Genomic surveillance of Clostridioides difficile transmission and virulence in a healthcare setting.

Clostridioides difficile infection (CDI) is a major cause of healthcare-associated diarrhea, despite the widespread implementation of contact precautions for patients with CDI. Here, we investigate strain contamination in a hospital setting and genomic determinants of disease outcomes. Across two wards over six months, we selectively cultured C. difficile from patients (n=384) and their environments. Whole-genome sequencing (WGS) of 146 isolates revealed that most C. difficile isolates were from clade 1 (131/146, 89.7%), while only one isolate of the hypervirulent ST1 was recovered. Of culture-positive admissions (n=79), 19 (24%) of patients were colonized with toxigenic C. difficile on admission to the hospital. We defined 25 strain networks at ≤ 2 core gene SNPs; 2 of these networks contain strains from different patients. Strain networks were temporally linked (p<0.0001). To understand genomic correlates of disease, we conducted WGS on an additional cohort of C. difficile (n=102 isolates) from the same hospital and confirmed that clade 1 isolates are responsible for most CDI cases. We found that while toxigenic C. difficile isolates are associated with the presence of cdtR , nontoxigenic isolates have an increased abundance of prophages. Our pangenomic analysis of clade 1 isolates suggests that while toxin genes ( tcdABER and cdtR ) were associated with CDI symptoms, they are dispensable for patient colonization. These data indicate toxigenic and nontoxigenic C. difficile contamination persists in a hospital setting and highlight further investigation into how accessory genomic repertoires contribute to C. difficile colonization and disease.