Background:
Polymyxins are currently used as the last-line therapy against multidrug-resistant (MDR) Acinetobacter baumannii. As resistance to polymyxins can emerge in A. baumannii with monotherapy, combination therapies are often employed in the clinic. Previous studies showed that polymyxin-rifampicin combination displayed synergistic killing against MDR A. baumannii; however, the synergistic mechanism remains unclear. In the present study, we employed metabolomics to investigate the synergistic mechanisms of polymyxin B-rifampicin combination against a model strain of MDR A. baumannii AB5075.
Methods:
Bacterial log-phase culture was treated with polymyxin B (0.75 mg/L), rifampicin (1 mg/L), and their combination, respectively. Samples were collected at 0, 1, 4 and 24 hr, and LC-MS was employed to analyse the metabolome. MzMatch, IDEOM and MetaboAnalyst were used for bioinformatic analysis. Metabolites with fold change > 2, FDR < 0.05 were subjected to pathway analysis.
Results:
Polymyxin B monotherapy only caused early (1 hr) perturbation of Phosphatidylethanolamines metabolism (e.g. sn-glycero-3-phosphoethanolamine). Rifampicin monotherapy induced significant perturbations in nucleotide and amino acid metabolism (14 metabolites) at 4 hr. More key metabolic pathways (e.g. energy, lipid, nucleotide, amino acid metabolism) were significantly perturbed by the combination at 1 and 4 hr (36 and 61 significant metabolites, respectively). Significant changes in the levels of glycerophospholipids and fatty acids were observed after the combination treatment for 1 and 4 hr. Of particularly interested is that the combination exclusively increased the intermediate metabolite pools in pentose phosphate pathway at 1 hr. Furthermore, the pyrimidine metabolism and histidine degradation pathways were significantly increased. Interestingly, metabolites in the nucleotide and amino acid biosynthesis pathways were significantly decreased at 4 hr. Compared to each monotherapy, most key metabolic pathways were disrupted by this combination.
Conclusions:
This is the first study to employ metabolomics to unveil the synergistic killing mechanisms by polymyxin-rifampicin combination against MDR A. baumannii. The time-dependent synergistic activity via disruption of PPP, nucleotide and amino acid metabolism will help design better polymyxin combinations in the clinic.