Multi-strain Clostridioides difficile infection: Increased Detection and Clinical Implications

Clostridioides difficile infection (CDI) is an antibiotic-associated diarrheal disease. Mixed infection has been previously reported. However, its clinical implications are not well defined. In this study, we evaluated the presence of multi-strain (MS) isolates in fecal specimens from patients diagnosed with CDI at a tertiary hospital in Virginia. C. difficile colonies (three per sample) were obtained from fecal specimen cultures (N=174) from patients diagnosed with CDI from April 2021 to February 2022. The toxin gene profile of each isolate was determined by PCR using specific primers for triose phosphate isomerase ( tpi ), toxin A ( tcdA ), toxin B ( tcdB ), binary toxin A ( cdtA), and binary toxin B ( cdtB ). A subset of fecal specimens (n=62) with Ct values ≤ 25 for tcdB underwent total bacterial isolation in TCCFA and were processed for whole-genome shotgun sequencing. Based on the toxin gene profile of each isolate, MS infection was identified in 32.8% (57/174) of patients. Among all the isolates (N=522), tcdA + tcdB + cdtA - cdtB - (32.38%), tcdA + tcdB + cdtA + cdtB + (32.18%), and tcdA + tcdB + cdtA + cdtB - (28.5%) were the most common toxin gene profiles identified. An analysis of clinical data of 160 patients showed that age, gender, recurrence, severity of disease, mortality, and being infected by a strain expressing the binary toxin gene did not distinguish between being infected by MS or SS (single strain). Among the MS cases (N=52), the presence of non-toxigenic strains decreased the risk of severe CDI (p=0.0098). Whole-genome shotgun sequencing analysis confirmed mixed infection in our cohort and identified that the C. difficile strains ST2 (clade 1, S-0352) and ST42 (clade 1, W0023a) are the most common sequence types in MS and SS cases, respectively. Overall, our findings indicated a high prevalence of MS infection among C. difficile -infected patients, and a non-toxigenic C. difficile strain association with less severe disease in MS cases. These results suggest that specific strains may differentially predominate in MS or SS infection and that the presence of non-toxigenic C. difficile strains in MS infection influences disease severity. The impact of MS infection on the outcomes of anti- C. difficile treatments warrant further investigation.