Ionomycin Exhibits Potent and Selective Bactericidal Activity Against Clostridioides difficile Through...

Clostridioides difficile represents a critical global health concern due to its high morbidity, mortality, and recurrent infections associated with current therapeutic options. There is an urgent need for novel, selective anti-C. difficile therapeutic agents. Screening the microbial metabolite library against C. difficile identified ionomycin, a calcium ionophore produced by Streptomyces conglobatus, as a potent inhibitor for C. difficile. Ionomycin exhibited potent activity against 30 C. difficile isolates, with minimum inhibitory concentrations of 1 μg/mL and 2 μg/mL against 50% (MIC₅₀) and 90% (MIC₉₀) of isolates, respectively. Time-kill assays revealed rapid bactericidal activity, achieving a ≥ 3 log₁₀ reduction within 8 h, surpassing the efficacy of vancomycin and fidaxomicin. At subinhibitory concentrations, ionomycin markedly reduced toxin production (~20%) and spore formation (~3 log₁₀ CFU/mL). Moreover, ionomycin exerted a potent activity against C. difficile spore germination and significantly prevented the toxin production from the germinating C. difficile cells. Importantly, ionomycin displayed limited activity against representative gut microbiota strains, indicating a favorable selectivity profile. Mechanistic investigations revealed a calcium-dependent mode of action, as exogenous calcium enhanced ionomycin-mediated bactericidal activity, whereas calcium chelation attenuated its effects. Consistent with this mechanism, ionomycin disrupted C. difficile membrane potential, an effect that was further potentiated by calcium supplementation. Collectively, these findings identify ionomycin as a potent and selective anti-C. difficile agent with a distinct calcium-dependent mechanism of action, supporting its potential as a promising therapeutic candidate warranting further investigation.