Molecular Detection of Aminoglycoside Resistance Mediated by Efflux Pump and Modifying Enzymes in Pseudomonas Aeruginosa Isolated From Iraqi Hospitals

2015 ◽  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Molecular Detection ◽  
Efflux Pump ◽  
Aminoglycoside Resistance

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 Expression of the MexXY Aminoglycoside Efflux Pump and Presence of an Aminoglycoside-Modifying Enzyme in Clinical Pseudomonas aeruginosa Isolates Are Highly Correlated

2020 ◽  
Vol 65 (1) ◽  
pp. e01166-20
Author(s):  
Alexander Seupt ◽  
Monika Schniederjans ◽  
Jürgen Tomasch ◽  
Susanne Häussler
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Aminoglycoside Resistance ◽  
Content Type ◽  
Main Driver ◽  
Gentamicin Resistance ◽  
Bacterial Cell Membrane ◽  
Highly Correlated ◽  
The Impact ◽  
Gene Expression Levels

ABSTRACTThe impact of MexXY efflux pump expression on aminoglycoside resistance in clinical Pseudomonas aeruginosa isolates has been debated. In this study, we found that, in general, elevated mexXY gene expression levels in clinical P. aeruginosa isolates confer to slight increases in aminoglycoside MIC levels; however, those levels rarely lead to clinically relevant resistance phenotypes. The main driver of resistance in the clinical isolates studied here was the acquisition of aminoglycoside-modifying enzymes (AMEs). Nevertheless, acquisition of an AME was strongly associated with mexY overexpression. In line with this observation, we demonstrate that the introduction of a gentamicin acetyltransferase confers to full gentamicin resistance levels in a P. aeruginosa type strain only if the MexXY efflux pump was active. We discuss that increased mexXY activity in clinical AME-harboring P. aeruginosa isolates might affect ion fluxes at the bacterial cell membrane and thus might play a role in the establishment of enhanced fitness that extends beyond aminoglycoside resistance.

scholarly journals Mutation-driven evolution of Pseudomonas aeruginosa in the presence of either ceftazidime or ceftazidime/avibactam

2018 ◽  
Author(s):  
Fernando Sanz-García ◽  
Sara Hernando-Amado ◽  
José Luis Martínez
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Multidrug Resistant ◽  
Beta Lactam ◽  
Two Component System ◽  
Aminoglycoside Resistance ◽  
Beta Lactamases ◽  
Chromosomal Deletions ◽  
Main Driver ◽  
Selection Of

ABSTRACTCeftazidime/avibactam is a combination of beta-lactam/beta-lactamases inhibitor, which use is restricted to some clinical cases including cystic fibrosis patients infected with multidrug resistant Pseudomonas aeruginosa, in which mutation is the main driver of resistance. This study aims to predict the mechanisms of mutation-driven resistance that are selected for when P. aeruginosa is challenged with either ceftazidime or ceftazidime/avibactam. For this purpose, P. aeruginosa PA14 was submitted to experimental evolution in the absence of antibiotics and in the presence of increasing concentrations of ceftazidime or ceftazidime/avibactam for 30 consecutive days. Final populations were analysed by whole-genome sequencing. All evolved populations reached similar levels of ceftazidime resistance. Besides, all of them were more susceptible to amikacin and produced pyomelanin. A first event in the evolution was the selection of large chromosomal deletions containing hmgA (involved in pyomelanin production), galU (involved in β-lactams resistance) and mexXY-oprM (involved in aminoglycoside resistance). Besides mutations in mpl and dacB that regulate β-lactamase expression, mutations related to MexAB-OprM overexpression were prevalent. Ceftazidime/avibactam challenge selected mutants in the putative efflux pump PA14_45890-45910 and in a two-component system (PA14_45870-45880), likely regulating its expression. All populations produce pyomelanin and were more susceptible to aminoglycosides likely due to the selection of large chromosomal deletions. Since pyomelanin-producing mutants, presenting similar deletions are regularly isolated from infections, the potential aminoglycosides hyper-susceptiblity and reduced β-lactams susceptibility of pyomelanin-producing P. aeruginosa should be taken into consideration for treating infections by these isolates.

scholarly journals MexXY efflux pump overexpression and aminoglycoside resistance in cystic fibrosis isolates of Pseudomonas aeruginosa from chronic infections

2017 ◽  
Vol 63 (12) ◽  
pp. 929-938 ◽  
Author(s):  
Manu Singh ◽  
Yvonne C.W. Yau ◽  
Shirley Wang ◽  
Valerie Waters ◽  
Ayush Kumar
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Multidrug Resistant ◽  
Regulatory Genes ◽  
Multidrug Efflux ◽  
Chronic Infections ◽  
Aminoglycoside Resistance ◽  
Resistance Nodulation Division ◽  
Lung Infections

In this study, we analyzed 15 multidrug-resistant cystic fibrosis isolates of Pseudomonas aeruginosa from chronic lung infections for expression of 4 different multidrug efflux systems (MexAB–OprM, MexCD–OprJ, MexEF–OprN, and MexXY), using quantitative reverse transcriptase PCR. Overexpression of MexXY pump was observed in all of the isolates tested. Analysis of regulatory genes that control the expression of these 4 efflux pumps revealed a number of previously uncharacterized mutations. Our work shows that MexXY pump overexpression is common in cystic fibrosis isolates and could be contributing to their reduced aminoglycoside susceptibility. Further, we also identified novel mutations in the regulatory genes of the 4 abovementioned Resistance–Nodulation–Division superfamily pumps that may be involved in the overexpression of these pumps.

scholarly journals Global reprogramming of virulence and antibiotic resistance in Pseudomonas aeruginosa by a single nucleotide polymorphism in the elongation factor-encoding gene, fusA1

2019 ◽  
Author(s):  
Eve A. Maunders ◽  
Rory C. Triniman ◽  
Taufiq Rahman ◽  
Martin Welch
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Large Scale ◽  
Efflux Pump ◽  
Elongation Factor ◽  
Virulence Gene ◽  
Mutant Gene ◽  
Opportunistic Pathogen ◽  
Aminoglycoside Resistance ◽  
Virulence Gene Expression

AbstractPseudomonas aeruginosa is a common opportunistic pathogen. The organism displays elevated intrinsic antibiotic resistance and can cause life-threatening infections. The gene encoding an elongation factor, FusA1, is frequently mutated in clinical isolates of P. aeruginosa from patients with cystic fibrosis (CF). Recent work has shown that fusA1 mutants often display elevated aminoglycoside resistance due to increased expression of the aminoglycoside efflux pump, MexXY. In the current work, we isolated a spontaneous gentamicin-resistant fusA1 mutant (FusA1P443L) in which mexXY expression was increased. Through a combination of proteomic and transcriptomic analyses, we found that the fusA1 mutant also exhibited large-scale but discrete changes in the expression of key pathogenicity-associated genes. Most notably, the fusA1 mutant displayed greatly increased expression of the Type III Secretion system (T3SS), widely considered to be the most potent virulence factor in the P. aeruginosa arsenal, and also elevated expression of the Type VI Secretion (T6S) machinery. This was unexpected because expression of the T3SS is usually reciprocally coordinated with T6S system expression. The fusA1 mutant also displayed elevated exopolysaccharide production, dysregulated siderophore production, elevated ribosomal protein synthesis, and transcriptomic signatures indicative of translational stress. Each of these phenotypes (and almost all of the transcriptomic and proteomic changes associated with the fusA1 mutation) were restored to levels comparable to that in the PAO1-derived progenitor strain by expression of the wild-type fusA1 gene in trans, indicating that the mutant gene is recessive. Our data show that in addition to elevating antibiotic resistance through mexXY expression (although we also identify additional contributory resistance mechanisms), mutations in fusA1 can lead to highly-selective dysregulation of virulence gene expression.

Aminoglycoside resistance mechanisms in Pseudomonas aeruginosa isolates from non-cystic fibrosis patients in Thailand

2013 ◽  
Vol 59 (1) ◽  
pp. 51-56 ◽  
Author(s):  
Kanchana Poonsuk ◽  
Chanwit Tribuddharat ◽  
Rungtip Chuanchuen
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
High Resistance ◽  
Amino Acid Change ◽  
Efflux Pump ◽  
Resistance Mechanisms ◽  
Aminoglycoside Resistance ◽  
Resistance Rates

This study aimed to examine aminoglycosides (AMGs) resistance mechanisms, including the AMG-modifying enzyme genes, mexXY, rplY, nuoG, and galU, in the Pseudomonas aeruginosa non-cystic fibrosis (CF) isolates in Thailand. One hundred P. aeruginosa isolates from non-CF patients were examined for susceptibility to AMGs and for the presence of 10 AMG-modifying enzyme genes. Thirty randomly selected isolates were tested for transcription of mexXY and nuoG and mutations in rplY and galU. All the P. aeruginosa isolates exhibited simultaneous resistance to at least 4 AMGs. High resistance rates to amikacin (92%), gentamicin (95%), streptomycin (99%), and tobramycin (96%) were observed, and all isolates were resistant to kanamycin, neomycin, and spectinomycin. Nine AMG-modifying enzyme genes were detected, including aadA1 (84%), aadB (84%), aadA2 (67%), ant(2″)-Ia (72%), strA-strB (70%), aph(3′)-IIb (57%), aac(3′)-Ia (40%), and aac(6′)-IIa (27%). None of the isolates harbored aac(6′)-IIb. Of 30 isolates tested, all but 1 isolate expressed MexXY. Two isolates did not express nuoG. Six isolates carried an amino acid change in RplY, but none of the isolates harbored mutation in galU. The results indicated that the AMG-modifying enzyme genes were widespread among the P. aeruginosa non-CF isolates. The MexXY efflux pump and inactivation for rplY played a role in AMG resistance but disruption of nuoG or galU did not.

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.

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