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PubMed1977

Characterization of the Chinese hamster ovary cell assay for the enterotoxins of Vibrio cholerae and Escherichia coli and for specific antisera, and toxoid.

The morphologic change in Chinese hamster ovary (CHO) cells that is associated with increased intracellular concentrations of adenosine 3':5'-cyclic phosphate (cyclic AMP) provided a highly sensitive assay for the heat-liable enterotoxins of Vibrio cholerae and Escherichia coli. The similarity between the responses of CHO cells and those of intestinal mucosa to enterotoxins was demonstrated by the inhibition of these effects by the ganglioside galactosyl-N-acetylgalactosaminyl [sialosyl] lactosyl ceramide (GM1), as well as by specific antisera. The antisera against either enterotoxin were more effective in inhibiting the effects of homologous toxin than those of heterologous toxin, but cross-neutralization also occurred in both cases. When choleragenoid, the natural toxoid of cholera toxin, was incubated with the CHO cells, the subsequent effects of the toxin on the morphology of the cells were prevented as were the effects of the toxin on intestinal secretion. However, choleragenoid did not prevent the effects of E. coli toxin. The similarity between the action of enterotoxins on CHO cell morphology and on secretion by the small intestine suggests that there are similar binding sites in these tissues. The potential usefulness of the CHO cell assay in quantitative measurement of antitoxic immunity is thus demonstrated.

Animals↗

PubMed1983

Acquisition and maintenance of enterotoxin plasmids in wild-type strains of Escherichia coli.

Enterotoxigenic strains of Escherichia coli (ETEC) may produce a heat-labile enterotoxin (LT), a heat-stable enterotoxin (ST) or both enterotoxins. Certain serogroups are represented more frequently than others in ETEC isolated from humans. The transfer of three plasmids encoding enterotoxin production (Ent) to 22 non-toxigenic E. coli strains of many different O:H serotypes was studied. The Ent plasmids encoded ST (TP276), or LT (TP277), or ST + LT (TP214), and all carried antibiotic-resistance determinants. Twenty-one recipient strains acquired TP214, 18 acquired TP277 and 14 acquired TP276. Strains of those serotypes to which ETEC in diarrhoeal studies commonly belong neither acquired nor maintained Ent plasmids with a higher frequency than strains of those serotypes to which ETEC rarely belong. The recipient strains, with one exception, all expressed ST, or LT, or ST and LT, when they had acquired the appropriate plasmid; a non-motile strain belonging to O serogroup 88 expressed LT but failed to express ST when it acquired TP214 or TP277.

Bacterial Toxins↗

PubMed1979

Characterisation and neutralisation of Aeromonas hydrophila enterotoxin in the rabbit ileal-loop model.

Cell-free culture filtrates and crude enterotoxin preparations from six strains of Aeromonas hydrophila caused the accumulation of fluid in rabbit ileal loops. This activity was due to a non-dialysable, heat and acid-labile antigenic protein and was lost when culture filtrates and crude enterotoxin preparations were heated at 60 degrees C for 20 min. or 56 degrees C for 30 min. respectively. Maximum activity was observed at pH 8.0-10.0; there was a gradual loss at lower pH and activity was abolished in culture filtrates held at pH 3.0 and crude enterotoxin preparations held at pH 4.0. Titration of the crude enterotoxin preparations in rabbit ileal loops showed that the ED50 (equivalent to 1 unit of toxin) was contained in 25 microgram of protein; a logarithmic plot of the neutralisation coefficients against antiserum concentrations showed that one unit of antitoxin was contained in 42 x 10(-4) ml of the antiserum.

Adult↗

PubMed1987

Purification of cytotoxic enterotoxin of Aeromonas sobria by use of monoclonal antibodies.

Cytotoxic enterotoxin of Aeromonas sobria was purified by affinity chromatography with monoclonal antibodies. The purified enterotoxin gave a single protein band in polyacrylamide gradient gel electrophoresis and its mol. wt estimated by this technique was 63,000; it had a pI of 6.2. The purified enterotoxin caused fluid accumulation in rat ileal loops and in infant mice, was cytotoxic to cultured cells, was haemolytic to human erythrocytes, and was lethal to mice after intravenous injection. The relative concentrations of enterotoxic, cytotoxic and haemolytic activities were approximately the same in a culture filtrate and in purified, electrophoretically homogeneous enterotoxin. The three activities were also inactivated to the same extent after incubation for 10 min at 56 degrees C. There was no immunological cross-reactivity with cholera toxin (CT) nor did antiserum to CT neutralise the biological effects of the toxin.

Aeromonas↗

PubMed1993

Isolation and purification of Aeromonas sobria cytotonic enterotoxin and beta-haemolysin.

Aeromonas sp., grown in tryptone soya broth supplemented with yeast extract, 0.6%, pH 7.5, and incubated with agitation at 100 oscillations/min for 15 h at 37 degrees C produced optimal amounts of beta-haemolysin and cytotonic enterotoxin. More prolonged incubation resulted in the loss of enterotoxic activity and anion exchange chromatographic analysis indicated the presence of a moiety capable of breaking down the toxin. Anion exchange fast protein liquid chromatography resulted in a single peak of haemolytic activity and two peaks with enterotoxic activity. The cytotonic enterotoxin was purified from the fraction most active in the infant mouse assay; the second peak, which did not cross-react immunologically, may represent a second cytotonic enterotoxin. Neither peak was observed in the chromatographic fractions of filtrates from strains devoid of activity in the infant mouse assay. Purified enterotoxin, estimated to have a mol. wt of 15 kDa by SDS-PAGE, caused fluid accumulation in the infant mouse assay, was non-haemolytic to rabbit erythrocytes, caused an increase in cAMP activity in tissue culture cells and did not cross-react immunologically with components of cholera toxin or the whole toxin. Purified beta-haemolysin had an estimated mol. wt of 55 kDa, lysed rabbit erythrocytes and did not cause fluid accumulation in the infant mouse test.

Aeromonas↗

PubMed1997

Enterotoxin production by Staphylococcus aureus clones and detection of Brazilian epidemic MRSA clone (III::B:A) among isolates from food handlers.

Staphylococcus aureus is a major bacterial pathogen involved in a wide range of diseases varying from infections to toxaemia. Staphylococcal food-poisoning syndrome is caused by ingestion of bacterial enterotoxins. These toxins are microbial superantigens and may also be virulence factors involved in staphylococcal infection. This study determined the enterotoxin types and pulsed-field gel patterns found among S. aureus isolates obtained from food handlers in community or hospital-located kitchens. Staphylococcal enterotoxin C was the most frequent enterotoxin produced. The data also suggested horizontal spread of ent genes among isolates belonging to the Brazilian epidemic MRSA clone III::B:A. A subclone of MRSA clone III::B:A was isolated from two hospital kitchen workers. This was the first report of this clone from a hospital in Teresina, Piaui State, although the presence of this MRSA clone has already been reported in six other Brazilian cities.

Brazil↗

PubMed1999

Enterotoxin production by coagulase-negative staphylococci in restaurant workers from Kuwait City may be a potential cause of food poisoning.

Staphylococcus aureus and coagulase-negative staphylococci (CNS) were isolated from the hands of food handlers in 50 restaurants in Kuwait City and studied for the production of staphylococcal enterotoxins, toxic shock syndrome toxin-1, slime and resistance to antimicrobial agents. One or a combination of staphylococcal enterotoxins A, B or C were produced by 6% of the isolates, with the majority producing enterotoxin B. Toxic shock syndrome toxin-1 was detected in c. 7% of the isolates; 47% produced slime. In all, 21% of the isolates were resistant to tetracycline and 11.2% were resistant to propamidine isethionate and mercuric chloride. There was no correlation between slime and toxin production or between slime production and antibiotic resistance. The detection of enterotoxigenic CNS on food handlers suggests that such strains may contribute to food poisoning if food is contaminated by them and held in conditions that allow their growth and elaboration of the enterotoxins. It is recommended that enterotoxigenic CNS should not be ignored when investigating suspected cases of staphylococcal food poisoning.

Anti-Bacterial Agents↗

PubMed1986

Growth and enterotoxin A production by Staphylococcus aureus S6 in Manchego type cheese.

Milk (from cow, goat and sheep) was inoculated with Staphylococcus aureus strain S6, which is generally considered to be a strong enterotoxin B producer and a weak enterotoxin A producer. It was then used to make Manchego type cheese as prepared industrially. Two concentrations of starter culture (1% and 0.1%) were tested. Staphylococcal growth was good in both but better in the more dilute culture. Staphylococcal enterotoxin B was not detected at any stage of the ripening process of any cheese tested. However enterotoxin A was detected in both starter concentrations, reaching as high as 769 ng/100 g of cheese in the 0.1% starter batches.

Animals↗

PubMed1993

Potential problems in the use of oligonucleotide probes for staphylococcal enterotoxin genes.

Oligonucleotide probes unique to the five major enterotoxin genes of Staphylococcus aureus were synthesized and used to detect DNA sequences homologous to these genes in 27 non-clinical isolates of Staph. aureus isolated from nasal swabs of 74 healthy human volunteers. Genomic DNA from all 27 isolates reacted with at least one of the probes. In a phenotypic assay for toxin production by a reverse passive latex agglutination test however, only 15 of the 27 isolates produced enterotoxin in culture. The results raise the possibility that a number of Staph. aureus isolates harbour DNA sequences that are apparently silent or mutant copies of the enterotoxin genes. This complicates the identification of enterotoxin producers by tests which depend on oligonucleotide or DNA hybridization.

Base Sequence↗

PubMed1994

Staphylococcal growth and enterotoxins (A-D) and thermonuclease synthesis in the presence of dehydrated garlic.

The inhibition of Staphylococcus aureus growth and enterotoxin and thermonuclease production by various concentrations of garlic (Allium sativum) was studied in BHI broth. The growth of Staph. aureus was inhibited by dehydrated garlic at levels of 1.5% (w/v) and over. Enterotoxins A, B and C1 were only detectable in broth containing < 1% of garlic while enterotoxin D was produced at a level of 2%. Garlic also inhibited thermonuclease (TNAse) production, complete inhibition being observed at levels > or = 1.5%. TNAse was not always detected when enterotoxin was present.

Bacterial Toxins↗

PubMed1988

Molecular cloning of a Salmonella typhi LT-like enterotoxin gene.

Diarrhoea is a common event during typhoid fever; nevertheless, the possible participation of a diarrhoea-inducing enterotoxin has not been described (Roy et al., 1985). Recombinant bacteriophage lambda FDC1 was isolated from a genomic library of Salmonella typhi, the causal agent of typhoid fever, by screening with a probe for the B subunit gene of the heat-labile, cholera-like, Escherichia coli enterotoxin (LT). Lambda FDC1 codes for an enterotoxin that causes secretion in rat ileal loops, that elongates Chinese hamster ovary (CHO) cells, that is recognized by antibodies against LT, and does not bind in vitro to ganglioside GM1. These results should allow further studies towards elucidating a possible role for the S. typhi enterotoxin in the pathogenesis of typhoid fever.

Amino Acid Sequence↗

PubMed1976

Neutralizing antibodies against Escherichia coli and Vibrio cholerae enterotoxins in human milk from a developing country.

By means of the adrenal cell assay and the rabbit small-bowel loop technique enterotoxin-neutralizing antibodies were demonstrated in all investigated milk samples from severely undernourished Pakistani women but, with a single exception, not in milk from Swedish mothers. The antibodies belonged to the IgA and IgG classes as observed in an enzyme-linked immunosorbent assay, which also revealed secretory component on specific enterotoxin antibodies, showing that the IgA antibodies were primarily of the secretory type. The epidemiological stituation and experimental data strongly indicate that Escherichia coli enterotoxin rather than immunologically cross-reactive Vibrio cholerae toxin in most cases constituted the antigenic stimulus for the milk antibodies. The presence in the milk of the Pakistani mothers of secretory IgA antibodies against an important diarrheogenic agent may have practical importance for protection of the breast-fed baby and probably also illustrates the frequent intestinal exposure of these women to enterotoxinogenic bacteria. Since enterotoxin antigen is presumed not to enter deeper tissues, the demonstration of secretory antibodies in milk agrees with a proposed homing of intestinally triggered lymphocytes to the mammary gland.

Antibodies, Bacterial↗

PubMed1994

Comparison of the Tecra VIA kit, Oxoid BCET-RPLA kit and CHO cell culture assay for the detection of Bacillus cereus diarrhoeal enterotoxin.

Two commercial serological kits (Oxoid BCET-RPLA and Tecra VIA) and a Chinese hamster ovary (CHO) cell cytotonicity assay for the detection of Bacillus cereus diarrhoeal enterotoxin were compared. Eleven B. cereus strains and one enterotoxigenic B. thuringiensis strain were evaluated. Both kits and the CHO cell assay yielded positive toxin responses for cell-free culture filtrates from eight out of 11 diarrhoeal enterotoxigenic strains. An emetic enterotoxin producing strain was negative with all three assays. Two B. cereus strains were negative using the BCET-RPLA kit, but positive with the Tecra VIA kit and CHO cell assay. The BCET-RPLA indicated significant levels of enterotoxin after samples were boiled, whereas the CHO cell and Tecra assays were negative. Overall, the cell culture assay was the most sensitive. However, the Tecra VIA kit provided similar results and was better suited for the rapid detection of B. cereus diarrhoeal enterotoxin.

Animals↗

PubMed2006

The profiles of enterotoxin genes in Staphylococcus aureus from nasal carriers.

AIMS: To evaluate the occurrence of enterotoxin genes in Staphylococcus aureus recovered from nasal carriers. METHODS AND RESULTS: Eighty S. aureus strains were tested for the presence of 17 new enterotoxin genes using multiplex-PCR. Sixty-one isolates were found to carry enterotoxin genes. The majority of the enterotoxigenic isolates carried enterotoxin gene cluster (egc) genes, namely seg, sei, sem, sen and seo. The egc type containing the seu gene was found in 19 of the 47 isolates with egc-like genes. Interestingly, no seu-containing egc coexisted with sec and sel, as was the case for a considerable portion of the isolates carrying a seu-negative egc. The tst gene was detected in two isolates carrying sec and sel only and in eight isolates carrying seu, but not in the isolates containing the seu-negative egc type. CONCLUSIONS: The genes forming an egc were found to be predominant in S. aureus from nasal carriers. The coexistence of a seu-positive egc with tst in contrast to an egc lacking the seu gene apparently is not associated with the presence of tst and can reflect a difference between these gene groupings. SIGNIFICANCE AND IMPACT OF THE STUDY: The egc types carried by the analysed isolates seem to have an influence on the distribution of other genes located on staphylococcal pathogenicity islands, which may modulate the repertoire of virulence factors carried by a single S. aureus strain.

Carrier State↗

PubMed1993

Stimulation of rat spleen cells by staphylococcal enterotoxins.

There is much interest in staphylococcal enterotoxins as T cell mitogens in humans, mice and rabbits. Rat spleen cells were shown to proliferate in response to staphylococcal enterotoxins A and B and toxic shock syndrome toxin-1 at concentrations (5 to 500 ng ml-1) which also stimulate mouse spleen cells. The proliferative response to all these enterotoxins was inhibited by cyclosporin A, indicating the response to be predominantly that of T cells. These results indicate that the rat provides another convenient model for the analysis of T cell responses to enterotoxins.

Animals↗

PubMed1991

Detection of antibodies to staphylococcal enterotoxins in the serum and milk of healthy goats.

A study was made of the presence of antibodies (Ab) to staphylococcal enterotoxins A to E (SEA-SEE) in the serum and milk of 133 healthy goats, using a competitive ELISA method. Antibodies to some enterotoxins were detected in 83 sera (62.4%) and in 41 (30.8%) milk samples. In serum, antibodies to all SE types were detected, the most frequent being antibodies to SEA (24.8%). Milk contained antibodies to SEA, SEB and SEC, the latter being the most frequent (24.8%). A statistical study was performed to correlate the number of animals harbouring antibodies to a given enterotoxin with the presence in these animals of staphylococci producing that enterotoxin.

Animals↗

PubMed1993

Production of heat-stable enterotoxin II by chicken clinical isolates of Escherichia coli.

Enterotoxigenic Escherichia coli isolated from diarrhea stools of chickens were examined for production of heat-stable enterotoxin II which is considered to be implicated only in diarrhea of pigs. Seven out of 38 strains examined were found to contain heat-stable enterotoxin II gene, determined by colony hybridization and the polymerase chain reaction. The culture supernatants of these strains caused fluid accumulation in the mouse intestinal loop test. This fluid accumulation activity was not lost by heating at 100 degrees C and was neutralized by anti-heat-stable enterotoxin II antiserum. Purified heat-stable enterotoxin II caused fluid accumulation in the chicken intestinal loop assay. These results indicate that STII-producing E. coli is implicated in chicken diarrhea.

Animals↗

PubMed1997

Bacillus cereus may produce two or more diarrheal enterotoxins.

Bacillus cereus strains were tested for production of diarrheal enterotoxin by the reverse passive latex agglutination test and for presence of B. cereus enterotoxin gene (bceT) by polymerase chain reaction. About 50% of 56 B. cereus strains reacted positive in broth culture in the reverse passive latex agglutination test, while the bceT gene was detected in 41.1%. Sixteen percent of the strains were positive for both diarrheal enterotoxin and bceT gene. A 741 bp probe prepared from the polymerase chain reaction product detected bceT gene in all strains that were positive with the polymerase chain reaction. This study indicates a likelihood of two or more enterotoxins being produced by B. cereus which may be involved in causing diarrheal type food poisoning.

Bacillus cereus↗