Clonal dissemination of a toxin-A-negative/toxin-B-positive Clostridium difficile strain from patients with antibiotic-associated diarrhea in Poland

Objective To determine the incidence of toxin-A-negative/toxin-B-positive Clostridium difficile strains and their genetic relatedness in the feces of patients suffering from antibiotic-associated diarrhea (AAD) in Polish hospitals. Methods C. difficile strains were cultured from patients' stool samples. The present study characterises these strains with respect to their cytopathogenicity on McCoy cell's and the absence of toxin A despite a functional toxin B as determined with commercial test kits (Culturette Brand Toxin CD-TCD toxin A test and C: difficile Tox A/B test). In addition, PCR using different primer pairs aiming at non-repeating or repeating regions of the toxin A and B genes were used to confirm the findings. All toxin A(-)B(+) strains were genetically characterised by random amplification of polymorphic DNA (RAPD) analysis, PCR ribotyping and, in part, pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments. Results We here present the presence of 17 toxin A(-)B(+) strains among, 159 C. difficile strains (11%) isolated from fecal samples from 413 patients with antibiotic-associated diarrhea. All 17 strains possessed the toxin B gene, demonstrated a cytopathogenic effect on the McCoy cells, and were positive in the Tox A/B test. Molecular typing of these 17 C. difficile strains, revealed that 7 of 17 (41%) toxin A(-)/B+ C. difficile strains could not be discriminated. It appeared that these strains had a genotype that could not be distinguished from that of a Japanese control strain. Conclusion Our observations imply that a particular genotype of toxin A-B+ C. difficile has spread extensively, not only in Poland but possibly even worldwide.