scholarly journals Differential selection of multidrug efflux systems by quinolones in Pseudomonas aeruginosa.

1997 ◽  
Vol 41 (11) ◽  
pp. 2540-2543 ◽  
Author(s):  
T Köhler ◽  
M Michea-Hamzehpour ◽  
P Plesiat ◽  
A L Kahr ◽  
J C Pechere
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Fluorine Atom ◽  
Efflux Pump ◽  
Resistance Mechanisms ◽  
Multidrug Efflux ◽  
Efflux System ◽  
In Vitro Exposure ◽  
Structural Determinant ◽  
Selection Of

Resistance mechanisms selected after in vitro exposure to 12 quinolones were analyzed for Pseudomonas aeruginosa. Efflux-type mutants were predominant. Quinolones differed in their ability to select a particular efflux system. While the newer fluoroquinolones favored the MexCD-OprJ system, the older quinolones selected exclusively the MexEF-OprN or MexAB-OprM systems. A protonable C-7 substituent in combination with a C-6 fluorine atom is a structural determinant of quinolones involved in efflux pump substrate specificity.

scholarly journals Expression of Pseudomonas aeruginosa Multidrug Efflux Pumps MexA-MexB-OprM and MexC-MexD-OprJ in a Multidrug-Sensitive Escherichia coli Strain

1998 ◽  
Vol 42 (1) ◽  
pp. 65-71 ◽  
Author(s):  
Ramakrishnan Srikumar ◽  
Tatiana Kon ◽  
Naomasa Gotoh ◽  
Keith Poole
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Crystal Violet ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Multidrug Efflux ◽  
Efflux System ◽  
E Coli ◽  
Multidrug Efflux Pumps

ABSTRACT The mexCD-oprJ and mexAB-oprM operons encode components of two distinct multidrug efflux pumps inPseudomonas aeruginosa. To assess the contribution of individual components to antibiotic resistance and substrate specificity, these operons and their component genes were cloned and expressed in Escherichia coli. Western immunoblotting confirmed expression of the P. aeruginosa efflux pump components in E. coli strains expressing and deficient in the endogenous multidrug efflux system (AcrAB), although only the ΔacrAB strain, KZM120, demonstrated increased resistance to antibiotics in the presence of the P. aeruginosa efflux genes. E. coli KZM120 expressing MexAB-OprM showed increased resistance to quinolones, chloramphenicol, erythromycin, azithromycin, sodium dodecyl sulfate (SDS), crystal violet, novobiocin, and, significantly, several β-lactams, which is reminiscent of the operation of this pump in P. aeruginosa. This confirmed previous suggestions that MexAB-OprM provides a direct contribution to β-lactam resistance via the efflux of this group of antibiotics. An increase in antibiotic resistance, however, was not observed when MexAB or OprM alone was expressed in KZM120. Thus, despite the fact that β-lactams act within the periplasm, OprM alone is insufficient to provide resistance to these agents. E. coli KZM120 expressing MexCD-OprJ also showed increased resistance to quinolones, chloramphenicol, macrolides, SDS, and crystal violet, though not to most β-lactams or novobiocin, again somewhat reminiscent of the antibiotic resistance profile of MexCD-OprJ-expressing strains ofP. aeruginosa. Surprisingly, E. coli KZM120 expressing MexCD alone also showed an increase in resistance to these agents, while an OprJ-expressing KZM120 failed to demonstrate any increase in antibiotic resistance. MexCD-mediated resistance, however, was absent in a tolC mutant of KZM120, indicating that MexCD functions in KZM120 in conjunction with TolC, the previously identified outer membrane component of the AcrAB-TolC efflux system. These data confirm that a tripartite efflux pump is necessary for the efflux of all substrate antibiotics and that the P. aeruginosa multidrug efflux pumps are functional and retain their substrate specificity in E. coli.

scholarly journals Overexpression of the MexEF-OprN Multidrug Efflux System Affects Cell-to-Cell Signaling in Pseudomonas aeruginosa

2001 ◽  
Vol 183 (18) ◽  
pp. 5213-5222 ◽  
Author(s):  
Thilo Köhler ◽  
Christian van Delden ◽  
Lasta Kocjancic Curty ◽  
Mehri Michea Hamzehpour ◽  
Jean-Claude Pechere
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Homoserine Lactone ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Efflux System ◽  
Sensing Systems ◽  
In The Wild ◽  
Type Mutant ◽  
Regulator Genes

ABSTRACT Intrinsic and acquired antibiotic resistance of the nosocomial pathogen Pseudomonas aeruginosa is mediated mainly by the expression of several efflux pumps of broad substrate specificity. Here we report that nfxC type mutants, overexpressing the MexEF-OprN efflux system, produce lower levels of extracellular virulence factors than the susceptible wild type. These include pyocyanin, elastase, and rhamnolipids, three factors controlled by the las and rhl quorum-sensing systems of P. aeruginosa. In agreement with these observations are the decreased transcription of the elastase genelasB and the rhamnosyltransferase genesrhlAB measured in nfxC type mutants. Expression of the lasR and rhlR regulator genes was not affected in the nfxC type mutant. In contrast, transcription of the C4-homoserine lactone (C4-HSL) autoinducer synthase gene rhlI was reduced by 50% in the nfxC type mutant relative to that in the wild type. This correlates with a similar decrease in C4-HSL levels detected in supernatants of the nfxC type mutant. Transcription of an rhlAB-lacZ fusion could be partially restored by the addition of synthetic C4-HSL andPseudomonas quinolone signal (PQS). It is proposed that the MexEF-OprN efflux pump affects intracellular PQS levels.

scholarly journals Toxic Electrophiles Induce Expression of the Multidrug Efflux Pump MexEF-OprN in Pseudomonas aeruginosa through a Novel Transcriptional Regulator, CmrA

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Paulo Juarez ◽  
Katy Jeannot ◽  
Patrick Plésiat ◽  
Catherine Llanes
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Redox Homeostasis ◽  
Transcriptional Regulator ◽  
Significant Loss ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Content Type ◽  
Constitutive Overexpression

ABSTRACT The multidrug efflux system MexEF-OprN is produced at low levels in wild-type strains of Pseudomonas aeruginosa. However, in so-called nfxC mutants, mutational alteration of the gene mexS results in constitutive overexpression of the pump, along with increased resistance of the bacterium to chloramphenicol, fluoroquinolones, and trimethoprim. In this study, analysis of in vitro-selected chloramphenicol-resistant clones of strain PA14 led to the identification of a new class of MexEF-OprN-overproducing mutants (called nfxC2) exhibiting alterations in an as-yet-uncharacterized gene, PA14_38040 (homolog of PA2047 in strain PAO1). This gene is predicted to encode an AraC-like transcriptional regulator and was called cmrA (for chloramphenicol resistance activator). In nfxC2 mutants, the mutated CmrA increases its proper gene expression and upregulates the operon mexEF-oprN through MexS and MexT, resulting in a multidrug resistance phenotype without significant loss in bacterial virulence. Transcriptomic experiments demonstrated that CmrA positively regulates a small set of 11 genes, including PA14_38020 (homolog of PA2048), which is required for the MexS/T-dependent activation of mexEF-oprN. PA2048 codes for a protein sharing conserved domains with the quinol monooxygenase YgiN from Escherichia coli. Interestingly, exposure of strain PA14 to toxic electrophilic molecules (glyoxal, methylglyoxal, and cinnamaldehyde) strongly activates the CmrA pathway and upregulates MexEF-OprN and, thus, increases the resistance of P. aeruginosa to the pump substrates. A picture emerges in which MexEF-OprN is central in the response of the pathogen to stresses affecting intracellular redox homeostasis.

scholarly journals In vitro dynamics and mechanisms of resistance development to imipenem and imipenem/relebactam in Pseudomonas aeruginosa

2020 ◽  
Vol 75 (9) ◽  
pp. 2508-2515 ◽  
Author(s):  
María A Gomis-Font ◽  
Gabriel Cabot ◽  
Irina Sánchez-Diener ◽  
Pablo A Fraile-Ribot ◽  
Carlos Juan ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Resistance Mechanisms ◽  
Infection Model ◽  
Mechanisms Of Resistance ◽  
Resistance Development ◽  
C Elegans ◽  
Imipenem Resistance ◽  
High Level

Abstract Objectives We analysed the dynamics and mechanisms of resistance development to imipenem alone or combined with relebactam in Pseudomonas aeruginosa WT (PAO1) and mutator (PAOMS; ΔmutS) strains. Methods PAO1 or PAOMS strains were incubated for 24 h in Mueller–Hinton Broth with 0.125–64 mg/L of imipenem ± relebactam 4 mg/L. Tubes from the highest antibiotic concentration showing growth were reinoculated in fresh medium containing concentrations up to 64 mg/L of imipenem ± relebactam for 7 days. Two colonies per strain, replicate experiment and antibiotic from early (Day 1) and late (Day 7) cultures were characterized by determining the susceptibility profiles, WGS and determination of the expression of ampC and efflux-pump-coding genes. Virulence was studied in a Caenorhabditis elegans infection model. Results Relebactam reduced imipenem resistance development for both strains, although resistance emerged much faster for PAOMS. WGS indicated that imipenem resistance was associated with mutations in the porin OprD and regulators of ampC, while the mutations in imipenem/relebactam-resistant mutants were located in oprD and regulatoras of MexAB-OprM. High-level imipenem/relebactam resistance was only documented in the PAOMS strain and was associated with an additional specific (T680A) mutation located in the catalytic pocket of ponA (PBP1a) and with reduced virulence in the C. elegans model. Conclusions Imipenem/relebactam could be a useful alternative for the treatment of MDR P. aeruginosa infections, potentially reducing resistance development during treatment. Moreover, this work deciphers the potential resistance mechanisms that may emerge upon the introduction of this novel combination into clinical practice.

scholarly journals Efflux Unbalance in Pseudomonas aeruginosa Isolates from Cystic Fibrosis Patients

2009 ◽  
Vol 53 (5) ◽  
pp. 1987-1997 ◽  
Author(s):  
Lucie Vettoretti ◽  
Patrick Plésiat ◽  
Cédric Muller ◽  
Farid El Garch ◽  
Gilles Phan ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Natural Resistance ◽  
Loss Of Function ◽  
Multidrug Efflux ◽  
Efflux System ◽  
Lactam Antibiotic ◽  
Rnd Transporter ◽  
First Time ◽  
Selection Of

ABSTRACT Retrospective analysis of 189 nonredundant strains of Pseudomonas aeruginosa sequentially recovered from the sputum samples of 46 cystic fibrosis (CF) patients over a 10-year period (1998 to 2007) revealed that 53 out of 189 (28%) samples were hypersusceptible to the β-lactam antibiotic ticarcillin (MIC ≤ 4 μg/ml) (phenotype dubbed Tichs). As evidenced by trans-complementation and gene inactivation experiments, the mutational upregulation of the efflux system MexXY was responsible for various degrees of resistance to aminoglycosides in a selection of 11 genotypically distinct strains (gentamicin MICs from 2 to 64 μg/ml). By demonstrating for the first time that the MexXY pump may evolve in CF strains, we found that a mutation leading to an F1018L change in the resistance-nodulation-cell division (RND) transporter MexY was able to increase pump-promoted resistance to aminoglycosides, cefepime, and fluoroquinolones twofold. The inactivation of the mexB gene (which codes for the RND transporter MexB) in the 11 selected strains showed that the Tichs phenotype was due to a mutational or functional loss of function of MexAB-OprM, the multidrug efflux system known to contribute to the natural resistance of P. aeruginosa to β-lactams (e.g., ticarcillin and aztreonam), fluoroquinolones, tetracycline, and novobiocin. Two of the selected strains synthesized abnormally low amounts of the MexB protein, and 3 of 11 strains expressed truncated MexB (n = 2) or MexA (n = 1) polypeptide as a result of mutations in the corresponding genes, while 7 of 11 strains produced wild-type though nonfunctional MexAB-OprM pumps at levels similar to or even higher than that of reference strain PAO1. Overall, our data indicate that while MexXY is necessary for P. aeruginosa to adapt to the hostile environment of the CF lung, the MexAB-OprM pump is dispensable and tends to be lost or inactivated in subpopulations of P. aeruginosa.

scholarly journals Role of the MexEF-OprN Efflux System in Low-Level Resistance of Pseudomonas aeruginosa to Ciprofloxacin

2011 ◽  
Vol 55 (12) ◽  
pp. 5676-5684 ◽  
Author(s):  
Catherine Llanes ◽  
Thilo Köhler ◽  
Isabelle Patry ◽  
Barbara Dehecq ◽  
Christian van Delden ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Reference Strain ◽  
Efflux Pump ◽  
Resistance Mechanisms ◽  
Efflux System ◽  
Content Type ◽  
Clinical Strains ◽  
Major Mechanism ◽  
Level Resistance

ABSTRACTIn this study, we investigated the resistance mechanisms to fluoroquinolones of 85 non-cystic fibrosis strains ofPseudomonas aeruginosaexhibiting a reduced susceptibility to ciprofloxacin (MICs from 0.25 to 2 μg/ml). In addition to MexAB-OprM (31 of 85 isolates) and MexXY/OprM (39 of 85), the MexEF-OprN efflux pump (10 of 85) was found to be commonly upregulated in this population that is considered susceptible or of intermediate susceptibility to ciprofloxacin, according to current breakpoints. Analysis of the 10 MexEF-OprN overproducers (nfxCmutants) revealed the presence of various mutations in themexT(2 isolates),mexS(5 isolates), and/ormvaT(2 isolates) genes, the inactivation of which is known to increase the expression of themexEF-oprNoperon in reference strain PAO1-UW. However, these genes were intact in 3 of 10 of the clinical strains. Interestingly, ciprofloxacin at 2 μg/ml or 4 μg/ml preferentially selectednfxCmutants from wild-type clinical strains (n= 10 isolates) and from first-step mutants (n= 10) overexpressing Mex pumps, thus indicating that MexEF-OprN represents a major mechanism by whichP. aeruginosamay acquire higher resistance levels to fluoroquinolones. These data support the notion that thenfxCmutants may be more prevalent in the clinical setting than anticipated and strongly suggest the involvement of still unknown genes in the regulation of this efflux system.

scholarly journals Constitutive Activation of MexT by Amino Acid Substitutions Results in MexEF-OprN Overproduction in Clinical Isolates ofPseudomonas aeruginosa

2018 ◽  
Vol 62 (5) ◽  
Author(s):  
Paulo Juarez ◽  
Isabelle Broutin ◽  
Christophe Bordi ◽  
Patrick Plésiat ◽  
Catherine Llanes
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Regulatory Gene ◽  
Clinical Isolates ◽  
Amino Acid Substitutions ◽  
Multidrug Efflux ◽  
Efflux System ◽  
Content Type ◽  
Constitutive Activation ◽  
Multidrug Efflux System

ABSTRACTWhen overproduced, the multidrug efflux system MexEF-OprN increases the resistance ofPseudomonas aeruginosato fluoroquinolones, chloramphenicol, and trimethoprim. In this work, we demonstrate that gain-of-function mutations in the regulatory genemexTresult in oligomerization of the LysR regulator MexT, constitutive upregulation of the efflux pump, and increased resistance in clinical isolates.

scholarly journals Development of in vitro resistance to fluoroquinolones in Pseudomonas aeruginosa

2020 ◽  
Author(s):  
Lei Zhao ◽  
Shiqi Wang ◽  
Xiaobing Li ◽  
Xiaojing He ◽  
Lingyan Jian
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Target Genes ◽  
Efflux Pump ◽  
Resistance Mechanisms ◽  
Fluoroquinolone Resistance ◽  
Relative Quantitation ◽  
Evolutionary Trajectories ◽  
Polymerase Chain ◽  
Quantitation Method

Abstract Fluoroquinolone resistance in Pseudomonas aeruginosa typically arises through site-specific mutations and overexpression of efflux pumps. In this study, we investigated the dynamics of different resistance mechanisms in P. aeruginosa populations that have evolved under fluoroquinolone pressure, as well as the interactions between these mechanisms in evolutionary trajectories. Bacteria of strain ATCC27853 were selected under different concentrations of ciprofloxacin and levofloxacin for six parallel lineages, followed by amplification of four target genes in the quinolone-resistance determining region (QRDR) and Sanger sequencing to identify the mutations. The expression of four efflux pump proteins was evaluated by real-time polymerase chain reaction using the relative quantitation method, with the ATCC27853 strain used as a control. We found that ciprofloxacin killed P. aeruginosa sooner than did levofloxacin. Further, we identified five different mutations in three subunits of QRDRs, with gyrA as the main mutated gene associated with conferring fluoroquinolone resistance. Additionally, we found a larger number of mutations appearing at 2 mg/L and 4 mg/L of ciprofloxacin and levofloxacin, respectively. Moreover, we identified the main efflux pump being expressed as MexCD-OprJ, with initial overexpression observed at 0.25 mg/L and 0.5 mg/L of ciprofloxacin and levofloxacin, respectively. These results demonstrated gyrA83 mutation and MexCD-OprJ overexpression as the primary mechanism conferring ciprofloxacin and levofloxacin resistance in P. aeruginosa. In addition, we also show that ciprofloxacin exhibited a stronger ability to kill the bacteria while potentially rendering it more susceptible to resistance.

scholarly journals Role of the MexAB-OprM Efflux Pump of Pseudomonas aeruginosa in Tolerance to Tea Tree (Melaleuca alternifolia) Oil and Its Monoterpene Components Terpinen-4-ol, 1,8-Cineole, and α-Terpineol

2008 ◽  
Vol 74 (6) ◽  
pp. 1932-1935 ◽  
Author(s):  
Chelsea J. Papadopoulos ◽  
Christine F. Carson ◽  
Barbara J. Chang ◽  
Thomas V. Riley
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antimicrobial Agent ◽  
Efflux Pump ◽  
Multidrug Efflux ◽  
Efflux System ◽  
Melaleuca Alternifolia ◽  
Tea Tree ◽  
Multidrug Efflux System

ABSTRACT Using a series of efflux mutants of Pseudomonas aeruginosa, the MexAB-OprM pump was identified as contributing to this organism's tolerance to the antimicrobial agent tea tree (Melaleuca alternifolia) oil and its monoterpene components terpinen-4-ol, 1,8-cineole, and α-terpineol. These data show that a multidrug efflux system of P. aeruginosa can extrude monoterpenes and related alcohols.

Conservation of the multidrug resistance efflux gene oprM in Pseudomonas aeruginosa.

1997 ◽  
Vol 41 (4) ◽  
pp. 853-856 ◽  
Author(s):  
N Bianco ◽  
S Neshat ◽  
K Poole
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Multidrug Resistance ◽  
Pseudomonas Syringae ◽  
Genomic Dna ◽  
Resistance Mechanisms ◽  
Pseudomonas Chlororaphis ◽  
Multidrug Efflux ◽  
Efflux System ◽  
Multidrug Resistance Gene ◽  
Multidrug Efflux System

An intragenic probe derived from the multidrug resistance gene oprM hybridized with genomic DNA from all 20 serotypes of Pseudomonas aeruginosa and from all 34 environmental and clinical isolates tested, indicating that the MexA-MexB-OprM multidrug efflux system is highly conserved in this organism. The oprM probe also hybridized with genomic DNA from Pseudomonas aureofaciens, Pseudomonas chlororaphis, Pseudomonas syringae, Burkholderia pseudomallei, and Pseudomonas putida, suggesting that efflux-mediated multidrug resistance mechanisms may be somewhat broadly distributed.

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