Escherichia coli remains the leading cause of urinary tract infections worldwide, while antimicrobial resistance (AMR) poses a growing public health threat, contributing to over 1.2 million deaths annually in 2019 and projected to cause 10 million deaths per year by 2050. Extensive and often inappropriate use of fluoroquinolones, particularly ciprofloxacin, has accelerated resistance development in E. coli, largely driven by target-site mutations and efflux pump overexpression, thereby severely limiting therapeutic options. Innovative strategies that enhance antibiotic efficacy are therefore urgently required to address this challenge.
In this study, the antibacterial activities of antimicrobial peptides (AMPs)—Batroxicidin (BatxC), Crotalicidin (Ctn), Latarcin-2a (Ltc-2a), and Tat-2—were evaluated individually and in combination with ciprofloxacin against ciprofloxacin-resistant clinical E. coli isolates. Minimum inhibitory concentrations (MICs) were determined using the broth microdilution method. Drug–drug interactions were assessed by checkerboard assays and interpreted using the fractional inhibitory concentration index (FICI) according to EUCAST/CLSI criteria. The contribution of efflux mechanisms was examined using the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Cytotoxicity was evaluated in HeLa cells using the MTT assay.
Ciprofloxacin combinations with BatxC, Ctn, and Ltc-2a resulted in fourfold reductions in ciprofloxacin MICs with additive interactions, whereas the ciprofloxacin–Tat-2 combination produced a 16-fold MIC reduction, consistent with partial synergy. Notably, triple combinations of ciprofloxacin, AMPs, and CCCP reduced ciprofloxacin MICs to <1 µg/mL (≥64-fold), highlighting efflux inhibition-mediated antibiotic resensitization. Cytotoxicity analysis showed that Ctn exhibited excellent biocompatibility, with ciprofloxacin–Ctn combinations maintaining ≥75% cell viability.
Overall, AMP-based combination therapy represents a promising, mechanism-driven strategy to restore ciprofloxacin activity against resistant E. coli. Further in vivo validation and mechanistic studies are required to support clinical translation.
