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Antimicrob Agents Chemother. Aug 2008; 52(8): 2992–2993.
Published online May 19, 2008. doi:  10.1128/AAC.01686-07
PMCID: PMC2493129

Coprevalence of Plasmid-Mediated Quinolone Resistance Determinants QepA, Qnr, and AAC(6′)-Ib-cr among 16S rRNA Methylase RmtB-Producing Escherichia coli Isolates from Pigs[down-pointing small open triangle]

Jian-Hua Liu, Yu-Ting Deng, Zhen-Ling Zeng, and Jun-Hua Gao
College of Veterinary Medicine
South China Agricultural University
Guangzhou 510642, People's Republic of China
Lin Chen
College of Jiangsu Animal Health and Veterinary Science
Taizhou 225300, People's Republic of China
Yoshichika Arakawa*
Department of Bacterial Pathogenesis and Infection Control
National Institute of Infectious Diseases
Tokyo, Japan

Plasmid-mediated quinolone resistance determinants, including Qnr peptides and AAC(6′)-Ib-cr, are increasingly identified worldwide among various clinical isolates of Enterobacteriaceae (7, 9, 10). Very recently, a novel plasmid-mediated fluoroquinolone-resistant determinant, QepA (quinolone efflux pump), which showed a considerable similarity to the major facilitator superfamily-type efflux pumps, was first identified in an Escherichia coli clinical isolate from Japan (13) and later found also in an E. coli isolate in Belgium (6). Interestingly, both of the two qepA-harboring E. coli isolates also contained the rmtB gene encoding a 16S rRNA methyltransferase, an emerging new molecular mechanism responsible for high-level pan-aminoglycoside resistance among gram-negative pathogens (3, 4, 6, 13, 14).

Our previous study showed that rmtB was highly prevalent among E. coli isolates from pigs in China (1). The aim of this study was to investigate the prevalence of plasmid-mediated quinolone resistance determinants among rmtB-producing E. coli isolates from pigs in China and to identify the association of the qepA gene with rmtB.

One hundred fifty-one E. coli isolates were obtained from pig feces sampled at two pig farms. These isolates were collected from 2005 to 2006, and 48 of them were identified as producers of RmtB. (Some of these data were published previously [1].) Screening for qepA, qnrA, qnrB, qnrS, and aac(6)-Ib-cr genes was carried out by PCR amplification among the 48 rmtB-positive isolates. For qepA, the following primers were used to produce a 218-bp amplicon: qepA-F (5′-GCAGGTCCAGCAGCGGGTAG-3′) and qepA-R (5′-CTTCCTGCCCGAGTATCGTG-3′). Positive results were confirmed by direct sequencing of PCR products. qnrA, qnrB, qnrS, and aac(6)-Ib-cr genes were detected by PCR using specific primers (the used qnrB primers were able to detect almost all known qnrB alleles except qnrB8), as previously described (5, 8, 11), and were finally confirmed by sequencing of each PCR product.

Overall, qepA, qnrB, qnrS, and aac(6)-Ib-cr were detected in 28 (58.3%), 1 (2.1%), 9 (18.8%), and 6 (12.5%) of 48 RmtB-producing E. coli isolates, respectively (Table (Table1).1). The qnrB genes were identified as qnrB6 alleles by sequencing. The qnrS genes were confirmed as qnrS1 (four isolates) and qnrS2 (five isolates) alleles by sequencing. Four isolates with uniform pulsed-field gel electrophoresis (PFGE) patterns harbored qepA, qnrS2, and aac(6)-Ib-cr genes concurrently.

Characteristics of E. coli isolates and transconjugants harboring rmtB, as well as qnr, qepA, and/or aac(6)-Ib-cr

To investigate the association of rmtB and qepA, rmtB-positive E. coli transconjugants described previously (1) were subjected to PCR amplification of qepA, and all transconjugants that originated from the 28 qepA-positive isolates selected with aminoglycoside resistance were positive for the qepA gene except one, suggesting an strong linkage of qepA with rmtB. Two rmtB-positive transconjugants also harbored qnrS1 or qnrS2.

MICs of ciprofloxacin, enrofloxacin, levofloxacin, nalidixic acid, and norfloxacin for the 27 qepA-positive and 2 qnrS-positive transconjugants were determined by the agar dilution method according to CLSI guidelines (2). The increase (fold) in quinolone MICs for transconjugants compared with those of recipients is shown in Table Table1.1. The MICs for transconjugants strongly indicated that qepA as well as qnrS conferred quinolone resistance, with a 4- to 32-fold increase in norfloxacin MICs and 1- to 32-fold increase in enrofloxacin and ciprofloxacin MICs. However, variations in the quinolone MICs for different transconjugants suggested that the QepA may be expressed at variable levels. Xu et al. (12) recently reported that different promoter strengths may cause the differences in qnrA expression levels and in ciprofloxacin MICs of different transconjugants. Further studies are needed to find out whether the wide range of MICs of quinolones for different qepA-harboring transconjugants depends on the diversities in qepA expression levels due to different promoter strengths. MICs of enrofloxacin for all isolates were also determined by the agar dilution method according to CLSI guidelines. As indicated in Table Table1,1, most isolates were resistant to enrofloxacin (MIC, ≥2 μg/ml), but six isolates were susceptible to enrofloxacin.

This study shows the high prevalence of plasmid-mediated quinolone resistance determinants among E. coli isolates recovered from food-producing animals. A total of 58.3% (28/48) of rmtB-positive E. coli isolates harbored qepA gene, indicating a close relationship between qepA and rmtB, which has been reported in the previous studies (6, 13). This is also the first time three different plasmid-mediated quinolone resistance determinants (QepA, Qnr, and AAC(6′)-Ib-cr) were identified in an E. coli strain. Coproduction of QepA, Qnr, AAC(6′)-Ib-cr, and RmtB may well facilitate the survival of bacteria under selective pressure of antimicrobial agents in both veterinary and human clinical environments, and the resistance determinants in food-producing animals could be transmitted to humans via the food chain. Further spread of these resistance determinants among pathogenic microbes may occur in the near future. Thus, it is necessary to monitor and minimize the spread of such resistance determinants among hazardous bacteria in both humans and animals.


We are grateful to Ming-Gui Wang (Division of Infectious Diseases, Huashan Hospital, Fudan University) and Sheng Chen (Department of Microbiology and Molecular Genetics, Medical College of Wisconsin) for critically reading the manuscript.

This work was supported in part by research grants from the National Natural Science Foundation of China (30500373 and 30130140) and National Key Technology R&D Program of China (2006BAK02A03-5).


[down-pointing small open triangle]Published ahead of print on 19 May 2008.


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