Combination of mitomycin C and low-dose metronidazole synergistically against <i>Clostridioides difficile</i> infection and recurrence prevention

<i>Clostridioides difficile</i> is an anaerobic, spore-forming pathogen responsible for illnesses ranging from mild diarrhea to life-threatening colitis. Current treatments rely on antibiotics such as vancomycin and metronidazole (MTZ), but high doses can disrupt gut microbiota, contributing to recurrent infections. Mitomycin C (MMC), a Food and Drug Administration-approved anticancer agent, is known to induce prophage activation in lysogenic bacteria. Given that over 70% of <i>C. difficile</i> strains harbor prophages, we evaluated MMC's potential to enhance antibiotic efficacy against <i>C. difficile</i> infection (CDI). <i>In vitro</i>, MMC synergized with MTZ to inhibit strain R20291 and clinical isolates of RT027 and RT078 while reducing the minimum bactericidal concentration of MTZ against biofilm-associated cells. Ex vivo assays using mouse fecal suspensions confirmed the enhanced killing effect of the combination. In a murine recurrence model, low-dose MTZ + MMC treatment significantly improved survival and reduced fecal spore counts compared to monotherapies or vancomycin. Importantly, the combination did not cause greater liver or kidney toxicity than other antibiotics and resulted in less colonic epithelial damage. Microbiota profiling revealed that MTZ + MMC better preserved gut microbial composition than standard treatments. These findings suggest that low-dose MTZ combined with MMC enhances antimicrobial efficacy while reducing toxicity and microbiota disruption, offering a promising strategy for CDI management.