Dogs in Rio de Janeiro as reservoirs of Clostridioides difficile ribotypes causing CDI in humans

Introduction: In the past decade, the incidence of community-acquired Clostridioides difficile infection (CA-CDI) has increased, suggesting a role for community reservoirs such as animals in its spread. Objective: This study aimed to isolate and characterize C. difficile strains from domestic dogs at veterinary clinics to enhance our understanding of C. difficile epidemiology in Rio de Janeiro. Material and methods: For this study 90 stool samples from dogs were collected and cultured in a selective medium (Clostridioides difficile Brucella agar - CDBA) for isolation. Species were identified by MALDI-TOF MS, with confirmation provided by PCR targeting the tpi gene. The antibiotic susceptibility test of the strains was performed using five antibiotics: vancomycin, metronidazole, moxifloxacin, rifampicin, and erythromycin. Strains resistant to metronidazole were further analyzed for the presence of the plasmid pCD-METRO using PCR. The presence of toxin genes (tcdA, tcdB, and cdtB) was investigated, alongside ribotyping and tcdC sequencing analyses. The strains were also tested for biofilm formation and motility. Results: C. difficile was isolated in 15.5 % (14/90) of the samples. Among the strains analyzed, 87.71 % (12/14) tested positive for both toxin genes tcdA and tcdB and belonged to ribotypes 106 (10/14) and 014/020 (2/14). The remaining 14.3 % (2/14) were non-toxigenic and were identified as ribotype 010. Regarding the antibiotic profile, 42.85 % (6/14) of the strains exhibited resistance to at least one antibiotic, including vancomycin (1/14) and metronidazole (1/14). The metronidazole-resistant strain was also positive for the plasmid pCD-METRO. All strains exhibited both biofilm formation and motility. Among the 12 toxigenic strains sequenced for the tcdC gene, two exhibited a deletion in the same region as the epidemic strain, NAP1 (RT027). Conclusion: Our study found some overlap between C. difficile ribotypes isolated from dogs and from cases of CDI in humans, and the C. difficile prevalence was higher in dogs with diarrhea (p = 0.034).