JAC Advance Access originally published online on July 7, 2008
Journal of Antimicrobial Chemotherapy 2008 62(3):479-483; doi:10.1093/jac/dkn244
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Original research |
Occurrence and mechanisms of amikacin resistance and its association with β-lactamases in Pseudomonas aeruginosa: a Korean nationwide study
1 Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea 2 Department of Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea 3 Division of Food Bioscience and Technology, College of Life Science and Biotechnology, Korea University, Seoul, Korea
* Corresponding author. Tel: +82-2-590-1604; Fax: +82-2-592-4190; E-mail: yjpk{at}catholic.ac.kr
Received 2 March 2008; returned 25 March 2008; revised 21 May 2008; accepted 23 May 2008
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Abstract |
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Objectives: We investigated the occurrence and mechanism of amikacin resistance and its association with various β-lactamase genes in Pseudomonas aeruginosa isolates.
Methods: Of the total 250 consecutive, non-duplicated isolates of P. aeruginosa, 55 isolates showed amikacin resistance. PCR amplification of genes for aminoglycoside (AG)-modifying enzymes [aac(3)-I, aac(3)-II/VI, aac(3)-III/IV, aac(6')-I, aac(6')-II, ant(2'')-I, ant(4')-II and aph(3')-VI], 16S rRNA methylases (rmtA, rmtB, rmtC and armA) and class 1 integrons was performed. In addition, we analysed the association of AG resistance genes with various β-lactamase genes.
Results and conclusions: In Korea, the amikacin resistance rate in P. aeruginosa was high (22%), and it varied among provinces (3.8% to 40%). Four types of AG-modifying enzyme genes [aph(3')-VI, ant(2'')-I, aac(6')-I and aac(3)-II/VI] were found in 48 isolates. Thirty-six strains harboured two or more types of enzymes, of which a combination of aph(3')-VI and ant(2'')-I was the most frequent (24/36 isolates, 66.7%). None harboured aac(3)-I, aac(3)-III/IV, aac(6')-II, ant(4')-II, rmtA, rmtB, rmtC or armA. Forty-two isolates co-harboured β-lactamase genes (mostly blaOXA-10). A class 1 integron was detected in all but one, and all the ant(2'')-I and 26/29 blaOXA-10 were found in it. In contrast, aph(3')-VI was not found to be associated with the class 1 integron. Considering the possibility of co-selection and dissemination, constant monitoring of resistance evolution is necessary.
Keywords: aminoglycoside-modifying enzymes , aph(3')-VI , β-lactamases , integrons
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Introduction |
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Aminoglycosides (AGs), in combination with β-lactams, are commonly used as antipseudomonal agents because they may exhibit synergy with β-lactams.1 Amikacin is the AG most frequently used for pseudomonal infections, because Pseudomonas aeruginosa strains with resistance to amikacin also exhibit a relatively high level of resistance to other AGs such as gentamicin, netilmicin, tobramycin and isepamicin.2
AG resistance arises via enzymic modification of the AGs, impermeability and multidrug-active efflux systems.1,3 Among them, inactivation of drugs by plasmid- or chromosome-encoded AG-modifying enzymes (AMEs) is the main mechanism of resistance.1 More recently, 16S rRNA methylase genes (rmtA, rmtB, rmtC, rmtD and armA) were identified in several nosocomial pathogens, including P. aeruginosa, conferring a high level of resistance (MIC > 512 mg/L) to all of the clinically important AGs, including amikacin.4,5 Because of the spread of resistance genes and considerable geographic variability,1,3,4 periodic monitoring of the resistance rate and pattern is required.
In this study, we performed the first nationwide survey to investigate the occurrence and mechanism of amikacin resistance and its association with β-lactamases.
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Materials and methods |
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Bacterial isolates and antimicrobial susceptibility
A total of 250 consecutive, non-duplicated isolates of P. aeruginosa was collected between April and June 2002 from a nationwide distribution of 12 university hospitals (Seoul, 6; Kyungki, 2; Kangwon, 1; Choongchung, 1; Chulla, 1; Kyungsang, 1) and one commercial laboratory. All the isolates had been studied for the presence of Ambler class A and D β-lactamases in a previous study.6 An amikacin susceptibility test was performed by the disc diffusion method according to the CLSI (formerly the NCCLS) guidelines,7 and 55 amikacin-resistant isolates were selected. The specimens consisted of sputum (27 isolates), urine (13 isolates), fluid/drainage (5 isolates), wound (4 isolates) and others (6 isolates). Twenty-two isolates were from patients in intensive care units, 19 from inpatients and 14 from outpatients.
For these 55 amikacin-resistant isolates, MICs of amikacin, gentamicin, tobramycin, netilmicin, isepamicin and arbekacin were determined by the agar dilution method. The MIC breakpoints for amikacin, gentamicin, netilmicin and tobramycin were determined by the CLSI guidelines. For isepamicin and arbekacin, breakpoints were determined according to the CA-SFM guidelines8 and Yokoyama et al.,4 respectively. The MICs corresponding to high-level resistance to amikacin, gentamicin, netilmicin, tobramycin, isepamicin and arbekacin were defined as being above 512 mg/L. Antimicrobial susceptibility profiles for piperacillin, piperacillin/tazobactam, ticarcillin, ticarcillin/clavulanic acid, ceftazidime, aztreonam, cefepime, ciprofloxacin, imipenem and meropenem were applied from a previous study.6
The DNA template for PCR amplification was obtained from the supernatant of a boiled extract of P. aeruginosa cells. PCR amplification was carried out for each gene in a total volume of 50 µL containing 50 ng of DNA, 25 pM of each primer, 100 µM dNTPs and 2 U of Taq DNA polymerase (Takara Shuzo, Shiga, Japan), using various annealing conditions for each primer set. Because amikacin-resistant P. aeruginosa isolates frequently exhibit resistance to other AGs,1,2 detection of various AME genes [aac(3)-I, aac(3)-II/VI, aac(3)-III/IV, aac(6')-I, aac(6')-II, ant(2'')-I, ant(4')-II and aph(3')-VI] was included. On the basis of AG resistance phenotypes,1 possible AMEs were presumed to be active, and primers specific for AME genes were selected. For isolates showing high-level resistance to amikacin or arbekacin, 16S rRNA methylase genes (rmtA, rmtB, rmtC and armA) were investigated.4,5
The presence of blaIMP-1 and blaVIM-2 was also investigated by PCR amplification, and the data for class A (blaPER-1, blaVEB-1, blaPSE-1, blaTEM, blaSHV, blaCTX-M and blaGES-1) and class D (blaOXA-10, blaOXA-17, blaOXA-2, blaOXA-4 and blaOXA-30) β-lactamases were applied from a previous study with the same isolates.6 The association of AMEs and β-lactamases with integrons was investigated using PCR, with primer sets comprising class 1 integron-specific primers binding to the 5'-CS and/or 3'-CS and primers binding to the resistance genes.
All primers used for PCR techniques are listed in Table S1 [available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/)].
Statistical analysis was carried out using the 
2 test in the SPSS program (Version 11.5, SPSS Inc., Chicago, IL, USA). A P value of 0.05 was considered significant.
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Results and discussion |
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The amikacin resistance rate in P. aeruginosa in this study was higher (22.0%) than that in Europe, North America or Taiwan (13.7%, 4.6% and 10%, respectively).1 It varied greatly among the provinces (from 3.8% to 40%): in the south-western part of Korea (Chulla and Choongchung) the resistance rate was very high (40%), whereas in the south-eastern part (Kyungsang) it was very low (3.8%). Resistance rates also varied among hospitals (from 0% to 45%). These findings might be due to the difference in selective pressure exerted by antibiotic usage or the quality of infection control practice.
Among the 55 amikacin-resistant isolates, the overall resistance rates to gentamicin, netilmicin, tobramycin, isepamicin and arbekacin were 98.2%, 98.2%, 90.1%, 87.3% and 76.4%, respectively. The rates of high-level resistance to AGs were 76.4% for gentamicin, 43.6% for netilmicin, 23.6% for tobramycin, 7.2% for amikacin, 3.6% for isepamicin and 3.6% for arbekacin, respectively (Figure 1). For 35 strains showing pan-AG resistance, meropenem was the most active β-lactam (24/35 isolates, 68.6%), followed by ceftazidime (20/35, 57.1%), imipenem (19/35, 54.3%), cefepime (11/35, 31.4%) and aztreonam (9/35, 25.7%). However, nine isolates that were resistant to both imipenem and ceftazidime were also resistant to other β-lactams, including meropenem.
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The detection of AG resistance genes is a useful tool in an epidemiological setting. Four types of AME genes [aph(3')-VI, ant(2'')-I, aac(6')-I and aac(3)-II/VI] were found in 48 (87.3%) isolates, and aph(3')-VI was the most frequently found gene (37 isolates). Thirty-six isolates (36/48, 75%) harboured two or more kinds of AME genes, and a combination of aph(3')-VI and ant(2'')-I was the most common (24/36, 66.7%), followed by aph(3')-VI and aac(6')-I (6/36, 16.7%). They were distributed among various provinces. This finding is in contrast to studies conducted in Europe and the USA, where the aac(6')-I gene was the most frequent, and most of the genes were present as a single AG-modifying gene.1,3
Compared with the antimicrobial susceptibility test (AST) results, the PCR results did not correlate well (Table 1). Only nine isolates were concordant between the AST and PCR results. Thirty-seven isolates revealed unexpected resistance phenotypes, and nine isolates did not show resistance in spite of the presence of the resistance gene. We presume that the reason for the former might be other resistance mechanisms such as multidrug efflux, impermeability or rare types of AMEs, but we could not clearly understand the latter phenomenon.
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Co-production of β-lactamase genes and AME genes was observed in 42 isolates (Table 1). The blaOXA-10 gene was the most prevalent β-lactamase gene (29/42 isolates, 69.0%), and it was usually associated with aph(3')-VI and ant(2'')-I. In addition, all the six isolates harbouring blaPSE-1 co-harboured aac(6')-I, as reported previously.1 IntI1 was detected in 97.6% (41/42 isolates) of the strains, whereas it was detected in only one of the five isolates without any AME or β-lactamase genes. The association with a class 1 integron was confirmed in all blaVIM-2 and ant(2'')-I, 26 of 29 blaOXA-10 and 6 of 13 aac(6')-I, but none of the aph(3')-VI or blaOXA-4 was found to be associated with it. This finding supports the fact that blaOXA-10 is frequently associated with ant(2'')-I as part of an integron9 and explains why it is the most prevalent β-lactamase gene and associated with ant(2'')-I. Although aph(3')-VI was located in Tn1528 in a previous study,10 it could not be determined in this study.
Recently, high-level AG resistance determinants, rmtA and rmtD, were reported in P. aeruginosa from Japan1,4 and Brazil.5 In this study, although four isolates showed high-level resistance to arbekacin or amikacin, they did not harbour 16S rRNA methylases. This finding is in line with a previous Korean study, in which none of the 27 P. aeruginosa isolates showing arbekacin or amikacin resistance harboured the 16S rRNA methylase genes.11 Because arbekacin and amikacin are substrates for the MexXY-OprM efflux pump,1,9 we assume that a combination of AMEs and efflux pumps might be the reason for the high AG MICs for these isolates.
In conclusion, the amikacin resistance rate in P. aeruginosa was high (22.0%) in Korea, and most (87.3%) of the isolates harboured AG resistance genes. aph(3')-VI was the most frequent and was frequently found in combination with ant(2'')-I and blaOXA-10. A class 1 integron was detected in a high percentage (97.6%) of the isolates harbouring both AMEs and β-lactamases. Considering the possibility of co-selection and dissemination of multidrug resistance, constant monitoring of AG and β-lactam resistance evolution is necessary.
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Funding |
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This work was supported by a grant from the Korea Food and Drug Administration in 2007 (FD07052).
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Transparency declarations |
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None to declare.
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Supplementary data |
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TopAbstractIntroductionMaterials and methodsResults and discussionFundingTransparency declarationsSupplementary dataReferences |
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Table S1 is available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
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Acknowledgements |
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We thank all the contributing laboratories that provided isolates for this study and also are indebted to J. J. Park, K. G. Park and K. H. Cha for excellent technical assistance.
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References |
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Doi Y, Ghilardi A, Adams J, et al. High prevalence of metallo-β-lactamase and 16S rRNA methylase coproduction among imipenem-resistant Pseudomonas aeruginosa isolates in Bazil. Antimicrob Agents Chemother (2007) 51:3388–90.
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7 National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Disk Susceptibility Tests—Seventh Edition: Approved Standard M2-A7. (2002) Wayne, PA, USA: NCCLS.
8 Members of the SFM Antibiogram Committee. Antibiogram Committee of the French Society of Microbiology. Report 2003. Int J Antimicrob Agents (2003) 21:364–91.[CrossRef][Web of Science][Medline]
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Poirel L, Naas T, Guibert M, et al. Molecular and biochemical characterization of VEB-1, a novel class A extended-spectrum β-lactamase encoded by an Escherichia coli integron gene. Antimicrob Agents Chemother (1999) 43:573–81.
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Lambert T, Gerbaud G, Courvalin P. Characterization of transposon Tn1528, which confers amikacin resistance by synthesis of aminoglycoside 3'-O-phophotransferase type VI. Antimicrob Agents Chemother (1994) 38:702–6.
11 Lee H, Yong D, Yum JH, et al. Dissemination of 16S rRNA methylase-mediated highly amikacin-resistant isolates of Klebsiella pneumoniae and Acinetobacter baumannii in Korea. Diagn Microbiol Infect Dis (2006) 56:305–12.[CrossRef][Web of Science][Medline]
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