Clostridium difficile Ribotype 027 Virulence Phenotype Influences Disease Severity when Compared to Ribotype Non-027 Strains in the Hamster CDAD Model

Introduction: C. difficile ribotype 027 (RT027) is the epidemic strain found primarily in North America, and, recently, a strain of C. difficile has been named a superbug by the CDC. Studies have suggested an enhanced virulence phenotype for RT027 such as increased toxin production, but the impact on disease severity on in vivo models is not well understood. This study describes the in vitro characterization of important virulence factors for several RT027 and non-RT027 C. difficile clinical isolates, and how these characteristics may impact disease severity in the hamster C. difficile associated disease (HCDAD) model. Materials and Methods: Six RT027 and six non-RT027 clinical isolates were evaluated in vitro for total spore counts and Toxin A/B titers in 72H broth cultures. Spore counts were generated from heat/ethanol shock culture samples and plated onto CCFA containing 0.1% taurocholate, and toxin A/B titers were determined from spent broth with the tgcBIOMICS ELISA assay. The HCDAD studies involved infecting male Golden Syrian hamsters with 72H broth cultures of two RT027 and two non-RT027 isolates, followed by subcutaneous administration of 10 mg/kg clindamycin 24H post-infection. One group of infected hamsters was orally treated with 20 mg/kg vancomycin once a day for 3 days following clindamycin administration, while the other group remained untreated. Survival was monitored for 11 days after infection and 3 hamsters were euthanized at set time points to determine cecal fluid associated the CFU/spore counts and Toxin A/B titers. Results: The RT027 and the non-RT027 strains generated similar mean CFU/mL in 72H broth cultures, while the mean spore counts were 548 spores/mL for the RT027 strains and 273 spores/mL for the non-RT027 strains. In addition, the 72H broth-associated mean toxin A/B titers were 2.8-fold higher for RT027 strains when compared to the 72H titers of non-RT027 strains. In the HCDAD studies 14% of the non-027 infected hamsters became moribund, while 71% of the hamsters infected with the RT027 isolates became moribund. The mean cecal fluid Toxin A/B titers for RT027 infected hamsters were 2.3 to 9-fold higher than the titers for non-RT027 infected hamsters. Conclusion: The results highlight that C. difficile RT027 isolates, when compared to non-RT027 clinical isolates, have enhanced virulence that corresponds to a more severe disease in the HCDAD model. Understanding why these strains have this phenotype, in vivo as compared to in vitro, is important for treatment as new ribotypes, with varying virulence, are continuously emerging.