The Identification and Characterization ofClostridium perfringensby Real-Time PCR, Location of Enterotoxin Gene, and Heat Resistance

Abstract

Clostridium perfringens carrying the enterotoxin gene is an important cause of both foodborne and non-foodborne diarrheal disease. Rapid identification of isolates carrying the enterotoxin gene is invaluable for outbreak investigation whilst information on the genomic location of the enterotoxin (cpe) gene can improve our understanding of disease transmission. This paper describes the validation of a real-time polymerase chain reaction (PCR) assay for the identification of C. perfringens and assessment of the potential to cause diarrhea, together with an investigation into the genomic location of the cpe genes in isolates from confirmed incidents of C. perfringens diarrhea. The real-time assay was shown to be specific for the identification of 253 C. perfringens cultures and gave results concordant with those from motility nitrate and lactose gelatine media, the Nagler reaction, and a conventional block-based PCR assay. The cpe gene was detected in 223 of 253 C. perfringens cultures isolated in association with human gastrointestinal disease. A subset of cpe-positive C. perfringens isolates associated with separate incidents of diarrheal disease were investigated further for plasmid or chromosomal location of the cpe gene using a multiplex PCR assay. The cpe gene was plasmid encoded in two isolates from cases of sporadic diarrhea and six isolates from cases of food poisoning. The cpe gene from the remaining 11 isolates from different food poisoning outbreaks was found to be chromosomally encoded. One of the C. perfringens strains with a plasmid encoded cpe gene formed spores of high heat resistance and five formed spores that were sensitive to heating. Eight of the isolates with a chromosomal cpe gene formed heat-resistant spores, and two formed spores with an intermediate heat resistance.

Toxin Plasmids of Clostridium perfringens

“…As mentioned above, in 75 to 80% of type A food poisoning isolates, the cpe gene is chromosomal (1,170,187,237) and located near an upstream IS1469 sequence and flanking IS1470 sequences (Fig. 3).…”

“…C. perfringens type A food poisoning usually develops when meat or poultry products become heavily contaminated with a CPEpositive type A strain. In ϳ75 to 80% of characterized cases, the causative type A strain carries a chromosomal, rather than a plasmid-borne, cpe gene (1,170). The specific association of type A chromosomal cpe isolates with food poisoning likely involves the exceptional resistance properties of their spores (171)(172)(173)(174)(175).…”

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

“…A duplex TaqMan quantitative PCR was performed as described by Albini et al (1), targeting the following two genes: plc (also named cpa), encoding alpha-toxin, which is ubiquitously present in C. perfringens (15,29), and cpe, encoding CPE. Strains SM101 and ATCC 13124 were included as positive and negative controls (37).…”

“…Interestingly, the C. perfringens strains that carry the cpe gene on the chromosome seem to have a different specific preferred growth niche than the strains that carry the cpe gene on a plasmid. Both vegetative cells and spores of representative C-cpe strains showed overall higher resistance to heat or other environmental stresses than the P-cpe strains (15,27,28,30,43). This may result in better survival and potential outgrowth of the C-cpe strains during food processing, storage, and inappropriate handling by consumers, resulting in a more frequent association with food poisoning.…”

Clostridium perfringens type A–E toxin plasmids

“…Additionally, CPE-producing type A strains are associated with 5–15% of nonfoodborne human intestinal diseases, including antibiotic-associated diarrhea (AAD) and sporadic diarrhea (SD) [5]. The enterotoxin gene ( cpe) can be located chromosomally or on plasmids, with ~70% of food poisoning strains harboring a chromosomal copy of cpe, whereas the remaining ~30% of food poisoning strains, and virtually all AAD/SD strains, carry a plasmid-borne cpe gene [6, 7]. All of these strains cause disease when C. perfringens sporulates in the intestine and produces CPE (see below).…”