The suitability of Tórtora's medium for the production of enterotoxin in Clostridium perfringens strains

Examination of 200 samples from soil and the same number of samples from healthy human feces yielded 49 (24.5%) and 105 (52.5%) strains of heat-resistant Clostridium perfringens respectively. Fourteen (7.0%) strains isolated from soil and 37 (18.5%) from feces synthesized enterotoxin, as demonstrated by Tórtora's method, at sufficient levels to permit its detection by mouse lethality, microslide double gel diffusion or counterimmunoelectrophoresis tests. By using the Duncan-Strong (DS) method, only four (2%) enterotoxigenic strains from soil and 14 (7.0%) from feces were obtained. The supernatant fluid from two enterotoxigenic-negative strains grown in DS medium gave a false-positive reaction when they were injected intravenously into mice. Tórtora's medium was preferable because a larger number of isolated strains produced spores and enterotoxin to permit their recognition as enterotoxigenic strains.     

Sporulation of Clostridium perfringens Type A in Vacuum-Sealed Meats

Type A strains of Clostridium perfringens inoculated into vacuum-sealed packages of hamburger, roast beef, ground beef plus cream sauce, and turkey roll produced high levels of spores.     

DETECTION of ENTEROTOXIGENIC CLOSTRIDIUM PERFRINGENS IN GROUND BEEF

Clostridium perfringens is widely distributed in foods. This experiment was performed to assess occurrence of C. perfringens cultures and toxigenic strains isolated from ground beef Samples (118) were collected from 20 locations in Northeast Kansas and the number of C. perfringens was enumerated in these samples by Fung's Double-tube method with tryptose sulfite cycloserine agar medium. Out of 118 samples, 54 (46%) were found positive for C. perfringens. Pure isolates of C. perfringens were further grown in cooked meat medium for 24 h at 42C then heat shocked at 75C for 20 min and inoculated into modified Duncan and Strong medium for production of C. perfringens enterotoxin. Presence of enterotoxin was tested by the reverse passive latex agglutination test (Oxoid), which can detect enterotoxin up to a minimum level of 2 ng/mL. the data indicate that 46% of the beef samples harbored C. perfringens , but only 32 (6%) of 525 isolates were found to produce enterotoxin. This study emphasized the importance of continued surveillance of C. perfringens in meats and meat products and assessment of the toxigenesis of isolates.     

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.     

Clostridium perfringens type A: certain characters of epidemiologic significance.

Survival of spores of Cl. perfringens type A was significantly greater than that of vegetative cells in acid pH (pH 1.2). Survival of spores in soil varied from strain to strain. Time required for 5 million spores to be reduced to 500 per gram of soil varied from 2 to 8 months with an average of 4.5+/-2.3 months. Quantitative and qualitative heat resistance studies revealed that a majority of the Indian and all the American strains tested were heat-sensitive. These characters of Cl. perfringens have an important bearing on the epidemiology of food poisoning due to this agent.     

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.     

Identification and Typing of Clostridia in Raw Milk in Egypt

S ummary : Three spp. of clostridia were isolated from samples of raw cow and buffalo milk obtained near Cairo. Of 150 isolates of anaerobes, 108 were Clostridium perfringens , 30 were Cl. butyricum , and 12 were Cl. sporogenes. The Cl. perfringens isolates comprised 100 nonhaemolytic and 8 pathogenic haemolytic strains. The latter strains typed by neutralization tests, were of type A. Fifteen of the nonhaemolytic strains were also of type A; of these, 6 strains produced heat resistant spores and 9 strains produced heat susceptible spores. Feeding mice with these 15 nonhaemolytic strains caused marked reduction in intestinal passage time.     

Prevalence and characterization of Clostridium perfringens from spices in Argentina

Spices can present high microbial counts and Clostridium perfringens, Bacillus cereus, Salmonella and Shigella, among others have been isolated from spices. C. perfringens is an important pathogen agent causing, among other diseases, enteritis in humans caused by C. perfringens enterotoxin (CPE) which causes human food poisoning and enterotoxemia in domestic animals. The aims of the present work were (i) to establish the hygienic sanitary quality of some spices in San Luis, Argentina; (ii) to determine the presence of C. perfringens in these spices by means of the most probable number (MPN) and count on plate methods; (iii) to characterize the enterotoxigenic strains of C. perfringens by PCR and immunological methods such as reverse passive latex agglutination (RPLA) and (iv) to type by PCR C. perfringens strains isolated. A total of 115 samples of spices, 67 of which were purchased in local retail stores and 48 domestically collected were analysed. Total aerobe counts on tryptone glucose yeast extract agar medium of the 115 samples were between <10 and 10(6) CFU/g. The colifecal counts using Mac Conkey broth of the 115 samples were <4-10(3)CFU/g, with 28 samples (24.34%) exceeding the limit established by the Spanish Alimentary Code (10 CFU/g) while 2 samples (1.73%) had a sulfite reducing anaerobe load above standard limits. A total of 14 C. perfringens strains (12.17%) were isolated and characterized from 115 samples by the standard biochemical tests. Four of which (28.60%) turned out to be enterotoxigenic by PCR and RPLA. In order to type C. perfringens strains based on their main toxins, the 14 strains were analysed by PCR. All strains belonged to type A. All RPLA positive strains were cpe(+) by PCR. The percentage of enterotoxigenic strains was more elevated that those reported in other studies for this type of sample. These results indicate that sanitary conditions in different production stages of species must be improved to reduce health hazards. The high percentage (24.34%) of samples with colifecal values above standard limits is an indication of deficient sanitary conditions. These results suggest the need to provide legislation on the sanitary and hygienic quality of spices in our country.     

Antisense-RNA-mediated decreased synthesis of small, acid-soluble spore proteins leads to decreased resistance of clostridium perfringens spores to moist heat and UV radiation

Previous work has suggested that a group of alpha/beta-type small, acid-soluble spore proteins (SASP) is involved in the resistance of Clostridium perfringens spores to moist heat. However, this suggestion is based on the analysis of C. perfringens spores lacking only one of the three genes encoding alpha/beta-type SASP in this organism. We have now used antisense RNA to decrease levels of alpha/beta-type SASP in C. perfringens spores by approximately 90%. These spores had significantly reduced resistance to both moist heat and UV radiation but not to dry heat. These results clearly demonstrate the important role of alpha/beta-type SASP in the resistance of C. perfringens spores.     

Nonradioactive colony hybridization assay for detection and enumeration of enterotoxigenic Clostridium perfringens in raw beef.

A DNA probe endolabeled with digoxigenin by PCR was developed to detect and enumerate enterotoxigenic Clostridium perfringens in raw beef. After 2 h of hybridization, membranes were developed by using an anti-digoxigenin-alkaline phosphatase conjugated antibody. The resulting chromogenic reaction allowed us to detect and enumerate < or = 10 CFU of C. perfringens per g.     

Heat resistance, spore germination, and enterotoxigenicity of Clostridium perfringens

Heat resistance at 95 C, heat activation at 75 C, and germination response were determined for spores of 10 serotype strains of Clostridium perfringens type A, including five heat-resistant and five heat-sensitive strains. The D95-values ranged from 17.6 to 63.0 and from 1.3 to 2.8 for the heat-resistant and the heat-sensitive strains, respectively. The heat-activation values, the ratios between the heated and unheated viable counts of spore suspensions, ranged from 0.0035 to 0.65 and from 6.5 to 60.0 for the heat-sensitive and the heat-resistant strains, respectively. Spores of these strains were divided into two distinct germination types on the basis of their germination response; spores of the heat-resistant strains germinated in KC1 medium after heat activation (K-type), and spores of the heat-sensitive strains germinated in a mixture of L-alanine, inosine, and CaCl2 in the presence of CO2 without heat activation (A-type). The strains were tested for enterotoxigenicity by a reversed passive latex-agglutination (RPLA) test. All the heat-resistant strains were RPLA-positive, whereas the heat-sensitive strains were all RPLA-negative. A total of 37 strains of the organism isolated from food-poisoning outbreaks were tested for spore germination and enterotoxin formation. All of the 20 heat-resistant strains showed K-type spore germination and, except for three strains, were RPLA-positive, whereas all of the 17 heat-sensitive strains showed A-type spore germination and, except for only one strain, were RPLA-negative.     

Detection of enterotoxigenic Clostridium perfringens in food and fecal samples with a duplex PCR and the slide latex agglutination test.

A duplex PCR procedure was evaluated for the detection of Clostridium perfringens in food and biological samples and for the identification of enterotoxigenic strains. This method uses two sets of primers which amplify in the same reaction two different DNA fragments simultaneously: the 283-bp C. perfringens phospholipase C gene fragment and the 426-bp enterotoxin gene fragment. Internal primers within the two primer sets confirmed the specificity of the method by DNA-DNA hybridization with the PCR products. No cross-reaction was observed with other Clostridium species or with other bacteria routinely found in food. The detection level was approximately 10(5) C. perfringens cells per g of stool or food sample. When overnight enrichment culture was used, 10 C. perfringens cells per g was detected in 57 artificially contaminated food samples. The duplex PCR is a rapid, sensitive, and reliable method for the detection and identification of enterotoxigenic C. perfringens strains in food samples. A slide latex agglutination test was also evaluated as a rapid, simple technique for the detection of C. perfringens enterotoxin in stool samples.     

The induction of trehalose and glycerol in Saccharomyces cerevisiae in response to various stresses

Trehalose and glycerol have been implicated as potential stress protectants that accumulate in yeasts during various stress conditions. We investigated the levels of glycerol and trehalose and the expression profiles of genes involved in their metabolism to determine their involvement in the response of Saccharomyces cerevisiae XQ1 to thermal, sorbitol and ethanol stresses. The results showed that the genes involved in the synthesis and degradation of trehalose and glycerol were stress induced, and that trehalose and glycerol were synthesized simultaneously during the initial stages (a sensitive response period) of diverse stress treatments. Trehalose accumulated markedly under heat treatment, but not under sorbitol or ethanol stress, whereas glycerol accumulated strikingly under sorbitol stress conditions. Interestingly, extracellular trehalose seemed to be involved in protecting cells from damage under unfavorable conditions. Moreover, our results suggest that the stress-activated futile ATP cycles of trehalose and glycerol turnover are of general importance during cellular stress adaptation.     

Factors contributing to heat resistance of Clostridium perfringens endospores

The endospores formed by strains of type A Clostridium perfringens that produce the C. perfringens enterotoxin (CPE) are known to be more resistant to heat and cold than strains that do not produce this toxin. The high heat resistance of these spores allows them to survive the cooking process, leading to a large number of food-poisoning cases each year. The relative importance of factors contributing to the establishment of heat resistance in this species is currently unknown. The present study examines the spores formed by both CPE(+) and CPE(-) strains for factors known to affect heat resistance in other species. We have found that the concentrations of DPA and metal ions, the size of the spore core, and the protoplast-to-sporoplast ratio are determining factors affecting heat resistance in these strains. While the overall thickness of the spore peptidoglycan was found to be consistent in all strains, the relative amounts of cortex and germ cell wall peptidoglycan also appear to play a role in the heat resistance of these strains.     

Evaluation of a Clostridium perfringens predictive model, developed under isothermal conditions in broth, to predict growth in ground beef during cooling

Proper temperature control is essential in minimizing Clostridium perfringens germination, growth, and toxin production. The U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) offers two options for the cooling of meat products: follow a standard time-temperature schedule or validate that alternative cooling regimens result in no more than a 1-log(10) CFU/g increase of C. perfringens and no growth of Clostridium botulinum. A mathematical model developed by Juneja et al. (Food Microbiol. 16:335-349, 1999) may be helpful in determining if the C. perfringens performance standard has been achieved, but this model has not been extensively validated. The objective of this study was to validate the Juneja 1999 model in ground beef under a variety of changing temperature and temperature abuse situations. The Juneja 1999 model consistently underpredicted growth of C. perfringens during exponential cooling of ground beef. The model also underpredicted growth of C. perfringens in ground beef cooled at two different rates. The results presented here show generally good agreement with published data on the growth of C. perfringens in similar products. The model error may be due to faster-than-expected exponential growth rates in ground beef during cooling or an error in the mathematical formulation of the model.     

反向间接胶乳凝集试验法检测粪便A型产气荚膜梭菌肠毒素的结果及评价

本文以１０种５２株供试菌分别与７个不同年龄组的健康人粪便混合,共配成３６４份模拟标本,采用反向间接胶乳凝集（ＲＰＬＡ）试验法与生物学试验法（小鼠致死试验、豚鼠皮肤血管透性因子试验,Ｖｅｒｏ细胞毒性试验）检测各标本中的Ａ型产气荚膜梭菌肠毒素（简称Ｃｐ－Ｅｎｔ）。除产气荚膜梭菌之外的其他菌种培养液２３８份标本（３４株）,ＲＰＬＡ与生物学试验结果完全一致,均为阴性。产气荚膜梭菌１２６份标本（１８株）中有７０份标本的ＲＰＬＡ同生物学诸法完全一致地检出了Ｃｐ－Ｅｎｔ．有１株７份标本（ＣｐＮＣＴＣ８７９７）的ＲＰＬＡ为阳性,而各生物学试验却均为阴性,该菌株经ＰＣＲ检查证明确为肠毒素原性产气荚膜梭菌,表明ＲＰＬＡ比生物学试验法更灵敏。有５株（ＣｐＮＣＴｃ８２３８,ＣｐＮＣＴＣ１０６１１,ＣｐＮＣＴＣ１０６１４,ＣｐＮＣＴＣ１０６１２,ＣｐＬ－５２）３５份标本ＲＰＬＡ与各生物学试验结果均为阴性,但经ＰＣＲ检吉证明该５株菌均为肠毒素原性产气荚膜梭菌,后经超声波破碎菌体提取物对其中部分菌株进行试验的结果仍然显示了ＲＰＬＡ与生物学法的一致性。有２株（ＣｐＮＣＴＣ８６８６,ＣｐＮＣＴＣ８４４９）１４份标本的所有结果均为阴性,ＰＣＲ     

Enterotoxin formation by Clostridium perfringens type A studied by the use of fluorescent antibody.

Fluorescein isothiocyanate-conjugated antibody to purified enterotoxin of Clostridium perfringens was used to study the intracellular formation of enterotoxin by this organism. Enterotoxin was detected at 4 h of growth at the end of the cell containing forespore. With the development of the spore, enterotoxin accumulation continued and involved the entire length of the cell until its lysis with the release of enterotoxin and mature spore. The spores did not contain demonstrable enterotoxin. Only a certain number of the sporulated cells of the enterotoxigenic strains studied produced this toxin. The amount of enterotoxin produced varied with sporulation percentage, and between strains and individual cells.     

Carbohydrate Metabolism During Ascospore Development in Yeast

Carbohydrate metabolism, under sporulation conditions, was compared in sporulating and non-sporulating diploids of Saccharomyces cerevisiae. Total carbohydrate was fractionated into trehalose, glycogen, mannan, and an alkali-insoluble fraction composed of glucan and insoluble glycogen. The behavior of three fractions was essentially the same in both sporulating and non-sporulating strains; trehalose, mannan, and the insoluble fraction were all synthesized to about the same extent regardless of a strain's ability to undergo meiosis or sporulation. In contrast, aspects of soluble glycogen metabolism depended on sporulation. Although glycogen synthesis took place in both sporulating and non-sporulating strains, only sporulating strains exhibited a period of glycogen degradation, which coincided with the final maturation of ascospores. We also determined the carbohydrate composition of spores isolated from mature asci. Spores contained all components present in vegetative cells, but in different proportions. In cells, the most abundant carbohydrate was mannan, followed by glycogen, then trehalose, and finally the alkali-insoluble fraction; in spores, trehalose was most abundant, followed by the alkali-insoluble fraction, glycogen, and mannan in that order.     

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.     

Effect of Lysozyme on Ionic Forms of Spores of Clostridium perfringens Type A

H spores of Clostridium perfringens type A (two strains) were more sensitive to germination by lysozyme than native spores. Resistance to lysozyme of H spores was restored by calcium loading.