A multisite genomic epidemiology study of Clostridioides difficile infections in the U.S. supports differential roles of healthcare versus community spread for two common strains

Clostridioides difficile is the leading cause of healthcare-associated infectious diarrhea. However, it is increasingly appreciated that healthcare-associated infections derive from both community and healthcare transmission, and that the primary sites of C. difficile transmission may be strain dependent. We conducted a multisite genomic epidemiology study to assess differential genomic evidence of healthcare vs. community spread for two of the most common C. difficile strains in the U.S.: sequence type (ST) 1 (associated with Ribotype 027) and ST2 (associated with Ribotype 014/020). Isolates recovered from stool specimens collected during standard clinical care at three geographically distinct U.S. medical centers between 2010 and 2018 underwent whole genome sequencing and phylogenetic analyses. ST1 and ST2 isolates both displayed some evidence of phylogenetic clustering by study site, but clustering was stronger and more apparent in ST1, consistent with our healthcare-based study more comprehensively sampling local transmission of ST1 compared to ST2 strains. Analyses of pairwise single nucleotide variant (SNV) distance distributions were also consistent with more evidence of healthcare transmission of ST1 compared to ST2, with 44% of ST1 isolates being within 2 SNVs of another isolate from the same geographic collection site compared to 5.5% of ST2 isolates (p-value = <0.001). Conversely, ST2 isolates were more likely to have close genetic neighbors across disparate geographic sites compared to ST1 isolates, further supporting non-healthcare routes of spread for ST2 and highlighting the potential for misattributing genomic similarity among ST2 isolates to recent healthcare transmission. Finally, we estimated a lower evolutionary rate for the ST2 lineage compared to the ST1 lineage using Bayesian timed phylogenomic analyses, and hypothesize that this may contribute to observed differences in geographic concordance among closely related isolates. Together, these findings suggest that ST1 and ST2, while both common causes of C. difficile infection in hospitals, show differential reliance on community and hospital spread. This conclusion supports the need for strain-specific criteria for interpreting genomic linkages and emphasizes the importance of considering differences in the epidemiology of circulating strains when devising interventions to reduce the burden of C. difficile infections. DATA SUMMARY All whole genome sequence data was uploaded to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under Bioproject accessions PRJNA595724, PRJNA561087, and PRJNA594943. Metadata that comply with patient privacy rules are included in the Supplementary Materials. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by the National Institutes of Health via U01AI12455 (A.M-J., E.S., V.B.Y.), the Molecular Mechanisms of Microbial Pathogenesis Training Grant (T32AI007528, A.M-J.), U01AI124290 (T.C.S., K.W.G.), and U01AI124275 (E.G.P., M.K.). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study was approved by the University of Michigan Institutional Review Board, the University of Houston Committee for the Protection of Human Subjects, and the Memorial Sloan Kettering Cancer Center Institutional Review Board. All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All whole genome sequence data was uploaded to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under Bioproject accessions PRJNA595724, PRJNA561087, and PRJNA594943. Metadata that comply with patient privacy rules are included in the Supplementary Materials.