Synergy of PMBN and closantel against mcr-1-carrying multidrug- resistant E. coli and Salmonella via membrane permeabilization- driven accumulation and metabolic disruption

Background The rapid dissemination of plasmid-mediated colistin resistance gene mcr-1, especially within livestock settings over the past decade, has progressively undermined colistin, a cornerstone antimicrobial in certain veterinary indications. Drug repurposing and antimicrobial combination therapy offer promising approaches to enhance the activity of existing antimicrobials. Here, we investigated whether polymyxin B nonapeptide (PMBN)-mediated outer membrane (OM) permeabilization could restore the antibacterial activity of closantel (CLO) against multidrug-resistant (MDR) GNB and sought to elucidate the underlying mechanism. Results The CLO-PMBN exhibited potent synergistic bactericidal activity against all of 30 tested strains, including mcr-1-carrying MDR isolates, with fractional inhibitory concentration indices (FICI) ranging from 0.012 to 0.250. Time-kill assays demonstrated a > 3 lg CFU/mL reduction within 4 h and complete eradication by 24 hours (h) for all four E. coli strains tested, whereas single agents were ineffective. Notably, this combination effectively curtailed resistance development and, more importantly, significantly improved therapeutic outcomes in vivo, while maintaining a favorable hemolytic safety profile. LC-MS/MS demonstrated that PMBN markedly enhanced intracellular accumulation of CLO by overcoming the OM barrier of E. coli. Elevated intracellular CLO exposure led to adenosine triphosphate (ATP) depletion, efflux impairment, reactive oxygen species (ROS) accumulation, and widespread metabolic reprogramming, including energy metabolism, redox balance, and membrane-associated pathways. Conclusions PMBN permeabilizes the OM to drive intracellular CLO accumulation, culminating in bioenergetic and metabolic collapse that restores CLO's bactericidal activity against mcr-1-carrying MDR E. coli and Salmonella.