Anti-Germinants as a New Strategy to Prevent Clostridium Difficile Infections

Clostridium difficileinfections (CDI) have emerged as a leading cause of hospital-associated complications. CDI is the major cause of antibiotic-related cases of diarrhea and nearly all cases of pseudomembranous colitis. The infective form of C. difficileis the spore, a dormant and hardy structure that forms under stress. Germination of C. difficile spores into toxin producing bacteria in the GI tract of susceptible patients is the first step in CDI establishment. Patient susceptibility occurs with a disruption of the natural gut microbiota by broad-spectrum antibiotics. Antibiotic treatments usually resolve CDI but refractory cases are on the rise. Of great concern is the high incidence of recurrence due to persistence of spores in the gut following antibiotic treatment and/or spore re-ingestion. Besides surface decontamination there are currently no protocols for prevention of CDI. C. difficile spores must germinate to cause disease. Therefore, a logical approach to preventing CDI is to prevent spore germination. Unlike other Bacillus and Clostridia, the genome of C. difficile does not encode for any known germination binding site(s). Small molecules are typically required to activate spore germination in Bacillus and Clostridia.C. difficile germinates in the presence of taurocholate, a natural bile salt, and glycine, an amino acid. The natural bile salt, chenodeoxycholate, has been shown to inhibit spore germination in vitro. We used structure activity analysis to define the microenvironment of the putative C. difficile germination binding site(s). Amino acids and amino acid analogs were analyzed for activation or inhibition of C. difficile spore germination. To determine which functional groups of bile salts are necessary and sufficient to activate or inhibit spore germination, we prepared bile salt analogs of taurocholate and chenodeoxycholate. This analysis elucidated specific functional groups recognized by C. difficile spores. Furthermore, many bile salt analogs are able to bind but are not recognized by the putative C. difficile germination binding site(s). During this structure analysis, we discovered that a meta-benzene sulfonic acid derivative of taurocholate (CamSA) was a strong inhibitor of spore germination in vitro. CamSA is stable and non-toxic based on pharmacokinetic in vitro studies. CamSA showed no acute toxicity at the highest concentrations tested. More importantly, a single dose of CamSA prevents CDI in mice. Ingested C. difficile spores were quantitatively recovered from feces and intestines of CamSA-protected mice. Using CamSA as a probe, we were able to establish when onset of disease occurs in mice after infection with C. difficile spores. The results presented in this dissertation project support a mechanism whereby the anti-germination effect of CamSA is responsible for preventing CDI signs