Antibacterial and antivirulence activities of abafungin against Clostridioides difficile: in vitro and in vivo studies.

Clostridioides difficile infection (CDI) remains a major cause of healthcare-associated diarrhea worldwide, and is classified as an urgent public health threat by the Centers for Disease Control and Prevention. Current frontline therapies, including vancomycin and fidaxomicin, are associated with disruption of the intestinal microbiota, recurrence, and emerging reduced susceptibility, underscoring the need for additional therapeutic strategies. Using a drug-repurposing approach, we identified the antifungal agent abafungin as a potent inhibitor of C. difficile. Abafungin is an arylguanidine antifungal with reported sporicidal activity against fungal pathogens, a property of particular relevance to CDI, given the central role of spores in disease persistence and recurrence. Herein, abafungin exhibited potent in vitro activity against a diverse panel of clinical C. difficile isolates, inhibiting 50% (MIC5₀) and 90% (MIC₉₀) of the tested strains at the concentrations of 0.25 μg/mL and 0.5 μg/mL, respectively, while demonstrating minimal activity against representative gut commensal bacteria. At sub-inhibitory concentrations, abafungin significantly reduced toxin production (31.8%), decreased spore formation (~4-log₁₀ reduction), and inhibited spore germination. Abafungin showed favorable gastrointestinal stability and demonstrated synergistic activity with vancomycin and metronidazole. Transcriptomic analysis revealed that abafungin disrupts metal ion transport and intracellular metal homeostasis in C. difficile, a mechanism supported by the antagonistic effect of Mg²+ supplementation on its antibacterial activity. These observations suggest a potential association between abafungin activity and metal-responsive cellular pathways. In a murine model of recurrent CDI, a combination therapy of abafungin and vancomycin significantly improved survival (83.3%) compared with vancomycin monotherapy (16.6%). Collectively, these findings establish abafungin as a potential anti-C. difficile inhibitor that suppresses C. difficile growth and virulence-associated processes and enhances the therapeutic efficacy of vancomycin in vivo. Also, these observations provide a basis for further studies exploring abafungin as a useful lead scaffold for anti-C. difficile drug discovery.