Evidence that the enterotoxin gene can be episomal in Clostridium perfringens isolates associated with non-food-borne human gastrointestinal diseases

Abstract

Clostridium perfringens enterotoxin (CPE) is responsible for the diarrheal and cramping symptoms of human C. perfringens type A food poisoning. CPE-producing C. perfringens isolates have also recently been associated with several non-food-borne human gastrointestinal (GI) illnesses, including antibiotic-associated diarrhea and sporadic diarrhea. The current study has used restriction fragment length polymorphism (RFLP) and pulsed-field gel electrophoresis (PFGE) analyses to compare the genotypes of 43 cpe-positive C. perfringens isolates obtained from diverse sources. All North American and European food-poisoning isolates examined in this study were found to carry a chromosomal cpe, while all non-food-borne human GI disease isolates characterized in this study were determined to carry their cpe on an episome. Collectively, these results provide the first evidence that distinct subpopulations of cpe-positive C. perfringens isolates may be responsible for C. perfringens type A food poisoning versus CPE-associated non-food-borne human GI diseases. If these putative associations are confirmed in additional surveys, cpe RFLP and PFGE genotyping assays may facilitate the differential diagnosis of food-borne versus non-food-borne CPE-associated human GI illnesses and may also be useful epidemiologic tools for identifying reservoirs or transmission mechanisms for the subpopulations of cpe-positive isolates specifically responsible for CPE-associated food-borne versus non-food-borne human GI diseases.

FIG. 1

RFLP analysis of NruI-digested total DNA from selected C. perfringens disease isolates demonstrating presumptive evidence for episomal versus chromosomal distribution of cpe in isolates from different disease sources. Southern blots were probed with a 639-bp DIG-labeled cpe-specific fragment. VET, veterinary. All samples except 8239/EcoRI were cut with NruI; 8239/EcoRI was cut with EcoRI and the results are shown for comparison. Molecular sizes (in kilobase pairs) of the DNA markers are given to the left of each blot. See Table 1 for full strain designations.

FIG. 2

PFGE evidence supporting the localization of cpe to episomes in isolates from some disease sources. PFGE and Southern hybridization analysis of undigested and I-CeuI-digested genomic DNA from select C. perfringens disease isolates. (A) pulsed-field gel stained with ethidium bromide. (B) Southern blot of the gel shown in panel A. The blot was probed with a 639-bp DIG-labeled cpe-specific fragment. FP, food poisoning; VET, veterinary; U, undigested, intact genomic DNA; C, I-CeuI-digested DNA. The gel was calibrated with Saccharomyces cerevisiae chromosomal DNA. Molecular sizes of the DNA markers are given in the center. See Table 1 for full strain designations.