Co-prevalance of PMQR and 16S rRNA methylase genes in clinical Escherichia coli isolates with high diversity of CTX-M from diseased farmed pigeons

Veterinary Microbiology 178 (2015) 238–245

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Co-prevalance of PMQR and 16S rRNA methylase genes in clinical Escherichia coli isolates with high diversity of CTX-M from diseased farmed pigeons Ling Yang a,1, Lei Yang a,1, Dian-Hong Lü b , Wen-Hui Zhang a,b , Si-Qi Ren a , Ya-Hong Liu a , Zhen-Ling Zeng a , Hong-Xia Jiang a, * a College of Veterinary Medicine, Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development and Safety Evaluation, South China Agricultural University (SCAU), Guangzhou 510642, China b Laboratory of Clinical Microbiology, Institute of Veterinary Medicine, Guangdong Academy of Agriculture Sciences, Guangzhou 510640, China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 4 March 2015 Received in revised form 8 May 2015 Accepted 11 May 2015

In the present study, we determined the molecular epidemiology of extended-spectrum b-lactamases (ESBLs) in Escherichia coli isolated from diseased farmed pigeons in China. A total of 71 E. coli isolates were collected from three pigeon farms from 2011 to 2012 and screened for the presence of the ESBL genes. The ESBLs producers were further tested for the presence of PMQR-encoding genes as well as the 16S rRNA methylase gene using PCR and DNA sequence analysis. Co-transfer of plasmids encoding for ESBLs, PMQR determinants and/or 16S rRNA methylase gene was performed by conjugation into E. coli. The genetic relatedness and plasmid replicon type were determined. A total of 41 ESBLs producers were identified. Only CTX-M type ESBLs were detected, with the most common CTX-M types being CTX-M-65 (n = 17), CTX-M-27 (n = 11), CTX-M-55 (n = 10). Thirty-eight CTX-M-positive isolates were found to harbor at least one PMQR gene, with aac(60 )-Ib-cr (n = 32) and oqxAB (n = 21) being the most prevalent. The rmtB was the only prevalent 16S rRNA methylase gene detected in 24 (58.1%) CTX-M-positive isolates. Although most of the CTX-M producers had distinct pulsotypes, clonal transmission in the same farm was observed. blaCTXM genes were carried by IncF alone or in combination with IncK plasmids with three different sizes, including 76.8 Kb (n = 20), 194 Kb (n = 5), 104.5 Kb (n = 2). PFGE profiles of CTX-M-positive E. coli isolates indicated potential horizontal spread of these multidrug resistant strains along with those CTX-M encoding genes. Our findings highlight the importance of pigeons as a reservoir of multiple antimicrobial resistance genes. ã2015 Elsevier B.V. All rights reserved.

Keywords: E. coli CTX-M Multi-drug resistance

1. Introduction Development of antimicrobial resistance by microbial pathogens and commensals represents a major threat to animal and public health (Clarke, 2006). The emergence of antimicrobial resistance in food-producing animals is of major public health significance arising from the risk of these bacteria entering the food chain (Van den Bogaard and Stobberingh, 2000). Growing evidence indicates that intensive agricultural and veterinary usage of antimicrobial compounds contributes to the emergence and dissemination of antimicrobial resistance in bacteria derived from food-producing animals (Molbak, 2004).

* Corresponding author. Tel.: +86 20 85283934; fax: +86 20 85284896. E-mail address: [email protected] (H.-X. Jiang). These authors contributed equally to this work.

1

http://dx.doi.org/10.1016/j.vetmic.2015.05.009 0378-1135/ ã 2015 Elsevier B.V. All rights reserved.

b-lactams, fluoroquinolones and aminoglycosides continue to play important roles in the treatment of serious infections caused by Gram-negative pathogens. However, Enterobacteriaceae with multidrug resistance phenotypes of these antibiotics have been increasing worldwide (Canton and Ruiz-Garbajosa, 2011). The most common cause of bacterial resistance to b-lactams is the production of b-lactamase (especially extended-spectrum b-lactamases, ESBLs). A dramatic increase in the number of class A and class D b-lactamases has been described since the 1980s (Bush and Jacoby, 2010). ESBLs of class A mainly include enzymes of TEM, SHV, CTX-M, VEB, and GES, which represent public health concerns because of their ability to hydrolyze expanded-spectrum cephalosporins (such as cefotaxime, ceftriaxone) (Pitout and Laupland, 2008). Studies over the last ten years have revealed that the CTX-M enzymes have nearly displaced other ESBL enzymes in Enterobacteriaceae (Bush, 2010; Canton et al., 2012). One reason for this displacement is the extraordinary dissemination of the

L. Yang et al. / Veterinary Microbiology 178 (2015) 238–245

corresponding blaCTX-M genes in highly mobilizable genetic platforms, including plasmids and transposons (Canton et al., 2012; Woodford et al., 2011). Another reason for this increase is the coresistance phenomenon in CTX-M-producing bacteria, particularly to fluoroquinolones and aminoglycosides, which might facilitate co-selection processes from which multidrug resistant organisms arise (Canton and Ruiz-Garbajosa, 2011; D’Andrea et al., 2013). The presence of different resistance genes undoubtedly gives advantages to the bacteria under antibiotic selective force and increases the opportunity for persistence of the bacteria and resistance genes (Canton et al., 2003). In China, high antimicrobial resistance rates of b-lactams, fluoroquinolones and aminoglycosides in Enterobacteriaceae have been reported frequently in a variety of food-producing animals, which are considered to be reservoirs of commensals carrying various resistant determinants (Jiang et al., 2012; Jiang et al., 2011; Liu et al., 2013; Ma et al., 2012). Pigeons are normally considered as food-producing animals in China, some countries of the Western Europe and Southeast Asia, in which case the same restrictions on antimicrobial usages are applied as previously discussed for other food-producing animals (Maron et al., 2013). For these reasons the pigeon industry has also been considered a potential reservoir of resistance-gene-producing Gram-negative bacteria that may be acquired by humans through handling or consumption of contaminated products. However, the carriage of these bacteria by pigeons in China has not been well characterized. This study was designed to determine the extent to which antimicrobial resistant E. coli strains occur in pigeons in China in order to determine whether this animal species might serve as a reservoir or vehicle for the dissemination of antimicrobial resistant strains into food-chains. We performed a pilot study to gain insight into the occurrence of antimicrobial resistance in E. coli isolated from pigeons. Our studies reported co-prevalence of CTX-M-type extended-spectrum b-lactamases (ESBLs) genes with plasmidmediated quinolone resistance (PMQR) genes and those encoding 16S rRNA methylase that may display reduced susceptibility to multi-antimicrobial agents in E. coli isolates of pigeon origin. 2. Materials and methods 2.1. Bacterial isolates A total of 97 samples of intestinal tract (n = 46), viscera (n = 31), blood (n = 16) and other sites (n = 4) were collected from 97 pigeons aged between 20 and 25-days from three different pigeon farms (named Farm 1–3: 34 samples from Farm 1, 32 from Farm 2, 31 from Farm 3) in Guangdong Province in China during the period of February 2011 and December 2011. Many of the pigeons appeared very sick with poor appetite, going to die or already dead at the time received at the Institute of Animal Health. Samples were collected on-site when the birds in the herds presented symptoms before being transported to the Institute of Animal Health. Diagnosis of New Castle disease was made by PCR. The sampling was carried out in a sterile room using sterile cotton swabs. All samples were dispatched within 12 h to the Veterinary Research Institute, Guangdong Academy of Agricultural Science, where they were seeded onto MacConkey agar and incubated at 37  C for 18 h. One colony with typical E. coli morphology was selected from each sample and was identified with API20E systems (BioMerieux, Beijing, China). All isolates were stored at 80  C in Luria–Bertani broth containing 30% glycerol. 2.2. Antimicrobial susceptibility The minimal inhibitory concentrations (MICs) of 16 antibiotics, including ampicillin (AMP), cefotaxime (CTX), cefoxitin (CXT),

239

ceftiofur (CTF), ceftazidime (CAZ), ceftriaxone (CTR), nalidixic acid (NAL), ciprofloxacin (CIP), kanamycin (KAN), gentamycin (GEN), amikacin (AMK), tetracycline (TET), chloramphenicol (CHL), florfenicol (FFC), olaquindox (OLA), were determined by a agar dilution method according to the standards described by Clinical and Laboratory Standards Institution (M100-S25 and VET01-A4/ VET01-S2). Isolates were classified as either susceptible or resistant according to the interpretative criteria recommended by the CLSI (M100-S25) (ampicillin, cefotaxime, ceftiofur, ceftriaxone, cefoxitin, ceftazidime, nalidixic acid, ciprofloxacin, kanamycin, amikacin), veterinary CLSI (gentamycin, enrofloxacin, tetracycline, chloramphenicol, florfenicol) (VET01-S2) and DANMAP 98 (olaquindox  64 mg/mL) (DANMAP, 1999). E. coli ATCC strain 25922 was used as quality control in the antimicrobial susceptibility testing. 2.3. Detection of ESBL genes E. coli isolates that displayed resistance to cefotaxime or ceftiofur were screened for ESBL-genes using PCR as described previously for blaCTX-M genotype groups 1, 2, 8, 9 and 25, blaTEM, blaSHV, blaOXA (Jiang et al., 2012). The obtained DNA amplicons were submitted to BGI Life Tech Co., Ltd. (Beijing, China) for sequencing and sequences were compared with those included in the GenBank database by using the BLAST algorithm (www.ncbi. nlm.nih.gov) and at www.lahey.org/Studies/ in order to identify the specific b-lactamase genes. ESBL confirmatory testing was not included in this study. 2.4. Chromosomal mutation, PMQR genes and 16srRNA genes in CTXM-positive isolates All blaCTX-M-positive isolates were screened for PMQR genes [aac(60 )-Ib-cr, qnrA, B, C, D, S, qepA and oqxAB and mutations in the quinolone resistance-determining regions (QRDR) of gyrA, gyrB, parC and parE using previously described primers and protocols (Shaheen et al., 2013). The presence of 16S rRNA methylase genes (armA, rmtA, rmtB, rmtC and rmtD, npmA) were also analyzed by PCR in blaCTX-Mpositive isolates, using specific primers and conditions described previously (Doi and Arakawa, 2007). 2.5. Strain typing Genetic relatedness of all isolates containing CTX-M-encoding genes was analyzed by pulsed-field gel electrophoresis (PFGE) of XbaI-digested chromosomal DNA (CHEF Mapper1, Bio-Rad Laboratories, Hercules, CA) as described previously(Jiang et al., 2014). Resulting PFGE patterns were interpreted according to the method of Tenover et al. (1995). 2.6. Conjugation assay and plasmid analyses E. coli isolates positive to blaCTX-M were selected to perform conjugation experiments by the broth-mating method using E. coli C600 as the recipient. Transconjugants were selected on MacConkey agar plates containing streptomycin (1000 mg/ml) and cefotaxime (2 mg/ml) as described previously (Jiang et al., 2014). Gene analyses of the blaCTX-M positive transconjugants were confirmed by PCR as described above. Plasmid replicon typing of all transconjugants was done by a PCR based method using 18 pairs of primers described previously (Carattoli et al., 2005). PFGE with S1 nuclease (Takara Biotechnology, Dalian, China) digestion of whole genomic DNA was performed for all transconjugants, as described previously (Barton et al., 1995). Primers used to design

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L. Yang et al. / Veterinary Microbiology 178 (2015) 238–245

blaCTX-M-probes were the same with those used to amplify CTX-Mencoding genes. After Southern transfer to a Hybond-N+ membrane (GE Healthcare, Little Chalfont, United Kingdom), the plasmids were probed with the blaCTX-M gene according to the instructions of the commercial detection kit. (DIG High Prime DNA Labeling and Detection Starter Kit I, Roche Applied Science, Mannheim, Germany). 3. Results 3.1. Antimicrobial susceptibility analysis Seventy-one (34 from Farm 1, 26 from Farm 2 and 11 from Farm 3) consecutive and unique E. coli isolates were detected. The distributions of MIC for the 16 antimicrobial agents tested against the isolates are shown in Table 1. Susceptibility testing showed that the resistance rates of ceftiofur, cefotaxime, ceftriaxone and cefoxitin were 56.3%, 53.5%, 45.1% and 12.7%, respectively. Only two isolates showed resistance to ceftazidime. Thirty three (46.5%) of the isolates showed co-resistance to three tested third generation cephalosporins (ceftiofur, cefotaxime and ceftriaxone). The majority of the isolates were highly resistant to nalidixic acid (98.6%), enrofloxacin (90.1%) and ciprofloxacin (90.1%). Seven of the isolates had reduced susceptibility to ciprofloxacin (MIC, 0.06– 1 mg/L). The antimicrobial resistance rates to the aminoglycosides were as follows: kanamycin (83.1%), gentamycin (81.7%), amikacin (42.3%). A high proportion of resistance to other antimicrobial agents commonly used on animal farms was also demonstrated in this study, including tetracycline (98.6%), ampicillin (95.8%), chloramphenicol (93.0%), olaquindox (81.7%) and florfenicol (69.0%). All of the isolates were resistant to three or more classes of antimicrobials tested. Several (28.2%) isolates were resistant to 14 antimicrobial agents, and the most frequently observed pattern of multidrug resistance was AMP- CTX- CTF- CTR- NAL- ENR- CIPGEN- KAN- AMI- TET- CHL- FFC- OLA, which was the phenotype of 17 (23.9%) isolates from this study.

Table 1 Numbers of drug resistant E. coli isolates from sick pigeons in China. Antimicrobial

Number(%) of resistant isolates

b-Lactams Ampicillin Ceftiofur Cefotaxime Ceftriaxone Cefoxitin

68(95.8) 40(56.3) 38(53.5) 32(45.1) 9(12.7)

Fluoroquinolones Nalidixic acid Enrofloxacin Ciprofloxacin

70(98.6) 64(90.1) 64(90.1)

Aminoglycosides Kanamycin Gentamicin Amikacin

59(83.1) 58(81.7) 30(42.3)

Others Tetracycline Chloramphenicol Olaquindox Florfenicol

70(98.6) 66(93.0) 58(81.7) 49(69.0)

3.2. Detection of ESBL genes in third-generation- cephalosporinresistant E. coli Genes encoding CTX-M-type extended spectrum b-lactamase were identified in 41 (97.6%) of 42 isolates showing resistance to cefotaxime or ceftiofur and no other-type extended spectrum b-lactamases were detected. Sequencing of the amplicons obtained from the PCR for the different blaCTX-M subgroups revealed that blaCTX-M encoding CTX-M-65 was the most prevalent (41.5%, n = 17), and blaCTX-M encoding CTX-M-27, CTX-M-55, CTXM-14, CTX-M-15 and CTX-M-13 were detected in 23.8% (n = 10), 23.8% (n = 10), 9.5% (n = 4), 4.8% (n = 2) and 2.38% (n = 1) of the total isolates, respectively. Three strains harboured both CTX-M1 cluster and CTX-M-9 cluster genes: one strain co-harboured blaCTX-M-55 and blaCTX-M-65, one strain co-harboured blaCTX-M-15 and blaCTX-M-65 and one strain co-harboured blaCTX-M-55 and blaCTX-M-27. Sequencing of blaTEM gene revealed blaTEM-1b (n = 36), blaTEM-135 (n = 2) and blaTEM-1 (n = 1). OXA-30, SHV-11 and SHV-1 were detected in 6, 2 and 1 blaCTX-M-positive strains, respectively (Table 2). 3.3. Characterization of CTX-M-positive strains PMQR determinants were detected in 38 (92.7%) of the 41CTXM-positive strains. The predominant PMQR genes were aac(60 )-Ibcr (78.0%, n = 32), followed by oqxAB (51.2%, n = 21), qnr (41.5%, n = 17) and qepA (4.9%, n = 2). Among qnr-positive strains, qnrD, qnrS and qnrB were detected in 14 (34.1%), 9 (21.9%) and 7 (17.1%) isolates, respectively. No qnrA and qnrC genes were detected in any of the E. coli isolates (Table 2). Almost all (95.1%) CTX-M-positive isolates (except for strain 123 and 124, which showed reduced susceptibility to all tested FQs and carried aac(60 )-Ib-cr gene) had at least two amino acid substitutions in QRDR of gyrA, parC and parE. Double mutations of gyrA (S83L/D87N) were detected in 37 isolates, and two strains showed a single mutation at position 83 in gyrA (S83I and S83L, respectively). All strains with gyrA mutations had one or two substitutions in the QRDR of parC (S80I/R, A56T, E84G), 12 of which also contain a mutation in parE (with S458A in 10 and L416F in 2 isolates, Table 2). No mutations were found in the QRDR of gyrB. Three out of 39 isolates with QRDR mutations harbored no PMQR genes (data not shown). Twenty-four (58.1%) blaCTX-M containing strains carried rmtB, and 22 of them also carried PMQR genes (Table 2). We did not identify rmtA, rmtC, rmtD, armA and npmA in this study. 3.4. Strain typing Twenty-four different XbaI-PFGE patterns (with similarity percentage of 90%) were obtained from 33 available PFGE profiles of blaCTX-M-positive E. coli; no PFGE patterns were obtained from the other 8 isolates. No clonal relationship was observed between E. coli isolates from different farms (Fig. 1.). A clonal relationship was not only sporadically observed between E. coli strains harboring the same CTX-M-encoding gene (e.g., strains 1243-2 and 1245-2 from Farm 1, strains 123 and 124 from Farm 2 Fig. 1), but also observed between isolates harboring different CTXM-encoding genes (e.g., strains 1239 and 1245-1. Fig. 1). However, most of the CTX-M-65- or CTX-M-55-positive isolates from the same farm had no major clonal relationship based on PFGE profiles. 3.5. Characteristics of CTX-M positive transconjugants Twenty-seven (65.8%) blaCTX-M-gene transconjugants were obtained, in which 16 isolates co-harbored blaCTX-M, PMQR and rmtB gene (9 blaCTX-M + aac(60 )-Ib-cr/rmtB, 6 blaCTX-M + qnrD/qnrS/ rmtB,1 blaCTX-M + aac(60 )-Ib-cr/rmtB/oqxAB); 6 co-harboured blaCTX-

Table 2 Characteristics of CTX-M-producing Escherichia coli isolated from sick pigeons in China. Strain

Farm no.

Tissue source

105

Farm 3

106

Farm 3

107

Farm 3

123

Farm 2 Farm 2

125

Farm 2

444 551 552

Farm 1 Farm 1 Farm 1

553 554

Farm 1 Farm 1

556

CTX CIP

AMK

128

64

>512

64

32

64

32

64

0.06 >512

ESBL gene (s)

Other resistant gene(s)

Plasmid transfer

MIC of transconjungant-s to FQs (mg/ml) NALa CIPb

gyrA

parC

parE

S83I

S80I

–

–

64

>512

blaCTX-M-55 blaTEM-1b,blaSHV-11, qnrD, qnrS, oqxAB, rmtB blaCTX-M-65 blaCTX-M-65 blaTEM-1b, blaSHV-11, qnrD, qnrS, oqxAB, rmtB

S83L D87N

S80I

–

–

>512

blaCTX-M-55 blaTEM-1b, qnrD, qnrS, rmtB

S83L D87N

S80I

–

blaCTX-M-27 blaTEM-135, aac(60 )- Ib-cr, rmtB

–

–

–

0

Information of CTX-M-positive plasmids Co-transferred resistant gene(s)

Size (Kb)

Replicon type(s)

0.125

qnrD, qnrS, rmtB

194

F, FIA, K

64

0.125

76.8

F, K

–

64

0.125

76.8

F, K

–

4

<0.03

blaTEM-1b, qnrD, qnrS, rmtB blaTEM-1b, qnrD, qnrS, rmtB blaTEM-135, rmtB

76.8

F

blaTEM-135, aac(6 )Ib-cr, rmtB rmtB

76.8

F, N

76.8

F

– – 194

– – F, FIA, K

– 76.8

– F, K

76.8

F,K

– –

– –

0

–

–

–

–

4

0.06

blaTEM-1b, blaOXA-30, rmtB

S83L D87N

S80I

–

–

4

<0.03

1 1 >512

blaCTX-M-14 blaTEM-1b, aac(60 )-Ib -cr, oqxAB blaCTX-M-65 blaTEM-1b, blaOXA-30, aac(60 )-Ib-cr,qnrD blaCTX-M-65 blaTEM-1b, qnrD, qnrS, oqxAB, rmtB

S83L D87N S83L D87N S83L D87N

S80I S80I S80I E84G

– – –

– – –

– – 64

– – 0.125

64 256

>512 >512

blaCTX-M-65 blaTEM-1b, blaOXA-30, aac(6 0 )-Ib-cr, rmtB blaCTX-M-65 blaTEM-1b aac(60 )-Ib -cr, qnrB, oqxAB, rmtB

S83L D87N S83L D87N

S80I S80I

– – S458A +

– 4

– 0.06

256

32

>512

blaCTX-M-65 blaTEM-1b, aac(60 )-Ib -cr, qnrD, qnrS, rmtB

S83L D87N

S80I

–

+

4

0.06

Viscera Viscera

32 32

32 32

2 2

S83L D87N S83L D87N

S80I S80I

– –

– –

– –

– –

Intestinal tract Viscera Viscera Intestinal tract Intestinal tract Intestinal tract Blood Viscera Viscera Intestinal tract Intestinal tract Viscera Intestinal tract Intestinal tract Intestinal tract Intestinal tract

32

256

>512

blaCTX-M-65 blaTEM-1b, aac(60 )-Ib -cr, qnrD, qnrS, oqxAB blaCTX-M-65 blaTEM-1b, aac(60 )-Ib -cr, qnrD, qnrS, oqxAB, qepA blaCTX-M-65 blaTEM-1b, aac(60 )-Ib -cr, rmtB

– – blaTEM-1b, qnrD, qnrS, rmtB – blaTEM-1b, aac(60 )-Ib -cr, rmtB blaTEM-1b, aac(60 )-Ib -cr, rmtB – –

S83L D87N

S80I E84G

–

+

4

<0.03

rmtB

104.5

F, K

16 8 8

32 32 32

2 2 2

blaCTX-M-14 aac(60 )-Ib-cr, qnrB blaCTX-M-27 blaOXA-30, aac(60 )-Ib -cr, oqxAB blaCTX-M-27 blaTEM-1b, aac(60 )-Ib -cr, oqxAB

S83L D87N S83L D87N S83L D87N

S80I S80R S80R

– + S458A – S458A –

4 – –

0.06 – –

aac(60 )-Ib-cr – –

76.8 – –

F, K – –

128

64

2

blaCTX-M-27 blaTEM-1b, aac(60 )-Ib -cr, oqxAB

S83L D87N

S80R

S458A –

–

–

–

–

–

128

32

2

blaCTX-M-27 blaTEM-1b, aac(60 )-Ib -cr, qnrB, oqxAB

S83L D87N

S80R

S458A –

–

–

–

–

–

128 128 8 64

32 64 0.5 64

>512 >512 1 4

blaCTX-M-27 blaCTX-M-27 blaCTX-M-14 blaCTX-M-65

S83L D87N S83L D87N S83L S83L D87N

S80R S80I S80I S80I

S458A S458A – –

– – – –

– – – –

– – – –

– – – –

– – – –

– – – –

16

16

2

blaCTX-M-13

S83L D87N

S80I

L416F

–

–

–

–

–

–

8 256

64 32

2 >512

S83L D87N S83L D87N

S80I S80I

L416F –

+ +

4 4

0.06 0.06

F, K F, K

16

>512

S83L D87N

S80I

+

4

0.06

76.8

F, K

128

32

>512

S83L D87N

S80I

–

+

64

0.125

76.8

F, FIA, K

256

32

>512

blaCTX-M-65 blaTEM-1b, blaOXA-30, blaSHV-1, qnrD, qnrS, oqxAB, rmtB blaCTX-M-65 blaTEM-1b, qnrD, qnrS, rmtB

S83L D87N

S80I

–

+

64

0.125

aac(60 )-Ib-cr blaTEM-1b, aac(60 )-Ib -cr, rmtB blaTEM-1b, aac(60 )-Ib -cr, rmtB blaTEM-1b, qnrD, qnrS, rmtB qnrD, qnrS, rmtB

104.5 76.8

128

blaCTX-M-14 blaTEM-1, aac(60 )-Ib -cr, qnrB, qnrD, oqxAB blaCTX-M-65 blaTEM-1b, aac(60 )-Ib -cr, qnrD, rmtB blaCTX-M-15 blaCTX-M-55 blaTEM-1b, aac(60 )-Ib -cr, qnrB, qnrD, rmtB

76.8

F, FIA, K

64

0.06 >512

blaCTX-M-27 blaTEM-135, aac(6 )- Ib-cr, rmtB

256

64

>512

blaCTX-M-15

8 16 64

16 32 32

Viscera Viscera

16 64

Farm 1

Viscera

557 558

Farm 1 Farm 1

559

Farm 1

597 601 602

Farm 1 Farm 2 Farm 2

603

Farm 2

604

Farm 2

760 762 763 1202-1

Farm Farm Farm Farm

1202-2

Farm 2

1208-2 1234-1

Farm 2 Farm 1

1234-2

Farm 1

1235

Farm 1

1236

Farm 1

2 2 2 2

QRDR mutations

blaTEM-1b, blaTEM-1b, blaTEM-1b, blaTEM-1b, qepA blaTEM-1b

aac(60 )-Ib -cr, oqxAB, rmtB aac(60 )-Ib -cr, oqxAB, rmtB aac(60 )-Ib -cr, qnrB, qnrD, oqxAB blaOXA-30, aac(60 )-Ib-cr, oqxAB,

L. Yang et al. / Veterinary Microbiology 178 (2015) 238–245

124

Intestinal tract Intestinal tract Intestinal tract Intestinal tract Intestinal tract Intestinal tract Viscera Viscera Viscera

MIC (mg/ml)

241

>512 32 256 Farm 1 1245-2

Viscera

Farm 1 1245-1

MIC, minimal inhibitory concentration; ESBL, extended-spectrun b-lactamase; FQs, fluoroquinolones; QRDR, quinolone resistance determining region; CTX, cefotaxime; CIP, ciprofloxacin; AMK, amikacin; NAL, nalidixic acid.

F, K 76.8 blaCTX-M-55 blaTEM-1b, aac(6 )-Ib -cr, oqxAB, rmtB

S83L D87N

A56TS80I

+

4

0.06

F,K 76.8 4 + S80I S83L D87N blaCTX-M-55 blaTEM-1b, aac(60 )-Ib -cr, rmtB >512 64 512

Farm 1 1244

Viscera

0

0.06

F, K 194 S83L D87N

Farm 1 1243-2

Viscera

64

64

2

blaCTX-M-65 blaTEM-1b aac(60 )-Ib -cr, qnrB, oqxAB

S80I

S458A +

8

0.125

F, K 194 4 S458A + S80I S83L D87N

Farm 1 1243-1

Viscera

256

32

>

blaCTX-M-65 blaTEM-1b, aac(60 )-Ib -cr

S80I S83L D87N blaCTX-M-55 blaTEM-1b, aac(6 )-Ib -cr, oqxAB, rmtB >512 32 256

Farm 1 1242-2

Viscera

A56TS80I S83L D87N

0

blaCTX-M-55 blaTEM-1b, aac(60 )-Ib -cr, rmtB >512 16 256

Farm 1 Farm 1 1241 1242-1

Blood

>512 >512 32 32 128 256

Farm 1 Farm 1 1239 1240

Blood Blood

0.06

F, K 76.8 +

4

0.06

F,K 76.8 +

4

0.06

F, K F, K 76.8 76.8 S83L D87N S83L D87N blaTEM-1b, aac(60 )-Ib -cr, rmtB blaTEM-1b, aac(60 )-Ib -cr, rmtB

S80I S80I

–

+ +

4 8

<0.03 0.125

blaTEM-1b, rmtB aac(60 )-Ib-cr, oqxAB, rmtB blaTEM-1b, aac(60 )-Ib -cr, rmtB blaTEM-1b, aac(60 )-Ib -cr, rmtB blaTEM-1b, aac(60 )-Ib -cr blaTEM-1b, aac(60 )-Ib -cr, oqxAB blaTEM-1b, aac(60 )-Ib -cr, rmtB blaTEM-1b aac(60 )-Ib -cr, rmtB

F,FIB,K,P F, K 194 76.8 blaTEM-1b blaTEM-1b, rmtB S83L D87N S83L D87N 64 128

64 16

2 >512

blaCTX-M-27 blaCTX-M-55 blaCTX-M-27 blaCTX-M-65 blaCTX-M-55

blaTEM-1b blaTEM-1b, aac(60 )-Ib -cr, rmtB

S80I A56TS80I

S458A + – +

4 4

<0.03 <0.03

76.8 blaTEM-1b, rmtB A56TS80I Farm 1

Intestinal tract Blood Blood

128

16

>512

blaCTX-M-55 blaTEM-1b, aac(60 )-Ib -cr, oqxAB, rmtB

S83L D87N

parC gyrA AMK CTX CIP

1238

Tissue source Farm no. Strain

Table 2 (Continued)

MIC (mg/ml)

ESBL gene (s)

Other resistant gene(s)

QRDR mutations

parE

+

4

<0.03

Size (Kb) Co-transferred resistant gene(s)

Plasmid transfer

MIC of transconjungant-s to FQs (mg/ml) NALa CIPb

Information of CTX-M-positive plasmids

F,K,P

L. Yang et al. / Veterinary Microbiology 178 (2015) 238–245 Replicon type(s)

242

M and rmtB gene; 4 co-harboured blaCTX-M and PMQR gene (3 blaCTX-M + aac(60 )-Ib-cr and 1 blaCTX-M + aac(60 )-Ib-cr/oqxAB) (Table 2). The plasmids present in the transconjugants were assigned to the following incompatibility groups: IncF, IncK, IncFIA, IncFIB, IncP and IncN. All transconjugants contained the IncF plasmid. 92.6% (25/27) transconjugants showed existence of at least two incompatibility groups, and the combination of IncF and IncK was the most frequent (Table 2). Southern blot hybridization revealed that the plasmid on which blaCTX-M genes were located could be separated into three groups with different sizes: 76.8 Kb (20), 194 Kb (5) and 104.5 Kb (2) (Table 2).

4. Discussion Frequent and improper application of antimicrobials as treatment or feed additives in animals, resulting in high antimicrobial resistance, is becoming a serious concern in China. Many studies have been performed to investigate the antimicrobial resistance of E. coli isolated from various kinds of food-producing animals, such as pigs, poultry and fish (Jiang et al., 2012; Jiang et al., 2011 Zheng et al., 2012), however, antimicrobial resistance profiles of E. coli from pigeons are still unknown. As China’s largest producer of pigeon for food, and export, understanding the incidence and composition of the antibiotic resistance reservoir in pigeons in Guangdong, China, is of significance. Seventy-one E. coli isolates were multidrug-resistant strains and most (66.2%) of them were resistant to the third-generation cephaloporins, fluoroquinolones and aminoglycosides tested in this study. Most of the third-generation cephaloporins- and fluoroquinolones-resistant strains were from Farm 1. In pigeon farms of China, ciprofloxacin or norfloxacin were usually added in drinking water or feed to prevent paratyphoid fever, and intramuscular injection of ciprofloxacin would be administered to treat diseased pigeons; cefotaxime, ceftiofur and ceftriaxone would be used to treat pigeon colibacillosis. 90.1% resistance rates of E. coli to ciprofloxacin and enrofloxacin reported here is higher than previously reported findings in other food-producing animals in China, where 78% of E. coli from pigs (healthy or diseased) were resistant to ciprofloxacin (Liu et al., 2013), 73.6% and 69.5% of E. coli from diseased food-producing animals (pigs and poultry) were resistant to ciprofloxacin and enrofloxacin respectively (Jiang et al., 2011), and only 4.1% of E. coli from farmed fish showed resistance to ciprofloxacin (Jiang et al., 2012). Resistance rates to the third generation cephalosporins (especially ceftotaxime, 53.5%) and amikacin (42.3%) were also higher than that reported previous studies mentioned before. Our results may indicate that these important clinical antimicrobial agents were used more frequently in pigeon farms in China. Until now, blaCTX-M was the most predominant ESBL-encoding gene among Enterobacteriaceae isolated from both animals and humans worldwide (Canton et al., 2012; D’Andrea et al., 2013). In this study, 97.6% (41/42) of the third-generation- cephalosporinresistant isolates contained blaCTX-M and seven different CTX-M types were detected, indicating a high diversity of CTX-Mencoding genes in E. coli isolates from pigeons in China. A much lower frequency of CTX-M-type ESBLs was observed in E. coli from farmed animals in 2003–2005 (2.4%) (Liu et al., 2007), food animals (pigs, cattle and poultry) in 2007–2009 (12.4%) (Zheng et al., 2012) and farmed fish in 2010 (1.5%) (Jiang et al., 2012) in China. Among the detected CTX-M-type genes, blaCTX-M-65 and blaCTX-M-27 were the two most dominant in the present study, which is different from previous reports that CTX-M-14 and CTX-M-55 enzymes were the two most frequent types detected in all animal species from different regions in China (Sun et al., 2010 Zheng et al., 2012). Consistent with the results of this study, all these blaCTX-M variants

[(Fig._1)TD$IG]

L. Yang et al. / Veterinary Microbiology 178 (2015) 238–245

243

Fig. 1. Pulsed-field gel electrophoresis fingerprinting patterns of Xbal-digested total DNA preparations from Escherichia coli isolates harboring CTX-M-encoding genes.

had been detected recently in E. coli strains among food animals and humans in China (Jiang et al., 2012; Li et al., 2011 Zheng et al., 2012). Interestingly, CTX-M-13 which was first identified in one Klebsiella pneumoniae strain from a patient in the First Municipal People’s Hospital of Guangzhou in China, between 1997 and 1998 (Chanawong et al., 2002), was detected in one E. coli isolate. This gene was once detected as the most predominant ESBL gene in Proteus mirabilis isolates from humans in Hong Kong (Ho et al.,

2005). To the best of our knowledge, the current study is the first report about CTX-M-13 existence in E. coli from food-producing animals. High fluoroquinolone-resistance rates were observed in blaCTXM-positive isolates. As descried before, high resistance levels to quinolones are mostly attributable to point mutations in the QRDR of the target genes gyrA, gyrB, parC, parE (Shaheen et al., 2013). In this study, mutations occurred in both gyrA (at position 83 and 87)

244

L. Yang et al. / Veterinary Microbiology 178 (2015) 238–245

and parC (at position 80 and/or 84 or 56) of all high-level ciprofloxacin- or enrofloxacin-resistant strains, which was in accordance with the report that high level fluoroquinolone resistance requires double mutations in gyrA at position 83 and 87 and at least one parC mutation at either position 80 or 84 (Shaheen et al., 2013). Similar mutations were reported in broadspectrum-cephalosporin-resistant E. coli isolates recovered from companion animals in the USA (Shaheen et al., 2013). Although PMQR genes are supplementary mechanisms that produce low levels of fluoroquinolone resistance, they can present a higher level resistance by interacting with genomic determinants (Jiang et al., 2012). The present study demonstrated high prevalence of aac(60 )Ib-cr (78.0%) and oqxAB (51.2%) among blaCTX-M-positive E. coli isolates from pigeons. This is different from previous studies that reported qnrB and qnrS were the most prevalent PMQR genes in E. coli from farmed fish in China (Jiang et al., 2012) and qnrS and oqxAB were the most prevalent PMQR genes in E. coli from pigs in China (Liu et al., 2013). However, our findings are similar to recent reports that aac(60 )-Ib-cr and oqxAB were the most prevalent PMQR genes in the E. coli isolates from poultry in China (Chen et al., 2012). The co-prevalence rate of ESBL-PMQR genes was higher than that (15.9%) in E. coli isolated from pigs in China (Liu et al., 2013), and also higher than that (45%) in E. coli isolated from zoo animals (Wang et al., 2012). Plasmid-mediated 16S rRNA methylases, first detected in K. pneumoniae isolated from a patient in 2003, confers high level resistance to various clinically important aminoglycosides (Yang et al., 2011). According to previous studies, rmtB appears to be the most widespread and has been detected primarily in Asia and Europe. In 2007, the emergence of rmtB was first reported in Enterobacteriaceae isolated from pigs in China (Chen et al., 2007). After that, several studies have reported the presence of 16S rRNA methylase genes in Enterobacteriaceae isolated from food-producing animals (Deng et al., 2011; Du et al., 2009). rmtB was detected in 58.5% CTX-M-producing isolates in this study, demonstrating a similar prevalence trend of 16S rRNA methylase genes in E. coli isolated from food-producing animals and humans (Du et al., 2009; Liu et al., 2013; Yang et al., 2011). An increasing number of studies have reported the presence of plasmids carrying b-lactamase genes (especially genes encoding CTX-M and TEM-1), PMQR determinant genes, and/or 16S rRNA methylases genes among E. coli from both animals and humans (Chen et al., 2012; Deng et al., 2011; He et al., 2013; Jiang et al., 2012). In this study, 27 blaCTX-M-positive transconjugants were obtained, 59.3% of which co-transferred blaCTX-M with qnr or aac (60 )-Ib-cr and/or oqxAB and rmtB, indicating that these different resistant determinants can be transfered horizontally along with the transfer of CTX-M-encoding genes. Hybridation of the blaCTX-Mprobe with plasmid in transconjugants revealed that blaCTX-M genes were located on three groups of plasmids with different sizes. Most of the transcojugants carried the IncF replicon alone or in combination with IncK replicons, suggesting the horizontal dissemination of blaCTX-M genes by a IncF or IncK conjugative plasmid. It was reported that CTX-M-encoding genes were always associated with IncF plasmids varying in size from 50-200Kb (Carattoli, 2009 Zheng et al., 2012). Typical examples are the pandemic dissemination of CTX-M-15 in E. coli isolated from humans favored by IncFII plasmids(Coque et al., 2008) and the dissemination of CTX-M-65 in E. coli isolated from animals in China contributed by F33:A-:B-type plasmids (He et al., 2013). Previous studies also demonstrated the rapid dissemination of genetically unrelated E. coli producing CTX-M-14, and the diffusion of the blaCTX-M-14 gene were related to the spread of a common plasmid in the IncK group (Stokes et al., 2012). In addition, 12 (44.4%) and 9 (33.3%) of 27 transconjugants were blaCTX-65- and blaCTX-M-55positive, respectively. This may explain the predominant

prevalence of these two genes in E. coli isolated from pigeons in China and also indicates their significance in producing multidrug resistance in those bacteria. Moreover, strains grouped into different PFGE patterns harboring the same CTX-M-type gene (especially among strains harboring blaCTX-M-65 and among strains harboring blaCTX-M-55), indicating that in horizontal plasmids carrying blaCTX-M genes dissemination was occurring in addition to the dissemination of strains. In conclusion, high percentages of resistance to ciprofloxacin, cefotaxime or cefoxitin and amikacin, were detected in E. coli isolated from pigeons in China. As plasmid-associated resistance elements, genes encoding CTX-M exhibited a strong association with PMQR determinants and RmtB conferring multidrug resistant phenotypes, which indicates that E. coli isolates of pigeons could serve as a reservoir of multiple antimicrobial resistance genes. Horizontal spread of blaCTX-M genes along with other clinical relevant resistant genes indicated the high risk of CTX-M harboring E. coli strains among farmed pigeons, and more attention should be paid to antimicrobial resistance in E. coli from these overlooked food-producing animals. Acknowledgments This work was supported in-part by the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT13063) and the Natural Science Foundation of Guangdong Province (No. S2012030006590). References Barton, B.M., Harding, G.P., Zuccarelli, J., 1995. A general method for detecting and sizing large plasmids. Anal. Biochem. 226, 235–240. Bush, K., Jacoby, G.A., 2010. Updated functional classification of b-lactamases. Antimicrob. Agents Chemother. 54, 969–976. Bush, K., 2010. Alarming b-lactamase-mediated resistance in multidrug-resistant Enterobacteriaceae. Curr. Opin. Microbiol. 13, 558–564. Canton, R., Ruiz-Garbajosa, P., 2011. Co-resistance: an opportunity for the bacteria and resistance genes. Curr. Opin. Pharmacol. 11, 477–485. Canton, R., Coque, T.M., Baquero, F., 2003. Multi-resistant Gram-negative bacilli: from epidemics to endemics. Curr. Opin. Infect. Dis. 16, 315–325. Canton, R., Jose, M.G.A., Galan, J.C., 2012. CTX-M enzymes: origin and diffusion. Front. Microbiol. 3, 1–19. Carattoli, A., Bertini, A., Villa, L., Falbo, V., Hopkins, K.L., Threlfall, E.J., 2005. Identification of plasmids by PCR-based replicon typing. J. Microbiol. Methods 63, 219–228. Carattoli, A., 2009. Resistance plasmid families in Enterobacteriaceae. Antimicrob. Agents Chemother. 53, 2227–2238. Chanawong, A., M’Zali, F.H., Heritage, J., Xiong, J.H., Hawkey, P.M., 2002. Three cefotaximases, CTX-M-9, CTX-M-13, and CTX-M-14, among Enterobacteriaceae in the People’s Republic of China. Antimicrob. Agents Chemother. 46, 630–637. Chen, L., Chen, Z.L., Liu, J.H., Zeng, Z.L., Ma, J.Y., Jiang, H.X., 2007. Emergence of RmtB methylase-producing Escherichia coli and Enterobacter cloacae isolates from pigs in China. J. Antimicrob. Chemother. 59, 880–885. Chen, X., Zhang, W., Pan, W., Yin, J., Pan, Z., Gao, S., Jiao, X., 2012. Prevalence of qnr, aac(60 )-Ib-cr, qepA, and oqxAB in Escherichia coli isolates from humans, animals, and the environment. Antimicrob. Agents Chemother. 56, 3423–3427. Clarke, C.R., 2006. Antimicrobial resistance. Vet. Clin. North Am. Small Anim. Pract. 36, 987–1001. Clinical and Laboratory Standards Institute, 2013. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated From Animals; Approved Standard, fourth edition and supplement. Documents VET 01-A4 and VET01-S2. CLSI, Wayne, Pennsylvania, USA. Clinical and Laboratory Standards Institute, 2015. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-fifth Informational Supplement. CLSI document M100-S25. CLSI, Wayne, Pennsylvania, USA. Coque, T.M., Novais, A., Carattoli, A., Poirel, L., Pitout, J., Peixe, L., Baquero, F., Canton, R., Nordmann, P., 2008. Dissemination of clonally related Escherichia coli strains expressing extended-spectrum b-Lactamase CTX-M-15. Emerg. Infect. Dis. 14, 195–200. D’Andrea, M.M., Arena, F., Pallecchi, L., Rossolini, G.M., 2013. CTX-M-type b-lactamases: a successful story of antibiotic resistance. Int. J. Med. Microbiol. 303, 305–317. DANMAP, 1999. 98-consumption of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from food animals, food and human in Denmark. Statens Serum Institut, Danish Veterinary and Food Adiministration. Danish Medicines Agency, Danish Veterinary Laboratary, Copenhagen, Denmark.

L. Yang et al. / Veterinary Microbiology 178 (2015) 238–245 Deng, Y., He, L., Chen, S., Zheng, H., Zeng, Z., Liu, Y., Sun, Y., Ma, J., Chen, Z., Liu, J.-H., 2011. F33: A-: B- and F2: A-: B- plasmids mediate dissemination of rmtB-blaCTXM-9 group genes and rmtB-qepA in Enterobacteriaceae isolates from pets in China. Antimicrob. Agents Chemother. 55, 4926–4929. Doi, Y., Arakawa, Y., 2007. 16S ribosomal RNA methylation: emerging resistance mechanism against aminoglycosides. Clin. Infect. Dis. 45, 88–94. Du, X.D., Wu, C.M., Liu, H.B., Li, X.S., Beier, R.C., Xiao, F., Qin, S.S., Huang, S.Y., Shen, J. Z., 2009. Plasmid-mediated ArmA and RmtB 16S rRNA methylases in Escherichia coli isolated from chickens. J. Antimicrob. Chemother. 64, 1328–1330. He, L., Partridge, S.R., Yang, X., Hou, J., Deng, Y., Yao, Q., Zeng, Z., Chen, Z., Liu, J.H., 2013. Complete nucleotide sequence of pHN7A8, an F33:A- :B- type epidemic plasmid carrying blaCTX-M-65, fosA3 and rmtB from China. J. Antimicrob. Chemother. doi:http://dx.doi.org/10.1093/jac/dks369. Ho, P.L., Ho, A.Y., Chow, K.H., Wong, R.C., Duan, R.S., Ho, W.L., Mak, G.C., Tsang, K.W., Yam, W.C., Yuen, K.Y., 2005. Occurrence and molecular analysis of extendedspectrum b-lactamase-producing Proteus mirabilis in Hong Kong, 1999–2002. J. Antimicrob. Chemother. 55, 840–845. Jiang, H.X., Lu, D.H., Chen, Z.L., Wang, X.M., Chen, J.R., Liu, Y.H., Liao, X.P., Liu, J.H., Zeng, Z.L., 2011. High prevalence and widespread distribution of multi-resistant Escherichia coli isolates in pigs and poultry in China. Vet. J. 187, 99–103. Jiang, H.-X., Tang, D., Liu, Y.-H., Zhang, X.-H., Zeng, Z.-L., Xu, L., Hawkey, P.M., 2012. Prevalence and characteristics of b-lactamase and plasmid-mediated quinolone resistance genes in Escherichia coli isolated from farmed fish in China. J. Antimicrob. Chemother. 67, 2350–2353. Jiang, H.X., Song, L., Liu, J., Zhang, X.H., Ren, Y.N., Zhang, W.H., Zhang, J.Y., Liu, Y.H., Webber, M.A., Ogbolu, D.O., Zeng, Z.L., Piddock, L.J., 2014. Multiple transmissible genes encoding fluoroquinolone and third-generation cephalosporin resistance co-located in non-typhoidal Salmonella isolated from food-producing animals in China. Int. J. Antimicrob. Agents 43, 242–247. Li, B., Sun, J.-Y., Liu, Q.-Z., Han, L.-Z., Huang, X.-H., Ni, Y.-X., 2011. High prevalence of CTX-M b-lactamases in faecal Escherichia coli strains from healthy humans in Fuzhou. China Scand. J. Infect. Dis. 43, 170–174. Liu, J.H., Wei, S.Y., Ma, J.Y., Zeng, Z.L., Lu, D.H., Yang, G.X., Chen, Z.L., 2007. Detection and characterisation of CTX-M and CMY-2 b-lactamases among Escherichia coli isolates from farm animals in Guangdong Province of China. Int. J. Antimicrob. Agents 29, 576–581. Liu, B.T., Liao, X.P., Yue, L., Chen, X.Y., Li, L., Yang, S.S., Sun, J., Zhang, S., Liao, S.D., Liu, Y. H., 2013. Prevalence of b-lactamase and 16S rRNA methylase genes among clinical Escherichia coli isolates carrying plasmid-mediated quinolone resistance genes from animals. Microb. Drug Resist. 19, 237–245. Ma, J., Liu, J.H., Lv, L., Zong, Z., Sun, Y., Zheng, H., Chen, Z., Zeng, Z.L., 2012. Characterization of extended-spectrum b-lactamase genes found among Escherichia coli isolates from duck and environmental samples obtained on a duck farm. Appl. Environ. Microbiol. 78, 3668–3673.

245

Maron, D.F., Smith, T.J., Nachman, K.E., 2013. Restrictions on antimicrobial use in food animal production: an international regulatory and economic survey. Global Health 9 doi:http://dx.doi.org/10.1186/1744-8603-9-4. Molbak, K., 2004. Spread of resistant bacteria and resistance genes from animals to humans—the public health consequences. J. Vet. Med. 51, 364–369. Pitout, J.D.D., Laupland, K.B., 2008. Extended-spectrum b-lactamase-producing Enterobacteriaceae: an emerging public-health concern. Lancet Infect. Dis. 8, 159–166. Shaheen, B.W., Nayak, R., Foley, S.L., Boothe, D.M., 2013. Chromosomal and plasmidmediated fluoroquinolone resistance mechanisms among broad-spectrumcephalosporin-resistant Escherichia coli isolates recovered from companion animals in the USA. J. Antimicrob. Chemother. 68, 1019–1024. Stokes, M.O., Cottell, J.L., Piddock, L.J., Wu, G., Wootton, M., Mevius, D.J., Randall, L.P., Teale, C.J., Fielder, M.D., Coldham, N.G., 2012. Detection and characterization of pCT-like plasmid vectors for blaCTX-M-14 in Escherichia coli isolates from humans, turkeys and cattle in England and Wales. J. Antimicrob. Chemother. 67, 1639–1644. Sun, Y., Zeng, Z., Chen, S., May, J., He, L., Liu, Y., Deng, Y., Lei, T., Zhao, J., Liu, J.H., 2010. High prevalence of blaCTX-M extended-spectrum b-lactamase genes in Escherichia coli isolates from pets and emergence of CTX-M-64 in China. Clin. Microbiol. Infect. 16, 1475–1481. Tenover, F.C., Arbeit, R.D., Goering, R.V., Mickelsen, P.A., Murray, B.E., Persing, D.H., Swaminathan, B., 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis criteria for bacterial strain typing. J. Clin. Microbiol. 33, 2233–2239. Van den Bogaard, A.E., Stobberingh, E.E., 2000. Epidemiology of resistance to antibiotics: links between animals and humans. Int. J. Antimicrob. Agents 14, 327–335. Wang, Y., He, T., Han, J., Wang, J., Foley, S.L., Yang, G., Wan, S., Shen, J., Wu, C., 2012. Prevalence of ESBLs and PMQR genes in fecal Escherichia coli isolated from the non-human primates in six zoos in China. Microb. Drug Resist. 159, 53–59. Woodford, N., Turton, J.F., Livermore, D.M., 2011. Multiresistant Gram-negative bacteria: the role of high-risk clones in the dissemination of antibiotic resistance. FEMS Microbiol. Rev. 35, 736–755. Yang, J., Ye, L., Wang, W., Luo, Y., Zhang, Y., Han, L., 2011. Diverse prevalence of 16S rRNA methylase genes armA and rmtB amongst clinical multidrug-resistant Escherichia coli and Klebsiella pneumoniae isolates. Int. J. Antimicrob. Agents 38, 348–351. Zheng, H., Zeng, Z., Chen, S., Liu, Y., Yao, Q., Deng, Y., Chen, X., Lv, L., Zhuo, C., Chen, Z., Liu, J.H., 2012. Prevalence and characterisation of CTX-M b-lactamases amongst Escherichia coli isolates from healthy food animals in China. Int. J. Antimicrob. Agents 39, 305–310.