scholarly journals Resistance to β-Lactam Antibiotics inPseudomonas aeruginosa Due to Interplay between the MexAB-OprM Efflux Pump and β-Lactamase

1999 ◽  
Vol 43 (5) ◽  
pp. 1301-1303 ◽  
Author(s):  
Taiji Nakae ◽  
Akira Nakajima ◽  
Toshihisa Ono ◽  
Kohjiro Saito ◽  
Hiroshi Yoneyama
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Efflux System ◽  
High Level

ABSTRACT We evaluated the roles of the MexAB-OprM efflux pump and β-lactamase in β-lactam resistance in Pseudomonas aeruginosa by constructing OprM-deficient, OprM basal level, and OprM fully expressed mutants from β-lactamase-negative, -inducible, and -overexpressed strains. We conclude that, with the notable exception of imipenem, the MexAB-OprM pump contributes significantly to β-lactam resistance in both β-lactamase-negative and β-lactamase-inducible strains, while the contribution of the MexAB-OprM efflux system is negligible in strains with overexpressed β-lactamase. Overexpression of the efflux pump alone contributes to the high level of β-lactam resistance in the absence of β-lactamase.

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 Characterization of a Pseudomonas aeruginosa Efflux Pump Contributing to Aminoglycoside Impermeability

1999 ◽  
Vol 43 (12) ◽  
pp. 2975-2983 ◽  
Author(s):  
Shannon Westbrock-Wadman ◽  
David R. Sherman ◽  
Mark J. Hickey ◽  
Silvija N. Coulter ◽  
Ya Qi Zhu ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Molecular Mechanisms ◽  
Efflux Pump ◽  
Subtractive Hybridization ◽  
Hybridization Technique ◽  
Efflux System ◽  
Aminoglycoside Resistance ◽  
Molecular Events ◽  
Genes Encoding ◽  
The Many

ABSTRACT Pseudomonas aeruginosa can employ many distinct mechanisms of resistance to aminoglycoside antibiotics; however, in cystic fibrosis patients, more than 90% of aminoglycoside-resistantP. aeruginosa isolates are of the impermeability phenotype. The precise molecular mechanisms that produce aminoglycoside impermeability-type resistance are yet to be elucidated. A subtractive hybridization technique was used to reveal gene expression differences between PAO1 and isogenic, spontaneous aminoglycoside-resistant mutants of the impermeability phenotype. Among the many genes found to be up-regulated in these laboratory mutants were the amrABgenes encoding a recently discovered efflux system. TheamrAB genes appear to be the same as the recently describedmexXY genes; however, the resistance profile that we see inP. aeruginosa is very different from that described forEscherichia coli with mexXY. Direct evidence for AmrAB involvement in aminoglycoside resistance was provided by the deletion of amrB in the PAO1-derived laboratory mutant, which resulted in the restoration of aminoglycoside sensitivity to a level nearly identical to that of the parent strain. Furthermore, transcription of the amrAB genes was shown to be up-regulated in P. aeruginosa clinical isolates displaying the impermeability phenotype compared to a genotypically matched sensitive clinical isolate from the same patient. This suggests the possibility that AmrAB-mediated efflux is a clinically relevant mechanism of aminoglycoside resistance. Although it is unlikely that hyperexpression of AmrAB is the sole mechanism conferring the impermeability phenotype, we believe that the Amr efflux system can contribute to a complex interaction of molecular events resulting in the aminoglycoside impermeability-type resistance phenotype.

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 Identification and Characterization of TriABC-OpmH, a Triclosan Efflux Pump of Pseudomonas aeruginosa Requiring Two Membrane Fusion Proteins

2007 ◽  
Vol 189 (21) ◽  
pp. 7600-7609 ◽  
Author(s):  
Takehiko Mima ◽  
Swati Joshi ◽  
Margarita Gomez-Escalada ◽  
Herbert P. Schweizer
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Membrane Fusion ◽  
Fusion Proteins ◽  
Efflux Pump ◽  
Divalent Cations ◽  
Constitutive Expression ◽  
Multiple Copies ◽  
High Level ◽  
Triclosan Resistance ◽  
Membrane Fusion Proteins

ABSTRACT Pseudomonas aeruginosa achieves high-level (MIC > 1 mg/ml) triclosan resistance either by constitutive expression of MexAB-OprM, an efflux pump of the resistance nodulation cell division (RND) family, or expression of MexCD-OprJ, MexEF-OprN, and MexJK-OpmH in regulatory mutants. A triclosan-resistant target enzyme and perhaps other mechanisms probably act synergistically with efflux. To probe this notion, we exposed the susceptible Δ(mexAB-oprM) Δ(mexCD-oprJ) Δ(mexEF-oprN) Δ(mexJK) Δ(mexXY) strain PAO509 to increasing triclosan concentrations and derived a resistant strain, PAO509.5. This mutant overexpressed the PA0156-PA0157-PA0158 pump, which only effluxed triclosan, but not closely related compounds, antibiotics, and divalent cations, and was therefore renamed TriABC. Constitutive expression of the triABC operon was due to a single promoter-up mutation. Deletion of two adjacent genes, pcaR and PA0159, encoding transcriptional regulators had no effect on expression of this operon. TriABC is the only P. aeruginosa RND pump which contains two membrane fusion proteins, TriA and TriB, and both are required for efflux pump function. Probably owing to tight transcriptional coupling of the triABC genes, complementation of individual mutations was only partially achievable. Full complementation was only observed when a complete triABC operon was provided in trans, either in single or multiple copies. TriABC associated with OpmH, but not OprM, for assembly of a functional triclosan efflux pump. TriABC is the fifth RND pump in P. aeruginosa shown to efficiently efflux triclosan, supporting the notion that efflux is the primary mechanism responsible for this bacterium's high intrinsic and acquired triclosan resistance.

scholarly journals The MexJK Efflux Pump of Pseudomonas aeruginosa Requires OprM for Antibiotic Efflux but Not for Efflux of Triclosan

2002 ◽  
Vol 184 (18) ◽  
pp. 5036-5044 ◽  
Author(s):  
Rungtip Chuanchuen ◽  
Craig T. Narasaki ◽  
Herbert P. Schweizer
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Efflux Pump ◽  
Resistant Mutant ◽  
Efflux System ◽  
Membrane Fusion Protein ◽  
Single Nucleotide Change ◽  
Antibiotic Efflux

ABSTRACT Using the biocide triclosan as a selective agent, several triclosan-resistant mutants of a susceptible Pseudomonas aeruginosa strain were isolated. Cloning and characterization of a DNA fragment conferring triclosan resistance from one of these mutants revealed a hitherto uncharacterized efflux system of the resistance nodulation cell division (RND) family, which was named MexJK and which is encoded by the mexJK operon. Expression of this operon is negatively regulated by the product of mexL, a gene located upstream of and transcribed divergently from mexJK. The triclosan-resistant mutant contained a single nucleotide change in mexL, which caused an amino acid change in the putative helix-turn-helix domain of MexL. The MexL protein belongs to the TetR family of repressor proteins. The MexJK system effluxed tetracycline and erythromycin but only in the presence of the outer membrane protein channel OprM; OprJ and OprN did not function with MexJK. Triclosan efflux required neither of the outer membrane protein channels tested but necessitated the MexJ membrane fusion protein and the MexK inner membrane RND transporter. The results presented in this study suggest that MexJK may function as a two-component RND pump for triclosan efflux but must associate with OprM to form a tripartite antibiotic efflux system. Furthermore, the results confirm that triclosan is an excellent tool for the study of RND multidrug efflux systems and that this popular biocide therefore readily selects mutants which are cross-resistant with antibiotics.

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 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 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.

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