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PubMed1986

Diagnostic importance of Clostridium perfringens enterotoxin analysis in recurring enteritis among elderly, chronic care psychiatric patients.

A series of Clostridium perfringens-related gastrointestinal outbreaks occurred over a period of several months among elderly, chronic care patients in a psychiatric hospital. Several serotypes of C. perfringens and many nontypeable isolates were found. The distribution of certain serotypes and the incidence of detection of enterotoxin in fecal extracts were related to wards on which patients were resident (six wards were involved). Several patients were reported to have chronic or recurring fecal incontinence or diarrhea or both. With a background of elevated spore counts of several serotypes and chronic diarrhea, only detection of enterotoxin could provide definitive evidence of C. perfringens etiology in gastoenteritis cases.

Aged↗
PubMed1986

Competitive enzyme-linked immunosorbent assay for cholera-related enterotoxins in Salmonella typhimurium.

We report a rapid competitive enzyme-linked immunosorbent assay to screen Salmonella typhimurium strains for cholera-related enterotoxin antigens. Polymyxin B extracts of bacterial cells from syncase-glucose broth cultures of 7 of 15 strains gave positive results. The specificity of the test was confirmed with known heat-labile-enterotoxin-positive and -negative Escherichia coli strains which gave significantly different values.

Antigens, Bacterial↗
PubMed1988

Characterization of an outbreak of Clostridium perfringens food poisoning by quantitative fecal culture and fecal enterotoxin measurement.

Published criteria for implicating Clostridium perfringens as the cause of food-poisoning outbreaks include finding a median fecal C. perfringens spore count of greater than 10(6)/g among specimens from ill persons. We investigated a food-poisoning outbreak with the epidemiologic characteristics of C. perfringens-related disease in a nursing home in which the median fecal spore count for ill patients (2.5 X 10(7)/g) was similar to that for well patients (4.0 X 10(6)/g), making the etiology of the outbreak uncertain. All ill and well patients tested had eaten turkey, the implicated food item. C. perfringens enterotoxin was detected by reverse passive latex agglutination in fecal specimens from six of six ill and none of four well patients who had eaten turkey (P = 0.005), suggesting that this organism had caused the outbreak. This investigation suggests that detection of fecal C. perfringens enterotoxin is a specific way to identify this organism as the causative agent in food-poisoning outbreaks.

Aged↗
PubMed1988

Comparison of two GM1-erythrocyte assays to detect heat-labile Escherichia coli enterotoxin in stool specimens.

Two erythrocyte immunoassay techniques to detect the presence of Escherichia coli heat-labile enterotoxin (LTh) in stool supernatants and cell-free culture supernatants were compared. In the competitive assay, GM1 ganglioside was coated onto V-shaped-well microdilution plates and enterotoxin was coupled to sheep erythrocytes. As little as 0.8 ng of LTh per ml was detected by this method, which was based on the competition between the LTh of the test sample and the sensitized erythrocytes. The second assay made use of chimera antibody prepared by coupling polyclonal anti-LTh antibody to a monoclonal antibody specific for sheep erythrocytes. In this case, LTh, which was specifically bound to a GM1 ganglioside-coated plate, was detected by successively adding the chimera antibody and sheep erythrocytes. The limit of detection of the chimera antibody erythrocyte immunoassay was 0.2 ng/ml. Stool samples were collected from 167 infants hospitalized for diarrhea in the hospital of Noumea, New Caledonia. False-negative reactions due to proteases present in the stool samples were avoided by the addition of phenylmethylsulfonyl fluoride.

Bacterial Toxins↗
PubMed1988

Chromatofocusing in the purification of staphylococcal enterotoxin D.

A chromatofocusing procedure for the purification of staphylococcal enterotoxin D was developed. The purification included the removal of the toxic protein from culture supernatant fluids of Staphylococcus aureus 1151m by batch adsorption with CG-50 resin, chromatofocusing on Polybuffer Exchanger 94, and gel permeation chromatography on Sephacryl S-200. The purity of the staphylococcal enterotoxin D obtained was approximately 98%.

Chromatography, Gel↗
PubMed1989

Detection with synthetic oligonucleotide probes of nucleotide sequence variations in the genes encoding enterotoxins of Escherichia coli.

We examined variations in the genes encoding heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT) in 88 strains of Escherichia coli isolated from individuals with traveler's diarrhea to find suitable sequences for use as oligonucleotide probes. Four oligonucleotide probes of the gene encoding ST of human origin (STIb or STh), one oligonucleotide probe of the gene encoding ST of porcine origin (STIa or STp), and three oligonucleotide probes of the gene encoding LT of human origin (LTIh) were used in DNA colony hybridization tests. In 15 of 22 strains possessing the STh gene and 28 of 42 strains producing LT, the sequences of all regions tested were identical to the published sequences. One region in the STh gene examined with a 18-mer probe was relatively well conserved and was shown to be closely associated with the enterotoxicity of the E. coli strains in suckling mice. This oligonucleotide, however, hybridized with strains of Vibrio cholerae O1, V. parahaemolyticus, and Yersinia enterocolitica that gave negative results in the suckling mouse assay.

Animals↗
PubMed1989

Assay of heat-labile enterotoxins by their ADP-ribosyltransferase activities.

Simple enzymatic assays to detect heat-labile enterotoxins whose modes of action are similar to that of cholera toxin were evaluated. The assays are performed by using an artificial substrate, diethylamino benzylidine-aminoguanidine, which is an ADP-ribose acceptor. The product, formed in the presence of NAD+, can be quantitated by spectrofluorometric, spectrophotometric, or high-performance liquid chromatographic (HPLC) methods. As little as 25 ng (spectrofluorometry) or 125 ng (spectrophotometry or HPLC) of cholera toxin can be detected in an assay volume of 250 microliters. The detection limit for heat-labile enterotoxin by either the spectrophotometric or HPLC methods was 125 ng/250 microliters. Because the results are quantitative, the enzymatic methods can be used for medium development, determination of factors that influence toxin production, and other applications that heretofore could be accomplished only with difficulty. The enzymatic methods add a new dimension to the assay of toxins that ribosylate arginine residues of proteins. Sensitivities of the assays might be improved by developing better synthetic substrates, and applications could be broadened by the development of artificial substrates containing other functional groups.

Animals↗
PubMed1989

Rapid purification of staphylococcal enterotoxin B by high-pressure liquid chromatography.

The Staphylococcus aureus enterotoxins represent a group of proteins that cause emesis and diarrhea in humans and other primates. We have developed a rapid two-step high-pressure liquid chromatography (HPLC) procedure for purification of staphylococcal enterotoxin B (SEB). Sterile filtrates (2.5 liters) of strain 10-275 were adsorbed directly onto a reversed-phase column (50 mm by 30 cm Delta Pak; 300 A [30 nm], 15 microns, C18). SEB was obtained by using a unique sequential gradient system. First, an aqueous ammonium acetate to acetonitrile gradient followed by an aqueous trifluoroacetic acid (TFA) wash was used to remove contaminants. A subsequent TFA to acetonitrile-TFA gradient eluted the bound SEB. Further purification was obtained by rechromatography on a cation-exchange column. From 35 to 45% of the SEB in starting filtrates was recovered. Analysis by immunoblotting of samples separated on sodium dodecyl sulfate-polyacrylamide gels indicated that HPLC-purified SEB exhibited immunological and biochemical properties similar to those of the SEB standard. Induction of an emetic response in rhesus monkeys showed that the HPLC-purified toxin also retained biological activity.

Animals↗
PubMed1991

Detection of genes for enterotoxins, exfoliative toxins, and toxic shock syndrome toxin 1 in Staphylococcus aureus by the polymerase chain reaction.

Eight pairs of synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) protocol to detect genes for staphylococcal enterotoxins A to E, exfoliative toxins A and B, and toxic shock syndrome toxin 1 in Staphylococcus aureus strains isolated from clinical specimens and contaminated foods. Primers were targeted to internal regions of the toxin genes, and amplification fragments were detected after the PCR by agarose gel electrophoresis. Unequivocal discrimination of toxin genes was obtained by the PCR by using nucleic acids extracted from 88 strains of S. aureus whose toxigenicity was established biologically and immunologically. In immunological assays, two strains of S. aureus produced equivocal results for production of enterotoxin C or toxic shock syndrome toxin 1, giving an overall concordance between phenotypic and genotypic identification of 97.7%. Primer specificity was established in the PCR by using nucleic acids from known toxin-producing bacterial pathogens and from nontoxigenic S. aureus. Strains of Streptococcus spp., including some producers of pyrogenic exotoxin A carrying the speA gene, were negative by the PCR designed to detect staphylococcal toxins. The detection limits were established for all the staphylococcal toxin genes within their respective PCR protocols. The identification of staphylococcal toxin genes in strains of S. aureus by the PCR offers a very specific, sensitive, relatively rapid, and inexpensive alternative to traditional immunological assays which depend on adequate gene expression for reliability and sensitivity.

Bacterial Toxins↗
PubMed1992

Development of an immunoassay using recombinant maltose-binding protein-STa fusions for quantitating antibody responses against STa, the heat-stable enterotoxin of Escherichia coli.

A set of fusion proteins containing heat-stable enterotoxin (STa) and maltose-binding protein were engineered. These molecules were readily purified and used as solid-phase antigens in an enzyme-linked immunosorbent assay to monitor anti-STa responses in mice immunized with a recombinant vaccine composed of STa and the B subunit of heat-labile enterotoxin.

ATP-Binding Cassette Transporters↗
PubMed1993

Production of enterotoxins and toxic shock syndrome toxin by bovine mammary isolates of Staphylococcus aureus.

The production of staphylococcal enterotoxin A (SEA), SEB, SEC, SED, and SEE and toxic shock syndrome toxin 1 by bovine mammary isolates of Staphylococcus aureus was evaluated. Enterotoxin secretion was detected by immunodiffusion using specific polyclonal antisera. Of 262 isolates examined, 75 (28.6%) produced one or more toxins. The most common pattern was secretion of both SEC and SED and toxic shock syndrome toxin 1. No isolates secreted SEE, one produced SEA, and seven secreted SEB.

Animals↗
PubMed1997

Detection and sequences of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 gene in enterotoxigenic E. coli strains isolated from piglets and calves with diarrhea.

Enterotoxigenic Escherichia coli (ETEC) strains isolated from piglets and calves with diarrhea were tested for the presence of the enteroaggregative E. coli enterotoxin 1 (EAST1) gene sequences by PCR and colony hybridization. The EAST1 gene was found in most porcine ETEC strains with adherence factor K88, especially in those elaborating heat-labile enterotoxin. One porcine ETEC strain with adherence factor K99 was also positive for the EAST1 gene. In contrast, 987P-positive (987P+) ETEC strains from piglets, K99+ ETEC strains from calves, and K99+ F41+ or F41+ ETEC strains from piglets and calves were negative for the EAST1 gene. The K88ab+ or K88ac+ ETEC strains tested possessed the EAST1 gene on a plasmid that was distinct from a K88-encoding plasmid. The EAST1 gene sequences of the K88+ ETEC strains were identical to each other and 99.1 and 98.3% homologous to the previously reported sequences of ETEC strains colonizing humans and enteroaggregative E. coli strains, respectively. The data indicate that the EAST1 gene is distributed among porcine ETEC strains in association with the adherence factor type.

Amino Acid Sequence↗
PubMed1997

Genes coding for enterotoxins and verotoxins in porcine Escherichia coli strains belonging to different O:K:H serotypes: relationship with toxic phenotypes.

Seventy-four E. coli strains isolated from piglets with diarrhea or edema disease in Spain were serotyped and examined for production of heat-labile (LT) and heat-stable (ST) enterotoxins (LT-I, LT-II, STaH, STaP, and STb) and verotoxins (VT1, VT2, and VT2v = VTe) by phenotypic (Vero cell assay and infant mouse test) and genotypic (colony hybridization and PCR) methods. In general, an excellent correlation was found between the results obtained with a PCR approach and those determined with biological assays. DNA probes used in the hybridization also showed a very good agreement with phenotypic results, with the exception of a VT1 probe that initially produced 10 false-positive reactions. The gene coding for STb (58 strains) was the most prevalent gene detected by PCR, followed by those coding for STa (46 strains), LT (19 strains), VT2v (11 strains), and VT1 (1 strain). Apparently, in Spain three seropathotypes are predominant: (i) O149:K91:H10 K88+ LT-I+ STb+, (ii) O141:K85ab:H- P987+ STaP+, and (iii) O138:K81:H14 or H- STaP+ VT2v+. We conclude that PCR is a fast, specific, and practical method for the identification of enterotoxin and VT genes in clinical and epidemiological studies.

Animals↗
PubMed2004

Multiplex PCR genotyping assay that distinguishes between isolates of Clostridium perfringens type A carrying a chromosomal enterotoxin gene (cpe) locus, a plasmid cpe locus with an IS1470-like sequence, or a plasmid cpe locus with an IS1151 sequence.

Clostridium perfringens type A isolates carrying the enterotoxin (cpe) gene are important causes of both food poisoning and non-food-borne diarrheas in humans. In North America and Europe, food poisoning isolates were previously shown to carry a chromosomal cpe gene, while non-food-borne gastrointestinal (GI) disease isolates from those two geographic locations were found to have a plasmid cpe gene. In this report, we describe the development of an economical multiplex PCR cpe genotyping assay that works with culture lysates to distinguish among type A isolates carrying a chromosomal cpe gene, a plasmid cpe gene with a downstream IS1470-like sequence, or a plasmid cpe gene with a downstream IS1151 sequence. When this multiplex PCR assay was applied in molecular epidemiologic studies, it was found that (i) all 57 examined type A isolates with a plasmid cpe gene have either IS1470-like or IS1151 sequences downstream of the plasmid cpe gene; (ii) an IS1470-like sequence, rather than an IS1151 sequence, is more commonly present downstream of the plasmid cpe gene (particularly in North American non-food-borne human GI disease isolates); and (iii) as previously shown in the United States and Europe, isolates carrying the chromosomal cpe gene also appear to be the major cause of C. perfringens food poisoning in Japan. The superiority of this new multiplex PCR assay over existing cpe genotyping approaches should facilitate further molecular epidemiologic investigations of C. perfringens enterotoxin-associated GI illnesses and their associated cpe-positive type A isolates.

Bacterial Typing Techniques↗
PubMed2004

Rotavirus enterotoxin NSP4 binds to the extracellular matrix proteins laminin-beta3 and fibronectin.

Rotavirus is the most important cause of viral gastroenteritis and dehydrating diarrhea in young children. Rotavirus nonstructural protein 4 (NSP4) is an enterotoxin that was identified as an important agent in symptomatic rotavirus infection. To identify cellular proteins that interact with NSP4, a two-hybrid technique with Saccharomyces cerevisiae was used. NSP4 cDNA, derived from the human rotavirus strain Wa, was cloned into the yeast shuttle vector pGBKT7. An intestinal cDNA library derived from Caco-2 cells cloned into the yeast shuttle vector pGAD10 was screened for proteins that interact with NSP4. Protein interactions were confirmed in vivo by coimmunoprecipitation and immunohistochemical colocalization. After two-hybrid library screening, we repeatedly isolated cDNAs encoding the extracellular matrix (ECM) protein laminin-beta3 (amino acids [aa] 274 to 878) and a cDNA encoding the ECM protein fibronectin (aa 1755 to 1884). Using deletion mutants of NSP4, we mapped the region of interaction with the ECM proteins between aa 87 and 145. Deletion analysis of laminin-beta3 indicated that the region comprising aa 726 to 875 of laminin-beta3 interacts with NSP4. Interaction of NSP4 with either laminin-beta3 or fibronectin was confirmed by coimmunoprecipitation. NSP4 was present in infected enterocytes and in the basement membrane (BM) of infected neonatal mice and colocalized with laminin-beta3, indicating a physiological interaction. In conclusion, two-hybrid screening with NSP4 yielded two potential target proteins, laminin-beta3 and fibronectin, interacting with the enterotoxin NSP4. The release of NSP4 from the basal side of infected epithelial cells and the subsequent binding to ECM proteins localized at the BM may signify a new mechanism by which rotavirus disease is established.

Animals↗
PubMed1992

Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin.

Cholera and the related Escherichia coli-associated diarrheal disease are important problems confronting Third World nations and any area where water supplies can become contaminated. The disease is extremely debilitating and may be fatal in the absence of treatment. Symptoms are caused by the action of cholera toxin, secreted by the bacterium Vibrio cholerae, or by a closely related heat-labile enterotoxin, produced by Escherichia coli, that causes a milder, more common traveler's diarrhea. Both toxins bind receptors in intestinal epithelial cells and insert an enzymatic subunit that modifies a G protein associated with the adenylate cyclase complex. The consequent stimulated production of cyclic AMP, or other factors such as increased synthesis of prostaglandins by intoxicated cells, initiates a metabolic cascade that results in the excessive secretion of fluid and electrolytes characteristic of the disease. The toxins have a very high degree of structural and functional homology and may be evolutionarily related. Several effective new vaccine formulations have been developed and tested, and a growing family of endogenous cofactors is being discovered in eukaryotic cells. The recent elucidation of the three-dimensional structure of the heat-labile enterotoxin has provided an opportunity to examine and compare the correlations between structure and function of the two toxins. This information may improve our understanding of the disease process itself, as well as illuminate the role of the toxin in studies of signal transduction and G-protein function.

Amino Acid Sequence↗
PubMed1985

Detection of Clostridium difficile enterotoxin in neonates by latex agglutination.

Clostridium difficile enterotoxin (D-1) was detected in 13 symptomatic and nine asymptomatic neonates in the neonatal intensive care unit by latex agglutination test but was not found in 18 healthy neonates in two other newborn nurseries. Environmental contamination in the intensive care unit may have been the cause. An association between the presence of enterotoxin and clinical symptoms is discussed.

Clostridium↗