Carbadox, a veterinary growth promoter exhibits anticlostridial activity through induction of oxidative stress in Clostridioides difficile

BACKGROUND: Clostridioides difficile is the leading cause of antibiotic-associated diarrhea in hospitalized patients and is classified as an urgent public health threat. Current therapies, while effective, are limited by antibiotic-induced dysbiosis, treatment failure, and high recurrence rates, underscoring the need for novel therapeutics. Carbadox (CRX), an antibacterial growth promoter, has recently been identified as a potential anti-C. difficile inhibitor. This study aimed to comprehensively evaluate the anti-C. difficile activity, mechanism of action, and in vivo efficacy of CRX. METHODS: CRX activity was assessed against a diverse panel of clinical C. difficile isolates using MIC determination, time-kill kinetics, and post-antibiotic effect assays. Its effects on toxin production were evaluated at subinhibitory concentrations, while post-germination vegetative growth was assessed under germination-permissive conditions at bactericidal concentrations. Mechanistic studies included macromolecular synthesis assays, reactive oxygen species (ROS) quantification, and antioxidant rescue experiments. Potential drug interactions were tested in combination assays with standard-of-care agents. Finally, the efficacy of CRX was examined in both acute and recurrent C. difficile infection (CDI) mouse models. RESULTS: CRX demonstrated potent in vitro activity, inhibiting 50% of clinical isolates at an MIC₅₀ of 1 µg/mL. In time-kill assays, CRX rapidly cleared high bacterial inoculum within 4 h and displayed a prolonged post-antibiotic effect of up to 14 h. At subinhibitory concentrations, CRX significantly reduced toxin production compared with vancomycin, while at bactericidal concentrations it suppressed vegetative growth following spore germination. Mechanistic analysis revealed marked inhibition of DNA synthesis and increased oxidative stress-associated DNA damage signals, which was reversed by N-acetyl-L-cysteine supplementation. In vivo, CRX showed no recurrence among mice surviving to the end of therapy in both CRX treatment regimens, outperforming vancomycin in relapse prevention. CONCLUSION: CRX exhibits strong anti-C. difficile activity through DNA synthesis inhibition and increased oxidative stress-associated DNA damage, coupled with suppression of toxin production and inhibition of vegetative growth post-spore germination. Its efficacy in both acute and recurrent CDI mouse models highlights CRX as a useful proof-of-concept scaffold for the development of safer analogs targeting C. difficile. Further optimization and safety evaluation will be essential to advance its translational potential.