Plant Volatile Nanoemulsion Resensitize Pseudomonas aeruginosa to Benzalkonium Chloride

The rising resistance of Pseudomonas aeruginosa to benzalkonium chloride (BAC) represents a major challenge in infection control. This study developed a benzalkonium chloride-essential oil nanoemulsion (BAC-EO-NE) using Litsea cubeba and Melaleuca alternifolia (tea tree) oils to overcome BAC resistance through terpene-mediated synergism. GC-MS profiling identified citronellal (17.3%), citronellol (13.8%), and 1,8-cineole (44.5%) as dominant membrane-active constituents. The optimized nanoemulsion displayed nanoscale droplet size (132.2 nm), narrow PDI (0.162), and stable zeta potential (-13.5 mV) with excellent thermodynamic stability. Biocompatibility studies revealed markedly reduced hemolysis (4.7 ± 0.21%) versus free BAC (9.2 ± 0.32%) and high fibroblast viability (> 77%) after 72 h. The BAC-EO-NE exhibited enhanced antibacterial activity, producing up to 2.7-fold larger inhibition zones and 4-sixfold lower MIC/MBC values compared to BAC. Notably, resistance in BAC-resistant P. aeruginosa was reversed, lowering MIC from > 350 to 65 µg/mL. The formulation effectively disrupted biofilms (MBEC 400-750 ppm) and achieved superior contact-killing kinetics on treated textiles. SYTOX Green assays confirmed increased membrane permeability in P. aeruginosa and Methicillin-resistant S. aureus (MRSA). In an ex vivo porcine-skin disinfection model, BAC-EO-NE completely eradicated S. aureus within 96 h, outperforming mupirocin. These findings demonstrate, for the first time, that plant volatile nanoemulsion-assisted membrane disruption enhances bacterial susceptibility to BAC and improves antibacterial activity. The BAC-EO-NE represents a promising, biocompatible strategy for next-generation disinfectants and topical antimicrobials targeting resistant pathogens.