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PubMed1999

Competitive immunoassay for staphylococcal enterotoxin A using capillary electrophoresis with laser-induced fluorescence detection.

Staphylococcal enterotoxins are a family of toxic proteins secreted by S. aureus. Using capillary electrophoresis (CE) linked with laser-induced fluorescence, a highly sensitive and selective assay using antibody-antigen recognition was developed for the determination of Staphylococcal enterotoxin A. Staphylococcal enterotoxin A (SEA) was chemically labeled with fluorescein and the product was used as a fluorescent tracer. A competitive assay was developed to detect SEA at concentrations between 0.3 nM and 6.5 nM with standard deviations of less than 5%. The detection limit was found to be 3 amol with the potential improvement by further optimization of the assay. No cross-reactivity between staphylococcal enterotoxin B and the SEA antibody was found at the concentrations used for the CE immunoassay.

Electrophoresis, Capillary↗

PubMed1988

Prevalence of serum antibodies to toxic-shock-syndrome-toxin-1 and to staphylococcal enterotoxins A, B and C in West-Germany.

Sera of 152 healthy blood donors and 43 infants 9 to 12 months of age were tested for serum antibodies to TSST-1 and staphylococcal enterotoxins A, B and C by a microtiter ELISA assay. Only 50% of the adult population had detectable antibody-titers to enterotoxin A, whereas 80% to enterotoxin C and 90% to enterotoxin B and TSST-1, which is very similar to the prevalence of TSST-1 antibodies among USA-residents. The "protective" titer of TSST-1-antibodies can be estimated to be 1:100 in the test system used by comparison with anti-TSST-1-titers in five acute phase sera from confirmed menstrual TSS cases.

Adult↗

PubMed1991

Coagglutination test for rapid detection of Salmonella enterotoxin.

The Staphylococcal Coagglutination (CoA) test using Staphylococcus aureus (Cowan type-1) cells coated with antiserum against purified Salmonella enterotoxin was standardized and carried out to screen Salmonella serotypes for their enterotoxigenicity. Of the 101 cell-free culture supernatants from an equal number of Salmonella strains belonging to 15 different serotypes tested, 76 were found to be enterotoxigenic. All of the 24 strains which gave positive reactions in the rabbit ligated ileal loop test were also positive with this test. As little as 7.5 ng of purified Salmonella enterotoxin could be detected by the CoA test. The presence of enterotoxin in polymyxin B extract of cells and cell-free preparation of a S. typhimurium strain was detected as early as after 1.5 and 3.0 h of incubation, respectively. Besides the test standardized with the antiserum to purified V. cholerae toxin (CT) could detect purified toxin in a quantity as little as 5.0 ng and also enterotoxigenicity in an E. coli strain (LT+). The usefulness of the test as a routine, rapid and economic one for the detection of enterotoxin of Salmonella, Vibrio cholerae and E. coli is discussed.

Agglutination Tests↗

PubMed2000

Enterotoxins and the enteric nervous system--a fatal attraction.

Although there has been extensive investigation of the biochemical consequences of the interactions between bacterial enterotoxins and intestinal epithelial cells and the mechanisms by which they induce intestinal secretion, relatively little attention has been given to other aspects of the host response to these enterotoxins. There is now compelling evidence that the enteric nervous system has a major role in enhancing the secretory state induced by cholera toxin, the E. coli enterotoxins and possibly C. difficile toxin A. Cholera toxin for example is thought to activate a neural reflex via the release of 5-hydroxytryptamine from enterochromaffin cells. Neurotransmitters involved in the reflex include substance P and vasoactive intestinal polypeptide. Delineation of these neural pathways may offer new possibilities for the pharmacological control of enterotoxin-mediated secretion.

Animals↗

PubMed1972

Serological detection of enterotoxin in foods implicated in staphylococcal food poisoning.

Two methods are described for the extraction of enterotoxin from foods incriminated in incidents of staphylococcal food poisoning. Enterotoxin was detected serologically in 12 of 24 food samples from 20 separate incidents: eight samples contained enterotoxin A, three contained D and one both A and B. The amount of enterotoxin in nine foods, based on 100% recovery, varied from 0.02 to 0.09 mug./g.Data are also given on the numbers of Staphylococcus aureus isolated from samples of food from 39 food poisoning incidents. Colony counts varied between 7.5 x 10(5) and 9 x 10(9)/g. with a median value of 7 x 10(7)/g.

Cell Count↗

PubMed1993

Enterotoxins and toxic-shock syndrome toxin-1 in non-enteric staphylococcal disease.

Over the 7 years 1985-91, 997 strains of Staphylococcus aureus from 962 patients with diseases other than food poisoning have been tested for the production of enterotoxins and toxic shock syndrome toxin-1 (TSST-1) and phage typed. In all, 128 cases could be classified as confirmed or probable toxic shock syndrome (TSS) but a further 199 cases were classified as possible or unconfirmed TSS. In 219 cases, an alternative diagnosis could be supported and 45 cases were classified as sudden infant death syndrome. In 371 cases, insufficient information for classification was available. Strains of phage group I producing TSST-1 were associated with menstrual TSS. Many menstrual TSS cases were aged less than 20 and were using non-introducer tampons. When all strains were reviewed, strong associations were observed between TSST-1 production and phage group I strains, enterotoxin B production and group V strains, enterotoxin C and phage-type 95 strains and between enterotoxin A without TSST-1 and phage group III strains.

Adolescent↗

PubMed1998

The Clostridium perfringens enterotoxin from equine isolates; its characterization, sequence and role in foal diarrhoea.

During a survey of foal diarrhoea between 1991 and 1994, Clostridium perfringens was significantly associated with disease with 56% of cases infected [1]. The contribution of enterotoxigenic C. perfringens to this association, was assessed by use of the reverse passive latex agglutination test for enterotoxin (RPLA; Oxoid Unipath) and vero cell toxicity neutralized by antitoxin on stored faecal samples and sporulated faecal isolates of C. perfringens. Polymerase chain reaction (PCR1) based on the DNA sequence for the whole enterotoxin gene [2] yielded a fragment from an equine isolate of the anticipated size which, cloned into plasmid M13 phage, had a sequence essentially identical to the published sequence. Consequently, all faecal isolates were also tested by PCR1 and for a part of the enterotoxin gene (PCR2). Significant association with diarrhoea (controls not in contact with cases) was found with positive RPLA tests on faeces (OR = 13, P = 0.002) and isolates (OR = 4.57, P = < 0.0001), vero cell toxicity of isolates (OR = 1.78, P = 0.026), and PCR1 (OR = nd, P = 0.029) but not PCR2 or vero cell toxicity of faeces. Significant association with diarrhoea was also found for isolates negative by RPLA (OR = 3.91; CI 2.05-7.57; P < 0.0001) or PCR1 (OR = 4.81; CI 2.84-8.20; P < 0.0001). Many of the isolates from RPLA positive faeces and verotoxic isolates were PCR negative and no evidence could be found for the presence of the enterotoxin gene in a random selection of RPLA positive/PCR negative isolates by gene probe on chromosomal DNA and PCR reaction product or vero cell toxicity neutralized by specific antiserum. Failure of the vero cell toxicity on faeces to be associated with diarrhoea or for cytotoxicity of cultures and RPLA on cultures to agree with the PCRs was believed to be related to the presence of other cytotoxins, the inherent cytotoxicity of equine faeces and to the poor specificity of the commercial antiserum used in the test. Enterotoxigenic C. perfringens could not account for the overall association of C. perfringens with foal diarrhoea because (a) cultures positive by PCR, RPLA or cytotoxicity were not significantly more common amongst isolates from cases than controls; and (b) the proportion of isolates from cases positive by PCR (PCR1 or PCR2) was too small at 9.7%.

Animals↗

PubMed2002

Inactivation of staphylococcal enterotoxin-A with an electrolyzed anodic solution.

Electrolyzed anodic NaCl solutions [EW+], prepared by the electrolysis of 0.1% NaCl, have been shown to instantly inactivate most pathogens that cause food-borne disease. Elimination of food-borne pathogens does not necessarily guarantee food safety because enterotoxins produced by pathogens may remain active. We have tested whether EW+ can inactivate Staphylococcal enterotoxin A (SEA), one of the major enterotoxins responsible for food poisoning. Fixed quantities of SEA were mixed with increasing molar ratios of EW+, and SEA was evaluated by reversed-phase passive latex agglutination (RPLA) test, immunoassay, native polyacrylamide gel electrophoresis (PAGE), and amino acid analysis after 30 min incubations. Exposure of 70 ng, or 2.6 pmol, of SEA in 25 microL of PBS to a 10-fold volume of EW+, or ca. 64.6 x 10(3)-fold molar excess of HOCl in EW+, caused a loss of immuno-reactivity between SEA and a specific anti-SEA antibody. Native PAGE indicated that EW+ caused fragmentation of SEA, and amino acid analysis indicated a loss in amino acid content, in particular Met, Tyr, Ile, Asn, and Asp. Staphylococcal enterotoxin-A excreted into culture broth was also inactivated by exposure to an excess molar ratio of EW+. Thus, EW+ may be a useful management tool to ensure food hygiene by food processing industries.

Amino Acids↗

PubMed1990

Specific recognition of staphylococcal enterotoxin A by human T cells bearing receptors with the V gamma 9 region.

T cells bearing the alpha beta receptor can specifically react with target cells coated with staphylococcal enterotoxin and expressing major histocompatibility complex class II molecules; these responses depend on which variable region (V) of the receptor's beta-subunit is used. We have now examined whether a similar situation exists for human T cells bearing the gamma delta receptor. We found that reactivity to staphylococcal enterotoxin A is strictly dependent on the presence of the V gamma 9 variable region in the gamma delta T-cell receptor (TCR). These cytotoxic responses required the expression of HLA class II molecules by the target cell and could be inhibited by anti-gamma delta TCR and by anti-HLA-class-II monoclonal antibodies. In contrast to alpha beta TCR+ cell clones, no proliferative response of V gamma 9+ T-cell clones towards stimulator cells coated with enterotoxin A was observed in vitro. These results indicate that the gamma delta TCR repertoire might be influenced by enterotoxin A produced during staphylococcal infections in vivo. This could provide a molecular basis for the observation that V gamma 9+ T cells form the large majority of peripheral gamma delta TCR+ cells but only a small proportion of thymic gamma delta TCR+ cells.

Cell Line↗

PubMed1995

Residues defining V beta specificity in staphylococcal enterotoxins.

The three-dimensional structure of staphylococcal enterotoxin C2 has been determined at 2.7 A resolution by x-ray diffraction, while the structures of enterotoxins A and E have been modelled based on their sequence homology to other staphylococcal enterotoxins. The T-cell receptor-binding sites of staphylococcal enterotoxin (SE) B and SEC2 are compared and the stereochemical interactions likely to be responsible for their differing V beta specificities are identified. A similar comparison is made between SEA and SEE.

Amino Acid Sequence↗

PubMed1998

Growth and enterotoxin production of Staphylococcus aureus during the manufacture and ripening of Camembert-type cheeses from raw goats' milk.

Tests were carried out to determine the effect of manufacturing procedures for a Camembert-type cheese from raw goats' milk on the growth and survival of Staphylococcus aureus organisms added to milk at the start of the process, and to study the possible presence of staphylococcal enterotoxin A in these cheeses. The initial staphylococcal counts were, respectively, 2, 3, 4, 5 and 6 log cfu ml-1. Cheese was prepared following the industrial specifications and ripened for 41 d. Detection of enterotoxins was done by the Vidas SET test and by an indirect double-sandwich ELISA technique using antienterotoxin monoclonal antibodies. Generally, numbers of microbes increased at a similar rate during manufacture in all cheeses until salting. During the ripening period, the aerobic plate count population and Staph. aureus levels remained stable and high. There was an approximately 1 log reduction of Staph. aureus in cheeses made with an initial inoculum of Staph. aureus greater than 10(3) cfu ml-1 at the end of the ripening period (41 d) compared with the count at 22 h. The level of staphylococcal enterotoxin A recovered varied from 1 to 3.2 ng g-1 of cheese made with an initial population of 10(3)-10(6) cfu ml-1. No trace of enterotoxin A was detected in cheeses made with the lowest Staph. aureus inoculum used in this study.

Animals↗

PubMed2003

Identification of a new putative enterotoxin SEU encoded by the egc cluster of Staphylococcus aureus.

AIMS: This paper reports on a new putative enterotoxin SEU encoded by the enterotoxin gene cluster egc from Staphylococcus aureus and on a real-time polymerase chain reaction (PCR) assay for detecting the seu gene. METHODS AND RESULTS: PCR and sequencing revealed a new putative enterotoxin SEU encoded by some egc clusters. The seu gene resulted from sequence divergence in the psient1 and psient2 pseudogenes previously described in the egc cluster (Jarraud et al. [2001] Journal of Immunology166, 669). The presence of the seu gene was investigated in a collection of S. aureus strains by conventional PCR and by a specific 5'-nuclease PCR assay. Among the 24 strains harbouring the egc cluster, four tested positive for the seu gene. CONCLUSIONS: The existence of the seu gene adds to the number of newly described se genes and underlines the need for a better understanding of their role in the pathogenesis of S. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: A thorough study of the seu gene should provide further insight into the phylogenetics of the staphylococcal enterotoxins.

Amino Acid Sequence↗

PubMed1979

Escherichia coli heat-labile enterotoxin: DNA-directed in vitro synthesis and structure.

Escherichia coli heat-labile enterotoxin was synthesized in a cell-free system directed by DNA of the plasmid P307. Synthesis of the toxin, assayed by the elongation induced in Chinese hamster ovary cells, was strongly stimulated by cyclic AMP and occurred at physiological levels of Mg2+ only when the polyamine spermidine was present. Activity was abolished by heat and antisera prepared against the enterotoxins of both E. coli P263 and Vibrio cholera. Tritium-labeled enterotoxin was purified by immunoprecipitation and examined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. When gel slices were assayed for the ability to stimulate adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity in erythrocyte ghosts, two peaks were found, one at Mr 26,000 and frequently, but not always, another at Mr 23,000. Detection of radiolabeled protein by fluorography and scintillation counting of gel slices revealed three prominent polypeptides, two corresponding to the peaks having adenylate cyclase-stimulating activity and a further one of Mr 11,500, identical to that of the cholera subunit B. The data suggest that the E. coli heat-labile enterotoxin synthesized in the cell-free system has a subunit structure.

Animals↗

PubMed1993

Enteroaggregative Escherichia coli heat-stable enterotoxin 1 represents another subfamily of E. coli heat-stable toxin.

Enteroaggregative Escherichia coli (EAggEC) are associated with persistent diarrhea in young children. Some of these organisms produce a low-molecular-weight, heat-stable, plasmid-encoded enterotoxin that has been named EAggEC heat-stable enterotoxin 1 (EAST1). We have cloned a 4.4-kb DNA fragment from the virulence plasmid of prototype EAggEC strain 17-2, which expresses enterotoxic activity as measured by electrogenic response in Ussing chambers mounted with rabbit ileal tissue. DNA-sequence analysis of this fragment identified an open reading frame (ORF) encoding a cysteine-rich polypeptide of 38 amino acids (M(r), 4100). Insertional and deletional mutations in this ORF resulted in loss of enterotoxic activity. The ORF was cloned into a T7 expression vector, and postinduction culture filtrates exhibited enterotoxic activity and increased ileal tissue cGMP levels. A synthetic peptide consisting of predicted amino acid residues 8-29 also showed enterotoxic activity. These data indicate that this ORF, named astA (EAggEC heat-stable enterotoxin), represents the EAST1 structural gene. EAST1 shows significant homology with the enterotoxic domain of heat-stable enterotoxin a (STa) of enterotoxigenic E. coli and with guanylin, a mammalian analog of STa. Unlike STa, which requires six cysteines and three disulfide linkages for full biological activity, both EAST1 and guanylin contain four cysteine residues. Based on the cGMP data and the sequence homology to STa and guanylin, it is predicted that EAST1 stimulates the particulate form of guanylate cyclase through the same receptor-binding region as STa and guanylin.

Amino Acid Sequence↗

PubMed2004

Enterotoxin B is the predominant toxin involved in staphylococcal scarlet fever in Taiwan.

This study retrospectively reviewed all pediatric cases of staphylococcal scarlet fever (SSF) that occurred during a 10-year period in a 1400-bed tertiary medical center in northern Taiwan. All 20 cases of SSF occurred in previously healthy individuals. Skin and soft-tissue infections predominated among children from whom Staphylococcus aureus was isolated. Polymerase chain reaction testing was used to detect known staphylococcal toxin genes, and of the isolates studied, most (18 [90%] of 20) contained only the staphylococcal enterotoxin B. One of the other strains was positive for staphylococcal enterotoxin A only, and the last strain was positive for both staphylococcal enterotoxin G and staphylococcal enterotoxin I. Pulsed-field gel electrophoresis identified a small cluster of isolates (6 [30%] of 20) that were genetically related, but these strains came from epidemiologically unrelated patients during a 3-year period.

Adolescent↗

PubMed1975

Classification of enterotoxins on the basis of activity in cell culture.

Two cell culture systems were used in a study of the biological properties of several bacterial enterotoxins in vitro. By means of one model, in which HeLa cell monolayers were used, cytotoxic effects, interms of detachment of cells from a glass surface due to cell death, were assayed. By means of the second model, activation of the adenyl cyclase-cyclic adenosine 3', 5'-monophosphate (AMP) system, in terms of increased steroidogenesis by Y-1 adrenal cells (an effect which we have termed cytotonic), was assayed. None of the four toxins manifested both properties. Rather there was a clear segregation into two cytotoxic enterotoxins (Shigella dysenteriae type 1 and Clostridium perfingens) and two cytotonic products (Vibrio cholerae and Escherichia coli). These data raise the possibility that some enterotoxins may not interact with the adenyl cyclase-cyclic AMP system; this possibility has also been suggested by studies of the toxin of S. dysenteriae type 1 in the rabbit small bowel. These cell culture systems may therefore serve as convenient models for the study of the mechanism of action of both classes of enterotoxin.

Adenylyl Cyclases↗

PubMed1975

Immunogenicity of formaldehyde-inactivated enterotoxins A and C1 of Staphylococcus aureus.

Quantitative precipitation of antisera specific for native enterotoxin revealed that 70% and 60%, respectively, of the antigenic determinants of enterotoxins A and C1 of Staphylococcus aureus were inactivated by formaldehyde at pH 5.0 or 7.5 contained large polymers (excluded by Sepharose 2B) and induced strong humoral immune responses in rhesus monkeys. Enterotoxin A inactivated at pH 5.0 or 7.5 was composed mostly of small polymers (excluded by Sephadex G-100 but included by Sepharose 2B); it provoked a poor immune response in monkeys (about equivalent to the response obtained with weakly reactive toxin inactivated at alkaline pH). It was concluded that potent enterotoxoids were formed by extensive cross-linking of enterotoxin C1 into large polymers in acidic or neutral formaldehyde solution.

Animals↗

PubMed1976

Purification of the polymyxin-released, heat-labile enterotoxin of Escherichia coli.

The heat-labile enterotoxin of Escherichia coli strain H-10407 has been purified by use of a commercially available affinity gel (Affi-Gel 202). This gel possesses a strong and highly specific affinity for the enterotoxin released from intact E. coli cells by polymyxin B. The polymyxin-release technique could be used with fermenter-size batches of E. coli cells grown in a casamino acids-yeast extract medium. With a simple (NH4)2SO4 back-extraction step prior to affinity chromatography, large batches of E. coli enterotoxin could be processed rapidly. Affi-Gel 202-purified E. coli enterotoxin produced a single precipitin band in the presence of several different antisera against crude preparations of the toxin. The same antigen produced a precipitin band in the presence of both cholera antitoxin and antiserum to choleragenoid.

Antigens, Bacterial↗