Sub-inhibitory concentrations of metronidazole alter the proteomic profile and biological effects of extracellular vesicles from Clostridioides difficile

BACKGROUND: Colonization with C. difficile is common in patients with inflammatory bowel disease (IBD). The use of antibiotics can trigger the transition from colonization to active infection, complicating IBD management and presenting challenges for the diagnosis of C. difficile infection (CDI). This study aims to investigate the effects of metronidazole-exposed outer membrane vesicles (EVs) derived from a toxigenic C. difficile strain with no detectable production of toxins A (TcdA) and B (TcdB) under the tested conditions on both the parental strain and host cells. METHODS: groups. Functional assays included: biofilm formation (crystal violet staining), host cell viability and migration (proliferation assays, flow cytometry, scratch assay), and pro-inflammatory cytokine secretion (ELISA). RESULTS: ) significantly inhibited Caco-2 cell migration. Meanwhile, Mi-EVs induced the secretion of TNF-α and IL-6 by macrophages in a dose-dependent manner. CONCLUSIONS: Sub‑inhibitory concentrations of metronidazole promote the secretion of C. difficile EVs, which in turn enhance bacterial biofilm formation, host cell viability, and inflammatory responses in a concentration‑dependent manner, while also inhibiting cell migration in a concentration‑dependent manner. These findings offer insights into the complex interplay between antibiotics, colonizing C. difficile strains, and the host in the context of IBD.