Clostridium difficile recently has been linked to antibiotic-associated enterocolitis and pseudomembranous colitis which are severe and sometimes fatal diarrheal syndromes. This toxin-producing, strictly anaerobic bacterium was first described in 1935, but has only been recognized as a clinically significant pathogen within the last 8 or 9 years. Evidence exists that the pathogenic effect of this organism is mediated by two toxins and that adherence of C. difficile to the host may possibly play a role in pathogenicity. Conclusive studies on the genetic determinant(s) responsible for the expression of C. difficile toxins have not been reported. However, plasmids and bacteriophage mediated production of toxin and other extracellular enzymes have been previously demonstrated in other clostridial species such as Clostridium perfringens, Clostridium tetani, Clostridium botulinum and Clostridium novyi. Because of the previously reported linkage of toxin genes to extrachromosomal DNA and the documented occurrence of adhesive or invasive genes being coded for by plasmids in other bacteria, the high molecular weight extrachromosomal DNA of C. difficile will be characterized. Preliminary work in our laboratory has demonstrated a large extrachromosomal DNA element in 16 out of 16 strains of C. difficile tested. Also bacteriophage particals were identified with electron microscopy to be associated with C. difficile. The major objectives of this two year pilot study is to determine if the large extrachromosomal DNA observed is plasmid or phage in nature and to determine if this DNA codes for assayable determinants responsible for pathogenicity.
