The in vitro Virulence Phenotype of Clostridium difficile Ribotype 027 Impacts Disease Severity in the Mouse and Hamster CDAD Models




Pulse, Mark
Vitucci, John
Simecka, Jerry


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C. difficile ribotype 027 (RT027) is the epidemic strain found primarily in North America. 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 characteristics for several RT027 and non-RT027 C. difficile clinical isolates, and how these factors may impact disease severity in the hamster and mouse C. difficile associated disease models. 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 mouse C. difficile model involved being administered for 5 days through oral gavage or drinking water. The mice were then placed on a non-antibiotic supplemented water for 48 hours IP administered 10 mg/kg of clindamycin, and orally inoculated with C. difficile spores 24H later. Survival was monitored for 10 days and fecal samples were taken each day to be processed for CFU/spore counts. The HCDAD studies involved inoculating 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 post-mortem cecal fluid samples were taken from 3 hamsters at set disease-associated time points to determine the CFU/spore counts and Toxin A/B titers. 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 mouse model, 100% of the animals infected with the non-027 isolate survived regardless of how antibiotics were administered. In contrast, 13-26% morbidity was associated with mice infected with the RT027 isolate after being given antibiotics by oral administration or through supplemented water. 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.