Role of 7alpha-dehydroxylated secondary bile acids in Clostridioides difficile colonization resistance

Secondary bile acids, generated through microbial transformation of primary bile acids secreted in bile, play a significant role in shaping lower 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 7a-dehydroxylating bacteria such as Clostridium scindens protect against C. difficile infection by producing secondary bile acids like deoxycholic acid (DCA). However, due to the genetic intractability of known 7a-dehydroxylating species, direct experimental validation of this causal relationship has been challenging. In this study, we leveraged the first available 7a-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 OligoMM12 mice with isogenic wild-type or baiH-deficient mutant strains of the recently described 7a-dehydroxylating species Faecalicatena contorta and included wild-type C. scindens colonized OligoMM12 mice as a positive control. Wild-type F. contorta intestinally accumulated 7-dehydroxylated bile acids at levels equivalent to C. scindens, and 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 microbial 7a-dehydroxylation alone is not sufficient for 7-dehydroxylating bacteria to confer colonization resistance against 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