Integrated metabolomic and transcriptomic analyses of the synergistic effect of polymyxin–rifampicin combination against Pseudomonas aeruginosa

Background: Understanding the mechanism of antimicrobial action is critical for improving antibiotic therapy. For the first time, we integrated correlative metabolomics and transcriptomics of Pseudomonas aeruginosa to elucidate the mechanism of synergistic killing of polymyxin–rifampicin combination. Methods: Liquid chromatography-mass spectrometry and RNA-seq analyses were conducted to identify the significant changes in the metabolome and transcriptome of P. aeruginosa PAO1 after exposure to polymyxin B (1 mg/L) and rifampicin (2 mg/L) alone, or in combination over 24 h. A genome-scale metabolic network was employed for integrative analysis. Results: In the first 4-h treatment, polymyxin B monotherapy induced significant lipid perturbations, predominantly to fatty acids and glycerophospholipids, indicating a substantial disorganization of the bacterial outer membrane. Expression of ParRS, a two-component regulatory system involved in polymyxin resistance, was increased by polymyxin B alone. Rifampicin alone caused marginal metabolic perturbations but significantly affected gene expression at 24 h. The combination decreased the gene expression of quorum sensing regulated virulence factors at 1 h (e.g. key genes involved in phenazine biosynthesis, secretion system and biofilm formation); and increased the expression of peptidoglycan biosynthesis genes at 4 h. Notably, the combination caused substantial accumulation of nucleotides and amino acids that last at least 4 h, indicating that bacterial cells were in a state of metabolic arrest. Conclusion: This study underscores the substantial potential of integrative systems pharmacology to determine mechanisms of synergistic bacterial killing by antibiotic combinations, which will help optimize their use in patients. Graphical Abstract: [Figure not available: see fulltext.]