Graduate Thesis Or Dissertation
 

Genotypic and phenotypic characterization of enterotoxigenic Clostridium perfringens type A fecal isolates associated with human gastrointestinal diseases in the United Kingdom

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  • Clostridium perfringens type A isolates producing enterotoxin (CPE) are an important cause of food poisoning and non-food-borne human gastrointestinal (GI) diseases, including antibiotic-associated diarrhea (AAD), and spontaneous diarrhea (SD). In enterotoxigenic type A isolates, the cpe gene is found on the chromosome in food poisoning isolates, but is present on a large virulence plasmid in AAD and SD type A isolates. Food poisoning cases typically exhibit shorter duration of infection and less severe GI symptoms than AAD or SD. Since previous epidemiological evidence has linked the newly discovered beta2-toxin (CPB2) to gastroenteritis in pigs, horses, and chickens, we hypothesize that the CPB2 toxin may be an accessory toxin when cpe positive type A isolates cause human AAD or SD. In the current study, the presence and expression of CPE and CPB2 were assessed in 44 C. perfringens type A human fecal isolates associated with GI diseases in the United Kingdom. Polymerase chain reaction (PCR) and restriction fragment length polymorphisim (RFLP) confirmed the presence of the cpe (32%) and cpb2 (39%) genes. Furthermore, pulsed field gel electrophoresis (PFGE) and I-CeuI RFLP PFGE Southern blot analysis was used to show the localization of the cpe and cpb2 genes, as well as to determine that there was no clonal relationship between the isolates. All surveyed cpb2-positive isolates were determined to carry their cpb2 gene on a large plasmid that was estimated to be the similar size of the cpe large plasmid. Finally, CPE and CPB2 Western blotting demonstrated that all cpe-positive isolates expressed CPE and that all cpb2-positive isolates expressed CPB2. This study identified, for the first time, the C. perfringens non-food-borne human GI disease isolates carrying both the cpe and cpb2 genes (18%), and these isolates all actively expressed both CPE and CPB2. It was also shown that, although CPE expression occurs only under sporulation conditions, CPB2 expressed both in vegetative and sporulation conditions. The CPB2 made by two of these cpe /cpb2 - positive isolates was determined to be very (-99%) similar to the deduced amino acid sequence of the biologically-active CPB2 made by the original type C isolate CWC245. Finally, the expression of CPB2 by only type A isolates carrying the cpe gene on a plasmid and not the isolates carrying a chromosomal cpe gene, could possibly explain the increased GI symptoms and disease duration associated with these non-food-borne GI diseases. Collectively, the current results support a significant association between cpb2-positive C. perfringens isolates and non-food-borne GI disease in human.
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