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

<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

Stability of CamSA and taurocholate towards bile salt hydrolases.

<p>CamSA (white bar) and taurocholate (black bars) were incubated with cultures of <i>B. longum</i> or <i>L gasseri</i>. Percent conjugated bile salts were derived by dividing the intensity of TLC spots obtained at different times by the intensity of the TLC spot obtained at the beginning of incubation (time 0). Time 0 was set at 100% and is not shown for clarity. Standard deviations represent at least five independent measures.</p

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

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

Enhanced germination rate of B. cereus spores germinated in conditioned supernatants.

<p>Wild-type <i>B. cereus</i> spores were germinated with a 0.2 mM inosine solution (•) or in conditioned supernatants containing 0.2 mM inosine (▪). <i>B. cereus</i> spores were also germinated with 0.2 mM inosine and 20 µM alanine (X).</p

<i>B. cereus</i> spore germination at low spore concentrations.

<p>(A) <i>B. cereus</i> spores were diluted in 10 or 4000 ml (OD₅₈₀ of 1 or 0.0025, respectively) of germination buffer supplemented with 0.2 mM inosine and 0 or 40 µM alanine. Thirty min post-inosine exposure, spores were collected by centrifugation, and pellets were treated with malachite green to stain resting spores and safranin-O to stain germinated cells. Samples were placed under a microscope and a field selected at random. (B) <i>B. cereus</i> spores were diluted in germination buffer (OD₅₈₀ of 1 to 0.0025) in the presence of 0.2 mM inosine. Stained samples were placed under a light microscope and the amount of resting spores and germinated cells were counted on three different fields selected at random. The percentage of germinated spores was plotted against the initial spore optical density.</p

7-AMC adducts detected in <i>wt B. cereus</i> conditioned supernatants.

<p>Wild type <i>B. cereus</i> 569 spores were resuspended in 200 µl TMB buffer to OD₅₈₀ = 1. Spores were treated with 0.2 mM inosine and supernatants were collected 30 min post-inosine addition. Collected supernatants were labeled with 7-AMC. 7-AMC adducts sere separated by RP-HPLC and identified by mass spectrometry. Concentrations were calculated by fluorescence spectroscopy.</p