Journal of Antimicrobial Chemotherapy (2008) 61, 1003 1006 doi:10.

1093/jac/dkn063 Advance Access publication 25 February 2008

Plasmid-mediated quinolone resistance determinants qnr and aac(60 )Ib-cr in extended-spectrum b-lactamase-producing Escherichia coli and Klebsiella pneumoniae in China
Yan Jiang1,2, Zhihui Zhou1, Ying Qian1, Zeqing Wei1, Yunsong Yu1*, Songnian Hu2,3 and Lanjuan Li1
State Key Laboratory for Diagnosis and Treatment of Infectious Disease, First Afliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, China; 2James D. Watson Institute of Genome Sciences, Zhejiang University, Hangzhou, Zhejiang 310008, China; 3Beijing Genomics Institute, Chinese Academy of Sciences, Beijing 101300, China
Received 22 October 2007; returned 20 November 2007; revised 25 January 2008; accepted 26 January 2008 Objectives: To characterize the prevalence of plasmid-mediated quinolone resistance determinants qnr and aac(60 )-Ib-cr in extended-spectrum b-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae. Methods: qnrA, qnrB, qnrS, aac(60 )-Ib-cr and ESBL-encoding genes were detected by PCR. MICs of 10 antimicrobial agents were determined by Etest. PFGE was used to investigate the clonality of qnr- and aac(60 )-Ib-cr-producing isolates. Conjugation and Southern hybridizations were used to conrm whether qnr, aac(60 )-Ib-cr or ESBL-encoding genes were located on plasmids. Results: Twenty-nine (8.0%) of 362 isolates were positive for qnr genes, and the qnrA-, qnrB- and qnrS-type genes were detected alone or in combination in 13 (3.6%), 8 (2.2%) and 9 (2.5%), respectively. Sixty-two (17.1%) isolates were positive for aac(60 )-Ib, of which 36 (9.9% of all) had the -cr variant. Conjugation and Southern hybridization revealed that qnrA, aac(60 )-Ib-cr and ESBL-encoding genes were always located on the same plasmids. Conclusions: qnr and aac(60 )-Ib-cr genes were detected in 8.0% and 9.9% of ESBL-producing E. coli and K. pneumoniae, respectively. The plasmids carrying the qnr gene could be transferred by conjugation together with ESBL-encoding genes and aac(60 )-Ib-cr. Keywords: conjugation, PFGE, Southern hybridization
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Introduction
The rst plasmid-mediated quinolone resistance (PMQR) determinant, qnr (later termed qnrA), was described in a Klebsiella pneumoniae strain from the USA in 1998.1 Three major groups of qnr determinants, qnrA, qnrB and qnrS, have since been identied in different species of the Enterobacteriaceae.2,3 The Qnr peptides are pentapeptide repeat proteins, which protect DNA gyrase and topoisomerase IV from quinolone inhibition, leading to an 8- to 32-fold increase in MICs of quinolones. Recently, two other PMQR mechanisms have been described. AAC(60 )-Ib-cr, a variant aminoglycoside acetyltransferase

capable of modifying ciprooxacin and reducing its activity, is now recognized to be widely prevalent and circulated together with qnr genes.4 The other PMQR determinant is the quinolone efux pump gene, qepA, and is mediated by a probable transposable element anked by two copies of IS26.5 Rates of quinolone resistance in clinical isolates of Escherichia coli and K. pneumoniae are unusually high in China, especially in strains producing extended-spectrum b-lactamases (ESBLs).6 Since PMQR genes have often been reported to be co-associated with genes encoding ESBLs or other b-lactamases,7,8 the aim of this study was to investigate the prevalence of qnr and aac(6 0 )-Ib-cr genes among ESBL-producing isolates.

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*Corresponding author: Tel: 86-571-8723-6421; Fax: 86-571-8723-6423; E-mail: yvys119@163.com

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Materials and methods

Bacterial isolates and susceptibility testing
Ninety-nine isolates of K. pneumoniae and 263 of E. coli producing ESBLs were collected from six provinces or districts (Zhejiang, Henan, Jiangsu, Xinjiang, Liaoning, Macao) in China between September 1998 and November 2002, each isolated from a sputum or urine specimen of a separate patient. Screening for the presence of ESBLs in K. pneumoniae and E. coli was performed according to the CLSI (formerly NCCLS) guidelines. MICs of ciprooxacin, ooxacin, noroxacin, moxioxacin, cefotaxime, ceftazidime, streptomycin, amikacin, gentamicin and ampicillin were determined using Etests (AB Biodisk, Solna, Sweden), following the manufacturers instructions. E. coli ATCC 25922 was used as the quality control strain.

Results
Prevalence of qnr-group and aac(6 0 )-Ib-cr genes
Twenty-nine (8.0%) of 362 phenotypic ESBL-producing isolates were positive for qnr genes, with qnrA-, qnrB- and qnrS-type alleles detected in 5 (1.9%), 4 (1.5%) and 5 (1.9%) E. coli isolates, and in 8 (8.1%), 4 (4.0%) and 4 (4.0%) K. pneumoniae isolates, respectively. These were found to be qnrA1, qnrB4, qnrB6, qnrB8 and qnrS1 alleles by sequencing of PCR products (Table 1). The rate of qnr carriage among K. pneumoniae (16.2%) was higher than in E. coli (5.3%). Interestingly, one isolate of K. pneumoniae carried both qnrB and qnrS. Sixty-two (17.1%) of 362 isolates were positive for aac(6 0 )-Ib, of which 36 (9.9% of all) isolates carried the -cr variant. Twenty-one (48.8%) of 43 aac(6 0 )-Ib-positive E. coli isolates and 15 (78.9%) of 19 aac(6 0 )-Ib-positive K. pneumoniae isolates, respectively, carried the -cr variant. Notably, the aac(6 0 )-Ib-cr gene was detected in 55.2% (16/29 isolates) of qnr-positive isolates, compared with only 6.0% (20/333 isolates) of qnr-negative isolates.

PCR amplication and sequencing

The qnrA, qnrB and qnrS genes were detected by PCR in clinical isolates or transconjugants using the following primers: for qnrA, qnrA_up (50 -AAG GAA GCC GTA TGG ATA TT-30 ) and qnrA_dw (50 -AGC TAA TCC GGC AGC ACT AT-30 ) to give a 670 bp product; for qnrB, qnrB_up (50 -CGA CCT GAG CGG CAC TGA AT-30 ) and qnrB_dw (50 -TGA GCA ACG ATG CCT GGT AG-30 ) to give a 515 bp product; and for qnrS, qnrS_up (50 -ACC TTC ACC GCT TGC ACA TT-30 ) and qnrS_dw (50 -CCA GTG CTT CGA GAA TCA GT-30 ) to give a 571 bp product. Primers for aac(6 0 )-Ib were aac_up (50 -TGA CCT TGC GAT GCT CTA TG-30 ) and aac_dw (50 -TTA GGC ATC ACT GCG TGT TC-30 ), generating a 509 bp fragment. Amplication and identication of ESBL-encoding genes were performed with previously described primers9 for blaCTX-M-1-like, blaCTX-M-2-like, blaCTX-M-8-like, blaCTX-M-9-like, blaSHV and blaTEM. PCR experiments were carried out according to standard conditions with an annealing temperature of 588C. Both strands of amplicons were sequenced using an ABI 3730 sequencer (Applied Biosystems, Foster City, USA) with the same primers used for PCR amplication.

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Pulsed-eld gel electrophoresis

The molecular characterization of qnr-positive and aac(6 0 )-Ib-cr-positive isolates by PFGE showed great genomic diversity among them. The 49 isolates gave 42 major patterns. Twenty-four patterns in 29 qnr-positive isolates and 32 patterns in 36 aac(6 0 )-Ib-cr-positive isolates showed that most of these strains were not clonal.

ESBL genotype and association with qnr

The CTX-M-9-group was the major ESBL type, being detected in $90% (325 isolates) of the ESBLs-producing isolates. A blaCTX-M gene was detected in 27 of 29 qnr-positive isolates; most were blaCTX-M-9-like (13 isolates produced CTX-M-14 and 12 isolates produced CTX-M-24). Moreover, TEM-1-type b-lactamase was detected in 16 qnr-positive isolates, and SHV-12- or SHV-1-type b-lactamase in 9 qnr-positive isolates. blaSHV genes were only detected in nine qnrB-positive or qnrS-positive strains but not in qnrA-positive isolates (Table 1).

Pulsed-eld gel electrophoresis

Genomic DNA of each qnr-positive and aac(6)-Ib-cr-positive isolate was analysed by PFGE after digestion with XbaI (Sangon, China). DNA fragments were separated by electrophoresis in 1% agarose III (China) in 0.5 TBE buffer with CHEF apparatus (CHEF MAPPER XA, Bio-Rad, USA) at 148C, 6 V/cm and with alternating pulses at a 1208 angle in a 5 35 s pulse time gradient for 20 h. A phage lambda DNA ladder (New England BioLabs, Beverly, USA) was used as a size marker.

Plasmid analysis and susceptibility tests

The plasmids containing qnrA alleles were $60 kb in size (Figure 1), and probes for aac(6 0 )-Ib-cr and blaCTX-M genes were hybridized with the same plasmids (data not shown). These plasmids were transferred by conjugation from all of 13 isolates used as donors. The MICs of ciprooxacin for 13 transconjugants were 0.06 2 mg/L, representing an increase of 4- to 125-fold compared with the recipient (MIC 0.016 mg/L).

Plasmid manipulations and analysis

The QIAGEN Large-Construct Kit (Qiagen, Germany) was used to extract plasmids from qnr-positive isolates according to the manufacturers instructions. E. coli V517 was used as a standard for plasmid size analysis. Southern hybridization using a DIG DNA labelling and detection kit (Roche Applied Science, Mannheim, Germany) was performed with PCR-generated probes specic for qnr, ESBL-encoding genes and aac(6 0 )-Ib-cr. Conjugation experiments using the azide-resistant E. coli strain J53AzR as the recipient were performed as previously described.10 Transconjugants were selected on Mueller Hinton (MH) agar containing sodium azide (100 mg/L) and cefotaxime (2 mg/L).

Discussion
Plasmid-mediated genes, such as qnr and aac(6 0 )-Ib-cr, may facilitate spread and increase the prevalence of quinolone-resistant strains. To date, qnr genes have been widely detected in southern and eastern Asia, North and South American and Europe. The present study demonstrated a

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qnr and aac(60 )-Ib-cr in E. coli and K. pneumoniae

Table 1. PCR amplication results for 29 qnr-positive isolates qnr type A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 A1 B4 and S1 B6 B4 B4 B4 B4 B8 B4 S1 S1 S1 S1 S1 S1 S1 S1 Isolate number 14-2 151 152 153 156 25 31 96 142 140 63 Z8 Z11 kw042 kw041 kw040 k005 364 319 Z19 236 kw070 kw073 032 S-Z8 23 S17 123 22-4 K. K. K. K. K. K. K. K. Species pneumoniae pneumoniae pneumoniae pneumoniae pneumoniae pneumoniae pneumoniae pneumoniae E. coli E. coli E. coli E. coli E. coli pneumoniae pneumoniae pneumoniae pneumoniae E. coli E. coli E. coli E. coli pneumoniae pneumoniae pneumoniae E. coli E. coli E. coli E. coli E. coli blaCTX-M CTX-M-14 CTX-M-24 CTX-M-24 CTX-M-24 CTX-M-24 CTX-M-24 CTX-M-24 CTX-M-24 CTX-M-9 CTX-M-14 CTX-M-14 CTX-M-14 CTX-M-14 CTX-M-24 CTX-M-14 CTX-M-14 CTX-M-14 CTX-M-14 CTX-M-3 CTX-M-24 CTX-M-14 CTX-M-14 CTX-M-14 CTX-M-14 CTX-M-24 CTX-M-24 CTX-M-24 blaTEM TEM-1 TEM-1 TEM-1 TEM-1 TEM-1 TEM-1 TEM-1 TEM-1 TEM-1 TEM-1 TEM-1 TEM-1 TEM-1 TEM-1 TEM-1 TEM-1 blaSHV SHV-12 SHV-12 SHV-1 SHV-1 SHV-12 SHV-12 SHV-12 SHV-12 SHV-1 aac(6 0 )-Ib aac(6 0 )-Ib-cr aac(6 0 )-Ib-cr aac(6 0 )-Ib-cr aac(6 0 )-Ib-cr aac(6 0 )-Ib-cr aac(6 0 )-Ib-cr aac(6 0 )-Ib-cr aac(6 0 )-Ib-cr aac(6 0 )-Ib aac(6 0 )-Ib-cr aac(6 0 )-Ib-cr aac(6 0 )-Ib-cr aac(6 0 )-Ib-cr aac(6 0 )-Ib-cr aac(6 0 )-Ib-cr aac(6 0 )-Ib aac(6 0 )-Ib aac(6 0 )-Ib aac(6 0 )-Ib aac(6 0 )-Ib-cr aac(6 0 )-Ib-cr

K. K. K. K.

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K. K. K.

Figure 1. Plasmid DNAs from 13 qnrA-positive clinical isolates (a) and Southern hybridization of plasmid DNAs with the qnrA-specic probe (b). Lane 1, E. coli V517 (used as a standard for plasmid size); lane 2, 151; lane 3, 152; lane 4, 153; lane 5, Z8; lane 6, Z11; lane 7, 142; lane 8, 140; lane 9, 156; lane 10, 96; lane 11, 63; lane 12, 31; lane 13, 14_2; and lane 14, 25.

prevalence of 8.0% for qnr genes among 362 ESBL-producing isolates of E. coli and K. pneumoniae collected from 1998 to 2002 in parts of China. The prevalence of qnr was higher among K. pneumoniae than in E. coli, as noted by other authors,11 but the three known qnr groups, qnrA-, qnrB- and qnrS-type, were all found at similar frequencies, despite the low rates of qnrA observed in recent surveillance studies.9 A high prevalence of qnr among ESBL-producing enterobacterial species has been described in several reports.7,8 Our study showed that qnr gene could co-exist with blaCTX-M, blaTEM and blaSHV alleles. The major types of blaCTX-M were blaCTX-M-14 and blaCTX-M-24 (both encoding group 9 CTX-M enzymes), which are the most prevalent types in China.6 AAC(60 )-Ib-cr, a novel PMQR protein, was rst reported in 2003, but is now recognized to be widely disseminated. This variant of the aminoglycoside acetyltransferase gene aac(6 0 )-Ib confers resistance to certain quinolones and to aminoglycosides. Others have found aac(6 0 )-Ib-cr in clinical isolates from the USA and in CTX-M-15-producing isolates from different continents, albeit not associated with qnr genes.12 In our study, the rate of aac(6 0 )-Ib carriage among qnr-positive isolates (72.4%) was much higher than that among all isolates (17.1%); furthermore, the rate of the aac(6 0 )-Ib-cr variant was higher among qnr-positive isolates (55.2%) than among qnr-negative isolates (6.0%). Southern hybridization indicated that qnr genes were

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always co-resident with aac(6 0 )-Ib or aac(6 0 )-Ib-cr gene on the same plasmids. Indeed, determination of the complete sequence of one qnrA-carrying plasmid (designated pKP96, 67 850 bp) conrmed that qnrA, aac(6 0 )-Ib-cr and blaCTX-M-24 were all located in the same plasmid (data not shown). Such co-transmissibility will contribute the spread of many antimicrobial resistances. In contrast with qnrA, there is, as yet, little information about the genetic background of qnrB and qnrS in our isolates and this needs further study.
2. Jacoby GA, Walsh KE, Mills DM et al. qnrB, another plasmidmediated gene for quinolone resistance. Antimicrob Agents Chemother 2006; 50: 117882. 3. Hata M, Suzuki M, Matsumoto M et al. Cloning of a novel gene for quinolone resistance from a transferable plasmid in Shigella exneri 2b. Antimicrob Agents Chemother 2005; 49: 8013. 4. Robicsek A, Strahilevitz J, Jacoby GA et al. Fluoroquinolone-modifying enzyme: a new adaptation of a common aminoglycoside acetyltransferase. Nat Med 2006; 12: 83 8. 5. Yamane K, Wachino J, Suzuki S et al. New plasmid-mediated uoroquinolone efux pump, QepA, found in an Escherichia coli clinical isolate. Antimicrob Agents Chemother 2007; 51: 335460. 6. Yu Y, Ji S, Chen Y et al. Resistance of strains producing extended-spectrum b-lactamases and genotype distribution in China. J Infect 2007; 54: 53 7. 7. Lavigne JP, Marchandin H, Delmas J et al. qnrA in CTX-M-producing Escherichia coli isolates from France. Antimicrob Agents Chemother 2006; 50: 42248. 8. Poirel L, Leviandier C, Nordmann P. Prevalence and genetic analysis of plasmid-mediated quinolone resistance determinants QnrA and QnrS in Enterobacteriaceae isolates from a French university hospital. Antimicrob Agents Chemother 2006; 50: 39927. 9. Wu JJ, Ko WC, Tsai SH et al. Prevalence of plasmid-mediated quinolone resistance determinants, QnrA, QnrB, and QnrS, among clinical isolates of Enterobacter cloacae in a Taiwanese hospital. Antimicrob Agents Chemother 2007; 51: 12237. 10. Wang M, Tran JH, Jacoby GA et al. Plasmid-mediated quinolone resistance in clinical isolates of Escherichia coli from Shanghai, China. Antimicrob Agents Chemother 2003; 47: 22428. 11. Robicsek A, Strahilevitz J, Sahm DF et al. qnr prevalence in ceftazidime-resistant Enterobacteriaceae isolates from the United States. Antimicrob Agents Chemother 2006; 50: 28724. 12. Karisik E, Ellington MJ, Pike R et al. Molecular characterization of plasmids encoding CTX-M-15 b-lactamases from Escherichia coli strains in the United Kingdom. J Antimicrob Chemother 2006; 58: 6658.

Acknowledgements
We are grateful to Minggui Wang, PhD, Huashan Hospital, Fudan University, for kindly providing the E. coli strain J53AzR.

Funding
This work was supported in part by the National Basic Research Programme 973 of China (no. 2005CB523101) and the Natural Science Foundation of Zhejiang Province (no. Y206123).

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Transparency declarations
None to declare.

References
1. Martinez-Martinez L, Pascual A, Jacoby GA. Quinolone resistance from a transferable plasmid. Lancet 1998; 351: 7979.

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