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Abstract
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.
Highlights
Clostridium perfringens, an anaerobic Gram-positive bacterium, is ubiquitous in the intestinal flora of human and animals, and is commonly isolated from environmental materials such as soil and water [1,2]
To assess genetic diversity amongst enterotoxigenic C. perfringens isolates, a PCR survey was first performed to evaluate the carriage of selected genes including chromosomal toxin genes, several chromosomal housekeeping genes, plasmid maintenance genes, and genes related to plasmid transfer (Table 1)
For eight representative housekeeping genes, PCR amplified a product of the expected size from all investigated C. perfringens strains
Summary
Clostridium perfringens, an anaerobic Gram-positive bacterium, is ubiquitous in the intestinal flora of human and animals, and is commonly isolated from environmental materials such as soil and water [1,2]. In a commonly used classification scheme, C. perfringens is divided into five toxinotypes (A to E) based on the production of four toxins (alpha, beta, epsilon, and iota); this bacterium produces ten other toxins such as C. perfringens enterotoxin (CPE), beta toxin, and theta toxin ( known at perfringolysin O or PFO) [2]. Of these many toxins, CPE is extremely important for human gastrointestinal diseases such as food poisoning and antibioticassociated diarrhea [2]. There are several reports of biological differences, such as heat resistance and other traits [6,7,8], between chromosomal cpe isolates and plasmid cpe isolates that may suggest these two types of strains possess different genetic backgrounds
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