Clostridioides difficilecolonization is not mediated by bile salts and requires Stickland fermentation of proline in anin vitromodel of infection

Abstract

ABSTRACTTreatment with antibiotics is a major risk factor forClostridioides difficileinfection, likely due to depletion of the gastrointestinal microbiota. Two microbiota-mediated mechanisms thought to limitC. difficilecolonization include conversion of conjugated primary bile salts into secondary bile salts toxic toC. difficilegrowth, and competition between the microbiota andC. difficilefor limiting nutrients. Using a continuous flow model of the distal colon, we investigated how treatment with six clinically-used antibiotics influenced susceptibility toC. difficileinfection 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 withC. difficilesusceptibility. Antibiotic treatment also disrupted microbial bile salt metabolism, increasing levels of the primary bile salt, cholate, and decreasing levels of the secondary bile salt, deoxycholate. However, decreased levels of deoxycholate did not correlate with increasedC. difficilesusceptibility. Further, bile salts were not required to inhibitC. difficilecolonization. We tested whether amino acid fermentation contributed to persistence ofC. difficilein antibiotic-treated communities.C. difficilemutants unable to use proline as an electron acceptor in Stickland fermentation due to disruption of proline reductase (ΔprdB) had significantly lower levels of colonization than wild-type strains in four of six antibiotic-treated communities tested. This data provides further support for the importance of bile salt-independent mechanisms in regulating colonization ofC. difficile.IMPORTANCEC. difficileis one of the leading causes of hospital-acquired infections and antibiotic-associated diarrhea. Several potential mechanisms through which the microbiota can limitC. difficileinfection have been identified and are potential targets for new therapeutics. However, it is unclear which mechanisms ofC. difficileinhibition represent the best targets for development of new therapeutics. These studies demonstrate that in a complexin vitromodel ofC. difficileinfection, colonization resistance is independent of microbial bile salt metabolism. Instead, the ability ofC. difficileto 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 regulateC. difficilecolonization.