IDENTIFICATION OF TOXIN A-NEGATIVE, TOXIN B-POSITIVE CLOSTRIDIUM-DIFFICILE BY PCR

Toxigenic strains of Clostridium difficile have been reported to produ ce both toxins A and B nearly always, and nontoxigenic strains have be en reported to produce neither of these toxins. Recent studies indicat e that it is not always true. We established a PCR assay to differenti ate toxin A-negative, toxin B-positive (toxin A-, toxin B+) strains fr om both toxin-positive (toxin A+, toxin B+) strains and both toxin-neg ative (toxin A-, toxin B-) strains as an alternative to cell culture a ssay and enzyme-linked immunosorbent assay (ELISA). By using the PCR p rimer set NK11 and NK9 derived from the repeating sequences of the tox in A gene, a shorter segment (ca. 700 bp) was amplified from toxin A-, toxin B+ strains compared to the size of the segment amplified from t oxin Af, toxin B+ strains (ca. 1,200 bp), and no product was amplified from toxin A-, toxin B- strains. We examined a total of 421 C. diffic ile isolates by PCR. Of these, 48 strains showed a shorter segment by the PCR, were negative by ELISAs for the detection of toxin A, and wer e positive by cell culture assay. Although the cytotoxin produced by t he toxin A-, toxin B+ strains ws neutralized by anti-toxin B serum, th e appearance of the cytotoxic effects on Vero cell monolayers was dist inguishable from that of toxin A+, toxin B+ strains. By immunoblotting , the 44 toxin A-, toxin B+ strains were typed to serogroup F and the remaining four strains were serogroup X. Pulsed-field gel electrophore sis separated the 48 strains into 19 types. The PCR assay for the dete ction of the repeating sequences combined with PCR amplification of th e nonrepeating sequences of either the toxin A or the toxin B gene is indicated to be useful for differentiating toxin A-, toxin B+ strains from toxin A+, toxin B+ and toxin A-, toxin B- strains and will contri bute to elucidation of the precise role of toxin A-, toxin B+ strains in intestinal diseases.