Genetic characterization of toxin A-negative, toxin B-positive Clostridium difficile isolates by PCR.

Toxin-specific enzyme immunoassays, cytotoxicity assays, and PCR were used to analyze 48 toxin A-negative, toxin B-positive Clostridium difficile isolates from various geographical sites around the world. All the isolates were negative by the TOX-A TEST and positive by the TOX A/B TEST. A deletion of approximately 1.7 kb was found at the 3' end of the toxA gene for all the isolates, similar to the deletion in toxinotype VIII strains (e.g., C. difficile serotype F 1470). Additional PCR analysis indicated that the toxin B encoded by these isolates contains sequence variations downstream of the active site compared to the sequence of reference strain VPI 10463. This variation may extend the glucosylation spectrum to Ras proteins, as observed previously for closely related lethal toxin from Clostridium sordellii and toxin B from toxin A-negative, toxin B-positive strain F 1470. Toxin A-negative, toxin B-positive isolates have recently been associated with disease in humans, and they may be more common than was previously supposed.