<i>Clostridioides difficile</i> colonization is not mediated by bile salts and utilizes Stickland fermentation of proline in an <i>in vitro</i> model

Treatment with antibiotics is a major risk factor for <i>Clostridioides difficile</i> infection, likely due to depletion of the gastrointestinal microbiota. Two microbiota-mediated mechanisms thought to limit <i>C. difficile</i> colonization include the conversion of conjugated primary bile salts into secondary bile salts toxic to <i>C. difficile</i> growth and competition between the microbiota and <i>C. difficile</i> for limiting nutrients. Using a continuous flow model that simulates the nutrient conditions of the distal colon, we investigated how treatment with 6 clinically used antibiotics influenced susceptibility to <i>C. difficile</i> infection in 12 different microbial communities cultivated from healthy individuals. Antibiotic treatment reduced microbial richness; disruption varied by antibiotic class and microbiota composition, but did not correlate with <i>C. difficile</i> susceptibility. Antibiotic treatment also disrupted microbial bile salt metabolism, increasing levels of the primary bile salt, cholate. However, changes in bile salt did not correlate with increased <i>C. difficile</i> susceptibility. Furthermore, bile salts were not required to inhibit <i>C. difficile</i> colonization. We tested whether amino acid fermentation contributed to the persistence of <i>C. difficile</i> in antibiotic-treated communities. <i>C. difficile</i> mutants unable to use proline as an electron acceptor in Stickland fermentation due to disruption of proline reductase (<i>prdB</i>-) had significantly lower levels of colonization than wild-type strains in four of six antibiotic-treated communities tested. The inability to ferment glycine or leucine as electron acceptors, however, was not sufficient to limit colonization in any communities. The data provide further support for the importance of bile salt-independent mechanisms in regulating the colonization of <i>C. difficile</i>.IMPORTANCE<i>Clostridioides difficile</i> is one of the leading causes of hospital-acquired infections and antibiotic-associated diarrhea. Several potential mechanisms through which the microbiota can limit <i>C. difficile</i> infection have been identified and are potential targets for new therapeutics. However, it is unclear which mechanisms of <i>C. difficile</i> inhibition represent the best targets for the development of new therapeutics. These studies demonstrate that in a complex <i>in vitro</i> model of <i>C. difficile</i> infection, colonization resistance is independent of microbial bile salt metabolism. Instead, the ability of <i>C. difficile</i> to colonize is dependent upon its ability to metabolize proline, although proline-dependent colonization is context dependent and is not observed in all disrupted communities. Altogether, these studies support the need for further work to understand how bile-independent mechanisms regulate <i>C. difficile</i> colonization.