Resource-access breadth marks protective strains against toxigenic Clostridioides difficile

Abstract Antibiotic disruption of gut microbiota creates opportunities for toxigenic Clostridioides difficile . One strategy is to occupy this niche with a non-toxigenic or weakly virulent strain, but even strains that cause little disease may fail to compete during pathogen challenge. We screened 21 sequence type 1 (ST1) isolates for their ability to protect mice co-colonized with toxigenic VPI10463, separating disease potential from protection. Some strains, such as ST1-75, caused little disease and protected, whereas others, such as ST1-68, caused little disease but failed to protect. Metabolomics showed that ST1-75 depleted more proline than ST1-68, whereas another protective strain, ST1-6, had a different metabolic profile; both protective strains nevertheless responded to many substrates. Substrate-utilization profiling revealed that protection across the 21 isolates correlated with amino-acid overlap with VPI10463, but not overall nutrient overlap or shared genome-encoded protein families. Relative amino-acid access explained variation beyond mono-colonization disease potential, and a consumer-resource model showed how nutrient overlap together with additional resource access can support persistence during competition. Additional experiments support that ST1-mediated protection occurs even in the absence of mature B and T cells. These findings distinguish low disease potential from protection, link protection to strain competition, and identify relative resource access as an informative phenotype for prioritizing strains for biotherapeutic testing. Importance Clostridioides difficile causes antibiotic-associated intestinal disease, but beneficial C. difficile strains could prevent this disease by competing with harmful strains. Selecting such strains requires more than identifying strains that cause little disease on their own: they must also compete effectively against a virulent one. We show that related C. difficile strains vary in their protective ability, and we linked this to their capacity to access amino acids and other resources used by the pathogen. Our findings highlight resource access as a key trait for selecting candidate strains and offer an ecological framework for developing live biotherapeutics against C. difficile disease.