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PubMed1985

Purification and properties of an enterotoxin from a coatless spore mutant of Clostridium perfringens type A.

A method is described for isolating an enterotoxin from a coatless spore mutant (8-6) of Clostridium perfringens type A. The characteristics of this enterotoxin only slightly resembled those of previously isolated enterotoxins of C. perfringens. The type A (8-6) enterotoxin was found to be composed of two subunits of Mr 18 000 with isoelectric points of 3.8 and 4.3. The LD50 for mice was 39 micrograms/kg with 0.10 micrograms corresponding to one erythemal unit. The type A (8-6) enterotoxin was inactivated by heating for 10 min at 60 degrees C. The amino acid composition data of type A (8-6) and delta toxins was similar, but type A (8-6) and type A enterotoxins showed less similarity. This lack of similarity between type A and type A (8-6) enterotoxins was confirmed by the failure of anti-sera to type A enterotoxin to neutralize the type A (8-6) enterotoxin, in both the mouse and erythemal tests.

Amino Acids↗

PubMed1993

[Identification of enterotoxin-producing Clostridium perfringens by the polymerase chain reaction].

Polymerase chain reaction was applied to identify enterotoxin-producing Clostridium perfringens by amplifying a segment of the C. perfringens enterotoxin gene. All of the four enterotoxin-positive reference strains tested were PCR positive while an enterotoxin-negative strain was PCR positive. All 17 clostridial strains (16 species) other than C. perfringens were PCR negative. With clinical strains isolated from various clinical specimens in Japan, Korea, and Thailand, all three enterotoxin-positive isolates were PCR positive and all 82 enterotoxin-negative isolates were PCR negative. PCR results for amplifying a region containing the initiation codon of the C. perfringens enterotoxin gene also demonstrated complete agreement with enterotoxin producibility. These results suggested that the PCR assay is a rapid and simple test for identifying the enterotoxin-producing C. perfringens without using any cultures and spore treatments.

Base Sequence↗

PubMed1969

Effect of meat and isolated meat proteins on the thermal inactivation of staphylococcal enterotoxin B.

The thermal inactivation of staphylococcal enterotoxin B was studied in a phosphate-saline buffer, in the presence of two meat proteins, myosin and metmyoglobin (MetMb), and in a ground-beef slurry. When enterotoxin B was incubated at temperatures from 60 to 110 C, it was shown that the initial thermal inactivation at 80 C was faster than at 100 or 110C. The heating of enterotoxin B at 60, 80, and 100 C in the presence of either myosin or MetMb resulted in a rapid loss of the enterotoxin. Thermal loss of the enterotoxin B molecule in the presence of meat proteins was more pronounced at 80 C than at either 60 or 100 C. Thermal loss of enterotoxin B molecule in the presence of meat proteins was more pronounced at 80 C than at either 60 or 100 C. Thermal loss of enterotoxin B in a ground round slurry was rapid when compared to inactivation in a phosphate-saline buffer. The rapid loss of enterotoxin B in the slurry may be due to a combination of thermal inactivation and the binding of enterotoxin molecules to meat proteins.

Buffers↗

PubMed1969

Immunofluorescent detection of enterotoxin B in food and a culture medium.

Both Staphylococcus aureus strains 243 and S-6 cells producing enterotoxin B and free enterotoxin in food and culture medium were rapidly demonstrated by using the fluorescent-antibody technique. Comparison of cell fluorescence and enterotoxin B production determined by double gel diffusion showed that an estimation of enterotoxin production could be made by observing the degree of cell fluorescence. The fluorescent-antibody technique was used to determine whether cells were producing enterotoxin under varying nutritional and environmental conditions: NaCl concentration, culture aeration, and time and temperature of incubation in Brain Heart Infusion broth and shrimp slurries. At the various NaCl concentrations, the fluorescence of cells was found positively associated with enterotoxin B production only during the first 12 hr of growth. As the NaCl concentration was increased from 0 to 10%, the fluorescence of cells and toxin production decreased. Maximum for cell fluorescence and enterotoxin production was observed at 37 C. Little or no difference in cell fluorescence and enterotoxin production with both strains was found between Brain Heart Infusion broth and shrimp slurry cultures. All results obtained with the fluorescent-antibody technique were verified with double gel diffusion for enterotoxin detection and quantitation.

Culture Media↗

PubMed1974

Comparison of the action of Escherichia coli enterotoxin on the thymocyte adenylate cyclase-cyclic adenosine monophosphate system to that of cholera toxin and prostaglandin E1.

Mouse thymocytes were used to compare mechanisms by which Vibrio cholerae and heat-labile Escherichia coli enterotoxins activate the adenylate cyclase-cyclic adenosine monophosphate (AMP) system. Both enterotoxins had their time-delayed increase in cyclic AMP neutralized by antisera to V. cholerae or E. coli enterotoxin, blocked by low concentrations of ganglioside G(M1), and destroyed by prior heating. Enterotoxin activation of adenylate cyclase was similarly affected. By contrast, prostaglandin E(1)-mediated increases in cyclic AMP were not affected by specific antitoxins or gangliosides. Combination of maximal stimulatory doses of both enterotoxins did not produce additive increases in cyclic AMP. Wash experiments suggested that both enterotoxins bind rapidly and tightly to thymocytes at 37 C. However, lowering the incubation temperature to 8 C reduced the affinity of E. coli enterotoxin but not cholera toxin for thymocytes. Results suggest that heat-labile E. coli enterotoxin and cholera enterotoxin may activate the same adenylate cyclase enzyme by similar mechanisms.

Adenosine Triphosphate↗

PubMed1971

Biological characteristics of Clostridium perfringens type A enterotoxin.

An enterotoxin with the ability to induce fluid accumulation in rabbit ileal loops, erythema in the skin of guinea pigs, and lethality in mice appears in cell extracts (CE) and culture filtrates (CF) of sporulating cells of some Clostridium perfringens type A strains. All activities in CE and CF were eluted simultaneously from a Sephadex G-200 column. Different elution patterns were obtained for these activities present in CE and CF. Rabbit immune serum against CF and the active CE fractions eliminated the three biological activities in CE and CF. These activities present in CF and CE were not eliminated by any of the known antitoxins present in diagnostic serum against C. perfringens types A, B, C, D, and E. Immunodiffusion studies with immune serum against active CE fractions and CF indicated a precipitin line of identity between CF and CE of NCTC 8798 and other enterotoxin-positive strains but not enterotoxin-negative strains. Disc electrophoresis of active G-200 fractions on 7.0% polyacrylamide gels revealed a single area containing erythemal activity and mouse lethality. Immunodiffusion with acrylamide gels, containing crude fractionated enterotoxin, and immune serum against partially purified enterotoxin revealed a single precipitin band in the same area as the biological activities. Immunoelectrophoresis of CE of enterotoxin-positive and enterotoxin-negative strains also showed one precipitin band which occurred only with enterotoxin-positive strains. These findings suggest that one component is responsible for the biological activities attributed to the enterotoxin.

Animals↗

PubMed1988

Cholera enterotoxin-induced mucus secretion and increase in the mucus blanket of the rabbit ileum in vivo.

The in vivo rabbit ileum was used to study the relationship of cholera enterotoxin-induced water and electrolyte secretion and mucus secretion and to determine whether the enterotoxin influenced the intestinal mucus blanket. In experiments in which luminal fluid viscosity was used to assess mucus secretion, it was found that while cholera enterotoxin induced a sustained secretion of water and electrolytes, the toxin-induced mucus hypersecretion was short lived (3 to 5 h) and subsequent exposure of the mucosa to cholera enterotoxin or prostaglandin E1 did not stimulate mucus secretion further. Dibutyryl cyclic AMP and theophylline caused a modest mucus secretion in ileal loops which differed from that of cholera enterotoxin in both magnitude and in the fact that the mucus secretion occurred 2 to 3 h after the onset of water and electrolyte secretion. An oral replacement solution was used in the ileum to reduce the enterotoxin-induced loss of water and electrolytes into the lumen. While such a solution slowed the appearance of acidic glycoprotein in the intestinal lumen, it did not change the amount of glycoprotein secreted over a 7-h period, suggesting that toxin-induced mucus secretion was not simply due to a flushing action of the experimentally caused diarrhea. To assess mucus blanket thickness, neutral glycoprotein was recovered from the blanket of rabbit ileal loops 7 h after exposure to cholera enterotoxin and the thickness of the mucus blanket was measured directly 4 and 18 h after toxin exposure. Both methods indicated that even though cholera enterotoxin-induced mucus hypersecretion had subsided and there was histological evidence of goblet cell mucin depletion, there was a sustained increase in mucus blanket thickness that was detectable for at least 18 h after mucosal enterotoxin exposure.

Animals↗

PubMed1993

Cloning, expression, and sequence analysis of a cytolytic enterotoxin gene from Aeromonas hydrophila.

The structural gene and regulatory element for a cytolytic enterotoxin of a diarrheal isolate, SSU, of Aeromonas hydrophila was cloned and its DNA sequence was determined. A complementary, mixed synthetic oligonucleotide based on the first 10 NH2-terminal amino acid residues of the Aeromonas cytolytic enterotoxin was used as a probe to screen a genomic library constructed in bacteriophage EMBL3. Cell lysates of Escherichia coli (lambda CH4), containing the cytolytic enterotoxin gene, lysed rabbit red blood cells and destroyed Chinese hamster ovary cells, caused fluid secretion in rat ileal loops, and were lethal to mice when injected intravenously. All biological activities associated with the cytolytic enterotoxin were neutralized by rabbit homologous polyclonal antibodies. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent Western blot analysis of the cell lysate of E. coli (lambda CH4) revealed a protein band of approximately 52 kDa, using antisera to the cytolytic enterotoxin or antibodies generated against a synthetic peptide to the toxin. DNA sequence analysis of a 2.8-kb SalI-BamHI fragment revealed the presence of one large open reading frame (1479 bp) that would encode a protein of 54.5 kDa, a precursor form of the cytolytic enterotoxin, with a 23 amino acid leader peptide. Despite a significant amount of homology at the DNA and amino acid levels between our cytolytic enterotoxin and two aerolysins of Aeromonas species, variation in the restriction maps of these three toxin genes was prominent. Likewise, considerable divergence in DNA sequence was observed upstream of the structural genes for the reported aerolysins and our cytolytic enterotoxin, suggesting that these structurally similar toxin molecules may be regulated differently. Finally, our data showed that the cytolytic enterotoxin from a diarrheal isolate, SSU, of A. hydrophila exhibited characteristics that were unique compared with those of the reported aerolysins.

Aeromonas hydrophila↗

PubMed2002

[Occurrence and characteristics of staphylococcal enterotoxin antibodies in gynecologic patients].

OBJECTIVE: To control the occurrence of the antibodies against Staphylococcal type A and type B enterotoxin in gynaecological patients and in selected patients to determine the thermodynamic parameters of antibodies against Staphylococcal enterotoxins in their blood samples. DESIGN: Retrospective clinical study. SETTING: 2nd Department of Obstetrics and Gynaecology, P. J. Safárik University Kosice, Slovak Republic; Research Institute of Veterinary Medicine, Kosice, Slovak Republic; Department of Microbiology and Immunology, University of Veterinary Medicine, Kosice, Slovak Republic; Department of Food Microbiology and Toxicology, Food Research Institute, University of Wisconsin, Madison, USA. METHODS: The occurrence of antibodies against Staphylococcal type A and type B enterotoxin was determined in 68 patients hospitalized in Department of Obstetrics and Gynecology in Kosice. RESULTS: The occurrence of antibodies against Staphylococcal enterotoxins was determined by radioimmunoassay (RIA) in blood samples of 45 (66%) patients. The antibodies against Staphylococcal type A enterotoxin were determined in 36 (53%) patients and the antibodies against Staphylococcal type B enterotoxin were determined in 9 (13%) patients. The antibodies against both type A and type B enterotoxins were determined simultaneously in blood samples of 10% of all patients. The thermodynamic parameters of the antibodies were determined in 5 patients with positive serum findings. CONCLUSION: With regard to the existence of heterogeneous clinical findings in large amount of patients with antibodies against Staphylococcal enterotoxins, the next study of Staphylococcal enterotoxins role in pathogenesis of wide spectrum of diseases is necessary.

Adult↗

PubMed1978

The effect of cholera toxin and heat labile and heat stable Escherichia coli enterotoxin on cyclic AMP concentrations in small intestinal mucosa of pig and rabbit.

The effect of cholera toxin, heat labile and heat stable Escherichia coli enterotoxin on mucosal cyclic AMP concentrations was determined on the proximal jejunum of weanling pigs and young rabbits. Ligated loops were injected with solutions containing no enterotoxin for control and either cholera toxin, heat labile or heat stable E. coli enterotoxin. The loops were drained after either two, four or six hours incubation at which time accumulated fluid was recorded and mucosal samples removed for determination of cyclic AMP concentration. In the rabbit, cholera toxin and heat labile, but not heat stable E. coli enterotoxin stimulated intestinal secretion while in the pig all three enterotoxins induced net fluid accumulation. Cholera toxin and heat labile, but not heat stable E. coli enterotoxin elevated rabbit mucosal cyclic AMP concentrations. In the pig these enterotoxins had no significant effect on mucosal cyclic AMP concentrations. The results are inconsistent with the hypothesis that the adenyl cyclase system is an essential step for enterotoxin induced intestinal secretion. The activation of intestinal adenyl cyclase by bacterial enterotoxins may only be an associated and not a necessary event for the stimulation of intestinal secretion.

Animals↗

PubMed1970

Synthesis of Enterotoxin by L-Forms of Staphylococcus aureus.

After prolonged treatment with methicillin and penicillin, L-forms were prepared from 12 of 22 strains of Staphylococcus aureus. These L-forms were obtained from five of nine strains which produced enterotoxin A, from three of six strains that produced enterotoxin B, and from three of six strains that produced enterotoxin C. An L-form was also prepared from S. aureus strain AH, which produces both enterotoxins A and C. The synthesis of enterotoxin A by parent strains was least affected by the conditions required for L-form growth. Detectable amounts of enterotoxin A were produced by all of the revertants tested and by five of the six L-forms which were derived from type A-producing cells. Detectable amounts of enterotoxin B were produced by four of the five revertants but by none of the L-forms derived from type B-producing cells. Revertants and L-forms from cells which produced enterotoxin C did not produce enough enterotoxin C to be detectable on microslides.

Journal Article↗

PubMed1995

Frequency of toxic shock syndrome toxin- and enterotoxin-producing clinical isolates of Staphylococcus aureus.

In 183 clinical isolates of Staphylococcus aureus, toxic shock syndrome toxin was detected in 13.7%, enterotoxin A in 20.2%, enterotoxin B in 7.7%, enterotoxin C in 5.5% and enterotoxin D in 3.3%. Seventy-three (39.9%) of the strains were found to produce one or more toxins. Multiple secretion of toxins was rare (< 1% of all strains) except for the combinations enterotoxin A with toxic shock syndrome toxin and enterotoxin A with enterotoxin D. These combinations were observed at significantly higher frequencies (4.9% and 2.2%, respectively) than would have been expected from values calculated from respective individual frequencies (0.88% and 0.06%) (p < 0.0001). In comparison with Staphylococcus aureus strains isolated from miscellaneous material, Staphylococcus aureus blood culture isolates produced enterotoxin D in significantly larger amounts and at higher frequencies (p = 0.01 in both cases). These toxins might play an important pathogenic role in Staphylococcus aureus infections.

Bacterial Toxins↗

PubMed1992

5-HT receptor antagonists and heat-stable Escherichia coli enterotoxin-induced effects in the rat.

The effect of heat-stable E. coli enterotoxin on intestinal fluid secretion is commonly considered to be mediated by stimulation of mucosal cyclic guanosine monophosphate (cGMP). It was demonstrated recently that 5-hydroxytryptamine (5-HT) acts as an important mediator in cholera toxin-induced fluid secretion. To elucidate the possible involvement of 5-HT in the secretory response to heat-stable E. coli enterotoxin, in vivo experiments were performed in the rat jejunum. The inhibitory effects of the 5-HT2 receptor antagonist ketanserin, the 5-HT3 receptor antagonist tropisetron and indomethacin were studied in heat-stable E. coli enterotoxin-induced fluid secretion. Tropisetron and ketanserin (100 micrograms/kg each) alone only partially reduced the secretory effect of the toxin. However, in combination, the two blockers (100 plus 100 micrograms/kg) significantly reduced and at 200 plus 200 micrograms/kg totally abolished heat-stable E. coli enterotoxin-induced secretion without influencing the enterotoxin-induced increase in cGMP. Pretreatment with indomethacin (10 mg/kg) reduced the secretory response to the enterotoxin by about 50%. These results support the concept that 5-HT is an important mediator in intestinal fluid secretion induced by heat-stable E. coli enterotoxin. The enterotoxin may use 5-HT to stimulate prostaglandin formation via 5-HT2 receptors and to activate neuronal structures via 5-HT3 receptors.

Animals↗

PubMed1982

Cytotonic enterotoxin from Aeromonas hydrophila.

Aeromonas hydrophila produces two hemolysins and an enterotoxin during growth. Enterotoxin, separated from the hemolysins, gave positive reactions in the rabbit intestinal loop test, the rabbit skin test and the adrenal Y1 cell test. Neutralization experiments in the rabbit loop, rabbit skin and Y1 cell tests failed to demonstrate any immunological relationship between Aeromonas enterotoxin and cholera toxin or Escherichia coli heat-labile enterotoxin. Prior incubation of Aeromonas enterotoxin with gangliosides did not inhibit the positive test results in these systems. A co-agglutination test with antiserum to purified cholera toxin was negative for Aeromonas enterotoxin, which therefore seems to be immunologically distinct from cholera toxin. The Aeromonas enterotoxin induced steroid secretion in adrenal Y1 cells and increased the intracellular cyclic adenosine monophosphate (cAMP) content of Y1 cells as well as of rabbit intestinal epithelial cells. It thus seems to act via the adenylate cyclase-cAMP pathway and should be classified as a cytotonic enterotoxin according to the classification of Keusch and Donta (1975).

Adenylyl Cyclases↗

PubMed1988

Effect of environmental conditions during soya-bean fermentation on the growth of Staphylococcus aureus and production and thermal stability of enterotoxins A and B.

During tempe manufacture, Staphylococcus aureus was able to survive in newly started soya-bean soaks (final pH 4.7), whereas it died during soaks with accelerated souring (final pH 4.0). No S. aureus were detected after subsequent heat treatment of soaked beans. S. aureus strain S6 could produce enterotoxins A and B if inoculated on cooked soya-beans. Its growth and enterotoxin production were not influenced by inoculation rate nor by the presence of actively growing Rhizopus oligosporus, the fungus responsible for tempe formation. Enterotoxin levels were highest after 48 h fermentation. Presence of lactobacilli (L. brevis or L. plantarum) during the tempe fermentation did not influence staphylococcal growth, but enterotoxin levels were reduced. Reduced oxygen pressure did not affect staphylococcal growth, but resulted in lower enterotoxin levels after 48 h incubation. The thermal stability of enterotoxin A in tempe was significantly lower than in Brain Heart Infusion broth of comparable pH. Heating of enterotoxin-containing tempe mash reduced enterotoxin A by 99.7% as measured with ELISA and animal feeding methods.

Animals↗

PubMed1991

Growth, sporulation and enterotoxin production by Clostridium perfringens type A in the presence of human bile salts.

The effect of human bile juice and bile salts (sodium cholate, sodium taurocholate, sodium glycochenodeoxycholate and sodium chenodeoxycholate) on growth, sporulation and enterotoxin production by enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens was determined. Each bile salt inhibited growth to a different degree. A mixture of bile salts completely inhibited the growth of enterotoxin-positive strains of this organism. Human bile juice completely inhibited the growth of all the strains at a dilution of 1:320. A distinct stimulatory effect of the bile salts on sporulation was observed in the case of C. perfringens strains NCTC 8239 and NCTC 8679. The salts also increased enterotoxin concentrations in the cell extracts of the enterotoxin-positive strains tested. No effect on enterotoxin production was detected when an enterotoxin-negative strain was examined.

Bile Acids and Salts↗

PubMed2004

Comparison between ultrafiltration and trichloroacetic acid precipitation method for concentration of Staphylococcus aureus enterotoxin in dairy samples.

Staphylococcus aureus enterotoxin that may be contained at low concentrations in milk and dairy products can cause food poisoning. To detect this enterotoxin at low concentrations, samples should be concentrated. We evaluated the performance of centrifugal ultrafiltration method (UF) in comparison with trichloroacetic acid precipitation method (TCA) for the concentration of S. aureus enterotoxin in milk and dairy products. S. aureus enterotoxin A (SEA) were added at various concentrations to ultra high-temperature heating process (UHT) milk, UHT concentrated skim milk, UHT skim milk powder, low heat-treated (LH) skim milk powder, and raw milk. SEA was concentrated by TCA and UF once a day on a total of 3 days by different researchers to prepare test solutions. The fluorescence value (TV) of test solutions was determined using an immunofluorescence autoanalyzer (miniVIDAS), and the linearity and slope of the regression line, relative standard deviation (RSD(RW)) at each added concentration, detection limit (DL), quantification limit (QL), and the recovery rate by each concentration method were obtained according to the guidelines of the International Conference on Harmonization (ICH). The slope of the regression line obtained by UF was steeper than that by TCA for all dairy samples excluding LH (74 degrees C, 20 s) skim milk powder. RSDRW, DL, and QL obtained by UF were comparable to or more excellent than those obtained by TCA. The procedure of UF was simpler than that of TCA. The recovery rate and rapidity were similar between the two methods. The DL and QL of enterotoxins other than SEA in dairy products by UF or TCA were estimated based on the DL and QL of SEA. In this estimation, consideration was given to reactions between each enterotoxin and its antibody, and also to the immunoactivity maintenance rate of each enterotoxin after addition of trichloroacetic acid in TCA. The estimated values were similar to those obtained by experiments using enterotoxin C1 (SEC1). UF using a centrifugal ultrafiltration membrane can be more readily performed and similar to or more reliable than TCA. UF combined with a miniVIDAS can be used for quantitative routine analysis.

Animals↗

PubMed1995

Importance of staphylococci that produce nanogram quantities of enterotoxin.

Many staphylococcal strains produce enterotoxin, the toxin that is the cause of staphylococcal food poisoning. If a strain is enterotoxigenic it is possible for it to be involved in food poisoning. The gel diffusion methods were the first methods developed for detection of the enterotoxins and were thought adequate to detect their production. However, they were not adequate to detect enterotoxin in foods involved in food poisoning. When researchers began using the sensitive methods, such as enzyme-linked immunosorbent assay (ELISA) and reversed passive latex agglutination (RPLA), to check strains for enterotoxin production, some strains produced nanogram quantities of enterotoxin. When it was reported that several coagulase-negative species produced less than 10 ng/ml of enterotoxin, it was imperative to determine whether these strains produced enough enterotoxin in foods to cause food poisoning. At the present time research is under way to determine whether these strains produce enough enterotoxin in foods to cause food poisoning.

Animals↗