Abstract Clostridioides difficile (C. difficile) strains belonging to the epidemic BI/NAP1/027 (RT027) group have been associated with increased transmissibility and disease severity. In addition to the major toxin A and toxin B virulence factors, RT027 strains also encode the CDT binary toxin. Our lab previously identified a toxigenic RT027 isolate, ST1-75, that is avirulent in mice despite densely colonizing the colon. Here, we show that co-infecting mice with the avirulent ST1-75 and virulent R20291 strains protects mice from colitis due to rapid clearance of the virulent strain and persistence of the avirulent strain. Although avirulence of ST1-75 is due to a mutation in the cdtR gene, which encodes a response regulator that modulates the production of all three C. difficile toxins, the ability of ST1-75 to protect against acute colitis is not directly attributable to the cdtR mutation. Metabolomic analyses indicate that the ST1-75 strain depletes amino acids more rapidly than the R20291 strain and supplementation with amino acids ablates ST1-75’s competitive advantage, suggesting that the ST1-75 strain limits the growth of virulent R20291 bacteria by amino acid depletion. Since the germination kinetics and sensitivity to the co-germinant glycine are similar for the ST1-75 and R20291 strains, our results identify the rapidity of in vivo nutrient depletion as a mechanism providing strain-specific, virulence-independent competitive advantages to different BI/NAP1/027 strains. They also suggest that the ST1-75 strain may, as a biotherapeutic agent, enhance resistance to CDI in high-risk patients. Importance Clostridioides difficile infections (CDI) are prevalent in healthcare settings and are associated with high recurrence rates. Therapies to prevent CDI, including recent FDA-approved live biotherapeutic products, are costly and have not been used to prevent primary infections. While a nontoxigenic C. difficile strain (NTCD-M3) protects against virulent CDI in animals and reduced CDI recurrence in a phase 2 clinical trial, protection against CDI recurrence in humans was variable and required high doses of the nontoxigenic strain. Here we show that an avirulent C. difficile isolate, ST1-75, efficiently outcompetes virulent C. difficile strains in mice when co-infected at a 1:1 ratio. Our data suggest that inter-strain competition results from ST1-75’s more rapid depletion of amino acids than the virulent R20291 strain. Our study identifies inter-strain nutrient depletion as a potentially exploitable mechanism to reduce the incidence of CDI.

Protection against Clostridioides difficile disease by a naturally avirulent C. difficile strain