Linked mutations within the pathogenicity locus of Clostridioides difficile increase virulence

Abstract

Background The clinical manifestations of Clostridioides difficile infections range from diarrhoea to pseudomembranous colitis (PMC) and death. We evaluated the association between gene content in C. difficile clinical isolates and disease severity. Methods Fifty-three C. difficile isolates were subjected to Sanger sequencing, clinical data were used to analyse the association of gene content with disease severity, and 83 non-duplicate isolates were collected to confirm the results. Virulence was further examined by functional in vitro and in vivo experiments. Results Among the 53 C. difficile isolates, ribotypes 017 (n = 9, 17.0%) and 012 (n = 8, 15.1%) were predominant. Fifteen strains exhibited a correlation between mutations of pathogenicity locus genes (tcdB, tcdC, tcdR, and tcdE) and were named linked-mutation strains. Ribotypes are not associated with clinical PMC and Linked-mutation strains. The proportion of patients with PMC was higher in the group infected with linked-mutation strains than in the non-linked-mutation group (57.14% vs. 0%, p < 0.001). The linked-mutation rate of C. difficile was higher in patients with PMC than in patients without PMC (89.47% vs. 7.8%, p < 0.0001). Linked-mutation strains showed greater cytotoxicity in vitro and caused more severe tissue damage in a mouse model. Conclusions Linked-mutation strains are associated with high virulence and PMC development. This result will help monitor the clinical prognosis of C. difficile infection and provide key insights for developing therapeutic targets and monoclonal antibodies.