Mechanisms Underlying Synergistic Killing of Polymyxin B in Combination with Cannabidiol against Acinetobacter baumannii: A Metabolomic Study

Abstract

Polymyxins have resurged as the last-resort antibiotics against multidrug-resistant Acinetobacter baumannii. As reports of polymyxin resistance in A. baumannii with monotherapy have become increasingly common, combination therapy is usually the only remaining treatment option. A novel and effective strategy is to combine polymyxins with non-antibiotic drugs. This study aimed to investigate, using untargeted metabolomics, the mechanisms of antibacterial killing synergy of the combination of polymyxin B with a synthetic cannabidiol against A. baumannii ATCC 19606. The antibacterial synergy of the combination against a panel of Gram-negative pathogens (Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa) was also explored using checkerboard and static time-kill assays. The polymyxin B-cannabidiol combination showed synergistic antibacterial activity in checkerboard and static time-kill assays against both polymyxin-susceptible and polymyxin-resistant isolates. The metabolomics study at 1 h demonstrated that polymyxin B monotherapy and the combination (to the greatest extent) significantly perturbed the complex interrelated metabolic pathways involved in the bacterial cell envelope biogenesis (amino sugar and nucleotide sugar metabolism, peptidoglycan, and lipopolysaccharide (LPS) biosynthesis), nucleotides (purine and pyrimidine metabolism) and peptide metabolism; notably, these pathways are key regulators of bacterial DNA and RNA biosynthesis. Intriguingly, the combination caused a major perturbation in bacterial membrane lipids (glycerophospholipids and fatty acids) compared to very minimal changes induced by monotherapies. At 4 h, polymyxin B-cannabidiol induced more pronounced effects on the abovementioned pathways compared to the minimal impact of monotherapies. This metabolomics study for the first time showed that in disorganization of the bacterial envelope formation, the DNA and RNA biosynthetic pathways were the most likely molecular mechanisms for the synergy of the combination. The study suggests the possibility of cannabidiol repositioning, in combination with polymyxins, for treatment of MDR polymyxin-resistant Gram-negative infections.

Keywords: MDR Gram-negative; antimicrobial peptides; antimicrobial resistance; cannabidiol; metabolomics.

Figure 1

Time-kill curves for polymyxin B (PMB) and cannabidiol (CBD) monotherapies and in combinations against polymyxin B-susceptible A. baumannii strain ATCC 19606 (polymyxin B MIC = 1 mg/L, cannabidiol MIC > 64 mg/L) and polymyxin B-resistant A. baumannii strain FADDI-AB144 (polymyxin B MIC = 8 mg/L, cannabidiol MIC > 64 mg/L); against polymyxin B-susceptible K. pneumoniae FADDI-KP002 (polymyxin B MIC = 0.5 mg/L, cannabidiol MIC > 64 mg/L) and polymyxin B-resistant K. pneumoniae FADDI-KP012 (polymyxin B MIC = 32 mg/L, cannabidiol MIC > 64 mg/L); against polymyxin B-resistant P. aeruginosa strains FADDI-PA070 (polymyxin B MIC = 128 mg/L, cannabidiol MIC > 64 mg/L) and FADDI-PA006 (polymyxin B MIC = 8 mg/L, cannabidiol MIC > 64 mg/L). Data are mean values of three independent cultures, and vertical bars represent the standard deviations. Error bars are too small to appear in the graphs.

Figure 2

Perturbations of bacterial lipids. (A) Heatmap and (B) bar charts for significantly perturbed lipids in A. baumannii ATCC 19606 following treatment with polymyxin B (PMB, blue), cannabidiol (CBD, red) and the combination (COM, green) at 1 h. (C) Heatmap for significantly perturbed lipids in A. baumannii ATCC 19606 following treatment with polymyxin B (PMB, blue), cannabidiol (CBD, red) and the combination (COM, green) at 4 h. Lipid names are putatively assigned based on accurate mass (≥0.59-log₂-fold, p ≤ 0.05). Control, untreated samples; PE, phosphoethanolamines; PG, glycerophosphoglycerols; PS, glycerophosphoserines; PC, glycerophosphocholines; PA, glycerophosphates; LysoPE, lysophosphatidylethanolamines; PGP, glycerophosphoglycerophosphates; FA, fatty acids. * ≥0.59-log₂-fold, p ≤ 0.05 (one-way ANOVA).

Figure 3

(A) Schematic diagram depicting significantly impacted metabolites and bar charts of amino-sugar and nucleotide-sugar metabolism and downstream peptidoglycan biosynthesis for A. baumannii ATCC 19606 treated with polymyxin B (PMB) or cannabidiol (CBD) monotherapy and the combination (COM) after 4 h exposure. (B) Bar charts for the significantly impacted metabolites of pentose phosphate pathway (PPP) and lipopolysaccharide (LPS) biosynthesis following polymyxin B (PMB) or cannabidiol (CBD) monotherapy and the combination treatment at 4 h (≥0.59-log₂-fold, p ≤ 0.05). Blue rectangles: significantly inhibited metabolites; Red rectangles: Significantly increased metabolites.

Figure 4

(A) Graph showing the impacted pyrimidine and purine metabolism for A. baumannii ATCC 19606 after 4 h treatment with polymyxin B (PMB), cannabidiol (CBD) and their combination (COM). (B) Bar charts for the significantly impacted metabolites of pyrimidine and purine metabolism after treatment with polymyxin B (PMB) or cannabidiol (CBD) monotherapy and their combination (COM) at 1 and 4 h (≥0.59-log₂-fold, p ≤ 0.05). Blue rectangles: significantly inhibited metabolites; Red rectangles: Significantly increased metabolites.