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PubMed1993

Analysis of enterotoxin production by Bacillus cereus from dairy products, food poisoning incidents and non-gastrointestinal infections.

Of 85 strains of Bacillus cereus isolated in Norway from dairy products, 59% were found to be enterotoxigenic, and 15% were psychrotrophic. Six of the isolates (7%) were identified as potential psychrotrophic food-poisoning strains as they were both enterotoxigenic and exhibited good growth at 6 degrees C. Enterotoxin production was detected using the Western immunoblot technique, and a commercially available reversed passive latex agglutination (RPLA) assay (Unipath BCET-RPLA TD950). Both methods gave essentially the same results. In a separate study, the Western immunoblot and RPLA assays were used in a conjunction with the in vivo vascular permeability reaction (VPR) assay to determine enterotoxin production among 25 isolates of Bacillus cereus referred to the PHLS Food Hygiene Laboratory from incidents of diarrhoeal- and emetic-syndrome food poisoning and non-gastrointestinal infections. Eighty-four percent of these isolates were found to be enterotoxigenic by the Western immunoblot and the RPLA assays, and these results were in good agreement with those obtained by the VPR assay. In both studies, the BCET-RPLA kit proved to be a simple and reliable means for determining enterotoxin production by strains of Bacillus cereus.

Animals↗

PubMed1984

Production of enterotoxin(s) by Staphylococcus hyicus.

Five strains of Staphylococcus hyicus (3 of subspecies hyicus and 2 of chromogenes) were tested serologically for their ability to produce known and unknown enterotoxin(s) and were also examined using monkey bioassays. None of the 5 strains produced any detectable level of the known enterotoxins A--E. However, all of them produced emetic responses in 2 or more of 6 cynomologus monkeys when culture growth was fed intragastrically. One of the 5 strains (S. hyicus subspecies hyicus, VII 76) produced emetic responses in 3 of 6 monkeys with 50 ml of culture growth. The other 4 strains required 1 l of culture broth (concentrated 20-fold) to produce an emetic response in at least 2 of 6 monkeys. Enterotoxin production is, therefore, not unique to S. aureus species. Since some of these organisms do not produce coagulase and thermonuclease, they would be ignored in food hazard evaluation.

Animals↗

PubMed1992

Evaluation of three different STb assays and comparison of enterotoxin pattern over a five-year period in Swedish porcine Escherichia coli.

The pig intestinal loop (PIL) assay, inhibition enzyme-linked immunosorbent assay (ELISA), and DNA hybridization assay were compared for analysis of Escherichia coli heat-stable enterotoxin b (STb) on 201 porcine E. coli strains. The DNA hybridization had a 95% correlation with the STb ELISA and was therefore chosen as the method for subsequent screening of enterotoxin genes: heat labile (LT), heat-stable a (STa), and/or STb. In contrast to the PIL assay, both the STb ELISA and DNA hybridization assays were more sensitive, reliable, reproducible, and showed good correlation with each other. Consequently, the STb ELISA is preferable for analysis of toxin preparations and screening of E. coli, whereas the DNA hybridization is better for large-scale epidemiologic screening. Escherichia coli strains (n = 437) associated with porcine diarrhea isolated in Sweden during 1989 were investigated. Of the strains, 135 (31%) were positive for at least one of these toxins and, therefore, designated enterotoxigenic E. coli (ETEC). Our results were compared with the enterotoxin pattern found in earlier studies of Swedish porcine strains. The only change in occurrence of toxins was found in strains isolated from piglets less than 1 week of age. LT- and STb-producing ETEC had decreased, and STa-producing ETEC had increased in prevalence. The occurrence of STb among ETEC of weaned pigs was 93%. This toxin was also found to be more common than STa when strains from all age groups were considered.

Animals↗

PubMed1989

Inhibition by the protein kinase inhibitors, isoquinolinesulfonamides, of fluid accumulation induced by Escherichia coli heat-stable enterotoxin, 8-bromo-cGMP and 8-bromo-cAMP in suckling mice.

The effects of isoquinolinesulfonamides, which inhibit protein kinase, on fluid accumulations induced by heat-stable enterotoxin of enterotoxigenic Escherichia coli (STh), 8-bromo-cGMP and 8-bromo-cAMP in suckling mice were studied. Both N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) and N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9) inhibited the fluid accumulation induced by STh. Fluid accumulation induced by four mouse units of STh (four-fold the minimum effective dose, 10 ng) was completely inhibited by 0.4 mumol of H-8 or H-9. H-8 and H-9 also inhibited fluid accumulation induced by 8-bromo-cGMP. On the other hand, H-8 and H-9 only partially inhibited fluid accumulation in suckling mice induced by 8-bromo-cAMP, probably because their affinities to cAMP-dependent protein kinase were lower than their affinities to cGMP-dependent protein kinase. From these results, it is concluded that the activation of cGMP-dependent protein kinase by increase in cGMP by ST or by 8-bromo-cGMP, and very probably the activation of cAMP-dependent protein kinase by increase in cAMP by cholera enterotoxin and heat-labile enterotoxin of enterotoxigenic E. coli and by 8-bromo-cAMP are necessary steps in signal transduction following increases in concentrations of cGMP and cAMP in intestinal brush border cells.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2006

Detection of enterotoxin genes seg, seh, sei, and sej and of a novel aroA genotype in Jordanian clinical isolates of Staphylococcus aureus.

The presence of staphylococcal enterotoxin (SE) genes (seg, seh, sei, and sej) and the correlation of their prevalence with the genotypes were studied in 100 clinical isolates of Staphylococcus aureus. Polymerase chain reaction (PCR) of SE genes indicated that 39% of the isolates were enterotoxigenic. Thirty-seven percent of the total isolates were seg positive, whereas 24% and 4% were sei and seh positive, respectively. All isolates containing sei were positive for seg, whereas sej gene was not detected. Genotyping by PCR-restriction fragment length polymorphism analysis of the aroA gene revealed that 39% of the isolates were type A and 11% were type B, and 50% displayed a novel (N) genotype. The presence of the enterotoxin genes was independent (P < 0.05) of the genotypes of the tested S. aureus isolates. This study has demonstrated that the seg was the most dominant enterotoxin gene and that the enterotoxigenic Jordanian S. aureus isolates belong to different genotypes, and N genotype was predominant.

Enterotoxins↗

PubMed1988

The effect of a Clostridium perfringens 8-6 enterotoxin on viability and macromolecular synthesis in Vero cells.

Clostridium perfringens type A 8-6 enterotoxin causes gross morphological damage to Vero cells grown in tissue culture. Damage was observed to occur after only 30 minutes exposure at concentrations as low as 20 ng/ml, and within 60 minutes 95% of the cells had detached. Concentrations as low as 0.01 ng were able to cause detectable inhibition of plating efficiency. The enterotoxin inhibited DNA, RNA and protein synthesis and caused the reversal of glucose transport. Heat inactivated enterotoxin had no effect on cell function or morphology.

Animals↗

PubMed1998

Inhibition of the adherence of cholera toxin and the heat-labile enterotoxin of Escherichia coli to cell-surface GM1 by oligosaccharide-derivatized dendrimers.

The adherence of either cholera toxin or the heat-labile enterotoxin of Escherichia coli to monosialoganglioside gal(beta1-3)galNAc(beta1-4)[sialic acid (alpha2-3)]gal(beta1-4)glc(beta)1-ceramide (GM1) present on the surface of epithelial cells lining the intestine is the first step of a series that results in the induction of a watery diarrhea. While cholera is more severe, both can lead to death as a result of dehydration. To determine the potential of defined multivalent oligosaccharides, synthesized by the covalent attachment of multiple phenylisothiocyanate (PITC) derivatives of gal(beta1-3)galNAc(beta1-4)[sialic acid(alpha2-3)]gal(beta1-4)glc (oligo-GM1) to the arms of a poly(propylene imine) dendrimer, as therapeutic agents for these diseases, their ability to inhibit adherence of the toxins to cell surface-associated GM1 was determined. They not only inhibited choleragenoid (binding subunit of cholera toxin) binding to GM1-treated NCTC-2071 cells (chemically transformed murine fibroblasts) at 5 degrees, but also inhibited adherence of the choleragenoid, cholera toxin, and heat-labile enterotoxin of E. coli to GM1-treated NCTC-2071 cells at 37 degrees. Inhibition was observed whether the toxin was preincubated with the oligo-GM1-PITC-derivatized dendrimer prior to addition to cells or given just after the addition of the derivatized dendrimer to cells. The derivatized dendrimer had no effect on cell viability, as monitored by trypan blue exclusion. Blue-shifts in tryptophan fluorescence emission spectra maxima induced by adherence of either choleragenoid, cholera holotoxin, or the heat-labile enterotoxin of E. coli to oligo-GM1-PITC-derivatized dendrimers were similar to those induced by adherence to GM1 or oligo-GM1. Comparable shifts were not observed when the toxins were incubated with gangliosides that fail to function as receptors.

Adhesiveness↗

PubMed1997

Guanylin-, heat-stable enterotoxin of Escherichia coli- and vasoactive intestinal peptide-induced water and ion secretion in the rat intestine in vivo.

The heat-stable enterotoxin of Escherichia coli binds to an intestinal receptor, guanylyl cyclase-C, and produces cGMP to induce diarrhea. Guanylin is an endogenous ligand of this receptor. In the present in vivo study, the intestinal water and ion secretion induced by mucosal application of 2 nmol/ml guanylin or 5 or 10 units/ml heat-stable enterotoxin into closed loops was compared in the rat. The characteristics of secretion induced by cAMP following intravenous perfusion of 1.2 nmol/100 g per h vasoactive intestinal peptide were compared to those induced by cGMP. Unidirectional Na+ and Cl- fluxes were estimated by addition of 22Na into the loop and i.v. injection of 36Cl. Guanylin induced less water and ion secretion than that produced by heat-stable enterotoxin in the colon, confirming the results of in vitro studies, and also in duodenum and ileum. The cAMP- or cGMP-mediated response had a similar pattern, i.e., an inhibition of Na+ absorption and an increase in anion secretion.

Animals↗

PubMed2002

Trypan blue uptake by chinese hamster ovary cultured epithelial cells: a cellular model to study Escherichia coli STb enterotoxin.

The thermostable enterotoxin b (STb) produced by enterotoxigenic Escherichia coli strains is responsible for diarrheal diseases mainly in weaning piglets. For now, the only available assay for biological activity of STb toxin was in the animal host (i.e. piglet) or in an animal model (i.e. rat, mouse). In this study, we developed a cellular model for the study of the biological activity of STb enterotoxin. Using a trypan blue vital stain method, we showed that STb-treated cells of three out of the five cell lines tested absorbed more vital stain than their controls. Of all the cell lines tested, the chinese hamster's ovary derivated cells (CHO) were the most sensitive, absorbing 50% more trypan blue than their control. Maximal stain uptake was observed after 2h. We then evaluated the trypan blue uptake for 16 STb mutants, produced in a previous work, on the CHO cell lines in order to compare it with the in vivo rat loop assay data. Interestingly, we observed a good correlation between the two bioassays. In fact, the biological activity observed in the rat could be correlated with the trypan blue uptake by the CHO cells (R(2)=0.78) for STb toxin and the 16 mutants. Using the variance analysis statistical test, we determined that the correlation between the two bioassays is significant (F(c)> or =F(0.005)). These results suggest that the trypan blue uptake bioassay could represent a new method to evaluate the biological activity and facilitate the elucidation of the mechanism of action of E. coli STb enterotoxin.

Amino Acid Sequence↗

PubMed2002

Evidence for the involvement of a macrophage-derived chemotactic mediator in the neutrophil recruitment induced by staphylococcal enterotoxin B in mice.

Staphylococcus aureus secretes enterotoxins which are superantigens and the major cause of food poisoning in man. Staphylococcal enterotoxins types A and B can induce neutrophil migration into the peritoneal cavity of mice through sensory C-fiber neuropeptides, lipoxygenase or cyclooxygenase metabolites, nitric oxide, histamine, platelet-activating factor and resident macrophages. In this work, we examined the influence of macrophage-derived products on neutrophil migration during peritonitis induced by staphylococcal enterotoxin type B (SEB) in mice. Macrophages stimulated with SEB released a thermolabile neutrophil chemotactic protein with a molecular weight of 1,000-3,000 (by ultrafiltration). This release was inhibited 30% by dexamethasone (an inhibitor of cytokine synthesis and phospholipase A(2) activity), but not by indomethacin (a cyclooxygenase inhibitor) or BW755C (a dual cyclo- and lipoxygenase inhibitor). Dexamethasone also inhibited (100%) the neutrophil migration induced by the chemotactic protein. Similar inhibition occurred in mice pretreated with BWA4C (lipoxygenase inhibitor; 90%), BW755C (99%), BN52021 (platelet-activating factor-acether receptor antagonist; 93%), cimetidine (histamine H(2) receptor antagonist; 76%), capsaicin (a depletor of sensory C-fiber neuropeptides; 82%) and the neurokinin-1 receptor antagonist SR140333 (71%), but not by indomethacin or the neurokinin(2) receptor antagonist SR48968. These results confirm that macrophages are involved in the neutrophil recruitment induced by SEB, and that the chemotactic protein apparently induces neutrophil migration by a mechanism mediated by platelet-activating factor, histamine H(2) receptors, lipoxygenase products and substance P.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗

PubMed1976

Secretory IgA against enterotoxins in breast-milk.

A pool of colostrum from Guatemalan mothers (Guatemalan colostrum)) obtained 2-4 days post partum inhibited the induced fluid accumulation in rabbit ileal loops when incubated with Vibrio cholerae or Escherichia coli enterotoxin. There was a linear relationship between the quantity of colostrum used and the protection achieved. Pools of Guatemalan breast-milk obtained 15-30 days post partum and North American breast-milk had the same effect when tested with E. coli and V. cholerae enterotoxins, respectively. The antitoxic activity of a given pool correlated with its IgA content but not with the concentration of IgG or IgM. Guatemalan colostrum globulins were precipitated by ammonium sulphate. The globulins were filtered through a 'Biogel A5' column and fractions obtained. When tested in rabbit ileal loops the antienterotoxin activity in these fractions closely paralleled their IgA but not their detectable IgG or IgM content. We hypothesise that IgA antibody to enterotoxin, present in breast-milk of normal mothers, is probably a manifestation of natural immunity. The passive transfer of these antibodies to the infant may explain why breast-milk prevents E. coli diarrhoea in the neonate.

Animals↗

PubMed1978

Antimicrobial resistance and enterotoxin production among isolates of Escherichia coli in the Far East.

The frequency of association between transferable extrachromosomal D.N.A. (plasmid) mediated antibiotic resistance and enterotoxin productin is unknown. The antimicrobial susceptibility of 176 enterotoxigenic Escherichia coli from 57 children and adults in the Philippines, Korea, Taiwan, and Indonesia has been examined. 126 isolates (72%) were resistant to one or more antibiotic(s); 77 (44%) were resistant to four or more antibiotics. 43 E. coli which produced both heat-labile and heat-stable toxin, 110 isolates which produced only heat-labile toxin, and 23 which produced only heat-stable toxin were frequently resistant to multiple antibiotics. 25 of 31 resistant isolates tested, 80% transferred antibiotic resistance in bacterial mating experiments. In 35% of the matings transferring antibiotic resistance, the ability to produce enterotoxin was also conferred on the recipients. This in-vitro observation suggests that the widespread use of antibiotics could increase the distribution of enterotoxigenic E. coli, as genes coding for antibiotic resistance and enterotoxin production are frequently transferred together.

Adult↗

PubMed2002

Effects of staphylococcal enterotoxin A on pituitary-adrenal activation and neophobic behavior in the C57BL/6 mouse.

Bacterial superantigens, such as the staphylococcal enterotoxins (SE), exert a strong capacity for in vivo stimulation of T cell proliferation and cytokine production. Among these superantigens, staphylococcal enterotoxin B (SEB) has been shown to promote anxiety-like properties, possibly mediated by activation of central corticotropin-releasing hormone. In the present study, using male C57BL/6J mice, it was shown that challenge with another prominent superantigen, staphylococcal enterotoxin A (SEA), produced a dose-dependent (0.2-50 microg/mouse) increase in corticosterone and ACTH levels. Interestingly, while the adrenocorticoid response to SEA persisted in cyclosporine A-pretreated mice, it was completely abolished in RAG-1 deficient mice that lack functional T and B lymphocytes. The latter is consistent with the need for cellular interactions involving T cells and B cells (probably in an antigen-presenting capacity) that will initiate events leading to pituitary-adrenal activation by SEA. Since pituitary-adrenal activation typically alters "emotional" reactivity in animals, a final set of experiments assessed behavioral responses to an open field, exposure to a novel object, and a novel appetitive stimulus. These tests revealed a significant augmentation of reactivity to the novel object in SEA-challenged mice, although activity in the open field was not affected. Furthermore, consumption of a novel solution was reduced only if testing involved unfamiliar contextual circumstances. This suggested that anorexic effects per se were not induced by SEA at the dose used, but that attentional mechanisms focused on novelty were enhanced.

Adrenocorticotropic Hormone↗

PubMed2000

Staphylococcal enterotoxins.

Staphylococcus aureus is a major human pathogen that produces a wide array of toxins, thus causing various types of disease symptoms. Staphylococcal enterotoxins (SEs), a family of nine major serological types of heat stable enterotoxins, are a leading cause of gastroenteritis resulting from consumption of contaminated food. In addition, SEs are powerful superantigens that stimulate non-specific T-cell proliferation. SEs share close phylogenetic relationships, with similar structures and activities. Here we review the structure and function of each known enterotoxin.

Amino Acid Sequence↗

PubMed2001

Influence of holding temperature on the growth and survival of Salmonella spp. and Staphylococcus aureus and the production of staphylococcal enterotoxin in egg products.

In this study, growth and survival of Salmonella spp. and Staphylococcus aureus in steamed egg and scrambled egg held at 5, 18, 22, 37, 55 and 60 degrees C are investigated. The production of staphylococcal enterotoxin in steamed egg is also examined. Results reveal that Salmonella spp. and Staph. aureus in the egg products multiply best at 37 degrees C, followed closely by 22 and 18 degrees C. Neither pathogen showed growth in the egg products held at 5 degrees C. Initial inoculation dose, holding temperature and holding time affected the population of both organisms found in the egg products. Staphylococcal enterotoxin A (SEA) and B (SEB) are detected only in the egg products held at 37 or 22 degrees C. After holding at 37 degrees C for 36 h, scrambled egg inoculated with ca. 5.0 log cfu/g Staph. aureus contains the highest levels of SEA (> 64 ng/g) and SEB (> 64 ng/g). Although Salmonella spp. and Staph. aureus grow better in steamed eggs than in scrambled eggs, production of staphylococcal enterotoxin, in general, was higher in scrambled eggs than in steamed eggs. On the other hand, a repaid destruction of the test organisms in steamed eggs held at 60 degrees C was observed. Holding the steamed eggs at 60 degrees C, Salmonella spp. and Staph. aureus with an initial population of ca. 5.9 and 5.6 log cfu/g, respectively, reduced to a non-detectable level in 1 h.

Colony Count, Microbial↗

PubMed1987

Enterotoxin production by strains of Staphylococcus intermedius and Staphylococcus aureus isolated from dog infections.

Sixty-six strains of S. intermedius and 10 of S. aureus isolated from infected dogs were examined for enterotoxin production. 39.5% of the strain (37.9% of S. intermedius and 50% of S. aureus) produced one or more enterotoxins. The predominant types produced by S. intermedius were C1 and C2, and only two of the strains synthesized enterotoxin A. One of the S. aureus strains produced the toxic shock syndrome toxin 1.

Animals↗

PubMed2001

Prevalence of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) gene in isolates in weaned pigs with diarrhea and/or edema disease.

A total of 476 Escherichia coli isolated from weaned pigs with diarrhea and/or edema disease were screened for the presence of the enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1) gene by polymerase chain reaction (PCR). E. coli strains that carried EAST1 genes were also tested by PCR for the presence of genes for five fimbriae (F4, F5, F6, F18 and F41), two heat-stable (STa and STb) and one heat-labile (LT) enterotoxin, and Shiga toxin 2e (Stx2e). One hundred and forty nine (31.3%) of the 476 E. coli isolates carried the gene for EAST1. Of these 149 isolates, 66 (44.3%) carried the east1 gene only and 83 (55.7%) carried genes for the fimbrial adhesins or enterotoxins. E. coli which carried east1 gene also possessed genes for STa or F4 frequently. EAST1 may represent an additional determinant in the pathogenesis of E. coli diarrhea in weaned pigs.

Animals↗

PubMed2003

Detection of Escherichia coli Shiga toxin (stx) and enterotoxin (estA and elt) genes in fecal samples from non-diarrheic and diarrheic greyhounds.

Virulence factors responsible for acute diarrhea in greyhounds have not been well established. The objective of this study was to determine if a correlation exists between disease and the presence of the Escherichia coli toxin genes in non-diarrheic and diarrheic greyhound feces. DNA extracted from broth cultures was evaluated for the presence of Shiga toxin and enterotoxin genes and broth samples were evaluated for Shiga toxin and heat-labile enterotoxin. Shiga toxin (stx1 and stx2) and enterotoxin (et and estA) genes were identified in both non-diarrheic and diarrheic samples after in vitro cultured of swabs at 37 degrees C for 16-24h. The stx1 gene was present in 3% of non-diarrheic and 15% diarrheic samples and the stx2 gene was identified in 36 and 23%, non-diarrheic and diarrheic samples, respectively. Shiga toxin was present in 48% diarrheic and 25% of the non-diarrheic in vitro cultured samples. The elt gene was detected in vitro cultured swabs in 12% of the non-diarrheic and 7% of the diarrheic samples. Labile toxin was present in the feces of small numbers of both groups of dogs. A significant correlation existed between the presence of both stx1 genes and Shiga toxin in feces, and lack of disease in non-diarrheic (P=0.01) and presence of disease in diarrheic (P=0.024) greyhounds. Correlation between production of Shiga toxin and detection of stx1 or stx2 was significant in both the diarrheic and non-diarrheic feces (P=0.03); however, only the presence of stx1 correlated with diarrhea in both groups of samples (P<0.008). The incidence of toxigenic E. coli in both non-diarrheic and diarrheic greyhounds indicates a zoonotic potential from dogs to humans and requires further study.

Acute Disease↗