We aimed to investigate the prevalence, genotypic diversity, and clonality of Clostridioides difficile strains from household composters and faeces of small mammals captured nearby.

Compost piles from three locations were sampled, with three compost samples collected from each bin. Additionally, 5–6 small mammals were trapped around each compost pile. C. difficile was isolated from faecal and compost samples and characterized with PCR ribotyping, toxinotyping, and genome sequencing.

C. difficile was detected in all nine compost samples, while only two (12.5 %) of 16 small mammal faecal samples tested positive. Ten PCR ribotypes (RTs) were identified, with only one, RT014/020, being toxigenic. The remaining nine RTs belonged to two cryptic clades, C-II and C-III. Some of the isolates from cryptic clades i.e. RT SLO 308 carried a divergent tcdA gene and yielded weakly positive results with C. diff Quik Chek Complete immunoassay. Clonal C. difficile isolates of RT SLO 308 were found at the same site in both a compost sample and a small mammal, suggesting potential transmission. Clonality was also observed among isolates from different compost samples within a single pile.

While our study could not establish the exact direction of C. difficile transmission between compost and mice, it highlights the role of small mammals within the One Health framework of C. difficile transmission pathways.