Polymyxin B combinations with FDA-approved non-antibiotic phenothiazine drugs targeting multi-drug resistance of Gram-negative pathogens

Abstract

The status quo for combating uprising antibacterial resistance is to employ synergistic combinations of antibiotics. Nevertheless, the currently available combination therapies are fast becoming untenable. Combining antibiotics with various FDA-approved non-antibiotic drugs has emerged as a novel strategy against otherwise untreatable multi-drug resistant (MDR) pathogens. The apex of this study was to investigate the mechanisms of antibacterial synergy of the combination of polymyxin B with the phenothiazines against the MDR Gram-negative pathogens Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa. The synergistic antibacterial effects were tested using checkerboard and static time-kill assays. Electron microscopy (EM) and untargeted metabolomics were used to ascertain the mechanism(s) of the antibacterial synergy. The combination of polymyxin B and the phenothiazines showed synergistic antibacterial activity in checkerboard and static time-kill assays at clinically relevant concentrations against both polymyxin-susceptible and polymyxin-resistant isolates. EM revealed that the polymyxin B-prochlorperazine combination resulted in greater damage to the bacterial cell compared to each drug monotherapy. In metabolomics, at 0.5 h, polymyxin B monotherapy and the combination (to a greatest extent) disorganised the bacterial cell envelope as manifested by a major perturbation in bacterial membrane lipids (glycerophospholipids and fatty acids), peptidoglycan and lipopolysaccharide (LPS) biosynthesis. At the late time exposure (4 h), the aforementioned effects (except LPS biosynthesis) perpetuated mainly with the combination therapy, indicating the disorganising bacterial membrane biogenesis is potentially behind the mechanisms of antibacterial synergy. In conclusion, the study highlights the potential usefulness of the combination of polymyxin B with phenothiazines for the treatment of polymyxin-resistant Gram-negative infections (e.g. CNS infections).

Keywords: Antimicrobial peptides; Antimicrobial resistance; Gram-negative; MDR; Metabolomics; Phenothiazines.

Graphical abstract

Fig. 1

The chemical structures for polymyxins and the phenothiazines drugs (prochlorperazine, chlorpromazine and thiethylperazine) employed in this study.

Fig. 2

Time kill curves for polymyxin B and prochlorperazine monotherapies and in combinations against polymyxin-resistant P. aeruginosa strains, FADDI-PA070 (polymyxin B MIC = 64 mg/L, prochlorperazine MIC = 128 mg/L) and FADDI-PA006 (polymyxin B MIC = 8 mg/L, prochlorperazine MIC > 128 mg/L); against polymyxin B-susceptible A. baumannii strains, ATCC 19606 (polymyxin B MIC = 1 mg/L, prochlorperazine MIC > 128 mg/L) and ATCC 17978 (polymyxin B MIC = 0.5 mg/L, prochlorperazine MIC = 128 mg/L); and against polymyxin B-resistant A. baumannii strains FADDI-AB148 and FADDI-AB144 (polymyxin B MIC = 8 mg/L, prochlorperazine MIC > 128 mg/L for both strains).

Fig. 3

Scanning and transmission electron microscopy images of polymyxin-resistant P. aeruginosa isolate FADDI-PA070 (polymyxin B MIC = 64 mg/L, prochlorperazine MIC = 128 mg/L) treated with polymyxin B or prochlorperazine monotherapy and in combination.

Fig. 4

Perturbations of bacterial lipids. Significantly perturbed lipids in A. baumannii ATCC17978 following treatment with polymyxin B (PMB, red), prochlorperazine (PCH, green) and the combination (COM, purple) for (A) 0.5 and (B) 4 h. Lipid names are putatively assigned based on accurate mass (≥0.58553-log₂-fold, p ≤ 0.05; FDR ≤ 0.05). Control, untreated samples; PE, phosphoethanolamines; PG, glycerophosphoglycerols; PS, glycerophosphoserines; PC, glycerophosphocholines; PA, glycerophosphates; LysoPE, lysophosphatidylethanolamines; LysoPC, lysophosphatidylcholines; LPA, lysophosphatidic acid FA, fatty acids. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

The schematic diagram depicting significantly impacted metabolites of amino-sugar and nucleotide-sugar metabolism, peptidoglycan biosynthesis, lipopolysaccharides biosynthesis and pentose phosphate pathway for A. baumannii ATCC 17978 treated with polymyxin B (PMB) or prochlorperazine (PCH) monotherapy and the combination (COM) after 0.5 h exposure (A). Bar charts for the significantly influenced metabolites of amino-sugar and nucleotide-sugar metabolism, peptidoglycan biosynthesis, lipopolysaccharides biosynthesis and pentose phosphate pathways at 0.5 h and 4 h (B), and (C) Lysine metabolism at 0.5 and 4 h (≥1.0-log₂-fold, p ≤ 0.05; FDR ≤ 0.05).

Fig. 6

The graph shown the significantly impact metabolites of fatty acids metabolism for A. baumannii ATCC 17978 after 0.5 h treatment with polymyxin B (PMB), prochlorperazine (PCH) and the combination (COM) (A). Bar charts for the significantly impacted metabolites of fatty acid metabolism after treatment with polymyxin B (PMB) or prochlorperazine (PCH) monotherapy and the combination (COM) at 0.5 h (B) and 4 h (C) (≥1.0-log₂-fold, p ≤ 0.05; FDR ≤ 0.05).

Fig. 7

(A) Schematic diagram depicted the significantly impacted arginine and proline pathways of A. baumannii ATCC 17978 following treatment with polymyxin B (PMB), prochlorperazine (PCH) and the combination (COM) at 0.5 h (B) Bar charts for the significantly impacted of A. baumannii ATCC 17978 treated by polymyxin B (PMB), prochlorperazine (PCH) and the combination (COM) in arginine and proline pathways at 0.5 h and at 4 h (C) (≥1.0-log2-fold, p ≤ 0.05; FDR ≤ 0.05).

Fig. 8

Schematic summary for the main significantly impacted pathways in A. baumannii ATCC 17978 following treatment with polymyxin B (PMB) prochlorperazine (PCH) monotherapy and the combination (COM).