Co-carriage of qnrS1,floR,and bla CTX-M-14 on a Multidrug-Resistant Plasmid in Escherichia coli Isolated from Pigs

Abstract

Plasmid-mediated quinolone resistance (PMQR) determinants were widely distributed among Enterobacteriaceae. The objectives of the present study were to analyze PMQR-positive Escherichia coli isolates from pigs, and to investigate the association between these determinants and other resistant genes. A total of 129 porcine E. coli isolates were included in this study. The presence of PMQR, floR, bla _CTX-M-14, and bla _TEM-1 genes were detected by polymerase chain reaction (PCR) amplification and confirmed by subsequent sequencing. The PMQR-positive isolates were subjected to plasmid profiling, and transformation experiments were conducted to identify the quinolone resistance plasmids. The qnrS1 region of a quinolone resistance plasmid was cloned and sequenced. Among the 129 E. coli isolates, the positive rate for PMQR determinants was 42.6%, and the prevalence of qnr genes, aa(6')-Ib–cr, and qepA were 23.3%, 18.6%, and 0.8%, respectively. A qnrS1-carrying plasmid of 81 kb, named plasmid T078 (pT078), was detected from one multidrug-resistant isolate. Hybridization and PCR analysis confirmed that floR, bla _CTX-M-14, and bla _TEM-1 genes were also located on this plasmid. Sequence analysis identified the qnrS1 gene flanked by a truncated transposase gene. Moreover, complete tetracycline resistance genes tet(A) and tet(R) were found upstream of the qnrS1 gene, and floR gene was found downstream of the qnrS1 gene on the plasmid pT078. To our knowledge, this is the first study demonstrating the occurrence of qnrS1, floR, bla _CTX-M-14, bla _TEM-1, and tet(A) on one plasmid in E. coli isolated from food animals.

Introduction

S usceptible bacteria become resistant to antimicrobial drugs through a variety of mechanisms, such as chromosomal mutations, DNA uptake through transformation, or the process of conjugation (Jacoby, 2005). The bla _CTX-M genes, which mediate extended-spectrum β-lactamase (ESBL) resistance, are often carried on transferable plasmids. CTX-M-type enzymes, such as bla _CTX-M-14 and bla _CTX-M-15, are a group of class A ESBLs that are particularly active against cefotaxime and ceftriaxone. These enzymes are emerging in members of the family Enterobacteriaceae, where they can cause resistance to ceftriaxone and other ESBLs. Quinolone resistance can also be conferred by plasmid-mediated determinants, including the qnr genes (qnrA, qnrB, qnrS, qnrD, and qnrC) (Cavaco et al., 2009; Hata et al., 2005; Jacoby et al., 2006; Martinez-Martinez et al., 1998; Wang et al., 2009), aac(6′)-Ib-cr, and qepA (Robicsek et al., 2006; Yamane et al., 2007). The qnr gene products usually confer resistance to nalidixic acid and reduce susceptibility to fluoroquinolones (Martinez-Martinez et al., 1998). qnr genes may support the selection of bacterial clones with resistance-conferring chromosomal mutations (Jacoby et al., 2003; Martinez-Martinez et al., 1998; Poirel et al., 2006; Rodriguez-Martinez et al., 2007). The emergence of qnr among Klebsiella pneumoniae and Enterobacter spp. coincided with a rapid increase in resistance to fluoroquinolones within these bacteria (Strahilevitz et al., 2007). The gene floR codes for a membrane-associated efflux protein of a major facilitator superfamily that mediates high-level resistance to chloramphenicol and florfenicol (Braibant et al., 2005). This gene was previously reported as part of the Salmonella genomic island 1–associated multidrug-resistance gene cluster, but was also detected in various Gram-negative enteric bacteria on plasmids (Kehrenberg et al., 2006b).

The ability of plasmids to evolve independently of their hosts has allowed numerous resistance genes from diverse species of bacteria to be assembled within a single plasmid and transformed into a wide variety of organisms (Galani et al., 2010). There have been many reports on the association of the qnrA determinants with ESBLs (VEB-1, SHV-7, and CTX-M) (Ferreira et al., 2010; Nordmann and Poirel, 2005). The qnrB gene was also found to co-occur with the carbapenemase gene bla _KPC-2 on the same plasmid (Chmelnitsky et al., 2008; Endimiani et al., 2008). However, other resistance genes were seldom reported to co-occur with the qnr genes. Our previous study showed that the bla _CTX-M genes, floR and qnr genes, were highly prevalent in E. coli isolated from animals in China (Huang et al., 2011; Xia et al., 2011).

The objectives of the present study were to examine the emergence of potential plasmid-mediated quinolone resistance (PMQR) determinants coexistent with bla _CTX-M genes and floR gene in E. coli isolates from pigs in China and to identify the association between qnr genes and other resistance genes.

Methods

Bacterial isolates and susceptibility testing

Bacterial strains were obtained from three pig farms in North China by the China Institute of Veterinary Drug Control and five pig farms in South China by Sichuan Agricultural University from 2005 to 2007. Samples were collected from feces of pigs with diarrhea using sterile cloacal swabs. The samples were inoculated in sterile nutrient broth (Becton Dickinson Difco Laboratories, Detroit, MI) and incubated for 12 h at 37°C. The strains from the same pig were grown on one chromogenic medium plate for selecting E. coli (CHROMagar, Paris, France). Only one colony per plate was selected, and blue colonies on the culture plates were regarded as presumptive E. coli colonies. A total of 129 E. coli isolates were confirmed using the API-20E system (bioMerieux, Marcy l'Etoile, France). Minimum inhibitory concentrations (MICs) of isolates and transconjugants were performed by the broth microdilution method in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2007, 2008). E. coli ATCC 25922 was used as a quality control strain (Lu et al., 2011).

PCR amplification and DNA sequencing

Rapid DNA preparation was performed by heating a single colony at 100°C in a volume of 100 μL of distilled water, followed by a centrifugation step to remove cell debris. Based on sequence alignments of the qnrA-, qnrB-, qnrS-, qnrC-like genes; qepA; and aac(6')-Ib, polymerase chain reaction (PCR) primers were designed to amplify internal fragments of the target genes with sizes of 580, 264, 428, 307, 218, and 482 bp, respectively (Table 1). PCR conditions were as follows: (i) 5 min at 95°C and (ii) 35 cycles, with 1 cycle consisting of 30 s at 94°C, 40 s at 56°C, 1 min at 72°C, and (iii) 10 min at 72°C. For the PMQR-positive isolates, amplification and identification of ESBL-encoding genes (bla _TEM , bla _CTX-M) and floR gene were performed with previously described primers (Table 1) (Blickwede and Schwarz, 2004; Huang et al., 2009). PCR experiments were carried out using the above conditions with an annealing temperature of 56°C, 58°C, or 60°C, respectively. Both strands of amplicons were sequenced using an ABI 3730 sequencer (Applied Biosystems, Norwalk, CT) with the same primers used for PCR amplification.

Table 1.

Primers Used in This Study

Primer	Sequence	Size of product (bp)	Source or reference
qnrA F	5′-AGAGGATTTCTCACGCCAGG-3′	580	Cattoir et al. (2007)
qnrA R	5′-TGCCAGGCACAGATCTTGAC-3′
qnrB F	5′-GGMATHGAAATTCGCCACTG-3′	264	Cattoir et al. (2007)
qnrB R	5′-TTTGCYGYYCGCCAGTCGAA-3′
qnrS F	5′-GCAAGTTCATTGAACAGGGT-3′	428	Cattoir et al. (2007)
qnrS R	5′-TCTAAACCGTCGAGTTCGGCG-3′
qnrC F	5′-GGGTTGTACATTTATTGAATCG-3′	307	Kim et al. (2009)
qnrC R	5′-CACCTACCCATTTATTTTCA-3′
aac-Ib F	5′-TTGCGATGCTCTATGAGTGGCTA-3′	482	Park et al. (2009)
aac-Ib R	5′-CTCGAATGCCTGGCGTGTTT-3′
QepA F	5′-GCAGTCCAGCAGCGGGTAG-3′	218	Liu et al. (2008)
QepA R	5′-CTTCCTGCCCGAGTATCGTG-3′
TEM F	5′-TCGCCGCATACACTATTCTCAGAATGA-3′	445	Wu et al. (2007)
TEM R	5′-ACGCTCACCGGCTCCAGATTTAT-3′
CTX-M F	5′-ATGTGCAGACCAGTAAGTATGGC-3′	593	Wu et al. (2007)
CTX-M R	5′-TGGGTAATAGTACCAGAACAGCGG-3′
FloR F	5′-GCATCCTGAACACGACGCCCGCT-3′	1031	Blickwede et al. (2004)
FloR R	5′-GCTGTGGTCGTGACGGTAACGGC-3′

Transfer of PMQR determinants

Conjugation experiments using the azide-resistant E. coli strain J53Az^R as the recipient were performed as described previously (Wang et al., 2003). Transconjugants were selected on trypticase soy agar (TSA) plates containing sodium azide (100 mg/L) and ampicillin (100 mg/L). Transformation of E. coli DH10B with the plasmid DNA of clinical E. coli isolates was performed using a standard electroporation technique. Transformants were selected on TSA plates containing ciprofloxacin (0.04 mg/L).

Southern hybridization analysis

The QIAGEN Large-Construct Kit (Qiagen, Hilden, Germany) was used to extract plasmids from floR-positive isolates, bla _CTX-M-positive isolates, and transformants according to the manufacturer's instructions. E. coli V517, E. coli J53 R27, E. coli J53 R1, and E. coli J53 Plac were used as plasmid size standards. Southern hybridization using DIG DNA labeling and detection kit (Roche Applied Science, Mannheim, Germany) was performed with PCR-generated probes specific for qnrS1, bla _TEM-1, bla _CTX-M-14, or floR probes.

Cloning and sequence analysis of the qnrS1 region of plasmid pT078

Plasmid pT078 extracted from transformant T078 was digested with EcoRI, ligated to the same restricted pBluescript II SK⁺ (Stratagence, Amsterdam, The Netherlands), and transformed into E. coli DH10B. Selection of clones carrying the qnrS1-containing fragment was performed on Luria agar plates containing ampicillin and nalidixic acid. Restriction enzyme analysis was used to determine the size of the cloned qnrS1-carrying fragments. The nucleotide sequences of the qnrS1-bearing fragments were first determined by using the commercially available standard primers M13 universal and M13 reverse. Additional sequences flanking the qnrS1-carrying region were then determined by primer walking using the non-digested plasmid as a sequencing template. The sequence of a 17575-bp segment of plasmid pT078 has been deposited in the GenBank database under the accession number GQ214053. Sequence comparisons were carried out using the BLAST program, available at the U.S. National Center for Biotechnology Information (www.ncbi.nlm.nih.gov).

Results

Screening and identification of resistance genes

Among the 129 E. coli isolates examined in this study, the prevalence of qnr genes, aac(6')-Ib–cr, and qepA were 23.3% (30), 18.6% (24), and 0.8% (1), respectively. Among the qnr determinants, qnrA-, qnrB-, and qnrS-type genes were detected in one (0.8%), seven (5.4%), and 22 (17.1%) strains, respectively, but no isolate was positive for qnrC. Among the 55 PMQR-positive E. coli isolates, the positive rate for bla _TEM-1 genes was 100%. Strain DS05, 1206-S23, and 1205-S13 contained both qnrS gene and floR gene. Strain 1213-S18 and 1207-SE51 contained both qnrS gene and bla _CTX-M-15 gene. Interestingly, strain 1205-S13 also contained bla _CTX-M-14 gene (Table 2).

Table 2.

Characteristics of the Five Donor Strains and Their Escherichia coli DH10B Transformants

								MIC(mg /L)
Strain	Plasmid (size in kb)	Resistant genes	NAL	CIP	OFL	ENR	AMP	CTF	CAZ	AMK	GEN	FFC	DOX
DS05	MP	qnrS, floR,,bla_TEM-1	>512	1	1	1	>512	2	0.5	>256	>256	256	2
T 045^a	67.3	qnrS, bla_TEM-1	8	0.5	0.5	0.25	>512	0.5	0.0625	4	2	2	1
1206-S23	MP	qnrS, floR, bla_TEM-1	>512	32	16	32	>512	4	1	8	4	256	16
T075	57.4	qnrS, bla_TEM-1	8	0.5	0.5	0.25	>512	1	0.125	4	2	1	1
1205-S13	MP	qnrS, floR, tetA,bla_TEM-1 bla_CTX-M-14,	>512	32	32	4	>512	128	0.5	16	256	256	8
T 078	81.2	qnrS, floR, tetA bla_TEM-1, bla_CTX-M-14,	8	0.5	0.5	0.25	>512	64	0.125	4	128	256	8
1213-S18	MP	qnrS, bla_CTX-M-15,bla_TEM-1	>512	32	16	32	>512	128	0.5	8	4	4	2
T 065	66.2	qnrS, bla_TEM-1	8	0.5	0.5	0.25	>512	1	0.0625	1	2	2	1
1207-SE51	MP	qnrS, bla_CTX-M-15,bla_TEM-1	>512	32	16	32	>512	128	0.25	8	4	8	2
T 073	62.3	qnrS, bla_TEM-1	8	0.5	0.5	0.25	>512	1	0.0625	1	2	2	1
J53	NA	NA	1	0.008	0.008	0.008	1	0.125	0.0625	0.5	1	1	1

The strains starting with “T” are transformants.

NAL, nalidixic acid; CIP, ciprofloxacin; ENR, enrofloxacin; NOR, norfloxacin; OFL, ofloxacin; CAZ, ceftazidime; CTF, ceftiofur; GEN, gentamicin; AMK, amikacin; FFC, florfenicol; DOX, doxycycline; AMP, ampicillin; NA, not applicable; MP, multiple plasmids.

Transferability of resistance determinants

For the PMQR-positive isolates, such as strains DS05, 1206-S23, 1205-S13, 1213-S18, and 1207-SE51, conjugation experiments were performed with E. coli J53 as the recipient strain by liquid and solid mating-out assays as described previously (Wang et al., 2003). Transformation of E. coli DH10B with the plasmid DNA extracted from clinical E. coli isolates was performed using a standard electroporation technique, and the five transformants were named T045, T075, T078, T065, and T073, respectively. Results of antimicrobial susceptibility testing for the five donors and five transformants were shown in Table 2, and the antimicrobial susceptibility of the five transconjugants was similar to the five transformants (data not shown). Molecular analysis revealed that all of the five donors had multiple plasmids, and each of the transformants carried only one transferable plasmid (Table 2). The floR gene in 1205-S13 was cotransferred to T078 with qnrS1 gene, whereas the floR genes in DS05 and 1206-S23 were not. The bla _CTX-M-14 gene in 1205-S13 was also cotransferred to T078 with qnrS1 gene, whereas the bla _CTX-M-15 genes in 1213-S18 and 1207-SE51 were not. Electrophoresis of the plasmid (pT078) extracted from transformant T078 was followed by Southern hybridization as described previously. The result showed that the four genes (qnrS1, bla _TEM-1, bla _CTX-M-14, and floR) were located on the same plasmid (see Fig. 2 below). The ciprofloxacin MICs of the transconjugants and transformants were 0.5 mg/L, representing a 64-fold increase over that of the recipient strain (MIC 0.008 mg/L). Increases in the MICs of multiple classes of antimicrobials were observed in the transformants, including a 32-fold increase in the MICs of three FQs from 0.25 to 0.5 mg/L (Table 2).

Analysis of the qnrS1-carrying plasmid pT078

The 81.2-kb plasmid pT078 mediated resistance to florfenicol, ciprofloxacin, and ceftiofur as well as to doxycycline, which was confirmed by in vitro susceptibility testing of transformants. PCR for the qnrS, floR, bla _CTX-M-14, and bla _TEM-1 genes showed that all of these genes were present on the pT078 plasmid. A 17575-bp segment of pT078, which comprised the tet(A), qnrS1, and floR regions, was cloned and sequenced. The open reading frames (ORFs) detected in this segment are displayed in Figure 1. The sequence consisted of three different segments; the initial 5515-bp exhibited 99% nucleotide sequence identity to the plasmid pWSH1 (accession no. DQ464880). Within this region, tet(A) resistance gene, tet(R) gene, pecM gene, and an incomplete tnpA gene were detected. The adjacent 4614 bp showed 100% identity to plasmid pINF5 from Salmonella Infantis (Kehrenberg et al., 2006a) and included the qnrS1 resistance gene area, two truncated CS12 insertion sequences, and an incomplete Tn5058-related resolvase gene. Further downstream, another 7096-bp region with 98% identity to plasmid pAR060302 (accession no. FJ621588) was detected. This region included floR resistance gene, a truncated IS26 insertion sequence, and three ORFs. Between the last two segments, a 350-bp region without significant homology to database entries was detected (Fig. 1).

FIG. 1.

Comparison of the sequenced parts of plasmids pWSH1, pINF5, and pAR060302 with the fragment of pT078 from Escherichia coli. The reading frames are shown as arrows, with the arrowhead indicating the direction of transcription. The arrows marked as hP and cP in the map of pAR060302 indicate reading frames for hypothetical proteins and a conserved protein, respectively. The areas of homology are shown by using the same shape.

Restriction analysis of pT078 was performed using three endonucleases, respectively. Restriction fragments generated with EcoRI, XbaI, or BamHI were chosen for Southern hybridization with qnrS1, bla _TEM-1, bla _CTX-M-14, or floR probes. In the case of restriction with EcoRI, qnrS1, and floR probes hybridized to the same fragment (18kb) (Fig. 2), for the lines which were digested by XbaI, all of the probes hybridized to the same fragment (23 kb), and the undigested plasmid (Fig. 2).

FIG. 2.

Restriction and hybridization analysis of the plasmid pT078. (A) Plasmid restriction profiles generated by EcoRI (E), XbaI (X), and BamHI (B). Lane M, λHind III DNA Marker (New England Biolabs, Beverly, MA). (B) Hybridizations of digested DNA with the qnrS1 probe. Two bands were detected in Lane X; the upper band is the undigested plasmid. (C) Hybridizations of digested DNA with the floR probe. Two bands were detected in Lane X; the upper band is the undigested plasmid. (D) Hybridizations of digested DNA with the bla _CTX-M-14 probe. Two bands were detected in Lane X; the upper band is the undigested plasmid. (E) Hybridizations of digested DNA with the bla _TEM-1 probe. Two bands were detected in Lane X; the upper band is the undigested plasmid.

Discussion

The use of antibiotics in the prophylaxis of diseases as well as their effect on growth promotion of healthy animals is common in livestock production in China. The emergence of antibiotic resistance of pathogens is a growing concern in veterinary medicine. Antibiotic-resistant pathogens pose the threat of severe and costly animal health problems. Furthermore, the increasing level of resistance to frontline antimicrobial agents in treating human diseases, such as extended-spectrum cephalosporins and fluoroquinolones, is a significant public health concern (Krueger et al., 2011).

The widespread dissemination of PMQR could potentially fuel the rapid development of fluoroquinolone resistance, because PMQR reduces the susceptibility to antibiotics and supports the occurrence of resistance-associated mutations on bacterial chromosomes. To date, these PMQR genes have been widely reported in Southern and Eastern Asia, North and South America, and Europe (Amabile-Cuevas et al., 2010; Dahmen et al., 2010). These PMQR determinants are usually carried on mobile elements and can be transferred among different bacterial strains or species. In isolates from Canada, France, Thailand, and Turkey, qnrA1 was associated with VEB-1β-lactamases (Poirel et al., 2005), while in samples from other countries, qnrA1 and ESBL SHV-12 and various CTX-M enzymes were linked (Bado et al., 2010). The qnrB alleles had also been associated with, or linked to the ESBLs SHV-12, TEM-52, and various CTX-M (Cattoir et al., 2007; Jiang et al., 2008). Unlike qnrA and qnrB, qnrS was seldom associated with ESBL or AmpC β-lactamase genes. In the present study, we identified bla _CTX-M-14, bla _TEM-1, floR, and qnrS1 genes located on one plasmid. Then, we characterized a large fragment carrying the qnrS1 gene and floR gene. Unexpectedly, another resistance gene, tet(A), was also located on this fragment. To our knowledge, this is the first study showing the occurrence of qnrS1, floR, bla _CTX-M-14, bla _TEM-1, and tet(A) located on the same plasmid isolated from food animals. In addition to the potential of the strain to develop high-level resistance to quinolones, the coexistence of multiple resistance genes on the same plasmid, which may confer resistance to β-lactamases, tetracycline, florfenicol, and quinolones, poses a serious epidemiological, clinical, and public health threat. The horizontal dissemination of plasmids carrying multiple antimicrobial resistance genes is rapidly resulting in more bacterial strains that are resistant to commonly used antibiotics. Antibiotic resistance developed in food-producing animals can be transferred to humans through food consumption or direct contact, and this has been shown in several studies (Manian, 2003; Molbak et al., 1999). The continuous and widespread use of florfenicol, fluoroquinolones, and β-lactamases in animal production will maintain the long-term presence of these resistance gene linkages. Since E. coli has a broad host range, a more large-scale appearance of these resistance gene linkages among different strains in both humans and animals is predicted for the future.

Footnotes

Acknowledgments

We would like to thank George A. Jacoby, David C. Hooper, and Minggui Wang for kindly providing the E. coli strains J53Az^R, V517, R1, R27, and plac. We thank Dr. Thomas Vorup-Jensen for assisting with the writing of this manuscript. This study was supported by the Program for Cheung Kong Scholars and the Innovative Research Team at the University of China (grant IRT0866), and Exclusive Research Fund for Public Welfare from Ministry of Agriculture of the People's of China (grant 200903055).

Disclosure Statement

No competing financial interests exist.

References

Amábile-Cuevas

, Arredondo-García

, Cruz

, Rosas

. Fluoroquinolone resistance in clinical and environmental isolates of Escherichia coli in Mexico City. J Appl Microbiol, 2010; 108:158–162.

Bado

, Cordeiro

, Robino

, García-Fulgueiras

, Seija

, Bazet

, Gutkind

, Ayala

, Vignoli

. Detection of class 1 and 2 integrons, extended-spectrum beta-lactamases and qnr alleles in enterobacterial isolates from the digestive tract of Intensive Care Unit inpatients. Int J Antimicrob Agents, 2010; 36:453–458.

Blickwede

, Schwarz

. Molecular analysis of florfenicol-resistant Escherichia coli isolates from pigs. J Antimicrob Chemother, 2004; 53:58–64.

Braibant

, Chevalier

, Chaslus-Dancla

, Pagès

, Cloeckaert

. Structural and functional study of the phenicol-specific efflux pump FloR belonging to the major facilitator superfamily. Antimicrob Agents Chemother, 2005; 49:2965–2971.

Cattoir

, Poirel

, Rotimi

, Soussy

, Nordmann

. Multiplex PCR for detection of plasmid-mediated quinolone resistance qnr genes in ESBL-producing enterobacterial isolates. J Antimicrob Chemother, 2007; 60:394–397.

Cavaco

, Hasman

, Xia

, Aarestrup

. qnrD, a novel gene conferring transferable quinolone resistance in Salmonella enterica serovar Kentucky and Bovismorbificans strains of human origin. Antimicrob Agents Chemother, 2009; 53:603–608.

Chmelnitsky

, Navon-Venezia

, Strahilevitz

, Carmeli

. Plasmid-mediated qnrB2 and carbapenemase gene bla(KPC-2) carried on the same plasmid in carbapenem-resistant ciprofloxacin-susceptible Enterobacter cloacae isolates. Antimicrob Agents Chemother, 2008; 52:2962–2965.

[CLSI] Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Seventeenth Informational SupplementCLSI Document M100-S17Wayne, PA: CLSI, 2007.

[CLSI] Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals: Approved Standard, 3^rd ed. Informational SupplementCLSI Document M31-A3Wayne, PA: CLSI, 2008.

10.

Dahmen

, Poirel

, Mansour

, Bouallègue

, Nordmann

. Prevalence of plasmid-mediated quinolone resistance determinants in Enterobacteriaceae from Tunisia. Clin Microbiol Infect, 2010; 16:1019–1023.

11.

Endimiani

, Carias

, Hujer

, Bethel

, Hujer

, Perez

, Hutton

, Fox

, Hall

, Jacobs

, Paterson

, Rice

, Jenkins

, Tenover

, Bonomo

. Presence of plasmid-mediated quinolone resistance in Klebsiella pneumoniae isolates possessing blaKPC in the United States. Antimicrob Agents Chemother, 2008; 52:2680–2682.

12.

Ferreira

, Toleman

, Ramalheira

, Da Silva

, Walsh

, Mendo

. First description of Klebsiella pneumoniae clinical isolates carrying both qnrA and qnrB genes in Portugal. Int J Antimicrob Agents, 2010; 35:584–586.

13.

Galani

, Souli

, Mitchell

, Chryssouli

, Giamarellou

. Presence of plasmid-mediated quinolone resistance in Klebsiella pneumoniae and Escherichia coli isolates possessing blaVIM-1 in Greece. Int J Antimicrob Agents, 2010; 36:252–254.

14.

Hata

, Suzuki

, Matsumoto

, Takahashi

, Sato

, Ibe

, Sakae

. Cloning of a novel gene for quinolone resistance from a transferable plasmid in Shigella flexneri 2b. Antimicrob Agents Chemother, 2005; 49:801–803.

15.

Huang

, Dai

, Xia

, Du

, Qi

, Liu

, Wu

, Shen

. Increased prevalence of plasmid-mediated quinolone resistance determinants in chicken Escherichia coli isolates from 2001 to 2007. Foodborne Pathog Dis, 2009; 6:1203–1209.

16.

Huang

, Zhu

, Wang

, Dai

, Wang

, Li

, Wu

, Shen

. Correlation of fluoroquinolone resistance with expression of qnrA gene in Kluyvera spp. Foodborne Pathog Dis, 2011; 8:1039–1044.

17.

Jacoby

. Mechanisms of resistance to quinolones. Clin Infect Dis, 2005; 41,Suppl 2:S120–S126.

18.

Jacoby

, Chow

, Waites

. Prevalence of plasmid-mediated quinolone resistance. Antimicrob Agents Chemother, 2003; 47:559–562.

19.

Jacoby

, Walsh

, Mills

, Walker

, Oh

, Robicsek

, Hooper

. qnrB, another plasmid-mediated gene for quinolone resistance. Antimicrob Agents Chemother, 2006; 50:1178–1182.

20.

Jiang

, Zhou

, Qian

, Wei

, Yu

, Hu

, Li

. Plasmid-mediated quinolone resistance determinants qnr and aac(6')-Ib-cr in extended-spectrum beta-lactamase–producing Escherichia coli and Klebsiella pneumoniae in China. J Antimicrob Chemother, 2008; 61:1003–1006.

21.

Kehrenberg

, Friederichs

, de Jong

, Michael

, Schwarz

. Identification of the plasmid-borne quinolone resistance gene qnrS in Salmonella enterica serovar Infantis. J Antimicrob Chemother, 2006a. 58:18–22.

22.

Kehrenberg

, Meunier

, Targant

, Cloeckaert

, Schwarz

, Madec

. Plasmid-mediated florfenicol resistance in Pasteurella trehalosi. J Antimicrob Chemother, 2006b. 58:13–17.

23.

Krueger

, Folster

, Medalla

, Joyce

, Perri

, Johnson

, Zervos

, Whichard

, Barzilay

. Commensal Escherichia coli isolate resistant to eight classes of antimicrobial agents in the United States. Foodborne Pathog Dis, 2011; 8:329–332.

24.

, Wu

, Zhao

, He

, Cao

, Dai

, Xia

, Qin

, Shen

. Prevalence of antimicrobial resistance among Salmonella isolates from chicken in China. Foodborne Pathog Dis, 2011; 8:45–53.

25.

Manian

. Asymptomatic nasal carriage of mupirocin-resistant, methicillin-resistant Staphylococcus aureus (MRSA) in a pet dog associated with MRSA infection in household contacts. Clin Infect Dis, 2003; 36:e26–e28.

26.

Martínez-Martínez

, Pascual

, Jacoby

. Quinolone resistance from a transferable plasmid. Lancet, 1998; 351:797–799.

27.

Mølbak

, Baggesen

, Aarestrup

, Ebbesen

, Engberg

, Frydendahl

, Gerner-Smidt

, Petersen

, Wegener

. An outbreak of multidrug-resistant, quinolone-resistant Salmonella enterica serotype Typhimurium DT104. N Engl J Med, 1999; 341:1420–1425.

28.

Nordmann

, Poirel

. Emergence of plasmid-mediated resistance to quinolones in Enterobacteriaceae. J Antimicrob Chemother, 2005; 56:463–469.

29.

Poirel

, Pitout

, Calvo

, Rodriguez-Martinez

, Church

, Nordmann

. In vivo selection of fluoroquinolone-resistant Escherichia coli isolates expressing plasmid-mediated quinolone resistance and expanded-spectrum beta-lactamase. Antimicrob Agents Chemother, 2006; 50:1525–1527.

30.

Poirel

, Van De Loo

, Mammeri

, Nordmann

. Association of plasmid-mediated quinolone resistance with extended-spectrum beta-lactamase VEB-1. Antimicrob Agents Chemother, 2005; 49:3091–3094.

31.

Robicsek

, Strahilevitz

, Jacoby

, Macielag

, Abbanat

, Park

, Bush

, Hooper

. Fluoroquinolone-modifying enzyme: A new adaptation of a common aminoglycoside acetyltransferase. Nat Med, 2006; 12:83–88.

32.

Rodríguez-Martínez

, Velasco

, García

, Cano

, Martínez-Martínez

, Pascual

. Mutant prevention concentrations of fluoroquinolones for Enterobacteriaceae expressing the plasmid-carried quinolone resistance determinant qnrA1. Antimicrob Agents Chemother, 2007; 51:2236–2239.

33.

Strahilevitz

, Engelstein

, Adler

, Temper

, Moses

, Block

, Robicsek

. Changes in qnr prevalence and fluoroquinolone resistance in clinical isolates of Klebsiella pneumoniae and Enterobacter spp. collected from 1990 to 2005. Antimicrob Agents Chemother, 2007; 51:3001–3003.

34.

Strahilevitz

, Jacoby

, Hooper

, Robicsek

. Plasmid-mediated quinolone resistance: A multifaceted threat. Clin Microbiol Rev, 2009; 22:664–689.

35.

Wang

, Guo

, Xu

, Wang

, Ye

, Wu

, Hooper

, Wang

. New plasmid-mediated quinolone resistance gene, qnrC, found in a clinical isolate of Proteus mirabilis. Antimicrob Agents Chemother, 2009; 53:1892–1897.

36.

Wang

, Tran

, Jacoby

, Zhang

, Wang

, Hooper

. Plasmid-mediated quinolone resistance in clinical isolates of Escherichia coli from Shanghai, China. Antimicrob Agents Chemother, 2003; 47:2242–2248.

37.

Xia

, Tao

, Shen

, Dai

, Wang

, Chen

, Wu

. A survey of beta-lactamase and 16S rRNA methylase genes among fluoroquinolone-resistant Escherichia coli isolates and their horizontal transmission in Shandong, China. Foodborne Pathog Dis, 2011; 8:1241–1248.

38.

Yamane

, Wachino

, Suzuki

, Kimura

, Shibata

, Kato

, Shibayama

, Konda

, Arakawa

. New plasmid-mediated fluoroquinolone efflux pump, QepA, found in an Escherichia coli clinical isolate. Antimicrob Agents Chemother, 2007; 51:3354–3360.