Decoupling bile acid 7α-dehydroxylation from colonization resistance to Clostridioides difficile
Secondary bile acids, generated through microbial transformation of primary bile acids secreted in bile, play a role in shaping intestinal microbial communities, modulating host immunity, and regulating energy metabolism. In vitro studies have shown that the balance between primary and secondary bile acids strongly affects spore germination, growth, and cellular physiology of Clostridioides difficile, a major nosocomial gut pathogen. In vivo correlations between microbiome composition, bile acid metabolome, and colonization resistance have led to the hypothesis that 7-dehydroxylating bacteria such as Clostridium scindens protect against C. difficile infection by producing secondary bile acids like deoxycholic acid. However, due to the genetic intractability of known 7-dehydroxylating species, direct experimental validation of this causal relationship has been challenging. In this study, we leveraged the first available 7-dehydroxylation-deficient baiH mutant to test the direct role of 7-dehydroxylated bile acid production in C. difficile colonization resistance in vivo. We colonized gnotobiotic mice with isogenic wild-type or baiH strains of the recently described 7-dehydroxylating species Faecalicatena contorta, including wild-type C. scindens-colonized mice as a positive control. Wild-type F. contorta accumulated 7-dehydroxylated bile acids at levels equivalent to C. scindens, in a strictly baiH-dependent manner. However, despite equivalent bile acid profiles, wild-type F. contorta failed to replicate the C. difficile-restrictive phenotype observed with C. scindens. These findings demonstrate that commensal clostridial 7-dehydroxylation alone is not sufficient for enhancing colonization resistance to C. difficile. Our results highlight the existence of additional, potentially bile acid-independent mechanisms by which certain commensals mediate protection, with important implications for microbiota-based therapies. Importance7-dehydroxylated secondary bile acids, including deoxycholic acid and lithocholic acid, produced by commensal clostridia are widely assumed to inhibit the important nosocomial pathogen Clostridioides difficile, yet their precise role in colonization resistance remains unresolved. Using a defined mouse microbiota and an isogenic Faecalicatena contorta strain pair differing in a single 7-dehydroxylation gene (baiH), we show that restoration of secondary bile acid production is not sufficient to delay C. difficile colonization in vivo. This contrasts with the protective effect of Clostridium scindens, which generates a similar bile acid profile. Our findings uncouple bile acid metabolism from protection and suggest that additional, strain-specific functions - such as nutrient competition or antimicrobial production - play a critical role. Understanding these mechanisms is essential for the rational design of next-generation microbiota-based therapies to prevent or treat recurrent C. difficile infection.