Molecular characterization of clostridium difficile PCR ribotypes R-002 and R-017 causing outbreaks and sporadic diarrhea

Abstract

Introduction: Clostridium difficile infection (CDI) is the major bacterial agent causing hospital-acquired diarrhea. The incidence and severity of CDI has increased significantly in recent decades. The distribution of PCR ribotype varies among the countries. PCR ribotype 017 and 002 are the predominant ribotypes in China and Hong Kong respectively. Molecular characterization of C. difficile isolates is useful for outbreak investigation and surveillance. Both PCR ribotyping and Multilocus variable-number tandem-repeat analysis (MLVA) are reliable molecular tools for characterization of C. difficile from outbreak and sporadic case. MLVA allows subtyping of genetically related C. difficile isolates of the same ribotype by their distinctive MLVA patterns. The aim of this study was to characterize the outbreak and sporadic C. difficile isolates of PCR ribotype 002 and 017 by using capillary gel electrophoresis-based PCR ribotyping and MLVA. Their cytotoxicity, sporulation rate and germination efficiency were also investigated in the study.

Results: A total of the 26 C. difficile isolates were identified by PCR ribotyping including fifteen isolates of PCR ribotype 002, eight isolates of ribotype 017, and three isolates of other ribotypes. The isolates of the same ribotypes were further sub-typed into outbreak and sporadic cluster. All isolates showed toxin-producing capability. The sporualtion rates of outbreak isolates of PCR ribotype 002 were significantly higher than that of sporadic isolates of PCR ribotype 002. Statistically higher sporulation rate was also observed in the outbreak isolates of PCR ribotype 017. The germination rate was also statistically higher in the outbreak isolates of PCR ribotype 002 than the sporadic cases.

Conclusion: Capillary electrophoresis-based PCR ribotyping identified fifteen C. difficile PCR ribotype 002 and eight PCR ribotype 017 in this study. MLVA then refined these isolates into two corresponding sub-groups of outbreak and sporadic isolates.

Higher sporulation and germination rates were observed in the outbreak isolates. Sporadic isolates demonstrated relatively lower sporulation and germination rates. The current evidences correlate the genotypic characterization to sporulation activity and germination efficiency.