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 ΔbaiCD 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 ΔbaiCD 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 ΔbaiCD 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 ΔbaiCD 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 ΔbaiCD strain could be utilised as a control strain in future bile acid studies.