Toxin gene analysis of a variant strain of Clostridium difficile that causes human clinical disease

A toxin variant strain of Clostridium difficile was isolated from two patie nts with C. difficile-associated disease (CDAD), one of whom died from exte nsive pseudomembranous colitis. This strain, identified by restriction endo nuclease analysis (REA) as type CF2, was not detected by an immunoassay for C, difficile toxin A. Culture supernatants of CF2 failed to elicit signifi cant enterotoxic activity in the rabbit ileal loop assay but did produce at ypical cytopathic effects in cell culture assay. Southern hybridization, PC R amplification, and DNA sequence analyses were performed on the toxin A (t cdA) and toxin B (tcdB) genes of type CF2 isolate 5340, Type CF2 5340 tcdA exhibited a 1,821-bp truncation, due to three deletions in the 3' end of th e gene, and a point mutation in the 5' end of the gene, resulting in a prem ature stop codon at tcdA position 139, Type CF2 5340 tcdB exhibited multipl e nucleotide base substitutions in the 5' end of the gene compared to tcdB of the standard toxigenic strain VPI 10463, Type CF2 5340 toxin gene nucleo tide sequences and deduced amino acid sequences showed a strong resemblance to those of the previously described variant C. difficile strain 1470, a s train reported to have reduced pathogenicity and no association with clinic al illness in humans. REA of strain 1470 identified this strain as a distin ct type (CF1) within the same REA group as the closely related type CF2, A review of our clinical-isolate collection identified five additional patien t's infected with type CF2, three of whom had documented CDAD, PCR amplific ation of the 3' end of tcdA demonstrated identical 1,8-kb, deletions in all seven type CF2 isolates. REA type CF2 is a toxin variant strain of C. diff icile that retains the ability to cause disease In humans but is not detect ed in clinical immunoassays for toxin A.