Characterization of the Polymyxin B Resistome of Pseudomonas aeruginosa

ABSTRACTMultidrug resistance inPseudomonas aeruginosais increasingly becoming a threat for human health. Indeed, some strains are resistant to almost all currently available antibiotics, leaving very limited choices for antimicrobial therapy. In many such cases, polymyxins are the only available option, although as their utilization increases so does the isolation of resistant strains. In this study, we screened a comprehensive PA14 mutant library to identify genes involved in changes of susceptibility to polymyxin B inP. aeruginosa. Surprisingly, our screening revealed that the polymyxin B resistome of this microorganism is fairly small. Thus, only one resistant mutant and 17 different susceptibility/intrinsic resistance determinants were identified. Among the susceptible mutants, a significant number carried transposon insertions in lipopolysaccharide (LPS)-related genes. LPS analysis revealed that four of these mutants (galU,lptC,wapR, andssg) had an altered banding profile in SDS-polyacrylamide gels and Western blots, with three of them exhibiting LPS core truncation and lack of O-antigen decoration. Further characterization of these four mutants showed that their increased susceptibility to polymyxin B was partly due to increased basal outer membrane permeability. Additionally, these mutants also lacked the aminoarabinose-substituted lipid A species observed in the wild type upon growth in low magnesium. Overall, our results emphasize the importance of LPS integrity and lipid A modification in resistance to polymyxins inP. aeruginosa, highlighting the relevance of characterizing the genes that affect biosynthesis of cell surface structures in this pathogen to follow the evolution of peptide resistance in the clinic.

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Alterations of Metabolic and Lipid Profiles in Polymyxin-ResistantPseudomonas aeruginosa

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02656-17 ◽

2018 ◽

Vol 62 (6) ◽

Cited By ~ 15

Author(s):

Mei-Ling Han ◽

Yan Zhu ◽

Darren J. Creek ◽

Yu-Wei Lin ◽

Dovile Anderson ◽

...

Keyword(s):

Lipid A ◽

Therapeutic Option ◽

Polymyxin B ◽

Multidrug Resistant ◽

Wild Type ◽

Metabolomics Data ◽

Content Type ◽

In The Wild ◽

High Level ◽

Acyl Coenzyme A

ABSTRACTMultidrug-resistantPseudomonas aeruginosapresents a global medical challenge, and polymyxins are a key last-resort therapeutic option. Unfortunately, polymyxin resistance inP. aeruginosahas been increasingly reported. The present study was designed to define metabolic differences between paired polymyxin-susceptible and -resistantP. aeruginosastrains using untargeted metabolomics and lipidomics analyses. The metabolomes of wild-typeP. aeruginosastrain K ([PAK] polymyxin B MIC, 1 mg/liter) and its pairedpmrBmutant strains, PAKpmrB6and PAKpmrB12(polymyxin B MICs of 16 mg/liter and 64 mg/liter, respectively) were characterized using liquid chromatography-mass spectrometry, and metabolic differences were identified through multivariate and univariate statistics. PAKpmrB6and PAKpmrB12, which displayed lipid A modifications with 4-amino-4-deoxy-l-arabinose, showed significant perturbations in amino acid and carbohydrate metabolism, particularly the intermediate metabolites from 4-amino-4-deoxy-l-arabinose synthesis and the methionine salvage cycle pathways. The genomics result showed a premature termination (Y275stop) inspeE(encoding spermidine synthase) in PAKpmrB6, and metabolomics data revealed a decreased intracellular level of spermidine in PAKpmrB6compared to that in PAKpmrB12. Our results indicate that spermidine may play an important role in high-level polymyxin resistance inP. aeruginosa. Interestingly, bothpmrBmutants had decreased levels of phospholipids, fatty acids, and acyl-coenzyme A compared to those in the wild-type PAK. Moreover, the more resistant PAKpmrB12mutant exhibited much lower levels of phospholipids than the PAKpmrB6mutant, suggesting that the decreased phospholipid level was associated with polymyxin resistance. In summary, this study provides novel mechanistic information on polymyxin resistance inP. aeruginosaand highlights its impacts on bacterial metabolism.

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The Efflux Pump MexXY/OprM Contributes to the Tolerance and Acquired Resistance of Pseudomonas aeruginosa to Colistin

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02033-19 ◽

2020 ◽

Vol 64 (4) ◽

Author(s):

Hélène Puja ◽

Arnaud Bolard ◽

Aurélie Noguès ◽

Patrick Plésiat ◽

Katy Jeannot

Keyword(s):

Pseudomonas Aeruginosa ◽

Lipid A ◽

Efflux Pump ◽

Acquired Resistance ◽

Intrinsic Resistance ◽

Drug Induced ◽

Functional Link ◽

Content Type ◽

Regulatory Systems ◽

Early Drug

ABSTRACT The intrinsic resistance of Pseudomonas aeruginosa to polymyxins in part relies on the addition of 4-amino-4-deoxy-l-arabinose (Ara4N) molecules to the lipid A of lipopolysaccharide (LPS), through induction of operon arnBCADTEF-ugd (arn) expression. As demonstrated previously, at least three two-component regulatory systems (PmrAB, ParRS, and CprRS) are able to upregulate this operon when bacteria are exposed to colistin. In the present study, gene deletion experiments with the bioluminescent strain PAO1::lux showed that ParRS is a key element in the tolerance of P. aeruginosa to this last-resort antibiotic (i.e., resistance to early drug killing). Other loci of the ParR regulon, such as those encoding the efflux proteins MexXY (mexXY), the polyamine biosynthetic pathway PA4773-PA4774-PA4775, and Ara4N LPS modification process (arnBCADTEF-ugd), also contribute to the bacterial tolerance in an intricate way with ParRS. Furthermore, we found that both stable upregulation of the arn operon and drug-induced ParRS-dependent overexpression of the mexXY genes accounted for the elevated resistance of pmrB mutants to colistin. Deletion of the mexXY genes in a constitutively activated ParR mutant of PAO1 was associated with significantly increased expression of the genes arnA, PA4773, and pmrA in the absence of colistin exposure, thereby highlighting a functional link between the MexXY/OprM pump, the PA4773-PA4774-PA4775 pathway, and Ara4N-based modification of LPS. The role played by MexXY/OprM in the adaptation of P. aeruginosa to polymyxins opens new perspectives for restoring the susceptibility of resistant mutants through the use of efflux inhibitors.

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Adaptive and Mutational Responses to Peptide Dendrimer Antimicrobials in Pseudomonas aeruginosa

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02040-19 ◽

2020 ◽

Vol 64 (4) ◽

Cited By ~ 3

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.

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

mSystems ◽

10.1128/msystems.00149-18 ◽

2019 ◽

Vol 4 (1) ◽

Cited By ~ 13

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.

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Resistance and Heteroresistance to Colistin in Pseudomonas aeruginosa Isolates from Wenzhou, China

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00556-19 ◽

2019 ◽

Vol 63 (10) ◽

Cited By ~ 8

Author(s):

Jie Lin ◽

Chunquan Xu ◽

Renchi Fang ◽

Jianming Cao ◽

Xiucai Zhang ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Lipid A ◽

Population Analysis ◽

Colistin Resistance ◽

Content Type ◽

Maldi Tof Ms ◽

Expression Levels ◽

Maldi Tof ◽

Broth Microdilution Method ◽

Tof Ms

ABSTRACT The goal was to investigate the mechanisms of colistin resistance and heteroresistance in Pseudomonas aeruginosa clinical isolates. Colistin resistance was determined by the broth microdilution method. Colistin heteroresistance was evaluated by population analysis profiling. Time-kill assays were also conducted. PCR sequencing was performed to detect the resistance genes among (hetero)resistant isolates, and quantitative real-time PCR assays were performed to determine their expression levels. Pulsed-field gel electrophoresis and multilocus sequence typing were performed. Lipid A characteristics were determined via matrix-assisted laser desorption–ionization time of flight mass spectrometry (MALDI-TOF MS). Two resistant isolates and 9 heteroresistant isolates were selected in this study. Substitutions in PmrB were detected in 2 resistant isolates. Among heteroresistant isolates, 8 of 9 heteroresistant isolates had nonsynonymous PmrB substitutions, and 2 isolates, including 1 with a PmrB substitution, had PhoQ alterations. Correspondingly, the expression levels of pmrA or phoP were upregulated in PmrB- or PhoQ-substituted isolates. One isolate also found alterations in ParRS and CprRS. The transcript levels of the pmrH gene were observed to increase across all investigated isolates. MALDI-TOF MS showed additional 4-amino-4-deoxy-l-arabinose (l-Ara4N) moieties in lipid A profiles in (hetero)resistant isolates. In conclusion, both colistin resistance and heteroresistance in P. aeruginosa in this study mainly involved alterations of the PmrAB regulatory system. There were strong associations between mutations in specific genetic loci for lipid A synthesis and regulation of modifications to lipid A. The transition of colistin heteroresistance to resistance should be addressed in future clinical surveillance.

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Activity of Ceftazidime-Avibactam against Fluoroquinolone-Resistant Enterobacteriaceae and Pseudomonas aeruginosa

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05136-14 ◽

2015 ◽

Vol 59 (6) ◽

pp. 3059-3065 ◽

Cited By ~ 14

Author(s):

C. Pitart ◽

F. Marco ◽

T. A. Keating ◽

W. W. Nichols ◽

J. Vila

Keyword(s):

Pseudomonas Aeruginosa ◽

Resistant Mutant ◽

Fluoroquinolone Resistance ◽

Cross Resistance ◽

Content Type ◽

E Coli ◽

Microdilution Method ◽

Broth Microdilution Method ◽

The Impact

ABSTRACTCeftazidime-avibactam and comparator antibiotics were tested by the broth microdilution method against 200Enterobacteriaceaeand 25Pseudomonas aeruginosastrains resistant to fluoroquinolones (including strains with the extended-spectrum β-lactamase [ESBL] phenotype and ceftazidime-resistant strains) collected from our institution. The MICs and mechanisms of resistance to fluoroquinolone were also studied. Ninety-nine percent of fluoroquinolone-resistantEnterobacteriaceaestrains were inhibited at a ceftazidime-avibactam MIC of ≤4 mg/liter (using the susceptible CLSI breakpoint for ceftazidime alone as a reference). Ceftazidime-avibactam was very active against ESBLEscherichia coli(MIC90of 0.25 mg/liter), ESBLKlebsiella pneumoniae(MIC90of 0.5 mg/liter), ceftazidime-resistant AmpC-producing species (MIC90of 1 mg/liter), non-ESBLE. coli(MIC90of ≤0.125 mg/liter), non-ESBLK. pneumoniae(MIC90of 0.25 mg/liter), and ceftazidime-nonresistant AmpC-producing species (MIC90of ≤0.5 mg/liter). Ninety-six percent of fluoroquinolone-resistantP. aeruginosastrains were inhibited at a ceftazidime-avibactam MIC of ≤8 mg/liter (using the susceptible CLSI breakpoint for ceftazidime alone as a reference), with a MIC90of 8 mg/liter. Additionally, fluoroquinolone-resistant mutants from each species tested were obtainedin vitrofrom two strains, one susceptible to ceftazidime and the other a β-lactamase producer with a high MIC against ceftazidime but susceptible to ceftazidime-avibactam. Thereby, the impact of fluoroquinolone resistance on the activity of ceftazidime-avibactam could be assessed. The MIC90values of ceftazidime-avibactam for the fluoroquinolone-resistant mutant strains ofEnterobacteriaceaeandP. aeruginosawere ≤4 mg/liter and ≤8 mg/liter, respectively. We conclude that the presence of fluoroquinolone resistance does not affectEnterobacteriaceaeandP. aeruginosasusceptibility to ceftazidime-avibactam; that is, there is no cross-resistance.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02111-19 ◽

2020 ◽

Vol 64 (6) ◽

Cited By ~ 2

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.

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A Screen for Antibiotic Resistance Determinants Reveals a Fitness Cost of the Flagellum in Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.00682-19 ◽

2019 ◽

Vol 202 (6) ◽

Cited By ~ 1

Author(s):

E. A. Rundell ◽

N. Commodore ◽

A. L. Goodman ◽

B. I. Kazmierczak

Keyword(s):

Pseudomonas Aeruginosa ◽

Outer Membrane ◽

Critical Role ◽

Fitness Cost ◽

Intrinsic Resistance ◽

Content Type ◽

Fitness Advantage ◽

Membrane Barrier ◽

Flagellar Assembly ◽

Cell Envelopes

ABSTRACT The intrinsic resistance of Pseudomonas aeruginosa to many antibiotics limits treatment options for pseudomonal infections. P. aeruginosa’s outer membrane is highly impermeable and decreases antibiotic entry into the cell. We used an unbiased high-throughput approach to examine mechanisms underlying outer membrane-mediated antibiotic exclusion. Insertion sequencing (INSeq) identified genes that altered fitness in the presence of linezolid, rifampin, and vancomycin, antibiotics to which P. aeruginosa is intrinsically resistant. We reasoned that resistance to at least one of these antibiotics would depend on outer membrane barrier function, as previously demonstrated in Escherichia coli and Vibrio cholerae. This approach demonstrated a critical role of the outer membrane barrier in vancomycin fitness, while efflux pumps were primary contributors to fitness in the presence of linezolid and rifampin. Disruption of flagellar assembly or function was sufficient to confer a fitness advantage to bacteria exposed to vancomycin. These findings clearly show that loss of flagellar function alone can confer a fitness advantage in the presence of an antibiotic. IMPORTANCE The cell envelopes of Gram-negative bacteria render them intrinsically resistant to many classes of antibiotics. We used insertion sequencing to identify genes whose disruption altered the fitness of a highly antibiotic-resistant pathogen, Pseudomonas aeruginosa, in the presence of antibiotics usually excluded by the cell envelope. This screen identified gene products involved in outer membrane biogenesis and homeostasis, respiration, and efflux as important contributors to fitness. An unanticipated fitness cost of flagellar assembly and function in the presence of the glycopeptide antibiotic vancomycin was further characterized. These findings have clinical relevance for individuals with cystic fibrosis who are infected with P. aeruginosa and undergo treatment with vancomycin for a concurrent Staphylococcus aureus infection.

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Identification of Novel VEB β-Lactamase Enzymes and Their Impact on Avibactam Inhibition

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00047-16 ◽

2016 ◽

Vol 60 (5) ◽

pp. 3183-3186 ◽

Cited By ~ 14

Author(s):

Sushmita D. Lahiri ◽

Richard A. Alm

Keyword(s):

Pseudomonas Aeruginosa ◽

Binding Pocket ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Content Type ◽

Class A ◽

Extended Spectrum ◽

Class C

ABSTRACTCeftazidime-avibactam has activity againstPseudomonas aeruginosaandEnterobacteriaceaeexpressing numerous class A and class C β-lactamases, although the ability to inhibit many minor enzyme variants has not been established. Novel VEB class A β-lactamases were identified during characterization of surveillance isolates. The cloned novel VEB β-lactamases possessed an extended-spectrum β-lactamase phenotype and were inhibited by avibactam in a concentration-dependent manner. The residues that comprised the avibactam binding pocket were either identical or functionally conserved. These data demonstrate that avibactam can inhibit VEB β-lactamases.

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Multidrug Adaptive Resistance of Pseudomonas aeruginosa Swarming Cells

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01999-19 ◽

2019 ◽

Vol 64 (3) ◽

Cited By ~ 2

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.

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