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PubMed1980

Enterotoxigenic Escherichia coli carrying plasmids coding for antibiotic resistance and enterotoxin production.

Nine strains of enterotoxigenic Escherichia coli that were resistant to antibiotics were tested for their ability to transfer both antibiotic resistance and enterotoxigenicity to E. coli K12. All nine isolates transferred antibiotic resistance in bacterial conjugation experiments, and in seven of these matings enterotoxigenicity was also transferred. To determine whether the genetic information coding for the production of enterotoxin and antibiotic resistance was located on the same plasmid in these strains, phage P1 transduction experiments were performed. P1 lysates prepared from enterotoxigenic antibiotic-resistant transconjugants were tested for their ability to transduce both phenotypes. Only P1 lysates prepared from transconjugants of E. coli 719B5 (O18ab:H27) were found to transduce resistance to streptomycin, sulfisoxazole, and tetracycline, as well as the ability to produce heat-labile and heat-stable enterotoxin. The results of these experiments strongly suggest that the genetic data coding for antibiotic resistance and enterotoxin production in E. coli 719B5 are carried on the same plasmid replicon.

Drug Resistance, Microbial↗

PubMed1986

In vivo and in vitro effects of a novel enterotoxin, STb, produced by Escherichia coli.

Escherichia coli may produce a heat-labile enterotoxin (LT) or two heat-stable enterotoxins (STa, STb). STb consistently causes secretion in vivo in 5-hr weaned-pig intestinal loops (P less than .0001). In Ussing chambers in vitro, crude culture filtrates of STb initiate a prompt increase in short circuit current (SCC; P less than or equal to .0001) and potential difference (P less than or equal to .0001) when compared with nontoxigenic culture filtrates. In bidirectional in vitro studies of ion flux (22Na and 36Cl), STb did not alter 22Na or 36Cl unidirectional or net fluxes. The calculated residual ion flux increased significantly (P less than .03), however, in tissues treated with STb and fully accounted for the STb-induced increase in SCC. Measurement of the electrolyte content of ligated intestinal segments in vivo further suggested that STb stimulated bicarbonate secretion. Relative to controls, significant accumulation of Na and Cl also occurred intraluminally in vivo. These data indicate that STb is a unique enterotoxin that causes net secretion in pig jejunum in vivo. In vitro and in vivo studies show that STb stimulates active secretion of nonchloride anion. We postulate that STb causes active bicarbonate secretion in weaned-piglet jejunum.

Animals↗

PubMed2000

A putative heat-labile enterotoxin expressed by strains of Aeromonas media.

Fifteen isolates of Aeromonas media (seven from diarrhoeal stools, four from water and four from superficial skin ulcers of catfish) were examined for enterotoxin production. Ten of these isolates (six diarrhoeal, one from water and three from fish) caused accumulation of fluid in the initial rabbit ileal loop (RIL) tests. Isolates from diarrhoeal stools and fish caused relatively more fluid accumulation than those from water. Those strains that caused little or no fluid accumulation in the initial experiments became enterotoxin producers after one passage through RILs, regardless of source, and also showed gradual enhancement of fluid outpouring after each subsequent passage. Inocula of c. 1 x 10(4) viable cells and 0.25 ml of culture filtrate (CF) caused fluid accumulation similar to that of toxigenic Vibrio cholerae 569B. The enterotoxic factor(s) were inactivated when held at 56 degrees C for 20 min or 65 degrees C for 10 min and showed biological activity over a wide range of pH values. These results suggest that strains of A. media, whether from diarrhoeal stools, water or infected fish, are potentially enterotoxigenic and may have the potential to produce a heat-labile and pH-stable diarrhoeagenic factor in the same way as other known heat-labile and pH-stable enterotoxins.

Aeromonas↗

PubMed1989

Coat and enterotoxin-related proteins in Clostridium perfringens spores.

Coat proteins from mature spores of two enterotoxin-positive (Ent+) and two enterotoxin-negative (Ent-) strains of Clostridium perfringens were solubilized using 50 mM-dithiothreitol and 1% sodium dodecyl sulphate at pH 9.7, and alkylated using 110 mM-iodoacetamide to prevent aggregation. The coat proteins and C. perfringens type A enterotoxin (CPE) were separated by SDS-PAGE and analysed by Western blotting using anti-CPE antibody. As previously reported, CPE aggregated in the presence of SDS, but no aggregation occurred at concentrations below 15 micrograms CPE ml-1. Two CPE-related proteins (34 and 48 kDa) were found in the solubilized spore coat protein of Ent+ strains while only the 48 kDa CPE-related protein was found in the spore coat fraction of Ent- strains. CPE-related proteins comprised 2.7% and 0.8% of the total solubilized coat protein of Ent+ and Ent- strains respectively. CPE-related proteins could be extracted from the spores with 1% SDS alone. They could also be released by disruption of whole spores, indicating that the CPE-related proteins may be in the spore core or trapped between the core and coat layers. The results suggest that CPE is not a major structural component of the coat fraction of C. perfringens spores.

Bacterial Proteins↗

PubMed1997

Comparison of biological effect of the two different enterotoxin complexes isolated from three different strains of Bacillus cereus.

The cytotoxicity of the two different enterotoxin complexes of Bacillus cereus was compared after isolation from three different strains. Protein components of non-haemolytic enterotoxin (NHE) of 39 kDa, 45 kDa and 105 kDa were isolated from all of the three strains, whilst proteins B, L1 and L2 of haemolysin BL (HBL) were isolated from supernatants of two strains (F837-76 and 1230-88). These proteins were not detected in strain 0075-95. Inhibition of protein synthesis in Vero cells was used as a measure of cytotoxicity. The HBL complex from strain F837-76 was highly toxic. This strain also produced the NHE complex. However, when purified, at least two of the components of NHE had to be present in higher amounts than those of the components of HBL to cause the same degree of toxicity. Both complexes purified from strain 1230-88 were cytotoxic. The amount required to cause the same degree of cytotoxicity was approximately equal for the components of the two complexes, except that higher amounts of the 105 kDa protein of NHE had to be present than for the other components. None of the purified complexes from strain 1230-88 was toxic in amounts comparable to those of the HBL complex of strain F837-76 and NHE of strain 0075-95. These results indicate that when measuring cytotoxic enterotoxins from B. cereus at least two different complexes and six different proteins have to be taken into consideration.

Amino Acid Sequence↗

PubMed1997

The Clostridium perfringens enterotoxin gene is on a transposable element in type A human food poisoning strains.

The Clostridium perfringens enterotoxin gene (cpe) is rarely found in naturally isolated strains. In human food poisoning strains, cpe is found on the chromosome, and is located episomally in animal isolates. Observations that the gene was somewhat unstable and could be gained or lost suggested that the gene was on a mobile element. An IS200-like element, IS1469, is almost always upstream of cpe. A new insertion element was identified, IS1470, a member of the IS30 family, which is found both up-an downstream of cpe in the type A strain NCTC 8239. PCR results confirmed that this configuration was conserved in type A human food poisoning strains. The enterotoxin gene was on a 6.3 kb transposon which, in addition to the two flanking copies of IS1470, included IS1469 and two 1 kb stretches, one on each side of cpe, with no open reading frames. Results indicated that 14 bp was copied from the genome during insertion. Details of the configuration of DNA in this transposon are presented, and the possible connection of this transposon with the movement of the enterotoxin gene is discussed.

Animals↗

PubMed1983

Detection of Clostridium perfringens enterotoxin by tissue culture and double-gel diffusion methods.

Culture filtrates of four enterotoxin-producing strains of Clostridium perfringens type A produced morphological changes in cells of 10 of 12 established mammalian cell lines tested, and particularly Vero (African green-monkey kidney) and MDCK (dog kidney) cells. Double-gel diffusion and Vero-cell tests detected enterotoxin in culture filtrates of, respectively, 29 (28%) and 56 (54%) of 103 strains of C. perfringens tested. The feasibility of cell-culture assays for the routine detection of C. perfringens enterotoxin is indicated.

Animals↗

PubMed1987

Aeromonas cytotonic enterotoxin cross reactive with cholera toxin.

Isolation by affinity chromatography from crude culture filtrate of Aeromonas sobria of protein that cross reacted with cholera toxin (CT) revealed a toxin that produced fluid accumulation in rat ileal loops and in infant mice and caused rounding of Y1 adrenal cells. All these activities were neutralised by antiserum to CT. There was no haemolytic or cytotoxic activity associated with this CT-cross reactive cytotonic enterotoxin. CT-cross reactive material detected in enzyme linked immunosorbent assay (ELISA) was produced by 25% of Aeromonas isolates from faeces of children with or without diarrhoea-26% of A. sobria, 20.0% of A. hydrophila and 24% of A. caviae tested gave positive ELISA results. Most strains that produced this cytotonic enterotoxin but no cytotoxic enterotoxin were isolated from children without diarrhoea. Toxin preparations from Aeromonas spp. that completely inhibited adenosine-5'-diphosphate-induced platelet aggregation, an effect related to elevation of intracellular cAMP, were, with one exception, cross reactive with CT in ELISA.

Aeromonas↗

PubMed1995

Prevalence of Vibrio cholerae with heat-stable enterotoxin (NAG-ST) and cholera toxin genes; restriction fragment length polymorphisms of NAG-ST genes among V. cholerae O serogroups from a major shrimp production area in Thailand.

A total of 148 Vibrio cholerae isolates from a major shrimp production area in Southern Thailand were examined by colony hybridisation for genes encoding heat-stable enterotoxin (NAG-ST) and cholera toxin (CT). Only non-O1 V. cholerae strains were found to harbour NAG-ST (14 of 146) whereas no strains hybridised with the CT probe. NAG-ST-positive V. cholerae non-O1 strains were isolated from shrimp farms situated close to urban areas. Five different O serogroups were found among NAG-ST positive non-O1 strains. Southern blot and restriction endonuclease analysis of NAG-ST-positive strains revealed a high degree of genetic divergence. A total of seven classes of enterotoxin gene patterns were found with HindIII and EcoRI restriction endonucleases. Enterotoxin gene patterns correlated with O-antigen expression in 84% of isolates tested. In combination with other molecular techniques Southern blot analysis with an NAG-ST oligonucleotide probe could be useful for studying the molecular epidemiology of V. cholerae non-O1 strains.

Animals↗

PubMed1996

Characterisation of an enterotoxin-negative, cytotoxin-positive strain of Clostridium sordellii.

In ileal loop assay, ELISA and anion-exchange column chromatography, Clostridium sordellii strain 6018 was shown to produce a cytotoxin, but no detectable enterotoxin. DNA sequence and polymerase chain reaction analyses indicated that the lack of enterotoxin activity is not due to a lack of gene transcription, but to lack of a major portion of the enterotoxin gene. This is the first characterisation of such a strain.

Amino Acid Sequence↗

PubMed1994

A complex array of Hpr consensus DNA recognition sequences proximal to the enterotoxin gene in Clostridium perfringens type A.

Enterotoxin production in Clostridium perfringens is both strain dependent and sporulation associated. Underlying these phenotypic observations must lie a genetic and molecular explanation and the principal keys will be held within the DNA sequence both upstream and downstream of the structural gene cpe. In accordance with the above we have sequenced 4.1 kbp of DNA upstream of cpe in the type strain NCTC 8239. A region of DNA extending up to 1.5 kb 5' to cpe is conserved in all enterotoxin-positive strains. This region contains a putative ORF with substantial homology to an ORF in the Salmonella typhimurium IS200 insertion element and, in addition, contains multiple perfect consensus DNA-binding sequences for the Bacillus subtilis transition state regulator Hpr. The detailed structural elements revealed by the sequence analysis are presented and used to develop a new perspective on the molecular basis of enterotoxin production in this important food-poisoning bacterium.

Base Sequence↗

PubMed2004

Influence of subinhibitory concentrations of plant essential oils on the production of enterotoxins A and B and alpha-toxin by Staphylococcus aureus.

The data presented show the ability of subinhibitory concentrations of plant essential oils to influence the production of enterotoxins A and B and alpha-toxin by Staphylococcus aureus. Subinhibitory concentrations of the oils of bay, clove, cinnamon, nutmeg and thyme had no significant effect on the overall quantity of extracellular protein produced. Haemolysis due to alpha-toxin was significantly reduced after culture with all five plant essential oils. This reduction was greatest with the oils of bay, cinnamon and clove. These three oils also significantly decreased the production of enterotoxin A; the oils of clove and cinnamon also significantly decreased the production of enterotoxin B.

Enterotoxins↗

PubMed2004

Characterization of the Bacillus cereus Nhe enterotoxin.

The non-haemolytic enterotoxin (Nhe) is one of two three-component enterotoxins responsible for the diarrhoeal food-poisoning syndrome caused by Bacillus cereus. Nhe is composed of NheA, NheB and NheC. The three genes encoding the Nhe components constitute an operon, and the transcriptional start site is located 61 bp upstream of the nheA translational start site. The nhe genes were cloned separately, and expressed in either Bacillus subtilis or Escherichia coli. Separate expression showed that all three components were required for biological activity. In addition, NheA and NheB were purified from B. cereus culture supernatants. As NheC seems to be expressed in only small amounts by B. cereus, NheC was expressed and purified as a histidine-tagged fusion protein. The maximum cytotoxic activity was obtained when the molar ratio between NheA : NheB : His6-NheC was 10 : 10 : 1, and it was shown that NheB was the binding component of the enterotoxin complex.

Animals↗

PubMed2002

Structural basis for abrogated binding between staphylococcal enterotoxin A superantigen vaccine and MHC-IIalpha.

Staphylococcal enterotoxins (SEs) are superantigenic protein toxins responsible for a number of life-threatening diseases. The X-ray structure of a staphylococcal enterotoxin A (SEA) triple-mutant (L48R, D70R, and Y92A) vaccine reveals a cascade of structural rearrangements located in three loop regions essential for binding the alpha subunit of major histocompatibility complex class II (MHC-II) molecules. A comparison of hypothetical model complexes between SEA and the SEA triple mutant with MHC-II HLA-DR1 clearly shows disruption of key ionic and hydrophobic interactions necessary for forming the complex. Extensive dislocation of the disulfide loop in particular interferes with MHC-IIalpha binding. The triple-mutant structure provides new insights into the loss of superantigenicity and toxicity of an engineered superantigen and provides a basis for further design of enterotoxin vaccines.

Amino Acid Substitution↗

PubMed2000

Apoptosis observed in murine peritoneal macrophages treated with interferon gamma through staphylococcal enterotoxin-dependent cell-mediated cytotoxicity.

The concept of superantigens is well-known and widely accepted. In this brief communication, we analyze the behaviour of antigen-presenting cells after T-cell activation by staphylococcal enterotoxin B, a representative superantigen. We tried to activate murine T cells by inflammatory mouse peritoneal macrophage in the presence of staphylococcal enterotoxin B, but no T-cell activation was observed. We, therefore, analyzed surface-specific antigens of the macrophages. They expressed insufficient amounts of MHC class II, CD80 and CD86 molecules on the surface of the cells. On the contrary, increased amounts of MHC class II and CD86 molecules on the cell surfaces were observed after incubation with interferon gamma. Interferon gamma-primed macrophages were found to be competent to activate T cells in the presence of staphylococcal enterotoxin B. To our surprise, these macrophages underwent apoptosis in parallel with T-cell activation.

Animals↗

PubMed1997

Enterotoxin and cytotoxin production by Salmonella enteritidis strains isolated from gastroenteritis outbreaks.

Seventy-six Salmonella enteritidis, three Salmonella virchow and one Salmonella bradenrup strains were screened for enterotoxigenicity by using the Chinese hamster ovary (CHO), Y1 adrenal, Vero and HeLa cell tests. All the strains gave positive reactions for enterotoxin production, except one, and the relative sensitivity to the toxin exhibited by the different cell lines was evaluated. An enterotoxic activity has been identified in sonicated extracts of Salm. enteritidis. This enterotoxin was purified on Agarose A-5m (Bio-Rad) and Superose 12 HR 10/30 column. The enterotoxic activity was eluted from the Superose column in the first peak. Like Vibrio cholerae toxin CT and Escherichia coli enterotoxin LT, it was blocked by GM1 ganglioside, but at a higher concentration. In addition, a cytotoxic factor has been partially identified. The procedure for isolating the cytotoxin included ammonium sulphate precipitation, size-exclusion chromatography and anion exchange chromatography. This cytotoxin factor caused inhibition of protein synthesis in cultured cells, as determined by flow cytometry and [3H]-leucine incorporation. Flow cytometry analysis also showed an activation of CHO cells when exposed to this cytotoxic factor resulting in a state of active growth. Cytotoxic activity was not blocked by gangliosides.

Animals↗

PubMed2004

Identification and prevalence of an enterotoxin-related gene, se-int, in Staphylococcus intermedius isolates from dogs and pigeons.

AIMS: To determine the prevalence of enterotoxin-producing Staphylococcus intermedius in dogs and pigeons. METHODS AND RESULTS: A total of 106 S. intermedius isolates from 44 dogs and 62 pigeons were tested for the production of enterotoxins A, B, C and D by reverse passive latex agglutination (RPLA) and for sec-canine by PCR. Only one isolate from dog was positive for SEC and sec-canine. Screening of sec-canine-negative strains by nested PCR led to the identification of a novel enterotoxin-related gene, se-int. SE-int showed a significant homology (59-61% identity) with SEC and (56.6% identity) SEB. All 44 isolates from dogs and five isolates (8.1%) from pigeons were se-int positive. CONCLUSIONS: While S. intermedius was isolated more frequently from pigeons than from dogs, se-int was more prevalent among the S. intermedius isolates from dogs, compared with the pigeon isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: Further characterization of the se-int-positive S. intermedius strains should clarify their pathogenic potential including enterotoxigenicity and zoonotic transmissibility to human beings.

Amino Acid Sequence↗

PubMed2004

PCR detection of staphylococcal enterotoxin genes in Staphylococcus spp. strains isolated from meat and dairy products. Evidence for new variants of seG and seI in S. aureus AB-8802.

AIMS: Evaluation of the occurrence of most known staphylococcal enterotoxin (SE) genes, egc (enterotoxin gene cluster) and TSST1 (toxic shock syndrome toxin 1) gene in both coagulase-positive (CPS) and coagulase-negative (CNS) staphylococcal strains isolated from meat and dairy products. METHODS AND RESULTS: Specificity and reliability of the PCR detection methods used were ascertained by using nine reference strains of Staphylococcus (S. aureus) harbouring SE genes (seA to seE; seG, seH, seI, seM, seJ, seN and seO) and egc (containing the following sequence of genes: seO, seM, seI, phient1, phient2, seN and seG). Of 109 wild Staphylococcus spp. strains analysed, only 11 S. aureus strains were SE and/or TSST1 PCR-positive. The last 11 strains also appeared to harbour the egc. Restriction endonuclease analysis of part of the egc of both reference and wild strains showed that different variants of the egc exist. Moreover, nucleotide sequences of seG and seI indicate that the egc of the strain AB-8802 is characterized by the presence of variants of these enterotoxins (seGv and seIv). CONCLUSIONS: The occurrence of SE genes in CNS and other non-S. aureus species isolated from Napoli-type salami, raw water buffalo milk and natural whey cultures used for mozzarella cheese manufacturing is very rare. SIGNIFICANCE AND IMPACT OF THE STUDY: During this study it was shown that at least five different egc may exist in S. aureus. A thorough study of egc polymorphism should provide further insight into the phylogenetics of the egc.

Bacterial Toxins↗