mark
Bacillus licheniformis is a microorganism widely used by
the enzyme industry in fermentations for the production of
enzymes. Prior to fermentation, the strain undergoes dif-
ferent propagation steps potentially a¡ecting the physiol-
ogy of the strain i.e. the inoculum used for the production
of enzyme. In order to minimize variations in inoculum
quality it is a prerequisite to know how the culture is
a¡ected by di¡erent propagation conditions. Methods ap-
plied for this purpose include the use of 5(6)-carboxy£uor-
escein diacetate succinimidyl ester in combination with
£ow cytometry or £uorescence ratio imaging microscopy
for determination of cell vitality. Using DNA arrays, ex-
aminations of the genetic stress response at changing
propagation conditions are carried out. Ongoing work
that examines the e¡ect on the vitality and genetic stress
response of B. licheniformis found when substituting an
agar substrate with a liquid growth medium in the prop-
agation process will be described. The vitality of the cul-
ture is examined after growth on the solid agar surface
respectively in the liquid medium and the genetic stress
response is investigated shortly after inoculation into the
subsequently used seed tank medium.
 1st FEMS Congress / Posters 103^505
P9^9
TRANSPOSITION OF F-FACTOR LEADS TO SUP-
PRESSION OF lon MUTATIONS IN ESCHERICHIA
COLI CA-154 HfrH
H. G. Hovhannisyan
S. S. Hovhannisyan Institute of Microbiology, NAS RA,
Abovyan City, 378510, Armenia
The Lon ATP-dependent protease of E. coli participates in
the regulation of heat-shock and SOS stress responses and
responsible for the degradation of a number of regulatory
proteins and of many abnormal proteins. E. coli K-12
strains lacking Lon display two characteristic phenotypes,
increased sensitivity to UV and overproduction of capsu-
lar polysaccharides. These phenotypes result from failure
to degrade the speci¢c Lon protease substrates, SulA ^ cell
division inhibitor whose expression is induced as part of
the SOS response and RcsA ^ positive regulator of expres-
sion of the colanic acid synthesis genes. For selection non-
mucoid revertants of lon mutant E.coli K-12 CA154 HfrH
we have used the virulent bacteriophage M59 speci¢cally
lysates the capsulated cells overproducing colanic acid.
Among more than two hundred UV sensitive revertants
one was UV-resistant, but still carrying mutation in lon
gene. Interrupted conjugation experiments showed that
in the UV-resistant revertant called NM144 have changed
of genes transfer gradient and polarity to the F- recipient
strains. Chromosome transfer was started from point of
origin 60 min in counterclockwise direction. Transposition
of F-factor back to former at zero point region abolishes
the lon suppression. The ability to suppress two indepen-
dent lon phenotypes was most easily explained as expres-
sion of an unknown protease activity which can substitute
for Lon-protease. In the new region of F-factor insertion
located ssrA gene which acts as a negative regulator of
alternative Lon-protease (Alp) synthesis (Gottesman,
1996). Thus it is possible that insertion of F-factor inacti-
vated ssrA and induced Alp activity.
P9^10
THE USE OF MICROBES AS INDICATOR FOR BIO-
AVAILABILITY OF MERCURY IN SOIL
P. Jaeckel and M. Schloter
GSF-National Research Center for Environmental and
Health, Institute of Soil Ecology, Ingolsta«dter Landstr.1,
D-85764 Neuherberg, Germany
Due to industrial activities in South East Germany until
the late 60th of the last Century, soils and sediments close
to the industrial sites were highly contaminated with heavy
FEMSLE Congress 2-6-03
307
metals, especially with mercury. As most of the Hg2+ is
either bound in soil minerals or adsorbed on solid surfa-
ces, the bioavailability of Hg is low. In the present study
the in£uence of farming on the bioavailability of mercury
was investigated. As bacteria are only a¡ected by bioavail-
able substances and have direct contact to the soil solu-
tion, they can be used as sensitive bioindicators for pollu-
tants. Therefore the structural diversity, the microbial
adaptation (Pollution Induced Community Tolerance,
PICT) and gene response could be a sensitive way to mea-
sure the bioavailability of mercury in the environment.
The structural diversity was studied by bacterial commu-
nity ¢ngerprints (ARDRA) and PICT by community level
physiological pro¢les (CLPP) with and without mercury.
Due to the Hg-stress a horizontal gene transfer of resis-
tance plasmids containing the mer-gene may occur. Thus a
correlation between mer-gene transfer and bioavailability
of mercury was investigated.
P9^11
Hsp104 INDUCTION IN THE YEAST CANDIDA IN-
TERMEDIA EXPOSED TO Cr(VI)
P. Jamnik and P. Raspor
Food Science and Technology Department, Chair of Bio-
technology, Biotechnical faculty, Jamnikarjeva 101, 1000
Ljubljana, Slovenia
The survival of yeast cells is dependent on their ability to
sense alternations in the environment and to appropriately
respond to the new situation. One of the most conserved
mechanisms of cellular protection is the expression of a
polypeptide family named heat shock or stress proteins.
Among them Hsp104 has drawn a great attention, because
it promotes survival under extreme stresses such as high
temperatures, severe oxidative damage and high concen-
trations of ethanol. These stresses cause protein aggrega-
tion and Hsp104 promotes survival by facilitating the re-
solubilization of protein aggregates. The aim of our work
was to investigate whether Hsp104 is induced in the yeast
Candida intermedia exposed to Cr(VI). Cr(VI) compounds
are widely used in many industrial processes and cause
environmental toxicity. Cr(VI) belongs to redox active
metals, which play an important role in the generation
of reactive oxygen species in the cell. In our previous study
we have already reported that Cr(VI), speci¢cally in con-
centration of 100 WM causes elevated intracellular oxidant
level. Herein, we demonstrate that Cr(VI) stress result in
variation in protein synthesis, which indicate that Cr(VI)
might regulate the expression of some genes. At 100 WM
Cr(VI) induction of Hsp104 was observed, which is con-
nected to formation of protein aggregates. Hsp104 assists
their resolubilization and so contributes to cell survival.
Therefore, we showed that Hsp104 plays an important
308 1st FEMS Congress / Posters 103^505
role in defence system of yeast Candida intermedia to
Cr(VI).
We are grateful to the Ministry of Education and Sport of
the Republic of Slovenia for grant (no.: 35/8) and the
Ministry of Science and Technology of the Republic of
Slovenia (Project no.: J4-7454-490) for ¢nancial support.
P9^12
ENDOGENOUS ADP-RIBOSYLATION OF THE G
PROTEIN L SUBUNIT MODULATES GLQ ACTIVITY
IN FUNGI
N. Jeraj, H. Lenasi and K. Breskvar
University of Ljubljana, Faculty of Medicine, Institute of
Biochemistry, Vrazov trg 2, 1000 Ljubljana, Slovenia
Progesterone is toxic for ¢lamentous fungus Rhizopus nig-
ricans and at higher concentrations leads to inhibition of
fungal growth. The fungus responds to toxic e¡ects of
progesterone by detoxi¢cation of this steroid via P450
11K-hydroxylation. One of progesterone signaling routes
includes membrane progesterone receptors coupled to G
proteins. We demonstrate here some of the subsequent
steps of this signaling route. A reduction of cAMP levels
was observed after stimulation with progesterone, but was
not blocked by pertussis (PTX) or cholera toxin (CTX).
This ¢nding suggests that the K subunits of classical Gi or
Gs proteins are not involved in the inhibition of fungal
adenylyl cyclase. These studies prompted us to explore
the possibility that G protein LQ subunits (GLQ) might
play an important role in the response to progesterone.
Using a combination of (i) ADP-ribosylation assay ^ la-
belling with [32P]NAD; (ii) ADP-ribosylation in the pres-
ence of cholera toxin and pertussis toxin; and (iii) Western
blotting with a panel of anti-G-protein antibodies, we
demonstrated that R. nigricans expresses homologues of
the L like subunits (35kDa) and K subunits of the Gi1
subfamily (45kDa) found in mammals. The activity of
GLQ can be modulated by mono-ADP-ribosylation in a
similar way to the regulation of some G protein K sub-
units. Here we present the ¢rst report of the endogenous
mono-ADP-ribosylation of the G protein L subunit in an
organism from the kingdom of fungi.
FEMSLE Congress 2-6-03
P9^13
INFLUENCE OF EXOGENOUS STRESS FACTORS
ON THE PRODUCTION OF CAROTENOIDS BY
RED YEAST
R. Koci, I. Marova, J. Pokorna, O. Koutny
Department of Food Chemistry and Biotechnology, Faculty
of Chemistry, Technical University of Brno, Czech Republic
Carotenoids are the most widespread natural lipid-soluble
pigments with many important biological activities and
industrial applications. The aim of this study is to com-
pare the composition and content of carotenoids produced
by several strains of carotenogenic yeasts in the presence
of exogenous stress factors. Three strains of red yeasts
(Rhodotorula glutinis, Sporidiobolus salmonicolor, Pha⁄a
rhodozyma) were grown aerobically on glucose medium.
Oxidative stress was induced by 2-5 mM H2O2, osmotic
stress by 2-5% NaCl during 40- and/or 80-hours exposi-
tion. In£uence of aeration, temperature and heavy metals
was studied too. Growth parameters, oxygen consump-
tion, concentration of carotenoids (HPLC/MS) and other
metabolites (e.g. glycerol) were analyzed. Stress factors
resulted in di¡erent responses according likely to original
environment. P. rhodozyma was more sensitive to all types
of stress than the other strains. Under salt stress only R.
glutinis produced higher amount of all carotenoids. Oxi-
dative stress led in S. salmonicolor and R. glutinis to an
induction of both growth and production of carotenoids
(4-8x higher concentration of L-carotene, 3-4x lycopene, 2-
3x phytoene ; 2-4x K-carotene). Aeration and temperature
led to an increase of L-carotene production in limited
range. Pretreatment of R. glutinis with low concentration
of NaCl and/or H2O2 led to further induction of L-caro-
tene (2.3x), lycopene (3.9x) and phytoene (5.7x) and to
higher tolerance to additional oxidative stress. Higher pro-
duction of carotenoids in yeast cells under stress condi-
tions can act as an adaptive mechanism. Because yeast
cells exposed cross-protection against di¡erent stress
agents, carotenoids probably take part in general stress
response of red yeast.
 1st FEMS Congress / Posters 103^505
P9^14
COLONY AND ULTRASTRUCTURAL DIFFEREN-
CES AND SIMILARITIES IN TWO TRIMMATOS-
TROMA SPP. ISOLATED FROM DIFFERENT
WATER-STRESS ENVIRONMENTS
T. Kogej(1), A. A. Gorbushina(2), A. Schulte(2) and N.
Gunde-Cimerman(1)
(1) University of Ljubljana, Biotech. Faculty, Dept. of Bi-
ology, Vecna pot 111, SI-1000 Ljubljana, Slovenia; (2)
Geomicrobiology, ICBM, Carl von Ossietzky Universita«t
Oldenburg, POB 2503, D-26111 Oldenburg, Germany
Black yeasts are rare known extremophilic eukaryotic mi-
croorganisms. The morphology of black yeasts is impor-
tant for their survival in extreme environments, and in-
cludes characteristics on a colony level: polymorphism,
meristematic growth, endoconidiation, and on a cell level:
melanization, thick cell walls. Common features of the
black-yeast genus Trimmatostroma are melanized thick-
walled muriform cells that develop by conversion from
undi¡erentiated hyphae. Genus Trimmatostroma is classi-
¢ed in ascomycetous order Dothideales and contains 15
species, which are inhabitants of phylloplane (conifer nee-
dles), wood, rock surfaces, and hypersaline water. All of
the mentioned environments are osmotically stressful due
to a relatively low water activity. We have chosen to study
the colony- and cell-level adaptations to saline environ-
ment of two di¡erent species of the genus Trimmatostro-
ma, which were previously isolated from two di¡erent en-
vironments with comparably low water activity. A
halophilic species Trimmatostroma salinum was isolated
from hypersaline water of a saltern. A xerophilic species
Trimmatostroma sp. was isolated from a glass-window sur-
face of a church. Several di¡erent non-saline and saline
growth conditions were chosen according to their ability
of growth in the presence of NaCl. Both species were then
studied microscopically. Their colony structure was
studied histologically, and their cell-wall structure was ex-
amined by transmission electron microscopy. The growth
patterns of both species were identi¢ed and compared on a
colony level. Cell-wall structures with emphasis on mela-
nization patterns were also compared in both species as
their response to osmotic conditions.
FEMSLE Congress 2-6-03
309
P9^15
CHANGES IN LIPID COMPOSITION AS ADAPTIVE
RESPONSE OF ROCK INHABITING ASCOMYCE-
TOUS BLACK YEASTS TO DEHYDRATION AND
REHYDRATION
E. R. Kotlova(1), A. A. Gorbushuna(2), A. Schulte(2)
(1) Komarov Botanical Institute, Prof. Popov Str. 2, St.
Petersburg, Russia ; (2) Carl von Ossietzky University, P.
O. Box 2503, 26111 Oldenburg, Germany
The representatives of rock dwelling ascomycetous black
yeasts are highly adapted to the existence in stressed con-
ditions such as prolonged dehydration. Among the most
principal features responsible for their tolerance the fol-
lowing could be mentioned: slow growth rate, protective
structures surrounding cells, internal and external com-
pounds (polysaccharides, proteins) binding water. In the
present work we attended to e¡ects of de- and rehydration
on colonial organization, ultrastructure and lipid compo-
sition. For this purpose one strain of black yeasts isolated
from a marble substrate and identi¢ed as the Phaeococco-
myces sp. was used. Developed colonies were subjected to
slow or fast desiccation followed by rehydratation. Under
desiccation, distinctive intracolonial strati¢cations were
detected, which were visible due to the discernible distri-
bution of di¡erent cell types. Three individual cell types
were described, including round yeast-like cells, dividing
meristematic cell clusters and cylindrical hyphae. In accor-
dance with our previous ¢ndings lipid droplets inside the
cells were noticed as well. Among substances which attrib-
ute to the reserve lipid particles, triacylglycerols, sterol
esters and free fatty acids formed the most part. The con-
tent of each was practically doubled during fast desicca-
tion and reverted to the initial one after rewetting. Slow
dehydration induced the considerable decrease of their
amount which was also reverted after rehydration. On
the contrary, the amount of membrane lipids (phosphati-
dylcholines, phosphatidylethanolamines, sterols) remained
unchangeable during dehydration. Such stability of mem-
brane components might be due to the reserved lipids
which are not only material for energy production and
water supply but also for membrane lipid synthesis.
310 1st FEMS Congress / Posters 103^505
P9^17
SPHERICAL NANOSTRUCTURES BY PSEUDOMO-
NAS AERUGINOSA DURING PHOSPHATE STARVA-
TION : COMPLEX VESICLES OR NANOCELLS ?
S. La Porta, A. Gio¡re', M. Nicolo', S. Guglielmino
Dept. of Microbiological, Genetic and Molecular Sciences,
University of Messina, sal. Sperone, 31, 98166 Messina,
Italy
Pseudomonas aeruginosa, like many Gram negative bacte-
ria such as Vibrio spp., Escherichia coli and other pseudo-
monads, is able to survive in starvation conditions thanks
to the activation of complex regulatory systems leading to
physiological and structural adaptation, including varia-
tion of cell shape. In 0.2 Wm-¢ltered culture supernatant
after ¢ve days of phosphate starvation, spherical nano-
structures (NSs) of about 200 nm diameter were visualised
by scanning electron microscopy. NSs structures were in-
vestigated by SDS-PAGE, enzyme activities and nucleic
acids content. Electrophoretic analysis showed a complex
protein pro¢le with more than 30 bands. Enzymatic activ-
ity analyses revealed the presence of malate dehydrogen-
ases and cytochrome-oxidases. DNA and RNA have also
been detected into NSs; further analysis of DNA was
performed and particularly REP-PCR showed a pro¢le
similar to generating-cells, suggesting that NSs contain
large amounts of the genome. These data and further
SEM observations on phosphate-starved cells address to
either asymmetric septation or cell debris release due to
structural alterations.
P9^18
EFFECT OF HEAVY CARBON ISOTOPE 13C ON
THE CYTOLOGY OF RHODOSPIRILLUM RUBRUM
N. N. Ladyguina
Pushchino State University, Pushchino, Moscow region,
142290, Russia
Photosynthesis of bacteria is accompanied with of carbon
isotope fractionation. Usually the isotope ratio of 12C and
13
C of natural substrate (CO2) is evaluated 99:1. The pa-
per presents study on e¡ect of initial isotopic composition
of the CO2 carbon on cytology of the photosynthetic pur-
ple bacteria Rhodospirillum rubrum VKM B-1621 (type
strain). Initial content of the carbonate carbon in experi-
ments was 20 mM NaHCO3, however, enrichment with
13
C in di¡erent experiments varied from 10 to 57 %. The
control (blank) variant contained 1 % 13C, i.e. natural
composition. Morphology of bacterial population in con-
trol variant was presented with long chains of spirillas.
FEMSLE Congress 2-6-03
The number of single cells increased with the increase of
the 13C concentration in medium. At the highest concen-
trations of 13C (45 and 57 %), the size of single cells de-
creased and was about 90 % of natural size in the blank
variant. Thus, concentration of heavy carbon in NaHCO3
a¡ects cytology of the photosynthetic purple bacteria. This
e¡ect is realable at high concentration of 13C and reveals
in two ways: increases the single cells number and dimin-
ishes their sizes. The data provide evidence that 13C con-
centration in substrate can be an a¡ecting agent for bac-
teria.
Author thanks Drs. N.E.Suzina, A.L.Mazanov and
M.B.Vainshtein for their kind support.
P9^19
RESPONSE OF SULFATE-REDUCING BACTERIA
DESULFOVIBRIO DESULFURICANS TO MERCURY
A. Lapanje(1), D. Drobne(1), B. Beaumont(2), R. J. M.
van Spanning(2), M. Rupnik(1)
(1) Biotechnical Faculty, Department of Biology, Vec›na pot
111, 1000 Ljubljana, Slovenia; (2) Vrije Universiteit, NL-
1081 HV Amsterdam, The Netherlands
Presence of mercury is unfavourable for growth of sulfate-
reducing bacteria (SRB) for at least two reasons: mercury
toxicity and mercury derived higher redox potential. SRB
detoxify mercury by sul¢de precipitation into HgS. Also,
SRB transform mercury into more toxic methylmercury.
Methylmercury is mostly toxic to multicellular animals
because it is lipid soluble compound which di¡uses
through the membrane. Methyl mercury is then trans-
formed into ionic form where persists and make severe
e¡ects. For single cell organism the methylation of mer-
cury can be the mechanism for detoxi¢cation. The SRB
biochemically transform the ionic form of mercury, which
enters the cell, into lipid soluble form after the methyla-
tion process. This produced methylmercury goes out from
the cell with di¡usion. Besides this the methylation of
mercury is known to be enzymatic process and SRB can
actively transform mercury into methyl mercury. These
two mechanisms can serve SRB to survive high concen-
trations of mercury. Because of that we tried to ¢nd phys-
iological e¡ects of mercury on Desulfovibrio desulfuricans.
This could help us to understand detoxifying mechanisms.
We added di¡erent amounts of mercury into the media for
fermentative and respiratory growth. We inspected growth
curves as well as protein synthesis at the beginning of the
stationary phase. We investigated whole concentration of
proteins as well as patterning proteins on SDS PAGE gels.
The changes in expression were observed in soluble
although we investigate both phases ^ lipid soluble as
well as water soluble proteins. We also found that the
 1st FEMS Congress / Posters 103^505
growth curve shape was changed and showed biphasic
growth.
P9^20
REDUCTION OF EXOGENOUS FERRIC IRON BY A
CELL SURFACE-ASSOCIATED REDUCTASE OF EN-
TEROCOCCUS SPP.
P. Lisiecki, P. Wysocki
Department of Pharmaceutical Microbiology, Medical Uni-
versity of Jo¤dz¤, 90-235 Jo¤dz¤, Pomorska 137, Poland
Over the past few years enterococci have become one of
the most important noscomial and community acquired
pathogens. For several bacteria, a correlation between
mechanisms of iron assimilation and virulence has been
demonstrated. Enterococci as facultative anaerobes have
an obligate iron requirement. For this reason, there is an
increasing interest in strategies of iron acquisition in enter-
ococci. We showed that enterococci produced hydroxa-
mate class siderophore. In this study we have tried to
estimate the ability of enterococci to reduction of exoge-
nous Fe3+ by means of cell surface-associated reductases.
A total of 122 strains genus Enterococcus belonging to ten
species and isolated from clinical and environmental sour-
ces were used. Ten iron compounds were tested: ferric
ammonium sulphate, ferric ammonium citrate, ferric
versenate, ferric nitrate, ferric pyruvate, ferrioxamine B,
horses ferritin, human transferrin, lactoferrin and haemo-
globin. The reduction of Fe3+ was assayed with ferrozine
test. The ability of enterococci to reduce of Fe3+ was de-
tected with agar plate technique and reductase activity was
estimated in liquid medium. All strain of enterococci used
3+
in this study were able to reduce Fe of all tested iron
sources. The iron in form of ferric ammonium citrate,
lactoferrin and ferrioxamine B were the best iron sources
for enterococcal reductases. We found that reduction of
iron was slightly stimulated in NADH presence but not in
NADPH, and was enhanced by addition of FMN. Addi-
tion of Mg2+ to the reaction mixture had no in£uence on
reduction of exogenous ferric iron. All the strains showed
no di¡erences in reductases activities when they grew
under iron-poor or iron rich conditions. Haemin or hae-
moglobin in the culture medium did not increased reduc-
tases activities. The cell surface-associated ferric reductases
may be one of the important way of iron-acquisition in
enterococci.
FEMSLE Congress 2-6-03
311
P9^21
IN VIVO ANALYSIS OF CHAPERONES-DEPEN-
DENT REFOLDING OF BACTERIAL LUCIFERASES
DIFFERING IN THE THERMOSTABILITY AND LU-
MINENCENCE DECAY RATE
I. V. Manukhov, M. M. Mazhul, V. U. Kotova, G. B.
Zavilgelsky
State Scienti¢c Center of the Russian Federation, GosNII-
genetika, 1_yi Dorozhnyi Proezd 1, 117545 Moscow, Russia
The role of chaperones Hsp70 and Hsp100 in refolding in-
vivo of thermoinactivated luciferases from marine bacte-
rium Vibrio ¢scheri, Vibrio harveyi, Photobacterium leiog-
nathi and the terrestrial bacteria Photorhabdus lumines-
cence in Escherichia coli cells has been studied. These
luciferases are homologous but di¡er greatly in the rate
of thermal inactivation and in the rate luminescence decay.
It was shown that refolding of thermoinactivated lucifer-
ases is completely determinate by the DnaKJ system.
However these luciferases markedly di¡er in rate and de-
gree of refolding. The degree of refolding of thermolabile,
‘‘fast’’ V. ¢sheri luciferases reaches 80% of the initial level
over several minutes, whereas renaturation of thermo-
stable, ‘‘slow’’ Ph. luminencence luciferases proceeds sub-
stantially slower. Intermediate parameters of refolding are
characterized for V. harvei and Ph. leoignathi luciferases.
The measurement of the rate of thermo inactivation of
luciferases in E. coli wild strain and strains containing
mutations in clpA, clpB and clpX genes showed that Ph.
luminencence luciferase revealed reduced thermostability in
mutant strains E. coli clpA or clpB. In E. coli clpA this
e¡ect is not connected with DnaK-dependent refolding, in
E. coli clpB this e¡ect is determinate by DnaK-dependent
refolding and connected with the ability of chaperone
ClpB to provide disaggregation of the proteins. The addi-
tion of uncoupler of oxidative phosphorylation, carbon-
ylcyanide-3-chlorophenylhydratione (CCCP) results in a
sharp decrease in thermostability of luciferase to the level
typical of the enzyme in vitro. We suggest that thermo-
stable luciferases have enhanced a⁄nity with respect to
chaperone ClpA in comparision with DnaK, whereas ther-
molabile luciferases are characterized by enhanced a⁄nity
with respect to chaperone DnaK.
312 1st FEMS Congress / Posters 103^505
P9^22
GENES INVOLVED IN STRESS RESPONSE AND RE-
SISTANCE OF ESCHERICHIA COLI TO THE LAC-
TOPEROXIDASE SYSTEM
J. Sermon, P. Despiegeleer, K. Vanoirbeek, A. Aertsen, and
C. W. Michiels
Department of Food and Microbial Technology, Katholieke
Universiteit Leuven, Leuven, Belgium
Haem peroxidases such as lactoperoxidase (LP) catalyze
the oxidation by hydrogen peroxide of a wide range of
substrates into oxidizing reaction products with antimicro-
bial properties, and play a role in host defence against
infection. While the e¡ects of, and bacterial response to,
other oxidative stress agents such as hydrogen peroxide
and superoxide has received considerable attention in re-
cent years, little is known about oxidative stress caused by
these peroxidase enzymes. We report here the initial re-
sults of a study into the stress response and resistance
factors of Escherichia coli MG1655 to the LP-thiocyanate
system. In a ¢rst approach, we have selected random
transposon insertion mutants with an increased resistance
to the LP system and identi¢ed the location of the insert.
In a second approach, we used a gfp reporter gene assay to
identify promotors that are speci¢cally induced by the LP
/ thiocyanate system but not by hydrogen peroxide alone.
These approaches resulted in the identi¢cation of three
classes of genes : (i) genes which have been previously im-
plicated in (oxidative) stress response, such as cysJ and
lrp; (ii) genes with a known function, but unrelated to
stress response such as rfaQ, rfaB, sgaT; (iii) genes with
no assigned function to date. Together, these results sug-
gest that response and resistance to the LP system involve
a unique set of genes in E. coli. Further analysis will pro-
vide insight in the cellular targets of the LP system and in
the relation of these genes to other forms of oxidative
stress.
P9^23
FUNCTIONAL ANALYSIS OF THE LYSOZYME IN-
HIBITOR PROTEIN IVY IN E. COLI
D. Deckers(1), A. Aertsen(1), M. Atanassova(1), B. Mas-
schalck(1), C. Abergel(2), and C. W. Michiels(1)
(1) Department of Food and Microbial Technology, Katho-
lieke Universiteit Leuven, Leuven, Belgium ; (2) CNRS In-
stitute of Structural Biology & Microbiology, 13402 Mar-
seille Cedex 20, France
The product of the E. coli ORF ykfE was recently shown
to be a potent inhibitor of C-type lysozymes. This inhib-
FEMSLE Congress 2-6-03
itor, designated as Ivy, is the ¢rst lysozyme-inhibiting pro-
tein to be reported, and the objective of this work was to
study its potential function in E. coli. First we studied the
role of Ivy in E. coli sensitivity to hen egg white lysozyme.
These experiments were conducted under high pressure to
permeabilize the outer membrane for lysozyme. Compared
to the wild-type strain MG1655, lysozyme sensitivity was
increased in an ivy knock-out mutant, and reduced in the
same mutant overexpressing Ivy from a plasmid. Further,
we found that lysates of the knock-out strain displayed
lytic activity on a Micrococcus lysodeikticus cell suspen-
sion. Lysates from the wild-type and overexpression strain
did not exhibit such activity but inhibited the lytic activity
of the lysate obtained from the knock-out strain. Finally,
we observed under some conditions an increased resistance
of the ivy knock-out mutant to ampicillin, a beta lactam
antibiotic that interferes with cell wall synthesis and that
can induce autolysin activity. Together, these results sug-
gest a possible role of Ivy in (i) protecting pathogenic or
commensal E. coli against lysozyme produced by its host;
and (ii) regulating peptidoglycan metabolism.
P9^24
INFLUENCE OF THE ENVIRONMENTAL CONDI-
TIONS ON THE GROWTH OF AEROPYRUM PER-
NIX
I. Milek, B. Cigic¤, L. Pogac›nik, M. Skrt, N. Poklar Ulrih
University of Ljubljana, Biotechnical Faculty, Chair of
Chemistry, Jamnikarjeva 101, 1000 Ljubljana, Slovenija
Aeropyrum pernix is the ¢rst obligately aerobic and neu-
trophilic hyperthermophilic archaeon to be isolated. It be-
longs to Desulforococcales order in Crenarchaeota king-
dom. The fact that the majority of hyperthemophilic
Archaeas are anaerobic makes A. pernix very interesting
for laboratory experiments. Its optimum growth temper-
ature is between 90 and 95‡C and the optimum pH for
growth around 7. With the purpose to obtain high cell
densities in liquid culture, we systematically studied the
e¡ect of the medium composition and culture conditions
on growth of A. pernix. For cultivation 300 mL Nephelo
culture £asks were used. We have found out that the high-
est cell density was achieved by using 50 mL of marine
medium (Bacto Marine broth 2216, Difco) at pH 7.0 in
reciprocating water bath at 92‡C. We discuss di¡erent
types of cultivation (liquid medium, solid medium) and
the in£uence of the environmental conditions including
oxygen availability and pH on growth curve.
 1st FEMS Congress / Posters 103^505
P9^25
BEYOND THE STATIONARY-PHASE : LONG-TERM
SURVIVAL OF PSEUDOMONAS AERUGINOSA
M. Nicolo', S. Carnazza, M. Capone, S. Guglielmino
Dept. of Microbiological, Genetic and Molecular Sciences,
University of Messina, sal. Sperone, 31, 98166 Messina,
Italy
In the last decades, much attention has been focused on
survival of bacterial populations under nutrient limitation,
such as nutrient starvation and prolonged stationary
phase. Bacterial response to starvation is generally homeo-
static, but in some cases, such as phosphate starvation, the
emergence of ¢tter mutants, called PSOM, that take over
the culture has been described for Pseudomonas putida. On
the other hand, studies on prolonged stationary phase re-
vealed that, in Escherichia coli, mutants with higher ¢t-
ness, so-called ‘‘GASP’’ phenotypes, appear weekly. These
mutants, showing mutations in rpoS, are unable to enter
stationary phase and the continuous emergence of ¢tter
mutants seems to be the strategy which ensures the surviv-
al of the population. It should also be remarked that the
terms ‘‘starvation’’ and ‘‘stationary phase’’ have been used
as synonyms, by the aprioristic assumption that nutrient
starvation should be one of the limiting conditions in pro-
longed stationary phase, but little is known about the true
role of nutrient starvation in a prolonged stationary phase.
In this work, the survival mechanisms of Pseudomonas
aeruginosa have been investigated. The study has been
conducted in two parallel ways. First, as holistic criterion,
the phenomenon has been observed in a batch culture,
incubated for 24 months. Second, from a cartesian point
of view, the incidence of single variables has been ana-
lysed. The data suggest that, di¡erently from the increase
of ¢tness in E. coli, P. aeruginosa survival is based on the
modulation of cell lysis which, in turn, releases nutrients
recycled by remaining cells.
P9^26
HEAT SHOCK PROTEIN EXPRESSION IN BALB/C
(H-2d) MICE AT COURSE OF MOUSEPOX
J. Cymerys and M. G. Niemialtowski
Immunology Laboratory, Department of Preclinical Scien-
ces, Faculty of Veterinary Medicine, Warsaw Agricultural
University, ul. Ciszewskiego 8, 02-786 Warszawa, Poland
Ectromelia virus (ECTV) can develop a mousepox. It is
well known from studies with some other viruses that ex-
pression of heat shock proteins (hsp) may be modulated
during viral infections. To assess the role of hsp at di¡er-
FEMSLE Congress 2-6-03
313
ent clinical phases of mousepox (up to 20 days p.i.), liver,
spleen, brain and conjunctivae were collected and the lev-
els of hsp27, hsp70, and hsp90 were detected in studied
tissues of BALB/c mice infected with Moscow strain of
ECTV (ECTV-MOS). Results. In all examined organs
hsp27, hsp70, and hsp90 were expressed not only during
primary viraemia, but also during the peak (10-20 days
p.i.) of disease. In liver highest level of hsp27 and hsp90
was found at 1 day p.i,, whereas at 15 day p.i hsp70
predominated ( s 60% and s 80% positive cells, respec-
tively). In spleen hsp90 reached maximal expression at 5
day p.i., hsp27 at 10 day p.i., and hsp70 at 15 day p.i.
( s 80%, 65% and s 80% positive cells, respectively). Our
results con¢rmed that primary viraemia in parenchymal
organs during the course of mousepox is strongly associ-
ated with hsp expression. In the brain level of hsp90 was
signi¢cantly elevated between 1 and 5 d.p.i., then dimin-
ished to 20% positive cells. Similarly, hsp27 and hsp70
levels in brain cells were highest at 5 day p.i. In conjunc-
tivae expression of hsp accompanied the onset of clinical
sings of mousepox. Hsp27 expression reached maximum
value at 15 d.p.i., hsp70 signi¢cantly increased between 10
and 15 d.p.i., whereas hsp90 level between 1 and 15 d.p.i.
( s 80%). Conclusion. Our studies demonstates that
ECTV-MOS can modulate hsp levels at course of mouse-
pox.
This work was supported by the Foundation for Polish
Science [grant no 10/2000].
P9^27
CHARACTERIZATION OF STREPTOCOCCUS
PNEUMONIAE SERINE / THREONINE PROTEIN KI-
NASE StkP AND PROTEIN PHOSPHATASE PhpP
AND THEIR PHYSIOLOGICAL FUNCTION
L. Novakova, L. Prenosilova, J. Echenique, M.-C. Trombe
and P. Branny
Institute of Microbiology, Czech Academy of Sciences of
the Czech Republic, Videnska 1083, 142 20 Prague 4, Czech
Republic
Transmission and ampli¢cation of a signal by a network
of Ser/Thr/Tyr protein kinases plays a principal role in
di¡erentiation and cell-to-cell communication in eukary-
otes. During the last several years, however, a considerable
number of eukaryotic-type Ser/Thr protein kinases
(STKPs) were identi¢ed in prokaryotes. Being absent in
E. coli and present only in a few examples in many bac-
teria they are represented by a multigene families in Strep-
tomyces, Myxococcus, and Mycobacterium. All these bac-
teria display developmental characteristics comparable to
multicellular eukaryotes. Alternatively, STPKs have been
shown to be required for the full virulence of some patho-
gens in mouse models. Searching in the genome sequence
314 1st FEMS Congress / Posters 103^505
of S. pneumoniae revealed presence of a single eukaryotic-
like Ser/Thr protein kinase gene associated with a gene
encoding protein phosphatase. The genes were named
stkP and phpP, respetively. We expressed, puri¢ed and
characterized PhpP, StkP proteins. In vitro kinase assays
demonstrated that StkP is a functional kinase that is de-
pendent on either magnesium or manganese ions. A two-
dimensional phosphoamino acid analysis revealed strong
phosphorylation at a threonine residues. The PhpP phos-
phatase is an Mn2+-, but not a Ca2+- or a Mg2+, depen-
dent phosphatase. Its activity is inhibited NaF, but not by
okadaic acid, and is similar to that of PP2C phosphatase.
In S. pneumoniae both proteins are likely functionally
coupled as PhpP e⁄ciently dephosphorylates autophos-
phorylated form of StkP. Analysis of S. pneumoniae stkP
deletion mutant showed that protein kinase is likely in-
volved in the control of cell wall biosynthesis.
P9^28
OXIDATION STRESS AND SYSTEMS OF ANTIOXI-
DANT PROTECTION IN HETEROTROPHIC COL-
ORLESS SULFUR BACTERIA
D. A. Podkopaeva(1), M. Yu. Grabovich(1), G. A. Dubi-
nina(2)
(1) Voronezh State University, University square 1,394006,
Voronezh, Russia ; (2) Institute of Microbiology,Prospect
60-let Oktyabrya 7/2, 117811, Moscow, Russia
The in£uence of the oxygen regime of Spirillum winograd-
skii strain D-427, DSMZ 12756 cultivation on biosynthesis
processes, enzyme activity and role of di¡erent systems of
antioxidant cell protection from oxidation stress, are de-
termined. Under aerobic conditions (21% O2 in gas phase)
in contrast to microaerobic ones (2% O2 in gas phase),
bacteria growth is accompanied by changes in metabolism
^ lower activity of constructive metabolism processes and
more intense synthesis of exopolysaccharides as a way of
external cell protection from excess O2. This brings to two-
fold reduction of economic growth factor and cellular har-
vest. In spite of the fact that low activity of catalase is
compensated by a many-fold increase in the activity of
other cytoplasmic enzymes protecting from toxic O2
forms, mass lysis of cells begins in the middle of exponen-
tial phase growth and brings to the destruction of culture
in the stationary phase because of the accumulation of
H2O2 in periplasm up to 10 Wm (mg of protein)-1. No
cytochrome-c peroxidase, which is the main periplasmic
enzyme removing H2O2, is detected. The addition of thi-
osulfate to the medium results in cessation of cell lysis, 3 ^
fold increase of Qmax, and stabilization of culture as a
result of removing H2O2 in aerobic conditions. During
bacteria growth with thiosulfate, the activity of sulfur me-
tabolism enzymes of dissimilatory type is not observed,
FEMSLE Congress 2-6-03
which is indicative of microorganisms inability to lithohe-
terotrophic growth. Thus, we suggest that the main reason
of oxidation stress in cells is the spatial separation of ac-
tive systems of antioxidant protection from O2 localized in
cytoplasm and accumulation of H2O2 in periplasm be-
cause of cytochrome-c peroxidase absence.
This work was supported by the Russian Foundation of
Basic Research, grant no. 02-04-49185.
P9^29
CHANGES OF METABOLIC ACTIVITY OF ERWI-
NIA SP. GROWN UNDER SOME TYPES OF EXTER-
NAL STRESS
J. Pokorna, I. Marova, R. Koci, M. Drabkova, M. Knop-
pova
Department of Food Chemistry and Biotechnology Faculty
of Chemistry, Technical University of Brno, Czech Republic
The genus Erwinia represents nonphotosynthetic bacteria
pathogenic mostly for higher plants. The strains E. herbi-
cola and E. carotovora, respectively, are of increasing in-
terest as industrial microbes producing pectinolytic, cellu-
lolytic and proteolytic enzymes and antileukaemic
asparaginase. Cultures of Erwinia are producents of caro-
tenoids, which caused yellow-orange coloured phenotype.
The aim of this work was to study in£uence of exogenous
stress factors on genome integrity, metabolic activity and
carotenoid production by E. herbicola and E. carotovora.
Bacteria were grown aerobically (28‡C, permanent light-
ing). Exogenous stress was induced by addition of 3%
NaCl (osmotic stress), 5 mM NiCl2 and/or 5 mM H2O2
(oxidative stress). During the experiment cell morphology,
amount and composition of carotenoids (HPLC/MS), pro-
duction of neutral lipids, glycerol and extracellular pecti-
nases and proteases were followed. Chromosomal DNA
changes were followed by PFGE using macrorestriction
enzymes SmaI and NotI. Bacteria were more sensitive to
osmotic stress, a signi¢cant decrease of biomass (2.2x),
lipids (2.3x), œ- and œ-carotene (1.8x), lutein (1.8x) and
extracellular enzymes (2-3x) was observed. Osmotic stress
led to some changes in set of macrorestriction fragments
too. On the other hand, addition of H2O2 led to increased
production of lipids (1.4x), enzymes (1.1x), carotenes
(1.3x) and lutein (1.6x). Production of glycerol was 1.8x
higher under salt stress, but 1.6x lower under oxidative
stress when compared with control cultivation. Carote-
noids produced by Erwinia sp. could play a role in pro-
tection of cells from both oxidative stress in nature and
reactive oxygen species (superoxide, H2O2) produced as a
¢rst response by plant-pathogen interaction.
 1st FEMS Congress / Posters 103^505
P9^30
ROLE OF CCPA IN REGULATION OF CENTRAL
METABOLISM AND IN RESPONSE TO STRESS
CONDITION IN LACTOBACILLUS PLANTARUM
C. Castaldo(1), R. Marasco(2), L. Muscariello(1) and M.
Sacco(1)
(1) Dipartimento di Scienze Ambientali, Seconda Universi-
ta' di Napoli, Caserta, Italy ; (2) Dipartimento di Scienze
Biologiche ed Ambientali, Universita' del Sannio, Benevento,
Italy
In Gram-positive bacteria of low G+C content, CcpA (ca-
tabolite control protein A) is a master regulator which can
function either as a repressor or as an activator of tran-
scription. We characterised the ccpA gene of Lactobacillus
plantarum LM3 and isolated the LM3-2 strain carrying a
ccpA null mutation (ccpA1). We analysed proteins from
membrane and cell wall fractions of the L. plantarum
LM3 and LM3-2 strains. Proteins were extracted from
cells grown on glucose or ribose up to exponential phase.
The electrophoretic pattern of membrane proteins ex-
tracted from exponential cells grown on glucose showed
a di¡erential protein expression in the two strains. The
presence of a 49 kDa protein only in the wild type strain
suggested a CcpA-mediated activation in the expression of
this protein. The expression of 5 proteins, ranging from 44
to 67 kDa, was detected only in the mutant strain while
the expression of two other proteins of 41 and 47 kDa
resulted increased in the mutant strain, suggesting a
CcpA-dependent negative control of their expression. Dif-
ferent electrophoretic patterns were also shown in the cell
wall fractions, where we found a 45 kDa protein whose
expression resulted CcpA repressed in the wild type strain.
We also analyzed the protein expression in the wild-type
and mutant strains grown in salt stress condition, ¢nding
at least two proteins whose expression is repressed by
CcpA. We are now identifying the di¡erentially expressed
proteins to study the CcpA-mediated regulation of the
corresponding genes. We are also investigating the role
of the CcpA protein in response to starvation and envi-
ronmental changes in temperature and carbon source in
Lactobacillus plantarum.
FEMSLE Congress 2-6-03
315
P9^31
RIBOFLAVIN ENHANCES YEAST RESISTANCE TO
CHROMIUM
D. Fedorovych(1), H. Ksheminska(1), L. Babyak(1), P.
Kaszycki(2), H. Koloczek(2), A. Jaglarz(2), and A. A.
Sibirny(1,3)
(1) Institute of Cell Biology, Drahomanov Street 14/16,
79005 Lviv, Ukraine; (2) University of Agriculture, Bio-
chemistry Department, al. 29 Listopada 54, 31-425 Krako¤w,
Poland ; (3) Institute of Biotechnology, Rzeszow Univer-
sity, ul. Rejtana 16C, 35-310 Rzeszow, Poland
Some yeast strains, for example, Pichia guilliermondii, re-
sponded to Cr(VI) by stimulation of ribo£avin (RF) bio-
synthesis (Fedorovych et al., 2001). The P. guilliermondii
strain UKD 1453 incapable of RF oversynthesis possessed
higher sensitivity to Cr(VI). We suggested that extensive
£avinogenesis is a response to Cr(VI) treatment and a
mechanism leading to higher yeast survival. The e¡ect of
Cr(VI) on growth of P. guilliermondii mutants defective in
the regulation of RF biosynthesis and possessing di¡erent
levels of £avinogenesis was studied. The sensitivity of yeast
strains to Cr(VI) did not correlate with the level of £avi-
nogenic activity. However, when Cr(VI) was added to the
cultures which actively synthesized RF, growth inhibition
was diminished. Exogenous RF decreased the toxicity of
Cr(III) and Cr(YI). In the presence of Cr(III) or Cr(VI)
the growth of £avinogenic strain UKD 66 of P. guillier-
mondii and strain UKD 1453, unable to RF oversynthesis,
was characterized by a prolonged lag phase. Culture me-
dium supplementing with exogenous RF (200 Wg/ml) led to
a decrease in lag phase. Diminished toxicity of Cr(III), but
not of Cr(VI), was caused by reduced chromium uptake.
These data suggest the existence of interaction between
RF metabolism and chromium toxicity. It was shown ear-
lier that RF can decrease the nephrotoxic e¡ect of chro-
mate in young and adult rats (Appenroth et al., 1996). Our
data on RF protection of chromium sensitivity in P. guil-
liermondii strains provide evidence for RF crucial role in
chromium detoxi¢cation.
316 1st FEMS Congress / Posters 103^505
P9^32
STRESS RESPONSE OF YEAST CANDIDA INTER-
MEDIA UPON CR(III) SUPPLEMENTATION
M. Skrt(1) and P. Raspor(2)
(1) University of Ljubljana, Biotechnical faculty, Depart-
ment of Food Science and Technology, Chair of Chemistry,
Ljubljana, Slovenia; (2) University of Ljubljana, Biotech-
nical faculty, Department of Food Science and Technology,
Chair of Biotechnology, Ljubljana, Slovenia
One of the possible metal ions detoxi¢cation mechanisms
in yeast is binding metal ions with low-molecular-weight
species e.g. metallothioneins, phytochelatins, chelation by
GSH. The scope of this study was to clarify if low-molec-
ular-weight chromium binding species (LMWCr) are result
of yeast stress response upon Cr(III) supplementation. For
this purpose LMWCr were isolated from yeast biomass,
supplemented with Cr(III) in 3 mmol/L ¢nal concentra-
tion. For comparison low-molecular-weight species
(LMW) were also isolated from yeast biomass not supple-
mented with Cr(III). LMWCr and LMW were isolated
from yeast cell extract with ethanol precipitation, identi-
¢ed with SDS-PAGE and further separated on anion-ex-
change CIM-DEAE disc and gel ¢ltration on Superdex
Peptide matrix. SDS-PAGE indicated that among
LMWCr species new species were formed with molecular
weight of 45 kDa, 29 kDa, 16,9 kDa and 14 kDa, which
were not detected in control samples of LMW. Also pro-
tein content in cell extract from Cr(III) supplemented
yeast biomass was 87% higher than from control cell ex-
tract. With further separation of ethanol LMWCr and
control LMW precipitates on anion-exchange CIM-
DEAE disc, anion LMWCr and anion control LMW
were isolated. From gel ¢ltration separation of anion
LMWCr on Superdex Peptide matrix only one fraction
with apparent molecular weight of 10300 contained chro-
mium and molecular weight of control gel ¢ltration frac-
tions were much below 1000. In conclusion yeast Candida
intermedia has detoxi¢cation mechanism in form of bind-
ing metal ions with low-molecular-weight species but also
some other stress response mechanisms must be present
due to the induced other non chromium low-molecular-
weight binding species.
We are grateful to the Slovenian Ministry of Science and
Technology for ¢nancial support to M.S. and our project
(Project no.: J4-7454-490).
FEMSLE Congress 2-6-03
P9^33
STRUCTURAL STABILITY OF BACTERIAL EXTRA-
CELLULAR POLYSACCHARIDES MEASURED BY
SMALL ANGLES X- RAY SCATTERING (SAXS)
I. Dogsa(1), P. Laggner(2), M. Kriechbaum(2), I.
Mahne(1), D. Stopar(1)
(1) University of Ljubljana, Biotechnical Faculty, Depart-
ment of Food Science and Technology, Vecna pot 111, 1000
Ljubljana, Slovenia ; (2) Institute of Biophysics and X-Ray
Structure Research, Schmiedlstrasse 6A 8042 Graz, Austria
Many bacteria produce extracellular polysaccharides
(EPS), which protect bacteria against hostile environment,
help bacteria in cell adhesion, aggregation, and bio¢lm
formation. EPS was isolated from broth culture of a na-
tive marine bacterial strain isolated from the northern
Adriatic Sea. The EPS was exposed to di¡erent physico-
chemical conditions (i.e. pH, temperature and salt concen-
tration). Scattering of X-Rays at small angles (SAXS), and
DSC calorimetry was measured to determine structural
and thermodynamical stability of the EPS. SAXS scatter-
ing curve at room temperature showed increased scattering
towards small angles, indicating the presence of relatively
big particles. Temperatures in the range from 10 to 60‡C
did not produce any signi¢cant di¡erence in scattering
plot. Heat exposure at 95‡C for 30 min induced only a
slight change in the scattering curve at very small angles
(h 6 0.010). This is consistent with the EPS phase transi-
tion at 90‡C obtained with DSC calorimetry. The addition
of di¡erent mono and divalent ions in a concentration
range between 0.1 and 2M did not produce any signi¢cant
change in the scattering plot. On the other hand, di¡erent
pH values (from 1 to 11 in bu¡ered solutions) had a sig-
ni¢cant impact on the structure of the EPS. The highest
variation of the scattering curve upon pH titration was
observed in the most inner part (h 6 0.010), whereas in
the outer part of the scattering curve the system remained
stabile. Neither extremes of pH nor heat alone could com-
pletely change the system. However, when EPS was simul-
taneously treated by the combination of heat and acidity,
the system collapsed. This data suggest that EPS forms
structurally and thermodynamically a stabile system that
may help bacteria to survive in an ever changing environ-
ment.
 1st FEMS Congress / Posters 103^505
P9^34
IN VIVO 1H-NMR SPECTROSCOPY MEASURE-
MENTS OF DIFFUSIONAL WATER PERMEABILITY
IN BACTERIAL CELLS
T. Danevcic(1), A. Sepe(2), G. Lahajnar(2), D. Stopar(1)
(1) University of Ljubljana, Biotechnical faculty, Depart-
ment of Food Science and Technology, Vecna pot 111, 1000
Ljubljana, Slovenia; (2) Jozef Stefan Institute, Jamova 39,
1000 Ljubljana, Slovenia
While water crosses the lipid bilayer of cell membranes
relatively slowly, certain bacterial cells (i.e. Escherichia
coli) possess aquaporins, that facilitate rapid transmem-
brane transport of water in response to osmotic gradients.
In this study, membrane di¡usional water permeabilities,
Pd, of Gram-negative bacteria E. coli K12 and Vibrio sp.
(DSM 14379), suspended in bu¡er solution (0.02 M Tris-
HCl, 0.13 M NaCl, pH=7.4) containing 19.2 mM MnCl2,
were measured by determining the transmembrane water
exchange time, exch, using a 1H-T2 NMR technique. Water
proton NMR transverse T2 relaxation curves were ob-
tained at 20‡C on a Tecmag Apollo 100 MHz NMR spec-
trometer. The NMR transverse relaxation data were mea-
sured by the standard Carr-Purcell-Meiboom-Gill
(CPMG) pulse method, with the ^ pulse separation of
0.6 ms. The echo decay envelope was ¢tted to a theoretical
two exponential curve which is used to model the two-side
exchange of water molecules across the membrane. The
short component of the decay curve is due to the para-
magnetically doped extracellular water while the long
component is governed by the exchange time exch which
is inversely proportional to the permeability coe⁄cient Pd.
In E. coli membrane di¡usional water permeability Pd was
found to decrease signi¢cantly upon addition of 3 % etha-
nol or 5 mM HgCl2 to the cell suspension, indicating their
inhibitory action on the aquaporin channel function.
Membrane water permeability was also followed during
prophage induction with mitomycin C in Vibrio sp. in
order to determine the initial molecular events that causes
lysis of the host cell. The results suggest that 1H-T2 NMR
technique is a powerful method for in vivo monitoring of
the membrane di¡usional water permeability.
FEMSLE Congress 2-6-03
317
P9^35
RESPONSE TO METAL STRESS IN YEAST DEBAR-
YOMYCES HANSENII
I. Sydorovych, D. Fedorovych
Institute of Cell Biology, Dragomanov Street 14/16, 79005,
Lviv, Ukraine
Supplementing of culture media with Zn2+, Co2+, Mn2+ or
Cr6+ provoked the increasing of ribo£avin biosynthesis
and iron content in D. hansenii cells. It was shown that
toxicity of these metals was depended on the iron concen-
tration in the medium. Mutant strains which characterized
by elevated iron transport and high iron content in cells
were selected. Such mutants were resistant to Zn2+, Cu2+,
Co2+, Mn2+, but demonstrated di¡erent response to Cr6+.
Mutant cells possessed decreased catalase and superoxid-
dismutase activities and were sensitive to hydrogene per-
oxide in comparison to parent strain. Growth wild type
cells in media supplemented with increased concentration
of Mn2+ and Cr6+ provoked degradation of linear plasmid
DNA. The mutant cultures in the stationary phase formed
pseudomycelium as well as cells of log-phase grown in
media supplemented with metals. Mutant cells were also
characterised by increasing of £avinogenic activity. Ob-
served features of mutant phenotype as derepressed iron
transport and changed oxidative cells status was like to
phenotype of bacterial mutants with damaged Fur-repres-
sor. Our results suggest existence of similar response to
metal stress in eukaryotic and prokaryotic cells.
P9^36
CLP/SSRA MEDIATED STRESS ADAPTATION IN
STREPTOCOCCUS THERMOPHILUS
M. Varcamonti, G. Naclerio, D. Fusco and M. De Felice
University of Naples ‘‘Federico II’’, via Mezzocannone 16,
80134, Napoli, Italy
Bacterial stress adaptation has been recently studied in
great detail in a few organisms. Many proteins are in-
volved in this phenomenon, included proteases and pro-
tease co-factors. One group of these proteins is known as
Clp, whose expression is regulated by the CtsR repressor.
The proteins devoted to degradation after stress receive a
signal sequence encoded by the ssrA gene, which tails them
at the C-terminal. It is not known whether this phenom-
enon plays a general role in the control of proteolysis
under stress conditions or instead is speci¢c to particular
organisms. We studied this phenomenon in Streptococcus
thermophilus, a lactic acid bacterium with optimal growth
at 42‡C in the absence of oxygen. We made deletions of
318 1st FEMS Congress / Posters 103^505
clp(E,C,P,S), ctsR and ssrA genes of S. thermophilus and
analysed the phenotype of these mutants during various
stress adaptations. The ssrA mutant was temperature-sen-
sitive only, while mutants in other genes acquired a multi-
stress phenotype, since they were impaired in their ability
to grow or to survive to NaCl, pH, cold and heat shocks.
Protein patterns in di¡erent conditions were analysed and
one protein, induced in some mutants or in response to
some stresses, was identi¢ed and characterized.
P9^37
RECEPTORS FOR ENDOGENOUS AND EXOGE-
NOUS HYDROXAMATE SIDEROPHORES IN
STAPHYLOCOCCUS AUREUS B47 STRAIN
P. Wysocki, P. Lisiecki, J. Mikucki
Department of Pharmaceutical Microbiology, Medical Uni-
versity of Jo¤dz¤, 90-235 Jo¤dz¤, Pomorska 137, Poland
Staphylococcus aureus strain B47 has produced an endog-
enous siderophore called staphylobactin which belongs to
hydroxamic class chelators. This strain utilizes ¢ve exoge-
nous chelators as Fe(III) sources: schizokinen, 2,3-dihy-
droxybenzoylglycine, 2,3-dihydroxybenzoylserine, aero-
bactin, and acinetoferrin. These siderophores should
cooperate with corresponding membrane proteins. 2,3-Di-
hydroxybenzoic acid, rhodotorulic acid and desferriox-
amine B were not utilized Fe(III) as iron carriers. The
aim of this study was to determine which of the membrane
proteins serves as a receptor of particular siderophores
and establishing the relation between their binding and
iron uptake into the cells. Six iron-regulated proteins
have been identi¢ed in the cytoplasmatic membranes of
strain B47 cells harvested from synthetic Fe(III)-de¢cient
medium. They occurred in the 96-14 kDa region. No new
proteins were found in cell membranes harvested from
medium with excess Fe(III) iron. One protein (14kDa)
has been bound two siderophores: 59Fe-labeled staphylo-
bactin and 59Fe-labeled acinetoferrin which were uptaken
into the cells, and stimulated growth in the bioassay plate
tests. Using 59Fe-labelled ferrioxamine B it was demon-
strated that this chelator has bound to the 43kDa mem-
brane protein. Ferrioxamine B did not stimulate growth of
strain B47. The complex of 59Fe-siderophor was not trans-
ported into the cell because it was not detected in the
cytoplasmic fraction. No radioactive bands occurred on
autoradiograms of membranes with 59Fe-labelled rhodo-
torulic acid.
FEMSLE Congress 2-6-03
P9^38
COLICIN K SYNTHESIS IS POSTTRANSCRIPTION-
ALLY REGULATED BY THE STRESS ALARMONE
ppGpp
D. Zgur-Bertok(1), I. Kuhar(1,2), J. Mulec(1), Z. Podle-
sek(1), W. Gaastra(2), B. J. A. M. Jordi(2) and J. P. M.
van Putten(2)
(1) Dept. of Biology, Biotechnical Faculty, University of
Ljubljana, Slovenia; (2) Bacteriology Division, Dept. of
Infectious Diseases and Immunology, Utrecht University,
the Netherlands
Colicins are plasmid-encoded bacteriocins, synthesized by,
and active against Escherichia coli cells and sometimes
cells of closely related species such as Shigella and Salmo-
nella. Colicin producing strains occur with high frequency
among natural isolates and they have been implicated in
intraspecies population dynamics. Colicin K belongs to
the group of ColE1 pore-forming colicins which destroy
the electrochemical potential of the cytoplasmic mem-
brane. Production of the bacteriocin is encoded by three
genes, cka encoding the colicin, cki encoding immunity
and ckl encoding lysis. Synthesis of colicin K is character-
istically regulated by the SOS response via the LexA re-
pressor. Additionally, synthesis is growth phase dependent
and induced by nutrient depletion due to an increase in
ppGpp. Comparison of speci¢c mRNA levels before and
after nutrient starvation demonstrated that ppGpp acts
posttranscriptionally. Synonymous replacement of rare co-
dons by more frequently used ones at the 5’ end of the cka
mRNA, as well as increasing the availability of the rare
leu tRNA, abolished ppGpp mediated regulation. ppGpp
thus regulates colicin K synthesis via a novel post-tran-
scriptional mechanism that is based on rare codon usage
and variable cognate tRNA availability. Colicins are re-
leased semi-speci¢cally by cell lysis therefore, genes of the
pore forming colicins should be expressed di¡erentially so
that only a part of the population expresses the activity
and lysis genes. To observe expression of the colicin K
activity and immunity protein genes at the single cell level
through the growth cycle, transcription fusions of the cka
and cki promoters and the promoterless gfp were prepared
on the natural colicin K encoding plasmid pColK-K235.
Fluorescence microscopy revealed that the cka gene is ex-
pressed in only 3% of the bacterial population upon in-
duction by nutrient starvation while the immunity gene cki
is expressed in the large majority of the cells.
 1st FEMS Congress / Posters 103^505
P9^39
IMPROVEMENT OF DELTA-ENDOTOXIN PRO-
DUCTION BY HEAT AND SALT STRESS IN THE
INSECT PATHOGEN BACILLUS THURINGIENSIS
D. Gribi, N. Zouari and S. Jaoua
Laboratory of Biopesticides, Centre of Biotechnology of
Sfax, P.O. Box H K g, 3038 Sfax, Tunisia
The Gram-positive bacterium, Bacillus thuringiensis (Bt), is
characterised by the production of insecticidal crystal pro-
teins termed delta-endotoxins which exhbit speci¢c larvici-
dal toxity upon ingestion by susceptible insect larvae. Syn-
thesis of delta-endotoxins occurs concomitantly with
sporulation. The success of use of Bt as insecticide, cover-
ing more than 90% of the bioinsecticides, was based on
their use as mixtures of crystals and spores, all easily har-
vested from low-cost fermentation broth. Previouly, we
developed cheap culture media and appropriate fermenta-
tion procedures as well as overcome of delta-endotoxin
synthesis limitations for the low-cost production. Here,
we report possibilities to improve delta-endotoxins pro-
duction as a consequence of responses of Bt strains to
low levels of heat and salt stress. Conditions were estab-
lished which allowed the cells to adapt to heat and salt.
Each stressor results di¡erently in the improvement of
delta-endotoxins production, but both were shown to be
more e⁄cient at the beginig or the mid-exponential phase
of the cultures which become resistant at the stationary or
the sporulation steps. Heat stress caused an increase of
84% in synthesis yields of the sporulating cells, leading
to 38% delta-endotoxins production improvement with
24% less spores. In contrast, salt caused increase of 25%
of spores counts, corresonding to 28% toxins production
improvement. The synthesis yields of the spores were
slightly increased. Combined e¡ects of both stressors
lead to toxins production improvement of 66%, yied im-
provement of 40% and similar spores counts t the un-
stressed culture. Similar results were obtained by using
cheap production medium already optimised for the low-
cost production of bioinsecticides.
FEMSLE Congress 2-6-03
319
P10^1
CHEMILUMINESCENCE-BASED DETECTION OF
ENTEROCOCCUS FAECALIS IN WATER SUPPLIES
Ph. Andre, S. Petey, C. Metzger and D. J.-M. Vidon
Universite¤ Louis Pasteur, Faculte¤ de Pharmacie, INSERM
U-392, Laboratoire de Bacte¤riologie et Cryptogamie, 74
route du Rhin, 67400 Illkirch, France
The genus Enterococcus comprises 24 di¡erent species in-
cluding E. faecalis, E. faecium, E. durans as most common
species. Enterococci are ubiquitous bacteria belonging to
the human and animal intestinal £ora and are found in
waste water, soil and various plants. They are present in
human faeces of about 90% of healthy adults. For that
reason Enterococci are direct or indirect markers of faecal
contamination of water. It was shown by Huycke M.M et
al. [1] that E. faecalis produces extracellular reactive oxy-
gen species (ROS) such as superoxide (O2-) ions. Several
assessment methods have been used to quantitate ROS,
particularly the chemiluminescence (CL) assay. We previ-
ously shown that addition of luminol resulted in a 10-fold
increase in CL intensity emitted by other ROS-producing
bacteria, Listeria spp. [2, 3]. We used this phenomenon to
detect and rapidly count microcolonies of E. faecalis in
water supplies. Twelve strains of E. faecalis, isolated
from di¡erent water samples, emitted a high chemilumi-
nescence, around 105 relative light units (RLU), in the
presence of luminol (10-2 M) and sodium carbonate (1
mM), compared to 103 RLU emitted by other bacteria
such as E. coli, P. aeruginosa, B. cereus or S. aureus under
the same conditions. Kinetic studies of CL by E. faecalis
show a close parallelism between CL and growth curves
during the exponential phase, with a maximum of CL
reached just before entrance of bacteria in the stationary
phase. Beyond this critical point, during the stationary
phase, CL decreases rapidly. CL o¡ers a promising rapid
new method to survey microbiological quality of water
supplies. [1] M.M. Huycke et al (1996) J. Infect. Diseases
173: 743-746. [2] D.J-M Vidon et al. (1994) FEMS Micro-
biol. Lett. 120: 225-230. [3] D.J-M Vidon et al. (2001) J.
Appl. Microbiol. 90: 988-993.
320 1st FEMS Congress / Posters 103^505
P10^2
ANTIBACTERIAL ABILITIES OF INTESTINAL MI-
CROFLORA IN CULTURED AND RIVER JUVENILE
ATLANTIC SALMON (SALMO SALAR L.)
V. Arbac›iauskiened
Institute of Ecology, Vilnius University, Akademijos St. 2,
Vilnius 2600, Lithuania
The ¢sh digestive tract is colonized by a great number of
heterotrophic bacteria: aerobes, facultative and obligate
anaerobes. The intestinal bacteria community takes an ac-
tive part in the metabolic processes required for vital ac-
tivities of both the very bacteria and the macro-organism.
As bacterio£ora synthesizes enzymes, vitamins, amino
acids and participates in infection-protective reactions,
the physiological state of the macro-organism is closely
related to the activities of intestinal micro£ora. Bacterial
strains were isolated from the intestinal tract of cultured
and river juvenile Atlantic salmon (Salmo salar L.) and
examined for their antibacterial abilities against Escheri-
chia coli, Staphylococcus aureus, Bacillus subtilis, Candida
sp. and Flavobacterium columnare, which is causing colum-
nare disease. A total 200 isolates were classi¢ed into Aci-
netobacter, Aeromonas, Enterobacteriaceae, Pseudomonas
and Micrococcus. Aeromonas and Enterobacteriaceae
were dominant taxonomic groups in the intestinal tract
of juvenile Atlantic salmon. All tested taxonomic groups
exhibited antibacterial activity against ¢ve target strains.
These results suggest that intestinal bacteria may inhibit
some ¢sh pathogenic bacteria by producing antibacterial
substances.
P10^3
PHOSPHATE LIMITATION IN CLOSTRIDIUM ACE-
TOBUTYLICUM
R.-J. Fischer, M. Mix, S. Oehmcke, T. Fiedler, K. Schwarz
and H. Bahl
Department of Biological Sciences, Division of Microbiol-
ogy, University of Rostock, Albert-Einstein-Str. 3, D-18051
Rostock, Germany
Limitation of growth by the amount of phosphate sup-
plied in the medium favours the onset of sporulation in
clostridia. In addition, in Clostridium acetobutylicum phos-
phate limitation in combination with pH values below 5
results in a metabolic shift from acid to solvent formation.
To elucidate the role of phosphate limitation within the
complex regulatory network of sporulation and solvento-
genesis, we have started to analyse the phosphate regulon
of C. acetobutylicum. The mRNA levels of two operons
FEMSLE Congress 2-6-03
encoding a two-component regulatory system (tentatively
designated phoPR) and an ABC-transport system (tenta-
tively designated pstSCABphoU), respectively, were found
to be up-regulated under phosphate limitation. Using the
lacZ gene of Thermoanaerobacterium thermosulfurigenes
EM1 as a reporter gene, a promoter which responds in
C. acetobutylicum to low external phosphate concentra-
tions could be located on a 165-bp fragment in front of
pstS. Putative Pho boxes will be con¢rmed using the pu-
ri¢ed regulator PhoP of C. acetobutylicum for gel shift and
footprint experiments. Interestingly, Northern blot analy-
sis revealed that in a phosphate-limited chemostat at a
pH-value of 4.5 (conditions optimal for solvent produc-
tion) the level of the pstS-speci¢c mRNA in C. acetobuty-
licum is decreased by a factor of eight and of the total pst
operon by a factor of two. Under the assumption that no
alternate high-a⁄nity phosphate uptake system becomes
active under these conditions, it might be that the im-
paired supply of cells with phosphate is crucial for the
initiation of solvent formation (and sporulation). In agree-
ment with our hypothesis we observed faster phosphate
consumption in cultures of C. acetobutylicum at higher
pH values.
P10^4
TOUR MEDIATED GRATUITOUS ACTIVATION OF
THE SIGMA54-DEPENDENT PTOMO PROMOTER :
GENETIC AND PHYSIOLOGICAL STUDIES
D. Solera(1), F. Arenghi(1), T. Woelk(1), E. Galli(1) and
P. Barbieri(2)
(1) Dept. Genetics and Biology of Microorganisms, Univer-
sity of Milan, 20100 Milano, Italy ; (2) Dept. Structural
and Functional Biology, University of Insubria, 21100 Var-
ese, Italy
Expression of the Pseudomonas stutzeri OX1 toluene-o-xy-
lene monooxygenase encoding operon is driven by the
sigma54-dependent Ptomo promoter and is controlled by
the activator protein TouR, which belongs to the XylR/
DmpR subgroup of NtrC-like proteins. Contrarily to oth-
er regulators belonging to the same subfamily, which need
to bind an e¡ector molecule to promote transcription, in
vivo TouR can activate transcription from the Ptomo in
the absence of e¡ectors. Interestingly, this gratuitous acti-
vation is not constitutive, but occurs only at the onset of
the stationary phase. This phenomenon is speci¢cally im-
posed by TouR also to other sigma54-dependent pro-
moters. Monitoring the physical form of TouR during
the growth ruled out that it undergoes major covalent
modi¢cations. Dissection of the touR gene suggested that
the AB domain encoding region is necessary and su⁄cient
to impose gratuitous activation capability to hybrid regu-
lators. As the TouR mediated gratuitous activation takes
 1st FEMS Congress / Posters 103^505
place at the transition phase, it is evident that a physio-
logical control is superimposed to the speci¢c regulatory
system. Investigation about the link between the physio-
logical status of the cells and gratuitous activation dem-
onstrated that it is speci¢cally triggered by carbon starva-
tion and ruled out an involvement of the stationary phase
sigma factor sigma s as well as of the alarmone ppGpp.
P10^5
ENZYMES OF PROPOSED ANAPLEROTIC ACE-
TATE OXIDATION PATHWAY, THE CITRAMA-
LATE CYCLE, IN PURPLE BACTERIUM RHODO-
SPIRILLUM RUBRUM
I. A. Berg(1), G. Fuchs(2), R. N. Ivanovsky(1)
(1) Department of Microbiology, Moscow State University,
Moscow 119899, Russia ; (2) Institut Biologie II, Universi-
ta«t Freiburg, Scha«nzlestrasse 1, D-79104 Freiburg, Ger-
many
A new cyclic anaplerotic pathway of acetate oxidation to
glyoxylate, the citramalate cycle, has been proposed for
isocitrate lyase-negative purple bacterium Rhodospirillum
rubrum. In this cycle, acetyl-CoA and pyruvate are con-
verted to glyoxylate and propionyl-CoA, and citramalate
is a characteristic intermediate of this process. Propionyl-
CoA is further converted to pyruvate (acetyl-CoA accep-
tor), completing the cycle. To further elucidate functioning
of the citramalate cycle, we studied reactions leading to
citramalate synthesis from acetyl-CoA and pyruvate and
subsequent conversion of citramalate to propionyl-CoA
and glyoxylate. Extracts of acetate-grown R. rubrum cells
catalyzed condensation of acetyl-CoA and pyruvate to cit-
ramalate. We have also detected formation of mesaconyl-
CoA from propionyl-CoA and glyoxylate with L-methyl-
malyl-CoA as an intermediate, as well as mesaconase cat-
alyzing conversion of L-citramalate to mesaconate. Based
on the above ¢ndings, the following sequence of reactions
can be proposed for this part of the citramalate cycle:
acetyl-CoA + pyruvate ! L-citramalate ! mesaconate
! mesaconyl-CoA ! 3-methylmalyl-CoA ! propionyl-
CoA + glyoxylate. These results provide additional evi-
dence for the operation of citramalate cycle in acetate-
grown R. rubrum cells.
I.B. was supported by FEMS Fellowship.
FEMSLE Congress 2-6-03
321
P10^6
THE MUR SYNTHETASES: SPECIFICITY AND
STRUCTURE-ACTIVITY RELATIONSHIPS
M. Herve¤, S. Dementin, A. Boniface, A. Bouhss, D. Men-
gin-Lecreulx and D. Blanot
Enveloppes Bacte¤riennes et Antibiotiques, UMR 8619
CNRS, Ba“timent 430, Universite¤ de Paris-Sud, 91405 Or-
say, France
The Mur synthetases are essential enzymes of the peptido-
glycan biosynthetic pathway. They catalyse the successive
additions of L-alanine (MurC), D-glutamate (MurD), dia-
minopimelate or L-lysine (MurE), and D-alanyl-D-alanine
(MurF) onto nucleotide precursor UDP-N-acetylmuramic
acid. A ¢fth (non-essential) enzyme, Mpl, catalyses the
direct addition of tripeptide L-Ala-D-Glu-diaminopime-
late released during the recycling process of peptidoglycan.
Owing to their occurrence only in eubacteria, the MurC,
D, E and F enzymes are good targets for the search for
new antibacterial agents. Although the Mur synthetases
share the same reaction mechanism and act on substrates
of comparable chemical structure, their speci¢city is very
strict. In order to better understand the reasons for their
speci¢city despite their functional similarity, we have tak-
en as models two couples of enzymes di¡ering only in the
nucleophilic substrate: 1‡) MurC and Mpl, which add L-
Ala and the tripeptide, respectively; 2‡) MurE from Es-
cherichia coli (addition of meso-diaminopimelate) and
Staphylococcus aureus (addition of L-lysine). In this com-
munication, we will report on the following aspects of this
study : i) the overproduction and the puri¢cation of Mpl
from E. coli and MurE from S. aureus; ii) the determina-
tion of the kinetic parameters of these enzymes; iii) the
preparation of analogues of the peptide substrate of Mpl
and their recognition by the enzyme; iv) the elucidation,
by site-directed mutagenesis, of the role of the amino acid
residues of the amino acid-binding pocket of the MurE
enzymes; v) the construction of chimeras (E. coli MurC/
E. coli Mpl; E. coli MurE/S. aureus MurE) as an attempt
to permute their speci¢city.
P10^7
REGULATION OF ACTIVITY AND amoA mRNA IN
NITROSOSPIRA BRIENSIS
A. Bollmann, A. M. Saunders and M. H. Nicolaisen
University of Aarhus, Department of Microbial Ecology, Ny
Munkegade Bld. 540, DK-8000 Aarhus C, Denmark
Ammonia-oxidizing bacteria generate their energy by oxi-
dizing ammonium to nitrite catalyzed by the ammonia
322 1st FEMS Congress / Posters 103^505
monooxygenase. In nature they are facing very often
phases of £uctuating ammonium availability. So they
have to react very quickly to the changing conditions. In
this study we want to investigate the relationship between
ammonia-oxidizing activity and expression of mRNA of
the ammonia monooxygenase in ammonia-oxidizing bac-
teria and the response of these parameters to ammonium
starvation.Therefore we used Nitrosospira briensis grown
under controlled conditions in batch culture. N. briensis
was cultivated in coculture with the nitrite-oxidizing bac-
terium Nitrobacter winogradskyi, to eliminate problems
due to nitrite toxicity. The ammonia-oxidizing activity
was followed continuously with a NOx-biosensor detecting
the nitrite and nitrate concentration after the addition of
fresh ammonium to washed and concentrated cells. The
presence of mRNA was detected by RT-PCR with amoA
speci¢c primers. Ammonia-oxidizing bacteria were active
as long as ammonium was available in the culture. When
adding new ammonium to a culture starved for ammo-
nium the ammonia-oxidizing bacteria regained activity
within 30-60 min. AmoA mRNA could be detected, if am-
monium was present in the culture. These results indicate
that ammonia-oxidizing bacteria are able to reactivate the
ammonia-oxidizing activity very fast after starvation for
ammonium.
P10^8
EFFECT OF TELLURITE ON MEMBRANE REDOX
COMPONENTS OF THE PHOTOTROPHIC BACTE-
RIUM RHODOBACTER CAPSULATUS
R. Borghese, F. Borsetti and D. Zannoni
Department of Biology, University of Bologna, 42 Irnerio,
40126, Bologna, Italy
Potassium tellurite (K2TeO3) has long been known for its
antimicrobial properties and for being toxic to eukaryotic
cells and microrganisms. Some Gram-positive bacteria
show an intrinsic low-level resistance to TeO32- (50-120
Wg/ml), while high-level resistance has been determined
in certain Gram-negative obligate aerobic photosynthetic
species (500-2500 Wg/ml). As a reference, Escherichia coli
growth is inhibited at a tellurite concentration as low as 1
Wg/ml. Growth in the presence of tellurite is often associ-
ated to reduction to elemental tellurium that leads to
blackening of the cells due to internal or periplasmic ac-
cumulation of Te. The photosynthetic gram-negative bac-
terium Rhodobacter capsulatus belongs to the purple non-
sulfur bacteria, which include several species with resis-
tance levels ranging from 50 to 800 Wg/ml. In this species
there is a growth mode dependence on tellurite : photo-
synthetic anaerobic cultures grow in the presence of up
to 250-300 Wg/ml TeO32- ; respiratory aerobic cultures are
inhibited at tellurite levels below 10 Wg/ml. Rba. capsulatus
FEMSLE Congress 2-6-03
cells grown in the presence of tellurite show a decreased
level of c-type cytochromes, which is paralleled by a low
cyt-c oxidase activity and with an NADH dependent res-
piration which is almost totally driven by the quinol oxi-
dase pathway. Preliminary results indicate that oxygen
may play a role in determining the response of the cell
to TeO32-, even when present at concentrations that are
compatible with ‘‘orthodox’’ laboratory light-dependent
anaerobic growth. The possibility that tellurite exerts its
indirect toxic action by altering the redox processes asso-
ciated with respiratory and photosynthetic electron trans-
port chains will be discussed.
Work was supported by MIUR (PRIN2001)
P10^9
DYNAMICS OF BENZENE DEGRADATION BY
PSEUDOMONAS PUTIDA F1 AND RALSTONIA
PICKETTII PKO1 IN THE PRESENCE OF AN AL-
TERNATIVE SUBSTRATE
M. Bucheli-Witschel, I. Ru«egg, T. Hafner and T. Egli
Swiss Federal Institute for Environmental Science and Tech-
nology (EAWAG), U º berlandstrasse 133, 8600-Du«bendorf,
Switzerland
Benzene, toluene, ethylbenzene, and the xylenes (BTEX)
are hazardous aromatic compounds contained in various
petroleum products. Collectively they are amongst the
most commonly reported groundwater contaminants.
Many strains were isolated for their ability to degrade
one or several BTEX compounds, among these is Pseudo-
monas putida F1 and Ralsonia pickettii PKO1, which uti-
lize benzene, toluene, and ethylbenzene as carbon and en-
ergy source for growth. Genetically and biochemically the
pathways for the degradation of these compounds have
been elucidated in both strains. However, little is known
on the regulation of BTEX degradation. Therefore, we are
investigating the degradation of benzene by the strains
grown under batch or continuous culture conditions in
the presence of an alternative substrate, i.e. succinate.
The main focus lies on the dynamics of regulation when
transients are imposed on continuously growing cultures.
These transients comprise switches of the medium feed,
e.g. from succinate as sole carbon and energy source to
mixtures of succinate and benzene or to benzene only.
Moreover transients in the oxygen supply will be investi-
gated. First experiments with P. putida F1 revealed se-
quential growth in batch culture when a mixture of succi-
nate and benzene was supplied and succinate was the
preferred substrate. During transients from succinate
only to a mixture of succinate and benzene up-regulation
of benzene degradation was observed within two hours
after the switch whereas when the medium feed was
switched from succinate to benzene only the lag period
 1st FEMS Congress / Posters 103^505
until up-regulation occurrred was markedly longer result-
ing in wash-out of the bacteria.
P10^10
IN VITRO CLONING OF ACTINOBACILLUS PLEU-
ROPNEUMONIAE APX II GENE IN ESCHERICHIA
COLI
R. Burdychova¤(1), R.Horva¤th(2), M. Dendis(2), M.
Rychtera(1), L. Baba¤k(1) and M. Bartos›(2)
(1) BUT Brno, Department of Food Chemistry and Bio-
technology, Faculty of Chemistry, Purkyn›ova 118, 612 00
Brno, Czech republic ; (2) Genex CZ, s.r.o., Podstra¤nska¤
74, 627 00 Brno, Czech Republic
Actinobacillus pleuropneumoniae is a Gram-negative bacte-
rium which is the ethiological agent of porcine pleuro-
pneumonia, an acute or chronic respiratory infection of
pigs. Epidemiological data suggest that virulence is
strongly correlated with the presence of Apx toxins (Ja-
cobsen et al, 1996). Discoveries concerning the immuno-
genicity of A. pleuropneumoniae Apx toxins in infected
pigs have stimulated studies trying to achieve protection
from A. pleuropneumoniae by use of these toxins (Cray et
al., 1993). This study presents the cloning and expression
of A. pleuropneumoniae Apx II toxin in the pCRT7 TOPO
TA expression system (Invitrogen) to produce puri¢ed
protein for immunological studies. The gene coding Ap-
x II toxin was ampli¢ed from A. pleuropneumoniae sero-
type 4 DNA by PCR with primers APXIIAF 5a¤-atgt-
caaaaatcactttgtc-3a¤ and APXIIAR 5a¤-
¤
ttaagcggctctagctaatt-3a. The resulting fragment
(2871 bp) was cloned into the pCRT7/NT TOPO cloning
vector under control of the T7 promoter and introduced
into E. coli cells. The presence of insert was con¢rmed by
PCR and sequencing. The pCRT7/NT TOPO cloning vec-
tor was constructed ny Invitrogen to allow foreign pep-
tides to be expressed in E. coli as fusion proteins. Expres-
sions were practised in the laboratory fermentor
(B.Braun). The gene coding the Apx II toxin was extended
with a segment encoding a polyhistidine tag linked to its
C-terminal sequence allowing a one-step a⁄nity puri¢ca-
tion of the complex. Expression of Apx II in E. coli re-
sulted in the formation of insoluble inclusion bodies. The
inclusion bodies were solubilized and mixed with Ni-NTA
resin (Qiagen, USA). Puri¢cation according to a standard
puri¢cation protocol and imidazole elution was success-
fully used. The ease of this expression system, the powerful
single step puri¢cation and low cost make it possible to
produce Apx II toxin in large amounts for further study of
its physiological role.
FEMSLE Congress 2-6-03
323
P10^11
THE FIRST PARTIALLY SEQUENCED ENDOXYLA-
NASE FROM RUMEN BACTERIUM PSEUDOBU-
TYRIVIBRIO XYLANIVORANS MZ5T
T. Cepeljnik(1), M. Zorec(1), I. Krizaj(2), F. V. Nek-
rep(1), R. Marinsek-Logar(1)
(1) University of Ljubljana, Biotechnical Faculty, Zootech-
nical Dept., Groblje 3, SI-1230 Domzale, Slovenia; (2) In-
stitute Josef Stefan, Jamova 39, SI-1111 Ljubljana, Sloven-
ia
Rumen bacterium Pseudobutyrivibrio xylanivorans Mz5T
has a very potent enzyme system for xylan decomposition.
It is highly inducible and includes at least 11 electrophor-
etically distinct, cell bound xylanases which are excreted
into the growth medium, too. The smallest 29,5 kDa xy-
lanase is very interesting because of representing the ma-
jority of the xylanolitical activity in the later growth
phases. It was isolated by ammonium sulphate precipita-
tion and further hydrophobic interaction chromatography
to the electrophoretic homogeneity. Its speci¢c endoxyla-
nase nature was demonstrated : it degraded only xylan
from di¡erent sources and the smallest hydrolysis product
was xylotetraose. No cleavage of xylan backbone substi-
tuting sugars was detected. The highest activity was ob-
served with soluble xylan, whereas it barely degraded in-
soluble xylan fraction. No carbohydrate binding ability
was detected. Its optimal activity was observed at 38‡ C
and at pH 5,5, and its pI was 5,1. Its partial sequence was
obtained by Edman degradation and then PCR primers
were designed based on the sequences of the most homol-
ogous xylanases. Our xylanase belongs to clan GH-C of
glycosyl hydrolases, family 11, group B and has some
conserved regions and amino acids, especially in active
site: E94 ^ the nucleophyle, E 184 ^ acid/base catalyst
and Y96 that links both catalytic glutamates, acting as a
charge stabilizing residue. Regarding its high xylan de-
grading ability at physiologic conditions in animal gastro-
intestinal tract it could be used as feed additive in order to
improve forage digestion and animal health.
324 1st FEMS Congress / Posters 103^505
P10^12
INVOLVEMENT OF AZOSPIRILLUM EXTRACEL-
LULAR ENZYMES IN THE BACTERIAL PENETRA-
TION OF WHEAT-ROOT TISSUES
M. P. Chernyshova, V. V. Ignatov
Institute of Biochemistry and Physiology of Plants and Mi-
croorganisms, Russian Academy of Sciences, Saratov, Rus-
sia
In order to enter the plant-tissue interior, bacteria need to
overcome the plant cell wall, which is composed of a com-
plex of polysaccharides. In this work, we wanted to study
the properties of the extracellular pectolytic and proteo-
lytic enzymes from Azospirillum bacteria and the possible
involvement of these enzymes in the bacterial penetration
of wheat-root tissues. A. brasilense Sp7, A. brasilense
Sp245, A. irakense KBC1, and seedlings of the wheat cul-
tivar Saratovskaya 29 were used as the objects of study. A
Ca2+-dependent pectate-lyase activity (with an optimum at
pH 7,5-9,5) and Ca2+-dependent proteolytic activity (with
an optimum at pH 5,5 and 8,8) were found in the culture
liquid of all strains studied. We showed by zymography
that the azospirilla had not less than six forms of proteo-
lytic enzymes (molecular masses, 32-172 kDa) and one
pectate-lyase (molecular mass, 45 kDa). Among the A.
brasilense Sp7 and Sp245 bacteria that had passed through
the plants and showed enhanced enzyme activities (a 10,5-
fold increase in pectolytic activity and 7,5-fold increase in
proteolytic activity), the number of cells capable of pene-
trating the root-tissue interior was higher than that of cells
of the parent strains. There was no statistically certain
di¡erence in numbers between the interior population of
A. irakense KBC1, whose enzyme activity had not changed
during passage through the wheat plants, and the popula-
tion of the parent strain. These facts point to the close
association of extracellular-enzyme activity with the ability
of the bacteria to colonize the wheat-root-tissue interior.
P10^13
MICROORGANISMS ABLE TO ACCUMULATE
IRON COMPOUNDS
N. V. Chertov
Astrakhan State Technical University, 16 Taticheva Street,
414025 Astrakhan, Russia
Processes of bacterial interaction with iron compounds
have been attracting microbiologists attention for more
than 100 years. Several bacteria are able to accumulate
ferromagnetic minerals ^ magnetite, greigite in environ-
ment and also in their cells ^ the last case stipulate their
FEMSLE Congress 2-6-03
capability to magnetotaxis. This research investigated nat-
ural water basins in the area of the Low Volga for mag-
netotactic bacteria (MTB) with the aim: to study physical
and chemical parameters that help compounds’ biominer-
alization; to investigate the species content of water and
silt microecosystems that have magnetic characteristics ; to
single out accumulating culture of MTB, determine its
magnetic characteristics and establish its identity. The fol-
lowing methods were used: photometrical, atomic and ab-
sorbing method, chromate-mass-spectrometric, phyloge-
netic and also analytical scanning and enlightening
electronic microscopy. Investigation showed that there
can be iron compounds’ biomineralization in the Low
Volga basin in the condition of low content of diluted
organic substance and higher iron concentration both in
water and silt and in low alkaline environment. Silt micro-
organisms with high parameters of magnetic repletion are
presented by 23 species of bacteria that belong to 19 fam-
ilies. Accumulating culture of MTB was determined in the
model system of Lake Belyachnoye, characterized by low
alkaline environment, low content of diluted organic sub-
stance, higher content of general iron and by high mag-
netic silt repletion. It was established that the MTB have
the capability to magnitotaxis and biomineralization of
intercellular iron compounds. Phylogenetic analysis
proved that the bacteria belong to K-subclass of proteo-
bacteria. Inside this group the bacteria was closest to the
Magnetospirillum family, showing not less than 96% sim-
ilarity of sequences. Identi¢cation as Magnetospirillum
magnetotacticum was proven by the high level of similarity
in 16s rRNA gene sequence between the MTB and this
type species (99.6%).
P10^14
SUPEROXIDE DISMUTASE BIOSYNTHESIS IN DE-
SULFOVIBRIO DESULFURICANS
M. N. Davydova, R. Z. Sabirova, N. B. Tarasova and M.
N. Stepanova
Kazan Institute of Biochemistry and Biophysics Russian
Academy of Sciences, P.O. Box 30, Kazan 420111, Russia
In recent years many intensive investigations devoted to
the mechanisms of antioxidant defense system in anaero-
bic bacteria have been performed. Some sulfate-reducing
bacteria are considered to be aerotolerant and can resume
anoxic growth even after prolonged exposure to oxygen.
Most of them contain superoxide dismutase and catalase
and in some of them piridindinucleotide-peroxidases have
been detected. At least two defensive mechanisms against
reactive oxygen species have been suggested for sulfate-
reducing bacteria now: (i) the dismutation of superoxide
radical anions to molecular oxygen and hydrogen peroxide
that catalyses by superoxide dismutase (SOD) and (ii) the
 1st FEMS Congress / Posters 103^505
reduction of of superoxide radicals by superoxide reduc-
tase. It has been shown that sulfate-reducing bacteria D.
desulfuricans B-1388 can eliminate an reactive oxygen spe-
cies from reaction medium due to its SOD. The activity of
enzyme was independent of the growth phase of cells and
estimated as 0.9-1.0 units per mg protein. The SOD activ-
ity in D. desulfuricans was mainly associated with periplas-
mic fraction. The enzyme was puri¢ed from a periplasm of
D. desulfuricans. The e¡ect of pH and temperature on
SOD stability was studied. SOD is an attractive object
for investigation in the light of its role in physiological
adaptation of the anaerobes to oxidative stress.
P10^15
IDENTIFICATION AND CHARACTERIZATION OF
NOVEL MYCOLOYLTRANSFERASES IN CORYNE-
BACTERIUM GLUTAMICUM
C. De Sousa-D’Auria(1), R. Kacem(2), M. Chami(3), P.
Gounon(4), V. Puech(2), M. Tropis(2), G. Leblon(1), C.
Houssin(1) and M. Da¡e¤(2)
(1) BMII, (UMR 8621), IGM ^ Ba“t 409, University of
Orsay, 91405 Orsay, France; (2) IPBS (UMR 5089), Uni-
versite¤ Paul Sabatier, 31077 Toulouse, France; (3) M.E.
Mu«ller Institute Biozentrum, University of Basel, CH-4056
Basel, Switzerland ; (4) INSERM (U 452), UFR de Me¤-
decine, 06107 Nice Cedex 02, France
Mycolic acids, long chain (C70-C90) K-alkyl, L-hydroxy
fatty acids, are characteristic cell envelope components
of mycobacteria; similar but shorter chain acids occur in
corynebacteria and related taxa. These compounds are be-
lieved to play an important role in the physiology of these
bacteria. We have previously shown that PS1, a protein
encoded by the csp1 gene of Corynebacterium glutamicum,
possesses a mycoloyltransferase activity. Disruption of the
csp1 gene has led to the decrease of cell wall-linked cor-
ynomycolate by 50%, suggesting the occurrence of other
mycoloyltransferases. By sequence comparison with the
genes fbpA-C, encoding the antigen 85 complex of Myco-
bacterium tuberculosis, six genes mytA ^ F were character-
ized in C. glutamicum. These genes were inactivated and
the mutants were biochemically characterized. Compari-
son of their content in mycolates linked covalently to ara-
binogalactan and acyltrehaloses demonstrated that MytA,
B, D and F, but not MytC and E, were involved in the
transfer of corynomycoloyl residues onto arabinogalactan
and trehalose. A cmytA-/cmytB-disrupted double mutant
was also constructed and analyzed. The mutant strain ex-
hibited a growth defect and formed segmentation-defective
cells. Biochemical analyses showed that the mutant elabo-
rated less cell wall-bound corynomycolates and produced
less cristalline surface layer proteins associated with the
cell surface than did the cmyt-inactivated single mutants.
FEMSLE Congress 2-6-03
325
Electron microscopy analyses showed that the double mu-
tation had a profound impact on the integrity of the out-
ermost part of cell envelope of the mutant strain.Together
the data indicated that mycoloyltransferases are crucial for
the physiology of Corynebacterinea.
P10^16
THE ROLE OF OXYGEN IN METABOLISM REGU-
LATION OF AEROTOLERANT SPIROCHETES
FORMING SULFUR MATS OF THIODENDRON
G. A. Dubinina and M. Y. Grabovich
Institute of Microbiology of Russian Academy of Sciences,
7/2 Prospect 60-letiya Oktyabrya, Moscow 117312, Russia
Bacterial sulfur mats of ‘‘Thiodendron’’ is spread widely in
bottom sediments of marine, oceanic (regions of volcanic
and hydrothermal activity) and sul¢de springs ecosystems.
Anaerobic spirochetes are the main structural and forming
component accumulating elemental sulfur intracellulary.
The development of Thiodendron mats take place in micro-
aerobic zones of simultaneous incoming of H2S and O2.
Some strains of anaerobic spirochetes were isolated from
sulfur mats of di¡erent saline ecosystems : strain ‘‘P’’ ^
from mineral spring and strain ‘‘WS’’ ^ from littoral of
White Sea. These strains have fermentative type of metab-
olism and are aerotolerant. They grow preferably under
microaerobic conditions at concentration about 0.2-0.5 mg
O2 per liter. Some parameters (biomass yields, end prod-
ucts of glucose utilization, ATP generation, H2O2 accumu-
lation as well as some enzyme activities) have been deter-
mined in cells suspension under microaerobic and
anaerobic growth conditions. Metabolic processes were
essentially diversed under di¡erent growth conditions.
More e¡ective growth yield, economic coe⁄cient of glu-
cose utilization and rate of ATP generation were twice as
much at microaerobic growth. Bacterial growth was ac-
companied by exopolysaccharide accumulation in the cul-
tures and H2O2 formation in cell suspensions. Fermenta-
tive products yielded mainly acetate and CO2 in
microaerobic growth opposite mainly ethanol, formate
and H2 at anaerobic growth. In ¢rst case speci¢c activity
of acetate kinase and NADH oxidase were substantially
higher and conserve ATP via the former enzyme. NADH
regeneration via the last enzyme for glycolysis of glucose
provides advantages under oxygen-limited conditions com-
pared to anaerobic ones. The ability of spirochetes to
overcome oxidative stress of H2O2 is related to activity
of NADH peroxidase and by interactions with exogenic
reduced sulfur compounds. Elemental sulfur accumulation
in cells is occurred from sul¢de and tetrathionate is orig-
inated from thiosul¢te. So alternative pathways for glu-
cose metabolism of aerotolerant spirochetes are regulated
by oxic-anoxic conditions of growth.
326 1st FEMS Congress / Posters 103^505
P10^17
METHYLOCELLA SILVESTRIS IS A FACULTATIVE
METHANOTROPH
P. F. Dun¢eld(1) and S. N. Dedysh(2)
(1) Max-Planck-Institute for Terrestrial Microbiology,
Karl-von-Frisch Str., 35043 Marburg, Germany; (2) Insti-
tute of Microbiology, Russian Academy of Sciences, Mos-
cow, Russian Federation
A novel species of methanotrophic bacterium (Methylocel-
la silvestris) was isolated from an acidic forest cambisol in
Germany. It is a moderate acidophile (growth optimum
pH 5.5) and appears to possess only a soluble rather
than a particulate form of methane monooxygenase. Un-
like all other known methanotrophs, Methylocella silvest-
ris is able to grow not only on the one-carbon compounds
methane and methanol, but also on the two-carbon com-
pounds acetate, pyruvate, and succinate. The metabolism
of C1 and C2 compounds is coregulated. Addition of 2
mM acetate to a culture growing on methane temporarily
inhibited methane oxidation, but methane oxidation re-
sumed after the acetate was fully consumed. Addition of
acetate also decreased the threshold mixing ratio to which
methane was oxidised by Methylocella silvestris, presum-
ably because some reducing equivalents produced from
acetate catabolism were used for methane monooxygenase
functioning. To rule out the possibility that a non-meth-
anotrophic contaminant was responsible for acetate con-
sumption, culture purity was ascertained after growth on
acetate alone as well as after growth on methane alone. A
variety of microbiological and molecular tests of purity
were applied, including whole-cell hybridisation with a
newly-designed strain-speci¢c £uorescent oligonucleotide
probe. The results suggest that the population and activity
of methanotrophs in soil is not controlled by methane
supply alone. However, it is unlikely that Methylocella
silvestris is responsible for the uptake of atmospheric
methane (at 1.7 ppmv) in soils because its threshold for
methane consumption was always greater than 100 ppmv.
FEMSLE Congress 2-6-03
P10^18
PHYSIOLOGICAL AND BIOCHEMICAL FEATURES
OF GROWTH OF SULFOBACILLUS THERMOSUL-
FIDOOXIDANS 41 UNDER MICROAEROBIC CONDI-
TIONS
M. A. Egorova(1), E. N. Krasil’nikova(1), I. A. Tsapli-
na(2), L. M. Zakharchuk(1)
(1) Moscow State University, Vorob’evy gory, Moscow,
119899, Russia ; (2) Institute of Microbiology, Russian
Academy of Sciences, pr. 60-letia Octyabrya 7, k.2, Mos-
cow, 117811, Russia
The moderately thermophilic acidophilic facultatively che-
molithotrophic bacterium Sulfobacillus thermosul¢dooxi-
dans 41 is characterized by highly versatile metabolism
and may rapidly adapt to changing environments. This
organism was shown to be able to grow as autotroph,
heterotroph and mixotroph under intensive aeration. The
strain can grow under microaerobic conditions on medium
containing ferrous iron and glucose. In the current study
we have investigated growth parameters and activities of
carbon metabolism enzymes. Sulfobacillus thermosul¢-
dooxidans 41 is able to grow slowly under restricted aera-
tion conditions accomplishing ferrous oxidation and glu-
cose consumption. During 50-80 hours there are no
changes in number of cells and nutrient compounds con-
centration. The doubling time in exponential phase are 40-
45 h, maximum number of cells (0,9*107cells/ml) is at-
tained by 170-200 hours. Approximately 50% (45mM) fer-
rous iron and 60% (0,66mM) glucose are not used by
bacterium after 350-370 hours of growth and incomplete
glucose oxidation products (acetate, propionate) are de-
tected. The metabolism of glucose is seems to be proceed-
ing via Embden-Meyerhof-Parnas and/or oxidative pen-
tose phosphate pathway. The activities of key enzymes
of former ^ glucokinase, 6-phosphofructokinase, fructose
1,6-bisphosphate aldolase, 3-phosphoglyceraldehide dehy-
drogenase, pyruvate kinase, and the latter ^ 6-phospho-
gluconate dehydrogenase are demonstrated in cell-free ex-
tracts of Sulfobacillus thermosul¢dooxidans 41. Obviously
Entner-Doudoro¡ parthway is not operate as since there
are no activities of glucose-6-phosphate dehydratase, 2-
keto-3-deoxy-6-phosphogluconate aldolase. Moreover ac-
tivities of carbon dioxide ¢xation enzymes (autotrophic
and heterotrophic) ^ ribulosebisphosphate carboxylase,
phosphoenolpyruvate (PEP)- carboxylase, PEP carboxy-
transphosphorylase, PEP carboxykinase, pyruvate carbox-
ylase are revealed.
 1st FEMS Congress / Posters 103^505
P10^19
SUBSTRATUM FROM A VEGETATIVE PART OF
TOPINAMBOUR IS A PERSPECTIVE RAW MATERI-
AL FOR OBTAINING PHB
T. N. Emelina, T. V. Ryazanova
Siberian State Technological University, Krasnoyarsk,
660049, Pr. Mira 82, Russia
It is possible use of substratum out of di¡erent kinds of
row materials for obtaining of polyhydroxyalkanoates.
Among them are individual compounds, waste of the spi-
rit, sugar, hydrolytic industry. The important trend of re-
searches de¢ning the strategy of industrial production
PHAs is bound up with search of cheap and available
kinds of row materials. One of the most perspective source
is a topinambour. The topinambour forms a huge crop of
underground and overground parts of biomass. The top-
inambour is characterized with the high contents of car-
bohydrates up to 70 %, which signi¢cant share is fructose
and its polymers, the highest homologue, which is the
inulin. For obtaining of carbohydrate content substratum
the vegetative part of topinambour has been subjected
two-stages processing. The low molecular carbohydrates
were extracted with water, and then a solid rest was sub-
jected to hydrolysis with the diluted sulphuric acid. The
obtained hydrolysate was subjected ennobling by neutral-
ization and defecation. The research of chemical compo-
sition substratum has shown that it contains all necessary
for propagation of microorganisms micro and macroele-
ments and other biochemical components, which basic
share composes fructose ^ 54 g/l. Cultivation of bacteria
Ralstonia eutropha carried out on substratums with the
contents reduce sugar 1 % and fructose 27 g/l. It is estab-
lished that in case of diluted substratums, is observed the
greatest crop of biomass-up to 6-7 g/l. In case of a mode
with adding of substratum the reception of a crop up to 50
g/l and above is provided. With accumulation of biomass
there is increase of concentration polyhydroxybutirate up
to 70 %.
FEMSLE Congress 2-6-03
327
P10^20
THE BACTERIUM THERMUS THERMOPHILUS,
LIKE HYPERTHERMOPHILIC ARCHAEA, USES A
TWO-STEP PATHWAY FOR THE SYNTHESIS OF
MANNOSYLGLYCERATE
N. Empadinhas(1), L. Albuquerque(1), A. Henne(2), H.
Santos(3) and M. S. da Costa(1)
(1) Department of Biochemistry and Centre for Neuroscien-
ces of Coimbra, University of Coimbra, 3004-517 Coimbra,
Portugal; (2) Goettingen Genomics Laboratory, Institut fu«r
Mikrobiologie und Genetik, Grisebachstr. 8, 37077 Go«ttin-
gen, Germany; (3) Instituto de Tecnologia Qu|¤mica e Bio-
lo¤gica, Universidade Nova de Lisboa, Rua da Quinta
Grande 6, Apartado 127, 2780-156 Oeiras, Portugal
The biosynthetic pathway for the synthesis of the compat-
ible solute K-mannosylglycerate (MG) in the thermophilic
bacterium Thermus thermophilus HB27 was identi¢ed
based on the activities of recombinant mannosyl-3-phos-
phoglycerate synthase (MPGS) and mannosyl-3-phospho-
glycerate phosphatase (MPGP). The sequences of homol-
ogous genes from the archaeon Pyrococcus horikoshii were
used to identify mpgs and mpgp genes in T. thermophilus
HB27 genome. Both genes were separately overexpressed
in Escherichia coli. The molecular masses were 43.6 and
28.1 kDa for MPGS and MPGP, respectively. The re-
combinant MPGS catalyzed the synthesis of K-mannosyl-
3-phosphoglycerate (MPG) from GDP-mannose and D-3-
phosphoglycerate, while the recombinant MPGP dephos-
phorylated MPG to MG. The recombinant MPGS had
optimal activity at 80‡C and a pH optimum near 7.0;
MPGP had maximal activity between 90 and 95‡C and
at pH 6.0. Both enzymes were strictly dependent on diva-
lent cations; Mn2+ was most e¡ective for MPGS, while
Mn2+, Co2+, Mg2+ and Ni2+ activated MPGP. The gene
organization in T. thermophilus HB27 is di¡erent from the
P. horikoshii operon-like structure since the genes involved
in the conversion of fructose-6P to GDP-mannose are not
immediately downstream of the contiguous mpgs and
mpgp genes. The biosynthetic pathway to MG in T. ther-
mophilus HB27, through a phosphorylated intermediate, is
similar to the system found in hyperthermophilic archaea.
The investigation of biosynthetic pathways for MG is es-
sential to our knowledge of the physiological role of MG
on thermal and osmotic stress responses of thermophilic
bacteria and hyperthermophilic archaea and to the under-
standing of the puzzling distribution of MG among pro-
karyotes.
328 1st FEMS Congress / Posters 103^505
P10^21
CYTOBIOCHEMICAL PECULIARITIES OF MODER-
ATELY HALOALKALIPHILIC METHANOTROPH
METHYLOMICROBIUM BURYATENSE 5B GROWN
AT HIGH METHANOL CONCENTRATIONS
B. Ts. Eshinimaev, V. N. Khmelenina, N. E. Suzina, Y. A.
Trotsenko
Institute of Biochemistry and Physiology of Microorganisms
RAS, Prospekt Nauki, 5, Pushchino, Moscow region,
142290, Russia
Methanotrophs utilize methane and in less extent metha-
nol as carbon and energy sources. Recently, we have iso-
lated haloalkaliphilic/tolerant methanotrophs from soda
lakes of di¡erent geographical regions. These bacteria
grow optimally at pH 8.5-9.5 showing high tolerance to
NaCI (up to 2M) and high methanol concentrations (up to
1.75 M). The growth rate of moderately haloalkaliphilic
methanotroph Methylomicrobium buryatense 5B (0.2 h-1)
on 0.25 M methanol was twice as high as than on methane
(0.1 h-1). The growth on methanol was accompanied by a
reduction of ICM, the appearance of glycogen granules in
cells, and by accumulation of formaldehyde, formate, and
extracellular glycoprotein in growth medium. The glyco-
protein was found to contain 23% protein and 77% carbo-
hydrates, the latter being dominated by glucose, mannose,
and aminosugars. The major amino acids were glutamate,
aspartate, glycine, valine, and isoleucine. The activities of
sucrose-6-phosphate synthase, glycogen synthase, and
NADH dehydrogenase were found to be higher in meth-
anol-grown cells than in methane-grown cells. The data
obtained suggest that the high methanol tolerance of M.
buryatense 5B is based on the rapid sink of toxic formal-
dehyde for the synthesis of sucrose, glycogen, glycoprotein
and to the oxidation of excess reducing equivalents
through the respiratory chain.
FEMSLE Congress 2-6-03
P10^22
METAL ION INFLUENCE IN SULPHATE REDUC-
ING BACTERIA METABOLISM
C. Farinha(1), A. R. Lino(1), J. J. G. Moura(2), I.
Moura(2), S. Bursakov(2)
(1) Departamento de Qu|¤mica e Bioqu|¤mica, Faculdade de
Cie“ncias, Universidade de Lisboa, Edif|¤cio C8, Piso 4, Cam-
po Grande, 1749-016 Lisboa, Portugal; (2) Departamento
de Qu|¤mica e Bioqu|¤mica, Centro de Qu|¤mica Fina e Tec-
nologia, Faculdade de Cie“ncias e Tecnologia, Universidade
Nova de Lisboa, 2829-516 Caparica, Portugal
The trace elements Mo and Cu are indispensable for
growth of the sulphate reducer D.gigas. Recently, a novel
orange coloured protein (ORP) from D.gigas was puri¢ed
and proved to contain an unknown and new type of
mixed-metal sulphide cluster containing molybdenum
and copper (J.Am. Chem.Soc. 2000, 122, 8321-8322).
The UV/Vis spectrum of the protein resembles the one
of the tetrathiomolybdate with a shift on the wavelength
of the bands due to the presence of copper. Indeed, thio-
molybdates are formed in the growth media of D.gigas
cells (identi¢ed by the formation of an orange coloured
product with a visible spectrum similar to the one of thi-
omolydate). Therefore, the purpose of this study is to
understand the chemistry and nature of Cu, Mo, S inter-
action during bacterial growth. The production of thiomo-
lybdate anions was followed during growth and correlated
with di¡erent factors (metals concentration). The spectra
of the culture media depends on the Mo, as well as Cu
concentration. It was found that Mo is toxic at concen-
trations higher than 1X10-3 M because it interferes with
the sulphate ion and blocks APS production by ATP sul-
furylase. Orange coloured products are formed between
concentration of Mo between 1X10-3 and 1X10-4 M. There
is a correlation between the amount of H2S and the for-
mation of the thiomolybdates. The possible role as well as
the importance of the tetrathiomolybdate anions produc-
tion out of cells is discussed.
 1st FEMS Congress / Posters 103^505
P10^23
REGULATION OF FORMATION OF THE INTRA-
CELLULAR B-GALACTOSIDASE ACTIVITY IN AS-
PERGILLUS NIDULANS
E. Fekete(1), E. Sandor(1), A. Szentirmai(1), C. P. Ku-
bicek(2) and L. Kara¡a(1)
(1) Department of Microbiology and Biotechnology, Fac-
ulty of Science, University of Debrecen, H-4010, P.O.Box
63, Debrecen, Hungary; (2) Section Microbial Biochemis-
try and Gene Technology, Institute of Chemical Engineer-
ing, TU Vienna, A-1060, Getreidemarkt 9/E166, Austria.
We have chosen the f-galactosidase of Aspergillus nidulans
as an object for investigating how lactose and galactose
metabolism are regulated in ¢lamentous fungi because (a)
f-galactosidase and the lactose-metabolizing enzymes are
coordinately regulated in the yeast K. lactis; (b) a single
intracellular enzyme appears to be present in A. nidulans;
(c) the enzyme assay is easy; and (d) mutants in the lac-
tose-metabolizing pathway of A. nidulans are available. f-
Galactosidase was not formed during growth on glucose
or glycerol, but was rapidly induced during growth on
lactose or D-galactose. L-arabinose, and D-xylose also
induced f-galactosidase activity. Addition of glucose to
cultures growing on lactose led to a rapid decrease in
the f-galactosidase activity. In contrast, in cultures grow-
ing on D-galactose, addition of glucose decreased the ac-
tivity of f-galactosidase only slightly. Glucose inhibited
the uptake of lactose, but not of D-galactose, and required
the carbon catabolite repressor CreA for this. In addition,
CreA also repressed the formation of basal levels of f-
galactosidase and partially interfered with the induction
of f-galactosidase by D-galactose, L-arabinose and D-xy-
lose. Phosphorylation of D-galactose was not necessary
for induction. Interestingly, a mutant in galactose-1-phos-
phate uridylyl transferase produced f-galactosidase activ-
ity at a low, constitutive level even on glucose and glycer-
ol, and was no longer inducible by D-galactose, whereas it
was still inducible by L-arabinose. We conclude that the
biosynthesis of the intracellular f-galactosidase of Asper-
gillus nidulans is regulated by CreA, partially repressed by
galactose-1-phosphate uridylyl transferase, and induced by
D-galactose and L-arabinose in independent ways.
FEMSLE Congress 2-6-03
329
P10^24
THE MECHANISM OF ACETATE ASSIMILATION
IN ISOCITRATE LYASE-NEGATIVE PURPLE BAC-
TERIUM RHODOBACTER SPHAEROIDES
L. V. Filatova, I. A. Berg, R. N. Ivanovsky
Department of Microbiology, Moscow State University,
Moscow 119899, Russia
The citramalate cycle has been proposed as a new anapler-
otic pathway of acetate oxidation to glyoxylate for the
isocitrate lyase-negative purple bacterium Rhodospirillum
rubrum. In this cycle, acetyl-CoA is condensed with pyru-
vate to form citramalate, which is then converted to pro-
pionyl-CoA and glyoxylate via mesaconate, mesaconyl-
CoA and L-methylmalyl-CoA. Propionyl-CoA is further
converted to pyruvate (acetyl-CoA acceptor), completing
the cycle. In R. rubrum this conversion proceeds via CO2-
dependent propionyl-CoA carboxylase (PCC) pathway.
Now we show that another ICL^ purple bacterium, Rho-
dobacter sphaeroides, also possesses key enzymes of this
cycle in cell extracts. Condensation rates of acetyl-CoA
with pyruvate and propionyl-CoA with glyoxylate were
36.0 and 166.6 nmol min (mg protein)-1, respectively.
Products of both reactions were identi¢ed using HPLC
and were shown to be citramalate for acetyl-CoA conden-
sation with pyruvate and L-methylmalyl-CoA and mesa-
conyl-CoA for propionyl-CoA condensation with glyoxy-
late. The activities of these enzymes were down-regulated
in malate-grown cells indicating their involvement in ace-
tate metabolism. The enzymes required for subsequent
steps of the cycle, i.e. for the conversion of propionyl-
CoA to pyruvate via PCC, were found in Rb. sphaeroides.
However, acetate assimilation in Rb. sphaeroides, unlike R.
rubrum, was CO2-independent. It could have been ex-
plained by the existence in Rb. sphaeroides of CO2-inde-
pendent pathway of propionate oxidation to pyruvate, but
the activities of key enzymes of the known pathways could
not be detected. Since the carboxylation of propionate in
citramalate cycle is counterbalanced by decarboxylation of
oxaloacetate, other possible explanations are that PCC has
high a⁄nity for bicarbonate or that the cells are capable
to concentrate CO2 at the carboxylation site. Although the
Km of PCC for bicarbonate in cell extracts was as high as
4 mM, the Km of whole cells (cell suspensions) for bicar-
bonate was relatively low (0.4-1.0 mM). The study of this
problem is in progress now.
330 1st FEMS Congress / Posters 103^505
P10^25
CHARACTERISATION OF L-ARABINOSE TRANS-
PORT IN YEASTS
C. Fonseca and I. Spencer-Martins
Centro de Recursos Microbiolo¤gicos (CREM), Biotechnol-
ogy Unit, Faculty of Sciences and Technology, New Univer-
sity of Lisbon, 2829-516 Caparica, Portugal
Saccharomyces cerevisiae has long been used to make etha-
nol, but it lacks the ability to ferment ¢ve-carbon sugars
like D-xylose and L-arabinose, the most abundant hemi-
cellulose-derived pentoses. Whereas the bioconversion of
the predominant xylose into ethanol has been extensively
investigated, arabinose fermentation has only recently
started to receive more attention. Very few yeasts are ca-
pable of producing traces of ethanol from arabinose, even
though many can use this pentose aerobically, to produce
biomass. An alternative solution is to develop e⁄cient
arabinose-fermenting recombinant S. cerevisiae strains. A
possible shortcoming in this process is the low capacity of
S. cerevisiae cells to transport arabinose. Looking for a
suitable gene donor, we have undertaken the study of
arabinose transport in yeasts. An investigation of the
transport properties in yeasts found to take up labelled
L-arabinose at very high rates, indicated that two types
of transport systems were operational: a very high ca-
pacity/low a⁄nity (Km = 115-125 mM) facilitated di¡u-
sion, and a low capacity/high a⁄nity (Km = 0.19-0.27
mM) arabinose/H+ symporter. Both uptake systems were
found to be present when the yeasts were grown in L-
arabinose but not in D-glucose. Inhibition studies indi-
cated that the facilitated di¡usion component is speci¢c,
arabinose apparently being the only substrate. This spec-
i¢city, allied to the expression pattern of the arabinose
transport systems under di¡erent growth conditions, is
currently being used to isolate the gene(s) involved in L-
arabinose uptake.
P10^26
REDUCTION OF Cr (VI) AND U(VI) BY ANAEROBIC
THERMOPHILIC BACTERIA
S. N. Gavrilov, A. I. Slobodkin, E. A. Bonch-Osmolovskaya
Institute of Microbiology, Russian Academy of Sciences,
Prospect 60-letiya Oktyabrya 7/2, 117312, Moscow, Russia
The removal or immobilization of toxic metals and radio-
nuclides as environmental pollutants is an important as-
pect of microbial bioremediation. To date there are only
few reports on Cr(VI) reduction by thermophilic micro-
organisms. We investigated the ability of washed cell sus-
FEMSLE Congress 2-6-03
pensions of three thermophilic anaerobic bacteria to re-
move soluble Cr(VI) and U(VI) by reducing them to
their less soluble Cr(III) and U(IV) forms respectively.
Suspensions of an iron(III)-reducer Themoanaerobacter
siderophilus were able to reduce Cr(VI) as K2Cr2O7 (ini-
tial Cr(VI) concentrations 250 ^ 1000 WM) with H2 or
pyruvate being electron donor. Suspensions of another
iron-reducing organism, Thermoterrabacterium ferriredu-
cens, reduced 18 or 26 % of supplied Cr(VI) with sorbitol
or peptone as electron donors respectively within 4.5 hours
of incubation. Both of these organisms reduced Cr(VI)
even in the absence of any electron donor at the rates
comparable to those detected in the presence of electron
donors. The capability of Cr(VI) reduction in the same
conditions was also shown for cell suspensions of a non
iron-reducing Moorella glicerini. Cr(VI)-reducing activity
of this organism was signi¢cantly lower and was unaf-
fected by the presence of electron donors. Thermoterrabac-
terium ferrireducens cell suspensions were also tested for
the ability to reduce U(VI). Within 24 hours of incubation
at 65 ‡C with molecular hydrogen, being electron donor,
Thermoterrabacterium ferrireducens cells reduced nearly 35
% of supplied U(VI) sulphate to U (IV), which completely
precipitated in the form, characteristic of U(IV) hydrox-
ide. All the experiments were carried out at temperature
optima of the organisms. Abiotic metals reduction and
reduction caused by cell suspensions at room temperature
were signi¢cantly lower than biotic processes at elevated
temperatures.
P10^27
UNBALANCED MEMBRANE PHOSPHOLIPID
COMPOSITIONS AFFECT EXPRESSION AND SE-
CRETION OF ESCHERICHIA COLI ALKALINE
PHOSPHATASE
V. V. Golovastov, E. V. Anisimova, N. I. Mikhaleva, M. A.
Nesmeyanova
Skryabin Institute of Biochemistry and Physiology of Mi-
croorganisms, Russian Academy of Sciences, Pushchino
142290, Moscow Region, Russia
In the current work, a mutant strain of E. coli AD93
lacking phosphatidylethanolamine (PE) was used to estab-
lish the requirement of PE for e⁄cient secretion in vivo of
E. coli alkaline phosphatase (PhoA). PhoA secretion in the
cells lacking PE and grown in the presence of bivalent
cations Mg2+ or Ca2+ was shown to signi¢cantly decrease
in contrast to the cells containing PE. The spectrum of
enzyme isoforms also changes in these conditions, indicat-
ing the e¡ect of PE depletion on posttranslocational mod-
i¢cation of PhoA by periplasmic protease, probably due to
a decrease of secretion e⁄ciency of the latter. Depletion of
PE a¡ects also the expression of PhoA controlled not by
 1st FEMS Congress / Posters 103^505
the PBAD ara promoter, but PPHO promoter which belongs
to the Pho regulon. PE depletion signi¢cantly decreases
the relative content of immunoprecipitated radioactive
PhoA expressed under its own promoter control in the
total pool of radioactive protein and the incorporation
of RNA precursor [14C]-uracil into a TCA insoluble frac-
tion after induction of the phoA gene controlled by its own
PPHO promoter in cells lacking PE. Unbalanced phospho-
lipid composition due to inactivation of pgsA gene encod-
ing phosphatidylglycerol biosynthesis in the strain HD30/
pHD102 also depressed the expression and secretion of
PhoA controlled by the PPHO promoter. The results sug-
gest unbalanced membrane phospholipid composition pro-
foundly a¡ect not only PhoA secretion as a membrane
function but also the central and apparently non-mem-
brane event ^ the expression of the protein which belongs
to the Pho regulon, a member of the two-component reg-
ulatory system of Pi signal transduction. The latter is
known to consist of membrane bound sensor protein
PhoR and cytoplasmic regulator (regulatory protein)
PhoB interacting with DNA operator. This allows us to
conclude that dramatic changes in the phospholipid com-
position may a¡ect the conformation of membrane bound
sensors or their assembly and thus a¡ect the signal trans-
duction and the function of regulatory proteins, which
determine the expression of de¢nite proteins.
P10^28
FLAVOCYTOCHROME b2 FROM THERMOTOLER-
ANT METHYLOTROPHIC YEAST HANSENULA
POLYMORPHA : REGULATION OF SYNTHESIS
AND ISOLATION
M. V. Gonchar, O. V. Smutok, G. Z. Gayda
Institute of Cell Biology, Drahomanov Street 14/16, 79005
Lviv, Ukraine
L-lactate cytochrome c oxidoreductase (EC 1.1.2.3; £avo-
cytochrome b2, FC b2) due to its unique catalytic proper-
ties has potential bioanalytic importance and can substi-
tute NAD-dependent lactate dehydrogenase or bacterial
lactate oxidase in determination of L-lactate in biological
£uids and foodstu¡s by enzymatic and biosensor’s ap-
proaches. A wide application of FC b2 from baker’s yeast
in bioanalytics is hampered by its instability and the di⁄-
culties during puri¢cation of the enzyme. We have aimed
to study the possibility to isolate FC b2 from thermotoler-
ant yeast Hansenula polymorpha. It has been shown that
the synthesis of FC b2 is regulated by carbon sources
through derepression mechanism (during growth of cells
on glucose, ethanol, or glycerol a speci¢c activity of the
enzyme in cell free extracts is increased) as well through
induction by L-lactate. A synthesis of the enzyme is regu-
lated positively by oxygen, because aeration pattern
FEMSLE Congress 2-6-03
331
greatly in£uences the level of enzyme activity in cells.
Under optimal cultivation conditions, speci¢c activity of
FC b2 in cell free extracts reached up 0.4 WmoleXmin-
1
Xmg-1 protein. It has been proposed an e⁄cient scheme
of isolation and puri¢cation of FC b2 from H. polymorpha
cells which includes lysis of the cells by butanol, fraction-
ation by ammonium sulfate and chromatography on
DEAE-Toyopearl 650M. It was obtained practically ho-
mogeneous preparation of the enzyme with speci¢c activ-
ity up to 30 WmoleXmin-1Xmg-1 protein. On the contrary
to the baker’s homologue, FC b2 from H. polymorpha is
apparently more stable that makes it very suitable for
bioanalytical application in enzymatic kits and biosensors.
P10^29
REGULATION OF CARBON AND SULFUR METAB-
OLISM IN FILAMENTOUS FRESHWATER BACTE-
RIUM BEGGIATOA LEPTOMITIFORMIS D-402
M. Yu. Grabovich(1), G. A. Dubinina(2), V. Yu. Patrit-
skaya(2), M. S. Muntyan(3)
(1) Voronezh State University, University Square 1,394006,
Voronezh, Russia; (2) Institute of Microbiology, Prospect
60-let Oktyabrya 7/2, 117811, Moscow, Russia ; (3) Belo-
zerskii Institute of Physicochemical Biology, Moscow State
University, Moscow, Russia
The regulatory role of oxygen in metabolism of ¢lamen-
tous freshwater sulfur bacteria is detected. The ability to
mixotrophic growth of bacteria occurs in the wide range
of oxygen concentrations but maximum cellular harvest is
observed at the oxygen concentration not higher than 0.15
mgl-1. Beggiatoa leptomitiformis D-402 is capable of lith-
oautotrophic growth only under microaerobic conditions
at the concentration of dissolved oxygen not exceeding 0.5
mgl-1. High activity of key enzymes of Calvin cycle, which
are induced only under strictly microaerobic conditions, is
found in cells. The composition of cytochromic part of
respiratory chain in Beggiatoa leptomitiformis is regulated
by the concentration of dissolved oxygen and the presence
of reduced sulfur compounds: during organoheterotrophic
growth in membrane fractions, cytochromes c, b and a are
present; during mixotrophic and lithoautotrophic growth,
the amount of cytochromes b and c increases, the bb3 ^
type oxidase is observed in the terminal parts instead of
aa3 ^ type oxidase. The activity of enzymes of TCA (tri-
carboxylic acid cycle) during mixotrophic and autotrophic
growth in contrast to organoheterotrophic ones decreases
2- to 3-fold but the activity of enzymes of glyoxylate cycle
increases. The relationship of structure and kinetic char-
acteristics of Malate Dehydrogenase ^ one of the enzymes
of TCA and glyoxylate cycle ^ with the type of carbon
metabolism of Beggiatoa, is revealed : during organotro-
332 1st FEMS Congress / Posters 103^505
phic growth, the enzyme has dimeric structure but during
mixotrophic one it is tetrameric.
This work was supported by the Russian Foundation of
Basic Research, grant no. 02-04-49185.
P10^30
CELL FATTY ACID THERMOADAPTATION IN
STRAINS OF BACILLUS SPP. ISOLATED FROM RE-
FRIGERATED COOKED FOODS
M. E. Guerzoni(1), A. Gianotti(1) and C. Chaves Lo¤-
pez(2)
(1) Dipartimento di Protezione e Valorizzazione Agroali-
mentare, Sezione di Chimica e Tecnologia degli Alimenti
Universita' degli Studi di Bologna Via Fanin, 46 40127 Bo-
logna, Italy; (2) Facolta' di Agraria, Universita' degli Studi
di Teramo, Mosciano Stazione, Teramo, Italy
In this study the thermoadaptative changes in the cell fatty
acids (FA) of sixteen strains of B. cereus, B. subtilis and B.
licheniformis, isolated from cooked chilled foods and se-
lected on the basis of their more or less broad temperature
span, were analysed. In order to ascertain whether the
strain’s ability to grow over an extended temperature
span re£ected a peculiar ability to modulate fatty acids
in response to temperatures, or whether, on the other
hand, the fatty acid thermoadaptation pattern depended
on the species itself. Di¡erent patterns of fatty acids adap-
tation to temperature changes were observed. Some strains
of B. cereus, B. licheniformis and B. subtilis responded to a
temperature rise up to 45‡C by increasing the proportion
of unsaturated and saturated branched chain FA. On the
other hand the majority of the strains of B. subtilis, char-
acterized also at optimal temperatures by an elevated pro-
portion of branched chain FA, the FA unsaturation in-
crease proved to be prevalently a low temperature
response mechanism. However the strains characterized
by the major ability to e⁄ciently grow both at refrigera-
tion temperatures and over 50‡C exhibited a £exible FA
thermoadaptation pattern. In fact they shared with ther-
motolerant strains of B. cereus the BFA desaturation in-
crease at 45‡C and with B. subtilis the straight fatty acid
desaturation at 12‡C. The comparison with the acclimati-
zation patterns exhibited by the considered strains, indi-
cated that the £exibility of some strains re£ects individual
phenotypic traits normally not encompassed by the array
of species characteristics.
FEMSLE Congress 2-6-03
P10^31
G-RICH REGION OF a-MPP IS PROBABLY ESSEN-
TIAL FOR THE RECOGNITION OF THE SUB-
STRATE PRESEQUENCES, C-TERMINUS INFLUEN-
CES THE SUBUNIT STABILITY
K. Hola, N. Parkhomenko, A. Matuskova, O. Gakh, E.
Kutejova, J. Spizek, J. Janata
Institute of Microbiology, Academy of Sciences of the
Czech Republic, Videnska 1083, 142 20 Prague-4, Czech
Republic
Mitochondrial processing peptidase (MPP), a metallopep-
tidase consisting of a- and L-subunits, speci¢cally cleaves
o¡ the N-terminal leader peptides of the mitochondrial
protein precursors. The substrate binding induces a con-
formational change of the a-subunit, but not of the cata-
lytic L-subunit. Our studies concern two distinct regions:
extreme C-terminus and glycine-rich region. We suggest
that the C-terminal a-helix is essential for subunit stability.
The absence of 30 terminal amino acid residues (forming
a-helix) completely abolished the enzyme activity, on the
other hand, deletion of 2 or even 17 amino acid residues
retains full activity but not stability. In addition, the C-
terminus is also interesting in particular with respect to
interaction with Lon-protease. We assumed that the con-
formational change of a-MPP is related just with the G-
rich region. By means of site-directed mutagenesis of the
glycine-rich region (positions 284-303) we found that dele-
tions of one or two glycines in positions 284, 285 and 292,
293 resulted in a complete loss of the activity, whereas
substitutions at Y303 did not a¡ect it. In order to obtain
the optimal model for £uorescence assays for subunit-sub-
strate interaction we substituted all tree natural present
tryptophan residues (W147N, W223M, W491Y) of a-
MPP and tested modi¢ed proteins functionaly. Addition-
ally we prepared cassette allowing for any modi¢cation of
the whole G-rich site sequence. We have used it for inser-
tion of reporter Trp residues (F289W, Y299W, Y303W) as
well as for deletion in this region.
P10^32
AEROBIC METHYLOTROPHIC BACTERIA AS PHY-
TOSYMBIONTS : METABOLIC ASPECTS
E. G. Ivanova, N. V. Doronina, Y. A. Trotsenko
Institute of Biochemistry and Physiology of Microorgan-
isms, RAS, Pushchino, Prospect Nauki 5, Moscow region,
142290, Russia
Aerobic methylotrophic bacteria are present in seeds,
phyllo- and rhizosphere of many plants. This is now ex-
 1st FEMS Congress / Posters 103^505
plained by operation of the methanol cycle including
methanol formation and release by plants and utilization
by methylotrophs. It is logically to suggest that methylo-
trophs not only colonize plants but are symbiotically re-
lated to them. Our study aimed at elucidation of metabolic
dialogue of methylotrophs with plants. By using TLC,
HPLC, MS and bioassays cytokinins and auxins (up to
120 Wg/ml) were detected in spent media of aerobic meth-
ylotrophic bacteria belonging to di¡erent taxa. Vitamin
B12 was also produced and accumulated intracellularly
(up to 3 Wg/ml) by methylotrophs. PCR analysis revealed
the presence of nucleotide clusters homologous to the
genes responsible for cytokinin synthesis in the genomes
of most tested methylotrophic bacteria. Enzymic analysis
and identi¢cation of the intermediates showed that these
bacteria synthesize indole-3-acetic acid via indole-3-pyr-
uvic acid. Besides, PCR analysis revealed the presence of
nucleotide clusters homologous to the nifH gene respon-
sible for nitrogen ¢xation in the genome of aerobic meth-
ylobacteria, thus implying their diazotrophic ability. Re-
markably, colonization of gnotobiotic plants with
methylotrophs resulted in stable trophic association and
higher growth rate, regeneration potential, and enhanced
rooting when compared to the non-colonized plants.
P10^33
DETERMINATION OF BIOCHEMICAL POTEN-
TIALS OF LIPASES FROM GM+VE BACTERIA BA-
CILLUS AND STAPH.
F. Aziz and N. Jamil
Department of Microbiology, University of Karachi, Kara-
chi 75270, Pakistan
Out of 124 di¡erent Gm+ve isolate including Bacilli and
Staph, 30 strong lipases were categorized into 7 groups
based on nutritional requirements by the help of dendro-
gram derived from hierarchical agglomerative clustering.
Initially di¡erent edible oils were used as substrate for
plate assay, TLC and GC/MS analysis of lipase activity.
On the basis of the results of TLC of reaction mixtures, 18
isolates (from 7 nutritional groups were classi¢ed into four
subgroups depending upon biochemical potentials includ-
ing enzyme activity & substrate speci¢city. Variety of cat-
alytic activities were i.e. hydrolysis, estri¢cation, substrate
speci¢city (including type of fatty acids, glyceride Sn spec-
i¢city) and biotransforming abilities of lipase were exam-
ined successfully by using standard substrates. Highly ver-
satile lipases have been ransacked from spore forming
Bacilli (5va), clinical isolates (C1.6 & C 1.16) and environ-
mental isolates (Rc.9) of Staphs. 5va lipase is active at
41‡C and can tolerate alkaline pH 9. It can transform
di¡erent substrates into products like alcohol e.g. cyclo-
hexanol and dicarboxylic acid e.g. adipic acid. Cyclization
FEMSLE Congress 2-6-03
333
and lactonization were also found to be the characteristic
features for organic synthesis of cyclopropane ring con-
taining compounds and oxiranes. Furthermore phospho-
lipids were also hydrolyzed. Lipases from Staph seem to
have the potential for the synthesis of arachidic acids and
cyclopentyle ring containing compounds. These lipases
have shown broad substrate speci¢city. Our studies have
indicated that such lipases can be used as valuable tool for
drug designing and in oleochemical industry.
P10^34
EXTRACTION AND DETERMINATION OF METAB-
OLITES FROM ASPERGILLUS NIGER MYCELIUM
DURING SUBMERGED CULTIVATION
G. Gril and K. Jernejc
Laboratory of Biotechnology, National Institute of Chemis-
try, Hajdrihova 19, P.O.B. 660, SI-1001 Ljubljana, Slovenia
The ¢lamentous fungus Aspergillus niger is able to accu-
mulate and excrete high concentrations of organic acids
and di¡erent extracellular enzymes. In studies on metabol-
ic regulation the knowledge about the concentration of
intermediary metabolites is required to determine metabol-
ic pathways involved in their biosynthesis. Metabolic rates
are usually high, so during sampling enzymes must be
inactivated as rapidly as possible. Fast sampling and fast
inactivation of metabolism and complete extraction of me-
tabolite should be accomplished. Di¡erent methods for the
quenching of metabolism and di¡erent methods of metab-
olite extraction with subsequent enzymatic determination
of metabolite concentrations were evaluated. Some of me-
tabolites of Krebs cycle, recognized to be of importance in
organic acid production were checked. Direct quenching
of fungal mycelia in liquid nitrogen or in 60% methanol at
-40‡C were examined, with later extraction with acid, al-
kali or bu¡ered ethanol. Preliminary experiments were
conducted with commercial metabolites. The determina-
tion of metabolites : pyruvate, malate and 2-oxoglutarate,
extracted from A. niger mycelia was performed next. Fi-
nally metabolites were followed in A. niger mycelium dur-
ing 6 days of cultivation in a laboratory fermenter. Results
obtained by boiling bu¡ered ethanol were noticable lower
compared to acid and alkaline extraction regardless
quenching method. The loss by ethanol treatment could
be accounted to instability of metabolites in ethanol rather
than because of incomplete extraction or loss of sample
during extraction, since with commercial metabolites 20-30
% lower results were also obtained. With metabolites we
are interested with, acid and alkali extraction proved to be
more accurate than extraction with boiling bu¡ered etha-
nol regardless the guenching method.
334 1st FEMS Congress / Posters 103^505
P10^35
A REDUCTIVE PATHWAY OF GALACTOSE CATAB-
OLISM IN ASPERGILLUS NIDULANS
L. Kara¡a(1), E. Fekete(1), E. Sandor(1), A. Szentir-
mai(1), and C. P. Kubicek(2)
(1) Department of Microbiology and Biotechnology, Fac-
ulty of Science, University of Debrecen, P.O.Box 63, De-
brecen, Hungary; (2) Section Microbial Biochemistry and
Gene Technology, Institute of Chemical Engineering, TU
Vienna, Austria
Galactose catabolism in yeast is known to proceed via the
Leloir-pathway, that involves phosphorylation by galacto-
kinase and subsequent conversion into glucose-6-phos-
phate, and yeast loss-of-function mutants in galactokinase
are unable to use D-galactose as a carbon source. How-
ever, in the ¢lamentous fungus Aspergillus nidulans mu-
tants in the galE (galactokinase-encoding) gene could
grow on D-galactose in the presence of ammonium ^
but not nitrate ions ^ as nitrogen source. Mycelia of the
wild-type strain of A. nidulans accumulated some intracel-
lular galactitol (50 mM), whereas the galE mutant accu-
mulated a 10-fold higher intracellular concentration The
accumulated galactitol was catabolized lateron in both
strains. An A. nidulans loss-of-function mutant in arabi-
tol-dehydrogenase, however, was unable to catabolize the
accumulated galactitol. Further, an A. nidulans mutant in
hexokinase (frA2) was unable to grow on galactitol, and a
galE / frA2 double mutant was unable to grow on either
galactose or galactitol. Mycelia of A. nidulans frA2, pre-
grown on glycerol and transferred into a galactitol-con-
taining medium, accumulated intracellular tagatose, indi-
cating it as an end-product of galactitol oxidation. Both
the frA2 and the galE / frA2 mutants were unable to grow
on tagatose, indicating that tagatose catabolism involves
phosphorylation by the hexokinase. The results therefore
provide evidence for a second pathway of D-galactose
catabolism in fungi, which involves reduction of the galac-
tose into galactitol, NAD+-dependent oxidation by an
arabitol dehydrogenase to tagatose and its phosphoryla-
tion by hexokinase.
FEMSLE Congress 2-6-03
P10^36
INTERACTION OF SECB AND SECA WITH ALKA-
LINE PHOSPHATASE IN ESCHERICHIA COLI
O. V. Khokhlova and M. A. Nesmeyanova
Skryabin Institute of Biochemistry and Physiology of Mi-
croorganisms RAS, 142290 Pushchino, Moscow Region,
Russia
The export signal has been assumed to be localized not
only in the signal peptide of a secreted protein precursor,
but also in the N-terminal region of a mature polypeptide
chain. Mutant alkaline phosphatases (PhoA) of Escheri-
chia coli with amino acid substitutions of two positively
charged residues, Lys or Arg, in this region at di¡erent
distances from the signal peptide have been studied to
verify this assumption. The e⁄ciency of secretion of the
mutant proteins with amino acid substitutions in the re-
gion enlarged to 16 amino acid residues has been shown to
decrease; the closer to the signal peptide is the substitu-
tion, the greater is the decrease. The e⁄ciency of PhoA
secretion was also ¢rst shown to depend on the presence in
cells of a cytoplasmic chaperone ^ protein SecB, which has
been assessed by the analysis of both PhoA activity and
the dynamics of conversion of its pulse labeled precursor
to mature protein. Secretion e⁄ciency increases in the
presence of this chaperone at 30‡C, which is most favor-
able for the interaction of SecB with the export initiating
domain, supporting the original model assuming the de-
pendence of the SecB interaction with precursors on the
rate of their folding. The results show that this interaction
most probably occurs in the region of the export domain
located near the signal peptide (positions +2+3) and in a
complex with translocational ATPase ^ protein SecA and
are con¢rmed by coimmunoprecipitations of PhoA, SecB
and SecA.
P10^37
THE INFLUENCE OF STRUCTURE OF A NUTRIENT
MEDIUM AND THE CONDITIONS OF CULTIVA-
TION ON THE PHYSIOLOGICAL PROPERTIES OF
OIL-OXIDIZING ACTINOBACTERIA
A. A. Khudokormov, N. N. Volchenko, D. A. Melnikov and
E. V. Karaseva
Kuban State University, Biology Faculty, Department of
Genetics and Microbiology, 149 Stavropolskaya Street,
350040 Krasnodar, Russia
In connection with the increasing use of actinobacteria
during the clearing of ecosystems from oil it seems to be
of great interest to study the in£uences of cultivation con-
 1st FEMS Congress / Posters 103^505
ditions on the growth, oil-oxidizing activity and the pro-
duction of biosurfactants by the given microorganisms.
The received knowledge will allow a speeding up of the
biorestoration of the territories polluted with hydrocar-
bons. It can be achieved by optimizing the conditions of
the existence of native oil-oxidizing microorganisms. With
the knowledge of the in£uence of factors of environment
on the speed of destruction of hydrocarbons by actino-
bacteria, it is possible to predict the rate of self-restoration
of the ecosystems polluted with hydrocarbons. Work was
carried out within the framework of project INTAS 01-
2151. In the course of investigation, 8 strains of actino-
bacteria species, i.e. Rhodococcus, Nocardia, Gordonia,
selected in the ground of Krasnodar territory polluted
with hydrocarbons have been studied. For allocated
strains we determined the in£uence of the nutrient medium
structure and conditions of cultivations on oil-oxidizing
activity, production of biosurfactants and biomass. Re-
search was carried using the methods of mathematical
planning. We found out that at a ratio of carbon of nitro-
gen and phosphorus in the environment that equals
0,5:3,3:1, the production of actinobacteria of the biomass
by the strains is maximal. The increase of the amount of
carbon in 3-30 times resulted in the intensive synthesis of
biosurfactants. It was established, that the destruction of
petroleum is maximum at neutral and sub acidic initial pH
values, and black oil ^ at alkaline initial pH values. We
have also found out that the use of vegetable oil as the
only source of carbon for getting the primary culture of
oil-degradation bacteria allows to receive a maximum
quantity of a biomass of bacteria with high oil-oxidizing
activity.
P10^38
THE CATABOLISM OF CARBOHYDRATES AND
THE CONTENT OF FATTY ACIDS IN CELLS OF
THE CRABTREE-POSITIVE AND CRABTREE-NEGA-
TIVE YEASTS
Ya. Kolisnyk, S. Gudz
Lviv National University, Lviv, Ukraine
A phenomenon of inhibition of cell respiration activity
under aerobic conditions at increased glucose concentra-
tions is known as Crabtree e¡ect. It was found in cancer
cells, leukocytes and some yeasts. The regulatory mecha-
nisms of this phenomenon are not elucidated. It was
shown that the Crabtree e¡ect is observed in the yeasts
S. cerevisiae and C. pseudotropicalis unlike D. occidentalis.
Its value enhances with the increase of glucose concentra-
tion in the medium. It was established that during growth
of S. cerevisiae in the medium with 0,1% glucose the level
of nonetheri¢ed fatty acids in cells of these yeasts is higher
in comparison with D. occidentalis. The content of fatty
FEMSLE Congress 2-6-03
335
acids in Crabtree-positive and Crabtree-negative yeasts
changes in opposite directions at the increase of glucose
concentration in the medium : it is reduced in S. cerevisiae
and C. pseudotropicalis and increased in D. occidentalis. In
this case the level of C18-unsaturated fatty acids in S. ce-
revisiae and C16 :1, C18-unsaturated fatty acids in C. pseu-
dotropicalis decreased and the amount of saturated and
unsaturated fatty acids in D. occidentalis increased. In
the medium with 0,1% glucose and ethanol the amount
of etheri¢ed fatty acids increased in both species of inves-
tigated yeasts. The fatty acid content in S. cerevisiae and
C. pseudotropicalis is reduced when ethanol was added to
the medium containing 2% of glucose or lactose. The ad-
dition of lactose (2%) to the medium with glucose (2%) did
not change signi¢cantly the content of fatty acids in the
cells of investigated yeasts.
P10^39
DEPLETION OF PHOSHATIDYLETHANOLAMINE
LEADS TO INCREASING THE PERMEABILITY OF
THE OUTER MEMBRANE AND SECRETION OF
PERIPLASMIC ALKALINE PHOSPHOTASE INTO
THE MEDIUM
I. A. Koriakina, A. O. Badyakina, M. A. Nesmeyanova
Skryabin Institute of Biochemistry and Physiology of Mi-
croorganisms, Russian Academy of Sciences, Pushchino,
Moscow Region, 142290, Russia
In the current work we have studied the in£uence of un-
balanced phospholipid composition of E. coli membranes
on the secretion under conditions of overproduction of
alkaline phosphotase (PhoA) encoded by the phoA gene
constituent of plasmids into the medium. The PhoA secre-
tion was compared in the strain E. coli AD93, lacking the
major phospholipid zwitterionic phospatidilethanolamine
(PE) due to mutation in the pssA gene encoding biosyn-
thesis of the PE precursor and in the same strain carrying
the plasmid pDD72 with pss gene. PE depletion was
shown to lead to increasing the e⁄ciency of the secretion
into the medium of periplasmic PhoA encoded by the
phoA gene both under own PPHO promotor and under
endogenous PBAD promotor. It shows the increase of per-
meability of the outer membrane under these conditions.
Lipoprotein and lipopolysaccharide (LPS) determine the
barrier properties of cell envelope. The Analysis of cell
envelope components shows that under unbalanced phos-
pholipid composition of E. coli membranes the permeabil-
ity of cell envelope increases due to decreasing content of
LPS in the outer membrane. This decreasing LPS in the
outer membrane corresponds to increasing secretion of
exopolysaccharide into cultural medium. Unbalanced
phospholipid membrane composition was found to in£u-
ence also on the transcriptional expression of phoA. Here
336 1st FEMS Congress / Posters 103^505
the expression of PhoA is reduced. Electron-microscopy
investigations of the thin layers show some peculiarities
of E. coli cells lacking PE. In contrast to the cells with
normal phospholipid composition of membranes, the cells
of PE-lacking strain have a vast periplasm with large in-
clusions and the outer membrane at a distance from cyto-
plasmic membrane. Phospholipids are known to be meta-
bolic precursors of the envelope components. The results
obtained support the previously assumed interrelation be-
tween protein secretion into the medium and cell envelope
biogenesis at the level of phospholipid metabolism.
P10^40
4-NITROCATECHOL AND 5-NITROGUAIACOL
DEGRADATION BY RHODOCOCCUS OPACUS C6-1
ISOLATED FROM SOIL
N echova¤ and
L. Kotouc›kova¤, J. Nec›a, M. Mora¤vkova¤, L. C
›
M. Nemec
(1) Department of Microbiology, Faculty of Science, Ma-
saryk University in Brno, Tvrde¤ho 14, 602 00 Brno, Czech
Republic; (2) Veterinary Research Institute, Hudcova 70,
621 32 Brno, Czech Republic
Nitroaromatic compounds are widely distributed environ-
mental pollutants. Microbial degradation is a di⁄cult way
of removal of this group of compounds from environment.
4-Nitrocatechol was detected as intermediate of degrada-
tion and product of transformation of 4-nitrophenol. 5-
Nitroguaiacol is compound with plant growth stimulating
activity and their degradation was not studied previously.
We report degradation of both compounds by a Rhodo-
coccus species isolated from soil. Degradation of 4-nitro-
catechol is not connected with capability of degrade 4-ni-
trophenol in the case of the strain C6-1, in contrast to
known bacteria degrading 4-nitrocatechol. 4-Nitrocatechol
is used as sole source of carbon and nitrogen in concen-
tration up to 0.5 mol.l-1. The yellow colour of substrate
allows the using of method of automatic spectrophotom-
etry (bioscreen c) for detailed determination of degrada-
tion pro¢le of 4-nitrocatechol. The use of bioscreen c
instrumentation together with simulation of growth of
cells for correction of in£uence of turbidity increase on ab-
sorbance of substrate represent a good tool for measure-
ment of utilization of nitroaromatics as growth substrates.
Resting cells experiments were used for determination of
basic kinetic parameters of reaction. Analyses of SDS-
PAGE pro¢les of whole cell proteins of Rhodococcus opa-
cus C6-1 have enabled us to reveal changes in proteom of
cells after induction by nitroaromatic substrate and to
perform subsequent characterization of proteins connected
with 4-nitrocatechol or 5-nitroguaiacol degradation.
FEMSLE Congress 2-6-03
P10^41
SPECIFIC RECOGNITION BY RPOS OF THE ES-
CHERICHIA COLI AIDB PROMOTER IS DETER-
MINED BY NUCLEOTIDES WITHIN AND UP-
STREAM OF THE -10 PROMOTER ELEMENT
S. Lacour(1), A. Kolb(2), P. Landini(1)
(1) EAWAG, Swiss Federal Institute for Environmental
Sciences, Du«bendorf, Switzerland ; (2) Pasteur Institute,
Paris, France
Little is known about the mechanism of promoter recog-
nition by RNA polymerase associated with sigmaS (Esig-
maS), a sigma factor expressed in stationary phase. In
Escherichia coli both sigmaS and sigma70, the main sigma
factor of the cell, recognise a similar hexamer called -10
element in the promoter region. We are currently investi-
gating what promoter features can be responsible for the
selective recognition by EsigmaS. For this purpose, we
performed site-directed mutagenesis on the sigmaS-depen-
dent aidB promoter. We studied the activity of each pro-
moter derivative in vivo both in a wild type strain and in a
rpoS mutant, unable to produce the sigmaS factor. The
e¡ect of the mutations was con¢rmed with in vitro tran-
scription assays using puri¢ed RNA polymerase and sigma
factors. We con¢rmed the importance of the canonical C
nucleotide at position -13 for EsigmaS-dependent tran-
scription and we showed that this residue is determinant
both for initial binding to the promoter and for open
complex formation by EsigmaS. We found that the nature
of the ¢rst residue of the Pribnow box (C or T at the -12
position) is determinant for promoter selectivity and
Open-Complex formation. A TG motif is also required
for optimal transcription initiation by EsigmaS even
when its location di¡ers from the canonical -14/-15 posi-
tion for Esigma70.
P10^42
ANALYSIS OF CHITINOLYTIC AND ELASTOLYTIC
ENZYME ACTIVITY AND REGULATION IN CHRO-
MOBACTERIUM VIOLACEUM
A. S. Leese and M. K. Winson
Institute of Biological Sciences, University of Wales Aber-
ystwyth, Aberystwyth SY23 3DD, Wales, UK
Chromobacterium violaceum is a Gram negative opportun-
istic bacterial pathogen known to produce a range of fac-
tors during stationary phase growth, including violacein
pigment, cyanide, chitinases and proteases. Although the
expression of many of these factors has been shown to be
modulated by acyl homoserine lactone quorum sensing
 1st FEMS Congress / Posters 103^505
signals, our understanding of the environmental stimuli
which induce transcription, the precise detail of the regu-
latory control mechanisms involved and the activities and
locations of many of the gene products is less clear. To
investigate these factors in more detail, C. violaceum genes
encoding protein products with chitinolytic activity and
elastolytic activity have been shotgun cloned into Escheri-
chia coli and nucleotide sequenced and the gene products
functionally characterised. Chitinases are involved in the
hydrolysis of chitin, an abundant biopolymer. Proteins
exhibiting chitinolytic activities have previously been
shown to be produced by C. violaceum. The cloned chiti-
nase from C. violaceum exhibits N-acetylglucosaminidase
(chitobiase) activity and shows greatest homology to a
chitobiase from Serratia marcescens. The deduced molec-
ular weight of the unprocessed protein is 97 kDa. Elasta-
ses are metalloproteases capable of degrading the connec-
tive tissue elastin and have been implicated in the virulence
of a number of pathogenic bacteria. The enzymatic activ-
ities of the chitinase and elastase, their locations in C.
violaceum and environmental conditions a¡ecting their ex-
pression have been investigated in di¡erent genetic back-
grounds. The results indicate that the regulation of these
genes is complex and a number of environmental factors,
in addition to quorum sensing control, are implicated in
controlling their expression.
P10^43
H+-ATPASE OF TWO ASPERGILLUS NIGER
STRAINS
K. Jernejc and M. Legis›a
Laboratory of Biotechnology, National Institute of Chemis-
try, Hajdrihova 19, P.O.B. 660, SI-1001 Ljubljana, Slovenia
In the early stages of Aspergillus niger growth a drop in
the extracellular pH could be recorded. Within 24 hours
after inoculation pH drop from2.5 to 1.8 was recorded,
suggesting Aspergillus niger to be acid tolerant microor-
ganism. Acidi¢cation appeared only when ammonium ions
were used as a nitrogen source, whereas with glutamate
such e¡ect was not observed. The reduced pH was due
mostly to proton excretion from the cells, which could
be attributed to the H+-ATPase activity. In order to ob-
tain additional knowledge we have followed H+-ATPase in
two A. niger strains, strain A60-MZKI recognized as citric
acid producing strain and wild type strain A158-MZKI.
Cultivation of both strains under the same conditions with
ammonium ions as a sole nitrogen source resulted in sim-
ilar pH drop with two times more biomass produced by
A158 strain. Regarding H+-ATPase of both strains we
have determined the same kM values and four times higher
vmax with nonproducing A158 strain. The two H+-ATP-
ases also di¡ered in their pH optima, being higher with
FEMSLE Congress 2-6-03
337
citric acid producing strain A60. Inhibition of ATPases
with vanadate and copper ions was followed in both
strains as well. On the basis of results obtained we could
o¡er some additional explanations about regulatory role
of low extracellular pH.
P10^44
THE SIZE OF YEAST SACCHAROMYCES CEREVI-
SIAE CELLS DEPENDS ON PRESENCE OF THE
CONSTITUTIVE ACID PHOSPHATASE
A. I. Leibo(1), A. M. Lomonosov(2), I. V. Yaminsky(2)
and S. N. Egorov(1)
(1) Department of Molecular Biology, Biological Faculty,
Moscow State University, Vorobiovigory, 119992 Moscow,
Russia ; (2) Scanning Probe Microscopy Group, Moscow
State University & Advanced Technologies Center, 119992
Moscow, Russia
Acid Phosphatases (AP) are widespread in all living or-
ganisms. AP are known as enzymes which participate in
the cell phosphor maintaince. In yeast Saccharomyces ce-
revisiae there are two types of AP: (1) Constitutive Acid
Phosphatase (cAP) which is expressed when there is a lot
of phosphate in the culture media; (2) Repressible Acid
Phosphatase (rAP) which occurs only under phosphate
starvation. In yeast AP were detected in organelles which
composed the eukariotic secretory pathway and in the
area of cells’ surfacial structures. Also AP enter into the
structure of outgrowths on yeast cells’ surface. The aim of
our work was to identify if there would be dependence
between the cells’ size and presence/absence of the Consti-
tutive Acid Phosphatase. cAP gene (PHO3) was ampli¢ed
by PCR. This gene then was cloned to vector pDp34
under the control of GAPHL promoter. The plasmid
was transformed to the yeast strain YMR4 (K his3-11, 3-
15 leu2-3,2-112 URA3 canR pho5-pho3: :ura3v1). We
made a calculation of the size of the cells which growth
on the reach phosphate media. It was shown that the
diameter of the cells contained PHO3 gene was approxi-
mately 72 % of the diameter of the cells lacking this gene.
So we can suggest that cAP have a structural function in
the yeast cell wall. This is in agreement with earlier data
for other organisms. For example ¢lamentous acid phos-
phatase has a structure function of £agellar pocket of
Leishmania mexicana Promastigotes. In recent investiga-
tion using atomic force microscopy of cAP, secreted to
media, structure formation of this enzyme was shown.
338 1st FEMS Congress / Posters 103^505
P10^45
CHARACTERIZATION OF HEME-UPTAKE OPER-
ONS IN THE FISH PATHOGENS PHOTOBACTE-
RIUM DAMSELAE SUBSP. PISCICIDA AND VIBRIO
ANGUILLARUM
M. L. Lemos, S. Ju|¤z-R|¤o, S. Mouri•o and C. R. Osorio
Departamento de Microbiolog|¤a, Instituto de Acuicultura,
Universidad de Santiago de Compostela, Santiago de Com-
postela, Spain
Photobacterium damselae subsp. piscicida and Vibrio an-
guillarum are two important bacterial ¢sh pathogens, caus-
ing economical losses in marine and freshwater aquacul-
ture worldwide. Ability of these bacteria to obtain heme
compounds as iron sources from host tissues is believed to
play a role in pathogenesis, but the molecular basis of
heme scavenging systems is poorly studied. We have
cloned and characterized a cosmid clone harbouring a
complete heme-uptake system in V. anguillarum serotype
O1, including an outer membrane receptor, a TonB sys-
tem, a periplasmic heme-binding protein and an associated
ABC transporter, as well as two genes of unknown func-
tion. All these genes are linked in the chromosome. An
homologous system has been characterized in P. damselae
subsp. piscicida, consisting of a TonB system, periplasmic
heme-binding protein, an associated ABC transporter, and
two unknown genes homologous to those found in V.
anguillarum. However, no outer membrane heme receptor
is linked to the rest of genes in Photobacterium. The spa-
tial organization of these genes in the two species is similar
to that reported for other heme-utilizing gram-negative
bacteria, as V. cholerae and Plesiomonas shigelloides. Con-
struction of deletion mutants and gene expression assays
of these genes are being currently carried out, and will
help to unravel their precise role in the iron uptake me-
tabolism of these two important ¢sh pathogens.
P10^46
INCLUSION BODIES IN ESCHERICHIA COLI AND
PICHIA PASTORIS
A. Lenassi(1), SN. Peternel(1), V. Gaberc-Porekar(1) and
V. Menart(1,2)
(1) National Institute of Chemistry, Hajdrihova 19, SI-
1000 Ljubljana, Slovenia; (2) LEK Pharmaceuticals d.d.,
Verovs›kova 57, SI-1000 Ljubljana, Slovenia
Escherichia coli is a well-known and already established
host microorganism for the production of heterologous
proteins. However, other organisms such as non-conven-
tional yeasts, especially methylotrophic yeast Pichia pasto-
FEMSLE Congress 2-6-03
ris, are becoming increasingly important. Production of
heterologous proteins in E. coli often results in formation
of inclusion bodies usually described as macro-molecular
aggregates composed of biologically inactive target protein
and various amounts of native cytoplasmic proteins as
well as other macro-molecules. Therefore, standard isola-
tion of biologically active protein requires solubilization in
strong denaturants followed by renaturation and chroma-
tographic puri¢cation steps. For optimization of down-
stream processes better knowledge of inclusion bodies
composition, their solubility in washing bu¡ers, hydropho-
bicity, etc. would be required. We studied intracellular
expression of several hydrophobic and hydrophilic pro-
teins in various E. coli strains. We focused our studies
on the expression of green £uorescent protein (GFP) in
E. coli BL21 (DE3) and DH5K strains as well as in yeast
P. pastoris. Although GFP is a hydrophilic protein, com-
pletely soluble when produced at low levels, a signi¢cant
fraction of GFP is also found in the form of inclusion
bodies when the protein is highly expressed, either in E.
coli or in P. pastoris. Obviously, with overproduction of
proteins in host organism even hydrophilic proteins can be
forced into the formation of inclusion bodies. We found
that the composition and solubility characteristics of in-
clusion bodies strongly depend on the growth temperature
and induction regime.
P10^47
TRAP TRANSPORTERS IN PSEUDOMONAS AERU-
GINOSA : FUNCTIONAL ROLES OF A NOVEL TYPE
OF BACTERIAL SOLUTE TRANSPORT SYSTEM
M. R. Leon-Kempis(1), G. H. Thomas(2) and D. J.
Kelly(1)
(1) The University of She⁄eld, Molecular Biology and Bi-
otechnology Department, Firth Court, Western Bank, S10
2TN She⁄eld, England ; (2) The University of York, De-
partment of Biology, PO Box 373, YO10 5YW York, Eng-
land
The TRAP transporters are secondary solute transport
systems found to be widespread in both eubacteria and
archaea, but not in eukaryotes. Their main feature is the
utilization of a periplasmic binding protein akin to the
primary ABC transporters but they are coupled to an
electrochemical ion gradient and not to ATP hydrolysis
as in conventional ABC transporters. The general struc-
ture of TRAP transporters is: two transmembrane pro-
teins, probably involved in the actual translocation of
the solute through the cytoplasmic membrane; one bind-
ing protein, located in the periplasm. By looking at the
genome of Pseudomonas aeruginosa, it was noticed that it
has a large variety of transporters for mono-, di-, and tri-
carboxylates, but it appears to be conspicuously de¢cient
 1st FEMS Congress / Posters 103^505
in sugar transporters. Four complete TRAP systems are
present, three of which might bind C-4 dicarboxylates
based on sequence homology to the Dct TRAP transport-
er in Rhodobacter capsulatus. One of these systems has
been shown to transport C4-dicarboxylates with high af-
¢nity (Wmolar Km) experimentally. It is not expressed in
batch culture with excess succinate as carbon source,
whereas in continuous culture, under succinate-limitation,
it is highly expressed as indicated by both uptake kinetics
and immunoblots of the binding protein (PA5167). This
protein was over-expressed, puri¢ed and shown to bind C-
4 dicarboxylates. Thus, the physiological role of this sys-
tem is to scavenge C4-dicarboxylates under severe sub-
strate starvation ; it has no role in conditions of substrate
excess, where other low-a⁄nity systems are responsible for
dicarboxylate uptake.
P10^48
SACCHAROMYCES CEREVISIAE ECM11 GENE IN-
FLUENCES MEIOSIS AND AFFECTS SPORE VIA-
BILITY
U. Les›nik, P. Bedina Zavec, R. Komel and A. Comino
National Institute of Chemistry, Hajdrihova 19, SI-1000
Ljubljana, Slovenia
As reported previously, ECM11 is involved in cell wall
biosynthesis. Since Ecm11 protein is located in the nucleus
throughout all stages of mitosis and has no transcription
activation capability, it was reasonable to believe that the
ECM11 has also other functions in the yeast cell and
probably a¡ects the cell wall biogenesis only indirectly.
We provide evidence that di¡erent strains with deleted
ECM11 sporulate more slowly and less e⁄ciently then
the parental strains with wild type copies of ECM11. By
FACS analysis we demonstrated that DNA synthesis is
slower and less e⁄cient in the knock out strain. The re-
sults of tetrad analysis show that in the absence of ECM11
gene product the viability of spores is reduced to 50% and
that predominantly two viable spores per tetrad are
formed. However, by DAPI staining of spore nucleus of
intact asci we observed that all four spores contain DNA.
PFGE analysis of cells from spores of 2:0 segregated asci
showed di¡erences in comparison to the wild type chro-
mosome pattern, indicating additionally that chromosome
segregation is a¡ected by the ECM11 deletion. Although
vegetative cells with deleted ECM11 gene are sensitive to
Zymolase, and their asci more sensitive to Lyticase in
comparison to the wild type, the spore wall formation
seems to be una¡ected.
FEMSLE Congress 2-6-03
339
P10^49
SUB-CYTOLYTIC ANTHRAX LETHAL TOXIN IN-
HIBITED THE PHAGOCYTOSIS OF MOUSE MAC-
ROPHAGES IS INDEPENDENT OF IMPAIRING THE
MAPK KINASE ACTIVITY
H.-C. Lin, H.-H. Huang, H.-F. Shyu, S.-S. Tang, H.-W.
Liu, and J.-H. Kau
Institute of Preventive Medicine, National Defense Medical
Center, Taipei 237, Taiwan, Republic of China
Lethal toxin, a mitogen activated protein kinase kinase
(MAPKK) inhibitor, produced by Bacillus anthracis, spe-
ci¢cally kills the macrophage cells in vitro. When using
cytolytic dosage treatment (10-2 Wg/mlV1Wg/ml), lethal
toxin strongly inhibited phagocytosis; IL-1L, TNF-Q secre-
tion and MAPKK activity within 2 hours. However, in
sub-cytolytic dosage (10-5-10-7Wg/ml), the anthrax lethal
toxin remain able to speci¢cally inhibit 30V50 % phago-
cytosis activity of mouse macrophage J774A.1 cells with-
out a¡ecting MAPKK. Meanwhile, the secretion levels of
IL-1L and TNF-K were elevated in this condition. In this
report, we demonstrate that macrophage might be medi-
ated through alternative pathway under the cytolytic or
sub-cytolytic lethal toxin treatment respectively. The dos-
age of Anthrax toxin in the blood, in the nature status,
may gradually be elevated accompanied B. anthracis
propagated constantly. In other words, these results may
implicate the stepwise pathogenesis and alternative inhibi-
tion pathway of anthrax lethal toxin during anthrax infec-
tion.
P10^50
MODULATION OF THE PROTEIN FOLDING CA-
PACITY OF E. COLI BY SIMULTANEOUS OVER-
PRODUCTION OF DIFFERENT MOLECULAR
CHAPERONES
A. Walker(1), N. Weir(2), P. A. Lund(1)
(1) School of Biosciences, University of Birmingham, Bir-
mingham, B15 2TT, UK; (2) Celltech Therapeutics Ltd,
216 Bath Road, Slough SL1 4EN, UK
When heterologous proteins are over-expressed in bacteri-
al cells, they often form inclusion bodies, which can be a
major barrier to the puri¢cation of proteins for further
study or for producing proteins of potential commercial
value. Cells deal with the problem of protein aggregation
in the crowded environment of the cytoplasm by express-
ing molecular chaperones, and when molecular chaperones
are depleted, large scale protein aggregation occurs. The
two major families of molecular chaperones in bacteria are
340 1st FEMS Congress / Posters 103^505
the GroE proteins GroES and GroEL, and the DnaK,
DnaJ and GrpE proteins. Some success has been reported
with over-expressing some of the components of one or
other of these molecular chaperone machines to improve
the solubility of heterologous proteins. We have looked at
the e¡ects of simultaneous co-expression of both systems,
by constructing plasmids that expressed di¡erent combi-
nations of all ¢ve molecular chaperone proteins under the
control of inducible promoters. Most of the resulting plas-
mids were stable and did not cause any change in the
growth pro¢le of the E. coli cells that carried them. The
e¡ects of expressing the proteins on the solubility and
yield of a model substrate protein (a fusion between thio-
redoxin and the catalytic domain of a protein kinase) were
investigated, and we con¢rmed that the presence of the
two chaperone machines at elevated levels has a greater
e¡ect than either of the two ‘‘chaperone machines’’ acting
alone This ¢nding may be generally useful in improving
the solubility of proteins that normally form inclusion
bodies when over-expressed in E. coli.
P10^51
YEAST COG COMPLEX, A YPT1P EFFECTOR RE-
QUIRED FOR RETROGRADE INTRA-GOLGI TRAF-
FICKING, INTERACTS WITH GOLGI SNARES AND
WITH COPI VESICLE COAT PROTEINS
V. V. Lupashin(1), E. S. Suvorova(1) and R. Duden(2)
(1) Department of Physiology and Biophysics, University of
Arkansas for Medical Sciences, Little Rock, Arkansas
72205, USA; (2) Department of Clinical Biochemistry,
Cambridge Institute for Medical Research, University of
Cambridge, Hills Road, Cambridge CB2 2XY, UK
The sorting and targeting of protein transport vesicles to
speci¢c docking and fusion sites is essential for the main-
tenance of a proper organization of membrane compart-
ments in the eukaryotic cell. To ensure the appropriate
directionality of membrane £ow, target organelles must
possess a molecular machinery allowing for the speci¢c
recognition and docking of incoming vesicles. The con-
served oligomeric Golgi (COG) complex was identi¢ed
as one of the evolutionary conserved protein complexes
that regulate a cis-Golgi step in intracellular vesicular
transport. We have identi¢ed three new Saccharomyces
cerevisiae COG complex subunits, COG1p, COG4p and
COG6p. In yeast cytosol these proteins are found to be
tightly associated with the previously identi¢ed Sec35p
(COG2p) and Sec34p(COG3p). Mutations in the COG
complex subunits result in defects in basic Golgi func-
tions : glycosylation of secretory proteins, protein sorting
and retention of Golgi resident proteins. Furthermore, the
COG complex interacts genetically and physically with the
small GTP-binding Rab protein Ypt1p, Golgi vesicle coat
FEMSLE Congress 2-6-03
complex COPI as well as with intra-Golgi SNARE mole-
cules. The puri¢ed COG complex speci¢cally binds to the
SNARE domain of cis-Golgi t-SNARE Sed5p. In addi-
tion, the COG complex e⁄ciently interacts with preformed
Sed5p-containing SNARE complexes. We propose that
the COG protein complex act as a tether that connects
cis-Golgi membranes and COPI-coated, retrogradely tar-
geted intra-Golgi vesicles.
P10^52
THE CORRELATION BETWEEN OIL-DEGRADING
ACTIVITY AND COLONY MORPHOTYPES IN
SOME STRAINS OF NOCARDIOFORM ACTINO-
BACTERIA
D. A. Melnikov, O. S. Makhlaeva, E. V. Karaseva
Kuban State University, Stavropolskaya st. 149 room 414,
350040 Krasnodar, Russian Federation
Actinobacteria strains are known to be dominant in oil
pollution ecosystems and e⁄cient in oil degradation. The
studies of attributes of biodegradation at actinobacteria is
very important for the reception of high e¡ective strains.
48 strains of nocardioform actinobacteria were selected
from oil contamination site in Krasnodar region. These
strains we examined for the ability to grow on mineral
medium with n-alkanes (C5-C18), aromatic hydrocarbons
(derivates of benzene), some PAHs and 8 carbohydrates as
the sole source of carbon and energy. All the strains were
grouped in three clusters as M-, S- and R-colony morpho-
types. Eight M-strains, twenty S-strains and twenty R-
strains. M-strains were able to grow on almost all of ex-
amined carbohydrates and n-alkanes (C5-C18 and of high-
er molecular weight) in contrast to the R-strains were able
to grow only on glucose and 1-2 carbohydrates and n-al-
kanes (C13-C18 and of higher molecular weight). S-strains
have occupied the intermediate position. The characteris-
tics of biodegradation of PAHs and derivates of benzene
were established from all examined strains evenly. 40
strains were able to oxidize the derivates of benzene and
only six strains PAHs. All M-strains grew faster than S-
strains and especially R-strains on nutrient broth medium.
These data were used for the choice of strains introduced
in oil contaminated sites with di¡erent qualitative struc-
ture of hydrocarbon pollutant.
This work was carried out within the framework of project
INTAS 01-2151.
 1st FEMS Congress / Posters 103^505
P10^53
THE USE OF MULTI-PARAMETER FLOW CYTO-
METRY (MPFC) TO MONITOR VIABILITY OF A BI-
OPROCESS BY THE METHYLOTROPIC YEAST PI-
CHIA PASTORIS
J. M. Mercer(1), C. J. Hewitt(1) and B. V. Kara(2)
(1) Centre for Bioprocess Engineering, Dept. Chemical En-
gineering, University of Birmingham, Edgbaston, Birming-
ham, B15 2TT ; (2) Avecia Life Science Molecules, Belasis
Avenue, Billingham, Cleveland, TS23 1YN
The use of multi-parameter £ow cytometry has been used
over the years for monitoring clinical samples, cell cycle,
and cell sorting of various mammalian cells. Only recently
has the application of this technique been used for char-
acterising microbiological samples to gain an insight into
the heterogeneity of a sample that is generally overlooked.
The viability of a fermentation process by the methylo-
tropic yeast, Pichia pastoris, has been used as an alterna-
tive to traditional E. coli fermentations, as this organism
has the bene¢ts of potential high cell density fermentation,
high expression levels and product titre due to the tightly
regulated alcohol oxidase gene in the presence of metha-
nol. A typical Pichia pastoris fermentation is a three-stage
process, batch, fed-batch, and induction of the alcohol
oxidase gene. Other bene¢ts include post-translational
modi¢cations that are not seen in bacterial expression sys-
tems. Here the viability of Pichia pastoris in a complex
media and basal salts media, has been investigated with
the use of a ratiometric counting technique to determine
the total number of cells in the presence of cell speci¢c
£uorophores to detect various physiological states includ-
ing cell de-polarisation and cell death. It has been found
that regardless of the media used, the reproductive viabil-
ity, (CFU/ml), of Pichia pastoris increases to a maximum,
and then a sudden drop is observed whereas a correspond-
ing drop in viability with respect to cytoplasmic membrane
permeability and polarisation as measured by MPFC, did
not follow as expected. Therefore multi-parameter £ow
cytometry has been found to be an important tool mon-
itoring cell physiology during a fermentation process.
FEMSLE Congress 2-6-03
341
P10^54
SECRETION CAPACITY LIMITATIONS OF THE Sec
PATHWAY IN ESCHERICHIA COLI
F. J. Mergulha‹o, J. M. S. Cabral and G. A. Monteiro
Centro de Engenharia Biolo¤gica e Qu|¤mica, Instituto Supe-
rior Te¤cnico, Av. Rovisco Pais, 1049-001 Lisbon, Portugal
The secretion capacity of two E. coli strains (JM109 and
AF1000) was evaluated through the expression of two
human proinsulin fusion proteins using the translocation
signal sequence from Staphylococcal protein A. Although
a 5-fold di¡erence in the expression levels was attained by
the use of di¡erent promoters (protein A and malK pro-
moters) and copy-number vectors (700 and 50 copies per
cell), the maximum translocation rates for all the systems
were around 7.7x10e5 amino acids/cell.min. The secretion
capacity was found to be independent from the size of the
exiting peptide and from its translational rate. Several
strategies to partially overcome this secretion bottleneck
in E. coli cells are also presented.
P10^55
PH AFFECTS THE BUTYRATE PRODUCTION BY
HUMAN INTESTINAL FLORA THROUGH MODU-
LATION OF LACTATE METABOLISM
C. Michel, C. Bourriaud, M. C. Alexandre, F. Kozlowski
and C. Cherbut
UFDNH-INRA, BP71627, 44316 Nantes cedex 03, France
Butyrate, resulting from carbohydrates fermentation by
human intestinal bacteria is bene¢cial for mucosal health.
Parameters which govern its biosynthesis are poorly
known. Butyrate production is stimulated at slightly acidic
pH [1] and partly results from lactate metabolism [2].
Since slightly acidic environment favours lactic acid pro-
ducing bacteria [3] and a¡ects butyrate production from
lactate by some ruminal lactate-users [4], we have inves-
tigated the pH impact onto lactate production during the
fermentation of a butyrogenic substrate and on lactate
metabolism by human intestinal micro£ora. Kinetics stud-
ies of lactate and short-chain fatty acids (SCFA) concen-
trations were carried out from incubations of 3 faecal £ora
in the presence of FOS (10g.L-1) or lactate (30mM), at pH
5.8 or 6.5. pH 5.8 did not a¡ect the total amount of SCFA
produced but modi¢ed their relative proportions, as typi-
¢ed by an increased butyrate ratio (+ 4,2% in average). In
both pH conditions, lactate transitorily accumulated dur-
ing the ¢rst 5 hours of the incubation. The area under
these curves were increased at pH 5.8 for £ora A & B
(+ 17,8 and 12,1, respectively) but not for C (+ 0,4). Pre-
342 1st FEMS Congress / Posters 103^505
liminary results about pH e¡ect on lactate metabolism
suggested that pH 5.8 did not a¡ect the intensity of its
conversion but induced changes in end products with in-
creased proportion of butyrate. This study con¢rms that
butyrate production by human intestinal £ora could be
controlled by pH and suggests that this e¡ect results
from both the enhancement of lactate production and
the modi¢cation of its metabolism pathways.
[1] Michel et al (1998), Ananerobe 4: 257. [2] Bourriaud et
al (2003), in preparation. [3] Veilleux and Rowland (1981),
J. Gen. Microbiol, 123:103. [4] Counotte et al (1981),
AEM. 42: 649
P10^56
CHARACTERIZATION OF SIDEROPHORES PRO-
DUCED BY FLAVOBACTERIUM PSYCHROPHILUM
J. D. M\ller(1), A. C. Barnes(2,3), A. E. Ellis(2) and I.
Dalsgaard(1)
(1) Danish Institute for Fisheries Research, Fish Disease
Laboratory, Frederiksberg, Denmark; (2) Fisheries Re-
search Services, The Marine Laboratory, Victoria Road,
Aberdeen, Scotland, U.K.; (3) Employee of Novartis Ani-
mal Vaccines Limited
Flavobacterium psychrophilum is a Gram-negative rod
causing bacterial cold water disease (BCWD) and rainbow
trout fry syndrome (RTFS), both serious diseases a¡ecting
primarily rainbow trout fry and ¢ngerlings. Although
these diseases are of great importance in aquaculture
around the world, limited data are available on the way
this pathogen interacts with its host. An essential factor in
infections is the availability of iron, which is necessary for
the multiplication of the pathogen. Iron is often very lim-
ited within the host as free iron is readily bound to e.g.
transferrin in serum. Many pathogenic bacteria have, how-
ever, developed ways of chelating iron bound by the host.
One of these mechanisms is the utilization of siderophores,
small molecules released by the bacteria that will chelate
iron and subsequently transfer the iron to the pathogen.
We have screened strains of F. psychrophilum by growing
them on CAS-agar adjusted to F. psychrophilum growth
conditions. The type of siderophores was characterized as
well as the importance of siderophore release for bacterial
growth in iron-depleted media. Any relation between sid-
erophore expression by F. psychrophilum and certain plas-
mids was furthermore investigated. P10^57
FEMSLE Congress 2-6-03
REGULATION OF ENZYMES INVOLVED IN OR-
GANIC ACID BIOSYNTHESIS BY YEAST YARRO-
WIA LIPOLYTICA
I. G. Morgunov
G. K. Skryabin Institute of Biochemistry and Physiology of
Microorganisms, Russian Academy of Sciences, pr-t Nauki
5, Pushchino, Moscow region, Russia 142290
Yarrowia lipolytica produces organic acids ^ intermediates
of Krebs cycle from di¡erent carbon sources. The bio-
chemical mechanism of the organic acids overproduction
is not clearly understood. We studied the changes in the
enzymes activity levels and nucleotides contents of Yarro-
wia lipolytica cells grown under limitation by the nitrogen
sources or thiamine. The limitation of nitrogen in the cul-
tivation medium led to increase in the ATP/AMP and
NADH/NAD ratio and to excretion of citric acids. The
ATP/AMP ratio in the cells grown under thiamine limita-
tion decreased during the exponential phase to be constant
in the course of further cultivation, when the cells began to
excrete keto- acids. Changes in the nucleotides ratio of
nitrogen and thiamine-de¢cient cells are discussed in rela-
tions of the regulation of the Krebs cycle enzymes. Citrate
synthase and NAD-isocitrate dehydrogenase were isolated
and puri¢ed from Yarrowia lipolytica. The regulation of
the enzymes by nucleotides and Krebs cycle intermediates
were studied.
P10^58
SUCCINATE DEHYDROGENASE IS ESSENTIAL
FOR RESISTENS OF YEAST CELLS SACCHAROMY-
CES CEREVISIAE TO OXIDATIVE STRESS
N. Museykina(1), S. Victor(2), M. Kondrashova(2)
(1) Saratov State University, Saratov, Russia; (2) Insti-
tute of Theoretical and Experimental Biophysics, Moscow
Region, Pushchino, Russia
We investigated the role of succinate dehydrogenase
(SDH) in resistance of yeast S. cerevisiae against oxidative
stress. We have found that treatment of cells grown on
medium with ethanol up to mid ^ log phase, by 1mM
hydrogene peroxide led to 5 ^ ¢eld increases of SDH ac-
tivity. In the same time, we observed increasing of activity
of SOD and catalase. The survival of cells under these
conditions is 78%. In contrast, grown of cells in medium
which contains of 1 mM malonate (inhibitor of SDH), did
not lead to increase stress ^ induce activity of SDH,
whereas activity of SOD and catalase were on level which
typical for cells treated by hydrogene peroxide without
malonate. Despite, survival of cells were only 23%. We
have to emphasyse that SDH can play the key role in
 1st FEMS Congress / Posters 103^505
defence of S. cerevisiae cells against to oxidative stress,
because even high activity of SOD and catalase at low
activity of SDH cannot provide good survival cells after
treatment by 1mM hydrogene peroxide. We consider that
level of SDH activity can re£ect the degree of antioxidant
defence of cells. Probably, the role of SDH is realizated as
source of fast formation of ATP via respiratory chain of
mitochondria. In turn, fast in£ux of ATP is most impor-
tant factor in realization of adaptacion routs of the cells
against oxidative stress.
P10^59
PEROXISOME DEGRADATION IN THE ALKANE-
UTILIZING YEAST YARROWIA LIPOLYTICA
T. Y. Nazarko(1), J.-M. Nicaud(2) and A. A. Sibirny(1)
(1) Institute of Cell Biology, Drahomanov Street 14/16,
Lviv 79005, Ukraine; (2) Laboratoire de Microbiologie et
de Ge¤ne¤tique Mole¤culaire, UMR216, URA1925, INRA
Centre de Grignon, BP 01, F-78850 Thiverval-Grignon,
France
Extensive peroxisome proliferation during growth on oleic
acid, combined with the availability of excellent genetic
tools, made the dimorphic yeast Y. lipolytica a powerful
model system for studying peroxisome biogenesis. To de-
velop the conditions for studying the oppositely directed
process of peroxisome degradation (pexophagy) the fol-
lowing work on the H222 wild-type strain was carried
out. Conditions for monitoring the pexophagy in solid
medium were optimised. Strictly regulated peroxisomal
amine oxidase (AMO) was used as a reporter enzyme.
The best AMO induction/peroxisome proliferation was
achieved on ethanol/ethylamine plates and AMO inactiva-
tion/pexophagy ^ by overlaying the plates with top agar
containing glucose/ammonium sulfate mixture. The resid-
ual AMO was visualized by additional overlaying with top
agar AMO assay mixture. Conditions, optimal for perox-
isome proliferation and for pexophagy, appeared to be
rather di¡erent for plate and liquid cultures. In liquid
cultures, the highest AMO and peroxisomal isocitrate
lyase (ICL) induction was observed with oleic acid/ethyl-
amine, and fastest inactivation by glucose/ammonium
chloride, both media bu¡ered at pH 6.8. The rates of
AMO inactivation and peroxisomal thiolase degradation
in optimized conditions were comparable with the rates of
peroxisomal alcohol oxidase inactivation of the methylo-
trophic yeast Pichia pastoris and degradation of peroxi-
somal thiolase in Saccharomyces cerevisiae, respectively.
Surprisingly, we failed to induce ICL inactivation by glu-
cose under nitrogen limitation that indicates the existence
of pexophagy/autophagy switch in Y. lipolytica in response
to nitrogen starvation. Based on the developed conditions,
FEMSLE Congress 2-6-03
343
the monitoring of pexophagy in Y. lipolytica becomes pos-
sible.
P10^60
ENVIRONMENTAL REGULATION OF METHYL-
PHOSPHONATE DEGRADATION IN ESCHERICHIA
COLI
S. V. Matys, N. M. Kuzmina, K. S. Laurinavichius and M.
A. Nesmeyanova
Skryabin Institute of Biochemistry and Physiology of Mi-
croorganisms, Russian Academy of Sciences, Pushchino
142290, Moscow Region, Russia
Decomposition of inactivated carbon-phosphorus (C-P)
bond of alkylphosphonates, which is extremely resistant
to chemical actions, is an up-to-date problem of microbial
biochemistry and biotechnology. These compounds are
widespread both in nature and among xenobiotics (herbi-
cides, pesticides, toxic agents of chemical weapons) pollut-
ing the environment. However, they are degraded only by
microorganisms, mainly by gram-negative bacteria, due to
the function of the enzyme C-P lyase. The activity of this
enzyme has been revealed only in intact cells and up to
now has not been isolated and studied. There is no de-
tailed biochemical and physiological characterization of
alkylphosphanate degradation system either. In the
present work, the e¡ect of environmental factors on deg-
radation of methylphosphonate (Pn) by cells of Escheri-
chia coli has been studied. It was shown that Pn degrada-
tion begins after a long latent period (up to 48 h) of
adaptation to substrate, which proceeds more intensively
under cell growth at 30‡C and does not depend on sub-
strate concentration. Selection of adapted cells increases
the rate of Pn degradation by an order. Factors in£uenc-
ing the process were revealed. The optimal temperature of
Pn degradation by a growing cell culture is 30‡C. A de-
tailed analysis of the e¡ect of dosed portions of oxygen on
Pn degradation showed that the most favorable conditions
for C-P lyase reaction are microaerophilic conditions (low
partial pressure of oxygen), pH 8.0, and logarithmic
growth stage. Pn degradation by recombinant E. coli
strains containing plasmids with the genes of C-P lyase
complex, cloned under the own (pho) or heterologous
(lac) promoter, has been studied. It was shown that Pn
degradation by these strains does not require a long adap-
tation to Pn, however the enhanced synthesis of compo-
nents of C-P lyase complex, con¢rmed by the analysis of
proteins ^ products of the phn genes cloned in plasmids,
does not increase C-P lyase activity of cells and Pn degra-
dation. This indicates the presence of some factors of un-
known nature, limiting the process.
344 1st FEMS Congress / Posters 103^505
P10^61
GROWTH AND INDOLE-3-ACETIC ACID PRODUC-
TION OF AZOSPIRILLUM BRASILENSE SP245 AS
INFLUENCED BY PH, AMMONIA, PHOSPHATE
AND VARIOUS CARBON SOURCES
O. Ona(1), I. Smets(2), J. Van Impe(2), J. Vanderley-
den(1)
(1) CMPG, KULeuven, Kasteelpark Arenberg 20, B-3001
Heverlee, Belgium; (2) BioTeC, KULeuven, Kasteelpark
Arenberg 22, B-3001 Heverlee, Belgium
Worldwide inoculation experiments carried out with Azo-
spirillum brasilense have demonstrated the ability of this
bacterium to promote plant growth and enhance the yield
of crops in di¡erent soils and under di¡erent climatic con-
ditions. Soil factors such as temperature, pH, and the
availability of nutrients can a¡ect the synthesis of IAA
by bacteria associated with plant roots. This work presents
a ¢rst step towards the quanti¢cation of the e¡ect of dif-
ferent levels of ammonia, phosphate and various carbon
sources on growth and indole-3-acetic acid production by
Azospirillum brasilense sp245. Micro aerobic batch cul-
tures of Azospirillum were conducted in a bioreactor
with the following set points: pH 6.0, 6.3, and 6.8; dis-
solved oxygen 3%, and temperature 30‡C. Sulphuric and
phosphorous acid solutions (1N) were used as the pH
control solutions. Irrespective of the carbon source, the
growth pro¢le and IAA production pattern were similar
and no signi¢cant di¡erence in biomass accumulation was
observed. The carbon source in the fermentation medium
became depleted after 8-10 hours while IAA became sig-
ni¢cantly detectable only after a lag of several hours (i.e.
after about 16 hours). IAA levels in the medium were
highest with malate as the carbon source. Minimal
changes in the pH of culture were observed with fructose.
Phosphate is inhibits oxygen uptake at high concentra-
tions but the e¡ect of this on IAA accumulation is not
yet clear. Conclusion: Carbon source, pH, phosphates and
ammonia levels have an important impact on the IAA
biosynthesis by Azospirillum brasilense.
FEMSLE Congress 2-6-03
P10^62
IDENTIFICATION OF IRON REGULATED GENES
IN THE FISH PATHOGEN PHOTOBACTERIUM
DAMSELAE SUBSP. PISCICIDA BY A FUR TITRA-
TION ASSAY (FURTA)
C. R. Osorio(1,2), M. L. Lemos(1) and V. Braun(2)
(1) Depto. de Microbiolog|¤a, Instituto de Acuicultura, Uni-
versidade de Santiago, Santiago de Compostela, Spain; (2)
Mikrobiologie/Membranphysiologie, Universitat Tubingen,
Tubingen, Germany
Photobacterium damselae subsp. piscicida is the causative
agent of pasteurellosis, a ¢sh disease causing important
economical losses in marine aquaculture worldwide. Abil-
ity of this bacterium to obtain iron sources from host
tissues is believed to play a role in pathogenesis, but the
molecular basis of iron scavenging systems is poorly
studied. The Fur Titration Assay (furta) allows the
screening of gene libraries for the presence of clones con-
taining Fur boxes in their promoters, and thus is a valua-
ble tool for the identi¢cation of new putative iron regu-
lated genes. In order to identify genes belonging to the Fur
regulon in P. damselae subsp. piscicida, a plasmid gene
bank was screened by the furta assay. We identi¢ed 7
putative Fur-regulated genes in P. damselae subsp. piscici-
da. One of this genes, a putative tonB, has been described
previously in this species by our group. The remaining
clones include putative new genes with homologs in other
bacteria, and which have not been described to date in P.
damselae: A) two putative siderophore receptors, B) a sec-
ond tonB-exbB-exbD system, C) a ferritin gene, D) a pu-
tative siderophore biosynthetic protein, E) a putative tran-
scriptional regulator of siderophore biosynthesis.
Completion of DNA sequencing of these genes as well
as up- and downstream genes which may also be related
in function and constituting operons, is being accom-
plished. Construction of deletion mutants and expression
of these genes in Escherichia coli mutants for function
complementation, will help to unravel the role of the fur-
ta-positive genes in the iron uptake metabolism of P.
damselae subsp. piscicida.
 1st FEMS Congress / Posters 103^505
P10^63
ATTEMPT TO SILENCE THE murA GENE EXPRES-
SION IN ESCHERICHIA COLI BY ANTISENSE
STRATEGY
C. I. Pacot-Hiriart(1) and M. Skurnik(1,2)
(1) Department of Medical Biochemistry and Molecular
Biology, University of Turku, Turku, Finland ; (2) The
Haartman Institute, University of Helsinki, Finland
We have attempted to inhibit bacterial growth by interfer-
ing with peptidoglycan biosynthesis. The UDP-N-acetyl-
glucosamine enolpyruvate transferase (MurA) catalyses
the ¢rst committed step in peptidoglycan biosynthesis. In
Escherichia coli deletion of the murA gene is lethal. There-
fore, inhibition of the bacterial growth should be achieved
by silencing expression of the murA gene. Antisense RNA
strategy has proven to be very e⁄cient in silencing gene
expression. In order to silence the murA expression in E.
coli, we will clone ten di¡erent sequences of the murA gene
in antisense orientation under the control of the arabinose-
inducible promoter of the pBAD33oriT vector. We have
selected about 100, 200 and 300 bp long sequences from
the 5’-end, the middle and the 3’-end of the murA gene. In
addition one sequence will be a 180 bp long fragment
containing the Shine-Dalgarno region, the start codon
and the ¢rst 100 bp of the coding region of the murA
gene. The selected fragments were ampli¢ed by PCR and
are being cloned into pBAD33oriT vector to be expressed
under the control of the arabinose-inducible promoter. We
will test the di¡erent clones for the expression of their
murA antisense-RNA products and for their potential in-
hibitory e¡ect on the bacterial growth. These will be com-
pared to a vector control clone containing pBAD33-oriT
without any insert. Detailed results will be presented and
discussed in the poster.
P10^64
PEPTIDES, AMINO ACIDS AND BIOGENIC
AMINES IN LACTOBACILLUS ^ INTERACTIONS
AND METABOLISM
C. I. Pereira(1,2), C. Rodrigues(1), N. Magalha‹es(1), C.
Arraiano(2), M. V. San Roma‹o(1,2,3) and M. T. Barreto
Crespo(1,2)
(1) IBET- Apartado 12, 2781-901 Oeiras, Portugal; (2)
ITQB- Apartado 127, 2781-901 Oeiras, Portugal; (3)
EVN- INIA, Dois Portos, Portugal
Lactobacillus are lactic acid bacteria important for the
maturation of fermented food products such as cheese,
wine and meat. One of the central metabolic activities of
FEMSLE Congress 2-6-03
345
Lactobacillus in the hydrolysis of peptides to obtain the
amino acids needed for growth. The catabolism of the
resulting amino acids has implications in the quality and
safety of fermented foods because some strains can pro-
duce biogenic amines. In this work we have used as study
models two Lactobacillus strains isolated from meat tradi-
tionally fermented products. Peptidases Pep C, Pep V, Pep
V, PepX and Pep N were identi¢ed and their activities
quanti¢ed. The complete sequence of the genes pep C,
pep V and pep X is being performed based on PCR, hy-
bridisation and cloning strategies. Our results seem to be
quite relevant regarding the capacity of these Lactobacillus
strains to produce biogenic amines. Assays performed with
free amino acids as nitrogen sources have demonstrated
that upon exhaustion of the carbon source the production
of the respective biogenic amines can generate extra energy
for growth. The implication of this metabolism in a devel-
oped strategy to survive in an exhausted carbon source
stressed environment, like fermented meat, in these Lacto-
bacillus strains, is currently being investigated.
P10^65
CHARACTERISING OF THE LIPOPOPYSACCHAR-
IDE OUTER CORE BIOSYNTHESIS OF YERSINIA
ENTEROCOLITICA SEROTYPE O:3
E. Pinta(1), R. Venho(1), J. A. Bengoechea(2) and M.
Skurnik(1,3)
(1) Department of Medical Biochemistry, University of
Turku, Kiinamyllynkatu 10, 20520 Turku, Finland; (2)
Unidad de Investigacion, Hospital Son Dureta, Andrea Do-
ria 55, 07014 Palma Mallorca, Spain; (3) University of
Helsinki, The Haartman Institute, P.O. Box 21, 00014 Uni-
versity of Helsinki
Lipopolysaccharide (LPS) is an essential component of the
cell wall of Gram-negative bacteria. It has been shown
that LPS of Yersinia enterocolitica is a virulence factor
and therefore detailed information of its structure, biosyn-
thesis and genetics is needed to understand its role in the
disease process. In Y. enterocolitica O:3 (YeO3) the outer
core comprises of a hexasaccharide of two glucoses, two
N-acetyl-D-galactosamines, one galactose and one N-ace-
tyl-D-fucosamine. The YeO3 outer core gene cluster that
contains 9 genes has been cloned and sequenced. The func-
tions of the gene products were predicted based on amino
acid sequence similarities to known proteins, however
these functions are still putative. Six of the genes were
predicted to encode for glycosyltransferases (one for
each sugar residue in the hexasaccharide), two for enzymes
for NDP-sugar precursor biosynthesis and one for £ip-
pase. In order to de¢nitely assign the speci¢c functions
of the gene products biochemical and genetic analyses
were needed. To facilitate the analysis we constructed a
346 1st FEMS Congress / Posters 103^505
mobilizable plasmid pRV16NP in which the YeO3 outer
core gene cluster is expressed under the vector tetracycline
promoter and an outer core mutant host strain YeO3-c-
OC that has the complete outer core gene cluster deleted.
Mobilization of pRV16NP into YeO3-c-OC comple-
mented fully the outer core de¢cient phenotype. This al-
lows us to easily construct single and double mutants into
the outer core cluster genes on pRV16NP and analyse
their functions in the YeO3-c-OC background. At the mo-
ment we have constructed a few single gene mutants that
are under characterising.
P10^66
TWO NOVEL CELL WALL TEICHOIC ACIDS IN
TWO NOVEL BREVIBACTERIUM SPECIES
N. V. Potekhina(1), A. S. Shashkov(2), L. I. Evtushen-
ko(3) and I. B. Naumova(1)
(1) School of Biology, M. V. Lomonosov Moscow State
University, 119899 Moscow, Russia; (2) N. D. Zelinsky
Institute of Organic Chemistry, Russian Academy of Scien-
ces, Leninsky Prospect, 47, 119991 Moscow, Russia; (3)
Institute of Biochemistry and Physiology of Microorga-
nisms,Russian Academy of Sciences, Pushchino, 142290
Moscow Region, Russia
Two novel structures of cell wall teichoic acids were iden-
ti¢ed in environmental orange-colored strains belonging to
two new Brevibacterium species using chemical methods,
NMR spectroscopy and MS MALDI-TOF. An 1,6-poly(-
mannitol phosphate) polymer with phosphomonoester
groups at O-4(3) in the majority of mannitol residues
were found in B. anticuum, strains VKM Ac-2118T and
VKM Ac-2281, isolated from a frozen late Pliocene layer
(1.8-3 Myr, Kolyma lowland, Russia). B. permense VKM
Ac-2280T from salt-contaminated soil also possessed the
1,6-poly(mannitol phosphate) polymer, but its mannitol
residues had rhamnose at O-3(4) and pyruvic acid residues
at O-4 and O-5. The results obtained are in line with our
working hypothesis that the primary structure of cell wall
teichoic acid may serve as a taxonomic marker of the
intrageneric taxa of Brevibacterium. It is also noteworthy
that the side phosphate and pyruvic groups impart addi-
tional negative charge to the polymers and the cell surface,
and probably, along with other factors, convey the halo-
tolerant properties to the strains studied. It has been
shown, that the cell wall of an alkophilic bacillum com-
prised three polymers with markedly pronounced acidic
properties, viz., polyglucuronic, teichuronic, and polyglu-
tamic acids [R. Aono et al. (1993). J. Gen. Microbiol. 139.
2739-2744].
FEMSLE Congress 2-6-03
P10^67
CHARACTERIZATION OF ESCHERICHIA COLI
YgjG PROTEIN AS PUTRESCINE:2-OXOGLUTA-
RATE AMINOTRANSFERASE WITH BROAD SUB-
STRATE SPECIFICITIES
N. N. Samsonova, S. V. Smirnov, I. B. Altman and L. R.
Ptitsyn
Ajinomoto-Genetika Research Institute, Moscow, Russia
The ygjG open reading frame (ORF) from E. coli K12
encodes a protein with signi¢cant homology with pyridox-
al 5’-phosphate dependent g-aminotransferases. We ex-
pressed 1,380-bp ygjG ORF in E.coli and puri¢ed 50
kDa YgjG protein to apparent homogeneity. The puri¢ed
enzyme was able to transaminate putrescine, cadaverine
and in low extent spermidine with 2-oxoglutarate as a
preferable amino group acceptor. The enzyme activity
with agmatine, spermine or ornithine, as a donor of amino
group, was appreciably lower then that with putrescine.
Also, the enzyme exhibited signi¢cant activity toward a-
ketobutyrate and pyruvate as amino acceptors. The en-
zyme was active at alkaline pH with maximum at pH=9
and was almost completely inactive at pH=7. The kinetic
constants for putrescine and 2-oxoglutarate were Km = 2.4
mM, Vmax = 22.3 WM.s-1.mg-1 and Km = 6.5 mM, Vmax =
26.5 WM.s-1.mg-1, respectively. The Km values for cadaver-
ine and spermidine were 7,9 mM and 25,3 mM, respec-
tively. Comparison of kkat/Km ratios for putrescine (7,8 s-
1.
mM-1), cadaverine (1,9 s-1.mM-1) and spermidine (0,4 s-
1.
mM-1) demonstrates that putrescine was the best sub-
strate for this puri¢ed enzyme. Thus we concluded that
YgjG is the putrescine :2-oxoglutarate aminotransferase
(PATase; EC 2.6.1.29), which involved in arginine decar-
boxylase degradation pathway of E. coli.
P10^68
GENES AND ENZYMES OF ECTOINE BIOSYNTHE-
SIS IN HALOPHILIC / TOLERANT METHANO-
TROPHS
A. S. Reshetnikov, V. N. Khmelenina, Y. A. Trotsenko
Institute of Biochemistry and Physiology of Microorganisms
RAS, Prospekt Nauki, 5, Pushchino, Moscow re-
gion,142290,Russia
To cope with elevated external osmolarity haloalkaliphilic
methanotrophs synthesize low-molecular-weight compati-
ble solutes such as ectoine, (1,4,5,6-tetrahydro-2-methyl-
pyrimidine carboxylic acid), glutamate and sucrose. Ec-
toine synthesis in haloalkalitolerant methanotroph
M.alcaliphilum 20Z was shown to proceed from aspartate
 1st FEMS Congress / Posters 103^505
semialdehyde by three successive reaction catalyzed by
special enzymes: diaminobutyric acid transaminase
(EctB), diaminobutyrate acetyltransferase (EctB) and ec-
toine synthase (EctC). This enzymatic sequence being
analogous to that in the other Gram-negative eubacterial
halophiles (e.g. Halomonas elongata), however, aspartate,
but not glutamate, serves as amino donor for transamina-
tion of aspartate semialdehyde. The genes encoding EctA,
EctB and EctC proteins have been identi¢ed and charac-
terized. Analysis of the derived amino acid sequence of
ectABC showed low (no more 60%) similarity with the
appropriate enzymes in other halophilic bacteria (Marino-
coccus halophilus, Bacillus halodurans). The constructed
phylogenetic trees based on ectB and ectC sequences in-
dicated that among ectoine synthesizing halophiles, the
closest neighbors to methanotrophs were Gram-negative
bacteria.
P10^69
A NEW BIOASSAY METHOD FOR QUANTITATIVE
ANALYSIS OF TETRACYCLINE
N. Rodr|¤guez, J. G. Lore¤n and N. Rius
Facultat de Farma'cia, Universitat de Barcelona, Avda. Joan
XXIII s/n, 08028 Barcelona, Spain
Tetracyclines are broad-spectrum bacteriostatic antibiotics
produced by species of the genus Streptomyces. They are
active against a wide range of Gram-positive and Gram-
negative bacteria and they are especially useful for treat-
ment of acne, brucellosis, urinary tract infections caused
by Gram-negative bacteria, and infections caused by mi-
coplasmas, rickettsias, and chlamydias. Tetracyclines are
also used frequently in veterinary formulations to prevent
and control disease, as well as in feed additives to promote
weight gain. The agar di¡usion technique and turbidimet-
ric methods are used routinely for determining tetracycline
potency. These assays require a signi¢cant amount of ma-
terials and media and they are labour intensive and time
consuming. We have developed a new bioassay method for
determining the tetracycline potency of pharmaceutical
samples, based on the measurement of pH response of
bacterial suspensions after the addition of an aliquot of
a concentrated glucose solution. This method consisted of
preparing suspensions of Enterococcus faecalis ATCC
10541 in 0.85% NaCl sterile solution, which were magneti-
cally stirred at room temperature. After insertion of the
pH electrode the suspension was vigorously mixed and
exposed to the antibiotic. A glucose pulse was given and
changes in external pH were recorded. The decrease in
external pH of these suspensions depended on the concen-
tration of tetracycline. The less antibiotic added the higher
pH fall was observed. Correlation coe⁄cients of standard
curves derived from the assay were 0.99. The procedure is
FEMSLE Congress 2-6-03
347
rapid, precise and quantitative and requires minimal prep-
aration and minimal use of media, with savings in labora-
tory resources and time.
P10^70
TAURINE DEGRADATION IN RHODOCOCCUS
SPP: A REVERSE GENETIC APPROACH TO C-
AND S-LIMITED METABOLIC PATHWAYS
J. Ru¡, K. Denger, D. Schleheck and A. M. Cook
Department of Biology, The University, D-78457 Konstanz,
Germany
Taurine (2-aminoethanesulfonate) is the major organic sol-
ute in mammals. Its bacterial catabolism as sole carbon
and energy source for growth involves conversion to sul-
foacetaldehyde, which is desulfonated to acetyl phosphate
by sulfoacetaldehyde acetyltransferase (Xsc). Xsc is found
in many aerobic and anaerobic bacteria. Three Xsc-sub-
groups are known (Ru¡, 2003, Biochem. J.), and we con-
clude that Xsc from the Gram-positive Nocardiaceae Rho-
dococcus opacus ISO-5 and Rhodococcus sp. strain RHA1
belong to subgroup 1, which otherwise contains Xsc from
f-Proteobacteria (Cook, 2002, Arch. Microbiol.). Rhodo-
coccus spp. contained inducible taurine transaminase, Xsc
and phosphate transacetylase. Preliminary genomic se-
quence data from strain RHA1 (Microbial Envirogenetics,
Genome Canada, unpublished) indicated a genomic con-
text di¡erent from those known in the Proteobacteria.-
Taurine-sulfur is released for assimilation into the cell
material of aerobes by taurine dioxygenase, TauD (Ker-
tesz, 2000, FEMS Microbiol. Rev.; Nelson, 2002, Environ.
Microbiol.). Taurine was also used as sole sulfur source
for growth by strains ISO-5 and RHA1. A tauD-like gene
was found in the genome of strain RHA1. We thus antici-
pate being able to con¢rm in single organisms that, at the
physiological, biochemical and genetic levels, the dissim-
ilation of taurine carbon is catalyzed by di¡erent enzymes
under di¡erent regulation than the processes involved in
the assimilation of taurine-sulfur.
348 1st FEMS Congress / Posters 103^505
P10^71
REACTIONS OF TRANSAMINATION ARE ESSEN-
TIAL FOR GROWTH AND SURVIVAL OF YEAST
SACCHAROMYCES CEREVISIAE UNDER AEROBIC
METABOLISM
V. A. Samokhvalov(1,2), N. Yu. Museikina(2,4), D. I.
Ogorelyshev(1,3), M. N. Kondrashova(1)
(1) Institute of Theoretical and Experimental Biophysics
Russian Academy of Sciences, Pushchino, Russia ; (2) Sar-
atov State University, Saratov, Russia; (3) Pushchino
State University, Pushchino, Russia ; (4) Institute of Bio-
chemistry and Physiology of Microorganisms Russian Acad-
emy of Sciences
Transamination of glutamate and oxalacetate in mito-
chondria plays an essential role in rapid formation of suc-
cinate for acceleration of respiration and energy supply
(M.Kondrashova 1987, 1991). This leads to increase of
glycolysis inhibition by respiration, Pasteur E¡ect, and
to stimulation of glyconeogenesis in rat liver and in human
organism. In this work the role of transamination was
investigated in metabolism and vital activity of yeast Sac-
charomyces cerevisiae, grown on glucose, ethanol or galac-
tose, providing anaerobic, aerobic or aerobian fermenta-
tion metabolism respectively. To test transamination, 1
mM of aminohydroxy-acetate (AOA), transaminase inhib-
itor, was used. To test succinate participation, 1 mM of
malonate, succinate dehydrogenase inhibitor, was used.
AOA addition did not inhibit cell growth on glucose but
induced cell death at stationary phase when catabolite
repression of respiration was abolished. This suggested
that participation of transamination is essential only for
aerobic metabolism. Indeed, AOA inhibited growth on
ethanol per 62% and only per 23% on galactose. Survival
of cells after heat shock (37‡C, 1 hour) was also consid-
erably decreased by AOA under aerobic metabolism, 48%,
was not changed under anaerobic metabolism, and was
intermediate with galactose, 68%. In£uence of malonate
on cell survival after heat shock correlated completely
with sensitivity to AOA. These data support the view of
transamination is important for acceleration of respiration
and energy supply by rapid formation of succinate. We
showed that transamination is essential for adaptive,
stress-induced reactions. Finding this phenomenon also
in yeast evidences that this is general biological mecha-
nism.
FEMSLE Congress 2-6-03
P10^72
BIOCHEMICAL AND GENETIC CHARACTERIZA-
TION OF THE TWO-STEP PATHWAY FOR THE
SYNTHESIS OF MANNOSYLGLYCERATE IN THE
THERMOPHILIC BACTERIUM RHODOTHERMUS
MARINUS
N. Borges(1), J. D. Marugg(2), N. Empadinhas(3), M. S.
da Costa(3) and H. Santos(1)
(1) Instituto de Tecnologia Qu|¤mica e Biolo¤gica, Universi-
dade Nova de Lisboa, Rua da Quinta Grande 6, Apartado
127, 2780-156 Oeiras, Portugal; (2) Nestle¤ Research Cen-
ter, CH-1000 Lausanne 26, Switzerland; (3) Department of
Biochemistry and Centre for Neurosciences of Coimbra,
University of Coimbra, 3004-517 Coimbra, Portugal
Rhodothermus marinus accumulates mannosylglycerate
(MG) as a major compatible solute in response to growth
at supraoptimal temperatures and/or salinity. Two alter-
native pathways for the synthesis of MG have been iden-
ti¢ed: the single-step pathway, where, GDP-mannose is
condensed with D-glycerate to produce mannosylglycerate
in a single reaction catalysed by mannosylglycerate syn-
thase; in the two-step pathway, a mannosyl-3-phosphogly-
cerate synthase (MPGS) catalyses the conversion of GDP-
mannose and D-3-phosphoglycerate to a phosphorylated
intermediate, which is subsequently converted to MG by
the action of a mannosyl-3-phosphoglycerate phosphatase
(MPGP). MPGS was puri¢ed from R. marinus cells. The
N-terminal and internal a.a. sequences were used to design
speci¢c DNA probes, allowing the isolation of suitable
clones from a genomic library. A gene, designated mpgs,
encoding a protein of 427 amino acids and showing a
perfect matching with the amino acid sequence of the pu-
ri¢ed protein was found. Furthermore, a second open
reading frame located downstream of mpgs showed signi¢-
cant homology with the gene encoding MPGP in Pyrococ-
cus horikoshii. Taking into account the information of the
N-terminal and of the conserved sequence of the homolo-
gous phosphatases, a DNA probe was designed and the
complete sequence of mpgp was obtained. Both genes were
cloned and overexpressed in E. coli. The recombinant
MPGS and MPGP were characterised. MPGS was 14
times less active than that of P. horikoshii and possessed
29 additional residues in the C-terminal. Constructing a
gene lacking this extension resulted in a protein with a 10-
fold increase of MPGS activity. These results suggest a
post-translation regulatory process involving a speci¢c
protease.
 1st FEMS Congress / Posters 103^505
P10^73
DIFFERENCE IN THE REGULATION OF GENE EX-
PRESSION BY QUORUM SENSING-RELATED
GENES IN VIBRIO VULNIFICUS
N. R. Shin(1), K. S. Kim(2), S. J. Shin(1), D. Y. Lee(1),
S. G. Kang(1) and H. S. Yoo(1)
(1) Department of Infectious Diseases, College of Veteri-
nary Medicine and School of Agricultural Biotechnology,
Seoul National University, 151-742, Seoul; (2) Department
of Life Science, Sogang University, 121-742, Seoul, Korea
Quorum sensing is the regulatory mechanism of gene ex-
pression in response to the £uctuation of bacterial cell
population density. It has been known that many Gram
negative bacteria including Vibrio spp. regulate gene ex-
pression through quorum sensing system. Vibrio vulni¢cus
is also expected that use similar system as sure as many
other bacteria. Recently, luxS and smcR homologues were
found in Vibrio vulni¢cus. In this study, Vibrio vulni¢cus
mutants were constructed by deleting quorum sensing-re-
lated genes, luxS and smcR homologues, from a wild type
strain (ATCC29307) using homologous recombination. To
identify quorum sensing-regulated genes, we analysed
mRNA and the protein expression patterns of V. vulni¢cus
with DDRT-PCR and two-dimensional polyacrylamide
gel electrophoresis, respectively. Twenty proteins showing
di¡erential expression levels were identi¢ed by the gel elec-
torphoresis. Three di¡erent PCR products that belong to
range of 200-400bp were found in the comparison of wild-
type strain with mutants. We are identifying the proteins
and PCR products. Taken together, these results suggest
that quorum sensing-related genes of V. vulni¢cus might be
involved in regulating on expression of other genes.
P10^74
IDENTIFICATION OF INTAGENIC MUTATIONS IN
THE HANSENULA POLYMORPHA PEX6 GENE
THAT AFFECT PEROXISOME BIOGENESIS AND
METHYLOTROPHIC GROWTH
O. V. Stasyk(1), V. Y. Nazarko(1), T. Y. Nazarko(1), A.
Krikken(2), M. Veenhuis(2), A. A. Sibirny(1,3)
(1) Institute of Cell Biology Nat. Acad. Sci. of Ukraine,
Drahomanov Str. 14/16, Lviv 79005, Ukraine; (2) Gronin-
gen Biomolecular Sciences and Biotechnology Institute
(GBB), University of Groningen, Kerklaan 30, 9751 NN
Haren, The Netherlands; (3) Institute of Biotechnology,
Rzeszow University, Rejtana 16C, 35-310 Poland
Two interacting AAA ATPases, Pex1p and Pex6p, are in-
dispensable for peroxisome biogenesis. Mutations a¡ecting
FEMSLE Congress 2-6-03
349
corresponding genes are the most common cause of the
Zellweger syndrome in humans. By UV-mutagenesis of H.
polymorpha pex6 mutant de¢cient in peroxisome biogene-
sis, we isolated conditional cs strain with restored ability
to grow in methanol medium at 37‡C but not at 28‡C. It
exhibited conditional growth defect only in methanol, but
not in glucose or ethanol media. Electron microscopical
analysis revealed restored peroxisome biogenesis in meth-
anol-induced cspex6 cells at both temperatures. Genetic
analysis demonstrated that cs mutation is tightly linked
with the PEX6 locus. Parental pex6 and derivative cspex6
alleles were PCR ampli¢ed and sequenced. A point muta-
tion that leads to substitution of a conserved amino acid
residue (G737E) in the ¢rst AAA module of the PEX6
gene was identi¢ed for pex6 allele. An additional intra-
genic mutation for cspex6 allele was found to be a con-
served amino acid substitution in the second AAA domain
(R1000G). This second mutation does not lead to Pex-
phenotype independently. The isolated cspex6 strain was
more sensitive to elevated methanol concentrations rela-
tive to the wild-type strain. Studies are in progress to elu-
cidate molecular background of its conditional phenotype.
Our data suggest that H. polymorpha Pex6p may have a
dual function in peroxisome biogenesis: at its early stage
(impaired in the pex6), and at the later stage (impaired in
cspex6 at the restrictive temperature). Identi¢ed conserved
amino acid residues in the two AAA modules are involved
in these putative functions.
P10^75
rib1-86 MUTATION AS A TOOL FOR IDENTIFICA-
TION OF NEW GENES INVOLVED IN CONTROL
OF RIBOFLAVIN BIOSYNTHESIS IN YEAST PI-
CHIA GUILLIERMONDII
K. E. Kapustiak, M. M. Stenchuk, Y. R. Boretsky, O. V.
Stasyk, A. A. Sibirny
Institute of Cell Biology National Academy of Sciences of
Ukraine Dragomanov St., 14/16, 79005, Lviv, Ukraine
Yeast Pichia guilliermondii overproduces ribo£avin (vita-
min B2) in response to iron limitation. Molecular mecha-
nisms of such regulation are still unknown. To study this
phenomenon, it is necessary to select putative mutations
leading to altered regulation of ribo£avin biosynthesis and
identify genes involved. We have isolated P. guilliermondii
rib1-86 ribo£avin de¢cient mutant, which lacks activity of
GTP cyclohydrolase II catalyzing the ¢rst stage of ribo-
£avin biosynthesis. This strain spontaneously reverted to
ribo£avin prototrophy at a frequency 10-7 to 10-6. We
have cloned and sequenced RIB1 gene coding for GTP
cyclohydrolase from the mutant strain using PCR tech-
nique. In contrast to the wild-type gene, the mutant gene
did not complement ribo£avin auxotrophy neither in E.
350 1st FEMS Congress / Posters 103^505
coli nor in P. guilliermondii strains defective in GTP cyclo-
hydrolase II structural genes. The comparative analysis of
RIB1 and rib1-86 nucleotide sequences revealed a single
point mutation : substitution of G560 to A, that converts a
cysteine codone to tyrosine. The genetic analysis of a
spontaneous rib+ revertants of rib1-86 mutant revealed
six new genes RED1-RED6 (reduction) a¡ecting regula-
tion of ribo£avin biosynthesis as well as known before
genes RIB81, RIB80 and HIT1. Relative to the wild type
strain, red mutants possess : (i) increased activity of GTP
cyclohydrolase, ribo£avin synthase and elevated levels of
ribo£avin biosynthesis; (ii) enhanced ferric/cupric reduc-
tase activity and higher non-hemin iron content in cell;
(iii) increased sensitivity to transition metals, as well as
to hydrogen peroxide. Thus, rib1-86 mutation is a useful
tool for identi¢cation of genes involved in regulation of
ribo£avin biosynthesis.
P10^76
GENETIC ANALYSIS OF TREHALOSE AND MAN-
NOSYLGLYCERATE PRODUCING ENZYMES IN
THERMUS SPP. AND THEIR RELEVANCE FOR OS-
MOTOLERANCE
Z. Silva, S. Alarico and M. S. da Costa
Department of Biochemistry and Center for Neurosciences
of Coimbra, University of Coimbra, 3004-517 Coimbra,
Portugal
The organisms of the genus Thermus are thermophilic
(Top"70‡C) with di¡erent degrees of osmotolerance rang-
ing from 1 to 5% NaCl. Recently, the genes tps and tpp
from T. thermophilus RQ-1 were cloned and sequenced.
These genes are immediately followed by treS, and are
organized in an operon-like structure. The importance of
tps and tpp genes for osmoadaptation was also proven
since partial deletion of these genes greatly a¡ects salt
tolerance of T. thermophilus RQ-1 (our unpublished re-
sults). Interestingly, the screening for the presence of the
trehalose operon in other T. thermophilus strains allowed
us to conclude that the trehalose operon was absent in
HB-27 and that it was incomplete in strain GK-24, HB-
8, AT-62 (where only tpp and treS are present), whereas in
UT-2, PRQ-14, Fiji3A1 and B it shows the same organi-
zation as in RQ-1. The genes responsible for the synthesis
of trehalose were also absent in organisms belonging to T.
aquaticus, T. scotoductus, T. antranikiani, T. ¢liformis, T.
igniterrae, T. brockianus and T. oshimai. The presence of
mannosylglycerate synthesis genes (mpgs and mpgp) was
also screened, revealing that the presence of these genes
was exclusive of T. thermophilus strains. We believe that
the salt adaptation process of the T. thermophilus strains
lacking the trehalose operon involves transport of treha-
FEMSLE Congress 2-6-03
lose from the yeast-extract-based media. In the present
work, we have used a growth media deprived of external
trehalose to assess the salt tolerance limits of the organ-
isms and to establish a correlation between the presence of
the genes for the synthesis of the compatible solutes tre-
halose and mannosylglycerate and osmotolerance.
P10^77
PHYSIOLOGICAL ROLE OF TREHALOSE IN THER-
MUS THERMOPHILUS RQ-1
Z. Silva(1), S. Alarico(1), A. Nobre(1), J. Marugg(1,3),
R. Horlacher(2), W. Boos(2) and M. S. da Costa(1)
(1) Department of Biochemistry and Center for Neuroscien-
ces of Coimbra, University of Coimbra, 3004-517 Coimbra,
Portugal; (2) Department of Biology, University of Kon-
stanz, Universita«ttstrasse 10, D-78457 Konstanz, Germany;
(3) Nestle¤ Research Center, CH-1000 Lausanne 26, Swit-
zerland
In Thermus thermophilus a DNA fragment of 2.4 kb carry-
ing the tps (trehalose-phosphate synthase) and tpp (treha-
lose-phosphate phosphatase) and the amino terminal of
treS (trehalose synthase) was cloned from a gene library
and its complete nucleotide sequence was determined. Se-
quence analysis revealed that these genes are organized in
an operon-like structure. The deduced amino acid se-
quence of TPS (52.64 kDa) and TPP (26.91kDa), showed
35% and 36% identity to those of E. coli, respectively. T.
thermophilus RQ-1 responds to salt stress by accumulating
trehalose and mannosylglycerate, the former being the ma-
jor osmolyte. In order to understand the role of trehalose,
a T. thermophilus RQ-1 strain was constructed in which
the trehalose-phosphate synthase (tps) and trehalose-phos-
phate phosphatase (tpp) genes were disrupted. Analysis of
osmolytes accumulated by the mutated strain in response
to salt stress in de¢ned media, revealed the presence of
mannosylglycerate, but not trehalose. The e¡ect caused
by this mutation is a diminished salt tolerance (from 5%
for RQ-1 wt to 3% in the mutated strain). This negative
e¡ect on salt tolerance, observed in the mutant, is relieved
by the addition of trehalose to the growth media. In con-
trast to the e¡ect of trehalose, the addition of other osmo-
lytes (glycine betaine, mannosylglycerate, maltose and glu-
cose) caused no increase in salt tolerance. The results
presented here show that trehalose cannot be replaced
by other solutes to alleviate salt stress and, therefore, plays
a central role in the osmoadaptation of this organism.
 1st FEMS Congress / Posters 103^505
P10^78
ELECTRON TRANSPORT TO PERIPLASMIC NI-
TRATE REDUCTASE (NapA) OF WOLINELLA SUC-
CINOGENES IS INDEPENDENT OF A NapC PRO-
TEIN
J. Simon, M. Sa«nger and R. Gross
Institute of Microbiology, Johann Wolfgang Goethe Univer-
sity, Marie-Curie-Str. 9, D-60439 Frankfurt am Main, Ger-
many
The rumen bacterium Wolinella succinogenes grows by res-
piratory nitrate ammoni¢cation with formate as electron
donor [J. Simon (2002) FEMS Microbiol. Rev. 26: 285-
309]. A gene cluster encoding components of a putative
periplasmic nitrate reductase system (napAGHBFLD) was
sequenced. The napA gene was inactivated by inserting a
kanamycin resistance gene cassette. The resulting mutant
did not grow by nitrate respiration and did not reduce
nitrate during growth by fumarate respiration, in contrast
to wild-type. A NapA antigen detected by Western blot
analysis in the wild-type was absent in the napA mutant. It
is concluded that NapA is the only respiratory nitrate
reductase in W. succinogenes. The nap cluster of W. succi-
nogenes lacks a napC gene whose product is thought to
function in quinol oxidation and electron transfer to
NapA in other bacteria. The W. succinogenes genome en-
codes two members of the NapC/NirT family, NrfH and
FccC. Characterization of corresponding deletion mutants
indicates that neither of these two proteins is required for
nitrate respiration. A model of the electron transport
chain of nitrate respiration is proposed in which one or
more of the napF, G, H and L gene products mediate
electron transport from menaquinol to the periplasmic
NapAB complex. Inspection of the W. succinogenes ge-
nome sequence suggests that ammonia formation from
nitrate is catalysed exclusively by periplasmic respiratory
enzymes.
FEMSLE Congress 2-6-03
351
P10^79
IMPORT OF LHCPII PREPROTEIN INTO THE
COMPLEX CHLOROPLASTS OF EUGLENA GRACI-
LIS AND THEIR ENVELOPE MEMBRANES CHAR-
ACTERIZATION
S. Sla¤vikova¤(1), J. Krajc›ovic›(1), S. D. Schwartzbach(2)
(1) Institute of Cell Biology, Comenius University, Odbor-
a¤rske na¤m. 5, 811 07 Bratislava, Slovak Republic; (2) De-
partment of Microbiology and Molecular Cell Sciences,
University of Memphis, TN 38152, USA
Flagellate Euglena gracilis contains complex plastids en-
closed by three membranes which evolved through second-
ary endosymbiosis between a phagotrophic trypanosome
and eukaryotic alga. About 90% of plastid proteins are
nucleus encoded and synthesised with N-terminal prese-
quences in the cytosol of the cell. Preproteins are trans-
ported from the ER via Golgi apparatus to the chloro-
plasts integrated in the vesicle membranes. In vitro
transport of LHCPII was reconstituted using puri¢ed
chloroplasts, Golgi fraction, ATP and GTP incubated in
light at 26‡C. Signi¢cantly less matured LHCPII was
found when incubation was in the dark or without ATP
and GTP. GTP-QS completely inhibits import indicating a
requirement for GTP hydrolysis. Since LHCPII is not pre-
sented in the Golgi fraction, the appearance of LHCPII in
chloroplasts is indicative of fusion of pLHCPII containing
fraction with chloroplasts, import of pLHCPII into the
stroma and its processing to LHCPII by the processing
peptidase. For detailed determination where the imported
proteins are located, fractionation of chloroplasts into
stroma, envelope membranes and thylakoids was done.
The major components of stromal fraction are at about
12 and 52 kDa, corresponding to the small and large sub-
units of the enzyme Rubisco. The pro¢les of the envelope
membranes showed proteins in the regions corresponding
to the size of 30 to 65 kDa. The major components of
thylakoid fraction are found at about 25 to 30 kDa and
45 to 60 kDa. These groups of proteins are analogous to
the groups I and II polypeptides associated with photo-
systems I and II, respectively.
352 1st FEMS Congress / Posters 103^505
P10^80
LYTIC ENZYMES : SEQUENCING AND MOLECU-
LAR CLONING OF THE L-1,3-GLUCANASE GENE
FROM CELLULOMONAS CARTAE 191
G. A. M. Soares(1), H. H. Sato(2), P. A. Sullivan(3)
(1) Instituto de Biologia Experimental e Tecnolo¤gica/Insti-
tuto de Tecnologia Qu|¤mica e Biolo¤gica-Universidade Nova
de Lisboa, Apt. 12, 2781-901 Oeiras, Portugal; (2) Facul-
dade de Engenharia de Alimentos/UNICAMP, C.P. 6121
Campinas, Brazil; (3) Institute of Molecular BioScience-
Massey University, Private Bag 11222, Palmerston North,
New Zealand
The strain Cellulomonas cartae 191, which was previously
isolated from soil in an alcohol producing Plant in Brazil,
shows a good L-1,3-glucanase production. The L-1,3-glu-
canase enzyme has an important role in yeast cell lysis.
The aim of this work was to study the lytic L-1,3-gluca-
nase from C. cartae 191; its production, puri¢cation and
molecular cloning of this gene. The lytic enzyme was pu-
ri¢ed from the crude supernatant by ultra¢ltration, ion
exchange chromatography and SDS-PAGE electrophore-
sis. The N-terminal sequence of the lytic enzyme was ob-
tained and the L-1,3-glucanase gene was isolated from C.
cartae 191 genome and subsequently cloned in Escherichia
coli DH5K cells. The yeast cell wall was found to be the
best carbon source tested. It was obtained 75% more L-
1,3-glucanase activity using the optimized culture condi-
tions than the original one. The lytic enzyme showed a
molecular mass of 57 kDa in SDS-PAGE electrophoresis.
A 1.9 Kb fragment of DNA from C. cartae 191 containing
the gene for L-1,3-glucanase was successfully isolated and
its complete nucleotide sequence determined. The sequence
was found to contain an Open Reading Frame with 1650
bp that potentially encodes a protein of 549 amino acids.
A predicted cleavage site for the signal peptide showed a
mature enzyme containing 513 amino acids. The high se-
quence identity (92%) with the gene previously cloned
from a closely related bacterium Oerskovia xanthineolytica
essentially con¢rms that it was indeed the required gene.
P10^81
PURIFICATION AND KINETIC CHARECTERISA-
TION OF ALCOHOL DEHYDROGENASE OF RHI-
ZOPUS ORYZAE ATCC 9363
B. Sopaci, H. Hamamci , M. Yucel
Biotechnology Department, METU, P.O. Box 06531, An-
kara, Turkey
FEMSLE Congress 2-6-03
A lactic acid producing ¢lamentous fungus R. oryzae has
an advantageous properties that are selective production
of L(+) lactic acid and low cost of fermentation process.
Alcohol dehydrogenases are catalyses bi-directional H+
transfer reaction using NAD(P)H or NAD(P) as co-en-
zyme. There are three known isoenzymes in S. cerevisiea
and ¢ve structural gene have been isolated. S. cerevisiae
ADH shows an tetrameric structure, and this quarternary
structure changes according to organisms. The kinetic
mechanism however, is similar and obeys ordered bi-bi
kinetic mechanism in the direction of acetaldehyde reduc-
tion and random bi-bi kinetic mechanism in the direction
of ethanol oxidation. The aim of this study is to purify
and analyse kinetic properties of alcohol dehydrogenase
enzyme of R. oryzae ATCC 9363. The enzyme have been
partially puri¢ed with amonium sulphate fractionation
and size exclusion chromatography and further puri¢ca-
tion steps is being optimized. Puri¢ed enzyme going to be
characterized biochemically.
P10^82
TRAP TRANSPORTERS : A COMMON MECHANISM
OF LIGAND BINDING?
T. W. Southworth(1), G. H. Thomas(2) and D. J.
Kelly(1)
(1) Department of Molecular Biology and Biotechnology,
University of She⁄eld, Firth Court, Western Bank, Shef-
¢eld, S10 2TN, UK; (2) Department of Biology, University
of York, PO Box 373, York, YO10 5YW, UK
The TRAP (tripartite ATP-independent periplasmic)
transporters form a novel class of transport systems that
are secondary transporters but are dependent on an ex-
tracytoplasmic solute receptor (ESR) for activity. The best
characterised TRAP transporter is the Dct system from
Rhodobacter capsulatus, consisting of an extracytoplasmic
solute receptor protein, DctP, and two integral membrane
proteins, DctQ and DctM. Genome sequencing projects
have revealed a number of genes that potentially encode
TRAP systems in a variety of bacteria and archaea. We
propose that many TRAP systems transport carboxylate
anions of varying structure, and form high a⁄nity uptake
systems using substrates for which there are no or few
known ABC systems. Previous studies using stopped-
£ow tryptophan £uorescence spectroscopy have shown
that ligand-binding to DctP occurs by a mechanism in-
volving isomerisation of the closed form to the open
form, followed by rapid ligand binding. The closed con-
formation of DctP appears to be unusually stable, prob-
ably mediated by one or more salt-bridges. This mecha-
nism might be a general feature of TRAP-ESR’s. We have
investigated this by overexpression and puri¢cation of sev-
eral TRAP-ESR proteins from R. capsulatus and Pseudo-
 1st FEMS Congress / Posters 103^505
monas aeruginosa. Tryptophan £uorescence studies have
been employed to identify conformational changes in these
proteins on the addition of possible ligands. This method
is a useful way of identifying novel ligands without the use
of radio-labelled substrates. Investigations so far with a
DctP homologue of P. aeruginosa and the SmoM2 protein
of R. capsulatus protein have shown a similar mechanism
for binding of fumarate and several 2-oxoacids respec-
tively.
P10^83
THE ROLE OF THE TRANSCRIPTIONAL REGULA-
TOR WMPR IN PSEUDOALTEROMONAS TUNICA-
TA
S. J. Stelzer(1,2), S. Egan(1,2), S. Kjelleberg(1,2)
(1) School of Biotechnology and Biomolecular Sciences,
University of New South Wales, Sydney, New South Wales,
Australia; (2) Centre for Marine Biofouling and Bio-Inno-
vation, University of New South Wales, Sydney, New South
Wales, Australia
The marine, dark green-pigmented bacterium Pseudoalter-
omonas tunicata produces a range of extracellular com-
pounds, which inhibit a variety of common fouling organ-
isms. Production of pigment and antifouling compounds is
linked, and controlled by the transcriptional regulator
WmpR. Mutants in this gene are white, and have lost
both pigmentation and all antifouling properties. WmpR
has sequence similarity to the transcriptional regulator
ToxR, which is important in regulating genes involved in
bio¢lm formation, adaptive responses, and stationary
phase phenotypes in several bacteria. Using two-dimen-
sional polyacrylamide gel electrophoresis we showed that
there was no di¡erence in protein expression between the
wild type and wmpR mutant during log phase, however at
stationary phase 15 proteins were absent in the wmpR mu-
tant. The aim of this study was to determine if WmpR is a
speci¢c regulator of pigment and antifouling compounds
or if it also regulates the expression of more general sta-
tionary phase phenotypes, such as those involved in star-
vation and stress. The wild type and wmpR mutant were
starved of carbon, nitrogen or phosphate, and also ex-
posed to UV and hydrogen peroxide stress and their
growth responses to each were compared. The results in-
dicated that WmpR is likely to be a speci¢c regulator for
the expression of pigment and antifouling compounds and
does not appear to play a role in the general starvation
and stress response of Pseudoalteromonas tunicata. Cur-
rent mass spectrometry analysis will provide the identity
of the proteins controlled by WmpR. Evidence also sug-
gests a second level of pigment and antifouling regulation,
which is important during phosphate starvation.
FEMSLE Congress 2-6-03
353
P10^84
VISUALIZATION AND BIOCHEMICAL CHARAC-
TERIZATION OF THE INTERACTION BETWEEN
ALGINATE AND THE LECTIN CONCANAVALIN A
IN BIOFILMS OF PSEUDOMONAS AERUGINOSA
M. Strathmann, J. Wingender and H.-C. Flemming
University of Duisburg, Institute for Interface Biotechnol-
ogy, Aquatic Microbiology, Geibelstrasse 41, 47057 Duis-
burg, Germany
Fluorescently labelled lectins were used in combination
with confocal laser scanning microscopy to allow the vis-
ualization and characterization of carbohydrate-contain-
ing extracellular polymeric substances (EPS) in bio¢lms
of P. aeruginosa. Mucoid strains characterized by an over-
production of the exopolysaccharide alginate, and an iso-
genic, non-mucoid strain were compared. Model bio¢lms
grown on polycarbonate ¢lters were treated with the lectin
concanavalin A (ConA) that was £uorescently labelled
with £uorescein isothiocyanate or Alexa Fluor1 dyes.
Fluorescently labelled Con A yielded cloud-like regions
that were heterogeneously distributed within mucoid bio-
¢lms, whereas these structures were only rarely present in
bio¢lms of the non-mucoid strain. The bacteria visualised
with the £uorochrome SYTO 9 were localized both within
and between the ConA-stained regions. Sugar speci¢city of
lectins was veri¢ed by a competitive inhibition assay; lec-
tin binding was inhibited by the respective target saccha-
rides. ConA seemed to interact with the alginate compo-
nent of the EPS matrix, since (i) pre-treatment of bio¢lms
with an alginate lyase resulted in a loss of ConA bio¢lm
staining, and (ii) using an enzyme-linked lectin-sorbent
assay (ELLA), ConA was shown to bind to puri¢ed algi-
nates, but not to the same alginate that was degraded by
alginate lyase. Interaction between ConA and alginate was
further characterized by a⁄nity chromatography on
ConA-Sepharose, revealing three alginate fractions with
none, unspeci¢c and speci¢c binding to ConA, respec-
tively. The results suggest that alginate in bio¢lms of mu-
coid P. aeruginosa is heterogeneous with respect to (i) its
localization and (ii) its composition and/or its three-di-
mensional conformation.
354 1st FEMS Congress / Posters 103^505
P10^85
NOVEL STRUCTURE OF POLYSIALIC ACID AND
OTHER ANIONIC POLYMERS IN THE CELL
WALL OF PLANT PATHOGENIC STREPTOMY-
CETES
G. M. Streshinskaya(1), A. S. Shashkov(2), L. N. Kosma-
chevskaya(1), L. I. Evtushenko(3), I. B. Naumova(1), E.
Stackebrandt(4)
(1) School of Biology, M. V. Lomonosov Moscow State
University, Moscow 119899, Russia; (2) N. D. Zelinsky
Institute of Organic Chemistry, RAS; (3) Institute of Bio-
chemistry and Physiology of Microorganisms, RAS; (4)
DSMZ-Deutsche Sammlung von Mikroorganismen und
Zellkulturen GmbH, Mascheroder Weg 1b, D-38124
Braunschweig, Germany
The cell wall anionic polymers were studied in streptomy-
cetes, causative agents of potato scab, which are phyloge-
netically the closest to plant pathogenic species S. scabies
(group I) and S. setonii (S. caviscabies)(group II). The cell
wall of streptomycetes of group I contains several anionic
polymers among them poly(glycerol phosphate) teichoic
acids of di¡rent structure and polymers of 3-deoxy-D-
glycero-D-galacto-non-2-ulopyranosonic acid (Kdn) with
Glc, Gal or 3-O-MeGal substituents.. The cell wall of
streptomycetes of group II contains three anionic glyco-
polymers, viz., teichuronic acids of di¡erent structure, a L-
glucosylated polymer Kdn, and a L-glucosylated 1,5-poly(-
ribitol phosphate). Kdn was ¢rst discovered in the poly-
sialoglycoprotein rainbow trout eggs in the form of K-2,8-
linked oligomer. Later Kdn was found in glycoconjugates
of di¡erent animal and human tissues, as a constituent of
capsular heteropolysaccharide of Klebsiella ozaenae sero-
type K4 and in a polysaccharide isolated from Sinorhi-
zobium fredii. Until now polysaccharides with the main
chain composed of Kdn residues has not been found in
bacteria. The presence of Kdn-polymers might be charac-
teristic of plant pathogenic streptomycetes causing scab
diseases of potatoes and root crops. Probably, the local-
ization of Kdn-containing structures in the near-surface
regions of actinomycete hyphae is essential for their
growth taxis and their attachment to potato tuber.
The work was supported by INTAS grant No. 01-2040
and the Russian Foundation for Basic Research No. 01-
04-48769
FEMSLE Congress 2-6-03
P10^86
CHARACTERIZATION OF A NOVEL PLASMID AD-
DICTION SYSTEM OF pMTH4 PLASMID IDENTI-
FIED IN THE ALPHAPROTEOBACTERIAL SPECIES
PARACOCCUS METHYLUTENS
M. Szymanik, D. Bartosik and M. Wlodarczyk
Faculty of Biology, Warsaw University, 02-096 Warsaw,
Poland
One of the mechanisms ensuring a stable maintenance of
low copy number plasmids are plasmid addiction systems.
Plasmids encoding such systems produce a toxic protein
killing the cells while its action is not prevented by simul-
taneously produced antidote (protein or RNA). In the cell
cured of plasmid by aberrant segregation, neither the toxin
protein nor antidote are synthesized, but since antidote is
less stable than toxin its preventing action will be short
lasting and plasmid-less cells will be killed. Molecular
characterization of pMTH4 of Paracoccus methylutens al-
lowed to identify stabilizing system (sta) located upstream
of replication unit. The discovered system stabilizes unsta-
ble replicons in Paracoccus and Escherichia coli. In the sta
region two overlapping reading frames were identi¢ed :
ORF2 (294 bp) and ORF3 (276 bp). Their potential pro-
tein products (10.4 kDa and 10.1 kDa) do not show sim-
ilarity to the already described stabilizing systems. Muta-
tional inactivation of ORF3 destroyed stabilising potential
of entire system while our e¡ort to mutate ORF2 failed.
The above could suggest that the system acts as plasmid
addiction system with OFR3 product being toxin and
ORF2 antidote. Con¢rmation of such an assumption
came from our assay employing two-plasmid system one
of which (carrying cloned ORF2, potential antidote) was ts
vector. The presence of ORF3 product, while the produc-
tion of potential antidote was restricted by elevated tem-
perature resulted in growth inhibition and cell ¢lamenta-
tion. Further studies, comprising regulation of the system,
are in progress.
 1st FEMS Congress / Posters 103^505
P10^87
CLONING, SEQUENCING AND HETEROLOGOUS
EXPRESSION OF FAMILY 19 CHITINASE GENE
FROM ENDOPHYTIC STREPTOMYCES SP.
CMUAC130
T. Taechowisan(1), J. F. Peberdy(2) and S. Lumyong(1)
(1) Department of Biology, Faculty of Science, Chiang Mai
University, Chiang Mai 50200, Thailand; (2) School of
Life and Environmental Sciences, University of Nottingham,
University Park, Nottingham NG7 2RD, UK
A chitinase gene from endophytic Streptomyces sp.
CMUAc130 was cloned and expressed in Escherichia coli
JM109 using pUC18. The nucleotide sequence of this gene
was determined and compared its sequence with other
genes encoding chitinase. The recombinant protein could
be detected by Western-blot analysis. The antifungal ac-
tivity of the recombinant protein was estimated using the
hyphal extention-inhibition of Collectotrichum musae and
Fusarium oxysporum.
P10^88
CELLULASE GENES ORGANIZATION AND TRAN-
SCRIPTIONAL REGULATION IN STREPTOMYCES
E. Tamburini(2), V. Mascia(2), B. Perito(1), G. Mastro-
mei(1)
(1) Department of Animal Biology and Genetics ‘‘Leo Par-
di’’, University of Florence, Via Romana 17, 50125 Flor-
ence, Italy ; (2) Dipartimento di Biologia Sperimentale, Se-
zione di Microbiologia, University of Cagliari, Cittadella
Universitaria ss 554, 09042 Monserrato (ca), Italy
Streptomycetes are aerobic soil bacteria that degrade a
variety of insoluble organic material, such as cellulose.
The cellulolytic system comprises three classes of enzymes :
cellobiohydrolase, endo-1,4-f-glucanase and cellobiase.
Since S. coelicolor A3(2) genome project was recently com-
pleted, we analysed its cellulase genes organization at ge-
nomic level. Several gene clusters encoding cellulase ho-
mologous proteins and cellulose binding proteins were
identi¢ed. At present time, little is known about cellulase
regulation in Streptomyces. S. rochei A2 is a cellulolytic
strain isolated from termites gut. From this microorgan-
ism we cloned the eglS gene, encoding an endoglucanase.
The analysis of the eglS sequence showed that the fourth
codon is TTA. Similar genes are present in other Strepto-
myces, including S. coelicolor A3 (2), and all these genes
have at least one conserved TTA codon. This codon is
rare in Streptomyces and there is no known case of its
occurrence in genes required for vegetative growth. We
FEMSLE Congress 2-6-03
355
studied developmental regulation of eglS gene expression
in S. rochei A2. A CMC degrading activity is secreted in
the culture medium both during exponential and second-
ary growth. However, eglS transcripts can be detected
during the stationary phase, but not during the exponen-
tial growth. Therefore EglS production in S. rochei A2
seems to be growth phase dependent.
P10^89
METABOLISM OF NITROCELLULOSE BY DESUL-
FOVIBRIO DESULFURICANS
N. B. Tarasova, O. E. Petrova and M. N. Davydova
Kazan Institute of Biochemistry and Biophysics, Russian
Academy of Sciences, P.O. Box 30, Kazan 420111, Russia
For a long time the sulfate-reducing bacteria are consid-
ered as the group of microorganisms that use very limited
spectrum of substrates for their constructive and energetic
metabolism. It is the fact that the last years give the nu-
merous examples for the ability of these microbes to utilize
the unusual compounds in biomass production. D. desul-
furicans B-1388 demonstrates high activity in the degrada-
tion process of such complicated organic compound as
nitrocellulose (NC) that cannot be usually utilized by en-
vironmental micro£ora. The results obtained showed that
the culture transformed NC to the natural compound (cel-
lulose) due to nitroestherase activity (158 nmol NO3-/min
per mg of protein). The nitrate originated from NC was
reduced to ammonia in nitrate reductase and nitrite reduc-
tase reactions. In cell extracts the activities of nitrate and
nitrite reductases with benzyl viologen (BV) as electron
donor were 366 nmol BV/min per mg of protein and 197
nmol BV/min per mg of protein, respectively. These results
indicate that the ability of D. desulfuricans B-1388 to de-
nitrate NC with the following nitrate dissimilation is a
physiologically signi¢cant process for growth mainte-
nance.
P10^90
EXTRACELLULAR MEMBRANE LIKE STRUC-
TURES OF BIOFILMS
V. V. Tetz, V. P. Korobov, N. K. Artemenko, L. M. Lem-
kina, N. V. Panjkova and G. V. Tetz
Microbiology Department, St. Petersburg Pavlov State
Medical University, St. Petersburg, 197089, Russia
Membrane formations existing outside eukaryotic and
prokaryotic cells are poor investigated. A comparative
analysis of ultrastructure and composition of phospholip-
ids from extracellular membrane like structures of Gram-
356 1st FEMS Congress / Posters 103^505
negative and Gram-positive bacterial lawns (bio¢lms) was
made.Strains of Escherichia coli JC10240, B, K-12, ATCC
33527, Shigella £exneri VT100 and Staphylococcus aureus
VT- 209 were used for investigation. Mono and mixed
bacterial lawns were obtained by the same method, but
in last case the mixture of two di¡erent bacteria was plated
onto LB agar. Transmission electron microscopy investi-
gations were performed for the estimation of bio¢lms ul-
trastructure. Lipid spectrum of extracellular membrane,
like components was evaluated by the method of high-
e⁄cient thin-layer chromatography. The results of our in-
vestigations indicate the existence of di¡erent extracellular
membrane-like structures in bacterial lawns. These mem-
branes form vesicles and are a component of the surface
¢lm. These membranes have a structure which is nearly
identical in the communities of Gram-negative and Gram-
positive bacteria, and also in the mix variant of the joined
growth. Extracellular membranes have individual phos-
pholipid composition that re£ects the origin of the bacte-
ria that form this community. The increase in cardiolipin
content and decrease in the level of lysophospholipids in
¢lms coating the lawns should result in enhancing of
strength characteristics of these membrane-like forma-
tions.
P10^91
COMPARISON OF PROTEIN EXPRESSION PRO-
FILES OF STREPTOCOCCUS AGALACTIAE
GROWN ON STANDARD LABORATORY MEDIUM
AND IN AMNIOTIC FLUID OBTAINED FROM
HEALTHY VOLUNTEERS DURING ELECTIVE CAE-
SARIAN SECTION
C. A. Tierney, I. Sutcli¡e, D. Harrington, R. Koerner, H.
K. S. Hinshaw, M. Abu-Harb, J. Chamberlain.
Department of Microbiology, Sunderland Royal Hospital,
Sunderland, SR4 7TP, United Kingdom
Streptococcus agalactiae (GBS) is a leading aetiological
agent of potentially lethal neonatal infection. Factors
that contribute to GBS virulence have not been extensively
studied and consequently are not well understood. This
project investigated potential factors involved in foetal-
pathogen interactions, analysing protein expression pro-
¢les of GBS grown under conditions resembling those in
which these interactions take place in vivo. Method: Amni-
otic £uid (5-190ml) was collected from healthy volunteers
(with no previous evidence of GBS infection) during elec-
tive caesarean section. GBS clinical isolates and reference
strains were used to inoculate pooled samples of AF (neat
and supplemented) and Todd Hewitt broth. Growth of the
cultures was monitored at de¢ned time points using colony
counts. Cells were harvested then analysed by electropho-
resis. Protein pro¢les were examined by silver staining and
FEMSLE Congress 2-6-03
Western Blotting (WB) using a panel of antisera to known
GBS virulence factors. Results and Conclusion: Two
prominent protein bands (at 23kDa and 48kDa) were ob-
served in AF grown cells and were N-terminally se-
quenced. The ca. 23kDa band was a mixture of two pro-
teins, one of which probably derived from host
immunoglobulin. The 48kDa band showed N-terminal se-
quence similarity with the heavy chain variable region of
human immunoglobulin again suggesting the deposition of
antibodies on the surface of the bacterium. WB con¢rmed
the expression in AF of the known GBS proteins Sip and
Lbp. The results indicate that further experiments may be
used to improve the resolution of the protein pro¢ling
which could reveal other changes in GBS protein expres-
sion.
P10^92
ESCHERICHIA COLI F0F1-ATP SYNTHASE UNDER
FERMENTATION : ASSOCIATION WITH SECOND-
ARY SOLUTE TRANSPORTERS AND/OR ENZYMES
OF ANAEROBIC OXIDATION-REDUCTION
A. Trchounian
Department of Biophysics of the Biological Faculty, Yere-
van State University, 1 A. Manoukian Str., 375049 Yere-
van, Armenia
Escherichia coli F0F1-ATP synthase is the main membrane
protein complex of energetic relevance. The membrane
part, F0 contains three subunits, a, b, and c, the compo-
sition of which is probably a1b2c9-12. They form a channel,
through which H+ £ows to F1 down the proton electro-
chemical gradient (vWH+). Attached to F0 is F1, a soluble
complex of ¢ve distinct polypeptides that is capable of
catalyzing ATP hydrolysis and, in complex with F0,
ATP synthesis. This complex has a priority in maintaining
vWH+ in the context of ATP synthesis and hydrolysis.
However, under fermentative growth, in the absence of
oxidative phosphorylation, F0F1 seems to be implicated
as an essential part of ATP hydrolysis and H+ movement
associated with solute secondary porters, namely, the con-
stitutive low a⁄nity K+ uptake TrkA system. F0F1 might
be also involved in functioning of enzymes of anaerobic
oxidation-reduction such as formate hydrogenlyase
(FHL). A hypothesis on associations of F0F1 with TrkA
and/or FHL is advanced, evidences are collected and a set
of new results is reported. Can this hypothesis be consid-
ered as novel look at F0F1 under fermentation ? If yes, in
addition to generation of vWH+, due to associations with
relevant transport systems and/or enzymes, F0F1 is pro-
posed to have novel functions in creating a high K+ gra-
dient between the cytoplasm and the external medium and/
or oxidizing formate to carbon dioxide and fermentative
gas (H2). Under fermentation this ATP synthase may
 1st FEMS Congress / Posters 103^505
therefore play a more important role in bacterial physiol-
ogy.
P10^93
CELL WALL TEICHOIC ACID COMPOSITION IS A
CHEMICAL MARKER OF THE MYCELIUM-FORM-
ING NOCARDIOIDES SPECIES AND THE PRO-
POSAL OF NOCARDIOIDES PRAUSERII SP. NOV.
E. M. Tul’skaya(1), V. I. Krausova(2), E. Yu. Gavrish(3),
A. S. Shashkov(4), L. I. Evtushenko(2,3), I. B. Naumo-
va(1)
(1) School of Biology, Lomonosov Moscow State Univer-
sity, Moscow 119899, Russia; (2) G.K. Skryabin Institute
of Biochemistry and Physiology of Microorganisms, Russian
Academy of Sciences, Pushchino 142290, Moscow Region,
Russia; (3) Pushchino State University, Pushchino 142290,
Moscow Region, Russia; (4) Zelinsky Institute of Organic
Chemistry, Russian Academy of Sciences, Leninsky pros-
pect 47, Moscow117913, Russia
Data on the structures of cell wall teichoic acids have the
taxonomic value for genus Nocardioides. The type strain
N. luteus VKM Ac-1246T and six strains with white colo-
nies assigned previously to the species N. albus exhibited
from 63 to 74.2% DNA relatedness and their cell walls
contained identical 1,5-poly(ribitol phosphate) teichoic
acids completely substituted at C4 by K-D-galactopyrano-
syl residues carrying a 4,6-pyruvate ketal group in R-con-
¢guration. While the type strain N. albus VKM Ac-805T
possessed a poly (glycosylglycerol phosphate) polymer, in
which L-D-galactopyranosyl residues are substituted at C4
by L-D-glucopyranose carrying a 4,6-pyruvate ketal group
in S-con¢guration. Another strain, N. albus VKM Ac-806
(=DSM 43874), was found to distant from both N. albus
and N. luteus genomically (nearly 50 % DNA relatedness)
and supposedly contained at least two polymers of di¡er-
ent structure, teichoic acid closely related to teichoic acid
from cell wall of N. luteus and the polymer containing
rhamnose. On the basis of the data obtained, the new
species Nocardioides prauseri sp. nov. is proposed, with
the strain VKM Ac-806 (=DSM 43874 =ATCC 43083)
as the type. Further, the species descriptions of N. albus
and N. luteus are proposed to emend, with including of the
cell wall teichoic acid composition and their structural
components as the most important diagnostic characters
of these species. Respectively, the species N. luteus harbors
the strains characterized by white or yellow primary my-
celium.
This work was supported by INTAS grant No. 01-2040
and the Russian Foundation for Basic Research No. 01-
04-49854.
FEMSLE Congress 2-6-03
357
P10^94
REGULATION OF ARGININE CATABOLISM IN BA-
CILLUS LICHENIFORMIS BY THE REDOX SENS-
ING REGULATORY PROTEIN ARCR
A. Wohlko«nig(1), V. Stalon(1,2), C. Vander Wauven(2)
(1) Laboratoire de Microbiologie, Universite¤ Libre de
Bruxelles and (2) Institut de Recherches Microbiologiques
JM Wiame, Av. E. Gryzon, 1, B-1070 Brussels, Belgium
In the facultative anaerobe Bacillus licheniformis two dif-
ferent pathways allow L-arginine catabolism. Oxygen
pressure determines through which pathway breakdown
will occur, as arginase is induced in well-aerated cultures
and arginine deiminase in anaerobiosis. Our work on the
ArcR protein, a transcriptional activator of the arginine
deiminase operon arcABDC, has shown it to have proper-
ties of a redox sensor. In vitro studies on the wt protein
and mutant derivatives have shown that the cysteines lo-
cated on either side of the DNA binding motif play an
essential role in the redox sensing. The activator recog-
nized its DNA target only when reduced. The results sug-
gest that the DNA-binding activity could depend on the
cell redox status : in aerobiosis, ArcR would be oxidized
by a still unknown mechanism, and unable to recognize its
targets on the DNA. Moreover, gene expression studies in
mutant and wt strains, and identi¢cation of DNA targets
have shown that ArcR represses a gene encoding an argi-
nine repressor-like protein in anaerobiosis. This protein is
distinct from the true ArgR acting on the biosynthesis
genes and the arc operon. It is involved in the aerobic
induction of the arginase pathway. Our results support
the hypothesis that ArcR is a central regulatory protein
of the arginine catabolism in B. licheniformis: by promot-
ing simultaneously the induction of the deiminase operon
and the repression of the arginase pathway it is responsi-
ble for the aerobic/anaerobic switch in the arginine catab-
olism.
P10^95
THE MECHANISMS OF PHA SYNTHESIS BY CHE-
MOLYTHOTROPHIC BACTERIA
T. G. Volova
Institute of Biophysics of SB RAS, Krasnoyarsk, 660036,
Akademgorodok, Russia
Polyhydroxyalkanoates (PHAs) of various composition
and properties are reserve materials synthesized by micro-
organisms and thoroughly studied because of wide pros-
pects of their use. Their most valuable potential is in pro-
duction of various polymers (feasibility of controlling their
358 1st FEMS Congress / Posters 103^505
properties). To realize this potential it is essential to have
fundamental knowledge of synthesis mechanisms. Until
recently it has been assumed strains synthesizing short-
chain-length polymers cannot accumulate medium-chain-
length polymers due to PHA-synthases substrate speci¢c-
ity. The experiments with Ectothiorhodospira shaposhniko-
vii and Ralstonia eutropha have shown PHA-synthases has
a broader substrate speci¢city, suggesting a feasibility of
scl- and lcl- PHAs (C3/ C4/ C5/ C6) simultaneous synthesis.
The investigation of heteropolymer PHA synthesis mech-
anisms by Ralstonia eutropha and Seliberia carboxydohy-
drogena on mixed carbon substrate, involving the hydro-
carbon acids with C-chain length between C5 and C9 as a
co-substrate, has proved odd acids mostly stimulate the
hydroxyvalerate inclusion, while even acids ^ hydroxyhex-
anoate. These inclusions are not stable, so the production
of heteropolymer PHAs requires speci¢c biosynthesis con-
ditions. We have developed the cultivation conditions,
specifying the doses and acid amounts added to the me-
dium and regulating the subsequent cultivation period. As
a result, the PHAs with the C4 :C5 ratio from 9:1 to 1:9
can be obtained. However, it is problematic to obtain
three-component PHA (C4/C5/C6) as the C6 inclusion
‘‘lifetime’’ is shorter. We have managed to synthesize
three-component PHAs with hydroxyhexanoate inclusion
up to 6 mol% only. The C8 inclusion in the PHAs is even
less stable, thus the four-component PHAs (C4/C5/C6/C8)
are more di⁄cult to obtain.
P10^96
AN F1-ATPASE-DEFECTIVE MUTANT OF ESCHERI-
CHIA COLI K-12: NOVEL CELLULAR CHANGES
ASSOCIATED WITH THE ENHANCED GLUCOSE
METABOLISM IN A CHEMOSTAT
A. Yokota(1), K. Matsushita(2), Y. Takezawa(1), E.
Nishiumi(1), K. Onoe(1) and F. Tomita(1)
(1) Graduate School of Agriculture, Hokkaido University,
Sapporo 060-8589, Japan ; (2) Faculty of Agriculture, Ya-
maguchi University, Yamaguchi 753-0841, Japan
Enhancement of metabolic activity of the cell is important
for the e¡ective production of metabolites by fermenta-
tion. We have revealed that the introduction of F1-ATP-
ase defects, which abolishes oxidative phosphorylation,
leads to the enhanced glucose metabolism in industrially
important bacteria, E. coli and Corynebacterium glutami-
cum, with increased rates of respiration. We report the
important changes found in central metabolism of an
F1-ATPase-defective mutant of E. coli K-12. The £ux
analysis of the wild type and the mutant in a glucose-
limited chemostat revealed increased carbon £ux in path-
ways for both glycolysis and acetate formation from py-
ruvate in the mutant, while that in TCA cycle appeared to
FEMSLE Congress 2-6-03
be almost the same in both strains. The acetate formation
from pyruvate seemed advantageous for additional ATP
gain in the mutant where oxidative phosphorylation has
been impaired. Measurements of enzyme activities and
proteome analysis showed upregulation in pathways for
both glycolysis and acetate formation, and downregula-
tion in both TCA cycle and glyoxylate shunt. The most
striking changes were found in enzymes of respiratory
chain that consists of NADH dehydrogenases (NDH-
1+NDH-2) and terminal oxidases (Cyt bo+Cyt bd). The
total activity of NADH dehydrogenases and that of the
terminal oxidases were found to increase in the mutant,
accounting for its higher respiration rate. Furthermore,
NDH-2 and Cyt bd, minor components in the wild type,
became predominant in the mutant. As these are the by-
pass components, the mutant is able to recycle large
amounts of NADH avoiding generation of an excess pro-
ton motive force.
P10^97
THE INFLUENCE OF LOW MOLECULAR WEIGHT
AROMATIC COMPOUNDS ON BIOSYNTHESIS OF
MELANINS BY BLACK YEAST FUNGI
N. A. Yurlova
Department of Microbiology, State Chemico-Pharmaceuti-
cal Academy, Prof. Popova street 14, St.Petersburg,
197376, Russian Federation; Department of Microbiology,
State University, Universitetskaya nab., 7/9 St. Petersburg,
199034, Russian Federation
Melanins are high molecular weight pigments formed by
the oxidative polymerization of phenolic compounds. Mel-
anins are formed via the pentaketide pathway using the
natural precursor 1,8 ^ dihydroxynaphthalene (DHN) and
via the indole pathway using the precursor 3,4 ^ dihydrox-
yphenylalanine (DOPA). Wheeler and Bell (1986; 1987)
believed that the melanins in dematiaceous fungi are de-
rived from DHN rather than DOPA. Dark pigments are
produced by the action of polyphenoloxidases such as cat-
echolases (o ^ diphenoloxidases), laccases (p ^ diphenolox-
idases) and tyrosinase, which oxydise tyrosine. These en-
zymes can be confused with each other. We intended to
analyse what kind of polyphenoloxidases take part in bio-
synthesis of black yeast fungi (BYF) melanins. For that
purpose we used both substrates of o ^ diphenoloxidases
(4-hydroxyphenyl-pyruvic acid, L-phenyllactic acid, tyro-
sine, pyrocatechol, 3,4-dihydroxyphenilalanine) and sub-
strates of p ^ diphenoloxidases (syringaldazine, resorcinol,
p-phenylenediamine, phloroglucinol, homogentisic acid,
guaiacol, pyrogallic acid). 26 strains of BYF originating
from divergent natural biotopes were ionvestigated. Syrin-
galdazine, 4-hydrophenyl-pyruvic acid, L-phenyllactic
acid, pyrogallic acid optimally promoted melanin biosyn-
 1st FEMS Congress / Posters 103^505
thesis when compared to other groups of substrates inves-
tigated. Intensity of pigmentation of all BYF strains
studied was the lowest when guaiacol was used as a sub-
strate. Consequently, the present investigation con¢rms
the previously reported hypothesis for animal origin mel-
anins, indicating that the melanization process could in-
volve more enzymes and more substrates than those com-
monly recognized.
P10^98
LIPID AND HYDROCARBON COMPOSITION OF
THE GREEN ALGA BOTRYOCOCCUS
N. O. Zhila, G. S. Kalacheva, T. G. Volova
Institute of Biophysics of SB RAS, Krasnoyarsk, 660036,
Akademgorodok, Russia
The green unicellular colonial alga Botryococcus braunii
widespread in fresh and brackish waters is characterized
by a very large content of hydrocarbons; their content in
some strains amounts to 75% (of dry weight). B. braunii
appear capable of providing a renewable source of hydro-
carbons. Lipid composition and hydrocarbon structure of
two algae of the genus Botryococcus (entered into labora-
tory culture museum strain B. braunii Ku«tz IPPAS H-252
and a ¢eld sample collected for the ¢rst time from Lake
Shira (Khakasia, Siberia)) have been investigated. Several
di¡erent classes of compounds including hydrocarbons
have been identi¢ed among lipids in the laboratory cul-
ture. The dominant fraction in it was the polar lipids (up
to 50% of the lipids). Palmitic, oleic, C16 ^ C18 dienoic and
trienoic acids were main fatty acids of this alga. Aliphatic
hydrocarbons of the B. braunii Ku«tz IPPAS H-252
amounted maximally to about 1% of the dry biomass at
the end of exponential growth phase. The Botryococcus sp.
found in Lake Shira is characterized by a higher lipid
content ( 6 40% of the dry weight). The main lipids in
this sample were dienes and trienes (hydrocarbons 6 60%
of total lipid). Saturated, monounsaturated and very long
chain monounsatureted fatty acids, including C28 :1 and
C32 :1 acids, were identi¢ed in the Botryococcus found in
Lake Shira. The chemo-taxonomic criteria allow us to
unequivocally characterize the organism collected from
Lake Shira as Botryococcus braunii, race A.
FEMSLE Congress 2-6-03
359
P11^1
TOXICOLOGICAL ASPECTS OF TNT BIOTRANS-
FORMATION BY YEASTS
J. F. Abdrakhmanova, S. A. Zaripov and R. P. Naumova
Kazan State University, Dept. of Microbiology, Kremlyev-
skaya str., 18, Kazan, 420008, Russia
The most important aspect of the environmental pollution
with 2,4,6-trinitrotoluene (TNT) and its metabolites is ob-
viously the exposure of the wildlife, animals and humans
to the toxic e¡ects of these metabolites. Because of the
relatively unstable nature of TNT transformation products
^ hydroxylaminodinitrotoluenes (HADNT) and hydride
complex (H-TNT), we have chosen the Paramecium cau-
datum toxicity test, which allows testing within a reason-
ably short period of time, to estimate the relative toxicity
of TNT and the by-products of its degradation. Compar-
ative toxicological assessment of TNT, HADNT, and H-
TNT chemical standards revealed that HADNT are the
most toxic compounds and, supposedly, the supernatant
of the Saccharomyces sp. ZS-A1 strain, which converts
TNT almost exclusively to HADNT, must be the most
toxic for P. caudatum cells. Indeed, incubation of para-
mecium cells with the supernatant from this culture re-
sulted in the highest mortality rates in comparison with
other supernatants. The lowest mortality rates were ob-
served with the supernatants of Candida sp. AN-L13,
which converts TNT to H-TNT. The presence of both
metabolites in equimolar quantities in the incubation £uid
of Candida sp. AN-L14 stipulated the intermediate level of
toxicity. Our toxicological study suggests that the prefer-
ential pathway for TNT transformation could be via the
hydrogenation of the aromatic ring, thus, avoiding the
formation of highly toxic products, which may kill or in-
activate the members of TNT transforming consortia.
Thus, our results support the suggestion that the pathway
leading to H-TNT is the most attractive in terms of creat-
ing catabolic potential for the e¡ective TNT biodegrada-
tion.
360 1st FEMS Congress / Posters 103^505
P11^2
COMPARISON OF MOLECULAR AND CULTURE
DEPENDENT METHODS FOR THE DETECTION
OF PATHOGENS IN CONSTRUCTED WETLANDS
M. Alexandrino(1), E. Grohmann(1), I. Feuerpfeil(2), A.
Hummel(2) and U. Szewzyk(1)
(1) Technical University of Berlin, Faculty III, O⁄ce OE5,
Microbial Ecology Group, Franklinstr. 29, 10587 Berlin,
Germany ; (2) Federal Environmental Agency, Heinrich-
Heine-Str. 12, 08645 Bad Elster, Germany
The results obtained with a standard culture dependent
and a PCR based method were compared to assess the
removal of pathogens from wastewater in two constructed
wetlands. Campylobacter jejuni/coli and Yersinia enteroco-
litica serogroup 0:3 were selected as model organisms. Di-
rect cell counts and standard heterotrophic plate counts
were employed to characterise the elimination perfor-
mance of both wetlands for bacteria. For the detection
of both pathogens, PCR protocols were optimised for
whole DNA extracted from the pretreated and treated
water and results were compared with those obtained by
selective cultivation steps. In addition PCR detection of
Enterococcus faecalis as a standard indicator was per-
formed for each sample. All PCR protocols were success-
fully performed with a background of up to 1010 non-
target cells per reaction. Fifty cells of C. jejuni/coli and
less than 2.5 cells of Y. enterocolitica per ml treated water
were detected by PCR. The detection limit in the pre-
treated water was higher (200 cells C. jejuni/coli and less
than 10 cells Y. enterocolitica per ml). PCR detection lim-
its were in most cases in the same range or higher than
those of the culture dependent method. However, culture
methods did not achieve the same speci¢city and proved to
be much more time and work consuming than the molec-
ular method. The results obtained showed that during the
period of investigations there was no detectable formation
of VBNC states of Y. enterocolitica serogroup 0:3 and C.
jejuni/coli.
FEMSLE Congress 2-6-03
P11^3
NEW APPROACHES TO THE STUDY OF LITHO-
BIONTIC MICROORGANISMS, THEIR MICROBIAL
FOSSILS AND BIOMARKERS IN ANTARCTIC
ROCKS MICROHABITATS
C. Ascaso(1), A. de los R|¤os(1), J. Wierzchos(2)
(1) Centro de Ciencias medioambientales (CSIC) Serrano
115 dpdo. Madrid-28006, Spain; (2) Servei de Microsco-
pia, Universidad de Lleida, Lleida, Spain
The study of any ecosystem requires previous knowledge
of its components and the processes that take place within
it. If we are to understand the structure and function of
each component of the ecosystems that inhabit lithic sub-
strates, we need to be able to quantify and identify the
microorganisms present in each lithobiontic ecological
niche and to accurately characterise the mineralogical fea-
tures of these hidden microhabitats. Once we have estab-
lished and perfected the techniques that will allow us to
observe and identify these microorganisms and mineral
substrates in situ, as well as locating the presence of water
we must take into account that mechanical and chemical
changes in minerals and mineralisation of microbial cells
can give rise to physical and/or chemical traces (bio-
markers) and to microbial fossil formation. Scienti¢c in-
terest in these lithobionts may be ascribed to two principal
reasons. The ¢rst is that the Dry Valley desert zone of
Antarctica is one of the most inhospitable places on Earth
for life due to low light intensity, extremely low temper-
atures and the scare presence of water. The second reason
has become a challenging goal for geomicrobiology and
micropalaeontology since it is highly probable that Ant-
arctic rocks harbour relics and/or traces of endolithic mi-
crobial activity. Special attention was paid to new means
of identifying microorganism fossils based on the descrip-
tion of their preserved ultrastructural details. To this end,
live microorganisms were identi¢ed along with their bio-
markers and fossils and then microscopically and micro-
analytical characterised in situ by the simultaneous use of
SEM-BSE and energy dispersive X-ray spectroscopy
(EDS).
 1st FEMS Congress / Posters 103^505
P11^4
DIVERSITY AND FUNCTION OF BACTERIA IN A
FLUIDISED BED REACTOR TREATING URBAN EF-
FLUENTS
N. Babic, H. M. Cauchie and L. Ho¡mann
CRP-GL, CREBS, 162a, Avenue de la Fa|«encerie, L-1511-
Luxembourg, Luxembourg
The aim of this study is to optimise the treatment in £ui-
dised bed systems. The relationships between the nitrogen
removal rate and the composition of the consortia devel-
oping on the polyethylene carriers of a £uidised bed sys-
tem were studied in a wastewater treatment plant
(WWTP) near Luxembourg. DGGE and FISH were
used to detect changes in the bacterial community compo-
sition as a function of the e¥uent composition. Moreover,
nitri¢ers and denitri¢ers were monitored in the raw e¥u-
ent, the other compartment of the WWTP and the receiv-
ing river in order to determine the dispersal of these bac-
teria in the natural environment.
P11^5
GROWTH OF HALOALKALIPHILIC SULFUR-OXI-
DIZING BACTERIA IN CONTINUOUS CULTURE
AT HIGH SALT CONCENTRATION
H. Banciu(1), D. Y. Sorokin(2), R. Kleerebezem(1), G.
Muyzer(1) and J. G. Kuenen(1)
(1) Kluyver Laboratory for Biotechnology, TU Delft, 2628
BC Delft, The Netherlands; (2) Institute of Microbiology,
RAS, 117811-Moscow, Russia
Recently, a new group of chemolithoautotrophic sulfur-
oxidizing bacteria has been discovered in soda lakes.
They belong to 3 genera, Thioalkalimicrobium, Thioalkali-
vibrio and Thioalkalispira in the gamma subdivision of the
Proteobacteria [1,2]. These bacteria are halotolerant and
can grow at extremely high pH (up to 10.6). They use
thiosulfate, sul¢de, sulfur and polysul¢de as energy source
and HCO3- as the only carbon source. Few data are avail-
able on the growth kinetics of chemolithoautotrophic bac-
teria in continuous culture, especially under high pH or
salt conditions. Our results provide new information on
the eco-physiology of extremophilic organisms. A typical
representative of the Thioalkalivibrio, Tv. versutus ALJ 15,
is able to grow aerobically at high pH and within a broad
range of salt concentration (0.6 to 4 M total Na+). In
batch culture with thiosulfate as energy source, the strain
grows optimally at pH 10 and between 0.6 and 2 M of
total Na+. At 4 M Na+, the cells grow and oxidize thio-
sulfate at 50% of the maximum rates. Tv. versutus ALJ 15
FEMSLE Congress 2-6-03
361
was grown aerobically in thiosulfate-limited continuous
culture at pH 10 at 0.6, 2 and 4 M total Na+. The max-
imum speci¢c growth rates were 0.29, 0.21 and 0.1 h-1,
respectively. The highest molar yield (7.9 g protein/mol
thiosulfate) was observed at 0.6 M Na+. The yield de-
creased substantially at more extreme salt conditions. Sim-
ilar to that observed in batch culture, the maximum thio-
sulfate- and sul¢de-oxidizing potentials were higher at 0.6
and 2 M than at 4 M Na+. Elemental sulfur production
was prominent at 0.6 and 2 M, but not at 4 M Na+. The
apparent a⁄nity constant (Ks) for thiosulfate increased
with dilution rates from 3 to 10 WM. The oxidation rates
and biomass yields of the haloalkaliphilic bacteria de-
scribed here are comparable to those found under moder-
ate conditions. The extremophilic capacities of these bac-
teria can be exploited in industrial waste puri¢cation
technologies. The possible application of extremely salt-
tolerant Thioalkalivibrio strains to remove H2S from in-
dustrial o¡-gases is currently under investigation.
[1] D.Y. Sorokin et al (2001) Int J Syst Evol Microbiol,
51: 565-80. [2] D.Y. Sorokin et al (2002) Int J Syst Evol
Microbiol, 52: 2175-2182.
P11^6
INVESTIGATION OF ISOLATED PHOTOSYN-
THETIC SULFUR BACTERIA IN ORDER TO SOLVE
THE ECOLOGICAL PROBLEM OF H2S CONTAIN-
ING RESERVOIRS AROUND YAVORIV SULFUR
DEPOSIT
I. Baran, L. Kit, S. Hnatush, S. Gudz
Ivan Franko Lviv National University, Hrushevskoho ST 4,
79005, Ukraine
Some aspects of the way of H2S utilization by sulfur bac-
teria, which are present in the drain rivers, storage lakes
and reservoirs around Yavoriv invalid sulfur deposit are
investigated. The major problem is in the hydrogen sul¢de
water that permanently ¢lls the invalid sulfur deposit.
Photosynthetic purple and green bacteria are very attrac-
tive in our case because they use H2S during photosyn-
thesis. These bacteria are found at depths in the presence
of hydrogen sul¢de containing mud in the surrounding
aqueous biotopes of Yavoriv deposit. Several phototro-
phic sulfur bacteria were enriched, isolated and identi¢ed
through the cell morphology, photosynthetic pigment
composition after chromatography separation, substrate
utilization, evaluation of principal enzymes activity and
supported by the analysis of DNA. The pure cultures of
a greencolored strains (Pelodictyon sp., Chlorobium sp. and
consortium ‘‘Pelochromatium roseo-viride’’) and purple
sulfur bacteria (Chromatium sp., Lamprocistis sp., Thiocap-
sa sp.) are preliminary carried out. It has been con¢rmed
that these bacteria which are present exhibit a varied mor-
362 1st FEMS Congress / Posters 103^505
phology (by view under light and electron microscopes).
The phylogenetic relationship based on the 16sRNA anal-
ysis will be available too in order to complete the identi-
¢cation and compare the traditional classi¢cation system
of these bacteria with their phylogeny. Besides, the com-
petition for the sulphide, utilization of di¡erent parts of
spectrum in connection with the biological light utilization
between green and purple sulfur bacteria is investigated.
The current understanding and future perspectives of
water puri¢cation from H2S by using certain physiological
and metabolic peculiarities of phototrophic sulfur bacteria
are studied too.
P11^7
MICROBIAL COMMUNITY FROM SEDIMENT
CONTAMINATED WITH A HORMONALLY ACTIVE
SUBSTANCE
V. Barbosa and M. Schloter
Institute of Soil Ecology, GSF-National Research Center
for Environment and Health, Ingolsta«dter Landstrasse 1,
D-85754 Neuherberg, Germany
Trenbolone is a synthetic hormone used as a growth pro-
moter in beef cattle, with unknown e¡ects on aquatic en-
vironments. We investigated the response of benthic mi-
crobial communities to trenbolone in an outdoor
microcosm experiment. Steel containers (80 cm x 60 cm)
were ¢lled with a 10 cm layer of sediment, 230 l water and
planktonic organisms taken from an oligo-mesotrophic lit-
toral area of lake Ammersee (Germany). Technical tren-
bolone was constantly released into the water phase using
a semi-permeable membrane. Physico-chemical parameters
(pH, redox potential and temperature) as well as micro-
nutrients (ammonium, ortho-phosphate and nitrate) were
measured weekly. Analysis of bacterial (16S rDNA,
rRNA) and archaeal (16S rDNA) community structures
was performed by denaturing gradient gel electrophoresis
(DGGE). Clone library was established using bacterial
universal primers for 16S rRNA genes. The genotypic di-
versity of the community was assessed with ¢ngerprints of
PCR products obtained with random primers. Analysis of
the DGGE patterns showed very stable bacterial and
archaeal communities. Neither di¡erences between control
and treated microcosms nor seasonal variations were ob-
served. Fingerprints from random PCR products corrob-
orate the previous results, as no qualitative di¡erences
between control and treated microcosms were detected.
Most clones sequences matched with the proteo-bacteria
group, mainly belonging to Bulkolderia sp.
FEMSLE Congress 2-6-03
P11^8
EFFECT OF HYDROGEN SULPHIDE ON MICRO-
BIAL POPULATION DYNAMICS IN ACTIVATED
SLUDGE
V. L. Barbosa(1), D. A. Abaye(2), J. L. Callen(3) and R.
M. Stuetz(1)
(1) School of Water Science, Cran¢eld University, Cran-
¢eld, Bedfordshire, MK43 0AL, UK; (2) Agriculture and
Environment Division, Rothamsted Research, Harpenden,
Herts, AL5 2JQ, UK; (3) Anglian Water Services, Thorpe
Wood House, Thorpe Wood, Peterborough, Cambridge-
shire, PE3 6WT, UK
Activated sludge (AS) has been used e¡ectively for over 30
years as a bioscrubber to remove odours caused mainly by
hydrogen sulphide gas (H2S). The possibility of using an
existing AS process in a dual-role, for the treatment of the
wastewater and for odour control, has economic bene¢ts
with regard to space and operation. However, its use in
the UK has not been well established, since disagreement
exists in the literature regarding the e¡ect AS di¡usion of
odorous compounds has on the microbial population
treating the wastewater. This study used phospholipid
fatty-acid (PLFA) analysis to evaluate the e¡ects of H2S
AS di¡usion on the microbial population of AS. The com-
position of PLFAs provides a broad ¢ngerprint of the
microbial community, allowing for the estimation of bac-
terial and fungal biomass. A pilot plant comprising test
and control aeration basins was assembled and the test
basin received increasing H2S concentrations (25, 75 and
150 ppmv). The results showed an increase in the G-ve
bacterial group in the test, with increasing H2S concentra-
tion, while a slight decrease was observed in the control.
An increasing trend with increasing H2S concentration
was also observed for the fungal group. A decreasing
trend with increasing H2S dose occurred in the G+ve bac-
terial group, but the amount of PLFA indicative of G+ve
bacteria was higher in the test than in the control. The
wastewater treatment performance, measured as ammonia,
biological oxygen demand and chemical oxygen demand
removal was not a¡ected by H2S di¡usion.
P11^9
DEGRADATIVE PLASMIDS IN PAH AND XENOBI-
OTIC DEGRADING MEMBERS OF THE GENUS
SPHINGOMONAS
T. Basta and A. Stolz
Institut for Microbiology, University of Stuttgart, All-
mandring 31, 70569 Stuttgart, Germany
 1st FEMS Congress / Posters 103^505
Members of the bacterial genus Sphingomonas are able to
degrade a wide variety of polycyclic aromatic hydrocar-
bons (PAH) and xenobiotic compounds. Previous studies
demonstrated that the extraordinary degradative abilities
of Sphingomonads may be related to the presence of large
degradative plasmids. Therefore, in the present study it
was attempted to analyse 15 Sphingomonas-strains for
the presence of degradative plasmids and to obtain some
insight into the host range of these plasmids and their
conjugatability. Generally, in all strains 2-5 plasmids
were detected with sizes of 50-500 kb. In order to inves-
tigate the host range of these plasmids, the 184 kb con-
jugative plasmid pNL1 from Sphingomonas aromaticivor-
ans F199 was used. This plasmid, which encodes for the
degradation of naphthalene and biphenyl, was marked
with a kanamycin resistance gene. Using S. aromaticivor-
ans F199 pNL1 ORF363: :Tn5neo as donor strain in mat-
ing experiments, conjugative transfer of pNL1 to three
other aromatic degrading Sphingomonas-strains was dem-
onstrated. Furthermore, the existence of pNL1-similar
plasmids among three other deep-subsurface Sphingomo-
nas-strains isolated from the same location as S. aromati-
civorans F199, indicated that a horizontal gene transfer
among Sphingomonas-strains in environment can be medi-
ated by conjugative plasmids.
P11^10
YEAST CAPACITY FOR COPPER, ZINC AND SELE-
NIUM UPTAKE
M. Batic›(1), SN. Fujs(1), R. Milac›ic›(2), V. Stibilj(2) and
P. Raspor(1)
(1) Food Science and Technology Department, Biotechnical
Faculty, University of Ljubljana, Jamnikarjeva 101, 1000
Ljubljana, Slovenia; (2) Jozef Stefan Institute, Jamova,
1000 Ljubljana, Slovenia
Uptake of copper (5 mg L-1), zinc (50mg L-1) and selenium
(50mg L-1) added as CuSO4, ZnSO4 and Na2SeO4 with
yeasts Kluyveromyces marxianus, Saccharomyces cerevisi-
ae, Candida utilis and Candida intermedia was followed
24 hour at 28 ‡C in shaking culture on de¢ned medium
containing 10 g L-1 of glucose as a sole carbon source. At
the end of uptake process the concentration of copper and
zinc in the yeast biomass was determined by £ame atomic
absorption spectroscopy while selenium was determined
by atomic £orescence spectroscopy coupled hydride tech-
nique. Energy dependent uptake process provided yeast
cells with the essential elements for cellular metabolism.
Among yeasts tested K. marxianus showed the highest up-
take capacity for copper with 2.20 mg Cu g-1 of dry yeast
biomass followed S. cerevisiae with 6 %, C. utilis and C.
intermedia with 35 % lower values. Contrary, C. intermedia
with 3.21 mg Zn g-1 of dry yeast biomass exhibited highest
FEMSLE Congress 2-6-03
363
capability for zinc uptake. The yeasts tested demonstrated
similar selenium uptake capacity 0.06Y0.02 mg Se g-1 of
dry yeast biomass. On the obtained results the yeasts can
be classi¢ed in copper uptake cluster followed sequence S.
cerevisiae 6 C. intermedia 6 C. utilis 6 K. marxianus,
zinc uptake cluster with sequence S. cerevisiae 6 K. marx-
ianus 6 C. utilis 6 C. intermedia and selenium S. cere-
visiae 9 C. intermedia 9 K. marxianus 9 C. utilis. In the
process of metal uptake selected yeasts remove 77 % of
copper, 7% of zinc and 0.6 % of selenium from the media.
We wish to acknowledge the Ministry of Agriculture, For-
estry and Food and Ministry of Education, Science and
Sport of the Republic Slovenia for support project No.
V4-0402-00.
P11^11
MICROBIAL DIVERSITY IN ARABLE SOILS AS RE-
VEALED BY PHENOTYPIC AND GENOTYPIC FIN-
GERPRINTING TECHNIQUES
U. Bausenwein, A. Gattinger, A. Embacher, and M.
Schloter
GSF ^ National Research Centre for the Environment and
Health, Institute of Soil Ecology, Ingolsta«dter Landstr. 1,
85764 Neuherberg, Germany
We investigated the interactions between microorganisms
(biomass and composition) and organic matter (amount
and quality) in arable soils. Soil samples were taken at
three depths from two ¢eld sites (soil types : Gleyic Cam-
bisol and Haplic Phaezoem) with di¡ering cultivation
practices. The diversity of soil microbial communities
was assessed by using phenotypic (phospholipid fatty acids
(PLFA)) and genotypic (community DNA ampli¢ed with
a random primer) markers. Both approaches were used to
calculate diversity indices. The PLFA content of soil sam-
ples was further used for the quanti¢cation of total bio-
mass. Soil organic matter was quanti¢ed after cold water
extraction (water-extractable organic carbon, WEOC),
whereas humi¢cation indices were used for qualitative
evaluation. For both ¢eld sites we observed decreases in
biomass (up to 70%) with increasing soil depths. Total
biomass was strongly correlated with WEOC. Both phe-
notypic and genotypic markers showed that the ¢eld sites
had di¡erent microbial communities. These, however, were
only weakly in£uenced by sampling date, cultivation prac-
tice and soil depth. Changes in PLFA patterns were more
signi¢cant than changes in the DNA/PCR pattern, indicat-
ing a dynamic phenotype of the same soil microbes under
di¡erent environmental conditions. For one ¢eld site,
shifts in the community composition among di¡erent soil
depths were accompanied by changes in the humi¢cation
indices of the organic matter.
364 1st FEMS Congress / Posters 103^505
P11^12
SURVIVAL OF A NITROGEN-FILXING BACTERIUM
PSEUDOMONAS SP.418 AND ITS GENETICALLY
MODIFIED DERIVATIVES IN RHIZOSPHERE OF
DIFFERENT CROPS
A. A. Bazhanova and D. P. Bazhanov
Institute of Genetics and Cytology, NASB, Akademiche-
skaya St. 27, 200072, Minsk, Belarus
Survival of Pseudomonas sp.418 (Nif+), its Tn5-induced
Nif ^ mutant, and Nifc derivative, bearing constitutive
gene nifA of Klebsiella pneumoniae (plasmid pCK1), in
rhizosphere of barley, wheat, oats, blue lupine, yellow lu-
pine, and £ax was tested in laboratory and ¢eld experi-
ments. Seed inoculation by Pseudomonas sp.418 resulted in
colonization of seedling roots of all the crops tested. The
genetic modi¢cations were not found to a¡ect substan-
tially initial root colonization. Population of Pseudomonas
sp.418 in rhizosphere of barley, wheat, oats, blue lupine,
and yellow lupine grew during vegetation, while popula-
tions of Nif ^ mutant were stable or decreased. At the
£owering stage the number of Pseudomonas sp.418 cells
in rhizosphere of these crops was 10-100 times as high
as that of its Nif ^ mutant cells. Populations of the Nifc
bacterium declined during vegetation in rhizosphere of all
crops tested, being partially substituted by Nif + revertants
that lost the plasmid pCK1. So, nitrogen ¢xation contrib-
uted to survival and population growth of Pseudomonas
sp.418 during long-term root colonization.
P11^13
THE BIOEMULSIFIER ALASAN : CLONING AND
SEQUENCING GENES CODING FOR ALASAN PRO-
TEINS
R. Bekerman, G. Segal, and E. Rosenberg
Department of Molecular Microbiology and Biotechnology,
Tel Aviv University, Israel
Alasan, the bioemulsi¢er produced by Acinetobacter radio-
resistens KA53, is composed of three proteins (45 kDa, 31
kDa and 16 kDa) and an alanine-containing heteropoly-
saccharide. Previously, the gene (alnA) coding for the 45
kDa protein (AlnA) was cloned, sequenced and expressed
in Escherichia coli. The recombinant AlnA showed strong
emulsifying activity. The E. coli outer membrane protein
A (OmpA) has high sequence homology to the recombi-
nant AlnA, yet, it had no emulsifying activity. The 31 kDa
protein had low but signi¢cant emulsifying activity by it-
self, and greatly increased emulsifying activity of the 45
kDa protein. In order to understand the interaction be-
FEMSLE Congress 2-6-03
tween the 31 kDa and 45 kDa proteins, the gene coding
for the 31 kDa protein, referred to as alnB, has been
cloned and sequenced. The predicted protein, AlnB, has
a M.W of 20.7 kDa and pI of 4.85, and showed high
sequence homology (78% identity) to the alkyl hydroper-
oxide reductase C subunit (AHPc) of Pseudomonas putida,
suggesting a possible function. The cloned alnB is now
being expressed in E. coli in order to test whether the
recombinant protein will stimulate the emulsifying activity
of the 45 kDa protein and show similar enzymatic activity
as AHPc.
P11^14
FECAL BACTERIAL CONTAMINATION AND BE-
HAVIOUR OF E. COLI IN AN ESTUARINE ENVI-
RONMENT (SEINE, FRANCE)
T. Berthe(1), A. Touron(1), P. Servais(2), A. Ficht(3), F.
Petit(1)
(1) LMDF UPRES 2123 Groupe Biodiversite¤ et Environ-
nement, Rouen University, France; (2) ESA-ULB Brux-
elles; (3) SNS Rouen, France
A multidisciplinary research program (Seine aval) was
undertaken since 1996 to better understanding the micro-
bial contamination in the Seine estuary (France). This
macrotidal estuary is highly urbanised (30% of the french
population and 40% of french economic activity) and one
of the most contaminated by heavy metals. The bacterial
quality of this estuarine environment (water, mud£ats and
mussels) is classically evaluated by monitoring the abun-
dance of indicators of fecal contamination : [total coli-
forms, thermotolerant coliforms (fecal) and spores of
Clostridium perfringens (i.e.: sul¢te- reducing bacteria
that grow at 44‡C)]. In this environment, we investigated
relationships between the abundance of fecal indicator,
thermotolerant coliforms, and the presence of Escherichia
coli. The abundance of fecal coliforms exceeds the imper-
ative standards, at most sites, including the wastewater-
treatment plants and the tributaries. However, the propor-
tion of E. coli strains among the thermotolerant coliforms
at di¡erent sites, shows that the abundance of fecal coli-
forms is not always correlated with the presence of E. coli.
Typing of E. coli isolated from the Seine estuary was
monitoring by antibiotic and heavy metals resistance anal-
ysis. The proportion of bacteria that are resistant to at
least one antibiotic was highest in the treated e¥uents of
wastewater-treatment plants (50%), some tributaries and
in mussels (Mytilus edulis) from the mouth of the estuary
(52%) and lowest in the upstream part of the estuary.
Among them, some E. coli strains exhibit resistance to
numerous antibiotics (up to 8 di¡erent antibiotics).
 1st FEMS Congress / Posters 103^505
P11^15
CHARACTERIZATION OF RHODOCOCCUS OPA-
CUS R7, A STRAIN ABLE TO DEGRADE NAPHTHA-
LENE AND o-XYLENE
G. Bestetti(1), P. Di Gennaro(1), G. Sell(2)
(1) Department of Environmental Sciences, University of
Milano-Bicocca, Piazza Scienza,1 20126 Milan, Italy; (2)
Department of Organic and Industrial Chemistry, Univer-
sity of Milan, Via Venezian, 21, 20133 Milan, Italy
Naphthalene and methylbenzenes are examples of com-
mon hydrocarbons used in industrial processes and are
thus widespread environmental contaminants. Bacteria
which degrade these compounds under aerobic conditions
are widely distributed. This has led to extensive studies on
the metabolism of these compounds in Gram-negative
bacteria, such as Pseudomonas. In contrast, naphthalene
and xylene metabolism in Gram-positive nocardioform
bacteria has not been investigated to the same extent.
From sites contaminated with methylbenzenes and poly-
cyclic aromatic hydrocarbons (PAH), di¡erent Gram-neg-
ative and Gram-positive bacteria belonging to the Pseudo-
monas and Rhodococcus genus were selected. We
characterized Rhodococcus opacus R7 isolated from PAH
contaminated soil, the ¢rst strain so far described able to
degrade naphthalene and o-xylene, the isomer of xylenes
most recalcitrant to microbial degradation. The metabolic
studies suggest that the naphthalene degradation occurs
through the dioxygenation of the aromatic ring with the
formation of 1,2-dihydro-1,2-dihydroxynaphthalene, dehy-
drogenated to the corresponding 1,2-dihydroxyderivative
and further oxidized to salicyilic acid; this compound, a
key intermediate of naphthalene metabolism, is converted
to gentisic acid. The o-xylene pathway involves the mono-
oxygenation of the benzene nucleus leading to dimethyl-
phenol which is further metabolised to 3,4-dimethylcate-
chol, followed by a meta cleavage reaction. In
hybridisation experiments, no homology was evidenced
to catabolic genes involved in naphthalene and o-xylene
metabolism of Pseudomonas strains. By PCR a 2.0 kb
fragment from genomic DNA of R. opacus R7 was ampli-
¢ed. Sequencing analysis of the region allowed to identify
two genes encoding the two components of a naphthalene
dioxygenase, which showed high homology with narAa,
narAb genes of Rhodococcus sp. NCIMB12038.
FEMSLE Congress 2-6-03
365
P11^16
METAL RESISTANCE IN BACTERIA FROM THE
POLLUTED COASTAL SEAWATER AREAS
I. P. Bezverbnaya(1,2), L. S. Buzolyova(2)
(1) The Maritime State University, 50-a, Verchneportovaya
str., Vladivostok, Russia, 690059; (2) The Far National
State University, 27, Oktyabrskaya str., Vladivostok, Rus-
sia, 690600
The growing anthropogenic impact on the sea environ-
ment causes signi¢cant ecological changes, ¢rst of all in
coastal water areas. The pollution by heavy metals is a
general problem of many coastal seawater areas. Heavy
metals are widespread pollutants of water areas of the
Russian Far East, which is connected to natural features
of this area and to discharge of the crude sewage. We have
shown, that there was a signi¢cant amount of metal-resis-
tant bacteria in the coastal seawater areas communities of
cultivable heterotrophic bacteria. Bacteria were resistant
to several metals simultaneously. The quantity of metal-
resistant bacteria forms in community and a metal-resis-
tance level of each of bacteria culture were changing ac-
cording to character and density of environment’s pollu-
tion by heavy metals. Metal-resistant bacteria were
characterized by variety of individual responses to pres-
ence of heavy metals at the environment. Bacteria were
also resistant to a wide spectrum of antibiotics. Metal-re-
sistant bacteria di¡ered from each other in number and
size of plasmids. Metal-resistant bacteria isolated from
seawater have shown high abilities to remove of di¡erent
metals (Cd, Cu, Ni, Pb) without preliminary adaptation of
these cultures on metal containing media. The electron
microscopy analysis has shown, that bacteria ability to
metal immobilization has been connected not only to mor-
phological features of bacteria as capsule presence but also
to simultaneous realization of intracellular metal immobi-
lization mechanisms in extremely toxic conditions. Thus
the polluted coastal seawater areas are natural reservoir
for the directed selection of poly-resistant bacteria. Metal-
resistant bacteria from coastal water areas can be used for
metal removed from solutions.
366 1st FEMS Congress / Posters 103^505
P11^17
A NEW ENZYMATIC METHOD FOR THE DETACH-
MENT OF PARTICLE ASSOCIATED SOIL BACTE-
RIA
U. Bo«ckelmann, U. Szewzyk and E. Grohmann
Department of Microbial Ecology, Technical University,
Berlin, Germany
A new enzymatic technique for the detachment of bacteria
from soil particles was developed and applied to di¡erent
soil samples taken at various sampling sites and depths.
To characterize the polysaccharides, the main component
of the bacterial extracellular polymeric substances (EPS),
attached to the soil particles, a pre-staining of the soil
samples with di¡erent lectins was performed. Samples
from a sewage ¢eld, an urban park, a farmland, a forest
(mixed forest soil) and garden mold, were stained with a
set of FITC-labelled lectins from Triticum vulgaris, Ulex
europaeus, Concanavalin A and Pseudomonas aeruginosa.
According to the results, a combination of two enzymes,
K-glucosidase and L-galactosidase completed by a lipase
was chosen for degradation of the EPS structures, fol-
lowed by gentle ultrasonic treatment (ultrasonic bath)
and chemical dispersion in a modi¢ed sodium pyrophos-
phate bu¡er. Then the probes were ¢xed with formalde-
hyde and total cell counts were determined by DAPI stain-
ing. With the exception of the wheat ¢eld sample the new
technique revealed a considerable increase in the recorded
total cell counts for all investigated soil samples compared
to the conventional method, a simple dispersion of the
sample with a sodium pyrophosphate bu¡er. After the
new procedure DAPI-counts of the treated samples in-
creased up to 22-fold. We controlled the e⁄ciency of the
technique by scanning electron microscopy and could im-
pressively show that the enzymatic treatment followed by
soni¢cation e⁄ciently detached the bacteria and left the
soil particles almost blank.
FEMSLE Congress 2-6-03
P11^18
PHYSIOLOGIC STUDIES IN Cr(VI)-RESISTANT
AND Cr(VI)-REDUCING OCHROBACTRUM SPP. 5
bvl-1
R. S. Branco(1), M. C. Alpoim(2), V. M. Madeira(2) and
P. M. Morais(1)
(1) Instituto do Ambiente e Vida, 3004-517 Coimbra, Por-
tugal; (2) Departamento Bioqu|¤mica, Faculdade de Cie“n-
cias e Tecnologia da Universidade de Coimbra, Apartado
3126, 3001-401 Coimbra, Portugal
The increasing interest in the bioremediation potential of
bacteria lead us to characterized strain 5bvl-1, previously
isolated from a chromium-contaminated environment, for
its ability to resist and to reduce Cr(VI). The strain was
identi¢ed as Ochrobactrum tritici by comparative analysis
of the 16S rDNA gene sequence and DNA-DNA hybrid-
isation. The bacterium was resistant to a broad range of
antibiotics and to several metal ions such as Cr(VI), Ni2+,
Co2+, Cd2+ and Zn2+. The strain was able to grow, under
aerobic conditions, in as much as 10 mM Cr(VI). Cr(VI)
increasing concentrations in the medium decreased the
growth rate and the maximum growth yield. Strain 5
bvl-1 was also able to reduce Cr(VI). Although there is
a measurable Cr(VI) reduction during the exponential
growth phase of the culture, it only become more evident
when the culture reached the stationary phase. High
Cr(VI)-reduction yields and low Cr(VI)-uptake levels
were achieved on cultures obtained by using a starting
low cell density and a medium containing 1 mM Cr(VI).
Our results suggest that Cr(VI) reduction is an energy
demanding process and therefore depends on the metabol-
ic state of the cells and the energy available. The results
obtained also hold for the possibility that di¡erent Cr(VI)-
reduction mechanisms are ought to be operating. The ¢nd-
ings that the other strains of the genus in spite of not
being able to grow in the presence of Cr(VI) were able
to reduce Cr(VI), corroborate the idea that the Cr(VI)-
reduction ability does not confer Cr(VI)-resistance.
P11^19
ASSESSMENT OF THE BACTERIAL SOIL COMMU-
NITY IN THE URBAN PARK BERLINER TIERGART-
EN
B. Braun, U. Bo«ckelmann, E. Grohmann and U. Szewzyk
Department of Microbial Ecology, Technical University,
Berlin, Germany
Bacterial communities of urban soils are up to now little
investigated. In this study we want to characterize the
 1st FEMS Congress / Posters 103^505
microorganisms from di¡erent soil layers in two urban
soils (urban park, Berliner Tiergarten and sewage ¢eld,
Berlin Buch). Here we present the data of the sampling
¢eld Berliner Tiergarten. Changes in the bacterial commu-
nity structure and metabolic activity were reported as af-
fected by soil depth. We examined the in£uence of the soil
depth on bacterial numbers, allocation and community
composition. Traditional cultivation techniques and mo-
lecular methods, such as hybridization, ampli¢ed rDNA
restriction analysis (ARDRA) and denaturing gradient
gel electrophoresis (DGGE), were applied to investigate
the bacterial community distribution in three soil layers
of the urban park. We found a decrease in CFU in parallel
with the soil depth using ¢ve di¡erent culture media. The
total number of bacteria (DAPI counts) did not change
signi¢cantly. Investigating subclasses of Proteobacteria by
hybridization, we found gamma- Proteobacteria most
abundant in upper (10cm) and lower (90cm) soil layers,
whereas beta- Proteobacteria were dominant in the 35cm
layer. Isolates of all three soil layers were investigated with
the biolog system to obtain community ¢ngerprints based
on the metabolic potential of the microorganisms.
P11^20
BACTERIOPHAGE P22H5 AS A TRACER FOR
UNDERGROUND KARST WATERS
M. Bricelj
National Institute of Biology (NIB), Vec›na pot 111, 1000
Ljubljana, Slovenija
The P22H5 virulent bacteriophage of host bacterium Sal-
monella typhimurium was ¢rst introduced in combined
tracing experiment on the Central and Eastern part of
Peloponnesus. The phage of mouse typhoid bacterium
was chosen because the phages of Salmonella thyphiumu-
rium had been rarely encountred in surface waters. The
coliphages were avoided as tracers because of the high
bacteriophage background that could overnumber the
coliphage tracer in highly diluted water samples, when
they are faecaly polluted. In 14 tracer experiment on 10
locations in karstic regions, besides Greece, also in Austria
and Slovenia, the phage background for the host bacte-
rium never occured in traced waters. The tracer was in-
jected mostly in running water of sinking springs and in
three occasions in dry unsaturated karstic zone. In all
tracing experiments tracer reappeared with various veloc-
ities and recovery values. The average £ow velocity, based
on tg value of the phage tracer, varied from one injection
to another, within the range of 4,000 to 36 m/day, in the
regard to the traced distance and geological structure. The
greatest traced distance was 39 km and the smallest 800 m.
Although in the case of tracing in unsaturated zone the
tracer came out all three times. In the case of high and
FEMSLE Congress 2-6-03
367
middle water level of the outcoming springs, the average
time of tracer appearance based on tg data was 4.1 day
and in the season and low water level in the tested springs
the average time was 28.3 days.
P11^21
INFLUENCE OF HEAVY METALS ON THE BLACK
SEA BACTERIA
A. E. Bukhtiyarov and V. A. Ivanitsa
Department of Microbiology and Virology, I.I. Mechnikov
Odessa National University, ul. Dvoryanskaya 2, Odessa,
Ukraine 65026
Chemical pollution with heavy metals is one of the actual
problems of anthropogenic in£uence on marine environ-
ment. In laboratory conditions, we have studied how the
quantity, content of species, level of resistance to heavy
metals and potential of their accumulation changes in
dominant representatives of Odessa Gulf microbial com-
munities under in£uence of Hg2+, Cd2+ and Pb2+, which
are the most toxic ions for microbial coenoses. In inves-
tigated regions we have found cadmium and lead in less
than Maximum Allowable Concentration levels and trace
quantities of mercury. Bacteria isolated from the regions
with potential anthropogenic load have manifested signi¢-
cant levels of resistance to heavy metals. Isolated strains of
dominant bacteria relate to genera Vibrio, Pseudomonas,
Alcaligenes, Photobacterium, Aeromonas, Enterobacter and
Serratia. Representatives of genera Vibrionaceae prevail
among resistant bacteria. We have revealed changes of
MIC level of heavy metals for marine bacteria during 1-
year long storage both with the toxicant and without it.
The process of changing of initial MIC level in storage
with the toxicant has been less intensive then in storage
without one. The research has shown that marine bacte-
ria’s potential of accumulation of cadmium ranges from
3,6 mg
g-1 to 7,5 mg
g-1 of dry biomass. Most of marine
bacteria which are resistant to heavy metals have turned
out to be susceptible to 33 antimicrobial agents. Our fur-
ther investigations will be focused on study of genetic
mechanisms of resistance to the toxicants of representa-
tives of microbial communities of Odessa Gulf.
368 1st FEMS Congress / Posters 103^505
P11^22
THERIOGENIC FORMATION OF SOIL MICRO-
FLORA IN STEPPE WOODS OF UKRAINE
V. L. Bulakhov, O. Y. Pakhomov, O. A. Reva
Department of Zoology and Ecology, Dnipropetrovsk Na-
tional University, vul. Naukova 13, Dnipropetrovsk 49050,
Ukraine
One of the major factors, determining natural formation
of soil micro£ora, is theriogenic (mammalian). It is af-
fected by mammals in£uence on solidity, humidity, airing
properties, soil biological activity, organic substances. This
in£uence is realized in two ways ^ fossorial activity and
entering of excrements. In the ¢rst case, in steppe woods it
is exerted by European mole (Insectivora), mole rat (Ro-
dentia), small rodents (Rodentia: Muridae, Cricetidae),
wild boar (Artiodactyla). Under in£uence of mammals’
burrows in £ood-land oak-forests the numerical develop-
ment of total micro£ora increased from 4.49 up to 8.04
mln cells/g of soil. The similar results were obtained for
ravine oak-forests, pine woods and arti¢cial oak plantings.
For all period of burrows existence, the amount of heter-
otrophs in soil (20 cm depth) increased on 161.7 ^ 255 %
depending on forest type. Ammoni¢cators in speci¢ed for-
ests increased on 119.6 ^ 623 %; amylolitics ^ on 124 ^
211.6 %, bacteria spore ^ on 105 ^ 171 %; oligonitrophiles
^ on 137 ^ 355 %, oligotrophs ^ on 184 ^ 652 %, actyno-
mycetes ^ on 124 ^ 457 %. Acting excrements of mam-
mals-zoophagoes promote increase of total micro£ora by
73-176 %, small phytophagoes (Rodentia, Lagomorpha) ^
by 158-179 %; large phytophagoes (Suidae, Cervidae) ^ by
45-156%. The number of oligonitrophiles increased on 81-
319 %; oligotrophs ^ on 22-181 %; amylolitics ^ on 136-
417 %; actynomycetes ^ on 68-339%. So, theriogenic fac-
tor occupies 8 ^ 17 % in processes of biotic formation and
development of micro£ora in steppe forests. Thus, mam-
mals are the important ecological factors in natural for-
mation of soil micro£ora in forests.
P11^23
ICA A AND ICA D CO-EXPRESSION DETERMINES
BIOFILM PRODUCTION IN S. EPIDERMIDIS AND
ICA-OPERON DISTRIBUTION IS INDEPENDENT
OF THE SOURCE OF ISOLATION
V. Ca¢so, T. Bertuccio, F. Di Bassiano, M. Santagati, M.
L. Mezzatesta and S. Stefani
Department of Microbiology, University of Catania, Italy
Staphylococcus epidermidis is an catheter-related infections
important cause. IcaR-A-D-B-C genes are involved in the
FEMSLE Congress 2-6-03
polysaccaride-intercellular-adhesine (PIA) production,
necessary for bio¢lm accumulation in S. epidermis. PIA
synthesis has been reported to undergo a phase variation
regulation mediated by IS256 insertion and environmental
factors regulation. Thirty S. epidermis clinical strains were
tested for: i) antibiotics resistance; ii) quantitative and
qualitative bio¢lm production by spectrophotometric as-
say and Congo-Red-Agar; iii) ica-operon analysis and its
expression by PCR and RT-PCR with published and spe-
ci¢c primers designed on the Gene Bank sequence; iv)
genetic population and phylogenetic analysis (UPGMA)
by PFGE ; v) ica-operon probe hybridisations. All bio-
¢lm-producing strains were more resistant to antibiotics
tested with respect to bio¢lm-negative ones. Analysis of
ica-operon showed two di¡erent genetic organizations in
MR-MS S. epidermis derived from nosocomial or cathe-
ter-related infections : the ¢rst group showed expected size
amplicons for all ica genes; the second group showed a
hypothetical IS256 insertion in icaA and an icaB unex-
pected amplicon (700 versus 526 bp). Ica-operon sequenc-
ing, hybridisation and new speci¢c primers ampli¢cation,
revealed that this second group did not contained any ica-
operon. RT-PCR showed that icaA-D co-transcription
was necessary to determine a bio¢lm-positive-phenotype.
Genetic population analysis showed that catheter-related
strains are distributed in a homogenous cluster with re-
spect to nosocomial ones. In conclusion, all bio¢lm-form-
ing S. epidermidis are more resistant to antibiotics, possess
the ica-operon and its distribution within the sample is
independent of the source. These strains are a homogene-
ous cluster. icaA-D co-expression is necessary for bio¢lm-
forming ability. No strains contain IS256 in ica-operon.
P11^24
A MULTIPHASIC APPROACH TO EVALUATE
CRUDE OIL AND DIESEL BIODEGRADATION
WITH THERMOTOLERANT BACTERIA
A. Perfumo(1), F. Canganella(1), F. Manna(2)
(1) Department of Agrobiology and Agrochemistry, Univer-
sity of Tuscia, via C. De Lellis, 01100 Viterbo, Italy; (2)
Department of Chemical Studies and Technology of Biolog-
ically Active Substances, University La Sapienza, P.zzale A.
Moro, 00198 Roma, Italy
The biodegradation of crude oil and diesel is a largerly
investigated subject and research activities in this ¢eld
have been increasing during the past decades, due to en-
vironmental issues such as oil spilling and industrial resi-
dues disposal. As crude oil can be found either naturally
or following anthropic activities, it can be described as an
abundant substrate for microbial degradation. The are
mainly 3 problems to solve in order to achieve a signi¢cant
degradation by bacteria : 1) crude oil is found in di¡erent
 1st FEMS Congress / Posters 103^505
varieties with complex compositions and di¡erent struc-
tures; 2) the degradation of crude oil can be achieved by
bacteria but the toxicity of the substrate inhibits its meta-
bolic microbial exploitation; 3) experimental tests in vitro
can often lead to signi¢cant results but trials in vivo can
be unsuccessful. We report the isolation and characterisa-
tion of several strains capable to degrade crude oil and
diesel in a temperature range between 30 and 50‡C. More-
over, we have tested both microbial cultures and super-
natants for the degradation of crude oil, diesel, and oil-
derived waste. Following these studies, the activities of
surfactants were described and biochemical structures in-
vestigated. A di¡erent approach was also carried out with
mt biolog plates and isolated strains were tested for the
metabolic utilization of several substrates. Biochemical
and metabolic studies revealed interesting data and poten-
tial applications are also discussed.
P11^25
MICROBIAL METABOLISM OF FLUOROBEN-
ZENE : ISOLATION AND CHARACTERIZATION OF
A PURE SINGLE STRAIN BACTERIUM
M. F. Carvalho(1), P. De Marco(2), D. B Janssen(3) and
P. M. L. Castro(1)
(1) Escola Superior de Biotecnologia- Universidade Cato¤l-
ica Portuguesa, Rua Dr. Anto¤nio Bernardino de Almeida,
4200-072 Porto, Portugal; (2) Instituto de Biologia Molec-
ular e Celular, Universidade do Porto, Portugal; (3) Bio-
chemical Laboratory, Groningen Biomolecular Sciences and
Biotechnology Institute, University of Groningen, 9747 AG
Groningen, The Netherlands
The extensive use of haloaromatic compounds as solvents,
odorizers, ¢re retardants and pesticides has led to their
widespread release into the environment. Fluorinated
compounds are known to be more resistant to microbial
degradation than other halogenated chemicals and scant
information is available on its metabolic and cometabolic
fate in bacteria. A mono-species culture, capable of aero-
bic biodegradation of FB, was obtained from a microbial
consortium previously isolated, through selective enrich-
ment, from a contaminated drain in northern Portugal.
In batch cultures, the pure bacterium was able to degrade
FB up to concentrations of 480 mg l-1. In these experi-
ments liberation of £uoride was observed from the begin-
ning, indicating de£uorination of the parent compound.
Metabolic versatility studies were also conducted with
this bacterium revealing its ability to use other aromatic
compounds. The single strain culture has been used to
investigate the metabolic pathway of FB degradation. In
this way, enzymatic and metabolic studies are being con-
ducted. The results, so far achieved, revealed the forma-
tion of two peaks observed by HPLC analyses, corre-
FEMSLE Congress 2-6-03
369
sponding to two possible metabolites, in the supernatant
of FB degrading pure cultures growing in 145 mg l-1 of
FB. Identi¢cation of these metabolites was not yet con-
cluded and is still under way.
P11^26
FIELD RELEASES OF AZOSPIRILLUM BRASI-
LENSE Sp 245 GENETICALLY MODIFIED IN THE
SYNTHESIS OF INDOL-3-ACETIC ACID
M. Basaglia(1), U. Peruch(2), S. Poggiolini(2), J. Van-
derleyden(3), M. P. Nuti(4), S. Casella(1)
(1) Dipartimento di Biotecnologie Agrarie, Viale dell’Uni-
versita' 16, 35020, Legnaro (Padova), Italy; (2) Agronom-
ica, Ravenna, Italy ; (3) F.A. Janssens Lab. For Genetics,
Katholieke Universiteit Leuven, Belgium ; (4) Dipartimento
di Chimica e Biotecnologie Agrarie, Via del Borghetto 80,
56124 Pisa, Italy
It is known that Azospirillum brasilense may positively
a¡ect crop production parameters of Gramineae through
the production of Indole-3-acetic acid (IAA). From A.
brasilense Sp245, three GM strains were obtained, charac-
terized by di¡erent degrees of IAA production (IAA^,
IAAnormal, IAA+++) and luc tagged for monitoring pur-
pose. The strains were released in open environment as
seed inoculant for sorghum in a multiple ¢eld trial em-
bracing 96 single plots, covering a surface of 0.6 ha. The
performances of the 3 GM-Azospirilla were compared to
the respective untreated controls at three di¡erent levels of
nitrogen fertilization (0, 80, 160 kg N-1) in di¡erent water
supply conditions (irrigated at 100% restitution of the
evapo-traspiration versus non irrigated). The aim of this
lab-to-¢eld experiment was the study of the interaction of
GM-Azospirillum brasilense with some important biotic
and abiotic components of the ecosystem, assessing both
their agronomic bene¢t and the ecological impact of the
functional modi¢cations. The following parameters were
analysed at selected stages of sorghum growth cycle: soil
and rhizosphere colonization and survival of the released
strains, impact on selected culturable soil/rhizosphere mi-
crobial population, soil biomass, plant nitrogen uptake,
soil nitrogen at harvest time, potential denitri¢cation, yield
and early yield parameters. The agronomic implication of
the functional genetic modi¢cation, the insertion of the
reporter gene as monitoring tool, and the impact of both
modi¢cations on soil microbiota were then evaluated and
discussed.
370 1st FEMS Congress / Posters 103^505
P11^27
MOLECULAR ANALYSIS OF ENRICHMENT CUL-
TURES AND ISOLATES GROWING ON PHENAN-
THRENE
L. Cavalca, E. Dell’Amico, S. Bernasconi, M. Colombo, V.
Andreoni
Department of Food and Science Technology and Microbi-
ology, University of Milan, via Celoria 2, 20133 Milan,
Italy
The scarce bioavailability of non polar contaminants such
as PAHs compounds and their propensity to sorb to nat-
ural organic matter (NOM) are limits for their biodegra-
dation. We were interested in selecting microbial cultures
able of degrading phenanthrene in order to test their ac-
tivity towards the contaminant when sorbed to NOM.
Three enrichment cultures were selected on phenanthrene
as sole carbon and energy source from three di¡erent soils.
DGGE analysis of PCR-ampli¢ed V3 ribosomal region
revealed that the three consortia were composed by di¡er-
ent bacterial species. Some PAH-degrading isolates exhib-
ited bands co-migrating with bands detected in the enrich-
ments. The identi¢cation of the microorganisms was
performed by direct sequencing of single bands from
DGGE patterns. Degradation of 200 ppm solid phenan-
threne by the three enrichment cultures occurred at di¡er-
ent rates: culture C6, obtained from the most contaminat-
ed soil, depleted 95% of phenanthrene in 8 days (24.45mg/
day) ; culture C3 degraded the 78.5% in 21 days (7.48 mg/
day) and culture C2 degraded 28% of PAH in 15 days. We
present the results of DGGE analysis of enrichment cul-
tures examined during the time course of degradation ex-
periments. Investigations to determine the availability of
sorbed phenanthrene to the isolated cultures are in prog-
ress. The biodiversity of the selected cultures could ensure
the presence of degrading microorganisms with di¡erent
a⁄nity for PAH, which will be tested in model sorptive
phases and in soil.
FEMSLE Congress 2-6-03
P11^28
DISTRIBUTION AND DIVERSITY OF CONJUGA-
TIVE PLASMIDS AMONG SOME MULTIPLE ANTI-
BIORESISTANT E. COLI STRAINS ISOLATED
FROM FRESHWATERS
R. Cernat, V. Lazar, C. Balotescu, A. Cotar, C. Cojocaru
and D. Ene
Dep. Microbiology-Immunology, Faculty of Biology, Uni-
versity of Bucharest, Aleea Portocalelor 1-3, Sect.5, Bu-
charest 77206, Romania
In natural bacterial communities the microbial structure
and functions are subjected to dynamic environmental
and genetic adaptation. Plasmid-mediated horizontal
genes transfer has a major impact on the adaptability of
bacteria, exempli¢ed by the interspeci¢c and intergeneric
transfer of antibioresistance genes in a variety of aquatic
habitats (freshwaters, marine waters and chronically pol-
luted waters). The aim of this study was to establish the
distribution and diversity of plasmids and to study the
transfer of plasmids harboring multiple antimicrobial-re-
sistance determinants (R plasmids) belong to 14 multiple
antibiotic resistant E. coli strains isolated from fresh-
waters. Antimicrobial resistance patterns were performed
for aminoglycosides (amikacin, gentamycin, kanamycin
and tobramicin), L-lactams (ampicillin, imipenem), amox-
icillin/clavulanate, cephalosporins (ceftazidime, cefotax-
ime, cephalotin and cefamandole), quinolones (cipro£ox-
acin, nor£oxacin, o£oxacin and nalidixic acid),
tetracycline and chloramphenicol by disk di¡usion method
following NCCLS recommendations. Minimum inhibitory
concentrations (MICs) were performed using dilution
method. For the data analysis NCCLS breakpoints for
resistance and sensitivity were used. Bacterial plasmid iso-
lation was performed by an alkaline lysis method. R-plas-
mid transfer frequencies were estimated by conjugation of
drug-resistant E. coli strains used as donors with E. coli
DH5KF recipient marked with chromosomal resistance to
nalidixic acid (Nalr). The phenotypic data show the fre-
quency and dynamic £ow of multiple antibiotic resistant
and inducible L-lactamase-producing E. coli strains in
aquatic habitats. Electrophoretic patterns re£ect the high
incidence and diversity of plasmids in aquatic E. coli
strains. Plasmid-harboring E. coli strains transferred anti-
biotic resistance and, hence, possessed conjugative R plas-
mids. Of these, 80% transferred drug resistance at a fre-
quency of about 10-4.
 1st FEMS Congress / Posters 103^505
P11^29
CLONING AND CHARACTERISATION OF A 2-HAL-
OACID PERMEASE GENE FROM BURKHOLDERIA
CEPACIA MBA4
W. Y. K. Chung, H. P. S. Wong and J. S. H. Tsang
Molecular Microbiology Laboratory, Department of Bot-
any, The University of Hong Kong, Pokfulam Road,
Hong Kong S. A. R., China
Burkholderia cepacia MBA4 was isolated on its ability to
utilize 2-haloacids as carbon and energy source for
growth. It produces a single dehalogenase (DehIVa) in
batch culture condition in a regulatable manner. Dehalo-
genase associated permease has been proposed to mediate
the uptake of haloacid into the cell. In this paper, we
report the ¢rst cloning and expression of such a haloa-
cid-speci¢c transporter gene. The structural gene, desig-
nated as deh4p, was found located downstream of the
coding sequence of DehIVa. The nucleotide sequence of
deh4p was determined and characterized. An open reading
frame of 1,656 bp encoding for a putative peptide of 552
amino acids was identi¢ed. Deh4p has a putative molec-
ular weight of 59,414 and an isoelectric point of 9.88. The
nucleotide sequence of deh4p did not show any signi¢cant
homology with any genes in the standard databases. A
similar comparison with the assembled sequences of B.
cepacia J2315, however, identi¢ed a region that shows
74% identity. Comparison of the predicted amino acid se-
quence with the databases shows that Deh4p has the sig-
natures of sugar transport proteins and is an integral
membrane protein of the major facilitator superfamily.
P11^30
NICHE DIFFERENTIATION OF AMMONIA-OXIDIS-
ING BACTERIA COMMUNITIES ALONG A SALINI-
TY GRADIENT IN MICROCOSM EXPERIMENT
M. Coci(1,2), P. L. E. Bodelier(1), D. Riechmann(1), H.
J. Laanbroek(1)
(1) NIOO-KNAW Centre for Limnology, Rijsstraatweg 6,
3631AC, Nieuwersluis, The Netherlands; (2) University of
Catania, Department of Microbiology, Via Androne 81,
95124 Catania, Italy
Ammonia-oxidising bacteria convert ammonia to nitrite,
playing a key role in the nitrogen cycle. The functioning of
these bacteria in their natural habitat will mainly depend
on ammonia and oxygen availability. However, in the es-
tuaries also salinity may be a key factor driving their com-
munity composition and functioning. In this study, the
e¡ect of salinity was tested using samples from an inter-
FEMSLE Congress 2-6-03
371
tidal freshwater sediment of the river Schelde, Belgium.
Microcosms were set up allowing to sample sub-cores dur-
ing the incubation time and to obtain pore water samples
at di¡erent depths. The systems were ¢lled with freshwater
sediment (top 5 cm) and £ooded twice a day (6 hours
each) with a 1mM NH4+ medium of di¡erent salinity (
freshwater, brackish and marine) for a period of 5 weeks.
16S rRNA and amoA gene PCR-DGGE analyses of com-
munity composition showed that in the freshwater sedi-
ment micro-organisms related to Nitrosomonas ureae
were dominant. Flooding with freshwater medium resulted
in replacement of the N. ureae related ammonia oxidizers
by a strain a⁄liated with the Nitrosomonas 6a sequence
cluster. The shift occurred already in the ¢rst week of
incubation to a depth of 5 cm. Exposure to salt also re-
sulted in replacement by Nitrosomonas aestuarii, marina-
related species. This shift occurred after 4 and 3 weeks for
the brackish and marine treatment, respectively, and did
not extend deper than 1 cm. These experiment show that
changing the environmental condition lead to a rapid
shifts in the ammonia-oxidising community. This con¢rm
that di¡erent species inhabit di¡erent niches.
P11^31
LEGIONELLA SPP. IN GROUNDWATER
J. Costa(1), M. S. da Costa(2) and A. Ver|¤ssimo(1)
(1) Department of Zoology and Center for Neurociences of
Coimbra, University of Coimbra, 3004-517 Coimbra, Por-
tugal; (2) Department of Biochemistry and Center for Neu-
rociences of Coimbra, University of Coimbra, 3004-517
Coimbra, Portugal
We evaluated and demonstrated the persistence of Legio-
nella spp. in groundwater, used as water source in di¡erent
thermal spas. Legionella strains were detected, in 176
water samples, collected during several years, from four
boreholes, in two distinct geographical areas. FAME pro-
¢les were used to identify Legionella species, and isolates
were typed by Random Ampli¢ed Polymorphic DNA ^
PCR technique. Three major species were detected and
identi¢ed as L. pneumophila, L. oakridgensis and L. sain-
thelensi. The 127 isolates from one of the areas, identi¢ed
as L. pneumophila, had 6 distinct RAPD patterns, indicat-
ing the presence of di¡erent genetic groups. Two of these
six clones were persistent for at least ten years. In the
other area, L. oakridgensis constituted the major Legio-
nella specie isolated from the groundwater, during a three
years sampling period. These isolates had only one RAPD
type. The same was observed for the L. sainthelensi
strains. Despite all attempts to eliminate these strains
from the environment, their presence and persistence was
established, demonstrating that once groundwaters are
372 1st FEMS Congress / Posters 103^505
colonized by legionellae, those organisms are very di⁄cult,
if not impossible, to eradite.
P11^32
ATRAZINE DEGRADATION BY MICROORGAN-
ISMS ISOLATED FROM SWEET FLAG (ACORUS
CALAMUS) RHYSOSPHERE
R. Marecik, P. Kroliczak, K. Czaczyk, P. Cyplik
Department of Biotechnology and Food Microbiology, Agri-
cultural University of Poznan, Poland
Aquatic plants are well known tools to decontaminate
water and waste waters polluted with pesticides. Typha
species or Sweet Flag (Acorus calamus) can be used to
degradate the herbicide atrazine from aquatic systems
[Neralla 1998, McKinlay et al. 1999, Runes et al. 2001].
In most cases phytoremediation is following by microbial
degradation performed by micro-organisms present in the
rhyzosphere. The aim of this study was to isolate micro-
organisms from the rhysosphere of Sweet Flag (Acorus
calamus) and to evaluate their activity in the process of
atrazine degradation. Twenty di¡erent strains of psychro-
philic and mesophilic bacteria were distinguished among
isolated micro-organisms and atrazine-degradating experi-
ments were done. Three strains of psychro¢lic bacteria and
three strains of mesophilic bacteria were choosen to have
the highest activity in atrazine degradation. These strains
were then grown in the presence of atrazine in di¡erent
concentrations. Changes in atrazine concentration, atra-
zine degradetes concentration and number of growing mi-
crobial cells were observed during the ¢fteen days incuba-
tion. It was shown that both types, psychrophilic and
mesophilic bacteria can utilize atrazine from the medium.
In case of psychrophilic bacteria the level of atrazine uti-
lization was about 10-15% depends on the strain and the
highest reduction of atrazine was observed during the lat-
est growth phase. Mesophilic bacteria utilized atrazine on
the level of 15-30%. In all the cases only the trace amounts
of atrazine degradates were observed, but desisopropyla-
trazine was the most common degradete appeared during
the growth of psychrophilic bacteria. The growth kinetics
for tested strains were evaluated.
FEMSLE Congress 2-6-03
P11^33
FIELD AND LABORATORY STUDIES ON THE PO-
TENTIAL FOR TRANSFER OF ANTIBIOTIC RESIS-
TANCE IN STORED AND SPREAD FARM SLURRY
N. Cook(1), M. D’Agostino(1), S. Robinson(1) and K.
Kendall(2)
(1) DEFRA Central Science Laboratory (CSL), Sand
Hutton, York, YO41 1LZ, UK; (2) Askham Bryan Col-
lege, York YO23 3FR
Strains of E. coli and Salmonella enterica var. Typhimuri-
um which could receive transferable antibiotic resistances
were inoculated into non-sterile pig manure samples stored
in situ on a farm. E. coli strains possessing transferable
tetracycline resistance has previously been isolated from
the pig manure. The samples were monitored for persis-
tence of the recipient strains and for the occurrence of
antibiotic resistance transfer from indigenous micro£ora
to recipient strains. The recipient strains persisted for sev-
eral months. However no recombinants were isolated from
the in situ samples at any time. Two ¢eld lysimeters were
set up in situ to determine transfer from indigenous micro-
£ora to the recipient strains and the persistence of re-
combinants in manure amended soil. Again, potential re-
cipient strains persisted for several months without
acquisition of antibiotic resistance. In separate experi-
ments, manure microcosms were inoculated with E. coli
and Salmonella recipient strains. The recipient strains per-
sisted throughout the 5 weeks experiment duration at
varying levels, in£uenced mainly by incubation tempera-
ture. However no recombinants were isolated. This study
strongly indicates that the potential for dissemination of
antibiotic resistances among bacteria in stored and spread
slurry is negligible, probably through unfavorable environ-
mental conditions.
The work was supported by the UK Department for the
Environment, Food and Rural A¡airs.
P11^34
MICROBIAL ACTIVITY IN THE REGION OF THE
METHANE HYDRATES OF THE LAKE BAIKAL
O. Dagurova(1), B. Namsaraev(1), L. Gainutdinova(1), T.
Zemskaya(2), and O. Khlistov(2)
(1) Institute of General and Experimental Biology SD
RAS, Ulan-Ude, Russia ; (2) Limnological Institute SD
RAS, Irkutsk, Russia
In the region of the methane hydrates of South Baikal the
activity of bacteria participating in terminal stages of or-
ganic matter destruction were studied. The main product
 1st FEMS Congress / Posters 103^505
of the anaerobic destruction in the sediment of Lake Bai-
kal was methane. In the sediment of near methane hy-
drates the rate production methane were 5.0-341.6 mkl
CH4/ kg day. The biogenic and gas hydrates methane
use aerobic and anaerobic bacteria. In water of South
Baikal the rate of the aerobic methane oxidation was
0.59-1.78 mkl/ l day, in surfaces sediments ^ 37.8 ^ 273.1
mkl/ kg day. In the anaerobic condition (Eh from +70 to -
140 mV) the rate of the methane oxidation was 35,0 ^
243,7 mkl CH4/ kg day. In the region of gas hydrates
the rate of sulfate reduction was 0.78 ^ 54.6 mkg S/ kg
day. These data were higher the rate sulfate reduction in
other regions of Lake Baikal. The data on methane genesis
and methane oxidation show that metabolism of the mi-
crobial community is based on biogenic and gas hydrates
methane.
The work was supported by grants 01-05-97256 (RFBR)
and 16.7 (Lake Baikal ^ model of the Ocean).
P11^35
FINDING A LEAD-ABSORBING BACTERIUM, FOR
THE BIOLOGICAL TREATMENT OF INDUSTRIAL
WASTES
S. T. Dalir
Biology Group, Faculty of Science, Al-Zahra University,
Deh vanak, Tehran, Iran
This paper will discuss certain methods and processes to
segregate lead-absorbing bacteria from other micro-organ-
isms, for the purpose of ultimately identifying a bacterium
that could be used in the biological treatment of industrial
wastes containing the heavy metal, lead. To this end, the
wastes of some industries were tested. This research has
aimed at screening the lead-absorbing bacteria. Alto-
gether, this research has two phases of screening. Selecting
strains resistant to high lead density and screening selected
strains, based on absorption capability. Upon taking sam-
ple and its transfer to laboratory, suspension and serial
dilution were obtained. The serial dilution was cultured
on modi¢ed Luria-Bertani Agar (mLBA). After incuba-
tion and emergence of colonies, the puri¢cation stages,
two times on the said environment, was started and con-
tinued until single bacterial colonies on Nutrient agar en-
vironment was obtained. For achieving the goal in the
second phase, a fast screening method (Pu«mpel et al.,
1995) was employed. According to this method, the bac-
terial colonies, selected from the ¢rst phase, were cultured
on mLBA environment, free of lead, by punctual inocu-
lation (tooth-pick technique) and after incubation; mix-
tures of Agar-agar and Lead Nitrate (II) with di¡erent
concentrations were added on the grown colonies sepa-
rately. By proximating H2S to the colony, it became evi-
dent that in lead-contaminated regions, the interaction be-
FEMSLE Congress 2-6-03
373
tween H2S and lead would create the black sediment of
PbS and consequently, a thin and transparent layer occurs
around those colonies capable of absorbing lead. After
measuring the lead-absorption capacity in each of these
bacteria (Atomic absorption spectrophotometer), ulti-
mately, a particular strain with the absorption capacity
of 164 mg/gdw was selected to continue the research.
P11^36
DIVERSITY AND FUNCTION OF BACTERIOPLANK-
TON IN RESERVOIRS WITH CONTRASTING TRO-
PHIC STATUSES
S. Delgoulet(1,2), V. Jacquet(1), I. Thys(1), N. Babic(1)
and H. M. Cauchie(1)
(1) CRP-GL, CREBS, 162a, Avenue de la Fa|«encerie, L-
1511-Luxembourg Luxembourg ; (2) Universite¤ Blaise-
Pascal (Clermont-Ferrand II), Laboratoire de Biologie
des Protistes, UMR CNRS 6023, 63177 Aubie're Cedex,
France
The seasonal successions of bacterioplankton were studied
in reservoirs with four di¡erent trophic statuses : oligotro-
phic, mesotrophic, eutrophic and hypertrophic. Changes
in bacterial diversity were monitored using DGGE and
FISH and were related to changes in virioplankton, phy-
toplankton and zooplankton abundances as well as
changes in physico-chemistry. The metabolic activity of
the bacterioplankton was also monitored using the assim-
ilation rates of amino-acids and monosaccharides, the
abundance of bacteria stained with CTC and the exocel-
lular enzymatic activity as metabolic indicators. Prelimi-
nary results indicate that bacterioplankton dynamics are
mainly in£uenced by temperature and zooplankton devel-
opment in the mesotrophic reservoir whereas only physi-
co-chemical parameters seem to control bacterioplankton
dynamics in eutrophic lake. On the other hand, glucose
appears to be the main carbon source in the reservoirs
although other neutral sugars such as galactose are also
assimilated at a high rate. The variation of the abiotic and
biotic pressures on bacterioplankton along the trophic gra-
dient will be synthesised.
P11^37
METAL RESISTANT PGPR IN RHIZOSPHERE
COMMUNITIES OF AN ITALIAN WATER MEADOW
STUDIED BY DGGE ANALYSIS
E. Dell’Amico, L. Cavalca, and V. Andreoni
Department of Food and Science Technology and Microbi-
ology, University of Milan, Via Celoria 2 20133, Milan,
Italy
374 1st FEMS Congress / Posters 103^505
Heavy metal contamination of soil causes a variety of en-
vironmental problems and their remediation often involves
expensive restoration processes. Metal accumulating
plants have been used to remove toxic metal from soils
and their e⁄ciency of phytoaccumulation can be increased
by plant growth-promoting rhizobacteria (PGPR). The
bacterial community in the rhizosphere of graminaceous
plants grown on metal impacted soil was studied using
cultivation as well as cultivation-independent techniques.
Eubacterial community structures were examined by PCR-
DGGE of 16S rDNA to determine relative abundance and
specie diversity and concurrently, metal-resistant bacteria
were selected. On the basis of DGGE ¢ngerprintings, no
di¡erences in species diversity and abundance were ob-
served in rhizosphere samples collected in four di¡erent
sites of an Italian water meadow. Among 100 isolates
selected, numerous were metal resistant-bacteria with
PGPR characteristics. The majority hydrolyzed 1-amino-
cyclopropane-1-carboxylate (ACC) and/or synthesized in-
doleacetic acid via the indolepyruvic acid pathway; a
small number of isolates produced £uorescent sidero-
phores. Only in one strain with ACC deaminase activity,
an accD-like gene with 95% of homology with ACC de-
aminase gene of Pseudomonas strain 6G5 was found. Ge-
netic determinants for cadmium and zinc resistance have
been ampli¢ed in two strains of Ralstonia genus. The PCR
products were di¡erently homologous with czcC gene of
Ralstonia eutropha CH34, that codify for an outer mem-
brane e¥ux protein. These metal resistant PGPR will be
used in experiment of inoculation of seeds growing in
heavy metal contaminated soil in order to verify their
ability to protect seedlings against the hinibitory e¡ects
of heavy metals.
P11^38
COMBINED MOLECULAR AND MICROSCOPICAL
IDENTIFICATION OF MICROORGANISMS LIVING
IN ENDOLITHIC BIOFILMS
A. delos R|¤os(1), C. Ascaso(1) J. Wierzchos(2) and M.
Grube(3)
(1) Centro de Ciencias Medioambientales (CSIC), Serrano
115 dpdo, Madrid-28006, Spain; (2) Servei de Microscopia
electronica, Universidad de Lleida, Rovira Roure 44, Lleida,
Spain; (3) Institut fu«r Botanik Karl Franzens-Universita«t
Graz, Holteigasse 6, Graz-8010, Austria
At the interface formed between the lithic substrate and
atmosphere accumulations of microorganisms are orga-
nized as bio¢lms. These bio¢lms support life under a
broad range of environmental conditions, being often the
dominant and /or only biological components in extreme
environments. Microscopy shows that these bio¢lms har-
bour a great diversity of micro-organisms. Algae, cyano-
FEMSLE Congress 2-6-03
bacteria and fungi (free-living and lichenized) share the
microhabitat. Also, microscopy is essential for under-
standing bio¢lm structure and the interactions occurring
in it. However, to identify precisely the biological compo-
nents, we need to go beyond. This means that di¡erent
microscopy techniques such as transmission and scanning
electron microscopy and confocal scanning laser micros-
copy have to be combined with molecular techniques. The
combined use of both kind of techniques enabled the iden-
ti¢cation of the bio¢lma¤s biological components which
can be precisely localized in the lithic substrate. Molecular
methods need to be adapted to work with endolithic com-
munities. Samples are small because we need to di¡eren-
tiate microorganisms close to each other. We isolated
DNA from a small endolithic mass associated with the
rock. By subsequent PCR using speci¢c primers for fungi,
alga or cyanobacteria and sequencing of the PCR prod-
ucts, it was possible to identify some of the microbial
components of the bio¢lm. Direct PCR with very small
fragments of lithic substrate colonized by bio¢lms was
occasionally carried out and yielded results comparable
with those obtained after DNA isolation. The presence
of di¡erent genera of cyanobacteria and epilithic and en-
dolithic lichenised fungi in Antarctic endolithic bio¢lms
were detected with these methods.
P11^39
IDENTIFICATION OF MULTIPLE RING-HYDROXY-
LATING DIOXYGENASE GENES IN A CHRYSENE-
DEGRADING BACTERIAL STRAIN
S. Demaneche, J. Willison and Y. Jouanneau
Laboratoire de Biochimie et Biophysique des Systemes In-
tegres, DRDC CEA-Grenoble, 17 rue des Martyrs, 38054
Grenoble cedex 9, France
Mono and polycyclic aromatic hydrocarbons (PAH) are
widespread pollutants in the environment. Some bacteria
are able to degrade one or more PAH by oxidation,
thanks to speci¢c catabolic enzymes. Chrysene is a four-
ring PAH known to be mutagenic, carcinogenic, and re-
sistant to biodegradation. A few bacteria have been de-
scribed that could metabolise chrysene as a co-substrate,
but little is known about the biodegradation pathway in-
volved. A bacterial strain able to use this hydrocarbon as
sole carbon source has been isolated in our laboratory and
identi¢ed as a Sphingomonas species (strain CHY-1). A
PCR-based approach was developed to search for genes
responsible for PAH degradation in Sphingomonas sp.
CHY-1. Using degenerate primers designed after con-
served sequences of known ring-hydroxylating dioxyge-
nases, PCR products were ampli¢ed from genomic
DNA, then cloned and sequenced. Sequence analysis re-
vealed the existence of at least three potential dioxygenase
 1st FEMS Congress / Posters 103^505
genes. These genes were individually cloned by screening a
genomic library of Sphingomonas sp. CHY-1 using speci¢c
primers. Analysis of the resulting sequences based on com-
parison with dioxygenase alpha subunits available in the
data bases suggested that the three enzymes identi¢ed in
strain CHY-1 belong to the class II, class III and class IV
subfamilies of dioxygenases, respectively. As a means to
study the substrate speci¢city of each enzyme system, the
overexpression of the corresponding genes in suitable host
strains has been undertaken. The involvement of the se-
lected dioxygenases in PAH degradation will be investi-
gated with special care to the catabolism of chrysene.
P11^40
CHARACTERIZATION OF RHIZOBACTERIA FROM
TOMATO (LYCOPERSICON ESCULENTUM MILL.)
RHIZOPLANE
T. Dems›ar(1), SN. Kubik(1,2), M. Ravnikar(1) and M.
Rupnik(2)
(1) National Institute of Biology, Department for Plant
Physiology and Biotechnology, Vec›na pot 111, 1000 Ljubl-
jana, Slovenia; (2) University of Ljubljana, Biotechnical
Faculty, Department of Biology, Vec›na pot 111, 1000 Ljubl-
jana, Slovenia
Plant roots are associated with speci¢c microbial popula-
tion. Plant-associated bacteria are interesting as many
strains can have positive e¡ect on plant growth (PGPR;
plant growth-promoting rhizobacteria) or can protect
plants from pathogenic microorganisms. In this study bac-
teria were isolated with classical culturing techniques from
the rhizoplane of tomato plants. Among 40 strains £uo-
rescent pseudomonads and bacteria from genus Bacillus
were prevailing, but representatives of genus Arthrobacter,
Enterobacter and Stenotrophomonas were also found. All
strains were tested for in vitro inhibition of three common
pathogenic bacteria for tomato plants; Ralstonia solana-
cearum, Clavibacter michiganensis ssp. michiganensis and
Xanthomonas campestris pv. vesicatoria. Seventeen bacte-
rial isolates have inhibited at least one of the three tested
pathogenic strains and three of bacterial isolates have in-
hibited growth of all three pathogens. Strains were also
used in plant experiments to determine their PGPR e¡ect.
Tomato seeds were surface sterilised and inoculated with
bacterial isolates. Seeds were grown in sterile substrate for
5 weeks and watered with a bacterial suspension. Unino-
culated tomato seeds were used as a control. PGPR e¡ect
will be presented as measured wet and dry weight of
leaves, stems and roots of tomato plants. Some of isolated
strains from tomato rhizoplane have a potential for use in
agriculture.
FEMSLE Congress 2-6-03
375
P11^41
MICROCYSTIN: EFFECTS OF A CYANOTOXIN ON
THE GROWTH OF ESCHERICHIA COLI
R. A. Dixon(1), M. Al-Nazawi(2) and G. Alderson(2)
(1) Department of Biological Sciences, University of Lin-
coln, Lincoln LN6 7TS UK; (2) Department of Biomedical
Sciences, University of Bradford, Bradford, West Yorkshire
BD7 1DP, UK
Most Gram-negative bacteria are resistant to toxic agents
in the environment by virtue of the barrier e¡ects of their
cell envelope. We have shown that microcystin, a cyano-
toxin produced by Microcystis aeruginosa lacks antibacte-
rial activity. When tested in combination in vitro, inhibi-
tory values for a range of large molecular weight
hydrophobic antibiotics were signi¢cantly reduced in the
presence of at least 1/20 x MIC of microcystin. The degree
of inhibition was equivalent to that of the well-character-
ised permeabilising agent polymyxin B nonapeptide. The
permeabilising ability of sub-inhibitory concentrations of
microcystin to a¡ect the envelope of Escherichia coli was
demonstrated by a rapid and sustained reduction in ab-
sorbance values of lysozyme-treated cells and by enhanced
uptake of crystal violet in microcystin ^ treated cultures.
Further direct investigation of the e¡ects of microcystin on
cell envelope integrity of E. coli con¢rmed that following
treatment with microcystin, release of the speci¢c enzyme
marker of outer membrane disruption (Beta-lactamase) in
a marked strain occurred. Neither microcystin or poly-
myxin B nonapeptide a¡ected the integrity of the inner
membrane of E. coli as judged by the release of cytosolic
Beta-galactosidase. These, and other results are consistent
with microcystin causing signi¢cant permeability changes
to the enterobacterial envelope.
P11^42
FREE AND PLANKTON-ASSOCIATED HELICO-
BACTER PYLORI IN SEA WATER
G. Donelli(1), A. Del Vecchio(2), M. Di Candia(3), E. Di
Campli(3), M. Favaro(4) and L. Cellini(3)
(1) Department of Ultrastructures, Istituto Superiore di
Sanita', Rome, Italy ; (2) Agency for the Protection of En-
vironment, Pescara, Italy ; (3) Department of Biomedical
Sciences, University ‘‘G. D’Annunzio’’, Faculty of Medicine,
Chieti, Italy ; (4) Department of Biology, ‘‘Tor Vergata’’,
University Rome, Italy
Helicobacter pylori free and attached to planktonic organ-
isms was detected by nested-PCR from sea water samples
collected on the Italian coast of the Adriatic sea. The
376 1st FEMS Congress / Posters 103^505
study was conducted over one year in which sampling was
carried out once a month in a routinely monitored sam-
pling station. Dissolved oxygen, pH, salinity and chloro-
phyll ‘‘a’’ were the parameters recorded together with the
characterization of zooplanktonic organisms in the two
major groups of Cladocera and Copepoda. Plankton-asso-
ciated H. pylori DNA was searched for in water samples
¢ltered through 200 Wm and 64 Wm nylon nets. The ¢ltered
water was subsequently passed through 0.22 Wm pore-size
membranes to retain free bacteria. Nested-PCR using
primers for the urease C gene was performed to reveal
the presence of H. pylori. The sensitivity of the nested-
PCR assay for H. pylori detection was 62 CFU per 100
ml in spiked water samples containing also 4.5 x 102 total
coliforms, 0.2 x 102 faecal coliforms and 0.6 x 102 enter-
ococci. The DNA sequencing of ampli¢ed products con-
¢rmed the speci¢city of the assay. H. pylori either free or
bound to planktonic organisms was found in 7 out of 12
monthly samples. In particular, free bacteria were detected
during the summer sampling and in the months of No-
vember and December associated to planktonic cells.
These data suggest the presence of free and plankton-as-
sociated H. pylori cells in sea water indicating that it can
be a signi¢cant reservoir and a potential route of trans-
mission for this microbial pathogen.
P11^43
MICROBIAL INDICATION OF BASINS OF BAIKAL-
ANGARA-YENYSEI HYDROSYSTEMS
V. V. Drucker
The Limnological Institute of SB RAS, Irkutsk, Russia
At present the most powerful on our planet Baikal-An-
gara-Yenysei hydrosystem functions on the territory of
Siberia (Russia). It consists of four parts: the ¢rst one is
tributaries of Lake Baikal basin; the second one is Lake
Baikal, which became a large reservoir ; the third one is the
Angara river and its reservoirs (Irkutsk, Bratsk, Ust-
Ilimsk, Boguchanskoe reservoirs); the fourth one is the
Yenysei river and its reservoirs (Sayano-Shushenskoe,
Krasnoyarsk, Kureiskoe and Khantaisk reservoirs). Since
1972 investigations of microbial diversity, bacterial pro-
duction and destruction, microbiological monitoring and
evaluation of water quality, using standard and new meth-
ods of electronic and epi£uorescence microscopy, and mo-
lecular-biological investigations are done. Prognosis of
formation of bacterial plankton and water quality in arti-
¢cial reservoirs was developed, its zoning was carried out,
taking into consideration hydrochemical and hydrobiolog-
ical characteristics, an evaluation of trends of changes in
ecosystem in modern conditions is done.
FEMSLE Congress 2-6-03
P11^44
INFLUENCE OF WATER SAMPLES TRANSPORTA-
TION CONDITIONS ON RESULTS OF MICROBIO-
LOGICAL ANALYSES
R. Bitan, Z. Dveyrin, H. Ben-David
National PHL, Abu-Kabir, Israeli Ministry of Health,
P.O.B 8255, Tel-Aviv 61082, Israel
The primary goal of the survey was to determine the ex-
tent to which sampling and transportation conditions of
drinking water, a¡ect the results of microbiological anal-
yses. The secondary goal was to develop a method to
estimate the uncertainty in such microbiological testing.
Some international standards de¢ne the transportation
conditions (maximal time and temperature) for water sam-
ples. Uncertainty estimation is one of the requirements for
laboratory accreditation according to the ISO 17025.
However, in microbiology, even choosing the criteria and
methods for calculating uncertainty, encounters objective
di⁄culties, because of requirements for data collection
over a very long period, or alternatively, complex spiking
experiments. Combined uncertainty was estimated by a
modi¢ed ‘‘root sum square’’ method taking into account
duplicates analysis, international pro¢ciency testing and
intra-laboratory comparisons. The hypothesis that under
some conditions, duration and conditions of transporta-
tion may a¡ect the level of some microorganisms, and
thus, in£uence the ¢nal results, was tested in the sur-
vey.The results show that it is impractical to chill samples
down to the required temperature if transportation time is
shorter then the 6 hours (permitted by the standard meth-
od). However, incubation of ‘‘clean’’ water samples spiked
with microorganisms (in permitted levels) at various tem-
peratures for 6 hours (to simulate the transportation pro-
cess) did not cause signi¢cant changes in concentration of
indicator microorganisms. Thus, if transportation does not
extend the permitted period of 6 hours, temperature con-
ditions have little or no e¡ect on water quality.
 1st FEMS Congress / Posters 103^505
P11^45
EFFECTS OF HIGH CONCENTRATION OF ORGAN-
IC NUTRIENTS ON FUNCTIONAL ACTIVITY IN
AMMONIA OXIDIZER IN WASTEWATER TREAT-
MENT PROCESS
Y. Ebie(1), M. Matsumura(1), S. Tsuneda(2), A. Hira-
ta(2), and Y. Inamori(3)
(1) Institute of Applied Biochemistry, University of Tsuku-
ba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-0006, Japan ; (2)
Department of Chemical Engineering, Waseda University,
3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan ; (3)
National Institute for Environmental Studies, 16-2 Onoga-
wa, Tsukuba, Ibaraki 305-8506, Japan
Ammonia oxidation by chemolithoautotrophic ammonia
oxidizer is a rate-limiting step in biological nitrogen re-
moval process. However, ammonia-oxidizing activity
could not be estimated by conventional methods based
on rRNA and functional gene, to say nothing of cultiva-
tion-dependent method. In this study, RT-PCR technique
based on mRNA was combined with DGGE analysis to
investigate e¡ects of high concentration of organic nu-
trients on functional activity in ammonia oxidizer in
wastewater treatment process. To raise the concentration
of in£uent organic compounds, methanol was added inter-
mittently to the in£uent of actual domestic wastewater.
Total RNA was extracted from activated sludge collected
from aeration tank before and after methanol addition.
RT-PCR targeting mRNA coding a subunit of ammonia
monooxygenase (amoA) was combined with DGGE to
elucidate community structure based on ammonia-oxidiz-
ing activity. The RT-PCR-DGGE band patterns and the
subsequent sequence analysis revealed that the community
structure based on ammonia-oxidizing activity was com-
plicated and changed during this experiment. It is sug-
gested that the ammonia oxidizing activity of individual
species of ammonia oxidizer were di¡erent and shifted in
response to increasing organic compounds loading. In
conclusion, the combination of RT-PCR and DGGE pro-
vides new information that could not be available by con-
ventional methods based on rRNA and functional gene.
FEMSLE Congress 2-6-03
377
P11^46
DECOLORIZATION OF SYNTHETIC DYES BY FIL-
AMENTOUS FUNGI
I. Eichlerova, L. Homolka and F. Nerud
Institute of Microbiology AS CR, Videnska 1083, 142 20
Prague 4, Czech Republic
A wide spectrum of di¡erent species of ¢lamentous fungi
was screened for the overall hydrogen peroxide production
and synthetic dyes decolorization ability. To ¢nd relation-
ships between hydrogen peroxide secretion, ligninolytic or
other extracellular enzymes production and decolorization
abilities, several strains were more intensively studied and
their morphological, biochemical and physiological prop-
erties during cultivation on dye-containing media were
followed. We found that the increased ligninolytic activity
of the higher-producing strains must not necessarily lead
to higher decolorization capacity. On the other hand, the
low ligninolytic enzyme and overall H2O2 production was
mostly not su⁄cient for e⁄cient decolorization of the
studied dyes. Growth and biomass production play an
important role in the decolorization process. The presence
of individual synthetic dyes in the medium in£uenced
(mostly inhibited) the growth of the tested strains, which
caused incomplete degradation of tested dyes, but when
the biomass production was higher, the decolorization ca-
pacity increased.
This work was supported by grant no. 206/02/D119 from
the Grant Agency of the Czech Republic and by Institu-
tional Research concept no. AV0Z5020903.
P11^47
EVIDENCE FOR DEREPRESSION OF CONJUGA-
TION FROM STUDIES OF STATIONARY-PHASE
BACTERIA IN ARTIFICIAL SOIL MICROCOSMS
R. J. Ellis(1), M. J. Bailey(2) and H. C. J. Godfray
(1) NERC Centre for Population Biology, Imperial College
London, Silwood Park Campus, Ascot, Berks, SL5 7PY.
UK; (2) NERC Centre for Ecology and Hydrology, Mans-
¢eld Road, Oxford OX1 3SR, UK
An arti¢cial soil microcosm was developed which con-
sisted of sand, clay, humic acid and minerals. This system
allowed the study of microbial dynamics and interactions
under controlled conditions. Here the growth dynamics of
Pseudomonas £uorescens SBW25 derivatives (resistant to
di¡erent antibiotics) were investigated, together with a
study of the transfer of a natural plasmid, pQBR103
(330 kbp, Tra+, Hgr), between the variants. It was dem-
onstrated that the arti¢cial soil supported high numbers of
378 1st FEMS Congress / Posters 103^505
SBW25, equivalent to bacterial population densities in
natural soil. In competition, plasmid-bearing SBW25 do-
nors grew at a slower rate than plasmid-free recipients and
consequently achieved a lower population density. The
densities achieved were not limited by the availability of
nutrients. Plasmid transfer between the two variants was
only detected when populations had been in stationary
phase for at least 3 days. After this point plasmids moved
rapidly though the population to the point where, after 10
days, up to 80% of the original recipients had acquired the
plasmid and were classed as transconjugants. The transfer
frequencies attained in the model soil system were three
orders of magnitude greater than those recorded in stan-
dard ¢lter plate matings on minimal agar containing an
equivalent nutrient supply. A mass action model was de-
veloped to simulate the data. It revealed that the observed
dynamics could only be explained by an increased rate of
transfer from new transconjugants compared to the orig-
inal donors.
P11^48
GENETIC CONTROL OF ROOT COLONISATION
BY A NITROGEN-FIXING PSEUDOMONAS STUT-
ZERI
R. Carre•o-Lopez(1), N. Desnoues(2) and C. Elmer-
ich(2,3)
(1) Universidad Atonoma de Puebla, Puebla, Mexico ; (2)
Institut Pasteur, Paris, France; (3) ISV-CNRS, 91198 Gif
sur Yvette, France
It is now recognized that plant growth promoting rhizo-
bacteria (PGPR) play an important role in agriculture.
Numerous nitrogen-¢xing bacterial species have been iso-
lated from cereal crops and pasture grasses. In general,
nitrogen-¢xing root-associated diazotrophs are soil bacte-
ria able to colonize the root surface. Endophytes associate
more closely by colonizing the root and stem interior. The
Pseudomonas stutzeri strain A15 can ¢x nitrogen under
microaerobic conditions at the free-living state. The organ-
isation of the nitrogen ¢xation genes resembled that of
Azotobacter vinelandii. Characterisation of rpoN and asso-
ciated genes, ptsO and ptsN, as well as two-component
systems has been undertaken. Inactivation of rpoN led to
mutant strains impaired in nitrogen ¢xation, motility, car-
bon and nitrogen sources utilisation and colonisation of
the root system. It was shown that the defect of colonisa-
tion was due to the lack of £agella.
FEMSLE Congress 2-6-03
P11^49
RHIZOSPHERE BACTERIAL COMMUNITY INFLU-
ENCES BIOAVAILABILITY OF COPPER FOR
PLANT
E. E. Emnova(1), S. I. Toma(2), S. S. Lisnic(2)
(1) Institute of Microbiology, Moldovan Academy of Sci-
ences, MD-2028, Chisinau, MD; (2) Institute of Plant
Physiology, Moldovan Academy of Sciences, MD-2060,
Chisinau, MD
The ¢rst aim of this study was to compare the response of
bulk soil and rhizosphere bacterial associations to en-
hanced copper concentration in soil using £uorescein diac-
etate hydrolysis rate (FDHR) ^ proposed for tolerance
estimation of soil microorganisms to lead pollution. The
comparative research of soybean rhizosphere bacterial
community at low (5 and 30 mg/kg) and high (380 and
580 mg/kg) CuCl2 loads showed the shift of community
structure in favour of culturable gram-negative bacteria at
high levels of soil copper. The bacterial FDHR was in-
creased in last two treatments and correlated with the
gram-negative bacteria number, most likely pseudomo-
nades. No change of rhizosphere bacteria Cu-tolerance
was found after 45 days of exposition to high Cu concen-
trations; the parameters IC50 were the same in all studied
treatments. The survival of pseudomonades was supported
by plants, whereas the bulk soil bacterial communities lost
their FDHR activity almost completely at high Cu con-
centrations. The obvious negative impact of copper on
yield parameters of soybean was only observed at 580
mg CuCl2/kg of dry soil. Enhanced soil moisture in treat-
ments with high copper contents was revealed. Similar
observations have been reported for soil after Cu-treat-
ment. The relationship between exopolysaccharide produc-
tion by Gram-negative pseudomonades and soil water
contents have been published repeatedly. Our results sug-
gest a relation between soil copper concentration and exo-
polysaccharide synthesis by rhizosphere gram-negative
bacteria. Moreover, this microbial polymer appears to re-
duce the mobility and, thus, the bioavailability of copper.
 1st FEMS Congress / Posters 103^505
P11^50
COMPARISON OF MTBE AND TAME DEGRADA-
TION PATHWAYS IN MYCOBACTERIUM AUSTRO-
AFRICANUM IFP 2012
A. Francois, L. Garnier, H. Mathis, F. Fayolle and F.
Monot
Institut Francais du Pe¤trole, De¤partement de Biotechnologie
et Chimie de la Biomasse, 1 & 4, avenue de Bois-Pre¤au,
92852 Rueil-Malmaison Cedex, France
Fuel oxygenates such as methyl tert-butyl ether (MTBE),
ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether
(TAME) may be incorported to gasoline at high concen-
trations (up to 15%, vol/vol) to increase its octane index
and reduce air pollutant emissions from combustion. Be-
cause of their widespread use and the high frequency of
underground tank leakages, these compounds are poten-
tial contaminants of groundwater. Their fate in the envi-
ronment need to be documented and only few studies deal
with the biodegradation of TAME (worldwide production
of 1.5 Mt in 1999). TAME can be degraded under aerobic
conditions by cometabolism with ethanol and propane or
by a mixed culture. Tert-amyl alcohol (TAA) was the sole
metabolite identi¢ed during TAME catabolism. Using a
strain able to grow on MTBE, Mycobacterium austroafri-
canum IFP 2012, TAME was shown to be mineralized
more rapidly than MTBE and ETBE when used as a
sole carbon and energy source. A better understanding
of the TAME degradation pathway could provide new
information, especially about the initial attack on the ether
bond which was supposed to be very recalcitrant to enzy-
matic attack and responsible for the low biodegradability
of these ethers. Several metabolic intermediates of TAME
degradation were detected and a putative degradation
pathway proposed. It was then compared with that of
MTBE.
P11^51
EVALUATION OF TWO MOLECULAR FINGER-
PRINTING METHODS IN COMPARATIVE COMMU-
NITY STRUCTURE ANALYSIS OF AMMONIA OXI-
DIZER BACTERIA IN NITRIFICATION BASINS
T. Felfo«ldi, A. J. Sze¤kely, K. Ma¤rialigeti
Eo«tvo«s Lora¤nd University, Department of Microbiology,
Pa¤zma¤ny Pe¤ter se¤ta¤ny 1/c., H-1117 Budapest, Hungary
Molecular techniques based on the ammonia-monooxyge-
nase functional gene are useful tools to investigate the
diversity and phylogenetic relationships of autotrophic
ammonia-oxidizing bacteria. AmoA is a group-speci¢c
FEMSLE Congress 2-6-03
379
gene which encodes the active-site carrying subunit for
ammonia monooxygenase enzyme. At low ambient tem-
peratures e⁄ciency of nitri¢cation decreases which is a
common problem during biological sewage treatment.
Therefore the ammonia-oxidising community of nitri¢ca-
tion basins and their nitrifying capacity were examined in
order to increase the e⁄ciency of nitri¢cation in case of
extreme conditions such as low temperature. Furthermore,
terminal restriction fragment length polymorphism (T-
RFLP) analysis was improved and tested for rapid screen-
ing and compared with cloning-sequencing method as ¢n-
gerprinting technique of a special bacterial community.
NH4+, NO2- and NO3- levels were measured with the
aim of determining the rate of nitri¢cation of each basin.
An amoA clone library was created in order to reveal the
phylogenetic relationships of the bacteria. The compara-
tive sequence analysis of these clones assigned every se-
quence to the Nitrosomonas europaea branch within the
Nitrosomonas genus. The T-RFLP patterns of amoA
gene derived from direct DNA retrieval demonstrated
that at least two di¡erent OTUs were present in the sam-
ples. Beside the members of the Nitrosomonas europaea
branch the members of the Nitrosomonas oligotropha
branch were also detected. According to these results T-
RFLP proved to be a ¢ngerprinting technique of higher
resolution than the classical cloning-sequencing method.
By this complex examination of the ammonia-oxidizing
bacteria we were able to describe the community structure
of basins working appropriately also in low temperatures.
P11^52
FLUORESCENCE ACTIVATED CELL SORTING
AND THE MICROBIAL ECOLOGIST!
B. C. Ferrari(1,2), G. Oregaard(2) and S. J. Sorensen(2)
(1) Department of Biological Sciences, Macquarie Univer-
sity, Sydney, Australia ; (2) Institute of Molecular Biology,
University of Copenhagen, Copenhagen, Denmark
There is increasing need in microbial ecology to isolate
bacterial sub-populations from complex communities
within environmental samples. The ability to obtain highly
puri¢ed bacteria in real time enables further characterisa-
tion by culturing, £uorescent staining or molecular meth-
ods. Fluorescence activated cell sorting (FACS) can be
used to isolate such populations, however its application
in this ¢eld has been limited. Complex cell sorters such as
the BD FACSVantage or the Cytomation Mo Flo are
traditionally the £ow cytometers of choice for such anal-
yses, however these instruments are expensive, and com-
plex requiring specialist sta¡ of which many microbial labs
cannot support. We propose that a simple to use, less
complex instrument such as the BD FACSCalibur-sort
can be successfully applied in a research setting. These
380 1st FEMS Congress / Posters 103^505
instruments are limited in that they do not analyse samples
at a high event rate, however they do not require daily
alignment and they can be run by non-specialist operators.
With growing interest in isolating microbial populations
according to physiological or phylogenetic measurements,
made applicable by single cell analysis, we describe the
isolation of bacterial biosensors expressing GFP from a
complex microbial culture. Purities exceeding 99% were
achieved coupled with at least 55% recovery of target cells.
Additionally, 100 sorted E. coli can produce a PCR prod-
uct. Quanti¢cation of the culturable versus non-culturable
fraction of mixed populations can now be achieved using
this system by sorting rare GFP+ bacteria. This will allow
monitoring of horizontal gene transfer within indigenous
bacteria growing in complex environments including soil.
P11^53
Withdrawn.
FEMSLE Congress 2-6-03
P11^54
SEARCHING FOR PLANCTOMYCETES REVEALS
AN EVOLUTIONARILY DEEPLY BRANCHING
NEW BACTERIAL LINEAGE WITHIN THE MARINE
SPONGE APLYSINA AEROPHOBA
L. Fieseler(1), M. Horn(2), M. Wagner(2), U. Hent-
schel(1)
(1) Institut fuer Molekulare Infektionsbiologie, Universitaet
Wuerzburg, Roentgenring 11, D-97070 Wuerzburg, Ger-
many; (2) Lehrstuhl fuer Mikrobiologie, Technische Uni-
versitaet Muenchen, Am Hochanger 4, D-85350 Freising,
Germany
Sponges (Porifera) are evolutionarily ancient metazoans
that populate the tropical reefs in great abundance. Aply-
sina aerophoba, as well as many other demosponges, is
associated with large amounts of bacteria that exceed
those of seawater by 2-3 orders of magnitude. Transmis-
sion electron microscopy of A. aerophoba tissues revealed
an unusual bacterial morphotype containing a membrane-
enclosed cellular substructure. Because cell compartmenta-
tion has so far been reported only for the phylum Planc-
tomycetes we applied planctomycete-speci¢c 16S rDNA
primers for the construction of 16S rRNA gene libraries.
16S rRNA gene sequence analyses revealed a deeply root-
ing lineage that branches o¡ at the basis of the Verruco-
microbia and Planctomycetes. Fluorescence in situ hybrid-
ization (FISH) with newly designed probes targeted
against the respective microorganisms showed a high sig-
nal abundance in A. aerophoba tissues. The ring-shaped
appearance of FISH signals is consistent with cell compar-
timentation in the target microorganisms. Further analysis
of these unusual sponge-associated microorganisms could
provide new insights into the development of cellular orga-
nization.
 1st FEMS Congress / Posters 103^505
P11^55
HORIZONTAL TRANSFER OF BIODEGRADATIVE
PLASMIDS IN OPEN ENVIRONMENT
A. E. Filonov, L. I. Akhmetov, I. F. Puntus, T. Z. Esikova,
O. V. Volkova, S. L. Sokolov, A. B. Gafarov, T. Yu. Iz-
malkova, I. A. Kosheleva and A. M. Boronin
Institute of Biochemistry and Physiology of Microorgan-
isms, Russian Academy of Sciences, Pushchino, Moscow
Region, 142290, Russia
Naphthalene degradative plasmid pNF142 was labelled by
mini-transposon TnMod-OTc carrying tetracycline resis-
tance gene. The plasmid pNF142 : :TnMod-OTc resistant
to tetracycline was transferred into P. putida UT2442 car-
rying gfp and kanamycine resistance reporter genes in
chromosome. The phenotypic stability of plasmids
pNF142, pNF142 : :TnMod-OTc and pBS216 was studied.
The plasmids were stably maintained in strains P. £uores-
cens 142NF, P. putida BS394 cys and UT2442. The
TnMod-OTc insertion into the plasmid pNF142 does not
a¡ect plasmid stability and expression of naphthalene bi-
odegradation plasmid genes. P. putida strains UT2442
(pNF142) and BS394 (pNF142: :TnMod-OTc) cys ob-
tained by conjugation possessed growth parameters on
naphthalene similar to the speci¢c growth rate of a natural
isolate P. £uorescens 142NF (pNF142); transconjugant P.
putida UT2442 (pNF142: :TnMod-OTc) had a higher spe-
ci¢c growth rate in comparison with the naturally occur-
ring strains. The labelled naphthalene degrading strains
were used for monitoring plasmid horizontal transfer
among the introduced and indigenous microorganisms in
soil. It was demonstrated that in soil contaminated with
naphthalene the plasmid pNF142: :TnMod-OTc was trans-
ferred to indigenous soil bacteria (mainly £uorescent Pseu-
domonas) as well as to plasmid-free UT2442 (transfer fre-
quency 2X10-7 ^ 4X10-6). Plasmids from indigenous
bacteria had been transferred to plasmid-free P. putida
UT2442. Results showed that degradative plasmids are
able to disseminate among indigenous microbial popula-
tions enhancing their catabolic capabilities and therefore
may increase the e⁄ciency of hydrocarbon biodegrada-
tion. It can be suggested that not only the introduction
of degrading bacteria but also plasmid transfer lead to the
rise of microbial degradative potential.
This work was supported by the U.S. Civilian Research
and Development Foundation grant RB2-2377-PU-02 and
a grant from the Russian Federal Scienti¢c and Technical
Program, State contract 43.073.1.1.2502 ‘‘Environmental
Biotechnology’’.
FEMSLE Congress 2-6-03
381
P11^56
BACTERIAL DIVERSITY OF COMPOST AND VER-
MICOMPOST : A CULTIVATION-INDEPENDENT
COMMUNITY ANALYSIS
L. Fracchia(1), K. Trescher(2), M. G. Martinotti(3) and
C. C. Tebbe(2)
(1) Universita' del Piemonte Orientale, DISTA, Corso Bor-
salino, 54, 15100 Alessandria, Italy ; (2) Institut fu«r Agrar-
o«kologie, Bundesforschungsanstalt fu«r Landwirtschaft
(FAL), Bundesallee 50, 38116-Braunschweig- Germany;
(3) Universita' del Piemonte Orientale, DISCAFF, Viale
Ferrucci, 33, 28100 Novara, Italy
The bacterial diversity in a ¢nished compost and a ¢nished
vermicompost was studied by means of a cultivation-inde-
pendent approach based on polymerase chain reaction
(PCR)-ampli¢ed partial small subunit rRNA genes and
genetic pro¢ling by single-strand conformation polymor-
phism (SSCP). The community structures of three di¡erent
periods of storage of compost and of three di¡erent wind-
rows of vermicompost were compared with each other.
Liquid nitrogen and bead beating were used to homoge-
nise the samples and release DNA from the microbial
cells. PCR ampli¢cations were performed with primers
targeting the variable V4 and V5 regions of eubacterial
16S rRNA genes. PCR products were converted to sin-
gle-stranded DNA molecules by exonuclease digestion
and SSCP pro¢les were generated by electrophoresis on
polyacrylamide gel. The patterns generated from compost
and vermicompost samples were compared with each oth-
er by digital image analysis. We found di¡erences between
compost and vermicompost products and, for the compost
samples between the three di¡erent periods of storage. No
signi¢cant di¡erences of pro¢les were detected between the
three vermicompost piles. In order to characterise bacteria
typical for compost and vermicompost , bands from rep-
resentative SSCP patterns were isolated and sequenced.
Streptomyces-related sequences were detectable in com-
post, in particular, one sequence was closely related to
S. cattelya. Other sequences, especially from vermicom-
post were only distantly related to cultured organisms.
Some of these sequences could be grouped into the Acid-
obacteria phylum.
382 1st FEMS Congress / Posters 103^505
P11^57
ISOLATION, PURIFICATION AND CHARACTER-
IZATION OF A CHROMATE-REDUCTASE FROM
AN OCHROBACTRUM SPP. 5bvl-1
R. Francisco(1), P. Ver|¤ssimo(2), M. C. Alpoim(2) and P.
V. Morais(1)
(1) Instituto do Ambiente e Vida, 3004-517 Coimbra, Por-
tugal; (2) Departamento de Bioqu|¤mica, Universidade de
Coimbra, 3001-401 Coimbra, Portugal
Hexavalent chromium (Cr(VI)) is a strong oxidant and a
toxic pollutant. Bacterial reduction of Cr(VI) to the less
toxic and less water soluble Cr(III) has previously been
reported and there is accumulated evidence that bacterial
reduction of chromate can occur under both aerobic and
anaerobic conditions. Furthermore, microorganisms can
be a potential useful tool in the treatment of contaminated
soils and waters. Recently, we isolated several Cr(VI)-re-
sistant bacteria strains from a Cr(VI)-contaminated acti-
vated sludge. One of them, belonging to the species Ochro-
bactrum tritici (strain 5bvl-1), was found to show both
high Cr(VI)-resistance and reduction capacities. The iso-
lation and characterization of the enzyme(s) responsible
for reducing Cr(VI) would a¡ord information to imple-
ment or improve bioremediation strategies. The cells of
the strain 5bvl-1 were disrupted by sonication and each
fraction obtained was tested for chromate-reductase activ-
ity. Several preliminary tests were realized on the fraction
with Cr(VI) reduction capacity, preceding our attempt to
isolate, purify and characterize the chromate-reductase.
The preliminary work performed showed that the enzyme
chromate-reductase has di¡erent physiological character-
istics than the ones previously described. The role this
chromate reductase(s) plays in bacteria physiology has
not been explored yet.
FEMSLE Congress 2-6-03
P11^58
MICROBIAL ASSAY FOR RISK ASSESSMENT
(MARA) ^ A NEW TOOL FOR (ECO)TOXICITY
TESTING
J. Gabrielson(1), P. Colque-Navarro(1), M. Hart(2), I.
Ku«hn(1), D. McKenzie(3), R. Mo«llby(1)
(1) Microbiology and Tumorbiology Center (MTC), Kar-
olinska Institutet, Box 280, 171 77 Stockholm, Sweden; (2)
Dunsta¡nage Marine Laboratory, Oban, Argyll, Scotland
PA34 4AD, UK; (3) Integrin Advanced Biosystems, Ma-
rine Resource Centre, Barcaldine, Oban, Argyll, Scotland,
PA37 1SE, UK
Background. There is an emerging need for bioassays that
are more informative, more accurate and faster than ex-
isting assays. Animal tests that are available su¡er from
lack of standardisation, high costs and long test times.
There are some commercially available tests based on mi-
croorganisms, but all of them are based on one or a few
species. In the MARA-test, at least 11 strains of di¡erent
species will be studied in parallel. Results. MARA is a
bacteria-based method for risk assessment. The strains
are exposed to an appropriate concentration gradient of
the chemical to be tested, and as di¡erent strains exhibit
di¡erent sensitivities a growth inhibition pattern is ob-
tained. The assay is performed in 96-well microplates,
which are read with a £at-bed scanner. Numerical inhibi-
tion values for each strain are obtained by analysis of the
resulting images with a speci¢c software. The resulting
array of inhibition values is the ‘‘toxic ¢ngerprint’’ of
the chemical and it can be compared to other toxic ¢nger-
prints in a database. The toxic ¢ngerprint seems to be
speci¢c individual chemical compounds. If a single value
is desired, the result can be presented as e g the mean
value or the minimum value. The reproducibility and sen-
sitivity of MARA are comparable to other similar assays.
Conclusion. The MARA-method constitutes an alternative
to existing methods for (eco)toxicity testing. The strength
of the method is that a number of chemicals can be tested
on a large number of microbial strains simultaneously in a
cheap, simple and systemised way.
 1st FEMS Congress / Posters 103^505
P11^59
SEQUENCE ANALYSIS OF THE CLC ELEMENT OF
PSEUDOMONAS SP. STRAIN B13
M. Gaillard(1), V. Sentchilo(1), T. Vallaeys(2), F.-J. Vo-
rhoelter(3) and J. R. van der Meer(1)
(1) Swiss Federal Institute for Environmental Science and
Technology, CH-8600 Du«bendorf, Switzerland ; (2) Institut
Pasteur, 75724 Paris cedex 15, France; (3) IIT Biotech, D-
33615 Bielefeld, Germany
Pseudomonas sp. strain B13 was the ¢rst described Pseu-
domonas able to metabolize 3-chlorobenzoate (3-CBA).
The origin of this catabolic property comes from the pres-
ence of the clcABDE genes encoding chlorocatechol deg-
radation. This cluster of genes was located on a genomic
island, called the clc element. The clc element can transfer
from strain B13 to other bacteria. Critical to the transfer
process is a phage-like integrase gene (int-B13). This inte-
grase mediates the integration of the clc element into a
speci¢c target site, the 3’end of the glycine tRNA gene,
and also plays a role in the excision of the element. Inte-
grase expression is enhanced in bacteria growing on 3-
CBA. Evidence was obtained that regulation of integrase
expression and excision is mediated by factors encoded on
the clc element itself. Here we describe the sequence anal-
ysis of the complete clc element. In addition to the
clcABDE genes, the ¢rst 40 kb sequenced fragment lo-
cated on the right end revealed the presence of a cluster
of other metabolic genes for the 2-aminophenol degrada-
tion, as well as an aminophenol operon repressor and a
probable transcription regulator protein. On the left end,
seven conserved ORFs were discovered which probably
are implicated in regulatory functions.
P11^60
MICROBIAL AND BIOGEOCHEMICAL PROCESSES
IN PERENNIALLY ICE-COVERED ANTARCTIC
LAKES
V. F. Galchenko
Institute of Microbiology of RAS, Prospekt 60-let Oktjabr-
ja, 7/2, 117312 Moscow, Russia
It is believed that 4-4,5 billion years ago conditions were
similar and favorable for the beginnings of life on Mars
and Earth. However, more than 3 billion years ago, the
Martian temperature reduced considerably and surface
reservoirs froze. Apparently, life on Mars stopped at the
simple stage ^ prokaryotes. Antarctic perennially ice-cov-
ered lakes could serve as terrestrial analogues of Martian
frozen reservoirs. These lakes have existed under negative
FEMSLE Congress 2-6-03
383
average annual temperatures for 12-13,000 years and are
characterized by exceptionally prokaryotic communities.
Photo- and chemoautotrophic bacteria and archae realiz-
ing ‘‘biogeochemical’’ processes: cyanobacteriae, metha-
nogenes, sulfate reducers, methanotrophs, heterotrophs,
are probable terrestrial analogues of Martian lakes abo-
riginals. Antarctic perennially ice-covered lakes (Hoare,
Fryxell, Bonney-West, Polyanskii and White Smokes)
were investigated during expeditions of 1991-92 and
1995-96. Physicochemical parameters were analyzed: min-
eral salt composition, redox and hydrogen potentials, oxy-
gen, nitrogen, methane, and CO2 concentrations. The rates
of biogeochemical processes, namely oxygenous and anox-
ygenous photosynthesis, acetoclastic and autotrophic
methane formation, aerobic and anaerobic methane oxi-
dation, sulfate reduction, decomposition and oxidation of
acetate, lactate, glucose, thymidine inclusion in bacterial
cells were determined ; chlorophyll, carotenoids and ATP
contents were measured, the total number of bacteria and
species composition of methanotrophic micro£ora, stable
isotope compositions of sulphur and carbon were deter-
mined. Together with Dr. V. Fedorovich, a mathematical
model was created of biogeochemical processes of carbon
and sulphur cycles in Lake Fryxell. These processes are
described by di¡erential equations with partial derivatives
of the di¡usive type. The measurements of organic matter
photoproduction were used to calculate material and en-
ergy balances in connection with other biogeochemical
processes. The model describes the concentration pro¢les
of the main organic and mineral compounds and their
rates of biological transformation during the second
part of Antarctic day. The results indicate that the ecosys-
tem stability with space and time is due to a high conju-
gation of the microbiological processes investigated.
P11^61
ANALYSIS OF THE NORMAL MURINE ILEAL
FLORA
M. Galic, L. Sait, R. A. Strugnell, P. H. Janssen
Department of Microbiology and Immunology, University of
Melbourne, Royal Parade, Parkville, Victoria 3010, Austra-
lia
The resident microbial £ora of the gastrointestinal (GI)
tract of mammals is known to be crucial to the health
of the host. Past studies have relied on cultivation and
have often overlooked organisms, that are not readily cul-
turable. To gain a more comprehensive understanding of
the composition of the murine ileal £ora, we employed
Terminal Restriction Fragment Polymorphism (T-RFLP)
analysis of bacterial 16S rRNA genes. This technique re-
lies on variation in restriction sites among 16S rRNA gene
sequences and measures the sizes of £uorescently labelled
384 1st FEMS Congress / Posters 103^505
terminal restriction fragments (TRFs) by high-resolution
sequencing gel electrophoresis. T-RFLP was conducted in
combination with clone library analysis in order to deter-
mine the species origin of the TRFs, providing a more
comprehensive molecular analysis of microbial community
composition and diversity than clone library analysis or T-
RFLP alone. The study indicated that co-housed, geneti-
cally identical C57BL/6 mice have considerably di¡erent
ileal microbial community composition. Additionally, the
ilea of these mice were dominated by novel groups of
uncultivated organisms. Phylogenetic analysis of 16S
rRNA gene sequences originating from these organisms
revealed that a signi¢cant number form a coherent group
not a⁄liated with any recognised family, within the phy-
lum Bacteroidetes. These ¢ndings suggest there is a higher
than expected degree of variability in the composition of
the bacterial community of the ileum of co-housed, inbred
mice.
P11^62
FLOW CYTOMETRIC ANALYSIS OF MICROBIAL
COMMUNITIES IN HYPERSALINE SYSTEMS
A. Galotti, F. Jime¤nez-Go¤mez, R. Jime¤nez-Melero, F. Guer-
rero
Departamento de Biolog|¤a Animal, Biolog|¤a Vegetal y Eco-
log|¤a, Campus las Lagunillas, Universidad de Jae¤n, CP.
23071, Jae¤n, Spain
This study describes the size structure and the functional
composition of microbial components (heterotrophic bac-
teria and phytoplankton) of three aquatic saline ecosys-
tems situated in Jae¤n province, south of Spain (Honda
saline lake; San Carlos and Los Velez inland salines). A
combination of £ow cytometric techniques and epi£uores-
cence microscopy allowed the determination of the size
distribution and the apparent DNA content (high and
low signal) of the smallest components of this commun-
ities. The results shows that bacteria were extremely abun-
dant in all the cases, with values around 3.5 x 106 bacteria
ml-1 in Honda saline lake (16.8 g l-1); and ranged between
0.6 to 0.14 x106 bacteria ml-1 in the inland salines (175 and
93.7 g l-1, respectively). The High/Low DNA apparent
content ratio shifted dramatically between the saline sys-
tems, i.e 0.15 at Los Velez inland saline, and 3.16 in Hon-
da mesosaline lake. At the same time, the phytoplankton
abundance was higher in this last ecosystem, mainly dom-
inated by pico and nanoplankton what could estimulate
the growth of active heterotrophic bacteria.
FEMSLE Congress 2-6-03
P11^63
EFFECTS OF P. FLUORESCENS 92rk AND P190r,
AND OF THE AM FUNGUS G. MOSSEAE BEG12
ON TOMATO GROWTH, ROOT DEVELOPMENT
AND PHOSPHORUS UPTAKE
E. Gamalero(1), B. Pivato(1), N. Massa(1), A. Copet-
ta(1), A. Trotta(2) and G. Berta(1)
(1) University of Piemonte Orientale ‘‘Amedeo Avogadro’’,
Dept. of Science and Advanced Technologies, Alessandria,
Italy ; (2) University of Turin, Dept. of Vegetal Biology,
Turin, Italy
The increasing public concern about food and environ-
ment quality has resulted in research on alternative agri-
cultural practices characterized by low chemical inputs.
Microbial inoculations contribute to improve plant growth
and health. Among bene¢cial microorganisms proposed
for plant inoculation, £uorescent pseudomonads and ar-
buscular mycorrhizal fungi (AMF) received large atten-
tion. However, information on the e¡ect induced by rhi-
zobacteria and AMF, alone and in combination, on plant
growth and root architecture are scanty. We have focused
our attention on the impact of Pseudomonas £uorescens
92rk and P190r and of an AM fungus (Glomus mosseae
BEG12), alone or in combination, on tomato growth, root
architecture and phosphorus (P) uptake. P. £uorescens
92rk, P190r and G. mosseae BEG12, inoculated alone or
in combination, increased plant growth. The co-inocula-
tion of the three microorganisms, induced a synergistic
e¡ect on plant growth. Root architecture was also strongly
in£uenced, depending on the inoculum combination.
Plants inoculated with the two bacterial strains and G.
mosseae BEG12 showed the highest values of total root
length, total root area and volume, number of tips and
root branching degree. Moreover, P. £uorescens 92rk in-
creased the mycorrhizal colonization, showing a mycorrh-
ization helper e¡ect. Leaf P content was enhanced by the
bacterial strains and the AM fungus alone and in combi-
nation; the highest leaf P content was recorded in plants
inoculated with the three microorganisms. In conclusion,
our results support the use of these microorganisms as a
promising approach for the improvement of tomato
growth and for the development of a more environment-
friendly agriculture.
 1st FEMS Congress / Posters 103^505
P11^64
STRUCTURAL AND FUNCTIONAL DIVERSITY OF
BIOFILM LAYERS IN A SEQUENCED BATCH BIO-
FILM MINIREACTOR AS REVEALED BY EXTEN-
DEND PLFA AND 13C-PLFA ANALYSES
A. Gattinger(1), H. Eisenmann(2), W.-R. Abraham(3)
and M. Schloter(1)
(1) Institute for Soil Ecology and (2) Flow Cytometry
Group, GSF ^ National Research Center for Environment
and Health, Bodeno«kologie, D-85764 Neuherberg; (3) GBF
^ Gesellschaft fu«r Biotechnologische Forschung mbH, Abt.
Umweltmikrobiologie, D-38124 Braunschweig, Germany
Phospholipid fatty acids (PLFA) were determined to de-
scribe structural and functional diversity in a sequenced
batch bio¢lm minireactor. It contained a ¢xed bed with
about 3000 clay marbles (d=5.6 mm) that derived from a
longtime laboratory reactor and harboured a stable waste-
water bio¢lm. Samples for PLFA analyses were taken at 0,
1, 3, 6, 12, 24 and 48h after loading of the reactor with
arti¢cial wastewater (without a carbon source). For each
sampling date 3 sample treatments were generated. 50 clay
marbles were treated by ultrasonication, resulting in a liq-
uid subsample with detached PLFAs, and a bio¢lm carrier
subsample with still sticky PLFAs. Another 50 clay mar-
bles were kept untreated to determine total PLFAs on
carrier material. PLFA concentrations showed generally
a high chronological variation, but no signi¢cant indica-
tion of chronological biomass development, i.e. increase or
decrease of biomass. However, a trend for the increase of
PLFAs after one day of reactor processing is notable,
since 5 from 6 PLFA groups showed their maximum of
concentration at least after 24h. Following PLFA ¢nger-
printing, it can be assumed that the sticky fraction consists
of strictly anaerobic bacteria that live deep within the bio-
¢lm and are strongly adhered to the bio¢lm carrier, where-
as the outer bio¢lm layer comprises strictly aerobic, easy
detachable microbial species with markable amounts of
protozoans. To monitor potential synthesis of PLFAs dur-
ing the experiment, 13C-acetate was the only carbon source
added to arti¢cial wastewater. 13C-PLFA analyses re-
vealed di¡erences in acetate utilization between the inner
and the outer bio¢lm zones.
FEMSLE Congress 2-6-03
385
P11^65
LABELLING OF THE DELFTIA ACIDOVORANS
CA28 PLASMID PC1 WITH GFP AND TRANSFER
OF PC1 : : GFP TO THE BACTERIAL COMMUNITY
IN ACTIVATED SLUDGE
J. Goris(1), N. Boon(2), W. Verstraete(2), and P. De
Vos(1)
(1) Laboratory of Microbiology, Gent University, K. L.
Ledeganckstraat 35, B-9000 Gent, Belgium ; (2) Laborato-
ry of Microbial Ecology and Technology, Gent University,
Coupure Links 653, B-9000 Gent, Belgium
Genes encoding the degradation of haloaromatics are
often encoded on transmissible plasmids. The use of a
suitable marker gene, e.g. the gene encoding the green
£uorescent protein (GFP) of Aequorea victoria, facilitates
the tracking of such a catabolic plasmid within an ecosys-
tem. In this study, the 3-chloroaniline (3-CA) degrading
plasmid pC1 of Delftia acidovorans CA28 was tagged with
a mini-transposon containing the GFP gene and a kana-
mycin resistance gene. The labelled plasmid, designated
pC1: :gfp, was subsequently transferred to Pseudomonas
putida UWC3 and the plasmid transfer from this donor
to the bacterial community in activated sludge was
studied. Conjugation experiments were performed with
concentrated activated sludge on LB agar plates or di-
rectly in liquid activated sludge. Green £uorescent colonies
appearing on mineral medium containing 3-CA as sole
nitrogen source and kanamycin were picked up and veri-
¢ed to be true pC1: :gfp-harbouring transconjugants.
These isolates were subsequently identi¢ed using REP-
and BOX-PCR genomic ¢ngerprinting and partial 16S
rDNA sequencing. Repetitive element-PCR pro¢ling re-
vealed a large diversity in the transconjugant collection,
indicating the occurrence of multiple plasmid transfer
events. Remarkably, LB agar plate conjugations yielded
a di¡erent set of transconjugants compared to conjuga-
tions in liquid activated sludge. From the plate matings,
mainly Aeromonas sp. transconjugants were isolated, while
D. acidovorans strains dominated the transconjugant col-
lection from liquid activated sludge. Several pC1: :gfp-har-
bouring transconjugants were shown to perform a rapid
and complete 3-CA degradation, suggesting a potential for
bioaugmentation through dissemination of this catabolic
plasmid.
386 1st FEMS Congress / Posters 103^505
P11^66
SYMBIOTIC FUNGI ON LEAVES: FROM BIOFILMS
TO COMPLEX STRUCTURES
E. Baloch and M. Grube
Institute of Botany, Holteigasse 6, 8010 Graz, Austria
Leaves in tropical rainforests provide an ephemeral envi-
ronment for diverse microorganisms. The growing phyllo-
plane is rapidly colonized by diverse fungi and algae,
which may interact to form symbiotic systems known as
lichens. High competition on the restricted area of the
leave surface leads to particular colonization patterns
which we investigate in greater detail. Because the organ-
isms readily colonize any smooth surface in the natural
environment within a few months, it is possible to com-
pare the patterns on natural leaves and di¡erent arti¢cial
surfaces exposed in rain forests. Epi£uorescence microsco-
py was used to investigate the hyphal structures and initial
stages of lichenization. Lichens with coccale algae as pho-
tobionts initiate the symbiosis after single algal cells are
attached to the fungal hyphae, whereas lichens with tren-
tepohlioid photobionts usually invade already established
algal colonies on the leave surface. In other cases, pre-
formed lichen associations are colonized by other fungi
which may take over the algae of the host lichen. Direct
ampli¢cation of ribosomal DNA from both symbionts re-
veal the level of selectivity of fungi for their algal sym-
bionts, and indicate algal switching within lineages of fun-
gi.P11^67
CLONING, ISOLATION AND EXPRESSION OF A
TOXIC EXTRACELLULAR METALLOENDOPEPTI-
DASE, AsaP1, FROM AEROMONAS SALMONICIDA
IŁ. Hvanndal(1), V. Andre¤sdo¤ttir(1), A. C. Willis(2) and B.
K. Gudmundsdo¤ttir(1)
(1) Institute for Experimental Pathology, University of Ice-
land, Keldur, Reykjav|¤k, Iceland ; (2) MRC Immunochem-
istry Unit, Biochemistry, University of Oxford, UK
Infections of ¢sh by the bacterium Aeromonas salmonicida
often show typical symptoms of septicaemia causing sig-
ni¢cant mortality, but some strains of the species may also
cause ulcerative disease with more super¢cial symptoms.
A. hydrophila is an opportunistic pathogen of some mam-
malians (including humans) and ¢sh species, causing soft
tissue wound infections and diarrhea in the former and
fatal haemorrhagic septicaemia in the latter. A. salmonici-
da as well as A. hydrophila virulence may involve several
extracellular enzymes including acetylcholinesterase, pro-
teases and haemolysins. The major exotoxins of a group of
A. salmonicida strains has been isolated and described as a
FEMSLE Congress 2-6-03
20 kDa metalloendopeptidase termed AsaP1. The com-
plete asaP1 gene was isolated and sequenced. The se-
quence revealed an open reading frame of 1029 bp (343
aa) with 84% gene sequence identity to eprA1 of A. hydro-
phila. The encoded peptide has a predicted molecular mass
of V37 kDa. A possible ribosomal binding site was found
upstream of the start codon and a signal sequence was
identi¢ed with a possible cleavage site at aa position 21.
The mature protein is encoded by aa 172-343 and the
remaining part of the polypeptid, aa 22-171, form a pro-
peptide. The aa sequence of the active protein showed 87
% identity to the eprA1 protease of A. hydrophila and 45%
identity to a protease from the eatable mushroom Grifola
frondosa. Prediction of AsaP1 folding was made in silico,
using known 3D structures of two aspzincin peptidases,
the GfMEP from Grifola frondosa and the NPII from
Aspergillus oryzae.
P11^68
MOLECULAR AND PHENOTYPIC STUDIES OF
THE AsaP1 EXOTOXIN WITHIN A HETEROGE-
NEOUS COLLECTION OF AEROMONAS STRAINS
IŁ. Hvanndal(1), U. Wagner(2), V. Andre¤sdo¤ttir(1) and B.
K. Gudmundsdo¤ttir(1)
(1) Institute for Experimental Pathology, University of Ice-
land, Keldur, Reykjav|¤k; (2) Institute for Zoology, Univer-
sity of Leipzig, Germany
Infections due to various strains of the bacterium Aero-
monas salmonicida cause furunculosis and related diseases
of both feral and cultivated ¢sh stocks in freshwater and
marine environment. A major exotoxin of many A. salmo-
nicida, strains, is a metalloendopeptidase, AsaP1, with a
MW of V20kDa. The aim of this study was to investigate
the frequency of the asaP1 gene in a collection of 43 A.
salmonicida strains, including type strains for all the ¢ve
subspecies (salmonicida, achromogenes, masoucida, smithia
and pectinolytica) and compare to the secretion of AsaP1
of the same strains. Monoclonal antibody based ELISA
(Mab-ELISA) was used to detect AsaP1 in the strains
extracellular products. Two di¡erent PCR primer pairs
were used for amplifying the asaP1gene, one pair amplify-
ing the open reading frame and another a domain within
the gene containing the zinc-binding motif. All strains
tested were found to possess the sequence containing the
zinc-binding motif of AsaP1. According to the results the
A. salmonicida strains were divided into four groups. The
largest group (48%) contained strains possessing the whole
asaP1 gene, which were negative in the ELISA. Strains
possessing and expressing the asaP1 gene were 40%.
Only 4% of the strains were negative in genetic and phe-
notypic studies. One strain that was negative in the PCR
test amplifying the whole asaP1 gene was found to secrete
 1st FEMS Congress / Posters 103^505
the protease. Comparative sequencing of the asaP1 open
reading frame of 3 AsaP1 negative and 2 AsaP1 positive
strains revealed frame shift mutations, explaining the lack
of AsaP1 secretion by the same three strains.
P11^69
MICROBIAL TRANFORMATIONS OF N AND P AND
THE RISK OF EUTROPHICATION IN RESTORED
MARSHES
J. Hacin, P. Cadez, P. Kalan, D. Odic, J. Hladnik, D.
Skeledzija, I. Mahne
University of Ljubljana, Biotechnical Faculty, Dept. of Food
Science and Technology, Laboratory of Microbiology, Vec›-
na pot 111, 1000 Ljubljana, Slovenia
Agricultural use of most European peat soils over the past
centuries has resulted in peat degradation and enrichment
of soil with phosphorus, which is usually the plant growth
limiting nutrient in native peat soils. Raising the water
table is a common measure of peat conservation. Our
studies in the Ljubljana Marsh, Slovenia have shown
that re-wetting diminishes carbon and nitrogen mineralisa-
tion. We have recently joined the European project pro-
water, focused on increased mobilisation of phosphorus
in degraded and re-wetted peat soils and subsequent pol-
lution of surface and ground waters. A set of soil environ-
mental parameters (temperature, moisture content, water
and redox potential) has been measured continuously to
establish border conditions in the ¢eld consisting of two
peat soil types di¡ering signi¢cantly in organic matter con-
tent and maintained at di¡erent water table levels. Soil
data indicate that higher concentrations of soluble reactive
phosphorus are associated with higher water table and
higher carbon content. High water table lowers redox po-
tential which consequently leads to reduction of iron and
increased P mobilisation. The role of soil organic carbon
content in P mobilisation / immobilisation, however, is less
clear. In order to establish a relationship between soil
water content/water potential, soil organic matter content
and N and P mobilization/immobilisation, a series of in-
cubation experiments have been carried out under de¢ned
moisture and temperature conditions. Mineralization of N
and P are being followed and amounts of P and N immo-
bilized by microbial biomass assessed by fumigation-ex-
traction method. Changes in microbial biomass and activ-
ity are determined by substrate-induced respiration.
Increased P mobilisation at higher temperatures suggests
that microbial activity plays an important role in P release
from degraded peat soils. The e¡ect of inorganic (Fe3+)
and organic (corn/soybean residues) amendments on N
and P immobilisation is also being tested.
FEMSLE Congress 2-6-03
387
P11^70
NITROGEN DYNAMICS AND DENITRIFICATION IN
THE FENS OF LJUBLJANA MARSH, SLOVENIA
J. Hacin, T. Kovse, I. Mahne
University of Ljubljana, Biotechnical Faculty, Dept. of Food
Science and Technology, Laboratory of Microbiology, Vec›-
na pot 111, 1000 Ljubljana, Slovenia
Mineralisation of soil organic matter (SOM) is dependent
on the nature of SOM (C/N ratio, decomposability) and a
number of environmental factors (pH, temperature, soil
water status). Peat soils are extremely rich in organic mat-
ter, decomposition of which may result in pollution of
ground water with nutrients and ‘‘green-house’’ gas emis-
sions. Numerous studies indicate that pathways and rates
of SOM decomposition as well as gas emissions are con-
trolled by water level. In order to establish a compromise
water table level for peat and landscape preservation in
the fens of Ljubljana marsh, Slovenia, a ¢eld experiment
with controlled water levels has been set up on two soil
types di¡ering in SOM content but, not in pH and C/N
ratio. Seasonal dynamics of mineral nitrogen in the ¢eld
has been followed over the period of 3 years. Nitrogen
mineralization and denitri¢cation potentials were deter-
mined in samples collected and incubated under extreme
environmental conditions recorded in the ¢eld. Results
indicate that, nitrogen net mineralization in the ¢eld and
in samples incubated at 80% WHC was directly propor-
tional to soil organic C and N content. By contrast, N net
mineralization under anaerobic/waterlogged conditions
seems to be related to proportion of aerobic organisms
in samples and unrelated to soil C and N content. Deni-
tri¢cation potential appears to be a stable property of the
soil and an indicator of carbon available to microbes from
di¡erently degraded peat soils. This later hypothesis is
supported by a 30-100% conversion of nitrate to N2O in
high carbon soil compared to less than 5 % in low carbon
soil incubated in microcosms under the same conditions.
P11^71
EFFECTS OF TEMPERATURE ON THE ANAEROBIC
DEGRADATION OF PHENOL
L. Ha«gglund, A. Schnu«rer
Department of Microbiology, Swedish University of Agri-
cultural Sciences, P.O. Box 7025, SE-750 07 Uppsala, Swe-
den
E¡ects of temperature on the degradation of phenol were
investigated in small anaerobic batch systems with inocula
from two lab scale biogas digesters run at mesophilic
388 1st FEMS Congress / Posters 103^505
(37‡C) and thermophilic (55‡C) temperature. The digesters
have been run continuously since 1996 with the same
source separated organic household waste as substrate.
Both digesters have been stable with regard to pH, volatile
fatty acids, gas yield, methane content and volatile solid
reduction for the last ¢ve years. The only di¡erences in
process management are hydraulic retention time (19 days,
55‡C and 30 days, 37‡C) and temperature. Accordingly,
the di¡erences of the micro£ora in the digesters have
mainly been developed through di¡erences in temperature.
In the batch systems, the concentration of phenol was
measured with HPLC and the degree of mineralisation
was determined through measurement of methane produc-
tion. The results revealed that phenol was mineralised via
benzoic acid at 37‡C, but not at all at 55‡C. However, by
lowering temperature to 37-48‡C an activation of the phe-
nol degradation was observed in batch cultures from the
thermophilic digester. Degradation of phenol in batch cul-
tures from the mesophilic digester occurred between 20‡C
and 48‡C. These degradation results indicated the exis-
tence of the same phenol degrading microorganisms in
the mesophilic and thermophilic digesters. Microscopic
observations of phenol degrading enrichment cultures
from the digesters also support this hypothesis. Thus,
the variation in degradation potential appears to depend
on an inactivation of microbial enzymes by temperature,
rather than di¡erences in the microbial populations at the
mesophilic and thermophilic temperature.
P11^72
SOIL FUNGAL DIVERSITY AND POTENTIAL BIO-
CONTROL ACTIVITY OF ENDOGENE TRICHODER-
MA SPECIES UNDER THE INFLUENCE OF DIFFER-
ENT FARMING MANAGEMENT SYSTEMS
A. Hagn, K. Pritsch, M. Schloter and J. C. Munch
GSF ^ Research Center for Environment and Health, Insti-
tute of Soil Ecology, Ingolsta«dter Landstra_e 1, 85758 Neu-
herberg, Germany
Although fungi play a vital role for ecosystems, only few
studies were published so far showing fungal diversity and
dynamics in soil. We investigated the in£uence of conven-
tional farming techniques compared to precision farming
(a new agricultural management technique, focusing on
the heterogeneity of a ¢eld site and adapting the amount
of fertilizer given, to the yield expected in a particular
plot) on fungal communities in agricultural soil cropped
with winter wheat. Culture-independent and culture-de-
pendent methods were used. The most obvious in£uence
on fungal community structure were seasonal e¡ects. Es-
pecially after rainfalls an increased diversity could be ob-
served. Furthermore site-speci¢c e¡ects, which were due to
di¡erent levels of soil moisture contents of high- and low-
FEMSLE Congress 2-6-03
yield areas were detected with a higher diversity for high-
yield areas in September. Also minor in£uences which
could be due to di¡erent farming management systems
applied were found. At conventionally farmed sites some
species were abundant whereas at precision farming sites
an evenly distribution of species was found. Trichoderma
spp. and Fusarium spp. in this study were the most com-
monly isolated genera. As Trichoderma species can act as
potential biocontrol organisms, endogene Trichoderma
strains were tested for biocontrol properties against soil-
borne pathogens (Fusarium graminearum, Fusarium oxy-
sporum, Gaeumannomyces garminis var. tritici). Seasonal
e¡ects and minor in£uences of high-and low-yield areas
and management systems on the population structures of
Trichoderma spp. and functional aspects could be detected.
Their ability to act as potential biocontrol organisms
proved not to be species- but ecotype- and site-speci¢c
varying with the pathogenic fungus tested.
P11^73
REASSESSING PCR PRIMERS TARGETING nirK,
nirS, and nosZ GENES FOR MOLECULAR DIVER-
SITY SURVEYS OF DENITRIFYING BACTERIA
Y . Jarvis and S. Hallin
I. Throba«ck, K. Enwall, A
Department of Microbiology, Swedish University of Agri-
cultural Sciences, P.O. Box 7025, S-750 07 Uppsala, Swe-
den
Molecular diversity surveys of denitrifying bacteria in dif-
ferent environments were carried out using PCR-based
techniques. The nirK and nirS genes encoding for copper
and cd1 heme nitrite reductases, respectively, and the nosZ
gene encoding the nitrous oxide reductase in the denitri¢-
cation pathway were the targets. Primer design is the most
critical aspect of environmental diversity surveys and the
PCR primers that have been employed were based on a
limited number of sequences. In this study, sequence data
retrieved from the expanding databases was used to
reevaluate the speci¢cities and sensitivities of published
oligonucleotide primers for the detection of nirK, nirS,
and nosZ genes. Additionally, several new primers for
these genes were designed. All primers were evaluated by
screening de¢ned denitrifying strains and DNA from soil.
When using combinations of the published primers for
nirS and nirK the PCR frequently produced a noticeable
number of fragments apart from the one of the expected
size. This will cause trouble in di¡erent post-PCR appli-
cations. The sets of primers we suggest for nirS and nosZ
genes are new but published nirK primers still worked ¢ne.
All sets produced fragments less than 500 bp in size, which
is advantageous for DGGE analysis and makes sequenc-
ing faster and easier. The primer sets ampli¢ed most of the
strains that were tested, had a high speci¢city, and tar-
 1st FEMS Congress / Posters 103^505
geted the correct gene in di¡erent types of soil. Results
from sequencing of the PCR-ampli¢ed denitri¢cation
genes in soil also demonstrated that the primer sets were
relevant for environmental studies.
P11^74
GENES ENCODING A-TYPE FLAVOPROTEINS ARE
ESSENTIAL FOR PHOTOREDUCTION OF O2 IN CY-
ANOBACTERIA
Y. Helman(1), D. Tchernov(1), L. Reinhold(1), M. Shi-
bata(2), T. Ogawa(2), R. Schwarz(3),I. Ohad(1) and A.
Kaplan(1)
(1) Minerva Centre for Photosynthesis under Stress, The
Hebrew University of Jerusalem, Israel; (2) Bioscience
Center, Nagoya University, Chikusa, Nagoya 464-8601, Ja-
pan; (3) Faculty of Life Sciences, Bar-Ilan University,
Ramat-Gan 52900, Israel
Photoreduction of O2 by photosynthetic electron transfer,
the Mehler reaction, is observed in all groups of oxygenic
photosynthetic organisms. Although reported over 50
years ago the electron transport chain that mediates this
reaction has not yet been identi¢ed. Our study provides
the ¢rst evidence for the involvement of A-type £avopro-
teins. Mutants of Synechocystis sp. strain PCC 6803 de-
fective in genes encoding A-type £avoproteins, v£v1 and
v£v3, failed to exhibit O2 photoreduction but performed
normal photosynthesis and respiration. We show that the
light-enhanced O2 uptake was not due to respiration or
photorespiration. After dark acclimation, photooxidation
of P700 was severely depressed in mutants v£v1 and v£v3
(con¢rmed by steady state £uorescence measurements) but
recovered following light activation of CO2 ¢xation, which
provides P700 with an additional electron acceptor. Inhibi-
tion of CO2 ¢xation prevented recovery but scarcely af-
fected P700 oxidation in the wild type where the Mehler
reaction serves as an alternative route for electrons. We
conclude that the source of electrons for photoreduction
of O2 is PSI; and that A-type £avoproteins Flv1 and Flv3
are essential for this process in vivo. We propose that, in
contrast to the case in eukaryotes, the Mehler reaction in
cyanobacteria, mediated by highly conserved A-type £avo-
proteins which have been shown to reduce O2 directly to
water in vitro, does not produce reactive oxygen species
and that it may be evolutionarily related to the response of
anaerobic bacteria to O2.
FEMSLE Congress 2-6-03
389
P11^75
PHYLOGENETIC ANALYSIS OF BACTERIOPLANK-
TON COMMUNITY IN RIA DE AVEIRO
I. Henriques, M. J. Saavedra(1), A. Almeida(2), A. Cu-
nha(2), A. Correia
Centro de Biologia Celular, Campus Universita¤rio de San-
tiago, Departamento de Biologia, Universidade de Aveiro,
3810-193 Aveiro, Portugal. (1) Departamento de Higiene e
Sanidade, Universidade de Tra¤s-os.Montes e Alto Douro,
Vila Real, Portugal; (2) CESAM, Universidade de Aveiro
The estuarine system ‘‘Ria de Aveiro’’ is located in the
northwest coast of Portugal and is subjected to consider-
able inputs of industrial and domestic discharges. Re-
cently, we started to study the structure of bacterial com-
munities on this lagoon. This project has two main aims:
a) to acquire useful data for evaluation of future changes
associated with recently implemented water treatment pro-
grams; b) to compare the composition of bacterial com-
munities developing under di¡erent environmental condi-
tions. To perform this study, clone libraries of 16S rDNA
from di¡erent locations on the lagoon were constructed.
Here we report the results obtained by the analysis of the
library representing the community from a sampling site
under the in£uence of both fresh and marine water. A
total of 84 clones were recovered from this library and
characterized by HaeIII RFLP analysis. The partial se-
quence of 62 clones representative of unique restriction
patterns was determined. Phylogenetic analysis revealed
that 92% of the clones were a⁄liated with the proteobac-
teria and clustered with the K (38 clones), L (8 clones) Q (10
clones) and N (1 clone) classes respectively. Clearly the
dominant species, based on a genotype analyses, were
close relatives of Roseobacter spp. Clones a⁄liated with
some other classes including Verrucomicrobiae, Flavobac-
teria and Actinobacteria were also retrieved from the li-
brary. The results obtained strongly suggest that the bac-
terial community in this site of the estuary is a complex
community resulting from the juxtaposition of species
characteristic of marine water and more typical fresh
water species.
390 1st FEMS Congress / Posters 103^505
P11^76
A NEW RECORD OF TWO SPECIES OF THE CLASS
MONOGENEA (BENEDEN) BYCHOWSKY, 1937 AT
EEL (ANGUILLA ANGUILLA LINNAEUS, 1758)
FROM LAKE OHRID, MACEDONIA
S. Stojanovski(1), J. Hristovska(2), N. Hristovski(2), P.
Cakic(3), M. Hristovski(4), R. Nastova-Djordjiovska(5)
(1) Hydrobiological Institute, 6000 Ohrid, Macedonia; (2)
Faculty of Biotechnical Sciences, 7000 Bitola, Macedonia;
(3) Institute for Biological Researches ‘‘Sinisa Stankovic’’,
11000 Belgrade, Yugoslavia; (4) Faculty of Veterinary
Medicine, 1000 Skopje, Macedonia; (5) Institute of Animal
Science, 1000 Skopje, Macedonia
Parasitological examination from the Macedonian part of
the Lake Ohrid showed that of 191 specimens of eel (An-
guilla anguilla Linnaeus, 1758) 98 ¢shes (51.31%) were in-
fested with gill monogeneans. In our case study the pres-
ence of 2 dactylogyrid species was found:
Pseudodactylogyrus anguillae (Yin & Sproston, 1948) and
Pseudodactylogyrus bini (Kikuchi, 1929). Both species are
rather pathogenic to their hosts and can cause mortality of
heavily infected eels in ell farms in both Asia and Europe.
In the Lake Ohrid, 46 specimens of eel examined (24.08%)
were infested with Pseudodactylogyrus bini and 62 eels
(32.46%) were infested with Pseudodactylogyrus anguillae.
Mean intensity of infestation with Pseudodactylogyrus bini
was 2.54 and with Pseudodactylogyrus anguillae was 1.90.
Pseudodactylogyrus species have hitherto been reported
from eels in Central and North European countries. This
is the ¢rst record of Pseudodactylogyrus anguillae and
Pseudodactylogyrus bini for the ¢shes from Macedonia,
and with regard of available data ¢rst record for Balkan
Peninsula.
P11^77
CHARACTERIZATION OF ATRAZINE-DEGRADING
BACTERIAL COMMUNITIES
D. Hrs›ak(1), N. Udikovic¤(1), D. Filipc›ic¤(1) and F. Mar-
tin(2)
(1) Center for Marine and Environmental Research, Rudjer
Bos›kovic¤ Institute, P.O.Box 180, 10002 Zagreb, Croatia ;
(2) INRA-CMSE, Microbiologie des Sols, P.O.Box 86510,
21065 Dijon Cedex, France
The objective of this study was to evaluate biotransforma-
tion activity of atrazine degrading bacterial communities
originating from aquatic and soil ecosystems, especially to
assess their e¡ectiveness for treatment of e¥uents from
atrazine production. The enrichment of atrazine degrading
FEMSLE Congress 2-6-03
bacteria was carried out in continuous-£ow units under
the in£ow of mineral salts medium (AMS) with atrazine
as only carbon and nitrogen source. Three communities
(two enriched from e¥uents and one from soil of the
herbicide factory) were selected as the most e⁄cient since
they showed complete disappearance of atrazine under
continuous cultivation (monitored by HPLC analyses),
as well as substantial mineralizing activity during batch
cultivation in sealed £asks (60-70% atrazine released as
carbon dioxide, determined by TIC analyses). Plating on
selective AMS-agar plates suggested that during continu-
ous cultivation the community structure changed signi¢-
cantly, with appearance of morphologically new strains.
From these associations ¢ve strains were isolated express-
ing di¡erent atrazine mineralizing activity (17-53%). All
these strains were capable to transform atrazine to cyanu-
ric acid, but none of them could continue degradation, i.e.
the cleavage of s-triazine ring. The capacity of isolated
strains to degrade atrazine was further tested to the occur-
rence of known degrading genes. Enrichment of stable,
structurally new bacterial communities which were more
e⁄cient in atrazine degradation than any of the individual
populations alone suggested that the relationships between
community members, including those based on combined
metabolic capability and the transfer of catabolic genes,
are important for successful atrazine mineralization.
P11^78
INVESTIGATIONS ON THE METHANE PRODUC-
TION IN A MUNICIPAL COMPOSTING PLANT
U. Ja«ckel, K. Thummes, P. Ka«mpfer
Institut fu«r Angewandte Mikrobiologie, Universita«t Giessen,
Heinrich-Bu¡-Ring 26-32, D-35392 Giessen, FRG
The controlled composting process is supposed to guaran-
tee a processing of organic material under oxic conditions
by venting or turning of compost piles. Only few investi-
gations address the relevance of anoxic processes during
the aerobic composting process within microsites. How-
ever, the potential of methane production as ¢nal step of
anaerobic degradation of organic material is present in
heaps of di¡erent ages, which is indicated by instantane-
ously start of methane production after replacing the at-
mosphere by nitrogen in a laboratory experiment. The
maximum methane production was observed in a heap
at the age of 6 weeks with 36 Wmol CH4 gdw-1 d-1 at a
time were maximum total cell counts (DAPI method) of
4X109 cells gdw-1 were observed as well. Our experiments
show an optimum temperature for potential methane pro-
duction at 60‡C, indicating the activity of thermophilic
methanogens. The main ecological factors controlling
methanogenic activity in the composting pile may be at-
tributed to the water content and pH-value in the com-
 1st FEMS Congress / Posters 103^505
posting material. The initialisation of clear increased
methane production between pile ages from 4 to 6 weeks
correlates with increasing pH-value from 5.8 to 7.8. The
addition of water to compost material from 20% water
holding capacity (WHC) to 60% WHC led to a 10-fold
increase of methane production. Therefore, we assume
that the decreasing water content at the end of composting
process (6 ^ 11 weeks) is responsible for the observed
decrease in methane production.
P11^79
STUDY OF WATER MICROBIAL QUALITY IN
FRESHWATER LAKES IN TBILISI SURROUNDINGS
E. Jaiani, N. Janelidze, B. Lasareishvili, M. Tediashvili
G. Eliava Institute of Bacteriophage, Microbiology and Vi-
rology, 3 Gotua str., Tbilisi 380060, Georgia
Since 1995 the e¡ect of continuing urbanization on micro-
bial community of Lisi and Ku lakes has been evaluated.
These lakes are situated in a rapidly developing suburban
area of Tbilisi. In 2001-2002 the regular, monthly moni-
toring has been performed. Focus was made on Lisi lake,
because of drastically changes in hydrogeological and eco-
logical parameters of this lake in the summer 2001. Col-
lected water samples were analyzed for total bacterial
count, total coliforms, Escherichia coli count, aerobic
count, Enterococcus index, some waterborne pathogens
(Salmonella, Shigellla, Vibrio spp.) and content of somatic
bacteriophages. Physical properties such as temperature,
salinity, transparency, in parallel with biochemical oxygen
demand (BOD5), dissolved oxygen and chlorophyll a were
studied as well. Detection and identi¢cation of bacterial
strains were performed by standard culturing methods, as
well as by Petri¢lm plating ( 3M, USA) and API -tests (
Biomerioux, France). Phages were cloned and character-
ized by morphological and biological features. The studies
revealed that in Lisi lake during the last 6 years microbial,
physical and biochemical parameters of water pollution
have been changed. The most obvious deterioration of
the ecological status was registered in August-September
2001, that should be considered as a result of anthropo-
genic activity which led to drastic changes in hydrogeolog-
ical features of the Lisi Lake. Owing to adequate rehabil-
itating activities signi¢cant improvement of the ecological
state of the Lisi lake in Autumn 2002 was recorded. Ku
lake seems to be more stable ecosystem. Only in summer
seasons microbiological parameters exceeded accepted rec-
reational zone standards.
FEMSLE Congress 2-6-03
391
P11^80
DEGRADATION OF p-HYDROXYBENZOATE (PHB)
BY BACERIA: BIOCHEMICAL AND MOLECULAR
STUDIES
D. Paul, A. Chauhan, G. Pandey and R. K. Jain
Institute of Microbial Technology (IMTECH), Sector 39-
A, Chandigarh-160036, India
The use of microorganisms is expected to be an e¡ective
tool for remediation of polluted environments. Catabolism
of benzoate is thought to play an important role in the
degradation of complex xenobiotic aromatic compounds.
In soil microorganisms aromatic compounds are degraded
via many diverse ring-cleavage pathways. Two soil mi-
crobes isolated from pesticide-contaminated ¢elds and
identi¢ed as Arthrobacter protophormiae RKJ100 and Bur-
kholderia cepacia RKJ200 were found to be capable of
utilizing p-hydroxybenzoate as a sole source of carbon
and energy. Thinlayer chromatography and gas chroma-
tography of the ethyl extracts from the culture medium
showed the presence of protocatechuate (PC). Results of
gas chromatography-mass spectrometry of the extracted
metabolites agreed with the theoretical fragmentation pat-
tern of PC. Plasmid cured derivatives of RKJ100 and
RKJ200 were investigated to determine the location of
the genes involved in PHB degradation. In one of the
isolates, RKJ200, the degradation property was found to
be plasmid-encoded. A plasmid library of RKJ200 was
obtained in E. coli JM109 and two out of ¢ve putative
clones were screened for their ability to degrade PHB.
Biochemical tests performed on these two clones showed
that they could degrade PHB via the formation of PC as
an intermediate.
P11^81
BIOPROCESSING OF SELENOOXYANIONS BY
PSEUDOMONAS LUTEOLA STRAIN MGF-48
R. Jalali-Rad(1), M. R. Soudi(2), H. Ghafourian(1), M.
Malekzadeh(3), M. A. Ahmady(1), Y. Asef(1)
(1) Department of Biotechnology, Nuclear Research Cen-
ter, Atomic Energy Organization of Iran, Tehran, Iran; (2)
Department of Microbiology, Faculty of Science, Alzahra
University, Tehran, Iran; (3) Department of Microbiology,
Faculty of Science, Tehran University, Tehran, Iran
Strain MGF-48 was previously isolated from electroplat-
ing e¥uents. The strain was able to reduce selenooxyan-
ions to elemental selenium. Using AAS and Gamma Spec-
trophotometic Analysis, all Se measurements were
performed. The highest rate of selenite reduction took
392 1st FEMS Congress / Posters 103^505
place in medium containing 40 mg Se4+ l-1 and 50% of
Se4+ reduced to Se0. Simultaneously 20% of the total Se
content of the medium was volatilized by the strain. At
high concentrations the bacterium could tolerate selenate
much better than selenite. It was able to grow at concen-
trations as high as 8000 mg Se6+ l-1. Under optimum con-
ditions more than 50% of the initially added selenate was
volatilized. In addition, selenate was slightly reduced to
selenite and elemental Se when cultures were started at
high initial concentrations of the oxyanion (optimum ini-
tial concentration 6000 mg Se6+ l-1). Although these char-
acteristics are not unique to the strain MGF-48, but oc-
curring of these phenomena is not usual among the strains
of the species. The strain MGF-48 has been previously
introduced as a potent heavy metal biosorbing bacterium
in the literature. Thus, our ¢ndings are of interest regard-
ing bioremediation of both cationic and anionic pollu-
tions.
P11^82
THE EFFECT OF BACTERIZATION AND LIMING IN
THE PRODUCTION OF RED CLOVER IN ACID
SOILS
M. Jarak, M. Govedarica, S. Djuric
Faculty of Agriculture, Department of Microbiology, 21000
Novi Sad, Yugoslavia
Red clover (Trifolium pratense L.) is one of the most im-
portant legumes in the feeding of domestic animals.The
microsymbiont of clover is Rhizobium leguminosarum bv.
trifolii. In order to make the best use of nitrogen ¢xation,
the seed should be inoculated with highly e¡ective strains
of rhizobia before seeding.The aim of this experiment was
to examine the e¡ect of inoculation and liming on the
yield of red clover as well as on the microbiological activ-
ity in the rhizospheric soil.The lime (3 tha-1) was intro-
duced before ploughing.The seed of clover was inoculated
with Rhizobium leguminosarum bv. trifolii (1 ml 10 8 cells
on 1 g of seed) before seeding.The clover was grown on
soils with di¡erent pH values (5.1 and 6.2).The yield of
row forage, % of dry matter mass, the total number of
bacteria, the number of fungi and the number of azoto-
bacter were determined during the vegetation period. The
total yield of clover on the both localities was higher in
variants with liming (2% ^ 3.5 %) and inoculation (8 % ^
12%).The variant where both bacterization and liming
were applied gave best results.The total number of bacte-
ria and the number of azotobacter were higher in the soil
with pH value 6.2 whereas the number of fungi was ap-
proximately the same.The highest increase of the number
of azotobacter and the total number of bacteria occured in
the variant where both liming and inoculation were ap-
FEMSLE Congress 2-6-03
plied whereas these did not have in£uence on the number
of fungi.
P11^83
MOULDS AS AN INDICATOR OF ORIGIN OF FLU-
IDS IN THE FORMATION OF WULFENITE CRYS-
TALS IN MEZB ICA MINES, SLOVENIA
B. Jers›ek(1), M. Jers›ek(2), B. Mirtic›(3) and T. Dole-
nec(3)
(1) Biotechnical Faculty, University of Ljubljana, Jamni-
karjeva 101, 1000 Ljubljana, Slovenia; (2) Slovenian Mu-
seum of Natural History, Pres›ernova 20, 1000 Ljubljana,
Slovenia; (3) Faculty of Natural Sciences and Engineering,
University of Ljubljana, As›kerc›eva 12, 1000 Ljubljana,
Slovenia
Mez›ica mines are a part of the greatest metal mine in
North-Eastern Slovenia. The main ore minerals exploited
there were galena PbS and sphalerite ZnS. The mines are
well known because of famous crystals of wulfenite
PbMoO4. Nowadays the mines are closed and the wulfen-
ite specimens are a part of the natural heritage of Slovenia.
Formation of wulfenite crystals is still not clear. Normally
it is a product of reactions in the oxidation zone of Pb-Zn
minerals. There are many theories about formation con-
ditions of Pb and Zn minerals and therefore also at least
two theories about origin of £uids which caused transfor-
mation of primary minerals into secondary minerals in the
oxidation cone of ore deposit. Magmatic activities in the
surroundings of Mez›ica mines were intensive through geo-
logic history. Therefore some hydrothermal solution can
be an important source of molybdenum, but the £uids
enriched with molybdenum from upper levels of ore de-
posits may have origin also in earth surface. Investigated
crystals of wulfenite were collected in the Central part of
Mez›ica mines. Of nine fungal isolates two were identi¢ed
to species level, Syncephalastrum racemosum and Tricho-
derma harzianum. These results can demonstrate that for-
mation of wulfenite is connected to solutions from earth
surface. Identi¢ed moulds have temperature optimum
from 15 to 40‡ C that is characteristic for moulds from
subtropical and tropical climates. Regarding to tropical
climate in region of Mez›ica mines in Tertiary period we
could presuppose that moulds could be indicators of cli-
mate and paleoecological conditions on surface of the
earth millions years ago.
 1st FEMS Congress / Posters 103^505
P11^84
PATTERNS OF MICROBIAL GRAZING IN ANTARC-
TIC WATERS: EFFECT OF TEMPERATURE
F. Jime¤nez-Gomez, L. Zabala, M. Jime¤nez, A. Galotti, B.
Bautista
Departamento de Biolog|¤a Animal, Vegetal y Ecolog|¤a, Uni-
versidad de Jae¤n, CP. 23071, Jae¤n, Spain
Antarctic waters are typically inhabited by an inusual pi-
coplanktonic community, characterized by the absence of
procaryotic picoplankton. In these circumstances, the
grazing by heterotrophic nanno£agellates (HNF) is cen-
tered almost exclusively over heterotrophic bacteria. How-
ever, eukaryotic picophytoplankton is extremely variable
in this waters and, under some circumstances, like temper-
ature changes, it can proliferate and modify the described
microbial grazing pattern on bacteria. In this work, we
have analysed variations of grazing rates on microbial
components during ten onboard dilution experiments, car-
ried out in di¡erent waters around the Antarctic peninsu-
la. Changes on bacteria and picophytoplankton abundan-
ces were obtained from £ow cytometric analysis of
seawater sanples and syber-green-stained samples. Results
show that bacterial growth and nano£agellates grazing
rates were extremely variable and independent of picophy-
toplankton abundances.
P11^85
REGULATION OF PRTR, THE GENE ENCODING A
NOVEL PROTEINASE OF LACTOBACILLUS RHAM-
NOSUS BGT10
I. Tonic, I. Pastar, Lj. Topisirovic and G. Jovanovic
Institute of Molecular Genetics and Genetic Engineering,
Belgrade, Yugoslavia
A novel cell envelope proteinase PrtR from human isolate
L. rhamnosus BGT10 was identi¢ed recently in our labo-
ratory. The prtR gene and 5’ regulatory region were
cloned and sequenced. In this work the start of prtR tran-
scription and the position of promotor-like sequence were
determined. Deletion analysis of prtR regulatory region
and activities of di¡erent prtR-gusA operon fusions were
used to de¢ne the minimal promoter region. The GusA
assays showed that prtR promoter is controlled at the
transcriptional level and that the prtR expression is casi-
tone dependent. The maximal expression was obtained
with 0.1% casitone while gradual increase of casitone con-
centration proportionally decreased the prtR expression
implying the existance of negative regulation. We showed
that the negative regulatory element is situated down-
FEMSLE Congress 2-6-03
393
stream of prtR promoter like sequence. It has been shown
that expression of prtP gene in L. lactis is controlled by
the CodY repressor. We found that the gene of this regu-
lator is not present in L. rhamnosus BGT10 strain. The
proteolytic activity of L. rhamnosus BGT10 depends on
casitone as well and is in correlation with prtR-gusA oper-
on fusion experiments. Namely, the most e⁄cient proteo-
lytic activity was obtained when the cells were grown in
chemically de¢ned medium containing 0.1% of the casi-
tone.
P11^86
MEDIATED BIOSENSOR FOR DETECTION OF OR-
GANIC SUBSTANCES
A. A. Kalenyuk(1), A. N. Reshetilov(2)
(1) Institute for Sorption and Problems of Endoecology of
National Academy of Science, Kiev, Ukraine; (2) Institute
of Biochemistry and Physiology of Microorganisms of Rus-
sian Academy of Science, Puschino, Russia
The aim of this work was to investigate functional princi-
ples of mediated sensor on the basis of bacterial cells
(Gluconobacter oxydans, Pseudomonas putida, P.putida
BS3790, P.£uorescens, Bacillus subtilis) and the possibil-
ities of use active carbon as the bases of receptor elements
of sensors. A mediator electrode was constructed by ap-
plying the bacterial suspension to the surface of a syringe
whose tip was ¢lled with graphite paste of the following
composition: graphite powder Fluka No. 50870 of active
carbon powder, 1,1’-dimethylferrocene, para⁄n oil Fluka
No. 76235. The electrode volt-ampere characteristics with
1,1’-dimethylferrocene and without it in the range of
^750...+750 mV were recorded. The mediator e¡ect on
the character of electrode volt-ampere dependencies was
prominent with the appearance of a speci¢c maximum
within a potential range of 280^500 mV. The glucose
and ethanol calibration curves were plotted, indicating
that sensor responses were observed within the concentra-
tions of 0.01^10 and 0.1^10 mM for glucose and ethanol,
accordingly. The apparent Michaelis constant for glucose
and ethanol was Kgl=2.3Y0.4 and Ket=1.1Y0.1 mM. The
electrode operational stability was checked. Loss of activ-
ity (up to 50 and 25% of the initial response amplitude)
was registered at the 7-8th and 20th measurement, respec-
tively. The dependence of response amplitude on the mo-
lar concentration of potassium phosphate bu¡er was reg-
istered at pH 7.8. A change in the molar concentration
within 5^40 mM had no e¡ect on the response amplitude;
100 and 200 mM resulted in 20 and 50 % loss of the initial
response amplitude, respectively. Moreover, the viability
and high biochemical activity of immobilized microorgan-
isms on active carbon was con¢rmed, which proved the
394 1st FEMS Congress / Posters 103^505
possibility to use investigated materials as receptor ele-
ments for mediated biosensors.
P11^87
UV-RADIATION IMPACT ON MICROBIOLOGICAL
DEGRADATION OF OIL PRODUCTS
E. Karetnikova, L. Kondratyeva, V. Rapoport
Institute of Water and Ecological Problems FEB RAS,
Khabarovsk, 680000, Russia
In connection with ozone depletion UV-radiation gets the
important role as the ecological factor. In natural condi-
tions UV acts on microorganisms and on organic com-
pounds. It leads to change of character of processes of
destruction of organic compounds, including pollutants,
owing to their phototransformation and change of activity
microbocenoses. Oil and oil products is the important
class of pollutants. The intensity of microbiological degra-
dation of these compounds depends on their structure,
concentration and ability of microorganisms to growth
in various ecological conditions. The aim of the present
work is a study of peculiarities of microbiological degra-
dation of oil products (OP) after UV-irradiation. After 7
days incubation there was shown that, microbiological
degradation of OP was most intensive without UV-irradi-
ation. The rate of microbiological degradation of diesel
fuel (DF) decreased after UV-irradiation on microorgan-
isms of natural water and after phototransformation of
DF. Was shown, that the ¢lm, formed by fuel, carries
out a function ‘‘ of the absorber of UV ‘‘, and protects
bacterioplankton from UV-radiation, promoting preserva-
tion of activity of hydrocarbon-oxidizing bacteria. Thus,
the intensity and mechanisms of degradation of DF in the
interaction of photochemical and microbiological process-
es depend on a type of a combination of these factors. The
UV impact can result in decrease of rates of degradation
of oil and accumulation of some components, owing to
change of character of microbiological processes.
P11^88
MIGRATION OF LONG-LIVED RADIONUCLIDES,
INFLUENCED BY MICROORGANISMS
T. V. Khijniak, N. N. Medvedeva-Lyalikova
Institute of Microbiology RAS, 7/2 Prospect 60-letiya Ok-
tyabrya, Moscow 117312, Russia
The problem of radioactive wastes arises from experimen-
tal explosions of nuclear weapons, wastes from nuclear
fuel cycle reprocessing plants and the use of the isotopes
for medical purposes. The laboratory studies show that
FEMSLE Congress 2-6-03
during underground storage of waste 95-97% of radionu-
clides (Sr, Ru, Cs, Ce) precipitated. Remained solution
contains long-lived elements (Tc, Pu, Np, U), which can
migrate with pore water and therefore radioactive contam-
inated area enlarged. Microbial metal reduction results in
the precipitation of a low valence, reduced form of the
element, and has therefore been proposed as a strategy
to treat contaminated waters. We report that 99Tc and
237
Np were sorb by sediments of fresh water lakes.
Around 50% of 237Np was sorbed during 1 hour and
sorption completed after 1-2 months depending on type
of sediments. As for Tc, sorption was monotonic and ¢n-
ished after about 2 months. At the neutral pH, sulphate-
reducing bacteria reduce TcO4- and reduced technetium
was precipitated as TcIVO2. Under the anaerobic alkaline
conditions haloalkaliphilic bacteria Halomonas also reduce
Tc, but unlike neutral pH about 2/3 present in the highly
electronegative soluble Tc(IV) carbonate complexes. It
may be necessary to reassess current concepts of Tc trans-
port in anaerobic, carbonate enriched ground waters,
where Tc mobility has been considered to be controlled
by the low solubility of TcO2.
P11^89
DEVELOPMENT OF COMPLEX OF RADIOCHEMI-
CAL METHODS OF ANALYSIS OF POLYCHLORI-
NATED BIPHENYLS-DESTROYING ACTIVITY OF
SOIL BACTERIA STRAINS
A. A. Kim(1), G. T. Djuraeva(1), A. V. Khodiev(2), S. V.
Aleksandrov(2) and Kh. T. Yadgarov(3)
(1) Group of Biological Radiochemistry, Department of
Analysis and Informatization, Institute of Nuclear Physics,
Uzbekistan Academy of Sciences, Ulugbek, Tashkent
702132, Uzbekistan; (2) Division of Scienti¢c Develop-
ment, DIM Ltd.,Tashkent, Uzbekistan; (3) Laboratory of
Toxicological Genetics, Institute of Genetics and Experi-
mental Biology of Plants, Uzbekistan Academy of Sciences,
Uzbekistan
Polychlorinated biphenyls have a wide range of industrial
applications. In the USSR, industrial PCB mixtures were
manufactured under names Sovol and Sovtol. According
to the IUPAC nomenclature, more than 200 possible con-
geners were described while about 150 congeners have
been reported as present in the environment. Application
of tritium labelled PCBs in studies of their microbial deg-
radation permit tracing of minor quantities of the com-
pound and its degradation products in complicated sys-
tems with inexpensive methods for separation and
detection. A method for tritium labelling the complex
PCB isomer mixture of Sovol was developed. Simple, ef-
fective and inexpensive methods for screening for PCB
degradative activity of soil bacteria on the basis of tritium
 1st FEMS Congress / Posters 103^505
labelled Sovol was developed. The method of determina-
tion of PCB accumulation and degradation in soil bacteria
in culture allows determination of quantitative PCBs ac-
cumulation and degradation in bacteria during test of a
strains capability to use PCB as the sole source of carbon
and energy. The method of determination of Sovol degra-
dation by soil bacteria strains in model conditions in the
soil allows estimation of PCB-destructive activity of bac-
terial strains after introduction into the soil. Modi¢cation
of this method allows determination of PCB-destructive
activity of own soil microbiota. The collection of PCB-
degrading soil bacteria was investigated with the help of
developed method. The qualitative and quantitative pa-
rameters of 4 Bacillus strains were determined. It was
shown that all 4 strains possess ability to accumulate
and destroy PCB in both culture and soil conditions.
Thus, developed complex of radiochemical methods allows
determination of PCB-destructive bacterial activity during
primary screening, selection and testing of soil bacteria
strains.
P11^90
BENEFICIAL RHIZOBACTERIA PSEUDOMONAS
FOR THE BIOREMEDIATION OF CONTAMINATED
SOILS
V. V. Kochetkov, T. V. Siunova, O. I. Sizova, T. O. Ano-
khina, Sh. Z. Validov and A. M. Boronin
Institute of Biochemistry and Physiology of Microorgan-
isms, Russian Academy of Sciences, Pushchino, Moscow
region, 142290, Russia
Bioremediation of the contaminated soil can be improved
using combinations of plants and Plant Growth-Promot-
ing Rhizobacteria Pseudomonas (PGPRP) able to degrade
organic and (or) resistant to toxic compounds. Twenty-
two strains degrading phenanthrene, naphthalene, £uorene
and acenaphthene were isolated from the rhizosphere of
plants growing on oil-contaminated soil. Restriction anal-
ysis of 16S rRNA gene and partial 16S rRNA gene se-
quencing revealed among isolated strains Pseudomonas
spp., Sphingobacterium spp., Serratia spp. and Klebsiella
spp. Five Pseudomonas strains produce phenazine antibi-
otics and suppress the growth of phytopathogenic fungi.
These Pseudomonas strains decreased concentration of
phenanthrene in soil two or three times after 14 days in-
cubation as compared to control. Barley seed inoculation
by these strains promoted the height of plants as com-
pared with uninoculated controls in the soil with phenan-
threne. Some combinations of sorghum or sun£ower
plants with PGPRP and endomycorrhizal fungus Glomus
intraradices exhibit much promise for use in soil cleanup
from PAHs, arsenicals and heavy metals. The genetically
modi¢ed PGPRP strains acquired the naturally occurring
FEMSLE Congress 2-6-03
395
plasmids of the resistance to arsenites/arsenates, heavy
metals (Ni, Co, Zn, Cd) and degradative plasmids have
been obtained. Some of the constructed strains, which in
e¡ect were di¡erent combinations of host bacteria and
plasmids, degraded PAHs more e⁄ciently than natural
isolates. Moreover, such strains constructed on the basis
of native microorganisms using the naturally occurring
plasmids and the horizontal transfer of genetic informa-
tion inherent of living matter in nature, hardly fall under
restrictions imposed on the application of man-made mi-
croorganisms in the environment.
P11^91
DETECTION OF ANAMMOX MICROORGANISMS
FROM VARIOUS WASTEWATER TREATMENT
PLANTS BY PCR
T. Kohno(1), K. Sei(1), T. Nishino(1), K. Mori(1), and S.
Fukunaga(2)
(1) Department of Civil and Environmental Engineering,
University of Yamanashi, Kofu, Yamanashi, Japan ; (2)
Environmental and Chemical Process Department, Techni-
cal Development & Engineering Center, Ishikawajima-Har-
ima Heavy Industries Co., Ltd., Yokohama, Kanagawa, Ja-
pan
Anammox microorganisms, the novel nitrogen removing
microorganisms, produce nitrogen gas by oxidizing am-
monium with nitrite under anoxic conditions. As they
are autotrophic and nitrite is used as an electron acceptor,
ammonium can be biologically removed using less energy
in aeration and without any external carbon sources.
Anammox microorganisms are very attractive for applica-
tion in new nitrogen removal processes. However, their
distribution in natural environments or wastewater treat-
ment plants remains poorly understood. This study aimed
to detect Anammox microorganisms from various waste-
water treatment plants by PCR. Eleven activated sludge
and bio¢lm samples were collected from food processing
wastewater treatment plants, rural community sewerage,
night soil treatment plants, and leachate treatment plants
for municipal solid waste land¢ll sites. DNA was ex-
tracted by grinding, freeze-and-thaw, achromopeptidase
and SDS treatment, phenol-chloroform extraction, and
spin column puri¢cation. Anammox microorganisms
were detected by (i) conventional PCR with primer set
Pla46 and Amx820, (ii) semi-nested PCR with primer
sets Pla46 and EUBr-1387 followed by Pla46 and
Amx820, and (iii) nested PCR with primer sets Pla46
and EUBr-1387 followed by AMXN-F and AMXN-R
which we designed for Anammox microorganisms closely
related to Candidatus ‘Kuenenia stuttgartiensis’. Anam-
mox microorganisms were detected in ¢ve samples from
rural community sewerage and leachate treatment plants
396 1st FEMS Congress / Posters 103^505
for municipal solid waste land¢ll sites by conventional
PCR, and in all eleven samples by semi-nested PCR. On
the contrary, Anammox microorganisms closely related to
Candidatus ‘Kuenenia stuttgartiensis’ were only detected
in three samples from night soil treatment plants and
leachate treatment plants for municipal solid waste land¢ll
sites by nested PCR. It can be concluded that (i) Anam-
mox microorganisms were distributed in relatively various
wastewater treatment plants, though in low abundance
and (ii) Anammox microorganisms closely related to Can-
didatus ‘Kuenenia stuttgartiensis’ were less abundant than
those closely related to Candidatus ‘Brocadia anammoxi-
dans’.
P11^92
MICROBIAL COMMUNITY CAPABLE OF DEGRAD-
ING NAPHTHALENE UNDER HIGH SALT CON-
CENTRATION
I. A. Kosheleva, E. V. Akatova, O. V. Altyntseva, E. G.
Plotnikova, A. E. Filonov and A. M. Boronin
Institute of Biochemistry and Physiology of Microorganisms
RAS, Pushchino, Russia
Microbial community able to grow on naphthalene at the
presence of 15 % NaCl was isolated from high-mineralized
soil polluted by chemical wastes. It was determined that
the community consists of four microorganisms identi¢ed
as: Arthrobacter globiformis B1 and B45, the strain Brevi-
bacterium sp. nov. B4 capable of growing on naphthalene
and phenanthrene as a sole carbon and energy source at
the absence and presence (up to 7.5%) of sodium chloride,
and gram-negative halophilic strain Chromohalobacter sp.
B7 able to grow on LB medium at 3 ^ 29% NaCl but not
able to grow on naphthalene. The growth rates of Arthro-
bacter globiformis B1 and B45 on naphthalene increased
with increasing of NaCl concentration, but decreased for
Brevibacterium sp. nov. B4. By nuclear magnetic resonance
(NMR) it was shown that isolated halotolerant naphtha-
lene degrading strains were able to accumulate organic
osmoprotectants such as ectoine, betaine and threhalose
under cultivation at heightened NaCl concentrations. Hal-
ophilic strain Chromohalobacter sp. B7 stably maintain in
naphthalene degrading microbial community and able to
produce ectoine. The presence of pure ectoine or Chromo-
halobacter sp. B7 in mixed culture variously in£uence on
physiological parameters of degrader strains under di¡er-
ent salt concentration. All strains of the community were
maintained during growth on naphthalene and in£uence
on each other. It was proposed that Chromohalobacter sp.
B7 uses acetate and fatty acids produced by degrader
strains as the carbon and energy sources.
FEMSLE Congress 2-6-03
This work was supported by Russian Foundation for Ba-
sic Research, Grant N 02-04-49043, and by the U.S.
CRDF number RB2-2377-PU-02.
P11^93
‘‘ECONADIN’’ ^ A NEW BIOPREPARATION FOR
CONTINGENCY ELIMINATION OF OIL POLLU-
TION
G. A. Kozhanova, T. V. Gudzenko, V. I. Soloyov, T. A.
Belyayeva
I. I. Mechnicov Odessa National University, 2 Dvoryan-
skaya St. Odessa, 65026, Ukraine
The biopreparation ‘‘Econadin’’ (meaning ecological hope
in Ukrainian) is an immobilisation of non-pathogenic bac-
teria-destructors of Pseudomonas £uorescens on an organic
substrate (peat) according to special technology and de-
rived from the natural environment. The new generation
biopreparation has sorptive and destructive a⁄nity to oil
carbons. Russian patent No 20333975 ^ 30.04.1995, patent
of Ukraine No 43394 ^ 17.12.2001. ‘‘Econadin’’ is active
in a wide range of temperatures, buoyant, hydrophobic. It
does not lose its activity during long storage. Main advan-
tages of ‘‘Econadin’’ are rapid results, easy use and lack of
aggressive properties. It is used for cleaning the aquatic
environment of ports and coastal zone, for eliminating oil
spills and oil products on sediments, and also for thorough
¢ltering when treating industrial waste waters. The method
of using the ‘‘Econadin’’ biopreparation which block oil
pollution of the aquatic environment in the shortest pos-
sible time, prevents spreading and eliminates it with mini-
mum ecological loss. According to the method the prepa-
ration is applied to the polluted water surface as a thin
¢lm. The ¢rst sorptive e¡ect appears immediately. After
sorption of the oil products the ‘‘Econadin’’ bioprepara-
tion does not need to be collected and the destruction of
oil products occurs in natural conditions. ‘‘Econadin’’ is
produced by the Scienti¢c-Industrial Enterprise ‘‘Econad’’.
P11^94
ISOLATION, IDENTIFICATION AND CHARACTER-
IZATION OF A DENITRIFYING BACILLUS STRAIN
FROM A BIODENOX REACTOR
R. Kumaraswamy, G. Muyzer, M. C. M. Loosdrecht, and J.
G. Kuenen
Kluyver Laboratory for Biotechnology, TU-Delft, 2628 BC
Delft, The Netherlands
BioDeNOx is a process to remove nitrogen oxides (NOx)
from £ue gas at thermophilic conditions. It comprises two
 1st FEMS Congress / Posters 103^505
steps, (i) wet absorption of NOx to FeII(EDTA), and (ii)
biological reduction of NOx to N2 by denitrifying bacte-
ria. The following reaction takes place in the absorption
step, when the NOx-containing £ue gas is mixed with an
anaerobic FeII(EDTA)2- solution:
FeII(EDTA)2- + NO " FeII(EDTA)(NO)2-
The NOx absorbed is then reduced to di-nitrogen gas in a
process which uses ethanol as the electron donor:
6 FeIIEDTA(NO)2- + C2H5OH ! FeII(EDTA)2- + 3 N2 +
2 CO2 + 3 H2O
The main goal of the project is to isolate and characterize
the organism(s) responsible for NOx removal. Denitrifying
enrichments were grown with NO-2 as electron acceptor
and iron, ethanol, or acetate as electron donors. A mod-
erately thermophilic, denitrifying bacterium was isolated
from a BioDeNOx reactor running at 50‡C. The organism
is a Gram positive, spore-forming rod growing at 40 to
60‡C. Comparative sequence analysis of 16S rRNA indi-
cated a⁄liation with Bacillus azotoformans. The denitrify-
ing Bacillus used NO-2 and N2O as electron acceptors, and
ethanol or acetate as electron donors under chemoorgano-
heterotrophic conditions, while Fe2+ was used as an elec-
tron donor under mixotrophic conditions with 0.001% (w/
v) yeast extract. Molecular tools, such as PCR-DGGE and
FISH, were used to determine the dominance of this Ba-
cillus strain in the BioDeNOx process. Preliminary results
indicate the presence of this organism among others in
both laboratory and pilot scale bioreactors. Further iso-
lation and chemostat experiments will be conducted to
characterize the role of the Bacillus strain and other major
players in the BioDeNox process.
P11^95
AMMONIUM REMOVAL FROM ANAEROBIC FER-
MENTED WATER USING FUSARIUM SP. STRAIN
G8
Dz. Kungulovski and N. Atanasova-Pancevska
Institute of Biology, Faculty of Natural Sciences and Math-
ematics, P.O.Box 162, MKD-1001 Skopje, Republic of
Macedonia
The aim of this paper was to study ammonium removal
from highly loaded previously anaerobic fermented water
from pig manure, using Fusarium sp. strain G8. Three se-
ries of experiments were carried out; ¢rstly to con¢rm the
ability of our isolates of ¢lamentous fungi for heterotro-
phic nitri¢cation, secondly, to adapt our fungal isolates on
di¡erent concentration of ammonia (100, 500, 1000, 1500
mg L-1), and thirdly to compare and evaluate their indi-
vidual performances, in order of ammonia removal from
previously anaerobic fermented water from a pig manure.
In the third experiment, simultaneously, two series of
FEMSLE Congress 2-6-03
397
study were carried out, in order to compare and evaluate
individual performances of fungal isolates. In the ¢rst re-
actor the isolate Fusarium sp. strain G8 was used, as an
innocula, and in the second reactor Fusarium sp. strain
G1, G2, and G15 were used. It was found that Fusarium
sp. strain G1, G2, G8 and G15 showed the ability of
heterotrophic nitri¢cation and adaptability on high ammo-
nia concentrations. The study also showed that the e¡ect
of adding a fungal isolate Fusarium sp. strain G8 on an-
aerobic fermented water from pig manure was 95% in
reducing of N-NH4+, and 68% in reducing of COD. In
the second experiment, there was not any signi¢cant re-
ducing of COD, while the reducing of N-NH4+ was 82.5%.
P11^96
GROWTH OF AN ANAEROBIC RUMEN FUNGUS
STRAIN J3 ON VARIOUS CARBON SOURCES
Dz. Kungulovski and N. Atanasova-Pancevska
Institute of Biology, Faculty of Natural Sciences and Math-
ematics, P.O.Box 162, MKD-1001 Skopje, Republic of
Macedonia
Anaerobic fungi inhabiting the alimentary tract of most
herbivores are the primary colonizers of the plant material
ingested by host animals and they have been shown to
play a key role (alongside protozoans and bacteria) in
the degradation of ingested plant polymers. The growth
of rumen fungus strain J3 was evaluated in di¡erent car-
bon sources : glucose, maltose, cellobiose, starch and man-
ose. In this paper we report the carbon sources e¡ects on
the growth of rumen fungus strain J3. We used gas for-
mation as an indicator of growth of anaerobic fungus. The
organism had maximum on 6th day of incubation on me-
dium M10 supplemented with starch. The same organism
had maximum the ¢rst 5 days on maltose; autolysis oc-
curred in culture medium after maltose exhaustion. Strain
J3 was in stationary phase for the ¢rst 9 days on mannose,
and after that the gas formation was reduced. There were
similar growth curves obtained when strain J3 was incu-
bated on glucose and cellobiose, with the minimum reduc-
tion of gas formation. The present results demonstrate
that the J3 strain can utilize a wide range of disaccharides.
398 1st FEMS Congress / Posters 103^505
P11^97
GENOTOXICITY EVALUATION OF POLLUTED
WATERS BY COMET ASSAY ON EUCARYOTIC MI-
CROORGANISMS
B. Lah, R. Marinsek Logar and F. V. Nekrep
University of Ljubljana, Biotechnical faculty, Zootechnical
Department, Groblje 3, 1230 Domzale, Slovenia
The assessment of genotoxic potential in surface and waste
waters requires test methods among which are those that
detect DNA damage in organisms of aquatic biocenosis.
In this project the eukaryotic microorganisms were used
instead of laboratory or ¢eld animals. The comet assay or
single cell microgel electrophoresis was adapted to an
ubiquitous unicellular protozoon Tetrahymena thermophila
and yeast cells Saccharomyces cerevisiae, which are easily
and unexpensively grown in axenic laboratory cultures, to
detect DNA damage caused by complex mixtures like
waste waters. For this purpose, the original test protocol
described by Singh et al. (1988) was modi¢ed (lower con-
centrations of detergents in alkaline lysis bu¡er, reduction
of electrophoresis time). Short time exposures of immobi-
lised Tetrahymena thermophila and yeast cells to well-
known genotoxicants phenol and hydrogen peroxide led
to dose-dependent DNA damage with increasing concen-
tration of genotoxicant. Further we tested the genotoxicity
potential of the in£uent and e¥uent waters of the local
municipal waste water treatment plant. The results of both
modi¢ed protocols showed the genotoxic potential of the
in£uent samples and the reduction of genotoxicity in e¥u-
ent water. We conclude that both tests could be used as
relatively simple, sensitive and unexpensive genotoxicity
tests for polluted waters, especialy with Tetrahymena cells
having large nuclei. We intend to use other eucaryotic
microorganisms like unicellular algae for genotoxicity test-
ing in comet assay. As eucaryotic microorganisms possess
almost the same HmachineryH as animals, plants and
humans, they could be relatively successfully used instead
of animals for genotoxicity testing of waste waters, soil
and foods.
FEMSLE Congress 2-6-03
P11^98
USE OF YARROWIA LIPOLYTICA STRAINS FOR
THE TREATMENT OF OLIVE MILL WASTEWATER
R. Lanciotti(1), A. Gianotti(1), A. Paparella(2), G. Suz-
zi(2), M. E. Guerzoni(1)
(1) Dipartimento di Protezione e Valorizzazione Agroali-
mentare, Bologna University, via Fanin 46, 40127, Bologna,
Italy ; (2) Dipartimento di Scienze degli Alimenti, Univer-
sita' degli Studi di Teramo, via Spagna 1, Mosciano S’An-
gelo (Teramo), Italy
The principal aim of this work was to evaluate the ability
of di¡erent Yarrowia lipolytica strains, having di¡erent
origin, to grow in olive mill wastewater (OMW) and re-
duce its COD level. Veri¢ed the suitability of the di¡erent
strains for the required functions, the lipolytic activity as
well as the potential of these strains to produce citric acid,
and metabolise poliphenols were studied. All the strains
were able to grow in the hostile medium considered with-
out dilutions and nutrient supplementation. A great vari-
ability in the extra-cellular lipolytic activity of the strains
was observed. However, the comparison between the data
obtained in a semi-synthetic medium and in OMW sug-
gests that lipases with di¡erent speci¢city can be produced
in relation to the medium composition. Under the adopted
conditions, the reduction of the OMW COD values varied
from 1.47 and 41.22% of the initial value. Some strains
determined a signi¢cant reduction of polyphenol content
while other ones caused its apparent increase, attributable
to the production of brown pigments throughout a path-
way involving the accumulation and auto oxidation of
intermediates in tyrosine catabolism. Among the consid-
ered strains and under the adopted cultural conditions Y.
lipolytica Y9 and Y2, isolated from chilled foods, pro-
duced the highest citric acid concentrations. The results
obtained evidenced that some Y. lipolytica strains are
good candidates for the reduction of the pollution poten-
tial of OMW and for the production of enzymes and me-
tabolites such as lipase and citric acid.
 1st FEMS Congress / Posters 103^505
P11^99
GRANULATED BIOMASS OF THE MIXED MICRO-
BIAL CULTURES IN SIMULTANEOUS PROCESS
OF BIODEGRADATION WITH DENITRIFICATION
AND NITRIFICATION OF XENOBIOTICS IN PHAR-
MACEUTICAL WASTEWATER
T. Landeka Dragic›evic¤(1), V. SNoljan(2), M. Glancer-SNol-
jan(1), Z. SNmit(3), V. Matic¤(2)
(1) Faculty of Food Technology and Biotechnology, Uni-
versity of Zagreb, Pierotti Str. 6, 10 000 Zagreb, Croatia ;
(2) Eco-engineering, I. Andric¤a 76, 52 000 Porec›, Croatia ;
(3) Public Health of City Zagreb, Mirogojska 116, Zagreb,
Croatia
New European legislation on the quality of water released
into environment requires low levels of nitrogen- and
phosphorus-containing substances. Compared to chemical
quality of municipal wastewater, process wastewater, espe-
cially that from pharmaceutical industry, contains high
levels of nitrogenated substances. In addition to them,
wastewater contains very complex chemically structured
ingredients ^ xenobiotics -frequently occurring as nitri¢ca-
tion inhibitors. Their structure often contains nitrogen, to
which nitri¢cation ine⁄ciency of wastewater from phar-
maceutical industry is attributed. With granulated biomass
of the mixed microbial culture comprising nitrifying bac-
teria Nitrosomonas europaea, Nitrosococcus mobilis and
Nitrosolobus multiformis as well as denitrifying bacteria
Pseudomonas sp. and Xanthomonas sp. in a two-step pro-
cess (aerobic-anoxic) under de¢ned parameters of biodeg-
radation with denitri¢cation and nitri¢cation a high re-
moval level of nitrogen-containing substances has been
achieved from wastewater generated by the pharmaceuti-
cal industry that applies microbiological and chemical
drugs manufacture. Degradation of the ingredients from
xenobiotics has been achieved too and entire removal of
ammonium. Nitrate reduction level depended on the avail-
able carbon sources. The accomplished quality of treated
wastewater with respect to carbon-containing substances
(COD and BOD5 levels) and the ingredients with nitrogen,
complies with the e¡ective European laws.
FEMSLE Congress 2-6-03
399
P11^100
GLUCOSE AFFECTS CHROMATIC ADAPTATION
IN THE CYANOBACTERIUM CALOTHRIX SP. PCC
N. V. Lebedeva(1), L. R. Semenova(2), V. A. Boichen-
ko(3), N. A. Pronina(1), I. N. Stadnichuk(4)
(1) Institute of Plant Physiology, Russian Academy of Sci-
ences, Botanicheskaya 35, 127276 Moscow, Russia; (2)
Department of Biology, Moscow Lomonosov State Univer-
sity, 119899 Moscow, Russia; (3) Institute of Basic Bio-
logical Problems, Russian Academy of Sciences, 142290
Puschino, Moscow Region, Russia; (4) Institute of Bio-
chemistry, Russian Academy of Sciences, Leninski Prospekt
33, 117071 Moscow, Russia
Glucose signaling e¡ect on complementary chromatic
adaptation (CCA) was found in a ¢lamentous cyanobac-
terium Calothrix sp. PCC 7601. The transfer of Calothrix
from green to red light under photoautotrophic conditions
inversed the phycoerythrin (PE) to phycocyanin (PC) cell
ratio and led to a decrease of the total pigment content per
cell, including chlorophyll, phycobiliproteins and carote-
noids. In addition to the light quality action, glucose,
which is a substrate for photoheterotrophic growth of
Calothrix, a¡ected the relative amounts of PE and PC.
Under both, red and green light conditions, glucose inhib-
ited PE biosynthesis starting from the drop of cpeBA
mRNA (encodes PE apoproteins). In red light, when PC
biosynthesis was increased, glucose additionally stimulated
accumulation of this phycobiliprotein. In green light, when
PC formation was already reduced, glucose did not visibly
alter the light action on this process. The values of oppo-
site e¡ects of glucose on PE and PC contents did not
exceed 30% of their levels in chromatically adapted cells
that indicated pivotal role of light quality and modulating
role of glucose in the regulation of pigment composition of
Calothrix. The action of glucose in Calothrix was not
limited to the changes in the stoichiometry of phycobili-
proteins Simultaneously glucose diminished carotenoids
content, inhibited photosynthetic activity of PSII and al-
tered the cell morphology. Stereochemical analogue, 2-de-
oxy-D-glucose (2dDG), reproduced the e¡ects of glucose.
Basically, the glucose e¡ect coincided with e¡ect of red
light but green light and glucose acted in opposite direc-
tions. Action of glucose through known speci¢c comple-
mentary chromatic adaptation (CCA) phosphorelay path-
ways was supposed and cyanobacterial metabolism under
di¡erent light regimen is discussed.
400 1st FEMS Congress / Posters 103^505
P11^101
ANTIBIOTIC RESISTANCE MONITORING OF EN-
TEROCOCCI AND MESOPHILIC AEROMONADS
IN RIVERS GOUEºT AND LEFF (BRITTANY ^
FRANCE) : IMPACT OF ANTIBIOTIC USE IN ANI-
MAL HUSBANDRY
L. Delery(1), J. Minet(2), S. Morel(1) and J. Lesne(1)
(1) Laboratoire d’Etude et de Recherche en Environnement
et Sante¤, Ecole Nationale de la Sante¤ Publique, 35043 Ren-
nes cedex, France; (2) Laboratoire de Bacte¤riologie, Ho“pi-
tal Sud, C.H.U. de Rennes, 16 Bd de Bulgarie 35056 Rennes
cedex, France
Animal husbandry and medical treatment in hospital or in
the human community are powerful foci of antibiotic pres-
sure on intestinal bacteria. Among the imaginable routes
of communication between intestinal reservoirs of antibi-
otic resistance genes in humans and animal husbandry, the
role of riverine waters is still not well documented. Anti-
biotic resistance phenotypes of enterococci and mesophilic
aeromonads were monitored during summer 1999 in two
rivers, the Goue«t (33 and 47 isolates respectively) and the
Le¡ (58 and 75 isolates respectively) which drains larger
areas of intensive animal husbandry than the Goue«t does.
Most of the strains of enterococci were resistant to one
antibiotic at least (91% in river Goue«t, 52% in river Le¡),
and in river Le¡ multiresistant strains (3 resistances or
more) were predominant (64%). All the strains of aeromo-
nads in river Goue«t, and nearly all in river Le¡, were
sensible to all the tested antibiotics, except to f lactamins,
and thus belonged to the wild phenotype of aquatic aero-
monads. These results provide evidence for clonal spread
of resistant strains of the intestinal bacterial £ora (enter-
ococci) into the aquatic environment, especially under the
selection pressure of antibiotic use in animal husbandry.
Interestingly, resistance genes for tetracyclin and chloram-
phenicol, which are borne by mobile genetic elements, and
were frequent among the strains of enterococci in both
rivers, were also present among a few strains of aeromo-
nads, but only in river Le¡. This is consistent with the
dissemination of resistance genes by conjugational or
transformational transfer to the indigenous bacterial £ora
(aeromonads). Nevertheless, these data invalidate two
public health threats : (i) the continuous supply of resis-
tance genes to the human gut micro£ora directly by drink-
ing water via the aquatic micro£ora (i.e. aeromonads), and
(ii) the risk of cutaneous or systemic infection with anti-
biotic resistant strains of mesophilic aeromonads in au-
thorized natural bathing waters.
FEMSLE Congress 2-6-03
P11^102
EFFECT OF TETRACYCLINE ON TRANSFER OF
THE CONJUGATIVE TRANSPOSON TN916 BE-
TWEEN ENTEROCOCCUS FAECALIS CELLS IN
THE ANIMAL INTESTINE
M. I. Bahl(1,2), S. J. S\rensen(1), L. H. Hansen(1) and
T. R. Licht(2)
(1) Department of General Microbiology, University of Co-
penhagen, Denmark; (2) Danish Veterinary and Food Ad-
ministration, S\borg, Denmark
Transfer of conjugative transposons belonging to the
Tn916 family is known to be enhanced by the presence
of tetracycline. Still, a generally low frequency of transfer
impedes investigations of this e¡ect in environmental sys-
tems, as it can be di⁄cult to detect a very low number of
transconjugants in a non-sterile environment. We have
investigated the transfer of Tn916 among isogenic enter-
ococci colonizing in the intestine of gnotobiotic rats, as
this animal model allows a very low limit of detection.
The animals continuously received tetracycline in drinking
water. The concentrations of the drug were kept low
enough to allow colonization of a tetracycline-sensitive
recipient strain, before the resistant donor was introduced.
The numbers of transconjugants cultured from fecal sam-
ples were compared to the concentration of tetracycline in
drinking water. Furthermore, the bio available amount of
the drug in the intestinal environment was monitored in
situ using bacterial biosensors carrying transcriptional fu-
sions between tetracycline-regulated promoters and given
marker genes.
P11^103
ISOLATION AND CHARACTERISATION OF NOVEL
HALOTOLERANT IRON-OXIDISING ACIDOPHILES
FROM THE MARINE ENVIRONMENT
P. E. Linton and D. J. Jamieson
School of Life Sciences, Heriot-Watt University, Riccarton
Campus, Edinburgh, EH14 4AS, Scotland, United Kingdom
The occurrence of chemoautotrophic, acidophilic bacteria
in the marine environment has been widely noted and they
have been implicated in the biogeochemical cycling of iron
and biodeterioration of iron-containing structures in the
oceans. However, the isolation, molecular ecology, growth
pro¢les and physiological responses of these bacteria at
elevated salt levels have rarely been described, despite
widespread interest in their unique metabolic capacity
and potential application in the extraction of metals via
bioleaching of salt contaminated ores. In this study, three
 1st FEMS Congress / Posters 103^505
novel strains of halotolerant gram-positive, rod shaped,
acidophilic bacteria were isolated from estuarine and
coastal areas. Enrichment cultures were set up using pyrite
medium of di¡erent salinities with sediment and seawater
samples from a variety of metal contaminated areas ex-
posed to the sea or brackish water. These enrichment cul-
tures were then further puri¢ed using end-point dilution
culture methods. The growth characteristics, morphology
and growth pro¢les on metaliferrous ore samples of the
strains were characterised and the 16S rRNA genes were
sequenced and phylogeny assessed. The strains exhibit au-
totrophic growth on a variety of iron and sulfur com-
pounds, heterotrophic growth on yeast extract medium
as well as mixotrophic growth on a combination of these
substrates. The strains grow optimally at 30gl-1 sea salts,
in medium with a pH of 2 and at 37‡C. The growth char-
acteristics displayed by these strains highlight their poten-
tial application in high salinity bioleaching operations.
P11^104
BIODEGRADATIVE POTENTIAL OF ANTARCTIC
MARINE BACTERIA GROWN ON XENOBIOTIC
COMPOUNDS : DIESEL OIL AND POLYCHLORI-
NATED BIPHENYLS (PCBs)
A. Lo Giudice, L. Michaud, A. Allegra, V. Bruni
Dipartimento di Biologia Animale ed Ecologia Marina, Uni-
versita' di Messina, Salita Sperone 31, 98166 Messina, Italy
Biodegradation is the major natural mechanism in the re-
moval of pollutants from marine environments. The pur-
pose of this work was to assess the biodegradative poten-
tial of 126 psychrotrophic bacterial strains isolated from
seawater samples collected along the water column in a
sampling station located in Terra Nova Bay (Ross Sea,
Antarctica). Antarctic isolates were cultured at 4‡C and
20‡C up to a month into a mineral liquid medium con-
taining diesel oil or polychlorinated biphenyls (PCBs) at a
¢nal concentration of 1% (v/v) and 0.1% (v/v) respectively,
as the sole source of carbon and energy. Following the
incubation period, the strains showing a more evident ac-
tivity were selected and their biodegradation potential was
evaluated by quantitative gas-chromatographic analysis.
On the basis of the gas-chromatographic results, the bio-
degradation of both diesel oil and PCBs was higher at
20‡C than at 4‡C, as expected for psychrotrophic bacteria.
Diesel oil was strongly degraded by 7 and 40 bacterial
strains at 4‡C and 20‡C, respectively. Only two isolates
were able to utilize the substrate at both temperatures.
The percentage of diesel oil degradation ranged from
53.6% to 73.40% at 4‡C and from 77.88% and 99.60% at
20‡C. The utilization of PCBs was exclusively observed at
20‡C in 11 isolates whose diesel oil degradation was weak
or absent. The PCBs biodegradation ranged from 16.3%
FEMSLE Congress 2-6-03
401
to 73%. Our results con¢rm the possibility to utilize psy-
chrotrophic bacteria as xenobiotic degraders in the control
of marine pollution both in cold and temperate environ-
ments.
P11^105
INVESTIGATION OF BACTERIA INCLUDED IN NI-
TROGEN CYCLE IN THE WATER OF LAKE OHRID
AND ITS TRIBUTARIES
L. Lokoska
Hydrobiological Institute, Naum Ohridski 50, 6000 Ohrid,
Macedonia
The aim of this study has been to examine the amount of
bacteria included in nitrogen cycle (nitro¢xators, amoni¢-
cators, nitri¢cators and denitri¢cators) in the water of lake
Ohrid and its tributaries. The number of nitrogen-¢xing
bacteria, and amoni¢katros was determined by the selec-
tive method of plates, and the amount of nitrite-produc-
ing, nitrate-producing and denitryfying bacteria was deter-
mined by suitable liquid stock and were calculated
according Mac Credys statistic tables. Received results
support the well-known phenomenon that cooperation of
the biotic and abiotic factors of in the environment that
impact the life, dynamics and distribution of the micro-
organisms in the water. These results con¢rm that the bio-
logical productivity in the aqatorya is highly connected
with the rule of the bacterioplankton in the cycling of
the nutrients from nitrogen nature, from autohtonous
and allohtonous origin. Investigated groups of bacteria
are more present in the tributaries, with a maximum in
River Velgoska: nitrogen-¢xing bacteria according 3,024
bact. ml-1, urolitic 256,000 bact. ml-1, nitrite-producing
140 bact. ml-1, nitrate-producing bacteria 1,600 bact. ml-
1
and denitryfying 9,000 bact. ml-1. In the littoral region
most abundant were in the front of in£ow of River Vel-
goska : nitrogen-¢xing bacteria 1,020 bact. ml-1, urolitic
10,580 bact. ml-1, nitrite-producing 95 bact. ml-1, nitrate-
producing bacteria 400 bact. ml-1 and denitryfying 2,000
bact. ml-1. In the pelagic region maximal bacterial value
were: nitrogen-¢xing bacteria 89 bact. ml-1, urolitic 880
bact. ml-1, nitrite-producing 45 bact. ml-1, nitrate-produc-
ing bacteria (under 200) and denitryfying 150 bact. ml-1
are relatively lower.
402 1st FEMS Congress / Posters 103^505
P11^106
rRNA-BASED STABLE ISOTOPE PROBING OF
METHYLOTROPHIC BACTERIA AND FUNGI IN
SOIL UNDER LOW SUBSTRATE CONCENTRA-
TIONS
T. Lueders and M. W. Friedrich
Max-Planck-Institute for Terrestrial Microbiology, Karl-
von-Frisch Str., 35043 Marburg, Germany
Stable isotope probing (SIP) is currently one of the most
promising methods to bridge the gap between structure
and in-situ function of microbial populations in natural
habitats. We have enhanced the sensitivity of SIP by ap-
plying rRNA-based SIP to a 13C-methanol consuming
aerobic soil ecosystem incubated at very low methanol
concentrations of V200 WM. Isotopically labelled rRNA
was separated by density gradient centrifugation. Gradient
fractions were analysed by quantitative Real-Time PCR
for rRNA distribution, and by ¢ngerprinting and cloning
of SSU rRNA. Already after 6 days of incubation, the
appearance of 13C-labeled rRNA of methylotrophic bac-
teria was observed. At this early timepoint, Methylobacte-
rium-related SSU rRNA dominated in the labelled frac-
tions. After 13 days, labelling of rRNA increased, and a
shift within the heavy rRNA towards a dominance of
Methylobacillus-related organisms was observed. DNA-
based SIP conducted after a long-term incubation of 45
days revealed almost exclusively Methylobacillus-related
rDNA in the 13C-labeled fractions. Surprisingly, we also
detected fungal rRNA and rDNA in 13C-labelled gradient
fractions. SSU rRNA clones from these fractions were
related to Aspergillus- and Fusarium spp., indicating that
these fungi also contribute to the consumption of metha-
nol in soil, which has never been described before. These
results emphasize the potential of SIP, and represent the
¢rst results of rRNA-based SIP in a soil ecosystem. At the
same time, drawbacks of DNA-based SIP such as selective
enrichment of certain community members by long incu-
bation times are illustrated and overcome by the increased
sensitivity of rRNA-based SIP.
FEMSLE Congress 2-6-03
P11^107
MONITORING PHYSIOLOGICAL STATUS OF SPE-
CIFIC BACTERIAL POPULATIONS IN SOIL BY
FLOW CYTOMETRY
N. Maraha(1,2), A. Backman(1,2) and J. K. Jansson(1,3)
(1) Section for Natural Sciences, So«derto«rn University Col-
lege, 141 89 Huddinge, Sweden; (2) Department of Labo-
ratory Medicine, Karolinska Institute, Division of Clinical
Bacteriology F-82, Huddinge University Hospital, 141 86
Stockholm, Sweden ; (3) Department of Microbiology,
Swedish University of Agricultural Sciences, P.O. Box
7025, SE-75007 Uppsala, Sweden
The physiological status of speci¢c bacterial populations
in pure cultures and in soil was studied using £ow cyto-
metry in combination with di¡erent viability stains. One
gram- negative plant growth-promoting strain, Pseudomo-
nas £uorescens SBW25, and one gram-positive 4-chloro-
phenol-degrading strain, Arthrobacter chlorophenolicus
A6, were chromosomally tagged with the gfp gene, encod-
ing green £uorescent protein, and inoculated into non-ster-
ile soil. At speci¢ed periods, the soil bacterial community
was extracted using Nycodenz density gradients and either
CTC (cyano-ditolyl-tetrazoliumchloride) or PI (propidium
iodide) were added to stain viable or dead cells, respec-
tively. The total number of gfp-tagged cells were quanti-
¢ed by £ow cytometry. Furthermore, the proportion of
dead and viable cells within the gfp-tagged populations
were enumerated by gating gfp-tagged cells that also £uo-
resced red (due to the stains) by £ow cytometry. In addi-
tion, the number of culturable cells was determined by
selective plate counting and the CFU values were com-
pared to the results obtained by £ow cytometry to esti-
mate the number of viable but non-culturable cells .The
physiology of the populations was also studied in pure
cultures incubated in 1xPBS, 10%LB or LB. The A6 cell
population remained viable longer in low nutrient condi-
tions compared to rich nutrient conditions, opposed to
SBW25 cell populations. In soil, CFU-values of both
strains decreased while the total number of gfp-tagged cells
remained constant. Initially numbers of CTC-stained cells
for both strains decreased in soil while numbers of PI-
stained cells increased indicating a physiological adapta-
tion after inoculation into soil. After a short period both
populations experienced slow growth. Experiments using
viability stains under starvation conditions indicated that
the cells soon entered a dormant state. This ‘‘dormant’’
state was also detected for both populations in soil. These
results should be applicable to biotechnology companies
interested in monitoring the performance of microbial in-
oculants in the environment.
 1st FEMS Congress / Posters 103^505
P11^108
PREDATORY BACTERIA, BDELLOVIBRIO : SUR-
VIVAL STRATEGY AND POTENTIAL FOR USE
N. Y. Markelova
Institute of Basic Biological Problems, Russian Acad. Sci.,
Pushchino, Russia
Bacteria of the genus Bdellovibrio are predators, incapable
of axenic growth. Bdellovibrios play a main role into con-
trolling bacterial balance in nature, their life cycle includes
two phases: a motile free-living attack phase and growth
phase occurred in the periplasmic space of other bacteria
(bdelloplasts). The increasing level of ecologically hazard-
ous compounds in the environment has stimulated re-
search on their e¡ect on the activity of bdellovibrios.
The objective of this study was to evaluate the survival
ability cells of Bdellovibrio bacteriovorus 100 NCID 9529,
when environmentally common pollutants, such as urea
and phenol were added. Comparative evaluation of the
e¡ect of pollutants was followed in liquid cell-suspension
and in bio¢lm. In liquid free-living bdellovibrios were es-
timated by double-layer plaque-forming technique, in bio-
¢lm by examination under £uorescence microscope after
acridine-orange staining. Bio¢lm was produced by immo-
bilization of predator-prey system on transparent solid
surfaces. The results obtained showed that attachment of
Bdellovibrio to surfaces enhanced survival and indicated
that adhesive bdelloplasts are the primary modes of Bdel-
lovibrio-survival, protecting the bacteria in unfavorable
conditions. These ¢ndings support recent assumption of
surface-associated state of bdellovibrios in nature as a
survival strategy and contribute to a better understanding
of the role of these bacteria in the nature. Results may be
bene¢cial in development of the environmentally sound
biotechnology for water decontamination as well as bio-
terrorism preparedness.
P11^109
MICROBIAL COMMUNITY AND BIOLEACHING OF
HEAVY METALS OF WASTEWATER SLUDGE
L. S. Markosyan(1), N. S. Vardanyan(1), A. Kh. Paron-
yan(1), V. G. Nikoghosyan(1) and A. Delalio(2)
(1) Institute of Microbiology, NAS RA, Abovian City,
378510, Armenia; (2) Ecometal RD, Via Unione 11, San
Paolo, 25020, Italy
The sludge production in Europe is more than 9 million
ton dry sludge (tds) annually, which can be increased to
10.1 million tds annually by 2005. At present, the general
aim is to use the sludge in agriculture. The investigations
FEMSLE Congress 2-6-03
403
on microbial community, quantity of heavy metals and
their bioleaching in WWS from Armenia and Austria
have been carried out. The existence of various heavy
metals: As, Cd, Cu, Hg, Mg, Ca, Co, Pb, Sn, Zn, Fe,
Mn, Al in WWS in amount prevailing over the permissible
quantities to use as fertilizer have been shown. The exis-
tence of pathogens as well as di¡erent forms of other
microorganisms in sludge are very important not only
from hygienic point of view, but also for soil-fertility
and metabolic processes in the soils. The composition
and content of the existed pathogenic microorganisms of
di¡erent genera have been investigated. The results show
the existence of some pathogen microorganisms in WWS.
The studied WWS contains also chemolitotrophic bacteria
Tiobacillus ferrooxidans, Leptospirillium ferrooxidans, Tio-
bacillus thiooxidans, Sulfobacillus thermosulfooxidans, Tio-
bacillus thioparus, Thiobacillus denitri¢cans and sulfate-re-
ducing bacteria, which is important for bioleaching of
heavy metals. Some strains of phototrophic bacteria, be-
longing to the Rhodopseudomonas, Rhodobacter and Rho-
dospirillum genera and ammoni¢cators, denitrifying, nitri-
fying, nitrogen-¢xing also observed. The physiological and
biochemical properties of chemolitotrophic iron-oxidizing
bacteria: Acidithiobacillus ferrooxidans, Leptospirillum fer-
rooxidans, sulfo-oxidizing bacteria and bioleaching of
heavy metals in WWS have been studied. The results
show that, sulfo-oxidizing bacteria in contrast to iron-ox-
idizing are shown to cause intensive bioleaching in WWS.
P11^110
MICROBIAL BIODIVERSITY CONTAMINATING
MOTION PICTURE FILM STOCKS
A. Mart|¤n-Gonza¤lez, C. Abrusci and J. C. Gutie¤rrez
Dpto. Microbiolog|¤a-III. Facultad de Biolog|¤a. C/. Jose¤ An-
tonio Novais, 2 Universidad Complutense (UCM), 28040
Madrid, Spain
Motion picture ¢lms are made up of a cellulose triacetate
or cellulose nitrate base layer, coated by one or more
successive light-sensitive photographic emulsion layers.
Gelatine, silver salts and other chemical products compose
this emulsion. The main components of motion picture
¢lms (cellulose triacetate, cellulose nitrate, gelatine) may
be contaminated and degraded by di¡erent microorgan-
isms. From 17 samples of motion picture ¢lm stocks
(Spanish archives), we have isolated 32 microbial
strains ;18 ¢lamentous fungi strains, mainly included in
the genera Penicillium and Aspergillus, but also we have
isolated members of other genera, such as; Torula, Tricho-
derma and Alternaria. Among bacteria (a total of 14 iso-
lates), 4 strains of Gram-positive cocci , included in Micro-
coccus and Staphylococcus genera. Both Gram-positive
(Bacillus spp) and negative bacilli were also isolated
404 1st FEMS Congress / Posters 103^505
from this material. One member of the genus Streptomyces
was also detected in one sample. Ten of the isolated bac-
terial strains can hydrolyse gelatine and, therefore, they
can degrade the photographic emulsion. It has been con-
¢rmed by using di¡erent gelatine media and photographic
emulsions. The deteriorating capacity of these microbial
strains is analysed and discussed. Motion picture ¢lms
are quite susceptible material to undergo contamination
by both, prokaryotic and eukaryotic microorganisms. In
our opinion, to study and prevent the microbial contam-
ination of this type of materials is very important, because
they are really a valuable part of the historical and cul-
tural patrimony of any country.
This work was supported by Filmoteca Espa•ola (project
242-2002).
P11^111
PECTINOLYTIC BACTERIA INVOLVED IN THE
WATER RETTING PROCESS
A. Gordillo Leo¤n(1), E. Tamburini(2), B. Perito(1) and G.
Mastromei(1)
(1) Department of Animal Biology and Genetics ‘‘Leo Par-
di’’, University of Florence, via Romana 17, 50125 Florence,
Italy ; (2) Dipartimento di Biologia Sperimentale, Sezione
di Microbiologia, University of Cagliari, Cittadella Univer-
sitaria ss 554, 09042 Monserrato (ca), Italy
In the last years there has been a renewed interest in hemp
¢bre production. A major limitation to an e⁄cient and
high quality ¢bre production is the retting process.
Hemp retting involves immersion of the stalks in water
and it depends on production of pectic enzymes by a bac-
terial £ora, which develops during the process. Analysis of
the biodiversity of this micro£ora is important to improve
¢bre quality and to reduce production costs. Little is
known about the pectinolytic bacteria involved in water
retting and their enzymatic properties. We performed the
¢rst genotypic characterisation of the retting micro£ora
using the ardra method. Cultivable mesophilic anaerobic
and aerobic bacteria were isolated from unretted and ret-
ted material. A total of 104 anaerobic and 23 aerobic
pectinolytic strains were identi¢ed. Polygalacturonase ac-
tivity, the primary retting enzyme, was measured in the
supernatant of cell cultures; 24 anaerobic and 9 aerobic
isolates showed an enzymatic activity higher than the
reference strains. Anaerobic isolates were divided into
¢ve ardra groups and the aerobic isolates into three
groups. Partial 16S rRNA gene sequence was determined
for twelve strains, representative of each group. All anaer-
obic strains were assigned to the Clostridium genus, where-
as the aerobic isolates were assigned either to the Bacillus
or to the Paenibacillus genus. Anaerobic isolates with high
PG activity belong to two distinct phylogenetic clusters
FEMSLE Congress 2-6-03
related to C. acetobutylicum ^ C. felsineum and C. saccha-
robutylicum species. Aerobic isolates with high PG activity
belong to two distinct phylogenetic clusters related to B.
subtilisT and B. pumilusT.
P11^112
BIOLUMINESCENT BIOASSAYS BASED ON LUMI-
NOUS BACTERIA MARKER SYSTEM
S. E. Medvedeva, A. M. Kuznetsov, E. K. Rodicheva, N. A.
Tyulkova
Institute of Biophysics SB RAS, 660036, Krasnoyarsk, Rus-
sian Federation
High sensitivity of the luminescent system of luminous
bacteria to microquantities of various substances facili-
tates their employment as integral toxicity bioassay. Sim-
ple to measure luminescence, rapidity of the method, fea-
sibility of automating measurements and statistical
processing of data makes the luminous bacteria advanta-
geous over other biological assays. In Culture Collection
IBSO the basic integral bioassays are developed to analyze
the quality of di¡erent chemical solutions, natural waters
and e¥uents. They used the lyophilized bacteria with
marker lux-gene: Microbiosensor-B17-677F (based on ma-
rine luminous bacteria Photobacterium phosphoreum) and
Microbiosensor-ECK (based on Escherichia coli Z905 with
lux-gene) as well as the luminescent system isolated from
luminous bacteria. It takes not more than 30 min to do the
biotest (the other biotests take 48-96 h). The general char-
acteristics of biotests were studied to elucidate possibilities
of using in bioluminescent analysis. The analysis of model
toxic agents, PMW e¥uent waters and surface waters of
some region of Siberia was carried out. It was shown that
both bioluminescent Microbiosensors can be used to ana-
lyze the quality of natural waters and e¥uents of indus-
trial enterprises. A kit of reagents for analytical biolumi-
nescence (KRAB) based on the coupled enzyme system:
luciferase ^ NAD(P)H:FMN-oxidoreductase also can be
used in ecological monitoring and medicine.
This work was supported by the Russian Foundation for
Basic Research (grant 00-07-90111) and grant REC-002 on
program for Basic Research and Higher Education from
CRDF and the RF Ministry of General and Professional
Education.
 1st FEMS Congress / Posters 103^505
P11^113
POTENTIAL USE OF SPHINGOMONAS HERBICI-
DOVORANS MH TO DEGRADE PHENOXYALKA-
NOIC ACID HERBICIDES IN SOIL AND ANALYSIS
OF MICROBIAL COMMUNITY STRUCTURE
CHANGES FOLLOWING ITS INOCULATION
C. Meier, J. R. van der Meer
Swiss Federal Institute for Environmental Science and Tech-
nology, EAWAG, CH-8600 Du«bendorf, Switzerland
Bioremediation is an increasingly important technology
for the clean-up of contaminated areas, especially as it
may have little impact on soil compared to chemical or
physical methods. Usually, the capacity of indigenous bac-
teria is stimulated in order to achieve biodegradation.
However, speci¢c strains may also be inoculated to de-
grade target pollutants. Phenoxyalkanoic acids are com-
monly used weed killers. Despite their biodegradability,
residues are often found in ground- and surface water.
In this study we are focusing on using Sphingomonas her-
bicidovorans MH for degradation of the chiral pesticide 2-
(4-chloro-2-methylphenoxy)propanoic acid (mecoprop). S.
herbicidovorans MH can also degrade several other phe-
noxyalkanoic acid herbicides such as 2,4-dichlorophenoxy-
acetic acid (2,4-D) or 2-(2,4-dichlorophenoxy)propanoic
acid (dichlorprop). These features make this strain a useful
candidate for bioaugmentation. The possible e¡ects of the
introduction of Sphingomonas herbicidovorans MH strain
on the structure and function of the indigenous microbial
community were investigated. For this, Terminal-Restric-
tion Fragment Length Polymorphism (T-RFLP) was used.
T-RFLP analyses changes in the diversity of microbial
communities. The method is based on the isolation of total
DNA from soil samples followed by the PCR ampli¢ca-
tion of target genes. PCR ampli¢cation is performed with
£uorescently labeled primers. By digesting the end-labeled
PCR products DNA fragments with speci¢c lengths are
obtained. In our work we mainly used a conserved region
of the 16S rDNA as main target for diversity analysis.
Furthermore, speci¢c primers for certain groups of bacte-
ria were used to get a more detailed insight on the diver-
sity of the microbial soil community. Fingerprints were
compared between uninoculated soil and soils inoculated
with di¡erent amounts of Sphingomonas herbicidovorans
MH. The natural variation occurring in ¢ngerprints was
assessed by comparing di¡erent soil samples from the
same area collected over a period of one year.
FEMSLE Congress 2-6-03
405
P11^114
LIPOLYTIC BACTERIA FROM ANTARCTIC SEA-
SURFACE MICROLAYER: PHYSIOLOGICAL AND
MOLECULAR CHARACTERIZATION
L. Michaud, A. Lo Giudice, V. Bruni
Department of Animal Biology and Marine Ecology, Uni-
versity of Messina, Salita Sperone, 98166 Messina, Italy
In the marine environment a crucial role is played by the
surface microlayer representing the site of the energy and
matter transfer between the sea and the atmosphere. Many
bio-physico-chemical processes take place in this zone and
are involved in photochemical and biologically mediated
transformations of organic compounds. The aim of this
work was to investigate the lipolytic potential of Antarctic
bacteria inhabiting this transition environment. Twenty-
nine psychrotrophic bacterial strains, isolated from sea-
surface microlayer at Terra Nova Bay (Antarctica), were
screened for their ability to degrade di¡erent lipidic sub-
strates. Bacterial strains were grown at 4, 15 and 30‡C for
21 days on a basal medium supplemented with Tween 20,
40, 60, 80, 85, Triolein and Tributyrin. Lipolytic activity
was observed in twenty-three strains. Data obtained
showed that, excepted for Tween 40, the optimal temper-
ature for the utilization of several substrates was 15‡C.
Tween 85 and Tributyrin were not degraded at 4‡C.
Tween 20 was utilized by the majority of the isolates
(79%). Based on the results from the preliminary screen-
ing, the strains showing higher hydrolytic activity were
selected in order to study the individual and interactive
e¡ects of temperature, NaCl and pH on the lipolysis.
The only two strains able to use all substrates at, at least,
two incubation temperatures and particularly active in a
wide range of NaCl and pH, were ¢nally characterized at
phenotypical and molecular level. Further studies will be
undertaken for screening these strains for the hydrocar-
buroclastic activity on di¡erent n-alkanes and diesel oil.
P11^115
MICROBIAL LEACHING OF GOLD MINE PYRITE
BY THIOBACILLUS FERROOXIDANS
M. Mohseni
Department of Biology, Faculty of Sciences, University of
Mazandaran
Gold recovery from refractory ores, directly, has a low
yield and needs pretreatment. One of the best and useful
methods of pretreatment of refractory gold ores uses mi-
crobial leaching technology. In this process, microorgan-
isms with degradation of interferer minerals cause releas-
406 1st FEMS Congress / Posters 103^505
ing of gold. One of the most important microorganisms in
microbial leaching process is Thiobacillus ferrooxidans.
M4, M5 and M7, M8 strains isolated from Zirab coal
mine and Ramsar sulfuric stream, respectively, were used
in this research. The concentrated pyrite with sub 100 Wm
diameter was used in the microbial leaching process. After
an incubation period of 20 days at 30‡C, the percentage of
removed pyrite and the rate of microbial leaching were
determined. The results showed that isolated strains were
highly e¡ective in the removal of pyrite. The microbial
oxidation of pyrite has been a mixture of the direct and
indirect oxidation. Among of all strains that examined the
M8 strain was the most e¡ective agent in the removal of
pyrite, with a maximum of 93.6% pyrite removal in the
culture medium. With an increase of pulp density (pyrite),
the percentage of pyrite removal decreased, but the leach-
ing rate increased. The best rate determined in pyrite
leaching was that of the M7 strain, being about 22.4 mg/
l/h.
P11^116
METABOLISM OF CHIRAL PHENOXYALKANOIC
ACID HERBICIDES IN SPHINGOMONAS HERBICI-
DOVORANS MH
T. A. Mu«ller, S. M. Byrde, J. R. van der Meer, H.-P. E.
Kohler
EAWAG (Swiss Federal Institute of Environmental Science
and Technology), Du«bendorf, Switzerland
Sphingomonas herbicidovorans MH grows on the chiral
phenoxyalkanoic acid herbicides mecoprop and dichlor-
prop. The initial degradation step to the 4-chloro-2-methyl
phenol and 2,4-dichlorophenol is enantioselective and cat-
alyzed by two di¡erent K-ketoglutarate dependent dioxy-
genases. In S. herbicidovorans MH, further metabolism has
not been elucidated yet, but we hypothesized that it pro-
ceeds via the respective catechol and through the modi¢ed
ortho cleavage pathway. A part of the (R)-mecoprop di-
oxygenase was ampli¢ed by PCR and the whole gene (des-
ignated rdpA) was isolated and sequenced. The predicted
protein sequence showed 100% identity to RdpA from
Delftia acidovorans MC1, another dichlorprop degrader.
Homology to other K-ketoglutarate dependent dioxyge-
nases such as TfdA from Ralstonia eutropha JMP134
(pJP4) was about 30%. Enzyme activity measurements
con¢rmed the predicted enantioselectivity as only the
(R)-enantiomers were converted. Hybridization experi-
ments with rdpA under non-stringent conditions indicated
the presence of a homologous gene. The isolated gene
showed about 60% homology to sdpA from D. acidovorans
MC1 and 30% to other tfdA-like genes including rdpA. In
addition, two di¡erent chlorocatechol 1,2-dioxygenase
genes, designated clcAI and clcAII, were isolated. They
FEMSLE Congress 2-6-03
were 95% identical and showed about 50% homology to
other chlorocatechol 1,2-dioxygenases and 25-30% to cat-
echol 1,2-dioxygenases. The analysis of sequence align-
ments gave strong indication that ClcAI/II of S. herbicido-
vorans MH might belong to a new class of chlorocatechol
1,2-dioxygenases. Analysis of sequence homology and en-
zyme activity experiments provided evidence for the in-
volvement of a chlorocatechol 1,2-dioxygenase in the deg-
radation pathway of chloro- and methylcatechols in S.
herbicidovorans MH.
P11^117
DEGRADATION OF NITROAROMATIC COM-
POUNDS BY MEMBERS OF THE GENUS RHODO-
COCCUS
J. Navratilova(1), L. Kotouckova(1), M. Nemec(1), E.
Durnova(2), I. Sedlacek(3), J. Neca(4)
(1) Department of Microbiology, Faculty of Science, Ma-
saryk University Brno, Czech Republic ; (2) Department of
Bacteriology, Regional Institute of Hygiene, Ostrava, Czech
Republic; (3) Czech Collection of Microorganisms, Faculty
of Science, Masaryk University Brno, Czech Republic; (4)
Veterinary Research Institute, Brno, Czech Republic
Nitroaromatic compounds are widely distributed in the
environment. They are used as drugs, herbicides, pesti-
cides, explosives, dyes and solvents and can be found as
contaminants in waste waters, rivers and herbicide- or
pesticide-treated soils. Nitroaromatics and products of
their complete degradation have relatively high acute tox-
icity and some may be carcinogenic. A few microorgan-
isms are able to degrade these pollutants. Rhodococci,
which are widely found in soil and sludge, have been
shown to attack various xenobiotic compounds, including
nitroaromatics. We have isolated 4 bacterial strains from
soil by selective enrichment. Two strains (J2J and J3) were
isolated by enrichment with 4-nitrocatechol and two
strains (J6 and J7) by enrichment with 4-nitroguaiacol.
These strains were identi¢ed as members of the genus
Rhodococcus by a polyphasic taxonomic approach (mor-
phology, biochemical and physiological characterisation,
chemotaxonomic analysis, determination of partial se-
quence of genes for 16S rRNA). The nearest relatives
were R. opacus and R. percolatus. The strains were tested
for growth on a variety of nitroaromatic compounds in-
cluding 4-nitrocatechol (4-NC), 4-nitroguaiacol (4-NG), 5-
nitroguaiacol (5-NG), 2-nitrophenol (2-NP), 3-nitrophenol
(3-NP), 4-nitrophenol (4-NP), 2,4-dinitrobenzoic acid (2,4-
DNBA), 4,5-dimethoxy-2-nitrobenzoic acid (4,5-dime-
thoxy-2 NBA) and 2,3-diphluoro-6-nitrophenol (2,3-diP-6
NP). Established degradations were studied further. Dif-
ferences were seen in the degradation capability of strains
but none were able to degrade 2,4-DNBA, 4,5-dimethoxy-
 1st FEMS Congress / Posters 103^505
2 NBA and 2,3-diP-6 NP. Strain J3 was able to degrade 4-
NC, 5-NG and 3-NP. Strain J6 has the broadest degrada-
tion spectrum, being able to degrade 4-NC, 4-NG, 5-NG,
3-NP and 4-NP (all substrates at 0.05 mM). Degradation
of some nitroaromatic compounds (4-nitrocatechol, 4-ni-
troguaiacol and 4-nitrophenol) was monitored using the
automatic microbiological system bioscreen c. Degrada-
tion of these compounds was tested in the concentration
range 0.025 to 0.1 mM. Degradations of nitroaromatic
compounds were con¢rmed by HPLC.
P11^118
MICROBIAL MONITORING IN SPACECRAFT ENVI-
RONMENT
N. D. Novikova, N. A. Polikarpov, S. V. Poddubko and E.
A. Deshevaya
The RF SRC ^ Institute for Biomedical Problems of the
RAS, 76 A, Khoroshevskoye sh., 123007, Moscow, Russia
The Russian experience with long-operating spacecraft in-
dicates that the ecological problems of crew safety and
onboard hardware reliability become more serious as the
period of mission increases. Among them the microbial
factor is of particular implications. The base unit of the
orbital station (OS) MIR was launched on February 20,
1986, and on March 13 the ¢rst crew arrived on it. During
of the next 15 years the work on investigation of peculiar
evolution of micro£ora of OS MIR was being conducted:
regularly, when the changing crews returned back to
Earth, they brought microbiological samples with them;
the in-£ight bacteria strains and microscopic fungi were
isolated, identi¢ed and subject to the detailed research.
A total of 250 species of bacteria and fungi were found
onboard orbital station MIR, among which microorgan-
isms capable of resident colonization of the environment of
space objects as a unique anthropotechnological niche
were revealed. In such conditions the evolution of micro-
£ora is followed by the rise of medical and technical risks
that can a¡ect both sanitary-microbiological conditions of
the environment and the safety and reliability character-
istics of space equipment. The latter is caused by progress-
ing biological damage to the structural materials. Dynam-
ics of microbial loading does not have linearly progressing
character, but it is a wavy process of alternation of the
micro£ora activation and stabilization phases, on this
background there is a change of the species dominating
by quantity and prevalence.
FEMSLE Congress 2-6-03
407
P11^119
ORGANIC WASTE RESIDUES AFFECT THE ACTIV-
ITY OF AMMONIA OXIDISING BACTERIA IN SOIL
BUT NOT THE COMMUNITY STRUCTURE
Y . Jarvis and A. Schnu«rer
K. Nyberg, S. Hallin, I. Sundh, A
Department of Microbiology, Swedish University of Agri-
cultural Sciences, P.O. Box 7025, SE-750 07 Uppsala, Swe-
den
Autotrophic ammonia oxidising bacteria (AOB) consist of
a few specialised species and are often used as indicator
organisms of environmental disturbances. In this study,
the impact of organic waste residues, such as anaerobically
digested organic household waste, composted organic
household waste, swine manure and cow manure, on
AOB in soil was investigated. Soil, to with organic extracts
of the residues were added, were incubated during a three
month period under controlled conditions. The communi-
ty structure of AOB in the soil samples was determined
through denaturing gradient gel electrophoresis (DGGE)
followed by sequencing of 16S rDNA fragments, and the
potential ammonia oxidation (PAO) activity was mea-
sured. The di¡erent additions gave rise to variations in
PAO activity; the composted residue stimulated the activ-
ity whereas anaerobic digestion residue and swine manure
caused inhibitions. The DGGE band patterns, however,
were similar regardless of the di¡erences in activity and
the sequencings revealed the presence of the same AOB
species. Consequently, the organic compounds in the res-
idues a¡ected the AOB on a physiological level rather than
by causing community shifts. AOB community structure
may be more dependent on inherent soil properties than
on factors in added residues.
P11^120
SELECTION OF SOIL-BORNE PGPR BACTERIA AF-
FECTED BY HEAVY METAL COMPOUNDS
B. Oldal(1), I. Jevcsak(2) and M. Kecskes(2)
(1) Research Institute for Soil Science and Agricultural
Chemistry of the Hungarian Academy of Sciences, Depart-
ment of Soil Biology and Soil Biochemistry, Herman Otto
ut 15. MTA TAKI, 1022 Budapest, Hungary ; (2) Szent
Istvan University, Agricultural, Environmental Microbiol-
ogy and Soil Biotechnology Ph.D. Program, Budafoki ut
59. KEŁKI, 1111 Budapest, Hungary
47 rod-shaped bacterial strains were isolated from the (1)
rhizosphere and rhizoplane of lupine and vetch plants (18
strains) grown in the Westsik’s experiment on sandy soil
(Nyiregyhaza, Hungary), and from the (2a) soil of £ooded
408 1st FEMS Congress / Posters 103^505

area (8 strains), (2b) soil of river bank (9 strains) and (3)
surface water of the Upper-Tisza river (Hungary); and
tested for plant growth promoting e¡ect. 13 strains ^ iden-
ti¢ed by both 16S rDNA sequencing and biologtm method
^ were selected for further investigations among Pseudo-
monas, Aeromonas and Bacillus genera. Sensitivity tests
against di¡erent concentrations of some heavy metals
were carried out by measurement of bacterial growth in
liquid culture (turbidity). Among the investigated bacteria,
7 strains proved to have a signi¢cant resistance against the
heavy metal compounds compared to the other strains:
Aeromonas media W.1, Pseudomonas veronii W.7, Pasteur-
ella sp. W.30, Pseudomonas sp. W.36, Bacillus thu«ringiensis
C.69, and III.118, III.119 ; those total mean sensitivity did
not show a statistically reliable di¡erence. The inhibitory
e¡ect of heavy metal compounds on the bacterial growth
is the following (in a decreasing order) : (NH4)6Mo7O24,
CuSO4, ZnCl2, Cu(NO3)2, Zn(NO3)2, CuCl2, Pb(NO3)2,
Fe(II)SO4, Fe(III)Cl3, Fe(III)(NO3)3. Compounds of lead
had a weaker inhibition of strain growth than those of
copper and zinc, thus it is probable that the investigated
bacteria may be adapted to the pollution of their site of
origin. In case of ironic compounds, the ‘‘microelement-
e¡ect’’ is supposed, so the measure of inhibitory e¡ect
seems to be determined better by the anions dissociated
in the electrolyte.
P11^121
ISOLATION AND CHARACTERIZATION OF METH-
YLITROPHIC STRAINS FROM SEDIMENTS AND
SOIL SAMPLES
C. C. Pacheco(1), P. De Marco(1) and P. Moradas-Fer-
reira(1,2)
(1) Instituto de Biologia Molecular e Celular, Microbiolo-
gia Celular e Aplicada, Rua do Campo Alegre, 823, 4150-
180 Porto, Portugal; (2) Instituto de Cie“ncias Biome¤dicas
Abel Salazar,Universidade do Porto, Portugal
phomicrobium. With this work we have obtained a collec-
tion of bacteria that are able to grow in diverse challeng-
ing conditions and that may be used for bioremediation
purposes.
This work is supported by funding from the EC Fifth
Framework Programme.
P11^122
BIOFILM REMOVING EFFECT OF FIVE DESINFAC-
TANTS ON BACTERIA AND FUNGI ATTACHED TO
STAINLESS STEEL
K. Pap(1) and G. Kisko¤(2)
(1) Richter Gedeon Pharmaceutical Ltd, P.O. Box 27, H-
1475, Budapest, Hungary ; (2) Szent Istva¤n University, De-
partment of Microbiology and Biotechnology, Somloi ut 14-
16, H-1118, Budapest, Hungary
The bio¢lm removing e¡ect of ¢ve disinfectants (Nobactel,
Domestos, SU 392, Buraton, Descosal) was tested in vitro
against bacteria and fungi (including species of Staphylo-
coccus aureus ATCC 6538, Escherichia coli ATCC 8739,
Pseudomonas aeruginosa ATCC 9027, Candida albicans
ATCC 10231, Bacillus subtilis ATCC 6633 and Aspergillus
niger ATCC 16404). Two hours bio¢lms were made on
stainless steel surfaces. The exposure time of disinfectants
was 30 minutes. The stainless steel coupons were examined
under epi£uorescent microscope before and after the dis-
infection treatments. C. albicans, P. aeruginosa, E. coli and
S. aureus show great bio¢lm-producing capability. Every
disinfectant was bactericidal against test organisms but the
bio¢lm removal e¡ect was di¡erent. Domestos had a sub-
stantial bio¢lm removing e¡ect while SU 392 worked ef-
fectively on bio¢lm of B. subtilis, E. coli and S. aureus,
Descosal on the bio¢lm of B. subtilis and E. coli, Nobactel
on B. subtilis, P. aeruginosa, E. coli and S. aureus. Buraton
did not remove bio¢lm from test surface except E. coli.

More than 30 methylotrophic strains were isolated from

sediments and soil samples. These strains were morpho-
logically and physiologically characterized. Although all of
them were neutrophilic, 8 alkali-tolerant and 3 acid-toler-
ant were obtained. Temperature wise 15 strains could
grow at 8‡C. The majority of the strains were facultative
methylotrophs. Their resistance to heavy metals (cadmi-
um, chromium and mercury), arsenic and several antibi-
otics was tested. The tolerance to pollutants like naphtha-
lene, xylene, styrene, benzene, phenol, MTBE, TCE and
crude oil was also evaluated. 16S rRNA classi¢cation was
performed and revealed that most of the isolates were
members of the genera Methylobacterium and Methylophi-
lus, but there were isolates from the genera Pseudomonas,
Rhodococcus, A¢pia, Arthrobacter, Ancylobacter and Hy-

FEMSLE Congress 2-6-03

 1st FEMS Congress / Posters 103^505
P11^123
THE SYSTEMATICS AND ECOLOGY OF RUMINAL
PREVOTELLA AS REVEALED BY MOLECULAR
TECHNIQUES
M. Peterka(1,3), K. Teps›ic›(1), L. Lipoglavs›ek(1), J. Ko-
pecny(2) and G. Avgus›tin(1)
(1) University of Ljubljana, Biotechnical Faculty, Zootech-
nical Department, Groblje 3, 1230 Domz›ale, Slovenia; (2)
Academy of Sciences of Czech Republic, Institute of Anim.
Physiol. & Genetics, CR-10400 Prague 10, Czech Republic;
(3) present address : BIA separations d.o.o., Teslova 30,
1000 Ljubljana, Slovenia
The rumen microbial ecosystem is inhabited by a vast,
currently unknown number of bacterial species as revealed
by recent molecular investigations. It appears however
that a large proportion of rumen bacteria may be placed
within two phylogenetic groups i.e. low %G+C Gram-pos-
itive bacteria represented mainly by Cluster XIVa of the
Clostridiaceae, and on the other hand members of the
genus Prevotella, as the dominant representatives of
Gram-negative ruminal bacteria. The majority of available
16S rRNA sequences from ruminal supercluster of the
genus Prevotella form a separated phylogenetic group, dis-
tinct from prevotellas of other origins, isolated mainly
from human oral cavity. Sequences from the ruminal
supercluster were not found in the cattle and equine hind-
gut samples either. We have developed a series of prevo-
tella speci¢c molecular detection tools based on the com-
petititve PCR reaction and have analysed, in combination
with £ow cytometry, a number of rumen £uid samples
obtained from black-and-white Holstein cows during
four weeks lasting feeding trials. We demonstrated that
P. ruminicola represents by far the most abundant species
of the ruminal prevotellas, whereas the cell numbers of
another ruminal prevotella species P. bryantii remained
bellow c-PCR detection limit i.e. 106.ml-1. Large cell num-
ber £uctuations were observed too, resembling the popu-
lation £uctuations of the only other ruminal species moni-
tored by molecular means in a similar manner to date,
Fibrobacter succcinogenes. The total number of ruminal
bacteria remained fairly stable during the sampling period,
however, indicating that the £uctuation of di¡erent bacte-
rial populations in the rumen occurs in an a-synchronical
manner.
FEMSLE Congress 2-6-03
409
P11^124
DRINKING WATER SOURCE ‘‘RATNO OSTRVO’’
AND OIL POLLUTION ^ INFLUENCE OF THE DAN-
UBE AND CONTAMINATED SOIL ON MICROBIO-
LOGICAL WATER WELLS QUALITY
O. V. Petrovic(1), J. Simeunovic(1), D. Radnovic(1), M.
Matavulj(1), S. Gajin(1), B. Dalmacija(2)
(1) Department of Biology and Ecology, (2) Department of
Chemistry, Faculty of Natural Sciences and Mathematics,
University of Novi Sad, Trg Dositeja Obradovica 2, Serbia
‘‘Ratno Ostrvo’’ is the most important drinking water
source within the water supply system of Novi Sad.
With regard to natural potential, this water source has
promising development perspective. The Danube riparian
area provides adequate intake from the river direction and
the surrounding is suitable for the construction of water
intake structures like wells with horizontal drains. Consid-
ering environmental issues this water source is situated in
an unfavorable surrounding due to its vicinity to oil re¢n-
ery. After the NATO bombing of oil re¢nery large quan-
tities of crude oil and oil products leaked into the Danube
and nearby soil thus directly threatening this water source.
Microbiological and chemical analyses of ground water
and wells started short after the incident. Due to potential
danger of contamination by crude oil spillage, ground
water monitoring was continued during 2000-2002. Clas-
sical methods were applied in microbiological analyses,
including sanitary, ecological and biochemical aspects.
The group of hydrocarbon-oxidizing bacteria was deter-
mined on two di¡erent media with added crude oil of
para⁄n base. Both media were used with added reagent
threephenyltetrazolechlorid (TTC), which due to classes’
dehydrogenase visible activity enabled more reliable
counting of bacterial colonies. At present, microbiological
quality of drinking water could be described as satisfac-
tory. In¢ltration area of the Danube plays an important
role in the river bacterial £ora reduction. However, pres-
ence of oil-oxidizing and lipolytic bacteria in drinking
water indicates the contact with oil type substances by
means of in¢ltration from contaminated soil within the
re¢nery area.
410 1st FEMS Congress / Posters 103^505
P11^125
RESISTANCE AGAINST AND DEGRADATION OF
CHOLATE BY PSEUDOMONAS SP. STRAIN CHOL1
B. Philipp, B. Schink
Microbial Ecology, University of Konstanz, Fach M654,
78457 Konstanz, Germany
Pseudomonas sp. strain Chol1 was isolated from a soil
sample with the anionic detergent cholate as sole source
of carbon and energy. Strain Chol1 can grow with other
bile acids and their taurine-conjugated derivatives aerobi-
cally or anaerobically with nitrate as electron acceptor.
Aerobic growth with cholate was possible at concentra-
tions up to 50 mM. Suspensions of resting cells (109 cells
ml-1) grown with 2 mM cholate lysed rapidly when ex-
posed to cholate at concentrations higher than 10 Wmol
ml-1 (cholate/cell-ratio of 10-8 Wmol per cell). In our
growth experiments, cells have to survive much higher
cholate/cell-ratios at the time of inoculation. Therefore,
resistance mechanisms would be required to enable cells
to grow with cholate. The sensitivity of cells was increased
by addition of EDTA indicating the importance of the
outer membrane as a di¡usion barrier for the detergent.
At the beginning of growth strain Chol1 formed visible
aggregates. When grown with low shaking frequency,
these aggregates remained stable and signi¢cant bio¢lm
formation occurred in comparison to cells growing with
succinate. During growth, cholate was rapidly transformed
into 4 intermediates, which accumulated in the superna-
tant before they were degraded. According to HPLC-anal-
ysis, theses intermediates are more hydrophilic than chol-
ate and are likely to be aromatic compounds. The
formation of aggregates and bio¢lms as well as the trans-
formation of cholate to more hydrophilic compounds
could serve as mechanisms to protect strain Chol1 from
the lytic e¡ects of cholate. We are currently investigating
this hypothesis.
P11^126
SHIFTS IN THE NITRATE-REDUCING COMMUNI-
TY INDUCED BY THE MAIZE RHIZOSPHERE
S. Piutti, S. Hallet, F. Martin-Laurent, J. C. Germon and
L. Philippot
UMRA 111, INRA ^ CMSE, Microbiologie des Sols- Ge¤o-
sols, 17 rue Sully, B.V. 86510, 21065 Dijon Cedex, France
Microorganisms that use nitrate as an alternative terminal
electron acceptor play an important role in the global
nitrogen cycle. By modifying nitrate concentration, reduc-
ing the oxygen partial pressure and providing carbon sub-
FEMSLE Congress 2-6-03
strate via the exudation, the roots are an important micro-
biota for nitrate dissimitory microorganisms . The aim of
this study was to determine the in£uence of the rhizo-
sphere of maize on the nitrate reducing community in
soil. The narG gene encoding the membrane bound nitrate
reductase was selected as a functional marker for the dis-
similatory nitrate reducing community and a degenerated
primer set was designed after comparative sequence anal-
ysis. The use of narG is of special interest because the
phylogeny of the narG gene closely re£ects the 16S
rRNA phylogeny. The PCR-ampli¢ed fragments of narG
recovered by ampli¢cation of DNA extracted from maize-
planted- and bulk soil were cloned. Four hundreds and
seventy seven clones from six libraries were analysed by
restriction fragment length polymorphism to assess the
underlying narG diversity. In all, one hundred and twenty
two di¡erent RFLP types were identi¢ed and a least one
clone belonging to each RFLP type have then been se-
quenced. Rarefaction curves and diversity indexes indi-
cated that diversity was high. Our results shown that the
presence of the plant resulted in a shift in the structure of
the nitrate-reducing community with a signi¢cant increase
of clones clustering with the actinomycetes narG group.
P11^127
PROFILING OF A MEMBRANE SUPPORTED BIO-
FILM BY TGGE
A. Pires(1), G. Wolf(2), M. A. M. Reis(2), J. G. Cres-
po(2), M. T. Barreto Crespo(1)
(1) IBET/ITQB, Apartado 12, 2781-901 Oeiras, Portugal;
(2) Chemistry Department/CQFB, Faculdade de Cie“ncias e
Tecnologia, UNL, 2829-516 Caparica, Portugal
Discharges of volatile organic compounds (VOC) have
been subjected to increasing regulatory pressure over the
past decade due to their potential to cause environmental
harm. Membrane supported bio¢lm reactors were found
to be e¡ective in degrading VOC in wastewater while
keeping the loss of volatile compounds to the environment
to a minimum. The biological degradation of 3-chloro-4-
methylaniline (3C4MA) was achieved using an extractive
membrane bioreactor (EMB) inoculated with an unidenti-
¢ed mixed culture obtained from an industrial EMB pilot
plant that operated on e¥uent containing this compound.
The non-porous, selectively permeable membrane prevents
the contact between the e¥uent and the biological mixed
culture suspension. The pollutant in the wastewater dif-
fuses across the membrane where it is degraded by a bio-
¢lm that forms the membrane-biomedium interface. This
study focuses on the characterization of the bio¢lm under
di¡erent reactor operating conditions. Daily samples were
collected in three sections along the membrane, and also in
the biological compartment. At the end of each change in
 1st FEMS Congress / Posters 103^505
the reactor operation the bio¢lm of the corresponding
areas of the membrane was collected and the samples
were cultivated in a rich medium in order to isolate and
identify the culturable components of the total population.
Characterization and identi¢cation of the bio¢lm members
was also performed using a ¢ngerprinting approach that
consisted of total DNA extraction of the samples and
respective ampli¢cation using primers targeting the 16S
and 18SrDNA regions. The amplicons were then analysed
using thermal gradient gel electrophoresis. In£uence of
EMB operational parameters on the population was eval-
uated.
P11^128
THE ANTIOXIDANT VITAMIN PRETREATMENT
IMPROVES CHROMIUM(VI) REMEDIATION ABIL-
ITY OF YEAST SACCHAROMYCES CEREVISIAE
B. Poljs›ak(1,4), Z. Gazdag(2), M. Pesti(2), N. Far-
kas(3), S. Plesnic›ar(1) and P. Raspor(4)
(1) Polytechnic Nova Gorica, School of Environmental Sci-
ence, Vipavska 13, 5000 Nova Gorica, Slovenia; (2) De-
partment of General and Environmental Microbiology, Fac-
ulty of Sciences, University of Pecs, P.O.B. 266, H-7601
Pecs, Hungary; (3) Central Research Laboratory, Univer-
sity of Pe¤cs, P.O.Box 266, Hungary ; (4) University of
Ljubljana, Biotechnical Faculty, Food Science and Technol-
ogy Department, Chair of Biotechnology, Jamnikarjeva 101,
1000 Ljubljana, Slovenia
Chromium accumulation in yeasts o¡ers an alternative
technique for water decontamination. Chromate ions
have cytotoxic, mutagenic and carcinogenic e¡ects on all
eukaryotic cells tested. There are reports of Cr(VI) resis-
tance in yeasts, including S. cerevisiae. The resistance has
been attributed to a reduced Cr(VI) uptake resulting in
decreased intracellular chromium accumulation and better
cell viability. The objective of this study was to pretreat
yeast cells with two antioxidant vitamins (vitamin C and
vitamin E water soluble analogue Trolox) in order to in-
crease cell tolerance against reactive chromium intermedi-
ates and reactive oxygen species (ROS) formed during
Cr(VI) treatment. Intracellular oxidation was estimated
using two £uorescence indicators dihidro-2, 7-dichloro-
£uorescein and dihydrorhodamine 123. The role of anti-
oxidant pretreatment on chromium(VI) genotoxicity was
determined by measuring mitotic crossing over, mitotic
gene conversion and reverse mutations in S. cerevisiae
strain D7. Additionally, 8-OhdG, a marker of oxidative
damage to yeast DNA was measured using competitive
ELISA. Hydroxyl radical generation induced by Cr(VI)
was measured by electron spin resonance spectroscopy in
cell extract of S. cerevisiae. We found that vitamin pre-
treatment in£uenced cell viability due to decreased intra-
FEMSLE Congress 2-6-03
411
cellular oxidation and reduced Cr(VI) genotoxicity. Fur-
thermore, chromium content in biomass was determined
by £ame atomic absorption spectrometry (FAAS). Results
indicate the important role of cytosol reduction capacity
on increased Cr accumulation. Better cell viability, de-
creased intracellular ROS formation and increased Cr up-
take are properties, which signi¢cantly improve the use of
yeasts for environmental Cr(VI) cleanup.
We thank the Ministry of Science and Technology of the
Republic of Slovenia (Project no. : J4-7454-490) for ¢nan-
cial support.
P11^129
COMPARISON OF THE EFFECT OF THREE SUP-
PLEMENTARY COMPOUNDS ON THE ABILITY
OF A MICROBIAL COMMUNITY ISOLATED
FROM CONTAMINATED SOIL TO BIODEGRADE
DIESEL OIL
T. Po¤r, S. Re¤ve¤sz, K. Ma¤rialigeti
Eo«tvo«s Lora¤nd University, Department of Microbiology,
Hungary, 1117Budapest, Pa¤zma¤ny Pe¤ter se¤ta¤ny 1/C
Nowadays usage of hydrocarbons is general in transport
and industry. It brings about oil spills inevitably. The in-
tent of our investigations is to compare the e¡ect of dihy-
droxypropyl-L-cyclodextrin (diCD), Tween 80 and starch
on the ability of a microbial community isolated from soil
to degrade diesel oil. DiCD is a cyclic, nonreducing mal-
tooligosaccharide. It has the ability to form water-soluble
inclusion complexes by incorporating suitably sized low-
polarity molecules in its cavity. Tween 80 is a nonionic
surfactant. Our samples were taken from the environment
of a dismantled petrol station, and were maintained in oil
containing medium. The most e⁄cient diesel oil degrading
community was chosen for further investigation. The com-
munity was characterized and tested on Biolog0 GN2
plate. Its ability to utilize diesel oil as a carbon source
was examined in BHB (Bacto Buschnell-Hass) medium
containing diesel oil. The amount of CO2 originating as
a result of aerobic respiration was observed after giving
diCD, Tween 80 and starch to the medium with diesel oil.
DiCD was found to be considerably e⁄cient in the in-
crease of maximal rate of biodegradation and decrease
of adaptation period. According to our observations
Tween 80 is utilized as primary carbon source, so it does
not increase oil degradation. Starch can be only an alter-
native carbon source and it also had not e¡ect on biodeg-
radation.
412 1st FEMS Congress / Posters 103^505
P11^130
SEROLOGICAL DIFFERENTIATION OF SOME SI-
NORHIZOBIUM MELILOTI BACTERIOPHAGE ISO-
LATES
D. Radin
Faculty of Agriculture, Belgrade, Yugoslavia
Lytic bacteriophages infective for the root-nodule bacte-
rium Sinorhizobium meliloti, are wide spread almost in all
alfalfa ¢elds, where this plant is grown. Because of their
lytic activity, bacteriophages destroy the S. meliloti cells
and in this way in£uence the nitrogen ¢xation of these
cells in ¢elds. That was the reason why we started to in-
vestigate these phages, isolated from ecologicaly di¡erent
soils.Ten phage isolates were included in this investigation.
They were characterized by their morphology (complex
morphotype, consisting of a isometric, hexagonal head
and a short tail), host range, and % GC content. As sero-
logical studies are helpful in establishing phage groups
they were characterized by these properties, too. Selected
phages were tested against previously prepared antisera
for phage isolates LK1, LK2 and DN3. As it is usual,
neutralization tests were used in which speci¢c antisera
bind and inactivate the infective phage particles. The ini-
tial phage concentration of 106 particles (pfu)/ml was
tested with 1/1 to 1/512 diluted solutions of antisera. Ac-
cording to serological reactions and relationships two
groups of bacteriophages have been identi¢ed. One, repre-
senting serological related phages, consists of 8 typical
isolates, whose particles were neutralized immediately at
the begining of the test. Two phage isolates (SB1 and ML)
were resistant to the SLK1 antisera and because of that
identi¢ed as new serogroup among the phage tested. Sero-
logical properties are important for the phage determina-
tion, especially the detection of new serotypes, which
could be used in the selection of Sinorhizobium meliloti
resistant strains for the production of nitrogen fertilizers.
FEMSLE Congress 2-6-03
P11^131
DIVERSITY AND PERSISTENCE OF AEROMONAS
STRAINS DURING WASTE WATER RECYCLING
THROUGH DUCKWEED BASED AQUACULTURE
M. Rahman(1), I. Ku«hn(1), M. Rahman(2) and R. Mo«ll-
by(1)
(1) Microbiology and Tumor Biology Center (MTC), Kar-
olinska Institutet, SE-171 77 Stockholm, Sweden; (2) In-
ternational Center for Diarrhoeal Disease Research, GPO
Box 128, Dhaka 1000, Bangladesh
An attractive and economical way of aquaculture is to
feed ¢sh with aquatic plants, which have been grown on
waste water. The waste is thus transferred into high qual-
ity protein, decreasing the pollution problem. The aim of
the present study was to investigate the persistence and
diversity of individual Aeromonas strains surviving and
circulating in a duckweed based waste water recycling sys-
tem. Samples were collected from a ¢sh farm connected to
a treatment plant for hospital sewage. Samples were col-
lected from various sites of the cycle and grown on Les-
endo agar plates. Eight presumed Aeromonas colonies per
plate were subjected to biochemical ¢ngerprinting through
a computerised ¢ngerprinting system, the PhenePlate1
system, based on kinetic readings of selected highly dis-
criminating biochemical reactions. 132 samples yielded
1028 isolates that were typed into 247 di¡erent types, of
which 33 types occurred at several sites at several occa-
sions. These types/strains were mostly restricted to ¢sh,
human or environmental samples. Strains detected in all
kinds of samples were considered to be able to spread
from human to waste water to ¢sh to humans. It was
concluded that aquatic plants may serve as e⁄cient reser-
voirs of Aeromonas bacteria ; that a large variety of types/
strains of Aeromonas were found; that most of the types
were found in only limited habitats while a few strains
were able to spread through the waste water cycle. If the
latter strains are pathogenic to humans or may transfer
antibiotic resistance, they may cause problems to human
health.
 1st FEMS Congress / Posters 103^505
P11^132
EFFECT INOCULATION OF WHEAT AND DIFFER-
ENT TILLAGE SYSTEMS IN SOIL NITROGENASE
ACTIVITY
V. Raic›evic¤(1), D. Kovacevic(1), D. Micanovic(2), B. La-
levic(1)
(1) Faculty of Agriculture, Belgrade-Zemun, Nemanjina 6,
Yugoslavia; (2) ARI ‘‘SERBIA’’, Center for Small Grains
in Kragujevac, Yugoslavia
This paper deals with result of the e¡ect inoculation of
Azotobacter chroococcum and tillage systems on soils and
root nitrogenase activity.The trial was carried-out at the
experimental ¢elds Faculty of Agriculture- Zemun ‘‘Rad-
milovac’’ on the eutric combisol. The following tillage sys-
tems were included in investigations: 1. Conventional till-
age system-(CT), 2. Mulch tillage-(MT), 3. No-tillage
system (NT). The seeds were soaked for 30 min in an
Azotobacter chroococcum PS13 inoculum. Nitrogenase ac-
tivity was determined by gas chromatography, using Por-
apak N column (Hardy, 1968). The wheat root samples
were washed, the excess water was removed with ¢lter
paper, than, 1g of fresh weight were homogenized. The
homogenized root was transferred into 8,7 ml glass bottles
with 4 ml free-N substrate with mannitol as carbon source.
Samples of 0,2 ml were inserted with Hamilton syringe
into the injector and the area of the ethylene peak was
read on the display. The soils samples (1g) incubated at
48h and transferred into 8,7 ml glass bottles with 4 ml
substrate with glocose as carbon source. The ARA was
carried out on a gas chromatograph using Porapak N
column. Nitrogenase activity was derected in roots in soils
of both inoculated and noninoculated whear in cultivar
NS-Rana5. The obtained results indicate that the nitro-
genase activity very signi¢cantly in£uenced by the tillage
systems, inoculation, the phenophases wheat cultivars as
well as their interaction. The highest nitrogenase activity
in rhizosphere soil is found after mulch tillage. Maximum
nitrogenase activity roots was found during the heading
stage in mulch tillage.
FEMSLE Congress 2-6-03
413
P11^133
THE GROWTH OF MICROALGAE USING AN EF-
FLUENT FROM A BREWERY AS THE CULTURE
NUTRIENT MEDIUM
S. Oliveira, M. F. J. Raposo, P. M. L. Castro and R. M.
Morais
Escola Superior de Biotecnologia, Universidade Cato¤lica
Portuguesa, Rua Dr. Anto¤nio Bernardino de Almeida,
4200-072 Porto, Portugal
The treatment of e¥uents from Agro Food Industries is a
major issue in EU due to its industrial and economic im-
portance. Microalgae can be used in wastewater treatment
where they may be able to recycle nutrients. Besides, they
can reduce the nutrient load through stripping and precip-
itation, by producing oxygen for bacterial decomposition
of organic matter, by eliminating pathogenic bacteria
through bactericide action, and by solving odour prob-
lems. Treatment e⁄ciency and nutrient removal are man-
aged in function of algal growth, wastewater characteris-
tics (nutrient imbalance, chemical and biological toxicity),
and operational parameters. In this study, the e¥uent
coming from a brewery was used as the culture medium
for biomass production, which can be processed for valor-
isation or directly used as a biofertilizer. We evaluated the
growth of microalgae, either Chlorella vulgaris or the au-
tochthonous £ora, using the e¥uent of a brewery as the
nutrient medium. We also evaluated whether the micro-
algae used the compounds of the e¥uent as nutrients. The
microalgae were grown in di¡erent proportions of e¥uent,
33%, 50% and 100%. The e¥uent was diluted with distilled
water and a control experiment was established using BG
Medium (Blue Green). In addition, nitrogen of the brew-
ery e¥uent was brought to the BG medium concentration,
and pH was also corrected. Growth was enhanced when
using 50% e¥uent, with a signi¢cant decrease in the
amounts of ammonia, nitrates and phosphates of the ef-
£uent. Moreover, the malodour of the e¥uent has disap-
peared by the end of the experiment.
414 1st FEMS Congress / Posters 103^505
P11^134
STIMULATION OF MICROBIAL DEGRADATION
STIMULATION WITH CYCLODEXTRINS AT BTX
COMPOUNDS
S. Re¤ve¤sz, C. S. Romsics, T. Po¤r, R. Sipos, M. Palatinszky,
K. Ma¤rialigeti
Eo«tvo«s Lora¤nd University, Department of Microbiology,
Pa¤zma¤ny Pe¤ter se¤ta¤ny 1./C., Budapest, 1117-Hungary
A considerable amount of gasoline enters the environment
as a result of leakage from underground storage tanks,
accidental spills, or improper waste disposal practices.
These compounds of gasoline are some of the more com-
mon contaminants found in drinking water. BTX (ben-
zene, toluene, xylene) compounds are toxic, and their re-
moval from polluted environments is of special interest.
The hydroxypropyl-L-cyclodextrin (HPCD) and the dihy-
droxypropyl-L-cyclodextrin (diHPCD) are 7 unit cyclic
oligomers of 1,4-a-D-linked glucose. The most pertinent
property of cyclodextrins is having a hydrophilic shell
and a toroidal-shaped, apolar (hydrophobic) cavity. Cy-
clodextrins incorporate suitably low-polarity molecules in
their cavities forming molecular inclusion complexes. The
inclusion complex relies mostly on hydrophobic interac-
tions between cyclodextrins and organic molecules. How-
ever, cyclodextrins are extremely soluble in water and cer-
tain organic solvents. So the apparent solubility of the
quest molecule increases. We have isolated the strain
SM5T4 from a gasoline-contaminated soil in Budapest.
Using sequence analysis we were able to identify the
SM5T4 strain as Acinetobacter sp. The BTX degrading
capacity of SM5T4 was measured in the presence of
HPCD and diHPCD. The biodegradation was investigated
in closed vials with septum to prevent evaporation. The
quantity of BTX compounds was measured with GC-FID.
Both cyclodextrins were able to increase the apparent sol-
ubility of the organic compounds, so more benzene, tolu-
ene and xylene was degraded then without cyclodextrins.
The HPCD was more e¡ective to stimulation the degra-
dation, then diHPCD. The most e⁄cient result was given
by benzene, than toluene and xylene.
FEMSLE Congress 2-6-03
P11^135
MICROBIAL PROCESSES OF CARBON AND SUL-
FUR CYCLES IN MEROMICTIC BRACKISH SHIRA
LAKE (RUSSIA, SIBERIA)
D. Y. Rogozin(1), D. B. Kosolapov(2), N. V. Pimenov(3)
(1) Institute of Biophysics of Siberian Branch of Russian
Academy of Sciences, Krasnoyarsk, Russia; (2) Institute
for Biology of Inland Waters of Russian Academy of Sci-
ences, Borok, Russia ; (3) Institute of Microbiology of Rus-
sian Academy of Sciences, Moscow, Russia
The microbial and biogeochemical investigation of brack-
ish meromictic Shira Lake was curried out in the summer
of 2001 and 2002. Patterns of primary production, sulfate
reduction rate, methane production and methane oxida-
tion in water column and bottom sediments were ob-
tained. The lake is characterized by high contents of sul-
fate (91.3-116.3 mM). The hydrogen sul¢de accumulates in
the monimolimnion, where its concentration reaches 0.60
mM. Sulfate reduction rates in water, measured by radio-
metric technique, varied from 0.25 to 9.81 Wmol l-1d-1. The
process of sulfate reduction was recorded in the anoxic
water in an interval of depths from 12 m to the bottom.
There were two peaks of sulfate reduction rates: just be-
low the chemocline and near the sediment surface. In the
anaerobic water column sulfate reducing bacteria play the
main role in the terminal phase of organic material decom-
position. Based on our calculations, 73 % of daily pro-
duced organic carbon was consumed by sulfate reducing
bacteria. The water samples collected from this depth were
pink-colored due to the presence of purple sulfur bacteria
(6x105 cells/ml). These bacteria were identi¢ed as Lamp-
rocyctis purpurea (earlier Amoebobacter purpurea). The mi-
crobial structure of the chemocline was investigated by
thin-layer sampling. The abundance and biomass of geter-
otrophic bacteria, heterotrophic £agellates, ciliates, cyano-
bacteria and anoxic phototro¢c bacteria were determined
at depths from 11.3 to 12.3 m with 5 cm interval. Abun-
dance of purple sulfur bacteria and green sulfur bacteria
increased to 2x106 cells/ml at the depth of 11.85-11.95 m
forming a distinct layer of anoxic photosynthesis. Results
will be used in a model of carbon and sulfur cycles inter-
relation.
 1st FEMS Congress / Posters 103^505
P11^136
PETROLEUM DEGRADATION USING URIC ACID
AS AN INSOLUBLE NITROGEN SOURCE
O. Koren and E. Rosenberg
Department of Molecular Microbiology and Biotechnology,
The George S. Wise Faculty of Life Sciences, Tel Aviv
University, Ramat Aviv 69978, Israel
Bioremediation of petroleum contaminated sites is a prov-
en technology. However, the availability of nitrogen is
often the rate limiting factor in this process because petro-
leum contains only traces of nitrogen. Commercial N-fer-
tilizers are water-soluble and are ine¡ective in open sys-
tems, such as the sea, because they rapidly become too
dilute at the hydrocarbon/water interface where they are
needed for microbial growth. To overcome this problem,
we have examined the use of uric acid, a naturally-occur-
ing and water insoluble compound. Bacterium OK1, iso-
lated by enrichment culture, grew well on crude oil with
uric acid as the sole N source. Strain OK1 was classi¢ed as
Acinetobacter baumannii by 16S rDNA analysis and clas-
sical microbial tests. In addition to using crude oil, A.
baumannii OK1 grew on high molecular weight alkanes
with uric acid as the N source. The potential of uric
acid to serve as a N source in the bioremediation of hydro-
carbon pollution in open systems is now being investi-
gated.
P11^137
LEGIONELLA ISOLATED FROM ENVIRONMEN-
TAL SAMPLES IN SLOVENIA FROM 2000 TO 2002
N. Klun, K. Kastelic, T. Majstorovic¤, E. Modec, T. Rupel
Institute of Public Health of the Republic of Slovenia,
Centre for Environmental Health, Department of Sanitary
Microbiology, Grablovic›eva 44, 1000 Ljubljana, Slovenia
The Legionellaceae family recognizes at least 39 species
and 61 serogroups of legionella bacteria. More than half
of these species and serogroups have been associated with
human disease- legionellosis. Legionella pneumophila, the
¢rst legionella bacterial species identi¢ed, accounts for ap-
proximately 90% of infections, with illness most frequently
associated with serogroups 1, 4 and 6. Outbreaks of le-
gionellosis have been traced to a number of water sources
contaminated with Legionella; water in air conditioners
and cooling towers, showerheads, hot tubs, public foun-
tains and even a supermarket vegetable-misting machine.
Department of Sanitary Microbiology in Institute of Pub-
lic health started water testing on legionella bacteria in
1990. Since than the number of samples is increasing.
FEMSLE Congress 2-6-03
415
The water samples were mainly taken in public health ser-
vice buildings (hospitals, clinics, social institutions) and
hotel/thermal complexes. But they also arrived from indus-
try buildings, sport centers, private premises, water suppli-
ers and many other buildings. From year 2000 to 2002 714
samples were examined. Of these, 274 samples were pos-
itive for a Legionella species, in 160 samples Legionella
pneumophila serogroup 1 was detected. The highest per-
centage of positive samples on legionella bacteria is in
the group of water samples between 20 and 55‡C.
P11^138
OIL’S UTILIZATION WITH AN OILOXYGENIZING
MICROORGANISMS ASSOTIATION THOSE WERE
IMMOBILIZED ON FOAMSORBENT
T. V. Ryazanova and O. S. Fyedorova
Sibirian State Technological University, Krasnoyarsk
660049, Mira 82, Russia
Clean cultures of oiloxygenizing stamms were received
from soil of oil ¢elds. It was studied possibility of their
immobilization on foamsorbent created on the base of
carbamide resin. The foamsorbent has high oil-capacity
(60 kg of oil on 1 kg of sorbent). The foamsorbent con-
tains carbon, nitrogen, and phosphorus. It is not toxic, it
airs soil and in course of time it is decomposed. Immobi-
lization of microorganisms into the foamsorbent was made
of water suspension with a titre 10 7 cell/g with a drop
way. The results of researches and ¢eld tests have shown,
that immobilization on foamsorbent oiloxygenizing
stamms do not lose the activity in winter conditions and
are not washed away with foamsorbent by earth waters.
Petroleum’s analysis showed that to 90 % oil was utilized
especially in fractions with a temperature of boiling to 350
‡C with biodestruction. In consequence of self-decomposi-
tion the biosorbent does not require its cleaning out of
poorly accessible places. The tentative estimation of e⁄-
ciency of use biosorbent at liquidation of emergency £ood
of petroleum on a main oil pipeline has shown, that the
level of pollution of ground by petroleum at use biosorb-
ent has decreased for 1,5 week with 22 % till 1,7 -2,5 %.
Thus, the results of the carried out researches testify that
developed biosorbent on a basis of the specially picked up
associations oiloxygenizing stamms of microorganisms al-
located from soil micro£ora and immobilization on foam-
sorbent can be used for clearing soil and water from pe-
troleum.
416 1st FEMS Congress / Posters 103^505
P11^139
THE LECTIN WHEAT GERM AGGLUTININ AS A
GROWTH FACTOR FOR AZOSPIRILLUM BRASI-
LENSE
Yu. N. Sadovnikova, L. A. Bespalova, L. P. Antonyuk
Institute of Biochemistry and Physiology of Plants and Mi-
croorganisms, Saratov, Russia
Bacteria of the Azospirillum brasilense species colonize
plant root system establishing endophytic and associative
N2-¢xing symbioses with wheat, cotton, tomato as well as
other cultivated and wild plants. We studied the e¡ect of
the wheat lectin (wheat germ agglutinin, WGA), which is
known as a molecular signal for A. brasilense Sp245 [1], a
native endophytic symbiont of the wheat plant, on the
growth of azospirilla. WGA was shown to have no in£u-
ence on the growth azospirillum under aerobic (non-N2-
¢xing) conditions and in the case when action of the stim-
ulus was short-term. The wheat lectin in nanomolar con-
centrations was found to promote the growth of A. brasi-
lense Sp245 (an endophytic strain) and A. brasilense Sp7
(non-endophytic strain). Thus, the presence of WGA in
the growth medium of azospirillum brought about an in-
crease in (i) the biomass assessed after drying to the con-
stant weight and (ii) the number of viable cells in the cul-
ture estimated as colony forming units. The wheat lectin,
being available to bacteria, is supposed to stimulate
growth of A. brasilense under natural conditions that, in
its turn, may increase its population density and facilitate
the formation of the azospirillum-wheat symbiosis.
[1] L.P. Antonyuk and V.V. Ignatov. Russ. J. Plant Phys-
iol., 2001, 48: 427-433.
Supported by the Russian Foundation for Basic Research,
Project 01-04-48755, and the Russian Academy of Scien-
ces’ Commission, 6th Competition-Expertise, Grant 205.
P11^140
GENETIC BASIS OF THE ASSOCIATION OF
ERM(TR) GENE WITH OTHER MACROLIDE RESIS-
TANCE GENES AND WITH TET(O) IN STREPTO-
COCCUS PYOGENES
M. Santagati, C. Cascone, C. Fichera, G. Pozzi and S.
Stefani
Department of Microbiology, University of Catania and
Siena, Italy
Macrolides are important drugs for the treatment of strep-
tococcal infections. Methylation and drug e¥ux are the
major mechanisms involved in the resistance: the former
is responsible for MLSB phenotype mediated by two
FEMSLE Congress 2-6-03
genes, erm(B), and erm(TR) ; the latter is due to the pres-
ence of mef(A) gene conferring the M-phenotype. In a
previous study (Cascone et al), among 103 macrolide-re-
sistant S. pyogenes, we found 13 strains with the erm(TR)
gene in association with other macrolide resistance genes
and with tet(O). Our sample could be divided in 3 groups,
depending on the associated resistance genes: (i) erm(TR)/
erm(B)/tet(O), 2 strains ; (ii) erm(TR)/tet(O), 9 strains ; (iii)
erm(TR)/mef(A), 2 strains, all strains exhibited MLSB phe-
notype. The genetic linkage of the13 erm(TR)-carrying
strains was investigated by PFGE using ApaI. Less vari-
able background of PFGE pro¢les in our sample was
found, and erm(TR)-tet(O) and erm(TR)-erm(B)-tet(O)
strains appeared to be closely related, while two
erm(TR)/mef(A) strains were unrelated to the others and
between themselves. Southern Blot analysis indicated that
erm(TR) and tet(O) were always together on the same
PFGE fragment while mef(A) and erm(B) genes are lo-
cated in di¡erent fragments from erm(TR) and tet(O)
genes. In conjugation experiments, we found that erm(B)
and mef(A) ^ but not erm(TR) alone- could be transferred
to S. pyogenes KmR. Furthermore, the expression of all
resistance genes by RT-PCR, demonstrated that erm(TR),
erm(B), mef(A) were always co-transcribed. In conclusion,
the presence and expression of erm(TR) gene, could hide
other erythromycin genes conferring the MLS phenotype
in all cases.
P11^141
ANTIBIOTIC RESISTANCE AND GENE TRANSFER
POTENTIAL ON THE AVEIRO LAGOON
A. Santos(1), A. Almeida(2), A. Cunha(2), A. Correia(1)
(1) Centro de Biologia Celular, Campus Universita¤rio de
Santiago, Departamento de Biologia, Universidade de
Aveiro, 3810-193 Aveiro, Portugal; (2) CESAM, Universi-
dade de Aveiro
The presence of antibiotic-resistant bacteria in freshwater
sources has been found throughout the world. The Aveiro
lagoon constitutes a special environment for development
of bacteria. The quality of water is in£uenced by the dis-
charge of e¥uents from several sources. Monitorization
and surveillance of virulent organisms and determination
of antibiotic resistance pro¢les was never performed on
this particular habitat. Using di¡erential culture media
we isolated Enterobacteriaceae and Aeromonas spp. anti-
biotic-resistant bacteria, in freshwater samples, from 3
sites in the estuarine environment of the Aveiro lagoon.
The three sampling sites were chosen on the basis of their
water salinity characteristics and taking into consideration
the e¥uent discharges predominant on each site. Using
standard methods, the prevalence of organisms resistant
to beta-lactam and non-beta-lactam antibiotics was mea-
 1st FEMS Congress / Posters 103^505
sured. The resistance to ampicillin was highly predomi-
nant. Because of that, the ampicillin-resistant isolates
were further characterised. Horizontal transfer of resis-
tance genes is a successful mechanism for the transmission
and dissemination of multiple drug resistance among bac-
terial pathogens. Transfer of antibiotic resistance genes
between di¡erent species of bacteria can be facilitated by
mobile DNA elements, such as transposons and plasmids.
Integron have been identi¢ed on these mobile elements,
and they often contain one or more genes that encode
antibiotic resistance. In order to assess the potential trans-
fer of the resistance characteristics observed, the isolates
were analysed for their plasmid content by DNA extrac-
tion and gel electrophoresis, and for the presence of inte-
grons by PCR using primers speci¢c for class I integrons.
P11^142
MINERALIZATION OF INDIVIDUAL LINEAR AL-
KYLBENZENESULFONATE (LAS) CONGENERS (2-
C10-LAS, 2-C11-LAS AND 3-C12-LAS) BY DEFINED
PAIRS OF HETEROTROPHIC BACTERIA
D. Schleheck(1), T. Knepper(2), K. Fischer(1) and A. M.
Cook(1)
(1) Department of Biology, The University, D-78457 Kon-
stanz, Germany; (2) Institute for Water Research and
Water Technology, D-65201 Wiesbaden, Germany
Commercial linear alkylbenzenesulfonate (LAS) surfactant
is probably the major bulk chemical degraded in sewage
works, but little of the degradative microorganisms, their
community structure or the degradative process is under-
stood. Alpha-Proteobacterium strain DS-1 utilizes com-
mercial LAS quantitatively, and excretes many sulfophe-
nylcarboxylates (SPCs) or similar compounds. We found
that 2-(4-sulfophenyl)dodecane (2-C10-LAS) was con-
verted largely to 3-(4-sulfophenyl)butyrate (3-C4-SPC),
as were 2-C12-LAS and 2-C14-LAS. 2-C11-LAS was con-
verted largely to 3-C5-SPC and we con¢rmed that 3-C12-
LAS was converted largely to 4-C5-SPC. Traces of many
other SPCs were found (about 10 % of the carbon). We
isolated Comamonas testosteroni strains SPB-2 and KF-1,
which utilized 3-C4-SPC, and Delftia acidovorans SPH-1,
which utilized 4-C6-SPC enantioselectively, using the pu-
tative S-4-C6-SPC ¢rst. The degradative pathway(s) ap-
parently involved 4-sulfocatechol and 4-sulfocatechol-1,2-
dioxygenase. The organisms tolerated the surfactant LAS.
A two-member community of strains DS-1 and SPB-2
quantitatively converted commercial LAS to SPCs and
utilized three of them (3-C4-SPC, 3-C5-SPC and an un-
known). The community of strains DS-1 and SPH-1 quan-
titatively converted commercial LAS to SPCs and utilized
a di¡erent group of three compounds (4-C6-SPC, 4-C5-
SPC and an unknown). The 3-member community of
FEMSLE Congress 2-6-03
417
strains DS-1, SPB-2 and SPH-1 utilized those six SPCs.
We believe that this community mineralizes all eight 2-
and 3-substituted LAS congeners of the 20 congeners in
commercial LAS, and that several to many more organ-
isms are needed to complete a community able to miner-
alize all SPC-like compounds from commercial LAS.
P11^143
MOLECULAR MECHANISMS OF GENERAL ACCLI-
MATION RESPONSES IN PHOTOSYNTHETIC MI-
CROORGANISMS ^ LESSONS FROM CYANOBAC-
TERIA
R. Schwarz, A. Perelman, Y. Moskovitz, J. Shaltiel, R.
Lahmi, R. Ashwal, E. Sendarskaya, D. Hacohen and P.
Sigalevich
Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan,
52900 Israel
Photosynthetic organisms face the challenge of capturing
light e⁄ciently while avoiding the oxidative stress resulting
from excess absorbed light. To minimize the damaging
consequences of the generation of reactive oxygen species,
various antioxidant defence mechanisms have evolved. In
addition, photosynthetic organisms modulate their pig-
ment content to tune their light harvesting capacity to
various environmental changes to avoid excess excitation.
We use a genetic approach to study mechanisms that al-
low photosynthetic microorganisms to cope with oxidative
stress as well as the mechanisms underlying modulation of
pigment level in response to nutrient starvation and high
light intensity. Our recent studies on thioredoxin-peroxi-
dase and catalase mutants imply speci¢c role for each of
these enzymes; catalase is essential for detoxi¢cation of
high concentrations of externally added H2O2 while thio-
redoxin-peroxidase, though dispensable for the previous
role, is exclusively crucial for growth under excessive radi-
ation and may be required to cope with oxidative stress
resulting from high light illumination. A mutant lacking a
response regulator, which modulates pigment degradation
during nutrient starvation and light stress, appears highly
sensitive to externally added H2O2 and the superoxide-
producing agent, methyl viologen. Signaling pathways of
environmental stress and mechanisms of acclimation to
various environmental constraints will be discussed in light
of characterization of the response regulator-mutant, addi-
tional mutants impaired in modulation of pigment level
during starvation and oxidative stress-resistant mutants.
418 1st FEMS Congress / Posters 103^505
P11^144
A TOXICOLOGICAL STUDY OF FLUORANTHENE
AND ITS STABLE DEGRADATION METABOLITES
E. SNepic›(1), M. Bricelj(2) and H. Leskovs›ek(1)
(1)’’Joz›ef Stefan’’ Institute, Jamova 39, 1000 Ljubljana,
Slovenia; (2)’’National Institute of Biology’’, Vec›na pot
111, 1000 Ljubljana, Slovenia
The toxicity of nine stable metabolites of £uoranthene
remaining after a 10 day incubation period with a pure
bacterial strain of Pasteurella sp. IFA and £uoranthene
was studied. Toxicological tests, including growth inhibi-
tion and immobility were made for all 10 compounds us-
ing the organisms: Pseudomonas putida (bacteria), Scene-
desmus subspicatus (algae) and Daphnia magna and
Thamnocephalus platyurus (crustaceans). The tests revealed
that with the exception of 9-hydroxy£uorene ^ which is
only four times less toxic than £uoranthene ^ all the me-
tabolites were in the order of 1000 times less toxic towards
Scenedesmus subspicatus. P. putida cells are resistant to
£uoranthene and its primary metabolites, but are inhibited
by low molecular weight intermediates, especially benzoic
acid. Fluoranthene is not toxic to crustaceans, but its pri-
mary metabolites such as 9-£uorenone and 9-hydroxy-
£uorene are highly toxic to Daphnia magna, while the
low molecular weight metabolite 2-carboxybenzaldehyde
is toxic to Thamnocephalus platyurus. These toxicological
data indicate that the freshwater ecosystem food web
could be disturbed by enhanced bacterial degradation of
£uoranthene and therefore the ecotoxicological e¡ects of
its metabolites should be carefully studied before consid-
eration of its application as a bioremediation technique.
P11^145
ISOLATION AND SELECTION OF BIOSURFAC-
TANT-PRODUCING BACTERIA
M. Shavandi, G. A. Mohebali and A. Nouhi
Biology Department, Faculty of Science, Tehran University,
P.O. Box 14155-6455, Tehran, Iran
One hundred and sixty nine bacterial strains were isolated
from oil contaminated and uncontaminated soil and
water samples. Haemolysis was used as a criterion for
the primary isolation of biosurfactant producing-bacteria.
Forty-two strains had haemolytic activity, among them
twelve strains were good biosurfactant producers by mea-
suring surface tension and emulsi¢cation activity. Two
gram positive, rod shaped strains (RAY-1 and TA-6)
showed the highest biosurfactant production when grown
on kerosene and para⁄n as a sole source of carbon. As a
FEMSLE Congress 2-6-03
result of biosurfactant synthesis the surface tension of the
medium were reduced from 68mN/m to values below
30mN/m. The RAY-1 and TA-6 biosurfactants were
mainly glycolipids in nature and were stable during expo-
sure to high salinity (10% NACL) and elevated temper-
atures (100‡c for 15 min). The culture broths were e¡ective
in recovering up to 60 ^ 65% of the residual oil from oil-
saturated sand packs, indicating potential value in en-
hanced oil recovery.
P11^146
THE CHANGE OF TOXICOLOGICAL CHARACTER-
ISTICS OF WATER SAMPLES OF SURFACTANTS
AS A RESULT OF THE CLEANING PROCESSES
WITH PHOOZONATION AND BIODEGRADATION
E. V. Shtamm(1), N. B. Kozlova(1), E. V. Alexandro-
va(1), S. D. Razumovsky(1), M. L. Konstantinova(1), J.
Emne¤us(2), M. Winther-Nielsen(3)
(1) N.M. Emanuel’s Institute of biochemical physics of
Russian Academy of sciences, Moscow, Russia; (2) Lund
University, Lund, Sweden; (3) Institute for the Water En-
vironment, H\rsholm, Denmark
In this work as surfactants samples the solutions of non-
ylphenyldodecaetoxylateoxyl (NPE) and sodium salt of de-
cylphenylsulfonic acid (LAS) in distilled water were used.
Photoozonation reactor was equipped by photocell at the
bottom to avoid problems with foams. Optimal level of the
conversion for cleaning processes with biodegradation
(Pseudomonas sp. TD (for LAS) and Commamonas testos-
teroni TI (for NPE)) was established as V 25%. E⁄ciency
of detoxication of wastewaters samples was controlled by
di¡erent biotesting methods: on survival of crustaceous
(Ceriodaphnia dubia) (1), on inhibition of chemotaxic reac-
tion of infusoria (Tetrahymena pyriformis) (2) and alga
growth (Scenedesmus quadricauda) (3), and on suppression
of process of lipid peroxidation (LPO) of liposomes (4).
The most sensitive for surfactants were tests (1) and (2).
The values LC50 were 5mg/L for LAS and 13 mg/L for
NPE (1), IC50 were 2,5mg/L for LAS and 8,5mg/L for
NPE (2). It is shown, that after process of photoozonation
the toxicity of LAS and NPE samples are not increased
(tests 1, 2). Toxicological characteristics of LAS and
NPE e¥uents from bioreactors in dependence of concen-
trations of input surfactants were studied. For all concen-
trations (tests 1, 2) the toxicity of samples after biodegra-
dation process decreased in 2-3 times except the e¥uent
from the greatest NPE input concentration (200mg/L). Ac-
cording to tests 4, 5 only NPE-samples before (250 mg/L)
and after photoozonation (200 mg/L) are toxic. The results
are discussed from point of view of development of optimal
biotechnologies of wastewaters cleaning from surfactants.
This research was support by INTAS project 99-0995.
 1st FEMS Congress / Posters 103^505 419

P11^147 P11^148

Withdrawn. DETECTION OF SPIROCHETES IN, AND ISOLA-

TION FROM, HAEMATOPHAGOUS INSECTS IN
SOUTH MORAVIA, CZECH REPUBLIC

S. Sikutova(1,2), J. Halouzka(1) and J. Knoz(2)

(1) Laboratory of Medical Zoology, Institute of Vertebrate

Biology, Academy of Sciences of the Czech Republic, Klas-
terni 2, CZ-69142, Valtice, Czech Republic; (2) Depart-
ment of Comparative Animal Physiology and General Zo-
ology, Faculty of Science, Masaryk University, Kotlarska 2,
CZ-61137, Brno, Czech Republic

During the years 1999 ^ 2002, a total of 4898 individuals

of haematophagous insects (family Culicidae, Simuliidae,
Tabanidae) were collected in several localities in South
Moravia, Czech Republic. Content of individual specimen
midgut was homogenised in a drop of phosphate saline
bu¡er and examined by dark¢eld microscopy for the pres-
ence of spirochetes. Spirochetal microorganisms were
counted and samples with more than 100 spirochetes
were cultivated in BSK-H medium. 7 spirochetal strains
were isolated (6 from mosquitoes and 1 from black£ies).
The spirochetes were observed in all developmental stages
(except eggs) of mosquitoes and black£ies. The overall
positivity in family Culicidae was 11.40% (473/4149), in
Simuliidae 23.50% (137/583); in family Tabanidae were
not found spirochetes. The high positivity of larvae C.
pipiens was detected in 1999 (87.88%) and 2000 (87.33%)
on the locality Valtice ; the signi¢cant di¡erence in posi-
tivity of C. pipiens was documented at the same time on
locality Breclav, 37.60% and 0%, respectively. In Culex
pipiens was experimentaly proved transstadial transmission
of these spirochetes. The bacteriae were observed in over-
wintering mosquitoes both on the beginning and the end
of winter period. The highest positivity was found in C.
pipiens di¡erently from other examined mosquitoe species.
The results suggested species dominance in family Culici-
dae and Simuliidae on certain localities. Present experi-
ments with spirochetal genom indicate that isolated spiro-
chetal strains could be new bacterial species.

FEMSLE Congress 2-6-03

420 1st FEMS Congress / Posters 103^505
P11^149
DEVELOPMENT OF A SYSTEM FOR EVALUATION
OF ACCELERATED MICROBIAL BIODEGRADA-
TION OF CONCRETE USED FOR IMMOBILIZA-
TION OF RADIOACTIVE WASTE
A. Sivan(1), O. Aviam(1), M. Koresh(1), G. Bar-Nes(2)
and Y. Zeiri(2)
(1) Ben Gurion University of The Negev, The Institute for
Applied Biosciences, Beer Sheva, Israel; (2) Nuclear Re-
search Center Negev, Department of Chemistry, Beer She-
va, Israel
Low level radioactive waste containing the short-lived iso-
topes Cesium and Strontium is usually disposed of
(buried) following immobilization in concrete. It is impor-
tant that the radioactive elements will not leach from the
concrete at least for a period of 10 half-live times, approx-
imately 300 years. Acid producing bacteria biodegrade
concrete because the acid they release dissolves calcium
minerals, weakening the concrete matrix. Sulfur oxidizing
bacteria such as Thiobacillus spp. produce sulfuric acid,
and are considered a primary agent of concrete biodegra-
dation. Consequently, there is a need to understand fac-
tors that a¡ect concrete stability over long periods of time
in the laboratory, using controlled systems which acceler-
ate the biodegradation. We developed a semi-continuous
system that provide conditions for rapid the biodegrada-
tion of concrete. This facilitated to test the stability of
various cementitious mixtures to biodegradation, in less
than 30 days. Signi¢cant di¡erences were observed be-
tween concrete samples that were incubated with the bac-
terial cultures and those of the control. After 30 days of
incubation with the bacteria the concrete samples lost 9-
12% of their weight while almost no change was recorded
in the weight of the control samples. The leaching of the
various elements from the concrete (Sr, Cs, Ca and Si) was
greater in the presence of the bacteria than in sterile me-
dium. A second system that operates continuously, as a
chemostat, and exposes concrete samples to logarithmi-
cally growing cultures of sulfur oxidizing bacteria is cur-
rently being evaluated.
FEMSLE Congress 2-6-03
P11^150
THE EFFECT OF SALT CONCENTRATION ON CHI-
TINOLYTIC ACTIVITY OF A HALOTOLERANT BA-
CILLUS SP.
M. R. Soudi, P. Zangouei-nejad, and S. Sepehr
Department of Microbiology, Faculty of Science, Alzahra
University, Tehran, Iran
Many crustaceans live in marine environments and micro-
bial degradation of their chitin-containing exoskeletons
may occur at the shore land after their death.A halotoler-
ant Bacillus sp. that was isolated from the sands (Gaw-
Khooni marsh, Iran) showed chitinolytic activity when it
grew at di¡erent NaCl concentrations up to 100 g l-1.
While the strain could not grow at salt concentrations
higher than 100 g l-1, the exochitinase system partially
maintained its stability in media containing 150 g NaCl
l-1. The enzyme system showed the highest activity in the
range of 0-50 g NaCl l-1, but the activity declined toward
100 g NaCl l-1 and beyond this roughly remained un-
changed. The enzyme exhibited 64% of its original activity
after exposure to 150 g NaCl l-1. The highest amount of
enzyme (15 U ml-1) was produced in the salt-containing
nutrient medium in the range of 0-35 g NaCl l-1 during the
¢rst 48 h of the culture, but a delay in the phase of enzyme
production and a decline in amount of enzyme were ob-
served as the salt concentration was increased. Under the
natural condition, drying shells bring about the habitats
bearing di¡erent sodium chloride concentrations. The in-
creasing salt concentration may decrease the bacterial
growth, but chitinolytic activity of the secreted enzymes
will be preserved at higher salt concentrations. Although
chitinolytic activity of the halophilic archaea under hyper-
saline conditions has been known previously, but this ¢nd-
ing reveals the role of the Bacillus sp. in this environmen-
tal process. It also indicates the potentiality of the strain in
production of halotolerant chitinolytic enzymes.
 1st FEMS Congress / Posters 103^505
P11^151
KEY FUNCTIONS OF IRON-REDUCING AND IRON-
OXIDIZING BACTERIA IN INNOVATIVE BIOTECH-
NOLOGIES OF WASTEWATER TREATMENT
O. Stabnikova(1,2), V. Stabnikov(1), J.-Y. Wang(2), S.
T.-L. Tay(2), V. Ivanov(2,3), and J.-H. Tay(2)
(1) Department of Microbiology and Biotechnology, Ukrai-
nian State University of Food Technologies, 68 Vladimir-
skaya Str., Kiev 01033, Ukraine; (2) School of Civil and
Environmental Engineering, Nanyang Technological Univer-
sity, Singapore 639798; (3) Ukrainian Branch of World
Laboratory, 32A Turgenevskaya Str., Kiev 01054, Ukraine
Reduction of iron (III) of iron-containing clay minerals or
iron ores by iron-reducing bacteria (IRB) can signi¢cantly
enhance anaerobic treatment of wastewater. Iron (II), pro-
ducing by enrichment culture or diverse community of
IRB, precipitates or detoxicates ammonium, phosphate,
long-chain fatty acids, sul¢de, cyanides, and phenols. Fur-
ther aerobic / microaerophilic oxidation of iron (II) or its
chelates is an alternative method for the removal of am-
monium by nitri¢cation and denitri¢cation. Ammonium,
phosphate, potassium, heavy metals, and radionuclides co-
precipitate with negatively charged iron (III) hydroxides
produced in biooxidation. Microbiological monitoring of
iron-reducing, iron-oxidizing, sulfate-reducing, fermenting
bacteria, and methanogens in the anaerobic and aerobic
processes can be performed by most-probable number
method, but £ow cytometry or £uorescence spectrometry
measurement of £uorescence in situ hybridization with 16S
rRNA-targeted oligonucleotide probes are faster and more
convenient methods.
P11^152
SCREENING FOR BACTERIOCIN BETACIN IN BA-
CILLUS SP. FROM NATURAL HABITATS
S. Stankovic, M. Tadic, T. Beric-Bjedov, J. Knezevic-Vuk-
cevic, B. Vukovic-Gacic, D. Simic
Chair of Microbiology, Faculty of Biology, University of
Belgrade, Belgrade, Yugoslavia
Most representatives of the genus Bacillus produce sub-
stances with antibiotic properties, due to association
with di¡erent phages. Temperate phage SPL of B. subtilis
carries the gene for bacteriocin betacin. Seeking for iso-
lates that produces bacteriocin, we screened 208 samples
of soil, manure, hay and straw. For detection of bacter-
iocin production, we used betacin-negative B. subtilis 1050
as an indicator strain. B. subtilis 168 and B. subtilis 1147
were used as positive controls. 13 out of 62 isolates from
FEMSLE Congress 2-6-03
421
manure, 6 out of 13 isolates from hay and straw and 24
out of 123 isolates from soil were positive for bacteriocin.
The determination of isolates that produce betacin and
their identi¢cation to the level of species are in progress.
P11^153
EFFECTS OF OXYGEN LEVELS ON CHANGES IN
MICROBIAL COMMUNITY STRUCTURE DURING
COMPOSTING
Y . Jarvis, I. Sundh
K. Steger, A
Department of Microbiology, Swedish University of Agri-
cultural Sciences, Box 7025, 750 07 Uppsala, Sweden
In this study, the phospholipid fatty acid (PLFA) analysis
was applied to determine changes in microbial community
structure during composting of source-separated organic
household waste. The process was performed in a 200 L
laboratory compost reactor at three oxygen levels, 16%,
2.5% and 1% in the compost gas. After spontaneous heat-
ing of the material, the temperature was regulated to 55‡C.
The total concentration of PLFA, an estimate of the mi-
crobial biomass, peaked after 6 days at 16% O2, whereas
at 2.5 and 1% O2 highest concentrations were reached
after 8 and 12 days, respectively. This shows that the tran-
sition to growth of thermophilic organisms was delayed at
lower oxygen concentrations. Interestingly, 1% O2 led to
higher maximal PLFA concentrations (2.1 Wmol g-1 d.w.)
than 2.5 (1.5 Wmol g-1 d.w.) or 16% O2 (1.2 Wmol g-1 d.w.).
Initially, fatty acids typical of eukaryotic cells dominated,
e.g. 16:0, 18:2, 18:1g9 and 18:0, probably due to the
presence of fatty acids from organic matter in the original
waste. In all three processes, the transition to the thermo-
philic phase led to a shift in PLFA composition to strong
dominance by typical bacterial fatty acids, such as 14:0,
and iso-, anteiso-, and 10Me- branched fatty acids. In
conclusion, the initial mesophilic phase was prolonged
for composting at 2.5 and 1% O2 compared with 16%
O2. Although the maximum in total PLFA concentrations
also di¡ered with oxygen level, no dramatic di¡erences in
the fatty acid composition were obtained.
P11^154
BACTERIAL LYSATE QUALITY DETERMINES
GROWTH YIELDS OF HETEROTROPHIC BACTERI-
AL POPULATION
D. Odic, I. Mahne, D. Stopar
University of Ljubljana, Biotechnical Faculty, Department
of Food Science and Technology, Vecna pot 111, 1000
Ljubljana, Slovenia
422 1st FEMS Congress / Posters 103^505
Cell material that is released from lysed bacterial cells can
be immobilized by heterotrophic bacterial populations,
channeling a portion of bacterial production back to the
bacterial level and making it less available to the higher
trophic levels. In a model system Vibrio sp. (DSM14379)
cells isolated from the northern Adriatic Sea were lysed
either by sonication or autoclave and were used as a sole
carbon source for growth of di¡erent heterotrophic bac-
teria. In addition, we have established a closed growth
system in which we produced cell lysate by inducing Vibrio
sp. prophages with mitomycin C. Di¡erent phage morpho-
types were induced. Vibrio sp. cells were prior to the in-
duction grown under di¡erent temperature, salt and nu-
trient conditions. This in£uenced phage community and
quality of the released cell material. Results showed that
24 out of 26 bacterial isolates tested were able to grow in a
medium with bacterial lysate as the sole energy source.
Data also indicate that heterotrophic bacteria better utilize
thermally produced lysate as compared to the biologically
prepared lysate. This suggests that the quality of the pre-
pared lysate is an important factor that determines £ow of
the material in the microbial loop.
P11^155
EFFECTS OF MODIFIED Pb-, Zn- AND Cd- AVAIL-
ABILITY IN SOIL ON MICROBIAL COMMUNITY
AND ISOPROTURON DEGRADATION
M. Suhadolc(1), M. Schloter(2), R. Schroll(2), A. Gat-
tinger(2), J. C. Munch(2) and D. Lestan(1)
(1) Biotechnical Faculty, University of Ljubljana, Ljublja-
na, Slovenia; (2) GSF ^ National Research Center for
Environment and Health, Neuherberg, Germany
E¡ects of modi¢ed heavy metal availability on the micro-
bial community structure, biomass, and microbe-mediated
degradation of the 14C-labelled herbicide isoproturon were
evaluated in soil microcosms (15 cm diameter, 30 cm
high). Soil from Mez›ica valley, Slovenia with long lasting
history of heavy metal pollution and of a total content of
1800 mg kg-1 Pb, 600 mg kg-1 Zn, and 10 mg kg-1 Cd was
used. Availability of Pb, Zn, and Cd was estimated using
sequential extraction procedure. The transition of heavy
metals towards less available fractions, induced by apatite
addition, did not a¡ect microbial biomass (total PLFA),
DNA-¢ngerprint pattern, and degradation of the isopro-
turon, but to some extent a¡ected PLFA composition. The
increase in Pb-, Zn- and Cd- availability, induced by the
addition of water-soluble metal salts, resulted in biomass
reduction and shifts in community structure (PLFA com-
position and DNA ¢ngerprints), and it reduced the rate of
herbicide degradation and mineralization. 5 % of the total
initial 14C-isoproturon was mineralized over a period of 60
days in soils with increased heavy metal availability, com-
FEMSLE Congress 2-6-03
pared to 12 % in control soils. The concentration of iso-
proturon in the upper 0-5 cm soil depth was almost three
times higher in soils with increased heavy metal availabil-
ity than in control soil (605 Wg/kg and 227 Wg/kg, respec-
tively). Degradation pathways and kinetics were not in£u-
enced by changes in heavy metal availability.
P11^156
PHOSPHATE-REMOVING BACTERIA FROM DO-
MESTIC WASTE WATER
K. Rueangsaeng, M. Sukchotiratana
Department of Biology, Faculty of Science, Chiang Mai
University, Chiang Mai 50200, Thailand
Water pollution is at present. Phosphate is one of the main
nutrients for the growth of aquatic organisms. Excess
phosphate causes eutrophication. Removal of phosphate
in the waste water before discharge into the natural water
resource will decrease water pollution. Samples of waste
water were taken from Chiang Mai University Waste
water Treatment plant once a week for two months.They
were enriched in an enrichment medium containing pep-
tone 5 g/l, yeast extract 1 g/l. The enriched cultures were
then used to isolate bacteria from the waste water by
spreading on LB medium containing casein as nitrogen
source. The colonies obtained were then puri¢ed and
tested for their phosphate-removing ability by inoculating
in the synthetic waste water. Measurement of total phos-
phorus in the from of orthophosphate was done everyday
for ¢ve days by ascorbic acid method. One of the isolates
was found to reduce phosphate in the synthetic wastewater
from 25 mg/l by 39.13% , 39.13% , 43.48% , 56.52% ,
47.83% respectively. This isolate was identi¢ed to belong
to the Genus Serratia.
P11^157
BIOMASS DISTRIBUTION AND ACTIVITY OF
METHANE-OXIDISING BACTERIA IN LAKES
I. Sundh(1), D. Bastviken(2) and L. J. Tranvik(3)
(1) Department of Microbiology, SLU, P.O. Box 7025,
SE-750 07 Uppsala, Sweden ; (2) Department of Water
and Environmental Studies, Linko«ping University, SE-581
83 Linko«ping, Sweden ; (3) Department of Limnology,
Uppsala University, SE-752 36 Uppsala, Sweden
Methane-oxidising bacteria (MOB) in lakes mitigate emis-
sion of methane to the atmosphere, and represent a po-
tential pathway for reintroduction of carbon and energy
into pelagic food-webs. In this study, the abundance and
activity of MOB in the water column were investigated in
 1st FEMS Congress / Posters 103^505
three lakes having di¡erent contents of nutrients and hu-
mic substances. The abundance of MOB was determined
by analysis of group-speci¢c phospholipid fatty acids
(PLFA) from Type I and Type II MOB, and in situ activ-
ity was measured with a 14CH4 transformation method.
The PLFA analyses showed that Type I MOB made a
substantial contribution to the bacterial communities, at
most making up almost half the total bacterial biomass.
The fatty acid composition indicated that the Type I MOB
communities consisted of members most similar to species
of Methylomonas, Methylomicrobium and Methylosarcina.
In contrast, fatty acids from Type II MOB generally had
very low concentrations. The concentration of the fatty
acids from Type I MOB was positively correlated with
the in situ methane oxidation activity, further supporting
the use of these fatty acids as indicators of the biomass of
MOB in these lakes. During summer strati¢cation, the
MOB biomass and oxidation activity were highest in the
hypo- and metalimnion, whereas under ice during winter,
maxima occurred close to the sediments. Our results dem-
onstrate that Type I MOB is often a large component of
pelagic bacterial communities in lakes, and thus imply that
these bacteria should not be overlooked in lake carbon
budgets.
P11^158
ISOLATION AND CHARACTERIZATION OF TRI-
CHODERMA HARZIANUM MUTANTS WITH AL-
TERED COLONY MORPHOLOGY AND P-FLUORO-
PHENYLALANINE RESISTANCE
A. Szekeres(1), L. Szekeres(2), L. Manczinger(1) and F.
Kevei(1)
(1) Department of Microbiology, University of Szeged,
P.O. Box 533, H-6701 Szeged, Hungary; (2) Hungarian
Academy of Sciences and University of Szeged, Microbio-
logical Research Group, P.O. Box 533, H-6701 Szeged,
Hungary
Several Trichoderma strains have been reported to be ef-
fective in controlling plant diseases, and the action of fun-
gal hydrolytic enzymes has been considered as the main
mechanism involved in the antagonistic process. Some of
these enzymes, such as L-1,3-glucanases and low levels of
proteases are produced constitutively. Strain Trichoderma
harzianum T334 is parasitizing colonies of plant pathogen-
ic fungi, (e.g. Fusarium culmorum, Pythium debaryanum
and Rhizoctonia solani) at a medium level. This strain
was mutagenised by means of UV-irradiation to p-£uoro-
phenyl-alanine resistance and altered colony morphology.
We have undertaken a mutagenetic program for the con-
struction of protease enzyme overproducing Trichoderma
strains aiming the improvement of fungal antagonistic ca-
pacity. It was revealed by means of speci¢c chromogenic
FEMSLE Congress 2-6-03
423
protease substrates, that both trypsin-like and chymotryp-
sin-like protease secretion have been elevated in most of
the mutant strains. The elevation of constitutive enzyme
activities reached in some cases levels manifold of the wild
type strain. Intensity of protease production of the distinct
mutants highly varied in function of time. The pro¢les of
isoenzymes were also di¡erent when examined by gel ¢l-
tration chromatography. Some of the mutants proved to
be better antagonists against plant pathogens in in vitro
antagonism tests. This work suggests the possibility of
using mutants with improved constitutive extracellular
protease secretion against plant pahogenic fungi.
P11^159
EFFECTIVE WASTEWATER CLEANING TECHNOL-
OGY FROM SURFACTANTS
L. A. Taranova(1), G. V. Ivaschenko(1), I. A. Koshele-
va(2), S. L. Sokolov(2), E. V. Shtamm(3), S. D. Razu-
movsky(3), J. Emneus(4), M. Winter-Nielsen(5)
(1) Institute of Biocolloidal Chemistry of National Acad-
emy of Sciences,Ukraine ; (2) Institute of Biochemistry and
Physiology of Microorganisms of Russian Academy of Sci-
ences, Russia; (3) N. M. Emanuel’s Institute of Biochem-
ical Physics of Russian Academy of Sciences, Moscow, Rus-
sia; (4) Lund University, Lund, Sweden ; (5) Institute for
the Water Environment, H\rsholm, Denmark
E⁄cient detoxi¢cation of surfactant in waste water accom-
plished by the combination of photooxidation (ozone and
UV irradiation) and biological degradation using highly
active strains of surfactant degrading bacteria. Highly ac-
tive surfactant degrading strains capable to utilize linear
alkylbenzenesulphonate (LAS) and nonylphenyletoxylate
(NPE) as a sole carbon and energy source were selected.
Most active bacterial strains were identi¢ed as Pseudomo-
nas sp. TD (LAS) and Comamonas testosteroni TI (NPE)
by microbiological and molecular biological methods.
Both strains contain large plasmids involved in genetic
control of surfactant biodegradation. The rep-PCR ampli-
¢cation based on oligonucleotide primers corresponding to
genomic repetitive sequences (ERICIR, ERIC2 and BOX-
AIR) was proposed for continuous monitoring of selected
strains during wastewater treatment. The e⁄ciency of
wastewater puri¢cation processes from surfactants in
£ow conditions increase 2 times due to combination of
photoozonation and biodegradation. Photoozonation pro-
cess in comparison with dark ozonation generated anoth-
er, less toxic compounds, which don’t a¡ect on survival
and functional activity of formed bio¢lm. The photoozo-
nation process does not increase the toxicity of water sam-
ples with LAS and NPE, biodegradation decreases the
toxicity of these samples in 2-3 times. Only NPE samples
(250 mg/L) after photoozonation and biodegradation were
424 1st FEMS Congress / Posters 103^505
toxic in algae test and in test on the reaction of lipid per-
oxidation of liposomes. Continuous monitoring of bio-
reactor’s micro£ora revealed predomination of Pseudomo-
nadaceae species in all type of bioreactors during working
period. The appearing of facultative pathogenic species
caused by technological breaks of treatment requires per-
manent monitiring of bio¢lm composition.
P11^160
DEGRADATION OF NITROAROMATIC COM-
POUNDS BY BACTERIAL STRAINS P1P AND P3A
R. Tepla(1), E. Durnova(2), L. Kotouckova(1), M. Nem-
ec(1)
(1) Department of Microbiology, Faculty of Science, Ma-
saryk University, Tvrdeho 14, 602 00 Brno, Czech Repub-
lic; (2) Department of Bacteriology, Regional Institute of
Hygiene, Partyzanske nam. 7, 72892 Ostrava, Czech Re-
public
Nitroaromatic compounds are common pollutants pro-
duced in developed countries by the industrial manufac-
turing of dyes, explosives, pesticides, herbicides etc. For
example p-nitrophenol has been widely studied as a model
for the biodegradability of organic pollutants in both sur-
face and subsurface environments. Bacterial strains P1P
and P3A were isolated from soil by selective enrichment
with p-nitrophenol. Strain P1P was found to be a member
of the genus Arthrobacter (new species), strain P3A was
mostly related to Arthrobacter ureafaciens. These both
strains are capable of utilizing some of the nitroaromatics
as the sole sources of carbon, nitrogen and energy. At
present we are studying biodegradation of p-nitrophenol
and p-nitrocatechol. Degradation of other nitroaromatic
compounds e.g., p-nitrobenzoic acid, o-nitrobenzoic acid
or o,p-nitrobenzoic acid were not proved. However, p-ni-
trobenzoic acid evidently induces division of cells of the
strain P3A. Another interesting detection is that the com-
position of fatty acids is changed after cultivation in min-
eral medium enriched with nitroaromatic compounds (p-
nitrophenol, p-nitrocatechol, p-nitrobenzoic acid) and
yeast extract. Currently, studying of enzymatic activity
of the bacterial strain P1P is in the centre of our inves-
tigation.
FEMSLE Congress 2-6-03
P11^161
MICROBIAL DIVERSITY IN RARE ALKALINE EN-
VIRONMENT: HETEROTROPHIC AEROBIC POPU-
LATIONS
I. Tiago, A. P. Chung and A. Ver|¤ssimo
Departamento de Zoologia, Faculdade de Cie“ncias e Tecno-
logia da Universidade de Coimbra, 3001 Coimbra, Portugal
Heterotrophic populations were isolated and characterised
from a rare natural alkaline groundwater environment.
The alkalinity in this particular environment probably
generated by active serpentinization results in an
Ca(OH)2 enriched, extremely diluted groundwater with
pH 11.4. Eighty-nine strains were isolated at di¡erent
pH values. To achieve the degree of diversity present in
the environment the isolates were grouped based on sim-
ilarities determined by FAME analysis and whole-cell pro-
tein (SDS-PAGE) pro¢les. To select representative strains,
for further characterization of the populations present,
each group was screened using RAPD-PCR pro¢les for
the identi¢cation of clones. Phenotypic characterization,
determination of pH tolerance, G+C content and direct
sequencing of 16S rRNA genes were performed in the
selected isolates. At least twenty-two di¡erent populations
were identi¢ed. Despite the pH of the environment, the
vast majority of the isolates were alkali-tolerant and
none was able to grow at pH superior to 10.5. All the
isolates were Gram (+), and the majority were related
with high G+C group. The two more frequently isolated
populations were found to be phylogenetic related to Die-
tzia natrolimnae and Frigoribacterium spp. Others isolates
were related with genera Microbacterium, Nocardia, Bacil-
lus, Staphylococcus, Rhodococcus, Actinomyces, Leifsonia,
Janibacter, Kytococcus, Kocuria, Rothia and Micrococcus.
P11^162
DESIGN OF NEW PROMOTERS BASED ON PRO-
MOTER CROSS-REGULATION CAPACITY OF
TWO DISTANT XYLR-TYPE REGULATORS
D. Tropel and J. R. van der Meer
Swiss Federal Institute for Environmental Science and Tech-
nology, CH-8600 Du«bendorf, Switzerland
XylR-type regulators activate expression of aromatic com-
pound degradation pathways in the presence of the aro-
matic e¡ector. While it is known that highly homologous
regulators can cross regulate non-native promoters it was
not known whether distantly related proteins would. Re-
cently we described the HbpR protein which shares only
35% identity with other XylR-type regulators and thus it
 1st FEMS Congress / Posters 103^505
appeared interesting to investigate promoter cross activa-
tion between the HbpR/PhbpC and XylR/Pu systems.
HbpR and XylR were indeed able to activate each other’s
promoter although the transcription activity from the het-
erologous promoter was always lower than from the nat-
ural promoter. We discovered that the poor HbpR depen-
dent activation from Pu was caused by a poor binding on
the operator region. To change regulator binding we mu-
tated the operator region on PhbpC and tested the ability
of these mutants to become activated by HbpR and XylR.
Two mutants were obtained, which were e⁄ciently acti-
vated by both HbpR and XylR showing that promoters
can be created, which are permissive for both regulators.
Surprisingly a PhbpC promoter containing the XylR bind-
ing sites could be induced ¢ve-fold higher by XylR than
the native Pu promoter. Moreover, some mutants dis-
played a ten-fold lower basal HbpR-dependent transcrip-
tion activity than the native promoter while keeping max-
imum induction. These results showed that mutations in
the regulator binding sites allow to modify binding specif-
icities, to create hybrid promoters responsive to two dis-
tant related activator proteins and to increase the tran-
scription signal to noise ratio.
P11^163
ENHANCED BIODEGRADATION OF OIL SHALE
CHEMICAL INDUSTRY SOLID WASTES BY PHY-
TOREMEDITION AND BIOAUGMENTATION
J. Truu, E. Heinaru, E. Talpsep, L. Ka«rme, E. Vedler and
A. Heinaru
Institute of Molecular and Cell Biology, University of Tar-
tu, 23 Riia Str, 51010, Tartu, Estonia
The processed oil shale (semi-coke) contains several organ-
ic and inorganic compounds (oil fractions, sulphides, phe-
nolic compounds, polycyclic aromatic hydrocarbons).
Field experiments were carried out in order to test the
e¡ect of phytoremediation and bioaugmentation for reme-
diation of pollutants in semi-coke. Four pilot test plots (50
m2) were established at semi-coke depository in July 2001.
For bioaugmentation experiment the set of bacteria con-
sisting of three biodegradative strains isolated from nearby
area was selected. Several molecular microbiological meth-
ods (PCR-DGGE, RISA, RAPD) were used to assess and
compare the microbial community structure and diversity
as well as the presence and diversity of biodegradative
genes in collected samples. The dominant bacterial species
based on 16S rDNA sequences in semi-coke samples were
also identi¢ed. These analyses revealed that semi-coke mi-
crobial community is characterized by few dominant pop-
ulations and possesses low diversity. The phytomanipula-
tion increased the number of bacteria and diversity of
microbial community in semi-coke. In plots with plants
FEMSLE Congress 2-6-03
425
dominated two di¡erent multicomponent phenol hydrox-
ylases (LmPH) belonging to low- and moderate Ks kinetics
groups. Within a one and half year period starting from
establishment of test plots, the concentration of phenolic
compounds decreased up to 35% and oil products up to
three times at plots with vegetation compared to control.
Bioaugmentation increased biodegradation intensity of oil
products up to 50% compared to untreated controls.
P11^164
MICROBIAL ACTIVITY, BIOMASS AND COMMU-
NITY STRUCTURE IN SOIL-ROOT INTERFACE
AND BULK SOIL UNDER SCOTS PINE, NORWAY
SPRUCE AND SILVER BIRCH
M. Truu, J. Truu, K. Lo‹hmus, M. Ivask and A. Kanal
Environmental Protection Institute, Estonian Agricultural
University, 4 Akadeemia Str, 51003, Tartu, Estonia
Microbial biomass, respiration and community level phys-
iological pro¢les using Biolog microplates were determined
from four di¡erent soils under Scots pine, Norway spruce
and silver birch. Seedlings of these tree species were grown
for ¢ve months. The three soils originated from former
agricultural lands and one from open cast oils shale min-
ing. The aim of the project was to assess the impact of
di¡erent tree species on soil chemical and microbiological
parameters. CLPPs separated microbial communities from
di¡erent three species only in case of untransformed data,
indicating changes in overall microbial activity. While
bulk soil samples had practically similar microbial activity,
the soil-root interface (SRI) microbial communities exhibit
bigger di¡erences in activity values. Highest activities were
found in case of microbial communities in SRI of Scots
pine and Norway spruce. The structure of microbial com-
munities of bulk soil samples showed rather big variation
compared to SRI samples. Microbial biomass C and res-
piration activity did not di¡er under di¡erent seedlings.
The characterization of soil samples with molecular meth-
ods is in progress.
P11^165
PROMOTER ANALYSIS OF THE DEHALOGENASE
IVa GENE OF BURKHOLDERIA CEPACIA MBA4
J. S. H. Tsang and W. Y. K. Chung
Molecular Microbiology Laboratory, Department of Bot-
any, The University of Hong Kong, Pokfulam Road,
Hong Kong S. A. R., China
Burkholderia cepacia MBA4 can utilise 2-haloacids as the
energy and carbon source for growth. MBA4 produces a
426 1st FEMS Congress / Posters 103^505
haloacid dehalogenase (DehIVa) that can remove halogen
from 2-haloacids. DehIVa has been puri¢ed and charac-
terized biochemically. DehIVa is a dimeric protein of
45kDa and its expression was detected only in the presence
of halogenated substrate such as mono-bromoacetate and
2-mono-bromopropionate. This suggested that the expres-
sion of DehIVa is regulated. In this presentation, we re-
port the identi¢cation of the regulatory region for DehIVa
gene (deh4a). An in vivo reporter assay system has been
constructed to examine the putative promoter region. Plas-
mid pUCP28T, which contains a broad-host-range repli-
con (ori1600) and a trimethoprim resistance gene, is able
to replicate in many Gram negative bacteria including
MBA4. This plasmid was used as the vector backbone in
the reporter assay system. A green £uorescent protein gene
(gfpuv) was used as the reporter gene. The amount of
GFPuv produced in the cells was quanti¢ed by a £uores-
cent spectrophotometer. The £uorescent intensity is pro-
portional to the functional activity of the deh4a promoter.
Construct with 0.8kb promoter sequence of deh4a was
su⁄cient to express DehIVa-GFPuv fusion protein in a
regulatable manner. 5’-Deletion was carried out and de-
rivatives with shorter promoter sequences have been con-
structed and analysed. All the constructs except one with
no promoter sequence, were able to drive the regulatable
expression of the recombinant protein. The smallest con-
struct contains only 100 bp of upstream sequence.
P11^166
CHARACTERISATION OF THE PREDOMINANT BA-
CREX CLUSTER MEMBERS OF BACILLUS SPE-
CIES IN SOIL BY CULTIVATION AND 16S rDNA
ANALYSES
V. A. Tzeneva, Y. Li, A. Felske, W. M. de Vos, E. E.
Vaughan, H. Smidt and A. D. L. Akkermans
Laboratory of Microbiology, Wageningen University, Hes-
selink van Suchtelenweg 4, 6703 CT Wageningen, The
Netherlands
Culture independent molecular tools were developed for
16S rRNA-targeted detection of Bacillus species from the
bacrex-cluster that was recently discovered to be abun-
dant in soil samples. New primers were designed for ba-
crex-speci¢c ampli¢cation of the V6-V8 16S rRNA re-
gion. These primers are suitable for use in combination
with Denaturing Gradient Gel Electrophoresis (DGGE),
which allows for the rapid evaluation of composition and
activity of bacterial populations at moderately high tem-
poral resolutions. Convenient sources of information
about the microbial succession over time were archived
soil samples from the Dutch Wieringermeer polder, in
which the environment has changed from anaerobic to
aerobic and from saline to fresh water during the forma-
FEMSLE Congress 2-6-03
tion of the polder, since 1932. After each period of change
the bacterial ecosystem remained in relative equilibrium
over a long time. The in£uence of the di¡erent crops on
the bacterial community in the polder soils during the time
was also studied. DGGE results showed that the type of
planted crop in£uenced the total microbial community
composition in the samples, as well as the bacrex-cluster.
The molecular methods were complemented with cultiva-
tion experiments. Although some of the polder samples
were older than 50 years bacrex strains were isolated.
The comparison of the bacrex communities in the old
polder soil samples with those in the fresh soils indicated
that they were more abundant in the latter. The present
study contributes to our knowledge on the diversity and
abundance of this interesting group of microbes in soils
throughout the world.
P11^167
SELECTION OF 2,4-DICHLOROPHENOXYACETIC
ACID DEGRADING BACTERIA
N. Udompratyaporn(1), W. Sonthichai(1), S. Wang-
karn(2) and S. Bovonsombut(1)
(1) Department of Biology, Faculty of Science, Chiang Mai
University, Chiang Mai, 50200, Thailand; (2) Department
of Chemistry, Faculty of Science, Chiang Mai University,
Chiang Mai, 50200, Thailand
A selection of 2,4-dichlorophenoxyacetic acid (2,4-D) de-
grading bacteria were isolated from soil samples collected
at Doi Suthep and Doi Inthanon, Chiang Mai, Thailand.
The strains were isolated in basal medium containing 50
ppm of 2,4-D under aerobic condition at room temper-
ature. A total of 62 strains were isolated based on their
capabilities of degrading of 2,4-D. Bromocresol purple
was used as an indicator for detecting the hydrochloric
acid obtained from the pathway of degradation. Thirteen
isolates were found to have a high performance of degrad-
ing of 2,4-D.
P11^168
REDUCTION OF BROMATE TO BROMIDE
COUPLED WITH ACETATE OXIDATION BY A
MIXED MICROBIAL CULTURE
C. G. van Ginkel and B. van der Togt
Akzo Nobel Chemicals Research, P.O. Box 9300, 6800 SB
Arnhem, The Netherlands
Bromate, a genotoxic carcinogenic substance, is an oxi-
dized form of bromine. Bromate is found in water because
it is, for example, a disinfection byproduct of the ozona-
 1st FEMS Congress / Posters 103^505
tion of bromide-containing waters. Given the carcinogen-
ity of bromate, knowledge of its biological conversion is
essential to assess the impact on the environment. Bromate
is reduced by denitrifying and chlorate-reducing bacteria,
but these organisms cannot utilize bromate without prior
induction by nitrate or chlorate, respectively. We provide
the ¢rst example of microorganisms utilizing bromate as
sole electron acceptor for anaerobic acetate oxidation. In
enrichment cultures at initial bromate concentrations of
s 6 mM no reduction of bromate was observed. A con-
tinuous-£ow packed-bed column was therefore set-up en-
abling enrichment of microorganisms at low concentra-
tions. This column was packed with activated sludge
from a biological treatment plant and fed with bromate
and acetate for several months. Under anaerobic condi-
tions, complete reduction of bromate was demonstrated by
recovery of stoichiometric amounts of bromide from bro-
mate. The amount of acetate that is utilized for bacterial
growth without concurrent loss of bromate accounts for
about 30% of total acetate consumption. In addition a
logarithmic growth curve was obtained in a batch culture
inoculated with sludge from the column at an initial bro-
mate concentration of 0.12 mM. The growth rate derived
from this curve was 0.04 h-1. At high concentrations bro-
mate-utilizing microorganisms appear to su¡er from inac-
tivation by the formation of a reactive product. These
results prove the existence of microorganisms capable of
utilizing bromate as sole electron acceptor for growth.
P11^169
BIODEGRADATION KINETICS OF ALKYLAMINES
USED IN ENVIRONMENTAL RISK ASSESSMENT
C. G. van Ginkel, M. G. J. Geurts and B. van der Togt
Akzo Nobel Chemicals Research, P.O. Box 9300, 6800 SB
Arnhem, The Netherlands
Primary fatty amines contain a nitrogen atom attached to
one long alkyl chain. Commercial primary alkylamines
such as cocoamine are usually produced as a mixture of
homologs. The environmental risk of primary alkylamines
is assessed through PEC (predicted environmental concen-
tration) / PNEC (predicted no e¡ect concentration) ratios.
The key factor involved in controlling the environmental
concentrations of primary alkylamines is the e⁄ciency of
wastewater treatment systems. The EU Technical Guid-
ance Document (TGD), assigns rate constants based on
ready and inherent test data; these are not measured rate
constants but are ‘‘guesstimates’’. The models and rate
constants described in the TGD may be disputed during
the environmental risk assessment procedure. To study the
biodegradation of primary alkylamines in a proper way
modi¢ed ready biodegradability tests and simulation tests
of activated sludge treatment plants were carried out. Us-
FEMSLE Congress 2-6-03
427
ing data derived from these tests it could be demonstrated
that cocoamine is removed s 99% in conventional acti-
vated sludge treatment systems. Based on the broad sub-
strate speci¢city of microorganisms degrading alkylamines
with respect to the alkyl chain length and degradation via
L-oxidation, it is very likely that the biodegradation po-
tential of all primary alkylamines is comparable.
P11^170
CHARACTERIZATION OF THE BACTERIAL AND
ARCHAEAL COMMUNITIES OF TWO SYNTHESIS
GAS FED BIOREACTORS TREATING SULFATE
AND METAL RICH WASTEWATER
B. H. G. W. van Houten, K. Roest, A. P. H. M. Hermans,
V. A. Tzeneva, A. D. L. Akkermans and A. J. M. Stams
Laboratory of Microbiology, Wageningen University, Hes-
selink van Suchtelenweg 4, 6703 CT Wageningen, The
Netherlands
A new type of anaerobic wastewater treatment system has
been used e¡ectively for the treatment of wastewater rich
in sulfate and various metals but low in organic carbon.
Sulfate and metals are removed simultaneously as a result
of the sul¢dogenic activity of dissimilatory sulfate-reduc-
ing bacteria. The system is based on the retention of sul¢-
dogenic sludge in a gas-lift loop reactor that is fed with
hydrogen rich synthesis gas as the electron donor and an
organic carbon source like acetate. However, not much
was known about the microbial composition of the anaer-
obic sludge mediating this process. We have studied the
microbial communities of two separate systems by both
cultivation dependent and independent approaches. The
two systems were di¡erent in wastewater composition,
scale and set-up. Most Probable Number counts revealed
that the microbial communities of both systems were dom-
inated by chemolithoheterotrophic sulfate-reducing bacte-
ria. Chemolithoauthrophic sulfate-reducing bacteria,
methanogens and homoacetogens were present in much
lower numbers. Clone libraries of the 16S rDNA of both
sludges were constructed. The Bacterial clones of both
libraries showed high similarities with closely related spe-
cies of the genera Desulfovibrio and Desulfomicrobium. The
Archaeal clones showed high similarities with closely re-
lated species of the genera Methanobacterium and Metha-
nospirillum. DGGE analyses of 16S rDNA fragments
showed a low diversity in both the Bacterial and Archaeal
populations. The DGGE pro¢les of both sludges showed a
highly similar pattern. These results suggest that the mi-
crobial communities consist of specialised, phylogeneti-
cally related populations of sulfate-reducing bacteria and
methanogens.
428 1st FEMS Congress / Posters 103^505
P11^171
ENVIRONMENTAL MONITORING OF ANTAGO-
NISTIC TRICHODERMA
S. Fanti, A. Barbieri and G. Vannacci
Department of Fruit Science and Plant Protection ‘‘G. Scar-
amuzzi’’, Sect. Plant Pathology, University of Pisa, Via del
Borghetto, 80, 56124 Pisa, Italy
Isolate-speci¢c DNA ¢ngerprinting of antagonistic Tricho-
derma can be useful for monitoring their behaviour into
the environment. Genetic variability of two promising iso-
lates of Trichoderma e¡ective against Cytospora canker of
peach was assessed by DNA ampli¢cation with 10mer
random primer, microsatellite and M13 minisatellite.
M13 primer gave unique ¢ngerprinting for both isolates
if compared with those of 27 isolates belonging to 6 spe-
cies and with those of 65 Trichoderma collected from
peach trees and soil under their canopy where the release
of the antagonists was planned. The two antagonists were
then applied and Trichoderma isolates were collected by
selective medium at di¡erent time during the successive
12 months. The presence of antagonists was monitored
by M13 primer ampli¢cation after 4 and 12 months. The
65 isolates assessed before the release of the antagonists
came all but one from soil and gave 15 electrophoretic
pro¢les di¡erent from those of antagonists. After 4
months, 203 isolates were evaluated and gave rise to 23
pro¢les di¡erent from antagonists. Four of them were
present in pre-release. After 12 months 97 isolates were
assessed and gave rise to 13 pro¢les di¡erent from antag-
onists, three of them similar to three from pre-release as-
sessment and four already detected after 4 months. Only
one pro¢le was present in all the assessments and was one
of the few isolates coming from above soil plant parts.
Released antagonists were recovered after 4 months from
above plant parts but not from soil. After 12 months
released antagonists were not detected.
P11^172
INFLUENCE OF P. SYRINGAE PV. CORONAFA-
CIENS LIPOPOLYSACCHARIDE ON A. TUMEFA-
CIENS GALLS ON POTATO DISCS
L. M. Vashchenko, R. I. Gvozdyak, L. A. Pasichnyk
Institute of Microbiology and Virology of the National
Academy of Sciences of Ukraine, Kyiv, Ukraine
Lipopolysaccharides (LPS) of Gram-negative bacteria
have various biological properties. They can in£uence on
synthesize of antibodies, cytokines, play important role in
virulence. In these work we present results of our inves-
FEMSLE Congress 2-6-03
tigation of the in£uence of LPS of P. syringae. pv. coro-
nafaciens on tumor induced by A. tumefaciens on wound
surface of potato. LPS was extracted from strain Pseudo-
monas syringae pv. coronafaciens 9030 that was isolated
from injured tissues of oat in Ukraine. It have been estab-
lished that P. syringae. pv. coronafaciens 9030 LPS have
relatively high antitumor activity. P. syringae. pv. corona-
faciens LPS in concentration 10 mg/ml reduced induction
of tumors by A. tumefaciens strain 9052 on 84 %, and by
A. tumefaciens strain 9054 on 74%. Antitumor activity of
LPS decreased proportionally to reduction of concentra-
tion of its solution. LPS at concentration 0,1 mg/ml re-
duced induction of tumors on 23%. Antitumor activity did
not depend on time of processing of potato discs. The
mechanism of antitumor activity of P. syringae. pv. coro-
nafaciens LPS is not established yet. However antitumor
activity of this LPS is not associated with antibacterial
activity against A. tumefaciens. LPS of P. syringae. pv.
coronafaciens 9030 does not in£uence on attachment of
A. tumefaciens cells to potato.
P11^173
THE YEAST SACCHAROMYCES CEREVISIAE AS
AN EXPERIMENTAL EUKARYOTIC MODEL FOR
ASSESSING HERBICIDE TOXICITY
C. A. Viegas, M. G. Cabral, M. C. Teixeira, I. Sa¤-Correia
Centre for Biological and Chemical Engineering, Instituto
Superior Te¤cnico, Av. Rovisco Pais, 1049-001 Lisbon, Por-
tugal
The widespread application of herbicides may give rise to
considerable environmental concern. Simple and cost-ef-
fective screening methods are required to estimate their
toxicity. We have used the easy to cultivate and non-
pathogenic experimental eukaryotic model, Saccharomyces
cerevisiae, to compare the toxicity of herbicides of di¡er-
ent chemical classes and of 2,4-dichlorophenol (2,4-DCP),
an intermediate of the herbicide 2,4-dichlorophenoxyacetic
acid (2,4-D) degradation. Toxicity assessment was focused
on their inhibitory e¡ects on yeast growth, based on mi-
crotiter plate susceptibility assays. The relative toxicity
established, based on the No Observed E¡ect Concentra-
tion, estimated as the maximum concentration of the
tested compound having no e¡ect on yeast growth, was:
2,4-DCP s alachlor, metolachlor s s metribuzin s 2,4-
D, 2-methyl-4-chlorophenoxyacetic acid (MCPA). This or-
der of toxicity correlated with their lipophilicity and with
toxicity indexes reported in the literature for freshwater
organisms. Nevertheless, disruption of intracellular pH
homeostasis is also among the mechanisms underlying
the inhibitory action of 2,4-D and MCPA, at low pH,
di¡erently from 2,4-DCP. The deleterious e¡ects of the
acid herbicides MCPA and 2,4-D and of 2,4-DCP on S.
 1st FEMS Congress / Posters 103^505
cerevisiae growth and viability were examined having in
consideration the natural conditions usually present or
that may vary in natural ecosystems, a¡ecting their toxic-
ity. Results concerning the in£uence on the toxicity level
of external pH and of the phase of growth and the size of
the yeast cell population used as inoculum [1], will also be
presented.
[1] M.G. Cabral et al. Chemosphere (in press)
P11^174
THE STUDY OF THE MICROBIOLOGICAL ACTIV-
ITIES OF THE PEAT SOILS IN THE FOREST-
STEPPE ZONE OF RUSSIA
E. M. Volkova, N. V. Pokydyscheva, E. A. Rumyanzeva
Tula State Pedagogical University, pr. Lenina, 125, Tula
300026, Russia
The forest-steppe zone of Russia is characterized by the
low paludi¢cation. The mires occupy not more than 1 %
of the area, but play an important role in the conservation
of the biological diversity, in the carbon balance of the
region, in regulation of hydrological processes. The rate
of transformation of the organic matter of the peat is one
from the important attributes of the mires. The parameter
characterizes the potential fertile of the peat soils and de-
¢nes the directions of using it. The study of the peat de-
posits of the valley and karst mires in the northern part of
Middle-Russian height gives evidence about not high ac-
tivities of the microorganisms. The biggest biomass of
bacterium, actinomycetes and fungi we observed in the
top level of the peat (acrotelm: 0-1,5 m). This phenome-
non correlates with aeration and temperature of the peat.
It is the reason for the high rate of decomposition of the
plants remains. The maximum loss of the mass of the
cellulose material in that level of the peat constitutes 10-
12 % in the month. With increase in the depth of the peat
deposit we observed the reduction of the abundance of the
microorganisms and of the rate of the transformation of
organic substance. The intensity of the microbiological
processes may be shown in the rate of secretion of carbon
dioxide by the peat deposits. In our experiments we show
that the emission of the gas from the peat during the
season (May ^ September) was changing from 5 to 80
mg/m2 in hour. The parameter depends on the composi-
tion and structure of the community of the microorgan-
isms and the conditions which determinate the maximum
capable of living of them. The low intensity of the decom-
position processes of the plant remains in the peat ensures
the conservation of organic matter in the mire’s ecosys-
tems.
This research is supported by the Russian Fund of Fun-
damental Investigation (02-04-96011).
FEMSLE Congress 2-6-03
429
P11^175
EVALUATION OF DIFFERENT METHODS FOR OB-
TAINING AND STORAGE OF SAMPLES FROM MA-
RINE MACROORGANISMS TO ISOLATE MICRO-
ORGANISMS THEREFROM
K. Siebert(1), J. Freund(2), A. Muscholl-Silberhorn(2), R.
Wirth(1)
(1) Universita«t Regensburg, Mikrobiologie-NWFIII, Uni-
versita«tsstrasse 31, D-93053 Regensburg; (2) THETIS-
IBN GmbH, Notkestrasse 85, D-22607 Hamburg
Marine microorganisms are a highly interesting potential
source for new bioactive compounds: of all known
110,000 natural compounds only 7,000 are derived from
marine habitats, though 70% of our planet’s surface is
ocean. In many cases, microorganisms derived from ma-
rine macroorganisms are the actual producers of bioactive
compounds. thetis-ibn GmbH isolates marine microor-
ganisms at large scale, tests them for production of bio-
active compounds and determines the structures of the
latter by NMR, mass- and X-ray spectroscopy. As source
for the microorganisms, marine macroorganisms are used,
which are collected from all over the world. The macro-
organism material has to be stored on board the collection
vessels for several weeks in a way that allows maximal
recovery of microorganisms in the lab. Here we report
on our results of a comparison of 4 di¡erent methods to
store macroorganismic material. Our data indicate that
highest recovery of microorganisms is possible if samples
were collected and frozen by a new protocol (soaking
macroorganisms in 5% DMSO, followed by freezing and
storage in dry ice); the use of a commercial system (Roti-
Store; storage at ^ 20‡C) gave somewhat reduced survival.
Storage of material treated like bacterial glycerol cultures
(storage at ^ 20‡C) or covered by mineral oil (storage at
4‡C) resulted in unacceptable survival rates.
P11^176
MODELS OF 2,4,6-TRINITROTOLUENE (TNT) INI-
TIAL CONVERSION BY YEAST
S. A. Zaripov, J. F. Abdrakhmanova, A. V. Garusov and R.
P. Naumova
Kazan State University, Dept. of Microbiology, Kremlyev-
skaya str., 18, Kazan, 420008, Russia
The widespread distribution of nitroaromatic compounds
in the environment is related to their use as explosives.
2,4,6-Trinitrotoluene (TNT) is the major component of
military and industrial explosives. Diversity of microor-
ganisms and their powerful metabolic capabilities is a
430 1st FEMS Congress / Posters 103^505
huge reserve of exploitation in the developing technologies
in TNT-polluted areas remediation. This work is aimed to
investigate the metabolic capabilities of microeukaryotic
microorganisms ^ yeast. Independently of incubation con-
ditions, TNT undergoes the reductive transformation. Sac-
charomyces sp. ZS-A1 converted the parent compound
practically into isomers hydroxylaminodinitrotoluene
(HADNT) mixture only. On the contrary, the TNT trans-
formation by the Candida sp. AN-L13 strain resulted in
the stoichiometric accumulation of hydride Meisenheimer
complex (H-TNT). The Candida sp. AN-L14 strain dis-
played the combination of the two pathways, with accu-
mulation of almost equal quantities of both intermediates
of the TNT reductive attack. Three other Saccharomyces
strains transformed TNT to a greater or lesser extent into
HADNT, while the additionally tested Candida strains
produced the mixture of HADNT and H-TNT. No strains
analogous to Candida sp. AN-L13, which performs a prac-
tically unidirected reduction of the aromatic ring, were
found among other strains in our collection. The strain
Candida sp. AN-L13 deserves special attention because
of its ability to perform the initial TNT conversion steps
and also its capability to utilize crude oil and several in-
dividual aliphatic and aromatic hydrocarbons. This strain
as well as other microorganisms with comparable metabol-
ic capabilities is very interesting not only for academic
research but possess vast potential for bioremediation of
areas with complex contaminations.
P11^177
BACTERIA DEGRADING PHENOL AND ITS
CHLORINATED DERIVATIVES
N. V. Zharikova, V. V. Korobov, E. Yu. Zhurenko, L. R.
Ga¢yatova, A. A. Michalev, V. V. Madjar, E. G. Galkin, T.
V. Markusheva
Institute of Biology Ufa Scienti¢c Centre Russian Academy
of Sciences, 69, pr. Oktyabrya, 450054, Ufa, Russia
The bacterial strains have been isolated from modern bac-
terial community of soil ecosystem, exposed to long-time
e¡ect of a large group of petrochemical manufacture pol-
lutants such as phenol and its chlorinated derivatives. Ac-
cording to cultural, morphological, physiological, bio-
chemical features and 16S rDNA sequencing they have
been classi¢ed as the strains of Bacillus, Klebsiella, Serra-
cia and Pseudomonas genera. Conversion of phenol, 2,4-
dichlorophenol (2,4-DCP), 4-chlorophenoxyacetic acid (4-
CPA), 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-
trichlorophenoxyacetic acid (2,4,5-T) are investigated in
model systems and catabolism stages have been revealed.
The toxic intermediates have been not identi¢ed in course
of xenobiotics destruction. Genes of 2,4-D and 2,4,5-T
catabolism have been located on plasmids of the men-
FEMSLE Congress 2-6-03
tioned strains. The application of some Bacillus strains
for puri¢cation of chemical and petrochemical sewage
from phenol and its derivatives has been shown.
The study is supported by RFBR grant 02-04-97911 and
by grant of FSSTP HIntegration of Science and Education
of Russiag (n-0029).
P12^1
QUORUM-SENSING SIGNAL MOLECULE PRO-
DUCING PSEUDOMONADS FROM ARCTIC ICE
AND WATER SAMPLES
J. Ambroz›ic›(1), T. SNes›ok(1), M. Grabnar(1) and G. Av-
gus›tin(2)
(1) University of Ljubljana, Biotechnical Faculty, Depart-
ment of Biology, Vec›na pot 111, 1000 Ljubljana, Slovenia;
(2) University of Ljubljana, Biotechnical Faculty, Zootech-
nical dept., Groblje 3, SI-1230 Domz›ale, Slovenia
During an attempt to isolate fungi from arctic ice and
water samples on chloramphenicol containing growth me-
dia, growth of several bacterial colonies was observed too.
Approximately one third of isolated strains was found to
be quorum-sense positive as indicated by the production
of purple pigment by the Chromobacterium violaceum
CV026 biosensor strain. The phylogenetic analysis of the
quorum-sense positive isolates was undertaken by the
comparative sequencing of the small ribosomal sub-unit.
The isolates were phylogenetically a⁄liated to the group
of pseudomonads, and within this group to the phyloge-
netic subgroups P. tolaasii, P. amygdali and P. azotofor-
mans (as de¢ned by the ribosomal database project). N-
acylhomoserine lactone (AHL)-dependent quorum-sensing
system was already described in species from this groups,
however mainly in plant-associated strains and not from
psychrophilic or psychrotolerant isolates from arctic ice or
water samples. Since the pigmentation intensity of the
sensor strain varied substantially it appears that the tested
isolates produce di¡erent AHLs.
 1st FEMS Congress / Posters 103^505
P12^2
SENSITIVITY AND ACTIVITY OF BULGARIAN
VAGINAL LACTOBACILLI
P. M. Andreeva(1), A. D. Shterev(1), S. P. Diankova(2),
V. A. Bogdanova-Chipeva(3) and S. T. Danova(2)
(1) Medical University, Department of Obstetrics and Gy-
necology, University Hospital ‘‘Maychin Dom’’, So¢a, Bul-
garia ; (2) Institute of Microbiology, Bulgarian Academy of
Sciences, So¢a, Bulgaria ; (3) NBPMKK, So¢a, Bulgaria
Bacterial vaginosis (BV) is one of the most common vag-
inal infections. This condition can be characterized by a
decrease or elimination of Lactobacilli and an overgrowth
of mixed vaginal £ora (Gardnerella vaginalis, Mobiluncus,
Bacteroides, Prevotella, Mycoplasma). The factors that ini-
tiate the shift in the vaginal ecology are incompletely
understood. The present work was undertaken to study
the e¡ect of spermicidal contraceptives ^ Nonoxynol-9
(N-9) and Benzalkonium chloride, and di¡erent antibiotics
as a part of potential risk factors, promoting elimination
of lactobacilli. As a ¢rst stage, in a university hospital, at
131 reproductive age Bulgarian women was made special
enquire. Based on Amsel’s and Nugent’s criteria the diag-
nosis of BV was established in 54,96%. The percentage of
women using spermicides (12,5%) from this group was
fourfold higher in comparison with healthy women
(3,4%). In order to verify these results we tested in vitro
the e¡ects of the spermicides on a group of Bulgarian
vaginal lactobacilli. In therapeutic concentration Benzal-
conium de¢nitely depress lactobacilli, and N-9 ^ in 68,95%
Y 0,3. More than 50% from the lactobacilli were strongly
sensitive to the ten di¡erent antibiotics. It’s very possible
these factors to disrupt vaginal micro-ecology in-vivo. Fur-
thermore, at the examined lactobacilli we render an ac-
count of inhibition activity against Gardnerella vaginalis
ATCC 14018 in-vitro. Consequently the factors, which
have adverse e¡ect over the lactobacilli can be referred
to the risk factors for BV. To have knowledge of them
is a condition to develop the new approach for prophy-
lactics and treatment of BV.
P12^3
EFFECT OF INOCULUM SIZE ON MUTATION
RATES IN SALMONELLA TYPHIMURIUM
E. V. Babynin and F. Sh. Gizatullin
Department of Genetics, Kazan State University, Kazan,
Russian Federation
We have previously reported that His+ revertants in strain
Salmonella typhimurium BA13 occurred in response to his-
FEMSLE Congress 2-6-03
431
tidine starvation. Here we examined in£uence of numbers
of plated bacteria on mutation rates of His+ and Thy+
mutants in a thymine-requiring (thyA) derivative of BA13.
We showed that mutation frequencies of His+ and Thy+
revertants increased with a decrease of cell densities on
selective plates. The Thy+ and His+ mutation frequencies
were negatively correlated with numbers of plated cells (-
0.65 and -0.74, respectively, P 6 0.001). At the same time,
cell-free £uids obtained from starving populations of
BA13 had inhibiting e¡ect on the mutations under analy-
sis. These results suggest that mutagenesis is under control
of extracellular factors accumulating in environment. The
potential role of quorum sensing in adaptive mutagenesis
is discussed.
P12^4
THE EFFECT OF MICROORGANISMS ON FE-AS
PRECIPITATION IN HOT SPRING BIOMATS AT
CIRCUMNEUTRAL PH
N. Bel’kova(1,2), K. Tazaki(1), V. Parfenova(2), Ju. Za-
kharova(2) and V. Okrugin(3)
(1) Kanazawa University, Kanazawa, Japan; (2) Limno-
logical Institute SD RAS, Irkutsk, Russia ; (3) Institute of
Volcanology FED RAS, Petropavlovsk-Kamchatskii, Russia
Iron is fourth of the most abundant elements in the
Earth’s crust and biologically important for living organ-
isms. While arsenic is distributed in the upper crust mostly
at low concentration, tend to be found in the environ-
ments, which contain geologically young sediments and
is generally toxic to life. Some microorganisms exist which
can metabolize forms of these elements. Water and bio-
mats samples collected from hot springs at Vilyuchinskaya
hydrothermal system (Kamchatka Peninsula, Russia) were
analyzed using methods analytical chemistry and micro-
biology. Chemical analysis of hot spring water revealed
high concentration of iron, manganese and presence of
toxic elements, like arsenic and strontium. Enumeration
total bacteria number and enzymatically active bacteria
in hot spring water revealed low content of microorgan-
isms, but high percent of active bacterial cells. Low num-
ber of bacteria in hot spring waters could be resulted from
high mineralization and high concentration of toxic ele-
ments, detected in these waters; and average high percent
of enzymatically active bacteria in natural geosystem can
cause active processes of biochemical reactions. Iron and
arsenic were determined in bacterial cells by electron mi-
croscopy, equipped with an energy dispersive X-ray spec-
trometer. Iron-bacteria were isolated from iron-rich bio-
mats on the selective agar medium. Strain V2-1 was
described and used for laboratory experiments. Its cells
were gram-positive short rods with length of 2 to 3 Wm,
non-motile, and had growths on the cell walls. Bacterial
432 1st FEMS Congress / Posters 103^505
mats in modern geothermal areas are unique natural eco-
systems and microorganisms inhabitated here are interest-
ing in biochemical characteristics and using in bioremedi-
ation.
P12^5
MICROCOLONY FORMATION IN PSEUDOMONAS
AERUGINOSA BIOFILM AS A RESULT OF PROTO-
ZOAN GRAZING
T. Bergfeld(1,2,3), S. A. Rice(1,2) and S. Kjelleberg(1,2)
(1) School of Biotechnology and Biomolecular Sciences,
University of New South Wales, Sydney, Australia ; (2)
Centre for Marine Biofouling and Bio-Innovation, Univer-
sity of New South Wales, Sydney, Australia ; (3) Federal
Institute of Hydrology, Koblenz, Germany
The human pathogen Pseudomonas aeruginosa is com-
monly found in the environment growing as a bio¢lm.
Bio¢lms are believed to be adaptive strategies to protect
bacteria from stress. To investigate if bio¢lms also protect
bacteria against protozoan grazing, we performed grazing
experiments on preformed bio¢lms of P. aeruginosa. Two
types of bacterivorous heterotrophic nano£agellates (size 5
Wm) were used as grazers; the surface feeder Rhynchomo-
nas nasuta and the interception feeder Cafetaria sp. Pro-
tozoan predation on bio¢lms formed by di¡erent mutants
of P. aeruginosa was studied to better understand the bac-
terial-protozoan-interactions. In the assays without grazers
the bio¢lm remained £at and non-di¡erentiated. In the
assays with grazers, we observed that microcolonies
formed as a grazing defense mechanism after 48 hours.
There was no di¡erence in bio¢lm formation between
the wild type and the quorum sensing mutants lasI/rhlI
and lasR/rhlR under grazing pressure, while the rpoN mu-
tant had a toxic e¡ect on the grazers and the bio¢lm
remained non-di¡erentiated. In addition, the di¡erent re-
sponse of the pilA and £iM mutants as well as of an
alginate overproducing strain of P. aeruginosa to protozo-
an grazing pressure is presented.
FEMSLE Congress 2-6-03
P12^6
SERUM RESISTANCE DETERMINANTS OF YERSI-
NIA ENTEROCOLITICA SEROTYPE O:3
M. Biedzka(1,2), R. Venho(2) and M. Skurnik(1,2)
(1) The Haartman Institute, University of Helsinki, Helsin-
ki; (2) Department of Medical Biochemistry and Molecular
Biology, University of Turku, Turku, Finland
Similar to many other bacteria Yersinia enterocolitica uti-
lizes the strategy of complement resistance to neutralize
normal host defence mechanisms. The chromosomally en-
coded virulence factors Ail and lipopolysaccharide (LPS)
O-antigen and the virulence plasmid encoded adhesin
YadA have been associated with resistance to human se-
rum microbicidal activity. Speci¢c roles for these factors
have not been established yet. In the present work, serum
resistance of Y. enterocolitica serotype O:3 was analysed
using a collection of 24 strains expressing di¡erent combi-
nations of YadA, Ail, LPS O-antigen and LPS outer core.
The set of 23 mutants was constructed using di¡erent ge-
netic methods. Survival of the strains was tested in normal
serum, where both the classical (CP) and alternative (AP)
pathways of complement were functional, and in EGTA-
Mg serum, where only AP was functional. The results
showed that YadA was indispensable for the bacterial
survival. Ail, on the other hand, appeared to delay the
CP/AP-mediated killing. As to the roles of the O-antigen
and the outer core, our results suggested that they are
involved in serum resistance indirectly ^ strains deprived
of those factors (or one of them ^ especially the O-antigen)
and expressing Ail and YadA displayed increased resis-
tance to serum.
P12^7
IDENTIFICATION OF THE REGULON AND THE
BINDING SEQUENCE FOR CSGD, A TRANSCRIP-
TION REGULATOR INVOLVED IN ESCHERICHIA
COLI CURLI SYNTHESIS
E. Brombacher, A. J. B. Zehnder and P. Landini
Swiss Federal Institute for Environmental Science and Tech-
nology (EAWAG), 8600 Du«bendorf, Switzerland
Several bio¢lm-forming Escherichia coli strains produce
outer membrane structures called curli, ¢ber-like struc-
tures built up by the two proteins CsgB and CsgA. The
transcriptional regulator CsgD, triggers the expression of
these structural proteins. Curli formation is typically in-
duced by low temperature, low osmolarity and stationary
growth phase. To ¢nd out if CsgD controls other genes
involved in initial adhesion, we performed a gene array
 1st FEMS Congress / Posters 103^505
experiment comparing a wild type strain and an ompR234
mutant strain. The ompR234 mutation results in increased
csgD expression. We found 13 genes that were di¡erently
transcribed in the mutant compared to the wild type. In
the promoter regions of two of these genes (pepD and
yagS) we found an 11 bp sequence, also conserved in
the CsgD-dependent promoters csgBA and yaiC. Interest-
ingly, while csgB was found to be positively regulated,
pepD and yagS were downregulated in the gene array ex-
periment, indicating that CsgD can act both as an activa-
tor and a repressor. Some of these newly identi¢ed CsgD-
dependent genes are involved in bio¢lm formation: while
YaiC has a stimulatory e¡ect, cells expressing YagS show
reduced ability to form bio¢lms. Disruption of the 11-bp
sequence results in loss of CsgD activation at the csgBA
promoter, suggesting that this 11-bp sequence is the bind-
ing site for the CsgD protein.
P12^8
BIOCONTROL EFFECT OF KLUYVEROMYCES
LACTIS STRAINS AND PICHIA ANOMALA
AGAINST PENICILLIUM EXPANSUM
A. Bru«ckner(1), Cs. Moha¤csi-Farkas(1), Cs. Balla(2)
(1) Szent Istva¤n University, Department of Microbiology
and Biotechnology, Somlo¤i u¤t 14-16, 1118 Budapest, Hun-
gary ; (2)Szent Istva¤n University, Department of Refriger-
ation and Livestock Products Technology, Me¤nesi u¤t 45.
1118 Budapest, Hungary
Penicillium expansum is one of the most common moulds
attacking fruits and vegetables. It causes high economic
losses during the postharvest storage, furthermore, due
to its mycotoxin production it may induce human disea-
ses.Applying yeasts as antagonistic agents is one of the
possibilities of controlling mould growth. Pichia anomala
has been applied successfully on wheat. The inhibitory
e¡ect of some strains of Kluyveromyces lactis, a yeast ap-
pearing in dairy products, have been investigated and
compared with the biocontrol e¡ect of Pichia anomala.
Di¡erent culture media (e.g. malt extract medium, potato
dextrose medium) and di¡erent storage temperatures
(25‡C, 15‡C, 5‡C) have been applied. Yeast strains have
been diluted to di¡erent concentrations in order to ¢nd the
lowest concentration, which is still e¡ective against Peni-
cillium expansum. The trials have been carried out accord-
ing to the method of Bjo«rnberg and Schnu«rer.Mycelial
growth and/or conidia formation of Penicillium expansum
was inhibited by most of the applied yeast strains, but the
biocontrol e⁄ciency was di¡erent depending on the cul-
ture media and temperature. Pichia anomala inhibited
mould at lower cell concentrations than Kluyveromyces
lactis. Investigations on mode of action showed that the
FEMSLE Congress 2-6-03
433
presence of living yeast cells ensures the inhibition e¡ect of
Kluyveromyces lactis.
P12^9
BIOFILM FORMATION BY SALMONELLA SPP. IN
THE PRESENCE OF SERUM
I. Cirkovic(1), I. Dakic(1), M. Vivoda(1), M. Jova-
novic(2), S. Stepanovic(1)
(1) Institute of Microbiology and Immunology, School of
Medicine, Belgrade, Serbia; (2) Institute of Infectious and
Tropical Diseases ‘‘Dr Kosta Todorovic’’, Belgrade, Serbia
Although Salmonella spp. readily form bio¢lm on various
surfaces, including plastic, and could be isolated from the
blood of infected patients, infections of central venous
catheters caused by these bacteria have not been reported.
The objective of the present study was to test the hypoth-
esis that the ability of Salmonella spp. to form bio¢lm is
reduced in the presence of serum components. The in£u-
ence of human non-inactivated serum on growth and bio-
¢lm formation by 21 strains of Salmonella spp. was inves-
tigated in 96-well £at-bottomed plastic microplates. The
growth rates and bio¢lm formation were determined in
Trypcase-soy broth, supplemented with 0%, 5%, 10%
and 20% of serum, using an automated microtiter plate
reader. The bio¢lm formation was evaluated by the modi-
¢ed microtiter plate test. The growth of the Salmonella
isolates was not a¡ected by presence of serum, irespective
to the concentration. In contrast, the number of bio¢lm
producing strains was highest in broth with 0% of serum
(20, 95.2%), while 9 strains (42.8%) and only 5 strains
(23.8%) produced bio¢lm in broth supplemented with
5% and 10% of serum, respectively. When the Salmonella
isolates were grown in broth supplemented with 20% of
serum, no bio¢lm formation was noted. The obtained re-
sults showed that Samonella spp. readily form bio¢lm on
plastic surfaces when cultivated in broth only. However,
addition of serum signi¢cantly reduced the bio¢lm produc-
tion, and the decrease was dose depended which suggests
that bio¢lm formation by Salmonella spp. on plastic sur-
faces is impaired by serum components.
434 1st FEMS Congress / Posters 103^505
P12^10
COMPARATE EVALUATION OF LACTOBACILLI
SPECIES ISOLATES, PH LEVEL AND MATURA-
TION INDEX IN VAGINAL SMEAR OF POST MEN-
OPAUSAL WOMEN
V. Cvetkovic(1), R. Dragovic(2), A. Bezjak-Knezevic(3),
Lj. Ivanovic
(1) Institute of Immunology and Virology, Belgrade, Yugo-
slavia; (2) Medical Centre ‘‘Vozdovac’’, Belgrade, Yugo-
slavia; (3) Clinic for Gynaecology and Obstetrics ‘‘Narodni
Front’’, Belgrade, Yugoslavia
Oestrogen de¢ciency in postmenopausa causes changes
maturation of vaginal epithelial cells, pH level of vaginal
£uid and prevalence of Lactobacilli in vaginal £ora. Aim:
This study evaluated relation between presence of Lacto-
bacilli, pH level and maturation of epithelial cells in vag-
inal smear of postmenopausal women. Material and Meth-
ods: 76 postmenopausal patients attending gynaecological
clinic during 1997-2000, were assessed for atrophic vagini-
tis by history and vaginal examination. Vaginal specimens
were collected for screening Lactobacilli, measurement of
pH level and maturation index ( MI ). MI was expressed
as a number of super¢cial, intermediate and parabasal
cells as a percentage of the total cell count. Results : We
found that, in spite of similar ages of women according
menopausa occurrence, women with Lactobacilli had a
little more acid vaginal £uid and higher percent of super-
¢cial cells than women without Lactobacilli ; although
there were not a signi¢cant higher numbers. There was
no di¡erence between mean age of women with Lactoba-
cilli, and without them. Lactobacilli species isolates were
detected in only 28.94 % (22 women) and we found no
important associations between the presence of Lactoba-
cilli species and the patient’s age. Conclusions: For objec-
tive assessment of the vaginal epithelium of potmeno-
pausal women, maturation index and pH level can be
recommended. It is also important to examine of Lacto-
bacilli ‘s colonization in the vaginal epithelium, because
they denote a healthy vaginal milieu. In spite of oestrogen
de¢ciency’s unfavorable e¡ects to vaginal epithelium, Lac-
tobacilli can survive in vaginal £ora of postmenopausal
women and this fact will be examined in next study.
FEMSLE Congress 2-6-03
P12^11
CELL SURFACES HYDROPHOBICITY OF BACIL-
LUS SP. AND ITS ADHESION TO SOLID SURFACES
K. Czaczyk, W. Bialas, K. Trojanowska, A. Mueller
Department of Biotechnology and Food Microbiology, Agri-
cultural University of Poznan, Poland
The cell surfaces hydrophobicity (CSH) plays an impor-
tant role in an adhesion of bacteria to solid surfaces. Pre-
vious reports have suggested that attachment of bacterial
cells to surfaces may be enhanced when the cells are hy-
drophobic. The aim of this study was to investigate the
CSH properties of Bacillus sp. depended on the cultivation
conditions (time of culture, temperature, pH, and nutrient
concentrations) and its in£uence on adhesion to solid sur-
faces. The studies were carried out with 9 Bacillus sp.
strains (also isolated from natural environments) and
two kinds of surfaces : glass and stainless steel (304L and
316L). CSH was examined using bacterial adhesion to
hydrocarbons test (BATH). To compare the e¡ect of
each factor on CSH of Bacillus sp. cells, the experiments
were designed as a factorial search with three levels for
each variable (Box-Behnken scheme) and response surface
method was used. Adhesion of bacterial cells to solid sur-
faces was examined using direct £uorescence microscopy.
The obtained results indicate that the all examined factors
have an in£uence on CSH of Bacillus sp. cells. The stron-
gest e¡ect, for most strains, was observed in the case of
time of culture, peptone concentration and temperature.
The e¡ect of CSH on adhesion Bacillus sp. cells to solid
surfaces was observed only in the ¢rst stages of process
(till 4 hours). The increase of CSH caused the increase of
adhesion to hydrophilic surfaces as glass and stainless
steel.
P12^12
THE ROLE OF THE SATELLITE MICROFLORA AT
THE GREEN MICROALGA HAEMATOCOCCUS
PLUVIALIS FLOTOW CULTIVATION
T. I. Dudnicenco
Department of Biology, Moldova State University, 60 Ma-
teevici str., MD-2009, Chisinau, Republic of Moldova
This work presents the results obtained during the study
of the role of the satellite micro£ora of green microalga
Haematococcus pluvialis concerning hamatococcus produc-
tivity and biochemical composition of algal biomass. It is
well known that at the algae cultivation in laboratory and
industrial conditions together with them are developing
di¡erent satellite microorganisms. It is very important to
 1st FEMS Congress / Posters 103^505
obtain algae axenic cultures because only they re£ect the
real nature of algae growth and development. The micro-
alga H. pluvialis is a superproducer of ketocarotenoid as-
taxanthin. Astaxanthin is used as a dietary suppliment in
aquaculture for the production of salmon, trout, red sea
bream and shrimp, as a natural colorant in food industry
and as a bioactive remedy in pharmaceutics and cosmetics
manufacturing, owing to its antioxidant, anticancer and
immunomodulation properties. In the results of these ex-
periments were established that H. pluvialis algological
pure culture in laboratory conditions of cultivation repre-
sents association which consists from alga axenic culture
and bacteria belong to Pseudomonas genus. The produc-
tivity of haematococcus axenic cultures doesn’t di¡er from
nonaxenic culture. Between satellite bacteria Pseudomonas
sp. and algal cells are exists sintrophic relationships. In the
process of bioactive factors extracted from haematococcus
biomass, its quantity decrease, because of destructive ac-
tivity of bacteria’s enzymes. These facts condition the ne-
cessity of axenic haematococcus culture obtaining in bio-
technology.
P12^13
ORCHARD POPULATION DYNAMICS OF BACIL-
LUS SUBTILIS APPLIED FOR BIOCONTROL OF
APPLE FIRE BLIGHT
G. Broggini(1), B. Du¡y(2), E. Holliger(2), C. Gessler(1)
and A. Patocchi(1)
(1) Swiss Federal Institute of Technology (ETH), CH-
8092 Zu«rich; (2) Swiss Fedeal Research Center for Fruit
Production, Viticulture and Horticulture (FAW), CH-8820
Wa«denswil, Switzerland
Fire blight, caused by Erwinia amylovora, is the major
disease threat to apple, pear and other pome fruit world-
wide. The disease is now widespread in Europe and has
recently invaded Switzerland. Antibiotics (streptomycin
and oxytetracycline), which have been the most e¡ective
controls used in North America, are not permitted for
plant agricultural use in Switzerland. We investigated bio-
logical control as an alternative strategy for ¢re blight
management. A newly registered product Biopro0 based
on Bacillus subtilis strain BD170 was applied in several
orchards in Switzerland over 2 years. We developed mo-
lecular probes for monitoring the population dynamics of
this agent after ¢eld application. Bacillus was applied in
two ways, as a direct spray during apple £owering or using
honeybees as vectors. Direct spray resulted in e¡ective
primary colonisation of stigmas of £owers opened at
time of treatment. Secondary colonisation was also ob-
served for £owers that were closed or buds at the time
of treatment. Honeybees were poor vectors for the biocon-
trol agent, likely because of unsuitable bacterial formula-
FEMSLE Congress 2-6-03
435
tion. We also report detection of the biocontrol agent on
bees and in honey from treated hives.
P12^14
SUPPRESSION OF RHIZOCTONIA SOLANI DIS-
EASES OF SUGAR BEET BY ANTAGONISTIC N-1,3-
GLUCANASE AND ANTIBIOTIC-PRODUCING
YEASTS
K. A. El-Tarabily
Department of Biology, Faculty of Science, University of
United Arab Emirates, Al-Ain, 17551, United Arab Emi-
rates
Thirty-one yeasts isolated from a sugar beet rhizosphere
were screened for their ability to produce di¡usible and
volatile antifungal metabolites, chitinase and f-1,3-gluca-
nase active against Rhizoctonia solani AG2-2, the causal
agent of seedlings post emergence damping-o¡ and crown
and root rots of sugar beet. The three most promising taxa
were Candida valida, Rhodotorula glutinis, and Trichospor-
on asahii. The in vitro studies showed that R. glutinis and
T. asahii inhibited the growth of R. solani through the
production of volatile and di¡usible antifungal com-
pounds, respectively, whilst C. valida was active through
the production of f-1,3-glucanase. The three yeasts were
also able to colonize sugar beet roots and to produce
iodole acetic acid and gibberellic acid in their culture ¢l-
trates. All three antagonists individually or in combina-
tions, were suppressive of R. solani under controlled glass-
house conditions and signi¢cantly reduced the level of
disease severity. A positive association was evident with
their in vitro antagonism and disease reductions in each
case. The application of the three yeasts enhanced the
growth and development of sugar beet plants under glass-
house conditions. Glasshouse trials also showed that the
use of a mixture of the three yeasts, had a synergistic e¡ect
on the suppression of the root rots, and in the growth
promotion of sugar beet. This is the ¢rst published report
to use yeasts as a biological control agent of a soil-borne
plant pathogen.
436 1st FEMS Congress / Posters 103^505
P12^15
INTERACTIONS OF POLYSACCHARIDE-CONTAIN-
ING COMPLEXES OF AZOSPIRILLA WITH VARI-
OUS LECTINS OF SOIL BACTERIA
Yu. P. Fedonenko, S. A. Konnova, V. E. Nikitina, L. V.
Karpunina, V. V. Ignatov
Institute of Biochemistry and Physiology of Plants and Mi-
croorganisms, Russian Academy of Sciences, Saratov, Rus-
sia
Carbohydrate-containing surface components of azospiril-
la ^ the lipopolysaccharide^protein complex (LPPC) and
the polysaccharide^lipid complex (PSLC) ^ can interact
with lectins of Azospirillum and other soil bacteria. Use
of the immunodot method for studying the interaction
between the bacterial-surface structures (agglutinative pro-
teins and polysaccharidic complexes) allowed us to deter-
mine the concentration of the components participating in
this reaction. The interactions lectins of Azospirillum with
LPPCs, PSLCs, and PSs were both speci¢c and non-spe-
ci¢c. The LPSs that were isolated from the bacterial mem-
branes did not interact with the lectins of the azospirilla.
The cross-binding between the lectins and the polysaccha-
ride complexes of several Azospirillum strains was not ob-
served, although some polysaccharides contained lectin-
speci¢c carbohydrates. Immunodot analysis showed that
the Bacillus lectins (LI and LII) interacted with the
PSLC and LPPC of all the Azospirillum strains and with
the Rhizobium EPS and LPS. The data show the possibil-
ity of speci¢c binding of the bacillar lectins and rhizobial
agglutinins to the polysaccharidic complexes from certain
Azospirillum strains and, in the case of the agglutinins, to
their own polysaccharides. Such interactions are possibly
involved as an explanation for the establishment of vari-
ous biologically active, multicomponent, nitrogen-¢xing
bacterial associations in the near-root zone of plants. In
addition, the blocking of the bacterial-lectin-binding spe-
ci¢c sites by the Azospirillum and Rhizobium polysacchari-
dic complexes may prove e¡ective in the competition for
the sites of binding to the host-plant root surface.
This work was supported in part by the RFBR (grant no.
02-04-48224).
FEMSLE Congress 2-6-03
P12^16
INTERACTIONS BETWEEN LACTIC ACID BACTE-
RIA AND THE TEMPE FUNGUS RHIZOPUS OLIGO-
SPORUS
X. M. Feng, A. Eriksson, J. Schnu«rer
Department of Microbiology, Swedish University of Agri-
cultural Sciences, P.O.Box 7025, SE-750 07, Uppsala, Swe-
den
Rhizopus oligosporus has been used to ferment soybeans to
a white cake, tempe, used as food for many centuries in
Indonesia. Lactic acid bacteria (LABs) are considered a
part of the functional microorganisms in the tempe cake,
and are able to inhibit the growth of pathogenic bacteria.
It is unknown whether LABs a¡ect the growth of R. oli-
gosporus or not. A similar product, barley tempe, has been
developed in our department. The growth of LABs and
their a¡ects on the growth of R. oligosporus were inves-
tigated during the fermentation of barley tempe. Two iso-
lates of Lactobacillus plantarum, one of Lactobacillus reu-
teri and one of Lactobacillus fermentum grew well together
with R. oligosporus in barley, while ¢ve isolates of Lacto-
coccus lactis and one of Pediococcus pentosaceus had very
limited growth. Lactobacillus plantarum did not signi¢-
cantly reduce the growth of R. oligosporus, but Lactoba-
cillus reuteri, Lactobacillus fermentum and Lactococcus lac-
tis a¡ected the growth of R. oligosporus.
P12^17
CHARACTERISATION OF THE MENINGOCOCCAL
SECRETIN PILQ: ACTIONS AND INTERACTIONS
S. A. Frye, R. Assalkhou, Y. Esbensen, H. Tuven, A. V.
Benam, S. Balasingham and T. T\njum
Centre for Molecular Biology and Neuroscience and Insti-
tute of Microbiology, Rikshospitalet, University of Oslo, N-
0027 Oslo, Norway
Secretins are a large family of bacterial proteins associated
with membrane translocation of macromolecular com-
plexes. A subset of this family, termed PilQ proteins, are
required for type IV pilus biogenesis in Neisseria meningi-
tidis, the causative agent of meningococcal disease. Menin-
gococal PilQ is particularly interesting since it induces bac-
tericidal antibodies in its exclusive human host. PilQ is
found as a complex of approximately 900 kD and the
lipoprotein PilP is thought to be important for complex
stabilisation. Meningococcal PilQ is unique among secre-
tins because of its abundance in the outer membrane and
its N-terminally located polymorphic region containing
repetitive elements. We have puri¢ed the native PilQ com-
 1st FEMS Congress / Posters 103^505
plex from meningococcal outer membranes and shown
that it is a poreforming structure, organised as a ring of
12 identical subunits. Our data indicate that PilQ is the
pore through which the substrate, the growing pilus ¢bre
(polymerised PilE), is extruded to the bacterial surface.
New genetic techniques have allowed us to construct de-
¢ned mutants to characterise the domains within PilQ. In
particular, PilQ complex multimerisation and orientation
in the outer membrane, as well as surface exposure, are
being assessed. Furthermore, using far-western analysis we
have demonstrated that PilQ and PilE directly interact.
We also have identi¢ed the PilQ subunit domain that is
involved in the PilQ-PilE interaction. This work is critical
to understanding how PilQ functions; our aim is to detail
the dynamics of PilQ interactions with other components
such as PilE and PilP during pilus biogenesis and pilus
retraction.
P12^18
AN EVOLUTIONARY APPROACH TO UNDES-
TANDING BIOFILM FORMATION IN PSEUDOMO-
NAS FLUORESCENS SBW25
S. M. Gehrig(1) and P. B. Rainey(1,2)
(1) Department of Plant Sciences, University of Oxford,
Oxford OX1 3RB, UK; (2) School of Biological Sciences,
University of Auckland, Auckland, NZ
Pseudomonas £uorescens SBW25 rapidly diversi¢es when
propagated in a spatially structured micrcosm (static broth
culture), producing a range of morphologically distinct
niche-specialist genotypes. One of these morphs, termed
Wrinkly Spreader (WS) has a characteristic wrinkled mor-
phology on agar plates and forms a bio¢lm at the air-
broth interface. There are two operons required for ex-
pression of the WS phenotype, a structural locus wss,
and a chemosensory pathway wsp. A primary cause of
the morphology on plates and bio¢lm formation is over-
production of an acetylated cellulose polymer (ACP), trig-
gered by a single point mutation in the wsp regulatory
pathway, which controls ACP production encoded by
wss. In order to address questions relating to ecological
and evolutionary convergence the wss operon was deleted
from the ancestral (SM) genotype and this ACP-defective
mutant was allowed to evolve in a structured microcosm.
We were particularly interested to know whether this ge-
notype could, during the course of evolution, generate a
genotype that is ecologically equivalent to WS. Within 7
days of evolution (V50 generations) mutants had evolved
that were capable of colonising the air-broth interface. The
bio¢lm formed by these genotypes is not made of cellulose
and is very weak. This weakness however is not due to
slow growth as the strains grow normally. In addition the
SMvwss-derived bio¢lm forming genotypes overproduce
FEMSLE Congress 2-6-03
437
an attachment factor that allows them to attach strongly
to glass surfaces. This attachment factor is regulated by
the wsp operon, as is the case in SM and WS. Current
suppressor studies are directed at identifying the genetic
causes (structural and regulatory) of this newly manifest-
ing attachment factor.
P12^19
SUB-AERIAL ROCK BIOFILMS AS AN EVOLUTION-
ARY TEST GROUND: MICROBIAL SUCCESSIONS
AND INTERACTIONS OF HETEROTROPHIC AND
PHOTOTROPHIC ORGANISMS
A. A. Gorbushina
Geomicrobiology, ICBM, Oldenburg University, P.O. Box
2503, Oldenburg 26111, Germany
Rock surface colonisation is an important starting point in
the development of all terrestrial ecosystems on Earth. The
sub-aerial rock microbial community is quite diverse and
includes chemoorganotrophic fungi and bacteria and pho-
totrophic microorganisms like green algae and cyanobac-
teria. Bare rock surfaces ^ the oldest terrestrial habitats on
Earth ^ are commonly dominated by heterotrophic free-
living and symbiotic ascomycetes. Though energetically it
is easier to imagine that phototrophic organisms would
dominate such environments, our observations on sub-aer-
ial bio¢lms all over the world emphasise the role of fungi
in primary land colonisation especially on desert rock sur-
faces. The environmental hostility of sub-aerial rock sur-
face forces the phototrophs either to form mutualistic as-
sociations with fungi (epi- and endolithic lichens) or to
search protection from excessive sun irradiation between
the rock crystals. In the harshest desert rock environments
symbiotic lichens also often yield and only microcolonial
fungi (MCF) are present. Free-living MCF seem to be the
most stress-tolerant organisms in rock sub-aerial bio¢lms
and their dominance is usually associated with a longer
sub-aerial exposure of the rock. Nevertheless, MCF are
heterotrophic microorganisms and depend either on the
carbon input from atmospheric deposition or could form
symbiotic associations with phototrophs. MCF may thus
represent an interesting evolutionary line of community
development under extreme stress. Further they may
have acted as symbiotic partners for photobionts and oth-
er more sensitive sub-aerial microorganisms. Laboratory
analyses of associations between chemoorganotrophic
and phototrophic organisms are presented in terms of a
general review on potential evolutionary mechanisms in
sub-aerial bio¢lm development.
438 1st FEMS Congress / Posters 103^505
P12^20
MICROBIAL GENERATION OF ACID DRAINAGE IN
A RICH-IN-PYRITE ORE DUMP
V. I. Groudeva(1), S. N. Groudev(2) and A. S. Doyche-
va(1)
(1) Department of Microbiology, Faculty of Biology, Uni-
versity of So¢a, So¢a 1421, Bulgaria ; (2) Department of
Engineering Geoecology, University of Mining and Geology,
So¢a 1700, Bulgaria
An ore dump located in the proximity of an open-pit
copper mine in Central Bulgaria was for many years, after
rainfall, a source of acid drainage waters. The dump con-
sisted of about 500 000 tons of rich-in- pyrite low-grade
copper ores and contained 0.18% copper, 3.7% iron and
3.2% sulphur. The drainage waters, which £owed out from
the dump, had pH in the range of 1.5 ^ 2.3 and contained
about 0.1 ^ 0.5 g/l copper, 1-3 g/l iron and usually more
than 5 g/l sulphates. The generation of these polluted
waters was connected with the oxidative activity of acid-
ophilic chemolithotrophic bacteria, which inhabited the
dump. Acidithiobacillus ferrooxidans and Leptospirillum
ferrooxidans were the prevalent microbial species in the
dump but some moderately thermophilic chemolithotro-
phic bacteria related to Sulfobacillus thermosul¢dooxidans
and the genus Thiobacillus were also well present. The
distribution of chemolithotrophs was mostly con¢ned to
the upper layers (the top 0.5-1 m) of the dump with den-
sities as high as in excess of 108 cells/g of ore. Their num-
ber decreased with increasing depth and in layers deeper
than 5-7 m from the surface was negligible. It was found
that the microbial activity in situ depended on some essen-
tial environmental factors such as water, oxygen and nu-
trients contents in the dump. It was possible to change this
activity by suitable changes in the levels of these environ-
mental factors.
P12^21
OCCURRENCE OF KILLER YEAST STRAINS IN
FRUIT AND BERRY WINE YEAST POPULATIONS
G. Gulbiniene, T. Jokantaite, E. Serviene and V. Melvydas
Institute of Botany, Zaliuju ezeru 49, LT-2021, Vilnius
Lithuania
Killer yeast strains produce a protein toxin which is lethal
to other sensitive strains. Only three di¡erent types of kill-
er yeast, K1, K2 and K28 have been observed in Saccha-
romyces strains. The strains kill one another but are im-
mune to the killer toxin of their own class. Apples,
cranberries, rowan berries and black chokeberries wine
FEMSLE Congress 2-6-03
yeast populations from winery ‘‘Anyksciu vynas’’ were
used for the determination of killer yeast occurrence.
Spreading of killer yeast in wine yeast populations was
dependent on sort and quality of row material and in-
cluded 0,10 ^ 67,5 %. According to the interaction between
the standard killer yeasts and sizes of M dsRNA, isolated
killer strains were classi¢ed into K2, Kn and K2x types.
Kn type strains killed K1, K2 and K28 strains and it was
killed by K1, K2 and K28 strains. K2x type killed K1 and
K28 strains, but not K2 and it was killed by K1, K2, K28
or it was not killed by all killer strains. In the cases of Kn
and K2x sizes of M dsRNA, the spectrum of antagonistic
activity and resistance to the killer action is di¡erent from
those of all known killer strains of Saccharomyces. Our
results showed that these strains belongs to a new killer
type which had not been reported previously. In addition,
killer strains lacking M dsRNA have been isolated.
P12^22
COMMUNICATION WITHIN THE RHIZOPLANE
BACTERIAL COMMUNITY AND WITH ROOTS
VIA N-ACYLHOMOSERINE LACTONES
A. Hartmann(1), S. Gantner(1), C. Du«rr(1), V. Al-
brecht(1,4), M. Schmid(1), R. Schuhegger(2), F.B. Daz-
zo(3), A. Steidle(4), C. Langebartels(2) and L. Eberl(4)
(1) GSF-Institute of Soil Ecology, Department of Rhizo-
sphere Biology and (2) GSF-Institute of Biochemical Plant
Pathology, Ingolstaedter Landstr. 1, D-85764 Neuherberg,
Germany ; (3) Department of Microbiology, Michigan
State University, East Lansing, Mi 48824-1325, U.S.A.;
(4) Department of Microbiology, Technical University Mu-
nich, D-85354 Freising, Germany
Signal molecules of the N-acylhomoserine lactone (AHL)
type mediate quorum sensing in Gram-negative bacteria.
The root surface of plants is colonized by a diversity of
bacteria due to the substrate availability in the rhizo-
sphere. Rhizosphere isolates of Burkholderia spp. were ex-
amined for their AHL-production. Several di¡erent AHLs
could be detected in the Burkholderia isolates from the
rhizosphere of di¡erent plants. As it has been shown for
clinical isolates, AHL-production was most frequent with-
in the Burkholderia cepacia cluster. The in situ AHL-pro-
duction in the rhizoplane of tomato plants was examined
using rfp-labeled AHL-producer bacteria (Pseudomonas
putida IsoF pUT-Kan-rfp) and gfp-labeled AHL-reporter
bacteria (Pseudomonas putida F117, pKR-C12), de¢cient
for AHL-production. The distances of activation was de-
termined applying the CMEIAS-software and geostatisti-
cal methods using confocal laser scanning microscopic
mosaic images. A distance of 60 Wm was determined as
maximum ‘‘calling distance’’ between an AHL-producer
cell and an activated AHL-reporter cell. This clearly dem-
 1st FEMS Congress / Posters 103^505
onstrates, that AHLs can exert their action quite a dis-
tance away from their place of production in the rhizo-
plane. The involvement of AHL-production on the devel-
opment of plant resistance against the attack of
phytopathogenic fungi was investigated with tomato
plants and Serratia liquefaciens MG1 (AHL-wild type)
and S. liquefaciens MG44, de¢cient in AHL-production.
While tomato plants inoculated with S. liquefaciens MG1
developed systemic resistance towards Alternaria alternata,
the mutant failed to induce systemic resistance. S. liquefa-
ciens MG1 inoculated plants as well as plants treated with
10 WM N-hexanoyl homoserine lactone showed increased
levels of the signal compound salicylic acid, while the eth-
ylene precursor ACC was not increased. Using the Tom-
stressR microarray analysis, several pathogen-related pro-
teins appeared induced due to the treatment with HHL in
axenic plants.
P12^23
MOLECULAR CHARACTERISATION OF THE MI-
CROBIAL DIVERSITY IN ANAEROBIC WASTE-
WATER TREATMENT SYSTEMS
H. G. H. J. Heilig, C. Roest, H. Smidt, A. J. M. Stams and
W. M. de Vos
Laboratory of Microbiology, Wageningen University, Hes-
selink van Suchtelenweg 4, NL-6703 CT Wageningen, The
Netherlands
Up£ow Anaerobic Sludge Blanket (UASB) bio-reactors
are commonly applied with high e⁄ciency. However, the
microbial processes that take place inside the reactors re-
main largely unknown. DGGE analysis of the 16S rDNA
diversity revealed a largely stable microbial community
over a period of 3 years, during which the diversity within
the bacterial fraction was large, and within the archaeal
community rather small. This was con¢rmed by the anal-
ysis of a library of 333 clones, which was dominated by 7
de¢ned groups, among which were sequences that mostly
matched with non-cultured bacteria clustering in the Cy-
tophaga (9% of the clones) and Green Non-Sulphur (7%)
clades. Sequences of bacteria with a known function were
retrieved, including cellulose degradation (Cellulomonas
spp. (9%), Actinobacterium spp. (8%), Propionibacterium
spp. (6%)), sulphate reduction (Desulfovibrio, Desulfobul-
bus, Desulforhabdus spp. (9%)), and syntrophic fatty acid
degradation (Syntrophobacter spp. (3%)). The remaining
diversity originated from a variety of not yet cultured
bacteria. 16S rRNA targeted DNA oligonucleotide probe
hybridisation revealed that the bacterial fraction makes up
only 35% of the total microbial community, whereas the
remaining 65% was accounted for by archaeal sequences.
Preliminary studies on archaeal diversity showed three
dominant clusters, being Methanobacterium, Methanospril-
FEMSLE Congress 2-6-03
439
lum, and Methanosaeta spp. The characterised amplicons
provide a platform of 16S rDNA sequences, that will fa-
cilitate the construction of phylogenetic arrays. These can
be used to study the dynamics and function of prokaryotic
microbes in anaerobic bio-reactors and other ecosystems.
P12^24
DETECTION OF HOMOSERINE LACTONE-DE-
GRADING BACTERIA IN THE POTATO RHIZO-
SPHERE
S. Jafra(1,2), J. M. van der Wolf(1)
(1) Plant Research International, P. O. Box 16, 6700 AA
Wageningen, The Netherlands; (2) Department of Biotech-
nology, University of Gdansk, ul. Kladki 24, 80-822 Gdansk,
Poland
In bacterial populations numerous physiological processes
are regulated through the production of di¡usible signal
molecules. Communication between cells via such mole-
cules is known as ‘‘quorum sensing’’, as it depends on
the population density. In Gram-negative bacteria, these
molecules belong to the group of N-acyl homoserine lac-
tones (HSL). Also the control of soft rot diseases caused
by the plant pathogenic bacterium Erwinia carotovora
subsp. carotovora (Ecc) is quorum sensing dependent and
is regulated by HSLs. In our study, we analysed bacteria
isolated from the potato rhizosphere, for the production of
agents able to degrade HSLs from Ecc. We used a GFP-
based E. coli indicator strain for HSL detection. GFP
£uorescence produced by the indicator strain in the pres-
ence of HSL, was measured with the Molecular Imager
FX scanner. We detected 40 isolates (9 % of all tested
isolates), which were able to degrade synthetic HSLs
(OHHL, OOHL, OHL and HHL) and HSLs naturally
produced by Ecc. The GFP-scan showed quantitative dif-
ferences in HSL-degradation capacity of tested isolates.
According to 16S rDNA sequence analysis, the isolates
belonged, to di¡erent genera (Ochrobactrum, Rhodococcus,
Pseudomonas, Delftia). In planta experiments and analysis
of the HSL-degrading agent will show the possible appli-
cation of selected strains and compounds for biocontrol of
soft rot diseases caused by Ecc.
440 1st FEMS Congress / Posters 103^505
P12^25
CONSTRUCTION OF STABLE MIXED-CULTURE
SYSTEM
S. Kato, S. Haruta, M. Ishii and Y. Igarashi
Department of Biotechnology, University of Tokyo, Yayoi
1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan
In nature, many species of microorganisms are co-existing
with interacting each other. We consider that functional
and structural stability of the microbial communities are
very important for resisting various environmental distur-
bances. What is the factor determining stability of the
communities ? We bred microbial community capable of
e¡ectively degrading lignocellulose at 50‡C in a liquid me-
dium. Even after more than 20 times of subcultures, the
community maintained the same degradation e⁄ciency
and the same band patterns on DGGE gel. Furthermore,
its degradation ability was not a¡ected by pH changes
(5V9). These characters indicate that the community
has high stability in terms of the function and the struc-
ture. The analysis of the 16S rDNA sequences of the
DGGE bands indicated that anaerobic bacteria steadily
co-existed with aerobic bacteria. Although 10 strains of
aerobic isolates did not degrade cellulose, an anaerobic
isolate (Clostridium sp. strain CSK-1) had cellulose-de-
grading ability. Mixed culture with 3 strains of aerobic
isolates enabled CSK-1 to grow and degrade cellulose ef-
fectively under static (not strictly anaerobic) condition.
The mixed culture had almost the same cellulose degrad-
ing e⁄ciency and stability for subculture as those of the
original community. We are analyzing degradation e⁄-
ciency, metabolites and stability of mixed-culture systems
with a variety of combinations. It will lead us to clarify the
role of each bacteria and the mechanism of the stability,
and to obtain new knowledge for microbial ecology and
theoretical construction of functional microbial commun-
ities.
P12^26
Withdrawn.
FEMSLE Congress 2-6-03
P12^27
PARTIAL CHARACTERIZATION OF FC-1 KILLER
TOXIN ACTIVE AGAINST CRYPTOCOCCUS NEO-
FORMANS
J. Kucsera(1), M. Ohkusu(2), I. Pfei¡er(1), J. Litter(1)
and K. Takeo(2)
(1) Department of Microbiology, Faculty of Sciences, Uni-
versity of Szeged, P.O. Box 533, Szeged, Hungary ; (2)
Research Center for Pathogenic Fungi and Microbial Tox-
icosis, Chiba University, Chiba, Japan
Among yeasts killer phenomenon is widely spread, and
has been found in more than one hundred species. Killer
yeasts secrete killer toxins that kill sensitive strains. In
natural environment these toxins are weapons to kill the
competing yeast cells. These toxins have potential role in
medicine as well : can be used as potential therapeutic
agents or as prophylactic for the treatment of fungal dis-
eases. Cryptococcus neoformans is a leading cause of illness
and death among persons with AIDS. Previously we have
published that strain IFM 40078 of Filobasidium capsuli-
genum produces a killer toxin (FC-1) which is strongly
active against both clinical and environmental isolates of
C. neoformans. The toxin is also e¡ective on var. neofor-
mans, var. gattii and var. grubii (Kucsera et al. 2001). Here
we describe the main characteristics of the toxin and sug-
gest the mode of action by which it kills Cryptococcus
neoformans cells. According to our results the toxin has
proteinaceous nature, active in the range of pH 4.0 ^ 6.0,
and between 16.5 and 28 C. The molecular mass was esti-
mated above 30 kD by dialysis and ultra¢ltration. The
killing mechanism of FC-1 is not cell cycle-dependent,
and on the basis of propidium iodide staining involves
release of cellular components through ion channels
formed on citoplasma membrane.
 1st FEMS Congress / Posters 103^505
P12^28
INITIAL ADHESION OF POTENTIALLY PATHO-
GENIC BACTERIA TO ABIOTIC SURFACES
M.-H. Castonguay, P. Landini
Swiss Federal Institute for Environmental Science and Tech-
nology (EAWAG),
8600 Du«bendorf, Switzerland
We tested the ability of potentially pathogenic bacteria to
attach to surfaces when grown in di¡erent growth phase
and temperature conditions. Glass microbeads and sand
grains, two model surfaces of di¡erent nature, charge and
hydrophobic properties, were used as support material to
compare attachment. Gram positive bacteria like Staph-
ylococcus epidermidis e⁄ciently adhered to sand, a hydro-
phobic substrate, possibly due to a more hydrophobic na-
ture of their cell surface. The ability to attach appeared to
be variable among Gram negative bacteria: while Pseudo-
monas putida and Providencia stuartii attached e⁄ciently
to both hydrophilic and partially hydrophobic substrate,
other Gram negative species such as Escherichia coli and
Pseudomonas £uorescens displayed poor adhesion proper-
ties. Attachment of each individual species was strongly
a¡ected by growth at 25 or 37‡C and by harvesting in
either exponential or stationary phase. Since ionic strength
is reported to be an important factor for stimulation of
initial adhesion, we performed detachment experiments in
which the attached bacteria were washed with pure water
to cause a reduction of the ionic strength. However, we
observed that, while the bacteria with lower adhesion abil-
ity were easily eluted, bacteria strongly attaching to the
substrate (i.e. P. stuartii) could not be e⁄ciently detached
by lowering ionic strength. Detachment was obtained only
with the addition of an anionic detergent such as SDS.
This observation possibly suggests that, in addition to
physico-chemical interactions between cell surface and
the solid substrate, more speci¢c interactions, possibly
mediated by adhesins, might also take place.
FEMSLE Congress 2-6-03
441
P12^29
EXPRESSION OF THE PSEUDOMONAS AERU-
GINOSA RHAMNOLIPID BIOSYNTHESIS OPERON
IN BIOFILMS
Y. Lequette and E. P. Greenberg
Department of Microbiology and W. M. Keck Microbial
Communities and Cell Signaling Program, College of med-
icine, University of Iowa, Iowa City, Iowa USA
Bio¢lms of Pseudomonas aeruginosa can cause chronic in-
fections. Pseudomonas aeruginosa bio¢lms show a series of
developmental steps. Individual bacteria attach to surfa-
ces, the bacteria organize into microcolonies, and ¢nally
the microcolonies mature into large mushroom and pillar-
like entities with bacteria encased in an extracellular ma-
trix. Quorum sensing in£uences the last step in this devel-
opmental process and the quorum regulated rhamnolipid
synthesis genes, rhlAB are required for the development of
normal bio¢lm structure. Thus we sought to study the
expression of rhlAB in bio¢lms. We constructed a prhlA-
gfp transcriptional fusion and inserted into the att site on
the chromosome of a P. aeruginosa wild type and a rhlI,
quorum sensing signal synthesis mutant. In planktonic
cultures of the wild type or the rhlI mutant grown in the
presence (but not in the absence) of the quorum sensing
signal butyryl-homoserine lactone (butyryl-HSL) GFP
£uorescence was induced during the transition from loga-
rithmic growth to stationary phase. Bio¢lms were studied
by confocal scanning microscopy. In bio¢lms of the wild
type P. aeruginosa GFP was not expressed in attached
cells prior to microcolony formation. Cells in the centers
but not on the periphery of microcolonies showed £uores-
cence. In mushroom and pillar -like structures the most
£uorescent cells were at the base. Bio¢lms of the rhlI mu-
tant are not £uorescent without addition of butryryl-HSL.
The pattern of £uorescence in mature bio¢lms of the mu-
tant grown in the presence of butyryl-HSL is similar to
that observed in the wild-type strain. We do not under-
stand why cells in the middle of mushrooms and at the
upper surface do not show high levels of rhlAB induction.
The pattern of expression does not support the notion that
this is related to availability of oxygen or other nutrients
or levels of butyryl-HSL. Regardless of the mechanism of
control we believe this pattern of expression might be re-
lated to the control of bio¢lm structure by rhamnolipid
synthesis.
442 1st FEMS Congress / Posters 103^505
P12^30
INVERTED REPEATS: COMPARISON OF TWO ISO-
LATES OF MYCOBACTERIUM TUBERCULOSIS
WITH COMPLETE GENOME AND VISUALIZATION
OF CRUCIFORM STRUCTURE IN PLASMID DNA
BY ATOMIC FORCE MICROSCOPY
A. Limansky and O. Limanskaya
Mechnikov Institute of Microbiology and Immunology,
Pushkinskaya St., 14 Kharkov, 61057, Ukraine
Inverted repeats may regulate genetic processes by forma-
tion of hairpin secondary structures that block DNA pol-
ymerases. Long inverted repeats can be a source of ge-
nomic instability. Inverted repeats may adopt a linear
double stranded helix or a cruciform structure consisting
of two symmetrical hairpins. Theoretical and experimental
studies have shown that cruciform structures can exist in
negatively supercoiled DNA, as dependent upon such as
temperature and supercoil density. The present work com-
pared inverted repeats in the genome ( s 4000 kb) of two
isolates of Mycobacterium tuberculosis and direct visual-
ization of inverted repeats in plasmid pUC8 supercoiled
DNA (266b bp) by atomic force microscopy (AFM).
Analysis and modeling of the most thermodynamically
stable cruciform formations in M. tuberculosis and pUC8
plasmid DNA were performed. Computer analysis showed
that the two isolates of M. tuberculosis (H37Rv and
CDC1551) respectively contain 50 and 47 thermodynami-
cally stable inverted repeats (with 4-5 nucleotides (nt) loop
and stem length more 7bp). Both isolates have 8 long
palindromic sequences of 48-62 nucleotides with energy
from -56.2 kcal/mol to ^ 38.9 kcal/mol. 11 palindromic
sequences of 28-40 nt length were found for both isolates;
with -33.9 ^ -29.5 kcal/mol for CDC1551 and -35.5 ^ -
26.2kcal/ mol for H37Rv. These data show that long in-
verted repeats are highly stable for two di¡erent M. tuber-
culosis isolates. Plasmid pUC8 contains only one palin-
dromic sequence which can form a cruciform structure
in bu¡er solution. Cruciform is seen as clear-cut extrusions
on the DNA ¢laments with the length of the arms fully
consistent with the size of the hairpins expected from a 26
bp inverted repeat. The geometry of cruciform depends
upon ionic conditions. At low ionic strength cruciform
can adopt an extended conformation with an angle of
ca. 180‡ between the hairpins arms. AFM image shows
that hairpin of cruciform structure is formed by 13-14
bp. A search for self-complementary regions within the
pUC8 DNA sequence con¢rmed that the hairpin is formed
by the 11 bp stem and a loop of 4 nucleotides.
FEMSLE Congress 2-6-03
P12^31
CELL-CELL INTERACTIONS AMONG POPULA-
TIONS OF CLOSELY RELATED BACILLI
U. Cepon(1), J. Sabotic(1), P. Cadez(1), I. Mahne(1), D.
Dubnau(2) and I. Mandic-Mulec(1)
(1) University of Ljubljana, Biotechnical Faculty, Depart-
ment of Food Science and Technology, Vecna pot 111, 1000
Ljubljana, Slovenia; (2) Public Health Research Institute,
225 Warren Street, Newark, NJ 07103, USA. dubnau@-
phri.org
Natural genetic competence is controlled by the quorum
sensing (QS) system composed of ComP/ComA two com-
ponent regulatory pair, the ComX pheromone and the
ComQ processing enzyme. Previous studies showed that
an extensive polymorphism exists in the comQXP’ loci of
natural Bacillus isolates that determines the speci¢city of
QS response. It was observed that 13 isolates analyzed can
be grouped into 4 di¡erent pherotypes that do not induce
each other into competence. It was suggested that poly-
morphism of comQXP’ loci may thus represent a novel
mechanism of sexual isolation between population of
closely related Bacillus strains (Ansaldi et al., 2002). Se-
quencing of comQXP’ loci of 10 additional natural Bacil-
lus isolates and in vivo analysis of speci¢city of QS systems
in additional strains con¢rmed previous ¢ndings and sug-
gested that 16 so far analyzed isolates may group into 7
pherotypes. In addition we examined whether Bacillus iso-
lates included in the study a¡ect the growth of each other.
Condition media of 14 natural isolates grown to stationary
phase were prepared and their growth inhibitory activity
was tested against this set of isolates. No pattern of
growth inhibition that could be linked to the pherotype
speci¢city pattern was observed. However, two of the nat-
ural Bacillus isolates (RO-F-3 and RO-FF-1) exhibited a
potent killing activity towards almost all the other Bacillus
isolates tested. In both strains the activity was induced in
the late exponential phase and began to diminish 4 hrs
after the entry into the stationary phase. This activity
was also higher when producer strains were grown in
rich LB medium than in minimal competence medium
and, at least in the case of the RO-FF-1 strain, the activity
was thermolabile.
 1st FEMS Congress / Posters 103^505
P12^32
NAPHTAQUINONE METABOLITES OF FUNGUS
FUSARIUM DECEMCELLULARE: THE MECHA-
NISM OF CYTOTOXIC EFFECT
A. G. Medentsev, A. Yu. Arinbasarova, N. M. Smirnova, V.
K. Akimenko
G.K. Skryabin Institute of Biochemistry and Physiology of
Microorganisms, Russian Academy of Sciences, 142290,
Pushchino, Prospekt Nauki 5, Moscow region, Russia
Naphthazarin metabolites such as javanicin, fusarubine,
anhydrofusarubine, bostricoidine produced by fungus Fu-
sarium decemcellulare were shown to reveal antimicrobial
and phytotoxic features. The e¡ects on the rate of oxygen
uptake by mitochondrial, microsomal or soluble fractions
of etyolated pea seedlings, yeast Yarrowia lipolytica were
studied. It was found that all the autoxidable metabolites
under study were able to catalase NAD(P)H oxidation.
Besides, the naphthoquinones accepted reducing equiva-
lents from exogenous NAD(P)H dehydrogenases located
at the outer surface of the inner mitochondrial membrane
and directly transferred them to O2 bypassing the respira-
tory chain. In the presence of the pigments mitochondrial
respiration was insensitive to cyanide. The superoxide rad-
ical, O2- was identi¢ed as a product of O2 reduction. These
and literature data suggest that the cytotoxic action of
fungal metabolites is due to disturbances in energy metab-
olism and constructive exchanges resulting from the intra-
cellular oxidation of piridine nucleotides NAD(P)H or to
the formation of highly toxic species (O2-, H2O2) and semi-
quinone forms of the pigments responsible for the inhibi-
tion of macromolecule (DNA, RNA, etc.) exchange.
P12^33
EXPRESSION OF CKA IN ONLY 3% OF A COLICIN
K ENCODING POPULATION OF E. COLI IS CON-
TROLLED BY LEXA AT THE TRANSCRIPTIONAL
LEVEL
P. Mrak, J. Mulec, Z. Podlesek and D. Zgur-Bertok
Department of Biology, Biotechnical Faculty, University of
Ljubljana, Slovenia
In prokaryotes, only a few examples of di¡erential gene
expression in cell populations have been described. Colicin
production in natural populations of Escherichia coli,
while providing a competitive advantage in the natural
habitat, also leads to lysis of the toxin-producing cell.
Colicin K synthesis has been found to be induced due to
increase in ppGpp. Using two transcriptional fusions, cka-
gfp and cki-gfp and a translational fusion cka-gfp, we
FEMSLE Congress 2-6-03
443
show at the single cell level that the colicin K activity
gene cka is expressed in only 3 % of the bacterial popula-
tion upon induction by nutrient starvation. In contrast,
the immunity gene cki, is expressed in the large majority
of the cells throughout cell growth phase. Expression of
the cka-gfp fusion upon induction with mitomycin C was
observed in almost all cells in the population. Additional
experiments using the cka-gfp fusions in a lexA defective
strain and in a relA spoT mutant strain indicate that di¡er-
ential expression of cka is primarily established at the level
of transcription with derepression of the SOS repressor,
LexA. We also show a signi¢cant translational enhance-
ment of cka expression by the downstream box (DB) ele-
ment located at nucleotides 4 ^ 20 in the cka coding re-
gion. Using translational and transcriptional cka-lacZ
fusions with two di¡erent alterations in the DB sequence
we observed a 10 and 2-fold decrease in L-galactosidase
activity in translational and transcriptional fusions, respec-
tively compared to the wild type DB sequence. Although
the cka DB has only 69% homology to the consensus DB
sequence it obviously has a signi¢cant in£uence on cka
mRNA translation.
P12^34
VARIATIONS IN THE LuxR-HOMOLOGUE sdiA OF
DIFFERENT SALMONELLA WILD TYPE STRAINS
L. L. Nesse(1), L. Ravn Flodgaard(2), I. Olsaker(3), G.
Holstad(1)
(1) National Veterinary Institute, P.O. Box 8156 Dep, N-
0033 Oslo, Norway; (2) Danish Institute for Fisheries Re-
search, c/o DTU Building 221, DK-2000 Kgs. Lyngby, Den-
mark; (3) Norwegian School of Veterinary Science,
P.O.Box 8146 Dep, N-0033 Oslo, Norway
Bacteria may regulate the expression of speci¢c genes in
response to signals that they or other bacteria secrete
(quorum sensing). Many Gram-negative bacteria produce
N-acylated homoserine lactones (AHLs) as QS-signals. An
AHL receptor (SdiA) has been identi¢ed in Salmonella
enterica serovar Typhimurium, whereas production of
own AHLs has not been shown. In the present experiment,
the sdiA gene of 19 di¡erent wild type salmonella strains
was sequenced and compared with the corresponding se-
quences of S. Thyphimurium LT2 /ATCC 700740
(AE0087861.1) and S. Thyphimurium ATCC 14028
(U88651). The serovars Agona, Montevideo, Senftenberg
and Typhimurium were studied, and isolates from hu-
mans, animals, wild birds and feed factories were included
together with the national reference strains of each sero-
var. All strains displayed di¡erent pro¢les in pulsed-¢eld
gel electrophoresis. Furthermore, AHL-production in 11
strains representing all four serovars was tested in the
AHL-monitors Agrobacterium tumefaciens pZLR4 and
444 1st FEMS Congress / Posters 103^505
Chromobacterium violaceum CV026 (direct and reverse).
Supernatants or extractions from the strains grown in
LB5 were also tested in a well-di¡usion assay using A. tu-
mefaciens pZLR4, C. violaceum CV026 and a LasR-Lux
monitor (pMH297). The extractions were ¢nally tested on
TLC developed with the reversed C. violaceum CV026 as-
say and the LasR-Lux monitor.
Charlton et al. (2000). A novel and sensitive method for
the quanti¢cation of N-3- oxoacyl homoserine lactones
using gas chromatography-mass spectrometry: application
to a model bacterial bio¢lm. Environmental Microbiology
2, 530-541; Ravn, et al. (2001). Methods for detecting ac-
ylated homoserine lactones produced by Gram-negative
bacteria and their application in studies of AHL-produc-
tion kinetics. Journal of Microbiological Methods 44, 239-
251.
P12^35
LASER SCANNING MICROSCOPY OF MICROBIAL
COMMUNITIES ^ USING 1-PHOTON OR 2-PHO-
TON EXCITATION ?
T. R. Neu(1), U. Kuhlicke(1), J. R. Lawrence(2)
(1) Department of Inland Water Research Magdeburg,
UFZ Centre for Environmental Research Leipzig-Halle,
Brueckstrasse 3A, 39114 Magdeburg, Germany; (2) Na-
tional Water Research Institute, 11 Innovation Boulevard,
Saskatoon, Saskatchewan, Canada, S7N 3H5
Confocal laser scanning microscopy using 1-photon exci-
tation (1P-LSM) has become a routine technique for the
examination of microbial communities. More recently la-
ser scanning microscopes equipped with pulsed 2-photon
infra red lasers are o¡ered by most companies. So far this
new technique has been used for microbiological samples
in very few studies. In order to apply 2-photon laser scan-
ning microscopy (2P-LSM) the excitation cross section of
£uorochromes needs to be examined. For this reason the
tunable Ti/Saphire laser was adjusted from 760-900 nm to
record the emission signal of common stains used for mi-
crobiological samples. The stains tested included AO,
DAPI, SYTO, SYTOX, SYBR, SYPRO, CalcoFluor-
White, CellTracker and £uor-conjugated lectins. The re-
sults showed that most stains can be excited with the ap-
propriate infra red wave length. However, this wave length
is not necessarily double the wavelength used for 1-photon
excitation. Comparison of 1P-LSM with 2P-LSM were
carried out with a variety of dense microbial aggregates
and bio¢lms. The results veri¢ed the higher resolution of
2P-LSM in deep bio¢lm regions. Nevertheless, if signal
intensities from di¡erent samples were graphed against
each other, it was found that there was not always an
advantage of employing 2-photon excitation. A further
problem of damage to the specimen or to the substratum
FEMSLE Congress 2-6-03
may arise with samples having a high infra red adsorption
capacity. In conclusion, the decision to apply 1-photon or
2-photon LSM for examination of microbial communities
has to be made with respect to the sample properties in-
cluding : photosensitivity, absorption e¡ects, auto£uores-
cence, density and scattering.
P12^36
FULL RANGE LECTIN-BINDING-ANALYSIS OF EPS
GLYCOCONJUGATES IN LOTIC BIOFILMS
GROWN UNDER DIFFERENT SUBSTRATE CONDI-
TIONS
C. Staudt(1), U. Kuhlicke(1), H. Horn(2), D. C. Hem-
pel(3), T. R. Neu(1)
(1) Department of Inland Water Research Magdeburg,
UFZ Centre for Environmental Research Leipzig-Halle,
Brueckstrasse 3A, 39114 Magdeburg, Germany; (2) Hy-
drochemistry, Hochschule Magdeburg-Stendal (FH), Breit-
scheidstrasse 2, 39114 Magdeburg, Germany; (3) IBVT,
Institute for Biochemical Engineering, Technical University
of Braunschweig, Gau_strasse 17, 38106 Braunschweig,
Germany
Fluorescently labelled lectins have been suggested as in
situ probes to stain lectin-speci¢c glycoconjugates in the
extrazellular polymeric substances (EPS) of bio¢lms. In
this study the complete panel of all commercially available
lectins (63) were tested for their binding properties. For
this purpose lotic bio¢lms from the river Saale were grown
in rotating annular reactors with river water only and with
additional carbon sources e. g. glucose or methanol. Sam-
ples were analysed by standard Confocal Laser Scanning
Microscopy. The volume of the stained glycoconjugates
was calculated after thresholding by measuring the quan-
tity of voxels in the 3-dimensional image stacks. The var-
ious lectins showed di¡erential staining in bio¢lms grown
with di¡erent substrates and with respect to bacterial cell
morphology. Of the lectins tested, 40 showed a useful
binding pattern for bio¢lm glycoconjugate analysis. In glu-
cose grown bio¢lms the lectins Aleuria aurantia, Phaseolus
coccineus, Solanum tuberosum, and Triticum vulgaris
showed a strong binding pattern. Whereas in methanol
bio¢lms the lectins of Aleuria aurantia and Hippeastrum
hybrid proofed to be most suitable. Other lectins e.g. Can-
avalia ensiformis and Iberis amara had a strong a⁄nity to
all types of bio¢lms. Some of the lectins showed a weak
binding pattern or did not bind at all. In conclusion, there
is a range of potential lectins for glycoconjugate analysis
in microbial bio¢lms. Nevertheless, each type of bio¢lm
has to be evaluated using ideally the full range of available
lectins in order to select the most suitable panel of lectins
for lectin-binding-analysis in microbial communities.
 1st FEMS Congress / Posters 103^505
P12^37
IN SITU ANALYSIS OF BACTERIAL MICROCOL-
ONY STRUCTURE AND ACTIVITY USING LASER
SCANNING MICROSCOPY
J. R. Lawrence(1), G. D. W. Swerhone(1), T. R. Neu(2)
(1) National Water Research Institute, 11 Innovation Bou-
levard, Saskatoon, Saskatchewan, Canada, S7N 3H5; (2)
Department of Inland Water Research Magdeburg, UFZ
Centre for Environmental Research Leipzig-Halle, Brueck-
strasse 3A, 39114 Magdeburg, Germany
Fluorescence confocal laser microscopy was used to assess
the structure, composition and activity of bacterial micro-
colonies in natural river bio¢lms. Bio¢lms were cultivated
in rotating annular reactors with river water as sole source
of nutrients and inoculum. A variety of microcolonies with
extensive exopolymer matrices were present in these bio-
¢lms. Lectin staining indicated a three component exopol-
ymer matrix, i) cell associated, ii) intercellular and iii) an
outer coat covering the entire colony. The cell-associated
layer bound lectins with speci¢city for galactose, glucose,
mannose and N-acetyl glucosamine ; the intercellular layer
bound lectins with a⁄nity for glucuronic acids; and the
outer coat, lectins with speci¢city for fucose. Permeability
studies using size fractionated £uor conjugated dextrans
and modi¢ed surface chemistry £uorescent beads (20 nm,
40 nm 100 nm) indicated that all three layers were perme-
able to 20 nm beads. The intercellular exopolymer was
penetrated by 940 nm probes and the outer coat was
not permeable to v100 nm £uorescent beads. Phosphatase
activity was found to be most intense at the cell surface
and least within the intercellular exopolymer. Glucose ox-
idase activity was only detected in the cell-associated layer
of polymer. The membrane potential sensitive dye JC-1
formed £uorescent J-aggregates only within the outer
coat layer of the microcolony. A dual labelled (rhoda-
mine-£uorescein) pH reporter indicated a gradient of pH
from the cell surface to the microcolony exterior. In con-
clusion, exopolymers provide a structuring mechanism to
segregate extracellular activities at the microscale and may
provide a basis for unitary metabolism.
FEMSLE Congress 2-6-03
445
P12^38
MICROSCALE AND MOLECULAR ASSESSMENT
OF THE IMPACTS OF NICKEL ON RIVER BIOFILM
COMMUNITIES
J. R. Lawrence(1), M. Chenier(2), D. Beaumier(2), G. D.
W. Swerhone(1), T. R. Neu(3), C. W. Greer(2)
(1) National Water Research Institute, 11 Innovation Bou-
levard, Saskatoon, Saskatchewan, Canada, S7N 3H5; (2)
Biotechnology Research Institute, National Research Coun-
cil, 6100 Royalmount Avenue, Montreal, Quebec, Canada,
H4P 2R2; (3) Department of Inland Water Research Mag-
deburg, UFZ Centre for Environmental Research Leipzig-
Halle, Brueckstrasse 3A, 39114 Magdeburg, Germany
The in£uence of: nutrients, dissolved oxygen concentra-
tion (DO) and nickel (Ni) concentration on river bio¢lm
development, structure, function and composition was as-
sessed in rotating annular reactors. Treatments included,
river water at 0.5 mg l-1 or 7.5 mg l-1 DO, plus a combi-
nation of carbon, nitrogen and phosphorus nutrients
(CNP), with or without 0.01, 0.05, 0.1, or 0.5 mg l-1 Ni.
Nickel resulted in elimination of cyanobacterial popula-
tions and reduced photosynthetic biomass. Lectin binding
analyses indicated changes in exopolymer abundance and
glycoconjugate make up of the bio¢lms in response to all
treatments. Carbon utilization spectra were signi¢cantly
depressed at 0.1 and 0.5 mg l-1 nickel corresponding to
the drinking water and release rate guidelines respectively.
In the presence of CNP and at both DO levels, Ni neg-
atively a¡ected denitri¢cation but had no e¡ect on hexa-
decane mineralization or sulfate reduction. Analysis of
total community DNA indicated abundant eubacterial
16S rDNA, whereas Archaea were not detected. Ampli¢-
cation of the alkB gene indicated a positive e¡ect of nu-
trients and a negative e¡ect of Ni. The nirS gene was not
detected in samples treated with 0.5 mg l-1 Ni. DGGE
analyses indicated e¡ects of both nutrients and Ni, with
0.5 mg l-1 Ni resulting in the appearance of unique bands
in DNA from Ni, DO, and CNP treatments. FISH anal-
yses indicated a signi¢cant decrease in beta proteobacterial
and cytophaga-£avobacterial abundance. These observa-
tions indicate that guideline Ni concentrations may have
signi¢cant impacts on river microbial community diversity
and function.
446 1st FEMS Congress / Posters 103^505
P12^39
3-DIMENSIONAL ANALYSIS OF BIOFILM IMAGES
COLLECTED BY LASER SCANNING MICROSCOPY
A. Eitner(1), O. Buettner(1), S. Bergner(2), K. To«n-
nies(2), J. R. Lawrence(3), T. R. Neu(1)
(1) Department of Inland Water Research Magdeburg,
UFZ Centre for Environmental Research Leipzig-Halle,
Brueckstrasse 3A, 39114 Magdeburg, Germany; (2) De-
partment of Computing Science, Otto-von-Guericke-Univer-
sity, Universitaetsplatz 2, 39106 Magdeburg, Germany ; (3)
National Water Research Institute, 11 Innovation Boule-
vard, Saskatoon, Saskatchewan, Canada, S7N 3H5
Laser scanning microscopy has developed into an indis-
pensable tool for in situ analysis of microbial commun-
ities. It may be applied for three reasons: visualisation,
analysis and quanti¢cation. The latter however remains
di⁄cult as environmental microbial bio¢lms show a com-
plex composition and the software packages available
have a number of limitations. In order to analyse multi-
channel data of lotic bio¢lms four software packages were
compared. The image ¢les used for analysis were 3-chan-
nel images showing nucleic acid stained bacteria, lectin
stained glycoconjugates, chlorophyll auto£uorescence as
well as co-localisation of cyanobacterial auto£uorescence.
With all programs, the most important and critical step of
digital image analysis is the segmentation procedure.
scionimage is freely available, has the advantage that it
is widely used and allows macro programming e.g. for
semi-automated analyses of multiple data sets. voxelshop
is suitable for detailed analysis but is time consuming. It
has no macro option and cannot be automated. Co-local-
isation of signals is only available in combination with
another program. comstat is also freely available and
has been developed for analysis of pure or mixed culture
bio¢lms in £ow cells. The program includes the analyses of
structural parameters. microstat was developed for im-
ages of complex environmental bio¢lms with co-localised
signals. The algorithm takes advantage of a new segmen-
tation method combining a voxel-based classi¢cation with
low-level shape characteristics. In conclusion, the sample
properties as well as the purpose of the analysis will ¢nally
determine the decision to select a speci¢c program for
digital image analysis of laser microscopy data sets.
FEMSLE Congress 2-6-03
P12^40
CHARACTERISATION OF THE WOLBACHIA BAC-
TERIA ASSOCIATED WITH COLLEMBOLA SPE-
CIES COLLECTED IN HUNGARY
G. Nyirò, K. Ma¤rialigeti
Department of Microbiology, Eo«tvo«s Lora¤nd University,
Pa¤zma¤ny Pe¤ter stny. 1/C, H-1117, Budapest, Hungary
Wolbachia pipientis bacteria are obligate cytoplasmic al-
pha-Proteobacteria often found in invertebrates like ar-
thropods and nematodes. These symbiotic bacteria are
transovarially inherited and also able to establish mutual-
istic but also parasitic interactions with their host organ-
isms. Based on 16S rDNA sequence data obtained from
the isotomid collembolan host Folsomia candida and phy-
logenetic analysis a distinct group of Wolbachiae, group E
was formed. In this work further sequence data were ob-
tained from various Collembola host-species collected in
Hungary using 16S rDNA, ftsZ and wsp markers in order
to characterize the real phylogenetic diversity of group-E
Wolbachiae.
P12^41
INTERACTIONS IN SYNTROPHIC PROPIONATE-
OXIDIZING METHANOGENIC CONSORTIA
C. M. Plugge and A. J. M. Stams
Laboratory of Microbiology, Wageningen University, H.
van Suchtelenweg 4 6703 CT Wageningen, The Netherlands
Methanogenic environments are widespread in nature. In
these environments large amounts of the greenhouse gas
methane are released. Although the uncontrolled forma-
tion of methane is undesirable, there are controlled bio-
technological processes that generate methane as an en-
ergy source. A variety of microbial processes are
involved in the anaerobic methanogenic degradation of
organic matter. The ultimate end product from these mi-
crobial processes is methane and carbon dioxide. Interspe-
cies electron transfer plays a crucial role in methanogenic
environments. Fermentative and acetogenic bacteria pro-
duce reducing equivalents that are consumed by methano-
gens. Hydrogen consumption by methanogens leads to
enhanced growth rates and results in a shift in the metab-
olism of fermenting bacteria. Moreover, acetogenic bacte-
ria are strictly dependent on methanogens for the removal
of end products. E.g. propionate oxidation is only possible
at low product concentrations, and therefore cooperation
with H2-scavenging methanogens is obligatory. Syntropho-
bacter species are well-known gram-negative propionate-
degrading bacteria, clustering in the N-subdivision of the
 1st FEMS Congress / Posters 103^505
proteobacteria. In suspended co-cultures our model-organ-
ism Syntrophobacter fumaroxidans is only able to oxidize
propionate syntrophically with methanogens that use both
H2 and formate as electron donor for CO2-reduction. As a
consequence, all microbes have to tune their metabolism.
Information exchange takes place on a physiological level
by the exchange of compounds that may lead to the gen-
eration of speci¢c cell wall proteins. To test this, we com-
pared the cell wall protein composition of Syntrophobacter
fumaroxidans grown in pure culture and in coculture with
a methanogen using 2D-gelelectrophoresis. A selection of
spots was identi¢ed by mass spectrometry.
P12^42
YEAST TWO HYBRID APPROACH TO CHARAC-
TERIZE MOSQUITO-DENGUE VIRUS INTERAC-
TIONS
M. Porcar and A. Delecluse
Interaction Mole¤culaires Flavivirus-Ho“tes, Institut Pasteur,
25, rue du Dr. Roux 75724 Paris Cedex 15
Dengue is an emerging disease a¡ecting the tropical re-
gions worldwide. Dengue virus (DENv) is transmitted by
the mosquito Aedes aegypti, and the geographical distri-
bution of the disease overlaps that of the vector. The abil-
ity of mosquitoes to transmit the disease, known as vector
competence, varies among the di¡erent mosquito popula-
tions and is known to be genetically determined. The fac-
tors involved in DENv entry and replication in the mos-
quito are still unknown. In order to identify such factors,
we have developed a yeast two hybrid approach; the strat-
egy of choice consisted of detecting and characterizing
mosquito proteins interacting with the enveloppe (E) and
membrane (M) DENv proteins. Several fragments of these
proteins have been cloned into bait plasmid vectors. In
parallel, an expression cDNA bank from Aedes aegypti
has been constructed into prey vectors; the mRNA were
extracted from insect midguts, since this tissue is consid-
ered to be the ¢rst target for viral entry. The ¢rst inter-
action results obtained between preys and baits will be
presented.
FEMSLE Congress 2-6-03
447
P12^43
ROLE OF CYCLIC PEPTIDES IN INTERACTIONS
BETWEEN PSEUDOMONAS AND ZOOSPORIC
FUNGI
J. T. de Souza(1), M. de Boer(2), C. F. Geerds(1), P. de
Waard(3), T. A. van Beek(4), and J. M. Raaijmakers(1)
(1) Laboratory of Phytopathology, Wageningen University,
P.O. Box 8025, 6709 PG Wageningen; (2) Applied Plant
Research, section Flowerbulbs, Lisse ; (3) NMR Centre,
Wageningen University ; (4) Natural Products Chemistry
group, Laboratory of Organic Chemistry, Wageningen Uni-
versity, The Netherlands
Microbial compounds that alter the conditions prevailing
at a surface or interface are often referred to as bioemul-
si¢ers or biosurfactants. A variety of microorganisms, in-
cluding bacteria, fungi and yeasts, have been described to
produce biosurfactants. Several of these biosurfactants are
well-characterized chemically and catagorized into high-
and low-molecular mass compounds. Among the bacterial
genera, Pseudomonas species have been reported to con-
tain strains that produce biosurfactants. Fluorescent Pseu-
domonas spp. are common inhabitants of soil and rhizo-
sphere environments and have received considerable
interest in the areas of bioremediation and biological con-
trol of plant pathogenic fungi. In the area of biological
control, we have recently identi¢ed a strain of P. £uores-
cens, referred to as strain A, that produces biosurfactant(s)
with lytic activity against zoospores of a variety of plant
pathogenic fungi, including Pythium, Phytophthora, and
Albugo candida. RP-HPLC and NMR analyses indicated
that strain A produces at least ¢ve extracellular com-
pounds with lytic activity against zoospores. One of these
compounds was identi¢ed as a cyclic lipopeptide with nine
amino acids that reduces the surface tension of water to
approximately 29 mN/m. At concentrations of 5-25 Wg/ml,
these peptides caused cessation of motility of zoospores of
all three fungal genera and lysis of entire zoospore pop-
ulations in less than 1 minute. In bioassays and ¢eld-ex-
periments, the biosurfactant-producing Pseudomonas
strain A gave signi¢cant control of Pythium root rot of
£owerbulbs and Pythium damping-o¡ of cucumber. Muta-
tional analysis revealed that the biosurfactant(s) plays a
determinative role in interactions between Pseudomonas
strain A and Pythium species.
448 1st FEMS Congress / Posters 103^505
P12^44
FUNGAL DEFENSE MECHANISMS AGAINST AN-
TAGONISTIC BACTERIA
A. Schouten(1), G. van den Berg(1), V. Edel(2), C. Stein-
berg(2), N. Gautheron(2), P. Lemanceau(2) and J. M.
Raaijmakers(1)
(1) Laboratory of Phytopathology, Wageningen University,
P.O. Box 8025, 6709 PG Wageningen, Netherlands; (2)
UMR INRA/Universite¤ de Bourgogne ‘Microbiologie et
Ge¤ochimie du Sol’, INRA-CMSE, BP 86510 21065 Dijon
Cedex, France
In soil and plant-associated environments, interactions
within and among microbial communities are numerous
and range from synergistic and mutualistic to antagonistic
and parasitic. Antagonistic interactions have been ex-
ploited in the area of biological control of plant pathogen-
ic fungi. To date, biological control is typically viewed
from the perspective of how antagonists a¡ect pathogens.
This study examines the other face of this interaction, i.e.
how plant pathogenic and saprophytic fungi respond to
antagonistic bacteria. More speci¢cally, we studied the
variation in sensitivity of plant pathogenic and sapro-
phytic Fusarium oxysporum to the phenolic antibiotic
2,4-diacetylphloroglucinol (2,4-DAPG) produced by an-
tagonistic Pseudomonas £uorescens. Among seventy patho-
genic and twenty-seven non-pathogenic Fusarium oxyspo-
rum isolates, obtained from di¡erent host plants and
geographic locations, considerable variation in sensitivity
to 2,4-DAPG was found. For many F. oxysporum isolates,
growth was completely inhibited at 2,4-DAPG concentra-
tions of 25 to 50 Wg/ml. However, 18% of the pathogenic
and 25% of the non-pathogenic F. oxysporum isolates were
able to grow at 2,4-DAPG concentrations of 400 Wg/ml
and were classi¢ed as 2,4-DAPG resistant. HPLC analysis
showed that most of the resistant F. oxysporum isolates
degrade 2,4-DAPG. Preliminary phylogenetic analyses
showed that there is no clear relationship between 2,4-
DAPG resistance and geographic origin or formae spe-
ciales of F. oxysporum. In conclusion, this study shows
that just as microbial antagonists utilize a diverse arsenal
of mechanisms to dominate interactions with pathogens,
pathogens have surprisingly diverse responses to cope with
antagonism.
FEMSLE Congress 2-6-03
P12^45
IN VITRO INTERACTION BETWEEN YERSINIA EN-
TEROCOLITICA SEROTYPE O:3, ITS LIPOPOLY-
SACCHARIDE MUTANT STRAINS AND HUMAN
MACROPHAGES
S. Salmenlinna(1), J.-A. Bengoechea(2) and M. Skur-
nik(1)
(1) Haartman Institute, University of Helsinki, Helsinki,
Finland ; (2) Unidad de Investigacion, Hospital Son Dureta,
Palma Mallorca, Spain
Yersinia enterocolitica transfers from gut to underlying
tissue through M cells in Peyer’s patches. It is believed
that thereafter the local defence is mediated by macro-
phages. It has previously been demonstrated that Y. enter-
ocolitica lipopolysaccharide (LPS) components play a crit-
ical role in outer membrane properties relevant in
resistance against antimicrobial peptides. To investigate
the interaction between macrophages and invading bacte-
ria, we infected stimulated human macrophage cell line
U937 with Y. enterocolitica serotype O:3 wild type (wt)
bacteria and with several di¡erent LPS mutant bacteria.
The in£uence of presence or absence of virulence plasmid
pYV was also analysed. Di¡erent bacterial growth temper-
atures and post infection incubation temperatures were
compared. Preliminary results indicate that during the ¢rst
hours of incubation most of the wt bacteria are killed,
whereas outer core deleted mutants are more resistant to
killing. Subsequently, wt bacteria start to increase in num-
ber, but mutants are rapidly eliminated. Tissue culture
supernatants of macrophages infected with bacteria har-
bouring di¡erent LPS structures vary in their ability to kill
wt and LPS mutant bacteria. These results suggest that the
majority of the wt bacteria are phagocytosed and killed
rapidly, whereas killing of mutant bacteria, possibly re-
maining in the supernatant, occur later by a secreted anti-
microbial substance. Further analysis of the secreted sub-
stance will be performed.
P12^46
COMPETITION FOR WHEAT STRAW AND ANTAG-
ONISTIC BEHAVIOUR OF TRICHODERMA ISO-
LATES AGAINST RHIZOCTONIA SOLANI
S. Sarrocco, C. Bernardi, M. Forti and G. Vannacci
Department of Fruit Science and Plant Protection ‘‘G. Scar-
amuzzi’’, Sect. Plant Pathology, University of Pisa, Via del
Borghetto, 80-56124, Pisa, Italy
Rhizoctonia solani incites radish seed rot or death of in-
fected seedlings either before or just after their emergence
 1st FEMS Congress / Posters 103^505
from the soil. As plants grow they become resistant there-
fore biocontrol agents are required to defend plants for a
short period of time. Trichoderma is a genus including
well-known biocontrol isolates and in this work a large
collection has been screened to ¢nd isolates e¡ective in
reducing Rhizoctonia damping of in radish and to get in-
formation about their competition with the pathogen for
nutrients in soil. More than one thousand isolates of Tri-
choderma were screened on the basis of their ‘‘in vitro’’
growth rate at 24‡C. The best 15 were chosen and used
as inoculants in a not sterile pot mix screening to select
those that reduced Rhizoctonia disease incidence on radish
seedlings in simultaneous and not-simultaneous soil co-in-
oculation of pathogen and antagonists. The best 4 Tricho-
derma isolates were singularly coinoculated with Rhizocto-
nia in sterilized pot mix and after 24 hours pieces of straw
were buried. Every 2 days, straw pieces were collected and
plated on Rhizoctonia selective medium to evaluate the
percentage of colonization by the pathogen. At the same
time seeds of radish were sown on the same pots from
which straw was recovered. Emergence and disease prog-
ress on seedlings was recorded. Possession of straw by the
pathogen seems important for disease progress. Data are
presented that show the four Trichoderma isolates di¡ering
in reducing straw possession by the pathogen and are dis-
cussed in view of ruderal/combative/stress tolerant life
strategies of Trichoderma.
P12^47
IDENTIFICATION, TIMING AND SIGNAL SPECI-
FICITY OF PSEUDOMONAS AERUGINOSA QUO-
RUM-CONTROLLED GENES: A TRANSCRIPTOME
ANALYSIS
M. Schuster(1), C. P. Lostroh(1), T. Ogi(2), and E. P.
Greenberg(1)
(1) Department of Microbiology & W. M. Keck Microbial
Communities and Cell Signaling Program, University of
Iowa, Iowa City, IA 52242, U.S.A.; (2) Genome Damage
and Stability Centre, University of Sussex, Falmer, Brigh-
ton BN1 9QG, United Kingdom
There are two interrelated acyl-homoserine lactone quo-
rum-sensing-signaling systems in Pseudomonas aeruginosa.
These systems, the LasR-I system and the RhlR-I system,
are global regulators of gene expression. We have under-
taken a transcriptome analysis to identify quorum-sensing-
controlled genes and to better understand quorum-sensing
control of P. aeruginosa gene expression. We compared
gene expression in a LasI-RhlI signal mutant grown with
added signals to gene expression without added signals
and we compared a LasR-RhlR signal receptor mutant
to its parent. In all we identi¢ed 315 quorum-induced
and 38 quorum-repressed genes, representing about 6%
FEMSLE Congress 2-6-03
449
of the P. aeruginosa genome. The quorum-repressed genes
were activated in stationary phase in quorum-sensing mu-
tants but were not activated in the parent strain. The
analysis of quorum-induced genes suggests that the signal
speci¢cities are on a continuum, and that the timing of
gene expression is on a continuum (some are induced early
in growth, most are induced at the transition from loga-
rithmic to stationary phase, and some are induced during
stationary phase). In general, timing was not related to
signal concentrations. We suggest that the level of the
signal receptor, LasR, is a critical trigger for quorum-ac-
tivated gene expression. Acyl-homoserine lactone quorum
sensing appears to be a system that allows an ordered
expression of hundreds of genes during P. aeruginosa cul-
ture growth.
P12^48
QUORUM-SENSING AFFECTS ENTRY OF GROUP-
A STREPTOCOCCUS INTO EPITHELIAL CELLS
S. Sela(1) and M. Marouni(2)
(1) Department of Food Sciences, ARO, The Volcani Cen-
ter, P.O.Box 6, Beth-Dagan, 50250 Israel ; (2) Department
of Human Microbiology, Sackler school of Medicine, Tel-
Aviv University, Tel-Aviv, Israel
Many species of bacteria regulate gene expression in re-
sponse to cell population density through a mechanism
called quorum sensing. Intercellular communication con-
trols gene expression in response to population density.
Response to environmental stress enable bacteria to ac-
quire an adaptive response to provide signi¢cant bene¢ts
in the colonization of hosts, defense against competitors,
adaptation to varying physical conditions, as well as cel-
lular di¡erentiation. Streptococcus pyogenes is a major hu-
man pathogen that is responsible for a wide range of dis-
eases ranging from mild pharyngotonsillitis and
pyodermas to life-threatening invasive infections. Re-
cently, it was found that S. pyogenes possess a homologue
of the luxS gene, associated with the expression of auto-
inducer 2 (AI-2), which participates in quorum sensing of
both Gram-positive and Gram-negative bacteria. To test
whether streptococcal internalization is a¡ected by AI-2, a
LuxS deletion mutant was constructed and its internaliza-
tion e⁄ciency was determined in HEp-2 cell model. Inter-
estingly, the mutant was internalized by HEp-2 cells with
higher e⁄ciency compared to the wild type. To elucidate
the mechanism by which the mutation has a¡ected bacte-
rial internalization we have performed Northern blot anal-
ysis. The transcription of several S. pyogenes virulence
genes known to a¡ect bacterial uptake by epithelial cells
was determined. LuxS mutation signi¢cantly reduced ex-
pression of streptococcal pyrogenic exotoxin B, and in-
creased expression of M protein. These results indicate
450 1st FEMS Congress / Posters 103^505
that LuxS activity plays a central role in the expression of
streptococcal virulence factors involved in bacterial uptake
by epithelial cells.
P12^49
REGULATION OF THE LATERAL FLAGELLA COL-
ONISATION SYSTEM OF AEROMONAS CAVIAE
G. J. Horsburgh(1), S. Clark(1), S. M. Kirov(2) and J. G.
Shaw(1)
(1) University of She⁄eld Medical School, Division of Ge-
nomic Medicine, Beech Hill Road, She⁄eld, S10 2RX, UK;
(2) Discipline of Pathology, University of Tasmania, Ho-
bart, Tasmania 7001, Australia
Aeromonas spp. are ubiquitous inhabitants of aquatic en-
vironments that are increasingly being recognised as gas-
trointestinal pathogens. The ability to move over and col-
onise surfaces after initial attachment is thought to be an
important component of virulence and bio¢lm formation.
Swarming motility, a £agellum-dependent behaviour that
allows bacteria to move over surfaces, has been implicated
in the colonisation process. In liquid environments, Aero-
monas caviae utilise a polar monotrichous £agella for mo-
tility, but in viscous environments or on solid surfaces
some strains express a separate lateral £agella system for
swarming. Strains of an A. caviae isolate with speci¢c mu-
tations that knockout the expression of the lateral £agella
were shown to be unable to swarm, had reduced adherence
to tissue culture cells and bio¢lm formation. Unlike Vibrio
parahaemolyticus, the polar £agellum of A. caviae does not
appear to be the mechano-sensor for lateral £agella ex-
pression, as mutations of the polar £agella £a (¢lament,
hook) and £g (basal body, hook) loci do not a¡ect the
expression or control of lateral £agella production. Where-
as, transposon mutation of £iM and downstream genes
(switch, export) resulted in the loss of both polar and
lateral £agella systems. This suggests that the polar and
lateral £agella systems are mainly distinct but probably
share the same export apparatus. Analysis of the regula-
tion of the lateral £agella system is being under taken
through the use of chromosomal xylE transcriptional fu-
sions, transposon mutagenesis and direct genomic se-
quencing.
FEMSLE Congress 2-6-03
P12^50
NOVEL BACTERIA DEGRADING QUORUM-SENS-
ING SIGNAL MOLECULES : USE OF A RHODOCOC-
CUS ERYTHROPOLIS ISOLATE AS A BIOCON-
TROL AGENT DIRECTED TOWARDS PLANT
PATHOGENIC BACTERIA
S. Uroz(1), C. D’angelo(1), P. Oger(2), D. Faure(1) and
Y. Dessaux(1)
(1) Institut des Sciences du Ve¤ge¤tal, CNRS UPR2235, Gif-
sur-Yvette CEDEX, France; (2) Laboratoire de Sciences
de la Terre, Ecole Normale Supe¤rieure, 43 alle¤e d’Italie,
69364 Lyon CEDEX, France
The NAHL-mediated quorum-sensing signalization path-
way is common among Gram-negative bacteria, including
pathogenic (Agrobacterium, Erwinia, Pseudomonas) and
bene¢cial (Pseudomonas, Rhizobium) plant-associated bac-
teria. In a complex environment such as the rhizosphere,
chemical and enzymatic degradation of the NAHLs is a
key factor that may modulate the expression of the
NAHL-regulated functions. Because of the abundance of
the NAHL-producing strains in the root environment, we
hypothesized that the rhizosphere also contains bacteria
that degrade the quorum-sensing signal. Using enrichment
procedures, we investigated their occurrence, their taxo-
nomic position, their NAHL-degradation spectrum, and
also their use as biocontrol agent. Bacteria degrading the
quorum-sensing (QS) signal molecule C6-HSL were iso-
lated from tobacco rhizosphere. Twenty-¢ve isolates de-
grading C6-HSL felt into in 6 groups according to their
genomic REP-PCR and rrs PCR-RFLP pro¢les. Repre-
sentative strains from each group were identi¢ed as mem-
bers of the Pseudomonas, Comamonas, Variovorax and
Rhodococcus genera. All these isolates degraded acyl-
HSL other than C6-HSL, albeit with di¡erent speci¢city
and kinetics. One of these isolates, R. erythropolis strain
W2, exhibited a highly e⁄cient catabolic activity. Conse-
quently, it has been used in attempts to quench quorum-
sensing signals regulating various functions of other mi-
crobes. In vitro, W2 strongly interfered with violacein pro-
duction by Chromobacterium. In planta, R. erythropolis
strain W2 markedly reduced the pathogenicity of Pecto-
bacterium carotovorum subsp. carotovora. These results re-
veal the diversity of the QS-interfering bacteria in the rhi-
zosphere and demonstrate the validity of targeting
quorum-sensing signal molecules to control pathogens
with natural bacterial isolates.
 1st FEMS Congress / Posters 103^505 451

P12^51 P12^52

CELL-CELL CONTACTS ARE ESSENTIAL FOR THE Withdrawn.

GROWTH OF BACTERIAL CULTURES UNDER
POOR NUTRIENT CONDITIONS

S. A. Voloshin and A. S. Kaprelyants

Bach Institute of Biochemistry, RAS, 117091, Moscow,

Leninsky pr., 33, Russia

Intercellular communications in bacterial cultures medi-

ated by chemical signals have widespread importance for
many speci¢c events in bacterial life. Some studies also
revealed signi¢cant variation of gene expression and
change in cell metabolism when cell contact each other
physically (e.g. in bio¢lms). However, little is known
how formation of cell-cell contacts may in£uence cell
growth, in particular if such contacts provide some advan-
tage for cells when they grow under inappropriate (poor)
conditions? To answer this question we compared behav-
ior of two Gram positive, related bacteria- Rhodococcus
rhodochrous and Micrococcus luteus that form aggregates
stabilized by extracellular substance(s) when they grow in
liquid medium. We compared cell growth under normal
conditions (£ask shaking, 100 rpm) and conditions where
cell-cell contacts were signi¢cantly impaired (by intensive
agitation (IA)-250 rpm of a £ask contained glass protru-
sions to induce turbulence of the medium). When cells
grew in rich medium we did not observe any signi¢cant
di¡erence in growth curve either under normal or IA con-
dition. In contrast, cells of both strains were not able to
develop visible optical density on de¢ned minimal media
under IA condition. This inability was not connected with
degree of cell aeration. The observed e¡ect was reversible :
decrease in agitation speed resulted in start of visible cell
growth. Supernatant taken from log phase cells of R. rho-
dochrous grown on minimal medium stimulated growth of
the same cells under IA conditions. The stimulatory activ-
ity of the supernatant was heat-stable. Microscopy of R.
rhodochrous revealed transition of normal road-shaped
cells to coccoid cells under IA conditions. We suggest
that cell-cell contacts allow cells to multiply under inap-
propriate nutrient conditions when individual cells failed
to grow. Probably cells are able for dividing within aggre-
gates due to (local) cryptic growth.

FEMSLE Congress 2-6-03

452 1st FEMS Congress / Posters 103^505
P12^53
ACTIVATION OF NF-KB IN MONOCYTES BY
STAPHYLOCOCCUS EPIDERMIDIS
Y . Ljungh
N. Yanagisawa and A
Medical Microbiology, Dermatology and Infection, Lund
University, So«lvegatan 23, SE223 62 Lund, Sweden
Staphylococcus epidermidis is a major cause of opportun-
istic infections, including those associated with biomateri-
als. Many cell surface proteins of S. epidermidis have been
recognised to mediate microbes to biomaterial surfaces,
and to eukaryotic cells by binding proteins in plasma
and extracellular matrices. Cell surface proteins of S. epi-
dermidis were subjected to investigation on the potential to
activate NF-UB in monocytes. S. epidermidis J9P, isolated
from an infected graft, was cultured in Todd Hewitt broth
for 22 to 24 hours. Cell surface components were extracted
by using lithium chloride (LiCl) 1M, lysostaphin 200Wg/ml
or SDS 2%. U937 were cultured in RPMI media contain-
ing foetal bovine serum 10% and L-glutamine 2mM. Nu-
clear extracts of U937 cells were obtained 2 hours after
incubation of bacteria or cell surface extracts. Electropho-
retic mobility shift assay (EMSA) was made with 32P-la-
beled NF-UB consensus oligonuclear probe by using 4Wg
of nuclear extracts of U937 cells. NF-UB was activated in
U937 cells when live cells of J9P was incubated at 105 to
107cfu/ml but was not when 10cfu/ml was used. DNA-
binding activity of NF-UB was inhibited by addition of
1000-fold cold probe. SDS-PAGE of LiCl, lysostaphin
and SDS cell surface extracts of J9P contained 110, 60,
52, 38, 21 and 16kDa proteins, that bound vitronectin on
immunoblots. One Wg/ml of LiCl extract activated NF-UB
in U937 cells but cell surface components extracted by
lysostaphin and SDS did not. Activation of NF-UB in
U937 cells by J9P may involve components of the bacterial
cell surface.
P13^1
DEVELOPMENT OF NEW VECTOR SYSTEMS FOR
TRANSFORMATION OF ZYGOMYCETES
Ł cs, I. Nyilasi, T. Papp, and Cs. Va¤gvo«lgyi
K. A
University of Szeged Department of Microbiology, P.O.
Box 533 H-6701 Szeged, Hungary
Mucor circinelloides and Rhizomucor miehei are ¢lamen-
tous fungi belonging to the Zygomycetes. Their special
biochemical, morphological and physiological features es-
tablished a longstanding interest of both applied and the-
oretical research. Until now, the transformation systems
developed in Mucor circinelloides are based on comple-
FEMSLE Congress 2-6-03
mentation of (e.g. amin oacid) auxotrophy. In spite of
the e⁄ciency, such transformation systems have a serious
drawback: a de¢nite stable mutant has to be isolated from
each strain before the transformation. Therefore, it seems
desirable to establish transformation systems based on a
strong native promoter allowing e⁄cient expression of het-
erologous resistance gene as selection marker. In the
present study transformation systems based on selective
drugs have been tested for use in Zygomycetes. Transfor-
mation vectors have been constructed which contain hy-
gromycin B resistance gene under the control of the pro-
moter of the glyceraldehyde-3-phosphate dehydrogenase
(gpd) gene from M. circinelloides and R. miehei. In con-
trast to other transformation systems which rely on nutri-
tional auxotrophic markers for the selection of transform-
ants, the combination of a gpd promoter sequence and a
dominant selectable marker allows the transformation of
wild type strains. Optimal conditions for transformation
which increase the sensitivity of these fungi for hygro-
mycin have also been worked out.
P13^2
GENOME-WIDE ANALYSIS OF LACTIC ACID BAC-
TERIA: BIOINFORMATICS AND TRANSCRIPTOME
ANALYSIS
R. J. S. Baerends, S. A. F. T. van Hijum, H. A. Karsens, A.
de Jong, C. D. den Hengst, A. L. Zomer, N. E. Kramer, G.
Buist, J. Kok and O. P. Kuipers
Molecular Genetics, Groningen Biomolecular Sciences and
Biotechnology Institute, University of Groningen, P.O. Box
14, 9750 AA Haren, The Netherlands http://molgen.biol.-
rug.nl
The recent availability of the complete nucleotide sequen-
ces of the chromosomes of many micro-organisms, among
which important human pathogens such as Streptococcus
pneumoniae, and industrially important organisms such as
Bacillus subtilis and Lactococcus lactis, should allow re-
construction and understanding of the physiology of
each organism. Among the prokaryotes the Gram-positive
bacteria are a very prominent group that is characterised
by a strong evolutionary relatedness. Some species are of
great importance in the industrial agro-food chain. Our
research group has a long history in the research of the
Gram-positive bacteria B. subtilis and L. lactis. Recently,
DNA microarray analysis was implemented in our re-
search. DNA microarrays on glass slides are being con-
structed in-house that contain ampli¢ed DNA fragments
of the complete genomes of B. subtilis, L. lactis IL1403, L.
lactis MG1363 (genome sequencing consortium : M. Gas-
son [Institute of Food Research, Norwich, UK], D. van
Sinderen [University College, Cork, Ireland] and O.P.
Kuipers/J. Kok), S. pneumoniae (together with P. Her-
 1st FEMS Congress / Posters 103^505
mans, Pediatrics, Rotterdam) and, soon, B. cereus. Using
genome-wide transcriptome analysis we aim to reconstruct
the metabolic pathways and gene regulatory networks of
the bacteria under study, with a focus on stress response,
protein secretion, competence development, sporulation,
£avour formation, health risk assessment and food safety.
The poster will present the validation of the transcriptome
analysis procedure using L. lactis IL1403 and MG1363
DNA microarrays and software and bioinformatics tools
developed for analysis of the transcriptome data.
P13^3
DNA REPAIR IN NEISSERIA MENINGITIDIS:
CHARACTERISATION OF THE DNA GLYCOSY-
LASE MutY
T. Davidsen, Y. Esbensen, N.-M. Jahnsen, E. Seeberg and
T. T\njum
Centre for Molecular Biology and Neuroscience and Insti-
tute of Microbiology, Rikshospitalet, University of Oslo, N-
0027 Oslo, Norway
Genome alterations due to horisontal gene transfer and
abundant recombinational events, as well as DNA damag-
ing agents, are causes of constant strain on the gene pool
of Neisseria meningitidis, the causative agent of meningo-
cocccal disease. This naturally competent bacterium hosts
a genome which is highly recombinogenic and has an ele-
vated spontaneous mutation rate, representing unusual
challenges in terms of genome maintenance. However,
the meningococcus is well equipped for DNA repair and
expresses components representing all known DNA repair
pathways. The base excision repair pathway is one of the
major lines of defence against the detrimental e¡ects of
DNA damage. The enzymes initiating this pathway are
called DNA glycosylases, and they recognise and excise
di¡erent kinds of DNA damage. We have cloned and
over-expressed the meningococcal DNA glycosylase
MutY of the base excision repair pathway in E. coli. Pu-
ri¢ed meningococcal DNA glycosylase activity was moni-
tored towards a range of DNA substrates. The DNA
binding and enzyme activities of the MutY full-length
and partial domains were assessed. Meningococcal strains
with mutations in the genes encoding MutY and a range
of components representing all major DNA repair path-
ways were constructed, and wildtype and mutant strains
were compared with regard to mutagenicity and survival
under varying stress conditions. The DNA glycosylase mu-
tants displayed a decreased survival rate and an elevated
spontaneous mutation rate compared to the wild type
strain. Our ¢ndings show that meningococcal MutY plays
a de¢nite role in genome maintenance, alone and in its
interactions with other DNA repair mechanisms.
FEMSLE Congress 2-6-03
453
P13^4
DETECTION OF MYCOBACTERIUM AVIUM
SUBSP. PARATUBERCULOSIS IN GOATS MILK BY
IMMUNOMAGNETIC PCR
B. Dj\nne(1), M. R. Jensen(1), I. R. Grant(2), G. Hol-
stad(1)
(1) National Veterinary Institute, Post Box 8156 Dep., N-
0033 Oslo, Norway ; (2) Department of Food Science (Mi-
crobiology), The Queen’s University of Belfast, Belfast BT9
5PX, N. Ireland, UK
Mycobacterium avium subsp. paratuberculosis (M. paratu-
berculosis) has been suggested as an aetiological agent of
Crohn’s disease. The bacterium has been isolated from
cow’s milk and detected by immunomagnetic separation
combined by PCR (IMS-PCR) in goat’s milk. Milk sam-
ples from 340 individual dairy goats in 34 herds through-
out Norway were examined for M. paratuberculosis by
culture and IMS-PCR. The samples included; vaccinated
goats in herds with paratuberculosis, vaccinated goats in
herds with no history of paratuberculosis and unvacci-
nated goats in herds with no history of paratuberculosis.
Viable M. paratuberculosis were not detected by culture in
any sample, but 7.1% tested positive by IMS-PCR when
the PCR products were visualised by dot blot hybridisa-
tion. PCR products from ¢ve milk samples originating
from ¢ve di¡erent herds were sequenced ; all showed
99% homology with the IS900 sequence from M. paratu-
berculosis. The percentage of IMS-PCR positive samples
from herds where paratuberculosis had previously been
reported was signi¢cantly lower than from herds where
the infection had never been diagnosed (3.3% and 9.1%
respectively, P = 0.048). Similar proportions of milk sam-
ples from vaccinated and non-vaccinated goats tested pos-
itive for the presence of M. paratuberculosis. Vaccinated
goats older than four years tested positive more often than
vaccinated animals less than two years old. Samples col-
lected in May tested signi¢cantly more often positive than
milk sampled during February to March (13.8% and 2.9%
respectively, P = 0.001). This study showed that raw goats’
milk in Norway might be contaminated with M. paratu-
berculosis.
454 1st FEMS Congress / Posters 103^505
P13^5
PLASMID BORNE PATHOGENECITY ISLAND OF
F18+ ENTEROTOXIC ESCHERICHIA COLI
P. Zs. Fekete(1), Gy. Schneider(2,3), F. Olasz(4), G.
Blum-Oehler(2), J. Hacker(2,3) and B. Nagy (1)
(1) Veterinary Medical Research Institute of the Hungarian
Academy of Sciences, Budapest, Hungary ; (2) Institut fu«r
Moleculare Microbiologie, Universita«t Wu«rzburg Wu«rz-
burg, Germany; (3) Institute of Medical Microbiology
and Immunology, University of Pe¤cs, Pe¤cs, Hungary ; (4)
Agricultural Biotechnology Center, Go«do«llò, Hungary
Most virulence genes of enterotoxigenic Escherichia coli
(ETEC) are located on plasmids. The gene for heat-stable
enterotoxin I. (sta) is part of the Tn1681 (So and McCar-
thy, 1980), and the heat-stable enterotoxin II. (stb) gene
was described in the Tn4521 (Lee et al, 1983). In studies
presented here, one large (V120 kb) plasmid of a porcine
ETEC strain producing F18 ¢mbriae, was found to har-
bour both sta, and stb, and tetracycline resistance. In fur-
ther studies the nucleotide sequence of an approx. 10 000
bp fragment of the virulence plasmid (pTc) was deter-
mined, and results suggested that this fragment was part
of a pathogenicity island-like (PAI-like) gene cluster, hith-
erto unknown. Sequences in the £anking regions of the sta
indicated the presence of the known Tn1681, but £anking
sequences of the neighbouring stb gene were completely
di¡erent from the already known Tn4521. This ‘‘ST-
PAI’’ cluster was mobilized ^ as a 40 kb fragment ^ into
another plasmid (pACYC177). This 40 kb transposition
product (named AKR2) contained both sta and stb. and
the replication origin of pTc. Several F4(K88)+ and F18+
ETEC strains from weaned pigs of di¡erent geographical
origin (Hungary, Austria, and USA) were also PCR-ed to
see if they posses the ‘‘Tn4521-like’’ or the ‘‘pTc-like’’
£anking regions of stb. It turned out that ‘‘Th4521-stb’’
was present in K88+ ETEC, while ‘‘PAI-stb’’ was present
in F18 ETEC. These results suggest the existence and
worldwide spread of a new plasmid encoded pathogenecity
island (ST-PAI) in porcine postweaning ETEC strains,
producing F18 ¢mbriae.
FEMSLE Congress 2-6-03
P13^6
COMPLETE SEQUENCE ANALYSIS OF THE GE-
NOME OF EJ-1, A MYOVIRIDAE PNEUMOCOCCAL
BACTERIOPHAGE
P. Romero, R. Lo¤pez and E. Garc|¤a
Centro de Investigaciones Biolo¤gicas, CSIC, Vela¤zquez 144,
28006 Madrid, Spain
EJ-1 is an inducible prophage present in the 101/87 strain,
a nontypeable, optochin-resistant, and bile negative Strep-
tococcus pneumoniae isolate (D|¤az et al. 1992. J. Bacteriol.
174:5516^5525). This phage harbours a gene (ejl) very
similar (81% identity) to the lytA101 gene of the host bac-
terium that codes for the major autolytic amidase.
Although Siphoviridae (bearing long £exible tails) and Po-
doviridae (bearing short tails stubs) bacteriophages have
been reported, EJ-1 is the ¢rst Myoviridae (bearing con-
tractile tail) phage that has been studied in the genus
Streptococcus. The attB, attP, attL, and attR sequences
have been determined showing that EJ-1 DNA integrates
into the 3’ end of the SP1113/spr1020 gene of S. pneumo-
niae, which codes for a histone-like, DNA-binding protein,
but does not interrupt the reading frame of the gene. In-
tegration of temperate phages at an equivalent position
had been reported in Group A streptococci. The ca. 40-
kb, double-stranded linear DNA of EJ-1 has been com-
pletely sequenced and the gene products predicted from
the nucleotide sequence have been compared to those in-
cluded in the databases. Interestingly, the general organi-
zation of the EJ-1 genome is similar to that previously
reported for Siphoviridae phages infecting low-GC-content
Gram-positive bacteria (H. Bru«ssow (2001) Annu. Rev.
Microbiol. 55, 283^303). However, the proteins encoded
by genes belonging to the structural cluster exhibit signi¢-
cant similarities to those of the defective Bacillus subtilis
phage PBSX also showing a Myoviridae morphotype. Our
results favour a phage taxonomical system based on com-
parative genomics of the structural gene module (head
and/or tail genes).
P13^7
DRUG EXPORT PERMEASE INVOLVED IN MULTI-
DRUG RESISTANCE IN KLUYVEROMYCES LACTIS
Y. Gbelska, M. Takacova, D. Imrichova and J. Subik
Comenius University, Faculty of Natural Sciences, Depart-
ment of Microbiology and Virology, 842 15 Bratislava, Slo-
vak Republic
Several transport systems play an important role in con-
ferring multiple drug resistance, presumably due to the
 1st FEMS Congress / Posters 103^505
catalysis of energy-dependent extrusion of a large number
of structurally and functionally unrelated compounds out
of the cells. In the present work the gene encoding K. lactis
membrane permease was cloned by functional complemen-
tation of the cycloheximide hypersensitivity phenotype of
Saccharomyces cerevisiae mutant strain lacking functional
PDR1 and PDR3 genes. The isolated gene exhibits 48.9 %
identity with S. cerevisiae ATR1 gene and encodes a pro-
tein of 553 amino acids. When present in multicopy, it
e⁄ciently complemented the phenotype associated with
the pdr5 or pdr1pdr3 mutations in S. cerevisiae. Overex-
pression of the gene in K. lactis wild-type strains led to the
resistance to several cytotoxic compounds like 4-nitroqui-
noline-N-oxide, 3-aminotriazole and ketoconazole. The
gene has been assigned to K. lactis chromosome III. Based
on the phenotype of homologous and heterologous trans-
formants we propose, that the gene encodes a membrane-
associated component of the machinery responsible for
pumping several toxic compounds out of the cell.
P13^8
COMPARATIVE STUDIES ON GENES INVOLVED
IN FLAGELLA PRODUCTION IN DIFFERENT SAL-
MONELLA SEROVARS
A. Imre(1), F. Olasz(2) and B. Nagy(1)
(1) Veterinary Medical Research Institute of the Hungarian
Academy of Sciences, Budapest, Hungary; (2) Agricultural
Biotechnology Center, Go«do«llò, Hungary
Analysis of £agellin genes was carried out on wild-type
Salmonella Typhimurium (10), Salmonella Hadar (10)
and Salmonella Enteritidis (48) strains by Polymerase
chain reaction (PCR). The following genes were examined:
two di¡erent structural genes of £agellin (£iC, £jB), £jA,
wich is the repressor of £iC, the operator region of £iC
and the invertase system (hin, hixL, hixR), which is re-
sponsible for phase variation in Salmonella. We have
found that all of the examined genes (£iC, £iC-operator,
£jB, £jA, hin, hixL, hixR) were present in S. Typhimurium
and S. Hadar strains. However, the strains of S. Enter-
itidis are lacking the invertase system (hin, hixL, hixR) as
well as £jA and £jB, on the other hand the £iC and its
operator are present.The results con¢rm at DNA level the
serological knowledge concerning the H antigenes of Sal-
monella, which constitute one of the bases of the sero-
grouping of the Kaufmann-White typing-scheme. Accord-
ing to this the S. Typhimurium strains belonging to the B
serogroup and the S. Hadar strains belonging to the C2
serogroup possess two di¡erent H antigenes (2 phases).
Thus, PCR results showed that the invertase system and
both £agellin genes are present in the serovars mentioned
above. In contrast, the S. Enteritidis strains of the D se-
rogroup can only exist in one certain phase (g m). PCR
FEMSLE Congress 2-6-03
455
results con¢rm, that S. Enteritidis strains are lacking the
£jB gene responsible for the II. phase, the repressor pro-
tein FljA and the invertase system (hin, hixL, hixR). The
above results will probably be applicable in making live,
oral Salmonella markered vaccines.
P13^9
GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGE-
NASE AS A MARKER OF FUNGAL GROWTH DUR-
ING THE INTERACTION OF THE NECROTROPHIC
FUNGUS SCLEROTINIA SCLEROTIORUM WITH
ITS PLANT HOSTS
Zs. Kasza(1,2), P. Cotton(1), Cs. Va¤gvo«lgyi(2), M. Fe-
vre(1)
(1) Universite¤ Claude Bernard Lyon I, UMR 5122 Micro-
biologie et Ge¤ne¤tique, Laboratoire Biologie Cellulaire Fon-
gique,10 rue Dubois (bat ex 405), Villeurbanne 69622,
France ; (2) University of Szeged Department of Microbi-
ology, P.O. Box 533 H-6701 Szeged, Hungary
The gpd gene of Sclerotinia sclerotiorum was cloned from
genomic library. Analysis of the fragment subcloned re-
vealed an ORF of 1133 bp and a promoter of 735 bp.
The ORF contained two putative introns with convention-
al splice sites. Analysis of the promoter sequence revealed
the presence of several elements that could act as signals
required for transcription initiation and regulation. North-
ern blot analysis revealed that gpd is di¡erentially ex-
pressed during growth in the presence of di¡erent carbon
sources. GPD was highly expressed during the pathogen-
esis of four di¡erent plants. Northern analysis revealed
similar expression pro¢les. The intensity of gpd hybridisa-
tion signals increased and reached a maximum when the
plant tissues were completely invaded by the fungus. As
actin is frequently used as a marker of fungal growth
during plant-fungus interactions, we compared expression
pro¢les obtained with gpd and actin during pathogenesis.
The analysis revealed similar pro¢les for both genes. Our
results show that even if gpd is not a constitutively ex-
pressed gene its expression pro¢le re£ects the amount of
fungus during the interaction of Sclerotinia sclerotiorum
with its plant hosts.
P13^10
Withdrawn.
456 1st FEMS Congress / Posters 103^505
P13^11
TRANSPOSON MUTAGENESIS OF THE PHAGE
xYeO3-12
S. Kiljunen(1), H. Vilen(2), H. Savilahti(2) and M. Skur-
nik(1)
(1) Department of Medical Biochemistry and Molecular
Biology, University of Turku; (2) Institute of Biotechnol-
ogy, University of Helsinki
xYeO3-12 is a lytic bacteriophage that infects Yersinia
enterocolitica O:3 (YeO3). The phage is also able to infect
and proliferate in Escherichia coli strains expressing the
YeO3 O-antigen, which is the phage receptor. The genome
of xYeO3-12 is a 39 600 bp long linear, double-stranded
DNA molecule that codes for 58 putative genes. xYeO3-
12 belongs into T7 ^ group of phages, T3 being its closest
relative. Based on homologies to T7 and T3, the functions
of many of xYeO3-12 genes have been elucidated, but
there are still many open reading frames in the genome
whose role is not known. Here, we used e⁄cient trans-
poson insertion mutagenesis to study the role of the phage
genes. As the transposition mutagenesis system, we utilised
the MuA transposase-catalysed in vitro transposition reac-
tion, with lacZ as a marker gene in the transposon. Mu-
tant phages were identi¢ed by the blue color of the plaques
growing in a strain JM109/pAY100. Altogether 17 trans-
poson mutants were obtained, two of which contained
double-insertions. To study the phenotypes of the mu-
tants, EOPs (e⁄ciency of plating) and phage ¢tnesses
were measured in di¡erent bacterial hosts. Mutants having
insertions in genes coding for DNA ligase and lysozyme
propagated slower than xYeO3-12 in YeO3 but not in E.
coli. Also, in Yersinia but not in other bacteria tested,
mutants having insertion in phage gene 0.45 had clearly
lower EOP. Still, further studies are needed to understand
the biological basis for these phenotypes.
FEMSLE Congress 2-6-03
P13^12
ADAPTATION OF CAMPYLOBACTER DURING
OXYGEN STARVATION ^ PROTEOME ANALYSIS
L .M. Lawrance, C. Constantinidou, J. A. Cole and C. W.
Penn
School of Biosciences, University of Birmingham, Birming-
ham, B15 2TT, UK
Campylobacter is the major cause of bacterial food-poi-
soning in the developed world, where contaminated poul-
try is a major source of infection. Considered microaero-
philic, Campylobacter survives in the chicken caecum in
almost anaerobic conditions. However, traces of oxygen
are essential for growth. In this study C. jejuni 11168
was grown in di¡erent oxygen concentrations, and pro-
teins involved in adaptation to reduced oxygen were iden-
ti¢ed. Protein extracts from biomass obtained from each
of three levels of oxygenation were separated by 2D elec-
trophoresis. Spots showing substantial di¡erence in abun-
dance between cultures, when analysed using PDQuest
software, were excised from the gel, digested with trypsin
and identi¢ed using mass spectrometry. One of the pro-
teins identi¢ed was the large sub-unit of formate dehydro-
genase (Fdh), the abundance of which increased as the
oxygen level decreased. C. jejuni Fdh appears metabol-
ically isolated, as the genome lacks pyruvate-formate
lyase. Furthermore focA, a formate transport system, is
absent. However, genes for many enzymes involved in an-
aerobic metabolism are present. In Escherichia coli fdh is
controlled by the fumarate-nitrate reductase regulator
(FNR). However, there is no FNR binding motif up-
stream of the C. jejuni fdh. A signal motif for the twin
arginine secretion pathway (T-R-R-S-F-L-K) is present
in the N-terminal region of Fdh strongly suggesting it is
a periplasmic protein, and that exogenous formate is the
primary source of formate for C. jejuni, and a key electron
donor for anaerobic respiration.
 1st FEMS Congress / Posters 103^505
P13^13
MUREIN HYDROLASES OF STREPTOCOCCUS
PNEUMONIAE : STRUCTURE OF THE CHOLINE-
RECOGNIZING DOMAIN AND BIOLOGICAL
ROLES OF FOUR CHOLINE-BINDING PROTEINS
R. Lo¤pez, B. de las Rivas, J. L. Garc|¤a, and P. Garc|¤a
Centro de Investigaciones Biolo¤gicas, CSIC, Vela¤zquez 144,
28006 Madrid, Spain
Murein hydrolases are enzymes that degrade the cell. walls
of microorganisms and these enzymes exhibit both sub-
strate and bond speci¢city. In Streptococcus pneumoniae,
a human pathogen, these proteins display a modular orga-
nization and have for activity an absolute requirement for
the presence of choline in the teichoic acid, a structural
component of the cell wall, and are named as choline-
binding proteins (CBPs). Pneumococcus has evolved a
mechanism unique for anchoring and displaying proteins
with the cell wall. A recent step ahead in the detailed
knowledge of CBPs has been the elucidation of the crystal
structure of the binding module of the LytA amidase, the
major autolysin in pneumococcus (C-LytA): a novel sol-
enoid-like fold consisting exclusively of L-hairpins that
stack to form the left-handed superhelix. Choline mole-
cules at the hydrophobic interface of consecutive hairpins
maintain this unique structure. The active form of LytA
requires the formation of a dimer. Molecular and bio-
chemical analyses of two new CBPs have allowed to iden-
tify the ¢rst L-n-acetylglucosaminidase (LytB) and a phos-
phoryl choline esterase (Pce) of S. pneumoniae. The
combined use of mutants and the preparation of a trans-
lational fusion constructed between gfp and lytB supports
the notion that LytB recognizes speci¢c polar receptors
and is responsible for cell separation. Global analysis of
the four CBPs with enzymatic activity identi¢ed in pneu-
mococcus have permitted to draw a picture on the phys-
iological roles of LytA, the lysozyme LytC, LytB and Pce
in this pathogen.
FEMSLE Congress 2-6-03
457
P13^14
MOLECULAR CLONING AND SEQUENCE ANALY-
SIS OF THE GENE ENCODING 3-HYDROXY-3-
METHYLGLUTHARYL COENZYME A REDUCTASE
IN RHIZOMUCOR MIEHEI
Ł cs, M. Vastag and Cs. Va¤gvo«lgyi
Gy. Luka¤cs, K. A
Department of Microbiology, Faculty of Sciences, Univer-
sity of Szeged, P.O. Box 533, Szeged 6701, Hungary
Among thermophilic fungi, Rhizomucor miehei is a practi-
cally and theoretically signi¢cant member of the Mucor-
ales. Ocassionally, they are agents of opportunistic infec-
tions. Other strains are important in the food industry as
protease producers. The homothallic nature of this species
provides a good opportunity for studying sexual processes
of Zygomycetes. The aim of this study was to clone and
characterise the 3-hydroxy-3-methylglutharyl coenzyme A
reductase (HMG-CoA reductase) of Rhizomucor miehei.S-
peci¢c primers have been used to amplify a short con-
served region of the gene by polymerase chain reaction.
The resulting 314 bp length DNA segment proved to be
highly homologous to known HMG-CoA sequences. This
amplicon was labeled with non-radioactive dioxigenine
and used as a homologous probe to select positive clones
from a genomic library prepared from the strain Rhizomu-
cor miehei ATCC 46344 in Q FixII phage. These hybridi-
zation experiments resulted 6 clones which contain HMG-
CoA reductase gene sequences. One Q phage clone has
been selected and after puri¢cation steps it was used for
the further molecular analysis. Restriction endonuclease
digestions and hybridization experiments revealed that
this clone contains a genomic insert of about 8000 bp.
This insert was cloned into pBluescript plasmid with
XhoI restriction endonuclease. A plasmid construction
with a BamHI insert of about 5000 bp has been used for
further subcloning experiments. Data from the determined
DNA and the predicted aminoacid sequences have been
analysed. Further studies is in progress to determine the
complete sequence and to clarify its role in the sexual pro-
cesses of Zygomycetes.
458 1st FEMS Congress / Posters 103^505
P13^15
REGULATION OF THE PLASMID FUNCTIONS
THROUGH THE TOPOLOGY OF THE REPLICON
I. Muiznieks, N. Matjushkova, G. Dumpe, U. Malinovskis
and G. Makarenkova
Faculty of Biology, University of Latvia, 4, Kronvalda
Blvd., LV-1586, Riga, Latvia
The topology, spatial architecture, alongside with the lin-
ear sequence elements of the bacterial plasmids, play im-
portant role in the regulation of their replication and gene
expression. The maintenance stability of the pBR -group
plasmids in Escherichia coli is increased by in vivo inser-
tions of IS elements, which carry sequence -directed bent
DNA segments, into the 5’- portion of the tet resistance
gene. The sequence of the gene is identical in all the pBR
plasmids, but the insertion hot-spots, which produce the
e¡ect of the stabilisation, are di¡erent, although highly
speci¢c for every particular member of the plasmid family:
pBR313, pBR322, pBR327 or pBR329. Recloning of the
IS elements into di¡erent positions of the tet gene demon-
strated that the e¡ect of the maintenance stabilisation is
related not just with tet gene inactivation, but also with
the localisation and direction of the integrated sequences.
The plasmids, which exhibited the highest maintenance
stability started replication earlier, had higher supercoiling
density and copy number in the ¢rst half of the exponen-
tial phase of the host strain’s growth in comparison to
their less stable counterparts or to the plasmids without
IS elements. Similarly, the expression of cloned human
interferon alpha-5 gene was enhanced by the deletions in
the plasmid sequences, which were not directly involved in
the regulation of the gene transcription, but altered the
supercoiling density of the replicon. We suppose that the
enhanced supercoiling of the plasmid molecules facilitate
the availability of the promoters and ori region to the
transcription / replication machinery.
P13^16
AGROBACTERIUM TUMEFACIENS-MEDIATED
TRANSFORMATION OF MUCOR CIRCINELLOIDES
Ł cs, Gy. Luka¤cs, T. Papp, Zs. Kasza and Cs.
I. Nyilasi, K. A
Va¤gvo«lgyi
Department of Microbiology, Faculty of Sciences, Univer-
sity of Szeged, P.O. Box 533, Szeged 6701, Hungary
Mucor circinelloides is an important member of Zygomy-
cetes: it can cause serious postharvest losses in various
agricultural products and also well known as opportunistic
human pathogen. The development of an e⁄cient trans-
FEMSLE Congress 2-6-03
formation system would be signi¢cant for the investigation
of its genetic background. However, from the several
transformation methods tested for other fungi, only few
can be applied successfully in Zygomycetes. Agrobacterium
tumefaciens has the natural ability to transfer a segment of
DNA from its Ti plasmid -known as the T-DNA- into
plant cell genomes. The process of T-DNA transfer is de-
pendent on the expression of Agrobacterium tumefaciens’
vir genes. It was found recently, that fungi also could be
transformed by this approach; transformation of some
ascomycetes and basidiomycetes fungi has been reported
until now. Stimulated by these reports, we tested this
method for transforming Mucor circinelloides. Transfor-
mation was performed with the pBHt2 plasmid containing
the hygromycin B resistance gene under the Aspergillus
nidulans trpC promoter and terminated by the CaMV35S
poly (A) signal. Optimal conditions for the e⁄cient trans-
formation have been worked out. After the cocultivation
of the bacterium and the fungi, some transformant Mucor
colonies were isolated from the selective medium. The
presence of the gene in the genomic DNA of transformed
isolates was veri¢ed also by PCR reaction. Future studies
are progress to increase the transformation frequency.
P13^17
DESIGN, PRODUCTION AND APPLICATION OF A
DNA MICROARRAY FOR K-12 AND EHEC STRAINS
OF ESCHERICHIA COLI
C. Constantinidou, J. L. Hobman, S. J. W. Busby, J. A.
Cole, M. Patel, S. D. Minchin, C. W. Penn
School of Biosciences, University of Birmingham, Birming-
ham, UK
DNA microarray analysis of gene expression is a powerful
technique for monitoring simultaneously the expression of
thousands of genes. DNA microarrays are produced either
by in situ synthesis of oligonucleotides on silica (A¡yme-
trix), or by the immobilisation of PCR products or oligo-
nucleotides on to a solid support. To evaluate the merits
of di¡erent approaches, we spotted PCR products, and
50mer, 65mer and 70mer oligonucleotides, for the same
subset of genes, on to GAPS-coated glass slides. The prim-
ers and oligonucleotides were from commercial sources
and were based on manufacturers’ design criteria. Based
in part on the evaluation of the resulting data, we have
designed a whole genome microarray representing all
genes from the genome sequences of E. coli: K-12
(MG1655), O157 EDL933, and O157 Sakai. The array is
constructed from 70-mer oligonucleotides and includes a
comprehensive range of controls, and is available to aca-
demic researchers on a not-for-pro¢t basis. The data ob-
tained will conform to MIAME standards, through a col-
laboration with the BUGS group at St Georges Hospital
 1st FEMS Congress / Posters 103^505
Medical School, London (http://www.sghms.ac.uk/depts/
medmicro/bugs/). Evaluation of the array, including statis-
tical analysis of array and data quality to inform exper-
imental design, is in progress, using data from comparison
of global transcription in wild type and deletion mutants
of K-12 and Sakai strains. The mutations were made in
genes involved in global regulation of transcription, and
preliminary data on global transcription in these mutants
will be presented.
Authors 1 and 2 contributed equally to this work.
P13^18
TRANSCRIPTOME OF TREPONEMA PALLIDUM :
GENE EXPRESSION PROFILE DURING EXPERI-
MENTAL RABBIT INFECTION
D. Smajs(1), P. Matejkova(1), S. J. Norris(2) and G. M.
Weinstock(3)
(1) Department of Biology, Faculty of Medicine, Masaryk
University, Jostova 10, 662 43 Brno, Czech Republic; (2)
Department of Pathology and Laboratory Medicine, Uni-
versity of Texas-Houston Medical School, 6431 Fannin
Street, Houston, TX 77030, USA; (3) Human Genome
Sequencing Center, Baylor College of Medicine, One Baylor
Plaza, Alkek N1519, Houston, TX 77030, USA
DNA microarray technology was utilized to study gene
expression by the syphilis spirochete Treponema pallidum
subsp. pallidum (Nichols) during infection of rabbits. Mi-
croarrays containing all 1039 annotated ORFs of Trepo-
nema pallidum subspecies pallidum (Nichols) were printed
on glass slides. For 1034 ORFs (out of 1039), signals high-
er than the threshold (average of negative control spots +
3 SDs) were detected for both RNA and DNA probes.
Total RNA from T. pallidum isolated from rabbit testes
10 days post infection was labeled and standardized by
cohybridization of the same arrays with treponemal chro-
mosomal DNA labeled with a di¡erent £uorescent
marker. This internal standardization technique proved
to be highly reproducible and to decrease the impact of
variables such as host nucleic acid contamination or var-
iable target DNA lengths. The most highly transcribed
genes were found to correlate with the most conspicuous
spots identi¢ed by two dimensional gel electrophoresis,
indicating that the transcript levels generally corresponded
to the relative protein concentrations. Genes with high
transcript concentrations included those encoding £agellar
¢lament and cytoplasmic ¢lament proteins, prominent
lipoproteins and membrane proteins, chaperonins, pro-
teins involved in red-ox balance, chemotaxis regulatory
proteins, a V-ATPase operon, and certain metabolic en-
zymes such as glycolytic pathway enzymes. Independent
quantitation of the expression of 84 T. pallidum genes us-
ing real-time RT-PCR approach yielded a high degree of
FEMSLE Congress 2-6-03
459
correlation (r = 0.94). Characterization of the T. pallidum
transcriptome during experimental infection provides fur-
ther insight into the importance of gene expression levels
in the survival and pathogenesis of this bacterium in the
mammalian host.
P13^19
IDENTIFICATION OF PUTATIVE LIPOPROTEIN
GENES IN THE GENOME OF STREPTOCOCCUS
AGALACTIAE USING PATTERNS FOR THE RECOG-
NITION OF BACTERIAL LIPOPROTEINS
I. C. Sutcli¡e(1) and D. J. Harrington(2)
(1) Institute of Pharmacy, University of Sunderland, Sun-
derland, UK; (2) Department of Biomedical Sciences, Uni-
versity of Bradford, West Yorkshire, UK
N-terminal lipidation is a major mechanism by which bac-
teria can tether proteins to membranes and is of particular
importance to Gram-positive bacteria due to the absence
of a retentive outer membrane. Lipidation is directed by
the presence of a cysteine-containing ‘lipobox’ within the
lipoprotein signal peptide sequence and this feature has
greatly facilitated the identi¢cation of putative lipopro-
teins by analysis of translated gene sequences. The appro-
priate N-terminal sequence features are currently de-
scribed by the Prosite pattern entry PS00013 (see http://
ca.expasy.org/prosite/). As this is a frequently matched
pattern, we have designed a taxon-speci¢c pattern
(G+LPP) for the identi¢cation of Gram-positive bacterial
lipoproteins, based on the signal peptide features of 33
experimentally veri¢ed lipoproteins. Patterns searches
with the PS00013 and G+LPP patterns have been used
to identify probable lipoproteins in the genome of Strep-
tococcus pyogenes (I.C. Sutcli¡e and D.J. Harrington Mi-
crobiology 148, 2065-2077). This method is herein applied
to the recently published genomes of Streptococcus agalac-
tiae strains. The results con¢rm the likely abundance of
lipoproteins in Gram-positive bacterial proteomes, with at
least 30 probable lipoproteins identi¢ed in S. agalactiae.
As in other Gram-positive bacteria, the largest functional
category of the S. agalactiae lipoproteins is those predicted
to be substrate binding proteins of ABC transport sys-
tems. These include a homologue of pneumococcal PsaA
and we have Western blot data consistent with the expres-
sion and lipidation of this protein. Other lipoproteins ap-
pear to participate in protein export/maintenance. These
data suggest lipoproteins may have signi¢cant roles that
in£uence virulence of this important pathogen.
460 1st FEMS Congress / Posters 103^505
P13^20
FUNCTIONALITY OF INTEGRATION HOST FAC-
TOR IN PSEUDOMONAS PUTIDA : IN VIVO BIND-
ING AND GENOME-WIDE PREDICTION FOR TAR-
GET SEQUENCES
M. Valls, A. Munoz and V. de Lorenzo
Centro Nacional de Biotecnolog|¤a (CSIC) Campus UAM,
28049 Madrid, Spain
The integration host factor (IHF) is a small (20 kDa) basic
heterodimeric protein that binds and bends DNA speci¢-
cally. It belongs to the family of prokaryotic histone-like
proteins that includes HU, FIS and H-NS. Unlike other
HU-like proteins, IHF binds to speci¢c DNA sequences;
that in E. coli approximates to the asymmetric consensus
watcaannnnttr (where w is a or t; r is purine). IHF has
been implicated in nearly all major DNA functions, in-
cluding replication, transcription, site-speci¢c recombina-
tion, transposition, partitioning, transfer and packaging
into phage. Many promoters a¡ected by IHF are depen-
dent on RNA polymerase holoenzyme containing the al-
ternative factor c54. This holoenzyme requires the presence
of an activator protein that binds to the promoter up-
stream region to promote transcription. The Pu promoter
regulating the upper xyl operon of the TOL plasmid
pWW0 of Pseudomonas putida is a paradigmatic c54-de-
pendent promoter. IHF binding to Pu ¢xes the optimal
promoter geometry, facilitating contacts between distant
proteins and aiding the recruitment of RNA polymerase.
Understanding what determines IHF binding and its spe-
ci¢c targets in the genome is critical to better understand-
ing its physiological role. A thorough genome-wide esti-
mate of the binding sites has only been performed in the
E. coli chromosome, where the existence and location of
over 700 IHF sites was predicted using an extended con-
sensus and Hidden Markov Models. We used the Pu pro-
moter as a tool for de¢ning an in vivo functional consensus
for IHF binding in P. putida. We delineated the sequence
for IHF binding in P. putida by making single-base pair
substitutions within the cognate IHF binding site at the Pu
promoter and by examining promoter activity and IHF
binding in vivo with high-intensity UV footprinting on
live cells. The resulting information has been formatted
to generate an operative matrix for the genome-wide pre-
diction of IHF binding sites in the recently sequenced
chromosome of this bacterium.
G. Bertoni et al (1998). EMBO J. 17:5120-8. V. de Lor-
enzo et al (1991). EMBO J. 10:1159-67. J. Pe¤rez-Mart|¤n et
al (1994). J Biol Chem. 269:22657-22662. M. Valls et al
(2002). J Biol Chem. 277:2169-75.
FEMSLE Congress 2-6-03
P13^21
COMPLETE NUCLEOTIDE SEQUENCE OF THE 2,4-
DICHLOROPHENOXYACETIC ACID-DEGRADA-
TIVE PLASMID pEST4011 OF ACHROMOBACTER
XYLOSOXIDANS SUBSP. DENITRIFICANS STRAIN
EST4002
E. Vedler, M. Vahter and A. Heinaru
Institute of Molecular and Cell Biology, Tartu University,
23 Riia Street, Tartu 51010, Estonia
Many microorganisms are able to degrade herbicide 2,4-
dichlorophenoxyacetic acid (2,4-D). They usually contain
tfd-like (pJP4-like) genes for 2,4-D degradation, often en-
coded on transmissible plasmids. The 2,4-D-degradative
bacterium Achromobacter xylosoxidans subsp. denitri¢cans
strain EST4002, isolated in Estonia, was found to contain
the 70 kb plasmid pEST4011. The catabolic region of
pEST4011 is 39 kb long and it is lying between two copies
of insertion element IS1071-like sequences. IS1071 is a
class II (Tn3 family) insertion element, associated with
di¡erent catabolic genes and operons and globally distrib-
uted in recent past. We speculate that this insertion ele-
ment might have had a role in the formation of plasmid
pEST4011. The 2,4-D degradation genes of EST4002 are
all located on a catabolic region of pEST4011, they are all
tfd-like except for a tcbD (needed for degradation of 1,2,4-
trichlorobenzene by Pseudomonas sp. strain P51), coding
for chloromuconate cycloisomerase. The 7 kb region,
which contains the tcbD gene among other genes, is du-
plicated in pEST4011. The backbone of pEST4011 is
about 24 kb long and it contains genes necessary for plas-
mid maintenance and e¡ective transfer mechanisms re-
sponsible for dissemination of the catabolic functions in
bacterial populations of polluted sites. The backbone
genes are homologuous to the corresponding genes of
broad host range IncP1-alpha and IncP1-beta plasmids
R751, RP4, RK2, pADP-1 and several others. As only
few catabolic plasmids have been totally sequenced by
now, our study would give substantial information about
evolutionary mechanisms of formation of plasmids which
enable degradation of xenobiotic compounds.
 1st FEMS Congress / Posters 103^505
P13^22
THE PROPERTIES OF OVINE AND CAPRINE (MVV
AND CAEV) TAT PROTEINS ARE CLOSER TO HIV
AND SIV VPR PROTEINS
S. Villet, C. Faure, B. Bouzar, G. Verdier, C. Legras, and
Y. Chebloune
Laboratoire de Biologie Mole¤culaire, Cellulaire et Maladies
Emergentes, UMR 754 INRA/ENVL/UCBL, Universite¤
Claude Bernard, Bat B. 50 Avenue Tony Garnier, 69 366
Lyon cedex 07, France
Tat gene was shown to be present in all lentivirus genomes
and encodes a small protein whose main function is the
transactivation of LTR promoter. Based on the e⁄ciency
of transactivation activity we can distinguish between the
primate and the small ruminant lentivirus Tat proteins. In
a comparative study we have shown that MVV Tat pro-
tein upregulates only 2 to 3 fold the LTR activity, and no
upregulation was observed with CAEV Tat protein, in
contrast, 60 fold increase was observed with HIV-1 Tat
protein on HIV LTR promoter activity. The results of our
comparative study revealed the lack of any evidence of
function in small ruminant lentivirus Tat proteins similar
to that of primate lentivirus Tat proteins. Based on these
results we were interested to revisit the main function of
small ruminant lentivirus regulatory Tat protein. Consid-
ering that MVV and CAEV Tat proteins are structurally
more closely related to Vpr than to HIV Tat protein, we
looked for possible similarity of these proteins in term of
function. Like HIV Vpr protein, MVV and CAEV Tat
proteins seem to be localized in viral particles, to increase
a proportion of cells in G2/M phase of cell cycle, and to
induce apoptosis. Our ongoing studies aim to clearly dem-
onstrate that MVV and CAEV Tat proteins share proper-
ties of HIV and SIV Vpr proteins, and the absence of
transactivation system of small ruminant lentivirus LTRs
unlike the primate lentivirus group.
FEMSLE Congress 2-6-03
461
P13^23
CHARACTERIZATION OF A CLASS 1 INTEGRON
THAT CARRIES MULTIPLE ANTIBIOTIC RESIS-
TANCE GENES AND IS ASSOCIATED WITH A DE-
FECTIVE IS26 ELEMENT
S. Vourli(1), V. Miriagou(1), E. Lebessi(2), G. Sta-
mos(2), P. Giakkoupi(1), L. S. Tzouvelekis(3), E. Tzele-
pi(1) and N. J. Legakis(3)
(1) Laboratory of Bacteriology, Hellenic Pasteur Institute,
127 Vas. So¢as ave., Athens 115 21, Greece ; (2) Labora-
tory of Microbiology, P. & A. Kyriakou Children’s Hospi-
tal, Athens, Greece; (3) Department of Microbiology,
Medical School, University of Athens, M. Asias 75, Athens
115 21, Greece
A novel class 1 integron that carries an array of antibiotic
resistance genes including blaIBC-1 and its genetic environ-
ment was characterized, from an Escherichia coli clinical
isolate. Antibiotic resistance was transferred by conjuga-
tion via a plasmid of approximately 100 Mda. DNA map-
ping was facilitated by PCR using oligonucleotide primers
speci¢c for various segments of the class 1 integron and
IS26 structures. Detection of the antibiotic resistance
genes was also performed by PCR. Nucleotide sequencing
was performed directly on several overlapping amplicons.
Sequence similarity searches were carried out with the
blast program. The coding region of the integron consti-
tuted of an array of antibiotic resistance genes including
aac(6)-Ib, blaIBC-1, dhfrI, orf0 and aadA1. A P1 hybrid
promoter and a P2 inactive promoter located within the
intI1 gene preceded the gene cassettes region. Nucleotide
sequences similar to the IS26 insertion element were found
upstream the intI and downstream of the sulI genes. The
respective transposase genes facing the integron were trun-
cated at the N-terminus of the protein.
P13^24
FUNCTIONAL GENOMICS OF LACTOBACILLUS
SAKEI
S. Chaillou, M. Champomier-Verge's, A.-M. Crutz-Le Coq,
M. Cornet, P. Anglade, A.-M. Dudez, T. Me¤ra, S. Beau¢ls,
C. Belleanne¤e and M. Zagorec
Flore Lactique et Environnement Carne¤, INRA-CRJ, Do-
maine de Vilvert, F78350 Jouy-en-Josas, France
Lactobacillus sakei is a lactic acid bacterium commonly
found on meat products and used as starter for the pro-
duction of sausage in western Europe. In order to under-
stand the adaptation of this species to its environment and
the functions important for its use as starter, we have
462 1st FEMS Congress / Posters 103^505
sequenced the chromosome of a strain isolated from saus-
age. In total, some 15,000 sequencing reactions allowed to
obtain the whole genome sequence (1.880 Mb), with a
redundancy of about 4. The genome data will allow a
comparison with other related bacteria and with species
found in the same ecological environment. In parallel,
two dimensional gel electrophoresis and mass spectrome-
try analysis was performed on cellular extracts from cells
grown under various environmental conditions. From
about 250 spots detected on gels, approximately half could
be directly identi¢ed after query the genome sequences
with mass spectrometry data. The study of some proteins,
the expression of which varies depending on environmen-
tal conditions, will be presented. Genetic tools, to mutate
genes or operons, or to modulate gene expression, are
currently being developed in order to complement the
functional genomic study of L. sakei. The combination
of these tools, with genomics and proteomics data, will
be used to screen speci¢c functions important for the tech-
nological use of L. sakei in meat industry.
P13^25
FUNCTIONAL GENOMICS OF ASPERGILLUS NI-
GER STRAINS
R. Meulenberg, S. Breestraat, H. H. Menke, N. N. M. E.
van Peij, G. S. P. Groot and H. Stam
DSM Food Specialties, Research & Development, P.O. Box
1 (bag 624-0295), 2600 MA Delft, The Netherlands
The ¢lamentous fungus Aspergillus niger is a main micro-
organism used for enzyme production. Wild-type strains
of A. niger have the capacity of secreting large amounts of
various enzymes and are suitable hosts for homologous
and heterologous gene expression. Classical strain im-
provement programs and de¢ned genetic modi¢cations
have been used successfully to improve enzyme productiv-
ity. In order to rationalize and speed up the ongoing
strain- and process-improvement program, as well as to
identify potential new products, DSM sequenced the com-
plete 35.9 Mb genome of A. niger. The 7.5-fold coverage
random sequencing of carefully selected large insert bacs
allowed the assembly of the 8 linkage groups into 19 large
so-called supercontigs, each supercontig containing only
small sequence gaps. Over 14,000 open reading frames
(ORFs) were identi¢ed and functionally classi¢ed using a
combination of speci¢cally trained computer algorithms
and manual ORF veri¢cation and annotation. This pro-
gram was one of the largest commercial sequencing proj-
ects in Europe. Recently, A. niger GeneChips were de-
signed allowing the analysis of the genome-wide
transcriptome. The dynamics of A. niger strains at the
mRNA- and the DNA-level can be followed using these
DNA chips. Examples of Comparative Genomic Hybrid-
FEMSLE Congress 2-6-03
isation and controlled fermentations followed by whole
genome transcriptomic analysis will be shown. Especially,
the importance of a highly reproducible chip protocol in-
cluding fermentation sampling, RNA- and chip-processing
will be discussed. The A. niger genome was sequenced by
Gene Alliance (www.gene-alliance.com) and annotated by
Biomax Informatics (www.biomax.de). The GeneChips ex-
periments were performed in collaboration with the Mi-
croarray Department (www.microarray.nl).
P14^1
RAPID DELINEATION OF CLAVISPORA LUSITA-
NIAE CLINICAL ISOLATES BY PCR-SSCP ANALY-
SIS. A RETROSPECTIVE STUDY COMPARING
THREE MOLECULAR METHODS
M. Arabatzis(1), K. Kollia(1), P. Menounos(2), M. Log-
otheti(1), A. Velegraki(1) and N. J. Legakis(1)
Mycology Reference Laboratory, Microbiology Depart-
ment, Medical School, University of Athens, Mikras Asias
75-77, Goudi, Athens 11527, Greece; (2) Research Labo-
ratory, Military School of Nursing, Athens, Greece
Strain delineation of the emerging opportunistic pathogen
Clavispora lusitaniae was studied using 12 strains : Ten
clinical strains isolated between 1998 and 2001 from
bloodstream infections (6), pulmonary infection (1), oral
lesions (2) and vaginal infection (1) of compromised pa-
tients, and two strains, CBS 6936 and CBS 5094. This
retrospective study aimed to assess the occurrence of C.
lusitaniae subtypes within and among hospitals, and in
outpatients who were regularly screened for fungal infec-
tions in the course of radio-chemotherapy. Strain typing
was attained by single strand conformation polymorphism
(SSCP) analysis of the rRNA ITS 1 and 2 PCR ampli¢ed
regions. The results were compared with those of three
currently employed pulsed ¢eld gel electrophoresis
(PFGE) methods, and with minisatellite length polymorh-
ism (MLP) analysis. PFGE karyotyping separated 7-9
chromosomes, contrasting the 6-8 previously reported.
Electrokaryotyping of S¢ I digested chromosomes and
MLP analysis grouped isolates in 5 and 4 distinct clusters
respectively. All methods revealed strain heterogeneity,
though not as extensive as previously recorded. SSCP
analysis of ITS 1/2 ampli¢cation products (ITS 1 region)
generated ¢ve subtypes, with sequencing con¢rmed nucle-
otide polymorphism and with high discriminatory power.
All strains displayed a homogeneous SSCP pattern of ITS
3/4 ampli¢ed sequences (ITS 2 region). Although the ITS
1/2 PCR-SSCP protocol is evaluated for the ¢rst time in
typing C. lusitaniae, the results strongly suggest that it
allows rapid and reliable delineation of strains. Pending
examination of a larger sample size, this protocol can be
 1st FEMS Congress / Posters 103^505
recommended for rapid prospective identi¢cation of hos-
pital outbreaks.
P14^2
THE FIRST ISOLATION OF MALASSEZIA SP. IN
SERBIA
V. Arsic-Arsenijevic, D. Milobratovic, A. Dzamic, S. Mi-
trovic, A. Trpkovic, Lj. Petkovic
Institute of Microbiology and Immunology, School of Med-
icine, University of Belgrade, Dr Subotica 1, ll000 Belgrade,
Serbia and Montenegro
Today is known that genus Malassezia includes seven spe-
cies : M. furfur, M. sympodialis, M. obtusa, M. globosa, M.
restricta, M. sloo⁄ae and M. pachydermatis, but role of
each of the species in the pathogenesis of desease is not
eluciated yet, so futher laboratory isolation and identi¢ca-
tion are necessary. We report the ¢rst case of isolation of
Malassezia globosa in Serbia (Belgrade), in a patient sufer-
ring from Pityriasis versicolor. Identi¢cation of M. globosa
was based on macroscopic, microscopic and biochemical
characteristics. Isolation was done on Leeming-Notman
medium and on Dixona agar, at 350C, during 7 days in
aerobic conditions. Also the yeast biochemical phenotype
was determined: catalase (+), lipase (+), esculin degrada-
tion (-). M. globosa is a lipophilic yeast of the genus Ma-
lassezia and the common member of the skin £ora. In
concordance with some predisponing factors M. globosa
is implicated in the pathogenesis of several skin diseases
(pityriasis versicolor, malassezia foliculitis, seborrheic der-
matitis and some forms of atopic dermatitis). In immuno-
compromised patients and neonates this yeast can even
cause fatal systemic infections. Because the role of Malas-
sezia spp. in pathogenesis of skin desease is not still deter-
mined, we suggest laboratory diagnosis and identi¢cation
of these species as a routine diagnostic procedure.
P14^3
PURIFICATION AND CHARACTERIZATION OF
LACCASE FROM THE WHITE-ROT FUNGUS DAE-
DALEA QUERCINA
P. Baldrian
Laboratory of Biochemistry of the Wood-Rotting Fungi,
Institute of Microbiology ASCR, Videnska 1083, Prague
4, 14220, Czech Republic
The ligninolytic system of Daedalea quercina consists of
laccase and Mn-peroxidase, however, the latter enzyme
is produced only shortly and in low amounts. Laccase
(EC 1.10.3.2) was isolated as the principal lignin-modify-
FEMSLE Congress 2-6-03
463
ing enzyme present in the liquid cultures of Daedalea quer-
cina. The enzyme was puri¢ed to a homogeneous prepa-
ration using anion-exchange, and size-exclusion
chromatographies. SDS-PAGE analysis showed the puri-
¢ed laccase to be a monomeric protein of 69.0 kDa. The
enzyme had an isoelectric point of around pH 3.0 and
lacked the absorption maximum at 610 nm, typical for
blue laccases. The enzyme exhibited a strongly acidic op-
timum pH with 2,2’-azino-bis(3-ethylbenzothiazoline-6-
sulfonic acid) diammonium salt ^ bellow 2.0 (4.0 with
2,6-dimethoxyphenol, 4.5 with guaiacol, and 7.0 with sy-
ringaldazine) and the highest stability in neutral and alka-
line pH. The highest activity was obtained at 70 ‡C in
citrate-phosphate bu¡er (pH 5.0) and 55 ‡C in tartrate
bu¡er (pH 3.0). The enzyme was stable at temperatures
up to 55 ‡C. Among metal ions tested Mn, Hg and Cd
were the strongest inhibitors, whereas the enzyme activity
increased in the presence of copper. The puri¢ed laccase
was e¡ective in the decolorization of chemically di¡erent
dyes ^ Chicago Sky Blue 6B, Poly B-411, Reactive Black
5, Reactive Blue 2, Remazol Brilliant Blue R and Trypane
Blue ^ without any redox mediators.
This work was supported by the Grant Agency of the
Czech Academy of Sciences (B5020202).
P14^4
EFFECT OF COPPER AND CADMIUM ON THE
CELLULOLYTIC AND HEMICELLULOLYTIC EN-
ZYMES OF PLEUROTUS OSTREATUS
P. Baldrian and J. Gabriel
Laboratory of Biochemistry of the Wood-Rotting Fungi,
Institute of Microbiology ASCR, Videnska 1083, Prague
4, 14220, Czech Republic
The white-rot fungus Pleurotus ostreatus produces high
levels of ligninolytic enzymes (laccase and Mn-peroxidase)
and it is able to grow in non-sterile soil. It makes this
microorganism a promising tool for in situ biodegradation
of soils contaminated with PAH, PCB, synthetic dyes or
other organic pollutants. Cellulose and hemicelluloses are
the major source of energy for its growth in soil. During
the growth on wheat straw, Pleurotus ostreatus produced
the cellulolytic enzymes endo-1,4-beta-glucanase, exo-1,4-
beta-glucanase and 1,4-beta-glucosidase and the hemicel-
lulolytic enzymes endo-1,4-beta-mannanase, endo-1,4-
beta-xylanase, 1,4-beta-mannosidase and 1,4-beta-xylosi-
dase. Copper and cadmium a¡ect the overall degradation
of straw substrate as well as the activity of cellulolytic and
hemicellulolytic enzymes. Most cellulose-decomposing en-
zymes were positively regulated by the presence of 2 mM
Cd, whereas the activity of 1,4-beta-mannosidase was de-
creased. The e¡ect of 2 mM Cu on the enzymes was less
pronounced, but the loss of straw dry weight was signi¢-
464 1st FEMS Congress / Posters 103^505
cantly decreased. The toxicity of Cu increased during the
straw colonization by the fungus, since the metal bound to
the organic matrix was released during straw transforma-
tion by the fungus. Both Cd and Cu a¡ected the rate of
straw colonization by Pleurotus ostreatus. This work was
supported by the Grant Agency of the Czech Republic
(204/02/P100).
P14^5
LIPIDS IN cAMP-DEPENDENT PROTEIN KINASE
ASPERGILLUS NIGER MUTANTS
K. Jernejc and M. Benc›ina
Laboratory of Biotechnology, National Institute of Chemis-
try, Hajdrihova 19, P.O.B. 600, SI-1001 Ljubljana, Slovenia
The in£uence of cAMP-dependent protein kinase (PKA)
activity on lipid content in Aspergillus niger mutants with
overexpressed or deleted genes for either regulatory and/or
the catalytic subunit of PKA, was analyzed. Disruption of
the gene encoding for the PKA regulatory subunit resulted
in 20 % less total lipids, 30 % less neutral lipids, four times
more glycolipids and two-fold higher triacylglycerol lipase
activity compared with the control strain. The uncon-
trolled PKA activity was also linked to a ¢ve-fold decrease
in phosphatidylcholine, accompanied with apolar hyphal
growth and 1.5, 1.8 and 2.8 fold increases in phosphati-
dylethanolamine, lysophosphatidylethanolamine and
phosphatidylinositol, respectively, compared with the con-
trol strain. A decrease in total and neutral lipids accom-
panied with increased lipase activity were also determined
in mycelia producing citric acid. The lack of PKA activity
due to disruption of a gene encoding the PKA catalytic
subunit, resulted in a 1.6 times increase in total lipids with
two times more neutral lipids associated with lower tria-
cylglycerol lipase activity and a decrease in phospholipids.
The mutants with unrestricted PKA activity synthesized
twice as much citric acid as the control strain and three
times more than strains lacking PKA activity. These re-
sults indicate the involvement of cAMP mediated PKA
activity in regulation of triacylglycerol lipase activity ef-
fecting lipid content as well as morphogenesis and citric
acid synthesis.
FEMSLE Congress 2-6-03
P14^6
THE ROLE OF FUNGI IN THE PATHOGENESIS OF
CHRONIC POST-TRAUMATIC OSTEITIS
B. Beovic¤(1), N. Gunde-Cimerman(2), M. Cimerman(3),
P. Zalar(2), B. Gubina(3), M. Groznik(3)
(1) Department of Infectious Disease, University Medical
Centre, Ljubljana, Slovenia; (2) Dept. Biology, Biotechni-
cal Faculty, Univ. of Ljubljana, Slovenia; (3) Department
of Traumatology, University Medical centre, Ljubljana,
Slovenia
Chronic osteitis is a di⁄cult to treat and debilitating com-
plication of trauma. Low blood perfusion and bacteria are
known causes, but some recent reports suggest the role of
fungi in the in£ammatory process. Patients and methods.
Patients with chronic posttraumatic osteitis, hospitalised
at the Department of Traumatology from 1 July 2002 to
31 Dec 2003. Isolation of fungi from the bone specimens,
obtained during necrectomy was attempted in selective
media. Isolated fungi were analysed further at the Techni-
cal University in Lingby, Denmark and fungal species was
determined by the pro¢le of secondary metabolites deter-
mined by thin layer and high pressure liquid chromatog-
raphy. Routine histology and isolation of bacteria from
bone specimens was performed as well. 23 patients, 13
male and 10 female, with chronic posttraumatic osteitis
were included in the study. In 20 patients osteitis a¡ected
bones of the lower extremities, 8 of them had open bone
fractures. In 7 patients fungi were isolated (Neurospora
sp., Basidiomycetes sp., Aspergillus sp., Rhodotula sp., Pen-
icillium sp., Penicillium aurantiogriseum, Penicillium crus-
tosum). In two patients more than one species was iso-
lated. In one patient after open tibia and ¢bula fracture
Grocoft staining revealed fungal spores in the bone tissue,
and Neurospora sp. grew in the culture. In the other six
patients histology was inconsistent with fungal infection.
The results con¢rm previous data on the presence of fungi
in in£amed bone tissue. Further study is needed to identify
the pathogenic role of the fungi in chronic posttraumatic
osteitis.
 1st FEMS Congress / Posters 103^505
P14^7
SUBMERGED AND SOLID STATE BIOPROCESSING
OF SLOVENIAN GANODERMA LUCIDUM AND
EVALUATION OF ITS IMMUNOSTIMULATORY EF-
FECTS
M. Berovic›(1,2), B. Boh(3), J. Habjanic›(1), D. Hod-
z›ar(3), F. Pohleven(4), B. Wraber(5)
(1) National Institute of Chemistry, Hajdrihova 19, 1000
Ljubljana, Slovenia; (2) Department of Chemical, Bio-
chemical & Ecology Engineering, University of Ljubljana,
Askerc›eva 9, 1000 Ljubljana, Slovenija; (3) Faculty of Sci-
ence and Engineering, University of Ljubljana, Vegova 4,
1000 Ljubljana, Slovenija ; (4) Biotechnical Faculty, Uni-
versity of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana,
Slovenija; (5) Institute of Microbiology and Immunology,
Medical Faculty, University of Ljubljana, Zalos›ka 4, 1000
Ljubljana, Slovenija
Although Ganoderma mushrooms have been used for mil-
lennia in Chinese and Japanese traditional medicine for
treatment of several diseases, systematic research into their
pharmacological e¡ects started only about 25 years ago,
and has been focussed primarily on G. lucidum from Asian
habitats. As G. lucidum is scarce in nature, the amount of
wild mushroom is not su⁄cient for commercial exploita-
tion. Its cultivation on solid or liquid substrates has there-
fore become essential to meet the increasing demands on
the international markets. The Ganoderma lucidum strain
isolated from Slovenian forests (MZKI G97) was culti-
vated in a bioreactor by solid state cultivation on sawdust,
and by submerged method in a 10L stirred tank reactor
using a liquid substrate based on potato dextrose and olive
oil. The in£uences of inoculum and oxygen partial pres-
sure in batch and fed batch submerged cultivation were
studied. Up to 17.0 g L-1 of dry fungal biomass was pro-
duced. Extracellular (1,7 g L-1) and intracellular (0.45 g L-
1
) polysaccharide fractions were isolated, consisting mainly
of L-D-glucanes. The immunostimulatory e¡ects of iso-
lated polysaccharides were tested on induction of cytokine
(TNF-K, IFN-Q) synthesis in primary cultures of human
mononuclear cells isolated from a bu¡y coat. The TNF-K
inducing activity was comparable to romurtide, which has
been used as a supporting therapy in cancer patients
treated with radiotherapy and/or chemotherapy.
FEMSLE Congress 2-6-03
465
P14^8
CHARACTERIZATION OF ANTIFUNGAL COM-
POUNDS FROM THE LACTOBACILLUS RHAMNO-
SUS VT1 STRAIN ISOLATED FROM TARTAR
SAUCE
J. Chumchalova(1), P. Zachar(2), M. Giesova(1), H. Pet-
rakova(1), M. Plockova(1) and J. Lad(1)
(1) Department of Dairy and Fat Technology and (2) De-
partment of Analytical Chemistry, Institute of Chemical
Technology in Prague, Prague, Czech Republic
The potential of Lactobacillus strains to prevent the
growth of yeast was investigated and the active metabo-
lites of the strain Lactobacillus rhamnosus VT1 showing
highest inhibitory activity were puri¢ed and some of
them structurally identi¢ed. The antifungal activity of lac-
tobacilli was determined using modi¢ed milk agar plates.
Kluyveromyces marxianus var. marxianus DMF 1005 was
used as an indicator strain. The highest activity was
proved for the strain Lactobacillus rhamnosus VT1. The
produced antifungal compounds were puri¢ed from super-
natant adjusted to pH 2 prepared after cultivation of the
strain in MRS broth. Samples obtained after evaporation
of organic phase by twice extraction with ethyl acetate
caused totally inhibition of the yeast strain. The organic
extracts were esteri¢ed and chemical structures were eluci-
dated using gas chromatography ^ mass spectrometry
analysis. The compounds identi¢ed in the antimicrobial
fraction corresponded to palmitic acid and 2-methyl-5-hy-
droxy hexanoic acid. Two other peaks have not been com-
pletely resolved.
P14^9
Withdrawn.
466 1st FEMS Congress / Posters 103^505
P14^10
PHLEOMYCIN AND ZEOCIN AS SELECTIVE
AGENTS IN TRANSFORMATION OF SCHWANNIO-
MYCES OCCIDENTALIS
A. Dorosh, J. Hasek, I. Janatova
Laboratory of Cell Reproduction, Institute of Microbiology,
Academy of Sciences of the Czech Republic, Videnska 1083,
142 20 Prague 4, Czech Republic
In this work, we present development of the ¢rst dominant
selection system in S. occidentalis, which would be advan-
tageous to perform more sophisticated genetic manipula-
tions in this industrially interesting yeast. In our previous
work we have found that the Sable gene from Staphylo-
coccus aureus was able to confer the resistance to phleo-
mycin also to S. occidentalis cells when cloned down-
stream from the S. occidentalis invertase gene promoter.
However, no transformants could be obtained after direct
plating of transformants on phleomycin-containing plates.
Therefore, we isolated several promoters ^ SCR2, CYC1
of S. occidentalis and TEF1 of Ashbya gossypii and placed
them upstream from the Sable gene. When S. occidentalis
transformants were selected ¢rst for Ade prototrophy and
then tested for the resistance to phleomycin, clones har-
boring any of these promoters were resistant to both
phleomycin and Zeocin, however at various concentration.
The highest resistance level was observed in transformants
with the Sable gene under the SCR2 promoter. These
clones were able to grow on plates with more than
450Wg/ml of Zeocin. This expression cassette was also
the only one enabling direct selection of S. occidentalis
transformants on phleomycin or Zeocin containing plates
(5 Wg/ml). Post-incubation for 6 h in the complete medium
before plating was essential to reach a high e⁄ciency of
transformation. 104 transformants/Wg DNA was routinely
obtained that is only a slightly lower e⁄ciency than that
obtained with the auxotrophic marker ADE2.
This work was supported by the grant from the Ministry
of Education CR LN00B030.
FEMSLE Congress 2-6-03
P14^11
SCREENING OF DIFFERENTIALLY EXPRESSED
cDNA FRAGMENTS FOR THEIR IMPORTANCE
FOR OCHRATOXIN A BIOSYNTHESIS IN PENICIL-
LIUM NORDICUM
P. Fa«rber and R. Geisen
Federal Research Center for Nutrition, Institute of Hygiene
and Toxicology, Haid-und-Neu-Str. 9, D-76131 Karlsruhe,
Germany
Ochratoxin A (OTA) is an isocumarin derivative of the
secondary metabolism of ¢lamentous fungi belonging to
the genera Aspergillus and Penicillium. Despite the fact
that OTA is a genotoxic carcinogen for humans and ani-
mals, nearly nothing is known about the genetic basis of
OTA production by fungal producer strains. Structural as
well as regulatory genes remain unknown, they still have
to be identi¢ed. One possible method for the isolation and
the succeeding iden¢cation of genes involved in a meta-
bolic pathway is the Di¡erential Display Reverse Tran-
scription (DDRT-PCR) technique. Using this method we
could isolate about 70 di¡erent di¡erentially expressed
cDNA fragments which might be involved in the OTA
metabolic pathway. These di¡erentially expressed cDNAs
already have been cloned and sequenced, their importance
for OTA biosynthesis will be further investigated by gene
expression experiments. Genes involved in OTA biosyn-
thesis and their sequences respectively will be used for
the determination of OTA speci¢c probes and oligomeres
for OTA gene speci¢c PCR experiments. The ¢nal goal is
the implementation of OTA speci¢c oligomeres into a mi-
cro array for the screening of the expression of genes re-
sponsible for the biosynthesis of di¡erent important food
relevant mycotoxins.
P14^12
DEUTEROMYCETES: A SOURCE OF KERATINO-
LYTIC ENZYMES
J. Friedrich, H. Gradisar and R. Jerala
National Institute of Chemistry, Hajdrihova 19, SI-1000
Ljubljana, Slovenia
Keratinolytic enzymes are proteinases able to catalyse hy-
drolytic degradation of keratins. The later are resistant
natural scleroproteins found in skin and skin appendages
of vertebrates, such as hair, nail, feather, scale etc. In
nature, some insects and some microorganisms, including
¢lamentous fungi and yeasts, degrade keratins. Their en-
zymes could ¢nd application in di¡erent domains where
keratin should be eliminated: cosmetics, leather process-
 1st FEMS Congress / Posters 103^505
ing, waste degradation, and also where peptides from ker-
atins should be obtained. With the aim to investigate the
potential of non-pathogenic ¢lamentous fungi for the pro-
duction of keratinolytic enzymes several hundreds of
strains were screened in our previous experiments. It was
found that the most potent were deuteromycetous fungi.
Three selected fungi: Aspergillus £avus, Doratomyces mi-
crosporus and a wild isolate B639 were cultivated in shak-
en £asks and in a 5 l bioreactor under conditions inducing
synthesis of keratinolytic enzymes. These were isolated,
puri¢ed and characterized. All three enzymes are serine
proteinases with molecular masses from 22 to 34 kDa,
mostly active in alkaline environment (from 8 to 11) at
elevated temperatures (from 45 to 60‡C). They degrade
keratins of human stratum corneum at much higher degree
as the keratins of nail, hair and wool. Therefore, the ker-
atin of stratum corneum was prepared as soluble fraction
and the enzymatic degradation of keratin ¢laments was
followed by electrophoretic and spectroscopic methods.
We have characterized the time course of keratin degra-
dation and compared the properties of the obtained kera-
tinolytic enzymes.
P14^13
CHRYSONILIA SITOPHILA LIPASE ACTIVITY
F. B. Gaspar(1), S. Vitorino(1), E. Neves(1) and M. V.
San Roma‹o(1,2)
(1) Instituto de Biologia Experimental e Tecnolo¤gica/Inti-
tuto de Tecnologia Qu|¤mica e Biolo¤gica ^ Universidade
Nova de Lisboa, Apartado 12, 2781-901 Oeiras, Portugal;
(2) Estaca‹o Vitivin|¤cola Nacional, 2565-191 Dois Portos,
Portugal
Cork oaks, Quercus suber L., are indigenous to the Med-
iterranean region where they occur in open woodlands on
hills and lower slopes. They form a thick cork bark which
covers their trunks and branches. One of the best known
and valuable application of cork is the manufacturing of
cork stoppers for sealing wine bottles. The cork stopper
manufacturing process includes a stabilization period of
the cork slabs, after boiling, during which they are covered
by mould growth, Chrysonilia sitophila being the dominant
mould identi¢ed at this stage. The systematic development
of this mould on cork slabs points to its ability to enzy-
maticly degrade the main cork components. Suberin, an
aliphatic polyester where glycerol is the cross-linking
monomer, contributes to about 40% of the cell wall com-
position leading to the special properties of the cork tissue,
like insulation, elasticity and impenetrability to water. Li-
pases are able to hydrolyze glyceryl esters of long chain
fatty acids from suberin. The resulting action of lipase
activity on the wall of cork cells may lead to changes of
the physical and chemical properties of cork stoppers. In
FEMSLE Congress 2-6-03
467
this study, C. sitophila was grown in liquid media using
powdered cork as the only carbon source. The lipase ac-
tivity pro¢le of enzymatic crude extracts obtained at dis-
tinct mould growth times, was determined and showed
that C. sitophila is able to produce the enzymatic machin-
ery necessary to degrade the triacylglycerol-like substrates,
like suberin.
P14^14
CHARACTERIZATION OF A POLYKETIDE SYN-
THASE OF PENICILLIUM NORDICUM INVOLVED
IN OCHRATOXIN BIOSYNTHESIS
Z. Mayer, A. Karolewiez, P. Fa«rber and R. Geisen
Federal Research Centre for Nutrition, Institute of Hygiene
and Toxicology, Haid-und-Neu-Str. 9, 76131 Karlsruhe,
Germany
Ochratoxin is an important nephrotoxic mycotoxin pro-
duced by various Aspergillus species and two Penicillium
species, namely P. verrucosum and P. nordicum. The
ochratoxinogenic Penicillium species are responsible for
the occurrence of ochratoxin in cereals and ceral products.
The production of ochratoxin by these fungi is dependent
from environmental conditions like the presence of nu-
trients, the temperature or the water activity. This fact
indicates, that ochratoxin production is regulated at the
genetic level. A knowledge of this regulation would allow
the development of measures to minimize ochratoxin pro-
duction in plant derived foods. Ochratoxin is a polyketide
mycotoxin. We identi¢ed a polyketide synthase gene,
whose expression is correlated to ochratoxin production.
We were able to isolated a 750 bp fragment from all an-
alysed P. nordicum strains, by using general primers for
polyketide synthase genes. Interestingly this fragment was
not present in P. verrucosum, indicating that for ochratox-
in biosynthesis two di¡erent polyketid synthases are used
by both species. The sequence of the fragment has high
homology to other fungal polyketid synthases. An expres-
sion analysis by quantitative Real Time PCR revealed that
the polyketide synthase gene is induced in parallel with
ochratoxin A production. These results indicate that the
identi¢ed polyketide synthase gene is involved in ochratox-
in A biosynthesis.
468 1st FEMS Congress / Posters 103^505
P14^15
FUNGAL BIOREMEDIATION OF WASTE COPPER,
CHROMIUM AND BORON IMPREGNATED WOOD
M. Humar(1), F. Pohleven(1), M. SNentjurc(2) and S. A.
Amartey(3)
(1) Bio-technical Faculty, Department of Wood science and
Technology, University of Ljubljana, Slovenia; (2) Institute
Jozef Stefan, Ljubljana, Slovenija; (3) Forest Products Re-
search Centre, Buckinghamshire Chilterns University Col-
lege, High Wycombe, UK
The expected service life of copper, chromium and boron
(CCB) treated wood is about 20 ^ 50 years. After that
period, the treated wood is discarded as special waste.
Due to the toxic elements in such treated wood, burning
and land¢ll disposal are not considered as environmentally
friendly solutions. Extraction and recycling of the preser-
vatives from the waste wood is a much more promising
and environmentally acceptable. Method is based on the
conversion of the ¢xed biocides in the wood into soluble
forms, which can be subsequently leached out of the
wood. In order to elucidate the mechanism of this process,
copper sulphate, potassium dichromate and CCB treated
wood samples were leached after exposure to wood decay
fungi Antrodia vaillantii, Leucogyrophana pinastri, Gloeo-
phyllum trabeum and Poria monticola. The concentrations
of copper and chromium leached were determined. After-
wards, Electron paramagnetic resonance measurements of
leached and non-leached samples were also performed in
order to determine the paramagnetic complexes that were
formed. Copper tolerant wood decay fungi (A. vaillantii
and L. pinastri), increased Cu and Cr leaching from the
treated wood. The prime reason for this observation orig-
inates in oxalic acid excretion by copper tolerant fungi.
Oxalic acid reacts with heavy metals in wood, resulting
in de-¢xation and consequently higher biocide leaching.
P14^16
BIOCONVERSION OF CHITIN TO CHITOSAN : PU-
RIFICATION AND CHARACTERIZATION OF CHI-
TIN DEACETYLASE FROM RHIZOPUS NIGRICANS
N igon(2), V. Abram(2), H. Lenasi(1) and
N. Jeraj(1), U. Z
K. Breskvar(1)
(1) University of Ljubljana, Faculty of Medicine, Institute
of Biochemistry, Vrazov trg 2, 1000 Ljubljana, Slovenia;
(2) University of Ljubljana, Biotechnical Faculty, Jamni-
karjeva 101, SI-1111 Ljubljana, pp2995
Chitin deacetylase has been identi¢ed in di¡erent fungi.
The enzyme catalyses the hydrolysis of N-acetamido
FEMSLE Congress 2-6-03
bonds of chitin, converting it to chitosan. Chitosans
have several applications in areas such as biomedicine,
food ingredients, cosmetics and pharmaceuticals. In addi-
tion to harsh thermochemical procedure, chitosan can be
obtained by re¢ned enzymatic procedure leading to the
production of well-de¢ned chitosan oligomers and poly-
mers. For this purpose it is important to have at disposal
a suitable source of the enzyme. In the present report we
isolated and partially characterised chitin deacetylase from
¢lamentous fungus Rhizopus nigricans. The fungus was
grown for 18h, mycelia were homogenised and crude pro-
tein extract were subjected to enzyme isolation procedure.
Methods of (NH4)2SO4 precipitation, CM-Sepharose chro-
matography and DEAE-cellulose chromatography led to
enzyme homogeneity. The progress of enzyme puri¢cation
was followed by measurement of enzyme activity using
partially O-hydroxylated chitin (glycol chitin) radiola-
belled in N-acetyl groups as a substrate. Mr value of chitin
deacetylase obtained by SDS/PAGE was approximately
100 kDa.
P14^17
CONSTRUCTION OF A GENE BANK OF THE
OCHRATOXINOGENIC SPECIES PENICILLIUM
NORDICUM AND CHARACTERIZATION OF LAMB-
DA CLONES CARRYING OCHRATOXIN BIOSYN-
THETIC GENES
A. Karolewiez, P. Fa«rber and R. Geisen
Federal Research Centre for Nutrition, Institute of Hygiene
and Toxicology, Haid-und-Neu-Str. 9, 76139 Karlsruhe,
Germany
Ochratoxin is an important nephrotoxic mycotoxin pro-
duced by various Aspergillus species and two Penicillium
species, namely P. verrucosum and P. nordicum. The
ochratoxinogenic Penicillium species are responsible for
the occurrence of ochratoxin in cereals and ceral products.
To analyse and characterise genes which are involved in
ochratoxin A biosynthesis of Penicillium nordicum, we pre-
pared a Q gene bank with Penicillium nordicum genomic
DNA partially digested to a size of about 20kb. These
fragments were packed into Q phages. E.coli was trans-
fected with these phages and hybridizised with a probe
speci¢c for the polyketide synthase gene involved in ochra-
toxin biosynthesis. Of all phage clones 5 positive could be
selected. These clones have been analysed in more detail.
The results are presented here.
 1st FEMS Congress / Posters 103^505
P14^18
SEARCHING FOR THE GENE ENCODING STEROID
11L-HYDROXYLASE OF THE FILAMENTOUS FUN-
GUS COCHLIOBOLUS LUNATUS
E. Kus›c›er(1), R. Komel(2)
(1) Currently: Chair of Biotechnology, Food Science and
Technology Department, Biotechnical Faculty, University of
Ljubljana, Jamikarjeva 101, SI-1000 Ljubljana, Slovenia;
(2) Medical Center for Molecular Biology, Institute of Bio-
chemistry, Faculty of Medicine, University of Ljubljana,
Vrazov trg 2, SI-1000 Ljubljana, Slovenia
11L-hydroxylation of steroids is one of the key steps in the
production of corticosteroids. The enzymes responsible for
hydroxylation of steroids belong to the cytochrome P450
superfamily. Despite the substantial biotechnological im-
portance of these enzymes, the sequences of the genes
encoding any fungal steroid hydroxylase have not yet
been identi¢ed, neither has their primary protein structure
been described. The ¢lamentous fungus Cochliobolus luna-
tus is a phytopathogen, capable of 11L-hydroxylation of
steroids. Based on the conserved, cytochrome P450 speci¢c
regions, a 273 bp gene fragment has been ampli¢ed by
PCR. The fragment was sequenced, and the blast data-
base search clearly showed its cytochrome P450 nature.
According to the sequence of the obtained fragment,
new, cytochrome P450 speci¢c primers were designed. Us-
ing the method of rapid ampli¢cation of cDNA ends an
entire 3’-end of the gene, and a part of its 5’-end were
successfully ampli¢ed from the induced mycelium. The se-
quence of the 3’-end was determined, and with a blast
database search, a high homology with numerous cyto-
chromes P450 was shown. The obtained 3’-end fragment
will be used as a DNA probe for screening of genomic and
cDNA libraries. Thus the entire gene to which the 3’-end
belongs will probably be identi¢ed, as well as some other
cytochrome P450 genes present in the fungus. We estimate
that among these, steroid 11L-hydroxylase will be found
with a high probability as well. The acquired part of the
5’-end is currently being sequenced, and the information
obtained will be helpful in identifying the cloned gene
sequences.
FEMSLE Congress 2-6-03
469
P14^19
FRAGMENTATION AND ACTIVATION OF 6-PHOS-
PHOFRUCTO-1-KINASE IN ASPERGILLUS NIGER
S. Mesojednik, S. Smerkolj and M. Legis›a
National Institute of Chemistry, Hajdrihova 19, SI-1001
Ljubljana, Slovenia
Aspergillus niger is one of the most important industrial
microorganisms which is used for production of pimary
metabolite ^ citric acid and a number of commercially
important enzymes. The fungal cells can convert up to
80% of sucrose from the medium into a citric acid as a
product, which ranks A. niger among the most productive
microorganisms.For high yielding strains unrestricted met-
abolic £ow through glycolysis is characteristic where 6-
phosphofructo-1-kinase (PFK) seems to play a key regu-
latory role. While a high yielding strain was compared
with a wild type strain, signi¢cant drop of intracellular
pH was observed in former, but no such changes could
be detected under the same growth conditions in a wild
type strain. Under reduced pHi values, speci¢c proteases
were believed to be more active and serine proteases were
found to be able to cleave the native PFK molecule as
well. An inactive PFK fragment was therefore accumu-
lated in the cells as a result of proteolytic breakdown
which might be subsequently activated by phosphorylation
of protein molecule. The kinase responsible for phosphor-
ylation was found to be cAMP-dependent protein kinase
which became active only under the conditions that stim-
ulate cAMP synthesis, such as hypoosmotic shock or in-
tracellular acidi¢cation. Reactivated 48 kDa fragment
showed changed kinetics in respect to the native protein
and most importantly it was no longer inhibited by citrate
what is extremely important for a high citric acid yielding
strain.
P14^20
COMPARISON OF THE MITOCHONDRIAL DNA IN
TWO STRAINS OF CRYPTOCOCCUS NEOFOR-
MANS
J. Litter, Zs. Hamari, I. Pfei¡er, J. Kucsera
Department of Microbiology, Faculty of Sciences, Univer-
sity of Szeged, P.O. Box 533, H-6701 Szeged, Hungary
The opportunistic human pathogenic Cryptococcus neofor-
mans is a basidiomycetous fungi. As intact mitochondrial
respiration can be one of the most important factors of the
yeast’s successful survival in the body, our investigation
was aimed to characterize the mitochondrial DNA of
Cryptococcus neoformans. A few data are available about
470 1st FEMS Congress / Posters 103^505
the organization of the mitochondrial genome of the basi-
diomycetous fungi. In this study, physical map of two
varieties, C. n. var. neoformans and C. n. var. grubii was
constructed. Their mtDNAs were mapped with EcoRI and
EcoRV restriction enzymes and the correct order of sev-
eral mitochondrial genes was determined by Southern hy-
bridisations and partial sequencing. Most of the genes
important in the mitochondrial respiration (nad1, nad2,
nad3, nad4, nad4L, nad5, nad6, atp6, atp9, cox1, cox2,
cob) and the rns gene important in protein synthesis
were localised. We did not found any di¡erences in the
order of the genes. However, C. n. var. neoformans and
C. n. var. grubii di¡ered signi¢cantly in the size of their
mtDNA measuring 33,97 kb and 24,1 kb, respectively.
This di¡erence can be explained by the presence of introns
or alteration of the length of intergenic regions. Our DNA
sequencing data also supported this assumption. Di¡eren-
ces were found in the size of introns of cox1 and cob
genes.Detailed analysis of the sequences and their submis-
sion to database are in progress. This investigation was
supported by the Hungarian Scienti¢c Research Found
(OTKA) T035194, F032704 and Ministry of Health
(ETT 48/2000)
P14^21
THE SCREENING OF SIDERIAN TRICHODERMA
ISOLATES FOR THE CREATION OF BIOPREPA-
RATES WITH THE USAGE OF RESIDUALS OF
WOOD PROCESSING COMPLEX
E. G. Makhova, V. S. Gromovykh, A. S. Kandalenceva, T.
V. Ryazanova, T. I. Gromovykh
Siberian State Technological University, prospect Mira 82,
660049 Krasnoyarsk, Russia
The leading place among the agent fungi for the biological
activity against fungi diseases belongs to the fungi in genus
Trichoderma Pers. The amount of commercial prepara-
tion-trichodermins is made on the basis of di¡erent iso-
lates of the fungi, which are highly e¡ective in suppressing
the pathogenic microorganisms of agricultural plants.
However, the issues of using these biopreparations for
reforestation by growing hardwood seedlings have not
been examined yet. There are no recommendations for
bene¢cial strains, since the composition of the bene¢cial
strains of Trichoderma genus, inhabited in the roots found
in the surrounding soil of seedlings, has not been inves-
tigated. Attempts to use the recommended trichodermins
for the protection of agricultural plants in reforestation
have proven ine¡ective. The comparison estimation of Tri-
choderma isolates from soil microbiocenosis in Siberia has
been carried out. The most promising isolates against the
phytopathogenes of coniferous seedlings were selected
among the agent fungi. At present the original solid state
FEMSLE Congress 2-6-03
biotechnology has been created on the basis of these
strains with the usage of lignocarbohydrate residuals of
wood processing complex, in particular bark and lignin.
For the creation of biopreparates the comparative study of
features of growth on the larix bark was conducted. It was
established that aborigine Trichoderma’ strains ‘‘MG’’
(Trichoderma asperellum) and ‘‘MK’’(Trichoderma konin-
gii) had high biotechnological indexes. These strains can
be used as biocontrol agent as well as standard strain ‘‘U’’
Trichoderma harzianum).
P14^22
INFLUENCE OF ANTIOXIDATIVE STATUS ON
PROGRESSION OF FUNGAL SKIN INFECTIONS IN
DIABETICS
I. Ma¤rova¤(1), J. Za¤hejsky¤(2), K. Kan›kova¤(2)
(1) Faculty of Chemistry, Technical University of Brno,
Czech Republic; (2) Faculty of Medicine, Masaryk Univer-
sity, Brno, Czech Republic
Diabetes mellitus is one of the most frequent metabolic
distortions predisposing for infectious diseases. Fungal in-
fections of skin and soft tissues usually imply inadequate
long-term glucose control, but some of them may also
precede metabolic manifestation of diabetes. Moreover,
diabetes is accompanied by a suboptimal function of pro-
tective antioxidant mechanisms. The aim o this work was
to evaluate prevalence of skin fungal dermatoses (SFD) in
diabetics and to study in£uence of antioxidative status on
their presence a progression. A total of 221 unrelated Cau-
casian subjects treated for several dermatoses were en-
rolled in the study; 148 diabetic subjects (NIDDM ^
non-insulin dependent diabetes mellitus) and 73 non-dia-
betic subjects. Presence of super¢cial fungal (e.g. Mycosis,
Onychomycosis, Epidermophytia) and/or yeast (e.g. Can-
didosis, Intertrigo candidomycetica, Tinea) infections was
in diabetics 4.4-times and 2.8-times, respectively higher
than in a control group. Several SFD were diagnosed in
29.7 % of diabetics, but only in 6.8% of non-diabetics.
Plasma levels of all antioxidants tested were in diabetics
with SFD signi¢cantly lower than in diabetics without
SFD. Signi¢cant di¡erences of beta-carotene, lutein and
alpha-tocopherol (P 6 0.05, Kruskal-Walis ANOVA)
were found among diabetics and non-diabetics. Newly
identi¢ed intron polymorphisms 1704G/T and 2184A/G
in the RAGE gene (Receptor for Advanced Glycooxida-
tion End -products) were proved to be associated with the
antioxidative status in NIDDM. Complex impairment of
the phagocytic function of monocytes/macrophages due by
increased oxidative stress and glycooxidation is likely one
of the mechanisms, which are suggested to contribute to
the low resistance to infection in diabetic subjects.
 1st FEMS Congress / Posters 103^505
P14^23
A RELIABLE PCR-BASED METHOD FOR THE MO-
LECULAR IDENTIFICATION OF CRYPTOCOCCUS
NEOFORMANS
M. L. M. Martins(1,2) and I. Spencer-Martins(2)
(1) Institute of Hygiene and Tropical Medicine, New Uni-
versity of Lisbon, 1349-008 Lisbon ; (2) Centro de Recursos
Microbiolo¤gicos (CREM), Biotechnology Unit, Faculty of
Sciences and Technology, New University of Lisbon, 2829-
516 Caparica, Portugal
Cryptococcus neoformans is an ubiquitous encapsulated
yeast responsible for a frequently fatal infection, mainly
in AIDS and other immunocompromised patients. Cur-
rent laboratory diagnosis relies upon direct microscopic
examination, serological testing and culture of the etio-
logic agent from clinical specimens. The yeast is subse-
quently identi¢ed through the evaluation of conventional
phenotypic characteristics, a process which is laborious,
time-consuming and somewhat unreliable. The main ob-
jective of this work was to develop a PCR-based method
for the accurate and rapid routine identi¢cation of C. neo-
formans. A total of 51 strains were analysed, including 38
clinical isolates, phenotypically identi¢ed as C. neofor-
mans, and reference collection strains representing the
commonly recognised varieties of this species (neoformans
and gattii). The method under evaluation made use of a
species-speci¢c primer targeted at a variable region of the
26S rRNA gene, and was successfully applied to all
strains. Two strains failed to yield the 200 bp-band char-
acteristic of C. neoformans. The application of additional
molecular methods con¢rmed these strains were misla-
belled and actually represent di¡erent yeasts (Debaryomy-
ces hansenii and Cryptococcus albidus). The same PCR
method was further evaluated for the direct detection of
C. neoformans, using total DNA from the clinical material
as template. The high sensitivity observed constitutes a
strong asset for its utilization in clinical laboratories.
FEMSLE Congress 2-6-03
471
P14^24
HYDRODYNAMICS IMPACT ON THE MORPHOL-
OGY AND LiP ACTIVITY OF PHANEROCHAETE
CHRYSOSPORIUM
P. Milavec ZN mak(1), A. Podgornik(1), H. Podgornik(1)
and T. Koloini(2)
(1) BIA d.o.o., Teslova 30, SI-1000 Ljubljana, Slovenia;
(2) Faculty of Chemistry and Chemical Technology, Uni-
versity of Ljubljana, As›kerc›eva 5, SI-1000 Ljubljana, Slov-
enia
The basidiomycete fungus Phanerochaete chrysosporium
produces various extracelular peroxidases among which
lignin peroxidases (LiP) are the best studied. The enzymes
(LiP) are produced during secondary metabolism and are
excreted under nitrogen limitation. Since the fungus forms
pellets under shaking conditions, the agitation has a great
impact on the morphology and consequently on the LiP
production and stability. Since all the experiments of
growing the fungus in the bioreactor gave much lower
values of LiP activities then those obtained on a shaker,
mostly because of di¡erent hydrodynamics conditions, the
reason why the pellet morphology has to be considered in
the scale up approach is even more obvious. Therefore, we
have investigated the in£uence of the hydrodynamics
under shaking conditions on the pellet morphology. The
fungus was grown on a shaker in a vessels of di¡erent
diameters and in classic Erlenmeyer £asks under the
same conditions which are: inoculum size, medium com-
position, pH, agitation rate, temperature. The measuring
of the pellet size, pellet size distribution and number of
pellets, glucose and nitrogen concentration as well as LiP
activity and medium viscosity was performed. The results
have shown that the shape of the vessel has a great impact
on the hydrodynamics and consequently on the morphol-
ogy of the pellets. The pellets di¡er in size from 1mm up
to 20 mm depending on the vessel size. Furthermore all
the pellets were LiP active what is in a contrast to the
studies that have been reported in this area. Namely the
LiP activity appears with the delay in accordance to the
pellet size. In addition, number of the pellets can be as
well correlated with pellets size. The obtained results have
shown that the tested conditions signi¢cantly a¡ect the
morphology of the fungus and consequently the physio-
logical activity of the fungus.
472 1st FEMS Congress / Posters 103^505
P14^25
ISOLATION OF MALASSEZIA GLOBOSA AND MA-
LASSEZIA SYMPODIALIS IN PATIENTS WITH SE-
BORRHEIC DERMATITIS
D. Milobratovic¤, S. Dz›amic¤, S. Mitrovic¤ and V. Arsic¤-Ar-
senijevic¤
Institute of Microbiology and Immunology, University
School of Medicine, Dr Subotic¤a 1, 11000 Belgrade, Yugo-
slavia
Malassezia species consist of lipophilic yeasts that are op-
portunistic pathogen of human skin. The genus Malassezia
was recently shown to consists of seven species ^ six lipid
dependent: M. furfur, M. sympodialis, M. globosa, M. re-
stricta, M. sloo⁄ae and M. obtusa and ^ one lipid inde-
pendent species: M. pachydermatis, by molecular tech-
niques. Under speci¢c conditions Malassezia spp can
become pathogen and induce several skin diseases like
pityriasis versicolor (PV), seborrheic dermatitis (SD) and
some forms of atopic dermatitis and even systemic infec-
tions in immunocompromised patients and neonates re-
ceiving parental lipid alimentation. The role of Malassezia
spp in SD is controversial. Some authors report an over-
growth of Malassezia spp on the skin, but others were
unable to con¢rm that. Also that association of each se-
rovar of the genus Malassezia with SD has not been elu-
cidate yet. To determine whether the composition of Ma-
lassezia spp in patients with SD di¡ers from that of
patients with PV quantitative cultures were obtained by
stripping the lesional skin with a tape, placed on Leeming
and Notman medium (LNA) and on modi¢ed Dixon agar.
Plates were incubated at 35‡C and colonies were examined
after 10 days. The sample included 10 individuals with PV
and 10 with SD. High yeast density was de¢ned as s 100
cfu / tape. On LNA and on modi¢ed Dixon agar all cul-
tures were positive. Malassezia spp density observed
among patients with SD was not signi¢cantly di¡erent
from that of patients with PV. For identi¢cation to species
level cultural, microscopic and physiological characteris-
tics were recorded. M. sympodialis was the most common
species associated with PV patients, while M. globosa pre-
dominated on persons with SD.
FEMSLE Congress 2-6-03
P14^26
INFLUENCE OF FERMENTATION PARAMETERS
ON BIOTECHNOLOGICAL PRODUCTION OF MAN-
GANESE PEROXIDASES BY A WHITE ROT FUN-
GUS
M. Mohorc›ic›, J. Friedrich
National Institute of Chemistry, Hajdrihova 19, SI-1000
Ljubljana, Slovenia
White rot fungi attract more and more attention due to
their ability to synthesise powerful non-speci¢c oxidative
enzymes interesting for biotechnological application.
Among these enzymes there is manganese peroxidase
(MnP) which in presence of H2O2 and Mn2+ ions oxidases
resistant aromatic substances and may found application
in pulp and paper industry, decolourisation of coloured
industrial e¥uents or solids, exploitation of lignocellulo-
sics as well as detoxi¢cation of xenobiotics (DDT, dioxin
like compounds, anthracene) etc. Di¡erent wild fungal iso-
lates were screened for degradation of complex aromatic
compounds and one strain proved to be outstandingly
e¡ective. It was identi¢ed as Phanerochaete chrysosporium
and was selected for in vitro cultivation in submerged
aerobic conditions. The in£uences of cultivation parame-
ters were investigated for biotechnological production of
MnP. In shaken £asks the fungal mycelium formed pellets
whereas in stirred tank bioreactor the fungus tended to
adhere to surfaces. Regarding enzyme production it was
shown that inoculum concentration of 7 x 107 spores/ml
resulted in two to three times higher activity than at 3 x
107 spores/ml. The optimal concentration of Mn2+ ions
was 0.2 mM only in contrast to ¢ndings of other research-
ers reporting optimal concentrations of 1 to 5 mM The
enzyme production in shaken cultures was reproducible
while it was not easy to obtain the same activity in a
commonly used stirred tank bioreactor due to high sensi-
tivity of the system to aeration and agitation conditions.
P14^27
MICROMYCETES OF THE SEEDS OF THE VEGE-
TABLES CROPS OF THE CENTRAL PART OF RUS-
SIA
G. V. Pestsov, M. A. Chepurnova, S. V. Gorelova
Tula State Pedagogical University, Russia, 300026, Tula
As a result of mycological research of 35 vegetable crops
seeds it was revealed 75 fungi species. The most widely
distributed species of fungi were the representatives of
the following genus: Alternaria Nees ^ on the seeds of
25 agricultural crops; Fusarium Link ex Fr. ^ on 11; Cer-
 1st FEMS Congress / Posters 103^505
cospora Fress. ^ on 7, Cladosporium Lk. ^ on 18; Erysiphe
Lk. ^ on 13; Verticillium Nees ^ on 6 and Botrytis Mich. ^
on the seeds of 14 agricultural crops. The fungi of the
genus Aspergillus were found on the seeds of 30 crops,
the fungi of the genus Penicillium and Mucor on the seeds
of 29 vegetable crops of all learnt botanical families. The
most frequent ones are the fungi genus Aspergillus: Asp.
niger v. Tiegh., Asp. £avus Lk., Asp. fumigatus Fres., Asp.
£avipes (Bain. et Sart.) Thom et Church, Asp. chevalieri
(Mangin) Thom et Church; genus Penicillium: P. nigricans
(Bainier) Thom, P. viridicatum Westl., P. notatum Westl.,
P. chrysogenum Thom, P. variabile Sopp, P. expansum Lk.;
genus Mucor: M. mucedo Fres. emend Bref., M. racemosus
Fres. We revealed some species of fungi displaying de¢nite
relationship to the seeds of one species of the host-plant:
Ascochyta cucumis Fautr. et Roum - on Cucumis sativus
L.; A. pisi Lib.- on Pisum sativum L.; Cylindrosporium
melissae Mass. - on Satureja hortensis L.; Phyllosticta sp.
- on Scorzonera hispanica L.; Ph. sinapi Bond.-Mont. - on
Brassica juncea (L.) Czern.; Ramularia menthicola Sacc. -
on Melissa o⁄cinalis L.; Septoria cari Brezschn. - on Ca-
rum carvi L.; S. melissae Desm. - on Melissa o⁄cinalis L.
Under certain conditions these fungy can form di¡erent
toxic metabolites, which cause allergy and poisoning.
P14^28
SPECIFIC COMPOSITION OF CHAMPIGNON DIS-
EASE PATHOGENES AND ECOLOGICALLY SAFE
CONTROL MEASURES WITH THEM
G. V. Pestsov
Tula State Pedagogical University, Russia, 300026, Tula
Fungi are rather valuable power products. They contain
proteins, vitamins, mineral salts and other compounds
necessary for human organisms. The industrial production
became possible as a result of learning biology and work-
ing out the technology of cultivating some species, e.g
champignon ^ Agaricus bisporus (J. Lge) Imbach. For
growing this fungus the nutrient substratums and condi-
tions for cultivation were selected. Though these condi-
tions are favourable for di¡erent microorganisms develop-
ment, including pathogenic, insects and nematodes. They
cause damage to agaricus directly and are considered to be
carriers of disease pathogens. Without using proper con-
trol measures, the wermins can reduce fungus crops and
destroy them completely. As a result of carried out re-
searches, it was determined that the most widespread dis-
ease pathogens in cultivated constructions are the fungi
Papulaspora byssina Hoston, Fusarium oxysporum
(Schlecht.) Snyd. et Hans., F. solani (Mart.) App. et
Wr., Verticillium malthousei Ware, Mycogone perniciosa
Magnus and bacteria Pseudomonas sp. and Ps. tolaasii
Paine. In the series of the laboratory, ¢eld and industrial
FEMSLE Congress 2-6-03
473
tests the actions of 12 biological preparations in their dif-
ferent combinations resived an appraisal of e⁄ciency. A
high e⁄ciency against the pathogenic organisms complex
consisting of Bacillus thuringiensis and Streptomyces aver-
mitilis was established. The a¡ection of vermins and dis-
eases was reduced 33% and crop capacity increased 29%.
P14^29
FUSARIUM WILT AGENTS ON VEGETABLE ALIEN
CROPS IN RUSSIA
G. V. Pestsov, M. A. Chepurnova, S. V. Gorelova
Tula State Pedagogical University, Russia, 300026, Tula
Fusarium wilt on vegetables crops is wide spread in Rus-
sia. The disease develops on all stages of plant develop-
ment and reaches its maximal development in time of mass
bearing. Fusarium wilt agents are the following: on cab-
bage, Chinense cabbage ^ F. oxysporum Schl. f. sp. con-
glutinans (Wolenw.) Snyder et Hansen; on coriander - F.
oxysporum Schl. f. sp. coriandrii Narula et Joshi; on chrys-
anthemum ^ F. oxysporum Schl. f. sp. chrysanthemi; on
tomato ^ F. oxysporum Schl. f. sp. lycopersici (Sacc.)
Snyder et Hansen, F. solani (Mart.) App. et Wr., F. mon-
iliforme Sheld., F. gibbosum App. et Wr. emend Bilai ; on
eggplant ^ F. oxysporum Schl. f. sp. melongenae Matuo et
Ischigami ; on sweet pepper ^ F. oxysporum Schl. f. sp.
capsici Pestsov; on Chinese radish - F. oxysporum Schl.
f. sp. raphani Kendrick et Snyder; on spinach ^ F. oxy-
sporum Schl. f. sp. spinaciae ( Sherb.) Snyder et Hansen ;
on dill ^ F. oxysporum Schl. f. sp. anethi Gordon; on
stachys ^ F. oxysporum Schl. f. sp. stachydis Gordon. It
has been established, that these specialized forms have
high pathogenicity to host plant, but at favourable con-
ditions they are able to a¡ect other plants, mainly, in their
botanical family. In the process of pathogeny they form
metabolites. They can cause severe human diseases.
474 1st FEMS Congress / Posters 103^505
P14^30
ENZYME ACTIVITIES IN ECTOMYCORRHIZO-
SPHERES OF SPRUCE
K. Pritsch(1), S. Raidl(2), A. Hartmann(3), and M.
Schloter(3)
(1) Technical University Munich, Chair of Soil Ecology, D-
85758 Oberschleissheim, Germany ; (2) Ludwig Maximilian
University Munich, Department Biology I, Systematic My-
cology, Menzinger Str. 67, D-80638 Mu«nchen, Germany ;
(3) GSF-Research Center for Environment and Health, In-
stitute of Soil Ecology, Ingolstaedter Landstr. 1, D-85764
Neuherberg, Germany
Ectomycorrhizospheres are named in analogy to the ‘‘rhi-
zosphere’’ and comprise the compartment around ectomy-
corrhizal roots. Ectomycorrhizae occur on ¢ne roots of
important forest tree species within Fagales and Pinaceae
and are characterized by the presence of a hyphal mantle
formed by the respective ectomycorrhizal fungus. Besides
their known bene¢t for their host plants in terms of better
mineral nutrient and water uptake, many ectomycorrhizal
fungi also exhibit saprotrophic properties in organic car-
bon and nitrogen acquisition revealed by enzyme activities
in fruiting bodies and mycelia. Here we report from en-
zyme activities directly measured from ectomycorrhizae
using microplate systems for enzyme detection. Field
sampled mycorrhizal roots were assigned to di¡erent mor-
photypes. Hydrolytic enzyme activities were measured
with methylumbelliferone-labelled substrate analogues for
cellobiohydrolase, f-glucosidase, N-acetylglucosaminidase,
and phosphomonoesterase while oxidative enzymes (lac-
case, peroxidases) were measured with photometric tests.
Enzyme activities di¡ered among the di¡erent ectomycor-
rhizal species either by the overall occurrence or the level
of activity, revealing species-speci¢c patterns of enzyme
production. For example, Lactarius subdulcis mycorrhizae
showed higher laccase activities than all other species,
while Tomentellopsis submollis with high activities of all
hydrolytic enzymes tested did not exhibit any laccase ac-
tivity. Mycorrhizal roots surpassed non mycorrhizal roots
in enzyme activities, especially phosphomonoesterase and
N-acetylglucosaminidase, strongly suggesting a stimula-
tion of enzymatic processes in ectomycorrhizospheres by
the fungal partners.
FEMSLE Congress 2-6-03
P14^31
IMMUNOMODULATING AND ANTITUMOR EF-
FECTS OF FRUITING BODIES AND MYCELLIUM
OF PLEUROTUS MUSHROOMS
M. M. Shamtsyan(1) Y. O. Maksimova(1), V. G. Konu-
sova(2), A. S. Simbirtsev(2), N. N. Denisova(3), N. N.
Petrishchev(4)
(1) St. Petersburg State Institute of Technology (Technical
University), St. Petersburg, Russia ; (2) State Institute of
Highly Pure Preparations, St. Petersburg, Russia; (3) L.V.
Komarov Botanical Institute of Russian Academy of Scien-
ces, St. Petersburg, Russia; (4) St. Petersburg Pavlov
Medical University
The presence of biologically active substances with antitu-
mor, antiviral, antiallergenic activities in the fruiting
bodies of mushrooms and the prospects of their use in
the medical purposes attract wide interest of many re-
searchers. During the past years several publications out-
lined the positive e¡ect of frequent consumption of di¡er-
ent mushrooms, especially from genera Pleurotus as health
promotions and prevention of various diseases. Objects of
our study were liophilized aqueous extracts of fruiting
bodies and mycelium of mushrooms of genera of Pleuro-
tus: Pleurotus ostreatus and Pleurotus citrinopoleatus. Pri-
mary evaluation of their biological activities was carried
out in vitro by means of analyses of oxygen active forms
using luminoldependent chemiluminescence. Experiments
demonstrated that extracts possess of both mushrooms
expressed immunomodulating activity. Also it was shown,
that mentioned extracts are strong mitogens ^ in the test
of blast transformation of leukocytes, they signi¢cantly
stimulate cell ¢ssion of spleen of mouse genus CBA. The
ability of extracts of oyster mushroom to induce produc-
tion of anti in£ammatory cytokin interleukin -1L ^ a key
mediator of in£ammation reaction, was also demon-
strated. Experiments with mice feed by aqueous extract
of P. ostreatus mycelium shows pronounced immune en-
hancing action of their uptake in various dozes. Anti-tu-
mour action of aqueous extract of P. ostreatus fruiting
bodies was demonstrated on mice bearing melanoma
B16 cells. Daily uptake of 100 mg of extract per kg of
body weight for two weeks resulted in signi¢cant reduction
of tumor weight, and prolongation of lifetime of the tested
animals.
 1st FEMS Congress / Posters 103^505
P14^32
XYLOSE FERMENTATION TO ETHANOL BY THE
YEASTS HANSENULA POLYMORPHA AND PICHIA
STIPITIS
O. B. Ryabova(1), O. M. Chmil(1), D. Grabek(2), V. A.
Sibirny(2), Z. Kotylak(2), A. A. Sibirny(1,2)
(1) Institute of Cell Biology, NAS of Ukraine, Drahomanov
Str., 14/16, Lviv 79005 Ukraine; (2) Institute of Biotech-
nology, Rzeszow University, Rejtana 16C, Rzeszow 35-310
Poland
Wild-type strains of the thermotolerant methylotrophic
yeast Hansenula polymorpha are able to ferment glucose,
cellobiose and xylose to ethanol. It was found out that H.
polymorpha most actively fermented sugars to ethanol at
37‡ C, whereas well-known xylose-fermenting yeast Pichia
stipitis could not e¡ectively ferment carbon substrates at
this temperature. H. polymorpha also was able to ferment
both glucose and xylose at even higher temperatures, up to
45‡ C. This species appeared to be more ethanol tolerant
organism comparing with P. stipitis though more suscep-
tible one than Saccharomyces cerevisiae. It was found that
ribo£avin de¢cient mutant of H. polymorpha increases its
ethanol productivity from glucose and xylose under sub-
optimal supply with ribo£avin. It suggests that £avin def-
icit can redirect pyruvate £ux through pyruvate decarbox-
ylase to ethanol. Mutants of H. polymorpha defective in
alcohol dehydrogenase activity produced lower amounts
of ethanol from glucose, whereas levels of ethanol produc-
tion from xylose were identical for the wild-type strain and
the alcohol dehydrogenase defective mutant. Apparently
this enzyme does not limit ethanol production from worse
utilizing pentose substrate whereas is important for max-
imal accumulation of alcohol during utilization of more
favorable substrate, glucose. At the same time, alcohol
dehydrogenase defective mutants of P. stipitis accumulated
the same amounts of ethanol in the media with glucose
and xylose as the wild-type strain of this species.
FEMSLE Congress 2-6-03
475
P14^33
LIGNINOLYTIC ENZYME PRODUCTION IN CUL-
TURES OF IRPEX LACTEUS AND PHANERO-
CHAETE CHRYSOSPORIUM AND THEIR IMPLICA-
TION IN DYE DECOLORIZATION
K. Svobodova¤(1), H. Podgornik(2), E. Mus|¤lkova¤(1) and
N . Novotny¤(1)
C
(1) Institute of Microbiology, Academy of Sciences of the
Czech Republic, V|¤den›ska¤ 1083, 142 20 Prague 4, Czech
Republic; (2) Faculty of Chemistry and Chemical Technol-
ogy, University of Ljubljana, As›kerc›eva 5, 1000 Ljubljana,
Slovenia
White rot fungi are widely studied due to their ability to
degrade the natural biopolymer lignin as well as di¡erent
xenobiotics. Synthetic dyes are a speci¢c group of xeno-
biotics polluting the environment mostly by coloring sur-
face water. They are very recalcitrant to oxidative degra-
dation. Recently, di¡erent strains of white rot fungi were
isolated and tested for their ability to be used in potential
application in degradation processes. One of them is Irpex
lacteus capable of synthesis of extracellular manganese-de-
pendent peroxidase (MnP), lignin peroxidase (LiP) and
laccase (Lac). Its enzyme production and decolorization
capacity was compared with Phanerochaete chrysosporium.
I. lacteus was grown under stationary and submerged con-
ditions. In the former culture the level of MnP was sig-
ni¢cantly higher than in the latter culture. The levels of
Lac were similar in both cultures. In P. chrysosporium only
LiP and MnP were formed under submerged conditions.
Addition of surfactants had positive in£uence on lignino-
lytic enzyme production in submerged cultures. Di¡erence
in morphology as well as physiology was observed using
two di¡erent non-ionic surfactants, Tween 20 or Tween
80. In decolorization experiments azo-, anthraquinone-
and phthalocyanine dyes were mostly used and e⁄ciently
decolorized by both fungi. Growth medium from fungal
cultures was concentrated and dialyzed for further HPLC
analysis of the MnPs formed. Degradation of RBBR by
isolated MnP was tested to bring evidence of its involve-
ment in dye decolorization. Cultures of I. lacteus were also
shown to e⁄ciently decolorize dyes in textile-industry ef-
£uents.
The work was supported by projects: MSVMT 2001/031,
GA C V R 526/00/1303 and MSVZSV L4-3068-0103-01.
476 1st FEMS Congress / Posters 103^505
P14^34
SYNERGISM OF NON-COVALENTLY LINKED PRO-
TEINS IN BUILDING THE SACCHAROMYCES CE-
REVISIAE CELL WALL
V. Boric, R. Teparic, A. Mojzes, I. Slivac and V. Mrsa
Laboratory of Biochemistry, Faculty of Food Technology
and Biotechnology, University of Zagreb, Pierottijeva 6,
Zagreb, Croatia
Saccharomyces cerevisiae cell wall is composed of L-glu-
cans, mannoproteins and small amounts of chitin. Man-
noproteins can either be non-covalently (Scw proteins ^
soluble cell wall proteins), or covalently bound to glucan.
Their physiological role is mostly unknown. In order to
assess possible role of Scw proteins mutant strains were
constructed by disruption of SCW4, SCW10, SCW11, and
BGL2 genes. In£uence of these major Scw proteins on cell
integrity and protein secretion into the growth medium
was examined. scw4scw10, scw4scw10bgl2 and
scw4scw10scw11bgl2 secret less proteins in comparison to
wild type, single mutants and scw4scw10scw11. Sensitivity
of cell wall to L-1,3-glucanase was examined and
scw4scw10scw11 exhibited a signi¢cant increase in resis-
tance. However, when the outer mannoprotein layer was
removed by proteinase K all mutants showed wild type
level of sensitivity indicating that observed e¡ect is due
to changes in the mannan layer. Viability of mutants in
exponential and stationary phase of growth was moni-
tored. In the exponential phase scw4scw10 showed in-
creased fraction of dead cells (5%), while scw4scw10scw11
had less than 1% of dead cells. In the stationary phase the
fraction of dead cells increased to 22% in scw4scw10 and
to 17% in scw4scw10scw11. Number of dead cells was
reduced to 0.5-3% in all strains in both growth phases
when cells were grown in 1M sorbitol. Results indicate
that Scw proteins are involved in building and modifying
the cell wall and suggest a synergistic behavior of Scw4p,
Scw10p and Bgl2p, whereas the Scw11p acts antagonisti-
cally to those proteins.
P14^35
FUNGAL OTITIS EXTERNA IN ADULTS
A. Trpkovic, D. Milobratovic, Lj. Petkovic, V. Arsic-Arsen-
ijevic
Institute of Microbiology and Immunology, School of Med-
icine, University of Belgrade, Dr Subotica 1, 11 000 Beo-
grad
Otitis externa is most often caused by a bacterial patho-
gen, but the fungal infection of the ear (otomycosis) is not
FEMSLE Congress 2-6-03
uncommon clinical problem. It makes up to six percent of
all patients with symptoms of ear disease and up to 40% of
all external auditory canal infections. Local lesions create
favourable conditions for fungal growth and development
of mycosis. Most infections are present in patients who
underwent surgical procedures, in cases of previous med-
ical treatment of the external canal, and in patients with
impaired immunity. In 48 patients with a clinical diagnosis
of otomycosis 21 produced positive fungal isolates. The
majority of identi¢ed strains were Candida spp, whereas
the strains of Aspergillus spp. were less frequent. The iso-
lated fungal species were: Aspergillus fumigatus (6/21),
Candida parapsilosis (5/21), Aspergillus niger (2/21), Can-
dida famata (2/21), Candida guillermondii (2/21), Candida
albicans (2/21), Aspergillus niger (2/21) and Penicillium sp.
(2/21). In otitis which do not respond to conventional
therapy fungal infection should be considered. Mycologi-
cal examination should be performed in setting the accu-
rate diagnosis, also the underestimation of these pathogens
may lead to a prolonged or ine¡ective treatment.
P14^36
RECOMBINING POPULATION STRUCTURES AND
MATING TYPE GENE HOMOLOGUES IN THE
ASEXUAL FUNGUS ASPERGILLUS FUMIGATUS
J. Varga
Department of Microbiology, University of Szeged, Faculty
of Sciences, P.O.Box 533, H-6701 Szeged, Hungary
Aspergillus fumigatus is an important human pathogen
with no known sexual cycle. Our aim was to analyze the
reproductive mode of this fungus. Several methods have
been described for distinguishing recombination from
clonality in fungal populations. We applied the index of
association test and the parsimony tree length permutation
test on isoenzyme and sequence speci¢c DNA primer anal-
ysis data gathered from the literature. Both tests, together
with other observations, supported the premise that re-
combination played an important role in A. fumigatus
populations. As mating type genes govern sexual processes
in fungi, we also searched the TIGR un¢nished A. fumi-
gatus genomic database for mating type gene homologues.
Our search resulted in the identi¢cation of two ORFs ho-
mologous to the mat a-1 gene of Neurospora crassa. One
of these codes for a HMG box protein of 247 amino acids
and exhibits extensive similarities to MAT1-2 genes of a
number of ascomycetes (e.g. to those of Mycosphaerella
graminicola, Ceratocystis eucalypti), while the other ORF
encodes a HMG box protein of 434 amino acids, and was
found to be most closely related to the ste11 gene of
Schizosaccharomyces pombe. Homology to the MAT1-1
gene sequence of any of the tested fungi was not observed
in the TIGR A. fumigatus genomic database. We presume
 1st FEMS Congress / Posters 103^505
that A. fumigatus represents one mating type of a hetero-
thallic species. The other putative mating type either be-
came extinct, or exists in small endemic populations. Our
results indicate that either a cryptic sexual stage or past
meiotic processes are responsible for the recombining
structures observed in A. fumigatus populations.
P14^37
INFLUENCE OF MOULDS IN THE DEGRADATION
OF CHLOROPHENOLIC COMPOUNDS
S. Vitorino(1), F. Gaspar, L. F. Vilas Boas(1), M. R.
Bronze(1), A. S. Curvelo Garcia(2) and M. V. San Ro-
ma‹o(1,2)
(1) Instituto de Biologia Experimental e Tecnolo¤gica/Insti-
tuto de Tecnologia Qu|¤mica e Biolo¤gica-Universidade Nova
de Lisboa, Apartado 12, 2781-901 Oeiras, Portugal; (2)
Estaca‹o Vitivin|¤cola Nacional, 2565-191 Dois Portos, Por-
tugal
The industrial manufacturing of cork stoppers has a sig-
ni¢cant economic impact in Portuguese economy, since
Portugal is the largest producer of cork worldwide. The
manufacturing process of cork stoppers includes a matur-
ing stage after boiling the cork slabs. During this period,
moulds completely cover the slabs and Chrysonilia sitophi-
la is the dominant mould. Those moulds are claimed to be
responsible, among other causes, by the alteration of the
wine £avour or aroma. The most unpleasant of these ‘‘o¡
£avour’’ is the so-called cork taint in wine, clearly distin-
guishable of wine presenting a musty/moldy aroma. The
major compound responsible for cork taint is 2,4,6-tri-
chloroanisole (TCA), which is resultant of the chlorophe-
nols methylation as moulds detoxi¢cation process. Fungal
spores suspensions were prepared and inoculated into cul-
ture media containing cork dust and/or chlorophenol com-
pounds (2,4,6-trichlorophenol and pentachlorophenol) as
the only carbon source. After moulds growth, the com-
pounds were extracted and monitored. The quanti¢cation
of clorophenolic and methylated compounds (chloroani-
soles) were carried out using Gas Chromatography (GC)
and Gas Chromatography-Mass Spectrometry (GC-MS).
The phenolic compounds were quanti¢ed using High Per-
formance Liquid Chromatography (HPLC) and Capillary
Electrophoresis (EC). First results suggest that C. sitophila
is unable to contribute to the production of the com-
pounds at levels that usually are associated with the
cork taint in wine, even in the presence of chlorophenols
and it showed to have the capacity to restrict the growth
of other moulds species, that can compromise stopper
quality.
This work was partially supported by Program Sapiens
POCI/AGR/38940/2001 ^ Estudo dos mecanismos qu|¤mi-
cos e bioqu|¤micos de formac^a‹o de cloroaniso¤is.
FEMSLE Congress 2-6-03
477
P14^38
CLONING OF STRUCTURAL GENES INVOLVED IN
RIBOFLAVIN SYNTHESIS OF INDUSTRIAL FLAVI-
NOGENIC YEAST CANDIDA FAMATA
A. Y. Voronovsky(1,2), K. V. Dmytruk(1), O. P. Ish-
chuk(1), C. A. Abbas(2), A. A. Sibirny(1,3)
(1) Institute of Cell Biology, Drahomanov Street, 14/16,
Lviv 79005, Ukraine; (2) Archer Daniels Midland Co J.
Randall Research Center, 1001 Brush College Rd, Decatur
IL 62521 USA; (3) Institute of Biotechnology, Rzeszow
University, Rejtana 16C, Rzeszow 35-310 Poland
Ribo£avin overproducing mutants of the £avinogenic
yeast Candida famata isolated by conventional selection
methods are used for industrial ribo£avin production. Re-
cently, a transformation system was developed for this
species using LEU2 gene S. cerevisiae as a selectable
marker. Ribo£avin de¢cient mutants were isolated from
a previously selected C. famata leu2 strain and their bio-
chemical identi¢cation was performed. A C. famata gene
library was constructed and used for cloning of the corre-
sponding structural genes of ribo£avin synthesis by com-
plementation of the growth defects in the medium without
leucine and ribo£avin. As a result, DNA fragments con-
taining genes RIB1, RIB2, RIB5, RIB6 and RIB7 encoding
GTP cyclohydrolase, reductase, dimethylribityllumazine
synthase, dihydroxybutanone phosphate synthase and ri-
bo£avin synthase, respectively, were isolated and subse-
quently subcloned to the smallest possible fragments. Plas-
mids with these genes successfully complemented
ribo£avin auxotrophies of the corresponding mutants of
another £avinogenic yeast species, Pichia guilliermondii. It
suggested that C. famata structural genes of ribo£avin syn-
thesis and not some of suppressor genes were cloned. The
isolated fragments were sequenced. All C. famata genes
display high homology to previously cloned orthologs of
other organisms.
P14^39
THE DEVELOPMENT OF STABLE TRANSFORMA-
TION SYSTEM OF ZYGOMYCETES FUNGI, RHIZO-
PUS ORYZAE
T. V. Yuzbashev, S. P. Sineoky
Russian National Collection of Industrial Microorganisms,
FGUP GosNII genetika, 1-st Dorozhny proezd 1, 113545,
Moscow, Russian Federation
The genus Rhizopus is classi¢ed under the family Mucor-
aceae in the order Mucorales of the phylum Zygomycota.
Rhizopus secretes large number of enzymes such as glucoa-
478 1st FEMS Congress / Posters 103^505
mylase, aspartic proteinases, lipases and chemicals includ-
ing L-(+)-lactic acid, fumaric acid, and ethanol. Rhizopus
is also used for brewing and in production of various
fermentation foods in South-East Asia and China. For
the other hand Rhizopus is the most important and repre-
sentative agent of mucormycosis. The ability to investigate
this ¢lamentous fungus largely depends on the successful-
ness of developing the transformation methods. Like many
other Zygomycetous fungi, recombinant DNA introduced
into Rhizopus oryzae transformants is, however, very un-
stable. Since a sporangiospore of Rhizopus oryzae has sev-
eral nuclei, mycelia of transformants are heterokaryons
and the nuclei containing introduced DNA rapidly disap-
pear when selective pressure is removed. Here we present
results of our investigation directed to the stabilisation of
the transformants. We consider as perspective approach
based on the two-stage selection. The ¢rst stage includes
the use of dominant selectable marker allowing obtaining
resistant heterokaryon transformants. Then has been used
UV irradiation to get sporangiospore with one nucleus.
On the second stage recessive marker was used for direct
selection of homokaryotic clones.#P14^40
ANTIFUNGAL SUSCEPTIBILITY TESTING OF DER-
MATOPHYTES
I. Zdovc
University of Ljubljana, Veterinary Faculty, Institute of Mi-
crobiology and Parasitology, Gerbic›eva 60, 1000 Ljubljana,
Slovenia
Successful treatment of mycoses depends on many factors
and one of them is susceptibility of isolated strain to anti-
fungal drugs. Some useful systems are available for sus-
ceptibility testing in yeasts, but there is a lack of useful
methods for ¢lamentous fungi, especially for dermato-
phytes. Recently a commercial test for MIC value for ¢l-
amentous fungi was described. The purpose of this study
was to evaluate this method for testing the susceptibility of
dermatophytes to antifungal drugs. Altogether 30 strains
of di¡erent dermatophyte species (Microsporum canis, Mi-
crosporum gypseum, Microsporum equinum, Trichophyton
mentagrophytes) isolated from dogs, cats and other domes-
tic animals were examined regarding their susceptibility to
antimycotic activity. For comparison strains of Aspergillus
fumigatus, Aspergillus niger and Candida albicans were also
tested at the same time. Each strain was tested to Keto-
conazole, Fluconazole, Itraconazole, Amphotericin B and
Flucytosine (E test, AB Biodisk, Sweden). All strains pro-
duced detectable growth onto RPMI + MOPS + 2% glu-
cose agar within 48-96 hours. A single test for each anti-
fungal agent consists of a thin, inert plastic carrier with
prede¢ned exponential gradient of antibiotic. After 72
hours incubation at 25‡C the MIC value was read from
the scale at the point of intersection between the zone edge
FEMSLE Congress 2-6-03
and the test carrier. Preliminary results showed various
susceptibility of dermatophyte strains to di¡erent drugs
and are genus and species dependant. E test appears to
be a suitable procedure for testing the susceptibility of
dermatophytes but for ¢nal evaluation more tests should
be done. The weak point of E test for testing of moulds is
absence of tests for some other drugs that we generally
used for treatment of dermatophytosis.
P14^41
PELLETED GROWTH OF RHIZOPUS NIGRICANS
IN A STIRRED TANK BIOREACTOR
N nidars›ic›
P. Z
Faculty of Chemistry and Chemical Technology, University
of Ljubljana, As›kerc›eva 5, SI-1000 Ljubljana, Slovenia
A pelleted growth form of ¢lamentous fungus Rhizopus
nigricans has been proposed as naturally immobilized bio-
catalyst in a process of progesterone 11a-hydroxylation.
The control of submerged growth was already attained
in a shake-£asks system, while here we report on further
studies in a laboratory stirred tank bioreactor. The mor-
phological type and the related physiology strongly de-
pend on environmental conditions in the bioreactor, and
in turn a¡ect the rheological properties of the broth and
thereby bioreactor performance. By varying concentration
of spores from slants in shake £asks pre-cultures we have
obtained di¡erent types of mycelium for inoculation of
bioreactor, resulted in di¡erent submerged growth pattern
of Rhizopus nigricans in the reactor. Furthermore, the in-
£uence of energy dissipation rate on the morphology and
biomass yield was studied by the use of two types of im-
pellers, a classical Rushton turbine and a propeller, at
di¡erent agitation rates. Operating conditions had a sig-
ni¢cant e¡ect on the size distribution of pellets and their
structural density. The aim to obtain pelleted growth form
of Rhizopus nigricans in a laboratory-scale bioreactor and
to avoid problems associated with the high tendency of
this aerobic fungus to grow on the broth surface was ac-
complished.
P14^42
ON THE WAY TO SOLVENT CHOICE FOR BIO-
TRANSFORMATION BY RHIZOPUS NIGRICANS
N nidars›ic› and I. Plazl
U. Roglic›, P. Z
Faculty of Chemistry and Chemical Technology, University
of Ljubljana, As›kerc›eva 5, SI-1000 Ljubljana, Slovenia
Filamentous fungus Rhizopus nigricans is capable to cata-
lyse hydroxylation of C11 position on the steroid ring. The
 1st FEMS Congress / Posters 103^505
main problem of steroid biotransformations is the low
water solubility of these organic compounds. The applica-
tion of aqueous-organic media has been a promising tool
for improving these biotransformations. Therefore we
have investigated the in£uence of addition of various sol-
vents at di¡erent concentrations on the growing cells of
Rhizopus nigricans. We have shown that the solvent toler-
ance is in the correlation with the value of logarithm of the
partition coe⁄cient of a given organic solvent. Among
chosen solvents, the microorganism in this system mostly
tolerates iso-octane and heptane. The solubility of reactant
and product of progesterone 11K-hydroxylation in di¡er-
ent organic solvents was also determined. On the basis of
these results and further studies on the retention of en-
zyme activity in aqueous-organic media we intend to opti-
mise the investigated biotransformation.
P15^1
BEET NECROTIC YELLOW VEIN VIRUS IS THE
CAUSAL AGENT OF DANGEROUS DISEASE ‘‘RHI-
ZOMANIA’’ IN SUGARBEET CROP
J. Adel
The virus is in Genus Benyvirus (along with BSBMV).
Recent progresses in molecular plant virology has led to
appear high e⁄cient molecular detection methods for
plant pathogenic viruses, i.e BNYVV. RT-PCR method
is a PCR-based detection method by which a wide range
of phytopathogenic viruses can be detected. Spiegle and
Martin (1992) detected Potato leaf roll virus using PCR
method. Similar attempts have been made on various
plant viruses such as Potato virus Y,PVX,BSBMV,TMV
etc. RT-PCR detection of BNYVV was ¢rst reported by
Henry et al.(1995) using 5 speci¢c primers with need to
sequence of certain gel products. Morris et al.(2001) de-
tected BNYVV using RT-PCR, nested-PCR and DAS-
and TAS-ELISA methods without need to sequence. In
order to detect BNYVV, 50 suspicious and a¡ected sam-
ples of two susceptible cultivars ‘‘IC1 and G34’’ were col-
lected from Zarghan region (Fars prov.) and used as ex-
perimental sources. Samples are incubated at -32 to -
22‡C.In this paper, Beet necrotic yellow vein virus is de-
tected through PCR, one-step RT-PCR, immunocapture
RT-PCR and nested-PCR methods along with DAS- and
TAS-ELISA immunological methods. A 326 bp or 500 bp
fragment must appear on agarose-formaldehyde (denatur-
ing) gel to verify the infection of a sample. All PCR-based
methods are carried out using speci¢c primer designed
based on BNYVV RNA-2 triple gene block sequence by
Henry et al.(1995). The e⁄ciency of molecular methods is
compared to ELISA methods. It is to be expected that the
sensitivity of nested-PCR method is 800 times greater than
other PCR-based methods and 1200 times than DAS- and
TAS-ELISA methods.
FEMSLE Congress 2-6-03
479
P15^2
PREVALENCE OF ANTIBODIES TO BVD VIRUS IN
DAIRY COWS IN MONTENEGRO
M. Bojanic¤, L. Boz›aric¤, N. Pejovic¤
Biotechnical institute, Kralja Nikole bb, 81000, Podgorica,
Montenegro, Yugoslavia
Bovine viral diarrhea (BVD) is the disease with worldwide
distribution in domestic and wild ruminants and results in
severe economic losses to the cattle industry. The cause is
virus classi¢ed in the genus Pestivirus of the family Flavi-
viridae. E¡ect of BVDV infections on production losses
including reduced milk production, reduced conception
rate, abortions, congenital defects, stillbirths, growth re-
tardation, increased occurence of other diseases and death.
The acute form, characterised by fever and diarrhea, is
transient, with high morbidity rates and low mortality
rates. Adult animals can also present an asymptomatic
subclinical form. Mucosal disease has low morbidity rates
(1%), but high mortality rates. More often, it is character-
ised by seromucoid nasal secretions, ulcers at di¡erent
levels of the digestive tract, diarrhea that is often hemor-
rhagic. The serological antibody screening by ELISA (In-
stitut Pourquier) is used to detect the presence of the
BVDV in dairy cows by identi¢cation of the animals pre-
senting speci¢c antibodies. It does not allow identifying
immunotolerant persistently infected animals (seronega-
tive). Of 217 examined individual bovine milk samples,
85 have been positive (39,17%). Of 135 examined collective
bovine milk samples from farms, 34 (25,18%) have had
more than 30% seroprevalence on BVDV antibody. These
results are showing the need for examination and control
of BVD in the cattle in Montenegro.
P15^3
SOIL-BORNE CEREAL VIRUSES IN UKRAINE
I. Budzanivska, G. Snigur, F. Demyanenko, V. Polishuk
Virology Dept., Taras Shevchenko’ Kyiv National Univer-
sity, 64, Volodimirska str., Kyiv, 01033 Ukraine
Virus diseases of cereals are widely enough spread in Eu-
rope and in Ukraine. Soil-borne viruses were not carried
out in Ukraine. We have studied such viruses as barley
mild mosaic bymovirus (BaMMV), barley yellow mosaic
bymovirus (BaYMV), wheat soil-borne mosaic furovirus
(SBWMV) and wheat spindle streak mosaic bymovirus
(WSSMV) which are transmitted by of Polymyxa graminis
and can be kept in a ground untill 10-15 years in di¡erent
regions of Ukraine. According to the literary data only
one virus ^ BaYMV have been diagnosed in territory of
480 1st FEMS Congress / Posters 103^505
Ukraine. We have detected three viruses by biological
tests, electron microscopy and sandwich-ELISA. WSSMV
was identi¢ed by sandwich-ELISA only. We have created
map of soil-transmitted viruses distribution in di¡erent
agrarian regions of Ukraine. The questions of monitoring
and preventive maintenance of the further spreading of an
infection are discussed.
P15^4
MICROBIOLOGICAL ASPECTS IN WOMEN WITH
AND WITHOUT CYTOPATHOLOGICAL CERVICAL
CHANGES
N. Coman, O. Vizitiu, M. Ariciuc, A. Macovei
Cantacuzino Institute, Splaiul Independentei 103, Bucharest,
R-70.100, Romania
A group of 340 women has been cytologically investigated
by Papanicolau smear in order to evidentiate the cytopa-
thological changes speci¢c for HPV. From the patients
that suggested preneoplasic changes samples have been
prelevated for microbiological investigations. Among the
340 investigated women, 19% presented on smears cytopa-
thological changes speci¢c for HPV infection (binuclea-
tion, multinucleation, koilocytes, dyskeratosis, abnormal
mytoses). By microbiological investigation of vaginal sam-
ples of all studied subjects, the following microorganisms
were detected: Gardnerella vaginalis 20.6 %, Candida spp.
20.3%, Ureaplasma 16.7%, Group B Streptococcus 11.4%,
Chlamydia trachomatis 4.3%, Lactobacillus 29.7%. The mi-
crobiological investigation of the patients with cytopatho-
logical investigations showed the presence of: Gardnerella
vaginalis 26. 35 %, Ureaplasma 22.3%, Candida spp. 5%,
Trichomonas vaginalis 10.5%, Mycoplasma 12.3%, Lacto-
bacilus 12. 3%, Herpes simplex virus 5.2%. In the same
time, we noticed that there were cases with an association
between HPV induced lesions and the presence of anaer-
obes.
P15^5
PHAGOCYTIC CAPACITY OF POLYMORPHONU-
CLEAR CELLS IN RABBITS INFECTED WITH
TWO VARIOUS DOSES AND STRAINS OF VHD (VI-
RAL HAEMORRHAGIC DISEASE) VIRUS
W. DeptuTa, B. Hukowska, B. Tokarz-DeptuTa
Chair of Microbiology and Immunology, Faculty of Natural
Sciences, University of Szczecin, ul. Felczaka 3a, 71-412
Szczecin, Poland
The study aimed at monitoring one of the stages of phago-
cytosis process, i.e. the capacity of polymorphonuclear
FEMSLE Congress 2-6-03
granulocytes to ingest following infection of the rabbits
with the calculated 100% or 25% lethal dose of VHD
virus, strain Kr-1 and Fr-2. The studies were performed
on 60 clinically healthy rabbits, which were allocated to
four groups: Group I: rabbits immunized with 100% le-
thal dose of VHD virus, strain Kr-1 (10 rabbits), Group
II: 100% lethal dose of VHD virus, strain Fr-2 (10 rab-
bits); Group III: 25% lethal dose of VHD virus, strain Kr-
1 (10 rabbits) and Group IV: 25% lethal dose of VHD
virus, strain Fr-2 (10 rabbits). To each of the four groups
corresponded appropriate group of control rabbits (5 ani-
mals each), which received distilled water. Blood for tests
was sampled on hours 0, 4, 8, 12, 24, 36, 48, 52, 56, 60 and
every 24 h to the day 14 if the animals survived the period.
In the blood of rabbits, the capacity of neutrophilic gran-
ulocytes to ingest (ZP) standard strain of St. aureus 209P
was tested by determination of phagocytosis index (Ip)
and of the percentage of ingesting cells (phagocytes)
(%kp). Serum anti-VHD antibodies were established by
ELISA.Within the examined parameters (Ip, %kp), the
capacity to ingest was dependent upon the strain of
VHD virus and the employed dose of antigen. No anti-
VHD antibodies could be detected in the animals. In all
the examined groups of rabbits, mortality reached 100%
till the hour 60.
P15^6
DETECTION AND GENETIC CHARACTERISATION
OF TYPE 2 PORCINE CIRCOVIRUS (PCV-2) FROM
PIGS WITH POSTWEANING MULTISYSTEMIC
WASTING SYNDROME (PMWS) IN SLOVENIA
I. Toplak, P. Hostnik, J. Grom, D. Barlic›-Maganja
Department of Virology, Veterinary faculty, 1115 Ljublja-
na, Slovenia
Postweaning multisystemic wasting syndrome (PMWS) is
a new porcine disease that a¡ects mainly nursery and early
growing pigs, and clinicaly is characterisated by poor body
condition, dispnea, pallor of the skin. A porcine circovirus
type 2 (PCV-2) was ¢rst diagnosed from tissues of pigs
with postweaning multisystemic wasting syndrome
(PMWS) from eight pig farms and four small herds in
year 2002 in Slovenia. A rapid and convenient PCR-based
test was used for the detection of PCV-2 isolates. The 453
base pair (bp) nucleotide sequence, derived from the
ORF2, was aligned and compared to the corresponding
positions of published sequences of PCV-2. The phyloge-
netic analysis of Slovenian PCV-2 isolates showed rela-
tionship with PCV-2 from France, United Kingdom, the
Netherland, China, Germany and Canada. The phyloge-
netic tree, generated from those comparison, allowed the
PCV-2 isolates to be subdivided into at least two clusters,
which were labelled as a subtype PCV-2a and PCV-2b.
 1st FEMS Congress / Posters 103^505
The described method is not only suitable to con¢rm the
presence of PCV-2 in tissue samples of suspected PMWS
outbreaks, but also describes genetic di¡erences in ORF2
of PCV-2 isolates from di¡erent regions.
P15^7
PRODUCTION OF MAEDI-VISNA ANTIGEN USING
CELL LINE LCP
E. Hamzaraj(1) and K. Berxholi(2)
(1) Department of Biology, Faculty of Natural Sciences,
University of Tirana, Bulevardi Zog I, Tirane, Albania ;
(2) Faculty of Veterinary, Agriculture University, Kamez,
Tirane, Albania
Maedi-visna is a member of the lentivirus sub-family of
the family Retroviridae. Lentiviruses cause slow and per-
sistent infections. Introduction of virus is followed by the
infection of antigen presenting cells as macrophages, den-
trical cells and T helper (CD4+). Maedi-visna spreads by
direct contact between sheep, presumably by the respira-
tory route. Antibodies are produced very slowly in com-
parison to other infections. It is very important to produce
diagnostic antibodies for maedi to enable continuous mon-
itoring of £ocks. For production of maedi diagnostic anti-
bodies we used cell line LCP and the appropriate strain
that produces maedi antigen. The strain WCL of maedi-
visna, delivered by Teramo Institute, Italy, was used for
cell line infection. Antigen was produced using the main-
tenance media of our cell line, after freezing and thawing it
three times. Antigen control for speci¢city and a¡ectivity
was performed by the agar-gel test, using a commercial
antigen produced by reference institute, Pirbright, Eng-
land, as a standard. Formation of precipitation bands
shows that: (1) Antigen produced in our lab gives a pre-
cipitation band in the right time and of good quality. (2)
Precipitation line is a line of identity and continuous with
that of the reference antigen. (3) Precipitation line is
formed midway between serum well and antigen well. (4)
Precipitation line is sharp and appears at 18 hours, but
becomes stronger and identical to that of standard. These
results show that our antigen has good diagnostic power.
To prove this we undertook a preliminary testing in Alba-
nia during 1998-2000. Every serum sample collected was
checked in two parallel ID reactions: one with our antigen
and one with the reference. In all cases the result was the
same. We thereby con¢rmed for the ¢rst time the existence
of maedi-visna in Albania. We can conclude that the anti-
gen we have produced is of good quality, e¡ectivity, and
can be used by specialized laboratories for diagnosis of
maedi-visna disease.
FEMSLE Congress 2-6-03
481
P15^8
THE SITUATION OF ANIMAL RABIES CASES ES-
TABLISHED IN SLOVENIA
P. Hostnik, A. Bidovec, I. Toplak, D. Barlic›-Maganja, J.
Grom
University of Ljubljana, Veterinary Faculty, Gerbic›eva 60,
1115 Ljubljana, Slovenia
Red foxes are the main reservoir of rabies in Slovenia,
while rabies cases in other animal species are only sporad-
ic. Between 1988 and 1992 the vaccine baits were distrib-
uted manually. In 1995 air distribution of baits by plane
has been introduced. The program of oral vaccination of
wildlife on the territory of Slovenia is based on the expe-
riences of other countries. The rabies situation, the meth-
ods applied during the oral immunization of wildlife and
the results of vaccination have been summarised. The
number of positive rabies cases fell from 31.93% (1089/
3787) before the vaccination in 1995 to 0.59% (6/1195)
in 1999. In 2000, 115 (7.62%) cases of rabies (115/1509)
in di¡erent animal species were found. In 2001 135 pos-
itive rabies cases in 2153 examined animals (6.2%) were
found, and in 2002 from 1495 inspected animals 15 of
them (1%) were virus positive. In the last ¢ve years the
highest number of rabies virus positive animals were found
in the southern part of Slovenia near the Croatian border.
This ¢nding reveals that despite vaccination rabies is still
threatening Slovenia because not all the neighbouring
countries are vaccinating against rabies.
P15^9
ANTIPHYTOVIRAL ACTIVITY OF HETEROMETAL-
LIC COMPLEXES
A. V. Kharina, I. G. Bydzanivska, V. P. Polyschyk
Virology Dpt., Biology Faculty, Taras Shevchenko’ Kyiv
National University, 64 Volodymycrska st., Kyiv-01033,
Ukraine
Phytoviruses can be eliminated from infected plants
through techniques of in vitro meristem or tissue culture
coupled with chemotherapy. Antiviral activity of complex
compound of copper and cobalt was carried out by the
method tissue culture on TMV-Nicotiana tabacum model
system. Exsplants from TMV-infected plants were treated
with the heterometallic complexes and than transferred to
MS medium. Di¡erentiation of plantlets was realized on
medium supplemented with di¡erent concentration of
compounds. Under the research of complex compounds
antiviral activity on this model system it is established
that di¡erent compounds had di¡erent antiviral and phy-
482 1st FEMS Congress / Posters 103^505
totoxical activity. The compound N0 PO39 (Cu-
Co2(L)4Br2
HL) was not e¡ective inhibitor of viral infec-
tion in this model system. The substance N0221 (CuII2CoII-
CoIII2(Ac)4(H2L)2(L)2
2HAc) had severe toxic e¡ect and
decreased callus appearance from treated explants.
0,001% concentration of compounds N0436 (Cu2-
Co2(H2L)2(L)4Cl2
2H2O) demonstrated slight antiviral ef-
fect. Higher concentrations of this agent were toxic for
callus. The most e¡ective is the substance N0718 (Cu-
NiCl3(H2L)) that contains copper and nickel. The com-
pound stimulated the regeneration of plantlets from callus.
The plants obtained from treated explants had less inten-
sive symptoms of viral infection.
P15^10
VIRUS INFECTION OF SOME SPECIES FROM OR-
CHIDACEAE FAMILY IN NATURAL UKRAINIAN
FLORA
A. V. Korotyeyeva, V. P. Polischuk
Virology Dpt., Biology Faculty, Taras Shevchenko’ Kyiv
National University, 64 Volodymyrska st., Kyiv-01033, Uk-
raine
Virus infections a¡ect normal growth and reproduction of
orchids and could be one of the examples of exogenous
factor, changing plant population constancy in natural
landscapes. Majority of orchid species in natural £ora of
Europe belong to rare and disappearing species. Low level
of reproduction is typical for population of these plants.
Upon investigation of 20 terrestrial orchid species using
various types of ELISA and electron microscopy viruses
(TRV, TAV,CymMV and ArMV) circulating in popula-
tions of these plants in their inhabitance were determined.
Moreover, orchid viruses deriving from agrocenoses have
been detected in biocenoses of natural landscapes. It is
supposed that these viruses are the most dangerous for
preservation of orchids in their natural inhabitance. Nat-
ural inhabitances of Orchidacea plants with characteristic
higher frequency of viral infection were distinguished.
FEMSLE Congress 2-6-03
P15^11
MOLECULAR DETECTION OF CHICKEN ANEMIA
VIRUS IN DIFFERENT ORGANS OF INFECTED
CHICKENS
U. Krapez›, O. Zorman-Rojs, S. Mankoc›, D. Barlic›-Mag-
anja
University of Ljubljana, Veterinary Faculty, Gerbic›eva 60,
1115 Ljubljana, Slovenia
In Slovenia several outbreaks of chicken infectious anemia
occurred in broiler £ocks in September 2000 and February
2001. Direct detection of chicken anemia virus (CAV) in
di¡erent tissues from infected chickens was investigated by
a polymerase chain reaction (PCR) method. A set of prim-
ers covering 186 bp region on the VP2 gene was used for
the ampli¢cation. Ampli¢ed products were sized by ethid-
ium bromide-agarose gel electrophoresis. Results showed
that thymus, bone marrow, bursa of Fabricius, spleen,
liver, kidney and proventriculus obtained from infected
chickens were positive for CAV DNA by PCR. PCR prod-
ucts were also analyzed by PCR ELISA method using a
biotin labelled CAV DNA probe. CAV DNA was deteced
in the same tissues by PCR ELISA method as well as by
gel electrophoresis method. Direct nucleotide sequencing
of obtained PCR products was performed and the ob-
tained sequences were compared with nucleotide sequence
of CAV vaccinal strain Cuxhaven-1.
P15^12
MOLECULAR CHARACTERIZATION OF EQUINE
ARTERITIS VIRUS (EAV) FROM THE SEMEN OF
CARRIER STALLIONS
S. Mankoc›, D. Barlic›-Maganja, J. Grom, I. Toplak, I. Klo-
buc›ar, M. Kosec, P. Hostnik
Veterinarska fakulteta, Univerza v Ljubljani, Gerbic›eva 60,
1115 Ljubljana
The reverse transcription-polymerase chain reaction (RT-
PCR) assay was used to detect EAV in the semen of car-
rier stallions on the basis of the ampli¢cation of two viral
genome regions, which are most conserved between arter-
iviruses. Two di¡erent sets of oligonucleotide primers
complementary to the sequence located in the 3’ end of
the polymerase gene (open reading frame [ORF] 1b) and
to the sequence encoding for viral nucleocapsid protein
(ORF 7), were compared for their abilities to amplify
the target EAV sequences by the RT-PCR. The best re-
sults were obtained with oligonucleotide primers derived
from the ORF 7 region, while the ampli¢cation of the
ORF 1b region was less e⁄cient. In order to con¢rm the
 1st FEMS Congress / Posters 103^505
identity of the ampli¢cation products, the obtained DNAs
were detected by hybridization in microtiter plate using
biotinylated oligonucleotide probes in the PCR-ELISA as-
say. The ampli¢ed products were also sequenced on both
strands using the same sets of oligonucleotide primers as
for the RT-PCR.
P15^13
PCR-BASED MOLECULAR SYSTEMS DEVELOPED
IN THE ENTEROVIRUS 3D POLYMERASE CODING
REGION: APPLICATIONS FOR DIFFERENTIATION
BETWEEN ENTEROVIRUSES AND DETECTION OF
NATURAL HETEROTYPIC RECOMBINANTS
G. Oprisan(1), S. Guillot(2), V. Caro(2), L. Popa(1), M.
Combiescu(1) and A. Persu(1)
(1) Cantacuzino Institute, Splaiul Independentei 103, Bu-
charest, R-70.100 Romania; (2) Molecular Epidemiology
of Enteroviruses, Pasteur Institute, 25 rue du Dr Roux,
75724 Paris Cedex 15, France
In order to determine the molecular heterogeneity of enter-
oviruses in the 3’ part of the genome we developed three
di¡erent PCR systems designed to amplify polio- and non-
polio enteroviruses. The three overlapping fragments ob-
tained are covering almost the entire 3D-polymerase cod-
ing region and the 3’NCR. The proposed systems are able
to discriminate between di¡erent enterovirus strains after
digestion of the PCR products with restriction enzymes or
sequencing. Moreover, sequence analysis in two distant
genomic regions as capsid and polymerase-coding regions
can detect natural heterotypic recombinants.
P15^14
SEROLOGICAL AND MOLECULAR EVIDENCE OF
ENTEROVIRAL INFECTION IN PATIENTS WITH
MYOCARDITIS AND DILATED CARDIOMYOP-
ATHY
N. V. Paklonskaya(1), V. V. Dzyakanava(1), T. V. Amv-
rosieva(1), V. N. Kazinets(1), Z. Ph. Bogush(1), R. J.
Voilokova(3), D. G. Lazuk(2), C. Matskevich(1)
(1) Belarussian Research Institute for epidemiology and
microbiology, Minsk, Republic of Belarus; (2) Belarussian
Research Institute for cardiology, Minsk, Republic of Bela-
rus; (3) Institute for transplantation, Moscow, Russia
Enteroviruses are suspected to be etiologic agents in myo-
carditis and dilated cardiomyopathy (DCM). The aim of
this study was to investigate serological and molecular
evidence of enteroviral infection in patients with myocar-
ditis and DCM. Two hundred and fourteen serum speci-
FEMSLE Congress 2-6-03
483
mens from patients (n=38) with myocarditis, DCM
(n=60), other cardiac diseases (n=34) and healthy people
(n=50) were investigated by ELISA to detect enterovirus-
speci¢c immunoglobulins M (IgM). The presence of enter-
oviral genomic and antigenomic RNA was studied in my-
ocardium of patients with chronic myocarditis (n=8),
DCM (n=5) and other cardiac diseases by one-tube nested
polymerase chain reaction (PCR). Enterovirus-speci¢c
IgMs were detected in 85,2% of patients with acute myo-
carditis, in 47,8% of patients with chronic myocarditis, in
43% of patients with DCM but only in 4% of healthy
people. However, we detected a high background level of
enterovirus-speci¢c IgMs in patients with other cardiac
diseases (23,5%). Positive PCR results were obtained
from myocardial specimens in 25% of patients with
chronic myocarditis and in 60% of patients with DCM.
Among 5 positive for genomic RNA specimens, 2 exhib-
ited antigenomic RNA, whereas 3 were characterized by
the absence of antigenomic RNA. No enteroviral RNA
was detected in patients with other cardiac diseases.The
present study demonstrates that patients with myocarditis
and DCM had signi¢cantly higher level of enterovirus-
speci¢c IgMs. Our ¢ndings showed active enteroviral rep-
lication in myocardium for some patients with myocarditis
and DCM. These data support to the hypothesis about the
link between enteroviral infection and the pathogenesis of
chronic heart diseases.
P15^15
FROM NATURAL FUSION CO-RECEPTORS TO-
WARD SYNTHETIC INHIBITORS OF THE HIV-1
REPLICATION
A. V. Serbin(1,2), O. L. Alikhanova(1), I. V. Timofeev(3),
N. G. Perminova(3), N. Karbyshev(3), A. Bakunina(3)
(1) Biomodulators RC, Health RDF; (2) Topchiev Inst. of
Petrochem. Synth., RAS , Moscow; (3) SRC VB ‘‘Vek-
tor’’, Koltsovo, Russia
The cellular K-chemokine receptors CCR5 and CXCR4
play the key role, as co-receptors for the human immuno-
de¢ciency virus type 1 (HIV-1) fusion. We explore an ap-
proach to using of the virus-speci¢c functions of these
receptors for designing of water soluble polymer associ-
ated molecular simulators-antagonists (SPSA) which could
inhibit the HIV-1 replication, preventing earliest steps of
the virus penetration into cells. The submolecular regions
of CCR5 and CXCR4, most essential for interaction with
the viral envelope glycoproteins, were selected by SAR-
analysis and computer modeling. A series of peptides
(SP) reproducing the selected sequences from CCR5/
CXCR4, has been synthesized, modi¢ed toward terminal
Lys and via amino-groups incorporated onto anionic poly-
mer matrix (PM) which itself had moderate anti-HIV-1
484 1st FEMS Congress / Posters 103^505
activity, but low cytotoxicity (IC50 V 10-100 Wg/ml; CC50
v 1500 Wg/ml). We assumed that optimally constructed
SPSAs can accumulate the HIV-1-selective speci¢city of
SP in cooperation with PM mediated imitation of extra-
cellular CCR5/CXCR4 negative charge, electrostatic tar-
geting to gp120 (V3) and CD-4 (D1), as well probably,
CD-4 independent blocking of gp120-gp41 infective active
conformations. In vitro study showed that while low-mo-
lecular fragments from CCR5/CXCR4, SPs, are slightly
e¡ective against HIV-1 (IC50 v 100 Wg/ml), their poly-
mer-associated forms, SPSAs, manifest a strong synergetic
protection against HIV-1 strains. In particular, one of the
optimal SPSAs representatives, ASV.644, possessed the
IC50 V 0.1 and CC50 v 1,000 Wg/ml that correspond
the IS50 v 10,000. In view of wide antimicrobial activity
of polyanions, the novel substances are promising basis for
HIV/AIDS preventing microbicides (ISTC Project
#2175p).
P15^16
POLYMER-ASSOCIATED SYNTHETIC ANALOGS
OF BICYCLIC TERPENOIDS AS A NOVEL GENER-
ATION FOR ANTIVIRALS
A. V. Serbin(1,2), T. S. Grebinik(1,2), L. I. Kasyan(1), O.
L. Alikhanova(1), K. N. Kozeletskaya(3), M. Bour-
stain(4), A. G. Bukrinskaya(4), I. V. Timofeev(5), N. G.
Perminova(5)
(1) Biomodulators RC, Health RDF; (2) Topchiev Inst. of
Petrochem. Synth., RAS; (3) Ivanovsky Inst. of Virology,
Moscow ; (4) In£uenza Res. Inst., RAMS, St.-Petersburg;
(5) SRC VB ‘‘Vektor’’, Koltsovo, Russia
Bicyclic terpenoids, based on bicyclo[2,2,1]heptane (nor-
bornan), are widely represented in plants and possess ex-
pressed antiseptic activity that involves a heightened inter-
est of pharmacologists. On a frame molecular structure,
and biological activity these substances are close to tricy-
clic adamantan derivatives. So, the well-known anti-in£u-
enza drug, remantadine (Rm), is like on medical indica-
tions to the norbornan derived deitiforin (Df). However,
abiotic nature of adamantan with high ability to penetrate
through lipid-based protective barriers of human cells and
organs result in multiple by e¡ects. Earlier we demon-
strated that these negative properties may be e¡ectively
limited by macromolecular redesign of traditional low mo-
lecular drugs toward polymer-associated systems. Incorpo-
ration of adamantan onto water soluble non-toxic anionic
polymer matrixes via special spacer groups (amant series)
allow to enhance and essentially expand an antiviral po-
tentiality [Antivir Res (1999) 41, 135-44]. Now we inves-
tigate perspectives of this approach for the norbornan
analogue series. In parallel with Df, a line of norbornan
and norbornen derived endo- and exo- isomers with vari-
FEMSLE Congress 2-6-03
ous NH2-terminated spacer groups have been synthesized
and utilized for chemical conjugation with polymeric
anionic matrixes (PAM). The experimental in vitro evalu-
ation of the novel candidates for antivirals (norbent se-
ries) con¢rms the low cytotoxicity (CC50 v800-2000 Wg/
ml) and potent antiviral e⁄cacy not only against in£uenza
A, but also against various Rm/Df/PAM-resistant strains
of HIV-1, including tested AZT-resistant strains. In par-
ticular, one of the most active agent of the norbent series,
AS.504, has exhibited the anti-HIV-1, X794 LAI, protec-
tion with IS50V10,000 (10 fold more then analog of
amant series).
P15^17
CHANGES IN THE COURSE OF PLANT VIRAL IN-
FECTION UNDER THE INFLUENCE OF ELEVATED
LEVEL OF VARIOUS HEAVY METALS IN SOIL
A. V. Shevchenko, I. G. Budzanivska, T. P. Shevchenko, V.
P. Polischuk
Virology Dept., Biology Faculty, Taras Shevchenko’ Kyiv
National University, 64 Volodymyrska st., Kyiv-01033, Uk-
raine
Viral infection is a serious threat for plant growth and
development in arti¢cial cenoses because of the absence
of reliable controlling measures for most of them. They
cause signi¢cant crop losses inducing changes in plant or-
ganisms including malformation of leaves and fruits,
stunting and decease. In turn, increased content of di¡er-
ent heavy metals in soil severely inhibit development of
plants in natural inhabitance as well as in agrocenoses
a¡ecting enzyme and transport activities, photosynthetic
apparatus, resistance mechanisms and growth that gener-
ally lead to plant stunting and death, also resulting in yield
losses. Field experiments were conducted in order to study
the development of systemic tobacco mosaic virus infec-
tion in Lycopersicon esculentum L. plants under e¡ect of
separate heavy metal salts deposited in soil. As it is shown,
simultaneous e¡ect of viral infection and heavy metals in
tenfold maximum permissible concentration leads to de-
crease of total chlorophyll content in experiment plants
mainly due to the degradation of chlorophyll a. The re-
duction of chlorophyll concentration under the combined
in£uence of both stress factors was more serious compar-
ing to the separate e¡ect of every single factor. Plants’
treatment with toxic concentrations of lead and zinc
leaded to slight delay in the development of systemic
TMV infection together with more than twofold increase
of virus content in plants that may be an evidence of
synergism between these heavy metal’s and virus’ e¡ects.
Contrary, copper although decreased total chlorophyll
content but showed protective properties and signi¢cantly
reduced amount of virus in plants.
 1st FEMS Congress / Posters 103^505
P15^18
DISTRIBUTION OF VIRAL INFECTIONS OF SUN-
FLOWER IN UKRAINE
T. P. Shevchenko, G. M. Orlovska, S. M. Petrenko, A. L.
Boyko, V. P. Polischuk
Virology Dpt., Biology Faculty, Taras Shevchenko’ Kyiv
National University, 64 Volodymyrska st., Kyiv-01033, Uk-
raine
Sun£ower (Helianthus annuus) is the most widely spread
oil crop in Ukraine. Viral infections seriously a¡ect sun-
£ower ¢eld production causing worsening of yield quantity
and quality as well as favouring fast spreading of viruses
in ¢eld conditions through seed material. Therefore, atten-
tion was paid to non-seed-transmitted and seed-transmit-
ted viruses of sun£ower that is important for obtaining of
virus-free plant material. Tomato spotted wilt virus
(TSWV, Bunyaviridae), Alfalfa mosaic virus (AMV, Bro-
moviridae), Cucumber mosaic virus (CMV, Bromoviridae)
were chosen for further investigations as the most com-
mon sun£ower viruses detected in Ukraine. Here is repre-
sented the results of analysis of 11 sun£ower cultivars
growing in di¡erent regions of Ukraine on the presence
of TSWV, AMV and CMV antigens. Indirect ELISA was
conducted for detection of virus antigens in plant material,
and electrophoresis by Laemmli for studying of viral struc-
tural proteins as well. Following the obtained data, 6 sun-
£ower cultivars were mainly infected by TSWV, 1 cultivar
by AMV, and 5 cultivars by CMV. Moreover, more than
33% of all sun£ower plants belonging to 11 cultivars tested
were co-infected by TSWV and CMV con¢rming the pre-
vailing combined distribution of these two viruses infect-
ing sun£ower in agrocenoses of Ukraine. Demonstrated
results show high contamination level of sun£ower seeds
by de¢nite viruses and prove the necessity of seed testing
on the presence of viral antigens.
P15^19
EFFECT OF ZINC ON INFLUENZA A VIRUS IN-
DUCED CELL DEATH: A STUDY IN HELA CELLS
V. Srivastava(1), M. Khanna(1), S. Rawall(1), S. Shar-
ma(2)
(1) Dept. of Respiratory Virology and (2) Dept. of Path-
ology, Vallabhabhai Patel Chest Institute University of Del-
hi, Delhi 110 007 India
To investigate mechanism and mode of in£uenza virus
induced cell death, the associated morphological changes
and biological events were examined in HeLa cells infected
with a cell adapted pathogenic strain of in£uenza A virus
FEMSLE Congress 2-6-03
485
(A/Udorn/317/72/H3N2). In£uenza virus infection results
DNA fragmentation preceded by chromatin condensation.
Zinc an endonuclease inhibitor and Annexine V staining a
marker of phosphatidylserine (PS) externalization is used
to examine the mode of cell death. It was observed that
the cells pretreated with zinc have shown remarkable de-
crease in caspase3 activity and DNA fragmentation. The
cell pretreated with zinc has also shown low Annexin-V
staining and eventually low phosphatidylserine (PS) exter-
nalization. These results suggest that the zinc has its in-
hibitory e¡ect on caspase3 and endonuclease, it a¡ects on
endonucleases in a time and dose dependent manner.
Treatment of cells with zinc six hour of post infection
has shown no change in PS externalization and caspase3
activity as well. It was also evident that the PS external-
ization a process which precedes the DNA fragmentation
and con¢rmed by phagocytosis of infected HeLa cells at
various time intervals of post infection.
P15^20
DETECTION OF EPIDEMICS CAUSED BY NON-PO-
LIO ENTEROVIRUSES SENSITIVE AND RESIS-
TANT TO INTERFERENCE ISOLATED FROM
HEALTHY CHILDREN BETWEEN 1962 AND 1972
IN HUNGARY
B. Szebeni, T. Kubasova, E. Molnar, G. Nagy, Gy. Berenc-
si, I. Do«mo«k
Division of Virology, "Be¤la Johan’’ National Center for
Epidemiology, H-1097 Budapest, Gya¤li str. 2-6, Hungary
The recent eradication of wild polioviruses in the Euro-
pean Region has drawn our attention to the non-polio
enteroviruses circulating in the country at the early years
after the introduction of the oral poliovirus vaccination
campaigns. The e⁄cacy of oral poliovirus vaccines could
be followed up between 1962 and 1972 in Hungary, since
the monovalent poliovirus types have been distributed 6
weeks apart. Sampling from 3 to 5-hundred vaccinees have
been performed every year before and after the distribu-
tion of each monovalent viral dose. The poliovirus results
have been already published, but the analysis of the non-
polio enteroviruses could be done only recently in the
possession of the computer technologies. During 11 years
more than 15 thousand stool samples have been examined
using virus isolation on primary monkey kidney cells. The
serum samples also have been collected 6 weeks after the
last vaccination, but only poliovirus-speci¢c antibodies
could be examined. The circulation of 29 enterovirus se-
rotypes could be detected. Several viruses present in the
pre-vaccination samples have been eliminated by the inter-
ference of the oral poliovirus inocula. In certain years,
however, the virus seemed to be resistant to interferon,
since the post-vaccination samples contained signi¢cantly
486 1st FEMS Congress / Posters 103^505
higher proportion of echovirus 11 or 14 than that in the
pre-vaccination samples. All tested vaccinees were healthy
children from communities. The enteroviruses isolated
from samples of patients su¡ering from clinical diseases
will be also documented from the same period. The geo-
graphical distribution of the positive children and patient
shall be also documented.
P15^21
PLACENTAL VASCULAR CHANNEL CHANGES IN
HERPES SIMPLEX VIRUS (HSV) INFECTION
I. Torianik, L. Panchenko, I. Kuchma, N. Popova, S. Brus-
nik, L. Popova, and V. Kazmirchuk
Academy of Medical Sciences, Mechnikov Institute of Mi-
crobiology and Immunology, Kharkiv, Ukraine
The objective was to study morphologic placental vascular
channel changes in herpes simplex virus (HSV) infection in
women. In placental preparations stained with hematoxy-
lin-eosin, hematoxylin-picrofuchxin, silver impregnation
according to Koss and Rasskazova, vascular channel
and adjacent tissue areas were studied histologically. For
study objecti¢cation histo-topographic method was ap-
plied. The study has been carried out using the common
method: the slices of placentae were taken all over the
thickness of the organ both from central and peripheral
regions of placental disk. It was established that in all the
investigated cases vascular lesions took place. They were
accompanied by vascular wall disorganization and de-
struction, by development of stasis and thrombosis. Dis-
orders of the microcirculation led to numerous focal ane-
mic infarcts and hemorrhages. At the same time marked
in£ammatory in¢ltration was seen together with pro-
nounced destructive processes. This process mainly in-
cluded lymphoid cells. In¢ltrations were situated around
and in the vascular walls. Signs of focal sclerosis were
revealed in the basement membrane. Pictures of erythro-
cytic diapedesis were seen everywhere. Conclusions: HSV
leads to placental vascular channel reduction. Vascular
channel changes mainly include focal lymphoid in¢ltra-
tion, vascular walls destruction, stasis, thrombosis, disor-
ders of the mierocireulatiofa. Disorders of the microcircu-
lation lead to pale infarcts and hemorrhages in the organ.
FEMSLE Congress 2-6-03
P15^22
GREEN TEA CATECHINS INHIBIT ADENOVIRUS
INFECTION AND ADENOVIRUS PROTEASE IN VI-
TRO
J. M. Weber, A. Ruzindana-Umunyana, S. Sircar
Departement de Microbiologie et d’Infectiologie, Faculte de
Medecine, Universite de Sherbrooke, Sherbrooke, Quebec,
Canada, J1H 5N4
Green tea catechins have been reported to inhibit pro-
teases involved in cancer metastasis and infection by in£u-
enza virus and HIV. To date there are no e¡ective anti-
adenoviral therapies. Consequently we studied the e¡ect of
green tea catechins, and particularly the predominant
component, epigallocatechin 3-gallate (EGCG), on adeno-
virus infection and the viral proteinase adenain, in cell
culture. Adding EGCG (100 WM) to the medium of in-
fected cells reduced virus yield by two orders of magni-
tude, giving and IC50 of 25 WM and a therapeutic index of
22 in Hep2 cells. The agent was most e¡ective when added
to the cells during the transition from the early to the late
phase of viral infection suggesting that EGCG inhibits one
or more late steps in virus infection. One of these steps
appears to be virus assembly because the titer of infectious
virus and the production of physical particles was much
more a¡ected than the synthesis of virus proteins. Another
step might be the maturation cleavages carried out by
adenain. Of the four catechins tested on adenain, EGCG
was the most inhibitory with an IC50 of 109 WM, com-
pared with an IC50 of 714 WM for PCMB, a standard
cysteine protease inhibitor. EGCG and di¡erent green
teas inactivated puri¢ed adenovirions with IC50 of 250
and 245-3095, respectively. We conclude that the anti-ad-
enoviral activity of EGCG manifests itself through several
mechanisms, both outside and inside the cell, but at e¡ec-
tive drug concentrations well above that reported in the
serum of green tea drinkers.
 1st FEMS Congress / Posters 103^505
P15^23
PREVALENCE OF TYPE A AND B HUMAN INFLU-
ENZA VIRUS ANTIBODIES IN FREEBREEDING
DUCKS IN KOPACKI RIT, CROATIA
Z. Zupancic(1), N. Turk(1), Lj. Barbic(1), S. Kovac(1),
Z. Stojevic(2), V. Drazenovic(3), V. Staresina(1), Z. Mi-
las(1) and B. Jukic(1)
(1) School of Veterinary Medicine, University of Zagreb,
Department of Microbiology and Infectious Diseases with
Clinic, Heinzelova 55, 10000 Zagreb, Croatia ; (2) School
of Veterinary Medicine, University of Zagreb, Department
of Physiology, Heinzelova 55, 10000 Zagreb, Croatia; (3)
Institute of Public Health of Croatia, Rockefellerova 4,
10000 Zagreb, Croatia
The epidemiological role of animal species in the mainte-
nance and spreading of human in£uenza is still the subject
of interests throughout the world. A great many studies
obtained so far have shown that birds, especially wild
waterfowl, are of particular signi¢cance as a potential nat-
ural reservoirs of human in£uenza type A virus and a
possible transmitter of the virus by migration to far-
away places. In order to estimate the seroprevalence
against the human in£uenza virus in freebreeding ducks
we examined 64 duck sera using the hemagglutination in-
hibition method (HI) for the presence of antibodies
against two strains of type A human in£uenza virus ^
variant A/New Caledonia/20/99/VR-116 subtype H1N1
and variant A/Panama/2007/99 (RESVIR-17) subtype
H3N2 ^ and against the strain of type B human in£uenza
virus ^ variant B/Victoria/504/2000. The ducks were reared
in the experimental freebreeding system on the ¢sh-pond
in the area of Kopacki rit (Croatia) and were in close
contact with wild migratory birds during 4 weeks. Out
of the 64 duck sera a positive HI antibody titer (v 1:20)
was estimated to strain A/New Caledonia/20/99/VR-116
subtype H1N1 in 50 (78,1 %) samples; to strain A/Pana-
ma/2007/99 (RESVIR-17) subtype H3N2 in 53 (82,8 %)
samples and to strain B/Victoria/504/2000 in 62 (96,8 %)
samples. Results showed a high infection level among free-
breeding ducks in Kopacki rit drawing attention to a pos-
sible role of wild birds as a major natural reservoirs of
human in£uenza virus type A and type B. For the ¢rst
time we reported the seroprevalence against human in£u-
enza virus type B in birds in Croatia.
FEMSLE Congress 2-6-03
487
P16^1
NOVEL APPROACH FOR BIOSYNTHESIS OF A
NORFLOXACIN ANTIBIOTIC BY PSEUDOMONAS
AERUGINOSA
H. M. Atta
Botany and Microbiology Dept., Faculty of Science, Al-Az-
har University, Cairo, Egypt
A local bacterial culture could be isolated from a soil
sample collected from Cairo governorate, Egypt. The iso-
late AZ-C-22 are active against Gram positive and Gram
negative bacteria. From the taxonomic features, the bac-
terial isolate was likely belonging to Pseudomonas aeru-
ginosa in it’s morphological, physiological and biochemi-
cal characters. The active metabolites were extracted by
Chloroform (2-1, v/v). The separation of the active ingre-
dient and its puri¢cation was performed using both thin
layer chromatography (TLC) and column chromatogra-
phy techniques. The physico-chemical characteristics of
the puri¢ed antibiotic viz. color, melting point, solubility,
elemental analysis, spectroscopic characteristics and chem-
ical reactions have been investigated. This analysis indi-
cates a suggested impirical formula of C16H18FN3O3.
The biological activities i.e. MICs of the puri¢ed antibiotic
were also determined. A study the antiviral tests on both
poliomyelitis and measles were also determined. New ap-
proach for determination of Rf value and color developed
of Fluoroquinolone antibiotic by thin layer chromatogra-
phy (TLC). The collected data emphasized the fact that
the puri¢ed antibiotic compound was suggestive of being
belonging to Fluoroquinolone antibiotic (Nor£oxacin
antibiotic).
P16^2
COMPARABLE CAPABILITY OF DIFFERENT
TYPES OF ANAEROBIC BACTERIA TO HYDRO-
CARBONS SYNTHESIS
T. V. Bagaeva and E. E. Zinurova
Department of Microbiology, Kazan State University,
Kremlevskaya str. 18, 420008 Kazan, Russia
The capability to hydrocarbons synthesis, as cells compo-
nents was observed on the di¡erent, aerobic and anaero-
bic, types of bacteria. From the whole lipids contamina-
tion they contain maximum 3%, and introduced as one of
the cells components. However, our investigations showed
that bacteria cells in some special conditions such as: de-
creasing of SO4 in the cultural media for sulfate reducing
bacteria; absence in the cultural media to Clostridium sp. ;
decreasing of nitrogen containing salts to denitri¢cators,
488 1st FEMS Congress / Posters 103^505
are capable to synthesis an extracellular hydrocarbons.
These processes are more e¡ective if we add gas mixture
H2+CO2 to the atmosphere. In contrast to intracellular
hydrocarbons, alkans with the chain length C25-C35, near
80% of extracellular hydrocarbons are alkans C11-C24
area. The presence of isoforms depending on the type of
bacteria. the main role in the hydrocarbons synthesis pro-
cesses plays fat acids, CO2, gas H2 and water H2. It was
shown that any changes in the cultural media composition
or growth conditions must become stress for the cells and
in£uenced very much on the pathways of metabolism. Fi-
nally cells began to use another ways to fat acids produc-
ing and hydrocarbons synthesis.
P16^3
DULY DIAGNOSTICS OF PHYTOPATHOGENS ON
TOMATOES AND CUCUMBERS IN THE PRO-
TECTED GROUND ^ ONE OF MAJOR FACTORS
OF RECEPTION OF A HEALTHY CROP
L. A. Glukhova and M. N. Gorodkova
Institute of Genetics & Experimental Biology of Plant Uz-
bek Academy of Sciences, Yukori ^ Yuz, Qibray tumani,
Tashkent, 702151, Uzbekistan
Losses of a crop of vegetable cultures in protected ground
from diseases of microbiogenic character can achieve 40
%. Species composition and distribution of disease agents
are the basic components of phytopathologic monitoring.
Annual inspection of vegetable cultures in hothouse farms
of the Tashkent region are carried out from the time of
development of second pair leaves up to the mass matur-
ing of fruits. Alongside visual diagnosis, microbiological
examination of infected samples is undertaken under lab-
oratory conditions. During 2000-2002, mycobiota of to-
matoes was submitted by 51 species of 35 genera. Patho-
gens that can result in the destruction of plants or
signi¢cant losses of crop were revelaed : Phytophthora in-
festans, Geotrichum candidum, Diplodina destructyva,
Aphanomyces cladogamus, Pythium debarianum, Alternaria
solani ^ activators of rots of plant organs and loss of seed-
lings. Most widely distributed were micromycetes from
genus Fusarium, causing tracheomycosis withering and
dry rot of fruits. Also revealed were the mushrooms Co-
chliobolus sativus ^ causing root rot and black spot, Ver-
ticilliun dahliae and species of Acremonium ^ withering,
Phyllosticta infestans, Aureobasidium pullulans ^ spots
and anthracnoses of leafs, Rhizoctonia solani ^ brown
and dry rots. The rarely seen mushrooms ^ Cylindrocarpon
didymum, Colletotrichum atramentarium, Fulvia fulva, Ka-
batiella sp. were also detected. On cucumbers 25 species of
mushroom from 17 genera were found, including the
pathogens: Cladosporium cucumerinum ^ activator of
scab, Erisiphe cichoracearum ^ mealy dew, Phytophthora
FEMSLE Congress 2-6-03
parasitica ^ rots of various organs, Verticilliun albo ^
atrum ^ withering, Fusarium oxysporum ^ tracheomycosis
withering and root rot. Use of tomatoes and cucumbers
a¡ected by mushroom pathogens is unsafe for human
health. Duly diagnostics of activators of disease promotes
the realization of therapeutic and protective measures for
reception of a healthy crop. It helps to solve problems of
the foodstu¡s, ecology and protection of human health
from the harmful in£uence of toxic metabolites produced
by mushrooms.
P16^4
MUBARAK CITY FOR SCIENCE (MUCSAT): PRO-
PELLING EGYPT INTO THE WORLD OF AD-
VANCED TECHNOLOGY
A. A. M. Khalil
Department of Protein Research, Genetic Engineering and
Biotechnology Institute, Mubarak City for Science and Ap-
plied Technology, Research Zone, Borg Al-Arab, Post Code
21934, Alexandria, Egypt. On leave from : Department of
Biotechnology, Lund University, Lund, Sweden
Egypt is being pressed from below by the likes of China,
India, Mexico and Poland, all moving up the technology
ladder as their skills and know-how improve. As an in-
creasing amount of public money is invested in advanced
sciences research, a big challenge facing Egypt is how to
commercialize the results and expand companies to cap-
ture the economic bene¢ts, including good jobs. And how
do we develop the niches and build the industries that will
create opportunity and generate wealth for our country ?
Let’s start by looking at what’s happening in Mubarak
City for Science, so this was the ¢rst identi¢ed challenge.
The aim of building the City in Bourg el-Arab is to be
close to the wider industrial base in Alexandria, in which
40% of national industries concentrates. MuCSAT is the
pillar of The Egyptian Technology Coast. This project
aims at laying the base stone of north coast comprehensive
development and capitalizing on available potentialities
therein. The city buildings cover an area of 200 hectare
and the ¢rst phase includes the following: Genetic Engi-
neering and Biotechnology Institute, Information Technol-
ogy Institute, New Materials and Advanced Technology
Institute, Small-Scale Industries Development Center.
MuCSAT will enable Egypt to make progress in leaps
and bounds and cultivate home-grown technology. This
will be a prominent landmark if we ¢nish the job properly.
In the same time MuCSAT is convinced that the future of
both the North and the South are inseparably intercon-
nected. To achieve this, MuCSAT has enthusiastically a
propensity to construct long-term bilateral collaborations
with universities and research institutions in the North.
 1st FEMS Congress / Posters 103^505
P16^5
CATALYTIC FILAMENTOUS CARBONS (CFC) AND
MACROSTRUCTURED CFC-COATED CERAMICS
FOR IMMOBILIZATION OF MICROORGANISMS
G. A. Kovalenko, O. V. Komova, D. G. Kuvshinov, A. V.
Simakov, N. A. Rudina
Boreskov Institute of Catalysis, 630090 Novosibirsk, Russia
The problem to develop the e¡ective adsorbents for vari-
ous microorganisms is still relevant. On the one hand, the
e¡ective adsorbents as ¢lter materials are required for so-
lution of ecological problems, for example, in sewage pu-
ri¢cation. On the other hand, they may be used in bio-
technology as supports for immobilized bacterial cells,
which exhibit desired enzymatic activity to develop hetero-
geneous biocatalysts. Obviously, such adsorbents-supports
must meet certain criteria. First, they must have su⁄cient
adsorption capacity and ¢rmly hold the bacteria on the
surface. Second, they must retain and stabilize the biolog-
ical activity of immobilized microbial cells. Third, they
must possess high mechanical strength and resistance to
biological and chemical degradation. Finally, their cost
should be relatively low. Adsorption properties of sup-
ports based on bulk catalytic ¢lamentous carbons (CFC)
have been studied with respect to di¡erent non-growing
cells of microorganisms (E. coli, Bacillus subtilis, Rhodo-
coccus sp.). The factors in£uencing the adsorption e⁄-
ciency have been investigated. For bacteria, besides the
value of accessible surface area, roughness of the surface,
which is determined by the carbon yield, is a crucial factor
a¡ecting the e⁄ciency of the adsorption/desorption of bac-
teria on bulk CFC. Macrostructured CFC-coated ceramics
have been synthesized for the immobilization of the Rho-
dococcus sp. Foam-like ceramics, that has advanced mac-
rostructure and surface coated with catalytic ¢lamentary
carbon with low carbon yield, have been shown to be the
most e¡ective supports for adsorptive immobilization of
bacteria, in particular, Rhodococcus sp., and, actually,
these supports satis¢ed all requirements above.
FEMSLE Congress 2-6-03
489
P16^6
SCREENING OF THERMOTOLERANT ACTINOMY-
CETES WHICH PRODUCE D-MANNOSE ISOMER-
ASE
A. Tangjaiatitharn(1), V. Sahachaisaree(2), K. Izumori(3)
and S. Lumyong(1)
(1) Department of Biology, Faculty of Science, Chiang Mai
University, Chiang Mai 50200, Thailand; (2) Department
of Chemistry, Faculty of Science, Chiang Mai University,
Chiang Mai 50200, Thailand; (3) Department of Biochem-
istry and Food Science, Faculty of Agriculture, Kagawa
University, Kagawa, Japan
This research was to determine the new sources for D-
mannose isomerase production. Two hundred and nine-
teen thermotolerant actinomycetes isolates capable of
growth at 45 ‡C were isolated from various soil samples
of Thailand. All of the isolates were determined for D-
mannose isomerase activity by using D-mannose as a sub-
strate and ketose produced was measured by Cysteine-car-
bazol method. Only four strains of thermotolerant actino-
mycetes had positive results. The strain was grown
aerobically at 45 ‡C. The cells were sonicated and enzyme
was extracted. Isolate CMUB10 was found to give the
highest activity of 0.3057 unit/g.wet cell and the speci¢c
activity of 0.0140 unit/mg protein. This strain identi¢ed as
Streptomyces sp. according to morphology and amino acid
component of whole-cell extract.
P16^7
PRODUCTION OF RARE SUGAR FROM ACIDOTO-
LERANT AND THERMOTOLERANT ACETIC ACID
BACTERIA: ISOLATION AND SCREENING
W. Chittrong(1), Y. Yamada(2), K. Izumori(3) and S.
Lumyong(1)
(1) Department of Biology, Faculty of Science, Chiang Mai
University, Chiang Mai 50200, Thailand; (2) National
Center for Genetic Engineering and Biotechnology (bio-
tec), Bangkok 10400, Thailand; (3) Department of Bio-
chemistry and Food Science, Faculty of Agriculture, Kaga-
wa University, Kagawa, Japan
Acetic acid bacteria were isolated from 100 samples of
£owers and fruits collected in Chiang Mai, by an enrich-
ment culture approach for acetic acid bacteria invarious
acidic, pH 3.5-4.0; glucose-ethanol-acetic acid medium,
sorbitol medium, sucrose-acetic acid medium and metha-
nol medium, then incubated at room temperature for 2-3
days. Isolation of pure culture were carried out by streak
plate method on calcium carbonate ethanol agar plate in-
490 1st FEMS Congress / Posters 103^505
cubated at 30OC for 2-3 days. Among a large number of
acetic acid bacteria strains isolated, only six strains as-
sumed to be Asaia species, four strains assumed to be
Acetobacter species, and two strains assumed to be Gluco-
nobacter species. Fourteen isolates were thermotolerant
acetic acid bacteria. The isolated bacteria can be grown
on D-sorbitol, and during a study of taxonomic studied
and rare sugar production from various carbon sources.
Identi¢cation of product will be determined.
P16^8
CAN YOU TRUST THE AIR YOU BREATHE? YOU
CAN NOW!
P. Hall, R. Malyon
Microgenix limited, Wolfelands, High Street, Westerham,
Kent TN16 1RQ, UK
This, patented technology, quite simply kills airborne bac-
teria and viruses. Originally tested by scientists at the
highly accredited DSTL Laboratories, Porton Down in
the United Kingdom, eight billion spores of an antrax
simulant were passed through the Microgenix unit and
all eight billion were eradicated within one second! Fur-
ther scienti¢c tests carried out at the Centre for Applied
Microbiology and Research Centre (CAMR) at Porton
Down together with tests carried out at Leeds University
and extensive scienti¢c testing in Switzerland prove the
Microgenix system e¡ective against a huge range of bac-
teria and viruses ^ including MRSA and the deadly small-
pox.The Microgenix technology is based on a two-part
approach to killing deadly and infectious airborne patho-
gens. The Microgenix microbiological air puri¢cation sys-
tem employs an integrated 2-stage process involving pri-
mary ¢lter media treated with ‘Biogreen 3000’ together
with eradication utilising high powered germicidal ultra
violet light (UVC) at 254nm which, when applied together,
kills virtually all known bacteria and micro-organisms on
contact. The ‘Biogreen 3000’ ¢lter’ is a vital part of the
killing process ^ it is a high performance fabric ¢lter
coated with the Biogreen 3000 liquid. This liquid, when
dry, forms a microscopic spike-like structure creating a
deadly pathway in the ¢lter upon which the micro-organ-
isms are impaled. Biogreen 3000 is a kationic detergent
that disrupts the functionallity of a cell membrane.’Bio-
green 3000’ does not ‘poison’ the micro-organism, the
cell is physically ruptured upon contact.Microgenix kills
deadly airborne pathogens that pass through the system
and protects against the global problem of cross contam-
ination in the air that we breathe.
FEMSLE Congress 2-6-03
P16^9
DIFFERENTIAL PRESENTATION OF MULTICEL-
LULAR INTESTINAL PARASITES IN SCHOOL
CHILDREN IN THE REGION OF BIHAC Ł , IN THE
PERIOD 1973-1978, COMPARED TO SOME OTHER
FACTORS
H. Merdanic¤
Pedagogical Faculty, University of Bihac¤, Dz›anic¤a mahala
58, 77000 Bihac¤, Bosnia and Herzegovina
Parasitosis caused by multicellular intestinal parasites is a
problem in the pathology of children’s gastrointestinal dis-
orders. The goal of this research was to determine,
through sampling of feces and perianal print on multicel-
lular intestinal parasites among the primary and secondary
school pupils from the region of Bihac¤, their representa-
tion and frequency considering age, sex, village and town
living and social-economic state, healthcare and cultural
standards. The results were compared with research car-
ried out 25 years ago, a time when there was no organised
medical treatment of parasitosis. The proportion of infes-
tation averaged 78,58% in 1973 and ranged from 64,28%
to 88,77%. In 1998, the proportion of infestation averaged
56,28% and ranged from 26,31% to 74,50%. The di¡erence
in average infestation levels between these two researches
is statistically very important. Both researches showed that
children from villages were more infested than those from
towns, in total and gender-wise. The di¡erence between
the two researches at this level is very important. No sta-
tistically important di¡erence was found between the two
researches at the level of same sex and living standards,
but was found at the level of totality between town and
village children. There was no statistically important dif-
ference in infestation at 7-8 and 11-12 years, both in towns
and villages, but there was between the mentioned age and
15-16 years. The following multicellular intestinal parasites
were identi¢ed in both researches: Ascaris lumbricoides,
Trichiuris trichiura, Enterobius vermicularis, Hymenolepis
nana and Taenia sp. (Taenia solium et saginata). Due to
population migration, the following were expected to be
identi¢ed but were not: Ancylostoma duodenale, Strongy-
loides stercoralisi Trichostrongilus sp. Both tests identi¢ed
that parasites were more common in younger examinees
than older ones, and the di¡erence was statistically impor-
tant. The di¡erence in the proportion of infestation is
mainly caused by high economical, educational, social cul-
ture and healthcare levels, habits and behaviour in toilets
and towards fecal materials; prevention of the process of
fecal-oral way of spreading, practising control of meat
products and applying better personal hygiene.
 1st FEMS Congress / Posters 103^505
P16^10
ISOLATION OF RHIZOBIUM FROM THAI NATIVE
LEGUMES
S. Siri-Udom(1), B. Rerkasem(2) and S. Lumyong(1)
(1) Department of Biology, Chiang Mai University, Chiang
Mai 50200, Thailand; (2) Department of Agronomy, Fac-
ulty of Agriculture, Chiang Mai University, Chiang Mai
50200, Thailand
Grain legumes play an important role in cropping system
of Thailand. Many studies have shown that simultaneous
infection with Rhizobium can increase nodulation and
growth in legumes. The objectives of this research were
to isolate symbiotic and active Rhizobium from root nod-
ules of Thai native legumes and study host speci¢city to
determine the cross-inoculation grouping of these Rhi-
zobium. Fifty-two isolates of Rhizobium were isolated
from 21 Thai native legumes from The Forest Restoration
Research Unit (forru) at Suthep-Pui National Park and
Chiang Mai. There were 5 isolates from Dalbergia Eryth-
rina stricta, 3 isolates from Erythrina subumbrans, 3 iso-
lates from Archidendron clypearia, 8 isolates oliveri, 5 iso-
lates from from Albizia chinensis, 5 isolates from Albizia
odoratissima, 3 isolates from Albizia lebbeck, 3 isolates
from Dalbergia cultrata, 4 isolates from Acacia mearnsii,
5 isolates from Pterocarpus macrocarpus, 2 isolates from
Leucaena leucocephalade, 2 isolates from Mimosa pudica, 3
isolates from Millettia pubinervis and one isolate from
Mimosa pigra L. Twenty-three isolates were found to be
the fast-growing group and 29 isolates were found to be
the slow-growing group. Legume seeds were cultured in 10
soils from Chiang Mai, Nong Bau Lam Phu and Loei to
isolate Rhizobium and study in nodule formation. The iso-
lates of Rhizobium will keep for future identi¢cation and
cross-inoculation.
P16^11
ANTIBACTERIAL EFFECTIVENESS OF INTERFER-
ON PREPARATIONS
M. Ya. Spivak, O. V. Karpov, N. A. Tymoshok, N. M.
Zholobak, L. M. Lazarenko, V. M. Zotsenko, N. I. Grab-
chenko, L. A. Ganova, O. P. Mikhailenko
Danylo Zabolotny Institute of Microbiology & Virology,
National Academy of Sciences of Ukraine, Kyiv, Ukraine
We have designed the infection processes caused by Staph-
ylococcus aureus, Salmonella typhimurium, Esherishia coli,
Pseudomonas aeruginosa, Chlamidia trachomatis. It was
shown that the infection diseases cause reduce of an or-
ganism immunological reactivity and is accompanied by
FEMSLE Congress 2-6-03
491
suppress of phagocytes functional activity and natural kill-
er cells (NK) and also by disorder of an organism inter-
feron status. Reduce of phagocytes functional activity can
lead to decrease of their functions e¡ectiveness to an or-
ganism release from infection agents, and also to disorder
of phagocytes cooperative interrelations with lymphocytes
during the immune response formation.The protective ef-
fect of interferons (a/L, Q) preparations (IFN) under bac-
terial infections is determined to a great degree by capacity
to restore phagocytes, cells NK e¡ector functions. How-
ever, mechanisms taking part in realization of immunomo-
dulating activity and Q-IFN on these cells are not studied
well. We have shown that the protective action of a/L-
IFN recombinant as well as natural origin, is determined
to a great degree by their ability to reduce quantity of
persistence bacteria in in£ammation centres and also by
ability to increase an organism non speci¢c resistance: to
intensify phagocytes functional activity and NK, and also
execute the regulated in£uence on IFN production and
tumor necrosis factor (TNF). At the same time the intro-
ducing of recombinant processes of bacterial ethyology
didn’t in£uence pathogen elimination from an organism.
With this it was marked non-su⁄cient activation of
phagocytes system. It was shown that for the e¡ective
stimulation of phagocytes e¡ector functions it is necessary
to combine the use of other lymphokines, possessing the
trigger action, especially TNF. At the same time the nat-
ural Q-IFN preparations possess the complex of cytokines
(IL-1, TNF) which determine the cell immune response at
early stages of its realization, that promote to increasing
of pathogene elimination from an organism.
P16^12
HEAT SHOCK PROTEINS OF MYCOPLASMAS
D. Benc›ina, B. Slavec, M. Narat and P. Dovc›
Zootechnical Department, Biotechnical Faculty, University
of Ljubljana, 1230 Domz›ale, Slovenia
Heat shock proteins (Hsp) are found in virtually all life
forms, but only little is known about Hsp and their encod-
ing genes in Mycoplasma species. Using monoclonal anti-
body (mAb) Mhy3 to the DnaK protein (Hsp70) of M.
hyopneumoniae we detected its synthesis in more than 20
Mycoplasma species belonging to the phylogenetic groups
of the pneumoniae, hominis and spiroplasma. Their DnaK
proteins of about 63-67 kDa were also synthesized consti-
tutively. Sequencing of the dnaK gene homologues of avi-
an Mycoplasma species revealed that dnaK of M. gallisep-
ticum and M. imitans share certain sequence characteristics
with the dnaK of human pathogens, M. pneumoniae and
M. genitalium. Moreover, DnaK proteins of these four
species share epitopes recognized by mAbs to DnaK of
M. pneumoniae (mAb 5F7) and M. gallisepticum (mAb
492 1st FEMS Congress / Posters 103^505
MyG 004). Three other pathogenic avian Mycoplasma spe-
cies i.e. M. synoviae, M. meleagridis and M. iowae revealed
the dnaK sequences that were more similar to those of
species belonging to the hominis group. Intraspecies
dnaK polymorphisms were found in M. gallisepticum and
M. synoviae. Whereas many Mycoplasma species lack a
gene encoding the GroEL protein (Hsp 60), M. gallisepti-
cum and M. imitans have the groEL genes. Their groEL
sequences revealed over 80% sequence identity, but a con-
siderably lower identity (V60%) with the groEL sequence
of M. pneumoniae. In the infected poultry, hemagglutinins
of M. gallisepticum (pMGA) and of M. synoviae (VlhA)
induced signi¢cantly stronger antibody response than
DnaK proteins of these pathogenic Mycoplasma species.
P16^13
GENES ENABLING DNA RECOMBINATIONS IN
MYCOPLASMA SYNOVIAE
P. Dovc›, B. Slavec, A. Razpet and D. Benc›ina,
Zootechnical Department, Biotechnical Faculty, University
of Ljubljana, 1230 Domz›ale, Slovenia
Mycoplasma synoviae is a major poultry pathogen causing
great economic losses in poultry production. Whereas for
several Mycoplasma species, including another major poul-
try pathogen, M. gallisepticum, the complete genome se-
quence has been determined, only a few genes of M. syn-
oviae have been sequenced, so far. Gene families encoding
hemagglutinins in M. gallisepticum (pMGA genes) and M.
synoviae (vlhA gene and its pseudogenes) have arisen via
horizontal gene transfer. In M. synoviae site-speci¢c re-
combinations of the vlhA gene generate functional and
antigenic variants of its hemagglutinin. Generally, RecA
protein plays a central role in homologous recombina-
tions. We have sequenced the recA gene of di¡erent M.
synoviae strains. Their recA sequences revealed signi¢-
cantly higher % of identity with the recA of M. pulmonis
(V80 %) than with recA of M. gallisepticum ( 6 50%).
Intraspecies recA polymorphisms were higher in M. galli-
septicum than in M. synoviae. Sequencing of about 3 kbp
DNA fragment from the genomic DNA library of M.
synoviae type strain (WVU1853) identi¢ed an ORF encod-
ing a putative transposase similar to transposases of M.
hyopneumoniae (IS Mhp 1 tnp) and M. mycoides subsp.
mycoides SC (IS 1634). Site-speci¢c tyrosine recombinase
enable site-speci¢c DNA inversions and generate antigenic
variants of major lipoproteins in Mycoplasma bovis, M.
agalactiae and M. pulmonis. It seems that M. synoviae
has a gene related to the mbr gene which in M. bovis en-
codes the site-speci¢c recombinase. However, the nucleo-
tide sequence of the putative vlhA gene recombination site
is clearly di¡erent from sequences of the recombination
FEMSLE Congress 2-6-03
sites of M. bovis (vsp genes) and M. pulmonis (vrs box of
the vsa genes).
P16^14
GENETIC VARIABILITY OF MYCOPLASMA BOVIS
STRAINS IN BRITAIN
L. McAuli¡e(1), R. D. Ayling(1), B. Kokotovic(2) and R.
A. J. Nicholas(1)
(1) Mycoplasma Group, Department of Bacterial Diseases,
Veterinary Laboratories Agency (Weybridge), Surrey,
KT15 3NB, UK; (2) Department of Bacteriology, Danish
Veterinary Institute, Copenhagen V, DK-1790, Denmark
Mycoplasma bovis is one of the major aetiological agents
of bovine mycoplasma infections worldwide causing con-
siderable economic losses. M. bovis is an increasingly rec-
ognised cause of chronic pneumonia, polysynovitis, otitis
media and occasionally mastitis in cattle. Since there are
marked di¡erences in M. bovis prevalence across Europe,
there is a pressing need for monitoring animals during
commercial trade. In this study we describe the develop-
ment of molecular typing techniques for M. bovis. Sixty
M. bovis isolates (obtained in the UK between 1998 and
2002) were subjected to molecular typing using pulsed ¢eld
gel electrophoresis (PFGE) and ampli¢ed fragment length
polymorphism (AFLP) analysis. PFGE analysis demon-
strated that at least 12 distinct M. bovis pro¢les predom-
inate in the UK. AFLP analysis demonstrated much great-
er heterogeneity between isolates. This study represents the
¢rst attempt to type M. bovis in the UK and may have
important epidemiological implications.
P16^15
MICROECOLOGICAL ALTERATIONS OF THE
VAGINAL MICROFLORA IN PATIENTS WITH THE
UROGENITAL MYCOPLASMA INFECTIONS
E. V. Naumkina, N. V. Rudakov, L. V. Belkina, B. M.
Ivanova
Omsk State Medical Academy, Russia, Omsk, 9 Mira Pros-
pect
The role of opportunistic microorganisms in the gyneco-
logical pathology of the person in current conditions is
increasing. The complex study of the microecological al-
terations of the vaginal micro£ora in patients with urogen-
ital mycoplasmosis was carried out. 82 samples of vaginal
secret from women with various kind of vaginal secretion
were studied. Deviations in the vaginal micro£ora were
not detected in 10 cases (12,2 %). Urogenital mycoplasmo-
sis was diagnosed for 32 patients (39 %), U. urealyticum
 1st FEMS Congress / Posters 103^505
was isolated from 16 patients (19,5 %), urogenital myco-
plasma (M. hominis, M. genitalium) ^ from 12 (14,6 %).
The both ureaplasma and mycoplasma infection was ob-
served in 4 cases. Thus in 50 % of cases urogenital myco-
plasmosis was not accompanied by in£ammatory response
of the vaginal mucosa, so can be regarded as a dysbiosis.
In other cases the expressed in£ammation of a mucosa
(vaginitis) was marked. Urogenital mycoplasmosis were
accompanied by the following microecological alterations
of micro£ora. The de¢ciency of lactobacillus less than 10 4/
ml was marked in 59,4 % of cases. Candida spp. were
isolated from 46,8 % of the patients, the quantitative aug-
mentation of aerobic, facultative and obligate anaerobic
opportunistic microorganisms higher than 10 4/ml was
marked in 62,5 % of cases,and in 34,7 % they were selected
in di¡erent associations. Thus frequency and the expres-
siveness of microecological changes of micro£ora in pa-
tients with mycoplasma vaginitis was much above, than
when the in£ammatory response was not observed. These
facts show the important role of the opportunistic micro-
organisms as accompanying factor with urogenital myco-
plasmas.
P16^16
MYCOPLASMA FERMENTANS INTERACTION
WITH HELA CELLS
A. Yavlovich, A. Katzenell and S. Rottem
Department of Membrane Research, The Faculty of Medi-
cine, The Hebrew University of Jerusalem, Israel
Mycoplasma fermentans has been recently implicated as a
potential human pathogen. The adherence of this organ-
ism to host eukaryotic cells is an essential ¢rst stage of
infection and an absolute requirement for colonization
contributing to the pathogenic process. M. fermentans ad-
heres to HeLa cells in a time dependent manner with max-
imal binding obtained after 2h at 37‡C. Proteinase-K
treatment of M. fermentans reduced the capability to ad-
here to HeLa cells and a surface lipoprotein has been
shown to play a role in the adherence process. Nonethe-
less, the residual proteinase insensitive adherence was
stimulated by PEG whereas PEG has no e¡ect on the
proteinase sensitive adherence process. Furthermore, the
proteinase insensitive adhesion was markedly inhibited
by antibodies raised against the Choline containing phos-
phgolylipid of M. fermentans (MfGL-II) and by free Cho-
line-phosphate suggesting that M. fermentans possess two
adhesions, a surface lipoprotein and a Choline containing
plycolipid. Adherence of M. fermentans to HeLa cells was
markedly increased when M. fermentans were pre-incuba-
ted with plasminogen (Pg), a 92kDa serum and tissue gly-
coprotein. Furthermore, when Pg-bound M. fermentans
preparations were treated with a Pg activator (u-PA or
FEMSLE Congress 2-6-03
493
t-PA), we detected M. fermentans not only on the surface
but also within HeLa cells. It seems that the ability of M.
fermentans to invade host cells stems not from its potential
to bind Pg and to better adhere to host cells, but from the
activation of the bound Pg to plasmin, a protease that
may alter host cell surface proteins and thereby enable
invasion.
P16^17
ANTIOXIDANT ACTIVITY IN MYCOPLASMA FER-
MENTANS
A. Yavlovich(1), R. Kohen(2), I. Ginsburg(3) and S. Rot-
tem(1)
(1) Dept. of Membrane Research, (2) Dept. of Pharma-
ceutics, The Faculty of Medicine and (3) Dept. of Oral
Biology, The Faculty of Dental Medicine, The Hebrew Uni-
versity, Jerusalem, Israel
Mycoplasma fermentans was isolated from the human ur-
ogenital tract. This organism is considered to be a surface
parasite. However, recent studies showed that under cer-
tain condition M. fermentans can invade HeLa cells and
survive within them for prolonged periods of time (Infect.
Immun. 69, 1977-1982, 2001). It is expected that in order
to overcome the continuous exposure to oxidative stress
within the host cells this organism will develop an antioxi-
dant defense mechanism. Indeed, a high overall antioxi-
dant activity was detected in M. fermentans by the lumi-
nol-enhanced chemiluminescence (CL) assay
demonstrating that this organism quenched the reactive
oxygen species generated by combination of urea-hydro-
gen peroxide and Na2SeO3. The reductive capacity of M.
fermentans was than analyzed by cyclic voltametry show-
ing that this capacity resides preferentially in the cytosol
and is due to a low molecular weight antioxidant mole-
cule(s). We suggest that the enhanced antioxidant activity
of M. fermentans described in this study is a principal
defense mechanism playing a major role in the battle
against oxidative stress.
494 1st FEMS Congress / Posters 103^505
P16^18
CHARACTERIZATION AND METABOLIC PROPER-
TIES OF PROBIOTIC ENTEROCOCCI, ISOLATES
FROM DOGS FEED
A. Laukova¤(1), V. Strompfova¤(1), M. Marcin›a¤kova¤(1),
A. Ouwehand(2), M. Baele(3), L. Devriese(3)
(1) Institute of Animal Physiology, Slovak Academy of
Sciences, Kos›ice, Slovakia; (2) University of Turku, De-
partment of Biochemistry and Food Chemistry, Turku, Fin-
land; (3) University of Ghent, Faculty of Veterinary Med-
icine, Laboratory of Veterinary Bacteriology and Mycology,
Merelbeke, Ghent, Belgium
At present time, the use of probiotic micoorganisms is
common in the human nutrition. However, their use
started to be included more frequently also into the animal
nutrition. Nowdays, many people are pet owners. So, es-
pecially, the small animals (dogs, cats, rabbits etc.) repre-
sent the main group for that the use of probiotic can be
and/or is directed. In our study, the genotypic character-
ization by tDNA-PCR (involving capillary electrophore-
sis) of the target of enterococci, the isolates from dogs
feed was estimated as well as their other metabolic proper-
ties such as bacteriocin-production, adhesion ability, ure-
ase activity, antibiotic pro¢le, ability to grow in the pres-
ence of bile and presence of plasmids. The majority of our
isolates were taxonomically allotted to the species Entero-
coccus faecium, Ent. faecalis and Ent. hirae. Among 11
strains selected, all were kanamycin and gentamycin resis-
tant and 3 of them were also resistant to erythromycin. In
8 strains was also detected presence of plasmids. Adhesion
ability of our isolates to human as well as to dog mucus
was tested. And, high adhesion activity was detected to
human mucus (from 7.8 % up to 30.9%). Even higher
adhesion ability was found of these isolates to human
mucus than to canine mucus (3.7% -28.7%). Ent. faecium
AL3 was that with the highest adhesion ability. Entero-
cocci showed the ability to grow in the presence of oxgall
and they urease di¡ered from low to high values. They
were found without bacteriocin production to the target
strains used. However, testing is still in the progress. It
must be underlined that especially their adhesion ability
recommends these strains or several of them for their fur-
ther probiotic use. Moreover, adhesion ability of entero-
cocci to di¡erent mucus is the phenomenon which was
never before presented; mainly concerning new/wild
strains.
This work was supported by project 2/2043/22 of VEGA/
Slovak Scienti¢c Agency.
FEMSLE Congress 2-6-03
P16^19
BACTERIOCINOGENIC ENTEROCOCCUS FAECI-
UM EF55, A NEW POTENTIAL PROBIOTIC FOR
POULTRY
V. Strompfova¤ and A. Laukova¤
Institute of Animal Physiology Slovak Academy of Sciences,
SNolte¤sovej 4-6, 040 01 Kos›ice, Slovakia
Enterococci and other lactic acid bacteria probiotic prep-
arations have received more interest in animal manage-
ment as a result of gastrointestinal pathogens developing
antibiotic resistance and leaving chemical residues of anti-
biotics in meat. A potentially successful probiotic strain is
expected to have several desirable properties to be able
exerts its bene¢cial e¡ects. One of them is production of
antimicrobial compouds, e.g. bacteriocins. In the intestine
where other autochtonous bacteria grow as a competitive
micro£ora, the use of probiotic enterococci with bacterio-
cinogenic character may confer to gain their predominant
place. The purpose of this study was to determine some
probiotic criteria (acid and bile tolerance, adhesion activ-
ity, antibiotic pro¢le), especially bacteriocin-like activity,
by Enterococcus faecium EF55 ^ chicken isolate from con-
tent of crop. The antimicrobial spectrum of bacteriocin-
like substance was tested by agar spot test against a target
of Gram-positive and Gram-negative indicator bacteria.
The strain EF55 showed inhibitory activity towards other
enterococci as well as strains of staphylococci, streptococ-
ci, lactobacilli, lactococci, micrococci etc., but not against
Gram-negative bacteria. In adition, the e¡ect of enzymes,
pH and heat on bacteriocin activity was evaluated. This
antimicrobial substance was heat-stable (30 min at 60 ‡C,
80 ‡C and 100 ‡C) and stable over a pH range 4.0-9.0 at -
20 ‡C, 4 ‡C and 22 ‡C for 10 days tested, but it was
sensitive to proteolytic enzymes. It con¢rmed the protein-
aceous character of this substance. Further the crude bac-
teriocin extract of strain EF55 was used in the in vivo
experiment. By 3-day old conventional Japanese quials
the in£uence of daily orally administered extract on the
total counts of selected bacterial groups (enterococci,
staphylococci, lactobacilli, E. coli) in the faeces was inves-
tigated. The reduction of all bacteria mentioned was ob-
served with di¡erence 1.0-1.3 log cycles, especially after
¢rst crude extract application.
This work was supported by project 2/2043/22 of VEGA
Slovak Scienti¢c Agency.
 1st FEMS Congress / Posters 103^505 495

P16^20 P16^21

CANINE LACTOBACILLI AND THEIR PROBIOTIC Withdrawn.

CHARACTER

V. Strompfova¤(1), A. Laukova¤(1), A. C. Ouwehand(2)

and M. Fialkovic›ova¤(3)

(1) Institute of Animal Physiology, Slovak Academy of

Sciences, Kos›ice, Slovakia; (2) Department of Biochemis-
try and Food Chemistry, University of Turku, Turku, Fin-
land; (3) Clinic of diesases of horses, pets and birds, Uni-
versity of Veterinary Medicine, Kos›ice, Slovakia

Besides enterococci, bacilli and yeasts, lactobacilli are the

most frequently used probiotic microorganisms as canine
feed suplements [1]. Nowdays is a lack of information
concerning the isolates from dog origin which are studied
for their probiotic purpose. Therefore, our aim was to
isolate and to select the suitable strains of lactobacilli for
their potential use as probiotics. Our strains were isolated
from faeces of ten healthy dogs of di¡erent age, breed and
sex. Among properties tested by forty isolates of lactoba-
cilli (bile tolerance, antibiotic pro¢le, adhesion activity),
production of bacteriocin-like inhibitory substances
(BLIS) was a major focal point of our study. Bacteriocins
^ proteinaceous compounds with inhibitory activity
against more or less related bacterial genera to the pro-
ducer microorganism ^ are only one of metabolites with
antimicrobial activity produced by lactic acid bacteria,
which confer to an ecological advantage over competitors
present in the intestine. Isolated strains were screened for
production of these substances using the agar di¡usion
technique [2] on bu¡ered Schaedler agar (Becton & Dick-
inson, USA) against twenty indicator microorganisms. 31
isolates (77.5 %) were able to produce BLIS with activity
mainly against Gram-positive bacteria (Enterococcus sp.,
Lactobacillus sp., Staphylococcus sp., Streptococcus sp.,
Micrococcus sp., Lactococcus sp.), but also against Enter-
obacter sp. Strain AD1, one isolate of lactobacilli tested,
was choosen for application in vivo. It was daily orally
administered (3 ml/dog ; 109/ml cfu) to dogs with di¡erent
diseases and disorders of gastrointestinal tract. The strain
AD1 was able to survive and to establish in the canine
intestine. Several bacterial genera in the faeces as well as
biochemical parameters in blood were determined to eval-
uate the in£uence of used strain on health status of ill
dogs.
[1] O.B. Maia, et al (2001). Vet. Microbiol., 79, 183-189.
[2] B. Skalka et al. (1983) Zbl. Bakteriol. Hyg., A256, 168-
174.
This work was supported by project 2/2043/22 of VEGA
Slovak Scienti¢c Agency.

FEMSLE Congress 2-6-03

496 1st FEMS Congress / Posters 103^505
P16^22
BIOLOGICAL DECONTAMINATION OF AIR USING
A MULTISTAGE HIGH VOLTAGE REACTOR
V. Bergeron(1), M. First(2), A. Starinsky(3)
(1) Ecole Normale Supe¤rieure, Laboratoire de Physique, 46
alle¤e d’Italie, Lyon, France ; (2) Harvard School of Public
Health, 677 Huntington Avenue, Boston, USA; (3) Airin-
space, 112 Avenue du Ge¤ne¤ral de Gaulle, Rosny sous Bois,
France
Air quality is a major issue in our time, particularly for
immune-compromised individuals. Needed are e⁄cient,
cost e¡ective and versatile air treatment units to provide
protection against a broad range of bio-contaminates.
Here we present a novel technology capable of destroying
airborne fungi, bacteria, spores and viruses. This technol-
ogy was ¢rst developed for manned space-£ight and has
been transferred for use in earth-based applications (hos-
pitals, homes and public transportation). The Plasmer0
reactor relies on enhanced electrical ¢elds produced by
highly porous metal electrodes that envelope multistage
cold plasma chambers,. We provide recent data showing
that micro-organisms passing through the reactor are
completely destroyed. Under highly contaminated condi-
tions, a single pass through the Plasmer0 reactor, at air
£ow-rates of up to 100 cfm, led to a destruction rate that
was greater than 99 % for the following micro-organisms:
Serratia marcescens, Staphylococus aureus, Aspergillus ni-
ger, Bacillus subtilis. Furthermore, we present two novel
clinical applications of this technology, a mobile air de-
contamination device engineered for patient containment
(mobile biological clean-room) and a room air decontam-
inator. Both utilise these new electrostatic ¢eld reactors.
These new air-treatment devices have a marked advantage
over current ¢ltration units, due to their wide spectrum of
e⁄cacy combined with low maintenance and operating
cost.
FEMSLE Congress 2-6-03
P16^23
INCREASE OF PATATUBERCULOSIS IN WILD AN-
IMAL SPECIES IN STYRIA
A. Deutz(1), J. Spergser(2), P. Wagner(1), Th. Stei-
neck(3), J. Ko«fer(1), R. Rosengarten(2)
(1) Animal Health Service, Department of Veterinary Ad-
ministration, Styrian Provincial Government ; (2) Institut of
Bakteriology, Mykology und Hygiene, University of Veteri-
nary Medicine Vienna; (3) Research Institute of Wildlife
Ecology, University of Veterinary Medicine Vienna
This report deals with the increase in paratuberculosis in
six wild animal species (red deer, roe deer, chamois, mou-
£on, fallow deer, yellow-necked mouse) in spring/summer
2002 in Styria, a province in the south of Austria. Partic-
ularly striking was the occurrence of clinical symptoms
even in young animals. The paper also reports on the ¢rst
extraintestinal detection of M. avium subsp. paratubercu-
losis in wild animals and isolation of the pathogen from
roe deer, chamois and mou£on in the wild in Austria.
Possible causes of the increase in clinical cases, the clinical
picture of paratuberculosis in wild animals, diagnostic
possibilities as well as control and prevention measures
are discussed.
P16^24
CHROMOSOMAL CLASS C L-LACTAMASE IN EN-
TEROBACTER CLOACAE STRAIN IN A PORTU-
GUESE HOSPITAL
T. Conceica‹o(1), N. Faria(1), L. M. Lito(2), J. Melo
Cristino(2), M. J. Salgado(2), M. Pimentel(1), A.
Duarte(1)
(1) Faculty of Pharmacy, Av. Forcas Armadas, 1600-049,
Lisboa, Portugal; (2) Faculty of Medicine, Hospital Santa
Maria, Lisboa, Portugal
Several members of the genus Enterobacter are resistant to
cephalosporins due to the production of a chromosomally
encoded class C L-lactamase. The aim of this work was to
investigate the production of chromosomal L-lactamase
among nosocomial isolates of Enterobacter cloacae. E. clo-
acae FFUL2En was isolated in 1999, from the blood of a
patient hospitalised in a Medicine ward, at Hospital Santa
Maria, Lisboa. A routine antibiogram revealed resistance
to penicillins, aztreonam and broad-spectrum cephalo-
sporins, including cefoxitin, except to imipenem, amino-
glycosides and quinolones. By isoelectrofocusing the son-
icate extracts expressed a pI of 8.5, a presumptive
chromosomal cephalosporinase. Gene detection was per-
formed by PCR with ampC speci¢c primers designed ac-
 1st FEMS Congress / Posters 103^505
cording to the ampC sequences of E. cloacae strains, avail-
able in GenBank : EcloCHE, EcloGC1, EcloMHN,
EcloOUDh, EcloP99, EcloQ908R and EcloGN7471. Nu-
cleotide sequences were analysed using ClustalW and com-
pared with known sequences. The amplicon obtained from
E. cloacae FFUL2En was cloned into pPCR-Script1 Cam
SK(+) and the recombinant plasmid was transformed in
Epicurian Coli0 XL10-Gold0 Kan competent cells (Stra-
tagene). The E. coli harbouring the recombinant plasmid
pTN2 showed resistance to ampicillin, cefoxitine, cefurox-
ime, intermediate susceptibility to amoxicillin alone or in
combination with clavulanate and remained susceptible to
other L-lactams. On the basis of the protein alignments, an
AmpC type enzyme, MIR-2 was identi¢ed showing 98%
homology with the plasmid-mediated class C MIR-1 L-
lactamase produced by K. pneumoniae. This new enzyme
di¡ered from MIR-1 by four amino acid substitutions and
was chromosomally encoded as showed by Southern blot
analysis.
P16^25
AN EPIDEMIOLOGIC STUDY OF INTESTINAL
AMOEBIASIS IN BORDERS OF NORTH EAST IRAN
A. A. Karimi Zarchi, A. Mahmoodzadeh, H. Vatani and Sh.
Shirbazo
Medical Science University of Baghyatollah(a.s.), Iran
In descriptive epidemologic studies, it is very important to
determine rates of disease. In this research, we determined
the prevalence rate (PR) of intestinal amoebiasis (IA) and
related factors such as age and sex in borders of north east
Iran. The study was Cross-sectional. In total, 250 persons
(250 stool samples) were studied in three villages. (100, 88
and 61 persons from villages in the £at, slope and moun-
tain areas, respectively). No acute amoebiasis was seen.
PR of IA was 7.2 percent. Because all positive cysts
were in £at area, the PR of IA in this village was 18
percent. Proportion males infected was 11.9 % and females
22.4 %. Sex di¡erences of infected persons were not sta-
tistically signi¢cant. (P s 0.05) The highest proportion of
infected age group was 5-9 year. Mean of age of infected
persons was 22.7 years (standard deviationœ.3) and in
other was 24.1 years (standard deviation(.1). Di¡erences
in mean of age were not statistically signi¢cant (P s 0.05)
Discussion : Occurrence of Amebiasis is worldwide. The
most infected area in the world was Asia and Africa. PR
of amebiasis in cities of Iran is 6-8 % and in rural areas 5
to over 30 %. Total PR of IA was 7.2 %. All infected
persons were in £at area. Lack of infected person in other
villages for many reasons, especially sanitary water avail-
ability in these areas. That sex di¡erences of infected per-
sons were not statistically signi¢cant is consistent with
other studies. In this study, most cases were seen in the
FEMSLE Congress 2-6-03
497
5-9 year age group, whereas other studies showed that
most cases occur in 20-40 years. Mean age of positive
samples and negative were 22.7 years (SD= 17.3) and
24.1 years (SD(.1), a di¡erence that is not statistically sig-
ni¢cant. (P s 0.05). In conclusion, for control of disease,
water sources and other public ¢nances must be inten-
sively cared for.
P16^26
RIBOTYPING CORYNEBACTERIUM DIPHTHERIAE
ISOLATED IN RUSSIA, 1945 ^ 2002
S. Yu. Kombarova, V. G. Melnikov, O. Yu. Borisova, I. K.
Mazurova
Russian Federal Diphtheria Reference Laboratory, G.N.
Gabrichevsky Institute of Epidemiology and Microbiology,
Admiral Makarov Str 10, Moscow 125 212, Russia
Six hundred and thirty-¢ve toxigenic Corynebacterim diph-
theriae strains from throughout Russia, selected for tem-
poral and geographic diversity, were assayed by ribotyping
and biotyping. The use of the method of ribotyping made
it possible to register 22 C. diphtheriae ribotypes. The
study revealed that the genetic structure of C. diphtheriae
population varied in the dynamics of the epidemic pro-
cess: each epidemic cycle characterized by predominant
spread of epidemic strains of de¢nite biovars and ribo-
types. Thus, C. diphtheriae strains of biovar gravis, ribo-
type ‘Lyon’, dominated in the 40-60 years and C. diphther-
iae starins of biovar mitis, ribotype ‘Otchakov’, dominated
in 80 years. During last epidemic rise of diphtheria mor-
bidity in the 90s C. diphtheriae strains of biovar gravis,
ribotype ‘Sankt-Peterburg/Russia’, dominated among cir-
culating strains. Proportion of these ribotypes began to
increase 3 years before the rise of morbidity. After 1997,
during period of lower incidence, the structure of C. diph-
theriae population, homogeneous in the years of the epi-
demic, started showing a new trend. Between 1997 ^ 2002,
11 other ribotypes were registered : ‘Otchakov’, ‘Buzau’,
‘Cluj’, ‘Lyon’, ‘Vladimir’, ‘new 15’, ‘Vrancea’, ‘Lundi-
nium’, ‘Schawarzenberg’, ‘Pakistan’, ‘Ras-el-Ma’. Ribo-
types ‘Vrancea’, ‘Lundinium’, ‘Schawarzenberg’, ‘Paki-
stan’, ‘Ras-el-Ma’ were registered only in 2001 ^ 2002.
498 1st FEMS Congress / Posters 103^505
P16^27
MONITORING OF ANTIMICROBIAL RESISTANCE
OF NASOPHARYNGEAL STREPTOCOCCUS PNEU-
MONIAE IN CHILDREN FROM ORPHANAGES IN
RUSSIA: RESULTS OF SPARS STUDY
R. S. Kozlov(1), P. C. Appelbaum(2), K. Kosowska(2), O.
I. Kretchikova(1), J. A. Poupard(3) and L. S. Stratchoun-
ski(1)
(1) Institute of Antimicrobial Chemotherapy, Smolensk
State Medical Academy, P.O. Box 57, 28 Krupskaya
Street, Smolensk, 214019, Russian Federation; (2) Hershey
Medical Center, Department of Pathology, Hershey, USA;
(3) GlaxoSmithKline Pharmaceuticals, Collegeville, USA
Children from orphanages and day-care centers of an ad-
ditional risk of colonization by pneumococci, including
antibiotic-resistant isolates. It was previously shown that
monitoring of antimicrobial resistance of nasopharyngeal
strains is an excellent approach for prediction of resistance
in clinical isolates. We are reporting the results of the very
¢rst nation-wide study involving the same group of inves-
tigators sampling children in di¡erent cities using uni¢ed
methodology. Nasopharyngeal swabs were collected from
461 children less than 5 years old in 8 orphanages in 6
cities of European and Asian parts Russia (Moscow,
Saint-Petersburg, Smolensk, Volgograd, Ufa, Khabar-
ovsk) with immediate plating onto 5% Columbia blood
agar with 5 Wg/ml gentamicin. Susceptibility testing to
penicillin G (PEN), amoxicillin (AMO), amoxicillin/clavu-
lanate (AMC), cefotaxime (CTX), erythromycin A (ERY),
azithromycin (AZI), clarithromycin (CLA), clindamycin
(CLI), telithromycin (TEL), cipro£oxacin (CIP), levo£ox-
acin (LEV), gemi£oxacin (GEM), tetracycline (TET) and
co-trimoxazole (SXT) was performed by NCCLS micro-
dilution. Breakpoints were those of NCCLS (2002) except
for TEL (9 0.5; 1; v 2 mg/L), CIP (9 2; 4; v 8 mg/L),
GEM (9 0.25; 0.5; v 1 mg/L). A total of 238 S. pneumo-
niae were isolated with carriage rate varying from 26.4%
to 86.7% between di¡erent orphanages. Rate of non-sus-
ceptibility to PEN, TET and SXT in nasopharyngeal
pneumococci isolated from children from orphanages
was very high, 55.1%, 72.9% and 84.4%, respectively, sub-
stantially exceeding those from clinical isolates. The resis-
tance to macrolides and lincosamides was lower, but still
exceeded 25% for ERY (29.4%), AZI (25.8%), CLA
(26.7%) and CLI (24.6%). No resistance was determined
to AMO, AMC, TEL, LEV and GEM with the latter been
the most active in vitro among all tested antimicrobials.
FEMSLE Congress 2-6-03
P16^28
LONG-TERM SEROEPIDEMIOLOGY OF MYCO-
PLASMA PNEUMONIAE INFECTION IN POLAND
W. Rastawicki, S. KaTuzdewski, M. Jagielski, R. Gierczyn¤ski
National Institute of Hygiene, Chocimska Street 24, 00-791
Warsaw, Poland
In Poland, the epidemiological data on M. pneumoniae
infections have been collected since 1970. Investigations
were performed by 38 laboratories throughout the coun-
try, all using the same complement ¢xation test and the
same sonicated antigen containing the speci¢c M. pneumo-
niae proteins. To the year 2002 diagnostic serological tests
directed against infection with M. pneumoniae were per-
formed in 288.814 persons, mostly children at the pre-
school and school age with clinical symptoms of respira-
tory tract infections. The result of the complement ¢xation
test was accepted as positive when antibody titer was 60 or
higher, or at least a fourfold increase of the titer occurred
during the illness. During performance of the studies ¢ve
signi¢cant epidemics of mycoplasmosis were noted in Po-
land. The ¢rst four occurred regularly, at the 5 years in-
tervals, during the autumn-winter season in 1970/71, 1975/
76, 1980/81, 1985/86. The ¢fth epidemic started with a one
year delay, in 1991 and culminated, depending on the
region of the country, in 1992 or 1993. A high di¡erence
was found in mycoplasmosis incidence between the inter-
epidemic periods and the peaks of the epidemic (respec-
tively 2.1-4.1% and 23.0-38.0%). Since the last atypical
epidemic, in the years 1994 ^ 2001, there were not sharp
increases and decreases in frequency of mycoplasmosis.
The incidence was relatively high and oscillated between
12.0 % and 20.1 %. It seems that the last epidemic have
inaugurated a change from epidemic to endemic occur-
rence of M. pneumoniae in Poland.
P16^29
PREVALENCE OF ANTIBODIES TO YERSINIA EN-
TEROCOLITICA AND YERSINIA PSEUDOTUBER-
CULOSIS IN INFECTED AND NON-INFECTED SUB-
JECTS IN POLAND
W. Rastawicki, M. Jagielski, R. Gierczyn¤ski
National Institute of Hygiene, Chocimska Street 24, 00-791
Warsaw, Poland
Infections caused by Yersinia enterocolitica and Yersinia
pseudotuberculosis are common enteric diseases of humans
and animals. Enterocolitis, abdominal pain and arthritis
are the most common clinical manifestations. During the
period 1998-2002, 1744 sera from 1393 patients suspected
 1st FEMS Congress / Posters 103^505
for yersiniosis in the clinical investigations were tested by
enzyme linked immunosorbent assay (ELISA) in IgA, IgG
and IgM class of immunoglobulins. Additionally, 100 se-
rum samples from adult blood donors and 100 serum
samples from clinically healthy school children were
tested. The lipopolisacharyde antigens were prepared
from Y. enterocolitica serotypes O3, O5,27, O8, O9 and
Y. pseudotuberculosis I/III according to Boivina’s method.
The frequency of detection antibodies to particular sero-
types of Yersinia in serum samples from infected and non-
infected subjects is di¡erent. In the case of patients sus-
pected in clinical examination for yersiniosis, most fre-
quently, in all immunoglobulin classes, the positive results
were obtained with Y. enterocolitica O3 ^ 9,5 % in IgA,
12,8 % in IgG and 7,3 % in the IgM class. As much as 8.5
% of positive results were found in the IgA, 6.2 % in the
IgG and 4.0 % in the IgM with the antigen Y. pseudotu-
berculosis I. In serum samples obtained from clinically
healthy persons antibodies against all serotypes of Yersinia
were detectable signi¢cantly rarely ( p 6 0.001 ). Immu-
noglobulins IgA and IgG more frequently were found in
sera of adult persons and immunoglobulins IgM in sera of
children.
P16^30
EVALUATION OF THE PATHOGENICITY OF ENVI-
RONMENTAL KLEBSIELLA PNEUMONIAE ISO-
LATES
C. Struve and K. A. Krogfelt
Department of Gastrointestinal Infections, Statens Serum
Institut, Copenhagen, Denmark
Klebsiella pneumoniae is an important opportunistic patho-
gen accounting for up to 10% of all nosocomial bacterial
infections. K. pneumoniae infections can occur in nearly
any body site, however, urinary tract infections (UTI)
and infections of the respiratory tract predominate. Epi-
demiological studies have shown that K. pneumoniae in-
fections are frequently preceded by gastrointestinal (GI)
infection and the gastrointestinal tract is believed to be
the most important reservoir for transmission of the bac-
teria. In contrast to many other bacterial pathogens, K.
pneumoniae is ubiquitous in nature. The non-clinical hab-
itats include the mucosal surfaces of humans and animals
and environmental sources such as vegetation, soil and
surface waters. Several studies have described Klebsiella
isolates of environmental origin to be nearly identical to
clinical isolates with respect to several phenotypic proper-
ties. However, the pathogenic potential of environmental
K. pneumoniae isolates is unknown. We have evaluated the
pathogenicity of K. pneumoniae strains of environmental
and clinical origin directly by animal models of UTI and
GI colonization. Furthermore, the ability to adhere to and
FEMSLE Congress 2-6-03
499
invade human epithelial cells was examined. Although
strain to strain di¡erences were observed in the individual
infections models, overall strains of environmental origin
were found to be as virulent as clinical strains. Our results
hereby indicate that environmental strains of K. pneumo-
niae may constitute an important reservoir of potentially
pathogenic bacteria.
P16^31
A NEW APPROACH TO MICROBIOLOGICAL IN-
VESTIGATIONS OF SURFACE WATER QUALITY
IN LATVIA : MONITORING RESULTS 2002
A. Zandmane, S. Poikane, A. Grantins
Latvian Environment Agency, Jurmala, Latvia
The national environment monitoring program of surface
water quality in Latvia has until now mainly been based
on hydrological factors and results of physical, chemical
and biological analyses. Microbial parameters have not
been the focus of attention. Over the last decade, micro-
biological investigations have been carried out by the Lat-
vian Environmental Agency as small pilot projects in-
tended to solve speci¢c problems for public water use.
Microbiological parameters have been examined in coastal
recreational waters of the Baltic Sea and Gulf of Riga and
in numerous rivers and lakes (1997-1999). Special exami-
nations of public bathing water quality were carried out to
obtain the award of European Blue Flag. The microbio-
logical quality has been evaluated in compliance with both
Latvian and international normative documents and guid-
ance. Digital maps have been produced of microbiological
water quality. Monitoring of microbial parameters as in-
dicators of organic pollution in the surface waters are
recommended in International standard regulations
(drinking and bathing waters) and Eurowaternet guide-
lines that are binding for Latvia. To meet the EU require-
ments and provide the national standards and regulations
in the context of local environmental, social, economic
and cultural condition several activities were carried out
in the year 2002. National and international legislative
acts and guidelines concerning monitoring of microbiolog-
ical parameters in surface waters have been revised.
Screening the total station population of rivers and lakes
forms the new basic network for surface waters microbio-
logical monitoring. The microbial parameters (coliforms,
E. coli, intestinal enterococci, salmonella) as primary in-
dicators of organic (faecal) pollution have been selected
according to recommendations (Eurowaternet) and the di-
rect count of bacteria (epi£uorescent microscopy tech-
nique) and heterotrophic plate count (microbial colony
count, saprophytic bacteria) will be recommended for in-
clusion if necessary. International standard methods (ISO,
500 1st FEMS Congress / Posters 103^505
EN, APHA) must be used for the water quality testing in
laboratory practice.
P16^32
THE ENHANCEMENT OF PLANT GROWTH BY
RHIZOSPHERE BACTERIA IN SEMI ARID REGION
OF UZBEKISTAN
D. Juraeva, D. Egamberdiyeva, D. Qarshieva and K. Dav-
ranov
Institute of Microbiology, A. Qadiry str. 7 B, 700128 Tash-
kent, Uzbekistan
Agricultural mismanagement such as the inappropriate ap-
plication of mineral fertilizers and pesticides has resulted
in pollution and salinisation of agricultural lands and
water resources in developing countries central Asia. The
use of the non-hazardous biological methods in such re-
gions to increase plant production is an important ap-
proach to help sustainable development. In particular,
plant growth promoting bacteria (PGPR) have been re-
ported to be the key elements for plant establishment
and their use in agriculture can favour a reduction in
agro-chemical use and support ecological crop production.
The objectives of this study were to isolate rhizosphere
bacteria from di¡erent agricultural crops and to analyse
their plant growth promoting e¡ects on cotton, wheat,
maize and soybean in semi arid region of Uzbekistan.
The investigations were carried out in pot experiments
with calcareous Calcisol soil in Uzbekistan. After inocu-
lation with bacterial strains the root and shoot growth of
cotton, wheat, maize and soybean increased. A positive
e¡ect on yield of soybean in ¢eld experiments was ob-
tained after inoculation with Ps. radiobacter, P. putrifa-
ciens and Rhizobium simplex. Besides, growth-promoting
bacteria produced the phytohormon auxin, capable of ni-
trogenase activity. They are salt tolerant and temperature
resistance. In summary, the ¢nal results of our experi-
ments show that plant growth promoting bacteria can
play an essential role in helping plants establish and
grow in nutrient de¢cient, salinated soils, semi-arid re-
gions.
FEMSLE Congress 2-6-03
P16^33
DIGGING ACTIVITY RODENTS AS A FACTOR OF
INFLUENCE ON SOIL ‘‘BREATH’’EDAFOTOP
STEPPE WOODS OF UKRAINE
S. M. Kirienko
Department of Zoology, The Dnepropetrovsk National Uni-
versity, Dnepropetrovsk, Ukraine
The purpose of our work was not only the characteristic
of how heavy various metals in£uence a metabolism of
ground. But mainly it was necessary to estimate a role
of various kinds environment of forming in£uences mam-
mal on intensity of process of soil ‘‘breath’’ in conditions
of pollution of ground heavy metals. During the experi-
ment put by us on Prisamarsky a hospital the following
results were received. After 1 month of an exposition in
conditions of pollution of ground connections of cadmium
in burrow rodents observe appreciable increase of intensity
of soil ‘‘breath’’. Especially it is expressed on sites with a
weak and average degree of pollution Cd1 and Cd5 (in
2,36-3,41 times). On ZY5ecTByy the analysis of alloca-
tion by ground SO2 has shown three months, that in con-
ditions of pollution cadmium observes appreciable de-
crease (reduction) of a level of soil breath at weak and
average pollution (in 2,74-3,86 times). Some increase of
intensity of ‘‘breath’’ is marked also at pollution Cd10.
After of 12 months after the beginning of experiment,
from the site polluted with cadmium was begun with pro-
cess of restoration of a former level of a soil metabolism
that was accompanied by increase of size of ‘‘breath’’. In
relation to the control only parameter CO2 on site Cd1
appears above (in 1,22 times). Variants of concentration
Cd5 and Cd10 appear below control values in 1,14 and
2,05 times accordingly. Thus, digging activity rodents in
conditions of pollution edafotop heavy metals plays part
rodents of restoration of functions of ground. Especially it
is shown in 1-3 months at low and average levels of pol-
lution cadmium (increase of intensity of soil ‘‘breath’’ in
2,41-3,41 times.). At high levels of pollution the role bur-
row rodents appears insu⁄cient day of preservation of soil
functions at a former level.
 1st FEMS Congress / Posters 103^505
P16^34
BIOREMEDIATION OF SOIL MICROORGANISMS
IN FORESTS ECOSYSTEMS UNDER CONDITIONS
OF SOIL POLLUTION BY Cd
L. V. Grachova and A. Ye. Pakhomov
Dniepropetrovsk National University, Dept. Zoology and
Ecology, 13, Nauchny Lane, 49050, Dniepropetrovsk, Uk-
raine
We carried out experimental studying in£uence of mam-
mals digging and excretory activity on restoration of soil
microorganisms’ function in conditions of soil pollution
by Cd. The results show that soil pollution by Cd in con-
centration 100My/m2 reduces total of soil microorganisms
on 35,8 % in one month of in£uence of pollution and on
37,6 % in three months. The quantity of saprophytic bac-
teria is reduced on 16,1 % ^ 13 %, oligotrophic bacteria ^
on 33,2 % ^ 30,7 %, actinomycetes ^ on 30,2 % ^ 56,3 %
accordingly. The excretory activity of mammals promotes
augmentation of total of soil microorganisms by 41,8 % ^
on 33,2 %, on sites polluted by Cd. The quantity of sap-
rophytic bacteria increase on 23,6% ^ 71,9 %, oligotrophic
bacteria ^ on 24,3 % ^ 13,4 %, actinomycetes ^ on 20,7 %
^ 73,5 % accordingly. The digging activity of mammals
promotes augmentation of total of soil microorganisms
by 58,7 % ^ 15,2 %. The quantity of saprophytic bacteria
increase on 20,4%- 38,1 %, oligotrophic bacteria ^ on 24,7
% ^ 38,4 %, actinomycetes ^ on 38,7 % ^ 65,5 % accord-
ingly. The soil microorganisms directly participate in pro-
cesses bioremediation soils by means of accumulation of
heavy metals inside a cell or on its surface. Thus, improv-
ing physical and chemical properties of the soil, the dig-
ging and excretory activity of mammals renders positive
in£uence on ecological conditions of existence of micro-
organisms.
P16^35
YEAST DIVERSITY IN SOILS FROM THE REGIONS
WITH DIFFERENT CLIMATIC CONDITIONS
V. S. Podgorsky, S. S. Nagornaya, T. V. Babich
D.K. Zabolotny Institute of Microbiology and Virology,
National Academy of Science of Ukraine, Kiev, 03143, Uk-
raine
In connection with the problem of preservation of biodi-
versity has arisen the necessity of soil microorganisms in-
ventory making as well as yeasts. During 2000-2002 the
yeasts have been investigated in the soils of di¡erent cli-
matic zones of Ukraine: in regions with subtropical cli-
mate (Crimea), intercontinental climate of moderate
FEMSLE Congress 2-6-03
501
widths (Kiev, Kirovograd regions) and also in regions
which are under the constant in£uence of low tempera-
tures, and soils are formed on the permafrost (Yakut,
Antarctica). It has been determined di¡erences of yeasts
in quantitative and species composition of investigated
regions. The considerable number of yeasts is contained
in the Ukraine black earth ^ the middle zone of Ukraine ^
105-107 and more cells/g. Their species composition is
rather di¡erent. The species Williopsis californica, Debar-
yomyces hansenii, D.occidentalis, Cryptococcus albidus, C.
laurentii were predominated (in the order of their de-
crease). Kloeckera apiculata, Pichia anomala, P. membra-
naefaciens, Torulaspora delbrueckii, Saccharomyces cerevi-
siae, S. unisporus were rarely isolated. The Rhodotorula
glutinis, R. mucilaginosa occurred seldom. In the reddish-
brown alkaline Crimea soil the composition of yeasts was
less (103-105 cells/g). Here basidiomycetous species Cryp-
tococcus albidus, C. humicolus, C. laurentii, ascomycetous
yeasts Debaryomyces pseudopolymorphus were predomi-
nate. It often occured also the D. polymorphus, Trichospor-
on sp. The quantitative and species composition of yeasts
of cold regions is poor. The only basidiomycetous species
have been revealed ^ the representatives of coloured forms
of the species Cryptococcus, Filobasidium, Rhodotorula,
Leucosporidium. Among them there were yeasts with the
maximum temperature of 21‡C and less. These yeasts
didn’t ferment sugar, most of them assimilate nitrates,
formed a capsule. All these properties are characterised
for microorganisms of cold regions. Thus, the yeasts
form the peculiar associations, which are characteristic
for every of investigated regions.
P16^36
STANDARDIZING MEDIUM TO CULTURE A NI-
TROGEN FIXER AND A PHOSPHATE SOLUBIL-
IZER FOR BIO INOCULANT PRODUCTION
S. Poonguzhali and M. Thangaraju
Department of Agricultural Microbiology, Tamil Nadu
Agricultural University, Coimbatore 641 003, India
Crop productivity is usually limited by nitrogen and phos-
phorus availability in soils and it is estimated that 139
million tonnes of nitrogen is being added on to the soil
annually through biological nitrogen ¢xation. Azospiril-
lum, an associative symbiotic nitrogen ¢xing bacterium,
occurs with the roots of almost all plants of agricultural
importance. Yield responses to bacterization with Azospir-
illum inoculant was almost equivalent to that attainable by
application of 15-20 kg N ha-1. Similarly, e⁄cient and
economic use of phosphate fertilizers could be achieved
by using phosphate-solubilizing microorganisms (PSMs)
in legumes, cereals and other crops. Though nitrogen
¢xers and phosphate solubilizing bacteria are recom-
502 1st FEMS Congress / Posters 103^505
mended to farmers, presently they are being produced and
supplied as individual inoculants. Developing a technology
to culture the two organisms in a single medium and pro-
duce a common inoculant having a nitrogen ¢xer and a
phosphate solubilizer will certainly help promote biofertil-
izer technology. Experiments were carried out to standard-
ize a media in which both nitrogen ¢xer (Azospirillum lip-
oferum, Az 204) and phosphate solubilizing bacteria
(Bacillus megaterium, Pb1) could grow well. Glucose pep-
tone media with 0.5% yeast extract supported profuse
growth of both bacteria. It was found that the population
of both organisms was higher in the glucose peptone me-
dium compared to the population of Azospirillum in N-
free malic acid medium or B. megaterium in nutrient me-
dium. To have su⁄cient load of both organisms, B. mega-
terium was inoculated at di¡erent intervals after the inoc-
ulation of Azospirillum. Maximum population of both
organisms was obtained at 72h when B. megaterium was
inoculated after 48h of Azospirillum inoculation. The abil-
ity to grow a nitrogen ¢xer and a phosphate solubilizer
together in a common medium minimizes the cost of pro-
duction and saves considerable time. In addition, the han-
dling, distribution and application of the inoculant will be
much easier than for the individual inoculants.
P16^37
ISOLATION AND CHARACTERIZATION OF A BY-
PHENIL-DEGRADING BACTERIAL STRAIN IN THE
VENICE LAGOON
R. Marcon(1), F. Zecchini(2), M. Pepi(2) and F. Bal-
di(1,2)
(1) Ca’ Foscari University, Venice, Italy ; (2) InterUniver-
sity National Consortium ‘‘Chemistry for the Environment’’,
Marghera (Venice), Italy
Due to its naturalistic, artistic, historic, and economic im-
portance, at present days many research projects deal with
the environmental status of the Venice lagoon and its ca-
pabilities of self-depuration. Few literature data are avail-
able about aromatic hydrocarbon-degrading bacteria in
marine and lagoon environments. So our laboratory of
environmental microbiology is active in the ¢eld of isola-
tion and characterization of such bacteria in the Venice
lagoon. Several strains were isolated and SB1 is among the
best characterized ones. It is capable of using byphenil as
sole carbon and energy source. The metabolism of SB1
and similar bacteria may play a key role in the biologic
degradation of the PCBs present in high levels in the La-
goon, possibly participating to the aerobic phase of deg-
radation following the anaerobic dechlorination. SB1 is a
gram-negative rod, cathalase and oxydase positive, identi-
¢ed as Pseudomonas chloritidismutans by sequencing of the
rRNA-16S gene. Its metabolic pro¢le has been described
FEMSLE Congress 2-6-03
using the Biolog technique (a 96-well microtiter plate con-
taining 95 carbon sources and a redox indicator). Growth
pro¢les have been analyzed using a mineral medium
amended with byphenil and with a rich organic medium.
Investigated parameters were oxygen consumption (Oxy-
top, automated respirometric test), direct cell counts at
microscope, and optical density measured at 600 nm.
This led to assessment and comparison of doubling time
and oxygen consumption rate in the exponential phase of
growth in the two di¡erent media. Studies about the deg-
radation pathway of byphenil by SB1 are in progress.
P16^38
PLASMIDS OF A SIMAZINE-UTILIZING BACTE-
RIUM ISOLATED FROM MAIZE RHIZOSPHERE
D. P. Bazhanov, C. I. Zabenkova
Institute of Genetics and Cytology, NASB, Akademiche-
skaya St. 27, 200072, Minsk, Belarus
An obligately aerobic Gram-negative bacterium B601,
mineralizing simazine, was isolated from maize rhizo-
sphere. The bacterium carried two plasmids of approx.
210 and 60 kb in size. Southern blot analysis indicated
that genes homologous to atrazine utilization genes atzA,
-B and -C from Pseudomonas sp. ADP were located on the
210 kb plasmid, while 60 kb plasmid had regions homol-
ogous to type-4 ¢mbria subunit gene pilA from P. putida
WCS358. Spontaneous elimination of the capacity to uti-
lize simazine was observed during storage and growth of
the strain with easily utilized sources of nitrogen. It was
accompanied by deletions of the simazine degradation
gene cluster from the large plasmid. The capacity to utilize
simazine was transferred by conjugation from the wild
type strain to Smz^Rifr derivatives with the frequency of
10-5 per donor cell. Smz+ Rifr transconjugants had new
large plasmids, bearing both the genes for simazine utiliza-
tion and a region homologous to pilA. Approx. 50% of
transconjugants kept the deleted plasmid of the recipient,
while 60 kb plasmid was eliminated. It meant that genes
for simazine degradation were transferred and inherited in
the transconjugants on the replicon of 60 kb plasmid. So,
210 kb plasmid of the bacterium B601 was responsible for
simazine utilization, while 60 kb plasmid was transmissible
and mobilized simazine degradation genes.
 1st FEMS Congress / Posters 103^505
P16^39
ISOLATION OF LEAD-REMOVING MICROORGAN-
ISMS AND CHARACTERIZATION OF LEAD UP-
TAKE BY XANTHOBACTER MCD-58B
M. Dadashipour(1), S. Chinikar(2), S. Javadian(1) and F.
Malekzadeh(3)
(1) Biochemistry Department of Pasteur Institute of Iran,
Tehran 13164, Iran; (2) Biochemistry Department of Pas-
teur Institute of Iran; (3) Microbiology Division, Faculty
of Science, Tehran University, Tehran, Iran
In a screening experiment of 144 microbial strains isolated
from di¡erent environments, 7 isolates were selected and
among them, Xanthobacter MCD 58-B exhibited great po-
tential in Pb uptake (Isolated from soil in Shahre-ray gas
station, Tehran). Xanthobacter MCD 58-B is a Gram neg-
ative, polymorph, and slime producing bacterium in Glu-
cose Mineral Salts plus Yeast Extract medium. It’s poten-
tial in Pb removing from synthetic metal solution and
bioremediation was assessed by an Atomic Absorption
Spectrometer. In the cells harvested from nutrient broth,
the rate of Pb removing was 25 mg/g dry weight. The
bacteria harvested from GMS plus YE, Pb uptake ca-
pacity was greater (346 mg/g dry weight). Even at pH 2-
2.5 the Pb uptake was great. The bacterium was able to
uptake to 97% of the Pb from the initial solution (500
ppm). MIC and MBC values of Pb against the isolate
was equal (10 mM). Xanthobacter MCD 58-B is a fast
growing bacterium and the maximum growth rate ob-
tained at 8 hrs and the largest proportion of Pb uptake
was by it’s exopolymer slime when the bacterium was
grown in GMS plus YE medium. The maximum uptake
was recorded at ¢rst 45 min at pH 5.5 ( 346 mg Pb/g dry
weight ). The isolate was very e⁄cient in accumulation of
Pb from solution (97%). Morphological, cultural and bio-
chemical characteristics of the strain, placed it in genus
Xanthobacter. Further studies of the isolate are necessary
for determining the species and the role of exopolymer in
metal uptake.
FEMSLE Congress 2-6-03
503
P16^40
QUANTIFICATION OF ATZ GENE EXPRESSION IN
CHELATOBACTER HEINTZII AND PSEUDOMONAS
SP. ADP TWO ATRAZINE-DEGRADING STRAINS
ISOLATED FROM ADAPTED SOIL
M. Devers, G. Soulas and F. Martin-Laurent
UMR 111, INRA/Universite¤ de Bourgogne, INRA/CMSE,
Laboratoire de Microbiologie et de Ge¤ochimie des Sols, 17
rue Sully, 21065 DIJON cedex, France
Atrazine an herbicide mainly used on crop is degraded by
speci¢c telluric microbes. Atrazine-degrading (atz) genes
are plasmid borne, highly conserved and widely dispersed.
They have been found in several atrazine-degrading strains
belonging to Gram+ and Gram- bacteria collected all over
the world. It seems that atrazine-degrading rate depends
on bacterial strain suggesting that atrazine-degrading
pathway could di¡erentially be regulated upon the strain
considered. The aim of our work was to determine the
level of expression of atrazine-degrading genes in an K-
and Q-proteobacteria isolated from adapted soils Chelato-
bacter heintzii and Pseudomonas sp. strain ADP, respec-
tively. We developed RT-qPCR asssay, based on the use
of real-time PCR, in order to quantify atzABCDEF
mRNAs in Pseudomonas sp. strain ADP and atzABC
mRNAs in Chelatobacter heintzii. As a result, we showed
that atz genes are expressed at a basal level in Pseudomo-
nas sp. strain ADP con¢rming data issued from measure-
ment of atrazine-degrading activity indicating that its deg-
radation starts immediately after its addition to the the
medium. We also reported that atz gene expression in-
creased transitory in response to atrazine treatment. This
increase was observed only when no more atrazine was
remaining in the medium suggesting atrazine did not di-
rectly regulate the expression of atz genes. On the con-
trary, only atzA is expressed at a basal level in C. heintzii.
In addition, atzA and atzB gene expression was similarly
and signi¢cantly increased in C. heintzii response to atra-
zine treatment. However, atzC was not expressed at all in
C. heintzii neither treated nor non-treated with atrazine.
Therefore, we showed here that although atz genes are
expressed at a basal level their expression is transitory
up-regulated in response to atrazine treatment. In addi-
tion, we reported that almost 100% similar atz genes
hosted in di¡erent microbes are expressed di¡erently.
504 1st FEMS Congress / Posters 103^505
P16^41
DEVELOPMENT OF SULFATE-REDUCING AND
OIL-OXIDIZING BACTERIA IN OIL-POLLUTED
SOILS
I. Mammedova and A. Talibli
Lab. of Lithotrophic Microorganisms, Institute of Microbi-
ology of National Academy of Sciences (NAS), Patamdar
Sh. 40, Baku 370073 Azerbaijan
Investigation of microorganisms, which are resistant to the
toxic e¡ect of petroleum and are able to develop in con-
ditions of destroyed aeration regime has very important
scienti¢c and practical meaning, particularly in bioremedi-
ation of oil-polluted soils. A speci¢c microcenosis, in
which oil-oxidizing bacteria as well as represents of anaer-
obic and microaerophilic group of microorganisms and
particularly sulfate-reducing bacteria are dominant, gener-
ates in oil-polluted soils. We have investigated the devel-
opment of the oil-oxidizing and sulfate-reducing bacteria
strains under their collective and separate cultivation in
the arti¢cial model of the soil ecosystem, in which the
sterile soil was polluted with the sterile petroleum (10%).
Our results have demonstrated that (i) the cultures of the
sulfate-reducing bacteria are able to grow in 10% oil-pol-
luted soil and accumulate hydrosulfur in the environment
during their metabolism. (ii) Development and distribu-
tion of sulfate-reducing bacteria in oil-polluted soils are
limited due to accessible organic substrate. (iii) When the
oil-oxidizing microorganisms have utilized petroleum as
the only energetic source during their metabolism the in-
termediate products of biochemical oxidation of hydrocar-
bons are slowly accumulated in the soil in a period of year,
which are further involved in metabolism by sulfate-reduc-
ing bacteria. Based on our results, we can testify, that
there is a microcomplex forms in the soil, which takes
place in the autoremediation process of the soil, in which
the oil-oxidizing bacteria and microorganisms those are
resistant to the toxic e¡ect of the oil play the main role.
FEMSLE Congress 2-6-03
P16^42
GENETIC CHARACTERIZATION OF ATRAZINE-
DEGRADING BACTERIAL CONSORTIA ISOLATED
FROM MAIZE RHIZOSPHERE
F. Martin-Laurent(1), B. Barres(1), I. Wagschal(1), S.
Piutti(2), M. Devers(1), L. Philippot(1) and G. Soulas(1)
(1) UMR 111, INRA/Universite¤ de Bourgogne, INRA/
CMSE, Laboratoire de Microbiologie et de Ge¤ochimie des
Sols, 17 rue Sully, 21065 DIJON cedex, France; (2) UMR
INPL-ENSAIA-INRA, Agronomie et Environnement, 2
avenue de la Fore“t de Haye, BP 172, F-54500 Vandoeu-
vre-les-Nancy, France
Microbial communities degrading atrazine, a herbicide
mainly used on corn, have been studied in bulk or maize
rhizosphere soils treated or not with this herbicide. Sixty-
three atrazine-degrading consortia have been isolated.
Each consortium was physiologically and genetically char-
acterized via (i) an estimation of atrazine-degrading activ-
ity by HPLC, (ii) the determination of atrazine-degrading
genes present (atzABCDEF and trzDN) by both PCR and
colony hybridization, (iii) the phylogenic identi¢cation of
bacterial strains by amplifying, cloning and partial se-
quencing of the 16S rDNA gene. We showed that, based
on 16S rDNA RFLP analysis, atrazine-degrading consor-
tia could be sorted phylogenetically in 3 groups according
to their provenance (i.e. bulk or rhizosphere soil or inter-
mediary group). It suggests that maize plants in£uence the
structure of telluric atrazine-degrading communities. In
addition, analysis of atz gene presence showed that rhizo-
spheric consortia possessed a more complete and function-
al atrazine-degrading pathway than that of bulk soil.
These results indicate that maize rhizosphere may favour
atz and trz gene £uxes in adapted soil micro£ora. Indeed,
based on the analysis of 630 clones issued from libraries of
partial 16S rDNA generated for each consortia, 64 OTU
were identi¢ed. The sequencing of at least one clone of
each OTU showed that bacterial strains entering in atra-
zine-degrading bacterial consortia belong to K-, L-, Q-pro-
teobacteria, CFB group and actinomycetes. Rarefaction
curves as well as Shanon and Weaver index con¢rm data
obtained from community analysis. To conclude, our
work showed that the maize rhizosphere not only acts
on the structure of atrazine-degrading communities but
also probably promotes atrazine-degrading gene £uxes.
Further studies will aim to study atz gene £uxes in the
maize rhizosphere and to identify key mechanisms in-
volved in this phenomenon.
 1st FEMS Congress / Posters 103^505 505

P16^43

BIOREMEDIATION OF DISPERSE DYE AND DYING

EFFLUENTS BY MIXED CULTURE OF NEW ISO-
LATES OF BACILLUS AND STREPTOMYCES SP

A. A. Pourbabaee(1), M. N. Sarbolouki(2), F. Naja¢i(3)

(1) Department of Biology, (2) Institute of Biochemistry &

Biophysics and(3) Department of chemistry, Faculty Of
Science, University of Tehran, Tehran, Iran

We investigated the bioremediation of disperse dye (Ter-

asile Black) and dying wastewater containing this dye. In
between our isolates only two isolates were active in de-
colorization. Taxonomic identi¢cation including morpho-
logical and biochemical characterization indicated that
these isolates were strains of Bacillus and Streptomyces
that aerobically decolorized wastewater and Terasile black
dye respectively. Decolorization of Terasile Black dye oc-
curred in presence of just Streptomyces after 10 days of
incubation in 30:C,but no decolorization observed on
wastewater. Chemical analysis and growth measurement
shows that aerobic condition leads to the complete decom-
position of dye and increase of cell biomass. Decoloriza-
tion of wastewater occurred by Bacillus sp in presence of
additional carbon source. A mixed culture of Streptomyces
and Bacillus leads to removal of need to additional carbon
source. Decolorization of dying wastewater occurred after
15 days incubation in 30:C. Our results have suggested the
potential of these two strains in bioremediation of dye
contaminated water and wastewater sinergically.

FEMSLE Congress 2-6-03