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.