Repurposing azole antifungals as rapid bactericidal, membrane-disruptive therapeutics against <i>Clostridioides difficile</i>

ABSTRACT Clostridioides difficile is a leading cause of healthcare-associated infections and remains an urgent global public health concern. Current FDA-approved therapies for C. difficile infection (CDI) are limited and are frequently associated with treatment failure and high rates of disease recurrence. To address this unmet clinical need, we employed a drug repurposing strategy focused on FDA-approved compounds with established safety profiles and limited systemic absorption characteristics, which are desirable for localized gastrointestinal infections. Through this approach, we identified the antifungal azoles miconazole, econazole, and tioconazole as potent inhibitors of C. difficile . These compounds exhibited robust in vitro activity against a diverse panel of clinical C. difficile isolates, with MIC 50 values of 1, 2, and 2 µg/mL, respectively. Time-kill analyses demonstrated that all tested azoles exerted rapid bactericidal activity within 2 h, exceeding the killing kinetics of the current standard-of-care agents vancomycin and fidaxomicin, likely attributable to their membrane-disruptive mechanism that compromises cytoplasmic membrane integrity and leads to rapid cellular collapse. Additionally, the azoles displayed prolonged post-antibiotic effects (&gt;12 h). Furthermore, the azoles exhibited limited activity against two representative members of the commensal intestinal microbiota ( Bifidobacterium and Lactobacillus spp.) and retained antibacterial efficacy under physiologically relevant conditions, including high inoculum, variable pH, and simulated gastrointestinal fluids. Mechanistic studies supported membrane targeting as the primary mode of action. Azole exposure significantly disrupted bacterial membrane integrity, and scanning electron microscopy revealed marked morphological damage, including membrane distortion and cellular collapse, consistent with structural membrane damage. Notably, cholesterol supplementation increased C. difficile tolerance to azoles and other membrane-disrupting agents, suggesting that host cholesterol levels or dietary factors may influence CDI outcomes. Finally, in a Caenorhabditis elegans CDI infection model, all tested azoles significantly improved host survival following lethal C. difficile challenge. Collectively, our findings highlight the potential of azole antifungals as promising platforms that merit further investigation and modification in the pursuit of safer, more effective therapeutics for CDI treatment. IMPORTANCE Clostridioides difficile infection (CDI) is a serious and recurrent intestinal disease for which current treatment options remain limited and often disrupt the protective gut microbiota, increasing the risk of relapse. In this study, we show that the antifungal drugs miconazole, econazole, and tioconazole exhibit rapid and sustained activity against C. difficile under conditions that mimic the gastrointestinal environment. Unlike existing therapies, these agents display minimal activity against commensal gut bacteria and provide prolonged post-antibiotic effects. We also demonstrate that cholesterol, a common host-derived molecule, modulates bacterial susceptibility to these drugs, revealing a previously underappreciated link between host factors and therapeutic efficacy. Finally, these compounds confer significant protection in an in vivo Caenorhabditis elegans model of CDI, highlighting their potential as candidates for repurposing and further development.