Metabolic profiling unveils enhanced antibacterial synergy of polymyxin B and teixobactin against multi-drug resistant Acinetobacter baumannii

Abstract

This untargeted metabolomics study investigated the synergistic antibacterial activity of polymyxin B and Leu₁₀-teixobactin, a depsipeptide inhibitor of cell wall biosynthesis. Checkerboard microdilution assays revealed a significant synergy against polymyxin-susceptible and -resistant A. baumannii, excluding lipopolysaccharide-deficient variants. Time-kill assays confirmed bactericidal synergy, reducing bacterial burden by approximately 4-6-log₁₀CFU/mL. The combination (2xMIC polymyxin B and 0.5xMIC Leu₁₀-teixobactin) prevented bacterial regrowth after 24 h, indicating sustained efficacy against the emergence of resistant mutants. The analysis of A. baumannii ATCC™ 19606 metabolome demonstrated that the polymyxin B-Leu₁₀-teixobactin combination produced more pronounced perturbation compared to the individual antibiotics across all time points (1, 3 and 6 h). Pathway analysis revealed that lipid metabolism, cell envelope biogenesis, and cellular respiration were predominantly impacted by the combination, and to a lesser extent by polymyxin B monotherapy. Leu₁₀-teixobactin treatment alone had only a minor impact on the metabolome, primarily at the 6 h time point. Peptidoglycan assays confirmed the combination's concerted deleterious effects on bacterial cell envelope integrity. Electron microscopy further substantiated these findings, revealing pronounced cell envelope damage, membrane blebbing, and vacuole formation. These findings highlight the potential of the polymyxin B-Leu₁₀-teixobactin combination as an effective treatment in preventing resistance in A. baumannii.

Keywords: A. baumannii; Antimicrobial resistance; Metabolomics; Polymyxin B; Teixobactin.

Fig. 1

Time-kill curve for polymyxin B and Leu₁₀-teixobactin monotherapies, and their combinations againstA. baumanniiATCC™ 19606 (Polymyxin B MIC = 1 mg/L; Leu₁₀-teixobactin MIC = 8 mg/L) at 1, 3 and 6 h. Data are reported as mean values of three independent cultures, and the vertical bars represent the standard deviations. Error bars are too small to appear in the graphs. The grid line represents the lower detection limit for log₁₀CFU/mL at 1.3. PMB = Polymyxin B; TXB = Leu₁₀-teixobactin.

Fig. 2

Significantly perturbed lipids at 1, 3 and 6 h inA. baumanniiATCC™ 19606 following treatment with polymyxin B (PMB, Blue), Leu₁₀-teixobactin (TXB, Black) monotherapies and their combination (Com, Red). Lipid names are putatively assigned based on accurate mass (log₂-fold change (FC) ≥ 0.58 or ≤ -0.58, corresponding to a metabolite level change of approximately 1.5-fold; FDR-adjusted p-value < 0.05). PG, glycerophosphoglycerols; glycerophosphoglycerols; PS, glycerophosphoserines; PC, glycerophosphocholines; PA, glycerophosphates; PI; FA, fatty acids; CDP-DAG, Cytidine diphosphate diacylglycerol.

Fig. 3

Impact of polymyxin B, Leu₁₀-teixobactin monotherapies and their combination on bacterial cell envelope biogenesis. The bar charts illustrate the significantly impacted metabolites after 1, 3, and 6 h exposure. Perturbed metabolites were primarily involved in amino-sugar and nucleotide-sugar metabolism and downstream pathways, peptidoglycan and lipopolysaccharide (LPS) biosynthesis in A. baumannii ATCC™ 19606 in response to treatment with polymyxin B (PMB, blue) or Leu₁₀-teixobactin (TXB, black) monotherapy and their combination (COM, red). Log₂-fold change (FC) ≥ 0.58 or ≤ -0.58, corresponding to a metabolite level change of approximately 1.5-fold; FDR-adjusted p-value < 0.05.

Fig. 4

Impact of polymyxin B, Leu₁₀-teixobactin monotherapies and their combination on bacterial cellular respiration. Schematic diagram and heatmap depicting the significantly impacted metabolites involved in cellular respiration pathways at 1, 3, and 6 h post exposure, namely the tricarboxylic acid (TCA) cycle and electron transport chain pathways, of A. baumannii ATCC™ 19606. The alterations are shown following treatment with polymyxin B (PMB, blue) or Leu₁₀-teixobactin (TXB, black) monotherapies and their combination (COM, red) (log₂-fold change (FC) ≥ 0.58 or ≤ -0.58, corresponding to a metabolite level change of approximately 1.5-fold; FDR-adjusted p-value < 0.05). Blue rectangles: significantly inhibited metabolites; Red rectangles: Significantly increased metabolites. The figure was partly created with BioRender.com.

Fig. 5

Impact of polymyxin B, Leu₁₀-teixobactin monotherapies and their combination on bacterial peptidoglycan abundance. Detection of peptidoglycan from cultures of A. baumannii ATCC™ 19606 grown either untreated or treated with polymyxin B (1 µg/mL), Leu₁₀-teixobactin (4 µg/mL), or polymyxin B (1 µg/mL) + Leu₁₀-teixobactin (4 µg/mL) in combination, at 1 h, 3 h, and 6 h post-treatment. Data represent the mean ± standard deviation of three independent biological experiments. Statistical significance was determined by an unpaired t-test, *** = p < 0.001.

Fig. 6

Electron microscopy images. Scanning electron microscopy (Scale 500 nm) and transmission electron microscopy (Scale 200 nm) images of A. baumannii ATCC™ 19606, 1 h after treatment with polymyxin B (PMB, 2 µg/mL), Leu₁₀-teixobactin (TXB, 4 µg/mL), or polymyxin B (2 µg/mL) + Leu₁₀-teixobactin (4 µg/mL) in combination (COM).

Fig. 7

Schematic diagram. Summarizing the key metabolic perturbations in A. baumannii ATCC™ 19606 in response to polymyxin B and Leu₁₀-teixobactin combination treatment. The figure was created with BioRender.com.