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PubMed1994

Cloning and expression of putative cytotonic enterotoxin-encoding genes from Aeromonas hydrophila.

A genomic library from a diarrheal isolate, SSU, of Aeromonas hydrophila was constructed in a cosmid vector, pHC79, and in bacteriophage lambda EMBL3. Cell lysates from various Escherichia coli clones containing the recombinant cosmid were examined for their ability to elongate Chinese hamster ovary (CHO) cells, which is a typical enterotoxic response. Based on restriction analysis, a 4.0-kb SalI DNA fragment from one of the clones that exhibited enterotoxic activity was subcloned into a bacteriophage T7 RNA polymerase/promoter hyperexpression system. The cell lysate from this E. coli [pSL24] clone caused CHO cells to elongate and revealed the presence of a major 35-kDa polypeptide by [35S]methionine labeling and sodium dodecyl sulfate (SDS)-polyacrylamide-gel electrophoresis (PAGE). The toxin was biologically heat labile, losing all activity within 20 min at 56 degrees C. In addition, another enterotoxin-producing clone, E. coli[pSBS32], was isolated from cosmid and lambda bacteriophage libraries. We localized this heat-stable (56 degrees C/20 min) enterotoxin to a 4.8-kb SalI-BamHI fragment. Both enterotoxins caused elevation of cyclic adenosine monophosphate (cAMP) in CHO cells. The DNA fragments encoding these enterotoxins did not hybridize with each other. However, a 4.8-kb SalI-BamHI DNA fragment encoding a heat-stable enterotoxin hybridized to a 3.5-kb BamHI DNA fragment of a plasmid, pHPC100, that contained a cytotonic enterotoxin-encoding gene isolated from A. trota. Our data suggest Aeromonas species produce different structural types of cytotonic enterotoxins that are functionally similar.

Aeromonas hydrophila↗

PubMed1983

Hemagglutinating and hydrophobic surface properties of salmonellae producing enterotoxin neutralized by cholera anti-toxin.

Eleven Salmonella strains known to produce enterotoxin under aerobic culture conditions in deferrated (DF) medium at 37 degrees C were shown to produce enterotoxin with and without aeration at 22, 28, 37 and 42 degrees C. Heat-labile enterotoxin was generally produced with growth temperatures up to 37 degrees C irrespective of aeration. Heat-stable enterotoxin was produced up to 42 degrees C, mainly aerobically, as indicated by infant mouse assay (IMA), by six of the eleven strains tested. Nine strains produced heat-stable rapid permeability factor (RPF) in rabbit skin. Cholera anti-toxin neutralized reactivities of Salmonella heat-labile enterotoxin in four different biological assays. Mixed gangliosides also neutralized this activity in the cell-test systems. With guinea-pig erythrocytes, all strains underwent mannose-resistant hemagglutination (MRHA) irrespective of growth temperatures, i.e. 22 and 37 degrees C or medium, i.e., DF, tryptose soy broth (TSB) and colonization factor antigen (CFA) agar. At both growth temperatures, CFA agar-grown cells of each strain caused MRHA of bovine erythrocytes. Excepting three Salmonella typhimurium strains, DF broth-grown cells gave MRHA of bovine, chicken and human group A erythrocytes, CFA agar-grown cells caused MRHA of chicken and human blood, whereas TSB-grown cells caused few MRHA reactions. Salmonellae producing both heat-stable, (ST) and heat-labile, (LT) enterotoxins adsorbed to Phenyl Sepharose whereas salmonellae that produced only LT enterotoxin did not. The presence of MRHA adhesions did not correlate with cell-surface hydrophobicity. However, mannose-resistant hemagglutinins may occur more commonly among salmonellae than has been previously recognized.

Animals↗

PubMed1998

Behaviour and enterotoxin production by Staphylococcus aureus during the manufacture and ripening of raw goats' milk lactic cheeses.

To study the possible presence of staphylococcal enterotoxin A in raw goats' milk lactic cheese, milk was inoculated with an enterotoxigenic Staphylococcus aureus strain to a final concentration of 4, 5 and 6 log(cfu/ml). Cheese was prepared following industrial specifications and ripened for 42 d. Detection of the enterotoxins was by the Vidas Staph enterotoxin test (BioMérieux) and by an indirect double-sandwich ELISA technique using anti-enterotoxin monoclonal antibodies. Staphylococcal counts declined markedly after draining, and by the end of ripening they had disappeared from some cheeses. In contrast, aerobic mesophilic organisms grew well. The level of staphylococcal enterotoxin A recovered varied from 1 to 2.5 ng/g cheese made with an initial population of 10(5) or 10(6) cfu/ml. Only traces of enterotoxin A (0.5 ng/g) were detected in cheeses made with the lowest Staph. aureus inoculum used in this study. Enterotoxin A was also detected in cheeses from which Staph. aureus had disappeared.

Animals↗

PubMed1996

Bacteroides fragilis enterotoxin modulates epithelial permeability and bacterial internalization by HT-29 enterocytes.

BACKGROUND & AIMS: Enterotoxigenic Bacteroides fragilis has been associated with diarrheal disease, and the enterotoxin has a cytopathic effect on cultured HT-29 enterocytes. Experiments were designed to determine the effect of B. fragilis enterotoxin on bacteria-enterocyte interactions. METHODS: Confluent HT-29 enterocytes were incubated for 1 hour with B. fragilis enterotoxin, followed by 1 hour of incubation with pure cultures of enteric bacteria, namely, Salmonella typhimurium (two strains), Listeria monocytogenes (three strains), Proteus mirabilis, Escherichia coli (three strains), and Enterococcus faecalis. Enterocyte viability was assessed using vital dyes, epithelial permeability was measured using transepithelial electrical resistance, enterocyte morphology and bacteria-enterocyte interactions were visualized using light and electron microscopy, and bacterial internalization was assessed using a quantitative culture of lysed enterocytes. RESULTS: B. fragilis enterotoxin did not affect enterocyte viability but decreased transepithelial electrical resistance, and individual enterocytes pulled apart. Enterotoxin pretreatment decreased internalization of L. monocytogenes (P < 0.01) but increased (P < 0.01) internalization of the other strains of enteric bacteria. Augmented bacterial internalization was associated with preferential bacterial adherence on the exposed lateral surface of enterotoxin-treated enterocytes. CONCLUSIONS: B. fragilis enterotoxin was associated with HT-29 cell rounding and with augmented internalization of selected strains of enteric bacteria that were preferentially adherent on the exposed enterocyte lateral surface.

Bacterial Adhesion↗

PubMed1991

The CD4 molecule is not always required for the T cell response to bacterial enterotoxins.

T cells respond in a V beta-restricted fashion to bacterial enterotoxins bound to major histocompatibility complex (MHC) class II molecules. The requirement for CD4 in MHC class II-restricted T cell responses is very well established. We have assessed the role of CD4 in the T cell response to the bacterial enterotoxins Staphylococcal enterotoxin A (SEA), SEB, and toxic shock syndrome toxin 1. Three CD4- murine T cell hybridomas were transfected with the human CD4 molecule and assayed for interleukin 2 production in the presence of accessory cells bearing human MHC class II molecules and of the appropriate enterotoxin. The results clearly indicate that CD4- cells responded even to suboptimal concentrations of enterotoxin(s) equally well as CD4+ cells. Furthermore, expression of CD4 did not result in the acquisition of previously undetectable reactivity to enterotoxins. These results suggest that unlike the case with antigen-specific responses, formation of a T cell receptor-CD3/CD4 supramolecular complex is not always essential for T cell activation by bacterial enterotoxins.

Animals↗

PubMedDate not supplied

Epitopes of the cholera family of enterotoxins.

Hybridoma-derived monoclonal antibodies were raised to enterotoxins of the cholera family and to chimeric B-subunit proteins in which individual amino acid residues of a heat-labile, cholera-related enterotoxin from an Escherichia coli strain of porcine origin (P-LT) were substituted with corresponding residues from such an enterotoxin from an E. coli strain of human origin (H-LT). Single amino acid substitutions were found to have profound effects on the physicochemical behavior of the proteins and on their immunologic reactivity. With the use of enzyme-linked immunosorption assays (ELISAs) with and without the GM1 ganglioside receptor for these toxins, several distinct epitopes in GM1-binding domains were identified by different monoclonal antibodies. Polyclonal rabbit antisera to synthetic peptides of the cholera enterotoxin B subunit were cross-reactive to various degrees with the proteins in our library, which include two different cholera enterotoxins, two H-LTs, P-LT, and four chimeric proteins. Some of these reactions were blocked by GM1 ganglioside but not by the oligosaccharide of GM1, a finding suggesting that the peptides generated antibodies to epitopes near, but not in, a GM1-binding domain. A hypothetical evolutionary tree based on the reported amino acid sequences of the various enterotoxins is constructed. As additional enterotoxins are described, it will be interesting to determine if and where they fit in this scheme.

Amino Acid Sequence↗

PubMedDate not supplied

Antigenic determinants of the cholera/coli family of enterotoxins.

Hybridoma-derived monoclonal antibodies were raised to enterotoxins of the cholera family and to chimeric B-subunit proteins in which individual amino acid residues of a heat-labile, cholera-related enterotoxin from an Escherichia coli strain of porcine origin (P-LT) were substituted with corresponding residues from such an enterotoxin from an E. coli strain of human origin (H-LT). Single amino acid substitutions were found to have profound effects on the physicochemical behavior of the proteins and on their immunologic reactivity. With the use of enzyme-linked immunosorption assays (ELISAs) with and without the GM1 ganglioside receptor for these toxins, several distinct epitopes in GM1-binding domains were identified by different monoclonal antibodies. Polyclonal rabbit antisera to synthetic peptides of the cholera enterotoxin B subunit were cross-reactive to various degrees with the proteins in our library, which include two different cholera enterotoxins, two H-LTs, P-LT, and four chimeric proteins. Some of these reactions were blocked by GM1 ganglioside but not the oligosaccharide of GM1, a finding suggesting that the peptides generated antibodies to epitopes near, but not in, a GM1-binding domain. A hypothetical evolutionary tree based on the reported amino acid sequences of the various enterotoxins is constructed. As additional enterotoxins are described, it will be interesting to determine if and where they fit in this scheme.

Amino Acid Sequence↗

PubMed1976

Escherichia coli enterotoxin: purification, partial characterization, and immunological observations.

Enterotoxin, a diarrhea-inducing protein elaborated by pathogenic Escherichia coli strains, was isolated from the supernate of fermenter cultures of E. coli strain P263, a porcine enteropathogen. Purification involved chromatography and preparative isotachophoresis. The resulting product appeared to be pure according to immunoelectrophoretic, disc electrophoretic, ultracentrifugal, and immunologic criteria. The enterotoxin had an apparent molecular weight of 102,000 daltons, and its isoelectric point was 6.90. The isolated product was active in inducing experimental diarrhea in adult rabbits and piglets. In small dosage it also elicited a drastic increase in adenylate cyclase activity in broken-cell preparations of cat heart tissue. The enterotoxin activity was acid labile and was destroyed by heat (65 C for 30 min). It is suggested that the heat-stable enterotoxin was derived from heat-labile enterotoxin by complexing with endotoxin or with capsular material in the culture supernatant. The antigenic relations between the heat-labile enterotoxins of enteropathogenic E. coli strains of different serological types and different host adaptations, as well as between the E. coli enterotoxin and that of Vibrio cholerae, were investigated.

Animals↗

PubMed1982

In vivo effects of enterotoxin from Clostridium perfringens type A in the rabbit colon: binding vs. biologic activity.

Rabbit colonic loops were exposed to purified enterotoxin from Clostridium perfringens type A and perfused for analysis of the effects of the enterotoxin on transport and surface structure of the colon. The colon showed no apparent response to the enterotoxin in the transport of fluid and electrolytes as compared with untreated animals. There was a significant increase in levels of luminal fluid protein, corresponding to an observed increase in secretion of mucus. Histologic sections showed no epithelial damage, but scanning electron microscopy showed an increase in the number of mucous fields over the value in control animals. The colon showed little or no overt response to enterotoxin treatment, but isolated colonic epithelial cells bound 125I-labeled enterotoxin at levels even greater than those observed in cells from the ileum, where the enterotoxin has considerable biologic activity. Thus, although binding of enterotoxin is necessary for its biologic activity, biologic activity does not necessarily follow binding to specific receptors.

Absorption↗

PubMed2000

Production of an enterotoxin by a gastro-enteritis-associated Aeromonas strain.

The potential of motile Aeromonas species to cause human gastrointestinal infections has been recognised recently. Considerable worldwide epidemiological, microbiological and clinical investigations have shown that some strains of the different motile aeromonads are of increasing enteropathogenic significance, especially in children, the elderly and in immunocompromised individuals. Some of the diarrhoeal symptoms of Aeromonas-associated gastro-enteritis have been attributed to enterotoxins. In this study, 15 Aeromonas isolates from clinical and non-clinical sources, representing the three motile aeromonads commonly associated with gastro-enteritis (A. caviae, A. hydrophila and A. veronii biovar sobria), were tested for their ability to cause fluid accumulation in infant mice by the suckling mouse technique. Eight isolates were found to produce enterotoxin. Of these, an A. veronii biovar sobria strain (AS15), isolated from lamb kidney, was found to produce the highest enterotoxin score. An enterotoxin of c. 40 kDa produced by A. veronii biovar sobria AS15 was purified by Sephacryl S-100 gel filtration and high-performance liquid chromatography. This enterotoxin caused marked fluid accumulation in infant mice by the suckling mouse technique. The purified enterotoxin cross-reacted with cholera toxin antibodies and was readily inactivated by heating at 56 degrees C for 10 min. The production of a 'cholera-like' enterotoxin by Aeromonas isolates from samples of animal origin suggests that these organisms could be of public health significance in food products.

Aeromonas↗

PubMed1989

Enterotoxin production by Staphylococcus aureus isolates from cases of septicaemia and from healthy carriers.

In a prospective study, 52 Staphylococcus aureus isolates from individual patients with septicaemia and 27 nasal strains from separate, healthy carriers were compared for production of a range of extracellular proteins and toxins. Whereas there was no difference (p greater than 0.05) between septicaemic and nasal isolates with respect to incidence of alpha, beta, gamma and delta haemolysins, toxic shock syndrome toxin-1 or staphylokinase production, the incidence of enterotoxin A, B, and C production was higher among isolates from septicaemia (p less than 0.01). Of the isolates from septicaemia, 33 (63%) produced enterotoxins A, B, C or D alone or in combination. Only three (11%) of the nasal isolates produced a single enterotoxin, enterotoxin D. Of the isolates from septicaemia, 67% were hospital-acquired and greater than 25% of these were endemic, methicillin-resistant (MRSA) strains. All MRSA strains produced either enterotoxin A, or enterotoxin B, or both. These findings suggest a possible role for enterotoxins in the pathogenesis of S. aureus disease other than food poisoning.

Bacterial Toxins↗

PubMed1980

Heat-stable enterotoxin produced by Yersinia enterocolitica isolated from patients.

Twenty-three strains of Yersinia enterocolitica were isolated from children with gastrointestinal illness and examined for the production of enterotoxins by using both suckling mouse and Chinese hamster ovary (CHO) cell assay systems. Six strains were found to be enterotoxigenic in the suckling mouse assay, but all strains were negative in the CHO cell assay. Enterotoxin was detected in the culture supernatant when organisms were grown at 25 C but not at 37 C. Enterotoxin in a 15-fold concentrated culture supernatant was precipitated by adding absolute ethanol to a concentration of 90%. However, after being dialyzed against distilled water in Spectra/por 6 membrane tubing, it was soluble in 80% acetone. One unit dose of partially purified enterotoxin was 5.0 micrograms of protein/mouse in the suckling mouse assay. The molecular weight of enterotoxin was between 10,000 and 50,000 daltons as determined by ultrafiltration. It was stable to heat (121 C X 20 min or 100 C X 60 min). These observations indicated that Y. enterocolitica isolated in Japan also produce an enterotoxin similar to the heat-stable enterotoxin of Escherichia coli. However its physiochemical properties seem to be different from those of E. coli.

Child↗

PubMed1986

Scaled-up production and purification of Clostridium perfringens type A enterotoxin.

Methods for small-scale production of Clostridium perfringens type A enterotoxin were unsuitable for large-scale culture of this organism. Rapid, efficient harvesting of 40 1 batch culture of Cl. perfringens was achieved by tangential flow micro-filtration with the Millipore Pellicon cassette system. Enterotoxin-containing extracts were prepared by passing concentrated suspensions of the harvested cells through a French pressure cell. The overall yield of purified enterotoxin was 38.8%. The toxin gave a single band on native polyacrylamide gels but formed high molecular weight aggregates in the presence of sodium dodecyl sulphate. These aggregates frequently occurred during storage of non-sterile enterotoxin preparations but could be separated from the monomer toxin by gel filtration on Sephadex G-100. Purified monomer enterotoxin had biological activities of 119.3 micrograms/kg mouse lethal dose when injected intraperitoneally and 3333 capillary permeability increasing units/mg protein in guinea pig skin. Thirty micrograms of the enterotoxin caused fluid accumulation in ligated rabbit ileal loops. Aggregated enterotoxin had no demonstrable biological or immunological activity.

Animals↗

PubMed2004

Comparison of three immunological methods for detecting staphylococcal enterotoxins from food.

AIMS: Immunologically based assays for the detection of staphylococcal enterotoxins are numerous. These techniques include radio immunosorbent assays and enzyme-linked immunosorbent assays (ELISA), some of which are available as commercial kits. The purpose of this study was to compare the performances of three commercial immunoassays. METHODS AND RESULTS: Two automated detection systems, VIDAS SET bioMèrieux, VIDAS SET2 bioMérieux and an ELISA method, TRANSIA PLATE Staphylococcal Enterotoxins Diffchamb were compared for detecting different quantities of purified staphylococcal enterotoxins (A, B, C2, D and E) added to food. CONCLUSIONS: VIDAS SET2 had a greater specificity (100%) and sensitivity than VIDAS SET and TRANSIA PLATE Staphylococcal Enterotoxins. More precisely, VIDAS SET2 could detect <0.5 ng g(-1) of toxins A and B, <1 ng g(-1) of toxins C2 and E and 1 ng g(-1) of toxins D and E. SIGNIFICANCE AND IMPACT OF THE STUDY: Because staphylococcal food poisoning (resulting from ingestion of low levels of staphylococcal enterotoxins) is one of the most common forms of foodborne illness there is a need for specific and sensitive methods for detecting these enterotoxins. VIDAS SET2 appears to be suitable for detecting staphylococcal enterotoxins from food.

Bacteriological Techniques↗

PubMed1992

The effects of Escherichia coli heat-stable enterotoxin in renal sodium tubular transport.

To compare the function of sodium transport between intestine and renal tubule, we studied the effect of E. coli STa enterotoxin and 8-bromo cyclic GMP in perfused rat kidneys. Infusion of STa enterotoxin (0.017 and 0.1 micrograms/ml) caused a dose and time dependent decrease in total renal sodium tubular transport. The major site of STa effect was at the renal proximal tubule. Similar to Sta enterotoxin, 8-bromo cyclic GMP (10(-5) M) caused a significant decrease of fractional renal sodium proximal tubule transport. In contrast to STa enterotoxin, infusion of 8-bromo cyclic GMP resulted in a significant but short lasting (30 min.) increase in glomerular filtration rate. STa enterotoxin also decreased significantly the renal tissue potassium. This STa effect was related to a significant decrease in renal potassium tubular transport, resulting also in an increase of urinary potassium excretion. These studies demonstrate the specific functional effect of STa enterotoxin in promoting the decrease in renal proximal tubular sodium transport, similar to 8-bromo cyclic GMP. In perfused rat kidneys STa also decreased tissue potassium, mainly by a decrease in potassium transport and increase in urinary potassium excretion. These effects suggest the existence of an endogenous peptide resembling STa enterotoxin, that regulates the function of renal sodium tubular transport.

Animals↗

PubMed1981

Relationship of sporulation, enterotoxin formation, and spoilage during growth of Clostridium perfringens type A in cooked chicken.

Sporulation and enterotoxin formation were determined for 17 strains of Clostridium perfringens type A in autoclaved chicken dark meat and in Duncan-Strong sporulation medium. The mean numbers of heat-resistant spores detected after 24 h at 37 degrees C were log10 1.13 to log10 7.64/ml in Duncan-Strong medium and log10 4.93 to log10 6.59/g in chicken. Of 17 strains, 7 formed enterotoxin in Duncan-Strong culture supernatant (1.0 to 60 microgram/ml) and 8 produced enterotoxin in chicken (0.21 to 24 microgram/g). Additional studies with chicken were conducted with C. perfringens NCTC 8239. With an inoculum of 10(6) cells per g, greater than log10 7.99 vegetative cells per g were detected by 4 h in chicken at 37 degrees C. Heat-resistant spores occurred by 4 and 6 h and enterotoxin occurred by 8 and 6 h in autoclaved chicken dark meat and barbecued chicken drumsticks, respectively. Enterotoxin was detected in autoclaved dark meat after incubation at 45 degrees C for 1.5 h followed by 37 degrees C for 4.5 h, but not after incubation at 45 degrees C for 1.5 to 8 h. With an inoculum of 10(2) cells per g in oven-cooked or autoclaved chicken, greater than log10 8.00 vegetative cells per g were detected by 6 to 8 h at 37 degrees C, heat-resistant spores were detected by 8 h, and enterotoxin was detected by 12 h. A statistical analysis of odor determinants of chicken after growth of C. perfringens indicated that, at the 95% confidence level, the product was considered spoiled (off or unwholesome odor) by the time spores or enterotoxin were formed.

Animals↗

PubMed1988

A quantitative study of enterotoxin production by sheep milk staphylococci.

Of 124 staphylococcal strains isolated from sheep milk, 78 produced enterotoxin A, B, C, or D when evaluated by an enzyme-linked immunosorbent assay. Enterotoxins A and D, elaborated by 44 and 43 strains, respectively, showed the highest incidence. Enterotoxin production by coagulase-negative strains (one Staphylococcus cohnii, three S. epidermidis, five S. haemolyticus, and four S. xylosus) was detected. Linear and logarithmic-logarithmic regressions of optical density on enterotoxin concentration yielded the best-fitting equations for enterotoxin quantitation. A significantly higher incidence of enterotoxin producers and significantly higher levels of enterotoxins produced were recorded for coagulase-positive, thermostable nuclease-positive, hemolysis-positive, or mannitol-positive strains. Mannitol utilization was the best test for discriminating between enterotoxigenic and nonenterotoxigenic staphylococci.

Animals↗

PubMed1966

Thermal inactivation of staphylococcal enterotoxin B in veronal buffer.

The times and temperatures required to inactivate staphylococcal enterotoxin B were studied by use of the double-gel-diffusion technique to assay enterotoxin. Enterotoxin B (99 +% pure) was suspended in 0.04 M Veronal buffer, dispensed into borosilicate vials, and the vials were sealed and heated in an oil bath. An amount of 30 mug/ml of this toxin was reduced to less than 0.7 mug/ml in 103.0, 87.1, 70.5, 57.2, 39.1, 27.6, 16.4, and 12.0 min, respectively, at temperatures of 96, 99, 101.7, 104.4, 110, 115.6, 121, and 126.7 C. The end point for enterotoxin inactivation by gel diffusion was identical to that by intravenous injection of cats. Limited studies with crude enterotoxin B showed that the crude preparation was slightly more thermostable. The respective D values of crude and purified enterotoxin B were 64.5 and 52.3, 40.5 and 34.4, 29.7 and 23.5, 18.8 and 16.6, and 11.4 and 9.9 min at temperatures of 99, 104.4, 110, 115.6, and 121 C. The z value for purified enterotoxin B was 32.4 C. The experimental activation energy was 20,700 cal/g mole, standard enthalpy of activation at 120 C was 19,900 cal/g mole, standard entropy of activation at 120 C was -21.4 cal/g mole K, and the standard free energy of activation at 120 C was 28,200 cal/g mole.

Barbiturates↗