Background: Polymyxins are increasingly used as a last-resort therapy against extensive drug-resistant (XDR) Pseudomonas aeruginosa1. However, polymyxin resistance can rapidly emerge with monotherapy2. Synergistic antibiotic combination therapy holds great promise in maximising bacterial killing and minimise the emergence of resistance3. The combination of polymyxin B and enrofloxacin has been shown to display synergistic killing against XDR P. aeruginosa4. We employed a global metabolomic analysis to investigate the synergistic killing mechanism of polymyxin B (PMB) and enrofloxacin combination therapy against XDR P. aeruginosa.
Methods: An XDR isolate P. aeruginosa 12196 was treated with clinically relevant concentrations of PMB (2 mg/L) and enrofloxacin (1 mg/L) alone or in combination. Metabolome profiles were obtained with bacterial samples collected at 1, 4 and 24 h using LC-MS, and analysed using univariate and multivariate statistics via MetaboAnalyst (v 4.0). Significantly perturbed metabolites (q<0.05, Log2FC≥2) were subjected to pathway analysis using BioCyc.
Results: At 1 h PMB monotherapy induced potential membrane remodelling as indicated by perturbation of glycerophospholipid (e.g., sn-glycero-3-phosphoethanolamine) and fatty acid metabolites, whereas that with enrofloxacin monotherapy induced minimal metabolomics perturbations. Compared to PMB or enrofloxacin monotherapy, PMB/enrofloxacin combination therapy had dramatic effects on a greater number of metabolic pathways at 4 and 24 h. These included the pentose phosphate pathway, pyrimidine and purine ribonucleotide biogenesis, glycerophospholid biogenesis, peptidoglycan biogenesis, lipid and energy metabolism. Moreover, the increased glutathione (log2FC =5.01) and glutathione disulphide (log2FC = 2.41) levels indicated that PMB/enrofloxacin therapy induced oxidative stress to bacterial cells. Importantly, compared to PMB monotherapy the combination therapy significantly minimised PMB resistance via the inhibition of lipid A modification at 24h.
Conclusions: This is the first study to elucidate the synergistic mechanism of PMB/enrofloxacin against XDR P. aeruginosa. Metabolomic analyses provide crucial information to understanding mechanisms of activity, resistance and synergism of antibiotics at a systems level. Future integration of metabolomics data with pharmacokinetic/pharmacodynamic model will help to optimise dosing regimen in humans.