Antimicrobial susceptibility and virulence gene analysis of Clostridioides difficile from healthcare-associated infections in Southern Brazil

OBJECTIVES: Clostridioides difficile (CD) is the leading cause of antimicrobial-associated diarrhea (AAD) and causes a spectrum of disease with high recurrence, morbidity, and mortality. CD pathogenicity is primarily driven by toxins A and B, and, in some strains, by the binary toxin. This study evaluated antimicrobial susceptibility profiles and resistance and virulence genes profiles of isolates from patients with antimicrobial‑associated diarrhea in hospitals across southern Brazil. METHODS: Stool samples from 371 patients were analyzed by toxigenic cultures. Antimicrobial susceptibility to vancomycin, metronidazole, and ciprofloxacin was determined by gradient strip testing. Toxin production was confirmed by enzyme immunoassay and Polymerase Chain Reaction targeting toxin genes. Genomic DNA from selected isolates was sequenced on an Illumina MiSeq and analyzed using bioinformatics tools. RESULTS: Seventy-two CD isolates were recovered, of which 54 were toxigenic. All isolates were susceptible to metronidazole and vancomycin; all were resistant to ciprofloxacin. Multilocus sequence typing (MLST) identified 13 different sequence types (STs), with ST42 (23.5%), ST5 (20.6%), and ST2 (17.6%) most prevalent. Toxin genes and virulence factors related to adhesion and sporulation were detected. Although genes associated with vancomycin resistance were identified genotypically, no phenotypic resistance was observed. CONCLUSIONS: Clinical CD isolates from southern Brazil display substantial genomic diversity, with variable STs and distinct resistance and virulence gene profiles. An understanding of CD genomic diversity and toxin profiles is essential for guiding the development of new diagnostic tools, therapeutic strategies, and public-health interventions.