Bile acid-mediated inhibition of C. difficile by P. hiranonis revealed through a ΔbaiCD mutant

The biotransformation of primary to secondary bile acids via 7α-dehydroxylation is key in the onset of C. difficile- associated disease, due to their role in the germination and outgrowth of spores in the gut. Clostridium scindens , a gut commensal, has been implicated in bile acid-mediated colonisation resistance against C. difficile, but research has been hindered by its genetic intractability. Here we demonstrate the use of genetic tools in an alternative 7α-dehydroxylating species, Peptacetobacter hiranonis, for the generation of bile acid associated ( bai ) gene loss-of-function mutations. An appropriate vector backbone was first identified for successful conjugation into P. hiranonis , and the inducible CRISPR-Cas9 system RiboCas used to generate a baiCD deletion. Biotransformations of cholic acid (CA) by the Δ baiCD strain were assessed using UHPLC-HRMS. Bile acid-mediated colonisation resistance against C. difficile was then assayed using supernatant challenge and co-culture studies. The Δ baiCD strain was shown to be incapable of 7α-dehydroxylation, with no conversion of CA to deoxycholic acid (DCA). Supernatant challenge assays demonstrated that when incubated with CA the Δ baiCD strain did not result in the delayed germination of C. difficile spores observed with the wildtype strain. Co-culture assays also demonstrated that the reduction in vegetative growth of C. difficile when incubated with wildtype P. hiranonis in the presence of CA was not observed in the Δ baiCD strain. These results illustrate bile-acid mediated resistance to C. difficile by P. hiranonis, furthering our understanding of colonisation resistance by 7α-dehydroxylating bacteria. Moreover, as the first null mutant in any classical 7α-dehydroxylating species, the Δ baiCD strain could be utilised as a control strain in future bile acid studies. • An inducible CRISPR-Cas9 system was utilised for the first time in P. hiranonis , generating a bai gene mutant. • The Δ baiCD strain is incapable of converting cholic acid to deoxycholic acid. • Δ baiCD results in removal of colonisation resistance markers when P. hiranonis is co-cultured with C. difficile and cholic acid.