Comparative Metabolomics Revealed Key Pathways Associated with the Synergistic Killing of Multidrug-resistant by a Bacteriophage-polymyxin Combination

Resistance to the last-line polymyxins is emerging in multidrug-resistant and phage therapy is a promising alternative. However, phage monotherapy often rapidly causes resistance and few studies have examined antibiotic-phage combinations against . Here, we investigated the combination of polymyxin B with a novel phage pK8 against an -1-carrying polymyxin-resistant clinical isolate Kp II-503 (polymyxin B MIC, 8 mg/L). The phage genome was sequenced and bacterial metabolomes were analysed at 4 and 24 h following the treatment with polymyxin B (16 mg/L), phage pK8 (10 PFU/mL) and their combination. Minimal metabolic changes across 24 h were observed with polymyxin B alone; whereas a significant inhibition of the citrate cycle, pentose phosphate pathway, amino acid and nucleotide metabolism occurred with the phage-polymyxin combination at both 4 and 24 h, but with phage alone only at 4 h. The development of resistance to phage alone was associated with enhanced membrane lipid and decreased amino acid biosynthesis in Kp II-503. Notably, cAMP, cGMP and cCMP were significantly enriched (3.1-6.6 logfold) by phage alone and the combination only at 4 h. This is the first systems pharmacology study to investigate the enhanced bacterial killing by polymyxin-phage combination and provides important mechanistic information on phage killing, resistance and antibiotic-phage combination in .