Contact dependent suppression of Clostridioides difficile sporulation by enterococci requires the endocarditis and biofilm associated pilus.

Clostridioides difficile is a healthcare-associated infection that arises when broad-spectrum antibiotic treatment disrupts the gut microbiota and is transmitted by highly resistant spores. Vancomycin-resistant Enterococcus faecium (VRE) is an opportunistic pathogen frequently co-isolated from C. difficile patients. We found that C. difficile sporulation is significantly reduced in VRE-C. difficile co-culture. Physical separation of C. difficile and VRE in transwell co-culture restored sporulation. Mixed macrocolony culture assays on solid agar confirmed physical contact is necessary for sporulation inhibition. We screened a panel of enterococci and found that most strains reduce sporulation, except Enterococcus saccharolyticus, which lacks predicted surface displayed virulence factors in its genome. We performed a candidate gene screen using an Enterococcus faecalis OG1RF transposon library and found that an insertion in the major pilin ebpC partially restored C. difficile sporulation in co-culture. These data were confirmed with in-frame deletions in the ebpABC pilus operon and a clinical isolate of E. feacalis lacking ebpABC. These findings suggest enterococci modulate C. difficile sporulation through a contact-dependent mechanism involving the Ebp pilus.