A Single Amino Acid Substitution in <scp>CspA</scp> Increases Germination Sensitivity and Broadens Bile Salt Germinant Specificity in <i>Clostridioides difficile</i> Spores

Once regarded primarily as a healthcare-associated pathogen, Clostridioides difficile has increasingly been reported as a cause of community-acquired infection, raising questions about the contribution of animal and environmental reservoirs to persistence and transmission. Ribotype 033 (RT033) is predominantly associated with animal and environmental reservoirs, yet key traits underlying its ecology are poorly defined. Here we show that RT033 strains produce spores with enhanced germination sensitivity and expanded germinant specificity, enabling germination at low concentrations of bile-salts and in response to bile-salts that are typically inhibitory, including those more typical of animal hosts. Genetic analysis identified a single amino acid substitution (R1036I) in CspA, a component of the CspBAC germination apparatus, as the determinant of this phenotype. Expression of the RT033 cspBAC operon in a laboratory strain was sufficient to confer increased sensitivity and expanded bile-salt responsiveness. The R1036I substitution disrupts a conserved salt bridge at the CspA:CspC interface while not causing major destabilisation of the complex in the absence or presence of germinants and co-germinants. Together, our findings reveal a naturally occurring rewiring of bile-salt sensing in spores of RT033 strains that likely reflects adaptation to animal-associated bile-acid environments and may influence persistence, transmission, and zoonotic potential of this lineage.