Background: Clostridioides difficile infections have become more frequently diagnosed and associated with greater disease severity, which has resulted in an increase burden on the healthcare system. These increases are attributed to the increased prevalence of hypervirulent strains encompassing select ribotypes. These epidemic ribotypes were characterized as hypervirulent due to higher in vitro spore and toxin production, as well as increased incidence, severity and mortality within patients. However, it is unclear whether epidemic ribotypes are truly more virulent than non-epidemic ribotypes in vivo. Furthermore, there is conflicting evidence about the ability of a strain's in vitro phenotype to be predictive of their in vivo virulence. The goals of the current studies were to determine if epidemic ribotypes are more virulent than other ribotypes in animal models, and whether the in vitro virulence phenotype of an isolate or ribotype predict in vivo virulence. Results: To determine if epidemic strains were truly more virulent than other non-epidemic strains, the in vivo virulence of 13 C. difficile isolates (7 non-epidemic and 6 epidemic ribotype isolates) were determined in murine and hamster models of CDI. The isolates of epidemic ribotype of C. difficile were found to be more virulent in both the murine and hamster models than non-epidemic isolates. In particular, the group of epidemic ribotypes of C. difficile had lower LD 50 values in hamsters. The increased severity of disease was associated with higher levels of Toxin A and Toxin B production found in fecal samples, but not numbers of organisms recovered. The isolates were further characterized for their in vitro virulence phenotypes, e.g. toxin production, growth rates, spore formation and adherence of spores to intestinal epithelial cell lines. Although there were higher levels of toxins produced and greater adherence for the group of epidemic ribotypes, the in vitro profiles of individual isolates were not always predictive of their in vivo virulence. Conclusions: Overall, the group of epidemic ribotypes of C. difficile were more virulent in vivo despite individual isolates having similar phenotypes to the non-epidemic isolates in vitro.

C . difficile is an endospore-forming pathogen, which is becoming a common cause of microbial health-care associated gastrointestinal disease in the United States. Both healthy and symptomatic patients can shed C . difficile spores into the environment, which can survive for long periods, being resistant to desiccation, heat, and disinfectants. In healthcare facilities, environmental contamination with C . difficile is a major concern as a potential source of exposure to this pathogen and risk of disease in susceptible patients. Although hospital-acquired infection is recognized, community-acquired infection is an increasingly recognized health problem. Primary care clinics may be a significant source of exposure to this pathogen; however, there are limited data about presence of environmental C . difficile within clinics. To address the potential for primary care clinics as a source of environmental exposure to virulent C . difficile , we measured the frequency of environmental contamination with spores in clinic examination rooms and hospital rooms in Dallas-Fort Worth (DFW) area of Texas. The ribotypes and presence of toxin genes from some environmental isolates were compared. Our results indicate primary care clinics have higher frequencies of contamination than hospitals. After notification of the presence of C . difficile spores in the clinics and an educational discussion to emphasize the importance of this infection and methods of infection prevention, environmental contamination in clinics was reduced on subsequent sampling to that found in hospitals. Thus, primary care clinics can be a source of exposure to virulent C . difficile , and recognition of this possibility can result in improved infection prevention, potentially reducing community-acquired C . difficile infections and subsequent disease.

Between 2000-2007, Clostridium (now Closteroides) difficile infections have increased by a factor of 400% and have been associated with greater disease severity. These increases are associated to the increased prevalence of the NAP1/BI/027 ribotype (ribotype-027). This ribotype was characterized as hypervirulent, and one reason was the ability to produce greater numbers of spores in vitro. However, it is unclear whether the epidemic ribotype-027 are able to produce greater numbers of spores in vivo, and if this plays a role during clinically relevant treatments. To determine if epidemic strains are able to produce more spores during clinically relevant treatments, the growth and in vivo production of spores of four C. difficile isolates (2 non-epidemic and 2-epidemic) were determined in the hamster model of C. difficile infection (CDI). By using this model, the epidemic isolates of C. difficile were found to produce more spores than the non-epidemic isolates during treatment with vancomycin. The difference in spore numbers in response to the presence of vancomycin also occurred in in vitro cultures. These differences between the epidemic and non-epidemic isolates were consistent despite there being no difference to sensitivity to vancomycin in vitro. Thus, antibiotic treatment promoted higher levels of spores of epidemic isolates in vivo and in vitro than found