Prevalence of enterotoxigenic Clostridium perfringens Isolates in Pittsburgh (Pennsylvania) area soils and home kitchens

Abstract

In the United States and Europe, food poisoning due to Clostridium perfringens type A is predominantly caused by C. perfringens isolates carrying a chromosomal enterotoxin gene (cpe). Neither the reservoir for these isolates nor the point in the food chain where these bacteria contaminate foods is currently understood. Therefore, the current study investigated whether type A isolates carrying a chromosomal cpe gene are present in two potential reservoirs, i.e., soil and home kitchen surfaces. No C. perfringens isolates were recovered from home kitchen surfaces, but most surveyed soil samples contained C. perfringens. The recovered soil isolates were predominantly type A, but some type C, D, and E soil isolates were also identified. All cpe-positive isolates recovered from soil were genotyped as type A, with their cpe genes on cpe plasmids rather than the chromosome. However, two cpe-positive soil isolates did not carry a classical cpe plasmid. Both of those atypical cpe-positive soil isolates were sporulation capable yet failed to produce C. perfringens enterotoxin, possibly because of differences in their upstream promoter regions. Collectively these results suggest that neither soil nor home kitchen surfaces represent major reservoirs for type A isolates with chromosomal cpe that cause food poisoning, although soil does appear to be a reservoir for cpe-positive isolates causing non-food-borne gastrointestinal diseases.

FIG. 1.

Multiplex PCR genotyping results for C. perfringens soil isolates. (A) Toxin typing analysis of selected C. perfringens soil isolates, identifying cpe-negative type A isolates (S102-2, SS95-13); cpe-positive, cpb2-negative type A isolates (S292-3, S350-1, SS151-1); cpb2-positive, cpe-negative type A isolates (S37-1); type C isolates (SS32-1); type D isolates (S153-2, SS211-1); and type E isolates (S155-1 and SS372-2). (B) cpe genotyping results for cpe-positive type A isolates identifying type A soil isolates carrying a plasmid cpe locus with downstream IS1470-like sequences (S194-1, SS151-1), type A soil isolates carrying a plasmid cpe locus with downstream IS1151 sequences (S37-1, SS44-1), and non-genotypeable cpe-positive type A soil isolates S292-3 and S350-1. Isolate SS167-1 could not be genotyped using crude culture lysates, as shown, but (using purified DNA) was identified as carrying a cpe plasmid with downstream IS1151 sequences (not shown). For comparison, a type A food poisoning-causing derivative with a chromosomal cpe gene (SM101), a type A non-food-borne GI disease-causing isolate carrying a plasmid cpe locus with downstream IS1151 sequences (F5603), and a type A non-food-borne GI disease isolate carrying a plasmid cpe locus with downstream IS1470-like sequences (F4969) are also shown. M, marker lane.

FIG. 2.

PFGE Southern blot analysis of cpe gene location in selected cpe-positive C. perfringens type A soil isolates. The blot was hybridized with a cpe probe, and size markers are shown to the left of the blot. Also included for comparison are cpe-negative isolate ATCC 3624 (to demonstrate probe hybridization specificity) and type A non-food-borne GI disease-causing isolates F4969 and F5603, which have been shown by sequencing to carry cpe plasmids of ∼70 kb and ∼73 kb, respectively.

FIG. 3.

Western blot analysis of CPE production by cpe-positive C. perfringens type A soil isolates. Isolates were grown for 8 h in DS sporulation medium until phase-refractile spores were present in all cultures, as confirmed by phase-contrast microscopy. Culture lysates were then subjected to Western blot analysis using CPE antibodies. Shown are results for seven selected cpe-positive type A soil isolates (S37-1, SS44-1, S194-1, SS151-1, S292-3, S350-1, and SS167-1). Also shown for comparison are CPE Western blot results for sporulating culture lysates of known CPE-positive isolate F5603 and cpe-negative isolate ATCC 3624.