Targeting WHO-Priority MDR and XDR Gram-Negative Ocular Isolates by Combining Conventional Antimicrobials with Membrane-Active Peptides.

Background/objectives: Multidrug-resistant and extensively drug-resistant Gram-negative pathogens are a major cause of severe ocular infections, yet treatment options are increasingly compromised by escalating antibiotic resistance. Membrane-active peptides (colistin and Mel4) offer the potential to restore antibiotic susceptibility. This study investigated the efficacy of peptide- and colistin-based adjuvant strategies in enhancing the activity of conventional antibiotics. Methods: Antibacterial activity was assessed using MIC/MBC testing, checkerboard assays, time-kill kinetics, and biofilm disruption studies, supported by confocal microscopy. Toxicity was assessed using L929 fibroblasts and red blood cells. A mechanistic study was performed with a membrane permeability assay. Results: Clinical isolates of Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae showed high resistance to six antibiotic classes (up to MICs > 2048 mg/L), while polymyxin B and colistin remained active (MIC ≤ 2 mg/L). Mel4 displayed variable activity (MIC 7.8-500 mg/L). A total of 102 antibiotic-antibiotic, antibiotic-Mel4, and antibiotic-colistin combination tests were performed, of which 45 showed synergistic interactions. Combining traditional antimicrobials with either colistin or Mel4 restored antibiotic susceptibility, reducing MICs up to 2048-fold. Aminoglycoside-colistin combinations significantly lowered MICs, especially against K. pneumoniae. Time-kill assays confirmed rapid bactericidal effects (>3 log10 reduction in 3 h). The ciprofloxacin-Mel4 combination effectively disrupted biofilms (62-92%) with low toxicity and high cell viability. Membrane permeability assays showed that ciprofloxacin has limited activity, whereas both Mel4 and the ciprofloxacin-Mel4 combination showed enhanced activity across concentration gradients and over time. Conclusions: Membrane-active antimicrobials, colistin and Mel4, enhance conventional antibiotics against multidrug- and extensively drug-resistant ocular Gram-negative pathogens by restoring susceptibility, accelerating bactericidal effects, and disrupting biofilms. With low toxicity, these combinations represent promising therapeutic strategies for severe multidrug-resistant ocular infections.