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

Effect of the Synthetic Bile Salt Analog CamSA on the Hamster Model of Clostridium difficile Infection

Clostridium difficile infection (CDI) is the leading cause of antibiotic-associated diarrhea and has gained worldwide notoriety due to emerging hypervirulent strains and the high incidence of recurrence. We previously reported protection of mice from CDI using the antigerminant bile salt analog CamSA. Here we describe the effects of CamSA in the hamster model of CDI. CamSA treatment of hamsters showed no toxicity and did not affect the richness or diversity of gut microbiota; however, minor changes in community composition were observed. Treatment of C. difficile-challenged hamsters with CamSA doubled the mean time to death, compared to control hamsters. However, CamSA alone was insufficient to prevent CDI in hamsters. CamSA in conjunction with suboptimal concentrations of vancomycin led to complete protection from CDI in 70% of animals. Protected animals remained disease-free at least 30 days postchallenge and showed no signs of colonic tissue damage. In a delayed-treatment model of hamster CDI, CamSA was unable to prevent infection signs and death. These data support a putative model in which CamSA reduces the number of germinating C. difficile spores but does not keep all of the spores from germinating. Vancomycin halts division of any vegetative cells that are able to grow from spores that escape CamSA

Bacillus cereus Spores Release Alanine that Synergizes with Inosine to Promote Germination

spores germinate in the presence of a single germinant, inosine, yet with a significant lag period. spores. spores appear to have developed a unique quorum-sensing feedback mechanism to monitor spore density and to coordinate germination

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></div

CDI is established between 6 and 9 hours post-infection.

<p>(A) Survival of infected mice at 48 hours after challenge with <i>C. difficile</i> spores. Mice were treated with 300 mg/kg CamSA at 0, 6, 9, or 12 hours post-challenge. (B) Comparison of CDI severity after 24 hours (white bars) and 48 hours (black bars) for animals challenged with <i>C. difficile</i> spores and treated with 300 mg/kg CamSA at 0, 6, 9, or 12 hours post-challenge. Clinical endpoint was set as >6 in the CDI sign severity scale (dashed line). (C) <i>C. difficile</i> vegetative cell count in feces of untreated, diseased animals. Feces were collected from cages housing five untreated mice challenged with <i>C. difficile</i> spores. Open bars represent <i>C. difficile</i> vegetative cells. The amount of <i>C. difficile</i> spores excreted by untreated animals was negligible (<10% of vegetative cell counts). Standard deviations represent at least five independent measures. Recovered CFU and recovered spores represent mean values from pools of five animals.</p

Time line model for CDI onset in mice.

<p><i>C. difficile</i> spores (black circles) are ingested by the host. Spores rapidly transit through the upper GI tract and colonize the colon and cecum. Spore shedding begins less than 2 hours post-ingestion. Between 6 and 9 hours after ingestion sufficient numbers of spores germinate to establish infection. The outgrowing <i>C. difficile</i> cells (white circles) proliferate in the lower intestine, are shed, and can re-sporulate. A small amount of ingested spores remain in the lower intestine for more than 96 hours post ingestion.</p

Inhibition of <i>C. difficile</i> toxin production by CamSA treatment.

<p><i>C. difficile</i> spores were incubated overnight in media containing 0 µM CamSA (white bars) or 200 µM CamSA (black bars). The resulting spent media were added to Vero cell cultures and incubated for 24 hours. Cell viability was determined with the CellTiter Glo viability kit. The luminescence signal from untreated cells was set as 100% cell viability. Percent survival for other conditions was calculated relative to untreated cells. Error bars represent standard deviations from at least five independent measurements.</p

<i>C. difficile</i> spores accumulate in the cecum, colon, and feces of CamSA-treated animals.

<p>(A) Amount of <i>C. difficile</i> spores recovered at different time points following spore challenge from the cecum (white bars) and colon (black bars) of mice treated with 50 mg/kg CamSA. Student’s unpaired <i>t</i>-test was used to determine the significance of difference of means. *indicates recovered spores significantly below 72 hour levels (<i>P</i> = 0.019; Student's <i>t</i>-test). **indicates recovered spores significantly below 72 hour levels (<i>P</i> = 0.049; Student's <i>t</i>-test). (B) Feces were collected from cages housing five mice challenged with <i>C. difficile</i> spores and treated with 50 mg/kg CamSA. Closed bars represent <i>C. difficile</i> spores. The amount of <i>C. difficile</i> vegetative cells in CamSA-treated animals was negligible (<10% compared to spore counts). Standard deviations represent at least five independent measures. Recovered CFU and recovered spores represent mean values from a pool of five animals.</p

Cytotoxicity of CamSA.

<p>Vero cells (white bars) or Caco-2 cells (black bars) were incubated overnight with 10% DMSO, 10% EtOH, 50 µM CamSA or 200 µM CamSA. Cell viability was determined with the CellTiter Glo viability kit. The luminescence signal from DMSO-treated cells was undistinguishable from untreated cells and was set as 100% cell viability. Percent survival for other conditions was calculated relative to untreated cells. Error bars represent standard deviations from at least five independent measurements.</p