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Prevalence of resistance to aminoglycosides and fluoroquinolones among Pseudomonas aeruginosa strains in a University Hospital in Northeastern Poland


The present study was conducted to investigate the prevalence of genes encoding resistance to aminoglycosides and fluoroquinolones among twenty-five Pseudomonas aeruginosa isolated between 2002 and 2009. In PCR, following genes were detected: ant(2")-Ia in 9 (36.0%), aac(6')-Ib in 7 (28.0%), qnrB in 5 (20.0%), aph(3")-Ib in 2 (8.0%) of isolates.

Pseudomonas aeruginosa; plasmid-mediated resistance to aminoglycosides and fluoroquinolones; aminoglycoside-modifying enzymes


Prevalence of resistance to aminoglycosides and fluoroquinolones among Pseudomonas aeruginosa strains in a University Hospital in Northeastern Poland

Anna Diana Michalska* * Both authors contributed equally to this work. ; Pawel Tomasz Sacha* * Both authors contributed equally to this work. ; Dominika Ojdana; Anna Wieczorek; Elzbieta Tryniszewska

Department of Microbiological Diagnostics and Infectious Immunology, Medical University of Bialystok, Poland

Correspondence Correspondence: A.D. Michalska Department of Microbiological Diagnostics and Infectious Immunology Medical University of Bialystok Waszyngtona Str. 15A 15-269 Bialystok, Poland E-mail:


The present study was conducted to investigate the prevalence of genes encoding resistance to aminoglycosides and fluoroquinolones among twenty-five Pseudomonas aeruginosa isolated between 2002 and 2009. In PCR, following genes were detected: ant(2")-Ia in 9 (36.0%), aac(6')-Ib in 7 (28.0%), qnrB in 5 (20.0%), aph(3")-Ib in 2 (8.0%) of isolates.

Key words: Pseudomonas aeruginosa, plasmid-mediated resistance to aminoglycosides and fluoroquinolones, aminoglycoside-modifying enzymes.

Pseudomonasaeruginosa is a non-fermentative, Gram-negative bacterium widespread in the natural and artificial environment. Characteristic feature of this pathogen is a remarkable ability to develop antimicrobial resistance, thus infections caused by multidrug-resistant (MDR) strains are associated with high mortality rate and elevated treatment cost (Lister et al., 2009). Many studies report that selection of highly resistant mutants occurs in Intensive Care Units and P. aeruginosa is a main cause of nosocomial infections (Wolska et al., 2012). Resistance to antibiotics may be linked both with chromosomal mutations and acquisition of resistance genes located on mobile genetic elements, such as plasmids, integrons, and transposons (Lister et al., 2009). From variety of plasmid-mediated aminoglycoside resistance mechanisms, the most commonly encountered is the production of aminoglycoside-modifying enzymes (Tada et al., 2013). High level of resistance to aminoglycosides can also be mediated with production of 16S rRNA methyltransferases, which preclude disturbance of protein synthesis caused by aminoglycoside molecule (Doi and Arakawa, 2007). Currently ten genes encoding these enzymes were detected, of which the most common are armA and rmtB (Deng et al., 2013). Plasmid-associated resistance to fluroquinolones can be mediated by the production of Qnr proteins, which preserve DNA gyrase and topoisomerase IV from inhibition by quinolones (Poirel, 2012). This mechanism contributes to low-level fluoroquinolone resistance, but it is able to broadening the mutant selecting window (Drlica and Zhao, 2007).

The aim of this study was to determine the prevalence of plasmid-mediated genes encoding aminoglycoside-modifying enzymes, 16S rRNA methyltransferases, and Qnr-like proteins among MDR P. aeruginosa strains.

Twenty-five nonduplicated P. aeruginosa strains were obtained from patients hospitalized in two Intensive Care Units at University Hospital of Bialystok (northeastern Poland) between July 2002 and October 2009. Isolates were selected due to their reduced susceptibility to aminoglycosides, fluoroquinolones, third- and fourth generation cephalosporins, and/or carbapenems. Identification and susceptibility testing were conducted using an automated VITEK 2 system with AST-N093 cards (bioMérieux, Marcy l'Etoile, France). Susceptibility to antibiotics was interpreted according to the EUCAST criteria published on February 11, 2013 (The European Committee on Antimicrobial Susceptibility Testing, 2013). The minimal inhibitory concentrations (MICs) of gentamicin, amikacin, netilmicin, ciprofloxacin, imipenem, meropenem, ceftazidime, and cefepime were determined by Etest technique (bioMérieux). Plasmid material was isolated from overnight cultures by Plasmid Mini Kit (A&A Biotechnology, Gdynia, Poland). Screening of ampC gene was performed by polymerase chain reaction (PCR) with specific primer pair. Primers for amplification of aac(6')-Ib, aac(3)-Ia, ant(4')-IIa, ant(2")-Ia, aph(3")-Ib, armA, rmtB, qnrA, qnrB, qnrS genes were designed from sequences deposited in the National Center for Biotechnology Information website ( or were selected from the literature (Table 1). Conditions of each PCR reaction are listed in Table 1. All PCR assays were performed in the LabCycler Gradient (SensoQuest GmbH, Goettingen, Germany). Sequencing of genes encoding aminoglycoside-modifying enzymes was conducted with primers listed in Table 1, using the 3500 Genetic Analyzer (Applied Biosystems, Foster City, USA).

The genes encoding aminoglycoside-modifying enzymes were identified in plasmid material of 13 strains (52.0%). PCR assays revealed the presence of ant(2")-Ia gene in nine (36.0%), aac(6')-Ib gene in seven (28.0%), and aph(3")-Ib gene in two (8.0%) strains. Three isolates harbored two genes encoding aminoglycoside-modifying enzymes: aac(6')-Ib and ant(2")-Ia in two strains; ant(2")-Ia and aph(3")-Ib in one strain. One isolate carried three genes for resistance to aminoglycosides: aac(6')-Ib, ant(2")-Ia, and aph(3")-Ib. QnrB gene related with plasmid-mediated resistance to quinolones was detected in five (20.0%) strains. Sequencing of the PCR-positive products confirmed the presence of ant(2")-Ia, aac(6')-Ib, aph(3")-Ib, and qnrB1 genes in particular strains (GenBank accession numbers: ant(2")-Ia X04555.1; aac(6')-Ib JF901756.1; aph(3")-Ib M28829.1, qnrB1 DQ777878.1). Genes aac(3)-Ia, ant(4')-IIa, armA, rmtB, qnrA, and qnrS were not identified in plasmid DNA of tested strains. Characteristic of MDR strains with identified genes for resistance to aminoglycosides and quinolones are shown in Table 2. The highest efficiency among antimicrobials showed ceftazidime (68.0% of all tested strains were susceptible). The only aminoglycoside active against tested strains was amikacin (8.0% of all tested strains). Higher resistance rates were observed in strains carrying genes encoding aminoglycoside-modifying enzymes, than in strains without this genes detected. Level of resistance to ciprofloxacin was noticeably higher in strains harboring qnrB gene than in strains without this gene identified (MIC50: ≥ 32 vs. MIC50: 8). As for carbapenems, more isolates were susceptible to imipenem (28.0%) than meropenem (24.0%).

Over the years, numerous studies reported the increasing prevalence of MDR P. aeruginosa in hospital environments all around the world. The present study focused on the investigation of plasmid-mediated resistance to aminoglycosides and fluoroquinolones in hospital located in northeastern Poland. The most frequently detected gene was ant(2")-Ia (36.0%). Spanish research also revealed that ant(2")-Ia gene occurs most often among P. aeruginosa strains - it was identified in 65.0% (Fernandez et al., 2013), while in Iranian study it was observed in 28.0% of tested isolates (Vaziri et al., 2011). Our earlier investigation conducted on MDR P. aeruginosa reported the presence of aac(6')-Ib gene in 58.3% of isolates (Sacha et al., 2012), whereas in this assay it was detected in 28.0% of tested strains. PCR study performed to detect genes involved in the production of Qnr-like proteins revealed the presence of qnrB in 20.0% of tested strains. Among Enterobacteriaceae screened for production of plasmid-mediated fluoroquinolone resistance determinants, qnrB was reported as most prevalent gene (Kim et al., 2009). Earlier Polish study demonstrated that aminoglycoside and fluoroquinolone resistance rates were comparable to our results: amikacin (91.0% vs. 92.0%), gentamicin (98.0% vs. 100.0%), ciprofloxacin (98.0% vs. 100.0%). Percentage of strains resistant to beta-lactams was even higher: 93% were resistant to ceftazidime, 89% to cefepime, 41% to imipenem, 88% to meropenem (Paluchowska et al., 2012). Resistance rates of MDR isolates obtained from 10 Spanish hospitals were similar to those of our strains in the case of imipenem (66.67% vs. 72%), ceftazidime (40% vs. 32%), cefepime (73.33% vs. 88%) (Cabot et al., 2012).

This research focused on investigating the most commonly reported plasmid-mediated factors of aminoglycoside and fluoroquinolone resistance, and further assays are necessary to determine the other causes of antimicrobial resistance.

This study was partially supported by funds from Leading National Research Center in Bialystok (KNOW 50/2013).

Submitted: Septermber 30, 2013

Approved: April 17, 2014

All the content of the journal, except where otherwise noted, is licensed under a Creative Commons License CC BY-NC.

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  • Correspondence:
    A.D. Michalska
    Department of Microbiological Diagnostics and Infectious Immunology
    Medical University of Bialystok
    Waszyngtona Str. 15A
    15-269 Bialystok, Poland
  • *
    Both authors contributed equally to this work.
  • Publication Dates

    • Publication in this collection
      13 Feb 2015
    • Date of issue
      Dec 2014


    • Accepted
      17 Apr 2014
    • Received
      30 Sept 2013
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