Fate of Ingested <i>Clostridium difficile</i> Spores in Mice

<div><p><i>Clostridium difficile</i> infection (CDI) is a leading cause of antibiotic-associated diarrhea, a major nosocomial complication. The infective form of <i>C. difficile</i> is the spore, a dormant and resistant structure that forms under stress. Although spore germination is the first committed step in CDI onset, the temporal and spatial distribution of ingested <i>C. difficile</i> spores is not clearly understood. We recently reported that CamSA, a synthetic bile salt analog, inhibits <i>C. difficile</i> spore germination <i>in vitro</i> and <i>in vivo</i>. In this study, we took advantage of the anti-germination activity of bile salts to determine the fate of ingested <i>C. difficile</i> spores. We tested four different bile salts for efficacy in preventing CDI. Since CamSA was the only anti-germinant tested able to prevent signs of CDI, we characterized CamSa’s <i>in vitro</i> stability, distribution, and cytotoxicity. We report that CamSA is stable to simulated gastrointestinal (GI) environments, but will be degraded by members of the natural microbiota found in a healthy gut. Our data suggest that CamSA will not be systemically available, but instead will be localized to the GI tract. Since <i>in vitro</i> pharmacological parameters were acceptable, CamSA was used to probe the mouse model of CDI. By varying the timing of CamSA dosage, we estimated that <i>C. difficile</i> spores germinated and established infection less than 10 hours after ingestion. We also showed that ingested <i>C. difficile</i> spores rapidly transited through the GI tract and accumulated in the colon and cecum of CamSA-treated mice. From there, <i>C. difficile</i> spores were slowly shed over a 96-hour period. To our knowledge, this is the first report of using molecular probes to obtain disease progression information for <i>C. difficile</i> infection.</p>