Enumeration and Isolation of cpe -Positive Clostridium perfringens Spores from Feces

A hydrophobic grid membrane filter-colony hybridization (HGMF-CH) method for the enumeration and isolation of cpe gene-carrying ( cpe -positive) Clostridium perfringens spores from feces was developed. A 425-bp DNA probe specific for the cpe gene was sensitive and specific when tested with bacterial DNA and pure cultures. The enumeration of cpe -positive C. perfringens by the HGMF-CH method proved to be as sensitive as nested PCR combined with the most-probable number technique when tested with fecal samples from healthy individuals. With the aid of the HGMF-CH method, positive hybridization signals were detected from two out of seven fecal samples obtained from healthy individuals. Furthermore, cpe -positive C. perfringens was successfully isolated from both of these samples. The detection of cpe -positive C. perfringens by the HGMF-CH method is dependent on the ratio of cpe- positive C. perfringens colonies to total C. perfringens colonies growing on the HGMF-tryptose-sulfite-cycloserine plate. cpe- positive C. perfringens could be isolated if the ratio of cpe- positive C. perfringens spores to total C. perfringens spores was 6 × 10 −5 or higher. The HGMF-CH method provides an aid in the investigation of fecal samples of patients suffering from food poisoning or other diseases caused by cpe -positive C. perfringens . The method also offers a new approach in the investigation of the epidemiology of cpe- positive C. perfringens strains.