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Antimicrobial Agents and Chemotherapy, December 2008, p. 4268-4273, Vol. 52, No. 12
0066-4804/08/$08.00+0     doi:10.1128/AAC.00830-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

High Prevalence of Plasmid-Mediated Quinolone Resistance Genes qnr and aac(6')-Ib-cr in Clinical Isolates of Enterobacteriaceae from Nine Teaching Hospitals in China

Hong Yang,^1,2, Hongbin Chen,^1, Qiwen Yang,¹ Minjun Chen,¹ and Hui Wang^1*

Department of Clinical Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing 100730, China,¹ Department of Clinical Laboratory, Shenzhen Hospital of Peking University, Shenzhen 518036, China²

Received 24 June 2008/ Returned for modification 12 August 2008/ Accepted 14 September 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Quinolone resistance is an emerging problem in China. To investigate the prevalence of the plasmid-mediated quinolone resistance genes qnr and aac(6')-Ib-cr, a total of 265 clinical isolates of Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, and Enterobacter cloacae with ciprofloxacin MICs of 0.25 µg/ml were screened at nine teaching hospitals in China. The qnrA, qnrB, qnrS, and aac(6')-Ib genes were detected by PCR. The aac(6')-Ib-cr gene was further identified by digestion with BtsCI and/or direct sequencing. The qnr gene was present in significantly smaller numbers of isolates with cefotaxime MICs of <2 µg/ml than isolates with higher MICs (2.0 µg/ml) (20.6% and 42.1%, respectively; P < 0.05). aac(6')-Ib-cr was present in 17.0% of the isolates tested, and 7.9% of the isolates carried both the qnr and the aac(6')-Ib-cr genes. Among the isolates with cefotaxime MICs of 2.0 µg/ml, qnr and aac(6')-Ib-cr were present in 65.7% and 8.6% of E. cloacae isolates, respectively; 65.5% and 21.8% of K. pneumoniae isolates, respectively; 63.3% and 26.7% of C. freundii isolates, respectively; and 6.5% and 16.9% of E. coli isolates, respectively. The 20 transconjugants showed 16- to 128-fold increases in ciprofloxacin MICs, 14 showed 16- to 2,000-fold increases in cefotaxime MICs, and 5 showed 8- to 32-fold increases in cefoxitin MICs relative to those of the recipient due to the cotransmission of bla_CTX-M-14, bla_CTX-M-3, bla_DHA-1, bla_SHV-2, and bla_SHV-12 with the qnr and aac(6')-Ib-cr genes. Southern hybridization analysis showed that these genes were located on large plasmids of different sizes (53 to 193 kb). These findings indicate the high prevalence of qnr and aac(6')-Ib-cr in members of the family Enterobacteriaceae and the widespread dissemination of multidrug resistance in China.

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Plasmids carrying qnr genes have been found to mediate quinolone resistance (8). These genes encode pentapeptide repeat proteins that block the action of ciprofloxacin on bacterial DNA gyrase and topoisomerase IV (18-20). The plasmid-borne qnr genes currently comprise three families, qnrA, qnrB, and qnrS, whose nucleotide sequences differ from each other by 40% or more. The geographical distribution of qnrA genes is known to be wide (10), but that of the newer qnr types, qnrB (6) and qnrS (5), have seldom reported within China. Recently, a new mechanism of transferable quinolone resistance was reported: enzymatic inactivation of certain quinolones. The cr variant of aac(6')-Ib encodes an aminoglycoside acetyltransferase that confers reduced susceptibility to ciprofloxacin by N-acetylation of its piperazinyl amine (16).

Plasmids harboring qnrA may also encode extended-spectrum β-lactamases (ESBLs). Previous studies showed that qnr-positive strains frequently expressed ESBLs, such as CTX-M-15 and SHV-12 (6, 15). No previous nationwide survey has evaluated clinical isolates of Enterobacteriaceae with reduced susceptibility to ciprofloxacin and extended-spectrum cephalosporins in China for the presence of qnrA, qnrB, qnrS, and aac(6')-Ib-cr. Therefore, we investigated clinical isolates of Citrobacter freundii, Enterobacter cloacae, Escherichia coli, and Klebsiella pneumoniae collected from nine teaching hospitals in China for the presence of these genes and whether these genes are linked with ESBLs or plasmid-mediated AmpC genes, in order to more broadly characterize the epidemiology of these resistance elements in a population of clinical isolates.

(This work was orally presented in part at the 18th European Congress of Clinical Microbiology and Infectious Diseases, Barcelona, Spain, 19 to 22 April 2008, abstr. O85.)

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Bacterial isolates. The test isolates were drawn from the Chinese Meropenem Susceptibility Surveillance study collection, which consists of nonrepeat clinical isolates of specified enterobacterial genera annually collected by specified laboratories in each of the big nine cities in China. These cities are distributed in southern China (including Shanghai, Wuhan, Nanjing, Guangzhou, and Fuzhou) and northern China (including Beijing, Tianjin, Shenyang, and Jinan). A total of 265 isolates of C. freundii, E. cloacae, E. coli, and K. pneumoniae with ciprofloxacin MICs of 0.25 µg/ml were obtained from a screening of the 421 isolates of the four species mentioned above. The screened isolates were divided into two groups: group 1 had cefotaxime MICs of 2.0 µg/ml and ceftriaxone MICs of 2.0 µg/ml, and group 2 had cefotaxime or ceftriaxone MICs of <2.0 µg/ml. Group 1 included 30 isolates of C. freundii, 35 isolates of E. cloacae, 77 isolates of E. coli, and 55 isolates of K. pneumoniae.

qnr and aac(6')-Ib-cr detection. All 265 isolates selected were screened for the qnr (qnrA, qnrB, and qnrS) genes by multiplex PCR (15) and for aac(6')-Ib by PCR (11). All isolates positive for the aac(6')-Ib gene were further analyzed by digestion with BtsCI (New England Biolabs, Beverly, MA) and/or direct sequencing of the purified PCR products to identify aac(6')-Ib-cr, which lacks the BtsCI restriction site present in the wild-type gene.

Conjugation experiments. The transfer of quinolone resistance was studied by performing conjugation experiments, as described previously (21b). Conjugation experiments were performed with 34 isolates in group 1 (including C. freundii, E. cloacae, E. coli, and K. pneumoniae isolates) with qnr and/or aac(6')-Ib-cr as the donors and with azide-resistant E. coli J53 as the recipient. Transconjugants were selected on Trypticase soy agar plates containing sodium azide (150 µg/ml; Sigma Chemical Co., St. Louis, MO) for counterselection and sulfamethoxazole (180 µg/ml) to select for plasmid-mediated resistance. To determine if quinolone resistance was cotransferred, colonies were replica plated onto Trypticase soy agar plates with and without ciprofloxacin (0.05 µg/ml). The qnrA, qnrB, qnrS, and aac(6')-Ib-cr genes were detected in the transconjugants.

Antimicrobial susceptibility testing. The MICs of ciprofloxacin and the other antimicrobial agents tested were determined by Clinical and Laboratory Standards Institute (CLSI) agar dilution method M7-A7 (3) and were interpreted according to CLSI performance standard M100-S17 (4). The antimicrobials were supplied and stored according to the manufacturer's instructions. E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as reference strains for susceptibility testing.

PCR and sequencing of β-lactamase genes for transconjugants and respective donors. Genes coding for Ambler class A serine enzymes were detected by PCR with primers specific for bla_CTX-M, bla_TEM, and bla_SHV (21a). Plasmid-mediated AmpC β-lactamase genes were sought by use of a multiple PCR system, as described previously (12). The PCR products were purified by using a QIAquick PCR purification kit (Qiagen, Hilden, Germany). DNA sequencing of both strands was performed by the direct sequencing method with an ABI Prism 3100 genetic analyzer (Applied Biosystems, Foster City, CA).

PCR amplification and DNA sequencing of gyrA, gyrB, and parC. Mutations in the gyrA, gyrB, and parC genes were identified by DNA sequencing of their PCR products. PCR amplification of the quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, and parC was performed as described previously (16a, 21). Both strands of the purified PCR products were sequenced; and the DNA sequences of the QRDRs of gyrA, parC, and gyrB were compared with the DNA sequences of the QRDRs of E. cloacae, E. coli, C. freundii, and K. pneumoniae (GenBank accession numbers AF052256, AE000312, AF052253, and DQ673325, respectively, for gyrA; D88981, AE000384, AB003914, and NC009648, respectively, for parC; and AF302677, AE000447, AF071877, and NC009648, respectively, for gyrB).

Plasmid detection and Southern hybridization. Plasmid DNA was extracted with a Qiagen plasmid Miniprep kit (Qiagen), according to the manufacturer's recommendations. E. coli V517 (plasmid sizes, 54, 5.6, 5.1, 3.9, 3.0, 2.7, and 2.1 kb) and E. coli J53 containing plasmid R1 (92 kb) or R27 (182 kb) were used as standards. The sizes of the plasmids were calculated by using Quantity One software (Bio-Rad Laboratories, Hercules, CA). The qnrA, qnrB, qnrS, aac(6')-Ib-cr, bla_CTX-M, bla_SHV, and bla_DHA genes were purified by using a DNA and gel band purification kit (GFX PCR; Amersham Pharmacia) and were then labeled by supplementing the master mixture with digoxigenin-dUTP (Roche Applied Science, Mannheim, Germany). Southern hybridization and detection steps were accomplished with the digoxigenin-dUTP detection kit, as recommended by the manufacturer (Roche Applied Science).

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Prevalence of qnr and aac(6')-Ib-cr genes. The qnr gene was present in 83 (42.1%) of 197 isolates in group 1, and of these, 17 isolates carried qnrA (8.6%), 46 isolates carried qnrB (23.4%), and 24 isolates carried qnrS (12.2%) (Table 1). qnr was present in 19 (63.3%) of 30 C. freundii isolates, 23 (65.7%) of 35 E. cloacae isolates, 5 (6.5%) of 77 E. coli isolates, and 36 (65.5%) of 55 K. pneumoniae isolates (Table 1). aac(6')-Ib-cr was detected in 36 (18.3%) of 197 isolates. It was present in 26.7% of C. freundii isolates, 16.9% of E. coli isolates, 8.6% of E. cloacae isolates, and 21.8% of K. pneumoniae isolates. Two C. freundii isolates from sputum carried qnrA, qnrB, and aac(6')-Ib-cr; one K. pneumoniae isolate from sputum carried qnrB, qnrS, and aac(6')-Ib-cr; and one E. cloacae isolate from blood carried qnrB and qnrS.

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TABLE 1. Prevalence of qnr and aac(6')-Ib-cr genes in the selected Enterobacteriaceae isolates from nine teaching hospitals in China

The qnr gene was detected in 14 of 68 isolates in group 2, which was significantly less than the number of isolates in group 1 in which it was detected (20.6% and 42.1%, respectively; ² = 10.11; P < 0.05). However, there was no significant difference in the prevalence of aac(6')-Ib-cr between the two groups (18.3% in group 1 versus 13.2% in group 2; ² = 1.849; P > 0.05) (Table 1). For the E. cloacae isolates, the qnrA and qnrB genes were more common among isolates in group 1 than those in group 2. Unexpectedly, the qnrS gene was the most prevalent in the non-ESBL-producing E. cloacae isolates (six of eight isolates [75.0%]). For the K. pneumoniae and C. freundii isolates, the qnrB gene was more prevalent among isolates in group 2 than those in group 1. There was no significant difference between the two groups in the prevalence of isolates carrying both the qnr and the aac(6')-Ib-cr genes (9.1% and 4.4%, respectively; ² = 1.547; P > 0.05).

All qnr-positive PCR products from isolates in group 1 were sequenced. The qnr gene nomenclature proposed recently was used to define the subtypes of qnr (6a). The sequences of the qnrA-positive and qnrS-positive isolates were all shown to match those of qnrA1 and qnrS1, respectively. The sequences of qnrB-positive isolates were shown to match those of qnrB1, qnrB2, qnrB4, qnrB6, qnrB10, and qnrB16.

Conjugation experiments and antimicrobial susceptibility testing. Twenty transconjugants were obtained. Fourteen other qnr- and/or aac(6')-Ib-cr-bearing isolates failed to produce transconjugants, although multiple agents were used for selection. The qnr and aac(6')-Ib-cr genes can be cotransferred from different donors. Two isolates (isolates GZ40 and JS33) harboring different qnr genes transferred only a single qnr gene to the recipient, which indicated that different qnr genes were located on different plasmids (Table 2). The 20 transconjugants showed 16- to 128-fold increases in the MICs of ciprofloxacin and 16- to 64-fold increases in the MICs of levofloxacin relative to those of the recipient (Table 2). Fourteen of 20 transconjugants showed 16- to 2,000-fold increases in the MICs of cefotaxime and 4- to 128-fold increases in the MICs of ceftazidime. Clavulanic acid decreased greater than eightfold the MICs of extended-spectrum cephalosporins for these transconjugants. Five transconjugants showed 8- to 32-fold increases in the MICs of cefoxitin and seven showed more than a 256-fold increase in the MICs of gentamicin relative to those of the recipient. It is obvious that ciprofloxacin resistance was cotransferred with resistance to other antimicrobial agents, such as extended-spectrum cephalosporins, cephamycins, aminoglycosides, and sulfamethoxazole.

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TABLE 2. Plasmid-mediated quinolone resistance genes and MICs of antimicrobial agents for 20 donors and transconjugants

Identification of mutations in DNA gyrA, gyrB, and parC. DNA sequencing of the PCR product covering the entire QRDR of gyrA demonstrated the presence of mutations at codon 83 in 16 of 20 clinical isolates tested (Table 2). Point mutations in gyrA were found in E. cloacae, E. coli, C. freundii, and K. pneumoniae; and the resulting amino acid substitutions were Ser83Ile, Ser83Leu, Thr83Ile, and Ser83Ile, respectively. Two E. cloacae isolates had an additional mutation, Asp87Ala. In parC, the Ser80R or Ser80Ile substitutions were found in 12 of 20 clinical isolates. No mutations were found in the gyrB gene of any of 20 clinical isolates tested (Table 2). There was no mutation in three target genes among six transconjugants and the recipient.

Sequencing of β-lactamase genes for transconjugants. The properties of the 20 transconjugants are shown in Table 3. The qnr gene could be cotransmitted with aac(6')-Ib-cr, ESBLs, and plasmid-mediated AmpC in one conjugation experiment. CTX-M-14 and CTX-M-3 were the most prevalent ESBL types and DHA-1 was the most common plasmid-mediated AmpC type among the transconjugants. In addition to the qnr and aac(6')-Ib-cr genes, some transconjugants carried several β-lactamase genes. Strain SY26T, which had the qnrB4 and aac(6')-Ib-cr genes, produced DHA-1 and SHV-12; strain GZ51T, which had the qnrA1 gene, produced SHV-12 and CTX-M-14 (Table 3).

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TABLE 3. Properties of the transconjugants

Plasmid profiles and Southern blot analysis. Plasmid DNA was extracted from the 20 transconjugants and some of the respective donors. All the transconjugants and donors had different plasmid profiles. Twelve transconjugants carried one large plasmid of 53 to 195 kb (Table 3); eight carried two to three large plasmids of 65 to 200 kb. Southern hybridization analysis was performed with eight selected transcoujugants and their respective donors. Table 4 shows that the resistance genes detected were located on large plasmids of different sizes. We found that the genes for qnr, aac(6')-Ib-cr, bla_CTX-M, bla_SHV, and bla_DHA were located on a single large plasmid for each transconjugant. For example, the qnrB, aac(6')-Ib-cr, bla_CTX-M, and bla_DHA genes were on a plasmid of 177 kb in strain WH99T (Table 4). aac(6')-Ib-cr and bla_CTX-M were detected on two large plasmids of 172 and 107 kb, respectively, in strain JN1T. The results of Southern hybridization analysis with the donors were identical to those for their respective transconjugants.

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TABLE 4. Southern hybridization of plasmid DNAs from eight transconjugants with probes

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
This study showed that the prevalence of plasmid-mediated quinolone resistance due to the qnr and aac(6')-Ib-cr genes among clinical isolates of Enterobacteriaceae in China is high and that it is much higher than that reported in other areas, such as the United States (15) and Taiwan (22). In our study, the prevalence of qnr appeared to be much lower in E. coli isolates (4.8%) than in other species of clinical isolates of the Enterobacteriaceae. However, the prevalence of aac(6')-Ib-cr appeared to be lower in E. cloacae isolates (9.3%) than in isolates of the other species tested. qnrA was not found in relatively large numbers of nonfermenting bacilli, including 128 P. aeruginosa and 77 A. baumannii isolates (23). This implies that the mechanism for the incidence of qnr and/or aac(6')-Ib-cr is related to the particular species. Interestingly, we found that the qnrS gene was the most prevalent in non-ESBL-producing E. cloacae (75.0%), and qnrB and qnrA were more frequently found in the suspected ESBL producers. A study in Taiwan also found that the qnrS gene was more common than other qnr genes in non-ESBL-producing isolates of E. cloacae (22). The distributions of the different qnr genes in non-ESBL-producing Enterobacteriaceae need further study.

In this study, two C. freundii isolates carried qnrA, qnrB, and aac(6')-Ib-cr; one K. pneumoniae isolate carried qnrB, qnrS, and aac(6')-Ib-cr; and one E. cloacae isolate carried qnrB and qnrS, which indicated that these species could carry qnr genes of different subtypes. Conjugation experiments proved that the plasmid-mediated quinolone resistance was transferable. The transferable plasmid-mediated, low-level quinolone resistance associated with different qnr genes and aac(6')-Ib-cr was widespread among the isolates of the Enterobacteriaceae, and this perhaps contributed to the rapid increase in resistance to quinolones among bacteria in China. This study also indicated that chromosomal QRDR mutations in GyrA and ParC played an important role in mediating high-level quinolone resistance.

ESBLs are one of the most significant mechanisms of resistance to oxyimino-cephalosporins in the Enterobacteriaceae. In the 1980s, the ESBLs were predominantly TEM and SHV derivatives (2). However, since 2000, the CTX-M enzymes, originally described in South America, Asia, and Eastern Europe, have spread worldwide (1). In parallel, nosocomial outbreaks because of the expression of plasmid-mediated class C enzymes have increasingly been reported (9, 13). A statistical link between CTX-M production and nalidixic acid or fluoroquinolone resistance has been established, and this association can be explained at least in part by the high incidence of qnr genes in this ESBL type (7, 14). Another study showed a significant difference in the numbers of qnr-positive strains between the two time periods, 0 of 391 strains from 1991 to 1995 and 10 (3.5%) of 288 strains in 1996 to 2005 (P < 0.01), and suggested that ceftazidime resistance in qnr-positive Enterobacter strains was associated with a true ESBL-mediated mechanism (17). The information in Table 3 also suggests that the qnr and aac(6')-Ib-cr genes and certain ESBLs or AmpCs are frequently cotransmitted and coselected, and this study found that there is a genetic linkage between these resistance elements on plasmids. One horizontal transmission event can result in the acquisition of multidrug resistance genes by wild-type strains, so this has presumably contributed to the rapid increase in the prevalence of multidrug resistance among clinical bacteria. Despite these recent findings, the contributions of the qnr and aac(6')-Ib-cr genes to the increasing rates of quinolone resistance worldwide and the genetic association between quinolone resistance and ESBL- or AmpC-producing strains remain largely unknown, and further work is needed to examine the genetic environment and array of these resistance genes on plasmids.

In conclusion, transferable, plasmid-mediated quinolone resistance associated with qnr and aac(6')-Ib-cr is widely distributed in China. These genes are implicated in low-level fluoroquinolone resistance and may play a significant role in the generation of resistant mutants and therapeutic failure. qnr-mediated quinolone resistance associated with multidrug resistance has the additional effect of genetically linking low-level quinolone resistance with resistance to other antibiotics and thus promoting the coselection of resistance upon exposure to other antimicrobials to which resistance is also encoded on plasmids. The emergence of plasmid-mediated quinolone resistance may thus contribute to the rapid increase in bacterial resistance to quinolones in several ways. The cotransmission of qnr with aac(6')-Ib-cr, ESBLs, and plasmid-mediated AmpC genes speeds the formation of multidrug resistance in Enterobacteriaceae in China.

 
We are grateful to Wang Minggui and Xu Xiaogang, Institute of Antibiotics, Huashan Hospital, for kindly sending qnr-positive strains (strain UAB1 containing qnrA, strain PMG298 containing qnrB1, strain PSH7containing qnrB4, and strain PSH8 containing qnrS), E. coli V517, E. coli J53 containing plasmid R1, E. coli J53 containing plasmid R27, and E. coli J53 Az^R and for calculating the sizes of the plasmids.

 
* Corresponding author. Mailing address: Department of Clinical Laboratory, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, No. 1 Shuaifuyuan, Wangfujing, Beijing 100730, China. Phone and fax: 86-10-6529-5406. E-mail: wh_bj{at}tom.com

Published ahead of print on 22 September 2008.

H. Yang and H. Chen contributed equally to this paper.

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Antimicrobial Agents and Chemotherapy, December 2008, p. 4268-4273, Vol. 52, No. 12
0066-4804/08/$08.00+0     doi:10.1128/AAC.00830-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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