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.     

Inactivation of the gene (cpe ) encoding Clostridium perfringens enterotoxin eliminates the ability of two cpe-positive C. perfringens type A human gastrointestinal disease isolates to affect rabbit ileal loops

Previous epidemiological studies have implicated Clostridium perfringens enterotoxin (CPE) as a virulence factor in the pathogenesis of several gastrointestinal (GI) illnesses caused by C. perfringens type A isolates, including C. perfringens type A food poisoning and non-food-borne GI illnesses, such as antibiotic-associated diarrhoea and sporadic diarrhoea. To further evaluate the importance of CPE in the pathogenesis of these GI diseases, allelic exchange was used to construct cpe knock-out mutants in both SM101 (a derivative of a C. perfringens type A food poisoning isolate carrying a chromosomal cpe gene) and F4969 (a C. perfringens type A non-food-borne GI disease isolate carrying a plasmid-borne cpe gene). Western blot analyses confirmed that neither cpe knock-out mutant could express CPE during either sporulation or vegetative growth, and that this lack of CPE expression could be complemented by transforming these mutants with a recombinant plasmid carrying the wild-type cpe gene. When the virulence of the wild-type, mutant and complementing strains were compared in a rabbit ileal loop model, sporulating (but not vegetative) culture lysates of the wild-type isolates induced significant ileal loop fluid accumulation and intestinal histopathological damage, but neither sporulating nor vegetative culture lysates of the cpe knock-out mutants induced these intestinal effects. However, full sporulation-associated virulence could be restored by complementing these cpe knock-out mutants with a recombinant plasmid carrying the wild-type cpe gene, which confirms that the observed loss of virulence for the cpe knock-out mutants results from the specific inactivation of the cpe gene and the resultant loss of CPE expression. Therefore, in vivo analysis of our isogenic cpe mutants indicates that CPE expression is necessary for these two cpe-positive C. perfringens type A human disease isolates to cause GI effects in the culture lysate:ileal loop model system, a finding that supports CPE as an important virulence factor in GI diseases involving cpe-positive C. perfringens type A isolates.     

Identification of novel Clostridium perfringens type E strains that carry an iota toxin plasmid with a functional enterotoxin gene

Clostridium perfringens enterotoxin (CPE) is a major virulence factor for human gastrointestinal diseases, such as food poisoning and antibiotic associated diarrhea. The CPE-encoding gene (cpe) can be chromosomal or plasmid-borne. Recent development of conventional PCR cpe-genotyping assays makes it possible to identify cpe location (chromosomal or plasmid) in type A isolates. Initial studies for developing cpe genotyping assays indicated that all cpe-positive strains isolated from sickened patients were typable by cpe-genotypes, but surveys of C. perfringens environmental strains or strains from feces of healthy people suggested that this assay might not be useful for some cpe-carrying type A isolates. In the current study, a pulsed-field gel electrophoresis Southern blot assay showed that four cpe-genotype untypable isolates carried their cpe gene on a plasmid of ～65 kb. Complete sequence analysis of the ～65 kb variant cpe-carrying plasmid revealed no intact IS elements and a disrupted cytosine methyltransferase (dcm) gene. More importantly, this plasmid contains a conjugative transfer region, a variant cpe gene and variant iota toxin genes. The toxin genes encoded by this plasmid are expressed based upon the results of RT-PCR assays. The ～65 kb plasmid is closely related to the pCPF4969 cpe plasmid of type A isolates. MLST analyses indicated these isolates belong to a unique cluster of C. perfringens. Overall, these isolates carrying a variant functional cpe gene and iota toxin genes represent unique type E strains.     

Further comparison of temperature effects on growth and survival of Clostridium perfringens type A isolates carrying a chromosomal or plasmid-borne enterotoxin gene

Clostridium perfringens type A isolates can carry the enterotoxin gene (cpe) on either their chromosome or a plasmid, but food poisoning isolates usually have a chromosomal cpe gene. This linkage between chromosomal cpe isolates and food poisoning has previously been attributed, at least in part, to better high-temperature survival of chromosomal cpe isolates than of plasmid cpe isolates. In the current study we assessed whether vegetative cells and spores of chromosomal cpe isolates also survive better than vegetative cells and spores of plasmid cpe isolates survive when the vegetative cells and spores are subjected to low temperatures. Vegetative cells of chromosomal cpe isolates exhibited about eightfold-higher decimal reduction values (D values) at 4 degrees C and threefold-higher D values at -20 degrees C than vegetative cells of plasmid cpe isolates exhibited. After 6 months of incubation at 4 degrees C and -20 degrees C, the average log reductions in viability for spores of plasmid cpe isolates were about fourfold and about threefold greater, respectively, than the average log reductions in viability for spores from chromosomal cpe isolates. C. perfringens type A isolates carrying a chromosomal cpe gene also grew significantly faster than plasmid cpe isolates grew at 25 degrees C, 37 degrees C, or 43 degrees C. In addition, chromosomal cpe isolates grew at higher maximum and lower minimum temperatures than plasmid cpe isolates grew. Collectively, these results suggest that chromosomal cpe isolates are commonly involved in food poisoning because of their greater resistance to low (as well as high) temperatures for both survival and growth. They also indicate the importance of proper low-temperature storage conditions, as well as heating, for prevention of C. perfringens type A food poisoning.     

Phenotypic characterization of enterotoxigenic Clostridium perfringens isolates from non-foodborne human gastrointestinal diseases

Clostridium perfringens enterotoxin (CPE) has been implicated as an important virulence factor inC. perfringens type A food poisoning and several non-foodborne human gastrointestinal (GI) illnesses, including antibiotic-associated diarrhea (AAD) and sporadic diarrhea (SPOR). Recent studies have revealed genotypic differences between cpe-positive isolates originating from different disease sources, with most, or all, food poisoning isolates carrying a chomosomal cpe and most, or all, non-foodborne human GI disease isolates carrying an episomal cpe. To evaluate whether these genotypic differences cause phenotypic effects that could influence the pathogenesis of CPE-associated non-foodborne human GI illnesses, a collection of SPOR and AAD isolates has been phenotypically characterized in the current study. All cpe-positive non-foodborne disease isolates examined were found to express CPE in a sporulation-associated manner. The CPE made by these AAD and SPOR isolates was shown to have the same deduced amino acid sequence and toxicity as the classical CPE made by food poisoning isolates. All of the surveyed non-foodborne human GI disease isolates were found to classify as type AC. perfringens, since they produce alpha toxin, but not beta, iota, or epsilon toxins. Finally, no consistent clonal relationships were detected between the surveyed non-foodborne human GI disease isolates. Since, by the criteria examined, all non-foodborne human GI disease isolates examined in this study appear to be phenotypically similar to food poisoning isolates, the current results confirm that the examined AAD and SPOR isolates have enteropathogenic potential. However, given the phenotypic similarities between food poisoning, AAD, and SPOR isolates that have been demonstrated in this study, it remains unclear why the symptomology of non-foodborne human GI diseases is typically more severe and longer-lasting than that of C. perfringens type A food poisoning.     

Comparative experiments to examine the effects of heating on vegetative cells and spores of Clostridium perfringens isolates carrying plasmid genes versus chromosomal enterotoxin genes

Clostridium perfringens enterotoxin (CPE) is an important virulence factor for both C. perfringens type A food poisoning and several non-food-borne human gastrointestinal diseases. Recent studies have indicated that C. perfringens isolates associated with food poisoning carry a chromosomal cpe gene, while non-food-borne human gastrointestinal disease isolates carry a plasmid cpe gene. However, no explanation has been provided for the strong associations between certain cpe genotypes and particular CPE-associated diseases. Since C. perfringens food poisoning usually involves cooked meat products, we hypothesized that chromosomal cpe isolates are so strongly associated with food poisoning because (i) they are more heat resistant than plasmid cpe isolates, (ii) heating induces loss of the cpe plasmid, or (iii) heating induces migration of the plasmid cpe gene to the chromosome. When we tested these hypotheses, vegetative cells of chromosomal cpe isolates were found to exhibit, on average approximately twofold-higher decimal reduction values (D values) at 55 degrees C than vegetative cells of plasmid cpe isolates exhibited. Furthermore, the spores of chromosomal cpe isolates had, on average, approximately 60-fold-higher D values at 100 degrees C than the spores of plasmid cpe isolates had. Southern hybridization and CPE Western blot analyses demonstrated that all survivors of heating retained their cpe gene in its original plasmid or chromosomal location and could still express CPE. These results suggest that chromosomal cpe isolates are strongly associated with food poisoning, at least in part, because their cells and spores possess a high degree of heat resistance, which should enhance their survival in incompletely cooked or inadequately warmed foods.     

A Wide Variety of Clostridium perfringens Type A Food-Borne Isolates That Carry a Chromosomal cpe Gene Belong to One Multilocus Sequence Typing Cluster

Of 98 suspected food-borne Clostridium perfringens isolates obtained from a nationwide survey by the Food and Consumer Product Safety Authority in The Netherlands, 59 strains were identified as C. perfringens type A. Using PCR-based techniques, the cpe gene encoding enterotoxin was detected in eight isolates, showing a chromosomal location for seven isolates and a plasmid location for one isolate. Further characterization of these strains by using (GTG)(5) fingerprint repetitive sequence-based PCR analysis distinguished C. perfringens from other sulfite-reducing clostridia but did not allow for differentiation between various types of C. perfringens strains. To characterize the C. perfringens strains further, multilocus sequence typing (MLST) analysis was performed on eight housekeeping genes of both enterotoxic and non-cpe isolates, and the data were combined with a previous global survey covering strains associated with food poisoning, gas gangrene, and isolates from food or healthy individuals. This revealed that the chromosomal cpe strains (food strains and isolates from food poisoning cases) belong to a distinct cluster that is significantly distant from all the other cpe plasmid-carrying and cpe-negative strains. These results suggest that different groups of C. perfringens have undergone niche specialization and that a distinct group of food isolates has specific core genome sequences. Such findings have epidemiological and evolutionary significance. Better understanding of the origin and reservoir of enterotoxic C. perfringens may allow for improved control of this organism in foods.     

Complete sequencing and diversity analysis of the enterotoxin-encoding plasmids in Clostridium perfringens type A non-food-borne human gastrointestinal disease isolates

Enterotoxin-producing Clostridium perfringens type A isolates are an important cause of food poisoning and non-food-borne human gastrointestinal diseases, e.g., sporadic diarrhea (SPOR) and antibiotic-associated diarrhea (AAD). The enterotoxin gene (cpe) is usually chromosomal in food poisoning isolates but plasmid-borne in AAD/SPOR isolates. Previous studies determined that type A SPOR isolate F5603 has a plasmid (pCPF5603) carrying cpe, IS1151, and the beta2 toxin gene (cpb2), while type A SPOR isolate F4969 has a plasmid (pCPF4969) lacking cpb2 and IS1151 but carrying cpe and IS1470-like sequences. By completely sequencing these two cpe plasmids, the current study identified pCPF5603 as a 75.3-kb plasmid carrying 73 open reading frames (ORFs) and pCPF4969 as a 70.5-kb plasmid carrying 62 ORFs. These plasmids share an approximately 35-kb conserved region that potentially encodes virulence factors and carries ORFs found on the conjugative transposon Tn916. The 34.5-kb pCPF4969 variable region contains ORFs that putatively encode two bacteriocins and a two-component regulator similar to VirR/VirS, while the approximately 43.6-kb pCPF5603 variable region contains a functional cpb2 gene and several metabolic genes. Diversity studies indicated that other type A plasmid cpe+/IS1151 SPOR/AAD isolates carry a pCPF5603-like plasmid, while other type A plasmid cpe+/IS1470-like SPOR/AAD isolates carry a pCPF4969-like plasmid. Tn916-related ORFs similar to those in pCPF4969 (known to transfer conjugatively) were detected in the cpe plasmids of other type A SPOR/AAD isolates, as well as in representative C. perfringens type B to D isolates carrying other virulence plasmids, possibly suggesting that most or all C. perfringens virulence plasmids transfer conjugatively.     

Clostridium perfringens toxin genotypes in the feces of healthy North Americans

We investigated the frequency of Clostridium perfringens in the normal fecal flora of healthy North Americans. About half of 43 subjects were colonized with C. perfringens at levels of approximately 10(6)cfu/g feces. Only type A strains were recovered. Spores sometimes outnumbered vegetative cells. Several genotypes were found. Some donors carried two genotypes, some only one. We found no alpha, beta2 or enterotoxin in the stools of any donors. Though some isolates carried toxin genes (e.g. cpe and cpb2) on plasmids, we saw no indication that healthy humans are the reservoir for the chromosomally-borne cpe recovered from cases of C. perfringens food poisoning.     

Molecular characterization of Clostridium perfringens isolates from humans with sporadic diarrhea: evidence for transcriptional regulation of the beta2-toxin-encoding gene

Clostridium perfringens type A food poisoning is caused by C. perfringens isolates carrying a chromosomal enterotoxin gene (cpe), while non-food-borne gastrointestinal (GI) diseases, such as antibiotic-associated diarrhea (AAD) and sporadic diarrhea (SD), are caused by C. perfringens plasmid cpe isolates. A recent study reported the association of beta2 toxin (CPB2) with human GI diseases, and particularly AAD/SD, by demonstrating that a large percentage of AAD/SD isolates, in contrast to a small percentage of food poisoning isolates, carry the beta2-toxin gene (cpb2). This putative relationship was further tested in the current study by characterizing 14 cpe+ C. perfringens fecal isolates associated with recent cases of human SD in England (referred to hereafter as SD isolates). These SD isolates were all classified as cpe+ type A, and 12 of the 14 cpe+ isolates carry their cpe gene on the plasmid and 2 carry it on the chromosome. Interestingly, cpb2 is present in only 12 plasmid cpe isolates; 11 isolates carry cpe and cpb2 on different plasmids, but cpe and cpb2 are located on the same plasmid in one isolate. C. perfringens enterotoxin is produced by all 14 cpe+ SD isolates. However, only 10 of the 12 cpe+/cpb2+ SD isolates produced CPB2, with significant variation in amounts. The levels of cpb2 mRNA in low- to high-CPB2-producing SD isolates differed to such an extent (30-fold) that this difference could be considered a major cause of the differential level of CPB2 production in vitro by SD isolates. Furthermore, no silent or atypical cpb2 was found in a CPB2 Western blot-negative isolate, 5422/94, suggesting that the lack of CPB2 production in 5422/94 was due to low expression of cpb2 mRNA. This received support from our observation that the recombinant plasmid carrying 5422/94 cpb2, which overexpressed cpb2 mRNA, restored CPB2 production in F4969 (a cpb2-negative isolate). Collectively, our present results suggest that CPB2 merits further study as an accessory toxin in C. perfringens-associated SD.     

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.     

Detection of enterotoxigenic Clostridium perfringens in meat samples by using molecular methods

To prevent food-borne bacterial diseases and to trace bacterial contamination events to foods, microbial source tracking (MST) methods provide important epidemiological information. To apply molecular methods to MST, it is necessary not only to amplify bacterial cells to detection limit levels but also to prepare DNA with reduced inhibitory compounds and contamination. Isolates carrying the Clostridium perfringens enterotoxin gene (cpe) on the chromosome or a plasmid rank among the most important food-borne pathogens. Previous surveys indicated that cpe-positive C. perfringens isolates are present in only ～5% of nonoutbreak food samples and then only at low numbers, usually less than 3 cells/g. In this study, four molecular assays for the detection of cpe-positive C. perfringens isolates, i.e., ordinary PCR, nested PCR, real-time PCR, and loop-mediated isothermal amplification (LAMP), were developed and evaluated for their reliability using purified DNA. For use in the artificial contamination of meat samples, DNA templates were prepared by three different commercial DNA preparation kits. The four molecular assays always detected cpe when >103 cells/g of cpe-positive C. perfringens were present, using any kit. Of three tested commercial DNA preparation kits, the InstaGene matrix kit appeared to be most suitable for the testing of a large number of samples. By using the InstaGene matrix kit, the four molecular assays efficiently detected cpe using DNA prepared from enrichment culture specimens of meat samples contaminated with low numbers of cpe-positive C. perfringens vegetative cells or spores. Overall, the current study developed molecular assay protocols for MST to detect the contamination of foods with low numbers of cells, and at a low frequency, of cpe-positive C. perfringens isolates.     

Clostridium perfringens spore germination: characterization of germinants and their receptors

Clostridium perfringens food poisoning is caused by type A isolates carrying a chromosomal enterotoxin (cpe) gene (C-cpe), while C. perfringens-associated non-food-borne gastrointestinal (GI) diseases are caused by isolates carrying a plasmid-borne cpe gene (P-cpe). C. perfringens spores are thought to be the important infectious cell morphotype, and after inoculation into a suitable host, these spores must germinate and return to active growth to cause GI disease. We have found differences in the germination of spores of C-cpe and P-cpe isolates in that (i) while a mixture of L-asparagine and KCl was a good germinant for spores of C-cpe and P-cpe isolates, KCl and, to a lesser extent, L-asparagine triggered spore germination in C-cpe isolates only; and (ii) L-alanine or L-valine induced significant germination of spores of P-cpe but not C-cpe isolates. Spores of a gerK mutant of a C-cpe isolate in which two of the proteins of a spore nutrient germinant receptor were absent germinated slower than wild-type spores with KCl, did not germinate with L-asparagine, and germinated poorly compared to wild-type spores with the nonnutrient germinants dodecylamine and a 1:1 chelate of Ca2+ and dipicolinic acid. In contrast, spores of a gerAA mutant of a C-cpe isolate that lacked another component of a nutrient germinant receptor germinated at the same rate as that of wild-type spores with high concentrations of KCl, although they germinated slightly slower with a lower KCl concentration, suggesting an auxiliary role for GerAA in C. perfringens spore germination. In sum, this study identified nutrient germinants for spores of both C-cpe and P-cpe isolates of C. perfringens and provided evidence that proteins encoded by the gerK operon are required for both nutrient-induced and non-nutrient-induced spore germination.     

Molecular epidemiology of Clostridium perfringens related to food-borne outbreaks of disease in Finland from 1984 to 1999

From 1975 to 1999, Clostridium perfringens caused 238 food-borne disease outbreaks in Finland, which is 20% of all such reported outbreaks during these years. The fact that C. perfringens is commonly found in human and animal stools and that it is also widespread in the environment is a disadvantage when one is searching for the specific cause of a food-borne infection by traditional methods. In order to strengthen the evidence-based diagnostics of food poisonings suspected to be caused by C. perfringens, we retrospectively investigated 47 C. perfringens isolates by PCR for the cpe gene, which encodes enterotoxin; by reversed passive latex agglutination to detect the enterotoxin production; and by pulsed-field gel electrophoresis (PFGE) to compare their genotypes after restriction of DNA by the enzymes SmaI and ApaI. The strains were isolated during 1984 to 1999 from nine food-borne outbreaks of disease originally reported as having been caused by C. perfringens. In seven of the nine outbreaks our results supported the fact that the cause was C. perfringens. Our findings emphasize the importance of a more detailed characterization of C. perfringens isolates than mere identification to the species level in order to verify the cause of an outbreak. Also, to increase the probability of finding the significant cpe-positive C. perfringens strains, it is very important to isolate and investigate more than one colony from the fecal culture of a patient and screen all these isolates for the presence of the cpe gene before further laboratory work is done.     

Enterotoxic strains of Clostridium perfringens and sporulation

Clostridium perfringens strains of A type capable of enterotoxin (CPE) synthesis may be a potential source of food-poisoning. Suitability of methods for CPE detection on the protein level is limited by difficulties in inducing sporulation in vitro. A number of unknown facts concerning coregulation the sporulation processes and CPE synthesis are recognised. The goal of the work was to determine the level of correlation between CPE synthesis and spores formation. Enterotoxin and cpe gen were detected by RPLA after sporulation induction test and by methods based on amplification on the DNA and mRNA levels. Sixty-four C. perfringens strains of A type isolated from patients with food poisoning symptoms and from food samples were analysed. Collection of isolates was differentiated as not enterotoxic, enterotoxic, and potentially enterotoxic strains based on appropriate strain profile: plc(+), cpe(-), CPE(-); plc(+), cpe(+), CPE(+); and plc(+), cpe(-), CPE(-), respectively. No significant difference between expression of cpe mRNA in vegetative and sporulation phase was found. The obtained results indicate that sporulation is not an essential factor for cpe gene expression.     

A novel small acid soluble protein variant is important for spore resistance of most Clostridium perfringens food poisoning isolates

Clostridium perfringens is a major cause of food poisoning (FP) in developed countries. C. perfringens isolates usually induce the gastrointestinal symptoms of this FP by producing an enterotoxin that is encoded by a chromosomal (cpe) gene. Those typical FP strains also produce spores that are extremely resistant to food preservation approaches such as heating and chemical preservatives. This resistance favors their survival and subsequent germination in improperly cooked, prepared, or stored foods. The current study identified a novel alpha/beta-type small acid soluble protein, now named Ssp4, and showed that sporulating cultures of FP isolates producing resistant spores consistently express a variant Ssp4 with an Asp substitution at residue 36. In contrast, Gly was detected at Ssp4 residue 36 in C. perfringens strains producing sensitive spores. Studies with isogenic mutants and complementing strains demonstrated the importance of the Asp 36 Ssp4 variant for the exceptional heat and sodium nitrite resistance of spores made by most FP strains carrying a chromosomal cpe gene. Electrophoretic mobility shift assays and DNA binding studies showed that Ssp4 variants with an Asp at residue 36 bind more efficiently and tightly to DNA than do Ssp4 variants with Gly at residue 36. Besides suggesting one possible mechanistic explanation for the highly resistant spore phenotype of most FP strains carrying a chromosomal cpe gene, these findings may facilitate eventual development of targeted strategies to increase killing of the resistant spores in foods. They also provide the first indication that SASP variants can be important contributors to intra-species (and perhaps inter-species) variations in bacterial spore resistance phenotypes. Finally, Ssp4 may contribute to spore resistance properties throughout the genus Clostridium since ssp4 genes also exist in the genomes of other clostridial species.     

Multiplex PCR assay for toxinotyping Clostridium perfringens isolates obtained from Finnish broiler chickens

AIMS: The aim of the study was to determine the presence of genes coding for alpha (cpa), beta (cpb), epsilon (etx), iota (iA) and enterotoxin (cpe) from Clostridium perfringens broiler chicken isolates, using multiplex PCR assay established in the study. METHODS AND RESULTS: The multiplex PCR assay was shown to be specific when tested with 10 C. perfringens strains representing different toxin types, and 15 strains of other bacterial species. All 118 broiler chicken C. perfringens isolates were shown to carry the cpa gene but not cpb, etx, iap or cpe genes, signifying that all isolates represented type A and were cpe-negative. CONCLUSIONS: The assay established in the study enables the simultaneous detection of the major toxin genes and the cpe gene from C. perfringens isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study offers a new primer pair for detecting cpa, combined with a multiplex PCR assay. In addition, the study provides data of the presence of different toxin genes in C. perfringens isolates obtained from broiler chickens.     

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.     

Comparison of the levels of heat resistance of wild-type, cpe knockout, and cpe plasmid-cured Clostridium perfringens type A strains

An enterotoxin (cpe) plasmid was cured from a Clostridium perfringens non-food-borne gastrointestinal disease (NFBGID) isolate, and the heat resistance levels of wild-type, cpe knockout, and cpe plasmid-cured strains were compared. Our results demonstrated that (i) wild-type cpe has no influence in mediating high-level heat resistance in C. perfringens and (ii) the cpe plasmid does not confer heat sensitivity on NFBGID isolates.     

Molecular typing of Clostridium perfringens isolated from turkey meat by multiplex PCR

Aims:  To determine the presence of toxin genes in 22 Clostridium perfringens isolated from turkey meat samples by molecular typing.
Methods and Results:  For this purpose, alpha ( cpa ), beta ( cpb ), beta 2 ( cpb2 ), epsilon ( etx ), iota ( iA ) and enterotoxin ( cpe ) toxin genes were analysed by multiplex PCR. All 22 turkey meat Cl. perfringens isolates were found to carry the cpa , gene but in none of the isolates cpb , etx, iap or cpe genes were detected. Results showed that all isolates represented type A and were cpe negative.
Conclusions:  Our results indicate that Cl. perfringens type A is the most common type in turkey meat. Also multiplex PCR is effective and rapid method for typing of Cl. perfringens .
Significance and Impact of the Study:  It is the first study about molecular typing of Cl. perfringens using multiplex PCR in turkey meat samples in Turkey.