Synergistic antibacterial effect and mechanism of benzalkonium chloride and polymyxin B against Pseudomonas aeruginosa

Benzalkonium chloride (BAC) is widely employed as a broad-spectrum biocide and has emerged as a significant environmental pollutant. Polymyxin B (PB) serves as the last-line defense for the treatment of Gram-negative pathogens. Previous studies reported that BAC-adapted Pseudomonas aeruginosa increased the tolerance to PB. Herein, we present the novel finding that the combination of BAC and PB exhibited synergistic antibacterial effects against P. aeruginosa. Time-killing assay demonstrated a significant reduction in bacterial cell viability. Scanning electron microscopy, zeta potential analysis, hydrophobicity measurements, and fluorescence probe analyses collectively revealed severe disruption of the cell envelope and membrane potential induced by the combination of BAC and PB. Transcriptomic analysis revealed that the BAC-PB combination notably downregulated the expression of genes involved in lipid A modification and cell envelope production, including phoPQ, pmrAB, bamABCDE, lptABCDEG, lolB, yidC, and murJ. Additionally, the combination group exhibited augmented production of reactive oxygen species and diminished ATP synthesis. The expression of the genes associated with substance metabolism and energy generation was significantly impeded. This study provides significant implications for the interactions of biocides and antibiotics on Gram-negative pathogens, while also addressing antibiotic resistance and developing the external treatment strategy for Pseudomonas-infected wounds and burns.