The antiarrhythmic drugs dronedarone and amiodarone exhibit potent in vitro and in vivo activity against Clostridioides difficile.

Clostridioides difficile (C. difficile) is the most prevalent cause of healthcare-associated diarrhea. The limited number of FDA-approved antibiotics for C. difficile infection (CDI), along with increasing rates of treatment failure and recurrence, highlight the critical need for new therapeutic options. Here, we report the anti-C. difficile activity of the antiarrhythmic drugs, dronedarone (DRO) and amiodarone (AMD). Both drugs demonstrated strong efficacy against a panel of 25 clinical isolates of C. difficile, with MIC50 values of 4 µg/mL for DRO and 8 µg/mL for AMD. In a time-kill assay, DRO outperformed the approved drugs, vancomycin (VAN) and fidaxomicin (FDX), reducing the high bacterial inoculum below the limit of detection within 2 h. Additionally, both drugs were more effective than VAN at preventing the formation and germination of C. difficile spores. Mechanistic studies indicated that these drugs exert their effects by disrupting the bacterial cell membrane. Furthermore, in the C. difficile recurrence mouse model, both drugs showed a preventive effect comparable to that of VAN. Our findings suggest that these antiarrhythmic drugs have potential as compelling lead molecule for CDI drug discovery, warranting further investigation and potentially paving the way for new anti-C. difficile therapies.