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PubMed1973

Fluid secretory response of bovine Thiry jejunal fistula to enterotoxin of Clostridium perfringens.

The effect of enterotoxin of Clostridium perfringens type A was studied in Thiry fistula of the bovine jejunum. Accumulation of fluid as a pathological response was demonstrable within 30 min after introduction of enterotoxin into the fistula; the fluid volume increased rapidly within the first 2 hr and reached maximum in 7 hr. The enterotoxin was not destroyed by the jejunal fluid; 52% of it was absorbed in 2 to 3 hr, and all of the enterotoxin was taken up within 8 hr postinoculation. The accumulation of fluid by the fistula after 0.5 to 2 hr of exposure to enterotoxin ceased completely within 5 hr.

Animals↗

PubMed2002

Identification of a transcytosis epitope on staphylococcal enterotoxins.

Staphylococcal enterotoxins (SE) are exoproteins produced by Staphylococcus aureus that act as superantigens and have been implicated as a leading cause of food-borne disease and toxic shock. Little is known about how these molecules penetrate the gut lining and gain access to both local and systemic immune tissues. To model movement in vitro of staphylococcal enterotoxins, we have employed a monolayer system composed of crypt-like human colonic T-84 cells. SEB and SEA showed comparable dose-dependent transcytosis in vitro, while toxic shock syndrome toxin (TSST-1) exhibited increased movement at lower doses. Synthetic peptides corresponding to specific regions of the SEB molecule were tested in vitro to identify the domain of the protein involved in the transcytosis of SE. A toxin peptide of particular interest contains the amino acid sequence KKKVTAQELD, which is highly conserved across all SE. At a toxin-to-peptide ratio of 1:10, movement of SEB across the monolayers was reduced by 85%. Antisera made against the SEB peptide recognized native SEB and also inhibited SEB transcytosis. Finally, the conserved 10-amino-acid peptide inhibited transcytosis of multiple staphylococcal enterotoxins, SEA, SEE, and TSST-1. These data demonstrate that this region of the staphylococcal enterotoxins plays a distinct role in toxin movement across epithelial cells. It has implications for the prevention of staphylococcal enterotoxin-mediated disease by design of a peptide vaccine that could reduce systemic exposure to oral or inhaled superantigens. Since the sequence identified is highly conserved, it allows for a single epitope blocking the transcytosis of multiple SE.

Animals↗

PubMed1977

Evidence for stable messenger ribonucleic acid during sporulation and enterotoxin synthesis by Clostridium perfringens type A.

Stable messenger ribonucleic acid (mRNA) was shown to be involved in both enterotoxin synthesis and synthesis of other spore coat proteins in Clostridium perfringens. When used at a concentration that inhibited [14C]uracil incorporation, rifampin, a specific inhibitor of deoxyribonucleic acid-dependent RNA polymerase, prevented incorporation of a mixture of labeled amnoo acids by 3-h sporulating cells. At that time, enterotoxin protein was first detectable and cells were primarily at stage II or III of sporulation. When rifampin or streptolydigin was added to 5-h sporulating cells, which were primarily at stage IV or V and had significant toxin levels, incorporation of labeled amino acids continued through 30 min despite its presence. Rifampin also failed to prevent the specific synthesis of enterotoxin, a structural protein of the spore coat. The half-life of enterotoxin RNA was estimated to be at least 58 min. When cell extracts from 5-h sporulating cells that had been exposed to 3H-labeled amino acids for 10 min were subjected to electrophoresis on polyacrylamide gels and the gels were subsequently analyzed for radioactivity, two major peaks of radioactivity were obtained. The two peaks corresponded to enterotoxin and another spore coat protein(s). Similar results were obtained when the cells had been preincubated for 60 min with rifampin before label addition, indicating the functioning of stable mRNA.

Aminoglycosides↗

PubMed1983

Transport and processing of staphylococcal enterotoxin B.

A larger, membrane-bound form of staphylococcal enterotoxin B was shown by in vivo pulse-chase analysis to be the kinetic precursor to extracellular enterotoxin B. Processing of the enterotoxin B precursor molecules can apparently occur either cotranslationally or posttranslationally. Subcellular fractionation of cells revealed that all of the precursor toxin was associated with the membrane fraction. Once processed and released from the membrane, it was transiently associated with the cell wall before being released into the extracellular environment. The cell-wall-associated enterotoxin B was completely resistant to protease treatment and to extraction by high- or low-salt solutions at 0 to 2 degrees C, although it could be easily released from the cell by removal of the cell wall with lysostaphin. These data imply that newly formed enterotoxin B may be temporarily sequestered in specialized regions that require cell wall integrity before being released into the extracellular environment.

2,4-Dinitrophenol↗

PubMed1986

Cloning of genes that encode a new heat-labile enterotoxin of Escherichia coli.

The genes for a new enterotoxin were cloned from Escherichia coli SA53. The new toxin was heat labile and activated adenylate cyclase but was not neutralized by antisera against cholera toxin or E. coli heat-labile enterotoxin. Subcloning and minicell experiments indicated that the toxin is composed of two polypeptide subunits that are encoded by two genes. The two toxin subunits exhibited mobilities on polyacrylamide gels that are similar to those of cholera toxin and E. coli heat-labile enterotoxin subunits. A 0.8-kilobase DNA probe for the new enterotoxin failed to hybridize with the cloned structural genes for E. coli heat-labile enterotoxin.

Bacterial Toxins↗

PubMed1988

Cloning and nucleotide sequence of the type E staphylococcal enterotoxin gene.

The gene for staphylococcal enterotoxin type E (entE) was cloned from Staphylococcus aureus into plasmid vector pBR322 and introduced into Escherichia coli. A staphylococcal enterotoxin type E-producing E. coli strain was isolated. The complete nucleotide sequence of the cloned structural entE gene and the N-terminal amino acid sequence of mature staphylococcal enterotoxin type E were determined. The entE gene contained 771 base pairs that encoded a protein with a molecular weight of 29,358 which was apparently processed to a mature extracellular form with a molecular weight of 26,425. DNA sequence comparisons indicated that staphylococcal enterotoxins type E and A are closely related. There was 84% nucleotide sequence homology between entE and the gene for staphylococcal enterotoxin type A; these genes encoded protein products that had 214 (83%) homologous amino acid residues (mature forms had 188 [82%] homologous amino acid residues).

Amino Acid Sequence↗

PubMed1963

CULTURE MEDIUM FOR THE PRODUCTION OF STAPHYLOCOCCAL ENTEROTOXIN A.

Casman, E. P. (U.S. Food and Drug Administration, Washington, D.C.) and R. W. Bennett. Culture medium for the production of staphylococcal Enterotoxin A. J. Bacteriol. 86:18-23. 1963.-By use of Wadsworth's modification of the gel diffusion test of Ouchterlony for the assay of enterotoxin, cultivation on semisolid Brain Heart Infusion Agar of pH 5.3 permitted the detection of the production of staphylococcal Enterotoxin A with all of 49 strains without concentration of their toxins. The production of appreciably more potent enterotoxin was effected by using a cellophane sac culture technique. The use of the latter procedure, together with Brain Heart Infusion broth, permitted the detection of the production of trace amounts of Enterotoxins A and B.

Antitoxins↗

PubMed1979

Simplification of methods for the production and storage of specimens to be tested for heat-stable enterotoxin of Escherichia coli.

Experiments with the infant mouse test demonstrated that there is no need of shaking for heat-stable Escherichia coli enterotoxin production when low volume of medium per volume of flask ratios are used in stationary cultures. Centrifugation and filtration of the cultures to be tested are not necessary either, and Merthiolate (1:10,000) used as preservative has no deleterious effect on heat-stable enterotoxin activity. Based upon these findings, some modifications of the procedures for production and storage of heat-stable enterotoxin preparations are suggested. Standardized pieces of filter papers are wetted with Merthiolated stationary cultures which are to be assayed for heat-stable enterotoxin activity by the infant mouse test. From dried filter papers, heat-stable enterotoxin can be eulted unaltered up to 2 months after specimen preparation. With the proposed modifications, even modestly equipped laboratories will be able to carry out the infant mouse test or at least to prepare specimens to be assayed by more specialized laboratories.

Animals↗

PubMed1980

Antibodies to staphylococcal enterotoxin in laboratory personnel.

Eighty-five percent of laboratory personnel working with staphylococcal enterotoxin had antibodies to enterotoxin in their sera, whereas only 23% of the control group had antibodies specific for enterotoxin. Two persons who carried enterotoxin B-producing staphylococci in their noses, throats, or both, had antibodies to enterotoxin B in their sera.

Antibodies, Bacterial↗

PubMed1984

Rapid visual detection of Escherichia coli and Vibrio cholerae Heat-labile enterotoxins by nitrocellulose enzyme-linked immunosorbent assay.

A modification of the enzyme-linked immunosorbent assay for a sensitive and rapid visual detection of heat-labile enterotoxins from Escherichia coli and Vibrio cholerae is described. Small amounts of bacterial supernatant fluids are bound to nitrocellulose filters which are used as sorbents in the nitrocellulose enzyme-linked immunosorbent assay. The test is based on the immunological similarity between V. cholerae and E. coli heat-labile enterotoxins. Six isolates of V. cholerae and 48 isolates of E. coli were examined for heat-labile enterotoxins by the nitrocellulose enzyme-linked immunosorbent assay and the Vero cell bioassay. With some strains, the nitrocellulose enzyme-linked immunosorbent assay was found to be more sensitive for detection of E. coli heat-labile enterotoxin than the Vero cell test. A similar result was obtained by endpoint titration of heat-labile enterotoxin-positive E. coli H10407 culture fluid in both assays. The sensitivity of the nitrocellulose enzyme-linked immunosorbent assay for the detection of purified cholera toxin was at a total level of 1 ng, which is a good result when compared with other serological assays.

Animals↗

PubMed1987

Comparative study of synthetic oligonucleotide and cloned polynucleotide enterotoxin gene probes to identify enterotoxigenic Escherichia coli.

Escherichia coli isolated from 2,126 children in Thailand and the Philippines was examined for enterotoxin production and for DNA hybridization with synthetic oligonucleotide and cloned polynucleotide enterotoxin gene probes. A total of 233 infections with E. coli that were detected by one or more of these assays were identified. Of the infections, 75% (164/233) were identified by all three methods. An additional 18% (43/233) were identified by two of three methods. Isolates from 10% (19/183) of infections with E. coli that hybridized with both the oligonucleotide and cloned enterotoxin gene probes were nontoxigenic, as determined by the Y1 adrenal cell and suckling mouse assays. Although synthetic oligonucleotide probes to detect enterotoxigenic E. coli are more uniform and easier to use than cloned enterotoxin gene probes, the heat-labile toxin oligo probe used in this study did not identify 13% (11/87) of infections with E. coli that produced heat-labile toxin, as identified with the Y1 adrenal cell assay and the cloned enterotoxin gene probe. Synthetic oligonucleotide probes enable laboratories with only minimal equipment to use DNA hybridization assays to identify enterotoxigenic E. coli.

Bacterial Toxins↗

PubMed1987

Evaluation of immuno-dot-blot assay for detection of cholera-related enterotoxin antigen in Salmonella typhimurium.

Twenty-five strains of Salmonella typhimurium isolated in India were examined for the presence of cholera/coli-related enterotoxin antigen by a previously described latex particle agglutination test and by a newly developed immuno-dot-blot test using immunopurified goat antibody against the cholera-related enterotoxin isolated from an Escherichia coli strain of human origin. The immuno-dot-blot assay could detect 0.02 ng of purified enterotoxin. The amount of toxin antigen detected varied widely from strain to strain. Fourteen of the 25 polymyxin B-treated extracts of bacteria harvested from 6-h Casamino Acids-yeast extract broth cultures gave positive results in both serologic assays as well as in rabbit skin tests for delayed permeability factor. An additional strain was positive only in the immuno-dot-blot. Five of six stool isolates and six of seven blood isolates tested gave positive reactions. Two isolates of Salmonella enteritidis tested were also positive. The immuno-dot-blot test appears to be a simple, rapid, and reliable method for detection of cholera-related enterotoxin antigen in S. typhimurium. The demonstration of a cholera-related enterotoxin, even in small amounts, in a facultative intracellular pathogen raises interesting questions regarding its potential role in pathogenesis both of diarrheal disease and systemic infections caused by salmonellae.

Animals↗

PubMed1999

Involvement of enterotoxins G and I in staphylococcal toxic shock syndrome and staphylococcal scarlet fever.

We investigated the involvement of the recently described staphylococcal enterotoxins G and I in toxic shock syndrome. We reexamined Staphylococcus aureus strains isolated from patients with menstrual and nonmenstrual toxic shock syndrome (nine cases) or staphylococcal scarlet fever (three cases). These strains were selected because they produced none of the toxins known to be involved in these syndromes (toxic shock syndrome toxin 1 and enterotoxins A, B, C, and D), enterotoxin E or H, or exfoliative toxin A or B, despite the fact that superantigenic toxins were detected in a CD69-specific flow cytometry assay measuring T-cell activation. Sets of primers specific to the enterotoxin G and I genes (seg and sei, respectively) were designed and used for PCR amplification. All of the strains were positive for seg and sei. Sequence analysis confirmed that the PCR products, corresponded to the target genes. We suggest that staphylococcal enterotoxins G and I may be capable of causing human staphylococcal toxic shock syndrome and staphylococcal scarlet fever.

Enterotoxins↗

PubMed2000

Prevalence of enterotoxin genes in Aeromonas spp. isolated from children with diarrhea, healthy controls, and the environment.

Aeromonads are causative agents of a number of human infections. Even though aeromonads have been isolated from patients suffering from diarrhea, their etiological role in gastroenteritis is unclear. In spite of a number of virulence factors produced by Aeromonas species, their association with diarrhea has not been clearly linked. Recently, we have characterized a heat-labile cytotonic enterotoxin (Alt), a heat-stable cytotonic enterotoxin (Ast), and a cytotoxic enterotoxin (Act) from a diarrheal isolate of Aeromonas hydrophila. Alt and Ast are novel enterotoxins which are not related to cholera toxin; Act is aerolysin related and has hemolytic, cytotoxic, and enterotoxic activities. We studied the distribution of the alt, ast, and act enterotoxin genes in 115 of 125 aeromonads isolated from 1, 735 children with diarrhea, in all 27 aeromonads isolated from 830 control children (P = 7 x 10(-4) for comparison of rates of isolation of aeromonads from cases versus those from controls), and in 120 randomly selected aeromonads from different components of surface water in Bangladesh. Aeromonas isolates which were positive only for the presence of the alt gene had similar distributions in the three sources; the number of isolates positive only for the presence of the ast gene was significantly higher for the environmental samples than for samples from diarrheal children; and isolates positive only for the presence of the act gene were not found in any of the three sources. Importantly, the number of isolates positive for both the alt and ast genes was significantly higher for diarrheal children than for control children and the environment. Thus, this is the first study to indicate that the products of both the alt and ast genes may synergistically act to induce severe diarrhea. In 26 patients, Aeromonas spp. were isolated as the sole enteropathogen. Analysis of clinical data from 11 of these patients suggested that isolates positive for both the alt and ast genes were associated with watery diarrhea but that isolates positive only for the alt gene were associated with loose stools. Most of the isolates from the three sources could be classified into seven phenospecies and eight hybridization groups. For the first time, Aeromonas eucrenophila was isolated from two children, one with diarrhea and another without diarrhea.

Aeromonas↗

PubMed2004

Simultaneous analysis of multiple staphylococcal enterotoxin genes by an oligonucleotide microarray assay.

Staphylococcal enterotoxins (SEs) are a family of 17 major serological types of heat-stable enterotoxins that are one of the leading causes of gastroenteritis resulting from consumption of contaminated food. SEs are considered potential bioweapons. Many Staphylococcus aureus isolates contain multiple SEs. Because of the large number of SEs, serological typing and PCR typing are laborious and time-consuming. Furthermore, serological typing may not always be practical because of antigenic similarities among enterotoxins. We report on a microarray-based one-tube assay for the simultaneous detection and identification (genetic typing) of multiple enterotoxin (ent) genes. The proposed typing method is based on PCR amplification of the target region of the ent genes with degenerate primers, followed by characterization of the PCR products by microchip hybridization with oligonucleotide probes specific for each ent gene. We verified the performance of this method by using several other techniques, including PCR amplification with gene-specific primers, followed by gel electrophoresis or microarray hybridization, and sequencing of the enterotoxin genes. The assay was evaluated by analysis of previously characterized staphylococcal isolates containing 16 ent genes. The microarray assay revealed that some of these isolates contained additional previously undetected ent genes. The use of degenerate primers allows the simultaneous amplification and identification of as many as nine different ent genes in one S. aureus strain. The results of this study demonstrate the usefulness of the oligonucleotide microarray assay for the analysis of multitoxigenic strains, which are common among S. aureus strains, and for the analysis of microbial pathogens in general.

Base Sequence↗

PubMed1989

Heat stable enterotoxin produced by Escherichia coli in acute diarrhoea.

We screened 569 children with acute onset diarrhoea from five Italian towns for heat stable enterotoxin produced by Escherichia coli. We compared an enzyme linked immunoabsorbent assay (ELISA) with the standard suckling mouse assay for detection of the enterotoxin. A total of 31 (5.4%) children were found who had strains of E coli that produced heat stable enterotoxin: 26 strains were positive in both tests, four only in the suckling mouse assay, and one only in the ELISA. Compared with the suckling mouse assay the sensitivity of the ELISA was 87% and the specificity was 99.8%. The mean age of the children with E coli that produced heat stable enterotoxin was 22 months. Fifteen (48%) of the children had ingested potentially contaminated food and five (17%) had a previous contact with someone with diarrhoea. The clinical picture was milder than that described in developing countries. Faecal osmolality and the osmolal gap were consistent with a secretory diarrhoea in 12 out of 15 (80%) of these children. The mean duration of the diarrhoea was five days. Heat stable enterotoxin produced by E coli is a common cause of mild diarrhoeal illness in Italian children.

Adolescent↗

PubMed1978

Detection of Salmonella enterotoxin using rabbit ileal loops.

The presence of an enterotoxin produced by Salmonella in broth culture has been demonstrated by using the rabbit ileal loop model. Response by the animal to enterotoxin in sterile culture supernatant fluids is enhanced when the intestinal lumen is washed with a mucolytic agent prior to the administration of toxin. Fluid secretion is untreated intestinal loops was also observed if enterotoxin was administered with a live, invasive Salmonella strain which did not evoke a secretory response. A limited survey of Salmonella isolated for clinical and food sources indicated the common occurrence of enterotoxin production, and stock cultures maintained the ability to produce the toxin. The host-adapted species which were tested varied in their ability to produce enterotoxin.

Animals↗

PubMed1982

Effect of purine derivatives, papaverine hydrochloride, and imidazole on enterotoxin formation by Clostridium perfringens type A.

The percentage sporulation and enterotoxin specific activity were improved for all of five Clostridium perfringens strains, and numbers of heat-resistant spores were improved for four of five strains by replacing proteose peptone with peptone in Duncan-Strong (DS) medium. When raffinose replaced starch in DS, peptone was superior to proteose peptone in increasing percentage sporulation, numbers of heat-resistant spores, and enterotoxin formation for four of five strains. Enterotoxin levels for a strain varied when different lots of the same peptone were used. Additional experiments were conducted with three C. perfringens strains grown in DS medium with peptone. Enterotoxin specific activity was increased for three strains by adding papaverine (hydrochloride crystalline), for two strains by adding each of caffeine and 3-isobutyl-l-methylxanthine, for one strain by adding each of theophylline, 6-mercaptopurine, and 2-amino-6-mercaptopurine, and for none of the strains by adding imidazole. When enterotoxin formation was improved for a strain by one of the compounds, percentage sporulation increased, but growth decreased. Effective compounds also increased numbers of heat-resistant spores for strains H6 and R42, but slightly or not at all for strain E13. The action of these compounds was concentration dependent, with the optimal concentration differing between compounds and between strains grown in the presence of the same compound.

1-Methyl-3-isobutylxanthine↗