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 plasmidborne 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.

Clostridium perfringens type B and D isolates produce epsilon-toxin, the third most potent clostridial toxin. The epsilon-toxin gene (etx) is plasmid borne in type D isolates, but etx genetics have been poorly studied in type B isolates. This study reports the first sequencing of any etx plasmid, i.e., pCP8533etx, from type B strain NCTC8533. This etx plasmid is 64.7 kb, carries tcp conjugative transfer genes, and encodes additional potential virulence factors including beta2-toxin, sortase, and collagen adhesin but not beta-toxin. Clostridium perfringens is an important pathogen of humans and livestock (17). Isolates of this gram-positive, anaerobic bacterium are classified into one of five types (A to E), based upon their production of four typing toxins (␣-, ␤-, ε-, or -toxin). By definition, type B and D isolates must produce ε-toxin, which is the third most potent of all clostridial toxins and thus listed as a U.S. Department of Agriculture/CDC overlap class B select toxin. In addition to ε-toxin, type D isolates always produce alpha-toxin, while type B isolates consistently express both ␤-toxin and alpha-toxin. Besides their typing toxins, some type B or D isolates produce additional lethal toxins that are not used in the typing classification scheme. For example, some type B isolates produce perfringolysin O and/or beta2-toxin, while some type D isolates express perfringolysin O, beta2-toxin, and/or C. perfringens enterotoxin (8,21,22).Beyond their biodefense significance, epsilon-toxin-producing C. perfringens type B and D isolates are also important natural pathogens of domestic animals (17). Type B isolates cause enterotoxemias in sheep, and possibly other livestock, particularly in the United Kingdom (25). Type B enterotoxemias initiate with proliferation of these bacteria in the gut. Toxins are then produced that initially act on the intestines but are later absorbed through the intestinal mucosa to act on internal organs. Similar enterotoxemias are caused by type D isolates in sheep and goats, where circulating toxins lead to elevated blood pressure, fluid accumulation in body cavities, and edema in brain, heart, lungs, liver, and kidney. Studies using a mouse intravenous injection model have strongly suggested that ε-toxin is a major contributor to the lethality associated with both type B and D enterotoxemias (8,21,22).Recently, there has been increased research focus on the role of plasmids in C. perfringens virulence. It is now established that the genes encoding the enterotoxin (cpe), beta2-toxin (cpb2), ε-toxin (etx), iota-toxin (iap/ibp), and ␤-toxin (cpb) can each be carried by plasmids (5,15,16,22). To date, only a few of those toxin plasmids have been studied in any detail; for example, among etx plasmids, only those in type D isolates have received even partial characterization (22). Southern blot analyses of pulsed-field gels showed that type D etx plasmids exhibit considerable heterogeneity, ranging in apparent size from ϳ50 kb to ϳ110 kb (22). Those Southern blot analyses also indicate...

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.

Clostridium perfringens type A, is both a ubiquitous environmental bacterium and a major cause of human gastrointestinal disease, which usually involves strains producing C. perfringens enterotoxin (CPE). The gene (cpe) encoding this toxin can be carried on the chromosome or a large plasmid. Interestingly, strains carrying cpe on the chromosome and strains carrying cpe on a plasmid often exhibit different biological characteristics, such as resistance properties against heat. In this study, we investigated the genetic properties of C. perfringens by PCR-surveying 21 housekeeping genes and genes on representative plasmids and then confirmed those results by Southern blot assay (SB) of five genes. Furthermore, sequencing analysis of eight housekeeping genes and multilocus sequence typing (MLST) analysis were also performed. Fifty-eight C. perfringens strains were examined, including isolates from: food poisoning cases, human gastrointestinal disease cases, foods in Japan or the USA, or feces of healthy humans. In the PCR survey, eight of eleven housekeeping genes amplified positive reactions in all strains tested. However, by PCR survey and SB assay, one representative virulence gene, pfoA, was not detected in any strains carrying cpe on the chromosome. Genes involved in conjugative transfer of the cpe plasmid were also absent from almost all chromosomal cpe strains. MLST showed that, regardless of their geographic origin, date of isolation, or isolation source, chromosomal cpe isolates, i) assemble into one definitive cluster ii) lack pfoA and iii) lack a plasmid related to the cpe plasmid. Similarly, independent of their origin, strains carrying a cpe plasmid also appear to be related, but are more variable than chromosomal cpe strains, possibly because of the instability of cpe-borne plasmid(s) and/or the conjugative transfer of cpe-plasmid(s) into unrelated C. perfringens strains.

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