Pharmacology and Therapeutics - Research Publications
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ItemPolymyxins Bind to the Cell Surface of Unculturable Acinetobacter baumannii and Cause Unique Dependent Resistance(WILEY, 2020-08)Multidrug-resistant Acinetobacter baumannii is a top-priority pathogen globally and polymyxins are a last-line therapy. Polymyxin dependence in A. baumannii (i.e., nonculturable on agar without polymyxins) is a unique and highly-resistant phenotype with a significant potential to cause treatment failure in patients. The present study discovers that a polymyxin-dependent A. baumannii strain possesses mutations in both lpxC (lipopolysaccharide biosynthesis) and katG (reactive oxygen species scavenging) genes. Correlative multiomics analyses show a significantly remodeled cell envelope and remarkably abundant phosphatidylglycerol in the outer membrane (OM). Molecular dynamics simulations and quantitative membrane lipidomics reveal that polymyxin-dependent growth emerges only when the lipopolysaccharide-deficient OM distinctively remodels with ≥ 35% phosphatidylglycerol, and with "patch" binding on the OM by the rigid polymyxin molecules containing strong intramolecular hydrogen bonding. Rather than damaging the OM, polymyxins bind to the phosphatidylglycerol-rich OM and strengthen the membrane integrity, thereby protecting bacteria from external reactive oxygen species. Dependent growth is observed exclusively with polymyxin analogues, indicating a critical role of the specific amino acid sequence of polymyxins in forming unique structures for patch-binding to bacterial OM. Polymyxin dependence is a novel antibiotic resistance mechanism and the current findings highlight the risk of 'invisible' polymyxin-dependent isolates in the evolution of resistance.
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ItemSynergistic Combination of Polymyxin B and Enrofloxacin Induced Metabolic Perturbations in Extensive Drug-Resistant Pseudomonas aeruginosa(FRONTIERS MEDIA SA, 2019-10-03)Polymyxins are used as a last-resort class of antibiotics against multidrug-resistant (MDR) Gram-negative Pseudomonas aeruginosa. As polymyxin monotherapy is associated with potential development of resistance, combination therapy is highly recommended. This study investigated the mechanism underlying the synergistic killing of polymyxin B and enrofloxacin against extensive drug-resistant (XDR) P. aeruginosa. An XDR isolate P. aeruginosa 12196 was treated with clinically relevant concentrations of polymyxin B (2 mg/L) and enrofloxacin (1 mg/L) alone or in combination. Metabolome profiles were investigated from bacterial samples collected at 1-and 4-h posttreatment using liquid chromatography with tandem mass spectrometry (LC-MS/MS), and data were analyzed using univariate and multivariate statistics. Significantly perturbed metabolites (q < 0.05, fold change ≥ 2) were subjected to pathway analysis. The synergistic killing by polymyxin B-enrofloxacin combination was initially driven by polymyxin B as indicated by the perturbation of lipid metabolites at 1 h in particular. The killing was subsequently driven by enrofloxacin via the inhibition of DNA replication, resulting in the accumulation of nucleotides at 4 h. Furthermore, the combination uniquely altered levels of metabolites in energy metabolism and cell envelope biogenesis. Most importantly, the combination significantly minimized polymyxin resistance via the inhibition of lipid A modification pathway, which was most evident at 4 h. This is the first study to elucidate the synergistic mechanism of polymyxin B-enrofloxacin combination against XDR P. aeruginosa. The metabolomics approach taken in this study highlights its power to elucidate the mechanism of synergistic killing by antibiotic combinations at the systems level.
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ItemAnthelmintic closantel enhances bacterial killing of polymyxin B against multidrug-resistant Acinetobacter baumannii(NATURE PUBLISHING GROUP, 2016-06)Polymyxins, an old class of antibiotics, are currently used as the last resort for the treatment of multidrug-resistant (MDR) Acinetobacter baumannii. However, recent pharmacokinetic and pharmacodynamic data indicate that monotherapy can lead to the development of resistance. Novel approaches are urgently needed to preserve and improve the efficacy of this last-line class of antibiotics. This study examined the antimicrobial activity of novel combination of polymyxin B with anthelmintic closantel against A. baumannii. Closantel monotherapy (16 mg l(-1)) was ineffective against most tested A. baumannii isolates. However, closantel at 4-16 mg l(-1) with a clinically achievable concentration of polymyxin B (2 mg l(-1)) successfully inhibited the development of polymyxin resistance in polymyxin-susceptible isolates, and provided synergistic killing against polymyxin-resistant isolates (MIC ⩾4 mg l(-1)). Our findings suggest that the combination of polymyxin B with closantel could be potentially useful for the treatment of MDR, including polymyxin-resistant, A. baumannii infections. The repositioning of non-antibiotic drugs to treat bacterial infections may significantly expedite discovery of new treatment options for bacterial 'superbugs'.
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ItemComparative Metabolomics Reveals Key Pathways Associated With the Synergistic Killing of Colistin and Sulbactam Combination Against Multidrug-Resistant Acinetobacter baumannii(FRONTIERS MEDIA SA, 2019-07-04)Background: Polymyxins are a last-line class of antibiotics against multidrug-resistant Acinetobacter baumannii. However, polymyxin resistance can emerge with monotherapy, highlighting the need for synergistic combination therapies. Polymyxins in combination with β-lactams have shown remarkable synergy against multidrug-resistant A. baumannii. Methods: Liquid chromatography-mass spectrometry-based metabolomics was conducted to investigate the metabolic perturbations in an A. baumannii clinical isolate, AB090342, in response to colistin (1 mg/L), sulbactam (128 mg/L), and their combination at 1, 4, and 24 h. Metabolomics data were analyzed using univariate and multivariate statistics, and metabolites showing ≥2-fold changes were subjected to pathway analysis. Results: The synergistic activity of colistin-sulbactam combination was initially driven by colistin through perturbation of fatty acid and phospholipid levels at 1 h. Cell wall biosynthesis was perturbed by sulbactam alone and the combination over 24 h; this was demonstrated by the decreased levels of two important precursors, uridine diphosphate-N-acetylglucosamine and uridine diphosphate-N-acetylmuramate, together with perturbed lysine and amino sugar metabolism. Moreover, sulbactam alone and the combination significantly depleted nucleotide metabolism and the associated arginine biosynthesis, glutamate metabolism, and pentose phosphate pathway. Notably, the colistin-sulbactam combination decreased amino acid and nucleotide levels more dramatically at 4 h compared with both monotherapies. Conclusions: This is the first metabolomics study revealing the time-dependent synergistic activity of colistin and sulbactam against A. baumannii, which was largely driven by sulbactam through the inhibition of cell wall biosynthesis. Our mechanistic findings may help optimizing synergistic colistin combinations in patients.
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ItemMetabolic Analyses Revealed Time-Dependent Synergistic Killing by Colistin and Aztreonam Combination Against Multidrug-Resistant Acinetobacter baumannii(FRONTIERS MEDIA SA, 2018-11-16)Background: Polymyxins are a last-line class of antibiotics against multidrug-resistant Acinetobacter baumannii; however, polymyxin resistance can emerge with monotherapy. Therefore, synergistic combination therapy is a crucial strategy to reduce polymyxin resistance. Methods: This study conducted untargeted metabolomics to investigate metabolic responses of a multidrug-resistant (MDR) A. baumannii clinical isolate, AB090342, to colistin and aztreonam alone, and their combination at 1, 4, and 24 h. Metabolomics data were analyzed using univariate and multivariate statistics; metabolites showing ≥ 2-fold changes were subjected to bioinformatics analysis. Results: The synergistic action of colistin-aztreonam combination was initially driven by colistin via significant disruption of bacterial cell envelope, with decreased phospholipid and fatty acid levels at 1 h. Cell wall biosynthesis was inhibited at 4 and 24 h by aztreonam alone and the combination as shown by the decreased levels of two amino sugars, UDP-N-acetylglucosamine and UDP-N-acetylmuramate; these results suggested that aztreonam was primarily responsible for the synergistic killing at later time points. Moreover, aztreonam alone and the combination significantly depleted pentose phosphate pathway, amino acid, peptide and nucleotide metabolism, but elevated fatty acid and key phospholipid levels. Collectively, the combination synergy between colistin and aztreonam was mainly due to the inhibition of cell envelope biosynthesis via different metabolic perturbations. Conclusion: This metabolomics study is the first to elucidate multiple cellular pathways associated with the time-dependent synergistic action of colistin-aztreonam combination against MDR A. baumannii. Our results provide important mechanistic insights into optimizing synergistic colistin combinations in patients.
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ItemMechanistic Insights From Global Metabolomics Studies into Synergistic Bactericidal Effect of a Polymyxin B Combination With Tamoxifen Against Cystic Fibrosis MDR Pseudomonas aeruginosa(ELSEVIER SCIENCE BV, 2018)Polymyxins are amongst the most important antibiotics in modern medicine, in recent times their clinical utility has been overshadowed by nosocomial outbreaks of polymyxin resistant MDR Gram-negative 'superbugs'. An effective strategy to surmount polymyxin resistance is combination therapy with FDA-approved non-antibiotic drugs. Herein we used untargeted metabolomics to investigate the mechanism(s) of synergy between polymyxin B and the selective estrogen receptor modulator (SERM) tamoxifen against a polymyxin-resistant MDR cystic fibrosis (CF) Pseudomonas aeruginosa FADDI-PA006 isolate (polymyxin B MIC=8 mg/L , it is an MDR polymyxin resistant P. aeruginosa isolated from the lungs of a CF patient). The metabolome of FADDI-PA006 was profiled at 15 min, 1 and 4 h following treatment with polymyxin B (2 mg/L), tamoxifen (8 mg/L) either as monotherapy or in combination. At 15 min, the combination treatment induced a marked decrease in lipids, primarily fatty acid and glycerophospholipid metabolites that are involved in the biosynthesis of bacterial membranes. In line with the polymyxin-resistant status of this strain, at 1 h, both polymyxin B and tamoxifen monotherapies produced little effect on bacterial metabolism. In contrast to the combination which induced extensive reduction (≥ 1.0-log2-fold, p ≤ 0.05; FDR ≤ 0.05) in the levels of essential intermediates involved in cell envelope biosynthesis. Overall, these novel findings demonstrate that the primary mechanisms underlying the synergistic bactericidal effect of the combination against the polymyxin-resistant P. aeruginosa CF isolate FADDI-PA006 involves a disruption of the cell envelope biogenesis and an inhibition of aminoarabinose LPS modifications that confer polymyxin resistance.
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ItemUntargeted metabolomics analysis reveals key pathways responsible for the synergistic killing of colistin and doripenem combination against Acinetobacter baumannii(NATURE PORTFOLIO, 2017-03-30)Combination therapy is deployed for the treatment of multidrug-resistant Acinetobacter baumannii, as it can rapidly develop resistance to current antibiotics. This is the first study to investigate the synergistic effect of colistin/doripenem combination on the metabolome of A. baumannii. The metabolite levels were measured using LC-MS following treatment with colistin (2 mg/L) or doripenem (25 mg/L) alone, and their combination at 15 min, 1 hr and 4 hr (n = 4). Colistin caused early (15 min and 1 hr) disruption of the bacterial outer membrane and cell wall, as demonstrated by perturbation of glycerophospholipids and fatty acids. Concentrations of peptidoglycan biosynthesis metabolites decreased at 4 hr by doripenem alone, reflecting its mechanism of action. The combination induced significant changes to more key metabolic pathways relative to either monotherapy. Down-regulation of cell wall biosynthesis (via D-sedoheptulose 7-phosphate) and nucleotide metabolism (via D-ribose 5-phosphate) was associated with perturbations in the pentose phosphate pathway induced initially by colistin (15 min and 1 hr) and later by doripenem (4 hr). We discovered that the combination synergistically killed A. baumannii via time-dependent inhibition of different key metabolic pathways. Our study highlights the significant potential of systems pharmacology in elucidating the mechanism of synergy and optimizing antibiotic pharmacokinetics/pharmacodynamics.
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ItemComparative Metabolomics and Transcriptomics Reveal Multiple Pathways Associated with Polymyxin Killing in Pseudomonas aeruginosa(AMER SOC MICROBIOLOGY, 2019)Polymyxins are a last-line therapy against multidrug-resistant Pseudomonas aeruginosa; however, resistance to polymyxins has been increasingly reported. Therefore, understanding the mechanisms of polymyxin activity and resistance is crucial for preserving their clinical usefulness. This study employed comparative metabolomics and transcriptomics to investigate the responses of polymyxin-susceptible P. aeruginosa PAK (polymyxin B MIC, 1 mg/liter) and its polymyxin-resistant pmrB mutant PAKpmrB6 (MIC, 16 mg/liter) to polymyxin B (4, 8, and 128 mg/liter) at 1, 4, and 24 h, respectively. Our results revealed that polymyxin B at 4 mg/liter induced different metabolic and transcriptomic responses between polymyxin-susceptible and -resistant P. aeruginosa. In strain PAK, polymyxin B significantly activated PmrAB and the mediated arn operon, leading to increased 4-amino-4-deoxy-L-arabinose (L-Ara4N) synthesis and the addition to lipid A. In contrast, polymyxin B did not increase lipid A modification in strain PAKpmrB6. Moreover, the syntheses of lipopolysaccharide and peptidoglycan were significantly decreased in strain PAK but increased in strain PAKpmrB6 due to polymyxin B treatment. In addition, 4 mg/liter polymyxin B significantly perturbed phospholipid and fatty acid levels and induced oxidative stress in strain PAK, but not in PAKpmrB6. Notably, the increased trehalose-6-phosphate levels indicate that polymyxin B potentially caused osmotic imbalance in both strains. Furthermore, 8 and 128 mg/liter polymyxin B significantly elevated lipoamino acid levels and decreased phospholipid levels but without dramatic changes in lipid A modification in wild-type and mutant strains, respectively. Overall, this systems study is the first to elucidate the complex and dynamic interactions of multiple cellular pathways associated with the polymyxin mode of action against P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa has been highlighted by the recent WHO Global Priority Pathogen List due to multidrug resistance. Without new antibiotics, polymyxins remain a last-line therapeutic option for this difficult-to-treat pathogen. The emergence of polymyxin resistance highlights the growing threat to our already very limited antibiotic armamentarium and the urgency to understand the exact mechanisms of polymyxin activity and resistance. Integration of the correlative metabolomics and transcriptomics results in the present study discovered that polymyxin treatment caused significant perturbations in the biosynthesis of lipids, lipopolysaccharide, and peptidoglycan, central carbon metabolism, and oxidative stress. Importantly, lipid A modifications were surprisingly rapid in response to polymyxin treatment at clinically relevant concentrations. This is the first study to reveal the dynamics of polymyxin-induced cellular responses at the systems level, which highlights that combination therapy should be considered to minimize resistance to the last-line polymyxins. The results also provide much-needed mechanistic information which potentially benefits the discovery of new-generation polymyxins.