Detection of Enterotoxin in Faeces and Anti-enterotoxin in Serum after Clostridium perfringens Food-poisoning

Two outbreaks of Clostridium perfringens food-poisoning involving the same person were investigated. In the first, typical symptoms with diarrhoea and abdominal pain were observed. In the second, there were no classical signs of food-poisoning; the victim felt some flatulence and the faeces had a pasty appearance and an unpleasant smell. Counterimmunoelectrophoresis and the reversed passive haemagglutination test were rapid and reliable assay methods for enterotoxin in faeces. In the first outbreak, 13–16 μg enterotoxin/g faeces were detected, and 3–4 μg/g in the second. The detection of enterotoxin in faeces indicates the potential use of enterotoxin tests on diarrhoeal samples for diagnosing C. perfringens food-poisoning. No enterotoxin was detected in serum during the acute stage of the illness, but the antibody titre showed a considerable rise in the first two months after the food-poisoning outbreak.     

Experimental diarrhea in cynomolgus monkeys by oral administration with Clostridium perfringens type A viable cells or enterotoxin.

Purified C. perfringens type A enterotoxin fed orally in an amount of 5 mg caused both vomiting and diarrhea in the monkey only when the gastric juice had been neutralized. Exposure of enterotoxin to pH 4.0 or below rapidly destroyed the activity. All three monkeys receiving sodium bicarbonate and 2.4 X 10(10) viable cells grown in DS medium developed diarrhea, and only one of them vomited once. The diarrhea lasted for 13, 18 and 19 hr. The symptoms were similar to those reported in human cases of C. perfringens food poisoning. These results have verified the general notion that C. perfringens food poisoning should be categorized as a true "intravital intoxication". The reversed passive hemagglutination test detected enterotoxin directly in most fecal samples. This method may be applicable for diagnosis of human cases of C. perfringens food poisoning. Neither enterotoxin nor anti-enterotoxin was detected in serum samples taken from any monkey up to 21 days after the challenge. We are tempted to conclude, therefore, that no significant amount of C. perfringens enterotoxin is absorbed from the intestine.     

Enterotoxin Production by Lecithinase-positive and Lecithinase-negative Clostridium perfringens Isolated from Food Poisoning Outbreaks and other sources

Strains of Clostridium perfringens from a variety of sources were examined for their ability to produce enterotoxin in vitro. Fifty-six of 65 (86%) strains isolated from separate outbreaks of food poisoning were found to be enterotoxigenic, only two of 174 strains from other sources produced enterotoxin. The ability to produce this toxin was not confined to particular serotypes: types frequently encountered as the cause of outbreaks were also isolated as enterotoxin-negative strains from faeces, minced beef and meat carcasses. Loss of toxigenicity was also observed in different serotypes. Five strains of lecithinase-negative Cl. perfringens produced high levels of enterotoxin. Four strains of Clostridium plagarum failed to produce enterotoxin although they were serologically typable with the Cl. perfringens antisera.     

Multiple typing techniques applied to a Clostridium perfringens food poisoning outbreak.

Twenty-one stool specimens obtained from persons implicated in two food poisoning outbreaks at the same institution in Smith Falls, Ontario, were examined for Clostridium perfringens. Ninety-two colonies of Cl. perfringens (3-5 per stool specimen) were typed with antisera, bacteriocins and by plasmid analysis. They were also tested for the in vitro production of bacteriocin and enterotoxin. Sixteen of the 21 stool specimens were tested directly for enterotoxin. This was detected in 13, five of which were from individuals listed as 'asymptomatic' food handlers. The predominant strain isolated from 15 of the 21 stool samples produced bacteriocin and enterotoxin in vitro, contained no plasmids, and was of a common bacteriocin type and serotype.     

Multiple typing techniques applied to a Clostridium perfringens food poisoning outbreak

D.E. MAHONY, R. AHMED AND S.G. JACKSON, 1992. Twenty-one stool specimens obtained from persons implicated in two food poisoning outbreaks at the same institution in Smith Falls, Ontario, were examined for Clostridium perfringens. Ninety-two colonies of Cl. perfringens (3–5 per stool specimen) were typed with antisera, bacteriocins and by plasmid analysis. They were also tested for the in vitro production of bacteriocin and enterotoxin. Sixteen of the 21 stool specimens were tested directly for enterotoxin. This was detected in 13, five of which were from individuals listed as 'asymptomatic' food handlers. The predominant strain isolated from 15 of the 21 stool samples produced bacteriocin and enterotoxin in vitro , contained no plasmids, and was of a common bacteriocin type and serotype.     

Direct detection of Clostridium perfringens enterotoxin in patients' stools during an outbreak of food poisoning

An outbreak of diarrhoea in a hotel affected 25 time keepers attending the 1997 Mediterranean Games. Epidemiological investigation implicated a 'pasta al ragù' consumed at the hotel's restaurant and Clostridium perfringens food poisoning was identified by direct detection of C. perfringens enterotoxin in patients' stools. This report confirms that a careful evaluation of epidemiological features, together with the availability of direct and rapid laboratory methods, may lead to a prompt identification of C. perfringens food poisoning.     

Ribotyping of Clostridium perfringens from industrially produced ground meat

AIMS: Clostridium (Cl.) perfringens is a common cause of food poisoning outbreaks. Ribosomal DNA analysis (ribotyping), a method which analyses restriction fragment length polymorphisms in the chromosomal genes that encode rRNA, has been shown to be useful for microbial species identification and subtyping. METHODS AND RESULTS: The current study has used ribotyping to examine 111 Cl. perfringens isolates from industrially produced ground meat in order to collect a basis for a contamination survey. Among the 111 isolates 107 distinctly different ribopatterns were detected. In only four cases two Cl. perfringens isolates showed an identical ribopattern. The isolates gave identical ribotype patterns in three different runs, carried out 3-4 months apart from each other. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The discriminatory index for EcoRI ribotyping of the Cl. perfringens isolates was 0 x 99. Results showed that ribotyping is suitable for subtyping Cl. perfringens isolates from raw meat. Ribotyping appeared to be a useful tool for profound epidemiologic studies of Cl. perfringens-contamination in food production and processing.     

Molecular methods for the analysis of Clostridium perfringens relevant to food hygiene

Clostridium perfringens continues to be a common cause of food-borne disease. It produces an enterotoxin (CPE) which is released upon lysis of the vegetative cell during sporulation in the intestinal tract. Catering premises with insufficient cooling and reheating devices often seem to be the cause of outbreaks of C. perfringens food poisoning. Typing of C. perfringens is of great importance for investigating sources of food poisoning cases and for studying the epidemiology of this microorganism. This report describes the examination of 155 C. perfringens isolates by molecular methods. Isolates were taken from 10 food poisoning outbreaks and cases (n = 34, food and fecal isolates) and from meat and fish pastes (n = 121). Isolates were characterized by plasmid profiling, ribotyping, and/or macrorestriction analysis by pulsed-field gel electrophoresis (PFGE). Results show that all three methods are suitable for classifying C. perfringens isolates below the species level. Ribopatterns and PFGE patterns can be interpreted more easily than plasmid profiling results and can be recommended for contamination studies and epidemiologic investigation of food poisonings associated with C. perfringens.     

Comparative Studies of Bacillus Cereus Strains Isolated From Various Foods and Food Poisoning Outbreaks

Certain properties of 22 Bacillus cereus strains isolated from different foods and food poisoning episodes were investigated in order to evaluate possible différences between strains isolated from diarrhoeal and vomiting type food poisoning outbreaks. None of the strains isolated from vomiting type episodes produced acid from salicin and mannose, whereas 80 and 40 % of the strains from diarrhoeal type outbreaks were positive, respectively. No association between the antibiotic sensitivity pattern or the fatty acid composition and the source of a strain could be found, although some strains differed from the general pattern of B. cereus in some instances. No significant differences in the production of the skin factor between strains isolated from the two types of outbreaks were found either. The findings of this study support the observation that the food environment itself essentially affects the enterotoxin formation of B. cereus.     

A medium for the isolation, enumeration and rapid presumptive identification of injured Clostridium perfringens and Bacillus cereus

A blood-free egg yolk medium (BCP) containing pyruvate, inositol, mannitol and a bromocresol purple indicator in a nutrient agar base has been developed to initiate the growth of Clostridium perfringens . It is comparable to blood agar for the growth of normal, chilled stored vegetative cells and heat-injured spores of Cl. perfringens and Bacillus cereus . It has the advantage over blood agar in exhibiting presumptive evidence of Cl. perfringens (production of lecithinase and inositol fermentation) after an overnight incubation at 43°C-45°C. Pyruvate, catalase and other hydrogen peroxide degraders were found to remove toxins rapidly formed in media exposed to air and light. Free radical scavengers of superoxide, hydroxyl ions and singlet oxygen were ineffective. Without scavengers the formation of 10–20 μg/ml hydrogen peroxide in the exposed medium was indicated and found lethal to injured Cl. perfringens .
The BCP medium has been used successfully for the rapid identification and enumeration of Cl. perfringens in foods and faeces from food poisoning outbreaks and cases of suspected infectious diarrhoea. Greater recovery of severely injured vegetative Cl. perfringens could be obtained by pre-incubation at 37°C of inoculated media for 2–4 h followed by overnight incubation at 43°C-45°C. Tryptose-sulphite-cyclo-serine and Shahidi-Ferguson-perfringens agar base were found to inhibit the growth of several strains of injured vegetative Cl. perfringens . This was not completely overcome by the addition of pyruvate. The inclusion of mannitol also allows the medium to be used for the presumptive identification of B. cereus . Growth and lecithinase activity are profuse on BCP. Heat-injured spores are recovered equally well on BCP and blood agar. A scheme for the identification of some other clos-tridia on BCP is presented.     

Detection of enterotoxigenic Clostridium perfringens type A isolates in American retail foods

Currently there is only limited understanding of the reservoirs for Clostridium perfringens type A food poisoning. A recent survey (Y.-T. Lin and R. Labbe, Appl. Environ. Microbiol. 69:1642-1646, 2003) of non-outbreak American retail foods did not identify the presence of a single C. perfringens isolate carrying the enterotoxin gene (cpe) necessary for causing food poisoning. The present study revisited this issue, using revised methodology and food sampling strategies. In our survey, cpe-positive C. perfringens isolates were detected in approximately 1.4% of approximately 900 surveyed non-outbreak American retail foods. Interestingly, those enterotoxigenic isolates in non-outbreak foods appear indistinguishable from C. perfringens isolates known to cause food poisoning outbreaks: i.e., the enterotoxigenic retail food isolates all carry a chromosomal cpe gene, are classified as type A, and exhibit exceptional heat resistance. Collectively, these findings indicate that some American foods are contaminated, at the time of retail purchase, with C. perfringens isolates having full potential to cause food poisoning. Furthermore, demonstrating that type A isolates carrying a chromosomal cpe gene are the enterotoxigenic isolates most commonly present in foods helps to explain why these isolates (rather than type A isolates carrying a plasmid cpe gene or cpe-positive type C or D isolates) are strongly associated with food poisoning outbreaks. Finally, since type A chromosomal cpe isolates present in the surveyed raw foods exhibited strong heat resistance, it appears that exceptional heat resistance is not a survivor trait selected for by cooking but is instead an intrinsic trait possessed by many type A chromosomal cpe isolates.     

Comparison of latex agglutination and ELIS A for the detection of Clostridium perfringens type A enterotoxin in faeces

One hundred and thirty one faecal specimens from cases of suspected Clostridium perfringens food poisoning were examined by both a reverse passive latex agglutination test and a standard ELISA test for the presence of Cl. perfringens enterotoxin. The latex agglutination test proved as sensitive and specific as the ELISA, and required less time at the bench without the need for specialized equipment.     

Fluid accumulation in mouse ligated intestine inoculated with Clostridium perfringens enterotoxin.

Clostridium perfringens enterotoxin, when inoculated into the ligated intestinal loop of mice, caused marked distension due to fluid accumulation. The increase in weight of the intestinal loop was proportional to the log dose of enterotoxin within a range from 1 to 16 micrograms. The fluid accumulation was arrested by washing the loop with saline or by injection of the specific anti-enterotoxin serum into the loop 5 or even 30 min after inoculation of the enterotoxin. A significant increase in weight of the loop was found as early as 10 min after inoculation of the toxin. These results may suggest that entergotoxin is neither bound firmly to the mucosal membrane nor permeates into the cells of the intestinal wall. The mouse intestinal loop test is economical, simple to perform, and applicable for quantitative determination of the enteropathogenic activity of C. perfringens enterotoxin.     

Fluid accumulation in mouse ligated intestine inoculated with Clostridium perfringens enterotoxin.

Clostridium perfringens enterotoxin, when inoculated into the ligated intestinal loop of mice, caused marked distension due to fluid accumulation. The increase in weight of the intestinal loop was proportional to the log dose of enterotoxin within a range from 1 to 16 micrograms. The fluid accumulation was arrested by washing the loop with saline or by injection of the specific anti-enterotoxin serum into the loop 5 or even 30 min after inoculation of the enterotoxin. A significant increase in weight of the loop was found as early as 10 min after inoculation of the toxin. These results may suggest that entergotoxin is neither bound firmly to the mucosal membrane nor permeates into the cells of the intestinal wall. The mouse intestinal loop test is economical, simple to perform, and applicable for quantitative determination of the enteropathogenic activity of C. perfringens enterotoxin.     

Incidence of Enterotoxigenic Clostridium perfringens in Healthy Humans in relation to the Enhancement of Enterotoxin Production by Heat Treatment

Enterotoxin production was greatly enhanced in two of five food poisoning strains of Clostridium perfringens subjected to heat treatment prior to incubation in Duncan and Strong sporulation medium. Heating was carried out on three successive cultures of each strain, the optimum temperature for treatment being 85 °C for one strain and 95 °C for another: on each occasion cultures were heated for 20 min. The triple heat treatment procedure was used in testing strains of Cl. perfringens isolated from faeces of healthy human subjects for production of enterotoxin. Eleven of 35 (31%) individuals were found to be carriers of enterotoxigenic strains, the isolates producing more than 0·1 μ/ml of enterotoxin. Six of the 11 enterotoxigenic strains were killed by heating at 95 °C but one isolate produced more enterotoxin following treatment at this temperature than after heating at 75 °C.     

Ribotyping for strain characterization of Clostridium perfringens isolates from food poisoning cases and outbreaks.

Ribotyping was used to characterize 34 Clostridium perfringens strains isolated from 10 food poisoning cases and outbreaks over a 7-year period. Twelve different ribopatterns were generated by EcoRI digestion. In eight food poisoning cases and outbreaks, all of the ribotypes of each food and stool isolate were found to be identical. Two C. perfringens isolates showed unique patterns. Ribotyping was found to be a useful tool for determining the genetic relationship of C. perfringens isolates in the context of foodborne poisoning cases.     

Improved Reversed Passive Hemagglutination for Simple and Rapid Detection of Staphylococcal Enterotoxins A~E in Food

Detection and identification of staphylococcal enterotoxins in food or culture filtrates were performed using the reversed passive hemagglutination (RPHA) technique, with formalized sheep red blood cells (FSRBC) sensitized with immunoglobulins of anti-A, B, C, D, and E rabbit hyperimmune sera fractionated by affinity chromatography. The FSRBC sensitized with anti-A~E immunoglobulins showed a high level of reactivity and specificity in RPHA, against homologous types of purified enterotoxins and culture filtrates of toxin-producing strains. No non-specific reactions with various ingredients in foods nor cross-reactions among enterotoxin types were observed. The minimum amount of enterotoxins in foods detected by RPHA was calculated to be 0.01 μg/g without concentration, and the recovery rate of experimentally added toxins was calculated to be about 80%. Under routine laboratory practice, detection and identification of enterotoxins from incriminated foods of five food poisoning outbreaks were performed by RPHA within 3 hr after reception of the specimens. Among them, three were determined to be enterotoxin A food poisoning, one to be toxin C and the rest to be intoxication of A and D. The concentration of the toxins was between 0.014 and 3.65 μg per gram of food.     

A medium for the isolation, enumeration and rapid presumptive identification of injured Clostridium perfringens and Bacillus cereus

A blood-free egg yolk medium (BCP) containing pyruvate, inositol, mannitol and a bromocresol purple indicator in a nutrient agar base has been developed to initiate the growth of Clostridium perfringens. It is comparable to blood agar for the growth of normal, chilled stored vegetative cells and heat-injured spores of Cl. perfringens and Bacillus cereus. It has the advantage over blood agar in exhibiting presumptive evidence of Cl. perfringens (production of lecithinase and inositol fermentation) after an overnight incubation at 43 degrees - 45 degrees C. Pyruvate, catalase and other hydrogen peroxide degraders were found to remove toxins rapidly formed in media exposed to air and light. Free radical scavengers of superoxide, hydroxyl ions and singlet oxygen were ineffective. Without scavengers the formation of 10-20 micrograms/ml hydrogen peroxide in the exposed medium was indicated and found lethal to injured Cl. perfringens. The BCP medium has been used successfully for the rapid identification and enumeration of Cl. perfringens in foods and faeces from food poisoning outbreaks and cases of suspected infectious diarrhoea. Greater recovery of severely injured vegetative Cl. perfrigens could be obtained by pre-incubation at 37 degrees C of inoculated media for 2-4 h followed by overnight incubation at 43 degrees - 45 degrees C. Tryptose-sulphite-cycloserine and Shahidi-Ferguson-perfringens agar base were found to inhibit the growth of several strains of injured vegetative Cl. perfringens. This was not completely overcome by the addition of pyruvate. The inclusion of mannitol also allows the medium to be used for the presumptive identification of B. cereus. Growth and lecithinase activity are profuse on BCP. Heat-injured spores are recovered equally well on BCP and blood agar. A scheme for the identification of some other clostridia on BCP is presented.     

Association of beta2 toxin production with Clostridium perfringens type A human gastrointestinal disease isolates carrying a plasmid enterotoxin gene

Clostridium perfringens type A isolates carrying an enterotoxin (cpe) gene are an important cause of human gastrointestinal diseases, including food poisoning, antibiotic-associated diarrhoea (AAD) and sporadic diarrhoea (SD). Using polymerase chain reaction (PCR), the current study determined that the cpb2 gene encoding the recently discovered beta2 toxin is present in <15% of food poisoning isolates, which typically carry a chromosomal cpe gene. However, >75% of AAD/SD isolates, which usually carry a plasmid cpe gene, tested cpb2(+) by PCR. Western blot analysis demonstrated that >97% of those cpb2(+)/cpe(+) AAD/SD isolates can produce CPB2. Additional PCR analyses, sequencing studies and pulsed field gel electrophoresis experiments determined that AAD/SD isolates carry cpb2 and cpe on the same plasmid when IS1151 sequences are present downstream of cpe, but cpb2 and cpe are located on different plasmids in AAD/SD isolates where IS1470-like sequences are present downstream of cpe. Those analyses also demonstrated that two different CPB2 variants (named CPB2h1 or CPB2h2) can be produced by AAD/SD isolates, dependent on whether IS1470-like or IS1151 sequences are present downstream of their cpe gene. CPB2h1 is approximately 10-fold more cytotoxic for CaCo-2 cells than is CPB2h2. Collectively, these results suggest that CPB2 could be an accessory toxin in C. perfringens enterotoxin (CPE)-associated AAD/SD.     

Scaled-up production and purification of Clostridium perfringens type A enterotoxin

Methods for small-scale production of Clostridium perfringens type A enterotoxin were unsuitable for large-scale culture of this organism. Rapid, efficient harvesting of 40 1 batch culture of Cl. perfringens was achieved by tangential flow micro-filtration with the Millipore Pellicon cassette system. Enterotoxin-containing extracts were prepared by passing concentrated suspensions of the harvested cells through a French pressure cell. The overall yield of purified enterotoxin was 38·8%. The toxin gave a single band on native polyacrylamide gels but formed high molecular weight aggregates in the presence of sodium dodecyl sulphate. These aggregates frequently occurred during storage of non-sterile enterotoxin preparations but could be separated from the monomer toxin by gel filtration on Sephadex G-100. Purified monomer enterotoxin had biological activities of 119·3 μ g/kg mouse lethal dose when injected intraperitoneally and 3333 capillary permeability increasing units/mg protein in guinea pig skin. Thirty μg of the enterotoxin caused fluid accumulation in ligated rabbit ileal loops. Aggregated enterotoxin had no demonstrable biological or immunological activity.