Journal of Applied Microbiology 1997,82,19-31

Enterotoxin and cytotoxin production by Salmonella

enteritidis strains isolated from gastroenteritis outbreaks
M.T. Rumeu, M.A. Suarez, S. Morales’ and R. Rotger
Departamento de Microbiologia 11, Facultad de Farmacia, Universidad Complutense, Madrid, Spain
received 1 April 1996,revised 28 May 1996 and accepted 29 May 1996
5706/04/96:

M.T. RUMEU, M.A. SUAREZ, s. MORALES AND R . ROTGER. 1997.Seventy-six Salmonella

enteritidis, three Salmonella virchow and one Salmonella braedenrup strains were
screened for enterotoxigenicity by using the Chinese hamster ovary (CHO), Y 1 adrenal,
Vero and HeLa cell tests. All the strains gave positive reactions for enterotoxin production,
except one, and the relative sensitivity to the toxin exhibited by the different cell lines
was evaluated. An enterotoxic activity has been identified in sonicated extracts of Salrn.
enteritidis. This enterotoxin was purified on Agarose A-5m (Bio-Rad) and Superose 12
HR 10/30 column. T h e enterotoxic activity was eluted from the Superose column in the first
peak. Like Vibrio cholerae toxin CT and Escherichia coli enterotoxin LT, it was blocked
by GM, ganglioside, but at a higher concentration. In addition, a cytotoxic factor
has been partially identified. T h e procedure for isolating the cytotoxin included
ammonium sulphate precipitation, size-exclusion chromatography and anion exchange
chromatography. This cytotoxin factor caused inhibition of protein synthesis in cultured
cells, as determined by flow cytometry and [3H]-leucine incorporation. Flow
cytometry analysis also showed an activation of CHO cells when exposed to this
cytotoxic factor resulting in a state of active growth. Cytotoxic activity was not
blocked by gangliosides.

INTRODUCTION found to be thermostable whereas delayed PF was dem-

onstrated to be thermolabile. Delayed PF, like Escherichia coli
Salmonellosis includes several clinical manifestations such as
L T and CT, was shown to cause CHO cell elongation, and
gastroenteritis (food poisoning), enteric fever, bacteraemia
this cytopathic effect was inhibited by incubation with anti-
and carrier state. Gastroenteritis is the most common syn-
cholera toxin serum, by heating the supernatant fluids at
drome and it is predominantly caused by Salmonella enteritidis
100°C for 15 min or by adding mixed gangliosides (Koo and
in Europe, and by Salmonella typhimurium in other countries
Peterson 1982; Harihanan et al. 1986; Panigrahi et al. 1987).
(Garcia del Portillo and Finlay 1994). There is some contro-
The first report on the purification of an enterotoxin from
versy about the causes of the diarrhoea usually present in
Salm. typhimurium and subsequent characterization was done
these cases. It has been argued that the inflammatory response
by Finkelstein et al. (1983). This enterotoxin was reported to
induced by Salmonella is sufficient to provoke the fluid and
be structurally and immunologically similar to both C T and
electrolyte loss from the intestinal mucosa (Finlay and Falkow
the heat-labile enterotoxin (LT-I) of E. coli (Molina and
1988). However, several enterotoxic factors have been isolated
Peterson 1980 ; Finkelstein et al. 1983). Enterotoxin
from either culture supernatant fluids or broken cells of Sal-
production, as detected by means of diverse entero-
monella that could also be responsible for the diarrhoea.
toxigenicity assays, has been reported for a large number of
Two skin permeability factors (PF) detected in cultures of
Salmonella serotypes (Caprioli et al. 1981, 1982; Jiwa et al.
Salm. typhimurium have been described: rapid PF and
1981 ; Singh et al. 1985).The Salmonella enterotoxin may be
delayed PF (Sandefur and Peterson 1977). Rapid PF was
responsible, at least in part, for the loss of fluid and elec-
trolytes from small intestine, because rabbits immunized with
Correspondence to : Dr Rafael Rotger, Departamento de Mirrobiologia II,
heat-inactivated, purified cholera toxin are protected against
Facultad de Farmacia, Universidad Complutense, Ciudad Universitaria, 28040
Madrid, Spain. fluid loss when the intestinal loops are challenged with live
‘Current address : Centro Nacional de Biologin Celular y Retrovirus, Servicio Salm. typhimurium (Guianella et al. 1975). Partially purified
de Inmunologia, Majadahonda, 28220 Madrid? Spain. enterotoxin will result in a fluid accumulation in rabbit intes-
0 1997 The Society for Applied Bacteriology
20 M.T. RUMEU E T A L

tinal loops that is hlocked by monospecific cholera antitoxin shaking (200 rev min-I) for 20 h. The culture was then
(Peterson e/ ul. 1981). centrifuged at 10000 rev min-' for 8 min at 4°C. The cell
Attempts made to clone the enterotoxin encoding genes pellet was washed once with 5 ml of PBS (8 g 1-' NaCl, 0.2
i z ~ton ithe disco\-er!- of a new toxin,
from Salm. ~ ) ~ l ~ i n z i ~ rled '
g 1-' KCl, 1.44 g 1 Na2HPOJ, 0.24 g 1-' KH,P04, pH 7.4),
less related to L T and C T enterotoxins (Chopra rt ril. 1993). centrifuged and resuspended in 3 ml of PBS. Each washed
The cloned gene, stn, appears to be common in enterobacteria cell preparation was sonicated in a MSE Sonicator (MSE
(Chopra rt al. 1994a, b). Scientific Instruments, UK) for two separated 4 min periods.
In addition to the discoi-er!- of the enterotoxin, another After sonication, cell suspensions were centrifuged at 12 500
heat-stable toxic substance produced by S~iliii,enteritidis and re\ min-' for 20 min at 4°C and the supernatant fluids were
Salni. [yphzrnuviuin has been reported (Koupal and Deibel sterilized by filtration (Millipore, 0.22 pm). These cellular
1973 ; Koo and Peterson 1982 ; Reitmeyer et 01. 1986 ; Ash- extracts were used in cell toxicity assays.
kenazi et 01. 1989), and characterized as an outer membrane-
bound qtotoxin that inhibits protein s!-nthesis in cultured
cells as well as in the intestinal mucosa (Koo and Peterson Purification of the enterotoxic factor
1983 ; Koo et a / . 1984; Reitme!-er et 01. 1986; Ashkenazi et
For the purification and characterization of the enterotoxin,
rrl. 1988). Other c! totoxic products of Salmoriellrr have been
the strain Sahn. ententzdzs 1547 was grown in four 9.5-1 fer-
detected in L-forms of this bacteria (Kita e t a / . 1993) and two
mentors with Evan's medium, inoculated with approximately
genes, named r:yv and .s/y.4, expressed haemoll-tic acti\-ity
10' cells ml-' (Evans et ul. 1976), and incubated for 20 h at
when cloned in E. rdi, but not in their original S u h o n e i l ~ ~
37°C \\ith vigorous aeration. T h e bacteria were harvested by
hosts (Libby rt (11. 1990, 1994).
continuous flow centrifugation, and lysed in a French Pres-
The deduced sequence of salmolysin, the product of s(y+.l
sure cell press, at a pressure of 20 000-25 000 lb in-'. The
gene, showed significant homology n-ith regulator!. proteins,
effluent was centrifuged at 15 000 rev min-' to remove the
so the haemolytic activity of the clones could be due to a
cell debris. T h e precipitate obtained with a 60% saturated
regulator! e\-ent affecting the expression of an E. roli haemo-
solution of (NH+)2S0,, after dialysis against TEAN and cen-
l!-sin, rather than a haemol!-tic propert!- of the salmolysin
trifugation, was dissolved in TEAN and subsequently
(Ikhoux and Cossart 199.5).
purified. Then the sample was applied to a 2.5 x 25 cm col-
In this study, a representatii e group of Sniitz. eiiteritidis
umn of Agarose A-5m (Bio-Rad) and eluted with TEAN
from human sources was investigated by utilizing four widel!-
until the AZ8"of the column effluent reached baseline (Clem-
used cell tests, namel!- 1.1 adrenal, CHO, i'ero and HeLa cell
ents and Finkelstein 1979 ;Clements et al. 1980). The column
test to assess production of enterotovic and CJ totoxic factors,
\\as then eluted with 0.2 mol 1-' galactose in TEAN (Fin-
and to characterize piausible biological patterns of entero-
helstein et al. 1983).
toxigenicit!. among the salmonellae.
Size exclusion chromatography was performed on a Super-
ose 12 HR 10/30 column (Pharmacia, Sweden) with TEAN
MATERIALS AND METHODS
buffer at a flow rate of 0.8 ml min-'.
Bacterial strains
Seventv-six isolates of Scrl~n.enteritidis, one of Sohi. brrie- Partial purification of a cytotoxic activity
derzn~pand three of Srrlni. iirchaii, were kindly pro\-ided by
T h e strain Sabn. enteritidis 647 was grown in two 4.5 1 fer-
Dr M. ,4. Usera, Instituto de Salud Carlos 111, Majadahonda,
mentors, inoculated with loi cells ml-' of CAYE medium
Madrid, Spain. A11 of them had been isolated during various
(Koo and Peterson 1983) and incubated for 18 h at 37°C
diarrhoea outbreaks (Table 1). We obtained enterotoxin-pro-
nith vigorous aeration. T h e bacteria were lysed in a French
ducing Srzlni. pphitiwiiiin strains 1730 and 1980 from Dr R.
Pressure cell press, at a pressure of 20000-25 000 lb in-*.
.4. Finkelstein, Columbia, Ilissouri, t'S.4. Ft'e used E. roli
T h e precipitate obtained b j 30% saturation with (NHJ2S0.,,
strains carr!-ing the plasmids pEiVD299 (Dallas e/ al. 1979)
after dial! sis against PBS and centrifugation, was dissolved
and p J l l l 7 (Pearson ('/ a/. 1982), encoding respectively for
in PRS and subsequently purified. T h e sample was applied
L T and C T production, as well as the strain E. i d i H19B
to a size-exclusion chromatography column, on a Superose
(Caldera oocl ~ ' 1al. 1987) for SI,T-I production. Escherirhia
12 HR 10/30 column (Pharmacia) with PBS buffer at a flow
idi x-DHj !\as used as non-toxigenic negati\-e control.
rate of 0.8 ml min-'. Anion exchange chromatography was
performed on a Mono-QHR 5/5 (Pharmacia) with PBS
Toxin production
buffer, and proteins were eluted with PBS containing a linear
Culturcs \\ere grov 11 in 13 ml of Casamino acid-yeast extract gradient of 0-1 mol 1-' NaC1. Column eluates were moni-
(CAYE) medium (Koo and Peterson 1982) at 37°C with tored for u.\. absorbance a t 280 nm.
0 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 82, 19-31
 TOXIN PRODUCTION BY SALM. ENTERITIDIS STRAINS 21

Table 1 Salmonella isolates from human and food used in this study

Year of Sex and

Serotype Strain isolation City Case Sample age

Salm. enteritidzs 82134 86 Tenerife community faeces M-N

82139 86 Tenerife community faeces hl-N
82140 86 Tenerife community faeces F-N
86275 86 Tenerife community faeces F-N
86279 86 Tenerife community faeces F-N
86280 86 Tenerife community faeces F-N
366 86 Logroiio individual faeces M43
367 86 Logrofio individual faeces M-N
522 88 Barcelona family faeces hI42
523 88 Barcelona family faeces F-7
524 88 Barcelona family faeces M-10
607 87 N N faeces N
647 87 Zamora family faeces hi-12
648 87 Zamora family faeces F-8
724 87 Asturias community faeces F-54
725 87 Asturias community faeces F-54
729 87 Asturias community faeces M-53
73 1 87 Asturias community faeces M-68
740 87 Asturias family faeces N
743 87 Asturias community faeces F-27
846 87 Madrid individual blood F-6
847 87 Madrid individual faeces F-6
985 88 Madrid family faeces N-5
1224 87 Barcelona individual joint liquid M-N
3618 87 Toledo family faeces F-5
AG40 88 Barcelona N N N
AG41 88 Barcelona N N h
'
10149 88 Barce1ona community food N
10157 88 Barce1ona community faeces M-6
10169 88 Barcelona community faeces F-N
11364 88 Barcelona community faeces F-N
11365 88 Barcelona community faeces F-N
11366 88 Barcelona community faeces M-N
140 89 Gran Canaria N food N
267 89 Madrid N food N
27 5 89 Madrid N food N
280 89 Madrid N food N
487 89 Barcelona N food N
49 1 89 Barce1ona N food N
807 89 Madrid community faeces N
820 89 Madrid community faeces N
83 1 89 Madrid community faeces N
886 89 Barcelona community faeces N
898 89 Barcelona community faeces N
1086 89 Barcelona community faeces N
1174 89 N community faeces N
1180 89 N community faeces N
1182 89 N community faeces N
1183 89 N community faeces N
1364 89 N individual food N
1411 89 Vitoria N faeces M-N
1486 89 Malaga family faeces N
1487 89 Malaga family faeces N
0 1997 The Society for Applied Bacteriology,Journal of Applied Microbiology 82, 19-31
22 M.T. RUMEU E T A L .

Year of Sex and

Serotype Striin isolation Cit!- Case Sample age

1409 89 Castellbn family faeces N

1.i02 89 Castellbn family blood N
1.547 89 Bilbao S faeces M-14
I .548 88 Bilhao S faeces M-N
1.549 88 Bilbao N faeces F-6 1
16.50 89 z N faeces N
16-52 89 z S faeces N
2213 88 z N faeces N
2i)4 1 88 S S faeces N
2700 88 z N faeces N
2710 88 S N Faeces I\i
88-12 01 Sevilla individual faeces N
8902 91 I'alencia indidual blood N
9106 91 Sevilla individual blood N
11419 93 Huelva famill- faeces M-18
11420 93 Huelva familk- faeces hl-59
11421 93 Huelva family faeces F-62
1 1422 93 Huelva community faeces F-32
11423 93 Huelra community faeces F-N
11424 93 Huelva community faeces M
11426 93 Hueha communitl- faeces F
13517 93 Barcelona family faeces N
13718 93 Barcelona family faeces N
Snhn. br.iit~iit~lIr.lqJ __ 89 S N N N
.S"ili)i. ciri-tton? 117.5 93 Huesca famil!- faeces M-N
117.38 93 Huesca family faeces M-N
11739 93 Huesca family faeces M-N

X , 'iot hno\\n.

Characterization of cell toxicity I n order to localize the cytotoxic effect, the CHO assay
15 as used as described, but adding whole cells of Snlm. enter-
In x i [ w Chinese hamster ovary cell (Guerrant et d.197.1),
rtrdis 6.1'7 treated with chloroform vapours. Bacteria were
Y1 adrenal cell (Donta t't ( I / . 197.1), I-ero cell (Speirs et (11.
groiln in MacConkey agar glass Petri dishes overnight at
1977) and HeLa ccll assa!-s (Gentr!- c't al. 1980) were used to
37°C. T h r e e m l of chloroform were added to the inverted
test the enterotoxin and q t o t o x i n production of Salmnellri
Petri dishes for 1 h. After that, the Petri dishes were dried
strains. T h e CHO, \.era, HeLa and 1.1 cells were seeded in
u p in a laminar flow hood for 6 h to eliminate chloroform
96 well niicronietre plates at a density of 10' cells m1-l in
traces. To test that all bacteria were dead, samples were
Ham's F l 2 medium ( C H O cells) and ME11 medium (Vero,
seeded in liquid media. Dead cells were resuspended in PBS
HeLa and Y1 cells) supplemented with 10% fetal bovine calf
and added to CHO microtitre plates.
serum (Koo et ~ i / . 1981). -1different amount of each sample,
adjusted to I mp ni! ' of protein in a volume maximum of
100 id, itas added t o each nell, and all plates were incubated
Ganglioside binding assay
for 24-48 and 72 h d t 3'7°C. in a C 0 2 incubator. Sixfold serial
dilutions of all tesi samples I\ ere made in PBS buffer and T h e sonic extracts were combined with an aqueous solution
iverc addcd to each ~ ~ 1 'The 1 . reciprocal toxin titre was the of either mixed gangliosides (10 m g m l - ' ; Sigma Chemical
last dilution that shon-ed at least 50nu elongated culture cell. Co.) or purified GMI ganglioside (1 m g ml-I ;Sigma Chemi-
'I'hc sensitii it! of each cell line was defined as the percentage cal Co.), and incubated at 37°C for 30 min. Different con-
of cases in Mhich that line gave the highest reciprocal titre centrations of gangliosides and proteins were assayed, and
a-hcn compared with the rest of the cell lines. the toxic effect was always detected on CHO cells.
0 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 82, 19-31

Analytical procedures
The protein content of sonic extracts from the Salmonella
strains and resultant fractions in the purification procedure
were determined by the Bradford (1976) protein assay. SDS-
PAGE was performed in a modified Laemmli gel system, and
proteins were resolved in gels using silver staining (Merril et
al. 1982). Molecular weight standards, including rabbit mus-
cle phosphorylase b (97 400), bovine serum albumin (66 200),
hen egg white ovalbumin (45 000), bovine carbonic anhydrase
(3 1000) and soybean trypsin inhibitor (21 500) (Bio-Rad),
were used.
Flow cytometry analysis
CHO cells were maintained in Ham's F12 medium sup-
plemented with 10% fetal bovine serum. Each 25 ml flask
was incubated for 18-24 h in the presence of 5% CO, to
establish confluent monolayers. The culture medium was
aspirated and replaced with new complete medium with 1%
fetal bovine serum. Each flask was then exposed to different
amounts of each sample, adjusted to 1 mg ml-' of protein of
sterile sonicated extract of Salmonella. T h e cells were incu-
bated for 18 h, and then the monolayers were washed two
times with PBS buffer. Then 5 ml of a trypsin solution were
added. The flasks were maintained at room temperature until
cells were disrupted, and then cells were resuspended in 5 ml
of Ham's F12 medium. About 10' cells ml-' were used in
each assay.
The fluorochrome F I T C (fluorescein isothiocyanate) was
utilized in the flow cytometry protein analysis. Cells were
labelled by addition of 0.5 ml of F I T C (1 pg ml-' in PBS
buffer) followed by incubation (30 min) in darkness at 37°C
with mild agitation. In order to measure the DNA content,
cells were resuspended in order to be fixed and permeated in
1 ml of cold ethanol 70% in PBS. After 2 min the ethanol
was replaced by centrifugation. Cells were washed with PBS
and resuspended in 1 ml of RNAse (1 mg ml-') followed by
incubation (60 min) in darkness at 37°C with mild agitation.
Five minutes before starting the flow cytometry assay, 5 p1
of a solution of 0.5% IP (propidium iodide) in PBS was
added, in order to measure cell viability. Flow cytometry
analysis was carried out using a fluorescence-activated cell
analyser (FACScan ; Becton-Dickinson, San Jose, CA).
[3H]-leucine incorporation assay
A protein synthesis assay based on [3H]-leucine incorporation
was performed according to Koo and Peterson (1982) with
some modifications. Monolayers of CHO cells were estab-
lished by dispensing lo5 cells ml-' to each of a 24-well
microtitre dish and allowing incubation for 18 h in a CO,
incubator. The culture medium in each well was aspirated and
0 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology82, 19-31
TOXIN PRODUCTION BY S A L M . E N T E R l T l D l S STRAINS 23
replaced with 1 ml of leucine-deficient medium (InstamedB
MEM powdered medium with Earle's salts with non-essen-
tial amino acids, to which all the essential amino acids but
leucine were added) containing 10 pCi ml-' of L-[4-5 'HI-
leucine (250 pCi 250 pl-' ; Amersham Life Science).
These cells were treated either with sterile sonic cell-free
Salmonella extracts to a final concentration of I mg ml-' or
with PBS. After 18 h, the medium was removed and the
monolayer was washed four times with PBS.
Cells were disrupted in 0.1 ml of 0.5 mol I-' KOH for 30
min at room temperature. T h e microtitre dish was cen-
trifuged at 1000 rev min-' for 15 min to get rid of cell debris
that could interfere with the measurement. T h e supernatant
fluid of each well was collected and precipitated overnight
with cold 15% trichloroacetic acid at 4°C. T h e precipitates
were collected by filtering the solution through a PVDF filter
(ImmobilonB, Millipore). Each well was washed with 0.5
ml of cold 5% TCA to maximize the protein collection.
Radioactivity was determined by dissolving each filter disk in
1.5 ml of EcoliteB scintillation cocktail (Nuclear Iberica S.A.)
and then counted with a Beckman scintillation counter.
RESULTS
Detection of Salmonella enterotoxin and cytotoxin
by CHO, Vero, Y1 and HeLa cells in tissue cultures
As shown in Fig. 1, sonicated samples produced mor-
phological change in cell cultures that were morphologically
indistinguishable from those produced by cholera and LT
enterotoxin producing strains E. coEt (pJM17) and E. cofi
(pEWD299). Detection and titration of this toxic factor in
the presence of CHO, Vero, Y 1 and HeLa cells showed the
different production by different strains (see Table 2). All
Salmonella strains, except one (Salm. enteritidis 647), were
positive for production of enterotoxin in cultured cells.
The corresponding sensitivities of sonicated extracts in
CHO, Vero and Y1 cells were 68.2%, 59.2% and 34.1%
positive respectively. Observations in HeLa were not com-
pared with the ones obtained in other cell lines because the
toxic effects were not evident until 48 h. In the CHO cell
test, the majority of positive reactions were seen up to a 1 : 8
dilution of sonic extracts. Titres of sonic extracts on Y 1 and
Vero cells were, in general, similar when tested in parallel,
although the latter yielded higher titres in some cases. CHO
cells appear to be more sensitive to the toxins, and the
observed morphological changes were more evident. After
exposure to the enterotoxin for 18-24 h, the normal trap-
ezoid-shaped CHO cells were elongated to a spindle form, as
illustrated in Fig. lb.
On the other hand, sonic extracts from 53 strains (64.7%)
caused vacuole formation in cell cultures at 24 h, and were
lethal for Vero cells at 48 h. In CHO, Y 1 and HeLa the cell
24 M.T. RUMEU E T A L .

Fig. 1 Monola!-ers o f : (-1)Chinese hamster ovarl- (CHO) cells; (B) kidney ..\frican green monkey (Vero) cells; (C) adrenal human
(Y 1) cells ;(D) cervix carcinoma human (HeLa) cells. (a) Control ; (b) treated with preparations from Sulnznnellu enteritidis
1199 ; (c) treated with preparations from S o h . r n r r r i t i h 647

death occurred after '72 h. We compared this cytotoxic effect
with E. cali H19R, SLT-I toxin producer, but the latter
exhibited a rapid ccll detachment without \-acuole formation.
The enterotoxic activity was destroyed b!- heating to 100°C
for 1.5 rnin, but the c!-totoxic factor \vas heat stable (lOO°C
for 3W-5 min). K c used this characteristic to separate the
toxic effects. The cytotoxin caused rcunding and detachment
of CHO cells as described by Reitmeyer et nl. (1986). These
authors localized the cytotoxin in the outer membrane. This
was supported b! our observations that chloroform-treated
0 1997 The Society for Applied Bacteriology,Journal of Applied Microbiology 82, 19-31
bacteria produced the same characteristic effect on CHO
cells. Sonic extracts obtained from Salm. braedenrup or Salm.
rivchon, did not produce the cytotoxic effect.
Purification of an enterotoxic factor
As a result of the apparent similarities in the properties of
Salmonella enterotoxin and those of E. coli LT (Clements
and Finkelstein 1979 ; Clements et al. 1980) and Salnz. [yphi-

Fig. 1 (continued)
murium (Finkelstein et al. 1983), we applied the described
purification technique to the strain Salm. enteritidis 1547 as
described in Materials and Methods. Apparently, the heat-
labile peak behaved anomalously on the gel filtration because
of its affinity for the agarose matrix (Fig. 2a). One fraction
(C, see Fig. 2a), displayed the enterotoxic activity on cultured
cells. The active material, 130 ml, was concentrated by
ultrafiltration on YM-10 membranes. Five hundred pl (0.06
mg of protein) were applied to a Superose column. Each
fraction was tested by the CHO cell test. The enterotoxic
activity was detected in the first peak called A (Fig. 2b).
These observations were reproduced repeatedly. SDS-PAGE
0 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 82, 19-31
TOXIN PRODUCTION BY S A L M . ENTERlTlDlS S T R A I N S 25
and silver staining indicated purification of two different
protein subunits of a M , z 43 000 Da and M , z 11 000 Da
(Fig. 2c).
Partial purification of a cytotoxic factor
The purification of a cytotoxic factor of Salm. enteritidis 647 ,
involved ammonium sulphate precipitation of proteins pre-
sent in lysed cells, followed by gel filtration and anion ex-
change chromatography. Toxin activity from Salm. enteritidis
647 was purified as described in Materials and Methods. The
cytotoxic activity was detected in the peak A that eluted from
26 M . T . RUMEU E T A L .

Table 2 Determination of
Heat-stable enterotoxicity (heat-labile) and cytotoxicity
Serotxpe Strain CHO* \'ero* Yl* HeLa*t toxin (heat-stable) in cultured cells
-
Salnz. entzrattdis 82134 4 4 8 8 +
82 139 2 4 4 4 +
82140 2 2 4 2 +
86275 4 4 4 4 +
86279 4 2 4 4 +
86280 4 4 2 4 +
366 4 8 4 2 -

367 8 8 8 2 -
522 16 8 8 8 +
523 4 4 4 4 +
524 4 8 4 4 +
607 2 2 2 4 -

647 8 64 16 16 +
648 4 4 4 4 +
724 4 2 2 4 +
725 2 8 4 8 +
729 2 2 2 4 +-
73 1 2 2 8 2
740 8 8 2 4 -
743 2 4 4 8 +
846 4 2 4 2 -
847 2 2 2 2 +
985 2 8 4 2 +
1224 32 8 8 8 +
3618 4 2 4 8 +
.4G4O 2 2 2 8 +
AG41 2 2 2 8 +
10149 4 2 2 8 +
10157 2 2 4 8 +
10169 a 2 8 2 +
1 1364 16 8 2 4 +
11365 4 4 4 4 +
1 1366 4 2 8 4 -

140 2 2 2 4 +
267 8 8 4 4 -

27 5 16 8 8 8 +
280 16 8 2 4 +
487 8 4 2 2 +
49 1 8 16 4 2 +
807 8 8 4 4 -
820 32 16 4 2 -

83 1 16 8 2 2 -

886 16 2 2 2 +
898 2 8 4 4 -

1086 8 8 4 4 +
1174 4 8 8 2 +
1180 8 8 4 8 -

1182 16 16 4 4 +
1183 8 8 4 8 +
1364 8 16 4 4 -

1411 8 8 4 4 +
1486 2 4 2 8 +
1487 8 8 2 4 +-
1499 8 2 8 4

0 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology82, 19-31
 TOXIN PRODUCTION BY SALM. ENTERlTlDlS STRAINS 27

Table 2 (continued)
Heat-stable
Serotype Strain CHO* Vero* Y1* HeLa"? toxin
~ ~~ ~ ~-

1502 8 8 2 2 +
1547 8 16 16 8 +
1548 2 8 2 2
1549 8 16 2 4 +
1650 16 8 8 4 -
1652 16 8 8 16
2213 4 2 2 2 +
2641 8 8 4 8
2700 2 16 2 4
2719 8 8 8 16
8842 16 4 4 4 +
8992 2 8 2 2 +
9106 8 4 4 8 +
11419 8 4 4 8 +
11420 4 4 4 8 +
11421 4 4 2 8 +
11422 4 4 4 8 +
11423 4 4 2 4 +
11424 8 4 2 8 +
11426 4 4 2 8 +
13717 4 16 2 8
13718 8 16 2 4
Salm. bvaedenrup - 4 2 4 8
Salm. vtrchow 11737 2 8 2 16
11738 16 4 2 8
11739 8 4 2 8
Escherichza colt pEWD299(LT) 32 32 16 16
pJM17(CT) 32 32 16 16
H19B(SLT-I) 4 32 8 16
a-DH5 0 0 0 0
~

*Reciprocal heat-labile toxin titre, except in the case of Salm. enteritidis 647 in which the
heat-stable toxin is referred to.
tHeLa quantification was made at 48 h.

the column (Fig. 3a). This fraction was chromatographed on
a Mono-Q anion exchange column and toxin activity was
eluted with approximately 0.3 mol I-' NaCl (Fig. 3b). SDS-
PAGE, PAGE and silver staining indicated the localization
of the cytotoxic activity in one fraction that contained two
protein bands of M,E 29 000 and M,E 18000 (Fig. 3c).
Ganglioside binding assay
The partial purified enterotoxic activity usually elicited on
culture cells was blocked by the addition of 0.5 mg mg-'
protein of mixed gangliosides, and was neutralized by 0.05
mg mg-I protein of purified G M I ganglioside ; a 100-fold
amount of both gangliosides was required to neutralize the
enterotoxic activity when compared to LT toxin. The cyto-
0 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 82, 19-31
toxic activity was neither blocked by the addition of a mixture
of gangliosides (1 mg mg-I protein) nor by 0.5 mg mg-'
protein of purified GM, ganglioside.
Analysis of cell alterations caused by Salm.
enteritidis 647 cytotoxin
In this study we carried out a cytometric analysis of the cell
cycle and protein content in CHO cells. The results of the
cell cycle analysis of CHO cells treated with the sonic extracts
are shown on Table 3. CHO cells exposed to the sonic extract
of Salm. enteritidis 647 elicited a strong activation, clearly
switching to a synthesis and mitotic phase, showing that these
cells change to a state of active growth. The protein content,
28 M.T. RUMEU E T A L

0 10 20 30 40 50 60 70 80 90 100110
Fractions

E (b)
0
m
N
c
m
8
C
100.0
m
+?
z
A2
m
c
0 50.0
al
0,
A
m
c
C
0)
D~ G
2
al
Q
0.0
Protein fractions (min)
Fig. 2 (a) Chromarography of the enterotoxic component on a column (2.5 x 25 cm) of -4garose A-5m (Bio-Rad) equilibrated with
T L l X , 'fhe acti\-e component was eluted with 0.2 mol I-.' galactose. Fraction C collected (87-100) was active in CHO.cells
and was suhsequentl!- purified in a FPLC system. (b) The eluted denominated C was applied to a Superose 12 HR 10/30 column.
Elution \$as carried out xith TE.-\N buffer. 'The peak .\ shoned enterotoxic activitJ- in the CHO cell assay. (c) SDS-PAGE electrophoresis
(10% acqlamide) of Srzlnzotirlku mtrrifidis 1547 enterotoxin. Lane 1, Toxin partially purified obtained from peak A in Superose 12 HR
10/30 column heated and reduced with 2-mercaptoethanol. .I,Subunit ,If, 43 000 ; B, subunit M, z 11000. Lane 2, M , markers (Bio-Rad :
.V, 97 400, 66 200, 45 000, 31 000, 21 500). Silwr stained

as measured b>-the binding of FITC fluorochrome, was lower
when the cells u-ere exposed to the sonic extract of Salnz.
enteritidis 647. In order to pro\-e the protein synthesis inhi-
bition bv cytotoxin, we made another experiment that showed
how cytotoxic extracts affected incorporation of [.'H]-leucine
into protein. Siihnonellrr cytotoxin significantl!. inhibited pro-
tein synthesis in CHO cells; this inhibition was observed at
18 h. There were differences in the amount of ['HI-leucine
incorporated into protein by CHO cells when exposed to
negative control, enterotoxin isolated from Salnz. etireritidzs
740 or cytotoxic extracts of S o h . enwitidis 645 (Table 3).
DISCUSSION
In the present work, morphological changes in Y1 adrenal,
CHO, Vero and HeLa cells, typical of those induced by LT
0 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology82, 19-31
and CT, were demonstrated directly with sonic extracts of
Saltnonelln. CHO cells appear from this work to be more
sensitive than the Vero and Y1 adrenal cells. In HeLa the
quantification was recorded at 48 h, so these results were not
compared with the ones obtained from other cell lines. These
results are different to the ones reported by Jiwa (1981), who
found a higher sensitivity of Y1 with respect to CHO cells.
We have found that 79 (98.75%) of the 80 Snlmonella
strains investigated were producers of enterotoxin, as
detected in our biological assays. This is, to our knowledge,
the most extensive enterotoxin production assay of Salm.
ententidis carried out. T h e high frequency of enterotoxin
production seems logical since Sulrnonellu species are usually
associated with diarrhoea1 disease when present in the gas-
trointestinal tract (Guianella 1979).
The purification of the Salm. enterztzdis enterotoxin was
 TOXIN PRODUCTION BY SALM. ENTERITIDIS STRAINS 29

__
fl Protein fractions (rnin)
I?
B
r\ /

0.0 / Protein fractions (min)

Fig. 3 (a) Chromatography of the cytotoxic component on a FPLC system. Elution profile of Superose 12 HR 10/30. The cytotoxic
activity was evaluated by the CHO cell assay and it corresponded to the A peak. (b) Fractions with cytotoxin activity
corresponding to the eluted peak A from (a) were applied to a MonoQcolumn, and toxin activity was eluted by a linear gradient of @
1 mol I-' NaC1. The cytotoxic activity was evaluated by the CHO cell assay, and it corresponded to the B peak. (c) PAGE
electrophoresis of cytotoxic fractions. Lane 1, Proteins obtained from the concentrated cytotoxic fractions 1/ 10 dilution ; lane 2,
proteins obtained from the concentrated cytotoxic fractions not diluted. A, M , 29000; B, M , 18000. Silver stained

Table 3 Effect of different strain lysates in CHO cells in vitro

Cell cycle
~ ~

Strain Toxin GI(%) S(%) G2+M(%) FITC cpm x lo'*

Escherichia cola' or-DH5 None 72 23 5 977 19981f650

Salmonella enteritidis 1499 enterotoxin 49 44 7 1002 ND
Salm. enteritidis 740 enterotoxin ND ND ND ND 174075800
Salm. enteritidis 647 cytotoxin 42 40 18 357 7195 k680

Cell cycle was determined using propidium iodide. FITC fluorochrome was used for flow cytometry analysis of protein content. The
amount of [3H]-leucine incorporated, expressed as specific activity (in counts per min per pg of protein), was determined as described in the
text.
*Each value represents the arithmetic mean fstandard deviation of three determinations.
ND, Not determined.
0 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 82, 19-31
30 M.T. RUMEU E T A L .
attempted as described by Finkelstein et a/. (1983) for the
Salm. typhtnmrium S-1-T enterotoxin ; however, its affinity
to the agarose beads appeared to be lower, making more
difficult the purification because of low !-ields. These obser-
vations correlate with the affinity of the purified toxin for
GM, ganglioside, that we h a w measured as 100 times less
than observed with E. roli LT enterotoxin. T h e presence of
two prominent protein bands in SDS-P.%GE (Fig. 2c) sug-
gests a tno-subunit structure for the S~lnz.mteritidis entero-
toxin, as described for the S-LT from S a f t n . typhimurium
(Finkelstein et 01. 1983), with similar molecular sizes. On the
contrary, the S T N enterotoxin of Srrlni. typlzimurium has been
described as a polypeptide of 29 kDa, which actkit!- was also
blocked by GM, ganglioside, but with an affinitj- 3000 times
less when compared to CT toxin (Chopra et al. 1991a).
Cj-totoxin production has been emluated in 131 isolates of
Saiinonellii strains by Ashkenazi et nl. (1988), 94 of them
belonging to the Sulm. enteritidis serot!-pe. They found some
c!-totoxic activity in all the stains, but the amount of toxin
produced was \ ariable ; we detected c!-totoxic actii-it! only in
.53 of the 80 Smlmonelkr strains tested (61.7°,~)).As the!- e\-alu-
ated the toxic effect in I-IeI,a cells b!- ["HI-thynidine incor-
poration without previous heating of the sonic extracts, the
differences with our results could be due either to the higher
sensitivity of their c!-totoxicit!- ass!- or to the partial heat
inactivation of the c!-totoxin in our stud!-. Heat treatment of
the extracts is necessar!. in order to observe morphological
changes in the cells when the strains are also enterotoxin
producers.
T h e obseri ation that u-hole chloroform-killed cells of
Saltn. enteritidis exhibited the same cytotoxic activity as sonic
extracts did, corroborates the outer membrane localization of
the cytotoxin found by Reitme!-er et (ti. (1986). T h i s method
offers also the possihilit!- of a rapid screening of colonies for
cytotoxin production.
T h e use of flow- c!-tometry to evaluate the toxic effects o n
cultured mammalian cells offers new possibilities. O u r results
concerning protein content after exposure to the cpotoxic
extracts correlated well with the protein sl-nthesisassay mea-
sured by ["HI-leucine incorporation. As the cytometric a s s a y
are easj- and quick to carr!- out, they could be a good alter-
native to the visual observation of the cells, awiding the need
of heat treatment in order to exclude the enterotoxic activity.
T h e cell c!-cle analysis shoned a quite evident activation of
the cells treated either with ch-totoxin or enterotoxin. In the
first case, actiration is followed b! cellular death, while the
enterotoxin action is transiror! .
i'er!- little is k n o m about the nature and genetics of the
.7(ibno?ie/h c! totosin. T h i s report describes the first attempt
to purify it. .4t this moment, we cannot conclude if the toxic
activity is associated to one or both of the proteins present in
the actiw fraction. T h e production of antibod!- against each
of them should clarif!- this point. T h e resistance to heat
0 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 82, 19-31
~~
inactii-ation and to inhibition with gangliosides, together with
the vacuolating effect on the different cell lines, clearly dif-
ferentiates this toxin from enterotoxins.
T h e pathogenesis of salmonella-induced diarrhoeal dis-
eases is complex and may involve the interaction of several
mechanisms, including the CT-like enterotoxin(s), cyto-
toxin(s), and mucosal invasiveness, as well as the host
inflammatory response. T h e precise role of toxins, especially
in the pathogenesis of diarrhoeal diseases remains to be
solved.
ACKNOWLEDGEMENTS
T h i s work was supported by grant ALI94-0509 from Min-
isterio de Educacion y Ciencia. M T R and MAS were aided
by fellowships from Universidad Complutense and Min-
isterio d e Educacion y Ciencia, respectively. T h e authors
thank A. h a r e z for his help in flow cytometry.
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