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PubMed2001

In vitro effects of wood creosote on enterotoxin-induced secretion measured electrophysiologically in the rat jejunum and colon.

Secretory diarrhea occurs when the balance between intestinal absorption and secretion is disturbed by excessive secretion caused by enterotoxins produced by the pathogen. Wood creosote has long been used as a traditional antidiarrheal remedy. The goal of our study was to extend our knowledge about the antisecretory action of wood creosote against Escherichia coli enterotoxin-induced secretion in the small intestine and colon. Experiments were performed in mucosal sheets of rat jejunum and colon which were stripped of the external muscle layers to eliminate interactions with smooth muscle activity and local blood flow. Mucosal sheets were placed in modified Ussing chambers and hypersecretory conditions were induced by heat-labile (LT) or heat-stable (STa) E. coli enterotoxins added cumulatively (0.01-10 microg/ml) to the mucosal bathing solution. Intestinal secretion was monitored electrophysiologically as transmucosal short circuit current (Isc). LT induced a concentration-dependent increase in Isc in the rat jejunum, with no effect in the colon. In contrast, STa induced a significant increase in colonic Isc, without causing any change in Isc across the jejunum. In separate experiments the effects of increasing concentrations of wood creosote (0.1-50 microg/ml), added to the mucosal or serosal bathing solution, were examined against the secretory responses induced by LT or STa. In the small intestine the antisecretory activity of wood creosote against LT-induced secretion was more potent following serosal application, whereas in the colon wood creosote inhibited STa-induced secretion with equal potency following either serosal or mucosal addition. In summary, our findings demonstrate that wood creosote possesses antidiarrheal activity suppressing E. coli enterotoxin-induced secretion in both the small intestine and colon.

Animals↗

PubMed1998

A novel ELISA format for the rapid and sensitive detection of staphylococcal enterotoxin A.

Staphylococcal food poisoning is one of the leading causes of bacterial food poisoning each year. Detection kits for staphylococcal enterotoxins are commercially available and the assays can require from one and a half to twenty-four hours to complete with detection limits ranging from 0.5 to 2 ng enterotoxin per gram of food. We have successfully demonstrated a microsphere-packed capillary (MPC) ELISA for the detection of staphylococcal enterotoxin A (SEA) and have compared it to two commercially available kits. The MPC assay detected a lower amount of SEA in ham, chicken, cheese, and bean sprouts than either of the two commercially available kits. In addition, the novel MPC assay was completed in less than ten minutes, as compared to three and twenty-four hours for the two commercially available kits. This research also demonstrated that the MPC ELISA can contain integrated positive and negative controls and has the potential to simultaneously detect and identify multiple enterotoxins.

Enterotoxins↗

PubMedDate not supplied

Distribution of staphylococcal enterotoxin genes among Staphylococcus aureus isolates from poultry and humans with invasive staphylococcal disease.

Food poisoning by Staphylococcus aureus affects hundreds of thousands of people each year. Staphylococcus aureus also causes invasive diseases such as arthritis (in poultry) and septicemia (in poultry and humans). Foodborne disease is caused by the ingestion of a staphylococcal enterotoxin (SE). Enterotoxin has also been associated with other S. aureus illnesses in humans and domestic animals. In this study, polymerase chain reaction was used to detect the staphylococcal enterotoxin genes, SEA, SEB, SEC, SED, and SEE, in S. aureus isolates associated with invasive disease in poultry and humans. In the 34 poultry isolates, only one isolate was found to contain a SE gene, sec. In the 41 human isolates, over 51% tested positive for an SE gene with 12.2% positive for the gene for SEA, 2.4% for SEB, 22% for SEC, 24.4% for SED, and 0 for SEE. The disparity between the rates for SE gene(s) in poultry and human isolates suggests a lesser role for the enterotoxins in invasive poultry disease than in human disease.

Animals↗

PubMed1995

51Cr-labelled ethylenediaminetetraacetic acid and D-xylose absorption test in Escherichia coli enterotoxin-induced diarrhoea in mice.

BACKGROUND AND METHODS: D-Xylose and 51Cr-labelled ethylenediaminetetraacetic acid (EDTA) absorption tests were evaluated in control and Escherichia coli heat-stable, heat-labile, and both heat-stable and heat-labile enterotoxin-induced diarrhoea in mice. RESULTS: The absorption of D-xylose was greatest 45 min after its ingestion in both control and enterotoxin-treated groups. There was a significant (p < 0.01) decrease in xylose absorption at 45 min in all the three experimental groups as compared with the control group, which may be due to change in membrane fluidity and hence change in membrane permeability, whereas 51Cr-EDTA absorption was at its maximum 15 min after its ingestion in both control and experimental groups. And there was a significant (p < 0.05) increase in 51Cr-EDTA absorption in enterotoxin-treated groups as compared with the control group. CONCLUSIONS: These results suggest a change in tight junctional permeability which may be due to change in the cytoskeletal microfilaments. Both these tests hence show that E. coli enterotoxin-induced diarrhoea results in absorption and permeability changes.

Animals↗

PubMed1989

Growth and production of enterotoxin A by Staphylococcus aureus in cream.

Behavior of Staphylococcus aureus strains 100-A, 196-E, 254, 473, 505, and 521 in sweet (18 to 80% milk fat) and neutralized sour cream was studied. Cream was inoculated to contain approximately 10(3) to 10(4) S. aureus/ml, depending on milk fat content, and was incubated at 4, 22, or 37 degrees C. Determinations were made of aerobic plate count, S. aureus count, and pH. When growth in cream exceeded 10(7) S. aureus/ml, enterotoxin analysis was done. Sweet and neutralized sour cream supported growth of all strains of S. aureus tested. Strains 100-A, 196-E, 473, 505, and 521 grew sufficiently to produce enterotoxin in sweet cream of 18 or 32% milk fat held at 37 degrees C for 18 h or at 22 degrees C for 52 h. Populations of strains 100-A, 196-E, 505, and 521 exceeded 10(6) cells/ml in sweet cream of 36% milk fat held for 18 h at 37 degrees C. Strains 100-A and 521 grew to more than 10(6) cells/ml in sweet cream of 40% milk fat held for 18 h at 37 degrees C. No strain of S. aureus grew to levels associated with detectable enterotoxin production at 4 degrees C within 14 d in any cream. Incubation temperature, milk fat content of cream, and variation among strains influenced the ability of S. aureus to grow and produce enterotoxin.

Aerobiosis↗

PubMed2002

A superantigen bioassay to detect staphylococcal enterotoxin A.

Current detection methods for enterotoxins of Staphylococcus aureus are labor intensive and limited in sensitivity. Furthermore, these immunochemical protocols fail to adequately detect heat-treated enterotoxins. Staphylococcal enterotoxins cause severe gastrointestinal illness at relatively low concentrations and retain toxigenicity even after heat treatment. Presented here is a novel method to detect staphylococcal enterotoxin A (SEA). This method is a bioassay that exploits SEA's activity as a superantigen in that it induces in cytotoxic T lymphocytes a cytotoxic response against SEA-bound Raji cells. Target cell death is assayed colorimetrically with the CytoTox 96 cell lysis detection kit. In the experiments presented here, this bioassay was also able to detect heat-treated SEA, albeit with a slight compromise in sensitivity. This system detected SEA at picomolar concentrations. Because of the sensitivity of this assay, it is conceivable that it could be incorporated into current detection methods as a confirmatory test.

Biological Assay↗

PubMed1975

Evidence for cell-receptor activity in lymphocyte stimulation by staphylococcal enterotoxin.

A wide dose-response curve and the inhibitory effect on mitogenicity of specific antitoxin suggest that polyclonal lymphocyte activation by staphylococcal enterotoxin requires direct interaction of toxin with lymphocyte receptors of low avidity for the protein. Staphylococcal enterotoxins A, B, and C1 demonstrated equivalent mitogenic activity. Lymphocyte receptors involved in enterotoxin activation thus appear to be specific for nonantigenic regions of the toxin molecule. Monosaccharide (hapten) inhibiton data indicate that lymphocyte receptors for staphylococcal enterotoxin lack alpha-mannoside, galactose, acetylgalactosamine, acetylglucosamine, and fucose (or closely related saccharides) as determinant sugars and thus differ significantly in structure from lectin cell receptors.

Acetylgalactosamine↗

PubMedDate not supplied

Rapid and specific differentiation of enterotoxin-producing Escherichia coli strains from other gram-negative enteric bacteria using multiplex PCR.

Enterotoxigenic Escherichia coli (ETEC) may produce heat-labile (LT) and heat-stable (STI or STII) enterotoxins. Differentiation between ETEC and other pathogenic and non-pathogenic E. coli as well as other Gram-negative bacteria responsible for induction of diarrhoea, requires isolation, biochemical identification and determination of toxins (or their genes--elt, estI, estII). A multiplex polymerase chain reaction (PCR) system for the rapid and specific detection of enterotoxin-gene-positive E. coli was developed. The primers described by other authors, specific for the universal stress protein A (UspA) of E. coli and enterotoxin genes were used and allowed a simultaneous amplification of the E. coli-specific uspA and the respective toxin genes. The specificity of this multiplex PCR system was confirmed by testing ETEC, non-ETEC and other non-E. coli bacteria. The specific 884 bp uspA gene and 280 bp (eltI), 166 bp (estI) or 278 bp (estII) amplification products were generated with the respective ETEC strains whereas no amplification was detected with non-E. coli bacteria. The multiplex PCR developed allowed the rapid and specific identification of enterotoxin-producing E. coli colonies directly grown from faecal samples of pigs with diarrhoea. The test may be used as a method for the determination of ETEC among other pathogenic groups of E. coli and other Gram-negative enteric isolates.

Animals↗

PubMed2001

[Effect of clindamycin on stimulation of cell adhesion molecules by endotoxins and enterotoxin of Bacteroides fragilis strains].

The influence of clindamycin on expression of B. fragilis endotoxins (LPS) and enterotoxin stimulated cell adhesion molecules: ICAM-1, VCAM-1 and E-selectin on HMEC-1 (human microvascular endothelial cell line) was tested. Lipopolysaccharides from four Bacteroides fragilis strains: one nonenterotoxigenic (NTBF) and three enterotoxigenic (ETBF) were extracted by hot phenol-water method and purified. B. fragilis enterotoxin was prepared according to the method described by van Tassel et al. (1992). All bacterial preparations were used for stimulation at concentration 10 micrograms/ml. Clindamycin was used in concentration of 2 micrograms/ml. The influence of antimicrobial agent on the endotoxins and enterotoxin stimulation and expression of adhesion molecules was tested by ELISA, using monoclonal mouse anti-human antibodies (Genzyme, USA). Peroxidase-conjugated rabbit anti-mouse immunoglobulins (DAKO A/S Denmark) and OPD (Sigma USA) were used. The coloured reaction product was measured by reading the absorbance at 492 nm in SPECTRA II reader (SLT, Austria). It was observed that clindamycin influenced the expression of cell adhesion molecules on resting cell line. HMEC-1 cells stimulated with Bacteroides fragilis LPS preparations have suppressive effect on ICAM-1 expression. ICAM-1 expression was augmented when stimulated with Tox 1 and Tox 2 preparations. Clindamycin augmented the VCAM-1 expression in tests with all bacterial preparations. All used bacterial preparations of Bacteroides fragilis LPS and enterotoxin enhanced the expression of E-selectin with exception of LPS of NTBF strain.

Bacteroides fragilis↗

PubMed2002

PCR detection and prevalence of enterotoxin (cpe) gene in Clostridium perfringens isolated from diarrhea patients.

UNLABELLED: Clostridium perfringens isolated from patients with diarrhea (n=233) were analysed by a duplex PCR assay, in order to determine the prevalence of enterotoxin (cpe) gene and various factors involved in patients with cpe-positive isolates. This duplex PCR uses two sets of primers which amplify in the same reaction two different gene fragments: the phospholipase C (plc, alpha-toxin) and the enterotoxin (cpe) genes in C. perfringens. PCR analysis of 477 colonies of fecal spore isolates, from 159 patients who had a spore count > or = 10(3) cfu/g, gave positive plc gene detection in 436 colonies. The results were consistent with those obtained by using the standard method of C. perfringens species identification. 21 of 436 colonies gave positive results for both plc and cpe genes, indicating a prevalence of 4.8 per cent of C. perfringens that carried the cpe gene in cases of diarrhea. The majority of cases with cpe-positive isolates were women over 50 years of age (71.4%). These patients had diarrhea more than 6 times per day (71.4%) with a duration of 1-3 days (100%). Furthermore, 85.7 per cent of cases developed diarrhea after food consumption, 28.6 per cent had high spore counts of more than 106/g in their feces, and 71.4 per cent were co-infected with other enteric pathogens. The spore count should be interpreted with caution because not all isolates of C. perfringens from diarrhea patients with high fecal spore count carried the cpe gene, which encodes a sporulation-associated enterotoxin. CONCLUSION: The duplex PCR assay can thus become a tool for C. perfringens species identification together with the detection of enterotoxin gene. This PCR assay is faster, less expensive and more suitable for large-scale use in epidemiological studies than conventional procedures. The authors recommend this assay to screen for enterotoxigenic C. perfringens isolates from primary fecal spore isolation cultures, particularly in elderly patients with food-borne diarrhea and non-food related diarrhea.

Adolescent↗

PubMed1976

On detection of the Staphylococcal enterotoxin A by aggregate-hemagglutination technique and electron-microscopic data on this toxin.

Recently, great success has been attained in studies on agents responsible for food intoxication. The modern stage of research on food intoxication of bacterial etiology is qualitatively different from previous ones, since many investigators ignored the determination of food toxic agents and instituted direct investigation of biologically active substances produced by microorganisms which could cause food intoxications, i.e., enterotoxins. The data presented in this work concern the problem of Staphylococcus aureus enterotoxin, which was identified by Casman in 1960 (2). Our interest in enterotoxins of this kind originates from statistics on staphylococcal intoxication, since 49% of the latter is caused by St. aureus stamms, producing enterotoxin A.

Enterotoxins↗

PubMedDate not supplied

Enterotoxins and phage typing of Staphylococcus aureus isolated from clinical material and food in Libya.

Enterotoxin was detected in 22 (61.1%) of the 36 S. aureus strains isolated from clinical materials and in 3 (13%) of the 23 S. aureus strains from food samples (P < 0.05). On the basis of individual types of enterotoxin, staphylococcal enterotoxin A (SEA) was produced by 11.1%, SEB by 38.9% and SEC by 22.2% of SS. aureus strains from clinical material. Of the food S. aureus strains, SEC and SED produced by 8.7% and 4.3% respectively. Of the clinical and food S. aureus strains, 52.8% and 39.1%, respectively, were typeable by the 23 phages of International Phage Set. The majority of the typeable S. aureus strains from clinical and food sources belonged to group II being at 22.2% and 17.4% respectively. Furthermore, of the 14 SEB-producing S. aureus, 42.9% were of phage group II. In conclusion, the results obtained indicate that enterotoxin-producing S. aureus strains from clinical materials in Libya are not uncommon; however, certain foods appear not to be the source of such strains. Because of the low susceptibility to bacteriophages shown by S. aureus isolated in Libya, compared to reports from several countries, other methods of typing should be used in conjunction with phage typing in epidemiological investigations concerning this organism.

Antigens, Bacterial↗

PubMed1976

Activation of intestinal mucosal adenylate cyclase by Shigella dysenteriae I enterotoxin.

Because the mechanism whereby Shigella dysenteriae I enterotoxin induces intestinal secretion is unclear, the effect of this toxin on adenylate cyclase activity in rabbit ileal mucosa was studied under various in vitro and in vivo conditions. Activation of adenylate cyclase by Shigella enterotoxin was observed only when substrate (ATP) concentrations above the Km of adenylate cyclase were employed. These concentrations of ATP are greater than those required to demonstrate activation of adenylate cyclase by cholera toxin. Under optimal assay conditions, doses of Shigella toxin between 5.4 and 900 mug of toxin protein and in vivo incubation times between 6 and 18 hr all increased adenylate cyclase activity by about 100%. Shigella toxin produced significant but highly variable increases in mucosal cyclic AMP concentrations, which were less that the rises seen with a comparable dose of cholera toxin. This variability in cyclic AMP response to Shigella toxin and the disparity between Shigella and cholera toxins' effects on mucosal cyclic AMP are probably the result of the different kinetics of adenylate cyclase activated by these enterotoxins. Mucosal Na-K-ATPase activity was unaffected by Shigella toxin. These observations suggest that alterations in fluid and electrolyte transport induced by Shigella enterotoxin may, in part, be mediated by the adenylate cyclase-cyclic AMP system.

Adenosine Triphosphatases↗

PubMed1991

Evaluation of ELISA and GM1-ELISA for detection of Salmonella enterotoxin.

The ELISA and GM1-ELISA, by using antiserum to purified Salmonella enterotoxin (SE), were standardized and carried out to screen salmonellae isolated from foods of animal origin for enterotoxigenicity. Of the 101 strains of Salmonella belonging to 15 different serogroups tested, 76 (75.24%) strains from 13 serogroups were found enterotoxigenic. ELISA correlated well with rabbit ligated ileal loop (RLIL) test for the detection of enterotoxin producing salmonellae with 24 test strains. ELISA also yielded positive reaction with 7 of 13 RLIL negative strains. GM1-ELISA could not be carried out as none of the 101 cell free culture supernatants (CFCS) were able to bind with GM1-ganglioside. ELISA and GM1-ELISA were also standardized with antiserum to cholera toxin for the detection of salmonellae producing cholera related enterotoxin. None of the 101 strains was found to produce cholera related enterotoxin. ELISA could detect as low as 15 ng/100 microliters of purified SE and 10 ng/100 microliters of cholera toxin when tested with their homologous antisera.

Cholera Toxin↗

PubMed1991

Molecular structure of the toxin domain of heat-stable enterotoxin produced by a pathogenic strain of Escherichia coli. A putative binding site for a binding protein on rat intestinal epithelial cell membranes.

Heat-stable enterotoxins are a family of toxin peptides that are produced by enterotoxigenic Escherichia coli and consist of 18 and 19 amino acid residues (Aimoto, S., Takao, T., Shimonishi, Y., Hara, S., Takeda, T., Takeda, Y., and Miwatani, T. (1982) Eur. J. Biochem. 129, 257-263). A synthetic fully toxic analog of the enterotoxin, Mpr5-STp(5-17), where Mpr is beta-mercaptopropionic acid and which consists of 13 amino acid residues from Cys5 to Cys17 in a heat-stable enterotoxin but is deaminated at its N terminus (Kubota, H., Hidaka, Y., Ozaki, H., Ito, H., Hirayama, T., Takeda, Y., and Shimonishi, Y. (1989) Biochem. Biophys. Res. Commun. 161, 229-235), has been crystalized from water, and its crystal structure has been solved by a direct method and refined by least square procedures to give an R factor of 0.089. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1) with unit cell constants a = 21.010 (2) A, b = 27.621 (4) A, and c = 12.781 (1) A. The asymmetric unit of the crystals contains one peptide molecule with 13 water molecules. A right-hand spiral peptide backbone extends throughout the molecule. Three beta-turns are located along this spiral and fixed tightly by three intramolecular disulfide linkages. The actual structure predicts the biniding region on the enterotoxin to the receptor protein on the membrane of rat intestinal epithelial cells.

Amino Acid Sequence↗

PubMed1990

Paw oedema test for detection of Salmonella enterotoxin : modification and standardization.

Paw oedema test (POT) was standardized with modifications for the detection of Salmonella enterotoxin. Instead of measuring the weight of the inoculated paws after amputating the limbs at 48 hr post-inoculation, percent relative thickness of the order of 121 +/- 3.8% at 24-48 hr was found to be a better index. This test yielded parallel results to rabbit ligated ileal loop (RLIL) technique. The test was positive with enterotoxic crude cell lysates (CL) and cell free-culture-supernatants (CFCS) of S. newport and S. typhimurium, partially purified and purified enterotoxin of S. newport and purified cholera toxin. The test was found to be specific in that non-enterotoxic CFCS did not cause significant increase in the thickness. Minimum detection level of purified S. newport enterotoxin was estimated to be as low as 20 micrograms. Thus, the modified POT was considered to be an effective and economical bioassay model for the detection of Salmonella enterotoxin.

Animals↗

PubMed1979

NAD-dependent ADP-ribosylation of arginine and proteins by Escherichia coli heat-labile enterotoxin.

Escherichia coli heat-labile enterotoxin (labile toxin, LT) catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide and the ADP-ribosylation of arginine (Moss, J., and Richardson, S.H. (1978) J. Clin. Invest. 62, 281-285). Analysis of the product of the ADP-ribosylation of arginine by nuclear magnetic resonance spectroscopy indicated that the reaction was stereospecific and resulted in the formation of alpha-ADP-ribosyl-L-arginine. This reaction product rapidly anomerized to yield a mixture of the alpha and beta forms. In the presence of [adenine-U-14C]NAD, E. coli enterotoxin catalyzed the transfer of the radiolabel to proteins; the ADP-ribosylation of proteins was inhibited by arginine methyl ester, an alternative substrate. Digestion of the 14C-protein with snake venom phosphodiesterase released predominantly 5'-AMP. No product was obtained with a mobility similar to that of 2'-(5''-phosphoribosyl)-5'-AMP. This result is consistent with the covalent attachment by the enterotoxin of ADP-ribose rather than poly(ADP-ribose) to protein. Thus, LT is catalytically equivalent to choleragen, an enterotoxin of Vibrio cholerae, and activates adenylate cyclase through a similar stereospecific ADP-ribosylation reaction.

Adenosine Diphosphate Sugars↗

PubMed1990

[Detection of staphylococcal enterotoxins in milk and milk products].

For food evaluation the determination of the number of Staphylococcus aureus (hereinafter S. aureus) colonies is insufficient in view of present scientific knowledge. The results, advantages and shortcomings of diagnostic methods are demonstrated on an example of three methods of detection of staphylococcal enterotoxins in milk and milk products. 133 strains were investigated by the method of biotyping of S. aureus strains. Four strains of S. aureus were included in biotype A, seven xin-producing strains were isolated seventeen times by detection of 96 S. aureus strains were not included in any biotype, the other strains belonged to biotypes C and E. This method can be used as an auxiliary method of evaluation of foods containing S. aureus bacteria. The agar-gel precipitation method of enterotoxin detection in isolated strains of S. aureus has just restricted validity. The enteroto-strains. The main shortcoming of this method is a fact that the result concerning the isolated strains need not be identical with the result of enterotoxin detection in food. Direct assays of staphylococcal enterotoxins in milk and milk products using an enzymoimmunological method seem to be the most promising, mainly due to their high sensitivity (0.0001-0.001 micrograms.ml1-) and other advantages. Positive and negative results are presented on an example of two model trials with winter sheep milk cheese.

Animals↗