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Introduction 1

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INTRODUCTION

Food microbiology is the study of the microorganisms which


inhabit, create or contaminate food. Of major importance is the
study of microorganisms causing food spoilage. However "good"
bacteria such as probiotics are becoming increasingly important
in food science. In addition, microorganisms are vital for the
manufacture of cheese, yoghurt, other fermented foods, bread, beer
and wine.

FOOD SAFETY
Food safety is a major focus of food microbiology. Pathogenic
bacteria, viruses and toxins produced by microorganisms are all
possible contaminants of food. However, microorganisms and their
products can also be used to combat these pathogenic microbes.
Probiotic bacteria, including those which produce bacteriocins
can kill and inhibit pathogens. Alternatively, purified bacteriocins
such as nisin can be added directly to food products. Finally,
bacteriophage, viruses which only infect bacteria, can be used to
kill bacterial pathogens. Thorough preparation of food, including
proper cooking will kill most bacteria and viruses. However, toxins
produced by contaminants may not be heat-labile, and some will
not be eliminated by cooking.

FERMENTATION
Fermentation is one way microorganisms can change a food.
Yeast, especially S. cerevisiae, is used to leaven bread, brew beer
and make wine. Certain bacteria, including lactic acid bacteria,
are used to make yogurt, cheese, hot sauce, pickles and dishes
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such as kimchi. A common effect of these fermentations is that the disease can be much more severe and prolonged in
food product is less hospitable to other microorganisms, including immunocompromissed individuals.
pathogens and spoilage-causing microorganisms, thus extending
the food's shelf-life. Mycotoxins
Some cheese varieties also require mold microorganisms to Molds produce mycotoxins, which are secondary metabolites
ripen and develop their characteristic flavors. that can cause acute or chronic diseases in humans when ingested
from contaminated foods. Potential diseases include cancers and
Foodborne Pathogens tumors in different organs (heart, liver, kidney, nerves),
Foodborne pathogens are the leading causes of illness and gastrointestinal disturbances, alteration of the immune system,
death in less developed countries killing approximately 1.8 million and reproductive problems. Species of Aspergillus, Fusarium,
people annually. In developed countries foodborne pathogens are Penicillium, and Claviceps grow in agricultural commodities or
responsible for millions of cases of infectious gastrointestinal foods and produce the mycotoxins such as aflatoxins,
diseases each year, costing billions of dollars in medical care and deoxynivalenol, ochratoxin A, fumonisins, ergot alkaloids, T-2
lost productivity. New foodborne pathogens and foodborne toxin, and zearalenone and other minor mycotoxins such as
diseases are likely to emerge driven by factors such as pathogen cyclopiazonic acid and patulin. Mycotoxins occur mainly in cereal
evolution, changes in agricultural and food manufacturing grains (barley, maize, rye, wheat), coffee, dairy products, fruits,
practices, and changes to the human host status. There are growing nuts and spices. Control of mycotoxins in foods has focused on
concerns that terrorists could use pathogens to contaminate food minimizing mycotoxin production in the field, during storage or
and water supplies in attempts to incapacitate thousands of people destruction once produced. Monitoring foods for mycotoxins is
and disrupt economic growth. important to manage strategies such as regulations and guidelines,
which are used by 77 countries, and for developing exposure
Enteric Viruses assessments essential for accurate risk characterization.
Food and waterborne viruses contribute to a substantial
Yersinia Enterocolitica
number of illnesses throughout the world. Among those most
commonly known are hepatitis A virus, rotavirus, astrovirus, enteric Yersinia enterocolitica includes pathogens and environmental
adenovirus, hepatitis E virus, and the human caliciviruses strains that are ubiquitous in terrestrial and fresh water ecosystems.
consisting of the noroviruses and the Sapporo viruses. This diverse Evidence from large outbreaks of yersiniosis and from
group are transmitted by the fecal-oral route, often by ingestion of epidemiological studies of sporadic cases has shown that Y.
contaminated food and water. enterocolitica is a foodborne pathogen. Pork is often implicated as
the source of infection. The pig is the only animal consumed by
Protozoan Parasites man that regularly harbours pathogenic Y. enterocolitica. An
Protozoan parasites associated with food and water can cause important property of the bacterium is its ability to multiply at
illness in humans. Although parasites are more commonly found temperatures near to 0°C, and therefore in many chilled foods. The
in developing countries, developed countries have also experienced pathogenic serovars (mainly O:3, O:5,27, O:8 and O:9) show
several foodborne outbreaks. Contaminants may be inadvertently different geographical distribution. However, the appearance of
introduced to the foods by inadequate handling practices, either strains of serovars O:3 and O:9 in Europe, Japan in the 1970s, and
on the farm or during processing of foods. Protozoan parasites in North America by the end of the 1980s, is an example of a
can be found worldwide, either infecting wild animals or in water global pandemic. There is a possible risk of reactive arthritis
and contaminating crops grown for human consumption. The following infection with Y. enterocolitica.
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Vibrio Campylobacter
Vibrio species are prevalent in estuarine and marine Campylobacter spp., primarily C. jejuni subsp. jejuni is one of
environments and seven species can cause foodborne infections the major causes of bacterial gastroenteritis in the U.S. and
associated with seafood. Vibrio cholerae O1 and O139 serovtypes worldwide. Campylobacter infection is primarily a foodborne
produce cholera toxin and are agents of cholera. However, fecal- illness, usually without complications; however, serious sequelae
oral route infections in the terrestrial environment are responsible such as Guillain-Barre Syndrome occur in a small subset of infected
for epidemic cholera. V. cholerae non-O1/O139 strains may cause patients. Detection of C. jejuni in clinical samples is readily
gastroenteritis through production of known toxins or unknown accomplished by culture and non-culture methods.
mechanism.
Listeria Monocytogenes
Vibrio parahaemolytitucs strains capable of producing
Listeria monocytogenes is Gram-positive foodborne bacterial
thermostable direct hemolysin (TDH) and/or TDH-related
pathogen and the causative agent of human listeriosis. Listeriae
hemolysin are most important cause of gastroenteritis associated
are acquired primarily through the consumption of contaminated
with seafood consumption. Vibrio vulnificus is responsible for foods including soft cheese, raw milk, deli salads, and ready-to-
seafoodborne primary septicemia and its infectivity depends eat foods such as luncheon meats and frankfurters. Although L.
primarily on the risk factors of the host. V. vulnificus infection has monocytogenes infection is usually limited to individuals that are
the highest case fatality rate (50%) of any foodborne pathogen. immunocompromised, the high mortality rate associated with
Four other species (Vibrio mimicus, Vibrio hollisae, Vibrio fluvialis, human listeriosis makes L. monocytogenes the leading cause of
and Vibrio furnissii) can cause gastroenteritis. Some strains of death amongst foodborne bacterial pathogens. As a result,
these species produce known toxins but the pathogenic mechanism tremendous effort has been made at developing methods for the
is largely not understood. The ecology of and detection and control isolation, detection and control of L. monocytogenes in foods.
methods for all seafoodborne Vibrio pathogens are essentially
similar. Salmonella
Salmonella serotypes continue to be a prominent threat to
Staphylococcus Aureus
food safety worldwide. Infections are commonly acquired by animal
Staphylococcus aureus is a common cause of bacterial to human transmission though consumption of undercooked food
foodborne disease worldwide. Symptoms include vomiting and products derived from livestock or domestic fowl. The second half
diarrhea that occur shortly after ingestion of S. aureus- of the 20th century saw the emergence of Salmonella serotypes
contaminated food. The symptoms arise from ingestion of preformed that became associated with new food sources (i.e. chicken eggs)
enterotoxin, which accounts for the short incubation time. and the emergence of Salmonella serotypes with resistance against
Staphylococcal enterotoxins are superantigens and, as such, have multiple antibiotics.
adverse effects on the immune system.
Shigella
The enterotoxin genes are accessory genetic elements in S.
aureus, meaning that not all strains of this organism are enterotoxin- Shigella species are members of the family Enterobacteriacae
producing. The enterotoxin genes are found on prophage, plasmids, and are Gram negative, non-motile rods. Four subgroups exist
based on O-antigen structure and biochemical properties;
and pathogenicity islands in different strains of S. aureus.
S. dysenteriae (subgroup A), S. flexneri (subgroup B), S. boydii
Expression of the enterotoxin genes is often under the control of
(subgroup C) and S. sonnei (subgroup D). Symptoms include mild
global virulence gene regulatory systems.
to severe diarrhea with or without blood, fever, tenesmus, and
6 Introductory Food Microbiology Introduction 7

abdominal pain. Further complications of the disease may be vegetative cells in the host intestine results in debilitating acute
seizures, toxic megacolon, reactive arthritis and hemolytic uremic diarrhea and abdominal pain. Sales of refrigerated, processed
syndrome. Transmission of the pathogen is by the fecal-oral route, foods of extended durability including sous-vide foods, chilled
commonly through food and water. The infectious dose ranges ready-to-eat meals, and cook-chill foods have increased over recent
from 10-100 organisms. Shigella spp. have a sophisticated years. Anaerobic spore-formers have been identified as the primary
pathogenic mechanism to invade colonic epithelial cells of the microbiological concerns in these foods. Heightened awareness
host, man and higher primates, and the ability to multiply over intentional food source tampering with botulinum neurotoxin
intracellularly and spread from cell to adjacent cell via actin has arisen with respect to genes encoding the toxins that are
polymerization. Shigellae are one of the leading causes of bacterial capable of transfer to nontoxigenic clostridia.
foodborne illnesses and can spread quickly within a population.
Bacillus Cereus
Escherichia Coli The Bacillus cereus group comprises six members: B. anthracis,
More information is available concerning Escherichia coli than B. cereus, B. mycoides, B. pseudomycoides, B. thuringiensis and B.
any other organism, thus making E. coli the most thoroughly weihenstephanensis. These species are closely related and should
studied species in the microbial world. For many years, E. coli was be placed within one species, except for B. anthracis that possesses
considered a commensal of human and animal intestinal tracts specific large virulence plasmids. B. cereus is a normal soil
with low virulence potential. It is now known that many strains inhabitant and is frequently isolated from a variety of foods,
of E. coli act as pathogens inducing serious gastrointestinal diseases including vegetables, dairy products and meat. It causes a vomiting
and even death in humans. There are six major categories of E. coli or diarrhoea illness that is becoming increasingly important in the
strains that cause enteric diseases in humans including the: industrialized world. Some patients may experience both types of
(1) enterohemorrhagic E. coli, which cause hemorrhagic colitis illness simultaneously. The diarrhoeal type of illness is most
and hemolytic uremic syndrome, prevalent in the western hemisphere, whereas the emetic type is
most prevalent in Japan. Desserts, meat dishes, and dairy products
(2) enterotoxigenic E. coli, which induce traveler's diarrhea,
are the foods most frequently associated with diarrhoeal illness,
(3) enteropathogenic E. coli, which cause a persistent diarrhea whereas rice and pasta are the most common vehicles of emetic
in children living in developing countries, illness.
(4) enteroaggregative E. coli, which provoke diarrhea in The emetic toxin (cereulide) has been isolated and
children, characterized; it is a small ring peptide synthesised non-
(5) enteroinvasive E. coli that are biochemically and genetically ribosomally by a peptide synthetase. Three types of B. cereus
related to Shigella species and can induce diarrhea, and enterotoxins involved in foodborne outbreaks have been identified.
(6) diffusely adherent E. coli, which cause diarrhea and are Two of these enterotoxins are three-component proteins and are
distinguished by a characteristic type of adherence to related, while the last is a one-component protein (CytK). Deaths
mammalian cells. have been recorded both by strains that produce the emetic toxin
and by a strain producing only CytK. Some strains of the B. cereus
Clostridium Botulinum and Clostridium Perfringens group are able to grow at refrigeration temperatures. These variants
Clostridium botulinum produces extremely potent neurotoxins raise concern about the safety of cooked, refrigerated foods with
that result in the severe neuroparalytic disease, botulism. The an extended shelf life. B. cereus spores adhere to many surfaces
enterotoxin produced by C. perfringens during sporulation of and survive normal washing and disinfection (except for
hypochlorite and UVC) procedures. B. cereus foodborne illness is
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likely underreported because of its relatively mild symptoms, which or probiotic cultures in the aquaculture industry has been
are of short duration. examined.
The genus Carnobacterium currently consists of nine species,
CARNOBACTERIUM: POSITIVE AND NEGATIVE EFFECTS
but only two of these, Carnobacterium divergens and C.
IN THE ENVIRONMENT AND IN FOODS
maltaromaticum (formerly C. piscicola) are frequently encountered
The genus Carnobacterium contains nine species, but only C. in the environment and in foods (Table 1). This review concerns
divergens and C. maltaromaticum are frequently isolated from the importance of these two species in dairy, meat and fish products
natural environments and foods. They are tolerant to freezing/ and in the aquatic environment. The taxonomy and methods for
thawing and high pressure and able to grow at low temperatures, isolation and identification of carnobacteria are not described. It
anaerobically and with increased CO2 concentrations. They is, however, important to note that older studies frequently used
metabolize arginine and various carbohydrates, including chitin, carbohydrate fermentation patterns to distinguish between C.
and this may improve their survival in the environment. divergens and C. maltaromaticum. These methods are now
Carnobacterium divergens and C. maltaromaticum have been considered less reliable than molecular methods, and results relying
extensively studied as protective cultures in order to inhibit growth on phenotypic criteria must be interpreted with caution.
of Listeria monocytogenes in fish and meat products. Several
carnobacterial bacteriocins are known, and parameters that affect DISTRIBUTION IN THE NATURAL ENVIRONMENT AND
their production have been described. Currently, however, no FOODS
isolates are commercially applied as protective cultures. Natural Environment
Carnobacteria can spoil chilled foods, but spoilage activity shows
Among the nine reported species of Carnobacterium, only two
intraspecies and interspecies variation. The responsible spoilage
species, C. divergens and C. maltaromaticum, are frequently isolated
metabolites are not well characterized, but branched alcohols and
from various sources. Four species, C. alterfunditum, C. funditum,
aldehydes play a partial role.
C. gallinarum and C. mobile, have been isolated a few times from
Their production of tyramine in foods is critical for susceptible at most three sources. Three species, C. inhibens, C. pleistocenium
individuals, but carnobacteria are not otherwise human pathogens. and C. viridans, have only been isolated from one source, but it
Carnobacterium maltaromaticum can be a fish pathogen, although should be noted that Carnobacterium spp. related to, for example,
carnobacteria are also suggested as probiotic cultures for use in C. inhibens have been described. Carnobacterium spp. appear to
aquaculture. Representative genome sequences are not yet available, have both the temperate and polar aquatic environments as habitats
but would be valuable to answer questions associated with including live fish, marine sponges, Antarctic lakes, Arctic and
fundamental and applied aspects of this important genus. Antarctic sea water as well as the deep sea, aquous alkaline tufa
Carnobacteria are ubiquitous lactic acid bacteria (LAB) isolated columns from Greenland, and freshwater habitats from the
from cold and temperate environments. More importantly from a temperate clima zone, including a Sphagnum pond and rivers in
practical viewpoint, they also frequently predominate in a range the northwest region of Spain.
of foods, including fish, meat, and some dairy products. In this Although C. maltaromaticum and/or C. divergens have been
regard, they have been extensively studied in the last two decades isolated from tropical fish products, including smoked surubim, a
as protective cultures, to inhibit pathogenic and spoilage Brazilian tropical freshwater fish, and from vacuum-packed tuna
microorganisms, and as potential spoilage bacteria in chilled caught in the Indian Ocean and processed in Sri Lanka, it cannot,
seafood and meat products. In addition, owing to their presence at least in the latter case, be excluded that this is due to
in the aqueous environment, their importance as fish pathogens contamination associated with repackaging in Denmark.
10 Introductory Food Microbiology Introduction 11

The presence of carnobacteria has also been demonstrated in and it may offer protection against acid stress, as described for
the terrestrial environment, including a field treated with whey, Enterococcus faecalis and related species. Finally, carnobacteria
Canadian winter soil, permafrost ice, a compost pile, a collapsed are able to catabolize a range of carbohydrates, although there are
horse manure pile, the larval midgut of a moth species, and other considerable interspecies and intraspecies heterogeneities.
sources. It appears that the temperate/polar aquatic and terrestrial Examples of sources of such carbohydrates include animals
environments are both natural habitats. (e.g. chitin and, to some extent, lactose, although the targets of the
Carnobacterium divergens and C. maltaromaticum possess lactose hydrolytic activity shown by carnobacteria may instead be
traits that may play a role in their survival in these surroundings. byproducts of plant sugar polymers and saccharides adsorbed to
One study has reported that a Carnobacterium sp. soil isolate humic acid substances in the soil), plants (e.g. salicin and sucrose),
related to C. maltaromaticum survived 48 serial freeze-thaw cycles fungi (e.g. chitin and trehalose), prokaryotes (N-acetylglucosamine),
better than Escherichia coli and an Enterococcus sp. soil isolate, and living organisms in general (ribose). The importance of these
but worse than a few other soil isolates, including an Acinetobacter abilities for growth and survival in the environment deserves
sp. Indeed, these organisms may survive freezing for considerable further study. The genome sizes of C. alterfunditum, C. divergens
periods of time, as witnessed by the isolation of a Carnobacterium and C. pleistocenium strains and a Carnobacterium sp. AT7 deep
sp. preserved in a permafrost ice wedge for 25 000 years. Additional sea isolate have been estimated to be 2.9, 3.2, 3.2 and 2.4Mb,
data on the abilities of carnobacteria to grow at low temperatures respectively, and for the AT7 isolate, the data are reported in a
and to survive frozen storage is available for food products. Also, database described by Liolios (2006). These sizes are relatively
a cold-active b-galactosidase from C. maltaromaticum and a cold- large in comparison to many other LAB, suggesting that
adapted alanine dehydrogenase from a Carnobacterium sp. related carnobacterial genomes may encode a range of genes that makes
to C. alterfunditum have been reported. Some carnobacterial isolates them well adapted to deal with environmental challenges.
originate from natural high-pressure habitats, and additional data Even if carnobacteria are well adapted to temperate or polar
on resistance to pressure have been reported for high-pressure- aqueous environments, this does not always provide improved
processed foods. It has not been described how other physical/ ability to survive as compared to other bacteria. Thus, the actual
chemical parameters such as salt content, atmosphere and pH abilities of strains of C. divergens and C. maltaromaticum isolated
affect survival and growth of these organisms in the natural from a Sphagnum pond to survive in water from this source were
environment. not improved as compared to related gram-positive bacteria
With respect to energy-yielding substrates, one species, C. originating from other sources. At present, therefore, the precise
maltaromaticum, expresses chitinase activity. This may facilitate mechanisms by which Carnobacterium spp. persist in the natural
adherence to and survival on zooplankton as suggested for environment and their underlying genetics are not known. Finally,
enterococci, and also explain the isolation of this species from the it should be noted that the environments from which carnobacteria
midgut of the larval stage of a species of moth. Interestingly, the can be isolated are not always as extreme as they appear at first
arginine deiminase pathway is expressed by the most frequently sight. Thus, Carnobacterium spp. isolated from Lake Vanda,
encountered species, C. divergens, C. gallinarum, C. Antarctica were found at a depth of 61 m, where the temperature
maltaromaticum and C. mobile but not by C. inhibens or C. viridans. was c. 15-20 1C.
Arginine is not a substrate that results in growth of C. alterfunditum
and C. funditum, and information is not available for C. Dairy, Fish and Meat Products
pleistocenium. This amino acid may represent an additional energy High concentrations of bacteria (4106-107 CFUg_1) in food
source for growth and survival when carbohydrates are scarce, are typically required before their activity is sufficient to influence
12 Introductory Food Microbiology Introduction 13

the sensory properties of a product. For this to happen, occurrence 20 min. In vacuum-packed coldsmoked or sugar-salted ('gravad')
and growth kinetics are the key parameters, and databases, seafood with 3-7% NaCl in the water phase and a pH of 5.8-6.5,
including ComBase, are available to determine the effect of food high concentrations of C. divergens and C. maltaromaticum are
storage conditions and product characteristics on growth of specific particularly common, as reported for halibut, rainbow trout,
bacteria. However, information on carnobacteria has not yet been salmon, surubim, and tuna.
included in such databases. High concentrations of C. maltaromaticum are also reported
Carnobacterium maltaromaticum and another Carnobacterium in salted lumpfish roe, and it has been isolated from frozen, smoked
sp. have been isolated from milk, and they, in addition to C. mussels. Finally, for cooked seafood, high concentrations of C.
divergens, have been detected in soft cheeses, including mold- maltaromaticum have been detected in MAP shrimp after storage
ripened brie, mozzarella, Camembert and other types. The high at 2-8 1C. Both C. divergens and C. mobile were isolated from
concentration of C. divergens in the curd of mozzarella, exposed cooked and brined MAP shrimps (with NaCl, benzoic acid, citric
to 10-37 1C during processing, is in agreement with the maximum acid and sorbic acid) after storage at 2-8 1C. Clearly, carnobacteria
growth temperature (40 1C) of this species. are common in chilled fresh and lightly preserved seafood, but at
The occurrence of Carnobacterium in dairy products (and higher storage temperatures (15-25 1C), other species, including
other foods) is most likely underreported. This is due to the common Enterococcus spp., more frequently dominate the spoilage microbial
use of acetate containing media, particularly MRS agar or Rogosa community of seafood.
agar, for enumeration of LAB. Growth of Carnobacterium is Carnobacterium divergens and C. maltaromaticum are able to
inhibited by acetate, and both MRS and Rogosa agar media grow in meat products at temperatures as low as 2 to _1.5 1C, and
significantly underestimate concentrations in food. they are frequently predominant members (up to 50% C. divergens
Carnobacterium divergens and C. maltaromaticum are present and up to 26% C. maltaromaticum of the gram-positive or LAB
in seafood and are able to grow to high concentrations in different isolates obtained) of the microbial community of raw meat (beef,
fresh and lightly preserved products. pork, lamb, and poultry).
Studies of naturally contaminated products suggest which The two species are found irrespective of whether products
storage conditions and product characteristics select for have been stored aerobically, vacuum packaged, or subjected to
carnobacteria as compared to the other bacteria present in seafood. modified atmospheres, including gas compositions of CO2/N2 (%)
For chilled fresh seafood, we have found no reports where C. ranging from 10 : 90 to 80 : 20. One study demonstrated the
divergens and C. maltaromaticum dominated the microbial presence of C. divergens in beef stored in air but not in MAP beef
communities in aerobically stored products, but this has been with increased concentrations of O2 (20-40%) in addition to CO2
reported for modified atmosphere-packed (MAP) coalfish, cod, (40%). The growth of C. funditum is impaired by oxygen, and it
pollack, rainbow trout, salmon, shrimp, swordfish and tuna. will be of interest to study further whether addition of O2 to the
gas composition of modified atmospheres consistently inhibits
After frozen storage (_20 to _30 1C for 5-8 weeks), C. divergens
growth of carnobacteria, as has been observed for some other
and C. maltaromaticum seem to be particularly prominent in
bacteria.
chilled MAP fish, as has been reported for cod, garfish, salmon,
and tuna. In addition to freezing, these carnobacteria are relatively Further studies are needed in order to determine whether
resistant to high-pressure processing and are found in high differences in the presence of carnobacteria in meat are due to
concentrations in vacuum-packed and chilled squid mantle and variations in storage conditions or variations in contamination
cold-smoked salmon previously treated with 200-400MPa for 15- levels at the processing plants. The source of carnobacteria in
meat products is most probably the processing plant, as these
14 Introductory Food Microbiology Introduction 15

organisms have not been isolated from the gastrointestinal system FUNCTIONAL PROPERTIES OF CARNOBACTERIA
or skin of chicken, cattle, pigs or sheep, except for one unpublished Bacteriocins and Antimicrobial Properties
study. Thus, the source of contamination of broiler carcasses by C.
Carnobacteria with the ability to produce antimicrobial
divergens and C. maltaromaticum was shown to be the air in the
peptides, bacteriocins, are commonly encountered in foods. The
processing plant and not incoming broiler chickens. In fact, two
characterized carnobacterial bacteriocins belong to class I and
studies confirmed that C. divergens and C. maltaromaticum present
class II [e.g. Drider et al. (2006) for bacteriocin nomenclature]. So
in the raw material were eliminated or reduced in number by
far, only one class I bacteriocin (lantibiotic), UI149, has been
cooking of a processed meat product, ham.
reported to be produced by Carnobacterium. In contrast, ten amino
Carnobacterium divergens and C. maltaromaticum have, acidsequenced bacteriocins belong to class II, and most of these
however, been detected in a variety of processed meat products, more specifically to class IIa, which comprises small pediocin- like
including the cured pork product bacon, a Danish processed pork peptides. The inhibition spectrum of carnobacterial class IIa
product ('rullep_lse'), various Spanish processed meat products, bacteriocins includes Listeria, and antimicrobial activity is exerted
cooked poultry meat, pressure-treated (408-888MPa) chicken, and by pore formation, dissipation of membrane potential, and leakage
irradiated pork and chicken. On a few occasions, C. of internal low molecular weight substances.
maltaromaticum has even been isolated from fermented sausages;
Class IIa bacteriocins are ribosomally synthesized as inactive
as noted by Hammes & Hertel (2003), this contrasts with the usual
prepeptides that are modified by posttranslational cleavage of the
nonaciduric habitats of carnobacteria. Other Carnobacterium spp.
N-terminal peptide leader at a doubleglycine site in order to release
isolated from processed meat products include C. gallinarium
mature and active cationic peptides. Carnobacterial class IIa
(irradiated chicken), C. mobile (cooked turkey) and C. viridans
bacteriocins have similar amino acid sequences, with a
(vacuum-packed Bologna sausage). Although these organisms are
YGNGV(X)C(X)4C motif (X denotes any amino acid) near the N-
frequently isolated from processed meat products, it has been
terminus of the mature peptide. Two cysteine residues form a
suggested that they are rarely present in high numbers, and that
disulfide bond in the N-terminal region, and there is an
terminal spoilage of cooked, cured meats is primarily caused by
amphipathic a-helix near the C-terminus. The N-terminal region
aciduric LAB.
is relatively hydrophilic and conserved, whereas the C-terminus
Finally, a Carnobacterium sp. has also been isolated from egg is hydrophobic and diverse. To establish the structure-activity
contents, and it was shown that this isolate had a similar ability relationships of carnobacterial bacteriocins, the structures of
to penetrate the eggshell and contaminate the whole egg as various carnobacteriocin B2 and divercin V41 were modified. Thus, amino
other gram-positive and gram-negative bacteria. acid substitutions closer to the N-terminus in carnobacteriocin B2
Transmission routes for carnobacteria from the natural drastically reduced or eliminated antimicrobial activity, whereas
environment into food-manufacturing plants and further on to this was not so for substitutions close to the C-terminal part.
dairy, fish and meat products are not known to any extent. Divercin V41 contains a second disulfide bond located in the C-
Knowledge on this topic is essential to evaluate if species and terminal region.
intraspecific clusters that differ in spoilage ability (as discussed When the C-terminal region of divercin V41 was separated
later) also have different colonization abilities. Such knowledge from the N-terminus by endoproteinase Asp-N, only the C-terminal
would also illuminate the feasibility of using the production of fragment was active. After trypsin cleavage next to lysine at position
metabolites, especially tyramine, by C. divergens and C. 42 or disulfide reduction, the C-terminus lost its inhibitory activity.
maltaromaticum as an index of microbial spoilage of specific fish These results suggested that both hydrophobicity and folding
and meat products. imposed by this second disulfide bond were essential for
16 Introductory Food Microbiology Introduction 17

antilisterial activity of the C-terminal hydrophobic peptide. bacteriocin expression and secretion have been sequenced. External
Chemical oxidation of tryptophan residues by N-bromosuccinimide parameters also affect bacteriocin production. Thus, increasing
showed that these residues were crucial for inhibitory activity, as concentrations of NaCl (2-7%) reduced production of A9b/B2
modification of any one of them rendered divercin V41 inactive. bacteriocin, increasing the temperature from below 19 to 25 1C
The three-dimensional structure of carnobacteriocin B2, its inhibited production of piscicolin 126, and reducing the pH from
precursor and the corresponding immunity protein have been 6.5 to 5.5 inhibited production of carnobacteriocins BM1 and B2.
solved by nuclear magnetic resonance. These are, at present, the Acetate (A9b bacteriocin) or the presence of a bacteriocin-sensitive
only reported carnobacterial protein structures. strain belonging to the same genus induced bacteriocin production.
Carnobacterial class II bacteriocins that contain a It is clear that, in order to apply carnobacterial bacteriocins for the
doubleglycine- type leader peptide are transported by a dedicated biopreservation of foods, detailed knowledge of the factors that
ATP-binding cassette (ABC) transport system. In contrast, divergicin influence production of the bacteriocin is necessary.
A does not require a secretion protein, as the transport depend on Finally, it should be noted that the bacteriocinogenic activity
the general cellular secretion (sec) pathway. The genes involved in may be lost, as shown for a C. divergens divercin V41- producing
carnobacterial bacteriocin production are generally clustered in strain that retained only partial activity after being subjected to
operons. In the simplest case, corresponding to the sec-dependent spray-drying. Interest in applying carnobacterial bacteriocins in
divergicin A, the bacteriocin operon is composed of only the foods and feeds has been directed towards inhibiting Listeria
structural gene followed downstream by the gene encoding an monocytogenes and spoilage microorganisms, to extend the shelf-
immunity protein that protects the cell from is own bacteriocin. life of lightly preserved seafood. These attempts have generally,
Production of class IIa bacteriocins requires four genes as a but not always [e.g. regarding prevention of spoilage], met with
minimum, including genes encoding bacteriocin, immunity protein, success, although diversity in sensitivity of the target organism
ABC of transport protein and its membrane-bound accessory has been observed.
protein. Genes for bacteriocin production may be encoded on the However, the occurrence of resistant L. monocytogenes target
chromosome or on plasmids. Carnobacterium maltaromaticum organisms has led to the suggestion that bacteriocin-negative LAB
LV17 produces at least three class II bacteriocins. may be more suitable for practical use as bioprotective agents
Carnobacteriocins A and B2 are encoded on different but against L. monocytogenes in ready-to-eat foods. Indeed, L.
compatible plasmids, pCP49 (72 kb) and pCP40 (61 kb), respectively. monocytogenes is inhibited by carnobacterial cultures that do not
The carnobacteriocin BM1 structural gene and its immunity gene produce bacteriocins, and this is partly due to glucose depletion.
are localized on the chromosome. Activation and export of One study also reported a successful application of cultures with
carnobacteriocin BM1 depend on genes located on plasmid pCP40 no demonstrated bacteriocinogenic activity for extension of the
encoding carnobacteriocin B2. For carnobacteriocins BM1 and B2, shelflife of vacuum-packed cold-smoked salmon. This effect was
a peptide-pheromone dependent quorum-sensing mode caused by not, however, observed for a C. divergens strain that was unable
the bacteriocin itself or an autoinducer peptide is involved in the to produce the class IIa divercin V41.
regulation of bacteriocin production by two- and three-component Resistance of L. monocytogenes to the class IIa divergicin M35
signal transduction systems. was probably due to modification of the cell wall fatty acid
The components of this regulatory system consist of an composition. Listeria monocytogenes strains resistant to the class
induction factor, a histidine protein kinase, and a response IIa divercin V41 also showed substantial differences in protein
regulator. Four carnobacterial bacteriocin operons including genes expressions as compared with the wild type strain. A s54-
for immunity proteins and regulatory proteins involved in dependent PTS permease of the mannose family, which belongs to
18 Introductory Food Microbiology Introduction 19

the phosphotransferase system (PTS), is responsible for sensitivity We will first focus on catabolic reactions with carbohydrates
of L. monocytogenes to class IIa bacteriocins. These results and/or organic acids as substrates and with pyruvic acid as an
suggested that EIIt Man encoded by the mptACD operon might be intermediate metabolite. Respiration might occur in the presence
a target molecule for class IIa bacteriocins. Recently, two genes of hematin, as shown for C. maltaromaticum, and, indeed, this
coding for a glycerophosphoryl diester phosphodiesterase (GlpQ) species consumes substantial proportions of oxygen during
and a protein with a putative phosphoesterase function (Pde) exponential growth under aerobic conditions. Carnobacterium spp.
were identified as also being involved in the class IIa sensitivity are, however, considered to be homofermentative organisms that
of En. faecalis. Finally, it is important to note that the susceptibility produce lactic acid from glucose.
of the target strain is affected by various environmental conditions
The presence of the glycolytic pathway in C. divergens has
such as NaCl and pH.
been demonstrated. It can be debated whether carnobacteria should
Another cause of concern when using carnobacteria for instead be considered facultative or atypical heterofermentative
biopreservation is that C. divergens and C. maltaromaticum both organisms, as C. divergens and C. maltaromaticum are able to
produce tyramine, as discussed further below. Therefore, it has utilize ribose and gluconic acid as substrates for growth, and may
been suggested that a C. divergens strain in which the tyrosine produce acetic acid, formic acid, and CO2 as end-products of some
decarboxylase gene is inactivated by mutagenesis could be used secondary decarboxylation/ dissimilation reactions of pyruvic acid.
as a protective culture to prevent growth of L. monocytogenes in Indeed, carnobacteria were initially described as heterofermenters.
cold-smoked salmon. Currently, no carnobacterial culture,
Acetic acid production by C. divergens and C. maltaromaticum
bacteriocin producing or not, is commercially applied for protection
can be substantial during growth in laboratory media under aerobic
against the growth of L. monocytogenes or other bacterial pathogens
conditions or in MAP shrimp, and quantitatively it can exceed
or spoilage organisms in food.
lactic acid production. Production of acetic acid by C.
Effect of Catabolic Activities on Sensory Characteristics and Safety maltaromaticum is also increased relative to lactic acid if glucose
of Foods is substituted by ribose. Furthermore, C. maltaromaticum can
As shown in Table 4, the catabolic activities of carnobacteria produce large amounts of ethanol from glucose and ribose during
may result in sensory spoilage of inoculated fish and meat products. growth in a shrimp extract under anaerobic conditions.
Whether this is so for cheese products is not clear. In naturally Acetoin can be generated by C. maltaromaticum from pyruvic
contaminated products, it seems other members of the bacterial acid, and this reaction is also found for C. divergens and C.
community are typically more important with regard to sensory gallinarum but not for C. alterfunditum, C. funditum, C. inhibens
effects, including spoilage. and C. viridance, according to reactions in the Voges-Proskauer
It is of interest that spoilage was enhanced if moderate-spoilage test. Carnobacterium mobile gives variable reactions, and
strains of C. maltaromaticum were inoculated with nonspoilage information is not available for C. pleistocenium.
Vibrio sp. strains or the moderate-spoilage organism Brochothrix The factors affecting acetoin production are not well known,
thermosphacta into cold-smoked salmon that was subsequently but production is increased by resting cells of C. maltaromaticum
vacuum-packaged. However, this was not so for combinations of in the presence of hematin. In addition, two out of four strains of
C. maltaromaticum and Photobacterium phosphoreum in this C. divergens and C. maltaromaticum isolated from mozzarella
product. In addition, a spoilage synergy effect was observed for cheese were reported to be able to metabolize citric acid, but the
combinations of B. thermosphacta and C. divergens, C. potential sensory role of this reaction, e.g. by production of acetoin
maltaromaticum or C. mobile in MAP shrimp. and diacetyl, was not examined.
20 Introductory Food Microbiology Introduction 21

Production of diacetyl and 2,3-pentanedione by C. not C. mobile were able to produce NH4 in MAP shrimp, but this
maltaromaticum during growth in cold-smoked salmon resulted was not the cause of spoilage of this product. Production of indole
in a butter-like odor but not in spoilage. In conclusion, even, if from the amino acid tryptophan is also a potential cause of spoilage,
carbohydrate catabolism by carnobacteria appears to result in a although C. divergens, C. maltaromaticum and C. mobile are all
diverse number of metabolites, these have generally a limited effect unable to carry out this reaction in standard laboratory media. It
on the sensory attributes of foods. H2O2 may be produced by C. has, however, been observed that C. divergens and strains
divergens, and formation of this compound by C. viridans has belonging to a major phenotypic cluster of C. maltaromaticum
been associated with spoilage in the form of green discoloration were able to use tryptophan as a substrate during growth in MAP
of cured Bologna ham. shrimp. Whether this resulted in generation of indole was not
Whether carnobacteria possess proteolytic activities of potential examined.
importance for taste in products such as cheese is not known, and Production of tyramine from tyrosine is the only known
this deserves further investigation. In contrast, metabolites resulting metabolic reaction by Carnobacterium spp. that constitutes a cause
from the degradation of amino acids certainly cause sensory effects of concern regarding food safety. Not all carnobacterial species
in foods. Generation of branched alcohols and aldehydes (2- possess this ability, as C. mobile does not produce tyramine during
methyl-1-butanal, 2-methyl-1-butanol, 3-methyl-1-butanal, 3-methyl- growth in shrimp, and variation exists among different strains
1-butanol, 2-methylpropanal, and 2-methylpropanol) by and phenotypic clusters of C. divergens and C. maltaromaticum
transamination, decarboxylation and reduction of the amino acids for amounts of tyramine produced. Tyramine production by one
valine, leucine and isoleucine appears to be particularly important. C. divergens strain was at its maximum during the stationary
Production of some of these compounds, such as 3-methyl-1- growth phase and at a low initial pH in the presence of 0.6%
butanal, is strain or species dependent. Production by a C. glucose, whereas NaCl (10%) was inhibitory. Regardless of strain
maltaromaticum strain of 3-methylbutanal, 3-methylbutanol and variation and the effects of environmental parameters, tyramine
3-methylbutanoic acid from leucine was, in general, increased at production by C. divergens and/or C. maltaromaticum has been
pH values of 6.5 or higher and in the presence of increased reported in a range of foods, including meat (up to 28 mg kg_1),
concentrations of a-ketoisocaproic acid and glucose. This strain a meat-fat mixture (up to 121 mg kg_1), cold-smoked salmon (up
affected the odor and reduced the leucine content of a sausage to c. 370 mg kg_1), frozen and thawed salmon (up to 40-60 mg
mince, but a clear causal relationship was not demonstrated. kg_1), and shrimp (up to 20-60 mg kg_1).
Production by C. maltaromaticum of alcohols and aldehydes from These levels have no adverse effects on most consumers, but
valine, leucine and/or isoleucine resulted in a malty, green aroma for sensitive individuals, e.g. with reduced monoamine oxidase
in skimmed milk and shrimp, and has also caused spoilage of activity due to medication or hereditary deficiency, very little
cured ham. The association between the presence of these tyramine can cause migraine headaches, and an intake of no more
compounds and spoilage is, however, not always straightforward, than 5mg of tyramine per meal has been recommended. Typical
as production of 3-methyl-1- butanal and/or 3-methyl-1-butanol consumption of fish and meat products is 50-150 g per meal. As
in shrimp by certain C. maltaromaticum strains did not result in described above, C. divergens and/or C. maltaromaticum are able
malty off-flavors. to form c. 20-370 mg tyramine kg_1 in these products,
Production of NH4 from arginine as a result of its catabolism corresponding to c. 1-55 mg of tyramine per meal. Consequently,
catalyzed by the arginine deaminase pathway may, in theory, tyramine formation by carnobacteria in specific foods can represent
cause spoilage of shrimp or squid that contain high levels of free a hazard for sensitive individuals who might suffer migraine
arginine. Carnobacterium maltaromaticum and C. divergens but headaches. This risk for sensitive individuals can be minimized
22 Introductory Food Microbiology Introduction 23

by restricting their consumption of fish and meat to products that described. Two clinical isolates of C. divergens have also been
are newly processed, i.e. that are not close to the declared shelf- obtained. Carnobacteria are not known members of the human
life expiry day. gastrointestinal microbial community, unlike several other LAB.
Growth of Carnobacterium spp. in food products may result The exceptions are an unpublished study reporting an uncultured
in accumulation of a range of volatile alcohols, ketones, Carnobacterium sp. clone from cow rumen and two studies
hydrocarbons, and other compounds. The metabolic reactions demonstrating the presence of carnobacteria in pig and horse
leading to these products have not been studied in detail for effluent-impacted environments. Nevertheless, it is safe to conclude
Carnobacterium, in contrast to the situation for other LAB, but that the presence of Carnobacterium spp. in food does not present
may be a result of NAD1-generating reactions or nonenzymatic a risk factor for human illness, except for the production of the
reactions. Several ketones have characteristic odors, but whether biogenic amine, tyramine, as discussed previously. Neither do
they contribute to spoilage off-flavors as a result of carnobacterial Carnobacterium spp. appear to present a risk for nocosomial
metabolic activity is not well understood. With regard to lipids, C. infections in hospitals and similar institutions, although they
maltaromaticum has been reported to hydrolyze tributyrin, a have, in one instance, been isolated from contaminated blood
triglyceride with butyrate as fatty acid, but it is not known whether plasma.
this lipase activity contributes to flavor changes of dairy, fish and The presence of virulence factors in carnobacteria is not well
meat products. Carnobacterium divergens and C.maltaromaticum documented. Thus C. maltaromaticum strains isolated from
were not able to limit oxidation of linoleic acid during growth. diseased fish lacked hemolytic and phospholipolytic activities.
This indicates that these species may not prevent quality Carnobacterium viridans shows, however, b-hemolytic activity on
deterioration of meat products by lipid oxidation. Finally, C. sheep blood agar. Although several isolates of carnobacteria
maltaromaticum has been shown to cause a softer texture of salmon produce bacteriocins, none of these compounds has been reported
fillets when inoculated in high numbers. to exert a cytolytic activity as shown for the En. faecalis class I-
It is worth noting that interspecies and intraspecies differences related bacteriocin cytolysin. Regarding chemotherapeutic agents,
exist regarding carnobacterial spoilage capacity. Laursen et al. fish pathogenic strains of C. maltaromaticum were resistant to
(2006) showed that C. mobile and a minor phenotypic cluster of several of the agents widely used in aquaculture, such as
C. maltaromaticum did not spoil MAP shrimp, in contrast to what oxytetracycline, quinolones, nitrofurans and potentiated
was seen with C. divergens and the major phenotypic cluster of sulfonamides, but sensitive to erythromycin. This species is not
C. maltaromaticum. It would be of interest to examine whether resistant to lysozyme from salmonid eggs, which supports the
similar heterogeneities exist for Carnobacterium spp. growing in idea that it is not vertically transmitted, at least in this fish species.
other dairy, fish and meat products. As environmental parameters Finally, it should be noted that two clinical isolates of C. divergens
readily affect the metabolism of carbohydrates and amino acids, have been reported to possess a gene encoding a new class of
it is not surprising that product-specific and storage-specific penicillinase. It will be of interest to further explore the distribution
conditions also play important roles. The ability of two C. of this enzyme among carnobacterial species as well as its
maltaromaticum strains to relatively rapidly spoil meat during functionality.
growth under aerobic conditions at 7 1C but not or only after a
It has been documented that some strains of C. maltaromaticum
long storage period in vacuum packs at 2 1C serves as an example.
are pathogenic for several fish species, including Australian
Carnobacteria as Pathogenic Organisms and/or Probiotic Cultures salmonids, carp, rainbow trout, striped bass and channel catfish,
and salmon. Pathologic effects vary, and include, for example,
Two human clinical cases caused by opportunistic infection
septicemia, peritonitis, exophthalmia, accumulation of ascitic fluid,
with C. maltaromaticum and a Carnobacterium sp. have been
24 Introductory Food Microbiology Introduction 25

and hemorrhages. In most (but not all) instances, the virulence C. mobile or C. viridans) exerts chitinolytic acitivity, a feature that
appears, however, to be low and only causes problems in fish can be perceived as targeting the chitin-containing exoskeleton of
undergoing severe stress, e.g. due to spawning. It is, therefore, not insects. Indeed, insects may serve as a host for this species.
surprising that C. maltaromaticum and C. maltaromaticum-like
strains are also found in the intestine or gills of healthy fish, Genomics
including Arctic charr, Atlantic cod, Atlantic salmon, brown The complete chromosomes of many LAB species have now
bullhead, trout, and various freshwater fish. been sequenced. Currently, 19 complete genome sequences of
The presence of other carnobacterial species in healthy fish streptococci and 18 complete genome sequences of the
has also been reported, including C. divergens, Atlantic salmon, nonpathogenic LAB representing 14 species from the order
Atlantic cod and wolffish, trout, and freshwater fish, C. Lactobacillales are available. However, no genome sequence or
alterfunditum-like [rainbow trout], C. funditum-like [Arctic charr physical genetic map is available for Carnobacterium. The LAB
and Atlantic cod], C. gallinarum [Atlantic cod], C. inhibens have relatively small genomes for nonobligatory bacterial parasites
[Atlantic salmon], C. mobilelike [Arctic charr and Atlantic cod], or symbionts, ranging from c. 1.6 to c. 3Mb. For C. divergens and
and Carnobacterium spp. [Arctic charr and carp]. Clearly, further C. pleistocenium, the genome size was estimated to 3.2Mb, and for
research is necessary to clarify under what circumstances C. alterfunditum pf4T, it was estimated to be 2.9Mb. At the time
Carnobacterium spp. may be pathogenic for fish and whether the of writing (March 2007), there is only one Carnobacterium genome
infective capability is species and clone specific. sequencing project. Carnobacterium sp. AT7, a piezophilic strain
isolated from the Aleutian trench at a depth of 2500m, is being
The reported pathogenicity of C. maltaromaticum has not
sequenced for the Moore Foundation Marine Microbial Genome
hindered research into the possibility of using other carnobacteria
Sequencing Project with a grant to the J. Craig Venter Institute. The
as probiotic cultures in aquaculture, including Carnobacterium
data already available indicate that the Carnobacterium sp. AT7
sp., C. alterfunditum, C. divergens, and C. inhibens. Isolates of
genome contains 2.4Mb and encodes 2388 proteins.
these species, in addition to C. maltaromaticum, exhibit inhibitory
Carnobacterium sp. AT7 is closely related to C. alterfunditum and
activity towards bacterial fish pathogens. Carnobacterium
C. pleistocenium.
divergens and C. maltaromaticum enhance the cellular and
humoral immune responses and cytokine expression ratios of To date, knowledge on the genes and DNA sequences of
rainbow trout. Use of carnobacteria as probiotics leads to increased Carnobacterium is comparatively sparse, concerning mostly
survival of the fish in some instances [larvae of cod fry and bacteriocin-related genes in the species C. divergens and C.
Atlantic salmon fry, rainbow trout, and salmon], but not in others maltaromaticum and 16S rRNA and 16S-23S rRNA gene intergenic
[larvae of cod fry, and rainbow trout]. One study showed that in spacer sequences. Genes involved in some important carnobacterial
vitro exposure to C. divergens did not reverse the effects of bacterial metabolic traits have been sequenced. The genetic information
pathogens, although these were alleviated. Additional research is obtained is, for most sequences, derived from just one strain, and
necessary to validate the usefulness of carnobacteria as probiotic may therefore be strain specific, as shown for a number of L.
cultures. monocytogenes genes.
Carnobacterium maltaromaticum has also been reported to be Clearly, our knowledge of important phenotypes of strains
pathogenic for the fruit fly, Drosophila melanogaster, upon injection and species of Carnobacterium would benefit from the availability
into the thorax. This specific case of pathogenesis is probably best of a complete genome sequence for a strain representative of C.
understood as a result of opportunistic infection, although C. maltaromaticum, which is the species with most importance for
maltaromaticum (but not C. divergens, C. gallinarum, C. inhibens, the food and aquaculture industries. The most extensive study on
26 Introductory Food Microbiology Introduction 27

phenotypic variation of C. maltaromaticum revealed that the is desirable. In fact, very little research has been performed
majority of strains studied belonged to one phenotypic cluster so far concerning this aspect of carnobacteria in food.
(cluster H). Thus, in our opinion, a suitable candidate for a whole (3) Metabolic Activity: It is known that carnobacteria, or at
genomic sequence could be either C. maltaromaticum LMG 22901 least C. maltaromaticum and C. divergens, have the capacity
(isolated from pork meat), LMG 22899 (isolated from cod) or LMG to produce a wide range of metabolites in chilled food.
22898 (isolated from salmon), which all belong to this phenotypic Further studies on the importance of these metabolites
cluster. LMG 22901 does not harbor plasmids, and therefore with respect to positive and negative sensory attributes of
appears to be a prime candidate for such an endeavor. various foods are, however, needed.
(4) Diversity: We are beginning to appreciate how interspecies
CONCLUDING REMARKS
and intraspecies variation determine the positive and
We have come some way towards understanding important negative effects of the presence of carnobacteria in food
aspects of the presence of carnobacteria in foods and the and the environment, but we still do not understand how
environment, particularly concerning the genetics of carnobacterial such variation affects, for instance, their survival in food-
bacteriocins and their application. However, there are important processing plants and contamination of foods. The
areas where knowledge on these bacteria is limited. These include significance of such variation for their potential as fish
the following: pathogens also needs to be substantiated. This is also the
(1) Distribution and Quantitative Microbial Ecology: case for their role as spoilage organisms in meat and fish
Carnobacterium spp. have not been reported from a number products, with the exception of a few products such as
of habitats, such as plants, fermented vegetable foods and vacuum-packed smoked salmon and cooked MAP shrimps.
the gastrointestinal system of birds and mammals, (5) Genomics: Representative genome sequences would offer a
including humans, that have otherwise been associated very valuable road map in order to answer many of the
with several genera and species of LAB. This may, in some outstanding questions associated with this important
instances, be due to faulty methodology for detecting genus.
carnobacteria. Generally, however, our quantitative
understanding is limited regarding factors that influence Staphylococcus Aureus Growth Boundaries: Moving towards
inactivation, survival or growth of carnobacteria in various Mechanistic Predictive Models Based on Solute - Specific Effects
natural environments. In foods, more detailed information The formulation of shelf-stable intermediate-moisture products
is available, but mathematical models for quantitative is a critical food safety issue. Therefore, knowing the precise
prediction of processing, product and storage effects on boundary for the growth-no-growth interface of Staphylococcus
growth and metabolic activity, including bacteriocin and aureus is necessary for food safety risk assessment. This study
tyramine formation, awaits further development together was designed to examine the effects of various humectants and to
with recording of responses in databases. In addition, produce growth boundary models as tools for risk assessment.
further studies are needed to elucidate mechanisms The molecular mobility and the effects of various physical
underlying the relationship between environmental properties of humectants, such as their glass transition
parameters and kinetic responses of carnobacteria. temperatures, their membrane permeability, and their ionic and
(2) Routes of Food Contamination: To reduce the negative effects nonionic properties, on S. aureus growth were investigated. The
of carnobacteria in foods (spoilage metabolites and effects of relative humidity (RH; 84 to 95%, adjusted by sucrose
tyramine), further information on routes of contamination plus fructose, glycerol, or NaCl), initial pH (4.5 to 7.0, adjusted by
28 Introductory Food Microbiology Introduction 29

HCl), and potassium sorbate concentration (0 or 1,000 ppm) on minimum aw for microbial growth began with the review by Scott.
the growth of S. aureus were determined. Growth was monitored The important point is that the equality of aw with p/po is based
by turbidity over a 24-week period. Toxin production was on the assumption of the existence of thermodynamic equilibrium
determined by enterotoxin assay. The 1,792 data points generated and that the value is true only for specified conditions of
were analyzed by LIFEREG procedures, which showed that all temperature and pressure. Although dilute solutions obey the
parameters studied significantly affected the growth responses of laws of equilibrium thermodynamics, the same is not necessarily
S. aureus. Differences were observed in the growth-no-growth true for intermediate-moisture systems and is even less likely for
boundary when different humectants were used to achieve the low-moisture systems, the properties of which are determined
desired RH values in both the absence and the presence of mainly by slow reaction rates. The concept of aw then becomes
potassium sorbate. Sucrose plus fructose was most inhibitory at meaningless, because the measured rvp of water is no longer the
neutral pH values, while NaCl was most inhibitory at low pH same as the equilibrium vapor pressure. The equation aw = p/po
values. The addition of potassium sorbate greatly increased the therefore applies only in the range 0.95 <aw <1.0, where equilibrium
no-growth regions, particularly when pH was <6.0. Published is established rapidly. With many foods, this assumption of
kinetic growth and survival models were compared with boundary thermodynamic equilibrium is violated, so the appropriate term is
models developed in this work. The effects of solutes and differences rvp or relative humidity (RH) rather than aw.
in modeling approaches are discussed. For the past several decades, in the food science literature, the
Olsen et al. reported that Staphylococcus aureus was involved phenomenon denoted by the term "aw" has been widely used to
in 42 documented outbreaks of food-borne poisoning in the United predict microbial growth as well as the relationship between many
States, with 1,413 cases and 1 death occurring from 1993 to 1997. common food deterioration reactions and aw. Although RH is a
S. aureus has been estimated to cause approximately 185,000 better indicator of food stability and safety than the water content
illnesses, 1,750 hospitalizations, and 2 deaths per year in the of a system, it is not always a reliable predictor. For example,
United States, all from consumption of contaminated foods. Food- several authors have reported that the growth boundaries for
borne staphylococcal poisoning, caused by the ingestion of one or various genera of microorganisms differ depending on the type of
more preformed toxins in food contaminated with S. aureus, is one humectant used to depress RH rather than the absolute value of
of the most prevalent causes of gastroenteritis worldwide. Recently, RH. The literature on microbial osmoregulation via compatible
powdered milk produced in Taiki, Hokkaido, Japan, was linked solutes has also shown that the type of humectant, rather than the
to contaminated dairy products that made more than 14,700 people absolute value of RH, is critical.
in Japan ill in June and July of 2000. Health authorities determined In recent years, increasing evidence based on glass transition
that dry skim milk powder was contaminated with staphylococcal theory has shown that molecular mobility (Mm) may be an attribute
enterotoxin A. that deserves further attention, as it is related to many important
Scott related the relative vapor pressures (rvp's) of foods to the diffusion-limiting properties of foods. This is because the
thermodynamic activity of water, using the definition aw = p/po, temperature at which a viscous liquid changes to a "glass" (glass
where aw is the water activity as derived from the laws of transition temperature [Tg]), which governs Mm, is a solute-specific
equilibrium thermodynamics, p is the vapor pressure of the sample, property that is inversely linearly related to the measured RH of
and po is the vapor pressure of pure water. He showed a clear a system. Although the use of RH has served the food industry
correlation between the rvp of the growth medium and the rate of well for the past several decades since Scott introduced the concept
S. aureus growth. In the field of food science, the general acceptance of aw in 1953, further study to understand these solute-specific
and application of this concept relating the rvp's of foods to a effects on microbial growth should continue.
30 Introductory Food Microbiology Introduction 31

Microbial growth models are typically developed when the coagulase positive. Their identification as S. aureus was confirmed
objective is to understand the responses of microorganisms when by the use of a Riboprinter. Stock cultures were grown overnight
part of the range of conditions studied permits growth to occur. at 37°C in brain heart infusion (BHI) broth (Difco), suspended in
Such models can describe the increase in numbers with time solution containing a 2:1 BHI broth-glycerol solution, and stored
(kinetic models), the conditions allowing growth or no growth at -80°C until needed.
(boundary models), or the chance of growth (probabilistic models). For this study, BHI broth was reconstituted with the
Kinetic growth models are typically based on growth curves over appropriate ratio of water to sucrose-fructose (50:50, wt/wt) or to
a range of conditions and can allow growth rates to be predicted. NaCl to achieve the desired RHs for the study (84, 88, 92, or 95%).
In the 1980s, the United Kingdom Ministry of Agriculture, Fisheries, Appropriate amounts of 10% stock solutions of potassium sorbate
and Food and the U.S. Department of Agriculture (USDA) funded (Hoechst, Frankfurt, Germany) were added to achieve a final
programs to develop kinetic models for the growth, survival, and concentration of 1,000 ppm. The pH was adjusted to 4.5, 5.0, 6.0,
death of food-borne pathogens, including S. aureus. This work or 7.0 by using 0.1 or 1.0 N HCl. The final RH was determined by
resulted in suites of models which are currently available. NaCl using an Aqualab CX-2 aw meter. The RHs of the media measured
was the humectant chosen to control the RH in these models, and at ambient temperature differed by no more than ±0.003% relative
the resulting kinetic models do not allow the growth-no-growth to those measured at 37°C. Each of the 64 broths was filter sterilized
boundary to be estimated. and stored in screw-cap tubes at 4°C until needed.
The statistical effects and interactions of RH, initial pH, and
Bioscreen Microtiter Plate Preparation, Incubation, and
potassium sorbate concentration on the boundary for S. aureus
Measurement
growth were modeled in this study by using the approach
described previously. Additionally, the concepts of Mm and the The five stock cultures were removed from -80°C storage, and
effects of various physical properties of humectants, such as the one loopful of each was transferred separately into 9 ml of BHI
Tg, membrane permeability, and ionic and nonionic properties of broth. The inoculated broths were incubated for 18 h at 37°C. The
solutes, on S. aureus growth were investigated by controlling the five cultures were individually adjusted to optical densities (ODs)
RH with sucrose-fructose (50:50, wt/wt), glycerol, or NaCl. at 530 nm of 0.750 to 0.780 (measured with a Perkin-Elmer model
35 spectrophotometer) with 0.1% sterile peptone to achieve a
MATERIALS AND METHODS concentration of approximately 108 CFU/ml. Diluted cultures (10
Preparation of Bacteria and Media ml) were transferred into a single sterile test tube and vortexed to
create the S. aureus cocktail used in our experiments. Three 1/10
Five S. aureus strains were used as a cocktail in this study:
dilutions with sterile 0.1% peptone water were used to make a
ATCC 13565, ATCC 14458, and ATCC 27664, from the American
final working cocktail with a concentration of approximately 105
Type Culture Collection, Manassas, Va.; D-2, from Toxin
CFU/ml. This working cocktail was kept on ice until it was used
Technology, Inc., Sarasota, Fla.; and A-100, from the U.S. Army
to inoculate the various media used in the experiments. Samples
Natick Laboratories, Natick, Mass. A cocktail of strains was chosen
(1 ml) of the working cocktail were taken, serially diluted, spread
for use because it helps to overcome the variability between strains
plated onto Baird-Parker agar plates, and incubated at 37°C for 48
and increase the chance of detecting the fastest growth at different
h, and cells were counted to determine their initial concentrations.
points in the experimental matrix. These benefits of using a cocktail
help to create a fail-safe model without repeating the experiment Each of the 64 broths (10 ml) was aseptically transferred to a
many times with different strains. All strains showed typical growth sterile tube, and the S. aureus cocktail (100 µl) was added to the
on Baird-Parker agar (Difco, Detroit, Mich.) plates and were broth to achieve an initial concentration of approximately 103
32 Introductory Food Microbiology Introduction 33

CFU/ml. The inoculated broths (400 µl) were aseptically for initial analysis. Data were transferred to a Microsoft Excel
transferred into sterile 100-well microtiter plates, with eight replicate spreadsheet to determine TTG and then to SAS software for
wells per medium. The outer wells of the microtiter plates were modeling by the LIFEREG procedure.
filled with uninoculated medium to act both as a partial moisture An additional data set was also included in the development
barrier and as uninoculated controls. Each plate contained media of the model. This data set included information on TTG for the
at only one RH level. A piece of sterile thermaseal film was placed same S. aureus cocktail obtained by an identical experimental
on top of the open wells under the lid during incubation. The procedure, with the exception that the humectant used to achieve
plates were placed on a rack in a sealed plastic container that the desired RH of the BHI broth was glycerol, a glass-forming
contained the appropriate saturated salt slurries needed to membrane-permeable solute. The experimental design of Stewart
maintain the environmental RH and thereby ensure the stability et al. included three levels of potassium sorbate (0, 500, and 1,000
of the RHs of the media. The saturated salt solutions used were ppm). A total of 768 data points from the glycerol data set were
as follows: ZnSO4 · H2O (83% RH at 37°C), KNO (89% RH at included in the data analysis.
37°C), KPO4 (93% RH at 37°C), and K2Cr2O7 (96% RH at 37°C).
The containers were closed and incubated at 37°C. Plates were Statistical Analysis
periodically removed and shaken for 60 s, and the ODs of the The TTG data along with a censoring indicator, the percent
1,024 individual wells were measured for up to 24 weeks by using RH, the pH, the potassium sorbate level, and the humectant type
the wideband filter on the Bioscreen C system (Labsystems Oy, (sucrose-fructose, glycerol, or NaCl) were input data for the
Helsinki, Finland). The experiments were repeated on separate construction of the growth boundary model. RH, pH, and
dates. A total of 1,024 wells were monitored. potassium sorbate values were normalized [(RH - 89.75)/4.15],
[(pH - 5.625)/0.96], [(sorbate - 500)/500] prior to model development.
Data Collection
The TTG results from replicate test wells were averaged before
The S. aureus population was considered to have grown when modeling to minimize the effect of measurement error. The SAS
the Bioscreen OD measured with a wideband filter (ODwb) LIFEREG procedure was used to develop predictive models of the
increased from an initial reading between 0.200 and 0.220 to an natural logarithm of TTG (ln TTG) as a function of the factors
ODwb of 0.350 or higher. The time to growth (TTG) was determined percent RH, pH, preservative level, and humectant type. The
by calculating the geometric mean of the time of the last resulting model can easily be transformed into a regular time
measurement that showed no growth (ODwb < 0.350) and the first scale. It should be noted that the type of humectant is considered
time point that showed growth (ODwb > 0.350). In instances of no a categorical variable (i.e., nonnumeric) in the SAS procedure. In
growth, TTG was censored at the final measurement time of 168 growth modeling, there are often conditions under which no
days. A threshold ODwb value of 0.350 was used to score wells growth occurs; therefore, TTG may be missing or censored. Under
with S. aureus growth (ODwb ³ 0.350) and wells with no growth these conditions, ordinary least-squares regression is not applicable
(ODwb < 0.350). This threshold value was determined as discussed and special procedures are required.
in detail by Stewart et al. (40). In brief, ODwb measurements were
The LIFEREG procedure can accommodate such censored data
compared with plate counts, and an ODwb of 0.350 was identified
and uses maximum-likelihood estimation methods to find
as the lowest ODwb value at which an increase in turbidity
regression coefficients. Further details of this method of analysis
regularly correlated to an increase in plate counts.
are given by Allison (1). The LIFEREG procedure works best with
OD measurements were transferred from the Bioscreen C ASCII many replicates in which the intervals between growth parameters
file to a DOS text editor and then imported into Statistica software are equally spaced, with approximately 50% of conditions limiting
34 Introductory Food Microbiology Introduction 35

growth. The ranges of the parameters studied (RH, pH, and Model development is an iterative process. The first analysis
preservative levels) were chosen to satisfy these requirements. of the data created models which were all-inclusive; this allowed
for the determination of those factors which had a significant
Toxin Production effect on the growth of S. aureus. All of the main effects, quadratic
Toxin production for experimental conditions close to the effects, and two-factor interactions were significant (P < 0.005),
growth-no-growth boundary was measured by the VIDAS staph except for the potassium sorbate concentration quadratic term and
enterotoxin assay. The VSEA is a qualitative enzyme-linked the interaction between RH and potassium sorbate concentration.
fluorescent immunoassay performed in an automated mini-VIDAS The insignificant factors were dropped, and the data were
instrument. The media from the microtiter plates were removed for reanalyzed to develop the final models. SAS LIFEREG outputs a
assay after the final ODwb measurements were taken (24 weeks). table of regression coefficient estimates and approximate chi-square
The broth from each of the eight replicate wells was removed via distribution P values for each factor in the model. The relative
pipette, combined into two microcentrifuge tubes, and centrifuged importance of each factor can be judged by its P value: factors with
for 10 min. The supernatants were combined, and the pH values low P values have the greatest influence on and are most predictive
were adjusted with 1 N NaOH to 6.0 to 8.0. Samples (0.5 ml) were of the TTG.
placed into the appropriate wells in a test strip, and the assays The resulting model equation for the use of sucrose-fructose as
were run in the mini-Vidas. Positive and negative controls were the humectant was as follows (where TTG is measured in days,
run with each set of test strips. The VSEA is sensitive to 1 ng of RH is a percentage, and the potassium sorbate concentration is
toxin/ml of sample. measured in parts per million): ln TTG = 4.46 - (5.90 x RH) - (2.62
x pH) + (1.42 x sorb) + (1.78 x RH2) + (1.06 x pH2) + (0.48 x RH
RESULTS
x pH) - (0.94 x pH x sorb).
Model Development and Diagnostics
The resulting model equation for the use of glycerol as the
Models were developed from the 1,792 data points generated. humectant was ln TTG = 2.24 - (3.86 x RH) - (2.29 x pH) + (1.00
The models included all three main effects: percent RH, pH, and x sorb) + (1.78 x RH2) + (1.06 x pH2) + (0.48 x RH x pH) - (0.94
potassium sorbate concentration ("sorb" in the equations); their x pH x sorb).
quadratic effects (RH x RH = RH2; pH x pH = pH2; and sorb x
The resulting model equation for the use of NaCl as the
sorb = sorb2); and the three two-factor interactions (RH x pH, RH
humectant was ln TTG = 3.15 - (3.59 x RH) - (3.81 x pH) + (1.64
x sorb, and pH x sorb). The impact of the humectant type on the
x sorb) + (1.78 x RH2) + (1.06 x pH2) + (0.48 x RH x pH) - (0.94
main effects and interactions was assessed by including indicator
x pH x sorb).
variables in the model.
From the model equations, the TTG contour plots for sucrose-
These main effects, quadratic effects, and two-factor interactions
fructose, glycerol, and NaCl were created. The contour plots were
are collectively referred to as the factors. SAS LIFEREG allows the
created by generating a grid of evenly spaced points in the
user to specify the error distribution to account for the variation
experimental design space of pH, RH, and potassium sorbate
in TTG not explained by the regression model. For model
concentration. With a grid of 2,268 points, a predicted TTG was
development purposes, Weibull, lognormal, and loglogistic
calculated for each point and humectant by using the appropriate
distributions were considered. All three distributions gave very
model shown above.
similar models (the regression coefficients were similar in sign
and magnitude), and we selected the loglogistic distribution model, The grid of points and predicted TTG values was input into
which has been used in previous work. the SAS procedure "proc gcontour". Diagnostic methods to check
36 Introductory Food Microbiology Introduction 37

for departures from model assumptions can be used in a manner The resulting model equation for the use of sucrose-fructose as
that is similar to their use in ordinary regression analysis. However, the humectant was as follows (where TTG is measured in days,
when nonnormal distributions are being fitted, an appropriate RH is a percentage, and the potassium sorbate concentration is
standardization of residuals must be used (25). The SAS reliability measured in parts per million): ln TTG = 4.46 - (5.90 x RH) - (2.62
procedure was used for residual analysis. A probability plot of x pH) + (1.42 x sorb) + (1.78 x RH2) + (1.06 x pH2) + (0.48 x RH
standardized residuals was examined, and no unusual patterns x pH) - (0.94 x pH x sorb).
or anomalies were detected. The resulting model equation for the use of glycerol as the
humectant was ln TTG = 2.24 - (3.86 x RH) - (2.29 x pH) + (1.00
Model Development and Diagnostics
x sorb) + (1.78 x RH2) + (1.06 x pH2) + (0.48 x RH x pH) - (0.94
Models were developed from the 1,792 data points generated. x pH x sorb).
The models included all three main effects: percent RH, pH, and
The resulting model equation for the use of NaCl as the
potassium sorbate concentration (“sorb” in the equations); their
humectant was ln TTG = 3.15 - (3.59 x RH) - (3.81 x pH) + (1.64
quadratic effects (RH x RH = RH2; pH x pH = pH2; and sorb x sorb
x sorb) + (1.78 x RH2) + (1.06 x pH2) + (0.48 x RH x pH) - (0.94
= sorb2); and the three two-factor interactions (RH x pH, RH x
x pH x sorb).
sorb, and pH x sorb). The impact of the humectant type on the
main effects and interactions was assessed by including indicator From the model equations, the TTG contour plots for sucrose-
variables in the model. These main effects, quadratic effects, and fructose, glycerol, and NaCl were created. The contour plots were
two-factor interactions are collectively referred to as the factors. created by generating a grid of evenly spaced points in the
SAS LIFEREG allows the user to specify the error distribution to experimental design space of pH, RH, and potassium sorbate
account for the variation in TTG not explained by the regression concentration. With a grid of 2,268 points, a predicted TTG was
model. For model development purposes, Weibull, lognormal, and calculated for each point and humectant by using the appropriate
loglogistic distributions were considered. All three distributions model shown above. The grid of points and predicted TTG values
gave very similar models (the regression coefficients were similar was input into the SAS procedure “proc gcontour”. Diagnostic
in sign and magnitude), and we selected the loglogistic distribution methods to check for departures from model assumptions can be
model, which has been used in previous work. used in a manner that is similar to their use in ordinary regression
analysis. However, when nonnormal distributions are being fitted,
Model development is an iterative process. The first analysis
an appropriate standardization of residuals must be used. The
of the data created models which were all-inclusive; this allowed
SAS reliability procedure (SAS Institute, Inc.) was used for residual
for the determination of those factors which had a significant effect
analysis. A probability plot of standardized residuals was
on the growth of S. aureus. All of the main effects, quadratic effects,
examined, and no unusual patterns or anomalies were detected.
and two-factor interactions were significant (P < 0.005), except for
the potassium sorbate concentration quadratic term and the
MODEL PREDICTIONS AND COMPARISON WITH
interaction between RH and potassium sorbate concentration. The
PUBLISHED WORK
insignificant factors were dropped, and the data were reanalyzed
to develop the final models. SAS LIFEREG outputs a table of As conditions became increasingly unfavorable for growth,
regression coefficient estimates and approximate chi-square the contour lines drew closer together, indicating that conditions
distribution P values for each factor in the model. The relative were approaching those that do not allow growth. As the RH or
importance of each factor can be judged by its P value: factors with pH of the system decreased, a corresponding increase in TTG was
low P values have the greatest influence on and are most predictive seen and the no-growth area of the contour plot increased in size.
of the TTG. The addition of potassium sorbate at low pH values (4.5 to 5.5)
38 Introductory Food Microbiology Introduction 39

dramatically changed the contour plots in the low-pH area of the and cheeses. Unfortunately, these data cannot be used for model
plot. The effect of potassium sorbate decreased as the pH value validation, as RH was not reported and/or other parameters not
approached 7.0. included in our models (such as oxygen concentration or
Differences were observed in the growth-no-growth boundary temperature) were varied. Additionally, the pH and/or ingredient
when different humectants were used to achieve the desired RH lists of the products used in these studies were not reported.
values in both the absence and the presence of potassium sorbate. NaCl was most inhibitory at pH values below ca. 5.3 in the
The use of sucrose-fructose as the humectant resulted in the greatest absence of potassium sorbate and at pH values below ca. 6.1 in the
delay of S. aureus growth under the highest pH and RH combination presence of potassium sorbate at 1,000 ppm. For example, no growth
(i.e., pH 7.0 and 88% RH) either with or without potassium sorbate. was seen even at conditions of 95% RH. This finding disagrees
Even with the addition of potassium sorbate at 1,000 ppm at pH with predictions made by the USDA’s pathogen modeling program
7.0, glycerol and NaCl were less effective in delaying S. aureus (PMP) version 5.1, which indicates that at 95% RH and pH 4.5,
growth than sucrose-fructose with no added preservative. At pH S. aureus will grow from 103 to 107 CFU/ml in 1.65 days.
values above ca. 5.3 and in the absence of potassium sorbate, a Comparison of the sucrose-fructose and glycerol models developed
rank order was observed, with NaCl being the least inhibitory, in this study with other published work is difficult, as the majority
followed by glycerol, and with sucrose-fructose being the most of previously published studies have used NaCl to control the RH
inhibitory; by contrast, at pH values below ca. 5.3, NaCl showed of the system.
dramatically increased inhibition. As discussed previously, differences in the physical properties
With the addition of potassium sorbate at 1,000 ppm, the same of humectants can lead to various responses, even when RH is
rank order occurred at pH values above ca. 6.1. It has generally constant. The differences in modeling approaches also make
been accepted that the limits for the growth of S. aureus are 85% comparisons difficult. Neither the Food Micro Model of the United
RH when NaCl is used as the humectant and 89% when glycerol Kingdom Ministry of Agriculture, Fisheries, and Food nor PMP
is used. was created from data sets containing many replicated experiments
Our models showed the limiting RH for growth to be where the bacteria were subjected to stressed conditions. There is
approximately 88% (pH 7.0) when sucrose-fructose was used, 86% also the danger of extrapolation beyond the conditions under which
when glycerol was used, and 85% when NaCl was used. The data were actually collected. The PMP gives a prediction for growth
limiting RH with glycerol as the humectant was markedly different under the conditions of pH 4.5 and 95% RH (with NaCl as the
from that reported by Marshall et al., who found that the growth humectant), although data were not collected for this combination
limits were 89 and 86% RH when glycerol and NaCl were used of parameters. A full factorial design was utilized for the boundary
as the humectants, respectively. models presented in this paper, and the models were constructed
by using interpolation only.
This disagreement may be due to strain variation or to the fact
that only quarter-strength BHI broth was used in the work by Enterotoxin Assays
Marshall et al. while full-strength BHI broth was used in our
When the final ODwb was close to 0.350, the samples were
model system. Whatever the reason, this result suggests that if
tested for enterotoxin. Assays were also conducted for all samples
glycerol makes a large contribution to the humectant content of a
with combinations of 84% RH and pH 7.0 or 84% RH and pH 4.5.
food product, it would be wise to consider the target RH carefully.
A total of 94 assays were run, and in every case where the data
Additionally, there is published literature that describes the were scored as “no growth,” the toxin assay results were negative
ability of S. aureus to grow in various food systems, mainly meats for the presence of toxin, while in every case where the data were
40 Introductory Food Microbiology Introduction 41

scored as “growth,” the toxin assay results were positive for the polymer systems were formulated by Ferry. The importance of the
presence of toxin. This further supports the choice of an ODwb of role of glassy states in various sugar-containing foods was
0.350 as an appropriate cutoff value for TTG in the individual described by White and Cakebread, who suggested that these states
wells and greatly increases confidence in the practical value of the played an important role in the stability and processability of
models. many food systems.
The basic principles that apply to synthetic amorphous
DISCUSSION
polymers, as described by Ferry and others, also apply to the
Using RH without considering the effect of the type of behavior of glass-forming foods. Foods of this type include starch-
humectant has often led to difficulties in predicting the stability containing products like pasta and bread, boiled confections, protein-
and microbial safety of intermediate-moisture foods. For example, based foods, intermediate-moisture foods, and dried, frozen, or
S. aureus is highly salt tolerant and has been reported to grow at freeze-dried foods.
RHs as low as 85% in NaCl concentrations up to 25% (wt/wt);
The RH and Mm approaches to food stability are
however, RH values limiting growth are typically higher when
complementary, not contradictory. RH, referred to as aw, focuses
humectants other than NaCl are used to control RH.
on the availability of water in a food matrix, while Mm defines this
Notermans and Heuvelman showed that the RH limiting S. availability in terms of microviscosity and diffusibility, with the
aureus growth varied depending on whether sucrose (with which latter being dependent on the properties of water as a plasticizer
it was reported that an aw of 0.90 limited growth) or NaCl (with to depress the Tg of the food matrix.
which it was reported that an aw of 0.87 limited growth) was used
Humectants with various characteristics were employed in
as the humectant.
this study specifically to begin to explore the influence of various
Marshall et al. reported that the level of inhibition of the growth physical properties of humectants, such as their Tgs, on microbial
of S. aureus by glycerol at neutral pH was about 10% greater than growth under osmotically stressed conditions. When glass-forming
that caused by NaCl in the rvp range (reported as aw) from 0.96 solutes are used to achieve desired RH values in systems, the
to 0.90. underlying Tg and the relaxation behavior of the glasses become
Clearly, the choice of humectant used to depress the RH in a the controlling parameters of the system. Within each glass-forming
food system is critical and its effects on microbial survival and/ system, Tg is inversely linearly related to the measured RH of the
or growth depend not only on the RH but also on the chemical and system.
physical properties of the humectant, as well as the biological However, across different glass-forming solute systems, Tg
effects of the humectants on the microorganisms. increases as the weight-average molecular weight of the solutes
As stated previously, although RH is a better indicator for increases. As Tg increases, the viscosity of the system increases,
food stability and safety than water content, it is not always a which in turn decreases the mobility of molecules in the system
reliable predictor of stability. As described by Fennema, increasing (including the mobility of water molecules). This relative decrease
evidence has shown that the Tg and Mm may be important means in mobility between glass-forming systems may partially explain
to explain many diffusion-limiting properties of food. the varied responses of microorganisms in systems with matching
RH values but in which different humectants were utilized to
Interest in Mm was first generated by Luyet and associates in
achieve the targeted RH.
the United States as well as by Rey in France, who showed
relationships between Mm and biological materials, while basic The humectants used in this study included sucrose-fructose
concepts relating nonequilibrium Mm in synthetic amorphous (non-membrane-permeable glass-forming solutes with a reference
42 Introductory Food Microbiology Introduction 43

Tg of approximately -32°C), glycerol (a membrane-permeable glass- Additionally, the effects of osmotic stress on S. aureus cell size
forming solute with a reference Tg of approximately -65°C), and also differ depending on whether NaCl, sucrose, or glycerol is
NaCl (a non-glass-forming ionic solute which may affect various used as the humectant. Although these models are empirical in
ion channels in the cell). nature, they demonstrate that the limits of growth based on RH
The limits of microbial growth cannot be predicted by the RH would be different when various humectants were used to control
of the system alone, as seen by comparison of the growth RH, so comparing studies based on RH alone may lead to false
boundaries developed with the three types of humectants used in conclusions.
this study. Instead, the RH of the system should be considered in These results also allow us to begin to explore the possibility
conjunction with the physical properties of the solutes and their of moving towards a mechanistic approach to creating predictive
effects on the biological systems. Ionic solutes, such as NaCl, may models. Further work in this laboratory to study solute-specific
place both ionic and osmotic stresses on the cell. Of the three effects on cellular energy (ATP levels) is ongoing in an effort to
humectants studied, NaCl was the most inhibitory below pH continue to work towards a better understanding of the
values of ca. 5.3 in the absence of potassium sorbate. mechanism(s) of bacterial inactivation.
The no-growth region of NaCl was expanded to pH values Three statistical models describing the growth boundaries for
below ca. 6.1 with the addition of potassium sorbate at 1,000 ppm. S. aureus with respect to RH controlled by three types of humectants,
This exaggerated interaction between NaCl, low pH, and potassium pH, and preservative were developed, and results were confirmed
sorbate may suggest that the Na+ proton antiport may be the by toxin assays. Building on previous work that has established
critical ion channel involved. regions of growth and no growth, these mathematical models
These data suggest that growth inhibition may be due to predict actual TTG as a function of the humectant type, RH, pH,
overwhelming ionic stress. The membrane permeability of solutes and preservative level.
such as glycerol may influence the degree of osmotic stress These predictions can be used to develop much more detailed
experienced by the cell. and informative boundary surfaces. Good agreement between all
Additionally, the influence of the Tg of the solute on Mm three of these growth boundary surfaces and PMP growth kinetic
directly affects the degree of osmotic stress a specific solute (e.g., models was found in the relatively unstressed regions. Growth
sucrose-fructose or glycerol) places on the cell. Sucrose-fructose boundary predictions were outside the confidence limits of the
with a higher Tg than that of glycerol (and, therefore, a larger PMP growth kinetic model under more stressful conditions, even
constraint on Mm than that of glycerol) was most inhibitory at pH when NaCl was used as the humectant.
values above ca. 5.3 or 6.1 in the absence or presence of potassium Kinetic models and growth boundary models can be used as
sorbate, respectively. complementary tools for the development of microbially safe
Glycerol is also membrane permeable, and therefore, the cell products with short and long shelf lives. These models will allow
may experience less osmotic stress than when sucrose-fructose product developers to visualize the “safe space” for the formulation
(membrane impermeable) is utilized. Physiological effects on S. of shelf-stable intermediate-moisture foods by employing these
aureus cells have been shown to differ when various solutes are preservation factors, will allow microbiologists to assess risks
used to cause osmotic stress. Cellular transport systems responsible more effectively over a wide range of products, and will ultimately
for the uptake of compatible solutes by S. aureus during osmotic allow the consumer to have greater assurance of food safety.
stress have been shown to respond differently to the stresses caused The use of sucrose-fructose as the humectant results in the
by NaCl, sucrose, and glycerol. greatest delay of S. aureus growth when the highest pH and RH
44 Introductory Food Microbiology Role of Ctc from Listeria Monocytogenes in Osmotolerance 45

(i.e., pH 7.0 and 88% RH) are used either with or without potassium
sorbate. NaCl is the most inhibitory humectant at pH values below
ca. 5.3 in the absence of potassium sorbate and at pH values below
ca. 6.1 in the presence of potassium sorbate at 1,000 ppm. Solute-
specific effects on bacterial growth were evident, as growth
boundaries differed at the same RH values when different
humectants were used. 2
Over the almost 50 years since Scott first published his work
on the water relations of S. aureus, measurement techniques and ROLE OF CTC FROM LISTERIA
the fields of physical chemistry, microbial physiology, and microbial
modeling have progressed significantly. Scott was well aware that MONOCYTOGENES IN
aw would not necessarily be adequate to describe all of the OSMOTOLERANCE
properties of solutions that influence microbial growth, metabolism,
and survival. He suggested that, with further work to fill some of
the obvious gaps in our knowledge, we would discover whether Listeria monocytogenes is a food-borne pathogen with the ability
aw could be replaced by something more meaningful. With careful to grow under conditions of high osmolarity. In a previous study,
experimentation and understanding, we will continue to fill the we reported the identification of 12 proteins showing high
gaps and allow the food industry to produce the highest-quality induction after salt stress. One of these proteins is highly similar
products while ensuring food safety. to the general stress protein Ctc of Bacillus subtilis. In this study,
induction of Ctc after salt stress was confirmed at the
transcriptional level by using RNA slot blot experiments. To explore
the role of the ctc gene product in resistance to stresses, we
constructed a ctc insertional mutant. No difference in growth was
observed between the wild-type strain LO28 and the ctc mutant
either in rich medium after osmotic or heat stress or in minimal
medium after heat stress. However, in minimal medium after
osmotic stress, the growth rate of the mutant was increased by a
factor of 2. Moreover, electron microscopy analysis showed impaired
morphology of the mutant grown under osmotic stress conditions
in minimal medium. Addition of the osmoprotectant glycine betaine
to the medium completely abolished the osmotic sensitivity
phenotype of the ctc mutant. Altogether, these results suggest that
the Ctc protein of L. monocytogenes is involved in osmotic stress
tolerance in the absence of any osmoprotectant in the medium.

INTRODUCTION
Listeria monocytogenes is a food-borne pathogen widely
distributed in the environment. This microorganism is of particular
46 Introductory Food Microbiology Role of Ctc from Listeria Monocytogenes in Osmotolerance 47

concern in the food industry because of its ability to survive and expression of the ctc gene of B. subtilis occurs via the sB RNA
frequently to grow under a wide range of adverse conditions used polymerase subunit. The ctc promoter was one of the first sB-
to preserve food such as low temperature, low pH, and high dependent promoters identified and for this reason has been
osmolarity. Growth of L. monocytogenes has been reported at NaCl extensively studied. It is the best-characterized sB-dependent
concentrations as high as 10%. promoter and has become the promoter of choice in nearly all
Most bacteria cope with elevated osmolarity in the environment investigations of sB regulation. In contrast to the wealth of
by intracellular accumulation of compatible solutes, called information regarding the ctc promoter, the function of the ctc
osmolytes. Among the compatible solutes efficient in L. product itself in B. subtilis is less clear and seems to be dispensable.
monocytogenes, two quaternary amines, glycine betaine and Only reduced sporulation efficiency at high temperatures has been
carnitine, are the most effective. The accumulation of these observed in a ctc null mutant.
osmoprotectants in L. monocytogenes occurs through osmotic To investigate the function of the Ctc protein in L. monocytogenes,
activation of their transport from the medium rather than through especially with regard to the stress resistance of the bacterium, we
de novo synthesis. Accumulation of glycine betaine and carnitine analyzed the sequence of the corresponding gene and inactivated
occurs via at least two glycine betaine transporters encoded by the it by insertional mutation. Physiological studies indicate that the
betL gene and the gbu operon and one carnitine transporter encoded Ctc protein facilitates growth in minimal medium under conditions
by the opuC operon. A betL knockout mutant and a mutant of gbu of high osmolarity and in the absence of an osmoprotectant. This
obtained by transposition were significantly affected in their abilities is the first time that a role has been assigned to Ctc, which belongs
to accumulate glycine betaine and were unable to withstand to a family of unknown proteins.
concentrations of salt as high as the isogenic parent strain can
withstand. Similarly, a mutant with an insertional inactivation of MATERIALS AND METHODS
opuCA was defective in the uptake of carnitine and had impaired Bacterial Strains and Plasmids
growth at high osmolarity. Proline has been identified as an
L. monocytogenes LO28, a clinical isolate, was obtained from P.
osmolyte for L. monocytogenes. The proline transport mechanism
Cossart (Institut Pasteur, Paris, France). Bacterial plasmids were
has not been characterized yet. However, the proBA operon, coding
propagated in Escherichia coli TG1. Plasmid pHT315 was used as
for the enzymes that catalyze the two first steps of proline
a cloning vector for sequencing, and plasmid pAUL-A was used
biosynthesis, has recently been identified. Disruption of this operon
for gene disruption.
significantly reduced the growth of the corresponding mutant at
high salt concentrations. However, little information is available Culture Media and Stress Conditions
concerning other mechanisms that take place in L. monocytogenes
Cells were grown on complex culture media: brain heart
to enable the organism to cope with osmotic stress, especially
infusion (BHI) broth or agar (Difco Laboratories, Detroit, Mich.). A
when osmolytes are not available in the environment.
chemically defined minimal medium called Improved Minimal
In a previous study, by proteomic analysis and mass Medium, or IMM, was also used, but pyridoxal, which is not
spectrometry or N-terminal sequencing, 12 proteins showing high necessary for growth, was not added. Different stress conditions
induction after a salt stress were identified. One of these proteins used for growth rate or microscopy experiments were induced
is similar to the Ctc protein of Bacillus subtilis, a general stress according to the following procedure. Overnight cultures of strain
protein which belongs to the L25 family of ribosomal proteins. In LO28 or the ctc mutant were used to inoculate fresh medium at an
B. subtilis, the ctc gene is induced in response to osmotic, heat, and initial optical density at 600 nm (OD600) of 0.05. For heat stress,
oxidative stress and glucose limitation (14, 41). Regulation of the BHI medium or IMM was inoculated with a preculture grown in
48 Introductory Food Microbiology Role of Ctc from Listeria Monocytogenes in Osmotolerance 49

the same medium, and the cultures were incubated with shaking and OD2 with incorporation of two restriction sites, HindIII and
at 45°C. For osmotic stress, either BHI medium with or without BamHI. These primers were designed using the complete nucleotide
5.5% NaCl (final concentration, 6%) was inoculated with a sequence of L. monocytogenes strain EGDe. The PCR product was
preculture grown in BHI medium, or IMM with or without 3.5% digested with the HindIII and BamHI restriction enzymes and was
NaCl or with 0.6 M xylose was inoculated with a preculture grown cloned into similarly digested plasmid pHT315. Inserts of two
in IMM. Betaine (1 mM) or carnitine (1 mM) was added to IMM plasmids were sequenced.
containing NaCl when required. The cultures were incubated with
shaking at 37°C. Growth rate experiments were performed with a Construction of a Ctc Insertional Mutant
Microbiology Reader Bioscreen C (Labsystems, Helsinki, Finland) The ctc gene was insertionally inactivated by a simple
in 100-well sterile microplates, each well containing 300 µl of recombination event using the temperature-sensitive suicide vector
culture medium. The OD600 was monitored. Experiments were pAUL-A. A 435-bp internal ctc fragment was PCR amplified from
performed at least in duplicate and were repeated independently, chromosomal DNA with primers OD3 and OD4 with the
twice for heat stress experiments and three times for osmotic stress incorporation of two restriction sites, HindIII and BamHI. The
experiments. purified PCR product was digested with HindIII and BamHI, ligated
Antibiotics were used at the following concentrations: into similarly digested plasmid pAUL-A, and then transformed
ampicillin at 100 µg ml-1 for E. coli, and erythromycin at 5 µg ml- into E. coli. The resultant plasmid pAUL-CTC was used to create
1
and rifampin at 200 µg ml-1 for L. monocytogenes. a knockout in ctc by homologous recombination with the L.
monocytogenes chromosome as described previously. In the resulting
Cloning and Sequencing mutant, 135 bp in the 3' end of ctc was deleted. Southern blot
Plasmids were prepared using the Plasmid Midi kit (Qiagen, analysis and PCR (data not shown) confirmed the authenticity of
S.A., Courtabuf, France). Bacterial chromosomal DNA was isolated a single integration of pAUL-CTC in the chromosome of L.
as described previously. Restriction endonucleases, T4 DNA ligase, monocytogenes strain LO28. The stability of the pAUL-A insertion
and Taq polymerases were used as recommended by the was confirmed by PCR analysis of cultures grown in BHI medium
manufacturer (Roche Molecular Biochemicals, Mannheim, with or without erythromycin selection at 37°C.
Germany). DNA restriction fragments were purified from agarose
RNA Isolation and Analysis
gels by using the QIAquick gel extraction kit (Qiagen).
Oligonucleotides were synthesized by MGW-Biotech. Plasmids RNA samples were prepared from 10 ml of mid-logarithmic-
were introduced into E. coli by standard methods, while for L. phase cells (OD600, 0.5) grown in BHI medium or IMM, 30 min
monocytogenes, electroporation was achieved as described after the addition (or not) of NaCl (3.5% in IMM and 5.5% in BHI
previously. medium). After the shock, cells were harvested by centrifugation at
7,000 x g for 4 min at 4°C. Cells were treated with rifampin and
DNA sequencing was performed with the BigDye terminator
RNAprotect Bacteria reagent (Qiagen) and centrifuged at 7,000 x
cycle sequencing ready reaction kit (Applied Biosystems, Courtabuf,
g for 10 min at 4°C. The pellet was resuspended in 400 µl of buffer
France), and sequences were analyzed with an automatic DNA
S (10% glucose, 12.5 mM Tris [pH 7.6], 66 mM EDTA, 0.5% sodium
sequencer (ABI Prism 310 genetic sequencer; Applied Biosystems).
dodecyl sulfate) containing 10 mg of lysozyme ml-1, 200 µg of
Searches for sequence homology were performed with the FASTA
proteinase K ml-1, and 200 µg of rifampin ml-1. After the addition
program. Sequencing of the ctc gene of L. monocytogenes LO28 was
of glass beads, cells were subjected to mechanical disruption. RNA
performed as follows. A 1,185-bp chromosomal DNA fragment
was purified by a Trizol extraction, two chloroform-isoamyl alcohol
containing the ctc gene was amplified by PCR using primers OD1
extractions, and an isopropanol precipitation. The final RNA pellet
50 Introductory Food Microbiology Role of Ctc from Listeria Monocytogenes in Osmotolerance 51

was dissolved in water, treated with DNase, and quantified The ctc gene starts at an ATG codon, 9 nucleotides downstream
spectrophotometrically before storage at -70°C. The integrity and of a potential ribosome binding site (5' GGAG 3'), and ends with
the relative concentrations of RNA samples were checked by a TAA stop codon. The deduced polypeptide contains 207 residues
agarose gel electrophoresis and ethidium bromide staining. with a calculated molecular weight of 22,654 (pI 4.14). Only one
The mRNA of ctc was semiquantitatively analyzed by a slot amino acid differs between the Ctc sequences of L. monocytogenes
blot technique as follows. RNA samples were treated at 65°C in LO28 and Listeria innocua CLIP 11262 (Lys201® Asn substitution
20% formaldehyde for 15 min. The samples were then vacuum in L. innocua Ctc protein). Homology searches revealed a significant
blotted with a Bio-Rad slot blot apparatus onto positively charged degree of similarity with the Ctc protein of B. subtilis (42% identity),
nylon membranes. RNA was cross-linked to the membrane by UV the equivalent protein, TL5, from Thermus thermophilus (36%
radiation. Transcription of ctc was monitored using an intragenic identity), and all members of the Ctc protein family identified
digoxigenin (DIG)-labeled probe generated by PCR with primers during different genome-sequencing projects. Like those of other
OD3 and OD4. Detection of the labeled probe was mediated by Ctc proteins, the N-terminal part of the L. monocytogenes Ctc protein
addition of an anti-DIG alkaline phosphatase-conjugated enzyme presents similarities with members of the 50S ribosomal protein
and CDP-star substrate (Roche Molecular Biochemicals). Light L25 family, for instance, 28% identity with the L25 protein of E.
emission was captured by standard autoradiography (Hyperfilm; coli, RplY.
Amersham Biosciences). A 16S rRNA probe was used to control A putative sB-dependent promoter was found 45 bp upstream
the loading uniformity of RNA extracted under different conditions from the ATG start codon (5' GTTT-N15-GGGTAG 3') based on a
of stress. comparison with the s B-dependent consensus promoter (5'
GTTT[15/16 nt]GGGTAA3'). A stem-and-loop structure
Electron Microscopy (ATGGAAGATTCGCA TTTGTT TGCGATATCTTCCAT; DG = -77
Stationary-phase-grown bacteria were processed for kJ mol-1) followed by a T track is located 16 bp downstream from
observation by scanning electron microscopy (SEM). Bacteria were the TAA stop codon of the ctc gene. This palindromic structure is
fixed for 1 h with 4% glutaraldehyde in a 0.2 M cacodylate buffer. a putative transcriptional terminator. Analysis of the regions
Cells were dehydrated using a graded ethanol series (70, 95, and surrounding the ctc gene in strain EGDe revealed an upstream
100% ethanol, three times for 10 min each time) and subjected to gene (lmo00210) transcribed in the opposite direction and encoding
an acetone dehydration series of 30, 50, 70, and 100% acetone for a putative lactate dehydrogenase (67% identity with Lactobacillus
10 min each. One drop was spread on a microcover, coated with casei lactate dehydrogenase). A gene (lmo00212) encoding a protein
gold in an Emscope SC500, and observed with the Philips SEM with no significant similarities with any of the proteins recorded
505. in the databases is located downstream of the ctc gene and in the
same direction of transcription. This gene is separated from the ctc
RESULTS gene by the putative terminator mentioned above.
Sequence Analysis of the Ctc Gene of L. Monocytogenes LO28
Transcriptional Analysis of the Ctc Gene
Primers designed by using the sequences surrounding the ctc
In a previous study, the Ctc protein was identified as a protein
gene (lmo00211) of L. monocytogenes strain EGDe, whose genome
showing high induction after salt stress. Moreover, as described
has been entirely sequenced, were used to amplify the ctc gene of
above, we found a putative sB promoter in front of the ctc gene, and
strain LO28 by PCR. The PCR yielded one band, and the nucleotide B
transcription is strongly induced after an osmotic upshift.
sequence of this DNA fragment revealed 100% identity between
Therefore, semiquantitative analysis of the ctc gene transcription
the ctc sequences of strains LO28 and EGDe.
52 Introductory Food Microbiology Role of Ctc from Listeria Monocytogenes in Osmotolerance 53

was carried out using slot blots with RNA extracted from cultures compatible solutes such as glycine betaine or carnitine. In order to
of strain LO28 grown in rich or minimal medium before or after test if the ctc mutation still had an effect in the presence of
an osmotic shock. Similar results were obtained in both media. osmoprotectants, we added 1 mM glycine betaine or carnitine to
Under growth conditions without added NaCl, constitutive IMM supplemented with 3.5% NaCl. Addition of glycine betaine
expression of ctc was observed. Thirty minutes of exposure to NaCl nearly allowed strain LO28 and the ctc mutant to recover the
(3.5% in IMM and 5.5% in BHI medium [final concentration, 6%]) growth rate of strain LO28 cultivated in IMM without NaCl. The
resulted in an increase in the level of transcription. effect of the addition of carnitine was intermediate, but no
significant difference between the growth rates of the two strains
Role of Ctc in Osmotic and High-temperature Adaptation was observed in this situation.
To investigate the function of the ctc gene of L. monocytogenes In order to test if the Ctc protein was involved in general stress
LO28, it was mutated by insertional mutation by using the pAUL- tolerance, the growth of the ctc mutant versus LO28 was measured
A plasmid, a temperature-sensitive suicide vector, as described in under heat stress conditions at 45°C in BHI medium and IMM.
Materials and Methods. Phenotypic analysis did not show any Under these conditions, the growth of the mutant was similar to
difference between the ctc mutant and the wild-type strain with that of the wild-type strain. This suggests that the role of Ctc in
respect to the aspect of colonies, catalase, hemolysis on blood agar, stress tolerance is restricted to osmotic tolerance.
and metabolic profiles, as determined by using API-CH50
microplate assays. The Morphology of the Ctc Mutant is Impaired during Stationary
Phase in Minimal Medium Containing NaCl
The stress tolerance of the ctc mutant was compared with that
of strain LO28. Because the Ctc protein was primarily identified The wild-type strain LO28 and the ctc mutant were
as a protein induced by an osmotic upshift, we first examined subsequently examined using photonic microscopy during the
whether the absence of Ctc would have any effect on the ability exponential and stationary phases of growth at 37°C in BHI medium
of L. monocytogenes to grow under conditions of high osmotic and IMM with or without NaCl. No difference in morphology was
strength in a rich (BHI) or minimal (IMM) medium. Growth rates observed between the two strains during growth in BHI medium
(doubling times) were found identical for the wild-type strain and with or without NaCl or in IMM without NaCl (data not shown).
the ctc mutant in BHI medium (55 ± 5 min). No significant difference This was confirmed by transmission electron microscopy (TEM)
was found between the growth rates of the two strains after the using negative staining and by SEM by examining 48-h-grown
addition of NaCl to BHI medium (85 ± 10 min for the wild-type cultures (data not shown). In these three different media, bacteria
strain and 110 ± 20 min for the ctc mutant) or in IMM without appeared as small rods measuring 1 to 2 µm in length. In contrast,
added NaCl (100 ± 15 min for the wild-type strain and 125 ± 15 in IMM containing 3.5% NaCl, the ctc mutant displayed a different
min for the ctc mutant). However, in IMM supplemented with 3.5% morphology than the wild-type strain as observed by photonic
NaCl, the growth of the ctc mutant was significantly impaired. microscopy, TEM (data not shown), and SEM. After 48 h of growth
in this medium, the morphology of strain LO28 was characterized
The growth rate reached 620 ± 140 min, whereas it was 270
by a rod shape with a variable size ranging between 1 and 4 µm.
± 15 min for the wild-type strain. In order to test if the sensitivity
Approximately 1% of the cells displayed a bent rod shape. The ctc
of the ctc mutant was linked to salt stress or osmotic stress, growth
mutant also displayed a rod shape with a variable size ranging
was performed in IMM supplemented with 0.6 M xylose. An
between 1 and 6 µm, but 80% of the cells had a bent or twisted rod
average increase of 95% for the growth rate of the ctc mutant was
shape. The morphology of the ctc mutant did not differ significantly
obtained. The ability of L. monocytogenes to survive high salt
from that of the LO28 strain when 1 mM glycine betaine was
concentrations is attributed mainly to the accumulation of
added to the medium (data not shown).
54 Introductory Food Microbiology Role of Ctc from Listeria Monocytogenes in Osmotolerance 55

We have shown that the ctc gene is involved in the resistance controlled by mechanisms distinct from accumulation of compatible
of L. monocytogenes to high osmolarity in the absence of solutes. The last gene which has clearly been associated with
osmoprotectants such as glycine betaine and carnitine in the osmotolerance in L. monocytogenes is sB. The absence of sB impaired
medium. Thus, a ctc insertional mutant grew twice as slowly as the the ability of L. monocytogenes to use glycine betaine or carnitine as
wild-type strain LO28 under conditions of high osmolarity (0.6 M an osmoprotectant and impaired the transport of glycine betaine.
NaCl or xylose) in minimal medium. The transport of carnitine has not been studied. A potential sB-
Moreover, the morphology of the ctc mutant was impaired in dependent promoter has been identified upstream of the betL gene
this growth condition. Whereas the morphology of the wild-type and the opuC operon. This suggests that sB plays a key role in
strain LO28 was characterized by a rod shape, the ctc mutant osmotolerance of L. monocytogenes via regulation of the expression
morphology was characterized by a bent or twisted rod shape of two major osmoprotectant transport systems. We have identified
under osmotic stress conditions. When glycine betaine or carnitine, a putative sB-dependent promoter upstream of the ctc gene. In
known to be the most efficient osmoprotectants in L. monocytogenes, contrast to that of B. subtilis, the L. monocytogenes ctc gene does not
was added to this medium, the growth of the mutant became seem to belong to an operon. Moreover, expression of the ctc gene
identical to the growth of its isogenic parent strain. This can is strongly induced by an osmotic upshift, like that of the sB gene.
explain why the growth rates of the mutant and the wild-type Taken together, these observations suggest that the ctc gene may be
strain were identical in rich medium (BHI) supplemented with regulated, at least in part, at the transcriptional level by sB. This
5.5% NaCl. The BHI medium contains carnitine, which is relatively emphasizes the role of sB, which is probably a key regulator of
abundant in some mammalian tissues. The role of ctc in stress osmotolerance in L. monocytogenes in the presence or absence of
tolerance seems to be restricted to osmotic stress tolerance, since no compatible solutes in the environment.
difference between the wild-type strain and the ctc mutant was Currently, the function of the Ctc proteins is unknown. Our
observed under conditions of growth at high temperatures in rich results suggest that the Ctc protein of L. monocytogenes belongs to
or minimal medium. a novel system utilized by this bacterium to adapt to an osmotic
Few genes involved in salt stress tolerance have been identified upshift in the absence of an osmoprotectant. This is the first time
in L. monocytogenes until now. Survival of L. monocytogenes at high that a role has been assigned to the Ctc protein, whose gene is
salt concentrations is attributed mainly to the accumulation of widely distributed in bacterial genomes. The product of the ctc
three osmoprotectants, glycine betaine, carnitine, and proline. gene of L. monocytogenes, like other ctc gene products, presents
Independently of genes involved in the transport or biosynthesis similarities in its N-terminal part with the 50S ribosomal L25
of osmoprotectants, two genes encoding proteins of the Clp family protein and consequently belongs to the L25 ribosomal protein
have been identified, clpC and clpP. Inactivation of these genes family. According to the COG database, which compares the protein
confers a general stress sensitivity phenotype, including salt stress sequences encoded in 43 complete genomes, representing 30 major
sensitivity, on the corresponding mutants. These genes are known phylogenetic lineages, no L25 homologue is present in the archaeal
to encode general stress proteins, chaperones assisting the proper genomes, but L25 homologues are present in nearly all eubacterial
folding, refolding, or assembly of proteins and proteases processing genomes.
those that cannot be refolded. A recent study identified relA, a gene L25 homologues are found in all gram-negative bacteria and
encoding a (p)ppGpp synthetase, as a gene involved in in all gram-positive bacteria except Lactococcus lactis, Streptococcus
osmotolerance. The authors showed that (p)ppGpp is involved in pyogenes, Mycoplasma pneumoniae, and Mycoplasma genitalium. The
the growth of L. monocytogenes under high osmotic pressure and sequence homologies observed between the Ctc proteins and the
that the intracellular accumulation of (p)ppGpp is probably L25 proteins include many conserved residues, which the 5S rRNA-
56 Introductory Food Microbiology Identification of Listeria Monocytogenes Genes 57

L25 structure confirmed to be involved in the rRNA-protein


binding interaction, thereby confirming that these two groups of
proteins are strongly related. It is highly probable that the Ctc
proteins are associated at least in their N-terminal parts with the
ribosome and bind the 5S rRNA. Ribosomes have been implicated
as sensors of heat and cold shock in E. coli. Recent results implicated
the ribosome as a possible mediator of the activity of Obg, an 3
essential GTP-binding protein, and the stress induction of sB,
suggesting that ribosomes are also general sensors in B. subtilis. We
can hypothesize that the ribosome is also a sensor of salt stress,
IDENTIFICATION OF LISTERIA
at least in L. monocytogenes, through the activity of Ctc. Further MONOCYTOGENES GENES
investigations will be required to clarify the function of Ctc in L.
monocytogenes and in other bacteria.
The capacity of Listeria monocytogenes to tolerate salt and
alkaline stresses is of particular importance, as this pathogen is
often exposed to such environments during food processing and
food preservation. We screened a library of Tn917-lacZ insertional
mutants in order to identify genes involved in salt and/or alkaline
tolerance. We isolated six mutants sensitive to salt stress and 12
mutants sensitive to salt and alkaline stresses. The position of the
insertion of the transposon was located in 15 of these mutants. In
six mutants the transposon was inserted in intergenic regions, and
in nine mutants it was inserted in genes. Most of the genes have
unknown functions, but sequence comparisons indicated that they
encode putative transporters.

INTRODUCTION
Listeria monocytogenes is a food-borne pathogen that is widely
distributed in the environment. This microorganism is of particular
concern in the food industry because of its ability to survive, and
frequently to grow, under a wide range of adverse conditions used
to preserve food, such as low temperature, low pH, and high
osmolarity, or used to clean and sanitize equipment, such as high
pH. Growth of L. monocytogenes has been reported at NaCl
concentrations as high as 10% and at pHs as high as 9.
There is little information on the mechanisms that allow this
bacterium to cope with alkaline environments. Knowledge
concerning the mechanisms used by gram-positive bacteria for
adaptation and growth at alkaline pHs comes mainly from studies
58 Introductory Food Microbiology Identification of Listeria Monocytogenes Genes 59

of alkaliphilic strains of Bacillus species, such as Bacillus halodurans and a ClpP serine protease, respectively, have been identified.
C-125 or Bacillus pseudofirmus OF4. There is strong evidence that Inactivation of these genes conferred a general stress sensitivity
monovalent cation-proton antiporters are essential for maintaining phenotype, including sensitivity to salt stress, to the corresponding
a neutral cytoplasmic pH and, therefore, for growth under alkaline mutant. In a recent study workers identified relA, a gene encoding
conditions. In addition, the acidic cell wall polymers teichuronic a (p)ppGpp synthetase, as a gene involved in osmotolerance via
acid and teichuronopeptides contribute to pH homeostasis. These a mechanism different from the mechanism involving accumulation
wall macromolecules may provide a passive barrier to ion flux and of compatible solutes. It has also been shown that the general
elevation of the cytoplasmic buffering capacity at highly alkaline stress protein Ctc of L. monocytogenes is involved in osmotolerance
growth pHs. In Bacillus subtilis, monovalent cation/proton in the absence of any compatible solutes in the environment.
antiporters also seem to be important since the mrpA gene encoding In order to obtain a better understanding of the mechanisms
an Na + /H + antiporter and the tetA(L) gene encoding a involved in salt and alkaline tolerance, we used a library of
multifunctional tetracycline-metal/H+ antiporter that also exhibits transposon insertional mutants of L. monocytogenes LO28 to isolate
monovalent cation/H+ antiport activity are involved in Na+- mutants with decreased NaCl and/or alkaline tolerance. We
dependent pH homeostasis. succeeded in identifying different mutants that exhibit less
Most bacteria cope with elevated osmolarity in the environment resistance to salt and/or alkaline stress than the parental strain
by intracellular accumulation of particular osmolytes called and characterized the genes interrupted.
compatible solutes. These compatible solutes act in the cytosol by
counterbalancing the external osmolarity, thus preventing water MATERIALS AND METHODS
loss from the cell and plasmolysis without adversely affecting Bacterial Strains
macromolecular structure and function. Compatible solutes can be
The L. monocytogenes strains used were LO28, a clinical isolate
either transported into the cell or synthesized de novo. Survival of
of serotype 1/2C, and a library of Tn917-lacZ mutants of strain
L. monocytogenes at high salt concentrations is attributed mainly to
LO28. Bacterial plasmids were propagated in Escherichia coli strain
the accumulation of three compatible solutes: glycine betaine,
TG1.
carnitine, and proline. Accumulation of glycine betaine and
carnitine occurs via two glycine betaine transporters encoded by Culture Media and Stress Conditions
the betL gene and the gbu operon and one carnitine transporter
Cells were grown on complex culture media, including brain
encoded by the opuC operon. Disruption of these genes reduced
heat infusion (BHI) broth or agar (Difco Laboratories, Detroit, Mich.).
the osmotolerance of L. monocytogenes. Both betL and opuC have
Screening of the library of Tn917-lacZ mutants for sensitivity to
putative sB-dependent promoters. The absence of sB impaired the
salt and alkaline stresses was performed as follows. Wells of
ability of L. monocytogenes to use glycine betaine or carnitine as a
microplates containing 100 µl of BHI medium with erythromycin
compatible solute.
(BHI-erm) were inoculated with the different mutants. The
Proline transport has not been characterized yet. However, microplates were incubated at 37°C overnight and subsequently
disruption of the proBA operon (proline biosynthesis-encoding used to transfer the mutants, after 1:1 dilution with BHI-erm, onto
operon) reduced the growth of the corresponding mutant at high agar plates with a replicator. The plates used contained BHI-erm
salt concentrations. Little information is available concerning other with or without 5.5% NaCl (final concentration, 6%) and BHI-erm
mechanisms that L. monocytogenes uses to cope with salt stress, adjusted to pH 8.6 with NaOH. The growth of the mutants was
especially when compatible solutes are not available in the recorded after 48 h. Mutants selected after this first step were used
environment. Two genes, clpC and clpP, encoding a ClpC ATPase to perform liquid growth experiments with a Microbiology Reader
60 Introductory Food Microbiology Identification of Listeria Monocytogenes Genes 61

Bioscreen C (Labsystems, Helsinki, Finland) in 100-well sterile (Promega France, Charbonnières, France), a 3' T-end vector
microplates, and each well contained 300 µl of culture medium, as specifically designed for cloning PCR fragments, by following the
follows. manufacturer’s specifications. DNA sequencing was done with a
Overnight cultures of Tn917-lacZ mutants in BHI-erm were BigDye terminator cycle sequencing Ready Reaction kit (Applied
used to inoculate different media (BHI-erm with or without 5.5% Biosystems, Courtaboeuf, France). The reactions were performed
NaCl and BHI-erm with the pH adjusted to 8.5) at an initial optical with unlabeled primers and fluorescent dideoxynucleotides, and
density at 600 nm of ~0.1. The cultures were incubated with shaking then the reaction mixtures were analyzed with an automatic DNA
at 37°C. The optical density was monitored at 600 nm. Experiments sequencer (ABI Prism 310 genetic sequencer; Applied Biosystems).
were repeated independently at least twice. The LO28 strain was Blast sequence homology analyses were performed by using the
used as a control and was inoculated into BHI medium lacking National Center for Biotechnology Information network service.
erythromycin. For more detailed physiological characterization of The primers used for the sequence were RG1 (5'-
mutants sal5 and sal11, liquid growth experiments were performed CCCACTAAGCGCTCGGG-3'), RG7, and RG9. Oligonucleotides
by using the same procedure except that the pH of BHI medium were synthesized by MGW-Biotech (Courtaboeuf, France).
was adjusted to 8.5 with a glycine-NaOH-NaCl buffer. Growth
RESULTS
experiments were also performed in BHI medium supplemented
with 7% KCl or 15% xylose and were repeated independently at Selection of Salt and Alkaline Stress-sensitive Mutants
least four times. Growth curves were fitted by using a modified A library of approximately 2,500 Tn917-lacZ insertion mutants
Gompertz equation, and the generation time was calculated by was screened for salt and/or alkaline stress sensitivity. Each mutant
using nonlinear regression with the Statistica statistical software was grown on BHI-erm plates with or without 5.5% NaCl or with
(Statsoft, Tulsa, Okla.). the pH adjusted to 8.6. Twenty-three mutants showed a growth
Antibiotics were used at the following concentrations: 100 µg delay on at least one of the two stress media. Six mutants seemed
of ampicillin ml-1 for E. coli and 5 µg of erythromycin ml-1 for L. to be affected under salt stress conditions, nine mutants seemed to
monocytogenes. be affected under alkaline stress conditions, and eight mutants
seemed to be affected under both conditions. The phenotypes of
Identification of Transposition Target the mutants were further confirmed in liquid growth experiments
Inverse PCR was used to amplify the DNA fragment next to by comparing the growth curves to that of the LO28 wild-type
the region downstream from the Tn917-lacZ chromosomal strain. Five mutants were removed after this second step. The
insertions. Bacterial chromosomal DNA was isolated as described growth of two mutants sensitive to both stresses was impaired in
previously. Chromosomal DNA was digested with HindIII for BHI medium, and the alkaline sensitivity phenotype of three
mutants sal1, -2, -5, -6, -11, -17, -22, and -23 and mutants sl7, -10, mutants was not confirmed. Finally, we isolated six mutants
-13, -14, and -25 and with NdeI for mutants sl12 and sal21 and was sensitive to salt stress and 12 mutants sensitive to both salt stress
subsequently circularized by self-ligation. The region downstream and alkaline stress. The designations of mutants that were sensitive
from the Tn917-lacZ insertion was amplified by using primers only to salt stress begin with sl (for salt sensitivity locus), and the
RG7 (5'-ATTCCGTCTGAAGCAGTGGT-3') and RG9 (5'- designations of mutants that were that were sensitive to salt stress
GAACGCCGTCTACTTACAAG-3') for HindIII-digested DNA and and alkaline stress begin with sal (for salt and alkaline sensitivity
primers RG9 and RG11 (5'-GAATCACGTGTCCCTTTGCG-3') for locus). Southern hybridization with HindIII-digested chromosomal
NdeI-digested DNA. Amplification products were sequenced either DNA and, when required, EcoRI-digested chromosomal DNA with
directly or after they were cloned into the pGEM-T plasmid a digoxigenin-labeled DNA probe specific for the Tn917-lacZ
62 Introductory Food Microbiology Identification of Listeria Monocytogenes Genes 63

transposon revealed that the 18 mutants contained a single copy other organisms. lmo1443 has orthologues in different gram-
of the transposon and that the loci corresponded to 18 independent positive bacteria, and lmo2232 has a paralogue (lmo2399) and five
insertion loci (data not shown). The 18 remaining mutants were orthologues in B. subtilis yhdP, yrkA, yqhB, yugS, and yhdT and in
kept for further characterization. other eubacteria. Considering the orientation of the genes and the
presence of putative terminators, the phenotypes of these four
Identification of the Transposition Target mutants could not be linked to a polar effect of the mutation on
The inverse PCR method enabled us to clone and sequence the downstream genes. This was not the case for sal5, in which the
downstream transposon-chromosome junctions of 15 mutants. We transposon was inserted into lmo0992, a gene of unknown function
did not succeed in cloning the junctions of three mutants, sal3, - located upstream from lmo0991 which encodes a protein with
4, and -19. The sequences were compared with the complete similarities to NtpJ of Enterococcus hirae, a K+/Na+ transporter.
genome sequence of L. monocytogenes strain EGDe. In nine mutants, In these five mutants, similarity searches could not assign a
the transposon was inserted into open reading frames, whereas it putative function to the genes interrupted, but a search for
was inserted into intergenic regions in six mutants. transmembrane domains with the DAS program revealed that all
Genes Encoding Proteins with an Identified Function or a Putative corresponding proteins have putative transmembrane domains.
Function
Intergenic Regions
In the sl12 mutant, the transposon was inserted into the mutS
For six mutants, the position of the insertion of the transposon
gene, which is involved in DNA mismatch repair, whereas in
was located in intergenic regions.
mutants sal1 and -11 and sl14 it was inserted into transporters or
putative transporters. In sal1, the transposon was inserted into the The physiology of two mutants, sal5 and sal11, which were
ATPase subunit of an ATP binding cassette transporter, and in identified as sensitive to salt and alkaline stresses during the
sal11 it was inserted into the permease subunit of another ATP screening analysis, was characterized by quantifying the growth
binding cassette transporter. In sl14, the transposon was inserted of these organisms in different media.
into the mdrL gene, which encodes a multidrug efflux transporter Physiology of the sal5 and sal11 Mutants in Response to Osmotic
of the major facilitator superfamily. If the orientation of the genes and Alkaline Stresses
and the presence of putative terminators were taken into account,
Growth of the sal5 and sal11 mutants was examined by using
the phenotypes of mutants sal1 and -11 and sl14 could not be
BHI medium, BHI medium supplemented with 5.5% NaCl, 7%
linked to a polar effect of the mutation on downstream genes.
KCl, or 15% xylose, and BHI medium with the pH adjusted to 8.5.
However, the phenotype of mutant sl12 could be linked to a polar
For the growth experiments under osmotic stress conditions, the
effect of the mutation of a downstream gene, mutL coding for a
concentration of each of the three solutes (NaCl, KCl, and xylose)
DNA mismatch repair protein or lmo1405 coding for a putative
was approximately 1 M. The results showed that the phenotype
antiterminator regulatory protein.
identified during the screening analysis was confirmed. Both
Genes Encoding Proteins with Unknown Functions mutants were sensitive to NaCl stress and alkaline stress. However,
the sensitivity was far more pronounced with the alkaline stress
In sl7 and sal22, the transposon was inserted into the lmo1432
conditions; under these conditions the growth rate (doubling time)
gene at positions separated by 29 bp. The deduced protein encoded
of both mutants was approximately 300 min and was fivefold
by the lmo1432 gene is specific to Listeria. In sal2 and -17, the
greater than the growth rate of the wild-type strain. Under NaCl
transposon was inserted into two genes, lmo1443 and lmo2232,
osmotic stress conditions, the growth rates of mutants sal5 and
respectively, which encode proteins having unknown functions in
64 Introductory Food Microbiology Identification of Listeria Monocytogenes Genes 65

sal11 were 1.4- and 1.3-fold greater, respectively. Moreover, both protocol. In sal6, the transposon was inserted 109 bp upstream
mutants were also sensitive to KCl stress (but to a lesser extent) from the putative promoter of the gbu operon. This result indicates
and slightly sensitive to xylose stress. that regions upstream from the putative promoter are probably
involved in the transcription of this operon. In B. subtilis, two
DISCUSSION promoters have been identified in front of the opuA operon, and we
We isolated six Tn917-lacZ insertional mutants of L. hypothesize that this is also the case for L. monocytogenes.
monocytogenes strain LO28 sensitive to salt stress (sl mutants) and Surprisingly, the sal6 mutant was found to be sensitive to salt and
nine mutants sensitive to both salt and alkaline stresses (sal alkaline stresses. However, the alkaline phenotype is weak. Using
mutants) and located the positions of the insertions of the two-dimensional electrophoresis, we identified the GbuA protein,
transposons in the genomes of the different mutants. We used the the ATPase subunit of the Gbu transporter, as a protein induced
complete genome sequence of L. monocytogenes strain EGDe to by salt stress. In L. monocytogenes, expression of the sB factor is
rapidly identify the positions of the insertions of the transposons. strongly induced by salt stress, and consequently, expression of
We sequenced 15 fragments corresponding to a total of 6,249 the genes under the control of this sigma factor is also induced by
nucleotides and found only 4 nucleotides which differed in strains salt stress. However, no sB promoter recognition sequences are
LO28 and EGDe. These results suggest that the DNA sequences of present in front of the gbu operon, indicating that another salt
these two strains are very similar and justify utilization of the induction pathway is present in L. monocytogenes.
complete genome sequence of L. monocytogenes strain EGDe to
Genes with known Functions not Directly Linked to Salt Stress
study the L. monocytogenes strain LO28 genome.
In mutants sl12 and sl14 the transposon was inserted into the
On the basis of the functions or putative functions of the genes
mutS and mdrL genes, respectively. The mutSL locus of L.
disturbed by insertion of the transposons, we classified the mutants
monocytogenes is involved in both mismatch repair and homologous
in four categories.
recombination. The mdrL gene encodes a multidrug efflux
Genes with known Functions Directly Linked to Salt Stress transporter and is involved in the efflux of ethidium bromide. In
both cases, no salt stress sensitivity of the mdrL or mutSL mutant
In mutants sal6 and sl10, the transposon was inserted in front
has been mentioned previously; thus, this is the first time that
of the gbu operon. This operon, which is very similar to the opuA
these two loci have been associated with salt stress. MdrL extrudes
operon of B. subtilis, encodes an ATP-dependent transporter
different toxic components, and we hypothesize that this transporter
belonging to the ATP binding cassette transporter superfamily and
also extrudes Na+, perhaps in a nonspecific manner.
is involved in the transport of glycine betaine. A mutant with this
operon inactivated was found to be salt sensitive. Ko and Smith
GENES ENCODING PUTATIVE TRANSPORTERS
also identified sequences with significant similarities to the sA-
type -35 and -10 promoter recognition sequence TTGTGT-N15- In mutants sal1 and sal11, even though the function is unknown,
TATTGC. In the sl10 mutant, the transposon was located between sequence comparisons indicated a putative function. In both
the putative promoter and the coding DNA sequence of the gbu mutants, the transposons were inserted into genes encoding
operon. In this mutant, the promoter of the gbu operon is separated domains of two distinct ATP binding cassette transporters. In sal1,
from the gbu operon by the transposon. the gene interrupted, ykpA, encodes the ATP binding protein
domain of the transporter, and in sal11 the gene interrupted, lmo668,
Consequently, the gbu operon cannot be transcribed. The salt
encodes the permease protein domain. ykpA has orthologues in
stress sensitivity of the sl10 mutant confirmed the results of Ko and
gram-positive bacteria. The corresponding protein has been
Smith. This result provides good validation of our screening
identified in Staphylococcus aureus as an immunodominant antigen,
66 Introductory Food Microbiology Identification of Listeria Monocytogenes Genes 67

and antisense ablation of the ykpA gene led to a growth-inhibiting functions with the V-type Na+-ATPase at high pH and/or high
effect. Na+ concentrations in order to eliminate sodium ions and to drive
The lmo668 gene is located downstream from its putative potassium ion uptake. We found no similarities between the
associated ATP binding protein. Lmo668 has very significant sequences of lmo0990, lmo991, and lmo992 and the sequences of
similarities with Yadh of gram-negative bacteria. In these cases it the ntp genes encoding the V-type Na+-ATPase, and further work
is highly probable that ykpA and lmo0668 encode transporters, but is needed to check if there is an analogy of function among the
the transported solutes remain unknown. The transporters genes.
probably extrude Na+ and/or H+, perhaps in a nonspecific manner. Genes with Unknown Functions
Growth experiments with mutant sal11 indicated that the lmo0668
For mutants sal2, sl7, sal22, and sal17, the function of the gene
gene is probably involved in pH homeostasis because the sal11
interrupted is completely unknown, but the three genes interrupted
mutant was highly sensitive to alkaline pH, moderately sensitive
encode proteins with putative transmembrane domains. The
to NaCl stress, and slightly sensitive to KCl stress and xylose
transposon was inserted into the same gene, lmo1432, in mutants
stress. The differences observed among the effects of the three
sl7 and sal22. This interrupted gene seems to be specific to the
osmolytes, NaCl, KCl, and xylose, which were added at
genus Listeria. In sal2 the interrupted gene seems to be specific to
concentrations of approximately 1 M to the medium, were probably
gram-positive bacteria, whereas in sal17 it seems to be specific to
due to the fact that NaCl tends to be more stressful than KCl and
eubacteria. In mutants sal21 and sl25 the transposon was inserted
the fact that xylose provides only one-half the osmotic stress of
in front of putative operons encoding proteins with unknown
NaCl and KCl.
functions.
The last mutant in which expression of genes encoding putative
We were not able to identify sA-type or sB-type promoter
transporters are disturbed is mutant sal5. lmo0992, the gene
recognition sequences in front of these operons. However, we
interrupted by the transposon, belongs to an operon consisting of
hypothesize that in these two cases transcription of the operons
five genes. The first gene, lmo0989, encodes a putative
was eliminated by insertion of the transposon in the promoter
transcriptional regulator of the MarR family. The next three genes,
region and that the phenotypes of the mutants are linked to at least
lmo0990, lmo0991, and lmo0992, encode proteins whose functions
one of the genes of the operons.
are unknown but which are putative integral membrane proteins.
Lmo0991 and Lmo0992 are very similar to YkoY of Bacillus Finally, the salt sensitivity phenotype of mutant sl13 and the
megaterium, B. subtilis, and Bacillus anthracis. The last gene, lmo0993, salt and alkaline sensitivity phenotype of mutant sal23 are difficult
encodes a protein with similarity to the NtpJ protein of E. hirae to explain because in sl13 the transposon was inserted between
(33% identity). The ntpJ gene encodes a component of the KtrII two convergent genes and in sal23 it was inserted between the end
uptake system. NtpJ mediates K+ and Na+ cotransport. and the terminator of the lmo1140 gene.
The growth of an ntpJ-disrupted mutant is impaired at pH 10 We did not identify the relA gene during our screening
in K+-limited medium. The phenotypes of an ntpJ-disrupted mutant procedure. This gene encodes a (p)ppGpp synthase and was recently
and the sal5 mutant are similar; the sal5 mutant is highly sensitive identified during a screening procedure very similar to our
to alkaline pH in BHI medium. Thus, the phenotype of the sal5 procedure. However, this is not surprising because a library of
mutant is probably due to a polar effect of the mutation on lmo0993, transposons cannot be exhaustive. Moreover, we did not succeed
the ntpJ-like gene. In E. hirae, the ntpJ gene is the last gene of an in identifying the positions of insertion of the transposons in three
operon (ntp) encoding a V-type Na+-ATPase, which is important mutants, and we cannot exclude the possibility that one of these
for Na+ extrusion at high pH. The NtpJ K+/Na+ uptake system three mutants corresponds to a relA mutant.
68 Introductory Food Microbiology Identification of Listeria Monocytogenes Genes 69

We were interested in studying salt stress because it is one of cultures of S. thermophilus CNRZ 385 and its PrtS-negative mutant
the most commonly used methods for food conservation, and we were made in milk as well as mixed cultures of S. thermophilus and
were interested in studying alkaline stress because of the alkaline Lb. bulgaricus: S. thermophilus CNRZ 385 or its PrtS-negative mutant
nature of most of the detergents and some of the chemical sanitizers was associated with several strains of Lb. bulgaricus, including a
used to clean and sanitize equipment in food processing plants. PrtB-negative strain.
Little information is available concerning the physiology of L. The pH and growth of bacterial populations of the resulting
monocytogenes in response to alkaline stress. Growth of food factory mixed cultures were followed, and the Lactobacillus strain was
isolates was reported at pHs as high as 9, and this pathogen has found to influence both the extent of the benefit of Lb. bulgaricus/
been shown to be resistant to storage at pHs up to 12. It has also S. thermophilus association on milk acidification and the magnitude
been shown that alkaline pH induces cross-protection of L. of S. thermophilus population dominance at the end of fermentation.
monocytogenes against heat. Moreover, there is no information In all mixed cultures, the sequential growth of S. thermophilus then
concerning the mechanisms that take place in L. monocytogenes in of Lb. bulgarius and finally of both bacteria was observed. Although
order to cope with alkaline stress. It was interesting to study these proteinase PrtS was essential to S. thermophilus growth in milk in
two stresses in parallel because a combination of salt stress and pure culture, it had no effect on bacterial growth and thus on the
alkaline stress is more effective in decreasing the survival of L. final pH of mixed cultures in the presence of PrtB. In contrast,
monocytogenes than an individual type of stress. It is also known proteinase PrtB was necessary for the growth of S. thermophilus,
that homeostasis of Na+ and H+ ions is tightly linked, and in most and its absence resulted in a higher final pH. From these results,
cell membranes there are proteins that couple the fluxes of the two a model of growth of both bacteria in mixed cultures in milk is
ions via the Na+/H+ antiporter or the Ntp complex, for example. proposed.
During the screening procedure used in this study we identified
Streptococcus thermophilus is a thermophilic lactic acid bacterium
few genes involved in Na+ and/or alkaline stress tolerance. Further
(LAB), widely used as a starter to produce fermented dairy
work is needed to investigate the function of these genes, but the
products. It is generally used in association with other micro-
fact that they encode putative integral membrane proteins indicates
organisms, in particular with Lactobacillus delbrueckii subsp.
that they probably encode ion transporters.
bulgaricus (Lb. bulgaricus) for the manufacture of yoghurt. For this
CELL-WALL PROTEINASES PRTS AND PRTB HAVE A application, the fast-growing capacity of these bacteria in milk is
DIFFERENT ROLE IN STREPTOCOCCUS THERMOPHILUS / crucial to enable intense and rapid acidification of milk. LAB are
LACTOBACILLUS BULGARICUS MIXED CULTURES IN MILK fastidious micro-organisms, which have in particular several amino
acid auxotrophies. Most S. thermophilus strains are stimulated by
The manufacture of yoghurt relies on the simultaneous
the supply of two to five amino acids, whereas lactobacilli require
utilization of two starters: Streptococcus thermophilus and Lactobacillus
between three and 14 amino acids.
delbrueckii subsp. bulgaricus (Lb. bulgaricus). A protocooperation
usually takes place between the two species, which often results The optimal growth of LAB in milk thus depends on their
in enhanced milk acidification and aroma formation compared to proteolytic system, which hydrolyses milk caseins into peptides
pure cultures. Cell-wall proteinases of Lactococcus lactis and and amino acids. The cell-wall proteinases of LAB are of major
lactobacilli have been shown to be essential to growth in milk in importance in this process, as they are responsible for the first step
pure cultures. In this study, the role of proteinases PrtS from S. of casein breakdown. They belong to the same multi-domain
thermophilus and PrtB from Lb. bulgaricus in bacterial growth in proteinase family and show significant homologies, even though
milk was evaluated; a negative mutant for the prtS gene of S. differences in specificity, bacterial anchor and domain organization
thermophilus CNRZ 385 was constructed for this purpose. Pure have been described. The cell-wall proteinase of Lactococcus lactis
70 Introductory Food Microbiology Identification of Listeria Monocytogenes Genes 71

(PrtP), which is very frequent in this species, has been extensively The Escherichia coli strain was grown at 37 °C on Luria-Bertani
studied. In milk, Lc. lactis PrtP-negative strains only reach 10% of (Difco) medium with shaking, in the presence of erythromycin
the cell densities observed with PrtP-positive strains. In S. (Ery) (150 µg ml-1) when required.
thermophilus, the presence of a cell-wall proteinase, PrtS, recently Stock cultures of each strain of S. thermophilus and Lb. bulgaricus
characterized, is less common than in Lc. lactis. In this species, were prepared after growth at 42 °C on skim milk, supplemented
high cell-wall proteinase activities are associated with high milk- with yeast extract for proteinase-negative strains, from overnight
acidifying capacities. In Lb. bulgaricus, the cell-wall proteinase, PrtB, skim milk cultures, supplemented with yeast extract when required.
is also essential for optimal growth in milk; a proteinase-negative The pH was then measured, and bacterial numbers were estimated
strain reaches only 22% of the final biomass of a proteinase-positive by plating, with an automatic spiral plater, appropriate dilutions
strain when grown in milk. of the culture on agar medium: M17Lac was used for specific
In yoghurt, S. thermophilus and Lb. bulgaricus are grown in enumeration of S. thermophilus cells, and MRSLac pH 5·2,
association, which often results in a positive interaction. This supplemented with streptomycin when required, for specific
relationship, called protocooperation, has a beneficial effect on enumeration of Lb. bulgaricus cells.
growth of both species and on acid and aroma production. S.
For S. thermophilus strains and before dilution, chains of cells
thermophilus indeed produces pyruvic acid, formic acid and CO2,
were disrupted for 30 s in a mechanical blender. After 48 h
which stimulate the growth of Lb. bulgaricus. In turn, Lb. bulgaricus
incubation at 42 °C in anaerobic jars, cells were enumerated with
produces peptides and amino acids that stimulate S. thermophilus
the EC1 colony counter software. At the end of culture, bacteria
growth.
were directly frozen in liquid nitrogen and kept at -80 °C.
In the present study, we wished to determine the role of the
Growth rates of S. thermophilus 385 and 385-PrtS strains were
cell-wall proteinases PrtS from S. thermophilus and PrtB from Lb.
determined in M17 at 42 °C using a Microbiology Reader Bioscreen
bulgaricus in bacterial growth in milk. We therefore constructed a
C (Labsystems) in 100-well, sterile, covered microplates. Each well,
negative mutant for the cell-wall proteinase gene (prtS gene) of S.
containing 200 µl M17Lac, was inoculated at 1% with overnight
thermophilus CNRZ 385, which was recently sequenced and
M17Lac cultures of S. thermophilus and covered with one drop of
characterized in our laboratory. The latter mutant was used to
study the role of PrtS on the growth of S. thermophilus in pure paraffin oil. The optical density was measured at 600 nm every
culture in milk. We also took advantage of the availability of a 20 min, after gentle shaking. The apparent growth rate (µmax) was
PrtB-negative mutant of Lb. bulgaricus to evaluate the role of cell- defined as the maximum slope of semi-logarithmic representation
wall proteinases PrtS and PrtB on growth and thus on acidification of growth curves, assessed by optical density measurements.
in S. thermophilus/Lb. bulgaricus mixed cultures. Mixed cultures of S. thermophilus and Lb. bulgaricus strains
were performed at 42 °C by inoculating skim milk with 5x106
METHODS c.f.u. ml-1 of stock cultures of each strain. For proteinase-negative
Strains of S. thermophilus and Lb. bulgaricus were grown in strains, cells from stock culture were washed three times in 50 mM
three different media: reconstituted skim milk (Nilac Low Heat Tris buffer (pH 7) before inoculation to avoid peptides and/or
Milk powder, NIZO) heated for 10 min at 95 °C, supplemented amino acids being supplied in the mixed culture. Every 20 min, the
with yeast extract (3 g l-1; Difco) when required, M17 medium pH of the culture was measured, and bacteria were enumerated as
supplemented with 20 g lactose l -1 , and MRS medium described above. Total bacterial populations were estimated by
supplemented with 20 lactose g l-1 and acidified at pH 5·2, addition of data from enumerations of each bacterial species on
supplemented with streptomycin (Sigma) (2 mg ml-1) when required. specific medium to the others, as indicated above.
72 Introductory Food Microbiology Identification of Listeria Monocytogenes Genes 73

Proteinase Assay Preparation of Electrocompetent Cells of S. thermophilus and Lc.


The PrtS proteinase phenotype of S. thermophilus strains was lactis
determined on bacterial colonies in two ways. First, bacteria were Electrocompetent cells of S. thermophilus CNRZ 385 and Lc.
grown on FSDA medium (Fast Slow Difference Agar) (Huggins & lactis MG1363 were prepared according to the method of Holo &
Sandine, 1984 ). This milk-based agar medium made it possible to Nes (1989) , modified as follows. From an overnight culture in
differentiate bacteria exhibiting slow or limited growth in milk M17Lac, a culture was performed at 37 °C (S. thermophilus) or at
from those exhibiting rapid growth; in particular, bacteria 30 °C (Lc. lactis) by 1% inoculation of M17Lac containing DL-Thr
possessing a cell-wall proteinase activity appeared as white, (100 mM) for S. thermophilus or Gly (1·5%) for Lc. lactis until the
opaque, rounded colonies, whereas proteinase-negative colonies OD600 reached 0·6-1. Cells were collected by centrifugation at 5000
were small, flat and translucent. g for 5 min and washed four times in 0·5 M sucrose/10% glycerol
Second, bacteria from an overnight skim milk culture were solution. They were then resuspended in 10% glycerol/30%
diluted and plated on agar skim milk plates (cell culture dishes, PEG2000 solution for S. thermophilus or in 0·5 M sucrose/10%
35 mm in diameter). After 24-48 h incubation at 42 °C in anaerobic glycerol solution for Lc. lactis and immediately frozen in liquid N2
jars, colonies were covered by a solution containing Tris buffer and stored at -80 °C.
(50 mM, pH 7), a chromogenic substrate of proteinase PrtS (Suc- DNA Sequencing
Ala-Ala-Pro-Phe-ßNA, 10 mg ml-1; Novabiochem), 10 mg ml-1 Fast-
The Sanger method of DNA sequencing was carried out on
Garnet (GBC, Sigma) and 10-50 mM CaCl2. PrtS-positive clones
double-strand DNA plasmids and on PCR products with the
appeared as red colonies, whereas PrtS-negative clones remained
BigDye Terminator cycle sequencing ready reaction kit (370A DNA
white.
sequencer, Applied Biosystems).
Proteinase activity was measured on cellular suspensions
using [14C]casein as the substrate according to the method of Construction of a Negative Mutant for PrtS
Monnet et al. (1987) , modified as follows. Cell suspensions were A 3776 bp PCR product containing part of the prtS gene was
prepared from 4 ml overnight M17 cultures; cells were recovered amplified using oligonucleotides 1 (5' CAT CAC GGA AAG TCT
by centrifugation (20 min, 8000 g, 4 °C) and washed three times in AGG 3') and 2 (5' AAC GTA TTG ATA CTT ACC 3') from total
Tris buffer (50 mM, pH 7). The last pellet was suspended in 150 µl DNA of S. thermophilus CNRZ 385 strain. Streptococcal DNA
Bistris buffer (50 mM, pH 6·5) containing 10 mM CaCl2. Fifty (100 ng) was added to a PCR mixture containing 2·5 U of Taq
microlitres of cell suspension was incubated with 50 µl of 14C polymerase (Quantum Appligene) and 0·26 µM of each
casein solution (0·1%) at 37 °C for 15, 60 and 120 min. Enzyme oligonucleotide (Life Technology). After 5 min of denaturation at
reactions were stopped by the addition of 100 µl TCA (12%), left 94 °C, 30 cycles of 30 s annealing at 50 °C and 3 min of elongation
for 30 min at room temperature and centrifuged for 2 min at 10000 g, at 72 °C were carried out using a Perkin-Elmer DNA thermal cycler
and the radioactivity was then measured in the supernatants. (model 480). The amplified fragment was purified from 0·7%
Protease activity corresponded to the percentage of casein agarose gel with the QIAquick gel-extraction kit (Qiagen). It was
hydrolysis in 10 min. then ligated to pCR-XL-TOPO vector (Invitrogen) and cloned by
transformation of electrocompetent TOP10 E. coli cells (Invitrogen)
DNA Manipulations and Sequencing
according to the manufacturer’s protocol. The recombinant vector,
Total DNA Preparation pCR-XL-TOPO-DprtS-1, was purified with QIAprep Spin Miniprep
Total DNA of S. thermophilus CNRZ 385 was prepared as Kit (Qiagen) from the recombinant cells and digested with BsgI
described by Pospiech & Neumann (1995). (New England Biolabs).
74 Introductory Food Microbiology Identification of Listeria Monocytogenes Genes 75

A 5·4 kb fragment containing the TopoXL vector and part of enzyme. Only the region encoding the six C-terminal amino acids
the prtS gene was then purified from 0·7% agarose gel with among the 495 constituting the catalytic domain (PR domain) was
QIAquick gel extraction kit (Qiagen) and blunt-ended with T4 still present in the mutant and did not include the sequence
polymerase 3'® 5' exonuclease (Life Technologies) according to the encoding the residues involved in the catalytic activity of the
supplier’s protocol. It was then circularized by self-ligation with proteinase. Furthermore, protein exportation signals were no longer
Fast-link DNA ligation kit (Epicentre Technology); the resulting present in the mutant; the signal sequence was truncated, and the
plasmid, pCR®-XL-TOPO-DprtS-2, was produced by expected peptide cleavage site was excised.
transformation of electrocompetent TOP10 E. coli cells and purified Cell-wall proteinase activity of the wild-type and PrtS-negative
as described above. It was then digested with NotI and SpeI mutant of S. thermophilus. Using two different methods, we checked
(Eurogentec), and the resulting 2·078 kb fragment was purified as that the S. thermophilus PrtS-negative mutant lacked cell-wall
already described above. The 2·078 kb fragment (~200 ng) was proteinase activity. First, using 14C-labelled casein as a substrate,
ligated to pGhost9 vector (1~00 ng) (Maguin et al., 1996 ), digested we observed that cell suspensions of the PrtS-positive strain were
with NotI and SpeI. The ligation mix was used to electrotransform capable of hydrolysing casein (12·5% of total casein hydrolysed
100 µl of electrocompetent cells of Lc. lactis MG1363, as described within 10 min), whereas PrtS-negative cells had no detectable
by Holo & Nes (1989) . Recombinant clones were selected on caseinolytic activity. Second, we set up a rapid test on colonies
M17Lac Ery plates after incubation at 28 °C. The recombinant using a chromogenic substrate of PrtS. Three strains of S.
vector, pG+h9::DprtS, was purified as described above, and 20 µg thermophilus were used: the proteinase-negative strain CNRZ 302
was used to transform electrocompetent cells of S. thermophilus as negative control and the two proteinase-positive strains CNRZ
CNRZ 385, as previously described (Garault et al., 2000 ). Integration 385 and CNRZ 703, which have a high cell-wall proteinase activity.
of pG+h9::DprtS into the streptococcal chromosome was performed After growing on milk agar plates, colonies were covered with a
as described by Garault et al. (2000) with the following modification: solution containing the substrate Suc-A-A-P-F-ßNA, Fast-Garnet
to induce chromosomal integration of the plasmid, the culture was and different concentrations of CaCl2, the latter being an activator
diluted and plated on M17Lac Ery plates. of PrtS proteinase. Whatever the CaCl2 concentration (10, 20 or
Finally, the mutant for PrtS was obtained by successive 50 mM), colonies of strains 703 and 385 rapidly became red,
incubations of the culture containing the chromosomal integration whereas those of the negative strain 302 remained white. Using
at 37 °C to favour the excision of the pGhost9 plasmid. this test, we confirmed that the mutant strain was PrtS-negative,
as colonies remained white even after several hours of contact
PrtS is Essential to S. thermophilus Growth in Milk
with the substrate solution. This test functioned on milk plates but
S. thermophilus PrtS-negative mutant construction. We have not on rich medium M17 plates for strain 703, which confirmed a
described here for the first time the construction of a targeted probable regulation of prtS expression by the growth medium as
negative mutant for S. thermophilus cell-wall proteinase PrtS. This already observed for this strain. This test will be useful to screen
mutant of S. thermophilus CNRZ 385 was constructed by gene for S. thermophilus PrtS-negative strains in milk and also for PrtS-
replacement using a truncated copy of prtS gene cloned in pGhost9 deregulated strains in M17.
plasmid. DNA sequencing confirmed that this copy was inserted
Growth characteristics of the wild-type and PrtS-negative
at the prtS locus and that pGhost9 was subsequently excised,
mutant of S. thermophilus. By comparing the phenotypes of the
resulting in a truncated prtS gene. As expected, the truncated gene
parental strain 385 and its PrtS- mutant on FSDA, and their growth
was deprived of part of the signal sequence, all the pro-region
curves in liquid M17 and milk, we showed that proteinase PrtS
(removed after maturation of the protein in the parental strain),
was essential to the growth of S. thermophilus in milk.
and almost all of the region encoding the catalytic domain of the
76 Introductory Food Microbiology Identification of Listeria Monocytogenes Genes 77

The PrtS- mutant, plated on FSDA, appeared as flat and measuring the final pHs, final total bacterial populations and final
translucent colonies, as expected for PrtS- bacteria, whereas the individual populations of cultures performed with a strain of S.
PrtS+ parental strain appeared as white, opaque, rounded colonies. thermophilus PrtS+ (strain 385) or PrtS- (strain 385-PrtS) and a strain
In M17, both strains had similar growth curves with a µmax of Lb. bulgaricus PrtB+ (strains 397 and 1038) or PrtB- (strain 397-
of 0·89 and 0·85 h-1 for the parental strain and the mutant strain, PrtB).
respectively. In milk, streptococcal growth was determined The presence of proteinase PrtS had no effect either on the
indirectly by pH measurement. Growth of the PrtS- mutant was final bacterial populations or on the final pHs of mixed cultures
severely impaired in milk, as indicated by the reduced acidification involving PrtB+ Lactobacillus strains. The final total populations
of milk by this strain compared to the parental strain 385. For the were always similar in the presence or not of proteinase PrtS: 1·39
PrtS- strain, milk acidification, and thus bacterial growth, was and 1·36x109 c.f.u. ml-1, respectively, for cultures involving Lb.
restored to the same extent as that for the wild-type strain, after the bulgaricus strain 1038, and 7·05 and 6·27x108 c.f.u. ml-1, respectively,
addition of yeast extract to milk. with strain 397. The absence of any differences in final total
The Lactobacillus strain influences the extent of the positive populations corresponded to similar final individual populations
effect of S. thermophilus/Lb. bulgaricus association. of S. thermophilus and Lb. bulgaricus, regardless of the presence of
PrtS: 1·3x109 c.f.u. ml-1 for strains 385 and 385-PrtS and 8·9x107 c.f.u.
Mixed cultures of S. thermophilus and Lb. bulgaricus were made
ml-1 for strain 1038 for mixed cultures involving strain 1038,
using two different strains of Lb. bulgaricus. To choose the last two
5·5x108 c.f.u. ml-1 for strains 385 and 385-PrtS and 1·1x108 c.f.u. ml-
strains, we first determined the effect of the co-culture of Lb. 1
for strain 397 in mixed cultures involving strain 397. This
bulgaricus strains with the S. thermophilus CNRZ385 strain on milk
correlated well with the similar final pH obtained: 4·72 and 4·85 for
acidification, compared to the pure culture of Lb. bulgaricus. Among
mixed cultures involving, respectively, strain 1038 and strain 397.
the three strains of lactobacilli tested, the effect of adding strain
385 on the acidification was greatest with strains Lb. bulgaricus 397 It is noteworthy that both the final total bacterial populations
and 1038; indeed, for these two Lactobacillus strains, the addition and the acidification rates varied according to the Lactobacillus
of the Streptococcus highly enhanced the acidification rate compared strain associated with S. thermophilus strain 385. The final total
to the Lb. bulgaricus strain alone. Furthermore, the positive effect of population when using Lactobacillus strain 1038 (1·38x109 c.f.u. ml-
1
the bacterial association on milk acidification was more intense for ) was twice as high as that of strain 397 (6·67x108 c.f.u. ml-1),
strain 1038, as, for this strain, the acidification rate and the final because the Streptococcus populations were more than twice as
pH were higher and lower, respectively, in the mixed culture than high with strain 1038, Lactobacillus populations remaining constant.
in the pure culture. In contrast, addition of S. thermophilus strain Final pHs were not significantly different, but the time required to
385 had no significant effect on milk acidification by Lb. bulgaricus reach these pHs was shorter for mixed cultures, including strain
strain 752. Thus, strains 397 and 1038 of Lactobacillus were kept for 1038, than those including strain 397 (4·66 h with strain 1038
the following study. In addition, a proteinase-negative mutant of versus 5·66 h with strain 397).
strain Lb. bulgaricus CNRZ 397 was available and was used for the In contrast, the presence of proteinase PrtB affected both the
following experiments. final bacterial populations and the final pHs. Final total bacterial
In mixed cultures, proteinase PrtS has no effect on final pH and populations were threefold higher in the presence of PrtB than in
bacterial populations, but PrtB affects both. its absence (7·05x108 versus 2·8x108 c.f.u. ml-1). This resulted from
higher final populations of S. thermophilus in the presence of PrtB
The effect of proteinases PrtS and PrtB on acidification of
(6·1x108 versus 2·06x108 c.f.u. ml-1) and led to a significantly better
mixed cultures and bacterial populations was estimated by
acidification in the presence of PrtB (final pH 4·86 versus 5·42).
78 Introductory Food Microbiology Identification of Listeria Monocytogenes Genes 79

In our conditions of inoculation (Streptococcus/Lactobacillus ratio reduced acidification rate and an increased final pH (pH 5·42 in
of 1:1), S. thermophilus was systematically predominant in the total the absence of PrtB and 4·86 in the presence of PrtB).
final populations, regardless of the strain of Lactobacillus and the
presence of proteinases PrtS and PrtB. The magnitude of this DISCUSSION
predominance depended on the Lb. bulgaricus strain used: with The present work aimed at evaluating the role of proteinase
strain 1038, S. thermophilus populations were 15-fold higher than PrtS from S. thermophilus in the growth in milk of S. thermophilus
Lactobacillus populations and fivefold higher with strain 397, when in a pure culture. We also determined the effect of the presence of
PrtB was present. This predominance was less marked in the both PrtS and PrtB from Lb. bulgaricus on S. thermophilus/Lb.
absence of PrtB, as the S. thermophilus populations were threefold bulgaricus mixed cultures. For this purpose, we constructed a
lower (6·1x108 c.f.u. ml-1) than populations reached in the presence targeted negative mutant of proteinase PrtS from S. thermophilus
of PrtB (2·06x108 c.f.u. ml-1). CNRZ 385 and performed pure cultures of S. thermophilus and
Variation of individual populations of S. thermophilus and Lb. mixed cultures with Lb. bulgaricus in milk.
bulgaricus throughout mixed cultures in milk. In milk, the extent of the beneficial effect of the S. thermophilus/
Proteinase PrtS had no significant effect on the variation of pH Lb. bulgaricus association varies.
and of individual populations throughout the culture and, We observed that the effect of the co-culture of Lb. bulgaricus
regardless of the mixed culture considered (except that involving strains with S. thermophilus strain 385 on the acidification of milk,
strain PrtB-), the variation of these two parameters remained similar. and thus the benefit of the bacterial association, depends on the
Fig. 4a gives an example of this variation (a mixed culture made strain of Lb. bulgaricus used. In fact, with Lb. bulgaricus strain 752,
of S. thermophilus 385 and Lb. bulgaricus 1038); for mixed cultures we did not obtain a marked beneficial effect of the association with
including Lb. bulgaricus strain 397, we observed the same behaviour. S. thermophilus 385 as already observed by several authors with
During the first 60-90 min, which corresponded to the first other strains. In contrast, mixed cultures of strains 1038 and 397
acidification phase, S. thermophilus grew exponentially, whereas resulted in a higher acidification than pure cultures. Acidification
Lb. bulgaricus did not grow significantly. Then, as the pH remained was higher with strain 1038 than with strain 397 due to higher S.
constant, the streptococcal population stabilized for about 60- thermophilus populations, Lb. bulgaricus populations being similar.
90 min, whereas Lb. bulgaricus started to grow regularly and These higher S. thermophilus populations probably resulted from a
continuously. Finally, during the last 2 or 3 h of fermentation, better peptide and/or amino acid supply by one Lactobacillus strain
when the acidification rate was the highest, both the Lactobacillus compared to the other as these nitrogen compounds are growth-
and the Streptococcus grew regularly. limiting for S. thermophilus in milk.
In contrast, proteinase PrtB was clearly involved in the The two strains of Lb. bulgaricus thus probably differ in their
variation of bacterial populations and of pH, as demonstrated proteolytic potential, which is in agreement with the differences
with mixed cultures involving strain 397-PrtB. In fact, regardless observed in the final quantities of free amino acids and free NH2
of the presence of PrtB, the first two phases of acidification groups in the supernatants of mixed cultures including these two
corresponding to the sequential growth of S. thermophilus and Lb. strains (data not shown). Some authors have also reported a
bulgaricus were similar. However, during the third acidification variability in the Lb. bulgaricus proteolytic potential. This variability
phase, the growth of S. thermophilus slowed down in the absence could be related to the presence of one cell-wall proteinase in Lb.
of PrtB, the bacterial populations remaining almost constant during bulgaricus, which is the case of strain 397, or of two proteinases,
the last 2 h of fermentation. This reduced growth resulted in a as reported for other strains.
80 Introductory Food Microbiology Identification of Listeria Monocytogenes Genes 81

PrtS is essential to the growth of S. thermophilus in milk in of S. thermophilus in pure culture in milk has been characterized,
pure culture but not in mixed culture. in particular with regard to nitrogen nutrition; it consists of two
Proteinase PrtS is essential to the growth of S. thermophilus exponential growth phases, interrupted by a non-exponential
growth in milk as its PrtS- mutant was unable to grow efficiently growth phase. From these latter results and those of the present
in milk until a nutritional complement [yeast extract or work, we propose the following model of growth of S. thermophilus
bactotryptone (data not shown)] was added. This indicated that in mixed cultures with Lb. bulgaricus with three S. thermophilus
proteinase PrtS was involved in nitrogen supply to the cell, via growth phases corresponding to three acidification steps.
casein hydrolysis, which is consistent with data previously During the first acidification step, characterized by a small
obtained with cell-wall proteinases of other lactic acid bacteria. decrease in pH (<0·5 pH units), S. thermophilus grows exponentially,
However, we demonstrated here that proteinase PrtS had no whereas Lb. bulgaricus does not grow; S. thermophilus is thus
significant effect on the growth of S. thermophilus in mixed cultures responsible for this first acidification, as first observed by Pette &
in milk with Lb. bulgaricus; the growth of the parental strain 385 Lolkema (1950c ). The preferential growth of S. thermophilus can be
and of the PrtS- mutant in mixed culture was similar when Lb. explained first by the fact that S. thermophilus has fewer nutritional
bulgaricus proteinase PrtB was present. This indicates that requirements than lactobacilli in milk. In particular, S. thermophilus
assimilable nitrogen compounds necessary for S. thermophilus requires few amino acids and is capable of synthesizing branched-
growth are supplied by PrtB, as confirmed by the fact that the chain amino acids; its growth can probably be supported by free
absence of PrtB led to lower streptococcal populations. Furthermore, amino acids and peptides present in milk, as previously
as the streptococcal population was higher in the presence of PrtB demonstrated in pure culture, regardless of the presence of PrtS. In
than in the presence of PrtS, we can assume that PrtB is more contrast, Lb. bulgaricus is much more demanding from a nutritional
efficient in the supply of peptides to S. thermophilus than PrtS. This point of view than S. thermophilus (Letort, 2001 ); its optimal growth
can be explained by a more active proteinase PrtB compared to relies on the supply of essential factors (CO2, pyruvate, formate)
PrtS, as previous studies reported that the global proteolytic produced by S. thermophilus. Second, in our study, mixed cultures
activities of Lb. bulgaricus strains were 25-70 times higher than that were performed at 42 °C, a temperature more favourable for S.
of S. thermophilus strains. We cannot rule out the possibility that thermophilus, whose optimal growth temperature ranges between
PrtS and PrtB have different substrate specificity, which leads to 40 and 45 °C, versus 45-50 °C for Lb. bulgaricus.
the production of different peptides, some being more assimilable Then, the S. thermophilus exponential growth pauses and,
than others. Indeed, PrtS is capable of hydrolysing MS-Arg-Pro- concomitantly, the acidification, while Lb. bulgaricus begins to grow
Tyr-pNA, a substrate also hydrolysed by lactococcal proteinase slowly and regularly until the end of fermentation. This pause
PrtP but not by PrtB. Furthermore, when comparing the substrate- probably corresponds to depletion of amino acids and peptides in
binding region of proteinases PrtS and PrtB, in particular the milk, due to their consumption by S. thermophilus, as shown recently
residues 138, 166, 748, which have been identified as being directly by Letort et al. (2002) in pure culture, and the absence of
involved in substrate specificity in lactococci, we noticed that they compensatory production by cell-wall proteinases. These authors
are totally different in PrtS (Thr, Ala, Asp) and PrtB (Gly, Val, Thr). actually demonstrated that proteinase PrtS synthesis starts in the
Model of growth of S. thermophilus associated with Lb. middle of this phase and is maximal during the second exponential
bulgaricus and effect on acidification. growth phase in pure culture. Concerning the growth of Lb.
bulgaricus, we assume that as S. thermophilus reaches a high cellular
In all the mixed cultures performed in milk, we observed the
density during its first growth phase, it probably produces enough
sequential development of S. thermophilus and then of Lb. bulgaricus,
growth-stimulating factors to favour the growth of Lb. bulgaricus.
which is in agreement with previous studies. Recently, the growth
82 Introductory Food Microbiology Experimental Validation of Low Virulence in Field Strains 83

Finally, during the following acidification phase, which leads


to a high pH decrease (about 1·5 pH units), Lb. bulgaricus continues
to grow; at the same time, S. thermophilus starts a second exponential
growth phase. We suggest that this acidification results not only
from the growth of Lb. bulgaricus but also from that of S. thermophilus.
This acidification phase is indeed greatly improved by the addition
of S. thermophilus to a Lb. bulgaricus culture; furthermore, in the 4
absence of PrtB, acidification is reduced, while only S. thermophilus
populations significantly decrease. The growth of S. thermophilus
probably occurs because of the utilization of peptides produced by
EXPERIMENTAL VALIDATION OF
PrtS (when PrtB is absent) but also mainly by PrtB. No differences LOW VIRULENCE IN FIELD STRAINS
in the growth of S. thermophilus were observed in the presence or
absence of PrtS when PrtB was present, and S. thermophilus
populations were significantly reduced in the absence of PrtB, i.e. Several reports have described Listeria monocytogenes strains
when PrtS was the sole source of peptide production. which were nonpathogenic or weakly pathogenic, but little is
known about these low-virulence strains. We found that 9 field L.
In conclusion, we have determined the role of cell-wall monocytogenes strains were hypovirulent and 17 were avirulent,
proteinases PrtS and PrtB in the growth of S. thermophilus and Lb. based on the number of mice contaminated and the colonization
bulgaricus in mixed cultures. We have shown that PrtB is involved of their spleens after subcutaneous inoculation. All these strains
in the optimal growth of S. thermophilus, whereas PrtS does not possessed the known virulence genes. We have now assessed the
play a significant role when PrtB is present. Studies of the effect low virulence of these strains in other assays before determining
of these proteinases on the free amino acid and peptide contents how they differ from virulent strains. We have shown that the low-
as well as on the aroma profiles of mixed cultures are in progress. virulence strains exhibited a phenotypic stability and were not a
As precursors, amino acids are involved in the formation of aroma mixture of virulent and avirulent bacteria. They did not recover
in dairy products, and variations in their composition can affect virulence after many passages in mice and colonized the spleens
aroma development. However, the different pH values observed in of mice more poorly than virulent strains after i.v. inoculation.
the present study at the end of fermentation, when varying the Their lethal capacities, determined by 50% lethal dose (LD50), were
presence of proteinase PrtB, can modify the yoghurt flavour. lower than those of virulent strains. Like Listeria innocua, 14 of 17
avirulent strains had no LD50 and were eliminated by the lymph
nodes after subcutaneous inoculation. The virulent, hypovirulent,
and avirulent strains were always significantly different, whatever
the tests of virulence used, confirming the importance of these low-
virulence field strains in identifying the proteins involved in
virulence.

INTRODUCTION
Listeria monocytogenes organisms are ubiquitous gram-positive
bacteria. They are widespread in the environment and have been
isolated from many sources, including soil, sewage, decaying
vegetation, and food. They are responsible for human diseases
84 Introductory Food Microbiology Experimental Validation of Low Virulence in Field Strains 85

characterized by meningitis, meningoencephalitis, septicemia, is presently unknown. As a preliminary step toward understanding
abortion, and gastroenteritis. Through contaminated food, bacteria the cause of this low virulence, it seemed important to know
reach the gastrointestinal tract and can translocate the intestinal whether low virulence is a stable character over time which cannot
barrier to infect lymph nodes. Then, through lymph and blood, a be enhanced after in vivo passages. It was also important to know
fraction of the bacteria reach the spleen and liver. Apoptosis, whether these strains are also attenuated after intravenous (i.v.)
neutrophils, and phagocytic cells contribute to the rapid clearing inoculation and whether their lethality is modified.
of the bacteria before complete abolition by the specific immune
response. In some cases, such as the immunocompromised host, MATERIALS AND METHODS
bacteria multiply unrestrictedly in the hepatocytes from which Listeria Strains: The Listeria strains used and their
they disseminate through blood to the brain and placenta. Although characteristics are given in Table 1 . Virulence was estimated by
L. monocytogenes is also present in the environment and is probably the method of Roche et al. Different studies allowed the detection
frequently ingested by humans, listeriosis is very rare. The incidence of 9 hypovirulent strains and 17 avirulent strains. To analyze
is very low, around two to eight sporadic cases annually per these strains, 13 virulent L. monocytogenes strains and 1 Listeria
million people in Europe and the United States. If we exclude the innocua strain were added as control strains. The strains were
susceptibility of the host, another reason for this conflicting evidence maintained in storage medium at 4°C. For analysis, they were
may lie in the variability of virulence in the L. monocytogenes strains. cultured in brain heart infusion broth (3 ml) at 37°C for 8 h. BHI
Serotypes of L. monocytogenes could also be linked to the level of agar (BHIA; Difco) slopes were then seeded and incubated overnight
virulence, as only three serotypes (1/2a, 1/2b, and 4b) have been at 37°C. The colonies were suspended in 2 ml of phosphate-buffered
implicated in human cases. However, no bacterial genes related to saline (PBS) (pH 7.3), standardized turbidimetrically, and diluted
the serotype have yet been found. appropriately for each test.
Studies using different assays have shown that virulence varies
from one strain of L. monocytogenes to another. The mouse assays
Cell Line and Culture Conditions: The human adenocarcinoma
are extremely sensitive assays for evaluating the pathogenicity of
cell line HT-29 (no. 85061109; European Collection of Animal Cell
L. monocytogenes by the systemic route. The immunocompromised
Cultures, Salisbury, United Kingdom) between passages 27 and
mouse model has shown a considerable difference in the 50%
67 was used. Cells were grown in 75-cm2 plastic tissue culture
lethal doses (LD50s) of virulent and nonvirulent strains. In the
flasks in Dulbecco’s modified Eagle’s medium with glucose (4.5 g/
same way, subcutaneous (s.c.) inoculation of immunocompetent
liter) (Invitrogen) supplemented with 10% (vol/vol) fetal calf serum
mice is very sensitive and specific, depending on the clinical
(Invitrogen) and 2 mM L-glutamine (Invitrogen). Antibiotics (100
origin of the strains. Tissue culture assays, i.e., cytopathogenic
IU of penicillin per ml and 100 µg of streptomycin per ml; Sigma,
tests, have also been developed to distinguish pathogenic and
Saint-Quentin Fallavier, France) were routinely added to the culture
nonpathogenic L. monocytogenes strains. Certain genetic or
medium except for the virulence assays. Cells were maintained in
phenotypic markers have been linked to the virulence of the strains.
a humidified incubator (at least 90% relative humidity) at 37°C
In our previous paper, the virulence of L. monocytogenes strains under 5% (vol/vol) CO2.
was evaluated with a plaque-forming (PF) assay on HT-29 cells,
Phenotypic Stability: An initial PF assay was done on four
followed by s.c. injections of immunocompetent mice. We found 26
strains to demonstrate that hypovirulence was not the result of
low-virulence field L. monocytogenes strains identified as
two populations of bacteria, one infecting the cells and other unable
hypovirulent or avirulent. All these strains possessed the known
to infect the cells. The bacteria forming plaques were collected and
virulence genes and exhibited the same growth in nonselective
used for a second PF assay. The first PF assay was performed with
media by a bioscreen study. However, the cause of the low virulence
confluent monolayers of HT-29 cells in six-well tissue culture
86 Introductory Food Microbiology Experimental Validation of Low Virulence in Field Strains 87

plates. Cells were infected with 5 log CFU (105 CFU) per well inoculated s.c. in their left hind footpad with 50 µl of bacteria
suspended in Dulbecco’s modified Eagle’s medium for 2 h at 37°C. suspended in PBS. The inocula contained approximately 1 to 9 log
Incubation was continued for a further 1.5 h with 100 µg of CFU for the virulent strains, 2 to 9 log CFU for the hypovirulent
gentamicin (Sigma) per ml in the culture medium. Each well was stains, and 4 to 9 log CFU for the avirulent or nonpathogenic
then overlaid with an agarose gel containing 0.48% indubiose in strains. Each inoculum was checked by counting viable cells after
culture medium supplemented with 10 µg of gentamicin per ml. incubation on TSA plates for 48 h at 37°C. Mice were observed
The same medium was then added to prevent cell starvation, and every day for 15 days, and all deaths were recorded. All the mice
incubation was continued for 3 days. The cells including bacteria remaining on day 15 were killed. The LD50s were calculated using
around plaques were recovered and lysed, and the bacteria were a probit dose-response model, considering a log transformation of
used for a new PF assay (15). Bacteria maintained in the storage dose rates and the total number of mice that died. The percentage
medium were also tested. The results are expressed as the number of mice that died in the three groups were also analyzed by logistic
of plaques obtained for 7 log CFU deposited per well. regression.
Virulence Recovery after in Vivo Passages: Spleen colonization i.v. Injection: Female Swiss mice (6 to 9 weeks old) (Iffa-Credo)
by five strains was monitored during 10 successive passages in were injected with hypovirulent and avirulent strains. The mice
mice, and a PF assay was performed at the end of the experiment. were kept under controlled conditions (humidity, temperature, food
Groups of five 7-week-old conventional Swiss female mice (Iffa- delivery, and stress) during the experiments. Bacteria (4.5 log CFU)
Credo, Saint-Germain-sur-l’Arbresle, France) were injected s.c. in were suspended in 0.5 ml of PBS and injected i.v. The mice were
their left hind footpad with 6 log CFU suspended in 50 µl of PBS. killed with carbon dioxide, 2 days after inoculation. Their spleens
Each inoculum was checked by a viability count on tryptic soy were removed and homogenized in PBS using a glass homogenizer
agar (TSA) plates (Bio-Mérieux, Marcy l’Etoile, France). The plates with a loose-fitting pestle. Triton X-100 was added to a final
were incubated for 48 h at 37°C. Mice were killed 3 days after concentration of 0.001%, and dilutions were made immediately.
injection. Their spleens were removed aseptically, pooled, and Four mice were used for each strain. The viable bacteria in the
homogenized. Aliquots of each homogenate were used to assess inoculates and spleens was counted on Columbia agar. Results
the bacteria in the spleens or to prepare the inoculum for the were compared by analysis of variance and analyzed by the Tukey-
passage in the next mouse. Kramer multiple comparison method.
The spleen colonization assessed on TSA plates is expressed Kinetics of Colonization: Kinetics of colonization were analyzed
as the number of log CFU per homogenate (homogenates from the for four strains. Groups of five 6-week-old conventional Swiss
spleens from the five mice in the group were pooled). The inoculum female mice (Iffa-Credo) were injected s.c. in their left hind footpad
for the next passage was prepared by incubating 100 µl of with 4 log CFU suspended in 50 µl of PBS. The mice were killed
homogenate in BHI broth at 37°C for 32 h (step enrichment). The 1, 24, 48, and 72 h after injection.
incubated homogenate was then seeded on BHIA slopes and The left and right popliteal lymph nodes, lumbar lymph nodes,
incubated for 17 h at 37°C. The strains isolated after passage 10 spleen, liver, and lungs were removed from each mouse aseptically.
were compared to bacteria maintained in the storage medium in Samples were homogenized, and the homogenates were diluted in
a PF assay. The results are expressed as the number of plaques per BHI broth. Appropriate dilutions were plated onto TSA plates and
7 log CFU deposited per well. incubated at 37°C for 48 h. Viable bacteria were counted. The mean
Determination of Lethal doses in Mice: DBA/2 breeder mice log CFU per organ was calculated only for samples with bacteria.
were purchased from Iffa-Credo. The mice were kept in the animal Homogenates were kept overnight in BHI at 37°C for enrichment.
house of the laboratory in level 2 containment facilities, and the Enriched homogenates in which Listeria strains were not detected
mice reproduced. Groups of six 8- to 10-week-old female mice were were isolated on TSA plates and further incubated overnight at
88 Introductory Food Microbiology Experimental Validation of Low Virulence in Field Strains 89

37°C. The results are expressed as the number (log) of CFU per Lethality Study: Under our study conditions, L. monocytogenes
organ. strains were virulent, hypovirulent, or avirulent, depending on
their ability to colonize the spleens of mice. In order to know
RESULTS whether these strains exhibited the same lethality, the numbers of
Phenotypic Stability: All the low-virulence strains studied mice dying were recorded during the 15 days after inoculation
were cloned. We checked the possibility that low virulence could with increasing doses of Listeria strains. DBA/2 mice were chosen
be the result of expression of two phenotypes, one of which could for these study because they are more sensitive to L. monocytogenes
infect cells while the other could not. If this was the case, a small than the Swiss mice. Some LD50s were calculated from very few
subpopulation of bacteria would be able to form plaques and if we observations due to the few deaths caused by some strains. For
recovered these bacteria, the number of plaques in a second test some strains, none of the mice died after 15 days or there were not
should be higher. Among the low-virulence strains, we used the enough deaths to calculate an LD50. In that case, maximal injected
four strains that produced few plaques (strains BO34, CR282, 449, doses are used and indicate the difference in the virulence of the
and 442). Indeed, in order to observe a possible increase or decrease three groups (virulent, hypovirulent, and avirulent strains). An
in plaque number, we could not choose strains forming a high LD50 could be calculated for only 3 of the 17 avirulent L.
number of plaques or no plaque at all. We found no difference in monocytogenes strains; they were between 8.7 and 9.3 log CFU. The
the virulence of the bacteria recovered from the plaques and the 14 other strains were not lethal. The LD50s could be determined
control bacteria under the conditions we used, confirming that all for only five of the nine hypovirulent L. monocytogenes strains (8.3
bacteria within the population have the same level of virulence. to 9.0 log CFU). The LD50s for the 13 virulent strains were 4.1 to
7.9 log CFU. We also compared the percentages of dead mice in the
Lack of Recovery of Virulence after in Vivo Passages: In order
three groups by logistic regression. The difference was highly
to analyze a possible recovery of virulence in low-virulence strains
significant (P < 0.0001), depending on the injected dose. The
after 10 passages in mice, we chose the three strains that infected
difference between the hypovirulent and avirulent strains was
three of five mice (436, BO34, and 464) and the two strains that
also highly significant (P < 0.0001), with the hypovirulent strains
infected one of five mice (SO205 and BO43). As these strains were
being more lethal after injection of 9 log CFU.
hypovirulent, we pooled the five spleens in order to increase the
chance of recovering bacteria. The numbers of bacteria recovered i.v. injection: Differences in spleen colonization and lethal
from the spleens of mice infected with strains 436, 464, and BO34 capability were observed after s.c. injections. Bacteria were injected
were fairly constant during the experiment. The numbers of bacteria i.v. to determine whether their virulence was modified when the
found in the spleens of mice infected with strains SO205 and BO43 mode of inoculation changed. Only 4 of the 13 virulent strains
were equal to the threshold of detection, because they were not were tested, and their mean virulence ranged from 5.8 to 7.2 log
directly recovered from spleen homogenate but were recovered CFU per spleen homogenate, with a mean of 6.58 log CFU for the
after 1 and 10 passages, respectively, in spleen homogenates only 4 strains. The mean virulence of the hypovirulent strains was
after 32 h of growth in BHI medium. After four passages in vivo, lower than that of the virulent strains (4.0 to 6.3 log CFU per
the SO205 strain was not recovered from the spleens of the five spleen homogenate; mean, 5.07 log CFU). The avirulent strains
mice, despite the enrichment step. Thus, this value was below the included 11 strains with a virulence of 3.5 to 5.2 log CFU per
threshold. The number of plaques of bacteria recovered from the spleen homogenate, with a mean of 2.93 log CFU. The numbers of
spleens after 10 passages was then compared to the number of CFU recovered for six avirulent strains were below the threshold
plaques of bacteria maintained in storage medium. The bacteria for the four mice (2 log CFU per spleen homogenate). The percentage
that had been passaged in vivo formed 10 times fewer plaques of mice infected by the hypovirulent and virulent strains (100% of
than the control bacteria. 51 mice) was significantly different from those infected by the
90 Introductory Food Microbiology Experimental Validation of Low Virulence in Field Strains 91

avirulent strains (42% of 60 mice). Analysis of variance of the although several virulence assays have been described. We recently
means of log CFU indicates a highly significant difference (P < developed a virulence assay based on a PF assay and the s.c.
0.0001) between the three groups of strains. Analysis of the pairwise infection of mice. It allowed the detection of 26 low-virulence field
differences in mean numbers of bacteria per spleen homogenate strains of L. monocytogenes. However, it is important to better
was obtained by the Tukey-Kramer multiple comparison method. characterize this low virulence before undertaking genetic
All the different values compared, namely, the values for characterization of the strains. In 1987, Pine et al. reported that the
hypovirulent and virulent strains, hypovirulent and avirulent ATCC 35152 strain contains nonvirulent, nonhemolytic colonies
strains, and virulent and avirulent strains, were statistically that originated as spontaneous variants from the hemolytic parent
significant. All the simultaneous 95% confidence intervals by the strain. We therefore looked for such nonphenotypic stability, as
Tukey method exclude zero. any uncertainty could raise questions about the reliability of all
Rate of Colonization: The rates of colonization by the virulent data obtained with such strains. The bacteria recovered from and
L. monocytogenes strain EGDe, the hypovirulent strain BO34, the around a plaque formed the same number of plaques (of the same
avirulent strain 442, and L. innocua BUG499 were measured to size) as the parent bacterial culture, showing that low-virulence
determine how fast the bacteria spread in mice after s.c. inoculation strains did not consist of a mixture of virulent and avirulent
in their left hind footpad. Bacteria of the virulent strain were found bacteria.
in all the organs studied 1 h postinoculation. The number of mice Waseem et al. demonstrated that passaging the L. monocytogenes
infected and the degree of infection increased with time, so that all NCTC 7973 strain increases its virulence in rabbits, as evaluated
the mice were infected on day three. The hypovirulent strain spread by the recovery of viable bacteria from the infected organs. In the
more slowly than the virulent strain did. Bacteria were recovered same way, Wirsing von Koenig et al. have shown that mice became
in the spleens only on and after the second day and only in two more resistant after many passages as evaluated by LD50s. Thus,
or three of the five mice. The liver was never infected. The avirulent it is possible that the virulence of our strains also increases after
strain infected only the lymph nodes. The bacteria spread from the in vivo passages. Our data clearly show that bacteria conserved in
left popliteal lymph nodes to the lumbar lymph nodes but not to storage medium do not increase their virulence during successive
the spleen or liver, suggesting that the lymph nodes were sufficient subculturings. The number of Listeria per spleen after 10 passages
to eliminate the bacteria. No bacteria were found in the blood, even in mice was the same as those obtained at the first passage. In
after enrichment, but the lymph nodes were more severely infected addition, the virulence of the strains after 10 passages was not
than those of mice injected with the L. innocua strain. increased over that of their parent strains and was even diminished
for some strains.
DISCUSSION We also confirmed the low virulence of these strains by several
It is difficult to detect and characterize low-virulence L. classical tests. The lethality and the bacterial load of organs were
monocytogenes strains for several reasons. They grow at the same therefore used as criteria of pathogenicity after both s.c. and i.v.
rate as virulent strains on nonselective media (i.e., TSA and BHIA), inoculation. We did not use the oral inoculation route in mice,
but detecting low-virulence strains on some selective media is because the results were less reproducible. Moreover, it is not
problematic. Indeed, some of these strains could be detected on representative of human infection because mouse enterocytes do
Palcam medium only after 3 days of growth and were generally not express the same E-cadherin that human enterocytes do,
not detected or poorly detected on Rapid L’mono medium. according to the observations of Lecuit et al. Virulent strains have
Moreover, the low virulence of these strains is often ascertained by LD50s from 1 x 104 to 8 x 107 CFU, and the spleens are heavily
a single test, and there is no standard, well accepted method for colonized after both s.c. and i.v. inoculation. The hypovirulent and
identifying and defining low-virulence L. monocytogenes strains, avirulent strains were much less virulent than the virulent strains
92 Introductory Food Microbiology The Effect of Inoculum Size on the Lag Phase 93

in all the assays used. The hypovirulent strains were close to


avirulent strains in term of lethality, with an LD50 greater than 2
x 108 CFU. Mice inoculated with 109 CFU of L. innocua or avirulent
strains were only lethargic and had ruffled for the first few days
after infection and they recovered soon afterwards. However, the
hypovirulent strains colonized the organs better than the avirulent
strains, particularly the spleen. Taking into account the capacity 5
of the strains to colonize host organs, our results suggest that the
avirulent strains spread better than the nonpathogenic L. innocua
strain BUG 499 because L. innocua did not colonize the lumbar THE EFFECT OF INOCULUM SIZE
lymph nodes after inoculation into the footpad. ON THE LAG PHASE
The rate of infection after s.c. inoculation suggested that the
virulent strains spread quickly to heavily infect all the internal
organs and lymph nodes. The lumbar lymph nodes and liver seem The effect of inoculum size on population lag times of Listeria
to play a key role in the elimination of hypovirulent strains injected monocytogenes was investigated using the Bioscreen automated
into the hind footpad, whereas the lymph nodes alone are sufficient microtitre plate incubator and reader. Under optimum conditions,
to eliminate avirulent strains and L. innocua. These data agree with lag times were little affected by inoculum size and there was little
studies showing that Listeria bacteria are cleared rapidly from the variation between replicate inocula even at very low cell numbers.
lymph nodes by CD8+ T cells and from the bloodstream by the However, in media containing inhibitory concentrations of NaCl,
neutrophils and Kupffer’s cells in the liver. both the mean lag time and variation between replicate inocula
Thus, our data show that all the L. monocytogenes strains increased as the inoculum size became smaller.
screened by our PF assay are low-virulence strains and that this The variation in lag time of cells within a population was
low virulence is not an artifact. Although the virulence of the investigated in more detail by measuring the distribution of
avirulent L. monocytogenes strains is very similar to that of L. innocua, detection times from 64 replicate inocula containing only one or
the rates at which they colonize organs are different. They also two cells capable of initiating growth. The variance of the lag time
have the same in vitro and in vivo phenotypes as the strains distribution increased with increasing salt concentration and was
attenuated by deletion of actA/plcB and are no danger to human greater in exponential than in stationary phase inocula. The number
health. However, the hypovirulent L. monocytogenes strains have a of cells required to initiate growth increased from one cell under
low but real virulence that is confirmed by the clinical origin of optimum conditions to 105 cells in medium with 1.8 M NaCl.
two strains. All the virulence assays used (LD50, spleen colonization
The addition of spent medium from a stationary phase culture
after i.v. inoculation or s.c. inoculation in their left hind footpad)
reduced the variance and decreased lag times. The ability to initiate
clearly showed significant differences between the three levels of
growth under severe salt stress appears to depend on the presence
virulence established by our virulence assays. All these strains
of a resistant sub-fraction of the population, although high cell
have the main known genes of virulence, but in-frame mutations
densities assist adaptation of those resistant cells to the
could decrease their virulence. Genetic and phenotypic analyses of
unfavourable growth conditions by some unspecified medium
hypovirulent and avirulent L. monocytogenes strains are now in
progress in our laboratory. conditioning effect. These results are relevant to the prediction of
lag times and probability of growth from low numbers of stressed
cells in food.
94 Introductory Food Microbiology The Effect of Inoculum Size on the Lag Phase 95

INTRODUCTION statistical effect would only be expected at inoculum levels of


The lag phase of the microbial growth cycle represents the below about 102–103 cells. McKellar and Knight (2000) have also
time period needed for bacteria to adapt to a new environment presented a model of the lag phase of Listeria monocytogenes that
before cell multiplication commences. Recent work modelling the takes into account the variability of individual cells.
behaviour of bacteria in foods has shown that the lag phase is There have been relatively few experimental tests of the effect
more difficult to predict than is the specific growth rate. This of inoculum size on lag, but available data suggests that the effects
implies that certain relevant variables affecting lag time are not are small in broth cultures under optimum growth conditions and
taken into account, particularly the physiological state of the also in food. However more recently, it has been shown that the
inoculum and the inoculum size. lag time of L. monocytogenes was extended in stressed cells when
The physiological state of cells will be affected by their previous the inoculum size was small. Stephens et al. (1997) also showed
growth environment and by exposure to stress conditions. Cellular that the length of the lag phase is not only very variable in heat
injury caused by heating freezing or drying can extend the lag injured cultures of low cell density, but also on average, longer
time considerably and also increase its variability. Starved cells than those of high cell density.
may also have very extended lag phases. The temperature history The purpose of this study was to investigate the effect of
of the inoculum culture and its growth conditions have a profound inoculum size on the population lag time of L. monocytogenes
effect on lag. Some mathematical models used in predictive food under conditions of salt stress and to assess the heterogeneity in
microbiology contain a parameter related to the physiological state lag times of inocula containing very low cell numbers. During the
of the inoculum but it has not been possible so far to measure course of this work, an effect of population size on the probability
physiological state directly in terms that are useful for predicting of initiating growth became evident.
behaviour. This may become possible eventually with developments
in methods for measuring single cell metabolic activity. MATERIALS AND METHODS
Two classes of inoculum size effect on population lag may be Organism and Culture Conditions
envisaged (a) cooperative or inhibitory effects of high cell L. monocytogenes NCTC 11994 was used throughout these
concentrations or (b) statistical effects at low cell concentrations experiments. It was kept on glass beads at “70 °C and maintained
arising from the variability in individual lag times. There is little on tryptone soya agar slopes at 5 °C. Stationary phase inocula
specific information about the possible effects of cell–cell were prepared by inoculating 20 ml tryptone soya broth and
interactions on lag time although cell signalling has been shown incubating overnight at 37 °C. To produce an exponential phase
to affect the emergence of cells from dormancy and the lag time of inoculum, 0.1 ml of a stationary phase culture was inoculated into
populations in biofilms. The effect of substances produced in 20 ml TSB (pre-warmed to 37 °C) and incubated until the optical
culture supernatants on microbial growth was reviewed by density at 680 nm (OD 680 ) reached 0.15 measured in a
Kaprelyants et al. (1999) spectrophotometer with a light path of 1 cm. Viable counts were
Mathematical treatments of the statistical effects of inoculum measured by using spread plates. Tenfold dilutions of culture
size on population lag were provided by Baranyi (1998) and were made in Maximum Recovery Diluent (Oxoid) and duplicate
Baranyi and Pin (1999). These authors showed that as the cell 50 µl samples spread on TSA plates.
number in the inoculum decreases, the population lag increases
by an amount that depends on the distribution of individual lag Bioscreen
times and the maximum specific growth rate. The mathematical The effect of inoculum size on the variation in the population
analyses predict that under optimum growth conditions, this lag was investigated by preparing tenfold serial dilutions of
96 Introductory Food Microbiology The Effect of Inoculum Size on the Lag Phase 97

exponential and stationary phase cultures in either TSB, TSB with Mathematical Treatment of Data
1.2 M NaCl or TSB with 1.6 M NaCl. Data from the Bioscreen were analysed using the approach of
Ten replicate 300µl inocula from each dilution were placed in Baranyi (1998). Detection time is converted to a corresponding
the wells of a microtitre plate and the plates were then incubated ‘physiological state’ parameter (a) that is an expression of the
at 37 °C in the Labsystems Bioscreen C. The increase in turbidity ‘readiness of cells to grow’ using the following transformation
exp ( − µma x T det )
at 600 nm was monitored automatically every 10–30 min for 2–12
days. The plates were shaken between measurements. α=
r
Detection times obtained from the outermost wells of the
where µmax is the maximum specific growth rate, Tdet is the detection
microtitre plates were generally longer than those from the other
time, r is the ratio between the initial and the detected concentration
wells, particularly when the medium contained a high salt
reached at Tdet.
concentration. On further investigation, we found that for long
incubations (greater than 7 days), there was a decrease in the RESULTS
volume of media in these outer wells of up to 10%. We therefore
discarded the results obtained from all the outer wells in all The Effect of Inoculum Size on Lag Time
experiments. So, for the initial experiments, there were only eight To test whether there was an inoculum size effect, tenfold
replicates rather than 10 per dilution. serial dilutions of cultures were made in TSB containing different
To investigate variability in the length of the lag phase at very levels of NaCl, and the detection times recorded. Assuming that
low inoculum levels, cultures were diluted to a level that gave specific growth rate is unaffected by inoculum size, the difference
growth in approximately 50% of 64 wells. According to the Poisson in detection time between consecutive dilutions should be constant
distribution, 70% of positive wells would then contain a single and proportional to the maximum specific growth rate (µmax)
cell and 24% two cells. Three salt concentrations, 0, 1.2 and 1.6 M, provided there is no effect of inoculum size on population lag
were investigated. times. Hence, a plot of detection time versus the logarithm of the
inoculum size should give a straight line. If, on the other hand
The possible effect of medium conditioning (alteration of some
population lag times were affected by inoculum size, a deviation
physicochemical aspect of the growth medium by the previous
in linearity would be expected. Under optimum growth conditions,
growth of a culture) or cell signalling was checked by preparing
i.e. in TSB with no added NaCl, detection times were indeed
dilutions of log phase cells using 50:50 spent broth/fresh TSB,
linearly related to log inoculum size. The slopes of the
with added NaCl as before. The spent broth was prepared by
experimentally determined lines were close to those predicted
filtering a 24-h culture through a 0.2-mµ pore size nylon membrane from the growth rate estimated from viable count data from cultures
filter (Nalgene, Milton Keynes, UK). All experiments were repeated growing in the wells. The specific growth rates estimated from the
at least twice. detection times in Fig. 1a were 1.12 and 1.22 h”1 which is close to
Probability of Growth a growth rate of 1.27 h”1 estimated from viable count data from
cultures growing in the wells. There was little variability in the
The effect of inoculum size on the probability of initiating detection times of replicate inocula at each inoculum level although
growth in a culture was investigated by incubating 40 replicates slight scatter was observed at the lower inoculum levels (1–10
of a few critical inoculum levels, taken around the growth/no- cells). Under these conditions, there was no marked effect of
growth inoculum size boundary at three salt concentrations, 0, 1.7 inoculum size on population lag and no difference in the response
and 1.8 M. of exponential and stationary phase cells.
98 Introductory Food Microbiology The Effect of Inoculum Size on the Lag Phase 99

As conditions became more stressful and growth rate lag by a mechanism separate from the statistical effect due to the
decreased, i.e. in TSB containing 1.2 M NaCl, there was greater inherent lag time distribution.
variation in lag between replicates particularly for the exponential The distribution of detection times was studied in more detail
phase inoculum. The variability increased with decreasing for very small inocula. Sixty-four replicates were prepared at
inoculum size especially below 104 cells per well. The trend of the inoculum levels that gave growth in approximately half the
scatter was towards longer detection times rather than being replicates. The average number of cells per inoculum required to
distributed around the expected detection time. The net effect was give growth in half the replicates was determined independently
that as inoculum size decreased, the mean detection time deviated by plate counting.
more from that predicted assuming that lag time and growth rate
For TSB containing 0, 1.2 or 1.6 M NaCl, the mean inoculum
remain constant. Assuming that the maximum growth rate is
independent of inoculum size, the observed deviation represents sizes were 1.2, 5.0 and 2000 cells, respectively. However, since
an increase in lag at low cell numbers. An additional effect was only half the wells actually showed growth, the effective mean
that, with exponential phase cultures, no growth was seen from inoculum size according to the Poisson distribution would have
inocula containing less than 10 cells. Cultures inoculated with been 0.7 cells per well in each case. The data were normalised by
stationary-phase cells showed a linear relationship between setting histogram bin size equal to the doubling time for each salt
inoculum size and detection time, except at very low cell numbers, concentration, hence, the increase in variability in detection times
where detection times were longer than predicted and fewer wells shown in the figures was not simply an effect of growth rate. In
showed growth. TSB alone, there was very little variation in detection times (and
hence lag times) between inocula of approximately one cell.
The results from experiments with TSB containing 1.6 M NaCl
show a continuation of this trend, such that the relationship In medium containing 1.2 M NaCl, there was a pronounced
between inoculum size and mean detection time is no longer difference between the responses of exponential and stationary
linear. Less than 50% of the cultures grew when inoculated with phase cells. Stationary phase cells showed relatively little variation
104 cells, and none grew with inocula of less than 103 cells, in detection times a slight tailing towards longer detection times.
irrespective of the growth phase. Exponential phase inocula gave The range between shortest and longest detection time was
consistently longer detection times than stationary phase inocula. equivalent to about five doubling times. Under the same conditions,
The detection time data were transformed according to the exponential phase cells showed a much larger variation, with the
method of Baranyi and Pin (1999) to yield values. If the lag times longest detection times being approximately twice that of the
of the individual cells are not affected by the inoculum size, then shortest (a range of about 22 doubling times). A similar effect was
these physiological state values should be scattered around a observed at the higher salt concentration of 1.6 M.
constant, independently of inoculum size, but with increasing
Effect of Inoculum Size on the Ability to Initiate Growth
variance as the inoculum size decreases. When data from stationary
phase cells inoculated into 1.2 M NaCl were transformed in this When cells were inoculated into TSB with no added salt, the
way, values for stationary phase cells did scatter around a constant, percentage of wells showing growth was close to that predicted
except at the lowest inoculum level, indicating that inoculum size from the Poisson distribution, showing that growth was possible
did not influence readiness of cells to grow except at the lowest from single cells. However, with increasing salt concentration, the
inoculum level. number of cells required to initiate growth in 50% of the wells
However, with exponentially growing cells, the values deviated increased to more than 103 cells in media containing 1.7 M NaCl
progressively from the constant showing that inoculum size affected and around 106 cells the presence of 1.8 M NaCl.
100 Introductory Food Microbiology The Effect of Inoculum Size on the Lag Phase 101

Effect of Spent Medium THE EFFECT OF INOCULUM SIZE ON LAG TIME


To further investigate the cause of the inoculum size effects, Our basic assumption was that the maximum specific growth
log phase cells were inoculated into fresh TSB mixed with an rate (µmax), within the physiological range of any given organism,
equal volume of spent broth. The hypothesis was that inoculum is independent of cell history and is uniquely determined by its
size effects (a shortened lag phase or increased probability of environment. This is shown by the global constancy of reported
growth at high cell density) might be caused either by carry-over µmax values for particular organisms grown under the same
of substances with the inoculum, or the presence of a signal conditions. A recent report by Membré et al. (1999) describing an
chemical produced during the lag phase that may also be present effect of inoculum pre-treatment on growth rate of L. monocytogenes
in medium from stationary phase cultures. Spent medium had no is an exception to this rule, though the effect was very small.
effect at NaCl concentrations of 1.2 M or less. Therefore, for any set of conditions, a plot of the logarithm of
inoculum size versus detection time should yield a straight line
However, with 1.6 M NaCl detection times were shortened
whose slope is proportional to specific growth rate, provided lag
with inocula of 3000 cells per inoculum or less, and the variability
time is constant and unaffected by inoculum size. If the units on
between samples reduced. A simple ANOVA was run on the
the abscissa are Ln inoculum size, the slope will equal 1/µmax, if
detection times measured at the two lowest inoculum levels, where
the units are log2 inoculum size, the slope will be equal to doubling
the variabilty of the lag times of individual cells has the biggest
time. From the above, a deviation from linearity would imply that
effect on the population lag. An F-test showed that the probability
population lag was not independent of inoculum size.
that the shorter avarage lag times were observed only by chance
was less than 10"3. The standard deviation of the detection times The lag time of a population is less than the average lag time
decreased by more than 50% for both inoculum levels, confirming of individual cells because cells with shortest lags begin to multiply
the significance of the spent medium effect. soonest and their descendants dominate the population
numerically. This effect was analysed by Baranyi (1998) who
DISCUSSION derived a mathematical relationship between population lag and
the distribution of individual cell lag times and growth rate. This
Use of the Bioscreen
analysis revealed that as cell numbers in the inoculum increased
The Bioscreen proved to be a useful means of producing large from 1 to 103, the scatter of lag times from replicate inocula
quantities of growth curve data. It has been used for a number of decreased. The greatest variation in lag should thus occur in
different applications such as determining bacterial growth rates, inocula containing low numbers of cells from populations that
studying the effect of different conditions on growth, growth had a wide scatter of individual cell lag times. Any variation in
inhibition studies, enumeration of bacteria from food samples, lag time between replicate inocula will be reflected in a
and comparison of pre-enrichment media for resuscitation of corresponding variation in detection times.
injured salmonellae. However, the data produced need to be Because plots of inoculum size versus detection time were
analysed with care. The spurious readings obtained from the essentially linear for growth in TSB, we conclude that, under
outermost wells were most obvious when the cells were grown optimum conditions, there was no effect of inoculum size on
with high NaCl concentrations under conditions that required population lag, except that due to statistical variation which was
long incubation times before growth became detectable. This noticeable only at low cell numbers (100 cells per well or less). Our
probably resulted in evaporation from the outermost wells, a feature results obtained under optimum conditions agree with the work
that limits the incubation times that can be employed in these of Jason (1983) who found that lag and growth rate of Escherichia
studies. coli were independent of inoculum size. Duffy et al. (1994b) also
102 Introductory Food Microbiology The Effect of Inoculum Size on the Lag Phase 103

concluded from viable count data that the lag times of L. concentrations of 1.71 to 2.05 M NaCl, at least 105 cells were
monocytogenes in Listeria Selective Broth, Palcam broth or a mixture required for growth to occur, comparable with an inoculum size
of beef mince and broth, were unaffected by inoculum size. of 103 with 1.7 M and 106 with 1.8 M NaCl that we observed at
Although an increase in lag time was observed with decreasing 37 °C.
inoculum size this was statistically insignificant due to the wide
scatter of the data. Statistical Explanations of Population Size Effects
Under the more exacting growth conditions provided by TSB Studies with inocula containing predominantly single cells
containing 1.2 or 1.6 M NaCl, there was a wider scatter of detection suggested that the increase in mean population lag time at low
times as inoculum size decreased, and the mean value diverged cell number under stressful growth conditions could have been
increasingly from that predicted assuming a constant lag. explained simply by the corresponding increase in scatter in the
This was particularly noticeable with exponential phase lag times of single cells. In unstressed populations the variation
inocula. Exponential phase cells are generally more fragile and of lag times was only about ±2 doubling times. Part of that variation
susceptible to injury than those in the stationary phase and this can be accounted for by variation in the number of cells per well
may account for their longer and more variable lag times following as predicted from the Poisson distribution.
osmotic shock. Transformation of the data according to Baranyi Since the number of wells showing growth was around 50%,
and Pin (1999) showed that inoculum size had an effect on lag about 70% of positive wells would contain one cell and 24%
that was separate from the statistical effect caused by the would contain two cells. The detection times of wells containing
distribution of lag times of single cells, i.e. there was a cooperative two cells would be shorter by one generation time than those
population effect. containing only a single cell. Under stressful conditions, the scatter
An effect of inoculum size on lag has been shown previously increased to about ±10 doubling times implying that the variability
with L. monocytogenes inoculated into broth at pH 5.9 and an in detection time was largely due to greatly extended lags rather
incubation temperature of 14 °C, designed to simulate ripening of than different numbers of cells in the wells.
Camembert cheese. The biggest inoculum size effect was seen The question nevertheless arises whether inoculum size effects
when cells were grown at 30 °C then held for 4 weeks at 4 °C on lag time and the probability of initiating growth can be
before inoculation into broth at 14 °C, when an inoculum of 103 explained solely by the distribution of some resistance attribute
cells gave a lag of less than 21 h compared with 7.7 days for an within the population. In broth containing 1.2 M NaCl, a wide
inoculum of 10 cells. An extension of lag time and an increase in scatter of lag times was seen even with inocula containing 3000
variability between replicate inocula has also been reported for cells, a number which, from the theoretical results of Baranyi
heat-injured cells of Salmonella typhimurium. This is similar to the (1998), should have ensured that the lag times of replicates would
effect described here with salt-stressed cells of L monocytogenes. have converged close to the population mean.
However, a comparison of the dilution at which wells showed
Effect of Inoculum Size on the Probability of Initiating Growth
no growth with the known number of cells inoculated, showed
Under optimum conditions a single cell was able to initiate that the number of cells able to initiate growth was much less than
growth in TSB but, in the presence of high salt concentrations, the number present in the wells, and this may therefore bring the
much larger inocula were needed. A similar observation was made effective inoculum size within the range where statistical effects
by Razavilar and Genigeorgis (1988) who found that the number become apparent. The same argument applies with cells in 1.6 M
of cells of L. monocytogenes required to initiate growth at 30 °C was NaCl except the fraction of cells able to initiate growth was even
unaffected by NaCl concentrations up to 1.37 M. However, at smaller.
104 Introductory Food Microbiology The Effect of Inoculum Size on the Lag Phase 105

Cooperative Effects on Lag and the Ability to Initiate Growth RELEVANCE TO GROWTH IN FOOD
An alternative explanation for the inoculum size effects is that The results presented here predict that, under inhibitory
some kind of conditioning of the culture medium or chemical conditions, the mean lag time of cells that were sparsely distributed
signalling is required before growth can occur under stressful in food would be appreciably longer than that indicated from
conditions. There appears to be a critical threshold cell density broth experiments with large inocula. Mackey and Kerridge (1988)
below which growth initiation is not possible and this threshold found that lag times of salmonellae growing in minced beef at
is related to the severity of the culture conditions. temperatures between 10 and 35 °C were much more variable than
Cells may produce a chemical or physicochemical change in the corresponding growth rates. Considering all data, there were
situ, or there may be carry-over of substances from the inoculum. no statistically significant differences between the lag times of
The addition of spent medium to TSB containing 1.6 M NaCl large and small inocula (40 or 104 cells g”1, respectively) but at the
shortened detection times, and decreased lag time variability, but lowest most stressful temperature measured (10 °C), the lag time
no change was recorded in the probability of initiating growth. A was 50% longer (60 h compared with 40.6 h) for the lower inoculum
requirement for specific signal molecules in recovery from stress level.
has been reported in some bacteria. Cells of Nitrosomonas europaea The results presented here also suggest that, under inhibitory
recovered from starvation and commenced growth sooner when conditions, the probability that a pathogen could initiate growth
cells were present in a biofilm. in any single food item containing very low numbers of cells
Acylated homoserine lactones were shown to reduce the length would be less than that suggested by growth studies in broth
of the lag phase in a concentration dependent manner, suggesting inoculated with thousands of cells. The exact probability value
that these signal molecules responsible for this cooperative effect. per pack would depend on the distribution of resistance within
In the Gram positive Micrococcus luteus a peptide signal molecule the population and the actual number of cells present per pack.
that is necessary for the revival of dormant cells has been isolated The overall probability that growth would occur at least in some
from culture medium. packs would be the same as that calculated from broth studies
with large inocula. Any reduction of lag time by cooperative or
The growth stimulatory effects described here may also involve conditioning effects would depend on cell concentration and
signal molecules, but a non-specific conditioning effect could also proximity; for example whether cells were present as microcolonies
explain the phenomenon. For example, the protective effect of high or single cells. Predicting the behaviour of bacteria in food is often
cell densities on stressed bacteria may be due to non-specific based on experiments with initial inocula in excess of 103 cells
effects such as leakage of magnesium or other unspecified material ml”1. From our work, it would appear that beneath this level of
from dead and injured cells that protects the remaining cells. inoculation, the lag phase becomes longer and the probability of
Oxygen tension and redox potential are other examples of non- growth is less. This may have significance in estimating risk and
specific factors that can markedly effect the recovery of stressed calculating safe storage times for foods.
cells.
The results reported here support the view that the ability to
initiate growth under severe salt stress depends on the presence
of a resistant sub-fraction of the population, but that high cell
densities appear to assist the adaptation of those cells to the
unfavourable growth conditions by some unspecified medium
conditioning effect.
106 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 107

as mechanisms to prevent microbial growth and to ensure food


safety. Therefore, it is important to understand and to be able to
predict the responses of micro-organisms, more particularly
pathogenic micro-organisms as Listeria monocytogenes, in the
presence of variable environmental factors. In recent years, several

6 investigations which described the growth of micro-organisms in


the presence of temperature changes, were carried out. When
building these dynamic models, these authors made the hypothesis
that the transposition of results obtained from constant conditions
MODELLING THE GROWTH OF to variable conditions was possible. Cheroutre-Vialette et al. (1998)
LISTERIA MONOCYTOGENES IN demonstrated the importance of taking into account the variations
of environmental factors, which can occur during a food processing
DYNAMIC CONDITIONS and induce a stress situation for the micro-organisms. The objective
of this work was to produce a dynamic model that predicts the
A recurrent neural network for the prediction of Listeria growth of L. monocytogenes as a function of fluctuating conditions
monocytogenes growth under pH and aw variable conditions was of acid pH, alkaline pH and concentration of NaCl. To this end,
developed. The use of this model offered the possibility to take into an alternative approach to conventional methods of microbial
account the consequences of the variations of the factors on L. growth predictions, that is, a recurrent multilayer neural network
monocytogenes growth. The effects of solutions, such as NaCl, acetic approach, was used.
acid and NaOH, and their interactions on the response of L.
monocytogenes cells were studied. Furthermore, the results showed MATERIAL AND METHODS
the capacity of the recurrent neural network to predict growths Strain and Medium
carried out in different experimental conditions without using L. monocytogenes 14 (serotype 4b), obtained from an industrial
those used for its elaboration. environment, was used throughout the study. All growth
experiments were conducted in a tryptic meat broth (TMB). TMB
INTRODUCTION regulated at pH=7 and aw=1 was called standard medium.
Predictive microbiology combined the knowledge of bacterial
growth responses over a range of conditions with the power of Experimental Procedure
mathematical modelling to enable predictions of growth. An automated turbidimeter was used to follow the growth of
Mathematical modelling techniques can help to predict how food the strain in the micro-titer plates. The working volume in each
preservation systems may affect growth kinetics. Most mathematical well of the micro-titer plate was 400 l. Optical density (O.D.) was
models developed to simulate the growth of micro-organisms in read at a wavelength of 600 nm. According to the procedure
relation to environmental conditions, were elaborated from data expoused by Cheroutre-Vialette et al. (1998), for all experiments
coming from growth carried out in constant conditions of three conditions were studied: standard, limiting and shock
environmental factors, such as aw or pH. But, food-manufacturing conditions. Under standard and limiting conditions, bacteria were
processes can decrease the pH of food, produce organic acids, e.g., grown in TMB adjusted to the desired aw and pH values. The
pickling or fermentation, or reduce water activity, e.g., by addition osmotic variation was achieved by the addition of NaCl according
of an agent such as sodium. These processes are extensively used to Chirife and Resnik (1984). Acetic acid and NaOH (Prolabo)
108 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 109

were added to adjust low and high pH respectively. The shock • (O.D.ni)t, the mean of the O.D. of the 4 repetitions of non-
condition was defined as follows: the bacteria were grown in inoculated media for each combination;
standard medium until the beginning of the exponential phase • (DO.D.)t=(O.D.i)t–(O.D.ni)t;
and were then shocked by the abrupt addition of shock solutions.
• Yt=log10[(DO.D.)t/(DO.D.min)] where DO.D.min was the
These shock solutions were prepared in order to obtain a final
lowest DO.D. value above the detection threshold.
value similar to those indicated for the limiting conditions. The
temperature was 20°C. For each combination, six growth repetitions Recurrent Neural Network (RNN) Model
were carried out.
Neural network (NN) maps inputs to outputs. Supervised
Experimental Design neural networks are capable of learning from previous examples
through iteration without the requirement of a priori knowledge
The data of L. monocytogenes 14 growths at 20°C were taken
of the relationships between the process variables. NNs are made
from an experimental study using a combination of two central
of a number of simple, highly connected processing elements (PE)
composite designs and a factorial design. The ranges of the different
called neurones. The architecture of a NN describes how the NN
environmental parameters were as follows: NaCl: 0–8%; acid pH:
is constructed from layers of PEs. Each PE receives inputs from
5.6–7.0 or alkaline pH: 7.0–9.5. Each factor was studied at five
other PEs or from the outside. The PEs in the input layer only
levels and for each combination, the shock and limiting conditions
transfer scaled inputs to the appropriate PEs in the hidden layer
were studied. At least 50 experiments were performed according
through weighted connections. Each PE of the hidden layer and
to 25 growths in shock condition and 25 growths in limiting
output layer calculates the weighted sum of its inputs and passes
condition.
the result through a transfer function. Most often a non-linear
Additional Experiments sigmoid transfer function is employed. The NN is iteratively trained
by presenting to it representative exemplar input/output vectors.
A two-litre fermentor SET2M was used. O.D. of the growth
The weights of the neural connections are adjusted in order to
was measured with a spectrophotometer at 600 nm. The protocol
minimise a cost function equal to the mean square of the output
was similar to that in Bioscreen C adjusting the different volumes.
error. The weights are usually randomly initialised. It has been
Growths in shock conditions carried out in the fermentor allowed
shown that one hidden layer of neurones is sufficient to
the study of the osmotic shock effects by continuous addition
approximate any continuous non-linear function, although more
mode. Seven hundred and fifty milliliters of standard medium
complex networks may be employed in special applications.
were inoculated. At the beginning of exponential phase, 250 ml of
osmotic solution giving a final concentration of 8% NaCl were The distinction between feedforward and recurrent multilayer
added using a peristaltic pump. The addition mode of shock NNs is the following: for the feedforward multilayer NNs an
solution in Bioscreen C was by steps of 2%. element of a given layer can only receive information from previous
layers; on the contrary, for recurrent multilayer NNs, the values
Data Analysis calculated by the NN can be fed back to the NN input layer or any
Averages of the O.D. were calculated for the six repetitions of previous layers. The difference in structure induces a difference in
inoculated media and for the four repetitions of non-inoculated training: recurrent multilayer NNs have to be trained on a sequence
media. The data were then analysed using the procedure described of predictions, whereas training feedforward multilayer NNs
by Bégot et al. (1996). Four quantities were calculated at time t: involves only instantaneous predictions. Training a recurrent
neural network (RNN) is more time-consuming but it is able to
• (O.D.i)t, the mean of the O.D. of the 6 repetitions of
provide better results than a feedforward NN when simulating the
inoculated media for each combination;
110 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 111

evolution of phenomena with time. RNNs trained to learn dynamic the growth results carried out in fermentor or in Bioscreen C with
evolutions of a process are more constrained and are forced to find various modes of shock exposure were included into the validation
a greater stability as well as a representation of the dynamic links base.
between control and process variables. Therefore, RNNs provide
a better prediction confidence than feedforward multilayer NNs RESULTS
that only predict the outputs at a fixed horizon. Furthermore, this Growth Predictions of the Validation Base
technique is more interesting in an advanced control perspective.
The analysis of the growth predictions in the limiting
In the present study, a recurrent multilayer structure which conditions showed that the RNN represented satisfactorily the
contains one input layer, one hidden layer and one output layer, experimental data whatever the conditions tested (alkaline–osmotic
was used. The architecture of the RNN is presented in Fig. 2. It or acid–osmotic). The different characteristics of the L. monocytogenes
was designed to contain: response, i.e. induction of a lag time and growth recovery different
1. in the input layer, five input parameters: Yt–Dt, Yt–2.Dt, to those observed in the new environment, were taken into account
Yt–3.Dt, pHt–Dt and NaClt–Dt (%) by the RNN whatever the combination, alkaline–osmotic or acid–
2. in the output layer, one output parameter: Yt which osmotic. Furthermore, RNN was able to predict the effect of the
represented the predicted response. type of shocks and their combinations. There was a good agreement
between the experimental growth and the prediction.
A priori, there are no rules for the choice of the hidden
structure. It is determined empirically: the structure that gives the Growth Predictions of the Additional Experiments
best results is chosen. The optimum neurone number of the hidden The previous results of the validation base demonstrated the
layer was iteratively determined by developing several RNNs that ability of the RNN to predict growths under shock conditions
vary with the size of the hidden layer (3 to 10 neurones were when the pH and aw transitions were carried out abruptly by one
tested) and simultaneously observing the change in the mean step. The objective of additional experiments was to investigate
square of the output error. the capacity of the RNN to represent the response of L.
This was carried out with the training and testing data. Six monocytogenes cells shocked in exponential phase with 8% NaCl
neurones in the hidden layer was determined as the best structure. added by repeated steps of 2% NaCl or continuously during a
The sigmoid function f(x)=1/(1+exp(-x)) was chosen as an duration of one or four generation times. The effects of adding 8%
activation function for each neurone. The RNN was trained by NaCl in 7.7 h (four generation times). The extrapolation to new
iteration using a repeated presentation of representative exemplar experimental conditions (mode of shock exposure) was made with
input/output vector pairs. a good agreement by the RNN. It confirmed that the RNN has the
The weights of the neural connections, initially chosen capacity to predict growths carried out in different experimental
randomly, are adjusted by a non-linear optimisation technique: conditions from those used for its elaboration.
the quasi-newtonian formula of Shanno (1970) in order to minimise
a cost function equal to the mean square of the output error. The DISCUSSION
learning base was used to adjust the weights, the testing base to These results obtained at variable conditions showed that
provide over-learning during weights optimisation, and the neural networks can effectively be used to study the complex
validation base for validation of results. At least, 60% of experiments effects of fluctuating environmental conditions on micro-organism
were included in the learning and testing bases, the last 40% were behaviour. Such dynamic model could follow the microbial impact
in the validation base. In order to ensure the validity of the study, of different steps associated with production, distribution and
112 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 113

retailing of a food and so could be an important support to HACCP water activity (%NaCl), temperature. But envir-onmental factors,
and food safety systems. such as temperature, Aw, or pH which are often the most important
Growth of Listeria monocytogenes as a function of dynamic factors governing microbial behaviour in food, may vary extensively
environment at 10°C and accuracy of growth predictions with during food processing, throughout the complete production and
available models. dis-tribution chain of a food product. Indeed, a food product is
exposed to environmental var-iations during certain stages in a
A combination of a factorial design and two central composite
process, e.g. food cooling, natural product acidification or product
designs was used to assess quantita-tively the effects and
brining. In this way, it is important to be able to quantify
interactions of water activity (1-0.95) and pH (5.6-9.5) variations
undesirable micro-organism growths, like Listeria monocytogenes,
on the growth of Listeria monocytogenes in a meat broth at 10°C. At
in envir-onmentally variable conditions.
inoculation or at the beginning of the exponen-tial phase, the cells
were exposed to the addition of NaCI and acetic acid or NaCI and The purpose of this study was to determine the effects of the
NaOH. combined alkaline-osmotic and acid-osmotic conditions, constant
or vari-able during time, on the growth of a L. monocy-togenes
The effects of abrupt fluctuating conditions on the generation
strain at low temperature. Considering the temperatures likely to
and lag times were analysed using turbidity mea-surements. The
be used for refriger-ated storage (0-10°C) or for working meat
data indicated that the cells exposed to osmotic and acid or alkaline
pro-duct premises, the temperature of the study was established
variable conditions from the time of inoculation were less affected
at 10°C.
than cells exposed at the beginning of the exponential phase. In
this last case, a lag phase could be induced and the growth NaCl, acetic acid and NaOH were used to regulate water
recovery was different from those expected in the new environment. activity, acid pH and alkaline pH respectively. As the capacity of
Generation time values were estimated by three available predictive Bioscreen C for studying growth kinetics under variable conditions
models which describe the effects of temperature, salt concentration was ever established, this material was used in this study.
and pH on L. monocytogenes growth to highlight the potential Moreover, with the objective to illustrate the potential pro-blems
problems of variable conditions. associated with the modelisation of growth under fluctuating pH/
Aw, conditions, we proposed to analyse the accuracy of the
The aim of predictive microbiology lies in predicting the growth
gen-eration time predictions with available models and
kinetics of micro-organisms at a given moment during food
characterize these predictions with our ex-perimental data.
processing and so predicting the evolution of food during storage,
manufacturing or preservation accidents. One of the great interests Materials and Methods
of predictive micro- biology is to optimize the shelf life of a high
Strain and Media
risk food product by the use of appropriate predictions.
Mathematical models have been developed to simulate the growth Listeria monocytogenes 14 (serotype 4b, ob-tained from industrial
of micro-organisms in relation to given environ-mental conditions environment) was used throughout the study. Stock cultures were
and can be classified by the microbiological event studied, the maintained in TSA agar slopes and stored at 4°C.
modelling approach used or the variables considered. For preparation of inocula, cultures on TSA were subcultured
Most of these models were elaborated from data acquired in a meat medium (MM) which contained meat peptone 10 gl-1,
un-der constant environmental conditions as poly-nomial models, yeast extract (Dif-co) 5 gl-1 and glucose 5 gl-1. The culture medium
e.g. Food micromodel, Pathogen modeling program, which for growth studies was a tryptic meat broth (TMB). It was buffered
expressed bacterial growth as a function of factors such as pH, with a K2HPO4-KH2PO4 (Merck) 0.1 mol l-1 solution in proportion
1:1 (v/v) to pH 7.0.
114 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 115

Monitoring of Bacteria/growth possi-ble contamination. The well numbers were con-sidered as


An automated turbidimeter was used to follow the growth of replicates. The shock exposures were arranged as follows: 300 µl
L. monocytogenes 14 in the micro-titer plates. Optical density (OD) of inoculated and 300 µl of non-inoculated TMB were dispensed
was read at a wavelength of 600 nm. in each of six and four wells, respectively. An aliquot (100 µl) of
concentrated ( x 4) shock so-lutions or standard TMB was added
Experimental Procedure when the OD was near 0.2, corresponding to the begin-ning of the
The strain was incubated on TSA agar slopes for 7 h at 37°C exponential phase. The osmotic shock was achieved by the
and then transfered to MM that was incubated in a rotary shaker addition of NaCl (Prolabo) according to Chirife and Resnik (1984).
waterbath for 20 h at 20°C, by which time the strain had reached Acetic acid was added to adjust acid pH. The effect of high pH
the stationary phase. A proportion of the culture was inoculated stresses was studied with the presence of NaOH (Prolabo). The
in various media to give a con-centration of about 3.107 cfu ml-1, time which elapsed before adding the shock solutions to 90 wells
in order to be above the detection threshold of the Bio-screen C. was about approximately 10 min. These plates were placed in the
The viable number of bacteria was confirmed by TSA plate counts. Bioscreen C previously adjusted to 10°C.
Experimental Design
The growth of L. monocytogenes 14 at 10°C in the presence of
salt (0-8%), corresponding to Aw value (1-0.95) and in different
values of acid pH (5.6-7.0) or alkaline pH (7.0-9.5) was stu-died by
using an experimental design. This design was a combination of
two central com-posite designs and a factorial design. Each factor
was studied at five levels and for each combination, the shock and
limiting conditions were studied. At least 50 experi-ments were
performed.
Data Analysis
Averages of the OD were calculated for the six repetitions of
inoculated media and for the four repetitions of non-inoculated
Figure. Experimental plan showing the combination of osmotic and acid media. The data were then analysed using the procedure
stresses using NaCI and acetic acid respectively ((D); and the combination
1. (ODi)t, the mean OD of the six repetitions of inoculated
of osmotic and alkaline stresses using NaCI and NaOH respectively (Q).
The points of the factorial design are represented by n. The experiment
media;
numbers are indicated in italics. 2. (ODni)t, the mean of the OD of the four repetitions of non-
For all experiments, three conditions were studied: standard, inoculated media;
limiting and shock conditions. Under standard and limiting 3. (DOD)t=(ODi)t-(ODni)t;
conditions, bacteria were grown in TMB or in TMB ad-justed to 4. log10[(ÄOD)t/(ÄODmin)] where ÄODmin was the lowest
the desired AR, and pH values. For the inoculated TMB medium, DOD value above the detection threshold.
300 µl were dispensed in each of six wells and the same vo-lume
As indicated by Cheroutre-Vialette et al. (1998), a modified
of non-inoculated medium was dispensed in each of four wells in
Gompertz equation (Zwietering et al. 1990) was used to fit the
order to determine the OD of the growth medium and to detect
growth curves log10[(DOD)t/(DODmin)]=f(t). Growth parameters
116 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 117

such as A (logarithmic increase of population), LT (lag time) and acid-osmotic variations involved an unusual growth curve as
µ (maximal growth rate) were determined by non-linear regression shown by Fig. 3. Indeed, consecutively to these shocks, the growth
with STAT-ITCF statistic software. GT (generation time) was derived of micro-organism contin-ued and a rupture of the sigmoid curve
from µ using the relatio: GT=log10(2)/µ. was seen before the growth recovery. The Gompertz equation
In standard and limiting conditions, the time of inoculation fitting left this phenomenon out of account and considered the
was taken as zero time when considering the growth curve. In growth recovery cor-responding to the generation time values. In
shock conditions, the time of the addition of the shock solution exchange, large environmental conditions dur-ing exponential
was taken as zero time in the calculation of growth parameters. phase (i.e. shock condition) of the micro-organism induced a lag
phase before the growth recovery. This lag phase was more
Model Predictions pronounced especially as the concentrations of shock solutions
The ratio of predicted and observed generation times was were higher in case of alkaline-osmotic shocks. This induced lag
calculated for the studied combinations (predicted generation time/ period was really observed and cal-culated when a high
observed generation time). One complementary measure, proposed concentration of NaCl, e.g. 6.8 and 8%, was added with the organic
by Ross (1996) as simple index of the performance of models in acid in the culture.
predictive microbiology, was also evaluated. This value, termed Some growths were particularly affected by the exposure to
the ‘bias factor, may be interpreted as the average ratio of the the combined shock solutions in exponential phase. Indeed, no
predicted and observed values and is defined as follows: increase of op-tical density during the experimental period, i.e. 21
where y predicted is the predicted generation time, y observed days, underlying no increase of population, was noted for the
is the observed value, and n is the number of observations used combination pH 5.6 and 4 or 8% NaCl.
in the calcu-lation. Perfect agreement between predictions and An adaptation period to the new environ-ment (temperature,
observations will lead to a bias factor of 1. pH and water activity) was observed in limiting condition, i.e.
when the shock solutions were present at the begin-ning of the
Results
culture. This period reached 12 days if the cells were placed in
Growth kinetics of L. Monocytogenes presence of higher concentrations of NaCl and alkaline pH, 8%
The abrupt addition of 8% NaCl has a marked effect on the and 9.5, respectively (data not shown). An experimental condition
growth recovery of the micro-organism compared to the effect (pH 5.6, 8% NaCl) caused no increase of optical density during the
produced by the presence of NaOH to alkaline pH 9.5. The growth experimental period. The generation times calculated with the
curve asso-ciated to the combined alkaline-osmotic shock solutions Gompertz equation revealed that growth re-covery obtained in
differed from those of uncombined shocks suggesting an interactive limiting conditions was dif-ferent to those observed in shock
effect of NaOH and NaCl at 10°C. The effects of acid shock (pH conditions. Indeed, the shock condition pre-sented a generation
6.3), osmotic shock (8% NaCl) and combined acid (pH 6.3) - osmotic time value higher than calculated in limiting condition whatever
(8%) shocks at 10°C on the exponential phase of L. monocy-togenes the environmental variation.
14 growth. The acid-osmotic variable conditions have particu-larly In conclusion, two principal pheno-mena were observed when
affected the growth recovery of the strain. Indeed, the organic acid the bacteria sub-mitted to abrupt change of pH and Aw (i) large
and the solute pre-sented an interactive effect on the growth of environmental variations induced a lag phase following the
micro-organism. exposure to shock solutions, and (ii) the growth continued with
In shock condition, L. monocytogenes 14 responded a generation time value different from that observed before the
instantaneously to small changes of pH and Ate,. Small acid and change.
118 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 119

Polynomial Models Predictions The predictions were more especially conser-vative as the
The aim of this part was not to com-pare polynomial models, culture conditions were severe, i.e. low pH and high salt
but to show the position of their predictions with regard to variable concentration. Few plots were above the identity line indicating
environmental conditions. The range of predicted values was wide. that the predicted parameters were higher than the observed values
Considering the limiting condition, the ratio calculated was in in limiting conditions. This was observed for growth predictions
general less than 1.0, indicating that the pre-dicted value was associated to combined alkaline pH-osmotic conditions or
inferior to those observed. uncombined osmotic conditions. In cases of growth predictions
under low pH (pH < 6.3), the predictions values were in general
Table : Generation times, expressed in h, observed in shock or limiting three or four times be-low the observed values and might be 14
conditions. In brackets: asymp-
times inferior in presence of low pH and higher salt concentration,
Totic 95%
confidence i.e. pH 5.8 and 6.8% or pH 6.3 and 8%. Considering the shock
interval condition, the trend of models to have ‘fail safe’ predictions were
Experiment pH % NaCI Generation times confirmed and increased. All the points were below the identity
number line, representing predictions which were shorter than the observed
Limiting Shock generation time. These results were confirmed by the bias factors
conditions condition
which maybe interpreted as the geometric mean ratio of predicted
2 5. 6 0 26.6 (±0.4) 64 (±2)
2
and observed generation times. The bias factors calculated in
2 6. 3 0 9.6 (±0.1) 14.0 (±0.2) shock condition showed, on average, 1.5-fold greater bias than
1 5. 8 1. 2 19.9 (±0.3) 68 (±6) those observed in limiting condition.
5
1 6. 8 1. 2 6.8 (±0.1) 9.2 (±0.3) Discussion
4
5 5. 6 4 120 (±1) NI For several years, it was largely demonstrated that refrigeration
8 6. 3 4 19.9 (±0.3) 41(±1) in conjunction with other factors, e.g. salt or acid, may severely
9 6. 3 4 20.6 (±0.3) 39 (±1) retard or prevent the growth of micro-organisms. As L.
1 5. 8 6. 8 190 (±2) 215 (±3) monocytogenes is common in the food proces-sing environment and
9
1 6. 8 6. 8 10.2 (±0.1) 18.0 (±0.3) as a food product is often exposed to environmental variations, it
8 is important to evaluate the adapta-tion of this bacteria to variable
2 5. 6 8 NI NI conditions of environmental factors as pH and Aw at low
5
tem-perature. A previous study, Cheroutre-Vialette et al. (1998)
1 6. 3 8 56.8 (±0.6) 129 (±2)
1 has reported that the generation times calculated for the growths
20 (standard 7. 0 0 5.1(±0.1) of L. monocy-togenes in shock and limiting conditions under variable
condition) pH or Aw conditions at 20°C were sig-nificantly different.
3 7. 0 4 6.3 (±0.1) 9.5 (±0.1) Moreover, an additional lag time in shock condition could be
2 7. 0 8 9.3 (±0.1) 17.9 (±0.2)
4 observed. The results obtained in the present study car-ried out at
a: Growth parameter was calculated according to zero time considered as the low temperature and under com-bined pH-Aw conditions were in
time of inoculation. agreement with the conclusions established in this pre-vious study.
b: Growth parameter was calculated according to zero time considered as the

moment of shock.
The aim of this paper was to introduce more advances into the
NI: No increase in optical density during 21 days. field of predictive microbiology, by specifying the microbial
120 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 121

behavior under dynamic environment of pH and water activity investigations of bacterial growth pre-dictions with changes in
and collecting experimental data. In this way, the use of Bioscreen temperature by Zwie-tering et al. (1994) andVan Impe et al. (1995),
C allowed to rapidly ex-plore the microbial impact of varying pH or with changes in pH by Rosso (1995) admitted the possible
and Aw conditions. In recent years, the interest in developing transposition of results obtained from constant conditions to
dynamic mathematical models that describe the growth of variable condi-tions. Considering our results, such postulate is
microorganisms in the presence of environmental factor variations not suitable to take into account the physio-logical response of
has increased. With the objective to take into account the effects micro-organisms.
of temperature variations on microbial growth, Baranyi et al. (1993) The results of this study highlighted the pro-blems associated
observed that the abrupt transitions could lead to adjustment with the variable conditions and the available models established
periods, i.e. microbial cultures, when shifted abruptly from one in con-stant conditions. The results obtained with the predictions
temperature to another, may exhibit a transient growth rate before estimated with the models (FMM, PMP, Lm14) are not surprising
assuming the growth rate expected at the new temperature. taking into con-sideration that models were developed with strains
Table : Comparison of model predictions of the data of different origins, in media of various compositions and in
PMP FMM Lm14
different conditions of growth. For example, the pH was adjusted
Limiting Shock Limiting Shock Limiting Shock
with HCl in PMP or Lm14, lactic acid in FMM. Acetic acid was
na 15 14 15 14 9 8
Ratiob average 0.53 0.33 0.55 0.34 0.62 0.43
used for pH regulation in our growth experiments and it was well
Ratio minima 0.07 0.06 0.05 0.05 0.08 0.12 estab-lished that this organic acid presents a higher inhibitory
Ratio maxima 1.15 0.60 1.19 0.63 1.25 0.82
activity on L. monocytogenes. As can be pointed out by this study,
Bias factor 0.40 0.25 0.39 0.25 0.50 0.33
a: n ˆ number of observations used in the calculation.
the predictions of these models based on data generated under
constant conditions can reliably predict growth under fluctuating
b: ratio ˆ predicted generation time/observed generation time.
conditions of pH and Aw. But, a systematic over-prediction was
revealed under dynamic environment. Under a dynamic
Our data under higher pH and/or Aw, variations confirmed environment, the bias factor showed that the recovered growth
the pre-sence of adjustment period which was repre-sented by an rates caused much larger generation times than those under
induced lag phase. When these environmental changes were constant conditions. In consequence, the structure of these models
smaller, an unusual growth curve was observed. This seemed to be inappropriate to take into account the microbial
char-acteristic raised some questions about its origin. This could response to variable en-vironmental conditions.
be assigned to the material used, the Bioscreen C. The lateral
agitation of the plates in Bioscreen C could have been insufficient Moreover, the induced lag phase observed in dynamic
for homogenous distribution of the compound in the medium. At environment of pH and Aw could not be integrated by these models.
the moment of shock (acid or acid-osmotic), a time is needed for The necessity to determine a new way to model changing pH
the repartition of the shock solutions in the well. A study of values, varying Aw or other food paramenters was underlined.
Jorgensen et al. (1995) has also reported that the cells of L. Neuronal networks could represent a new approach to take into
monocytogenes incubated in media containing NaCl became rapidly account the varia-bility of cell response in variable conditions and
longer than cells grown without salt. Such morphological changes produce a dynamic model. A previous study provided a good
could also lead to variations in the turbidity of the medium. prediction of L. monocytogenes growth under variable conditions.
Such dynamic models could follow the microbial impact of the
The experiments of the present study showed also that the different steps associated with the production, distribu-tion and
recovered growth rate after the change of pH and/or Aw was retailing of a food.
usually less than those associated to the new environment. Other
122 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 123

EFFECTS OF PH OR AW STRESS ON GROWTH OF LISTERIA Monitoring of Bacterial Growth


MONOCYTOGENES An automated turbidimeter (Bioscreen C, Labsystem, Labsystem
The growth of three strains of Listeria monocytogenes at 20°C in France SA, Les Ulis, France) was used to follow the growth of
a meat broth of different pH or water activity was investigated. At Listeria strains in the micro-titer plates. Optical density (O.D.) was
inoculation or at the beginning of the exponential phase, cells read at a wavelength of 600 nm.
were exposed to stress by the addition of NaOH or NH4+, acetic A two-litre fermentor SET2M was used. The O.D. of the growth
acid, NaCl or KCl, in order to reach a pH of either 9.0 or 5.6, or was measured with a spectrophotometer (UV-160A, Shimadzu,
an aw of 0.950 or 0.965, respectively. Japan) at 600 nm.
The effects of the exposure to stress on the generation and lag
times of each strain were analysed by turbidity measurements for Microorganisms
cultures in micro-titer plates. Results were confirmed by conducting The strains used by Bégot et al. (1997) in the studies with
the same experiments in a fermentor, except for the maximal Bioscreen C were chosen for this work: L. monocytogenes CLIP
population reached. The three strains showed similar behaviour. 19804 isolated from meat products, L. monocytogenes 14 obtained
Cells were able to overcome the alkaline stress rapidly whereas from the food processing environment and L. monocytogenes CA
acid and osmotic shocks induced important changes of the growth Wisconsin associated with a cheese-borne listeriosis outbreak. All
parameters. Cells exposed to acid or osmotic conditions from the were serotype 4b which was involved in the recent epidemics in
time of inoculation were less affected than cells exposed at the France. L. monocytogenes 14 strain was also used in the fermentor
beginning of the mid-exponential phase. studies. Stock cultures were maintained on TSA agar slopes and
Listeria monocytogenes, a psychrotrophic bacteria, is considered stored at 4°C.
as an important food-borne pathogen. It can persist and grow at
Media
low pH, high pH, low water activity and at refrigeration
temperatures. For preparation of inocula, cultures on TSA were subcultured
in meat medium (MM) which contained meat peptone 10 g/l,
Work on predictive microbiology has been carried out in order
yeast extract (Difco) 5 g/l and glucose 5 g/l.
to improve the shelf life and safety of foods. Predictive models of
microbial growths were set out with respect to the main controlling The culture medium for growth studies was a tryptic meat
factors of the environment such as temperature, pH, and water broth (TMB) (Fournaud et al., 1973). It was buffered with a
activity. K2HPO4–KH2PO4 (Merck) 0.1 mol/l solution in proportion 1:1 (v/
v) and adjusted to pH 7.0 with NaOH (40 g/l).
These factors were often considered constant during growth.
But, environmental factors, particularly the temperature, may vary Types of Experiment
extensively throughout food processing. To take the environmental
For all experiments, three conditions were studied: standard,
variations during time into account, different authors have
limiting and shock conditions. Under standard and limiting
improved predictive models and proposed dynamic models which
conditions, bacteria were grown in TMB or in TMB adjusted to the
describe growths at varying temperature profiles.
desired aw or pH values. The following additions were made to
In this study, we report the effects of water activity and pH TMB: NaOH (solution of 200 g/l; Prolabo) 12.2 g/l or NH4+ (Merck)
shifts on the growth of three strains of L. monocytogenes using 7 g/l to obtain pH of 9.0, acetic acid (Carlo Erba, Nanterre, France)
different osmotic solutes (NaCl, KCl), organic acid (acetic acid) 4.1 g/l to obtain a pH of 5.6, 80 g/l of NaCl (Prolabo) or 80 g/
and bases (NaOH, NH4+). l of KCl (Prolabo) (Jakobsen et al., 1972) to obtain aw=0.95 and a
124 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 125

pH of 7.0 or aw=0.965 and pH of 7.0, respectively. The shock 1. (O.D.i)t, the mean of the O.D. for the 6 repetitions of
conditions were defined as follows: the bacteria were grown in inoculated media;
standard TMB until the beginning of the exponential phase and 2. (O.D.ni)t, the mean of the O.D. for the 4 repetitions of non-
were then exposed to the pH and aw values described above. inoculated media;
Growth in Microplates 3. (DO.D.)t=(O.D.i)t–(O.D.ni)t;
Each strain was incubated on TSA slopes for 7 h at 37°C and 4. log10 [(DO.D.)t/(DO.D.min)] where DO.D.min was the lowest
then transferred to MM that was incubated in a rotary shaker DO.D. value above the detection threshold.
waterbath (Aquatron, Infors, Switzerland) for 18 h at 20°C, by A modified Gompertz equation (Zwietering et al., 1990) was
which time growth of all strains had reached the stationary phase. used to fit the growth curves log10 [(DO.D.)t/(DO.D.min)]=f(t).
A proportion of the culture was inoculated in the various Growth parameters such as A (logarithmic increase of population),
media to give a concentration of about 5.107 CFU/ml in order to L (lag time) and (maximal growth rate) were determined by non-
be above the detection threshold of the Bioscreen C. For the linear regression with STAT-ITCF software. Tg (generation time)
inoculated TMB medium, 300 l were dispensed in 6 wells and the was derived from µusing the relation:
same volume of non-inoculated medium was dispensed in 4 wells log10 (2)
in order to determine the O.D. of the growth medium and to detect Tg =
µ
possible contamination. The well numbers were considered as
replicates. The same procedure was used for the pH and aw In standard and limiting conditions, the time of inoculation
adjusted TMB. The shock exposures were arranged as follows: 300 was taken as zero time when considering the growth curve. In
l of inoculated and 300 l of non-inoculated TMB were dispensed shock conditions, the time of the addition of the shock solution
in 6 and 4 wells, respectively. An aliquot (100 l) of concentrated was taken as zero time in the calculation of growth parameters.
(×4) stock solution or standard TMB was added when the O.D.
RESULTS
was near 0.2. The delay in adding the solutions to 70 wells was
approximately 7 min. These plates were incubated in the Bioscreen The growth curves of L. monocytogenes 14 in the presence of
C previously adjusted to 20°C. acetic acid and NaCl can be seen in Fig. 1a and b, respectively.
When the shock treatments (acid and osmotic) were applied after
Growth in Fermentor the beginning of growth of L. monocytogenes in standard media,
The protocol was similar to that in Bioscreen C except for the lags were shorter, but generation times increased, as compared
fermentor volume with additions adjusted accordingly. The with cells grown in limiting media. This phenomenon was
additions lasted about 10 min. The experimental conditions were particularly pronounced for the acetic acid example.
20°C with a shaking speed of 100 rpm and an aeration regulated In comparison with growth in standard media, growth of the
at 0.2 litre of sterile air per litre of medium per min. strain was not really affected by the presence of NaOH and NH4+
whatever the treatment, limiting or shock conditions. The growth
Data Analysis parameter values, i.e. generation and lag times, confirmed that
Averages of the O.D. were calculated for the six repetitions of cells adapted rapidly to the alkaline upshift to pH 9.0 with NaOH
inoculated media and for the four repetitions of non-inoculated or NH4+. Indeed, these values calculated in alkaline media were
media. The data were then analysed using the procedure described consistent with those found in standard media. Under acid and
by Bégot et al. (1996). Four quantities were calculated at time t: osmotic conditions, cell growth was more affected. Media
126 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 127

containing acetic acid was the most severe culture condition for demonstrated that the growth kinetics of L. monocytogenes were
the growth of the three strains. Under limiting conditions, although unaffected by the size of the initial inoculum i.e., the generation
the presence of acetic acid did not cause increasing lag times, and lag times derived from the Gompertz equation were not affected.
generation times were at least 3.5 times greater compared to the The L. monocytogenes strains showed similar responses
values in standard media. The solute used to control aw can following aw or pH stresses. Variations were generally larger in
influence the growth pattern of the microorganism. At the tested lag times than in generation times. There was very little difference
concentration (80 g/l), L. monocytogenes which is also considered in generation times between L. monocytogenes strains 14 and 19804
as a salt-tolerant microorganism, tolerated KCl better than NaCl. for most of the experiments. Any major variations were observed
Indeed, the generation and lag times in limiting and shock with the most severe culture conditions, i.e., in the presence of
conditions, were higher in the presence of NaCl 80 g/l. This acetic acid. L. monocytogenes strain CA was more affected by the
influence of NaCl can be due to a larger aw effect. different stress conditions and in particularly under limiting
Generally L. monocytogenes 14 and 19804 had similar conditions. These findings are consistent with those of
generation times whereas L. monocytogenes CA had longer Papageorgiou and Marth (1989) who also reported that L.
generation times under limiting conditions. Variations in lag time monocytogenes CA was less salt tolerant than strains in whey and
were noted among the strains in all tested conditions. skimmed milk containing 12% NaCl.
Generation and lag times of cultures in the fermentor are In recent years, the interest in developing dynamic
presented in Table 3. The values of generation times were similar mathematical models that describe the growth of microorganisms
to those obtained in Bioscreen C, but large variations in lag phases in the presence of temperature variations, has increased. When
were noted. The results obtained from the fermentor culture building dynamic models to predict bacterial growth with change
experiments confirmed the observations made with the Bioscreen C. in temperature, two hypotheses have been formulated: (i) the
bacteria show no additional lag phase due to a change in
DISCUSSION temperature during the exponential phase, and (ii) growth
In previous studies about growths in non-variable conditions, continues immediately at the specific growth rate associated with
the interest in using the Bioscreen C for analysis of microbial the temperature post-shift. More recently, for the elaboration of
growth parameters and its good reproductivity was established. dynamic models, Van Impe et al. (1995) and Rosso (1995) accepted
Our study confirmed these observations. The results obtained from as a postulate, that variable environmental conditions do not
the experiments carried out in the automated turbidimetric system induce stress conditions on a microbial population, i.e., they
were in agreement with those obtained in the fermentor. The increase considered that the microflora responds instantaneously to pH
in the number of L. monocytogenes was underestimated in the (Rosso, 1995) or temperature changes (Van Impe et al., 1995 and
Bioscreen C as compared to the fermentor. The homogeneous Rosso, 1995). Our tests carried out with the three L. monocytogenes
aeration and agitation which were controlled in the fermentor strains showed that growth under modified osmotic or acid
might be a possible explanation for this observation. Nevertheless, environmental conditions in exponential phase did not agree with
the differences found in the generation times between the two these hypotheses. Since 1992, Van Impe et al. (1992) have
methods were not significant. This suggests, therefore, that the recognized the need to take into account the previous history of
Bioscreen C is suitable for studying the growth of bacteria under a product, and therefore the previous history of a strain which
variable conditions. A high concentration of cells is required in could have an important impact on the lag time prediction of a
the Bioscreen C for a detectable change in absorbance, but microorganism. It is also necessary to quantify the ability of a
Buchanan and Phillips (1990) and Dalgaard et al. (1994) microorganism to grow under variable conditions. This could
128 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 129

allow the optimal combination of temperature, pH, aw and other predictive microbiology lies in predicting growth kinetics of micro-
factors with the purpose of increasing the shelf life of a given organisms at a given moment during food processing and thus
product. predicting the evolution of food during storage, manufacturing or
preservation accidents. Rosso et al. (1993, 1995) proposed, in their
VALIDATING THE USE OF GREEN FLUORESCENT- cardinal approach, a model in which the maximum microbial
MARKED ESCHERICHIA COLI O157 specific growth rate ( max) is a function of temperature or pH. The
Monitoring bacterial kinetics in food is of great importance in cardinal values of the considered strain are then determined. As
food safety. The targeted micro-organism has to be identified an example, for temperature values, the following parameters are
accurately among competitive flora. Using green fluorescent protein considered: T min (the temperature below which growth is no
(GFP)-transformed strains is a possible answer to such issues. longer observed), T max (the temperature above which no growth
However, quantitative studies require that this transformation does occurs) and T opt (the temperature at which the maximum specific
not alter the micro-organism behaviour: parent and transformed growth rate max equals its optimal value opt). The same procedure
organisms were thus compared. can be applied to pH and water activity a w. The minimal and
Three Escherichia coli O157:H7 strains were transformed using maximal values are important factors to food preservation, and
a GFP-plasmid expressing. Parent and transformed strains were the optimal value is a crucial parameter in predictive microbiology.
In this approach, for a given strain, opt is a food-dependent
compared according to their genetic characteristics and serotypes.
Growth ability was also assessed in constant and fluctuating parameter.
temperature profiles. Cardinal values of pH, water activity and The development of sensitive methods for monitoring bacteria
temperature were computed. No differences were observed between in foods, especially in presence of natural contaminating flora, is
parent and transformed strains for all these experiments. The of marked importance. Indeed, models have to be validated and it
plasmid was satisfactorily maintained within transformed strains is usually more appropriate to collect new data in food to have a
throughout the studies. Growth was eventually monitored in beef good validation. In this way, the objective of this work was firstly
meat. the construction of E. coli O157:H7 strains marked with the green
Although Escherichia coli is a common inhabitant of the human fluorescent protein (GFP) gene carried by a plasmid. The GFP from
and animal gastrointestinal tract, several pathogenic types of the the jellyfish Aequorea victoria, a versatile 27-kDa protein, has proved
to be valuable as a tool for studying a variety of biological questions,
species are able to cause a variety of human diseases. First
recognized as a human pathogen in 1982, E. coli O157:H7 [Shiga e.g. to study gene expression, to determine protein distribution or
toxin-producing E. coli (STEC)] has since been associated with to tag a cell lineage in vivo, in situ and in real time. The GFP emits
green light when excited with ultraviolet (u.v.) radiation and no
outbreaks of food-borne illness in countries across the world, with
many foods identified as vehicles of infection. The main virulence substrate or cofactor is required for fluorescence.
factor of STEC is the ability to form cytotoxic exotoxins. Two major Ajjarapu and Shelef (1999) used GFP-bearing E. coli O157:H7
categories of Shiga toxins have been distinguished, Shiga toxin 1 in ground beef and were able to monitor its evolution despite the
(stx1) and Shiga toxin 2 (stx2). The other virulence factors are the presence of a background microflora. It should yet be verified that
property of producing attachment-effacement lesions and the using GFP-transformed strains instead of nontransformed strains
presence of an entero-haemolysin gene. is a right way, i.e. transformed strains and parent strains should
Given the severity of the illness associated with E. coli O157:H7, behave in a similar manner. Fratamico et al. (1997) found no
it is necessary to provide the food industry with procedures for differences according to morphological or biochemical criteria or
using PCR. In a second part of the present work, the influence of
preventing its presence and controlling its growth. The aim of
130 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 131

temperature on the growth kinetics of both GFP-E. coli strains and Parent and transformed strains were compared according to
their parental strains was assessed in liquid medium. GFP their genetic characteristics (stx genes), serotypes and biochemical
expression was evaluated during growth. Cardinal values of pH, characteristics. Detection of stx genes was performed with
a w and temperature of GFP or parental strains were determined degenerate primers ES149 and ES151 as described by Read et al.
comparatively. Furthermore, experiments at varying temperature (1992). These primers amplified a conserved sequence of stx 1 and
profiles were carried out in order to simulate temperature-changing stx 2 genes. For DNA preparation, 1 ml of an overnight sample
conditions (food processing, food storage, etc.). The results obtained broth (BHI) was centrifuged at 12 000 g for 3 min. The pellet was
in liquid medium were complemented by experiments in food. washed in PBS buffer (pH 7·4; Sigma). The DNA from pelleted
cells was released by boiling and purified using Instagen DNA
Strains and Media purification matrix. Ten microlitres of the DNA were used as
Three E. coli O157:H7 strains were used in this study. One of template for PCR detection of stx genes. The conditions for the
the strains was isolated from bovine faeces and will be referred to PCR amplification were the same as those described by Uyttendaele
as strain 1, the other two were of clinical origin and will be et al. (1998).
referred to as strains 2 and 3. These strains were transformed with Serotyping of the different strains was performed for O157
a plasmid vector pBAD-GFPuv (BD Biosciences - Clontech Inc., and H7 determination with Difco antisera.
Palo Alto, CA, USA), adapted for visualization under u.v. light.
Parent or transformed strains were biochemically confirmed
Stock cultures were maintained at 80°C in cryobank.
by using the API 20E test strips.
All strains were resuscitated before use by inoculation into
10 ml of brain-heart infusion (BHI) broth (Oxoid Ltd, Basingstoke, Growth Kinetics as Function of Constant or Dynamic Temperature
Hampshire, UK), followed by incubation at 37°C for 8 h. The in CARY 100
subculture and culture medium was BHI (Oxoid) supplemented Behaviour of parent and GFP-expressing strains in function of
with yeast extract (Oxoid) 3 g l 1 and glucose 2 g l 1. The BHI temperature was compared in modified BHI. Parent and
supplemented medium was sterilized by filtration (0·20 m pore transformed strains were grown simultaneously in a
size). This will be referred to as modified BHI. spectrophotometer CARY 100. Three replicates were carried out
The count medium was plate count agar for the E. coli strains. for each condition.
Plates were incubated at 37°C for 24 h. After resuscitation (in modified BHI, 8 h at 37°C), the parent
or GFP-expressing strains were subcultured in modified BHI and
Construction and Identification of GFP-expressing E. coli
in modified BHI + ampicillin (50 g ml–1), respectively at the defined
Escherichia coli strains were transformed with pBAD-GFPuv temperature until the beginning of exponential phase. A 0·5% of
by the calcium chloride method. The pBAD-GFPuv plasmid vector this inoculum, corresponding to inoculum size of 106 CFU ml–1,
contains an arabinose-induced promoter and an ampicillin was then transferred to the culture medium (modified BHI) for
resistance gene. Transformants were selected by plating on PCA growth assessment. Growth was monitored in constant
supplemented with arabinose 2 mg ml–1 and ampicillin 50 µg ml– temperature conditions at 37, 20 and 10°C. Moreover, growth
1
(PCA + Ara + Amp). Colonies on PCA + Ara + Amp, that were evaluation was carried out in dynamic temperature conditions as
induced by arabinose and ampicillin resistant, were fluorescent follows: the strains were first placed at a start temperature until
under u.v. light. Stability of fluorescence was monitored. Indeed, beginning of exponential phase and then, a new temperature was
the percentage of fluorescent colonies before and after applied. Studied conditions were: 37°C followed by 20°C, 37°C
experimentations was also examined throughout the present study. followed by 10°C, 20°C followed by 10°C and 10°C followed by
132 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 133

20°C. The temperature gradient was usually achieved in <15 min. A growth curve was produced with 10-15 points and repeated
In order to check fluorescence, plate counts (PCA + Ara) were once. On each measurement time, one bag was opened for
performed at the beginning and at the end (cells in stationary enumeration. Meat was placed in 90 ml of tryptone salt broth, and
phase) of every experiment. then homogenized for 1 min. Dilutions were made in tryptone salt
broth to obtain appropriate levels. Plating was made on PCA for
Determination of Cardinal Values of pH, a w, Temperature in total bacterial count, on PCA + arabinose (0·2%) (PCA + Ara) for
Bioscreen C visualization of the GFP strain under u.v. light, and on
The methodology was defined in the framework of the French PCA + arabinose (0·2%) + ampicillin (50 g ml –1 )
Predictive Microbiology Project, Sym’Previus as described by (PCA + Ara + Amp) as a selective medium allowing expression of
Membre et al. (2002). The detection time method was used to fluorescence.
calculate max for different conditions of temperature (tested range:
8-45°C), pH (adjusted by HCl: pH 4-7) or a w (regulated by NaCl: Statistical Analysis of Data
0·5-8%) in a Bioscreen C. Briefly, this method is based on the fact Analysis of variance, regressions or confidence intervals
that successive binary dilutions produce growth curves that are calculations were performed using software S-Plus 2000. Graphical
shifted from each other by one-generation time value. outputs were also produced with Microsoft Excel 2000.
The obtained values of µmax allowed determination of cardinal The primary model chosen in this study was the model of
values (T min, T opt, T max, pHmin, pHopt, pHmax, a w min, a w opt, a w Rosso (1995). It was used for constant and dynamic temperature
) and µopt with Rosso secondary model. Cardinal values a w max studies and for food experiments, in order to calculate primary
max
were set to 1. Cardinal values pHmax were chosen so that pH model parameters (mainly lag time and growth rate max). A cardinal
model would be symmetric, i.e.: values model was chosen as secondary model.
pHopt = (pHmin + pHmax)/2
RESULTS
pHmax = 2pHopt – pHmin.
Construction and Identification of GFP-expressing E. coli
The cardinal values of pH, a w and temperature were obtained
pBAD-GFPuv was successfully introduced into the three E.
in modified BHI for the parent strain 2. For the transformed strain coli O157:H7 strains. Colonies of the transformed and parent strains
2, these cardinal values were determined in modified BHI + Ara. had identical morphological characteristics on PCA + Ara + Amp
The determination of fluorescence percentage was studied as (for GFP strains) and PCA (for parent strains), except that colonies
described before. of transformants appeared green under u.v. visualization, whereas
Experiments in Food those of the parent strains remained nonfluorescent.

In order to evaluate the potential utilization of GFP strain in The comparison of the parent and transformed strain genetic
food product, growth of GFP-transformed strain 2 was monitored characteristics, i.e. detection of stx genes, showed that virulence
in raw ground beef at 10°C. The strain was first subcultured in genes were present after the plasmidic transformation. Furthermore,
modified BHI + Amp at 37°C for 8 h; a second subculture was no serotypic (i.e. O157:H7) or biochemical difference was recorded
conducted in the same medium at 10°C for 4 days. between parent and transformed strains.

The suspension was diluted to produce an inoculation level Stability of fluorescence was also monitored. The
in beef of ca 2·5 103 CFU g 1. Inoculated meat was divided into transformants were cultured at 37°C in the nonselective liquid
10 g aliquots and placed in sterile bags. medium, i.e. without ampicillin. The plasmid was maintained in
more than 60% of colonies after 13 days of overnight cultures.
134 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 135

Growth Kinetics as Function of Constant or Dynamic Temperature concentration, the lowest acid pH or the highest temperature. But
GFP-expressing strains were compared with their respective the percentage never exceeded 33%.
parent strains according to growth rate and lag time values
Growth in Beef Meat
(calculated from Rosso primary model) under various temperature
conditions. Correct agreement between the strain results was The potentiality of use of GFP-expressing strain in food
observed. product was evaluated. A challenge-test with the transformed
strain 2 was carried out in raw ground beef at 10°C. High counts
Loss of GFP expression was also examined for transformed
of a competitive flora, up to 105 CFU ml–1, were initially found in
strains. Colonies plated on PCA + Ara were counted, and another
meat, which represented particularly hard condition to follow the
count was made under u.v. light in order to evaluate the proportion
growth of a specific E. coli strain. The total flora was counted on
of fluorescent colonies. The percentage of nonfluorescent colonies
PCA, and GFP strain on PCA + Ara and PCA + Ara + Amp (as a
corresponded to plasmid loss within the bacterial population.
selective medium). The evolution of the GFP strain was easily
Mean plasmid loss percentage was under 10% in all the cases.
distinguished from competitive flora thanks to its fluorescence: its
The maximal range of observed plasmid loss was 0-21%. These
survival could be monitored throughout the experiment. The results
observations indicated of a correct GFP expression, and therefore
obtained in the two plating media were similar.
a conservation of the plasmid at whatever the environmental
temperature conditions. A constant temperature of 10°C gave less DISCUSSION
favourable results, but experiments were longer (5 days). Strains
Using GFP transformation allowed for successful applications
seemed to keep their fluorescence better in dynamic conditions
in assessing various biological issues. In the present work, the
(rapid temperature changes) of growth.
construction and evaluation of E. coli strains carrying the GFP-
Cardinal values of a w, pH, Temperature expressing plasmid pBAD-GFPuv are reported.
Cardinal values of a w, pH and temperature for strain 2 are The presence of this plasmid does not affect the intrinsic
indicated in Table 2 with their confidence intervals. As shown by characteristics of the E. coli strains, i.e. the serotypic or biochemical
these results, the confidence intervals between the parent and the traits and virulence genes. No significant behaviour difference
transformed strains are overlapping. This trend confirmed the between marked strains and parent strains could be found, as
previous observations, showing no differences between the parent indicated by overlapping confidence intervals. A major limitation
strain and the GFP-expressing strain behaviour. It must be noted of the GFP marker is the small number of studies on the influence
that the pHmax was estimated by a symmetric model and no of environmental conditions on the GFP expression, and more
experiment was conducted to alkaline value. This could explain particularly, for a plasmid marker.
the fluctuation of calculated pHmax values. Our results have demonstrated the stability of the marker in
The loss of GFP-expression for transformed strain 2 was unfavourable environment, i.e. in presence of acid, salt and in
evaluated for all tested conditions of a w, pH and temperature. The cooling/heating environment. Indeed, the study of the transformant
means of the plasmid loss percentage, as well as the minimal and strain cardinal values showed that the GFP persists in large ranges
maximal percentages, were calculated and indicated in Table 3. of pH values (4·0-7·0), NaCl concentrations (0·5-8%) and
Whatever the environmental conditions, strain 2 exhibited a good temperature (8-45°C). Furthermore, the plasmid stability was
conservation of the plasmid. In general, the plasmid loss was confirmed in dynamic conditions of temperature, where the extent
below 10%. This value could be more pronounced for the most of variation could reach 27°C. It is well known that this factor may
severe culture conditions, e.g. in presence of the highest salt vary extensively throughout food processing. The stress induced
136 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 137

by the abrupt change of temperature did not increase the plasmid may be a valuable tool for microbial risk assessment, especially for
loss. foodborne pathogens. Thus, a number of Listeria monocytogenes
Most foods are complex systems with heterogeneous microbial strains responsible for human cases of listeriosis, in relation to the
populations. The introduction of the GFP marker provides a simple consumption of contaminated seafood, have been compared with
method to differentiate between inoculated strains and natural “natural” L. monocytogenes strains isolated from similar seafood
competitive flora of the product. Indeed, in the experiments on products. Complete factorial designs were used to assess
food described above, we could rapidly distinguish individual quantitatively the growth abilities of four clinical and four seafood
species within the natural flora of the product. GFP labelling in isolates of L. monocytogenes placed in various environmental
micro-organisms makes it a marker of choice for ecological studies. conditions. The cells were submitted to acid and osmotic stress as
Furthermore, the stability of the plasmid in the absence of selection they were in stationary phase (constant condition) or in
obviates the need for the addition of antibiotics to these systems exponential phase (dynamic condition). The effects and interactions
in short-time experiments (14 days in the tested food experiment). of pH (5–7) and NaCl concentration (0.5–8% v/v) were studied at
In laboratory medium, GFP-labelled strain could be detected on two growth temperatures (10 and 20 °C). Growth parameters (lag
the basis of green fluorescence, even after 13 days of inoculation. and generation times calculated with Gompertz equation) were
This observation is in accordance with the previus study of used to compare the behavior of the strains with respect to the
Bloemberg et al. (1997), who examined the stability of a GFP- conditions of culture. The results indicated an overall weak effect
containing plasmid in Pseudomonas spp. of acid stress alone, whereas osmotic stress clearly affected bacterial
growth and a synergic effect between these two factors was
Marking cells with GFP expressed from a plasmid provides
observed. Clinical strains displayed better adaptation than seafood
the flexibility to transform a wide range of strains. Indeed, the
strains in stationary phase, however, this difference was not verified
construction of such strains is relatively easy.
in exponential phase. Low temperature (10 °C) usually confirmed
Predictive food microbiology has been focusing on modelling the observations at 20 °C, and the differences between clinical and
the microbial responses to food environment, in the interest of food strains were more pronounced. Finally, a classification of the
food safety and for avoiding spoilage. Predictive models have eight strains, based on the collected data, showed three groups: (i)
been regularly published to describe the growth of a strain or a seafood strains, (ii) three clinical strains and (iii) the last clinical
mixture of strains as a function of the environmental factors of strain, alone due to its high resistance to adverse conditions.
food. A good way of validating a model is to compare its prediction
Listeria monocytogenes assumed public health significance as a
to real data obtained from food products. The standard method of
result of its presence in foods linked to several outbreaks of
data collection is the total viable count, which is a very labour-
listeriosis in Europe and North America. Most cases of human
intensive method. In this way, the use of GFP-marked strain of
listeriosis occur in immunocompromised individuals, pregnant
providing a useful system for monitoring the evolution of a defined
women and the elderly.
micro-organism and even developing among competitive ones,
presents strong advantages. L. monocytogenes is a widespread microorganism that has been
isolated from a variety offoods including fish. Although several
GROWTHS KINETICS COMPARISON OF CLINICAL AND kinds of food products have been incriminated as source of
SEAFOOD LISTERIA MONOCYTOGENES ISOLATES IN ACID sporadic or epidemic listeriosis cases, the involvement of fish and
AND OSMOTIC ENVIRONMENT fishery products is still very rare. In 1992, two perinatal listeriosis
Comparison of pathogenic bacterial strains of clinical origin cases, which occurred in Auckland, New Zealand, were associated
with strains of the same species isolated from the environment to the consumption of smoked mussels. Mussels were also involved
138 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 139

in cases occurred in Sydney, Australia. An outbreak caused by microbial risk assessment, especially for foodborne pathogens.
rainbow trout was reported in Sweden. Therefore, it was of interest to evaluate and compare the capacity
The prevalence of L. monocytogenes in naturally contaminated of growth of clinical and food strains, in order to determine whether
seafood was studied by Jorgensen and Huss (1998): the highest there was a relationship between strain origin and the growth
prevalence was found in cold-smoked fish (34–60%), while the potentialities in function of environmental factors.
lowest was found in heat-treated and cured seafood (4–12%). L. Consequently, the aim of this work was to investigate the
monocytogenes has been found in smoked salmon in several studies. growth behaviors of four L. monocytogenes strains responsible for
Ben Embarek (1994) reported a contamination rate of between 0% human cases of listeriosis in relation to consumption of
and 75%, with an overall prevalence of 10%, in cold-smoked contaminated seafood and four L. monocytogenes strains isolated
salmon samples. from seafood products, in function of temperature, pH and salt
In fresh as well as in several lightly preserved seafoods concentration. Furthermore, the effects of the pH and salt
including cold-smoked salmon, none of the processing steps concentration variations on the growth of the strains were taken
eliminate L. monocytogenes. Furthermore, cold-smoked fish products, into account, with the objective to simulate the variations which
which are typically consumed without cooking, are among the can occur during food processing and induce a stress situation for
ready-to-eat foods of particular concern due to the lack of a heat the microorganism.
inactivation step during processing. The ability of a microorganism
Monocytogenes Strains and Media
to survive in various foods depends on several combined
parameters in the foods such as temperature, pH, salt concentration Experiments were carried out with eight L. monocytogenes
(water activity). A range of seafoods, particularly the lightly strains. Four were clinical strains associated to fish or fish products
preserved products (<6% salt concentration, pH>5) such as smoked and four were isolated from seafoods.
fish products (hot and cold smoked), lightly salted products (brined Stock cultures were maintained at “80 °C in cryobank. All
cooked shrimp) or marinated products, are capable of supporting strains were resuscitated before use by inoculations into 10 ml of
the growth of L. monocytogenes (Huss et al., 2000). Moreover, L. Brain Heart Infusion (BHI) broth (Oxoid, Basingstoke, Hampshire,
monocytogenes is able to survive or grow at refrigeration UK), followed by incubation at 37 °C for 8 h.
temperatures. The pathogen growth was observed in vacuum- The subculture and culture medium was BHI (Oxoid)
packed smoked salmon stored at 10 or 2 °C. It is important to note supplemented with yeast extract (Oxoid) 3 g l”1, glucose (Prolabo,
that cold smoking is conducted at temperatures below 30 °C, most Fontenay sous bois, France) 2 g l”1 and buffered with a K2HPO4–
frequently 19–22 °C, for 2 to 3 h and, therefore, provides a possible KH2PO4 0.1 mol l”1 solution in proportion 1:1 (v/v) to pH 7.0.
environment for bacterial growth.
Several studies have shown that the virulence of individual L. Monitoring of Bacterial Growth
monocytogenes strains may differ. Furthermore, Datta (1994) An automated turbidimeter was used to follow the growth of
demonstrated that the pathogenicity of this microorganism can be L. monocytogenes strains in the micro-titer plates. Optical density
affected by various substrate factors, i.e. in foods. There are fewer (O.D.) was read at a wavelength of 600 nm. The working volume
published systematic studies in which growths of clinical and in each well of the micro-titer plate was 400µl.
food strains of L. monocytogenes under unfavourable conditions
(found in industrial environment) were compared. The comparison Experimental Procedure
of pathogenic bacterial strains of clinical origin to strains of the The ability of the eight L. monocytogenes strains to grow in
same species isolated from environment may be a valuable tool for osmotic and acid environment was determined as described by
140 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 141

Cheroutre-Vialette et al. (1998). After resuscitation, strains were For each combination, the constant and dynamic conditions
subcultured (1%) in the supplemented BHI 18 h at 20 °C, by which of pH and NaCl concentration were studied. At least, for each
time the strains had reached stationary phase. 0.5% of this strain, 36 experiments were performed according to 18 growths in
inoculum was then transferred in the culture medium for the constant condition and 18 growths in dynamic condition.
growth studies. The concentration of cells in the culture medium
was about 107 cfu ml”1, in order to be above the detection threshold Statistical Analysis of Data
of the Bioscreen C. The viable number of bacteria was confirmed Experimental data were statistically analysed by the use of S-
by PCA plate counts (AES). Plates were incubated for 24 h at 37 PLUS 2000 software.
°C and enumerated. Averages of the O.D. were calculated for the five repetitions of
For all experiments, three conditions were studied: control, inoculated media and for the two repetitions of non-inoculated
constant and dynamic conditions. Under control condition, cells media. The data were then analysed using the procedure described
in stationary phase (after subculture) were grown in culture by Bégot et al. (1996). Four quantities were calculated at time t:
medium BHI, without addition of salt and acid. In constant • (O.D.i)t, the mean of the O.D. of the five repetitions of
conditions, bacteria in stationary phase were grown in culture inoculated media for each combination;
medium adjusted to the desired aw and pH values. The osmotic
• (O.D.ni)t, the mean of the O.D. of the two repetitions of non-
variation was achieved by the addition of NaCl (Merck). A solution
inoculated media for each combination;
5 M of HCl (Merck, 32%) was added to regulate low pH. The
dynamic condition was defined as follows: the bacteria were grown • (DO.D.)t=(O.D.i)t–(O.D.ni)t;
in culture medium (control condition, i.e. without addition of • Yt=log10[(DO.D.)t/(DO.D.min)], where DO.D.min was the
NaCl and HCl) until the beginning of the exponential phase and lowest DO.D. value above the detection threshold.
were then shocked by the abrupt addition of shock solutions. As indicated by Cheroutre-Vialette et al. (1998), a modified
These shock solutions (osmotic and acid) were prepared in order Gompertz equation was used to fit the growth curves Yt=f(t).
to obtain final values similar to those indicated for the constant Growth parameters, A (logarithmic increase of population), l(lag
conditions. An aliquot (100 l) of concentrated (×4) shock solutions time) and µ(maximal growth rate), were determined by non-linear
was added when the O.D. was near 0.2, corresponding to the regression (S-PLUS software). GT (generation time) was derived
beginning of exponential phase. from musing the relation: GT=log10(2)/m.
For each combination, five growth repetitions were carried In control and constant conditions, the time of inoculation
out. was taken as zero time when considering the growth curve. In
dynamic condition, the time of addition of the shock solution was
Experimental Design
taken as zero time in the calculation of the growth parameters.
A factorial design was used to assess quantitatively the effects
To summarize the similarities in behavior of the strains, a
and interactions of NaCl and pH (HCl) conditions on the growth
hierarchical clustering method was used. A global data set,
of L. monocytogenes clinical or seafood strains at two temperatures.
including all the conditions of the experimental design, was built.
The combinations of the following conditions were used:
Lag and generation times for the 36 treatments (two temperatures,
Temperature (°C): 10, 20 °C three pH, three NaCl concentrations, static and dynamic
pH: 5, 6, 7 conditions) were used as variables measured on the eight strains.
NaCl (% v/v): 0.5, 4, 8 Data were standardized for each variable. The first stage of this
method is the computation of a distance measure between the
142 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 143

individuals. Each individual is considered as an initial cluster. At refrigerated temperature (10 °C), the strains were more
The two “closest” clusters are merged in a bigger one at each step sensitive to low pH. The interactive effect of the factors pH and
of the process. The distance between clusters is calculated using concentration of NaCl on the L. monocytogenes growth were more
the average distance method: the distance between clusters A and pronounced at 10 °C than at 20 °C. Furthermore, the differences
B is the average of the distances between the points of A and the between clinical and seafood strains were more underlined,
points of B. The algorithm stops when there is only one cluster left. especially as the culture conditions were severe, i.e. low pH and
The distances between merged clusters are retained: this will allow presence of NaCl. No food strains could grow in medium regulated
the program to produce a graph in the form of a classification tree. to pH 5.0 supplemented to 4% of NaCl, whereas the growth
Each node in the tree appears at a height equal to the distance at recovery of the whole clinical strains could be observed. The ability
which the clusters were merged. of Lm-C1 to grow in severe conditions was confirmed by these
results. No strains were able to grow, i.e. no increase of optical
Growth of L. monocytogenes Strains in Constant Acid and Osmotic density was observed during the experiment duration (14 days),
Environment at 10 °C, pH 5.0, 8% NaCl.
At 20 °C in control condition (i.e. in culture medium BHI), all
Comparison with Growths in Dynamic Conditions
the strains, clinical or food isolates, presented similar growth
parameters, i.e. lag and generation times. In dynamic condition, L. monocytogenes strains were subject to
abrupt variations of pH and salt concentration during the
The pH had negligible effect on the different strains. Indeed,
beginning of exponential phase at 20 or 10 °C. The stress induced
whatever the strain origin, the calculated lag and generations
by these variations could induce a lag phase, i.e. an adaptation
times at pH 6.0 were similar to those obtained with the control
period to new environment. The duration of this period was
growths. The growth parameters values of a clinical strain, Lm- variable and no relation between this period value (lag time) and
C4, and a food strain, Lm-E4 in control condition and in acid the strain origin or the environmental factors could be established.
constant condition (pH 6.0 and pH 5.0). The growths at pH 5.0
The generation times associated to growth recovery
have shown longer lag times. On the other hand, at 20 °C, the
consecutively to the environmental variation was different from
strains were more sensitive to NaCl. The presence of salt (8%)
those calculated in constant condition. For example, in Table 4,
induced longer lag phase and increased the generation time by a
the values of ratios of GT calculated in dynamic condition and GT
factor 2.5, on average, compared to the corresponding control
in constant condition for strains Lm-F4 and Lm-C4 are indicated.
condition. All the results obtained at 20 °C showed that no
In most cases, the growth recovery of the L. monocytogenes strains
differences were observed between clinical and seafood strains
exposed to environmental variations in exponential phase, i.e.
when testing acid environment alone or osmotic environment alone. dynamic condition, showed a generation time longer than in
On the other hand, at this growth temperature, it was observed constant condition, when the strains were in stationary phase.
differences between strains when the two factors were considered The conditions of culture in correlation with the presence of NaCl
simultaneously. On the whole, the calculated generation times of induced the higher ratios, i.e. the generation time observed in
the clinical strains were lower than those of food strains for dynamic condition was longer than in constant condition.
pHd”6.0 combined with 4% or 8% NaCl. Among the clinical
The better adaptation of clinical strains, as compared to food
strains, Lm-C1 was less affected by the combined acid and osmotic
strains observed in constant condition was not established in
conditions, more particularly in the most severe culture condition.
dynamic condition. The parameters values of the strains were
Variations in lag times were noted among the strains in the tested
comparable. It must be noted that, contrary to results in constant
conditions.
conditions, all the strains were capable to grow in the condition
144 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 145

pH 5.0, 4% NaCl, 10 °C. At this temperature, no growths were osmotic environment, at different temperatures. Indeed, the clinical
observed at the most severe condition, pH 5.0, 8% NaCl, as shown strains, in stationary phase, were revealed to be more resistant to
in constant condition. these environmental conditions, in comparison to environmental
Among the clinical strains used in this work, two strains, Lm- strains. These results are in accordance with the study of Dykes
C3 and Lm-C4, were implicated in the same outbreak, but isolated and Moorhead (2000) who examined the acid stress response of
from different patients and had different clonal types. Therefore, clinical or meat L. monocytogenes strains and demonstrated the
it was interesting to analyze their characteristics of growths. At 20 ability of clinical strains to survive an acid shock. Likewise, Avery
°C, Lm-C3 and Lm-C4 had a similar behavior in front of the and Buncic (1997) focused on another environmental factor, the
studied factors and their variations. The growth parameter values, temperature, and showed that 15 clinical strains of L. monocytogenes
i.e. lag and generation times, did not show significative difference. had higher resistance to the effects of unfavorable storage
As shown in Fig. 4 for 10 °C, few differences could be observed conditions, compared with 15 meat strains.
whatever the studied condition. In constant condition, at pH 6.0– Our results confirmed that more investigations were necessary
8% NaCl or pH 5.0–4% NaCl, Lm-C4 presented longer lag and to allow the evaluation of the growth characteristics of clinical
generation times, e.g. at the last combination the lag time of Lm- strains, compared to food strains, in particular in conditions found
C4 was elongated of 17 h compared to Lm-C3. in the food environment, with the objective to improve the risk
assessment. Eight strains were studied in the present work. The
Classification of the Different Strains behavior of a larger number of strains (implicated in the main
The data base collected in this present work allowed the listeriosis cases in the world or associated with different foods) in
constitution of a classification of the different strains, with the function of different environmental factors should now be explored
objective to verify the global behavior of the clinical and food to consolidate our conclusions. Indeed, when foodborne listeriosis
strains. The result, confirmed the previous conclusions. Three is considered, it could be hypothesized that a high resistance of
groups were created. One of the groups was constituted by all the some L. monocytogenes strains to environmental factors found in
seafood L. monocytogenes strains (Lm-F1, Lm-F2, Lm-F3, Lm-F4), a foods or industrial environment may contribute to the particular
second one contained three of the clinical L. monocytogenes strains capability of certain strains to cause illness and, consequently, to
(Lm-C2, Lm-C3, Lm-C4). This means that the food strains were in become clinical strains. The influence of environmental factors
general “closer” to each other than to the clinical strains. The (such as temperature, acid, etc.) on the expression of virulence
particular behavior of the clinical strain Lm-C1 underlined in our markers was also highlighted.
results was confirmed by this classification, since it was isolated The experiments of the present study, comparing the strains
from the other ones. Indeed, as indicated by the experimental behavior in constant and dynamic condition, showed differences
results, this clinical strain presented a better adaptation to on the calculated generation times. This observation is in agreement
unfavorable environment of temperature, acid pH and salt with the previous studies of Cheroutre-Vialette et al. (1998) and
concentration. Cheroutre-Vialette and Lebert (2000a). They observed that cells of
L. monocytogenes isolated from meat products or food processing
DISCUSSION environment submitted to environmental variations of pH/aw in
It is well known that L. monocytogenes is a microorganism exponential phase were usually more sensitive than cells in
which is able to survive, and frequently grow, under a wide range stationary phase. It was largely demonstrated that, phenotypically,
of adverse conditions such as low temperature, low pH and high bacteria in stationary-phase growth are more thermotolerant, acid-
osmolarity. In this work, significant differences in growth kinetics resistant and are better equipped to survive osmotic stress (Hill
were found between clinical and seafood strains in acid and/or and Rees). Nevertheless, in this work, for a studied combination
146 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 147

(pH 5.0, 4% NaCl, 10 °C), the food strains were able to overcome industrial sites (23%), were compared in four con-ditions of
the stress in exponential phase but not in stationary phase (this temperature, water activity (aw) and pH. Temperatures ranged
experiment was repeated three times with different inoculum). from 10-37 °C, pH from 5·6-7·0 and aw from 0·96-1. Growths were
The presence of L. monocytogenes in seafoods has been performed in a meat broth with an auto-mated turbidimeter
examined by several authors. The prevalence of this pathogen can (Bioscreen C, Labsystem). Growth curves were fitted using the
be associated to the light preservation process (salting, etc.), the Gom-pertz function, and growth parameters were calculated. The
extended shelf life at refrigerated temperatures, capable of differences between strains in lag phase duration were much
supporting the growth of L. monocytogenes, the consumption greater than in growth rate. The greatest differences occurred at 10
without further cooking. Therefore, it is important to evaluate the C, pH 7 and aw 0·96 : lag time values ranged from 4 h to 4 days.
adaptation of this microorganism in the food processing the Listeria population was separated into five groups, according
environment which could include variations (temperature, salting, to the lag time and maximal growth rate values using clustering
acidification, etc.) during time. Mathematical modeling techniques analysis. The majority of the strains isolated from industrial sites
are gradually becoming recognized and accepted as powerful were grouped together and showed faster growth than the others
tools for predicting the effects of food-preservation systems on in the four con-ditions studied. The serotype or the nature of the
bacterial growth kinetics. The applicability of available predictive meat from which the strains were isolated did not influence growth.
models to fish products, considering the products characteristics, The variability observed among strains raises questions about the
was approached. Recently, several studies have demonstrated the consequences in quantitative risk assessment and about the
significative interest to take into account the dynamic condition in construction of models in pre-dictive modeling.
the field of predictive microbiology, and to collect experimental Listeria bacteria are widespread in the environment. Among
data in this way. the different species, Listeria monocytogenes and Listeria innocua are
With the objective to introduce more advances into the field of the two most commonly isolated from food processing. L.
predictive microbiology and increase the safety of products, studies monocytogenes can cause the death of both the very young and
allowing correlations between clinical strains characteristics (such immunocompromised individuals. Most cases are traced to the
as growth kinetics, cell physiology, e.g. stress condition, expression contamination of raw or processed foods with L. monocytogenes.
of pathogenicity in function of particular environment found in Listeriosis is therefore a major threat to human health.
food) prove to be necessary. Another prospect is to study the Listeria monocytogenes can grow at low temperatures, low pH
genetical background of environmental strains in order to select and low water activity (aw); they are therefore able to survive and
the ones that may be most representative of the whole natural multiply in a wide range of food products.
population. The knowledge of typing data (serotype, ribotype, Work on predictive microbiology has been carried out in an
pulse field gel electrophoresis) for such strains and their attempt to improve the shelf life and safety of food. Predic-tive
comparison to the corresponding epidemiological data should models of microbial growths are set out with respect to the main
provide a useful tool for the Listeria risk assessment. controlling factors of the environment such as temperature, pH
and water activity. Despite variations in growth among strains
VARIABILITY OF THE RESPONSE OF 66 LISTERIA
numerous models have been constructed using only one or a
MONOCYTOGENES AND LISTERIA INNOCUA STRAINS TO
small pool of strains. In this work, we compared the growth of 66
DIFFERENT GROWTH CONDITIONS
strains of L. monocytogenes and L. innocua, isolated from meat, meat
The growth of 58 strains of Listeria monocytogenes and eight prod-ucts and industrials sites. Strains were clus-tered according
strains of Listeria innocua iso-lated from meat products (68%) and to their growth in a broth medium in four conditions of temperature,
148 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 149

pH and aw. Consequences on the construction of models are Bioscreen C. For each strain, eight successive wells of the same
discussed. column were filled. The last two wells received the same volume
of non-inoculated medium in order to determine the growth medium
MATERIALS AND METHODS optical density (OD) and controlled any possible contamination.
Strains Ws methodology is simi-lar to that used by Be got et al. (1996).
Fifty-eight strains of L. monocytogenes and eight strains of L. Experimental Design
innocua were studied: 45 strains were isolated from meat and meat
A Plackett Burman design (Plackett and Bur-man 1943) was
products, 15 from meat and dairy plants (materials, floors, walls)
used to test the effects of three factors: temperature, pH and water
four were involved in outbreaks. Five of 13 serotypes of L.
activity. Each factor was studied twice at two levels: high and low
monocytogenes were represented: 1/2a, 1/2b, 1/2c, 4b, 4d. Listeria
(Table 3). While a fac-torial design would have required eight
cultures were stored on tryptic soy agar slopes at 4 C.
experiments, four were sufficient with a Plackett Burman design.
Media
Curve Fitting
Culture inocula were prepared in a meat medium (MM): meat
OD data were transferred from the Bioscreen C to Excel
peptone (Merck) 10 g l 1, yeast extract (Difco) 5 g l 1, glucose
software (Microsoft Windows) and transformed for each
(Merck) 5 g l 1. The pH was adjusted to 7·0 with NaOH (Prolabo)
measurement. Four quantities were calculated at time t: (ODi)t, the
1 mol l 1. The medium was autoclaved at 120 C for 20 min.
average of the OD of eight replicates; (ODni)t, the average of the
Growth experiments were carried out in a tryptic meat broth OD of the non-inoculated medium
(TMB) which was steril-ized by filtration derived from tryptone
(DOD)t=(ODi)t (ODni)t
meat agar (GTV5p, Fournaud et al. 1973): meat extract (Merck) 10
g l 1, proteose peptone (Merck) 10 g l 1, tryptone (Difco) 5 g l 1, Log10[(DOD)t/DODmin]
glu-cose 5 g l 1. The medium was buffered with a K2HOP4-KH2PO4 where DODmin was the lowest DOD value above the detection
(Merck), 0·1 mol l 1 solution in proportion 1:1 (v/v) and adjusted threshold.
to pH 7·0 with NaOH 1 mol l 1. aw was set by adding NaCl In the linear range of the Bioscreen C ( DOD<1’2) (Begot et al.
(Merck) in accordance with Chirife and Resnik (1984). 1996), growth curves were fitted using the modified Gompertz
Tryptone sodium chloride medium (TSC) (tryptone 1 g l 1, equation:
NaCl 8·5 g l 1, pH 7·0) was used for dilutions, and TSA and APT Table : Serotypes, food sources and laboratory references of
agar (Difco) were used for plate counts and agar slope cultures. tested strains of Listeria
Growth Species Serotype Food source
Growths were measured by optical density in an automated a
1 Listeria monocytogenes P1 1/2c Pig carcass
turbidimeter, Bioscreen C. All strains were inoculated in MM and
2 L. innocua 2138 a 6a Minced meat
incubated for the same period of time (17 h) at 30 C. All cultures a
3 L. monocytogenes 24631 1/2b Rib of lamb
were therefore in stationary phase, thus avoiding disparities in
the subsequent growth kinetics. Nine millilitres of TMB were 4 L. monocytogenes 3670 a 1/2a Minced meat
inoculated with the subculture. The inoculum size was confirmed 5 L. monocytogenes 2141 a 1/2c Minced meat
a
by plate counts. The inocu-lated TMB was dispensed aseptically 6 L. monocytogenes 27795 1/2a Minced meat
in 300 µl volumes into honeycomb microplates (10 10) of the 7 L. monocytogenes 4133 a 1/2c Minced meat
150 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 151

8 L. monocytogenes 28423 a 1/2c Minced meat 46 L. monocytogenes 17e 1/2b Industrial sites
9 L. monocytogenes 2143 a 1/2a Minced meat 47 L. monocytogenes 18e 4b Industrial sites
10 L. monocytogenes 5602 a 4b Pig shoulder 48 L. monocytogenes 29e 4b Industrial sites
11 L. monocytogenes 880030b 1/2a Meat products 49 L. monocytogenes 39e 4b Industrial sites
12 L. monocytogenes 880390b 4b Meat products 50 L. monocytogenes 41 e
4b Industrial sites
13 L. monocytogenes 880398b 1/2c Meat products 51 L. monocytogenes 42 e 1/2c Industrial sites
14 L. monocytogenes 890307b 4b Meat products 52 L. monocytogenes 44 e 4b Industrial sites
15 L. monocytogenes 890313b 4d Meat products 53 L. monocytogenes 70e 4b Industrial sites
16 L. monocytogenes 890467b 4b Meat products 54 L. monocytogenes 99e 4b Industrial sites
17 L. monocytogenes CLIP 19908c 1/2c Meat products 55 L. monocytogenes Lo 28f 1/2a Human isolate
18 L. monocytogenes CLIP 19910c 1/2a Meat products 56 L. monocytogenes CDC Atlanta 9g 4b Milk
19 L. monocytogenes CLIP 19532c 1/2c Meat products 57 L. monocytogenes CA Wisconsing 4b Cheese
20 L. monocytogenes CLIP 19534c 1/2c Meat products 58 L. monocytogenes OH Wisconsing 4b Cheese
21 L. monocytogenes CLIP 19536c 1/2c Meat products 59 L. monocytogenes Scott A Wisconsing 4b Human isolate
22 L. monocytogenes CLIP 19712c 1/2a Meat products 60 L. innocua CLIP 20719 g
Meat
23 L. monocytogenes CLIP 19734c 1/2a Meat products 61 L. innocua CLIP 20728g Poultry
24 L. monocytogenes CLIP 19802c 4b Meat products 62 L. innocua CLIP 20741g Meat
25 L. monocytogenes CLIP 19804c 4b Meat products 63 L. innocua CLIP 20595g Meat
26 L. monocytogenes CLIP 19884c 1/2c Meat products 64 L. innocua CLIP 20600g Poultry
27 L. monocytogenes CLIP 19887c 1/2b Meat products 65 L. innocua CLIP 12511g Poultry
28 L. monocytogenes 925331d 1/2a Chicken 66 L. innocua CLIP 12512g Poultry
29 L. monocytogenes 925318d 1/2b Guinea-fowl Strains of Listeria were donated by:
30 L. monocytogenes 925321d 1/2c Sausages a
Dr Nicolas (Regional Laboratory of Haute-Vienne, France).
31 L. monocytogenes 925222d 1/2a Chicken b
Dr Marly (INRA of Nouzilly, France).
32 L. monocytogenes 925228d 4b Chicken c
Dr Rocourt (Pasteur Institute, France).
33 L. monocytogenes 925267d 1/2a Sausages d
Dr Courtieu (National Center of Listeria references, France).
34 L. monocytogenes 925257d 1/2c Minced meat e
Dr Jolivet (SOREDAB, France).
35 L. monocytogenes 925261d 1/2c Minced meat f
Dr Cossart (Pasteur Institute, France).
36 L. monocytogenes 925253d 1/2a Sausages g
Dr Richard (National Institute of Agronomic Research of Jouy-en-Josas,
37 L. monocytogenes 925201d 1/2a Sausages France).
38 L. monocytogenes ATCC 19111 1/2a Poultry
39 L. monocytogenes ATCC 19115 4b Human ⎛ N ⎞ ⎛ ( ∆OD )t ⎞
L og10 ⎜ ⎟ = L og10 ⎜⎜ ⎟⎟ (1)
cerebrospinal fluid ⎝N0 ⎠ ⎝ ∆ODmin ⎠
40 L. monocytogenes 1
e 4b Industrial sites
41 L. monocytogenes 4e 1/2b Industrial sites ⎛ ⎛ µ.e ⎞⎞
42 L. monocytogenes 10e 1/2a Industrial sites = A . exp ⎜ − exp ⎜ (L − t ) + 1 ⎟ ⎟
⎝ ⎝ A ⎠⎠
43 L. monocytogenes 13e 1/2b Industrial sites
44 L. monocytogenes 14e 4b Industrial sites where A is the logarithmic increase of bac-terial population, L, the
45 L. monocytogenes 16e 4b Industrial sites lag time, µ, the maxi-mal growth rate and t, the time.
152 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 153

Growth parameters were determined by non-linear regression Table: Plackett Burman design to study the effect of
with Statitcf software (Technical Institute of Cereals and Fodders, temperature, aw, pH on the growth of 66Listeria strains
France). Generation time (T) was derived from the maximal growth
rate (2): Conditions Name aw Temperature( C) pH

T = [Log10(2)]/m 1 010 0·96 37 5·6


2 100 1·00 10 5·6
Table: Conditions of culture incubation and growth
measurements of Listeria strains in anautomated turbidimeter 3 001 0·96 10 7·0
(Bioscreen C) 4 111 1·00 37 7·0

Temperature 37 C 10 C Results
Number of wells 100 200 The logarithmic increase of popu-lation, A, was maximal in
Wavelength 600 nm condition 111. Par-ameter A was higher in both conditions of pH
Measurement time 30 h 240 h 7·0 than those of pH 5·6.
4t 5 min 60 min The mean, below and above which there is an equal number
Shaking frequency 30 s per 1 min of strains, was below the average value for the parameters
Shaking intensity medium considered in all conditions. A proportion of 50-60% of the strains
was under the average for each parameter.
Statistical Analysis
Large variations in lag time and gener-ation time were noted
Two techniques were used to compare the growth of strains. among the strains in all conditions. The largest differences in lag
Lag times (L) and maxi-mum growth rates ( ) were both considered. time among strains were recorded in con-dition 001. In this
Average and standard deviation were calcu-lated for both variables. condition, the values were multiplied by a factor 25 from the
Each of the 66 values were then normalized: the average was minimum to the maximum value, taking the value from 4 h to 4
sub-tracted and the result divided by the stan-dard deviation. days. Larger variations among lag times were found successively
Each strain was depicted in a space of 2x4=8 dimensions. in condition 001, 100, 010 and 111.
Principal component analy-sis (PCA) was used to reduce the The more severe the growth conditions, the larger the number
dimension of the sample space. The method looks for the unique of disparities found in lag time. Fewer differences were found in
subspace of a given dimension which explains the major part of generation time. In condition 100, generation times tripled from
the variance in the original data. This analysis was performed the minimal to the maximal value. In the other conditions,
using Statitcf software. A cluster analysis was used and calculated generation times doubled when comparing the slowest and
with Statitcf software. Classification is achieved through successive fastest strain for each condition. Slight variations were found in
aggre-gations of individuals and then groups of individuals. The parameter A which corresponds to the logarithm increase of the
principle is to regroup indi-viduals according to their distance population density. We observed that strains exhibiting the longest
from the centre of gravity for the set of experimental points. Two generation time of the entire population did not show the maximal
individuals or two classes were aggregated when their fusion lag time.
involved a minimal increase of the intra-class dispersion. The
For example L. monocytogenes ATCC 19115 which has the
average value of each parameter was then calculated for each
longest generation time in condition 001, showed a lag time of
group and an overall average was calculated for the 66 strains.
50·8 h whereas the longest lag time was 97·9 h. Similarly, the
154 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 155

strain exhibiting the shortest generation time did not show the Cluster Analysis
shortest lag time. Results were identical in all conditions tested. The cluster analysis divided the population into five groups.
Table: Effect of temperature, aw and pH on the growth of 66 Group 1 (10 strains) was clearly separated from other groups.
Listeria strains It was comprised of strains which grew faster than the overall
average in all the conditions studied. The majority (66%) of the
Parameter aw Temp. pH Min. Max. Average Mean
strains belonging to this group originated from industrials sites.
A001 1·2 1·9 1·5 1·5
Group 2 (15 strains) was characterized by strains which grew
T001 0·96 10 7·0 8·9 20·1 13·3 13·0 more slowly than the overall average, which is in opposition to
L001 3·9 97·9 31·8 28·2 group 1. L. innocua was not present.
A010 1·1 1·6 1·4 1·4 Group 3 (28 strains) was intermediate. Strains exhibited lag
T010 0·96 37 5·6 1·3 2·9 1·7 1·7 times and maximal growth rates around the overall average. It is
L010 1·2 10·2 4·0 3·4 located at the centre of the PCA figures. All origins were represented.
A100 0·8 1·8 1·3 1·3 Group 4 (eight strains) clustered which grew more slowly
T100 1·00 10 5·6 8·4 24·2 12·9 11·8 than the overall average except for condition 100 where they grew
L100 2·4 40·2 17·4 16·6 faster. No epidemiological strains of L. monocytogenes were present
A111 1·1 1·9 1·7 1·8 in this group.
T111 1·00 37 7·0 0·5 0·9 0·7 0·7 Group 5 (five strains) was composed of strains which grew
L111 0·2 2·6 0·9 0·6 more slowly than the average of the strains, except for condition
001 where they grew faster. 80% of group 5 strains were L. innocua.
The variability of the growth response was expressed by the L. monocytogenes Scott A, which has been widely used to set up
following parameters: logarithm increase of population density models, belonged to this group.
(A), generation time (T) and lag time (L). T and L were expressed
The nature of the meat they were isolated from had no effect
in hours, A was an increase of Log10.
on the cluster analysis:
Strains were separated according to their ability to grow in
Table: Comparison of the generation times (h) and lag times (h)
each condition by using the variables L and µ. We chose three
averages calculated on the 66 Listeria strains and on each
axes which explained 68% of the initial information. Axis 1
group defined by the cluster analysis
separated strains according to the values of parameters L and µ.
Lag time and maximal growth rate were negatively and highly 10 C aw 0·96 37 C aw 0·96 10 C aw 1·0037 C aw 1·00
corre-lated in all conditions. pH 7·0 pH 5·6 pH 5·6 pH 7·0
T001 L001 T010 L010 T100 L100 T111 L111
Strains exhibiting fast growth (low lag time and high maximal
growth rate) were isolated from strains which grew slowly (high Overall average 13·3 31·8 1·7 4·0 12·9 17·4 0·7 0·9
lag time and low maximal growth rate). The second axis scaled Group 1 10·7 16·2 1·5 2·3 9·7 9·2 0·6 0·5
the conditions from worst to best: 001-100-010-111. Strains Group 2 13·9 44·8 1·9 6·6 13·5 23·2 0·6 0·6
separated again according to growth conditions. Axis 3 segregated Group 3 13·8 36·0 1·6 3·2 12·1 18·7 0·7 0·7
condition 001 from the others. Strains that grew well in this Group 4 13·3 28·2 2·1 4·2 10·8 10·1 0·8 1·8
condition, where temperature and water activity were low, were
Group 5 10·2 6·5 1·8 4·0 19·1 20·1 0·7 1·3
clustered.
156 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 157

Table: Distribution of the serotypes of L. monocytogenes strains difference between both strains is seven days and is reached in
in the five groups defined by the cluster analysis condition 001, when the inoculum is 1 cfu ml 1. Such results can
have great consequences in quantitative risk assessment. Listeriosis
Group 1/2a 1/2b 1/2c 4b 4d L. innocua Non- Total causes illness in certain categories of the population
determined
(immuno-compromised, old or pregnant people) but the conditions
1 2 3 1 3 0 1 0 10 of foodborne outbreaks are still not well known. The infectious
2 4 1 5 3 0 1 1 15 dose is supposed to be high but the minimal dose causing illness
3 8 2 5 11 1 1 0 28 depends on the vulnerability of the person who ingested the food,
4 1 0 3 3 0 1 0 8
the type and quality of the food, the level of the pathogen in the
food and the virulence of the strain. As a consequence, such
5 0 0 0 1 0 4 0 5
unknown data linked to the variability of the growth response
Total 15 6 14 21 1 8 1 66 show the difficulties than can be faced in estimating the probability
each group was composed of strains of diff-erent serotypes. of occurrence of listeriosis.
In our study, the strains were clustered according to their
Discussion
growth ability in the four conditions. They were not grouped
This study highlighted differences in growth among L. round their serotype nor the nature of the meat from which they
monocytogenes strains. Larger variations between the 66 strains were isolated. However it is interesting to note that group 1 was
were shown in lag times rather than in generation times. in majority composed of strains isolated from industrial sites.
Particularly wide variations were observed in condition 001 (10 C, These strains grew faster than the others in all the conditions
aw 0·96, pH 7·0) where lag times extended from 4 h to 4 days. tested. This suggests that they are accustomed to growing in a
These results tally with those of Barbosa et al. (1994) who reported wide range of harsh conditions (e.g. high osmolarities, low
that among 39 strains of L. monocytogenes, lag times ranged from temperatures). Studies have recently shown the ability of Listeria
1·5-2·9 days at 10 C, pH 7·2. Lag time is known to be the time to adapt their cellular physiology to harsh environments. Most of
required for the organ-isms to adapt their cellular components to the L. innocua studied were found in group 5, and grew more
the new environment. As a consequence, it is affected by the slowly than the average population, except in condition 001 (10
difference between subculture and culture condition, the C, aw 0·96, pH 7·0). These results concord with those of Barbosa
physiological state of the inoculum and by the strain. Indeed, in et al. (1994) but not with studies where modified or selective
our study, as we were careful to homogenize the conditions of the media were used. Listeria monocytogenes Scott A was also clustered
sub-cultures so as to avoid disparities in the sub-sequent growth in group 5; in condition 001, it has the fastest lag time (3·9 h) and
kinetics, the differences we obtained in the growth parameters the fastest generation time (9·4 h) of all the 66 strains. These
were attributed to the nature of the strain. results do not tally with those of Bar-bosa et al. (1994) who studied
Such variability in lag times added to the variability in this strain among 39 other strains and showed that it had the
generation times characterizes strains with slow or fast growth. longest lag time at 4 and 10 C. Other strains that were the causes
As the Inter-national Commission on Microbial Specifi-cation for of epidemic out-breaks were found in different groups: L.
Foods set the toler-ance limit in food at <100 L. monocytogenes per monocytogenes OH Wisconsin was clustered in group 1
gram at the point of consumption, it was possible to calculate the (characterized by fast growth), L. monocytogenes CDC Atlanta in
time needed to reach this limit in the four conditions studied for group 2 (slow growth) and L. monocytogenes CA Wisconsin, in
both ‘slow’ and ‘fast’ strains as the inoculum varied. The maximal group 3 (average growth).
158 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 159

Table: Prediction of time (in days) necessary to reach a limit We have shown the importance of studying a large number of
level of 102 cfu ml 1 as the inoculum varied from 1-10 cfu ml1 strains of various origins before constructing models to assess
strain variability. However, other factors should also be considered
Conditions aw Temp. pH Strains 1 cfu ml 1 10 cfu ml 1 to produce effective mod-els, for example inoculum level,
001 0·96 10 7·0 Fast 2,6 1,2 interactions between bacteria, interactive effects between strain
Slow 9,6 6,5 and product as shown by Rosenow and Marth (1987) and
010 0·96 37 5·6 Fast 0,4 0,2 comparison of growth in liquid media to growth on solid media.
Slow 1,2 0,8 Growth of Pseudomonas fluorescens and Pseudomonas fragi in a
100 1·00 10 5·6 Fast 2,4 1,1 meat medium as affected by pH (5.8 – 7.0), water activity (0.97
– 1.00) and temperature (7 – 25°C)
Slow 8,2 4,5
111 1·00 37 7·0 Fast 0,2 0,1 A total of 59 strains of Pseudomonas, isolated from meat products,
were grown in micro-titer plates in a meat medium over a range
Slow 0,4 0,2
of pH (5.8–7.0), aw (0.97–1.00) and temperature (7–25°C). Growths
The time was calculated in the four conditions of growth for a ‘slow’ were performed in a meat broth with an automated turbidimeter
‘ strain and a ‘fast’ ‘ strain. (Bioscreen C, Labsystem, France). The growth curves obtained in
Such results point out the difficulties in choosing a strain to this study did not have sigmoidal shapes making it impossible to
build a predictive model. Indeed numerous studies have involved calculate growth parameters using the Gompertz equation. The
the testing of one or a cocktail of three or five strains. L. medium was weakly oxygenated in the micro-titer plates and
monocytogenes Scott A was fre-quently used. The results of Barbosa reached 0%-dissolved oxygen at the beginning of the exponential
et al. (1994) along with those presented here raise some questions phase. Strains were separated into two groups: P. fragi and P.
as to predictive models: is it wise to construct models with only fluorescens. Strains of P. fragi had shorter lag times than those of
one strain? If so, which strain? That with an average, fast or slow P. fluorescens. The impact of such results is interesting in that these
growth? If not, how can we integrate the growth variability in could assist to explain the succession of flora that is observed
mod-els? If the fastest strain were considered, the model would during the processing of meat: P. fluorescens is the dominant bacteria
always predict a shorter lag time and lower growth rate than among Pseudomonas spp. at the beginning of a slaughter line and
those found for the majority of strains, and would thus always P. fragi becomes dominant during the chilling process.
provide safer growth estimates. But in this case, the industry Many flora of spoilage-bacteria have an effect on the shelf-life
could not support the costs involved in reducing the shelf life of of refrigerated food products. The main flora responsible for spoilage
a product. Furthermore, there is nothing to guarantee that a faster in fresh meat and milk products during aerobic storage however
strain will not be found in the future. For public health safety, the are the Pseudomonas species. These are dominant in poultry meat,
slowest strains should be avoided. In order to take the variability pork, and beef and lamb. In milk and cream the major spoilage
of the strains into account, two ways are proposed: the first bacteria have been identified as P. fluorescens and P. fragi. Widders
approach is the production of two predictive models using two et al. (1995) reported that the average contamination on whole
strains that are charac-terized by a fast and a slow growth; this carcasses at 4°C peaked at 3.90 log10 cfu/cm2 and 4.54 log10 cfu/
would result in giving a growth response interval. The second cm2 on meat surfaces at the same temperature. P. fragi and P.
approach would be a model integrating first the study of a fluorescens cause deterioration in quality of meat and milk products
strainhaving an average behavior in a large range of experimental due to the production of extracellular proteases and lipases at low
conditions, and second the results of this study. temperatures. Off-odours occur in milk when the population of
160 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 161

Pseudomonas spp. reaches 2.2×106 to 3.6×107 cfu/ml or on meat Materials


when the population reaches 107 to 108 cfu/cm2. P. fragi strains An automated turbidimeter was used to follow the growth of
produce fruity and putrid odours on beef and have a deleterious Pseudomonas spp. in the micro-titer plates. Optical density (OD) of
effect on the colour of meat stored at 1°C, resulting in a green and the growth was measured on a spectrophotometer. OD was read
slimy appearance. at a wavelength of 600 nm. The threshold of detection for the
Although many studies have been carried out on the occurrence Bioscreen C, and for the spectrophotometer, were determined at
and levels of contaminations in meat products, there has been 6.0×106 cfu/ml and 1.0×107 cfu/ml, respectively. The range where
little work on a comparison of relative growth of Pseudomonas OD was proportional to the bacterial population extended from
species and on the growth variations among these strains. The 0.010 to 0.8 OD for both apparatus.
aim of this study therefore was to compare the growth rates of A 2-l fermenter was used with pH monitored by a heat-
strains of P. fragi and P. fluorescens in a selected media over a sterilisable electrode. Dissolved oxygen was measured directly in
range of pH, water activity (aw) and temperature, and to attempt the fermenter with an oxygen probe and the zero calibration
to describe the resulting growth curves. determined with an oxygen-free medium provided by Setric Génie
Industriel. A full-scale reading was obtained by saturation of the
Strains
medium with air supplied continuously to the culture.
A total of 59 strains of Pseudomonas were selected from bacterial
counts made from meats of different origin. All were isolated from Inoculum
or grown on Pseudomonas Agar Base supplemented with Each strain was incubated on PCA slants at 25°C for 8 h and
Pseudomonas CFC Supplement (Oxoïd). The strains were identified then transferred to MM that was shaken in a rotary shaker
as P. fluorescens or P. fragi using standard tests. This involved: waterbath for 17 h at 25°C, by which time growth of all the strains
growing on Plate Count Agar at 4°C, and a range of tests including had reached the stationary phase. A proportion of the culture was
Gram stain, oxidase and catalase reaction, mobility, production of inoculated in TMB to give a concentration of about 107 cfu/ml in
acid in the presence of maltose and cellobiose, degradation of order to be above the detection threshold of the spectrophotometer
gelatine and production of pigment on King B medium. The strains and the Bioscreen C. The viable number of bacteria was determined
were stored at 4°C on PCA slants and transferred monthly. on PCA plates using standard microbiological practice immediately
after inoculation of the growth medium. Viable numbers for growth
Media in Bioscreen C ranged from 6.9–7.1 log10 cfu/ml and from 7.1–7.3
Two media were used for successive subcultures — these log10 cfu/ml for growth in fermenter.
were PCA slants and meat medium (MM) which contained meat
peptone (Merck) 10 g/l, yeast extract (Difco, OSI, Maurepas, France) Growth Experiments
5 g/l, and glucose (Prolabo, Fontenay sous Bois, France) 5 g/l. The Growth of the strains was obtained on micro-titer plates using
pH was adjusted to 7.0 by NaOH (40 g/l). The culture medium the protocol developed by Bégot et al. (1996): for each strain, 300
was a tryptic meat broth (TMB) (Fournaud et al., 1973) and was l of inoculated TMB was dispensed in eight successive cuvettes in
buffered with a K2HPO4-KH2PO4 (Merck), 0.1 mol/l solution to the micro-titer plates. Some cuvettes were filled with non-inoculated
adjust the pH. Water activity was controlled by adding NaCl TMB and were used as controls. OD was measured at 600 nm.
(Merck) according to Chirife and Resnik (1984). An aliquot of 0.5 The working volume of the fermenter was 1 l. The stirring
ml/l of anti-foam ANBIO 15 (1% v/v) was added for growth in speed was regulated to 70 rev./min. Prior to inoculation the
a fermenter. medium was saturated with oxygen, so that growth began at a
162 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 163

level between 90 and 95% of dissolved oxygen. Air could be RESULTS


supplied at the following volumetric rates: 0, 0.24, 0.40 and 0.80 Strains of both species grew at 4°C on PCA, were Gram-
l/l of medium/min. A fraction of the fermenter volume was negative, motile, gave positive oxidase and catalase reactions. P.
periodically removed to measure OD, however, no more than 10% fluorescens strains produced fluorescent pigments and gelatinase,
of the working volume was pipetted during growth. P. fragi strains did not. P. fragi strains produced acid in the
presence of maltose and cellobiose, P. fluorescens strains did not.
Growth Conditions
As shown in numerous studies, a decrease in temperature, pH
A fractional factorial design for each of the environmental
or aw increased the duration of both lag and generation times. Fig.
variables, aw, temperature, pH at three levels, gave nine conditions
1 shows for most growth conditions, growth was characterised by
for growth for each of the 59 Pseudomonas strains. These growth
an unusually shaped growth curve, i.e. the lag phase was clearly
conditions are given in Table 2. Growth in the fermenter was
visible with the growth phase generally reaching its maximal
carried out with P. fragi 103 in condition E111.
growth rate at the beginning of this phase. After the maximal
Growth Curve Treatments growth rate, there was a break in the curve leading to a decreasing
phase which did not reach a steady stationary phase, even after
Growth data obtained as a function of OD versus time were
long periods of incubation (up to 45 h in condition E111). These
transferred from Bioscreen C to Excel software (Microsoft Windows)
growth curves are thus characterised by an inflection point situated
and transformed for each measurement. Four quantities were
not long after the end of the lag phase and by a non-symmetrical
calculated at time t:
shape. The two parameters GT (generation time) and L (lag time).
1. (ODi)t, the OD average for the eight replicates It is seen that the interval between minima and maxima increased
2. (ODni)t, the OD average for the non-inoculated TMB as the conditions of pH, aw and temperature moved away from the
3. (DOD)t=(ODi)t”(ODni)t most favourable condition E111. It is to be noted that the gap
4. f(t)=Log10[(DOD)t/DODmin] where DODmin was the between minima and maxima increased more for L than for GT.
lowest DOD value above the detection threshold. P. fragi 103 rapidly metabolised dissolved oxygen when no air
The function f(t) was used to calculate two parameters for the was supplied, i.e. there is a break in growth as the dissolved
59 strains in the nine conditions: the maximal growth rate, Vmax, oxygen level reached 10% 1.5 h after inoculation. The time needed
and the lag time, L. The maximum growth rate of all the growth to reach 10% of the saturated value for dissolved oxygen was 1.5,
rates Vmax, was calculated for each time tn (Eq. 2). L corresponded 1.5, 2.5 and 3.5 h when the rates of air were respectively 0, 0.24,
to the intersection of the x-axis and the tangent in Vmax: 0.4 and 0.8 l per l of medium per min. Zero percent dissolved
oxygen was reached 1 h following these periods. Growth curves
f (t n +1 ) − f (t n −1 ) in these four conditions are shown in Fig. 3. For 2 h, growth
V tn = (1)
t n +1 − t n −1 curves are similar, then they separated and rose faster as the air
supply increased. The growth curve obtained in Bioscreen C was
Vmax = maximum (Vtn) (2)
close to that obtained in the fermenter at a flow rate of 0.24 l per
Statistical Analysis l of medium per min. It assumed confidently that conditions of
Average, mean, minimum and maximum were calculated for aeration in the Bioscreen C cuvettes are similar to this type of
the two variables L and GT. Strains were classified according to aeration.
their ability to grow in the different conditions. A cluster analysis The classification carried out on all the strains revealed two
was carried out with STAT-ITCF software. main groups. The first was comprised of all the P. fragi and four
164 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 165

P. fluorescens namely 51, 11.6A, M2L3 and P×10. The second only four strains of P. fluorescens were in the P. fragi group. These
comprised the remaining P. fluorescens. Statistical values for all were characterised by shorter lag times in condition E111 (25°C,
strains. L, averages, means, minima and maxima for each group. aw 1, pH 7). This explains their presence in the first group. The P.
It can be seen that shorter lag times were obtained for the first fragi group had on average shorter lag times than P. fluorescens.
group. Between each group differences were greater in conditions These differences have been observed on a great number of strains
E133 (aw 1.00, 7°C, pH 5.8), E232 (aw 0.985, 7°C, pH 6.4), E331 (aw and in a wide range of temperature, pH and aw; e.g. average L was
0.97, 7°C, pH 7.0) related to the lowest temperature. two-fold shorter in the P. fragi group at 7°C.
These results could help to explain the changes in the ecology
DISCUSSION of Pseudomonas flora shown by different authors who have studied
The growth curves obtained in this study did not have levels of contamination and areas of isolation during meat
sigmoidal shapes making it impossible to calculate growth processing and retailing. Lahellec and Colin (1981) observed the
parameters with the Gompertz equation which is usually used by spoilage strains in poultry: pigmented strains represented the
authors. A steady stationary phase was rarely obtained and for major population isolated in processing plants, but during storage
long experiments a biofilm could be seen on the top of the medium non-pigmented strains outgrew the former. On beef, lamb and
in the cuvettes. In addition to possible insufficient shaking of the pork, studies have shown the predominance of P. fluorescens from
micro-titer plates in Bioscreen C, it was supposed that the aeration the slaughter line to the chilling process. P. fluorescens is known
was also too weak. Growth in the fermenter confirmed this and to be largely present in the environment (floor, water), on animals
highlighted that the aeration in the cuvettes was equivalent to 0.24 (hide, skin) or also in water and surfaces in meat factories. On
l of air per l of medium per min. These conditions of aeration cutting lines and during storage and retailing, P. fragi was then
played too important a role for aerobic bacteria and became a found as the dominant flora on meat. Drosinos and Board (1995)
limiting factor for the growth of the Pseudomonas spp. A possible studied the evolution of the bacterial flora in minced lamb stored
explanation for the unexpected shape of the growth curves is that aerobically at 4°C and showed that P. fragi outgrew P. fluorescens.
the combined effect of the depletion of oxygen and the change in They tried to explain this succession of flora by differences in the
the metabolism: indeed, in some cases, Pseudomonas spp. can use metabolism of both species. In our study shorter lag times observed
nitrate as an alternate electron acceptor and can grow for P. fragi strains could be linked to their ability to adapt better
anaerobically. to new environmental conditions compared to P. fluorescens.
For many studies carried out in milk and milk products with Our findings make it possible to choose a limited number of
Pseudomonas spp. the generation time was the main parameter representative strains from the 59 Pseudomonas — so as to diminish
for comparison. Cox and MacRae (1988) reported that P. fragi the number of experiments needed to study these few strains with
grew faster than P. fluorescens in both U.H.T. and raw milk at 4°C. more precision in broth or on surface tissue of meat. A ‘low’,
In meat products, few studies have compared the behaviour of the ‘quick’ or ‘average’ strain characterised by a slow, rapid or average
species. This work carried out on 59 strains of Pseudomonas has growth in all or few conditions of the experimental design, could
shown small differences between calculated generation times that then be chosen in both species. Further studies should be
are confirmed by results of Pooni and Mead (1984) who worked undertaken: to substantiate the differences in lag times between
on strains isolated from poultry products and grown in heart the ‘average’ strains of both species and compare their generation
infusion broth. These authors showed that pigmented and non- times in better conditions of aeration; to study a ‘low’ strain and
pigmented Pseudomonas made little difference in average generation a ‘quick’ strain and observe the variability in their growth
times. The differences in lag time observed between both species responses; or to compare the growth parameters obtained in broth
are important. For the 59 strains separated into the two groups, and on the surface tissue of meat.
166 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 167

DIVERGICIN 750, A NOVEL BACTERIOCIN PRODUCED BY show significant sequence similar-ity and contain the -YGNGVXC-
CARNOBACTERIUM DIVERGENS 750 motif typical of bacteriocins active against Listeria. Recently,
Divergicin 750, a bacteriocin produced by Carnobacterium diver-gicin A, produced by C. dicergens LV 13 was char-acterised.
divergens 750, preferentially inhibited the growth of strains of It constitutes an interesting exception in being a small class II-like
Carnobacterium and Enterococcus. Selected strains of Listeria bacteriocin which uses the cells’ sec system for translocation. A
monocytogenes and Clostridium perfringens were also inhibited. lanthion-ine-containing bacteriocin, carnocin UI49 has been
The bacteriocin was purified to homogeneity by ammonium sulfate purified as well. Here we describe the purification, characterisation
precipitation and sequential S-Sepharose, hydrophobic interaction and cloning of divergicin 750, a novel bacteriocin from C. diuergens
and reversed phase chromatography. The complete amino acid 750.
sequence was determined by Edman degradation. The peptide
Growth of Bacteria
consisted of 34 amino acid residues. The calculated M(r) from the
peptide sequence, 3447.7, agreed well with that obtained by mass Thirty-seven strains of carnobacteria were screened for
spectrometry. Divergicin 750 did not show any sequence production of bacteriocin. Carnobac-terium dicergens 750 from the
similarities to other known bacteriocins. The plasmid-located laboratory stock of Bundesanstalt fur Fleischforschung, Kulmbach.
structural gene encoding divergicin 750 (dvn750) was cloned and Ger-many, produced divergicin 750. For production of bacteriocin,
sequenced. The gene encoded a primary translation product of 63 cells were grown at 25°C overnight in cMRS medium containing
amino acids with a deduced M(r) = 6789.4 which is cleaved Peptone proteose no. 3 (10 g l-1 ), yeast extract (5 g l-1), sucrose (20
between amino acid residues 29 and 30 to yield the mature g l-1 ), K HPO4 (2 g l-1 ), (NH4)2 citrate (2 g l -1 ), MgSO4 (0.02 g l-
1
bacteriocin. Lactic acid bacteria produce a variety of com- pounds ), MnSO4 (0.02 g l-1 ). pH was adjusted to 8.5 with NaOH and the
with antimicrobial activity. Some of these are proteins or peptides medium auto-claved for 20 min. In growth kinetic experiments C.
and are termed bacteriocins. Bacteriocins from lactic acid bacteria diuergens 750 was grown in D-MRS broth adjusted to initial pH
are cur- rently being divided into four classes. Class II bacteriocins 6.6 and pH 8.2. The broths were inoculated at a 0.1% level of
are small and have little post-translational modifications. They overnight cultures and incubated for 48 h at 25°C. At appropriate
are usually heat stable, hydrophobic and often act on the cell time intervals the number of colony-forming units and bacteriocin
membrane of susceptible target cells. Bacteriocins are commonly activity in the culture supernatant was determined. Growth of C.
secreted by a dedicated transport and maturation system. The dicergens 750 in D-MRS broth of different pH values was monitored
bacteriocins usually inhibit the growth of closely related species. using an automated turbidometer, BIOSCREEN C. 10 µl of a ten-
Some bacteriocins also inhibit the growth of pathogens and spoilage fold diluted 24-h culture were added to honeycomb wells
organ-isms and may thus be of interest for enhancing food safety con-taining 190 µl D-MRS broth of pH 4.5, 5.0, 6.0. 7.0, 8.0, 9.0,
and food hygiene. and 10.0. Al! inoculations were done in triplicate and incubation
was for 48 h at 30°C. After 14 h and 38 h, samples were withdrawn
Carnobacteria grow at low temperature, produce relatively from a second honeycomb plate and tested for bacteriocin activity.
little acid and volatile compounds and they generally also have To test the bacteriocin for pH stability, fractions of culture
low proteolytic and lipolytic activity. Little is known about supernatants were adjusted to pH values between 2 and 11 with
bacteriocins from carnobacteria. Some bacteriocin-producing 5 M NaOH or 2 N HCI. After 1 h of incubation at 20°C, residual
carnobacteria have been identified. We have previously purified activ-ity was examined by use of the agar spot assay.
and characterised piscicolin 61 from Carnobacterium piscicola LV61.
C. piscicola LV 17 produced three bacteriocins, termed Bacteriocin Activity Assays
carnobac-teriocin A, BM1 and B2 of which carnobacteriocin A Bacteriocin activity was quantified by using serial dilutions of
was identical to piscicolin 61. Carnobacteriocin BM 1 and B2 the bacteriocin in the agar spot test as described previously. One
168 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 169

arbitrary unit (AU) was defined as the reciprocal of the highest system. Cloning was done in Escherichia coli DH5 a using the
dilution yielding a definite zone of inhibition on the indicator cloning vector pGEM-7Zf( + ) (Promega). Positive clones were
lawn. Alternatively, bacteriocin activity was quanti-fied in a identified by colony hybridisation. The cloned DNA fragments
microliter plate assay system by measuring the growth of the were se-quenced completely on both strands using the Seque-nase
indicator organism in two-fold dilutions of bacteriocin in medium version 2.0 DNA sequencing kit (Amersham) and a primer walking
as described previously. strategy. Computer analyses were carried out on an IBM personal
When necessary, bacteriocin fractions were adjusted to pH 6.5 computer employing the DNASIS sequence analysis program and
and sterilised by filtration through Millipore filters (0.22 µm) prior on a UNIX computer employing the GCG programme package.
to activity measurements. One bacteriocin unit (BU) was defined
as the amount of bacteriocin which inhibited growth of the RESULTS
indicator organism by 50% as compared to a control culture Production and Inhibition Spectrum of Divergicin 750
without bacteriocin. Unless otherwise stated C. dicergens L66 was
Growth of C. divergens 750 at different initial pH values of the
used as an indicator strain.
growth media was measured. The bacteria grew well at high pH
Purification and Analysis of Divergicin 750 while growth was significantly retarded at pH 5. Bacteriocin was
pro-duced over a wide range of pH values and could be detected
Bacteriocin was purified essentially as described previously.
even down to pH 5. It appeared that diver-gicin 750 was produced
In short, a 2-1 culture of C. diuer-gens 750 was grown to the
stationary phase and cells were removed by centrifugation. The in the late exponential phase of growth as the cells entered the
bacteriocin pre-sent in the supernatant fraction was concentrated early stationary phase and remained fairly stable in the growth
by ammonium sulfate precipitation and subjected to ion exchange super-natant after production.
chromatography (S-Sepharose), hydropho-bic interaction When C. divergens 750 was grown at different temperatures,
chromatography (Octyl-Sepharose) and reversed phase FPLC maximum production of bacteriocin occurred at 25°C . Bacteriocin
(Pharmacia). The purified bacteriocin was stable in 40% (v/v) 2- production was de-tected down to growth at 4°C. At 37°C no
propanol containing 0.1% trifluoroacetic acid at - 20°C. activity was observed. Consequently, cells were grown at initial
Amino acid sequencing and mass analysis of puri-fied pH 8.5 and 25°C to enhance the yield of bacteriocin.
divergicin 750 were done as described previously. Table: Inhibition spectrum of purified divergicin 750

Recombinant DNA Techniques Indicator bacteria Number of sensitive/number tested


Basic cloning techniques were used. The probes were end- Carnobacterium dicergens 1 / 1
labelled with 32 P using a terminal transferase end-labelling kit. Carnobacterium piscicola I / 1
Plas-mid DNA from C. divergens 750 was digested with various
Carnobacteriumgallinarum 0/1
restriction endonucleases and the resulting fragments were
Enterococcus faecalis 2/2
separated on agarose gels, blotted onto nylon filters and hybridised
with a divergicin 750-specific degenerate oligonucleotide probe. Enterococcus faecium 0/
Lactobacillus sake 0/1
Two partly overlapping fragments, a 2-kb EcoRI frag-ment
and a 0.2-kb Mbol fragment, hybridising to the oligonucleotide Bacillus cereus 0/1
probe, were purified by excising them from a low-melting-point Brochothrix thermosphacta 0/1
agarose gel and subse-quently employing the MagicTM clean-up Clostridium butvricum 0/1
170 Introductory Food Microbiology Modelling the Growth of Listeria Monocytogenes... 171

Clostridium perfringens 1 / 1 Purification of Divergicin 750


Clostridium sporogenes 0/ 1
Purification stage Fraction Total Specific Yield
Listeria innocua 0/2 volume protein activity
Listeria icanocii 0/1 (ml) (mg) (BU mg-I X 10-4) (%)
Listeria monocytogenes 1 /5 Culture supernatant 2000 120 1.6 100
Listeria seeligeri 0/1 (NH4)>SO4 conc. (Fr I) 200 90 4.2 190
Listeria welshimeri 0/ 1 S-Seph. chrom. (Fr))) 50 21 0.2 2.5
Propionibacterium acnes 0/ t Octyl-Seph. chrom. (FrIIl) 9 1.4 100 70
Staphylococcus aureus 0/4 Rev. phase FPLC (FrIV) 2 0.07 1000 35
Streptococcus mutans 0/ 1
Inhibition was tested by using the purified bacteriocin (2048 AU ml-1) Cloning and DNA Sequencing of the Divergicin 750 Structural Gene
in the agar spot test assay described in Materials and methods. Hybridisation of a degenerate oligonucleotide probe to plasmid
Some lactic acid bacteria are known to produce more than one DNA from C. dirergens 750 gave one strong hybridisation signal
bacteriocin. To rule out interference from other inhibitory to a 2-kb EcoRI fragment and a partly overlapping 0.2-kb Mbol
substances, purified divergicin 750 was used in determination of frag-ment. The EcoRl and Mbol fragments were cloned and
sequenced. The gene encoded a primary translation prod-uct of 63
the inhibition spectrum. Divergicin 750 preferably inhibited strains
amino acids with a calculated Mr = 6789.4. Cleavage of this peptide
of Carnobacterium and Ente-rococcus. Selected strains of Clostridium
between amino acid residue 29 and 30 would give the mature
perfringens and Listeria monocytogenes were also inhibited.
divergicin 750. Uncertain amino acid residue determinations in
Purification of Dirergicin 750 posi-tions 1, 6, 11 and 26 of divergicin 750 were corrobo-rated by
deduction from the sequence of the cloned gene. Downstream of
Divergicin 750 was purified by ammonium sulfate
the structural gene, a region of dyad symmetry was found.
precipitation, sequential cation exchange, hydropho-bic interaction
and reversed-phase chromatography. The purified bacteriocin DISCUSSION
appeared homogeneous when subjected to FPLC-reversed phase
Divergicin 750 is a low molecular mass, hy-drophobic and
chromatography. Overall, a more that 600-fold increase in specific
basic peptide and thus shares charac-teristics with other
activity was observed (BU mg-1 protein). The purified bacteriocin
bacteriocins of lactic acid bacte-ria. Divergicin appeared to be
had an activ-ity of approx. 10 000 BU µg -1 protein.
produced only in the late exponential phase of growth . This is in
The complete amino acid sequence of the purified bacteriocin contrast to many other bacteriocins, for example sakacin A from
was determined by Edman degradation. The sequence was L. sake Lb706 and sakacin P from L. sake Lb674, which are
identical to that deduced from the structural gene after cloning. constitutively synthesized. Nothing is known about the mechanism
Divergicin 750 consisted of one polypeptide chain of 34 amino of regulation of divergicin 750. The bacteriocin was purified by a
acid residues. No unusual amino acid residues such as lanthionins procedure simi-lar to that used for other bacteriocins. During
were found. The calcu-lated Mr of 3447.7 was almost identical to purification a drop in activity was observed after elution from the
the value of 3448.5 Da obtained by PDMS. The extra 1 Da in the S-Sepharose column, whilst the activity was regained after the
mass analysis probably originated from protonation of the next purification step. The reason for this apparent drop is
polypeptide during desorption. unknown. The molecular mass of divergicin 750 as calcu-lated
from the deduced amino acid sequence was very close to that
172 Introductory Food Microbiology Epidemiological Typing of Bacillus spp Isolated from Food 173

determined by PDMS and is consistent with the proposed amino


acid sequence. Taken together with the information that no
lanthion-ins were detected upon amino acid composition anal-ysis,
divergicin 750 appeared not to be subjected to extensive post-
translational covalent modifications and thus belonged to the
class II bacteriocin group. No sequence similarities of divergicin
750 with other proteins encoded by sequences in the EMBL and 7
GenBank sequence databases were found. Divergicin 750 thus
differs from other bacteriocins of the lactic acid bacteria.
EPIDEMIOLOGICAL TYPING OF
A general mechanism of action for the class II bacteriocins has
been suggested. In this model the bacteriocin molecules are thought BACILLUS SPP ISOLATED FROM
to form am-phiphilic a-helices that combine to make a pore in the
cell membrane of susceptible cells. Nothing is known about the
FOOD
mechanism of action of divergicin 750. The region encompassing
amino acid residues 7-28 may be able to form an amphiphilic a- Biotypes, fatty acid profiles, and restriction fragment length
helix and may indicate a similar mode of action for divergicin 750. polymorphisms of a PCR product (PCR-RFLP of the cereolysin AB
Divergicin 750 is processed from a longer pri-mary translation gene) were compared for 62 isolates of the Bacillus cereus group.
product. The N-terminal 29 amino acid residues are cleaved off Eleven isolates originated from various foods, and 51 isolates
adjacent to a glycine doublet to yield the mature product. This were obtained from pasteurized milk which had been processed
leader sequence, although unusually long, shows typical by two different dairies. The isolates were clustered into 6 biotypes,
signatures of bacteriocin leader peptides of the dou-ble-glycine 10 fatty acid groups, or 7 PCR-RFLP clusters. Isolates with
type, for example a hydrophilic re-gion at positions - 8 to - 11 mesophilic or psychrotrophic characteristics were preferentially
flanked by hydropho-bic amino acid residues. Other bacteriocins distributed into specific fatty acid or PCR-RFLP groups (P = 0.004).
of lactic acid bacteria with this type of leader invariably have a Unique fatty acid clusters were predominantly found in milk
dedicated secretion system for their export. Indeed, a putative samples of each dairy (P < 0.0001), suggesting that certain dairy
open reading frame starting ap-prox. 1 kb downstream of the plants may harbor plant-specific B. cereus which might constantly
structural gene showed a high degree of similarity to bacteriocin contribute to postpasteurization contamination.
ABC ex-porter genes such as sapT. This indicates that dvn750 may
be part of a larger gene cluster involved in production and secretion Improving microbial safety and extending the shelf life of
of divergicin 750. Bacteriocins and bacteriocinogenic strains may pasteurized milk and related products have always been an
be used by the food industry as additives to prevent outgrowth of important concern to the dairy industry. A major factor limiting
pathogens and spoilage organisms, giv-ing an enhanced control realization of these goals is microorganisms surviving the
over production processes. The study of the structure of bacteriocins pasteurization process and/or contributing to postpasteurization
is an important step in establishing knowledge about the parts of contamination. Bacillus cereus, Bacillus thuringiensis, and Bacillus
the molecule that are responsible for the inhibitory activity. mycoides are frequently found in pasteurized milk, causing
Subsequently, it may be possible to increase the inhibitory spectrum spoilage because of the production of lipases and proteases. They
and enhance the activity by changing specific amino acid residues can also exhibit a health risk to the consumer since they produce
in the bacteriocin molecules. enterotoxins. It has been proposed to merge B. cereus, B.
thuringiensis, and B. mycoides into one single species. DNA-DNA
174 Introductory Food Microbiology Epidemiological Typing of Bacillus spp Isolated from Food 175

hybridization experiments and pulsed-field gel electrophoresis of processed by two different dairies. Isolation procedures followed
chromosomal DNA demonstrated a very close genetic relationship the methods described in the Association of Official Analytical
among the three species. Furthermore, the 16s rRNA sequences of Chemists’ Bacteriological Analytical Manual, except that B. cereus
these species are more than 99% similar. In the present article, B. selective agar supplemented with polymyxin B and egg yolk
cereus, B. thuringiensis, and B. mycoides are, therefore, not emulsion was used as selective plating medium.
distinguished and are referred to as B. cereus.
Growth of B. cereus at 8°C
Many dairy scientists believe that contamination of pasteurized
Growth curves for the 62 B. cereus isolates were determined
milk with Bacillus spp. results from spores that were present in
with the multiwell photometric plate reader Bioscreen C. Bacteria
the raw milk and survived the pasteurization process. However,
were grown for 18 h in brain heart infusion broth at 30°C and
other data in the literature suggest that postpasteurization
were diluted in fresh brain heart infusion broth to an optical
contamination might significantly contribute to Bacillus counts in
density at 600 nm (OD600) of 0.005, and the suspensions were
pasteurized milk. This controversy can partly be related to the lack
used to inoculate the multiwell plate of the Bioscreen C. The
of adequate typing methods for B. cereus. Ternstro¨m et al. described
turbidity development (OD600) of the suspended bacteria at 8°C
an attempt to use numerical phenotypic analysis for
was recorded for 144 h. Growth curves (i.e., turbidity development
characterization of dairy Bacillus spp. However, the authors did
curves) were subsequently generated from the OD readings by
not show any relationship between Bacillus isolates from raw and
using the computer software BIOLINK.
pasteurized milk. Va¨isa¨nen et al. used fatty acid analysis and
phages to type dairy B. cereus isolates. Although they found both Preliminary experiments had shown that growth velocity at
techniques useful for typing of B. cereus, the authors did not 8°C can be used to divide mesophilic and psychrotrophic B. cereus
systematically compare B. cereus isolated throughout the strains by recording the detection time for each strain. The detection
processing lines of single dairies. time is de- fined by the BIOLINK software as the time at which an
OD of 0.05 is reached. The detection time of psychrotrophic B.
Thus, their data do not allow conclusions about the origin of
cereus isolates is less than 100 h, whereas it is considerably
B. cereus found in pasteurized milk. The objective of the present
higher than 100 h for mesophilic isolates.
study was to evaluate the feasibility of three typing techniques for
B. cereus. The techniques chosen were biotyping, analysis of cellular Biochemical Properties
fatty acids (CFA), and restriction fragment length polymorphisms
Twenty-one biochemical variables of the 62 B. cereus isolates
of PCR products (PCR-RFLP). To allow direct comparison, the
were determined by using the VITEK Jr. system according to the
three techniques were applied to a defined collection of B. cereus
manufacturer’s instructions. From the biochemical patterns
strains. Because a large proportion of the Bacillus strains studied
obtained, a binary profile was established for each isolate. The
were isolated from pasteurized milk, additional epidemiological
distances between these profiles were calculated by Jaccard’s index
data for dairy Bacillus spp. were also acquired.
[d = 1 - c/(p + q + c)], where c is the number of variables present
Bacterial Strains in both strains, and p and q are the numbers of variables present
in each strain.
A total of 62 isolates of B. cereus were used. Eleven of these
isolates, originating from food-borne B. cereus outbreaks and from A dendrogram was constructed from the resulting distance
randomly collected milk samples, were obtained from our culture matrix by using the unweighted pair-group method with arithmetic
collection. The remaining 51 isolates were cultured from 23 retail averages (UGPMA). Calculations were made with the computer
cartons (250 ml and 500 ml) of pasteurized milk that had been programs MacDendro and MacMul.
176 Introductory Food Microbiology Epidemiological Typing of Bacillus spp Isolated from Food 177

PCR-RFLP composition, growth temperature, and growth rate. To determine


It has been demonstrated that the lecithinase and an ED value that is not affected by such variations in CFA and
sphingomyelinase genes (cerAB) of B. cereus display genetic that could therefore be used to obtain a reproducible grouping of
variations among different isolates. This diversity and its possible the B. cereus isolates within the dendrogram, the variability of the
application for typing of B. cereus were further explored in the fatty acid profile analysis was assessed by repeated testing of B.
present study. A PCR product encompassing a 1.5-kb fragment of cereus ATCC 14579. Repeated testing was conducted during a 3-
the cerAB gene was used to assess the genetic diversity of the 62 week period in the following manner. Two separate batches of
B. cereus isolates at that locus. PCR amplifications with primers medium plates were prepared independently and stored at 4°C.
specific for cerAB were performed as described previously. Ten- During each week, the test strain was subcultured daily onto fresh
microliter aliquots of each amplicon were digested for 2 h with 10 Trypticase soy agar plates.
U of the following restriction endonucleases: MnlI, HgaI, Sau96I, The second, fourth, and sixth subcultures were analyzed. Each
PvuII, and BstXI. Reaction conditions were set as recommended by of the subcultures tested was streaked onto duplicate plates of
the manufacturers of the enzymes. RFLP profiles were analyzed both batches of media. Each preparation of fatty acid methyl esters
by electrophoresis on 1.5% agarose gels stained with ethidium was split into two subsamples, and the fatty acid profiles were
bromide. A binary matrix of the profiles was used to calculate the analyzed separately with the MIS.
distances between strains by Jaccard’s index. A dendrogram was ED were calculated for the following datum pairs: (i) duplicate
constructed from the distance matrix by UGPMA as described readings of 33 identical sample preparations, (ii) sample
above. preparations from 33 identical subcultures streaked onto duplicate
Analysis of Whole-cell Fatty Acid Profiles with the MIDI System plates, (iii) sample preparations from 33 identical subcultures
grown on two different batches of media, (iv) 43 pairs of sample
Fatty acid methyl esters of B. cereus were analyzed by using
preparations obtained from common subcultures grown during
the Microbial Identification System. The analysis was conducted
each week (e.g., second, fourth, or sixth subculture obtained in the
according to the instructions given by MIDI (10). Briefly, isolates
1st and 2nd, 2nd and 3rd, or 1st and 3rd weeks), and (v) forty-
were grown on Trypticase soy agar (BBL) for 24 ±1 h at 28±1°C.
four pairs of sample preparations obtained from serial
At that time, 45 ±1 mg (wet weight) of cells was harvested from
subcultures grown during each week (e.g., second and fourth,
single colonies. Total fatty acids of the bacteria were converted to
fourth and sixth, or second and sixth subcultures obtained in each
fatty acid methyl esters which were subsequently separated and
week).
quantified with a Hewlett-Packard 5890 series II gas liquid
chromatography column equipped with a flame ionization detector. In this study, identical subcultures of the B. cereus isolates
A dendrogram based on the fatty acid profiles was established for were used to generate the fatty acid profiles. The critical value
137 B. cereus isolates (62 isolates described in this study plus 75 observed with two batches of media (ED, 6.7) could, therefore, be
B. cereus isolates isolated from various foods) by using the MIS used to group the 137 B. cereus isolates, resulting in 11 fatty acid
library generation software. This program calculates a similarity clusters. Although the MIS was originally designed for species
coefficient based on the Euclidean distances (ED) between pairs of identification, it has been suggested that it may also be valuable
isolates. for epidemiological typing of bacteria. Consequently, the datum
library generated with 137 B. cereus isolates was used to evaluate
Clustering of the isolates is determined by UGPMA. The
the relationships of the 62 B. cereus isolates. The fatty acid profiles
phenotypic expression of relative amounts of fatty acids within
of these isolates were attributed to 10 (Mi 2 to Mi 11) of the 11 B.
bacterial cells depends on various factors, which include medium
cereus fatty acid groups.
178 Introductory Food Microbiology Epidemiological Typing of Bacillus spp Isolated from Food 179

Correlations minutes. The test is a modification of one previously developed for


Comparison of growth characteristics and fatty acid profiles determining raw milk quality. In the latter test, milk samples were
of the 62 isolates showed that psychrotrophic isolates were treated with a detergent to lyse non-microbial cells present in the
preferentially (P , 0.0001) grouped into fatty acid clusters Mi 2 to milk. Microbial cells were removed by filtration and the ATP
Mi 6, whereas mesophilic isolates belonged to fatty acid groups present in the cells extracted and assayed using the luciferase-
Mi 7 to Mi 11. A similar tendency (P 5 0.004) was perceived when luciferin reaction. A number of problems were encountered when
growth temperature and PCR-RFLP groups were compared. No this protocol was applied to chicken carcass rinses. The primary
association could be observed among biotypes and growth problem was a quenching effect on the light emission by the
characteristics, PCR-RFLP groups, or fatty acid groups. Evaluation luciferase reaction caused by the presence of particulate and lipid
of the fatty acid groups for the 51 B. cereus isolates from pasteurized material in the rinse water. The quenching effect was substantially
milk revealed interesting tendencies. A majority of the isolates reduced by incorporating a pre-filtration step and an enzyme
found in samples processed by dairy A belonged to the subgroup treatment prior to filtration through the bacteria-retaining filter.
Mi 5, whereas isolates found in milk from dairy B belonged to Using the modified assay, an excellent correlation (r=0.9) was
groups Mi 6 and Mi 8 (P , 0.0001). Va¨isa¨nen et al. exploited obtained when the ATP count obtained on 150 chicken rinse
phage typing, minimum growth temperature, and analysis of CFA samples was compared with plate counts carried out on the same
for differentiation of dairy B. cereus isolates. rinse washes.
They found a correlation of fatty acid composition and The test can be used to make a rapid assessment of poultry
minimum growth temperature; however, they did not examine the carcass quality, based on selective cut-offs predetermined by the
fatty acid data with regard to further typing of the isolates. Our processor. If this level of count is exceeded, the carcass can be
results confirm the observations regarding composition of CFA subjected to a heat treatment before sale. The accuracy of the
and ability to grow at low temperatures. Additionally, we found prediction was high. Work was undertaken to compare methods
indications that psychrotrophic B. cereus strains with a specific used to enumerate bacteria in chicken carcass rinses. The methods
CFA profile were predominantly found in pasteurized milk studied included plate count (by the Spiral plater), ATP
processed by one dairy, while they were less frequently found in bioluminescence, impediometry (using the Bactometer), HGMF,
milk from the other processing plant. This plantspecific distribution and turbidiometry. This enabled comparison of the repeatability
of B. cereus in pasteurized milk suggests that the strains may and accuracy of the various methods. Only the ATP
originate from common reservoirs within dairy plants or within bioluminescence assay and HGMF correlated well with plate counts
certain farms supplying raw milk. This research was funded by for chicken carcass rinses and the repeatability of these test methods
the Ontario Food Quality and Safety Research Fund, by the Dairy was excellent.
Farmers of Ontario, and by the Natural Sciences and Engineering The ATP method was used to monitor critical control points
Research Council of Canada. H.S. was supported by a fellowship in the poultry processing plant. The CCP’s studied included the
from the Commission for the Advancement of Young Scholars, scald, the prechill and the chill tank waters. It was shown that the
Zu¨rich, Switzerland. ATP test could be used to give a reliable indication of the microbial
content of process waters during production and results could be
RAPID ASSESSEMENT OF THE QUALITY OF POULTRY obtained within 10 minutes. The microbial load in the scald tank
CARCASSES water was shown to increase throughout the working day and
An assay has been developed whereby it is possible to assess this was shown to be due to accumulation of microorganisms
microbial concentrations in poultry carcass rinses within 10 washed from the carcass rather than growth. Levels of microbial
180 Introductory Food Microbiology Epidemiological Typing of Bacillus spp Isolated from Food 181

contamination of the pre-chill and chill tanks were low in the The EPSs produced by LAB are either present as a capsule
plant studied on several sampling days. The microbial load in the attached to the cell surface, or secreted into the environment.
tanks was dependent on flow rate of water through the tanks and Based on their sugar compositions, the EPSs can be divided into
it was concluded that, based on microbial load in the chill tank, homopolysaccharides, composed of a single type of
water consumption at the plant could be reduced substantially. monosaccharide, and heteropolysaccharides, containing several
This will enable plant personnel to more efficiently respond to types of monosaccharide. Dextran is the most important
high microbial loads that may develop during chilling and result homopolysaccharide, which is a glucan produced, e.g. by
in more economical management of the water supply. Leuconostoc mesenteroides. The heteropolysaccharides produced by
LAB are generally composed of repeating units of up to eight
ANTIMICROBIAL COMPOUNDS AND EXTRACELLULAR monosaccharide residues; the chain length and degree of branching
POLYSACCHARIDES PRODUCED BY LACTIC ACID vary with the producing strains. The rheological properties of the
BACTERIA STRUCTURES AND PROPERTIES, polysaccharides depend on the monomeric composition, the
DISSERTATION number of side chains, the chain length and the charge (neutral
In a variety of ecological niches, microorganisms compete or anionic) of the polysaccharides, as well as the anomeric
with each other for survival and through evolution form unique configuration of the monosaccharides and the sequence in which
flora. In some food ecosystems, lactic acid bacteria (LAB) constitute they are arranged. The EPSs produced by LAB may act as
the dominant microflora. viscosifying agents to improve the texture and consistency of
These organisms are able to produce antimicrobial compounds fermented foods. Since LAB are food-grade microorganisms with
against competing flora, including food-borne spoilage and the GRAS status (Generally Recognized As Safe), the use of the
pathogenic bacteria. Under unfavorable environmental conditions secreted EPSs as natural alternatives to produce all-natural food
many species of LAB also produce exopolysaccharides (EPSs), products without additives has received increased attention. It
which protect themselves against desiccation, bacteriophage and has also been claimed that EPSs isolated from LAB cultures have
protozoan attack. antitumor activity.
Lactic acid bacteria provide the major preservative effects in Lactic acid bacteria are able to produce a large variety of
food fermentation which mankind has practiced for thousands of compounds which contribute to the flavor, color, texture and
years. The primary antimicrobial effect exerted by LAB is the consistency of fermented foods. However, the present study focuses
production of lactic acid and reduction of pH. In addition, LAB on two potentially important group of compounds, antimicrobial
produce various antimicrobial compounds, which can be classified compounds and EPSs, which differ largely in their chemistry and
as low-molecular-mass (LMM) compounds such as hydrogen functionalities. Attention has been paid to developing methods
peroxide (H2O2), carbon dioxide (CO2), diacetyl (2,3-butanedione), suitable for separation and purification of LMM antimicrobial
uncharacterized compounds, and high-molecular-mass (HMM) compounds aiming at inhibition of food-borne spoilage bacteria.
compounds like bacteriocins. In order to understand the relation between the structures and
rheological properties of the EPSs, a knowledge of their primary
Among bacteriocins so far characterized, nisin is best defined,
molecular structures is required. In this study, the primary
and the only purified bacteriocin produced by LAB that has been
molecular structures of the EPSs produced by Lb. helveticus strains
approved for use in food products. A LMM antimicrobial
have been studied by NMR spectroscopy. The EPSs produced by
compound, reuterin, has also been chemically identified, and the
several slime-forming Lactococcus lactis ssp. cremoris strains have
reuterin-producing Lactobacillus reuteri strain has been applied as
been characterized to understand the roles of the EPSs in the
a probiotic in dairy products.
rheology of fermented foods.
182 Introductory Food Microbiology Epidemiological Typing of Bacillus spp Isolated from Food 183

Antimicrobial Compounds Produced by Lactic Acid Bacteria large amount of lactic acid is in the undissociated form, and it is
Lactic acid bacteria are a physiologically diverse group of toxic to many bacteria, fungi and yeasts. However, different
organisms, which can be generally described as Gram-positive, microorganisms vary considerably in their sensitivity to lactic
nonsporing cocci or rods with lactic acid as the major product of acid. At pH 5.0 lactic acid was inhibitory toward spore-forming
carbohydrate fermentation. Traditionally, LAB comprise four genera bacteria but was ineffective against yeasts and moulds. It was
Lactobacillus, Leuconostoc, Pediococcus, and Streptococcus. However, possible to grow Aspergillus parasiticus NRRL 2999 in a medium
several new genera have been suggested for inclusion in the group containing 0.5 or 0.75% lactic acid at pH 3.5 or 4.5. Lindgren and
of LAB due to a recent taxonomic revision. The genus Streptococcus Dobrogosz (1990) showed that at different pH ranges the minimum
has been reorganized into Enterococcus, Lactococcus, Streptococcus inhibitory concentration (MIC) of the undissociated lactic acid
and Vagococcus. LAB are involved in the fermentation of a range was different against Clostridium tyrobutyricum, Enterobacter sp.
of milk, meat, cereal and vegetable foods. The antimicrobial and Propionibacterium freudenreichii ssp. shermanii. In addition, the
compounds produced by LAB can inhibit the growth of pathogenic stereoisomers of lactic acid also differ in antimicrobial activity, L-
bacteria of possible contaminants in the fermented products. In lactic acid being more inhibitory than the D-isomer.
the past two decades, there have been many reports on the Acetic and propionic acids produced by LAB strains through
bacteriocins produced by LAB. These bacteriocins are of a heterofermentative pathways, may interact with cell membranes,
proteinaceous nature and they have been grouped into class I, and cause intracellular acidification and protein denaturation.
lantibiotics which are small peptides (e.g. nisin), class II, small They are more antimicrobially effective than lactic acid due to
heat-stable peptides, class III, large heat-labile proteins, and class their higher pKa values (lactic acid 3.08, acetic acid 4.75, and
IV, complex bacteriocins which are not well defined. In the propionic acid 4.87), and higher percent of undissociated acids
following text, the LMM antimicrobial compounds produced by than lactic acid at a given pH. Acetic acid was more inhibitory
LAB will be discussed. than lactic and citric acids toward Listeria monocytogenes. Acetic
acid also acted synergistically with lactic acid; lactic acid decreases
Organic Acids
the pH of the medium, thereby increasing the toxicity of acetic
Fermentation by LAB is characterized by the accumulation of acid.
organic acids and the accompanying reduction in pH. The levels
and types of organic acids produced during the fermentation Hydrogen Peroxide and Carbon Dioxide
process depend on the species of organisms, culture composition Hydrogen peroxide is produced by LAB in the presence of
and growth conditions. The antimicrobial effect of organic acids oxygen as a result of the action of flavoprotein oxidases or
lies in the reduction of pH, as well as the undissociated form of nicotinamide adenine hydroxy dinucleotide (NADH) peroxidase.
the molecules. It has been proposed that the low external pH The antimicrobial effect of H2O2 may result from the oxidation of
causes acidification of the cell cytoplasm, while the undissociated sulfhydryl groups causing denaturing of a number of enzymes,
acid, being lipophilic, can diffuse passively across the membrane. and from the peroxidation of membrane lipids thus the increased
The undissociated acid acts by collapsing the electrochemical membrane permeability. H2O2 may also be as a precursor for
proton gradient, or by altering the cell membrane permeability the production of bactericidal free radicals such as superoxide
which results in disruption of substrate transport systems. (O2 -) and hydroxyl (OH.) radicals which can damage DNA.
Lactic acid is the major metabolite of LAB fermentation where It has been reported that the production of H2O2 by Lactobacillus
it is in equilibrium with its undissociated and dissociated forms, and Lactococcus strains inhibited Staphylococcus aureus, Pseudomonas
and the extent of the dissociation depends on pH. At low pH, a sp. and various psychotrophic microorganisms in foods. In raw
184 Introductory Food Microbiology Epidemiological Typing of Bacillus spp Isolated from Food 185

milk, H2O2 activates the lactoperoxidase system, producing Acetaldehyde at 10-100 ppm inhibits the growth of Staphylococcus
hypothiocyanate (OSCN-), higher oxyacids (O2SCN- and aureus, Salmonella typhimurium and E. coli in dairy products.
O3SCN-) and intermediate oxidation products that are inhibitory
Fatty Acids
to a wide spectrum of Gram-positive and Gram-negative bacteria.
Under certain conditions, some lactobacilli and lactococci
Carbon dioxide is mainly produced by heterofermentative LAB.
possessing lipolytic activities may produce significant amounts of
The precise mechanism of its antimicrobial action is still unknown.
fatty acids, e.g. in dry fermented sausage and fermented milk. The
However, CO 2 may play a role in creating an anaerobic
antimicrobial activity of fatty acids has been recognized for many
environment which inhibits enzymatic decarboxylations, and the
years. The unsaturated fatty acids are active against Gram-positive
accumulation of CO2 in the membrane lipid bilayer may cause a
bacteria, and the antifungal activity of fatty acids is dependent on
dysfunction in permeability. CO2 can effectively inhibit the growth
chain length, concentration, and pH of the medium. The
of many food spoilage microorganisms, especially Gram-negative
antimicrobial action of fatty acids has been thought to be due to
psychrotrophic bacteria. The degree of inhibition by CO2 varies
the undissociated molecule, not the anion, since pH had profound
considerably between the organisms. CO2 at 10% could lower the
effects on their activity, with a more rapid killing effect at lower
total bacterial counts by 50%, and at 20-50% it had a strong
pH.
antifungal activity.
Reuterin and Other Low-molecular-mass Compounds
Aroma Components
Reuterin is produced by Lb. reuteri, a heterofermentative species
Diacetyl is produced by strains within all genera of LAB by
inhabiting the gastrointestinal tract of humans and animals. It is
citrate fermentation. The antimicrobial effect of diacetyl has been
formed during the anaerobic growth of Lb. reuteri by the action of
known since the 1930s. It inhibits the growth of Gram-negative
glycerol dehydratase which catalyzes the conversion of glycerol
bacteria by reacting with the arginine-binding protein, thus
into reuterin. Reuterin has been chemically identified to be 3-
affecting the arginine utilization.
hydroxypropanal (â- hydroxypropionaldehyde), a highly soluble
Jay (1982) showed that Gram-negative bacteria were more pH-neutral compound which is in equilibrium with its hydrated
sensitive to diacetyl than Grampositive bacteria; the former was monomeric and cyclic dimeric forms. The biosynthesis pathway
inhibited by diacetyl at 200 ìg/mL and the latter at 300 ìg/mL. from glycerol to the three forms of reuterin is shown below.
Diacetyl at 344 ìg/mL inhibited strains of Listeria, Salmonella,
Yersinia, Escherichia coli, and Aeromonas. Since the production of Methods for Evaluation of Antimicrobial Activity
diacetyl during lactic fermentation is low, e.g. 4 ìg/mL produced Among many methods available for evaluation of antimicrobial
by Lc. lactis ssp. diacetylactis (Cogan 1980), and the acceptable activity, the methods described below have been used for
sensory levels of diacetyl are at 2-7 ìg/mL, its practical use as a determining the antimicrobial activity of compounds produced by
food preservative is limited. However, diacetyl may act LAB.
synergistically with other antimicrobial factors and contribute to
combined preservation systems in fermented foods. The Agar Diffusion Method
Acetaldehyde is produced by Lb. delbrueckii ssp. bulgaricus by The agar diffusion method has long been used for testing
the action of a threonine aldolase, which cleaves threonine into antimicrobial activity, and it was first used by Fleming in 1924.
acetaldehyde and glycine. Since Lb. delbrueckii ssp. bulgaricus and The method has been widely used for evaluation of antimicrobial
S. thermophilus in yoghurt cannot metabolize acetaldehyde, it activity, specially for biologically derived compounds. It includes
accumulates in the product at a concentration of about 25 ppm. agar well diffusion assay and disc assay. In this test, an
186 Introductory Food Microbiology Epidemiological Typing of Bacillus spp Isolated from Food 187

antimicrobial compound is applied to an agar plate on a paper antimicrobial activity of reuterin produced by Lb. reuteri, and the
disc or in a well. The compound diffuses into agar resulting in a activity of reuterin was expressed as MIC values or as the maximum
concentration gradient that is inversely proportional to the distance dilutions of the reuterin fraction.
from the disc or well. The size of the inhibition zone around the
The Automated Turbidometric Assay
disc or well is a measure of the degree of inhibition. The results
of the test are generally qualitative. The method requires that the A turbidometric assay based on automated systems determines
indicator organisms must grow rapidly, uniformly, and aerobically. the effect of a compound on the growth or death kinetics of a
Since highly hydrophobic antimicrobial compounds cannot diffuse microorganism. It provides information concerning the effect of an
in agar, they are not suitable for tests by this method used an agar antimicrobial that may cause a delayed lag phase or reduced
well diffusion assay for testing the antimicrobial activity of Lb. growth rate at concentrations below the MIC. Since the bacterial
rhamnosus GG by addition of a 10-fold concentrate of the GG strain growth is monitored by measuring the turbidity of the broth
or MRS broth to wells (diameter 4 mm) in agar against various medium, the method demands that the instrument be highly
anaerobic and facultative bacteria. The activity of an antimicrobial sensitive. Growth at levels below log 5.0 CFU/ml may not be
substance produced by Lb. delbrueckii ssp. bugaricus 7994 was detectable. Skyttä and Mattila-Sandholm (1991) described a
tested quantitatively with a disc assay procedure, using paper quantitative method based on automated turbidometry for assaying
assay discs 12.7 or 6.35 mm in diameter wetted with 30 or 10 ìl antimicrobial activity, which was expressed as area reduction
of sample against Pseudomonas fragi and Staphylococcus aureus. The percentage values measured under the growth curve. The method
assay methods used for determination of the antimicrobial activity has been used to test the antimicrobial activity of antimicrobial
of different species of LAB were slightly different with respect to compounds produced by P. damnosus and P. pentosaceus and Lb.
the sizes of the wells, discs and samples, and the incubation plantarum.
conditions were dependent on the indicator organisms used.
Exopolysaccharides Produced by LAB
Several modified procedures based on the agar diffusion method
have also been used for testing antimicrobial activity of LAB. Lactic acid bacteria produce polysaccharides as cell wall
These procedures include the agar spot test, deferred antagonism components and storage polymers, and also in many species, as
assay, and spot-onlawn assay. a capsule or slime. In the dairy industry, the slime-forming LAB
strains have traditionally been used in the production of fermented
The Agar and Broth Dilution Methods milk products, e.g. yogurts, Finnish ‘viili’ and Scandinavian
Agar and broth dilution methods are used as quantitative ‘långfil’. It has been generally acknowledged that the secreted
methods, suitable for microorganisms with variable growth rate EPSs by LAB play an important role in the rheological behavior
and for anaerobic, microaerophilic microorganisms. The results and texture of the products.
are expressed as MIC, which is the lowest concentration of an
Homopolysaccharides
antimicrobial that prevents growth of a microorganism after a
specific incubation period. In this test, an antimicrobial is serially Homopolysaccharides are a group of polysaccharides
diluted and a single concentration added to a culture tube or plate composed of one monosaccharide type. Several species of LAB are
added with nonselective broth or melted agar medium, which is able to utilize sucrose as a specific substrate to produce dextrans,
then inoculated with test organisms and incubated. The MIC is mutans, and levans. Dextrans are a large class of extracellularly
defined as the lowest concentration at which no growth occurs formed glucans produced by the genus Lactobacillus, Leuconostoc,
(absence of turbidity) in a medium following incubation. The broth and Streptococcus, of which Leuc. mesenteroides and Leuc. dextranicum
dilution assay has been used for the determination of the are the well-known dextran producers. Although each bacterial
188 Introductory Food Microbiology Epidemiological Typing of Bacillus spp Isolated from Food 189

strain produces a unique glucan, a common structural feature of heteropolysaccharides are synthesized intracellularly at the
all dextrans is a high percentage (up to 95%) of á- 1,6 linkages cytoplasmic membrane utilizing sugar nucleotides as precursors
with a smaller proportion of á-1,2, á-1,3, or á-1,4 linkages resulting for the assembly of polysaccharide chains.
in a highly branched molecule (Franz 1986). Dextrans are
Lactobacillus
synthesized outside the cell by dextransucrase, which catalyzes
sucrose to produce D-fructose and D-glucose, and transfers the The ability of lactobacilli to produce EPSs has been recognized
latter to an acceptor to form dextran. The reaction is as follows: for many years. In 1968, Kooiman first reported the structure of a
sucrose + glucan acceptor dextran or mutans + D-fructose Mutans heteropolysaccharide produced by a Lb. brevis strain isolated from
are synthesized in a similar way by S. mutans and S. sobrinus. kefir grains. This polysaccharide consists of a hexasaccharide
However, mutans differ from dextrans in containing a high repeating unit with D-galactose and D-glucose in the molar ratio
percentage of á-1,3 linkages, which are attributed to the insoluble 1:1. In the last decade, a number of heteropolysaccharides produced
nature of this type of polymers. Some S. salivarius strains are able by the Lactobacillus species have been investigated.
to produce fructans of the levan type with 2,6-linked â- Lb. helveticus strains produce several EPSs with varying
fructofuranoside residues. An extracellular enzyme levansucrase repeating units, though all containing galactose and glucose. The
is involved in hydrolyzing sucrose and transferring D-fructose to EPS produced by Lb. helveticus 776 has hexasaccharide repeating
growing fructan chains to form levans: sucrose + fructan acceptor units containing D-galactose and D-glucose. The EPS produced by
levan + D-glucose Lb. helveticus TY1-2 consists of heptasaccharide repeating units
Another type of homopolysaccharide is the galactan produced with Dgalactopyranosyl and D-glucopyranosyl, and 2-acetamido-
by Lc. lactis ssp. cremoris H414, which is composed of a branched 2-deoxy-D-glucopyranosyl residues. Recently, Stingele et al. (1997)
pentasaccharide repeating unit as shown below. showed that the EPS produced by Lb. helveticus Lh59 had an
identical primary molecular structure as the one produced by Lb.
A Pediococcus strain produced a â-D-glucan with a trisaccharide
helveticus TN-4, a presumed spontaneous mutant of the strain
repeating unit. Lactobacillus spp. G-77 has been shown to produce
TY1-2. This polymer is composed of a tetrasaccharide backbone
a 2-substituted- (1’!3)-â-D-glucan, identical to the EPS produced
with a lactosyl side-chain, and the molar ratio of D-galactose and
by P. damnosus 2.6. Lactobacillus spp. G-77 also produced a á-D-
D-glucose is 1:1.
glucan composed of a trisaccharide repeating unit. Recently, van
Geel-Schutten et al. (1998) reported for the first time the production Robijn et al. (1995b) reported the primary molecular structure
of a fructan by Lb. reuteri strain LB121 with raffinose as a sugar of a viscous EPS produced by Lb. sake 0-1 which was isolated from
substrate; this strain also produced both a glucan and a fructan fermented meat products. The EPS consists of a pentasaccharide
on sucrose. repeating unit of glucose, rhamnose, and glycerol phosphate. The
threedimensional structure of this polymer has been further studied
Heteropolysaccharides by molecular mechanics calculations. The helics generated by a
A wide range of LAB strains can produce polysaccharide builder program are highly extended, with either
heteropolysaccharides, which are composed of repeating units. 2-fold or 3- or 4-fold right-handed chiralities. Grobben et al. (1997)
The monosaccharide compositions of these EPSs are mostly showed that Lb. delbrueckii ssp. bulgaricus NCFB 2772 produced an
galactose and glucose, and also small amounts of rhamnose, EPS made up of galactose, and small quantities of glucose and
fructose, mannose, and galactosamine. In comparison with the rhamnose, and another EPS that, according to Sikkema and Oba
homopolysaccharides, the production of heteropolysaccharides (1998), was similar to the structure of the EPS produced by Lb.
by LAB is much lower (60 to 400 mg L-1). Generally, the delbrueckii ssp. bulgaricus rr. The enzymes involved in the production
190 Introductory Food Microbiology Epidemiological Typing of Bacillus spp Isolated from Food 191

of the sugar nucleotides of strain NCFB 2772 have been analyzed, The biosynthesis of the EPSs produced by Lc. lactis strains is
and based on this analysis a biosynthetic pathway for the EPS has generally associated with a plasmid. Transferring mucoidity
been proposed. Growth of the strain in a fructose-based medium plasmids from Lc. lactis ssp. cremoris ARH87 and MS to nonmucoid
led to the absence of the enzyme activities for the synthesis of the Lc. lactis strains proved the latter to be mucoid. van Kranenburg
rhamnose nucleotide, and accordingly no rhamnose was present et al. (1997) described a novel 12 kb EPS gene cluster located on
in the polysaccharide produced. The EPSs produced by Lb. a 40 kb plasmid, which was essential for the EPS synthesis of Lc.
acidophilus LMG9433, Lb. kefiranofaciens K1, and Lb. paracasei 34-1 lactis ssp. cremoris NIZO B40. Introduction of the EPS gene cluster
have also been structurally evaluated, the repeating units being from S. thermophilus Sfi6 to a non-EPS-producing Lc. lactis MG1363
pentamers, hexamers and tetramers, respectively. Recently, Lb. produced an EPS with a different structure from the EPS of the
rhamnosus strain C83 has been shown to produce an EPS composed native host (Stingele et al. 1999). The absence of the GalNAc residue
of a pentasaccharide repeating unit with a linear structure. This in the EPS of Lc. lactis MG1363 was probably caused by the lack
strain, as well as Lb. casei CG11 and Lb. sake 0-1, produced more of a UDP-N-acetylglucosamine C4- epimerase activity.
EPSs at lower temperatures, whereas several other Lactobacillus
Streptococcus
strains produced more EPSs at higher temperatures (compared
with the optimum temperatures of growth). S. thermophilus strains are used in combination with Lb.
delbrueckii ssp. bulgaricus strains in yoghurt starters. The EPSs
Lactococcus produced by several S. thermophilus strains have been found to
Among lactococci, only the slime-forming Lc. lactis ssp. cremoris have similar or identical primary molecular structures. Doco et al.
strains have been investigated. These strains producing EPSs play (1990) first reported the structure of an EPS produced by ropy S.
a role in the proper consistency of the fermented milk. The sugar thermophilus strains CNCMI 733, CNCMI 734 and CNCMI 735,
components of the EPSs are most frequently galactose, glucose, which consisted of a tetrasaccharide repeating unit of D-galactose,
and very often rhamnose. Nakajima et al. (1992a) reported a D-glucose, and N-acetyl-D-galactosamine in a molar ratio 2:1:1.
phosphate-containing heteropolysaccharide, named ‘viilian’, An EPS with an identical repeating unit structure has been reported
produced by Lc. lactis ssp. cremoris SBT 0495 which was isolated to be produced by S. thermophilus Sfi6. Lemoine et al. (1997) showed
from a Finnish ‘viili’ starter culture. The EPS consists of the that the EPSs produced by S. thermophilus Sfi12 and Sfi39 had
following repeating unit: molecular masses greater than 2 x 106, and both yielded a slimy
Lc. lactis ssp. cremoris B40 has also been found to produce a texture rather than a thickened one in yoghurt. However, they had
phosphopolysaccharide with an identical repeating unit as shown different sugar compositions and structures; the former consisting
above. Lc. lactis ssp. cremoris strain LC330 appeared to produce of a hexasaccharide repeating unit of galactose, glucose and
concurrently two EPSs; an anionic EPS composed of galactose, rhamnose, and the latter a tetrasaccharide repeating unit of
glucose, rhamnose, glucosamine and phosphate, and a neutral galactose and glucose.
EPS containing galactose, glucose and glucosamine with branched Recently, Faber et al. (1998) showed that S. thermophilus Rs
terminal galactose moieties. The mechanism for the biosynthesis and Sts produced EPSs of identical repeating units, but they had
of the EPS by Lc. lactis ssp. cremoris has not been investigated in different molecular masses, resulting in a difference in viscosity in
detail. Recently, Oba et al. (1996) proposed a biosynthetic pathway their milk cultures. Bubb et al. (1997) also showed that the EPS
for the production of ‘viilian’ by strain SBT 0495 with the following produced by S. thermophilus OR 901 had a similar repeating unit
steps: preparation of the membraneembedded lipid carrier; to the one of the strains Rs and Sts; all being branched
incorporation of the first monosaccharide with the phosphate on heptasaccharide repeating units of D-galactose and L-rhamnose
C-1; assembly of the intact repeating unit. in the same molar ratio: 5:2.
192 Introductory Food Microbiology Epidemiological Typing of Bacillus spp Isolated from Food 193

Isolation and Structural Elucidation of EPSs Nuclear Magnetic Resonance Spectroscopy


Isolation of EPSs NMR spectroscopy relies on the interaction of radio-frequency
EPSs produced by LAB can be isolated by precipitation with electromagnetic radiation with magnetically active nuclei in a
trichloroacetic acid (TCA) to remove proteins from the culture strong magnetic field. The radio frequencies used range from 200
media, and subsequently by ethanol and/or acetone to precipitate to 800 MHz, corresponding to magnetic fields from 4.7 to 18.8
polysaccharides. Gel filtration or ion exchange chromatogrphy is Tesla. 1H and 13C are the spin-active nuclei most frequently
often used for further purification of EPSs. Robijn et al. (1995a, encountered in carbohydrates. 1H and 13C NMR spectroscopy,
including one- (1D) and two-dimensional (2D), is a powerful tool
1995b, 1996a, 1996b) purified EPSs produced by several
for structural studies of carbohydrates, which also include
Lactobacillus strains by gel filtration with different columns (Sephacel
polysaccharides produced by LAB. 1D 1H NMR spectroscopy can
500, Sephacryl S-500, and Superrose-6).
be used for rapid identification or to check the purity of a
Gel filtration techniques were also used for purification of the polysaccharide sample. Signals in the anomeric region (about 4.3-
EPSs produced by Lc. lactis ssp. cremoris H414, S. thermophilus 5.5 ppm) of the spectrum and the coupling of the anomeric protons
Sfi16, and S. thermophilus CNCMI 733. Since many EPSs are (JH1,H2) may provide useful information about the number of
negatively charged, they can be bound to an anion exchanger. residues in a repeating unit, and the anomeric configuration,
This technique has been used for purification of the anionic EPSs respectively.
produced by Lc. lactis ssp. cremoris B40, and Lb. helveticus TY1-2
Yamamoto et al. (1995) recorded the 500 MHz 1H NMR
and TN-4. Marshall et al. (1995) showed that Lc. lactis ssp. cremoris
spectrum of the EPS produced by Lb. helveticus TN-4 in D2O at 70
strain LC330 produced at the same time both neutral and anionic
oC, and found six signals in the anomeric region with nearly
EPSs in the medium; these two EPSs were effectively separated
equal integrated intensities, suggesting there was a hexasaccharide
and purified by anion exchange chromatography.
repeating unit for this EPS. A study on the EPS produced by Lb.
Sugar and Methylation Analyses helveticus Lh59, by 400 MHz 1H NMR spectroscopy in Me2SO-d6
at 80 oC produced four anomeric protons signals in a molar ratio
In the analysis of monosaccharide compositions of EPSs
1:2:2:1, which also indicated a hexasaccharide repeating unit. The
produced by LAB, the EPSs are hydrolyzed with trifluoroacetic
1H NMR spectrum of the EPS produced by Lb. paracasei had four
acid (TFA), reduced and acetylated, and the acetate derivatives are
doublets in the anomeric region, and the coupling constants
analyzed with GC. For the methylation analysis, monosaccharides
(JH1,H2) of these signals (8.3 Hz, 7.7 Hz, 7.3 Hz, 7.7 Hz) were of
are usually derivatized into partially methylated alditol acetates,
the pyranoid ring form with all the residues in the â configuration.
which are introduced into the EI source of MS from a GC, or GLC
interface. Since the natural abundance of 13C is very low (1.1% relative
to 12C), the peak intensity of 13C has to be enhanced in 1D 13C
The substitution pattern of the monosaccharides can be NMR spectroscopy by using a large number of pulses, by taking
determined by comparing their fragmentation pattern with advantage of the nuclear Overhauser effect (NOE), or by using
reference EI-MS spectra. Yamamoto et al. (1994, 1995) studied the distortionless enhancement by polarization transfer (DEPT)
substitution pattern of the monosaccharides in the EPSs produced experiments. The values of 13C chemical shift and 13C-1H
Lb. helveticus TY1-2 and TN-4 by GLC-MS of the methylated and coupling (1JC,H) provide structural information of the
acetylated sugar residues. The EPS produced by Lb. helveticus 766 polysaccharides. Robijn et al. (1995a) found six signals in the
was analyzed by GLC-MS on DB-1 of the partially methylated anomeric region of the 13C NMR spectrum of the EPS produced
alditol acetates. by Lb. helveticus 766, confirming the suggested hexasaccharide
194 Introductory Food Microbiology Epidemiological Typing of Bacillus spp Isolated from Food 195

repeating unit by 1H NMR spectroscopy. Based on the C-1 chemical hydrodynamic volume of a molecule, is obtained by extrapolating
shifts in the 13C NMR spectrum of the EPS produced by Lb. the Huggins equation to zero concentration: çsp /c = [ç] + k’[ç]2c,
rhamnosus strain C83, Vanhaverbeke et al. (1998) assigned two where çsp is specific viscosity, c is polymer concentration, and k’
downfield signals (ä 110.12, 107.67) to two residues having â is a constant for a series of polymers of different molecular mass
configuration, and three signals at ä 99.73, 99.99 and 100.73 to in a given solvent. çsp /c is also defined as the reduced viscosity
three residues having á configuration. çred. For ionic polysaccharides in aqueous solutions, the value of
The 1D NMR techniques are often used for the assignment of çred increases with decreasing concentration, showing a
signals in the anomeric region. For detailed assignment for the polyelectrolyte effect. The behavior of the polyelectrolytes is
spin system of sugar residues, 2D techniques are needed. These influenced by intrachain Coulombic interaction, ionic strength,
techniques include 1H,1H-correlated spectroscopy (COSY), total pH and specific counterions. Oba et al. (1999) suggested that in a
correlation spectroscopy (TOCSY), homonuclear Hartmann-Hahn strain sweep test at very high dilution of the EPS produced by Lc.
spectroscopy (HOHAHA), gradient selected heteronuclear single lactic ssp. cremoris SBT 0495, the higher cross-over frequency of the
quantum coherence (gHSQC), gradient selected heteronuclear EPS in 0.1 M NaCl compared to that in pure water was due to the
multiple-bond correlation (gHMBC) experiment, and nuclear polyelectrolyte effect of this EPS. van den Berg et al. (1995) showed
Overhauser effect spectroscopy (NOESY). Bubb et al. (1997) used that over a wide range of shear rates, the viscosity of a 1% solution
a TOCSY experiment to further assign two signals of anomeric of the EPS produced by Lb. sake 0-1 decreased with increasing
protons in the 1H NMR spectrum of the EPS produced by S. shear rates, indicating a shear-thinning behavior, and the viscosity
thermophilus OR 901. By means of 2D COSY, HOHAHA, NOESY, was comparable to that of xanthan gum.
and 13C-1H HMQC (heteronuclear multiple quantum coherence), Dynamic Viscoelasticity
Robijn et al. (1996a) assigned almost all 1H and 13C resonances
In response to an applied stress, polysacharides may show a
in 1D 1H and 13C NMR spectra of the EPS produced by Lb.
viscoelastic behavior, i.e. a combination of truely viscous flow and
paracasei 34-1. The sequence of the monosaccharide residues in a
perfectly elastic response. In a dynamic test, the polysaccharide
repeating unit can be established by 2D NOESY and HMBC
sample is subject to sinusoidal shear oscillation with a wide range
experiments. The former experiment gives information about the
of frequencies (0.01-300 Hz). The relative magnitudes of G’ (storage
interresidue linkage from observation of the NOE between anomeric
modulus) and G” (loss modulus) vary with the state of the
protons and the protons at the substituted positions of
polysaccharide. For entangled solutions, where there is a greater
neighbouring sugar residues. The latter experiment gives rise to
contribution from the viscous element, G’ is low. When frequency
crosspeak between proton and carbon atoms that are long-range
decreases, there is a crossover in G’ and G”, and they flow as high
scalar coupled. Faber et al. (1998) used 2D NOESY together with
viscosity liquids at very low frequencies. For gel systems, G’ and
HSQC-NOE experiments to determine the sequence of the sugar
G” are parallel, with G’ > G” and largely frequency independent.
residues in the EPS produced by S. thermophilus Rs and Sts. The
Oba et al. (1999) showed that in a dynamic and steady shear
monosaccharide sequence in the EPS produced by Lb. paracasei 34-
measurement the aqueous solutions of the EPS produced by Lc.
1 was established by 2D NOESY experiments.
lactis ssp. cremoris SBT 0495 behaved as an entangled solution but
Rheological Characterization of EPSs not as a weak gel. Nishinari (1997) reported the frequency (0.01
- 10 ù/radÅ”s-1) dependence of G’ and G” for 1-3% solutions of
Solution Viscosity
gellan gum, an EPS produced by Pseudomonas elodea.
Viscosity ç is defined as the ratio between shear stress and
The 1% (0-30 °C) and 2% (30 °C) solutions had a typical dilute
shear rate. The intrinsic viscosity [ç], which measures the
solution behavior with G” > G’. The 2% (15 °C, 25 °C) and 3% (30
196 Introductory Food Microbiology Epidemiological Typing of Bacillus spp Isolated from Food 197

°C) solutions, however, had a concentrated solution behavior with As described ealier, S. thermophilus Rs and Sts produced EPSs of
a crossover of G’ and G” and G’ > G” at higher frequencies. the same structure, but had different viscosities in the milk cultures
Gelation occurs at 3% at 0-25 °C with G’ and G” being slightly due to their different molecular masses.
frequency dependent. The rheological behavior of the polysaccharides is also related
Gelling Properties to their three-dimensional structure. In addition to the viscosifying
effect of the polysaccharides, the interactions between the EPSs
Gels are defined as loose three-dimensional networks with
and the milk proteins, e.g. caseins, also play a role. Studies of a
structures ranging from homogeneous solutions (enthalphy-driven
yogurt gel with a scanning electron microscopy showed that the
aggregations) to heterogeneous rigid porous systems. Clark and
cells were attached to the protein coagulates by a network structure
Farrer (1995) described the mechanisms of gel formation in three
consisting of polysaccharide filaments. The microorganisms and/
main classes, firstly by point crosslinking with covalent bonds,
or the EPSs that they produce may affect the protein aggregation,
secondly by chain association driven by changes in temperature,
thereby affecting the physical properties of the milk gel. A recent
pH and ionic strength, and the presence of small molecules and
study showed that the rheological properties of stirred yoghurt
specific counterions, and thirdly by particle aggregation. Many
were affected by the type of EPSproducing strains used, suggesting
polysaccharide gels are formed by thermoreversable physical
an effect due to the interaction between the polymer and milk
associations, involving Coulombic, dipole-dipole, van der Waals,
proteins. Hess et al. (1997) proposed a model for shear-induced
charge transfer, and hydrophobic and hydrogen bonding
degradation of the microstructure of EPS-producing yogurt. Since
interactions, as well as double-helix formation and aggregation.
the associations of EPS with bacterial cells or casein micelles are
Gels can be characterized to be strong or weak based on their
stronger than the associations between the casein micelles, an
response to deformation. At large deformations, strong gels will
increase in shear will first disrupt the casein micelle network that
rupture and fail, while weak gels flow without fracture, and show
is not associated with EPS, subsequentely the associations between
recovery of solid character. Xanthan gum, the EPS produced by
the cells and EPS, and then the portion of the casein network that
Xanthomonas campestris, forms a weak gel, with large deformation
is associated with EPS.
fluid properties, but it also forms strong gel under extreme
conditions. Applications in Foods and Health Aspects
Rheological behaviors of EPSs in Fermented Milk Antimicrobial Compounds as Natural Food Preservatives
The use of EPS-producing LAB strains may improve the The quality of most foods deteriorates during storage. In
rheological properties of fermented milk. The gel structure and addition to physical, chemical and enzymatic factors which may
viscosity of the products are affected by the gel formation alter the sensory characteristics, the microbiological changes in
conditions, as well as the amount and the type of the EPSs foods may bring about a wide range of spoilage reactions, including
produced. Hammelehle et al. (1998) showed that fast warming food poisoning. Therefore, it is of significance to inhibit the growth
rates (20-50 °C) during acidification increased the gel firmness of spoilage microorganisms in foods. Due to a strong demand for
and storage modulus, and decreased the syneresis of a milk gel. natural and minimally processed foods, there has been a growing
Skim milk fermented by ropy EPS-producing strains exhibited interest in the use of antimicrobial compounds produced by LAB
similar rheological properties, and had greater viscosity than skim as a safe and natural way of food preservation.
milk fermented by non-ropy strains. Ropy EPS-producing strains
In addition to nisin which has been widely used in foods,
also increased the viscosity of yoghurt when compared to yoghurt
another antimicrobial compound that has been proposed for use
made with non-ropy cultures, and improved the texture of quarg.
in food preservation is reuterin produced by Lb. reuteri. Addition
198 Introductory Food Microbiology Epidemiological Typing of Bacillus spp Isolated from Food 199

of reuterin to ground beef was found to inhibit the growth of E. used as blood plasma extenders and as the basic component of
coli. Surface treatment of herring with a mixture of Lb. reuteri and many chromatographic stationary phases.
glycerol significantly improved the shelf-life of the product. Lb. Xanthan gum is the second microbial EPS which was approved
reuteri has been commercially used in combination with for use in foods in 1969. Although it is produced by the plant-
Bifidobacterium infantis and Lb. acidophilus in sweet and fermented pathogen Xanthomonas campetris, Sutherland (1998) described
milk under the trade name BRA-mjölk. xanthan as the “benchmark” product with respect to its importance
Antimicrobial compounds can be applied to foods either as in both food and nonfood applications, which include dairy
purified chemical agents, or as viable cultures in the case of products, drinks, confectionary, dressing, bakery products, syrups
fermented products. Novel purified antimicrobial compounds and pet foods, as well as the oil, pharmaceutical, cosmetic, paper,
require data to substantiate their lack of toxicity in order to obtain paint and textile industries. The production of xanthan is relatively
approval for their use in foods. Traditional fermented products inexpensive because of the high conversion of substrate (glucose)
that naturally contain antimicrobial compounds have been to polymer (60-70%). According to Becker et al. (1998), xanthan in
consumed for centuries, and starter cultures with selected solutions exhibits a high viscosity at low concentrations and strong
antimicrobial properties may be used to replace those used in pseudoplasticity, and it is stable over a wide range of pHs,
traditional fermented foods. However, problems may arise with temperatures and ionic strengths.
respect to retaining the flavor and texture of the products. Another probiotic Lb. reuteri also produced an antimicrobial
EPSs in Food Applications compound with a wide spectrum of activities. Studies on bacterial
adhesion showed that capsular polysaccharide might promote the
Polysaccharides may function in foods as viscosifying agents,
adherence of bacteria to biological surfaces, thereby facilitating the
stabilizers, emulsifiers, gelling agents, or water-binding agents.
colonization of various ecological niches. The EPSs were found to
The majority of the polysaccharides used in foods are of plant
be present in adherent biofilms; the EPSs might function as initial
origin. Most of them are chemically or enzymatically modified in
adhesion, and permanent adhesion compounds. As well as live
order to improve their rheological properties, e.g. cellulose, starch,
bacteria (probiotics) which can improve intestinal balance to
pectin, alginate and carrageenan. Therefore, their use is strongly
promote health, dietary carbohydrates may function as prebiotics,
restricted. EPSs of microbial origin have unique rheological
beneficially affecting the colonic microflora. These dietary
properties because of their capability of forming very viscous
carbohydrates include polysaccharides of plant origin (resistant
solutions at low concentrations and their pseudoplastic nature.
starch, â-glucan, cellulose, inulin), oligosaccharides (fructo-, gluco-
The EPSs produced by foodgrade LAB have been considered as a
, malto-, xylo- and soybean oligosaccharides), and lactose
new generation of food thickeners to improve the rheological
derivatives. There have been no reports of the use of EPSs produced
properties of foods. Dextran is the first industrial polysaccharide
by LAB as prebiotics. Although milk fermented with an EPS-
produced by LAB. It was discovered in 1880 in sugar cane or beet
producing strain Lc. lactis ssp. cremoris SBT0495 had cholesterol
syrups where dextran was found to be responsible for the
lowering activity, the mechanism is unknown.
thickening and gelation of the syrups. Due to their structural
differences, some dextrans are water soluble and others are Oda et al. (1983) reported an antitumor EPS produced by Lb.
insoluble. Dextran can be used in confectionary to improve moisture helveticus ssp. jugurti. The antitumor activity of the EPS was tested
retention, viscosity and inhibit sugar crystallization. In gum and against ascites Sarcoma-180 by injecting the EPS preparation
jelly candies it acts as a gelling agents. In ice cream it acts as a intraperitoneally. Mice given a 20 mg kg-1 dose for nine succesive
crystallization inhibitor, and in pudding mixes it provides the days had an increased life span value of 144%, and a value of
desirable body and mouth feel. In addition, dextran has also been greater than 233% corresponding to a 40 or 80 mg kg-1 dose. The
200 Introductory Food Microbiology Epidemiological Typing of Bacillus spp Isolated from Food 201

authors concluded that the antitumor activity of the EPS might be twice before use. The LAB strains examined for producing
based on its host-mediated actions. In order to understand the antimicrobial compounds were grown in MRS, KCA, and whey
antitumor activity, the effect of the EPSs or the EPS-producing cells permeate or whey media, and incubated at 30 °C or 37 °C. Food
on the immune system has been investigated. Forsén et al. (1987) spoilage bacteria and also LAB strains were used as indicator
showed that cell surface materials, possibly lipoteichoic acids, of organisms for antimicrobial tests.
Lc. lactis ssp. cremoris T5 produced Tcell mitogenic activity in
Separation and Purification of Antimicrobial Compounds
human lymphocytes. The slime produced by Bifidobacterium
adolescentis had immunomodifying effects on mouse splennocytes. After the growth of the LAB strains under proper conditions,
Kitazawa et al. (1992) showed that the slime-forming Lc. lactis ssp. cells in the culture broth were filtered, and the cell-free broth was
cremoris KVS20 had antitumor activity, and the slime contained concentrated 10-fold by lyophilization. The concentrate was then
strong B-cell dependent mitogenic substances. precipitated stepwise by ethanol from 30 to 97.5% with intermediate
centrifugation (30 min, 22 000g, 4 °C). The precipitates obtained
AIMS OF THE STUDY from each addition of ethanol and/or the final supernatants
One of the aims was to study the antimicrobial compounds showing antimicrobial activity were further purified by
produced by dairy lactic acid bacteria, particularly the low- chromatographic methods.
molecular-mass compound inhibitory toward various spoilage and Gel filtration was performed using a Bio-Rad Econo System.
pathogenic bacteria in foods. Another aim was to study the The sample (100 mg) was loaded onto a column (75 x 1.5 cm) on
extracellular polysaccharides produced by dairy lactic acid bacteria Bio-Gel P-2 polyacrylamide gel (M=100-1800, -400 mesh, Bio-Rad)
in view of the role of the exopolysaccharides in the improvement eluted with 0.05 M ammonium acetate (NH4OAc) at a flow rate of
of the texture and consistency of fermented foods. The specific 10 ml h-1, and the eluant was monitored at 280 nm. The active
aims of the study were: fractions were collected, lyophilized, and subjected to anion
1. To separate, purify and identify low-molecular-mass exchange chromatography using a Bio-Rad Econo system with a
column (25 x 1.5 cm) on weakly basic Fractogel TSK DEAE-650(S)
antimicrobial compounds produced by the lactic acid
gel. Elution was carried out at a flow rate of 1.0 ml min-1 using
bacterial strains, and to study the antimicrobial properties
a stepwise elution program: fractions 1-30 with water; fractions
of these compounds.
31-65 with 0.04 M NH4OAc adjusted to pH 5.5 with acetic acid
2. To isolate exopolysaccharides produced by the lactic acid (AcOH); fractions 66-90 with 0.5 M NH4OAc adjusted to pH 5.5
bacterial strains, to evaluate the primary molecular with AcOH.
structures of the exopolysaccharides, and to study the
rheological properties of the viscous exopolysaccharides. A fraction was collected every four minutes with monitoring
at 254 nm. The active fractions (except fractions containing lactic
MATERIALS AND METHODS acid) from anion exchange chromatography were further purified
by RP-HPLC using a model 600 E multisolvent delivery system
Antimicrobial Compounds Produced by LAB (I, II)
equipped with a Baseline 810 software. The mobile phase, 0.02 M
Bacterial Strains and Growth Conditions NH4OAc containing 1% AcOH (pH 3.80), was used after filtration
All bacterial strains used in the study of antimicrobial through a membrane filter (pore size, 0.2 ìm). Elution was performed
compounds were obtained from Valio Ltd, Research and isocratically from a Spherisorb S5 C8 column (250 x 4.6 mm, Phase
Development Service, Helsinki, Finland. The bacterial cultures Separations Ltd, Chester, England), fitted with a C8 precolumn
were maintained at -80 °C in glass beads and they were subcultured (Millipore) at a flow rate of 0.75 ml min-1, and at 40 °C for 30 min.
202 Introductory Food Microbiology Epidemiological Typing of Bacillus spp Isolated from Food 203

A fraction was collected each minute. The absorbance was the disc was measured. The spot test was done by spotting the
monitored at a range of wavelengths from 190 to 300 nm at an liquid sample (3 ìl) directly onto the surface of the solidified,
interval of 5 or 10 nm. seeded agar medium, and the diameter of the inhibition zone was
measured after incubation. Turbidometric assays were performed
Identification of Antimicrobial Compounds
using a Bioscreen C automated turbidometer equipped with a
1H and 13C NMR measurements were carried out on a Bruker Biolink software. The growth of indicator organisms in broth
AM 400 WB spectrometer (Karlsruhe, Germany), operating at 400.1 (300 ìl) containing antimicrobial compounds was studied in plates
MHz for 1H. Spectra were recorded with sample solutions in (100 wells). Each well was inoculated with 100 ìl broth culture
H2O/D2O (90/10) at ambient temperature and referenced to (grown overnight) of the test organism diluted to 106 to 107 CFU
sodium 3-trimethylsilyl- [2,2,3,3-2H4]propanoate. ml-1. The optical density was measured automatically at 30 min-
The electron impact (EI) and fast atom bombardment (FAB) interval, using a wideband filter (405-600 nm), and the plates were
mass spectra were recorded on a Jeol SX-102 double-focusing shaken at 3 min-interval for 20 s. The growth curves were
spectrometer. determined from the turbidity data.
EI: The sample was injected into the direct probe and the EPSs Produced by LAB (III, IV, V)
solvent (water) evaporated. The probe was inserted into the ion
source (250 °C). The filament was heated at a rate of 16 °C/min Bacterial Strains and Growth Conditions
up to 300 °C/min, the ionization current being 300 mA. The The LAB strains examined for producing EPSs and their
ionization energy was 70 eV and the accelerating voltage 10 kV. growth conditions are shown in Table 4. The source and methods
The spectra were recorded over the range 10-500 m/z. Calibration of maintainance of these strains were the same as described above
was based on PFK (perfluorokerosin, positive ion mode). for the LAB strains examined for producing antimicrobial
FAB: The sample was introduced on the target plate directly compounds in this study.
into the ion source (40 °C) in a glycerol matrix. The target was
Isolation of EPSs
bombarded with xenon atoms having a maximum of 6 kV energy.
The acceleration voltage of generated ions was 10 kV. The spectra For the isolation of the EPS produced by Lb. helveticus Äki4
were recorded at a scan range of 0-800 m/z. Calibration was based grown in MRS broth, bacterial cells were filtered from the medium,
on solid CsI (cesium iodide, positive ion mode). and the cell-free supernatant was concentrated 10- fold by
lyophilization. The concentrate was fractionally precipitated with
Antimicrobial assay ethanol from 40 to 95% with intermediate centrifugation. The
The agar diffusion method was performed using a disc test polysaccharide precipitated at 40% ethanol was washed, and
and a spot test. The disc test was performed according to a dissolved in water. After filtration through a syringe filter (0.8 ì/
procedure developed by Pulusani et al. (1979) with some 0.2 ìl), it was freeze-dried. The crude polysaccharide (20 mg) was
modifications: 10 ml of the melted agar medium was seeded with purified by anion-exchange chromatography with a column (25 x
100 ìl of an 18 ± 2 h old broth culture of the test organism in a 1.5 cm) on Fractogel TSK DEAE-650(S) gel using a Bio-Rad Econo
sterile petri dish. When the soft agar had hardened, an antibiotic system. The column was eluted at about 60 ml h-1 first with water
test disc (diameter 6 mm, Schleicher & Schuell) was placed on the for 80 min, and subsequently with 0.06 M NH4OAc adjusted to
agar surface, and 22 ìl of the sample was spotted onto the disc. pH 5.5 with AcOH for 120 min. A fraction was collected every
After incubation for 20 ± 2 h at the appropriate temperature for eight minutes with monitoring at 254 nm, and the presence of
each organism tested, the diameter of the inhibition zone around sugar was tested with a Molish reagent.
204 Introductory Food Microbiology Epidemiological Typing of Bacillus spp Isolated from Food 205

For the isolation of the EPSs produced by other LAB strains with (+)-2-butanol. Methylation analysis was performed according
grown in milk or whey medium, proteins and cells were initially to Hakomori (1964) using sodium methylsulfinylmethanide and
precipitated by addition of 4% (w/v) TCA (Merck) to the culture, iodomethane in dimethyl sulfoxide. The methylated compounds
and the mixture was stirred for 2 h. After centrifugation (35 min, were recovered by use of Sep-Pak C18 cartridges using the method
22 000 g, 4 °C), the supernatant was collected and filtered. Cold of Waeghe et al. (1983). The purified methylated sample was then
ethanol was then gradually added to the cell-free supernatant hydrolyzed (2 M TFA, 120 °C, 2 h), reduced, and acetylated.
from one to two, and three volumes of the supernatant with The partially methylated alditol acetates were analyzed by
intermediate centrifugation. The EPS precipitated was washed GLC-MS. NMR spectra of solutions in D2O were recorded at 65 °C
and dissolved in water. The aqueous solutions of EPS were filtered, and pD 5.5, using a Jeol GSX- 270, Jeol Alpha-400, or Varian Inova
and then extensively dialyzed against water overnight at 4 °C 600 or 800 MHz instrument. Chemical shifts are reported in ppm
with two changes of water, and finally lyophilized. relative to sodium 3-trimethyl-(2,2,3,3-2H4)propanoate (äH 0.00)
The purity of the EPS material was examined by gel filtration or acetone (äc 31.00) as internal references, or dioxan as an external
using a column (75 x 1.5 cm) of Bio-Gel P-30 polyacrylamide gel reference. Data processing was performed using standard Jeol
(exclusion limit 40 000 daltons, 100-200 mesh). The sample (1 mg) software, VNMR software, or Felix 2.3 (Biosym/MSI, San Diego,
was loaded onto the column and eluted with 0.05 M NH4OAc CA, USA). 1H,1H-COSY, relayed COSY, double-relayed COSY,
with UV monitoring at 280 nm. To check the ionic nature of the TOCSY, 13C,1H-COSY, gHSQC (Wilker et al. 1993) and HMBC
EPSs, anionexchange chromatography of the EPS solutions (Bax and Summers 1986) experiments were used to assign signals
(~1 mg mL-1) was performed using a column (25 x 1.5 cm) of and performed according to standard pulse sequences. For inter-
weakly basic Fractogel TSK DEAE-650(S) gel. Elution was carried residue correlations, 2D NOESY experiments with mixing times of
out at 1.1 mL min-1; first with water for 2 h, and subsequently 300 and 400 ms (III), or 75 and 150 ms (IV), and HMBC experiments
with NH4OAc from 0.1 to 0.5 M using a linear increasing gradient. with 60 and 90 ms (III), or 45 and 90 ms (IV) delays for the
evolution of long-range couplings were used.
Structural Elucidation of EPSs
GC analysis of alditol acetates was performed on a HP-5 Rheological Measurements of the EPSs Produced by Lc. lactis ssp.
fused silica column (0.20 mm x 25 m) using a temperature program Cremoris Strains
of 180 °C for 1 min followed by 3 °C min-1 to 250 °C. Hydrogen The viscosities of the dilute solutions of EPS at concentrations
was used as the carrier gas. The column was fitted to a Hewlett- of 0.01 up to 0.1 g dL-1 were measured at 25 °C with an Ubbelohde
Packard model 5890 series II gas chromatograph equipped with capillary viscometer (536 13/Ic, SCHOTT35 GERÄTE, Hofheim,
a flame ionization detector. GLC-MS analysis was performed on Germany), which allows the determination of the flow times with
a Hewlett-Packard model 5970 mass spectrometer equipped with an accuracy of 0.03 s. The aqueous solutions of EPS with or
an HP-5MS fused silica column (0.2 mm x 25 m). A temperature without the addition of salt were prepared by dissolving a
program of 170 °C for 3 min followed by 3 °C min-1 to 250 °C was measured amount of EPS in 0.1 M sodium chloride (NaCl) solution
used with helium as the carrier gas. or in deionized water. Sample dilution to the various required
In sugar analysis, the EPS samples were hydrolyzed with 2 M concentrations of EPS was done directly in the viscometer. After
TFA at 120 °C for 2 h. After reduction with sodium borohydride about 5 min for temperature equilibration, flow times were taken,
(NaBH4) and acetylation, the samples were analyzed by GC. The and each flow time was reproduced six times. The reduced viscosity
absolute configuration of the sugars present in the EPSs was and intrinsic viscosity of the EPS solutions were calculated from
determined essentially as devised by Leontein et al. (1978) but the collected data.
206 Introductory Food Microbiology Epidemiological Typing of Bacillus spp Isolated from Food 207

The effect of temperature, pH and salts on the rheological as pyroglutamic acid. Among the twenty LAB strains examined,
behavior of the EPS solutions (1%, w /v) was studied with a thirteen Lactobacillus and five Pediococcus strains were found to
Bohlin VOR rheometer (Bohlin Instruments Ltd, England) using produce PCA under the growth conditions used in this study.
concentric cylinders (C14) with a gap of 0.5 mm between the Four Lactobacillus strains produced, in addition to PCA, also HMM
upper and lower geometries. The viscometry measurements were antimicrobial compounds, as indicated by the presence of
performed at 5, 25, 40 or 60 °C, with increasing shear rates up to antimicrobial activity in the precipitates obtained from ethanol
291 s-1 in 29 steps. The pH of the EPS solutions was adjusted with precipitation. Since these HMM compounds exhibited a rather
lactic acid to 4.0, 5.0 and 6.5. The EPS solutions containing salts narrow range of activity they were not subjected to further studies.
were prepared by addition of EPS to 0.1 M NaCl or 0.1 M calcium On the basis of the results of this study, it seems that many LAB
chloride (CaCl2) solutions. The oscillation measurements were strains, particularly Lactobacillus strains, are able to produce PCA.
performed for the EPS (1%, w/v) in aqueous and salt solutions, Since LAB produce relatively large amounts of lactic acid,
and in skim milk at a frequency sweep from 0.01 to 15 Hz in 19 being antimicrobially active, it is important to remove the lactic
steps. For every temperature-dependent measurement a thermal acid in order to find other antimicrobial compounds, especially
equilibration time of about 60 min was used. those of low molecular mass. In the separation and purification
procedures developed in this study, both PCA and lactic acid
RESULTS AND DISCUSSION
were present in the culture supernatant obtained from ethanol
Antimicrobial Compounds Produced by LAB (I, II, unpublished precipitation, and they also appeared in almost the same range of
results) fractions after gel filtration. However, complete separation of lactic
Separation, Purification and Identification of Antimicrobial acid was achieved by anion exchange chromatography based on
Compounds a gel matrix (Fractogel TSK) suitable for separation of biomolecules.
Previously, size exclusion HPLC was used to separate lactic acid
After ethanol precipitation of the cell-free cultures of the LAB
from LMM antimicrobials, but all the fractions obtained were
strains, the obtained precipitates and the final supernatants were
found to contain antimicrobial activity because the mobile phase
tested for antimicrobial activity. The supernatants containing
(sodium phosphate) used was antimicrobially active.
antimicrobial activity were subjected to chromatographic separation
and purification. The active fractions appeared in a relatively Niku-Paavola et al. (1999) reported the separation of lactic
narrow part of the chromatogram on gel-filtration on Bio-Gel P-2 acid by gel filtration on Sephadex G-10 with water as an eluent
of the supernatants. Anion exchange chromatography of these and found several LMM antimicrobial compounds produced by
active fractions resulted in two different ranges of active fractions Lb. plantarum. Although there were some reports on the separation
(52-54 and 58- 63), fractions 58-63 containing lactic acid. Further and purification of LMM antimicrobial compounds produced by
purification by RP-HPLC of the fractions 52- 54 gave rise to one LAB, the techniques for seperation of lactic acid appeared not to
major peak at retention time 4.96 min and two small peaks at 3.65 be clearly demonstrated. In addition, there were reports on the use
and 5.78 min. The absorption maximum of the antimicrobial of a neutralization technique to eliminate the antimicrobial effect
compound was at 215 nm. Fractions of these peaks were collected of lactic acid, but lactic acid could not be separated with this
and tested for antimicrobial activity. Only the fraction of the peak technique, and the activity of acidic antimicrobials might be
at 4.96 min was found to contain antimicrobial activivity. suppressed due to neutralization. PCA is a natural constituent of
Identification of this active fraction by both NMR (1H and 13C) foods of plant origin, including vegetables and fruits, and
and MS (EI and FAB) spectra indicated that the antimicrobial fermented soybean and cereal products. Among LAB strains, only
compound was 2-pyrrolidone-5-carboxylic acid (PCA), also known S. bovis has previously been shown to produce PCA by conversion
208 Introductory Food Microbiology Epidemiological Typing of Bacillus spp Isolated from Food 209

of glutamine. PCA can also be synthesized by heating glutamic that PCA in broths was inhibitory against the test organisms at
acid using a dehydration process. It has been reported that PCA pH 5.0-5.89. At pH values when 1% PCA showed no activity, 2%
is able to increase cerebral blood flow and decrease the resistance PCA was still active. Earlier studies showed that neutralization
of brain vessels, which result in enhanced brain metabolism, i.e. caused the loss of the antimicrobial activity of Lb. acidophilus, since
increased glucose uptake and utilization by cerebral tissues and many inhibitory substances produced by lactic cultures were
decreased brain lactate dehydrogenase activity. Other biological relative stable in an acidic pH. In addition, the antimicrobial
functions of PCA are related to its presence as an amino-terminal activity of PCA, like other organic acids, may also be dependent
residue in many biologically significant peptides and proteins, on the undissociated form of the acid.
e.g., eisenine, LH-RH (luteinizing hormone releasing hormone), PCA was found to be less inhibitory than lactic acid. At the
and TRH (thyrotropin releasing hormone). same concentrations, lactic acid was more active than PCA against
The Antimicrobial Activity of 2-pyrrolidone-5-carboxylic Acid several indicator organisms tested. During the course of this study,
Produced by LAB we also observed that the production of PCA varied with strains
and it was generally small when compared with the amount of
Although LAB are able to produce a variety of antimicrobial
lactic acid produced. However, the significance of PCA is that, in
compounds, the present study, for the first time, reports that the
addition to its wide spectrum of antimicrobial activity, it is also
production of a certain cyclic amino acid (PCA) is involved in the
involved in many important biological functions as discussed
antimicrobial action of LAB. PCA has been considered, following
above. Among the PCA-producing LAB strains of this study, Lb.
the identification of reuterin, to be another well identified LMM
rhmnosus GG was found to produce relative large amounts of PCA.
antimicrobial compound produced by LAB.
Lb. rhamnosus GG has also been reported to produce a microcin-
The antimicrobial assay of PCA by agar diffusion method like LMM antimicrobial compound, and the strain has been
showed that PCA at 2% inhibited Bacillus subtilis, E. coli, Enterobacter, commercially applied in the production of probiotic milk products,
Klebsiella, and Pseudomonas strains. The most sensitive strains e.g. Gefilus in Finland. Considering the potential applications of
Enterobacter cloacae 1575, Pseudomonas fluorescens KJL G, and PCA, e.g. as PCA-producing starters in food preservation, further
Pseudomonas putida 1560-2 were inhibited by PCA at 0.5% and studies are needed on the optimal production of PCA by LAB
1.0%. Among all bacterial strains tested, the Gram-positive strains strains, the antimicrobial effects of PCA in foods and the effective
such as LAB strains, and several Listeria and Staphylococcus strains PCA concentrations, as well as the sensory quality of foods when
were not inhibited by PCA. An Enterococcus faecalis strain was PCA is used.
moderately inhibited by 0.5% PCA in BHI broth at 37 °C. It seemed
that Gramnegative bacteria were more sensitive to PCA than the EPSs Produced by LAB
Gram-positive ones. This is in agreement with previous studies Of the thirteen LAB strains examined, except for Lb. fermentum
which showed that organic acids (lactic and acetic acids) were G.1.2.1, Lb. rhamnosus LC705 and Lc. lactis ssp. cremoris SEPH 11
more inhibitory toward Gram-negative bacteria than Gram-positive which did not produce EPSs, ten strains produced neutral or
ones. PCA was heat stable and the antimicrobial activity did not anionic EPSs of varying amounts when they were grown under
change after heat treatments (63 °C 30 min, 72 °C 15 s or 121 °C the conditions of this study.
20 min). Although raising the pH of the PCA in water solutions
Previous studies have shown that the production of EPS is
reduced the antimicrobial activity, and completely destroyed the
growth-associated, and it is influenced by medium compositions,
activity at pH 3.8-4.0, the antimicrobial activity of PCA seemed to
culture temperature and pH. Gamar et al. (1997) showed that
be not solely due to the pH effect, but other factors may be possibly
variations in carbon sources in the growth medium resulted in
involved. The turbidometric assay of antimicrobial activity showed
210 Introductory Food Microbiology Epidemiological Typing of Bacillus spp Isolated from Food 211

different yields of the EPS produced by Lb. rhamnosus strain C83. The EPS produced by strain Lb161 was shown to be composed
Addition of glucose or sucrose to the milk medium stimulated the of a heptasaccharide repeating unit containing two terminal â-D-
EPS production and modified the sugar composition of the EPS glucose, one 2,3-substituted á-D-glucose, one 3- substituted á-D-
produced by Lb. casei ssp. casei NCIB 4114. galactose, one 4-substituted á-D-glucose, one 3,4-substituted â-D-
Several authors have also reported that more EPSs could be galactose and one 3-substituted â-D-glucose residue. The
produced by different LAB strains at lower temperatures, or with assignments of the spin systems for the seven sugar residues were
limited nitrogen sources. Although bacteria grow well, and more performed using 2D homo- and heteronuclear techniques. Each
cells may be obtained under optimized growth conditions, spin system with a specific sugar residue and substitution pattern
unfavourable culture conditions may stimulate EPS production by was identified on the basis of the JH,H values and the 1H and 13C
the cells as a form of bacterial self-protection. Therefore, these NMR chemical shifts. The sequence of the repeating unit was
factors discussed above need to be considered for optimizing EPS established using 2D NOESY and HMBC experiments with the
following structure:
production by the LAB strains in this study.
Structural Variations Among the EPSs Produced by Lb. Helveticus
EPSs Produced by Lb. Helveticus Strains
Strains
Lb. helveticus Äki4 grown in MRS broth, and strains Lb161
As shown above, the repeating units of the EPSs of Lb helveticus
and K16 grown in skim milk were found to produce EPSs.
Äki4 and Lb161 are a hexamer and a heptamer made up of a main
Viscometric measurements by Ubbelohdetype capillary viscometer
chain branched by one and two side chains, respectively. Structural
showed that at 0.5% (w/v) EPS in aqueous solutions, the EPSs of
studies on the EPS of strain K16 showed that the repeating unit
strains Lb161 and K16 were viscous, but the EPS of strain Äki was
of the EPS was composed of six sugars: one terminal D-galactose,
not viscous. The primary molecular structures of the EPSs produced
one terminal D-glucose, one 4- substituted D-galactose, one 4-
by strains Äki4 and Lb161 have been studied by sugar and
substituted D-glucose, one 2,4-substituted D-glucose and one 4,6-
methylation analyses, and 1D and 2D NMR spectroscopy.
substituted D-glucose.
Structural Elucidation of the EPSs Produced by Lb. Helveticus Aki4 Comparing with the so far published structures of the EPSs of
(III) and Lb161 (IV) other four Lb. helveticus strains, the common structural feature of
The EPS produced by strain Äki4 was shown to be composed the EPSs produced by all these Lb. helveticus strains is that the
of a hexasaccharide repeating unit containing one terminal â-D- EPSs contain either a hexa- or heptasaccharide repeating unit of
galactose, one 4- substituted á-D-galactose, one 6-substituted â-D- D-galactose and D-glucose. The EPS produced by Lb. helveticus
galactose, one 4-substituted â-D-glucose, one 3,6-substituted â-D- strain TY1-2 contains N-acetyl-D-glucosamine in addition to D-
galactose and one 6-substituted â-D-glucose residue. The galactose and D-glucose. Rhamnose and other monosaccharides
assignment of the spin systems for the six sugar residues was which have been found in the EPSs produced by many LAB
performed using 2D homo- and heteronuclear techniques. Each strains are not present in the EPSs of the Lb. helveticus strains.
spin system with a specific sugar residue and substitution pattern Since the rheological properties of polysaccharides are closely
was identified from the JH,H values, indicating the anomeric related to their primary molecular structures and threedimensional
configuration, and from the 1H and 13C NMR chemical shifts. structures, it would be of interest to study the relation between
The sequence of the sugar residues was determined using 2D structures and functional properties of the EPSs produced by LAB
NOESY and HMBC experiments. The structure of the repeating strains within the same species. The difference in the viscosities
unit of the EPS is as follows: of the EPSs of the Lb. helveticus strains of this study is probably
due to their different primary molecular structures (linkage and
212 Introductory Food Microbiology Epidemiological Typing of Bacillus spp Isolated from Food 213

degree of branching), molecular masses, three-dimensional lowered in the order of pH 6.5 > pH 5.0 > pH 4.0. The higher
structures and chain-chain interactions of the polysaccharides in viscosity of EPS in 0.1 M CaCl2 solutions than that in 0.1 M NaCl
solutions. solutions is due to different interactions between cations and
polyelectrolyte chains, Ca2+ being more effective than Na+ in this
EPSs Produced by Lc. Lactis ssp. Cremoris Strains and their
respect. Changing Na+ into Ca2+ ion was observed also to increase
Rheological Characterization (V)
the storage modulus of the sample. Samain et al. (1997) compared
The growth of Lc. lactis ssp. cremoris strains (ARH53, ARH74, the effect of salts on the viscosity of a gel-forming EPS produced
ARH84, ARH87, B40) in skim milk at 25 °C for 18-20 h resulted by Alteromonas sp. strain 1644 and showed that the divalent
in very viscous cultures as compared to the Lb. helveticus strains magnesium cation was more effective than the monovalent sodium
of this study. The EPSs (164-263 mg L-1) produced by these ‘viili’ cation.
strains were separated and purified from the cultures by treatment
The aqueous solutions of the EPS (1%, w/v) of strain ARH53
with 4% TCA and ethanol precipitation. The EPSs were shown to
behave viscoelastically. As shown in the oscillation measurements
be anionic in nature by anion exchange chromatography, and
at 5, 25 and 40 °C, the EPS solutions behaved as a viscoelastic
they contained the same monosaccharides rhamnose, glucose and
fluid at lower frequencies with G” > G’, and showed a predominant
galactose in similar molar ratios. The combined evidence from
elastic character (G’ > G”) at higher frequencies. The drastic decrease
sugar analysis and NMR spectroscopy indicates that the primary
in viscosity of the EPS aqueous solution with increasing shear rate
molecular structures of these EPSs are identical or closely related
also clearly shows the non-Newtonian behavior (shear-thinning)
to that from Lc. lactic ssp. cremoris SBT 0495. The underestimation
of the solution.
of the ratio of galactose resulted from incomplete release by acid
hydrolysis. The galactose residues were involved in the In skim milk to which the EPS of strain ARH53 was added,
phophodiester linkage in the polysaccharide and it was hard to the viscosity was much higher as compared with the EPS in
release them. aqueous solutions at 5, 25 and 40 °C. At shear rates lower than
9.21 s-1, an upward shift in viscosity was observed at 40 °C. The
In dilute aqueous solutions (up to 0.1 g dL-1), the EPSs
viscosity was higher at 40 °C than at 25 °C, and even higher than
produced by Lc. lactis ssp. cremoris strains exhibited a polyelectrolyte
at 5 °C at shear rates lower than 0.37 s-1. In the oscillation
effect. At very low concentrations ( 0.02 g dL-1), an increase in
measurements, at 5 °C there is a moderate frequency dependence
reduced viscosity with a decrease in concentration was observed.
of G’ with a crossover of G’ and G” at the lower end of the
In the presence of 0.1 M NaCl, the reduced viscosities of all the
frequency range, suggesting the formation of a weak gel. At 25 °C
EPS solutions were significantly lowered, and the polyelectrolyte
the system exhibited a viscoelastic behavior with a crossover of G’
effect disappeared as indicated by the straight lines of the Huggins
and G” at about 0.8 Hz. Increasing temperature to 40 °C led to the
plot. The intrinsic viscosities of the EPSs were therefore obtained
formation of a strong gel, as indicated by G’ » G”, and the parallel
by a linear extrapolation of the plots to zero EPS concentration.
curves of G’ and G” with only a slight frequency dependence, and
Since the EPS produced by strain ARH53 gave the highest intrinsic
the value of G’ being 198 Pascals at 15 Hz. The interactions
viscosity (19.62 dL g-1), this EPS might possess a relatively large
between EPS and casein micelles of skim milk were possibly
molecular size than the other Lc. lactis ssp. cremoris strains studied.
involved in the gelation. Hess et al. (1997) proposed that in yoghurt
At a higher concentration (1%, w/v), the viscosities of the aqueous
the interactions between EPS and casein micelles are stronger
solutions of EPS produced by strain ARH53 were temperature, pH
than those between the casein micelles. Intercalation of the EPSs
and salt dependent. At the same pH (4.0, 5.0 or 6.5), increasing
into the casein matrix was also involved in the improvement of the
temperature from 5 °C to 60 °C caused a decrease in viscosity. At
texture of quarg using ropy cultures. It seemed that the interactions
higher temperatures (40 °C and 60 °C), the viscosity was clearly
214 Introductory Food Microbiology Epidemiological Typing of Bacillus spp Isolated from Food 215

between the EPS and casein micelles increased at higher chromatographic methods. The technique of anion exchange
temperatures as observed in this study. chromatography developed in this study was essential for effective
The production of EPSs by Lc. lactic ssp. cremoris strains has separation of PCA from lactic acid, facilitating the identification
been reported earlier. However, the rheological properties of these of PCA by NMR and mass spectrometry. The chromatographic
EPSs were poorly understood. Recently, Oba et al. (1999) reported procedure based on this technique can be used for separation and
the viscoelastic properties of the EPS produced by strain SBT 0495. purification of other LMM antimicrobial compounds. To our
It is of interest that the rheological behavior of this EPS in aqueous knowledge, this is the first report of a cyclic amino acid, PCA,
solutions, e.g. polyelectrolyte effects and viscoelasticity, is similar which is involved in the antimicrobial action of LAB. PCA can be
to that of the EPS of strain ARH53. This is propably due to their considered as a well identified LMM antimicrobial compound
similar or identical structures as shown in this study. The similarity following the identification of reuterin produced by Lb.reuteri.
in rheological behavior was also noticed among the EPSs produced PCA was shown to be inhibitory toward many food-borne
by the five ‘viili’ strains of this study. The difference in viscosity spoilage bacteria such as Bacillus subtilis, Enterobacter, E. coli,
of the EPSs in dilute, as well as in concentrated solutions was Klebsiella and Pseudomonas. In antimicrobial tests against different
possibly due to their different molecular masses. organisms, the Gram-negative spoilage bacteria were found to be
It has previously been known that slime production is an more sensitive to PCA than the Gram-positive ones. Heat treatment
important factor contributing to the special slimy characteristic of of PCA did not change its antimicrobial activity. Although PCA
Finnish fermented milk ‘viili’. The increase in viscosity and gelling was active under acidic conditions, it appeared that the activity
at 40 °C resulted from the addition of the EPS produced by Lc. was not solely due to the pH effect. Regarding the mechanism of
lactis ssp. cremoris demonstrated in this study is of interest with antimicrobial action of PCA, more study is needed on the mode of
respect to the possible use of the EPS to improve the rheological action of PCA on sensitive bacterial cells and the MIC value, as
properties of milk products, for instance, yoghurts with well as the biosynthesis of PCA in different species of LAB. In
fermentation at about 42 °C. Considering the potential applications addition, further investigation on a wide range of LAB strains is
of the EPSs, future work on the optimization of the EPS production needed in order to find out whether production of PCA is a
and further physical characterization is required. common characteristic of LAB.
During the course of the studies on the antimicrobial
SUMMARY AND CONCLUSIONS compounds, we gradually precipitated HMM antimicrobial
Lactic acid bacteria are able to produce a large variety of compounds from culture supernatants by ethanol, while at the
compounds which give fermented foods their characteristic flavor same time following how polysaccharides were precipitated. The
and color, and also impart improved safety and rheology to the first EPS, which was precipitated at 40-60% ethanol, was found
foods. In this study, the production of antimicrobial compounds to be produced by Lb. helveticus Äki4 grown in MRS broth. NMR
and extracellular polysaccharides by LAB strains obtained from spectroscopic studies of the EPS showed that it was composed of
the dairy industry has been investigated in order to find the LAB a hexasaccharide repeating unit of D-galactose and D-glucose in
strains with potential applications in foods. a molar ratio of 2:1, and the main chain was branched with a side
chain of D-galactose. Although there were relatively large amounts
Studies on the twenty dairy LAB strains from Lactobacillus,
of the EPS (~500 mg L-1) produced by Lb. helveticus Äki4, this EPS
Lactococcus, Pediococcus and Streptococcus showed that eighteen
was found to be not viscous.
strains produced a LMM antimicrobial compound, 2-pyrrolidone-
5-carboxylic acid (PCA). Separation and purification of PCA from In the screening study of LAB strains producing viscous EPSs,
the growth media were achieved by ethanol precipitation and another Lb. helveticus strain, strain Lb161 grown in skim milk was
216 Introductory Food Microbiology Modelling the Growth Boundary of Staphylococcus Aureus 217

found to produce a viscous EPS. The EPS was isolated by the


treatment of the growth medium with TCA to precipitate the
proteins first, and subsequently by ethanol precipitation to obtain
the polysaccharide. The primary molecular structure of the EPS
has been elucidated by NMR spectroscopy. The EPS was shown
to consist of a heptasaccharide repeating unit of D-galactose and
D-glucose in a molar ratio 2:5 with two side chains, each consisting 8
of a D-glucose residue.
Further studies on slime-forming Lc. lactis ssp. cremoris strains MODELLING THE GROWTH
showed that growth of the strains ARH53, ARH74, ARH84, ARH87
or B30 in skim milk resulted in very slimy cultures. By using BOUNDARY OF STAPHYLOCOCCUS
basically the same methods as for the EPS of Lb. helveticus Lb161, AUREUS
polysacharides of varying amounts were isolated from these
cultures. Sugar analysis and NMR spectroscopy showed that the
EPSs had a rather similar or probably identical structure to the Knowing the precise boundary for growth of Staphylococcus
one reported earlier. Rheological studies showed that the EPSs in aureus is critical for food safety risk assessment, especially in the
dilute aqueous solutions behaved as polyelectrolytes, and the EPS formulation of safe, shelf-stable foods with intermediate relative
of strain ARH53 gave the highest intrinsic viscosity. Further humidity (RH) values. To date, most studies and resulting models
characterization of the EPS of strain ARH53 showed that the have led to the presumption that S. aureus is osmotolerant.
viscosity of the EPS in concentrated solutions was dependent on However, most studies and resulting models have focused on
the temperature, pH and ionic strength of the solutions. In skim growth kinetics using NaCl as the humectant. In this study, glycerol
milk, addition of the EPS of strain ARH53 resulted in a clear was used to investigate the effects of a glassforming nonionic
increase in viscosity and a gel was formed at 40 °C. humectant to avoid speci? c metabolic aspects of membrane ion
In our future studies of EPSs, we have planned to continue the transport. The experiments were designed to produce a growth
study of the EPSs produced by LAB, as well as bifidobacterial boundary model as a tool for risk assessment.
strains of food origins, and strains from the human intestine, The statistical effects and interactions of RH (84 to 95%
aiming at applications in food, functional food or clinical products. adjusted by glycerol), initial pH (4.5 to 7.0 adjusted by HCl), and
Although the present study provides data on the structures and potassium sorbate (0, 500, or 1,000 ppm) or calcium propionate (0,
rheological properties of EPSs, further studies are needed in order 500, or 1,000 ppm) on the aerobic growth of a ? ve-strain S. aureus
to understand the structure-function relations of the EPSs, the cocktail in brain heart infusion broth were explored. Inoculated
interaction of polysaccharides and proteins (e.g. casein), and the broths were distributed into microtiter plates and incubated at
roles of EPSs in their adhesion interaction with EPS-producing 37°C over appropriate saturated salt slurries to maintain RH.
strains in the human intestine. Growth was monitored by turbidity during a 24-week period.
Toxin production was explored by enterotoxin assay. The 1,280
generated data points were analyzed by SAS LIFEREG procedures,
which showed all studied parameters significantly affected the
growth responses of S. aureus with interactions between RH and
pH. The resulting growth/no growth boundary is presented.
218 Introductory Food Microbiology Modelling the Growth Boundary of Staphylococcus Aureus 219

INTRODUCTION Predictive models are typically broadly applied for the


Staphylococcus aureus is one of the leading causes of foodborne screening of products and/or processes before challenge or shelflife
illness and is ranked as one of the most prevalent causes of studies. However, it must be remembered that models are only
gastroenteritis worldwide, even though the occurrences of this tools to be used to help design products with food safety concerns
foodborne disease are grossly underreported. S. aureus was addressed at the beginning of the development process and that
determined to be the etiological agent in 367 (19.6%) of 1,869 there must be validation of the system through challenge or shelf-
documented bacterial foodborne outbreaks in the United States. life studies with the real product.
Approximately 25 major outbreaks of Staphylococcus food poisoning Both the U.S. Department of Agriculture Pathogen Modeling
occur annually in the United States. S. aureus has been estimated Program (PMP) and the Food MicroModel (FMM) developed in the
by the Centers for Disease Control and Prevention to cause 185,060 United Kingdom through the Ministry of Agriculture, Fisheries
illnesses, 1,753 hospitalizations, and 2 deaths per year in the and Food include S. aureus growth kinetic models. The PMP models
United States, all of which are via consumption of contaminated the effects of temperature, initial pH, NaCl concentration, and
foods. sodium nitrite concentration in a broth system on aerobic and
Because of its prevalence as a food poisoning organism, S. anaerobic growth kinetics of the organism.
aureus has been extensively studied to determine the physical and The FMM models the growth responses as affected by NaCl
chemical parameters that affect its growth and toxin formation. S. concentration, pH, and storage temperature in a model broth
aureus is ubiquitous to the mucous membranes and skin of warm- system. The PMP also includes a model for the survival of S. aureus
blooded animals. It is a poor competitor with other bacteria and in nongrowth conditions, which was developed from a model
is easily destroyed by cooking temperatures; however, its toxins broth system that includes the effects and interactions of pH
can survive heat processing equivalent to that given to low-acid controlled by lactate buffer, lactic acid concentration, NaCl
canned foods. Staphylococcal food poisoning frequently occurs concentration, sodium nitrite concentration, and varying
when food is contaminated after cooking by a person carrying the temperatures.
organism, and subsequently the food is temperature abused for For all of the above-mentionedmodels, NaCl was the humectant
several hours, which allows for toxin production before chosen to control the RH in the system, and the resulting models
consumption. It is the ingestion of the toxin that causes the are kinetic in nature and, therefore, do not allow the boundary for
foodborne disease. growth/no growth to be estimated. In contrast to kinetic modeling,
S. aureus is highly salt tolerant and has been reported to grow probability modeling focuses on determining if the microorganism
at relative humidities (RHs) as low as 85% in NaCl concentrations of concern will or will not grow, in other words, determining the
up to 25% (wt/wt) (11). The RHs limiting growth are typically growth/no growth interface.
higher when humectants other than NaCl are used to control RH This becomes increasingly important when pathogens are of
(11). Notermans and Heuvelman (19) reported that at a water concern, because the rate of growth may be less important than the
activity of 0.98 to 0.93, sucrose was more favorable for growth fact that the organism is present and may be able to grow to an
than NaCl, but at a water activity of 0.87, NaCl was more favorable. infectious dose or produce toxins.
Marshall et al. reported that glycerol inhibition of the growth rate
of S. aureus was about 10% greater than that caused by NaCl at In this study, the statistical effects and interactions of
water activity levels between 0.96 and 0.90. Empirical RH controlled by glycerol, initial pH, and potassium sorbate or
microbiological models can be designed to predict how calcium propionate on the boundary for S. aureus growth were
microorganisms relate to the environment. modeled
220 Introductory Food Microbiology Modelling the Growth Boundary of Staphylococcus Aureus 221

MATERIALS AND METHODS cocktail was kept on ice until used to inoculate the various media
Organisms and Media Preparation used in each experiment. A 1-ml sample of the working cocktail
was taken, serially diluted, spread plated onto Baird-Parker agar
The . ve S. aureus strains used to create the bacterial cocktail
plates, incubated at 37°C for 48 h, and counted to determine initial
used in this study were S. aureus ATCC 13565 (American Type
concentrations of cells.
Culture Collection, Rockville, Md.), which produces staphylococcal
enterotoxin A (SEA), ATCC 14458 (SEB), and ATCC 27664 (SEE); Ten milliliters of each of the 80 broths as described above was
D-2 (SED) obtained from Toxin Technology, Inc. (Sarasota, Fla.); aseptically transferred to sterile tubes, and 100 ml of the S. aureus
and A-100 (SEA) obtained from U.S. Army Natick Labs. All strains cocktail was added to the broth to achieve an initial concentration
showed typical growth on Baird-Parker agar plates and were of approximately 103 CFU/ml. The inoculated broth (400 ml) was
coagulase positive. Their identi. cation as S. aureus was con- . rmed aseptically transferred into sterile, 100-well microtiter plates in
by use of a Riboprinter. Stock cultures were grown overnight at eight replicate wells per medium. The outer wells of the microtiter
37°C in brain heart infusion broth, suspended in a 2:1 broth- plates were . lled with uninoculated medium to act as a partial
glycerol solution, and stored at -80°C until needed. The BHI broth moisture loss barrier and as uninoculated controls. Each plate
was reconstituted with the appropriate ratio of water to glycerol contained media at one RH level only. A piece of sterile Thermaseal
to achieve the desired RHs for the study (84, 88, 92, or 95% RH). . lm was placed on top of the open wells, and the lid was also in
Appropriate amounts of 10% stock solutions of potassium sorbate place during incubation. The plates were placed on a rack in a
or calcium propionate were added to achieve . nal concentrations plastic sealable container, which had the bottom . lled with
of 500 or 1,000 ppm. The pH was adjusted using 0.1 or 1.0 N HCl appropriate saturated salt slurries to maintain the environmental
(J. T. Baker, Inc., Phillipsburg, N.J.; pH 4.5, 5.0, 6.0, or 7.0). Final RH to ensure RH of the media in the plates remained as stable as
RH was determined using the Aqualab CX-2 water activity meter. possible. The saturated salt solutions used were as follows: ZnSO4
It was determined that the RH of the media did not change more · H2O (83% RH @ 37°C), KNO (89% RH @ 37°C), KPO4 (93% RH
than 60.003% when measured at ambient temperature versus 37°C. @ 37°C), and K2Cr2O7 (96% RH @ 37°C). The containers were
Each of the 80 broths was . lter sterilized and stored in screw- closed and placed into a 37°C incubator. The plates were
capped tubes at 4°C until needed. periodically removed from incubation, and the OD of the 640
individual wells was measured for up to 6 months using the
Bioscreen microtiter plate preparation, incubation, and wideband . lter on the Bioscreen C system (Labsystems Oy, Helsinki,
measurements. Finland). The experimentswere repeated on separate dates. A total
The . ve stock cultures were removed from -80°C storage, and of 1,280 wells were monitored.
one loopful was transferred into 9 ml of BHI broth, separately. The
Data Collection
inoculated broths were incubated for 18 h at 37°C. The . ve cultures
were individually adjusted to an optical density (OD) at 530 nm The S. aureus population was considered to have shown growth
of 0.750 to 0.780 (Perkin-Elmer 35 spectrophotometer) with 0.1% when the Bioscreen wideband OD (ODwb) measurement increased
sterile peptone to achieve a concentration of approximately 108 from an initial reading of 0.200 to 0.220 to 0.350 or higher. Time
CFU/ml. Two milliliters of each diluted culture was transferred to growth (TTG) was determined by calculating the geometric
into one sterile test tube and vortexed to create the S. aureus cocktail mean of the time of the last measurement that showed no growth
used in the studies. Three 1:10 dilutions with sterile 0.1% peptone (ODwb <0.350) and the . rst time point that showed growth (ODwb
water were used to make a . nal working cocktail with a >0.350). In instances of no growth, TTG was censored at the . nal
concentration of approximately 105 CFU/ ml. This working measurement time of 168 days.
222 Introductory Food Microbiology Modelling the Growth Boundary of Staphylococcus Aureus 223

OD measurements were made and transferred from the microtiter plates were removed for assay. The broth from each of
Bioscreen C ASCII . le to a DOS text editor and then imported into the eight replicate wells was removed via pipet and combined into
Statistica software for initial analysis. Data were then transferred two microcentrifuge tubes. The samples were spun down for 10
to a Microsoft Excel spreadsheet to determine TTG and then to min, and the supernatant from the two tubes was combined. The
SAS for modeling using LIFEREG. pH of the supernatant was adjusted with 1 N NaOH to a pH of
6.0 to 8.0. A half a milliliter of the sample was placed into the
Statistical Analysis appropriate well in a test strip, the strips were placed into the
The TTG data along with a censoring indicator and the RH, mini-VIDAS, and the assays were run. A positive and negative
pH, and potassium sorbate or calcium propionate levels were control were run with each set of test strips. The test is sensitive
input data for construction of two separate models: one based on to 1 ng of toxin per ml of sample.
the potassiumsorbate data set and one based on the calcium
propionate data set. These two data sets used a common block of RESULTS
data generated from the experimental conditions, which had no A threshold ODwb value of 0.350 was used to score wells
preservatives added. The SAS LIFEREG procedure was used to with S. aureus growth (ODwb $ 0.350) versus wells with no growth
develop predictive models of ln TTG as a function of the factors (ODwb, 0.350). This threshold value was determined in two ways.
RH, pH, and preservative level. By default, the procedure . ts a First, ODwb measurements were compared with plate counts, and
model to the log of the dependent variable. it was determined that an ODwb reading of 0.350 was
The resulting model can easily be transformed to a regular approximately the . rst ODwb value where an increase in turbidity
time scale. In growth modeling, there are often conditions where regularly correlated to an increase in plate counts, indicating the
no growth occurs; therefore, TTG is sometimes censored. Under approximate ODwb where the instrument was sensitive enough to
these conditions, ordinary least-squares regression is not applicable, accurately determine an increase in cell numbers by a change in
and special procedures are required. The LIFEREG procedure can turbidity. Second, the data were analyzed to determine TTG in
accommodate such censored data and uses maximum likelihood each well using threshold ODwb values for growth of 0.300, 0.350,
estimation methods to . nd regression coef. cients. When creating and 0.400, and models were created and compared. The models
models with the LIFEREG procedure, there is a need to have many created with the threshold value set at 0.300 did not make
replicates of conditions equally spaced out about the matrix, with biological sense. We suspect that this was because this ODwb
approximately 50% of the conditions allowing and 50% not corresponded to a level that is lower than the sensitivity of the
allowing growth for best-. tting purposes. Therefore, the ranges of instrument to measure an increase in cell numbers. When the
conditions studied (RH, pH, and preservative levels) were chosen threshold ODwb value was raised to 0.400, the models were similar
to satisfy these requirements. to those created with the TTG data when the threshold ODwb of
0.350 was used; however, the TTG data when the threshold ODwb
Toxin Production of 0.350 was used gave a slightly more conservative model. For
Toxin production for experimental conditions close to the these reasons, the models were developed with TTG data obtained
growth or no growth border was explored by enterotoxin assay by making the ODwb of 0.350 or more the lower limit for
using the VIDAS Staph Enterotoxin Assay. The VIDAS Staph determining that growth had occurred in a particular well.
Enterotoxin Assay is a qualitative enzyme-linked • uorescent The models included all three main effects: RH (rh), pH (ph),
immunoassay performed in the automated mini-VIDAS instrument and potassium sorbate (sorb) or calcium propionate (cal), their
(bioMerieux, Inc., Hazelwood, Mo.). After the . nal ODwb quadratic effects (RH·RH 5 rh2, pH·pH 5 ph2, and potassium
measurements were completed (24 weeks), the media from the
224 Introductory Food Microbiology Modelling the Growth Boundary of Staphylococcus Aureus 225

sorbate·potassium sorbate 5 sorb2 or calcium propionate· calcium Plots of residuals versus main effects were also examined, and no
propionate 5 cal2), and the three two-factor interactions (rh ·ph, rh unusual patterns or anomalies were detected.
·sorb, ph·sorb or rh ·ph, rh ·cal, ph· cal). These main effects, quadratic There was a slight difference in the curvature of the contour
effects, and interactions are collectively referred to as the factors. lines in the plots for the media with no preservatives data for each
LIFEREG outputs a table of regression coef. cient estimates and model, despite the fact that the same data for the no preservative
approximate chi-square distribution P values for each factor in the condition were used for developing each model. This difference
model. The relative importance of each factor can be judged by the occurs because two distinctly different mathematicalmodels were
P value: factors with small P values are most in• uential and produced by the analysis, and when different models are used to
predictive of the TTG. LIFEREG also allows the user to specify the predict TTG at a zero preservative level, they will produce slightly
error distribution to account for the variation in TTG not explained different predictions. It should be noted that the differences are
by the regression model. For model development purposes, Weibull, small and mainly in the low pH areas of the plots. In practice, the
lognormal, and log-logistic distributions were considered. It was target product formulation would be well away from the growth
found that all three distributions gave very similar models; the boundary to allow for inherent process variation.
regression coef. cients were similar in sign and magnitude. Since
All of the models make good biological sense. As conditions
the three distributions are not all from the same class of
become more and more unfavorable for growth, the contour lines
distributions, it is not possible to formally test for goodness of . t
are closer together, indicating conditions are approaching those
using likelihood ratio tests. However, since all models produced
that do not allow growth of the organism. As the RH or pH of the
similar regression equations, we selected the log-logistic
system decreases, a corresponding increase in TTG is seen, and
distribution model, because it had the largest sample log likelihood.
the no growth area of the contour plot increases in size. The
The model development process is an iterative process. The . addition of potassium sorbate at low pH (pH 4.5 to 5.5)
rst analysis of the data created models that were all inclusive; this dramatically changes the contour plots with steep curvature in the
allowed for the determination of those factors that had a signi. low pH area of the plots. The no growth boundary with respect
cant effect on the growth of S. aureus. All of the main effects, to RH is increased from approximately 89% to approximately 93%
quadratic effects, and two-factor interactions were signi. cant for when 1,000 ppm of potassium sorbate is present at pH 4.5. This
each model (P <0.005) except for the interaction between RH and effect is not as evident when calcium propionate is present; the no
potassium sorbate in the potassium sorbate model and the calcium growth boundary with respect to RH only increased approximately
propionate quadratic term in the calcium propionate model. For 1.5% from 88 to 89.5% when 1,000 ppm of calcium propionate is
this reason, in each model the insigni. cant factor was dropped present at pH 4.5. There is little change in the boundary with
and the data were reanalyzed to develop the . nal models. The respect to RH when the system is at pH 7.0 with the addition of
resulting model equation from the calcium propionate data was either preservative studied.
2.250 to 3.703· rh - 1.488·ph + 0.418· cal + 1.572· rh2 + 0.343·ph2
Various regions on the contour plots generated by the models
+ 0.814· rh·ph - 0.130·rh · cal - 0.221·ph·cal, and the resulting
were explored with enterotoxin assays for all conditions where
model equation from the potassium sorbate data was 2.624 to
the . nal ODwb measurement was borderline. Also, assays were
3.938·rh - 2.421·ph + 0.907· sorb + 1.638· rh2 + 0.887·ph2 - 0.190·
conducted for all samples with treatments at 84% RH, pH 7.0, and
sorb2 + 0.903·rh ·ph - 0.756·ph·sorb. From the model equations, the
84% RH, pH 4.5, although all ODwb measurements for treatments
TTG contour plots for calcium propionate and for potassium
at 84% RH were less than 0.350. The results are indicated by a
sorbate were created. For diagnostic purposes, plots of ln predicted
plus sign (toxin present) or minus sign (no toxin present) on the
time to growth versus ln observed time to growth were examined.
contour plots. A total of 71 assays were run, and in every case
226 Introductory Food Microbiology Modelling the Growth Boundary of Staphylococcus Aureus 227

where the data were scored as ‘‘no growth,’’ the toxin assay natural biovariability in the bacterial population, can be
results were negative for toxin, and in every case where the data meaningless under stressed conditions. The LIFEREG procedure
were scored as ‘‘growth,’’ the toxin assay results were positive for used to create the boundary models handles this by . tting the
the presence of toxin. This further supports the choice of an ODwb distribution to the error term, hence allowing probabilities to be
of 0.350 as the correct cutoff value for time to growth in the calculated. This is useful for risk assessment purposes and allows
individual wells. the model to be easily used in Monte Carlo simulations. One
consequence of modeling this way is that there is a need to have
DISCUSSION many replicates equally spaced about the matrix, with
Comparison of the models developed in this study with other approximately 50% of the conditions allowing and 50% not
published work is dif. cult, because most previously published allowing growth for best-. tting purposes. The log time to growth
studies have used NaCl to control the RH of the system, whereas is used to normalize the variance.
this study has used glycerol. The limits of S. aureus growth, or the It has generally been accepted that the limits for growth of S.
growth of any microorganism, cannot be predicted by the RH of aureus are 85% RH when NaCl is used as the humectant and 89%
the system alone; instead, both the RH of the system and the when glycerol is used. Our models, based on glycerol, show the
physical properties of the humectant used should be considered. limiting RH for growth to be approximately 86% (at pH 7.0),
For example, NaCl is a non–glass-forming ionic humectant that which disagrees with work published by Marshall et al., who
has specific effects on membrane transport systems. Therefore, found the growth limits were 89 and 86% RH when glycerol and
both the osmotic and ionic stresses placed on bacterial cells by NaCl were used as the humectants, respectively. This disagreement
NaCl could affect the ability of those cells to grow. Glycerol is a could be due to strain variation or to the fact that only quarter-
nonionic glass former that passively permeates the cell membrane. strength BHI broth was used in the work by Marshall et al.,
Therefore, glycerol would not have a direct effect on ion transport whereas full-strength BHI broth was used in our model system.
systems. Unlike NaCl, glycerol forms an aqueous glass. The glass Shapero et al. reported growth of S. aureus at 88% RH on tryptic
transition temperature (Tg) is a specific characteristic for each soy agar plates with RH adjusted with glycerol but did not conduct
glass-forming compound. The influence of a solute’s Tg on experiments with the system at any lower RH values.
molecular mobility directly affects the amount of osmotic stress a
Studies with sucrose (a glass former that is not cell membrane
specific solute places on cells. For these reasons, it should be
permeable and has its own Tg) used as the humectant showed
expected that the limits of growth based on RH would be different
growth at 90% RH but not at 87% RH, and Scott reported growth
when various humectants are used to control RH, and, therefore,
at 88 but not at 86% RH. Broughall et al. developed models for
comparing studies based on RH alone could lead to false
growth of S. aureus in UHT milk, with RH adjusted by the addition
conclusions.
of glucose (a glass former that is not cell membrane permeable and
The differences in modeling approaches also make has a different Tg). They found growth at 88% RH but did not
comparisons dif. cult. Both the FMM and PMP have very few explore the RH boundary further, because the additional glucose
kinetic curves generated in which cells would be under stressed caused the system to become saturated. There is published literature
conditions, the most dif. cult conditions in which to obtain kinetic that describes the ability of S. aureus to grow in various food
information. Also, there are few true replicates in these areas, with systems, mainly meats and cheeses, but most of these systems
most of the experimental design similar to a central composite used NaCl as the RH depressant and, therefore, cannot be used
design in which most of the replicates are in the middle of the to validate the models created with glycerol. Often in experiments
design. Kinetic models also model the mean value, which, due to conducted with food systems, the pH of the product is not reported,
228 Introductory Food Microbiology Modelling the Growth Boundary of Staphylococcus Aureus 229

nor is the list of ingredients published, which also leads to dif. Molecular methods for detection of probiotics and intestinal
culty when trying to compare data from various published studies. microbiota and evaluation of Lactobacillus brevis as a potential
The effectiveness of potassium sorbate to inhibit growth was probiotic dietary adjunct.
considerably greater compared with calcium propionate, which The human gastrointestinal (GI) tract harbours an extremely
was especially noticeable at low pH. Potassium sorbate and complex microbiota mainly composed of fastidious anaerobic
calcium propionate have some key similarities that may lead to organisms. Because these microbes have a profound impact on
the belief that they should act similarly as preservatives; potassium host’s health, modulation of microbiota with probiotic bacteria
sorbate has a pKa value of 4.74 and a molecular weight of 150.2, has been proposed. However, the mechanisms of action of both
whereas calcium propionate has a pKa value of 4.87 and molecular the GI microbes and the proposed probiotics remain obscure. To
weight of 186.2. gain more information, molecular identification methods for
Both have been reported in the literature to act as weak acids; members of the GI microbiota and the probiotic strains are needed.
the molecules remain undissociated at low pH, diffuse through Quick, robust methods are necessary for obtaining an overall image
the bulk lipids of the cell membrane, then enter and dissociate in of changes in GI microbiota, and more sophisticated methods are
the cytoplasm, where the pH is close to neutral. It is the dissociated needed for following up selected species or strains.
molecules that decrease the internal pH of the cell, which may In this study, new methods were tested for their applicability
prevent growth by perturbing metabolism. Recent studies have in monitoring GI microbiota and probiotic strains. Strain-level
shown that sorbic acid does have inhibitory effects in the genetic labelling without introduction of foreign DNA was
undissociated form, suggesting not only that it may act as a weak demonstrated with Lactobacillus helveticus CNRZ32 by inserting
acid but also that it may have a secondary mode of action associated a site containing silent mutations into the chromosomal pepX
with membrane-resident sorbic acid that acts as an uncoupler, gene. Intact phenotypic properties of the mutated strain were
which may disable active transport. It has also been reported that confirmed with a peptidase assay. The mutated and wild-type
for S. aureus the minimum inhibitory concentration of propionic strains could be detected from faeces and milk with the help of
acid is much greater than sorbic acid. This suggests that the levels specific primers. Thus, the labelling method could be used for
of calcium propionate used in this study that are in line with the specific marking of industrial or probiotic strains in a ‘food-grade’
typical use levels in foods may have been too low to observe the manner provided that a suitable target gene and genetic
inhibitory effect of calcium propionate on S. aureus growth. transformation tools are available.
In summary, two models describing the growth boundaries For analysis of GI microbes and selected intestinal or dairy
for S. aureus with respect to RH controlled by glycerol, pH, and lactic acid bacteria, several species- or group-specific
preservative were developed. The growth boundaries were explored oligonucleotide primers and probes were designed and tested
with toxin assays. For the . rst time, these models describe the with three different techniques. A polymerase chain reaction -
growth/no growth boundaries for S. aureus with respect to RH enzyme-linked immunosorbent assay (PCR-ELISA) application
controlled by glycerol and pH in addition to the preservatives with simultaneous utilisation of multiple species- or group-specific
potassium sorbate and calcium propionate. These models will oligonucleotide probes was suitable for detection of predominant
allow product developers to visualize the ‘‘safe space’’ for the members present in a mixed bacterial population. Sensitivity of
formulation of shelf-stable intermediate moisture foods using these the method was improved by using primers selective for the genus
preservation factors, will allow microbiologists to assess risks Bifidobacterium. Comparison of dot blot hybridisation and real-
more effectively over a wide range of products, and will ultimately time PCR demonstrated the superior properties of realtime PCR for
allow the consumer to have greater assurance of food safety. detection and quantification of bacterial ribosomal DNA targets.
230 Introductory Food Microbiology Modelling the Growth Boundary of Staphylococcus Aureus 231

The final part of this study comprised the evaluation of two of the GI tract microbiota and the effects of probiotic bacteria on
Lactobacillus brevis strains ATCC 8287 and ATCC 14869T as these microbes.
supplements in dairy products. The L. brevis strains showed
promising in vitro antagonistic properties towards selected REVIEW OF THE LITERATURE
potentially harmful microbes and were suitable as supplementary Intestinal Microbiota
strains in yoghurt, producing no undesirable side effects on the
An extremely complex microbiota which has a profound impact
quality or preservation of products. A small-scale feeding study
on host’s health. The normal gut bacterial population of an adult
demonstrated the survival of L. brevis ATCC 8287 in the human
is estimated to comprise more than 400 species, with a
GI tract, indicating, together with the favourable antagonistic
predominance of obligate anaerobes. The total number of microbes
properties, that this strain could be a candidate for use as a
present in one gram of intestinal content varies from less than 103
probiotic supplement in dairy products.
microbes in the stomach to 104 – 107 microbes in the small intestine
Molecular methods have facilitated culture-independent and 1010 – 1012 microbes in the colon. Indeed, the quantity of
studies of gastrointestinal (GI) tract microbes. The GI microbiota microbes present in the intestine (about 1014) exceeds 10-fold the
is mainly composed of anaerobic organisms and moreover, direct total number of all human cells. Until recently, analysis of intestinal
molecular approaches have confirmed the abundance of bacteria has mainly been based on cultivation-dependent methods.
uncultivable microbes in intestinal samples. The value of molecular Culture-independent studies have however confirmed that only a
methods for studying GI tract microbes is therefore immense. fraction of the organisms present in faeces are cultivable, therefore,
GI microbiota can be influenced by probiotic bacteria. The the results obtained by cultivation are likely to be biased.
probiotics, defined as “live microbial food supplements which Generally, Bacteroides, Eubacterium, Clostridium, Ruminococcus,
beneficially affect the host by improving the intestinal microbial Peptococcus, Peptostreptococcus, Bifidobacterium and Fusobacterium are
balance", have been proposed to possess several advantageous reported to constitute the majority of microbiota. Molecular analyses,
properties, such as antagonistic actions, production of however, suggest that most faecal bacteria belong to a few
antimicrobial substances, modulation of immune responses and phylogenetic lineages composed of organisms from several genera.
an impact on the metabolic activities of the gut. Bacteria related to Bacteroides, Prevotella and Porphyromonas seem
Probiotic bacteria seem to hold great promise for treatment of to represent one-third of bacteria present in faeces, whereas
gastrointestinal disorders, yet further studies are required to create Clostridium leptum subgroup and Clostridium coccoides groups both
a more scientific basis for the probiotic action. Furthermore, as no account for approximately one-fifth of the faecal bacterial
single strain is likely to have all the aforementioned beneficial populations. However, besides being an extremely diverse
properties, screening of new strains for probiotic potential is microbial ecosystem, the intestinal microbiota appears to be unique
necessary. for each individual. The normal microbiota of the GI tract works
as a barrier against pathogens, contributes to degradation of some
Although several applications, such as dot blot hybridisation,
food components, stimulates the host immune system, and
fluorescence in situ hybridisation (FISH), denaturing gradient gel
produces certain B vitamins, enzymes and short-chain fatty acids.
electrophoresis (DGGE) or thermal gradient gel electrophoresis
The microbes can also metabolise potentially carcinogenic
(TGGE), and polymerase chain reaction (PCR) with species- or
substances and drugs in either a beneficial or a disadvantageous
group-specific primers, are available for the detection and
way. However, the role and action of individual microbial species
semiquantitative analysis of GI microbiota, improvements are
or groups present in the GI tract are poorly known. Starting at
required, especially in relation to sensitivity, cost and quantification
birth, the microbiota develops in a successional manner. The first
power of the methods. This is fundamental for better understanding
232 Introductory Food Microbiology Modelling the Growth Boundary of Staphylococcus Aureus 233

colonising microbes include bifidobacteria, enterobacteria, are most common in probiotic products designed for human use;
clostridia, enterococci and ruminococci. Recent studies have probiotic properties of these bacteria are also the best studied.
suggested the importance of the type of colonising bacteria on the Lactobacilli belong to the lactic acid bacteria, which comprise
development of the gut immune system. A role in the development a diverse group of Gram-positive bacteria, most typically
of food allergies has been postulated for the intestinal microbiota represented by non-sporing, catalase-negative, devoid of
due to the occurrence of an adult-like bifidobacterial species cytochromes, non-aerobic but aerotolerant, fastidious and acid-
composition in the intestine of allergic infants. Significant structural tolerant cocci or rods producing lactic acid as the major end-
changes occur in the microbiota with age, including reduction of product during the fermentation of carbohydrates. Being of
bifidobacteria as well as an increase in the diversity of Atopobium fastidious nature, lactic acid bacteria require a rich environment
cluster species present in faeces. Nutritional aspects, however, for growth, such as decaying plant material, food products and a
have a profound effect on the composition of the intestinal microbial mammalian gastrointestinal tract or vagina. The genus Lactobacillus
population. is heterogeneous, containing species with 32-53% G+C of the
chromosomal DNA arranged into three groups based on differences
The Probiotics
in sugar metabolism caused by the presence or absence of fructose-
Probiotic bacteria have been defined as “live microbial food 1,6-diphosphate aldolase and phosphoketolase. Although
supplements which beneficially affect the host by improving the possessing some phenotypical features common for lactic acid
intestinal microbial balance." Probiotic bacteria are increasingly bacteria, the genus Bifidobacterium is actually related to the
utilised in human food as well as in animal feed products. Actinomycetes branch, having a high chromosomal G+C content.
However, composition of the intestinal microbiota is poorly known, Sugar metabolism of bifidobacteria differs from that of lactic acid
which hinders understanding of the probiotic functions. A probiotic bacteria; the bifidobacteria lack aldolase and glucose-6-phosphate
strain should be of host origin, non-pathogenic, technologically dehydrogenase, and hexose sugars are exclusively degraded by
suitable for industrial processes, acid- and bile-fast, adhere to the the fructose-6-phosphate pathway characterised by fructose-6-
gut epithelial tissue, persist in the gastrointestinal tract for short phosphate phosphoketolase. Bifidobacteria are predominant
periods, produce antimicrobial substances, modulate immune members of the human intestinal microbiota, with bacterial counts
responses and influence the metabolic activities of the gut. The of 109-1011 per gram of stool, with B. bifidum, B. longum, B. infantis,
properties of the strain should be well documented. Although B. breve, B. adolescentis, B. angulatum, B. catenulatum, B.
some criteria, such as the non-pathogenic status, technological pseudocatenulatum, and B. dentium reported as human isolates.
suitability and careful documentation of the probiotic effects of a
Several positive effects have been proposed for probiotic
microbial strain, are invariably required, no single strain is likely
lactobacilli and bifidobacteria. Antagonism towards intestinal
to carry all of the abovementioned properties. Moreover, probiotic
pathogens has been demonstrated for probiotics. Alleviation of
properties are considered strain-specific, and results obtained with
diarrhoea is a well-documented characteristic of some strains, and
one strain cannot therefore be claimed for another, even a closely
particularly the ability of Lactobacillus rhamnosus GG to shorten the
related strain. Microbes used in probiotic products include strains
duration of acute rotavirus diarrhoea has been established.
from several Lactobacillus and Bifidobacterium species, Enterococcus
Stimulation of the gut immune system by probiotic strains has
faecalis and Enterococcus faecium, Lactococcus lactis, Leuconostoc
been reported, and induction of cytokine profiles has been shown
mesenteroides, Pediococcus acidilactici, and Sporolactobacillus inulinus,
to be strain-dependent. Alleviation of allergic reactions in the
Streptococcus thermophilus, as well as Bacillus cereus, Escherichia coli,
gastrointestinal tract has also been suggested for some strains:
Propionibacterium freudenreichii, Saccharomyces cerevisiae and
using a mouse model, Murosaki et al. (1998) showed that heat-
Saccharomyces boulardii. However, lactobacilli and bifidobacteria
234 Introductory Food Microbiology Modelling the Growth Boundary of Staphylococcus Aureus 235

killed Lactobacillus plantarum L-137 suppressed production of Genetic Labelling of Lactic Acid Bacteria
antigenspecific IgE by stimulating the production of IL-12. In Insertion of an extra DNA label into a target strain genome
addition, Lactobacillus rhamnosus GG and Bifidobacterium lactis Bb- provides a way to monitor the specific strain in various
12 have been demonstrated to relieve symptoms of atopic eczema. environments. However, introduction of foreign DNA is often
Administration of L. rhamnosus GG has been shown to enhance required, and in many cases, genetic elements producing a
the production of IL-10, acting thus as an anti-inflammatory phenotypic change are used to distinguish the marked strain.
mediator in atopic children. Genetic elements encoding resistance to an antibiotic have been
Consumption of probiotics has also been observed to lower utilised for labelling of lactic acid bacteria, but the increasing
activities of harmful faecal enzymes. Some animal models have spread of antibiotic resistance factors between bacterial species or
even suggested the ability of probiotic strains to reduce the incidence genera make such approaches unsuitable for strains intended for
of cancer. Finally, probiotics may have significance for alleviation human or animal use. Therefore, usage of various ‘food-grade’
of intestinal disturbances. For example, ingestion of a mixture of markers has been suggested. Labelling of lactic acid bacteria with
lactobacillar and bifidobacterial strains together with a Streptococcus a plasmid-encoded green fluorescent protein gene placed under
salivarius ssp. thermophilus was observed to help maintain remission an inducible promoter has been reported for Lactococcus lactis and
status in ulcerative colitis patients. Lactobacillus plantarum, whereas Allison and Klaenhammer (1996)
suggested the use of a native Lactobacillus gene encoding immunity
Although lactobacilli and bifidobacteria are stated as GRAS
to Lactacin F as a food-grade genetic marker. A similar role has
(generally recognized as safe) organisms due to their long usage
also been demonstrated for ltnI conferring immunity to lacticin
and non-pathogenic status, safety issues have been investigated.
3147. Site-specific integration of desired genetic elements into
Isolation of these organisms from infections has been reported
bacterial chromosomes through phage attachment sites has been
with a low incidence, and in most cases, bifidobacteria and
described for Lactococcus lactis, Lactobacillus delbrueckii and
lactobacilli were associated with immunocompromised patients. Lactobacillus plantarum.
One major concern among lactic acid bacteria is, however,
resistance to vancomycin, especially within the genus Enterococcus Legislation as well as consumer acceptance limit the usage of
where the resistance has been shown to be transferable. genetically engineered organisms, and therefore, genetic changes
introduced to target organisms must be carefully considered.
Vancomycin resistance has been reported for lactobacilli by several
Maguin et al. (1996), for example, combined insertion sequence
authors, but it is generally considered to be an intrinsic property.
ISS1 with a thermosensitive replicon, enabling a high frequency of
Vancomycin resistance expressed by the probiotic strain L.
random insertion (about 1%) with Lactococcus, Enterococcus and
rhamnosus GG has been studied in detail.
Streptococcus thermophilus, while efficient excision of the plasmid
Strain GG was not observed to transfer vancomycin resistance generated stable mutants with no foreign markers, leaving only a
or receive other resistance elements from enterococci, nor were any single ISS1 copy at the mutated site. However, the smallest
genes resembling enterococcal vancomycin resistance genes detectable alteration introduced to a target organism is changing
found in Lactobacillus GG. Similarly, Klein et al. (2000) reported one or a few bases in a gene-coding sequence without affecting the
no indications for the presence of the vanA gene cluster, the vanB amino acid sequence of the corresponding gene product. In
gene or the vanC gene from five L. reuteri strains or L. rhamnosus principle, such a mutation could be caused by natural processes
GG, suggesting that the vancomycin resistance of the strains due to the degeneracy present in the genetic code.
studied is unrelated to the acquired resistance in the Enterococcus Silent mutations have been utilised for modification of a
species. Lactococcus lactis subsp. cremoris strain plasmid-encoded proteinase
236 Introductory Food Microbiology Modelling the Growth Boundary of Staphylococcus Aureus 237

prtP gene by in vitro mutation of the third positions of four adjacent Denaturing Gradient Gel Electrophoresis (DGGE) and Thermal
codons, thus providing a genetic label with no phenotypic effects. Gradient Gel Electrophoresis (TGGE)
Although technically more demanding, genetic marking should, Denaturing gradient gel electrophoresis (DGGE) and thermal
however, preferably be directed to gene replacement on a gradient gel electrophoresis (TGGE) have become popular methods
chromosomal locus to ensure maximal stability of the alterations for analysis of microbial populations present in various habitats
introduced. such as water ecosystems, microbial fermentations, and the GI
tract. These methods allow separation of nucleic acid molecules
Utilisation of Nucleic Acid Based Methods for Identification and
based on their size and sequential differences. Thus, a population
Monitoring of Bacteria in Population Samples
of DNA molecules, such as the 16S ribosomal RNA (rRNA) or
Utilisation of Methods Independent of Prior Knowledge on PCR-amplified 16S rDNA, can be studied and predominant
Sequence Data: Cloning and sequencing of the 16S ribosomal members of the population identified via sequencing of isolated
DNA (rDNA) pools in a population sample provides a method for nucleic acid bands. Sensitivity of gradient gel electrophoresis is
obtaining sequence-level information on uncultivable bacteria affected by the choice of PCR primers used. For example, utilisation
abundantly present in various parts of the gastrointestinal tract. of universal 16S primers limits the sensitivity to detection of 1%
Suau et al. (1999) analysed the sequence of 284 16S rDNA clones subpopulations. By contrast, utilisation of species- or group-
derived from one faecal sample and classified the clones into 82 specific primers may allow detection and identification of bacteria
molecular species, using 98% similarity criteria for a species. representing a minority of the total population.
Importantly, only 24% of the molecular species were derived from
Drawbacks of gradient gel electrophoresis include difficulty of
a described organism. Similarly, comparative 16S rDNA sequence
comparisons between individual gels, requirement of careful
analysis of the intestinal bacterial community in pigs revealed
adjustments, and the need for sequencing to confirm identities of
only a 17% fraction of previously described organisms among a
bands seen in a gel. Furthermore, some bacteria may remain
total of 375 phylotypes. In a large-scale study of subgingival plaque
unidentified due to low resolution of DNA bands. With both
samples, including analysis of a sequence of 2522 16S rDNA
DGGEand TGGE-based methods, sequencing is required for correct
clones, 347 species was observed among the clones studied, which
identification of individual bands seen in the gel. As stated by
correlated well with a previous estimate of expected species
Schmalenberger et al. (2001), intraspecies operon heterogeneities
diversity in the oral cavity. Thus, the direct cloning approach
may significantly contribute to genetic profiles in microbial
seems to give a good idea of the total microbiota present in a
community analysis, as amplification of one bacterial DNA may
complex population. It also facilitates determination of species-
yield several separate bands, which can then be wrongly
level differences between bacterial populations, as shown by the
interpreted as high microbial diversity. Depending on the 16S
discovery of novel phylotypes and species not previously
binding universal primer pairs used, single-strand conformation
associated with childhood caries by comparing of the oral
polymorphism analysis (SSCP) revealed an average of 1.7 – 2.3
microbiota of a healthy subject and a subject with early childhood
bands per pure cultured bacterial organism.
caries.
Approaches based on cloning are, however, rather tedious Terminal Restriction Fragment Length Polymorphism (T-RFLP)
and are not optimal for analysis of large numbers of samples. Like Terminal restriction fragment length polymorphism (T-RFLP)
all PCR-dependent methods, construction of clone libraries may is based on endonuclease digestion of PCR-amplified DNA and
be prone to biasing, possibly leading to falsification of the library capillary electrophoresis analysis of the terminal restriction
structure. Nevertheless, with the help of cloning, design of fragment (TRF) containing a fluorescent label. Terminal restriction
phylogenetically relevant oligonucleotide probes is enabled.
238 Introductory Food Microbiology Modelling the Growth Boundary of Staphylococcus Aureus 239

patterns have been used to analyse marine bacterioplankton enrich the target DNA. For example, 0.1 pg of B. distasonis or B.
communities as well as faecal bacteria. The method is, however, thetaiotaomicron DNA (approximately 10-20 cells) or 0.01 pg of B.
limited by the choice of primers, which can, with their different vulgatus DNA (1-2 cells) applied to a PCR was sufficient to produce
affinities, dramatically change the patterns observed, while another a positive hybridisation signal with specific digoxigeninlabelled
problem is the TRF length overlap by phylogenetically distant probes.
bacteria. An additional factor of concern for filter hybridisations is the
Analysis of Community DNA Profiles need to label each probe separately and to perform hybridisation
reactions after optimisations in separate vessels. However,
Analysis of total bacterial community structure can be
Ehrmann et al. (1994) described a reverse dot blot hybridisation
accomplished by measurement of guanosine-cytosine profiles of a
with several membrane-bound oligonucleotide probes for
population DNA sample with DNA reassociation and density
identification of lactic acid bacteria present in mixed populations.
gradient centrifugation. Density gradient fractionation of
Similarly, Becker et al. (2002) used simultaneous detection of PCR-
community DNA enables analysis of interesting fractions by
amplified rDNA of 23 oral bacterial species or groups with
cloning of PCR-amplified DNA pools, followed by sequencing.
oligonucleotide probes.
Density gradient centrifugation has been applied in analysis of
intestinal microbial guanosinecytosine profiles. However, because In the fluorescence in situ hybridisation (FISH) method, the
this approach requires sophisticated and expensive equipment, it bacterial cell samples to be studied are immobilised on microscope
is out of reach for most research laboratories. slides and made permeable for fluorescently labelled
oligonucleotides with subsequent microscopic observation of the
Utilisation of Specific Oligonucleotide Primers or Probes hybridisation signal intensities.
Hybridisation: Hybridisation offers a means for direct semi- FISH has been used for monitoring faecal microbiotas, and
quantitative monitoring of population samples. A very high detection of a 1 – 0.1% bacterial subpopulation has been obtained.
sensitivity for detection of DNA targets can be obtained with Automated analysis of fluorescent signals has also been utilised
radioactively labelled probes. However, because of its abundance
to facilitate objective interpretation of results. The major problem
in bacterial cells, rRNA provides for a more attractive target for
with FISH applications is the different penetration of probes in
hybridisation studies. Indeed, hybridisation assays targeting rDNA
bacteria with various cell wall types, resulting in a possible
have been verified as 10-fold less sensitive than assays for rRNA.
underestimation of Gram-positive bacteria.
Dot blot hybridisation with rRNA – targeted probes has been used
for semiquantitative analysis of ruminal microbes and intestinal Polymerase Chain Reaction (PCR): Amplification of target
microbiota. Comparison of hybridisation results of a specific probe nucleic acids with PCR is an effortless method for detection of
with a universal probe enables assessment of the target bacterial target DNA from various samples. In principle, the detection limit
proportion present in a sample. At best, detection of a 0.1 – 0.01% for a PCR assay, based on usage of one or two oligonucleotide
rRNA subpopulation has been reported, corresponding to primers specific for the target bacteria, is the presence of one copy
approximately 107 target cells if 1011 bacteria are considered to of the amplified DNA region. Although achievement of such an
be present in a gram of faeces. Use of radioactive isotopes, especially extremely sensitive assay is unlikely, the PCR remains a very
in large-scale and long-term analyses, is, however, complicated by powerful detection method, typically requiring a minimum of 10-
the short half-lives of labels. Nonradioactively labelled probes are 20 target copies for successful amplification. The high sensitivity
more convenient, but their sensitivity is limited. Preenrichment of is also somewhat problematic, as a simple aerosol contamination
the target DNA by polymerase chain reaction can be used to may lead to false amplification.
240 Introductory Food Microbiology Modelling the Growth Boundary of Staphylococcus Aureus 241

PCR-based detection of rRNA genes has been used for direct amplification of an internal standard, detection of Clostridium
detection of various bacteria present in faeces and pathogens from proteoclasticum was linear between 1 × 104 and 5 × 101 cells, with
clinical samples. Partial or full amplification of the 16S rDNA a detection limit of 50 fg or 25 cells, whereas detection of Oxalobacter
with primers specific for a genus or a few closely related genera formigenes from human faecal samples by competitive PCR was
has also been reported. In addition, utilisation of 16S-23S intergenic shown to be linear over a range of six logarithmic units, with a
sequences as targets of speciesspecific primers for different lactic detection limit of approximately 100 genomes.
acid bacteria has been described (Tilsala-Timisjärvi and Alatossava, Polymerase Chain Reaction – Enzyme-linked Immunosorbent
1997). Good sensitivities have been reported for the PCR detection Assay (PCR-ELISA) : Polymerase chain reaction – enzyme-linked
assays of faecal bacteria. For example, PCR detection sensitivity of immunosorbent assay (PCR-ELISA) combines utilisation of
five ruminococcal species with species-specific primers from faecal polymerase chain reaction for efficient multiplication of the target
samples spiked with the target species did not markedly differ DNA and hybridisation with a detection probe to ensure the
from the observed detection limit of 4-100 cells from pure cultures. specificity of the reaction. Figure 1 summarises the concept of
Although a highly potent detection method, results obtained PCR-ELISA detection. The PCR-amplified products are labelled
by conventional endpoint PCR should not be considered to be with digoxigenin during or after the amplification reaction and
directly quantitative. PCR may lead to differential amplification of hybridised with the specific biotinylated detection probe. The probe
target templates originally present in equal amounts. In addition, is immobilised in streptavidincoated microtitre plate wells, and
very low template concentrations may generate random fluctuations hybridised DNA products are detected via digoxigenintargeted
in priming efficiency of population DNA samples with universal antibodies linked with an enzyme capable of producing a
primers, leading to a bias in the end-product concentration. colorimetric or fluorimetric signal when brought together with a
Knowledge of the rRNA gene copy numbers and genome sizes of substrate. Usage of PCR-ELISA hybridisation for detection of single
bacteria in a mixed DNA sample has been observed to be pathogens of clinical importance and spoilage bacteria of food has
insufficient in predicting the final product ratio of a PCR been described. In comparison with filter hybridisation, PCR-ELISA
amplification. Suzuki and Giovannoni (1996) also noticed that the provides more easily standardised reaction conditions by
accumulation of end products during mixed-template PCR caused utilisation of commercially supplied microtitre plates. Other
biasing of the various end-product ratios towards a 1:1 situation, advantages of PCR-ELISA compared with several other methods
which was hypothesised to be caused by an increase in the are its relative simplicity and low costs.
homologous template hybridisation, decreasing the efficiency of Good sensitivities have been reported for PCR-ELISA assays,
primer annealing and subsequent amplification. and the results can in some cases be interpreted quantitatively.
Some improvements in the reliability of PCR quantification The limit for detection of Bordetella pertussis was 100 target
have been obtained by competitive PCR approaches. Hahn et al. organisms, starting from appliance of different target DNA amounts
(1995) created a quantitative PCR assay based on post- to PCR reaction. Less than ten Epstein-Barr virus genome copies
amplification differentiation of the internal standard and the added to 750 ng of background DNA were required for a positive
sample DNA by selective restriction analysis and digoxigenin- PCR-ELISA result. Similarly, a sensitivity of 5 cfu/ml blood was
based colorimetric detection. Quantitative detection of obtained for detection of C. albicans and A. fumigatus cells,
Mycobacterium tuberculosis PCR products was performed by enzyme- corresponding to the sensitivity of Southern blotting using
linked immunosorbent assay by comparison of hybridisation digoxigenin-labelled oligonucleotides. Fletcher et al. (1998) could
results with two probes and two IS6110 elements derived from detect Aspergillus fumigatus quantitatively by a PCR-ELISA method
either an internal control or a modified template. With co- on a log-scale between 100 and 1 pg of target DNA. Detection of
242 Introductory Food Microbiology Modelling the Growth Boundary of Staphylococcus Aureus 243

Escherichia coli in oysters was reported to be quantitative in the determination of larger bacterial groups have also been published.
range of 10-105 cfu/g. Detection of Campylobacter jejuni and C. coli Typically, sensitivities of 1 to 100 target genomes per reaction and
with PCR-ELISA was shown to be 10- to 100-fold more sensitive linearity ranges of 4 to 8 logarithmic units are obtained in assays
than a gel-based PCR method using the same primers, the smallest targeted to specific bacteria. Real-time PCR is also utilised as a
amount of C. jejuni template DNA giving a positive signal in the rapid diagnostic tool for detection of pathogenic bacterial species
assay being 1.5 fg. Some of the described PCR-ELISA applications or strains present in a sample. With pathogenic bacteria, the target
can be considered to be quantitative competitive PCR approaches. of choice for real-time PCR is generally a gene associated with the
Real-time PCR: Real-time PCR is based on on-line pathogenic traits.
measurement of the amplification reaction, enabling quantification Detection of PCR amplicons with specific probes is often
of the product during the logarithmic phase of PCR. The first and favoured over usage of intercalating dyes due to the former’s better
so far most commonly used real-time PCR approach is the 5´- sensitivity and lack of detection of falsely primed products.
nuclease (TaqMan) assay introduced by Holland et al. (1991). The However, SYBR Green I has become popular because of the
original assay operates using a radioactively labelled probe that possibility to use this intercalating dye in virtually any assay.
hybridises in the PCR template region to generate a specific, Real-time quantitative PCR with SYBR Green I has been reported
detectable signal from the amplification reaction. The detection to be 10-fold less sensitive than a corresponding TaqMan assay
probe becomes annealed to one of the DNA strands during the due to the formation of non-specific products in reactions starting
amplification and is cleaved by the Thermus aquaticus DNA with small amounts of template DNA. On the other hand, SYBR
polymerase 5´-exonuclease activity during the primer extension Green I has great potential in situations where a diverse target
step. Lee et al. (1993) further extended the 5´-nuclease assay by population is to be detected with PCR. A probe-based methodology
utilisation of doubly labelled oligonucleotide probes for fluorescent requires a binding site for the probe in the vicinity of one of the
measuring of formation of the specific PCR product during primer primers; however, such a conserved site is likely to be missing
extension. Introduction of an automated detection method enabled from a degenerate target DNA population. This should be taken
real-time monitoring of the PCR product formation during the into account, especially when a bacterial population containing
exponential amplification phase. Since the advent of real-time several hitherto unknown species is studied.
PCR, several techniques have been introduced, including primers
AIMS OF THE STUDY
with fluorescent dyes, molecular beacons, dual probes and
intercalating dyes such as SYBR Green I. Real-time PCR is a The aims of this study were to develop molecular methods for
superior technique for quantification of nucleic acids. While analysis and monitoring of faecal bacterial populations and
competitive PCR has been demonstrated to be as reproducible and putative probiotic strains as well as to characterise the technological
accurate as real-time PCR, the latter has the benefit of an easier properties of two Lactobacillus brevis strains. The following goals
methodology, reducing the need for sample DNA treatment. were set:
1. To create a genetic label by introducing silent mutations to
Although real-time PCR is a relatively new technique, several
adjacent amino acid codons of a genomic peptidase gene
detection or quantification assays targeting various bacteria have
of a selected Lactobacillus strain and to confirm the
already been described. Target bacteria include carious dentine
unchanged phenotype of the strain with an available
bacteria, Desulfotomaculum from soil, faecal bifidobacterial species,
peptidase assay.
Helicobacter hepaticus, Staphylococcus aureus, Borrelia burgdorferi sensu
lato, Campylobacter jejuni, Listeria monocytogenes, Rhodococcus 2. To develop oligonucleotide PCR primers or probes targeting
coprophilus and Mycoplasma genitalium. Some applications for the 16S ribosomal DNA as species- or group-specific
detection tools.
244 Introductory Food Microbiology Modelling the Growth Boundary of Staphylococcus Aureus 245

3. To exploit a PCR-ELISA application with multiple as outlined in the Materials and methods sections of original
oligonucleotide capture probes for analysis of artificial publications (I-V).
mixed DNA samples or faecal DNA preparations.
Basic DNA Techniques
4. To test suitability of real-time PCR for quantification of
selected intestinal or probiotic bacteria in faecal samples. Rapid isolation of genomic DNA from pure cultured bacteria
was performed by cell mill disruption of bacterial samples in the
5. To evaluate the applicability of two Lactobacillus brevis
presence of glass beads, followed by phenol-chloroform extraction
strains as supplementary strains with potential probiotic
and ethanol precipitation (I, II, III, V). For rapid and effective
actions in dairy products.
purification and isolation of DNA from faecal material, a method
described in Study I was used (I, III). A large-scale DNA isolation
MATERIALS AND METHODS
method described by Apajalahti et al. (1998) was used to produce
Microbial Strains, Plasmids, Human Cell Lines and Culture a sufficient amount of template DNA for real-time PCR (IV).
Conditions
Wizard Minipreps were used for isolation of plasmid DNA
The microbial strains, plasmids and human cell lines used in from E. coli clones (I), whereas the Qiagen Plasmid Protocol Kit
this study have been described in detail in the respective original was exploited for isolation of plasmid DNA from L. brevis (V).
publications I-V. Briefly, Lactobacillus helveticus CNRZ32 was chosen Restriction enzyme digestions and ligations were carried out
as a model organism for demonstration of genetic labelling (I). according to the enzyme manufacturer’s recommendations. The
Plasmids pUC19 and pSA3 were used to construct plasmids transformations of L. helveticus cells were performed as described
pKTH5052 and pKTH5053, respectively (I). The PCR-ELISA by Bhowmik and Steele (1993). Dot blot hybridisation was executed
application with universal primers targeting 16S and 23S rDNA using standard methods (IV). DNA concentrations were measured
was tested with a set of bacteria including 25 species, that with a Versafluor fluorometer (Bio-Rad). DNA sequencing was
represented type strains of lactobacilli, bifidobacteria and other performed with an ABI310 DNA sequencer (Applied Biosystems)
bacteria reported as members of human intestinal microbiota, or using BigDye Terminator chemistry (I).
lactobacilli species used in dairy fermentations (II). Extension of
the PCR-ELISA method with bifidobacteria-targeted 16S rDNA Design of Oligonucleotide Primers and Probes
amplification was tested with a set of ten bifidobacterial species Oligonucleotide primers and probes were designed with the
belonging to the human intestinal microbiota, suggested as help of published sequence data available in sequence databanks
potentially harmful oral microbes, or used in dairy products (III). (I, II, III, IV). Generally, the online Internet tools ClustalW and
With real-time PCR, six bacterial strains, each representing a Fasta provided by the European Bioinformatics Institute (EBI) were
bacterial species or group, present in the human intestinal tract or used for identifying the primers and probes with desired specificity
dairy products, were used (IV). towards intended target DNA. Oligonucleotides required for
Dairy technological and probiotic properties of two lactobacilli creation of the silent mutation site (I) were planned by utilising the
sequence of the L. helveticus CNRZ32 pepX gene (Accession number
strains, L. brevis GRL1 (ATCC 8287) and L. brevis GRL62 (ATCC
U22900). While creating the mutations, major changes in the codon
14869T), were determined using dairy starters, positive or negative
usage frequency were avoided to reduce the likelihood of changes
control strains or target strains for testing the antagonistic
in the expression level of the target gene. The Ribosomal Database
properties (V). The human intestinal cell lines Intestine-407 and
Project was utilised for designing the primers and probes used in
CacO2 were used in adhesion studies (V). Culturing of microbes
Study IV. In addition, signature oligonucleotides published
and human cell lines was carried out using media and conditions
246 Introductory Food Microbiology Modelling the Growth Boundary of Staphylococcus Aureus 247

previously by others were utilised or modified when necessary optical grade PCR plates and optical sealing tape. Optimal
(II, III, IV). concentrations for various reaction components were tested for
each primer set and chemistry with a dilution series of genomic
Polymerase Chain Reaction (I-V) DNA from the target test species. With SYBR Green I chemistry,
For end-point detection or production of DNA templates, effect of the polymerase type on PCR product formation was tested
polymerase chain reactions were carried out in reaction conditions with a standard polymerase, Dynazyme II, and hot-start
recommended by the manufacturer of Dynazyme DNA polymerase. polymerases AmpliTaq Gold® DNA Polymerase, BlueTaq and
In Study I, the PCR conditions used in detection of wildtype and FastStart Taq DNA Polymerase. The TaqMan assays were
mutant strains were as follows: the reaction mixture consisted of performed successfully with the Dynazyme II enzyme and therefore
50 mM Tris- HCl pH 9.0, 15 mM (NH4)2SO4, 0.1% Triton X-100, switching to the hot-start enzyme was not considered.
1.8 mM MgCl2, 360 µM of each deoxynucleotide triphosphate, 1
µM of each primer and 0.02 U/µl Dynazyme EXTTM Polymerase, Peptidase Activity Assays (I)
and 1 µl of template or water. Each sample was subjected to a Activity measurement of the PepX was determined from both
primary denaturation cycle at 95ºC for 2 minutes followed by 30 the wild-type and mutated L. helveticus strains according to the
cycles of denaturation at 95ºC for 30 seconds, annealing at 55ºC method of El Soda and Desmazeaud (1982).
for 30 seconds and elongation at 72ºC for 1.5 minutes. The reaction
was terminated at an elongation step for 5 minutes at 72ºC and Evaluation of L. brevis Strains as Dairy Adjuncts
followed by incubation at 4ºC. In Study IV, gradient PCR was used In vitro tolerance of the L. brevis strains to low pH, bile salts
to select optimal annealing temperatures for PCR primer pairs. and pancreatic fluid was examined as described in Study V.
Oligonucleotides were synthesised by commercial suppliers. Antimicrobial properties towards selected food spoilage bacteria
and dairy lactic acid bacteria starters were evaluated by following
PCR-ELISA (II, III)
the microbial growth in MRS broth supplemented with 10% filter-
PCR-ELISA was carried out as described in Studies II and III. sterilized L. brevis GRL1 or GRL62 supernatant at 37ºC for two
Briefly, the PCR products were labelled with the DIG-High Prime days with an automated turbidometer Bioscreen C Analysing
Kit according to the instructions of the manufacturer, and System. Adherence to Caco-2 and Intestine-407 cell lines was
concentrations of the PCR products were measured with the studied as described in Study V. Resistance to selected antibiotics
Versafluor fluorometer. For calculation of molecular quantities in
was tested with disc diffusion and microdilution methods, modified
the labelled end products, the amount of DNA (ng/µl) was divided
from instructions given by the antibiotic disc manufacturer and
by the length of the PCR product (base pair) multiplied by the
NCCLS. In vivo feeding trials with four healthy volunteers were
average weight of a base pair (ng/base pair). The hybridisations
performed to see whether the L. brevis strains could survive the GI
were performed in commercial streptavidin-coated microtiter plates
passage. Technological suitability of the strains for dairy processes
(Labsystems, Finland). Results were analysed by measuring
was also evaluated as described in Study V.
absorbance at 450 nm.
Real-time PCR (IV) RESULTS
Real-time PCR was tested with two different chemistries (5´- Strain-specific Genetic Labelling of Lactobacillus Helveticus
nuclease- and SYBR Green I assays) as described in Study IV. CNRZ32 (I)
Quantitative PCR was performed with an iCycler iQ and iCycler Silent mutation positions were introduced in adjacent codons
Optical System Interface software version 2.3. All PCR reactions of the Lactobacillus helveticus CNRZ32 chromosomal pepX gene
were performed in triplicate, in a volume of 25 µl, using 96-well without altering the amino acid sequence of the gene product,
248 Introductory Food Microbiology Modelling the Growth Boundary of Staphylococcus Aureus 249

creating a strain-specific tag which enabled direct nucleic-acid selected intestinal bacteria, lactobacilli and bifidobacteria. For this
level identification of the changed strain. The mutation site was purpose, species- or group-specific oligonucleotide probes for
introduced into the pepX gene with a PCR primer containing lactobacilli and selected intestinal bacteria were designed or
designed mismatched nucleotides. The target gene was amplified adapted from the literature. Specificity of the probes was tested
in two separate reactions to obtain a conserved fragment and a with 25 species. The 16S and 23S region of these test species was
mutation site-containing fragment, which were ligated together to first PCR-amplified with universal eubacterial primers, followed
form a single mutation site-containing fragment. The mutant gene by digoxigenin-labelling of the PCR products and addition of 2 ×
was first cloned with pUC19 to E. coli to create plasmid pKTH5052 1010 labelled molecules to each hybridisation reaction. For
and further subcloned to pSA3, resulting in the plasmid pKTH5053, hybridisation, the oligonucleotide probes to be tested were bound
which was then used to transform L. helveticus CNRZ32. to the microtiter plate wells via biotin-streptavidin linkage. Under
A L. helveticus CNRZ32 transformant strain GRL1021 the hybridisation conditions used, the specificity level expected
containing the plasmid pKTH5053 was grown at a restrictive was obtained with most of the oligonucleotide probes chosen.
temperature (45ºC) under antibiotic selection to find clones with Only the differentiation of closely related L. casei, L. paracasei and
the thermosensitive plasmid integrated into the bacterial L. rhamnosus was partly unsatisfactory. Furthermore, in addition
chromosome through homologous recombination between the wild- to its intended target DNA, the F. nucleatum probe, fuso16S, also
type and mutant forms of pepX. An erythromycin-resistant strain, recognized E. biforme. Sensitivity of the PCR-ELISA with the probes
observed to contain the integrated plasmid, was grown at tested varied between detection of 6.77 × 107 and 1.29 × 109 PCR-
permissive conditions (37ºC) for approximately 100 generations to amplified molecules in a hybridisation reaction. The sensitivity
enable a second homologous recombination event between the obtained was somewhat lower than expected, which was at least
two pepX genes, resulting in excision of the plasmid and one of partly due to compromises that had to be made in the hybridisation
the pepX genes. Samples were plated out and colonies that had conditions when using several probes simultaneously.
lost antibiotic resistance were screened with PCR for replacement PCR biasing was studied by combining genomic DNA from
of the wild-type pepX with the mutated pepX. A strain with the different test species into two mixed sample pools, each with
mutated gene was found and designated as GRL1023. Sequencing seven DNA targets, prior to PCR amplification. PCR was also
was used to confirm the correctness of the integration construct. performed using the pools in the same amplification.
The intact phenotypic properties of the GRL1023 were verified by The amplification products were hybridised with 14 different
measuring the activity of the target gene product PepX by utilising probes. Most of the probes tested performed well and could detect
its ability to break down L-glycyl-Lprolyl- p-nitroanilide, with their target DNA from mixed samples of both seven and fourteen
production of colour. End-point PCR with PCR primers targeting species, but the probes for B. adolescentis (ado440, b162), L. brevis
the mutated or original pepX sequence was successfully used to (lab86), L. paracasei (par160), L. plantarum (pla448) and L. curvatus
detect the wildtype and mutant strains added to faeces or milk. (cur150) failed to recognize their target DNAs. Failure in the
detection of B. adolescentis, L. paracasei and L. plantarum in particular
Suitability of PCR-ELISA for Analysis of Mixed DNA Populations
could have been due to either bias during multi-template PCR,
(II, III)
where some templates are amplified more efficiently as a result of
PCR-ELISA with Universal Primers Targeting the 16S and 23S differences in the specificity of eubacterial primers, or bias by
Genes (II) chance. Changing of the total template concentrations tested in the
A PCR-ELISA method was tested with 16 oligonucleotide PCR amplification was not observed to affect the hybridisation
probes to simultaneously detect under standardised conditions results.
250 Introductory Food Microbiology Modelling the Growth Boundary of Staphylococcus Aureus 251

PCR-ELISA with bifidobacterial primers targeting the 16S genes PCR could detect addition of 106 cells to one gram of faeces,
(III) The PCR-ELISA method was extended for detection of the provided that the target bacterium was not included in the original
most common Bifidobacterium species in humans and applied to a faecal sample. Addition of 109 cells of Bifidobacterium longum to
feeding trial including administration of Bifidobacterium lactis Bb- faeces resulted in no difference compared with control sample,
12 and galacto-oligosaccharide -containing syrup as probiotic and which had no added bacterial cells, due to the pre-existence of B.
prebiotic preparations, respectively. longum in the original sample.
Oligonucleotide probes based on 16S rDNA sequences were The two chemistries used in real-time PCR were equally
designed and tested for specificity and sensitivity with nine sensitive; however, successful usage of the intercalating dye SYBR
different bifidobacterial species, followed by analysis of faecal Green I required applying of a hot start polymerase to prevent
samples. Faecal bifidobacteria were monitored for fluctuations formation of primer dimers, whereas with 5´-nuclease chemistry,
during and after the feeding trial. Although the bifidobacterial a similar sensitivity level was reached with a conventional DNA
populations present in faeces were generally consistent between polymerase. Furthermore, the 5´-nuclease chemistry was faster to
samples taken from different time points, PCR-ELISA results perform because of its more straightforward incubation protocol,
suggested that Bifidobacterium longum was partly replaced by B. one run taking approximately 80 min.
lactis Bb-12 during the probiotic feeding. This proposition could
not, however, be verified with statistical methods. Generally, the Suitability of L. brevis Strains as Dairy Supplements (V)
bifidobacterial species composition of individual subjects was Two Lactobacillus brevis strains, ATCC 8287 and ATCC 14869T,
consistent and comprised 3-4 bifidobacterial species or groups. were evaluated for their applicability as putative probiotics in
The predominant species were Bifidobacterium longum and B. dairy products. The strains tolerated well low pH, bile acids and
adolescentis. pancreatic fluid under in vitro conditions. They also expressed
good in vitro adherence to human CacO2 and Intestine-407 cells.
Comparison of Real-time PCR and Dot Blot Hybridisation for Adherence of the L. brevis strains was especially strong to the
Quantification of the 16S Ribosomal DNA of Target Bacteria small intestinal cell line, Intestine 407. In antimicrobial activity
(IV) assays, strain ATCC 8287 showed inhibitory properties towards
Real-time PCR was tested with two chemistries: SYBR Green selected potentially harmful micro-organisms, particularly against
I and TaqMan probes. Six detection assays were tested for their Bacillus cereus. Antimicrobial resistance tests revealed that both L.
sensitivity in detecting target bacterial DNA from pure and mixed brevis strains were resistant to vancomycin as well as to several
samples with both chemistries. The same target DNAs were also other antibiotics, which is, however, typical for the genus
quantified with dot blot hybridisation probes. Real-time PCR was Lactobacillus. The L. brevis strains were unable to acidify milk to
shown to have sensitivity superior to dot blot hybridisation. The yoghurt but were suitable as supplement strains in yoghurts. This
linear range of amplification of pure cultured target DNA varied was demonstrated by producing a set of yoghurt products and
between 0.1-1 pg and 1-10 ng of specific target genome, which analysing their rheological and sensory properties during a cold
corresponds to approximately 20-400 to 2×105–4×106 genomes, storage period of 28 days. Despite its human origin, L. brevis
depending on the genome size of the target species. Furthermore, ATCC 14869T did not survive the gastrointestinal tract, whereas
a 1 pg addition of target DNA to 20 ng of mixed DNA samples L. brevis ATCC 8287 was detected in faecal samples taken during
could be detected reliably with both real-time PCR chemistries. A and immediately after ingestion of the strain. In conclusion, L.
reconstruction assay with addition of 109, 108, 107, 106 or 105 brevis ATCC 8287 was considered a promising candidate for use
target bacterial cells to faecal samples confirmed that real-time as a probiotic adjunct in dairy products.
252 Introductory Food Microbiology Modelling the Growth Boundary of Staphylococcus Aureus 253

DISCUSSION with groupspecific 16S rDNA primers, however, characterisation


Molecular biology tools are needed for better understanding of of diversity of smaller subpopulations has been reached. Similarly,
the composition and functions of the intestinal microbiota. In this the bifidobacteria-specific PCR-ELISA could successfully be used
study, different levels of detection were developed for lactic acid for species-level analysis of faecal bifidobacteria; previous DGGE
bacteria and faecal microbiota. analysis results of the same samples by Satokari et al. (2001b)
generally support results obtained with PCRELISA. One drawback
‘Probiotic studies require careful documentation of the
of PCR-ELISA identification has, however, been uncovered. When
proposed effects. For better monitoring of a potential strain, strain-
results were compared with PCR-ELISA analysis, it soon became
specific discrimination could be beneficial in, for example, feeding
evident that ado440, intended to be specific for B. adolescentis, also
studies. Furthermore, specific recognition of industrial strains with
recognized B. ruminantium observed in the faeces of some volunteers.
commercial value would help in monitoring the usage of these
New sequence alignments confirmed that the ado440 probe had
strains. Therefore, Lactobacillus helveticus CNRZ32 was chosen as
no mismatches with B. ruminantium 16S rDNA. While the
a model organism for demonstration of genetic labelling. Although
advantage of DGGE is the possibility for further sequence analysis
the strain in question does not fulfil the criteria set for a probiotic,
and identification of emerging new amplicons in DGGE profiles,
selection of this organism was considered justified for several
PCR-ELISA seems to be slightly more sensitive to changes in the
reasons. The strain is a starter strain used in the dairy industry,
relative amounts of target species.
its proteolytic system has been studied extensively and methods
for its transformation and gene disruption have been developed. Strain-level studies have confirmed the complexity, uniqueness
Various ‘food-grade’ markers for lactic acid bacteria have been and stability of bifidobacterial populations in the GI-tract. PCR-
suggested, often with introduction of new phenotypic properties. ELISA results were in accord with earlier reports. The observed
However, creation of a label without introduction of any species distribution correlated well with previous European
phenotypic changes into the target strain would be more easily studies, and species distribution usually remained unchanged
accepted by consumers and legislators. Hertel et al. (1992) labelled within the samples of each subject. However, reduction of intensities
a Lactococcus lactis strain by introducing silent mutations into a of B. longum - specific probes was observed frequently in the
plasmid-encoded gene. By contrast, to ensure the stability of the groups that ingested the probiotic B. lactis Bb-12, although this
created tag, we used a chromosomal gene for labelling of L. could not be verified with statistical analysis, mainly due to the
helveticus CNRZ32. As demonstrated in Study I, a silent mutation small size of the test groups. This suggests that B. lactis Bb-12 may
site is effortlessly detected from different matrices, such as faeces have partly replaced B. longum during the ingestion period.
or milk, by PCR. Alternatively, hybridisation probes could be Hybridisation allows direct semi-quantitative monitoring of
utilised for detection. population samples and thus avoids the problems caused by PCR
Direct detection of target nucleic acids present in population bias. Dot blot hybridisation with rRNAtargeted oligonucleotide
samples removes the need for cultivation steps. Here, PCR-ELISA, probes is a well-established method for studying faecal microbes.
dot blot hybridisation and real-time PCR were compared for their Ribosomal RNA is abundantly present in the bacterial cells, thus
suitability for analysis of population samples. Similar to PCRELISA increasing the sensitivity of the dot blot hybridisation to a
method described in this paper, DGGE- and TGGE-based reasonable level. However, radioactively labelled probes are
applications have been found to detect approximately 1-10% generally needed for successful detection of nucleic acid targets
subpopulations with PCR-amplified 16S rDNA fragments when from population samples. Here, dot blot hybridisation was
studying faecal samples, suggesting their use for analysis of performed with rDNA-targeted oligonucleotide probes to quantify
predominant microbes of population samples. By using DGGE the same starting material as with the real-time PCR applications
254 Introductory Food Microbiology Modelling the Growth Boundary of Staphylococcus Aureus 255

tested. Dot blot hybridisation could, at its best, detect a 3% DNA Furthermore, the relative sensitivities could not be extrapolated
subpopulation from mixed DNA samples. A similar sensitivity from DNA extractions performed directly from pure cultures.
level has been reported by Muttray and Mohn (2000) for Evaluation of possible probiotic properties of a bacterial strain
measurement of rDNA:rRNA ratios. Thus, the result was as requires intensive and carefully planned testing of the strain both
expected, being approximately 10-fold less sensitive than that in vivo and in vitro. Although L. brevis is not typically used as a
obtained with rRNAtargeted oligonucleotides. Real-time PCR was probiotic, Kishi et al. (1996) have shown that oral administration
tested as an alternative for a sensitive detection of target bacteria of live L. brevis ssp coagulans strain significantly stimulated the
in faeces. The sensitivity level obtained for the real-time PCR host immunity system by increasing IFN-á production in human
assays was 200-400 target bacteria in pure cultures or mixed DNA subjects. This result cannot, however, be directly extrapolated to
samples, depending on the genome size of a target bacterium. the L. brevis strains studied here. Adherence of lactic acid bacteria
Results obtained from artificial DNA mixtures were confirmed by to various components of human intestine has been studied with
a reconstruction assay; addition of 106 bacterial cells to faecal the human colon adenocarcinoma CacO2 cell line, Intestine-407
samples containing approximately 1011 bacteria could be detected cells derived from human embryonic jejunum and ileum, and
with real-time PCR in a quantitative manner. The acquired level, intestinal mucus. Both CacO2 and Intestine-407 cells were used in
providing a means for quantifying a 0.01% subpopulation in a the adhesion assays for evaluation of the adhesion ability of L.
DNA sample, was considered sufficient for studying the brevis. These cell lines offer complementary models for adhesion
gastrointestinal tract ecology. studies. When compared with the probiotic L. rhamnosus GG,
Although nucleic acid based detection methods offer a means adherence of the L. brevis strains was especially strong to the small
to avoid bias caused by cultivation or phenotypic testing of bacterial intestinal cell line, Intestine 407, suggesting that the small intestine
isolates, these techniques are not totally devoid of problems. The could be affected with the strains studied. L. brevis GRL62 has also
main issues for detection of nucleic acids in mixed samples are been shown to have intermediate adhesion properties to human
isolating DNA from different sources with constant efficiency, intestinal mucus.
minimising the occurrence of false-positive reactions due to The two L. brevis strains studied were antagonistic towards
contamination or poor planning of the primers or hybridisation some of the potentially harmful micro-organisms, while they did
probes used, and avoiding negative reactions due to inhibiting not significantly inhibit the growth of yoghurt-starter bacteria. In
substances. Also, any oligonucleotides used as detection probes or a previous study of Koga et al. (1998), L. brevis GRL1 (ATCC 8287),
primers should be considered specific only in relation to the current included in a panel of 41 Lactobacillus strains belonging to L.
sequence data. Several methods for faecal sample treatment for gasseri, L. reuteri, L. casei, L. helveticus, L. brevis, L. fermentum and L.
isolation of DNA have been described. Nevertheless, this step plantarum, weakly inhibited two Vibrio cholerae strains of the eight
remains somewhat problematic because of the presence of PCR- strains tested. In the present study, L. brevis GRL1 was shown to
inhibiting substances in the faeces and the overall complexity of strongly inhibit B. cereus and to some extent also the growth of S.
faecal microbial populations. Although over 90% recovery of the aureus and other harmful micro-organisms chosen for the assay. In
total bacterial DNA present in faeces has been claimed, non- vitro adhesion assays and testing for tolerance of low pH, bile
selective isolation of bacterial DNA from population samples is acids and pancreatic fluids have often been considered to be good
difficult to achieve or demonstrate. Commercial kits have recently indicators of the survival of a bacterial strain through the GI tract.
become available for isolation of faecal DNA. However, McOrist In this study, both L. brevis strains performed well in the in vitro
et al. (2002) observed differences in the relative efficacy of extraction tests, and survival through the stomach could therefore be
of bacterial DNA from faeces with four commercial kits. suggested for both strains, especially when simultaneous ingestion
256 Introductory Food Microbiology Modelling the Growth Boundary of Staphylococcus Aureus 257

of the bacteria with food, resulting in a higher pH in the stomach, silent labelling method should be the least unacceptable method
is taken into account. However, L. brevis GRL62 failed to survive for making genetic alterations to a bacterial strain used in food
under in vivo conditions. This was surprising as the strain has processing, especially as the phenotypic properties of the labelled
originally been isolated from human faeces. In contrast, L. brevis strain were demonstrated to remain unchanged, thus attesting to
GRL1, originally isolated from fermented olives, could tolerate the the safety of the method. PCR-ELISA with universal primers was
gastrointestinal conditions and was found to persist in the human shown to be suitable for detection of predominant bacteria present
gut. in a mixed population sample. Specificity of the PCR step proved
Although the strains studied were not suitable for fermenting to be critical for the sensitivity of the method; application of
yoghurt, supplementation of yoghurt with either of these strains bifidobacterialspecific 16S primers enabled species- or group-
had no negative effects on yoghurt taste, appearance, or specific detection of bifidobacteria present in human faeces. The
preservation. Therefore, L. brevis GRL1, which was able to survive overall sensitivity of PCR-ELISA matched DGGE approaches, with
in the GI tract, could be considered for use as a supplementary similar PCR primer specificities. Furthermore, being technically a
strain in yoghurt or other dairy products. To date, several rather simple method, PCR-ELISA is easily transferred between
commercial probiotics are already available and many others are different laboratories, and could, in principle, be used in a similar
being studied, but no single strain is likely to possess all of the manner as DGGE for analysis of large sample numbers.
beneficial properties suggested for probiotics. L. brevis GRL1, shown Real-time PCR was established to be a superior method for
to be antagonistic towards Bacillus cereus (this study) and Vibrio detection of single bacterial species or larger groups from mixed
cholerae, could be a valuable addition to the probiotics field. The DNA samples or faeces. Compared with dot blot hybridisation
surface (S)-layer of this strain is well-characterized and potential with radioactive probes, real-time PCR provided an improved
applications for the S-layer have been suggested. Recently, the S- sensitivity for detection, a means to avoid usage of radioactive
layer has been shown to mediate the ability of the L. brevis GRL1 labels, increased speed and volume of sample analysis, an easier
strain to adhere to human intestinal, urinary bladder and methodology and better reproducibility. Detection of target bacteria
endothelial cells. Good adhesion properties could assist in the added to faeces prior to the DNA isolation and purification steps
competitive exclusion of potentially harmful microbes by L. brevis was demonstrated. In future, multiplexing with different labels
GRL1, and usage of this strain as an effective mucosal vaccination and improvements in hardware and software to allow
tool because of its binding properties may be warranted. More simultaneous monitoring of a large number of PCR reactions are
studies are, however, required to confirm these hypotheses. likely to make real-time PCR an even more potent tool for
population analyses.
CONCLUSIONS
L. brevis GRL1 could be considered to be a potential probiotic
Creation of a strain-specific nucleic acid tag by labelling of a strain. Although several probiotics are already on the market, new
chromosomal target gene with a silent mutation and utilisation of strains with beneficial properties are continually being sought.
the label for strain-specific detection of the target strain was The strain in question has a well-characterized S-layer, and offers
demonstrated using Lactobacillus helveticus CNRZ32. Although the promise as a potential mucosal vaccination strain.
strain in question is not considered to be a probiotic, the method
described here could be used for probiotics. However, this requires
the pre-existence or development of suitable transformation tools
as well as sequence data of a suitable target gene. From the
viewpoint of current legislation and consumer acceptance, the
258 Introductory Food Microbiology Glossary 259

others cannot tolerate oxygen and are killed when exposed to air.
Such organisms are termed obligate anaerobes.
Antibody: An inducible immunoglobulin protein produced by
B lymphocytes of the immune system, in humans and other higher
animals, which recognizes and binds to a specific antigen molecule
9 of a foreign substance introduced into the organism. When
antibodies bind to corresponding antigens they set in motion a
process to eliminate the antigens.
GLOSSARY Antigen: Any foreign substance, such as virus, bacterium, or
protein, which after introduction into an organism (humans and
higher animals), elicits an immune response by stimulating the
Acid dyes: Dyes that are anionic or have negatively charged production of specific antibodies; or any large molecule which
groups such as carboxyls. Acid fast: Bacteria like the mycobacteria binds specifically to an antibody.
that cannot be easily decolorized with acid alcohol after being
Antimicrobial: A chemical agent that kills microorganisms or
stained with dyes such as basic fuchsin.
inhibits their growth.
Acid-fast staining: Staining procedure that differentiates
Apoptosis: Programmed cell death, the body’s normal method
between bacteria based on their ability to retain a dye when washed
of ending the lifecycle of cells through the cellular self-destruction.
with an acid alcohol solution.
When either heritable or somatic cell mutations cause malfunctions
Acidophile: Microorganism that has its growth optimum to occur in the apoptotic pathway, uncontrolled cell growth may
between about pH 0 and 5.5. proceed unchecked and cancer may result.
Actinobacteria: Group of gram-positive bacteria containing Bacterial growth: Can exhibit at least four different phases:
the actinomycetes and their high G 1 C relatives. lag phase, growth phase, stationary phase and death phase.
Actinomycete: Aerobic, gram-positive bacterium that forms Bacterial strain: Population of bacterial cells all descended
branching filaments (hyphae) and asexual spores. from a single pure isolate.
Aerobe: The descriptive name given to a microorganism that Base pair (bp): Two complementary nitrogenous bases in a
can grow in conditions where oxygen is present. Such organisms DNA molecule, such as the nucleotide coupling of adenine with
are capable of growing in normal air, equivalent to 20% oxygen, thymine (A:T) and guanine with cytosine (G:C); also, a unit of
or in aerated liquids containing dissolved oxygen. Many aerobes measurement for DNA sequences.
are equally able to grow in the absence of oxygen. These are Biofilm: Adherent layer of bacteria and/or other
termed facultative anaerobes. microorganisms on a solid surface bound together in a bacterially-
Allele: One of two or more alternative nucleotide sequences at derived polysaccharide matrix that is protective for the organisms;
a single gene locus which occurs on either of two homologous generally occuring at a liquid/solid interface and often developing
chromosomes in a diploid organism. into a complex ecological community (e.g., dental plaque bound
Anaerobe: A microorganism that is capable of growing in the tother by dextrans).
complete absence of oxygen. Some of these organisms may also be Bleaching: The loss of fluorescence usually due to
able to grow in oxygenated conditions (facultative aerobes), whereas photochemical reactions.
260 Introductory Food Microbiology Glossary 261

cDNA (complementary or copy DNA): DNA copies (e.g., with a fluorescent or radioactive tag) in order to detect its
synthesized from a messenger RNA template using the enzyme incorporation through hybridization with DNA in a sample.
reverse transcriptase; the single-stranded copy is often used as a Dot blot: A method for detecting proteins by the
probe to identify complementary sequences in DNA fragments or specific binding of an antibody or binding molecule to a
genes of interest. sample spot on nitrocellulose paper. The bound sample is
Chromosome: A single DNA molecule that is the self- visualized using an enzymatic or fluorimetric reporter conjugated
replicating genetic structure within the cell which carries the linear to the probe.
nucleotide sequence of genes. In humans (or eukaryotes), the DNA Doubling time: The time taken for a population to increase in
is supercoiled, compacted, and complexed with accessory proteins, number by a factor of two.
and organized into a number of such structures. Normal human
Enteric (entero-): Relating to the intestine.
cells contain 46 chromosomes (except the germ cells, egg and
sperm): 22 homologous pairs of autosomes and the sex-determining Enterotoxin: Proteins produced by bacteria that are either
X and Y chromosomes (XX for females and XY for males). ingested as pre-formed toxins or are produced by a pathogen that
Prokaryotes carry their entire genome on one circular chromosome has colonised the gastro-intestinal tract. Usually the toxin has
of DNA. specific targets and either disrupts cell function or kills the cell.

Coccus: (singular): Spherical-shaped cell; cocci (plural). Eukaryote: (meaning “true nucleus”) An organism which
possesses a nucleus with a double layer of membrane and other
Coliform bacteria (coliforms): Any fermentative (specifically membrane-bound organelles; includes such unicellular or
lactose-fermenting) Gram-negative anaerobic enteric bacilli (E. coli- multicellular members as all members of the protist, fungi, plant,
like). and animal kingdoms.
Commensal: Organisms existing in or on an animal or human Exons: The segment of a gene present in mature mRNA
without causing disease. transcripts that specify the amino acid sequence of a polypeptide
Conjugation (or bio-conjugation): The chemical joining of a during translation; exons of a gene are linked together by mRNA
biomolecule to another. splicing.
Death phase: The death phase occurs when cells are being Exotoxin: Potent toxic substance formed and released
inactivated or killed because conditions no longer support growth extracellularly by species of certain bacteria.
or survival. Some environmental factors such as temperature can Exponential phase: The period in which the cells of a defined
cause acute inactivation. Others may cause mild inactivation as bacterial population are growing and dividing continuously.
with growth in the presence of organic acids.
Extracellular: Produced, then excreted outside the organism.
DNA (deoxyribonucleic acid): The nucleic acid molecule
Facultative: Ability to adapt and live under various conditions.
consisting of deoxyribonucleotide building blocks that encode
genetic information. The genome of most organisms is contained Facultative anaerobe: Anaerobe that can survive with or
in a double-stranded, double-helical form held together with without oxygen.
chemical bonds between each strand of complementary nucleotide Family: Taxonomic level below order and above genus.
base pairs. Fastidious: Complex nutritional or cultural requirements,
DNA probe: A single-stranded piece of DNA that binds making isolation and culture of a fastidious organism more
specifically to a complementary DNA sequence; the probe is labeled difficult.
262 Introductory Food Microbiology Glossary 263

Fermentation: Enzymatic breakdown (catabolism) of Gene mapping: A linear map determining the relative position
carbohydrates generally in the absence of oxygen. of genes along a chromosome or plasmid. Distances are established
Fimbriae: Short, hair-like projections or appendages by linkage analysis and measured in linkage units.
(organelles) on the outer surface of certain bacteria composed of Gene: A nucleotide sequence of DNA that codes for a
protein subunits (pilin) extending outward from the surface that protein, or functional or structural RNA molecule; a locus
act as a virulence factor by promoting adherence; formerly known on a chromosome. The element that determines a trait in an
as pili; fimbria (singular). organism.
Flagellum: whip-like bacterial locomotory (provide motility) Genetic mutation: An alteration in the nucleotide sequence of
organelles anchored in the cell membranes that are composed of a DNA molecule; often from one allelic form of a gene to another
helically-coiled protein subunits (flagellin); flagella (plural). allele alternative.
FISH (Fluorescence In Situ Hybridization): A technique that Genome: The total amount of genetic material in a cell; in
employs fluorescent molecular tags to detect probes hybridized to eukaryotes the haploid set of chromosomes of an organism. The
chromosomes or chromatin; useful for genetic mapping and chromosome set is species-specific for the number genes and linkage
detecting chromosomal abnormalities. groups carried in genomic DNA.
Flow cytometer: Analytical instrument for flow cytometry. Genomics: The study of genes and their biochemical function
Flow cytometry: Automated analysis of cells or subcellular in an organism.
components by detection of the fluorescence or light-scatter of Genotype: The genetic constitution of an organism; or, a
sample fractions passing in narrow-stream droplets through a reference to an individual’s particular allele pair at a specific gene
laser beam. locus in the genome.
Fluorophores: Molecules that produce a fluorescent Genotyping: Analysis of genotype.
emission when irradiated with light at a suitable excitation
wavelength. Genus: Taxonomic level below Family and above Species.

Fomite: Inanimate object capable of transmitting infectious Gram-negative or Gram-positive: The classification given to
organisms to a host, e.g., soiled clothes, tissues and handkerchiefs, bacteria according to their staining properties as defined by the
food processing equipment, dishrags, etc. Gram stain procedure.

Gene amplification: The presence of multiple copies of a gene Growth curve: A graph displaying the behaviour of a bacterial
or segment of DNA; a mechanism by which proto-oncogenes are population over time.
activated in malignant cells. A tumor cell amplifies, or copies, Growth phase: During the growth phase, cells grow
DNA segments as a result of cell signals or the effects of exponentially and at a constant rate. The maximum slope of the
environmental insults. curve is the specific growth rate of the organism. Cell growth is
Gene expression: The process by which the encoded dependent upon the current environment (nutrients, temperature,
information of the genome is converted into cellular components. pH, etc.), but is not dependent upon the previous physiological
The DNA-coding sequences of expressed genes include those that state. In the field of predictive microbiology, growth rate is
are transcribed into mRNA and then translated into proteins, and commonly expressed as the change in cell number per time interval.
RNA that is transcribed from DNA, yet not translated into protein Growth rate: This is an expression of population increase in
(i.e., transfer and ribosomal RNAs). numbers expressed as log10 cfu/hour.
264 Introductory Food Microbiology Glossary 265

Haploid: A cell or individual with a genetic complement depends on the current environment as well as the previous
containing one copy of each nuclear chromosome. (Diploid refers physiological state of the cells. Cells that are from a very different
to the condition when a eukaryotic cell possesses two sets of environment or are damaged from their previous physiological
chromosomes.) state may require more time to adjust. In some foods a lag phase
Humectant: A solute that binds free water in a food, reducing does not exist which results in cells that are ready for immediate
the amount of water available to the microorganisms. growth.
Hybridize (or hybridization): The process where the hydrogen Lag time: The initial period in a bacterial population life
bonding of complementary DNA and/or RNA sequences forms a when cells are adjusting to a new environment before commencing
duplex molecule. growth.
Immunoassays: A technique that detects and measures a Legionella: Fastidious gram-negative rod is isolated from
specific antigen or biological substance by employing antibodies surface water, mud, and thermally polluted lakes and streams.
(e.g., dot blot, western blot, and ELISA). There is no known soil or animal source. It is pathogenic for
humans, causing pneumonia (Legionnaires’ disease) or a mild,
in situ hybridization (ISH): Use of a nucleic acid probe to
febrile disease (Pontiac fever).
detect and identify specific complementary sequences of DNA in
chromosomes or RNA in bacteria, eukaryotic cells, and tissue. Ligand: The molecule which binds to a protein molecule (e.g.,
receptor). As a ligand binds through the interaction of many weak,
in vitro: (“in glass”) Refers to the recreation of biological
noncovalent bonds formed to the binding site of a protein, the
processes in an artificial environment such as a test tube.
tight binding of a ligand depends upon a precise fit to the surface-
in vivo: (“in living”) Refers to biological processes within a exposed amino acid residues on the protein.
living organism or cell.
Listeria: Gram-positive rod widely distributed in the
Inoculum: A medium containing microorganisms to be environment. Some species are pathogenic for humans and animals
introduced into fresh media or food source in an experiment. (e.g. L. monocytogenes).
Intron: A nucleotide sequence intervening between exons Locus: (plural, loci) The specific site of a gene on a genetic
(coding regions) that is excised from a gene transcript during map or chromosome.
RNA processing.
Marker (genetic marker): Any genetically derived phenotypic
Kinetics: The properties of chemical agents or enzymes in the difference used in the analysis of inheritance patterns or to
efficiency and speed of their action upon a chemical reaction. differentiate between types of cells. An observable site on a
Klebsiella: Gram-negative rods occur in human feces and chromosome that is heritable and can be either a genetically-
clinical specimens, soil, water, grain, fruits, and vegetables. Some expressed region or noncoding segment of DNA (intron).
species are opportunistic pathogens. Kluyvera: Gram-negative rods Maximum population density: Point at which the maximum
occur in food, soil, sewage, and human clinical specimens. They number of bacterial cells can exist in an environment.
are infrequently opportunistic pathogens. Kurthia: Gram-positive
Meiosis: Process that allows one diploid celll to divide in a
rods are widely distributed in the environment, and are common
special way to generate haploid cells in eukaryotes.
in animal feces and meat products.
Mesophile: Microorganism able to grow well between 20°C
Lag phase: During the lag phase, cells increase in size but not
and 45°C, having an optima of 30°C to 40°C. Many can sustain
in number because they are adapting to a new environment, and,
growth at below 10°C albeit very slow growth.
synthesis and repair are taking place. The length of the lag phase
266 Introductory Food Microbiology Glossary 267

Metaphase: Stage in mitosis or meiosis during which Northern blot: Technique used to separate and transfer mRNA
the chromosomes are aligned along the equatorial plane of the from a gel to a filter in order to identify and locate mRNA sequences
cell. that are complementary to and hybridize with a labeled DNA
Methylobacterium: Mostly isolated from water and leaf surface probe.
microflora, and are facultative methylotrophs, that is capable of Novosphingobium: Gram-negative rods were originally
growing on one-carbon compounds such as formate, formaldehyde, included with Sphingomonas (see Sphingomonas).
and methanol as the sole source of carbon and energy, as well as Nucleic acid: Molecule composed of nucleotide subunits. See
on a wide range of multicarbon substrates. DNA and RNA.
Microaerophilllic environment: Environment with reduced Nucleotide: Basic building block of nucleic acids that is a
oxygen concentrations, often below 5%. Carbon dioxide levels monomeric molecule of DNA or RNA composed of: a pentose
may approach 10%. sugar (with 5-carbons such as deoxyribose in DNA, ribose in
Microbacterium: Gram-positive rod is found in dairy products, RNA), an organic nitrogenous base, and a phosophate group.
sewage, and insects. DNA consists of the four bases: adenine (A), guanine (G), cytosine
(C), and thymine (T); likewise for RNA, except for the substitution
Microbial load: Total number of living microorganisms in a
of uracil (U) for T.
given volume or mass of microbiological media or food.
Oligonucleotide: (“oligo” means few) A short length of DNA
Micrococcus: Gram-positive cocci occur primarily on
nucleotides, often used as primers for DNA synthesis or probes for
mammalian skin and in soil, but are commonly isolated from food
arrays and ISH/FISH; usually referred to as “oligo(s).”
products and the air.
Opportunistic: Microorganism that will only cause disease in
Microorganism: A living organism too small to be seen with
a patient with a poor or somehow weakened immune system.
the naked eye.
Organelle: Microscopic bodies in the cytoplasm of cells that
Mitosis: Process by which a cell separates its duplicated have distinctive functions (e.g., nucleus, mitochondria,
genome into two identical halves. It is generally followed endoplasmic reticulum, etc.).
immediately by cytokinesis which divides the cytoplasm and cell
membrane. This results in two identical daughter cells with a Pasteurisation: Mild heat treatment process given to foods.
roughly equal distribution of organelles and other cellular The process is designed to eradicate potential vegetative pathogens
(not bacterial spores) and reduce other microorganism numbers in
components.
an effort to decrease the rate of spoilage.
Moraxella: Gram-negative rod is parasitic on the mucous
Pantoea: Gram-negative rods are isolated from plant surfaces,
membranes of humans and other warm-blooded animals.
seeds, soil, and water, as well as from animals and human clinical
MRNA (messenger RNA): The RNA molecule, transcribed specimens. They are opportunistic human pathogens.
from the DNA of a gene, which serves as a template and encodes
Pathogen: Microorganism associated with disease in man.
the amino acid sequence of a protein.
pH: Measure of the acidity or alkalinity of a solution, defined
Multiplexing: Method by which many parameters are tested
as the - log10 of the hydrogen ion concentration.
and processed simultaneously.
Phenotype: Observable manifestation of a genetic trait,
Native microflora: Microorganisms that are normally found
resulting from a specific genotype and its interaction with the
within a food source (often referred to as spoilage organisms).
environment.
268 Introductory Food Microbiology Glossary 269

Physical map: Map indicating physical locations on a DNA Psychrobacter: Gram-negative rod is associated with fish,
molecule such as restriction enzyme recognition sites, RFLPs, and processed meat and poultry products. Some strains have been
genes; measured in base pairs (bp). isolated from pathological specimens from humans and animals.
Predictive microbiology: Area of food microbiology that uses Psychrotroph: Microorganism able to grow well between 0°C
mathematical models to define growth kinetics of microorganisms and 7°C, having an optima of 20°C to 30°C.
in food. Rahnella: Gram-negative rods occur in freshwater. They are
Primary model: Model that describes changes in microbial occasionally isolated from human clinical specimens, but are not
numbers in response to time. considered clinically significant.
Primer: Short segment of DNA or RNA that anneals to Ralstonia: see Pseudomonas.
a single strand of DNA in order to initiate template- Raoultella: see Klebsiella.
directed synthesis and extend a new DNA strand by the Rathayibacter: Some of these species are phytopathogens of
enzymatic action of DNA polymerase to produce a duplex- terrestial plants. Their main habitats are their respective plant
stranded molecule. hosts.
Probe: Single-stranded DNA or RNA molecule of specific base Receptor: Surface-exposed membrane protein on a cell which
sequence, either radioactively or fluorescently labeled, that is used binds to a specific ligand molecule with high affinity, in order to
to identify the complementary nucleotide sequence by transmit an extracellular signal and trigger intracellular
hybridization to the DNA fragment or gene of interest. biochemical events within the target cell.
Protein translocation: Spatial movement of protein within a Reporter: Gene which codes for an easily measured protein
cell (e.g., from the cytoplasm to nucleus, or into organelles). product and is fused downstream of the gene of interest in order
Protein: High-molecular weight biological molecule composed to assess the activity in the region upstream of the reporter gene.
of a polymer of amino acids linked via peptide bonds; may consist Colorimetric and fluorimetric reporters also can be conjugated to
of more than one polypeptide molecule that is folded into complex probes to monitor biological events.
shapes such as helices or sheet-like structures. Proteins are encoded Rhodococcus: Aerobic, Gram-positive actinomycetes. These
by the specific sequence of DNA nucleotides in a gene and give widely-occurring organisms are of considerable environmental
rise to the structure, function, and regulation of cells, tissues, and and biotechnological importance due to their broad metabolic
organs within the body. Protein classes include enzymes, diversity and array of unique enzymatic capabilities, plus their
antibodies, receptors, hormones, and growth factors. capacity to degrade hydro-carbons. They are able to survive for a
Proteome: Entire protein make-up of a particular organism. long time in soil. They are the most efficient in oil degradation
and, relatively speaking, the most abundant in soils and marine
Proteomics: Study of proteins and their biochemical function environments.
in an organism.
Rhizobium: Group of small, rod-shaped, gram-negative
Providencia: Gram-negative rods are isolated from human bacteria, which are able to produce nodules on the roots, or on
clinical specimens and from penguins. some cases the stems, of leguminous plants.
Pseudomonas: Gram-negative rod is widely distributed in RNA (ribonucleic acid): DNA-like organic molecule that
nature. Some species are pathogenic for humans, animals, or plants consists of nucleotide subunits—such as adenine, guanine,
(e.g. P. aeruginosa). cytosine, and uracil—which contain ribose sugars linked through
270 Introductory Food Microbiology Glossary 271

phosphodiester bonds. Different types of RNA function in the Staphylococcus: This gram-positive coccus is mainly
process of gene expression. associated with the skin and mucous membranes of warm-blooded
Saprophyte: Microorganism that normally grows on dead vertebrates, but they are often isolated from food products, dust
material. and water. Some species are opportunistic pathogens of humans
and animals, or produce extracellular toxins.
Secondary model: Model that predicts changes in primary
model parameters based on environmental conditions. Stationary phase: The stationary phase occurs at the maximum
population density, the point at which the maximum number of
Serratia: Gram-negative rods occur in human clinical
bacterial cells can exist in an environment. This typically represents
specimens, soil, water, plant surfaces, and other environmental
the carrying capacity of the environment. However, environmental
sites, digestive tracts of rodents, and insects. Some species are
factors such as pH, preservatives, antimicrobials, native microflora
opportunistic pathogens.
and atmospheric composition as well as depletion of growth-
SNP (Single Nucleotide Polymorphism): Variations in the limiting nutrients can affect the maximum population density.
sequence of DNA among individuals that are present in humans
Stenotrophomonas: see Pseudomonas.
with a frequency of about once in every 1000 bases, and useful in
assessing the patterns of inheritance in genetic linkage studies. Sub-lethal injury: This is cellular damage which results in
disruption of metabolic processes which, under ideal conditions,
Somatic cell mutation: Mutation in a cell that is acquired
is repairable. Such damage must be repaired before normal growth
during the lifetime of an organism and which cannot be genetically
can recommence.
inherited by offspring.
Tertiary model: Computer software routines that turn the
Somatic cell: Any cell in the body except the germ-line cells
primary and secondary models into “user-friendly” programs.
(sperm or egg cells).
Tsukamurella: Gram-positive rods are isolated from soil,
Southern blot: Procedure which transfers elecrophoretically
human sputum, and parts of bed bugs. Some strains can be
separated DNA fragments on an agarose gel to nitrocellulose
pathogenic.
filters for detection by hybridization with a labeled probe
complementary to the sequence of interest; the position on the Toxins: Compounds produced by a microorganism that are
filter of the probe, when exposed to x-ray film, appears as a band poisonous to other organisms.
on an autoradiogram. Vegetative cell: The vegetative cell state is the form in which
Sphingobacterium: Gram-negative rod found in soil, on plants, an organism is able to grow and divide continuously, given
foodstuffs, and in water sources. Sphingobium: Gram negative favourable conditions. Unlike endospores, vegetative cells are
rods were originally included with Sphingomonas (see relatively poor at surviving environmental stresses such as high
Sphingomonas). temperature and drying.
Sphingomonas: Relatively new genus derived from Water activity (aW): Water activity is a measure of the amount
Pseudomonas paucimobilis. These organisms are widely of free unassociated water molecules in a system. It runs on a scale
distributed, including having been found in water. Only of 0 to 1.0, where pure water equals 1.0. This parameter can also
Sphingomonas paucimobilis is considered clinically significant, be expressed as a percentage referred to as the relative humidity.
and has been isolated from a variety of clinical specimens It is influenced by dissolved solutes and insoluble food components
which act to bind water, thus reducing the available free water
Spoilage organisms: Microorganisms naturally found within
and hence aW value.
a food source that cause food spoilage.
272 Introductory Food Microbiology Bibliography 273

Weeksella: Gram-negative rod is not known in the general


environment. It is apparently a parasite, saprophyte, or commensal
of the internal surfaces of humans or other warm-blooded animals.
Western blot: A technique which transfers proteins
electrophoretically separated in a polyacrylamide gel to a
nitrocellulose membrane and uses specific antibodies to bind, BIBLIOGRAPHY
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Yersinia: Gram-negative rods occur in a broad spectrum of Anderson, Kenneth: International Dictionary of Food & Nutrition.
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Bernauer, Thomas: Genes, Trade, and Regulation: The Seeds of Conflict
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274 Introductory Food Microbiology Index 275

Hays, Judi Radice: Restaurant & Food Graphics. Glen Cove: PBC
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Hidellage, V.: Making Safe Food, IT Publications, London, 1992.
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Ihekoronye, A.I., and Ngoddy, P.O., : Integrated Food Science and INDEX
Technology for the Tropics, Macmillan Press Ltd., London, 1985.
Jay, J.M.: Modern Food Microbiology, D van Nostrand, New York,
A 202, 209, 211, 215, 216,
1978.
Accounts, 4. 219, 230, 231, 232, 233,
Low, J.: Fruit and Vegetables, IT Publications, London, 1984. 234, 235, 236, 237, 238,
Acid Dyes, 258.
Metting, Jr., F.B.: Soil Microbial Ecology: Applications in Agricultural Acidophile, 258. 239, 240, 241, 242, 243,
and Environmental Management, Marcel Dekker, New York, 1992. Actinobacteria, 258. 244, 245, 246, 248, 249,
Ngoddy, P.O., : Integrated Food Science and Technology for the Tropics, Administration, 234, 250, 255. 250, 251, 253, 255, 256,
Macmillan Press Ltd., London, 1985. Anaerobe, 258, 261. 257, 258, 259, 261, 262,
Apoptosis, 84, 259. 263, 264, 265, 271.
Roberts, D.: Food Poisoning and Food Hygiene, Edward Arnold Ltd,
Application, 17, 26, 28, 69, 176, Bioscreen, 31, 32, 48, 60, 71,
London, 1987. 84, 93, 95, 96, 97, 100,
229, 244, 257.
Sackler, Warren: Foodservice Cost Control Using Microsoft Excel for 108, 111, 113, 115, 120,
Approach, 30, 43, 97, 107, 112,
Windows. New York: Wiley, 1996. 121, 129, 158, 236, 238, 123, 124, 126, 132, 140,
Shetty, Kalidas: Food Biotechnology, New York, Dekker/CRC Press, 242, 266. 147, 148, 149, 152, 159,
2005. Association, 20, 69, 70, 76, 79, 161, 162, 163, 164, 167,
175, 178, 196, 272. 175, 203, 220, 221, 222,
Sims-Bell, Barbara: Career Opportunities in the Food and Beverage
Atmospheres, 13. 247.
Industry. New York: Facts on File, 1994.
Biotechnology, 61.
Slater, R.J.: Experiments in Molecular Biology, Clifton, Humana Press, B
1986. C
Bacteria, 1, 8, 11, 12, 13, 14,
Sprenger, R.A.: The Food Hygiene Handbook, Highfield Publications, 26, 30, 39, 46, 49, 50, Campylobacter, 5, 242.
Doncaster, 1996. 53, 55, 56, 57, 58, 63, Capacity, 22, 27, 57, 58, 69,
66, 67, 68, 70, 72, 73, 92, 106, 111, 113, 139,
Susan, R.: Biotechnology: An Introduction, Belmont, Thomson/Brooks/
76, 81, 84, 85, 86, 87, 269, 271.
Cole, 2005.
88, 89, 90, 91, 92, 93, Carbohydrates, 8, 10, 11, 19,
Thomas R.: Bountiful Harvest: Technology, Food Safety and the 22, 193, 199, 233, 262.
95, 101, 105, 107, 109,
Environment, Washington DC, Cato Institute, 2003. Carbon Dioxide, 87, 180, 183,
115, 116, 119, 120, 123,
Walden, Richard: Genetic Transformation in Plants, England, Open 124, 125, 127, 128, 130, 184, 266.
University Press, 1988. 141, 146, 148, 160, 162, Carnobacteria, 8, 9, 10, 11, 12,
Wallace, L.: Introduction to Professional Foodservice. New York: 165, 167, 168, 170, 171, 13, 14, 15, 18, 19, 20,
21, 22, 23, 24, 26, 27,
Wiley, 1996. 173, 176, 177, 178, 180,
181, 182, 183, 184, 185, 166, 167.
Zaccarelli, Herman E.: Food Service Management by Checklist: A Carnobacterium, 8, 9, 10, 11,
Handbook of Control Techniques. New York: Wiley, 1991. 186, 187, 188, 200, 201,
12, 13, 14, 15, 16, 19,
276 Introductory Food Microbiology Index 277

20, 21, 22, 23, 24, 25, Cultures, 8, 9, 17, 22, 24, 31, Emission, 50, 179, 262. Fluorophores, 262.
26, 166, 169, 170. 47, 48, 49, 52, 53, 60, Energy, 10, 43, 202, 266. Food Contamination, 26.
Chromosome, 16, 49, 62, 74, 68, 69, 70, 71, 72, 76, Enteric Viruses, 2. Food Microbiology, 1, 94, 136,
248, 260, 263, 264, 265. 77, 78, 79, 80, 81, 82, Enterotoxin, 4, 6, 28, 34, 39, 268.
Commission, 156, 178. 85, 95, 96, 97, 98, 100, 217, 220, 222, 225, 261. Food Safety, 1, 5, 21, 27, 43,
Community, 13, 18, 23, 236, 113, 120, 122, 123, 126, Environment, 4, 8, 9, 10, 11, 44, 107, 112, 128, 141,
237, 238, 259. 130, 133, 139, 148, 156, 14, 26, 27, 46, 55, 57, 172, 222, 223, 233.
Comparison, 11, 37, 39, 42, 51, 167, 181, 191, 196, 197, 58, 59, 83, 84, 94, 101, Foodborne Pathogens, 2, 137,
87, 90, 100, 103, 125, 198, 200, 206, 209, 212, 107, 111, 112, 113, 119, 139.
133, 136, 138, 143, 145, 213, 216, 220, 240, 254, 120, 121, 122, 123, 135,
146, 155, 159, 160, 164, 255. 136, 138, 139, 142, 143, G
174, 178, 179, 188, 226, 144, 145, 146, 147, 156, Genetic Mutation, 263.
229, 238, 240, 241, 250. D 165, 181, 184, 218, 233, Genomics, 25, 27, 263.
Conditions, 12, 13, 18, 19, 22, Department, 30, 219. 263, 264, 265, 266, 267,
29, 30, 33, 34, 37, 39, Development, 26, 33, 34, 35, 271, 272. I
41, 43, 45, 46, 47, 50, 36, 43, 80, 82, 129, 175, Escherichia Coli, 6, 10, 47, 59, Identification, 9, 31, 45, 57, 60,
52, 53, 54, 57, 58, 59, 200, 219, 224, 232, 256. 71, 101, 128, 130, 169, 62, 130, 133, 176, 177,
61, 63, 78, 85, 87, 88, Diagnostics, 34, 36. 184, 232, 242. 193, 202, 206, 208, 215,
89, 93, 94, 95, 97, 98, Distribution, 3, 9, 23, 26, 34, Eukaryote, 261. 229, 236, 237, 239, 248,
99, 100, 101, 102, 103, 35, 36, 93, 94, 96, 99, Evaluation, 131, 135, 145, 178,
253.
104, 105, 106, 107, 108, 101, 102, 103, 105, 111, 185, 229, 230, 247, 255.
120, 129, 156, 178, 224, Industry, 9, 29, 44, 46, 57,
111, 112, 113, 114, 115, Evolution, 2, 110, 112, 129, 135,
116, 117, 118, 119, 120, 227, 253, 266. 128, 158, 172, 173, 187,
136, 165, 180, 205.
121, 122, 123, 124, 125, Diversity, 17, 27, 176, 232, 236, 214, 252.
Experiments, 31, 32, 39, 45, 47,
126, 127, 130, 131, 132, 237, 253, 269. Infections, 4, 5, 23, 234.
48, 59, 60, 61, 63, 66,
134, 135, 137, 138, 140, DNA, 25, 48, 49, 50, 60, 61, Information, 6, 10, 12, 19, 25,
76, 87, 95, 96, 98, 105,
141, 142, 143, 144, 145, 62, 64, 72, 73, 74, 131, 26, 33, 46, 47, 57, 58,
107, 108, 110, 111, 114,
146, 147, 152, 153, 154, 168, 169, 171, 173, 174, 61, 68, 94, 109, 154, 172,
120, 121, 122, 123, 126,
155, 156, 157, 158, 162, 183, 229, 233, 235, 236, 187, 193, 194, 226, 229,
127, 128, 130, 132, 133,
163, 164, 165, 176, 180, 237, 238, 239, 240, 241, 134, 136, 139, 140, 141, 236, 260, 262.
182, 185, 186, 196, 200, 242, 243, 244, 245, 246, 145, 148, 149, 161, 164, Injection, 24, 86, 87, 89.
201, 203, 207, 209, 210, 247, 248, 249, 250, 251, 165, 167, 174, 175, 193, Inoculum, 86, 87, 93, 94, 95,
215, 219, 222, 225, 226, 254, 255, 257, 259, 260, 194, 205, 210, 211, 217, 96, 97, 98, 99, 100, 101,
227, 241, 244, 246, 248, 261, 262, 263, 264, 265, 227. 102, 103, 104, 105, 127,
249, 251, 256, 258, 260, 266, 267, 268, 269, 270. 131, 140, 146, 148, 156,
261, 270, 271. F 157, 158, 159, 161, 264.
Conjugation, 260. E Institute, 25, 37, 151, 152, 245.
Farmers, 178.
Conservation, 68, 134. Ecology, 4, 26, 165, 254. Fermentation, 1, 9, 69, 78, 81, Institutions, 23.
Constitution, 144, 263. Economic Growth, 2. 82, 106, 180, 182, 184, Instruments, 206.
Construction, 33, 49, 73, 74, Ecosystem, 231. 214, 233, 262. Integration, 49, 74, 235, 248.
129, 130, 133, 135, 136, Electron Microscopy, 45, 50, 53, Fermentor, 108, 111, 122, 123, Interests, 112.
147, 148, 222, 236. 197. Isolation, 5, 9, 10, 49, 165, 175,
124, 126.
278 Introductory Food Microbiology Index 279

192, 203, 204, 234, 245, Methylobacterium, 266. Osmotolerance, 45, 54, 55, 58, Products, 1, 3, 5, 7, 8, 9, 10,
254, 257, 261. Microbacterium, 266. 59. 11, 12, 13, 14, 18, 19,
Microbial Ecology, 26. 20, 21, 22, 27, 28, 39,
L Microbiology, 1, 48, 59, 71, 94, P 41, 43, 44, 55, 60, 69,
Laboratory, 19, 21, 43, 70, 86, 106, 112, 116, 119, 122, Pathogens, 1, 2, 3, 4, 5, 6, 8, 73, 82, 123, 136, 137,
92, 136, 149, 151, 167. 129, 132, 136, 146, 147, 18, 24, 27, 30, 137, 139, 138, 139, 145, 146, 147,
Legislation, 235, 256. 263, 268. 166, 172, 219, 231, 233, 148, 150, 159, 160, 164,
Listeria Monocytogenes, 5, 8, 17, Microorganisms, 1, 2, 8, 17, 240, 241, 264, 267, 270, 173, 174, 180, 181, 182,
45, 57, 83, 93, 95, 106, 29, 30, 40, 52, 130, 133, 271, 272. 184, 185, 187, 189, 196,
107, 112, 113, 122, 136, 137, 183, 189, 194, 195, Performance, 116. 198, 199, 207, 209, 214,
137, 146, 147, 149, 157, 197, 198, 200, 211, 212, Plants, 11, 13, 14, 26, 27, 68, 216, 219, 228, 230, 232,
166, 170, 183, 242. 232, 272. 148, 165, 173, 178, 268, 233, 240, 241, 243, 244,
Literature, 29, 38, 174, 227, Microplates, 48, 59, 60, 71, 124, 269, 270, 272. 246, 249, 251, 256, 264,
228, 231, 249. 148. Population, 6, 32, 69, 77, 78, 266, 269, 271, 272.
Low Virulence, 6, 83, 84, 85, Molecules, 41, 104, 172, 182, 80, 88, 93, 94, 95, 97, Project, 25, 132, 245.
88, 90, 91, 92. 196, 228, 237, 249, 262, 100, 101, 102, 103, 104, Protection, 11, 18, 68, 210.
271. 105, 116, 117, 122, 125, Proteins, 7, 16, 25, 45, 46, 47,
M Mycotoxins, 3. 127, 134, 141, 146, 147, 51, 54, 55, 56, 62, 63,
Majority, 26, 39, 147, 155, 157, 151, 153, 154, 155, 157, 66, 67, 68, 83, 166, 172,
158, 178, 198, 231. N 160, 161, 165, 221, 227, 182, 192, 197, 204, 208,
Management, 180. Natural Environment, 9, 10, 11, 229, 231, 232, 236, 237, 216, 260, 261, 262, 268,
Manufacture, 1, 68, 69. 14. 238, 240, 243, 252, 253, 272.
Measurement, 31, 32, 33, 44, Network, 61, 106, 107, 109, 254, 257, 259, 261, 263, Protozoan Parasites, 2.
76, 133, 149, 152, 162, 197. 265, 271.
195, 206, 221, 225, 238, Predictions, 37, 39, 43, 106,
R
242, 247, 254, 259. O 107, 109, 111, 112, 113, Relations, 44, 216.
Mechanism, 4, 6, 43, 46, 59, Observations, 55, 89, 91, 116, 116, 118, 119, 121, 225. Research, 24, 27, 151, 178, 200,
99, 171, 172, 184, 190, Preparation, 1, 30, 31, 72, 73, 238.
126, 134, 137, 178.
199, 215, 262. 113, 123, 131, 177, 190, Resolution, 237.
Opportunistic, 22, 24, 264, 267,
Media, 12, 19, 21, 30, 31, 32, 199, 220. RNA, 45, 47, 49, 50, 52, 237,
270, 271, 272.
34, 47, 52, 53, 59, 60, Prevention, 17, 218. 253, 260, 262, 263, 264,
Organelle, 267.
61, 63, 70, 84, 90, 93, Production, 3, 4, 8, 14, 16, 17, 266, 267, 268, 269, 270.
Organisms, 6, 10, 11, 14, 17,
96, 99, 100, 108, 109, 18, 19, 22, 27, 63, 69, 19, 20, 21, 23, 28, 34,
113, 114, 115, 120, 121,
S
83, 101, 106, 107, 112, 70, 80, 81, 82, 111, 113,
123, 124, 125, 126, 130, 121, 158, 159, 160, 167, Salmonella, 5, 102, 184, 185.
119, 121, 128, 129, 136,
135, 139, 141, 148, 157, 169, 172, 173, 179, 180, Species, 3, 4, 5, 6, 7, 8, 9,
172, 180, 182, 184, 186,
159, 160, 169, 177, 192, 183, 184, 187, 188, 189, 10, 11, 12, 13, 14, 19,
201, 203, 209, 215, 220,
201, 214, 220, 221, 222, 190, 199, 208, 209, 210, 20, 21, 22, 23, 24, 25,
229, 230, 231, 234, 235,
225, 244, 264, 266. 214, 215, 217, 218, 222, 26, 58, 68, 70, 71, 128,
236, 241, 251, 255, 258,
Methodology, 26, 132, 149, 242, 230, 234, 246, 248, 255, 136, 138, 147, 149, 159,
259, 260, 262, 266, 269, 160, 163, 164, 165, 166,
243, 257. 270, 271. 259.
280 Introductory Food Microbiology Introductory Food Microbiology 281

173, 174, 177, 180, 182, 72, 81, 87, 94, 102, 105,
185, 186, 187, 189, 211, 107, 108, 112, 113, 117,
215, 229, 230, 231, 232, 119, 120, 121, 122, 127,
233, 234, 235, 236, 237, 128, 129, 130, 131, 132,
239, 240, 242, 243, 244, 133, 134, 135, 136, 137,
247, 249, 250, 253, 257, 138, 139, 140, 142, 143,
261, 263, 264, 265, 268, 144, 145, 146, 147, 149,
CONTENTS
269, 270, 271, 272. 152, 153, 154, 159, 160,
Spoilage, 1, 2, 8, 13, 14, 17, 162, 163, 164, 165, 166,
18, 20, 21, 22, 26, 27, 177, 178, 196, 202, 204, Preface
136, 159, 164, 170, 171, 205, 206, 209, 212, 213, 1. Introduction 1
177, 178, 185, 186, 189, 216, 218, 219, 220, 226,
202, 205, 220, 246, 254, 248, 260, 263, 271. 2. Role of Ctc from Listeria Monocytogenes in
272. Treatment, 97, 101, 125, 179, Osmotolerance 45
Stress Conditions, 45, 47, 53, 54, 198, 212, 215, 216, 230,
59, 61, 63, 94, 127. 242, 254, 267. 3. Identification of Listeria Monocytogenes Genes 57
Symptoms, 4, 5, 8, 234.
V 4. Experimental Validation of Low Virulence in
T Validation, 39, 64, 83, 110, 111,
Field Strains 83
Technology, 30, 73, 74, 220. 129, 219. 5. The Effect of Inoculum Size on the Lag Phase 93
Temperature, 11, 12, 17, 29, Velocity, 175.
31, 39, 46, 49, 52, 57, Viruses, 1, 2. 6. Modelling the Growth of Listeria
Monocytogenes in Dynamic Conditions 106
‰‰‰
7. Epidemiological Typing of Bacillus spp Isolated
from Food 173
8. Modelling the Growth Boundary of
Staphylococcus Aureus 217
9. Glossary 258
Bibliography 273
Index 275
INTRODUCTORY
FOOD MICROBIOLOGY