High-Throughput Screening of FDA-Approved Drugs for Antibacterial and Antibiofilm Activities Against Multidrug-resistantPseudomonas aeruginosa

Background: Multidrug-resistant (MDR) Pseudomonas aeruginosa poses a critical clinical challenge because of its intrinsic resistance mechanisms and strong biofilm-forming capacity. However, effective therapeutic options remain limited. Drug repurposing offers a rapid and cost-effective strategy to identify novel antimicrobial candidates. Methods: A phenotype-based high-throughput screening of 2808 FDA-approved compounds was performed against MDR P. aeruginosa. Active compounds were further evaluated through antibacterial and antibiofilm assays; mechanistic characterization; and host safety assessment, including hemolysis and cytotoxicity assays. Results: Three repurposed drugs, namely, rifabutin, nafamostat, and tegaserod, exhibited notable antibacterial activity against MDR P. aeruginosa. Rifabutin and nafamostat demonstrated effective antibacterial and antibiofilm activities while maintaining favorable safety profiles. By contrast, although tegaserod showed strong antibacterial and antibiofilm effects, it exhibited significant hemolytic and cytotoxic liability at levels below its minimum inhibitory concentrations. Conclusions: By using a systematic screening–prioritization–safety filtering framework, this study identified rifabutin and nafamostat as prioritized repurposing candidates against MDR P. aeruginosa, highlighting the importance of early safety evaluation in antimicrobial drug repurposing.