Detection of Enterotoxigenic Clostridium perfringens in Meat Samples by Using Molecular Methods

Abstract

To prevent food-borne bacterial diseases and to trace bacterial contamination events to foods, microbial source tracking (MST) methods provide important epidemiological information. To apply molecular methods to MST, it is necessary not only to amplify bacterial cells to detection limit levels but also to prepare DNA with reduced inhibitory compounds and contamination. Isolates carrying the Clostridium perfringens enterotoxin gene (cpe) on the chromosome or a plasmid rank among the most important food-borne pathogens. Previous surveys indicated that cpe-positive C. perfringens isolates are present in only ϳ5% of nonoutbreak food samples and then only at low numbers, usually less than 3 cells/g. In this study, four molecular assays for the detection of cpe-positive C. perfringens isolates, i.e., ordinary PCR, nested PCR, real-time PCR, and loop-mediated isothermal amplification (LAMP), were developed and evaluated for their reliability using purified DNA. For use in the artificial contamination of meat samples, DNA templates were prepared by three different commercial DNA preparation kits. The four molecular assays always detected cpe when >10 3 cells/g of cpe-positive C. perfringens were present, using any kit. Of three tested commercial DNA preparation kits, the InstaGene matrix kit appeared to be most suitable for the testing of a large number of samples. By using the InstaGene matrix kit, the four molecular assays efficiently detected cpe using DNA prepared from enrichment culture specimens of meat samples contaminated with low numbers of cpe-positive C. perfringens vegetative cells or spores. Overall, the current study developed molecular assay protocols for MST to detect the contamination of foods with low numbers of cells, and at a low frequency, of cpe-positive C. perfringens isolates.Clostridium perfringens is an important pathogen of human gastrointestinal (GI) tract diseases such as food poisoning, antibiotic-associated diarrhea, and sporadic diarrhea as well as nosocomial diarrheal disease outbreaks (6, 10). The most important toxin made by this bacterium when it causes human GI tract diseases is Clostridium perfringens enterotoxin (CPE) (10,14). Although C. perfringens is a ubiquitous bacterium in the environment, only a small subpopulation of this bacterium, usually less than 5%, harbors the CPE gene (cpe) (10). Probably because of this rarity, previous surveys identifying cpepositive C. perfringens isolates in food, human feces, and the environment have reported various results (1,4,8,9,11,16). Moreover, the number of contaminated C. perfringens cells (cpe-positive and cpe-negative strains) present in most nonoutbreak food samples has been reported to be fewer than 3 using the most-probable-number method (9, 11, 16). Therefore, to prevent food poisoning and nosocomial outbreaks, a method able to detect cpe-positive strains with high sensitivity and applicability for the testing of a large number of samples is necessary for use in epidemiological surveys.Microbial source tracking (MST) methods allow...

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