scholarly journals Metabolomics Study of the Synergistic Killing of Polymyxin B in Combination with Amikacin against Polymyxin-Susceptible and -Resistant Pseudomonas aeruginosa

2019 ◽  
Vol 64 (1) ◽  
Author(s):  
Maytham Hussein ◽  
Mei-Ling Han ◽  
Yan Zhu ◽  
Qi Zhou ◽  
Yu-Wei Lin ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Acid Metabolism ◽  
Membrane Lipids ◽  
Cell Envelope ◽  
Polymyxin B ◽  
Mechanistic Study ◽  
Content Type ◽  
Susceptible Isolate ◽  
Metabolomics Study ◽  
Central Carbohydrate Metabolism

ABSTRACT In the present study, we employed untargeted metabolomics to investigate the synergistic killing mechanism of polymyxin B in combination with an aminoglycoside, amikacin, against a polymyxin-susceptible isolate of Pseudomonas aeruginosa, FADDI-PA111 (MIC = 2 mg/liter for both polymyxin B and amikacin), and a polymyxin-resistant Liverpool epidemic strain (LES), LESB58 (the corresponding MIC for both polymyxin B and amikacin is 16 mg/liter). The metabolites were extracted 15 min, 1 h, and 4 h following treatment with polymyxin B alone (2 mg/liter for FADDI-PA111; 4 mg/liter for LESB58), amikacin alone (2 mg/liter), and both in combination and analyzed using liquid chromatography-mass spectrometry (LC-MS). At 15 min and 1 h, polymyxin B alone induced significant perturbations in glycerophospholipid and fatty acid metabolism pathways in FADDI-PA111 and, to a lesser extent, in LESB58. Amikacin alone at 1 and 4 h induced significant perturbations in peptide and amino acid metabolism, which is in line with the mode of action of aminoglycosides. Pathway analysis of FADDI-PA111 revealed that the synergistic effect of the combination was largely due to the inhibition of cell envelope biogenesis, which was driven initially by polymyxin B via suppression of key metabolites involved in lipopolysaccharide, peptidoglycan, and membrane lipids (15 min and 1 h) and later by amikacin (4 h). Overall, these novel findings demonstrate that the disruption of cell envelope biogenesis and central carbohydrate metabolism, decreased levels of amino sugars, and a downregulated nucleotide pool are the metabolic pathways associated with the synergistic killing of the polymyxin-amikacin combination against P. aeruginosa. This mechanistic study might help optimize synergistic polymyxin B combinations in the clinical setting.

scholarly journals The Killing Mechanism of Teixobactin against Methicillin-Resistant Staphylococcus aureus: an Untargeted Metabolomics Study

mSystems ◽  
2020 ◽  
Vol 5 (3) ◽  
Author(s):  
Maytham Hussein ◽  
John A. Karas ◽  
Elena K. Schneider-Futschik ◽  
Fan Chen ◽  
James Swarbrick ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Membrane Lipids ◽  
Cell Envelope ◽  
Teichoic Acid ◽  
Bacterial Membrane ◽  
Gram Positive ◽  
Methicillin Resistant ◽  
Content Type ◽  
Metabolomics Study

ABSTRACT Antibiotics have served humankind through their use in modern medicine as effective treatments for otherwise fatal bacterial infections. Teixobactin is a first member of newly discovered natural antibiotics that was recently identified from a hitherto-unculturable soil bacterium, Eleftheria terrae, and recognized as a potent antibacterial agent against various Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci. The most distinctive characteristic of teixobactin as an effective antibiotic is that teixobactin resistance could not be evolved in a laboratory setting. It is purported that teixobactin’s “resistance-resistant” mechanism of action includes binding to the essential bacterial cell wall synthesis building blocks lipid II and lipid III. In the present study, metabolomics was used to investigate the potential metabolic pathways involved in the mechanisms of antibacterial activity of the synthetic teixobactin analogue Leu10-teixobactin against a MRSA strain, S. aureus ATCC 700699. The metabolomes of S. aureus ATCC 700699 cells 1, 3, and 6 h following treatment with Leu10-teixobactin (0.5 μg/ml, i.e., 0.5× MIC) were compared to those of the untreated controls. Leu10-teixobactin significantly perturbed bacterial membrane lipids (glycerophospholipids and fatty acids), peptidoglycan (lipid I and II) metabolism, and cell wall teichoic acid (lipid III) biosynthesis as early as after 1 h of treatment, reflecting an initial activity on the cell envelope. Concordant with its time-dependent antibacterial killing action, Leu10-teixobactin caused more perturbations in the levels of key intermediates in pathways of amino-sugar and nucleotide-sugar metabolism and their downstream peptidoglycan and teichoic acid biosynthesis at 3 and 6 h. Significant perturbations in arginine metabolism and the interrelated tricarboxylic acid cycle, histidine metabolism, pantothenate, and coenzyme A biosynthesis were also observed at 3 and 6 h. To conclude, this is the first study to provide novel metabolomics mechanistic information, which lends support to the development of teixobactin as an antibacterial drug for the treatment of multidrug-resistant Gram-positive infections. IMPORTANCE Antimicrobial resistance is one of the greatest threats to the global health system. It is imperative that new anti-infective therapeutics be developed against problematic “superbugs.” The cyclic depsipeptide teixobactin holds much promise as a new class of antibiotics for highly resistant Gram-positive pathogens (e.g., methicillin-resistant Staphylococcus aureus [MRSA]). Understanding its molecular mechanism(s) of action could lead to the design of new compounds with a broader activity spectrum. Here, we describe the first metabolomics study to investigate the killing mechanism(s) of teixobactin against MRSA. Our findings revealed that teixobactin significantly disorganized the bacterial cell envelope, as reflected by a profound perturbation in the bacterial membrane lipids and cell wall biosynthesis (peptidoglycan and teichoic acid). Importantly, teixobactin significantly suppressed the main intermediate d-alanyl-d-lactate involved in the mechanism of vancomycin resistance in S. aureus. These novel results help explain the unique mechanism of action of teixobactin and its lack of cross-resistance with vancomycin.

scholarly journals Efficacy of Antibiotic Combinations against Multidrug-Resistant Pseudomonas aeruginosa in Automated Time-Lapse Microscopy and Static Time-Kill Experiments

2020 ◽  
Vol 64 (6) ◽  
Author(s):  
Anna Olsson ◽  
Pikkei Wistrand-Yuen ◽  
Elisabet I. Nielsen ◽  
Lena E. Friberg ◽  
Linus Sandegren ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Time Lapse ◽  
Polymyxin B ◽  
Multidrug Resistant ◽  
Positive Interactions ◽  
Synergistic Activity ◽  
Content Type ◽  
Time Lapse Microscopy ◽  
Time Kill ◽  
Antibiotic Combination

ABSTRACT Antibiotic combination therapy is used for severe infections caused by multidrug-resistant (MDR) Gram-negative bacteria, yet data regarding which combinations are most effective are lacking. This study aimed to evaluate the in vitro efficacy of polymyxin B in combination with 13 other antibiotics against four clinical strains of MDR Pseudomonas aeruginosa. We evaluated the interactions of polymyxin B in combination with amikacin, aztreonam, cefepime, chloramphenicol, ciprofloxacin, fosfomycin, linezolid, meropenem, minocycline, rifampin, temocillin, thiamphenicol, or trimethoprim by automated time-lapse microscopy using predefined cutoff values indicating inhibition of growth (≤106 CFU/ml) at 24 h. Promising combinations were subsequently evaluated in static time-kill experiments. All strains were intermediate or resistant to polymyxin B, antipseudomonal β-lactams, ciprofloxacin, and amikacin. Genes encoding β-lactamases (e.g., blaPAO and blaOXA-50) and mutations associated with permeability and efflux were detected in all strains. In the time-lapse microscopy experiments, positive interactions were found with 39 of 52 antibiotic combination/bacterial strain setups. Enhanced activity was found against all four strains with polymyxin B used in combination with aztreonam, cefepime, fosfomycin, minocycline, thiamphenicol, and trimethoprim. Time-kill experiments showed additive or synergistic activity with 27 of the 39 tested polymyxin B combinations, most frequently with aztreonam, cefepime, and meropenem. Positive interactions were frequently found with the tested combinations, against strains that harbored several resistance mechanisms to the single drugs, and with antibiotics that are normally not active against P. aeruginosa. Further study is needed to explore the clinical utility of these combinations.

scholarly journals Elucidating the Regulon of Multidrug Resistance Regulator RarA in Klebsiella pneumoniae

2013 ◽  
Vol 57 (4) ◽  
pp. 1603-1609 ◽  
Author(s):  
Shyamasree De Majumdar ◽  
Mark Veleba ◽  
Sarah Finn ◽  
Séamus Fanning ◽  
Thamarai Schneiders
Keyword(s):  
Klebsiella Pneumoniae ◽  
Cell Envelope ◽  
Polymyxin B ◽  
Multidrug Resistant ◽  
Protein Synthesis Inhibitors ◽  
Content Type ◽  
Phenotypic Microarray ◽  
Genes Encoding ◽  
Clusters Of Orthologous Groups ◽  
Microarray Analyses

ABSTRACTRarA is an AraC-type regulator inKlebsiella pneumoniae, which, when overexpressed, confers a low-level multidrug-resistant (MDR) phenotype linked to the upregulation of both theacrABandoqxABefflux genes. IncreasedrarAexpression has also been shown to be integral in the development of tigecycline resistance in the absence oframAinK. pneumoniae. Given its phenotypic role in MDR, microarray analyses were performed to determine the RarA regulon. Transcriptome analysis was undertaken using strains Ecl8ΔrarA/pACrarA-2 (rarA-expressing construct) and Ecl8ΔrarA/pACYC184 (vector-only control) using bespoke microarray slides consisting of probes derived from the genomic sequences ofK. pneumoniaeMGH 78578 (NC_009648.1) and Kp342 (NC_011283.1). Our results show thatrarAoverexpression resulted in the differential expression of 66 genes (42 upregulated and 24 downregulated). Under the COG (clusters of orthologous groups) functional classification, the majority of affected genes belonged to the category of cell envelope biogenesis and posttranslational modification, along with genes encoding the previously uncharacterized transport proteins (e.g., KPN_03141,sdaCB, andleuE) and the porin OmpF. However, genes associated with energy production and conversion and amino acid transport/metabolism (e.g.,nuoA,narJ, andproWX) were found to be downregulated. Biolog phenotype analyses demonstrated thatrarAoverexpression confers enhanced growth of the overexpresser in the presence of several antibiotic classes (i.e., beta-lactams and fluoroquinolones), the antifungal/antiprotozoal compound clioquinol, disinfectants (8-hydroxyquinoline), protein synthesis inhibitors (i.e., minocycline and puromycin), membrane biogenesis agents (polymyxin B and amitriptyline), DNA synthesis (furaltadone), and the cytokinesis inhibitor (sanguinarine). Both our transcriptome and phenotypic microarray data support and extend the role of RarA in the MDR phenotype ofK. pneumoniae.

scholarly journals Multidrug Adaptive Resistance of Pseudomonas aeruginosa Swarming Cells

2019 ◽  
Vol 64 (3) ◽  
Author(s):  
Shannon R. Coleman ◽  
Travis Blimkie ◽  
Reza Falsafi ◽  
Robert E. W. Hancock
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Efflux Pump ◽  
Iron Acquisition ◽  
Polymyxin B ◽  
Rna Seq ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Content Type ◽  
Adaptive Resistance

ABSTRACT Swarming surface motility is a complex adaptation leading to multidrug antibiotic resistance and virulence factor production in Pseudomonas aeruginosa. Here, we expanded previous studies to demonstrate that under swarming conditions, P. aeruginosa PA14 is more resistant to multiple antibiotics, including aminoglycosides, β-lactams, chloramphenicol, ciprofloxacin, tetracycline, trimethoprim, and macrolides, than swimming cells, but is not more resistant to polymyxin B. We investigated the mechanism(s) of swarming-mediated antibiotic resistance by examining the transcriptomes of swarming cells and swarming cells treated with tobramycin by transcriptomics (RNA-Seq) and reverse transcriptase quantitative PCR (qRT-PCR). RNA-Seq of swarming cells (versus swimming) revealed 1,581 dysregulated genes, including 104 transcriptional regulators, two-component systems, and sigma factors, numerous upregulated virulence and iron acquisition factors, and downregulated ribosomal genes. Strain PA14 mutants in resistome genes that were dysregulated under swarming conditions were tested for their ability to swarm in the presence of tobramycin. In total, 41 mutants in genes dysregulated under swarming conditions were shown to be more resistant to tobramycin under swarming conditions, indicating that swarming-mediated tobramycin resistance was multideterminant. Focusing on two genes downregulated under swarming conditions, both prtN and wbpW mutants were more resistant to tobramycin, while the prtN mutant was additionally resistant to trimethoprim under swarming conditions; complementation of these mutants restored susceptibility. RNA-Seq of swarming cells treated with subinhibitory concentrations of tobramycin revealed the upregulation of the multidrug efflux pump MexXY and downregulation of virulence factors.

scholarly journals Multicenter Evaluation of Colistin Broth Disk Elution and Colistin Agar Test: a Report from the Clinical and Laboratory Standards Institute

2019 ◽  
Vol 57 (11) ◽  
Author(s):  
Romney M. Humphries ◽  
Daniel A. Green ◽  
Audrey N. Schuetz ◽  
Yehudit Bergman ◽  
Shawna Lewis ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antimicrobial Susceptibility ◽  
Susceptibility Testing ◽  
Polymyxin B ◽  
Antimicrobial Susceptibility Testing ◽  
Broth Microdilution ◽  
Content Type ◽  
Clinical Laboratories

ABSTRACT Susceptibility testing of the polymyxins (colistin and polymyxin B) is challenging for clinical laboratories. The Clinical and Laboratory Standards Institute (CLSI) Antimicrobial Susceptibility Testing Subcommittee evaluated two methods to enable accurate testing of these agents. These methods were a colistin broth disk elution (CBDE) and a colistin agar test (CAT), the latter of which was evaluated using two inoculum volumes, 1 μl (CAT-1) and 10 μl (CAT-10). The methods were evaluated using a collection of 270 isolates of Enterobacterales, 122 Pseudomonas aeruginosa isolates, and 106 Acinetobacter spp. isolates. Overall, 94.4% of CBDE results were in essential agreement and 97.9% in categorical agreement (CA) with reference broth microdilution MICs. Nine very major errors (VME; 3.2%) and 3 major errors (ME; 0.9%) were observed. With the CBDE, 98.6% CA was observed for Enterobacterales (2.5% VME, 0% ME), 99.3% CA was observed for P. aeruginosa (0% VME, 0.7% ME), and 93.1% CA was observed for Acinetobacter spp. (5.6% VME, 3.3% ME). Overall, CA was 94.9% with 6.8% VME using CAT-1 and improved to 98.3% with 3.9% VME using CAT-10. No ME were observed using either CAT-1 or CAT-10. Using the CAT-1/CAT-10, the CA observed was 99.4%/99.7% for Enterobacterales (1%/0.5% VME), 98.7%/100% for P. aeruginosa (8.3%/0% VME), and 88.5%/92.3% for Acinetobacter spp. (21.4%/14.3% VME). Based on these data, the CLSI antimicrobial susceptibility testing (AST) subcommittee endorsed the CBDE and CAT-10 methods for colistin testing of Enterobacterales and P. aeruginosa.

scholarly journals Adaptive and Mutational Responses to Peptide Dendrimer Antimicrobials in Pseudomonas aeruginosa

2020 ◽  
Vol 64 (4) ◽  
Author(s):  
Fatma Ben Jeddou ◽  
Léna Falconnet ◽  
Alexandre Luscher ◽  
Thissa Siriwardena ◽  
Jean-Louis Reymond ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Lipid A ◽  
Polymyxin B ◽  
Multidrug Resistant ◽  
Sensor Kinase ◽  
Loss Of Function ◽  
Adaptive Responses ◽  
Kinase Gene ◽  
Polyamine Biosynthesis ◽  
Content Type

ABSTRACT Colistin (polymyxin E) is a last-resort antibiotic against multidrug-resistant isolates of Pseudomonas aeruginosa. However, the nephro-toxicity of colistin limits its use, spurring the interest in novel antimicrobial peptides (AMP). Here, we show that the synthetic AMP-dendrimer G3KL (MW 4,531.38 Da, 15 positive charges, MIC = 8 mg/liter) showed faster killing than polymyxin B (Pmx-B) with no detectable resistance selection in P. aeruginosa strain PA14. Spontaneous mutants selected on Pmx-B, harboring loss of function mutations in the PhoQ sensor kinase gene, showed increased Pmx-B MICs and arnB operon expression (4-amino-l-arabinose addition to lipid A), but remained susceptible to dendrimers. Two mutants carrying a missense mutation in the periplasmic loop of the PmrB sensor kinase showed increased MICs for Pmx-B (8-fold) and G3KL (4-fold) but not for the dendrimer T7 (MW 4,885.64 Da, 16 positive charges, MIC = 8 mg/liter). The pmrB mutants showed increased expression of the arnB operon as well as of the speD2-speE2-PA4775 operon, located upstream of pmrAB, and involved in polyamine biosynthesis. Exogenous supplementation with the polyamines spermine and norspermine increased G3KL and T7 MICs in a phoQ mutant background but not in the PA14 wild type. This suggests that both addition of 4-amino-l-arabinose and secretion of polyamines are required to reduce susceptibility to dendrimers, probably neutralizing the negative charges present on the lipid A and the 2-keto-3-deoxyoctulosonic acid (KDO) sugars of the lipopolysaccharide (LPS), respectively. We further show by transcriptome analysis that the dendrimers G3KL and T7 induce adaptive responses through the CprRS two-component system in PA14.

scholarly journals Synergy of Silver Nanoparticles and Aztreonam against Pseudomonas aeruginosa PAO1 Biofilms

2014 ◽  
Vol 58 (10) ◽  
pp. 5818-5830 ◽  
Author(s):  
Marc B. Habash ◽  
Amber J. Park ◽  
Emily C. Vis ◽  
Robert J. Harris ◽  
Cezar M. Khursigara
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Silver Nanoparticles ◽  
Bacterial Infections ◽  
Antimicrobial Agents ◽  
Cell Envelope ◽  
Antimicrobial Properties ◽  
Planktonic Cell ◽  
Chronic Infections ◽  
Content Type

ABSTRACTPathogenic bacterial biofilms, such as those found in the lungs of patients with cystic fibrosis (CF), exhibit increased antimicrobial resistance, due in part to the inherent architecture of the biofilm community. The protection provided by the biofilm limits antimicrobial dispersion and penetration and reduces the efficacy of antibiotics that normally inhibit planktonic cell growth. Thus, alternative antimicrobial strategies are required to combat persistent infections. The antimicrobial properties of silver have been known for decades, but silver and silver-containing compounds have recently seen renewed interest as antimicrobial agents for treating bacterial infections. The goal of this study was to assess the efficacy of citrate-capped silver nanoparticles (AgNPs) of various sizes, alone and in combination with the monobactam antibiotic aztreonam, to inhibitPseudomonas aeruginosaPAO1 biofilms. Among the different sizes of AgNPs examined, 10-nm nanoparticles were most effective in inhibiting the recovery ofP. aeruginosabiofilm cultures and showed synergy of inhibition when combined with sub-MIC levels of aztreonam. Visualization of biofilms treated with combinations of 10-nm AgNPs and aztreonam indicated that the synergistic bactericidal effects are likely caused by better penetration of the small AgNPs into the biofilm matrix, which enhances the deleterious effects of aztreonam against the cell envelope ofP. aeruginosawithin the biofilms. These data suggest that small AgNPs synergistically enhance the antimicrobial effects of aztreonam againstP. aeruginosain vitro, and they reveal a potential role for combinations of small AgNPs and antibiotics in treating patients with chronic infections.

scholarly journals Comparative Metabolomics and Transcriptomics Reveal Multiple Pathways Associated with Polymyxin Killing in Pseudomonas aeruginosa

mSystems ◽  
2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Mei-Ling Han ◽  
Yan Zhu ◽  
Darren J. Creek ◽  
Yu-Wei Lin ◽  
Alina D. Gutu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Pseudomonas Aeruginosa ◽  
Lipid A ◽  
Therapeutic Option ◽  
Polymyxin B ◽  
Multidrug Resistant ◽  
Central Carbon Metabolism ◽  
Dynamic Interactions ◽  
Content Type ◽  
Comparative Metabolomics

ABSTRACT 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.

scholarly journals Determinants of Intrinsic Aminoglycoside Resistance in Pseudomonas aeruginosa

2012 ◽  
Vol 56 (11) ◽  
pp. 5591-5602 ◽  
Author(s):  
Thomas Krahn ◽  
Christie Gilmour ◽  
Justin Tilak ◽  
Sebastien Fraud ◽  
Nicholas Kerr ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cell Envelope ◽  
Acquired Resistance ◽  
Insertion Mutant ◽  
Deletion Mutants ◽  
Aminoglycoside Resistance ◽  
Content Type ◽  
Single Deletion ◽  
High Level ◽  
Increased Susceptibility

ABSTRACTScreening of a transposon insertion mutant library ofPseudomonas aeruginosafor increased susceptibility to paromomycin identified a number of genes whose disruption enhanced susceptibility of this organism to multiple aminoglycosides, including tobramycin, amikacin, and gentamicin. These included genes associated with lipid biosynthesis or metabolism (lptA,faoA), phosphate uptake (pstB), and two-component regulators (amgRS, PA2797-PA2798) and a gene of unknown function (PA0392). Deletion mutants lacking these showed enhanced panaminoglycoside susceptibility that was reversed by the cloned genes, confirming their contribution to intrinsic panaminoglycoside resistance. None of these mutants showed increased aminoglycoside permeation of the cell envelope, indicating that increased susceptibility was not related to enhanced aminoglycoside uptake owing to a reduced envelope barrier function. Several mutants (pstB,faoA, PA0392,amgR) did, however, show increased cytoplasmic membrane depolarization relative to wild type following gentamicin exposure, consistent with the membranes of these mutants being more prone to perturbation, likely by gentamicin-generated mistranslated polypeptides. Mutants lacking any two of these resistance genes in various combinations invariably showed increased aminoglycoside susceptibility relative to single-deletion mutants, confirming their independent contribution to resistance and highlighting the complexity of the intrinsic aminoglycoside resistome inP. aeruginosa. Deletion of these genes also compromised the high-level panaminoglycoside resistance of clinical isolates, emphasizing their important contribution to acquired resistance.

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