Effect of sub-minimum inhibitory concentrations of metronidazole, vancomycin, and fidaxomicin on growth rate and virulence of Clostridioides difficile.

Clostridioides difficile infection (CDI) is one of the most common health care-associated infections, primarily driven by the use of broad-spectrum antibiotics, which disrupts gut microbiota and facilitates bacterial overgrowth. This study aimed to investigate the effect of sub-minimum inhibitory concentrations (sub-MICs: 0.25× and 0.5×) of metronidazole (MTZ), vancomycin (VAN), and fidaxomicin (FDX) on growth rate, motility, toxin production, and sporulation of toxigenic C. difficile ribotype (RT001) and a non-toxigenic C. difficile strain (ATCC 700057, RT038). RT-qPCR was employed to assess the expression level of C. difficile genes encoding flagella (fliC, fliD), toxins (tcdA, tcdB), sporulating regulator (spo0A), and surface-associated proteins (fbp68, slpA, cwp84). Our findings showed that all antibiotics significantly delayed the growth rate of C. difficile strains, with FDX exhibiting the most substantial impact. Sub-MICs (0.5× MIC) reduced motility markedly, correlating with downregulated flagellar gene (fliC, fliD). Furthermore, sub-MICs of VAN and FDX suppressed both toxin production and sporulation. Notably, FDX uniquely downregulated the surface layer protein gene slpA across both strains. These results show that antibiotic sub-MICs can modulate bacterial virulence, highlighting the need to reassess the impact of antibiotic dosing on C. difficile pathogenesis during treatment.