Metabolism of bile salts in mice influences spore germination in Clostridium difficile

Abstract

Clostridium difficile, a spore-forming bacterium, causes antibiotic-associated diarrhea. In order to produce toxins and cause disease, C. difficile spores must germinate and grow out as vegetative cells in the host. Although a few compounds capable of germinating C. difficile spores in vitro have been identified, the in vivo signal(s) to which the spores respond were not previously known. Examination of intestinal and cecal extracts from untreated and antibiotic-treated mice revealed that extracts from the antibiotic-treated mice can stimulate colony formation from spores to greater levels. Treatment of these extracts with cholestyramine, a bile salt binding resin, severely decreased the ability of the extracts to stimulate colony formation from spores. This result, along with the facts that the germination factor is small, heat-stable, and water-soluble, support the idea that bile salts stimulate germination of C. difficile spores in vivo. All extracts able to stimulate high level of colony formation from spores had a higher proportion of primary to secondary bile salts than extracts that could not. In addition, cecal flora from antibiotic-treated mice was less able to modify the germinant taurocholate relative to flora from untreated mice, indicating that the population of bile salt modifying bacteria differed between the two groups. Taken together, these data suggest that an in vivo-produced compound, likely bile salts, stimulates colony formation from C. difficile spores and that levels of this compound are influenced by the commensal gastrointestinal flora.

Figure 1. An in vivo-produced factor can stimulate CFU recovery from spores of C. difficile.

A) CD196 spores were incubated for 30 min. with water, 0.1% taurocholate (TA) (gray bar), or extracts from mice that were untreated (black bars) or treated with clindamycin (striped bars), streptomycin (white bar), or ampicillin (cross-hatched bar), then diluted and spread to BHIS plates. The CFU/ml were compared to the CFU/ml on BHIS plates containing taurocholate, which was considered to reflect 100% germination. Data are the mean of at least three experiments, with error bars representing the standard error of the mean. B) Plates from a CFU recovery experiment in which spores were incubated with water, TA, or cecal extract from untreated or clindamycin-treated mice as in (A), then serially diluted ten-fold and spotted onto BHIS or BHIS + TA plates. A representative experiment is shown.

Figure 2. Investigation of properties of the germination factor.

Cecal extracts from clindamycin-treated mice were A) boiled for 10 min., B) dialyzed against H₂O in a 1 kDa membrane, or C) extracted with ethyl acetate (EtOAc) and were tested as in Fig. 1.

Figure 3. Cholestyramine treatment of small intestinal and cecal homogenates decreases their ability to induce spore germination.

CD196 spores were incubated with the indicated germinant for 30 min, then diluted and spread onto BHIS plates. The CFU/ml were compared to the CFU/ml on BHIS plates containing taurocholate (TA), which was considered to reflect 100% germination. Data are the mean of at least three experiments, with error bars representing the standard error of the mean. ND, not done.

Figure 4. Proportions of primary bile salts in small intestinal and cecal extracts.

Quantitation of bile salts in small intestinal and cecal extracts from untreated and clindamycin-treated mice was performed as previously described . Data are reported as the mean percentages of total bile salts, with levels of primary bile salts (µM) indicated in the bars and error bars representing the standard error of the mean.

Figure 5. Modification of taurocholate by cecal flora and its ability to stimulate CFU recovery from spores.

Contents of the ceca from freshly euthanized, clindaymin-treated (striped bars) or untreated (black bars) mice were isolated under anaerobic conditions, washed 5 times to remove any bile salts present, and incubated with 0.1% taurocholate for 24 hours at 37° C. Samples were pelleted, and the supernatant was boiled and used in A) bile salt quantitation and B) CFU recovery assays. The CFU/ml were compared to the CFU/ml recovered from spores incubated with taurocholate, which was considered 100% germination. Data are the mean of at least three experiments, with error bars representing the standard error of the mean. (* p<0.05, Student's t-test).