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Antimicrobial Agents and Chemotherapy, June 2005, p. 2522-2524, Vol. 49, No. 6
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.6.2522-2524.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Detection of qnr in Clinical Isolates of Escherichia coli from Korea

Jin-Yong Jeong, Hyun Jung Yoon, Eun Sil Kim, Yoola Lee, Sang-Ho Choi, Nam Joong Kim, Jun Hee Woo, and Yang Soo Kim*

Division of Infectious Diseases, Asan Medical Center and Asan Institute for Life Sciences, University of Ulsan College of Medicine, Center for Antimicrobial Resistance and Microbial Genetics, University of Ulsan, Seoul, Republic of Korea

Received 18 October 2004/ Returned for modification 19 November 2004/ Accepted 31 January 2005


   ABSTRACT
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qnr was detected in 2 of 260 Escherichia coli clinical isolates collected from a Korean hospital during the period 2001 to 2003. The two strains were not clonally related. qnr was located in In4 family class 1 integrons of original structure, downstream of orf513 and upstream from another resistance gene (dfrA3b) and a gene of unknown function (orf105). Transfer of the qnr determinant by conjugation could be detected from only one strain.


   TEXT
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Quinolone resistance is usually caused by various chromosomal mutations that alter the target enzymes, such as DNA gyrase and topoisomerase IV, or activate efflux systems (2). Plasmid-mediated quinolone resistance has only recently been discovered (5). The plasmid gene responsible for quinolone resistance, termed qnr, is carried on class 1 integrons of the In4 family, downstream of the conserved region containing the orf513 recombinase gene (4, 10, 12), and encodes a 218-amino-acid protein that belongs to the pentapeptide-repeat family of proteins (10). The presence of qnr increases the resistance to nalidixic acid and fluoroquinolones by four- to eightfold (5).

The presence of the qnr gene in clinical isolates from Korea has not yet been reported. Therefore, in this study we screened for the presence of the qnr gene in clinical isolates of Escherichia coli from patients in Korea and analyzed the transferability and the genetic context of the qnr gene.

The presence of the qnr gene was screened by PCR using specific primers in plasmid DNAs extracted from 260 consecutive nonduplicate E. coli isolates (194 ciprofloxacin resistant and 66 ciprofloxacin susceptible), which were obtained from patients admitted to the division of infectious diseases, Asan Medical Center, Seoul, Korea, during the period 2001 to 2003. Plasmid DNAs were extracted with a QIAGEN plasmid purification kit (QIAGEN, Valencia, CA), according to the manufacturer's recommendations. Plasmid DNA was amplified by PCR with primers 5'-GATAAAGTTTTTCAGCAAGAGG and 5'-ATCCAGATCGGCAAAGGTTA as described previously (3). DNA sequencing was performed directly on both strands of PCR products using a BigDye terminator sequencing kit and a 3700 DNA analyzer (Applied Biosystems, Foster City, CA).

The qnr gene was detected in two E. coli isolates from different patients: isolate 56 (from cultures of an open wound) and isolate 97 (from bile). Arbitrarily primed PCR (randomly amplified polymorphic DNA [RAPD]) fingerprinting was performed on DNA from both strains carrying qnr to assess the clonal relationship. RAPD analysis was performed according to procedures described previously (8) by using primers 254, 255, 256, and 257. A different DNA banding pattern was obtained from each isolate, indicating that they were not clonally related (Fig. 1). DNA sequencing showed that the nucleotide sequence of qnr in isolate 97 was identical to that of the originally reported qnr sequence (5), but a single-nucleotide change (A -> G at position 537), which did not change the amino acid sequence, was detected in qnr in isolate 56, as also detected in an earlier study (12).



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  		FIG. 1. Arbitrarily primed PCR (RAPD) fingerprinting patterns of strains 56 and 97. M, molecular marker (1 kb).

 
Antimicrobial susceptibility of the two E. coli isolates carrying qnr was determined by agar dilution in accordance with the guidelines of the NCCLS (7). MICs are reported in Table 1. The level of quinolone resistance was lower for isolate 56 than for isolate 97. Both isolates were investigated for the presence of mutations in the quinolone resistance-determining regions of gyrA, gyrB, parC, and parE genes as described previously (1). In isolate 97, a point mutation was identified in gyrA, leading to the replacement of Ser83 by Tyr, while no mutations were detected in the quinolone resistance-determining regions of gyrA, gyrB, parC, and parE of isolate 56.


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  		TABLE 1. Resistance profiles of E. coli isolates and transconjugant

 
To determine whether the qnr-carrying plasmid is transferable, conjugation experiments were performed using azide-resistant E. coli J53 as the recipient. With isolate 56, qnr-positive transconjugants were easily selected on Trypticase soy agar plates containing sodium azide (100 µg/ml) for counterselection and ampicillin (100 µg/ml) for selection of plasmid-encoded resistance. PCR tests confirmed that transconjugants carried qnr. The conjugation frequency (number of transconjugants/number of donor cells) was about 4 x 10–4. As shown in Table 1, the transconjugants obtained from isolate 56 were resistant to ampicillin, sulfamethoxazole, tetracycline, and trimethoprim and showed decreased susceptibility to quinolones compared with E. coli J53. However, no transconjugants were obtained using isolate 97 as a donor, in repeated experiments with ampicillin (100 µg/ml), trimethoprim (40 µg/ml), or sulfamethoxazole (300 µg/ml) as a selecting agent.

The genetic context of qnr in E. coli isolates 56 and 97 was determined by sequencing the flanking regions on plasmid DNA. Results showed that qnr was carried on class 1 integrons of the In4 family that were different from each other and from previously described qnr-containing integrons at the level of the gene cassette array and/or at the level of the unique region located downstream the conserved region containing orf513 (Fig. 2). In particular, unlike previously described qnr-containing integrons, in those from Korean isolates the qnr gene was followed by another resistance gene (dfrA3b) and by a putative gene of unknown function (named orf105) whose putative protein product exhibits 70% homology with the N-terminal sequence of thymidylate synthase of Salmonella enterica serovar Paratyphi A strain ATCC9150 (6).



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  		FIG. 2. Comparison of structures of the qnr-containing integrons from isolates 56 and 97 with those of In36 and In37 and that of the qnr-containing integron from plasmid pQR1 (4, 11).

 
In conclusion, although a previous study showed that qnr was present in 8% (6 of 78) ciprofloxacin-resistant clinical isolates of E. coli from Shanghai, China (12), qnr has rarely been found in clinical strains from other areas of the world (3, 4, 9, 11). In the present study, the qnr gene was found in less than 1% of E. coli isolates from a collection of clinical isolates from Seoul, Korea. Although the qnr gene is currently uncommon, its identification indicates the emergence of plasmid-mediated resistance to quinolones. This mechanism may contribute to the increase in fluoroquinolone resistance in Korea.

Nucleotide sequence accession numbers. The nucleotide sequences of the qnr-positive integrons in strain 56 and 97 have been submitted to GenBank and have been assigned accession numbers AY878718 and AY878717, respectively.


   ACKNOWLEDGMENTS
 
This work was supported by grant 2002-131 from the Asan Institute of Life Sciences, Seoul, Korea.


   FOOTNOTES
 
* Corresponding author. Mailing address: Division of Infectious Diseases, Asan Medical Center, University of Ulsan College of Medicine, 388-1 Pungnap-dong, Songpa-gu, Seoul 138-736, Korea. Phone: 82-2-3010-3303. Fax: 82-2-3010-6970. E-mail: yskim{at}amc.seoul.kr. Back


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Antimicrobial Agents and Chemotherapy, June 2005, p. 2522-2524, Vol. 49, No. 6
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.6.2522-2524.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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