C. DIFFICILE: A CHARACTERIZATION OF VIRULENCE FACTORS AND GROWTH BETWEEN EPIDEMIC VERSUS NON-EPIDEMIC STRAINS

Clostridium difficile (C. difficile) is a spore-forming, gram positive bacterium found naturally within a human’s intestinal flora, as well as in the environment. This organism is safe in small numbers as a part of the natural intestinal flora, but can pose problems when broad-spectrum antibiotics, such as clindamycin, kill off other natural flora. Presently, there are two different, over-arching categories of C. difficile, toxigenic strains, which cause disease, and non-toxigenic, non-disease causing strains. These toxigenic strains can be broken into sub-categories: non-epidemic and epidemic. These labels are given based on the prevalence of an isotype in a geographical location. The most prevalent isotype is classified as epidemic and the less prevalent isotypes are classified as non-epidemic. Previous research has produced in vitro studies of epidemic and non-epidemic C. difficile strains, and there have been no conclusive patterns determined from these studies to determine the difference of virulence factors between strains. Therefore, is there a difference with the growth characteristics and virulence factors contributing to the severity of infection between epidemic and non-epidemic C. difficile strains both in vitro and in vivo? In vitro characterization of five non-epidemic and five epidemic strains, starting with virulence factor characterization of viable and germinated cell counts, as well as Toxin A and B production quantification were found to be statistically different between strains. In contrast, observed differences were slight and suggest that, in vitro, C. difficile can be treated as a group from an non-epidemic versus epidemic standpoint. The next group of in vitro studies including Growth Curves, Minimum Inhibitory Concentration studies, and Inhibition Curves also showed little observed differences between the non-epidemic and epidemic strains continuing to support the observations from the first set of studies stating that: C. difficile can be characterized and treated as a group instead of individual strains during infection. Future in vivo studies will help to determine if these trends between non-epidemic and epidemic strains will remain viable when the environment contains additional factors during infection, such as p.H. shifts, nutrition influx, and treatment with antibiotics within a system. Purpose (a): Clostridium difficile (C. difficile) is a spore-forming, gram positive bacterium found naturally within a human’s intestinal flora capable of causing severe disease. Other research has focused on in vitro studies of epidemic and non-epidemic C. difficile strains, and these studies concluded no conclusive patterns between the difference in virulence factors between the individual strains. Therefore, an important question to ask is: for C. difficile, is there a difference with the growth characteristics and virulence factors contributing to the severity of infection between epidemic and non-epidemic C. difficile strains both in vitro and in vivo? Methods (b): For the in vitro studies, there are multiple protocols used including, viable cell counts, spore isolation and germinated cell counts, Toxin A or B ELISA assays, growth and inhibition curves, as well as minimum inhibitory concentration (MIC) determination. In vivo UNTHSC Pre-Clinical Services 21-day Recurrence Hamster Model and Next-Gen Sequencing for microbiome research will be utilized. Results (c): When non-epidemic and epidemic C. difficile strains were characterized for major virulence factors, statistically significant differences for both intrastrain and interstrain comparisons were observed. Growth curve data showed consistent growth patterns between the strains. MIC results were consistent between strains, with no more than a 100-fold difference between the MIC of any one drug for all the strains tested. Inhibition curve results also showed minimal variation between the different non-epidemic and epidemic strain behavior when growth was tested against metronidazole, moxifloxacin, and vancomycin. Conclusions (d): During characterization in vitro, between five non-epidemic and five epidemic strain’s virulence factors, the differences in results are small, yet statistically significant. Though statistically different, observed differences are minimal and not believed to affect the individual strain's overall virulence. Therefore, it is concluded, in vitro, different strains of C. difficile have similar growth patterns and have similar virulence characteristics as a group. When further study was conducted to compare growth patterns over 24-hours, MIC’s concentrations, and Inhibition Curves, interstrain comparisons once again showed small observed differences. The overall trends in antibiotic susceptibility and growth patterns when the media was without, and supplemented with, antibiotics were seen to be similar. This continues to support that C. difficile can be treated, in vitro, as a group, independent of the labels non-epidemic and epidemic.