scholarly journals Target (MexB)- and Efflux-Based Mechanisms Decreasing the Effectiveness of the Efflux Pump Inhibitor D13-9001 in Pseudomonas aeruginosa PAO1: Uncovering a New Role for MexMN-OprM in Efflux of β-Lactams and a Novel Regulatory Circuit (MmnRS) Controlling MexMN Expression

2018 ◽  
Vol 63 (2) ◽  
pp. e01718-18 ◽  
Author(s):  
Srijan Ranjitkar ◽  
Adriana K. Jones ◽  
Mina Mostafavi ◽  
Zachary Zwirko ◽  
Oleg Iartchouk ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Response Regulator ◽  
Heavy Metal Resistance ◽  
Metal Resistance ◽  
Rna Seq ◽  
Efflux Pump Inhibitor ◽  
Content Type ◽  
Regulatory Circuit ◽  
Outer Membrane Channel

ABSTRACT Efflux pumps contribute to antibiotic resistance in Gram-negative pathogens. Correspondingly, efflux pump inhibitors (EPIs) may reverse this resistance. D13-9001 specifically inhibits MexAB-OprM in Pseudomonas aeruginosa. Mutants with decreased susceptibility to MexAB-OprM inhibition by D13-9001 were identified, and these fell into two categories: those with alterations in the target MexB (F628L and ΔV177) and those with an alteration in a putative sensor kinase of unknown function, PA1438 (L172P). The alterations in MexB were consistent with reported structural studies of the D13-9001 interaction with MexB. The PA1438L172P alteration mediated a >150-fold upregulation of MexMN pump gene expression and a >50-fold upregulation of PA1438 and the neighboring response regulator gene, PA1437. We propose that these be renamed mmnR and mmnS for MexMN regulator and MexMN sensor, respectively. MexMN was shown to partner with the outer membrane channel protein OprM and to pump several β-lactams, monobactams, and tazobactam. Upregulated MexMN functionally replaced MexAB-OprM to efflux these compounds but was insusceptible to inhibition by D13-9001. MmnSL172P also mediated a decrease in susceptibility to imipenem and biapenem that was independent of MexMN-OprM. Expression of oprD, encoding the uptake channel for these compounds, was downregulated, suggesting that this channel is also part of the MmnSR regulon. Transcriptome sequencing (RNA-seq) of cells encoding MmnSL172P revealed, among other things, an interrelationship between the regulation of mexMN and genes involved in heavy metal resistance.

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 The rise and the fall of a Pseudomonas aeruginosa endemic lineage in a hospital

2021 ◽  
Vol 7 (9) ◽  
Author(s):  
Marie Petitjean ◽  
Paulo Juarez ◽  
Alexandre Meunier ◽  
Etienne Daguindau ◽  
Hélène Puja ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Infection Control ◽  
Water Distribution ◽  
Efflux Pump ◽  
Hospital Setting ◽  
Metal Resistance ◽  
Control Measures ◽  
Hospital Environment ◽  
Content Type ◽  
Over Time

The biological features that allow a pathogen to survive in the hospital environment are mostly unknown. The extinction of bacterial epidemics in hospitals is mostly attributed to changes in medical practice, including infection control, but the role of bacterial adaptation has never been documented. We analysed a collection of Pseudomonas aeruginosa isolates belonging to the Besançon Epidemic Strain (BES), responsible for a 12year nosocomial outbreak, using a genotype-to-phenotype approach. Bayesian analysis estimated the emergence of the clone in the hospital 5 years before its opening, during the creation of its water distribution network made of copper. BES survived better than the reference strains PAO1 and PA14 in a copper solution due to a genomic island containing 13 metal-resistance genes and was specifically able to proliferate in the ubiquitous amoeba Vermamoeba vermiformis. Mutations affecting amino-acid metabolism, antibiotic resistance, lipopolysaccharide biosynthesis, and regulation were enriched during the spread of BES. Seven distinct regulatory mutations attenuated the overexpression of the genes encoding the efflux pump MexAB-OprM over time. The fitness of BES decreased over time in correlation with its genome size. Overall, the resistance to inhibitors and predators presumably aided the proliferation and propagation of BES in the plumbing system of the hospital. The pathogen further spread among patients via multiple routes of contamination. The decreased prevalence of patients infected by BES mirrored the parallel and convergent genomic evolution and reduction that affected bacterial fitness. Along with infection control measures, this may have participated in the extinction of BES in the hospital setting.

scholarly journals Draft Genome Sequence of Pseudomonas aeruginosa Strain Hex1T Isolated from Soils Contaminated with Used Lubricating Oil in Argentina

2017 ◽  
Vol 5 (2) ◽  
Author(s):  
Adela M. Luján ◽  
Sofía Feliziani ◽  
Andrea M. Smania
Keyword(s):  
Energy Source ◽  
Pseudomonas Aeruginosa ◽  
Genome Sequence ◽  
Draft Genome ◽  
Heavy Metal Resistance ◽  
Metal Resistance ◽  
Draft Genome Sequence ◽  
Lubricating Oil ◽  
Content Type ◽  
Used Lubricating Oil

ABSTRACT Pseudomonas aeruginosa Hex1T was isolated from soils contaminated with used lubricating oil from a garage in Córdoba, Argentina. This strain is capable of utilizing this pollutant as the sole carbon and energy source. Here, we present the 6.9-Mb draft genome sequence of Hex1T, which contains many heavy metal-resistance genes.

scholarly journals The Biofilm Inhibitor Carolacton Enters Gram-Negative Cells: Studies Using a TolC-Deficient Strain of Escherichia coli

mSphere ◽  
2017 ◽  
Vol 2 (5) ◽  
Author(s):  
Jannik Donner ◽  
Michael Reck ◽  
Boyke Bunk ◽  
Michael Jarek ◽  
Constantin Benjamin App ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Streptococcus Pneumoniae ◽  
Carbon Atom ◽  
Efflux Pump ◽  
Gram Negative Bacteria ◽  
Rna Seq ◽  
Efflux Pump Inhibitor ◽  
Gram Negative ◽  
Content Type ◽  
E Coli

ABSTRACT The myxobacterial secondary metabolite carolacton inhibits growth of Streptococcus pneumoniae and kills biofilm cells of the caries- and endocarditis-associated pathogen Streptococcus mutans at nanomolar concentrations. Here, we studied the response to carolacton of an Escherichia coli strain that lacked the outer membrane protein TolC. Whole-genome sequencing of the laboratory E. coli strain TolC revealed the integration of an insertion element, IS5, at the tolC locus and a close phylogenetic relationship to the ancient E. coli K-12. We demonstrated via transcriptome sequencing (RNA-seq) and determination of MIC values that carolacton penetrates the phospholipid bilayer of the Gram-negative cell envelope and inhibits growth of E. coli TolC at similar concentrations as for streptococci. This inhibition is completely lost for a C-9 (R) epimer of carolacton, a derivative with an inverted stereocenter at carbon atom 9 [(S) → (R)] as the sole difference from the native molecule, which is also inactive in S. pneumoniae and S. mutans, suggesting a specific interaction of native carolacton with a conserved cellular target present in bacterial phyla as distantly related as Firmicutes and Proteobacteria. The efflux pump inhibitor (EPI) phenylalanine arginine β-naphthylamide (PAβN), which specifically inhibits AcrAB-TolC, renders E. coli susceptible to carolacton. Our data indicate that carolacton has potential for use in antimicrobial chemotherapy against Gram-negative bacteria, as a single drug or in combination with EPIs. Strain E. coli TolC has been deposited at the DSMZ; together with the associated RNA-seq data and MIC values, it can be used as a reference during future screenings for novel bioactive compounds. IMPORTANCE The emergence of pathogens resistant against most or all of the antibiotics currently used in human therapy is a global threat, and therefore the search for antimicrobials with novel targets and modes of action is of utmost importance. The myxobacterial secondary metabolite carolacton had previously been shown to inhibit biofilm formation and growth of streptococci. Here, we investigated if carolacton could act against Gram-negative bacteria, which are difficult targets because of their double-layered cytoplasmic envelope. We found that the model organism Escherichia coli is susceptible to carolacton, similar to the Gram-positive Streptococcus pneumoniae, if its multidrug efflux system AcrAB-TolC is either inactivated genetically, by disruption of the tolC gene, or physiologically by coadministering an efflux pump inhibitor. A carolacton epimer that has a different steric configuration at carbon atom 9 is completely inactive, suggesting that carolacton may interact with the same molecular target in both Gram-positive and Gram-negative bacteria.

scholarly journals Production of Norspermidine Contributes to Aminoglycoside Resistance in pmrAB Mutants of Pseudomonas aeruginosa

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Arnaud Bolard ◽  
Monika Schniederjans ◽  
Susanne Haüssler ◽  
Pauline Triponney ◽  
Benoît Valot ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Response Regulator ◽  
Cross Resistance ◽  
Aminoglycoside Resistance ◽  
Content Type ◽  
Uptake Pathway ◽  
Two Component Systems ◽  
Emergence Of Resistance

ABSTRACT Emergence of resistance to polymyxins in Pseudomonas aeruginosa is mainly due to mutations in two-component systems that promote the addition of 4-amino-4-deoxy-l-arabinose to the lipopolysaccharide (LPS) through upregulation of operon arnBCADTEF-ugd (arn) expression. Here, we demonstrate that mutations occurring in different domains of histidine kinase PmrB or in response regulator PmrA result in coresistance to aminoglycosides and colistin. All seventeen clinical strains tested exhibiting such a cross-resistance phenotype were found to be pmrAB mutants. As shown by gene deletion experiments, the decreased susceptibility of the mutants to aminoglycosides was independent from operon arn but required the efflux system MexXY-OprM and the products of three genes, PA4773-PA4774-PA4775, that are cotranscribed and activated with genes pmrAB. Gene PA4773 (annotated as speD2 in the PAO1 genome) and PA4774 (speE2) are predicted to encode enzymes involved in biosynthesis of polyamines. Comparative analysis of cell surface extracts of an in vitro selected pmrAB mutant, called AB16.2, and derivatives lacking PA4773, PA4774, and PA4775 revealed that these genes were needed for norspermidine production via a pathway that likely uses 1,3-diaminopropane, a precursor of polyamines. Altogether, our results suggest that norspermidine decreases the self-promoted uptake pathway of aminoglycosides across the outer membrane and, thereby, potentiates the activity of efflux pump MexXY-OprM.

scholarly journals Potentiation of Antibacterial Activity of the MB-1 Siderophore-Monobactam Conjugate Using an Efflux Pump Inhibitor

2015 ◽  
Vol 59 (4) ◽  
pp. 2439-2442 ◽  
Author(s):  
Andrew P. Tomaras ◽  
Jared L. Crandon ◽  
Craig J. McPherson ◽  
David P. Nicolau
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibacterial Activity ◽  
Efflux Pump ◽  
Efflux Pump Inhibitor ◽  
Content Type ◽  
Efflux Inhibition

ABSTRACTPreliminary enthusiasm over the encouraging spectrum andin vitroactivities of siderophore conjugates, such as MB-1, was recently tempered by unexpected variability inin vivoefficacy. The need for these conjugates to compete for iron with endogenously produced siderophores has exposed a significant liability for this novel antibacterial strategy. Here, we have exploited dependence on efflux for siderophore secretion inPseudomonas aeruginosaand provide evidence that efflux inhibition may circumvent thisin vivo-relevant resistance liability.

scholarly journals The Impact of an Efflux Pump Inhibitor on the Activity of Free and Liposomal Antibiotics against Pseudomonas aeruginosa

Pharmaceutics ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 577
Author(s):  
Douweh Leyla Gbian ◽  
Abdelwahab Omri
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Antimicrobial Activities ◽  
Dilution Method ◽  
Efflux Pump Inhibitor ◽  
Signal Production ◽  
Lung Infections ◽  
The Impact ◽  
Microbroth Dilution

The eradication of Pseudomonas aeruginosa in cystic fibrosis patients has become continuously difficult due to its increased resistance to treatments. This study assessed the efficacy of free and liposomal gentamicin and erythromycin, combined with Phenylalanine arginine beta-naphthylamide (PABN), a broad-spectrum efflux pump inhibitor, against P. aeruginosa isolates. Liposomes were prepared and characterized for their sizes and encapsulation efficiencies. The antimicrobial activities of formulations were determined by the microbroth dilution method. Their activity on P. aeruginosa biofilms was assessed, and the effect of sub-inhibitory concentrations on bacterial virulence factors, quorum sensing (QS) signals and bacterial motility was also evaluated. The average diameters of liposomes were 562.67 ± 33.74 nm for gentamicin and 3086.35 ± 553.95 nm for erythromycin, with encapsulation efficiencies of 13.89 ± 1.54% and 51.58 ± 2.84%, respectively. Liposomes and PABN combinations potentiated antibiotics by reducing minimum inhibitory and bactericidal concentrations by 4–32 fold overall. The formulations significantly inhibited biofilm formation and differentially attenuated virulence factor production as well as motility. Unexpectedly, QS signal production was not affected by treatments. Taken together, the results indicate that PABN shows potential as an adjuvant of liposomal macrolides and aminoglycosides in the management of lung infections in cystic fibrosis patients.

scholarly journals Curcumin as Efflux Pump Inhibitor Agent for Enhancement Treatment Against Multidrug Resistant Pseudomonas aeruginosa Isolates

2020 ◽  
pp. 59-67
Author(s):  
Sulaiman D. Sulaiman ◽  
Ghusoon A. Abdulhasan
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Inhibitory Concentration ◽  
Efflux Pump ◽  
Multidrug Resistant ◽  
Diffusion Method ◽  
Disc Diffusion Method ◽  
Efflux Pump Inhibitor ◽  
Before And After ◽  
Chromogenic Agar ◽  
Susceptibility Patterns

  Pseudomonas aeruginosa is considered as a developing opportunistic nosocomial pathogen and is well-known for its multidrug resistance that can be efficiently treated by a combination of antibiotics andefflux pump inhibitors (EPI). Therefore, the purpose of this study was to investigate the effect of curcumin as an EPI for the enhancement of the effectiveness of antibiotics against multidrug resistant (MDR) isolates ofP. aeruginosa. Susceptibility patterns of suspected bacteria was determined using the disc diffusion method andresistant bacteria were identified using chromogenic agar and 16S rDNA. The effectsof curcuminon the enhancement of antibiotics’s activity was evaluated usingthe broth microdilution method.The susceptibility patterns for 50 (67.6%) suspectedP. aeruginosaisolates showed that 36 (72%) of these isolateswere resistant to one of the used antibiotics,whereasonly 21 (42%) were MDR. The highest percentage of resistance was observedtoceftazidime (66%) followed by ciprofloxacin and levofloxacin (40%). Only 35 isolates were specified by chromogenic agar and 16S rDNAas P. aeruginosa.The minimal inhibitory concentration (MIC) of 35 isolates for ciprofloxacin resistant was between 4 and128 µg/ml while for ceftazidime was between 64and 512 µg/ml. After the addition of 50 μg/ml curcumin with ciprofloxacin, there wasa significant increase in the sensitivity (p≤ 0.01) of 13 MDR P.aeroginosa isolates whereas no differences in the sensitivity to ceftazidime were recorded before and after addition ofcurcumin. In conclusion, the results of this study show that curcumin can decrease the MIC value of ciprofloxacin in MDR isolates of P. aeruginosaand can be used as a native compound to enhance the treatment of resistant isolates with ciprofloxacin.

scholarly journals Assessment of Minocycline and Polymyxin B Combination against Acinetobacter baumannii

2015 ◽  
Vol 59 (5) ◽  
pp. 2720-2725 ◽  
Author(s):  
Dana R. Bowers ◽  
Henry Cao ◽  
Jian Zhou ◽  
Kimberly R. Ledesma ◽  
Dongxu Sun ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Clinical Utility ◽  
Efflux Pump ◽  
Polymyxin B ◽  
Intracellular Concentration ◽  
Bacterial Cells ◽  
Efflux Pump Inhibitor ◽  
Content Type

ABSTRACTAntimicrobial resistance amongAcinetobacter baumanniiis increasing worldwide, often necessitating combination therapy. The clinical utility of using minocycline with polymyxin B is not well established. In this study, we investigated the activity of minocycline and polymyxin B against 1 laboratory isolate and 3 clinical isolates ofA. baumannii. Minocycline susceptibility testing was performed with and without an efflux pump inhibitor, phenylalanine-arginine β-naphthylamide (PAβN). The intracellular minocycline concentration was determined with and without polymyxin B (0.5 μg/ml). Time-kill studies were performed over 24 h using approximately 106CFU/ml of each strain with clinically relevant minocycline concentrations (2 μg/ml and 8 μg/ml), with and without polymyxin B (0.5 μg/ml). Thein vivoefficacy of the combination was assessed in a neutropenic murine pneumonia model. Infected animals were administered minocycline (50 mg/kg), polymyxin B (10 mg/kg), or both to achieve clinically equivalent exposures in humans. A reduction in the minocycline MIC (≥4×) was observed in the presence of PAβN. The intracellular concentration andin vitrobactericidal effect of minocycline were both enhanced by polymyxin B. With 2 minocycline-susceptible strains, the bacterial burden in lung tissue at 24 h was considerably reduced by the combination compared to monotherapy with minocycline or polymyxin B. In addition, the combination prolonged survival of animals infected with a minocycline-susceptible strain. Polymyxin B increased the intracellular concentration of minocycline in bacterial cells and enhanced the bactericidal activity of minocycline, presumably due to efflux pump disruption. The clinical utility of this combination should be further investigated.

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