High Prevalence of Plasmid-Mediated Quinolone Resistance Determinants in Escherichia coli and Klebsiella pneumoniae Isolates from Pediatric Patients in China

Abstract

In this study, the prevalence of the plasmid-mediated quinolone resistance (PMQR) genes among pediatric clinical isolates of Escherichia coli and Klebsiella pneumoniae was investigated. A total of 243 nonduplicate clinical isolates of E. coli and K. pneumoniae collected between January 2010 and December 2012 from Beijing, China, were studied. In total, 55 isolates (22.63%) were positive for PMQR genes, the most frequently detected gene was qnrS (13.2%), followed by aac(6′)-Ib-cr (6.2%) and qnrB (3.7%). The qnrA and qepA genes were not detected. Furthermore, 92.73% (51/55) produced extended-spectrum β-lactamases (ESBLs) and 21.82% (12/55) were resistant to quinolones. DNA sequencing results showed that 14.55% (8/55) of isolates possessed gyrA mutations, while 1.82% (1/55) had parC mutations in the quinolone resistance-determining region (QRDR). Nalidixic acid or ciprofloxacin resistance of the transconjugants increased from 2- to 32-fold. Enterobacterial repetitive intergenic consensus sequence polymerase chain reaction typing indicated that most isolates were not clonally related. Our findings showed that the PMQR detection rate among pediatric clinical isolates of E. coli and K. pneumoniae was high in China. PMQR-positive strains were more common among ESBL-producing and ciprofloxacin-susceptible isolates. Conjugation experiments showed that these isolates could be transferred horizontally. The present study highlights the high prevalence of PMQR in Chinese pediatrics who are not treated with quinolones.

Introduction

Quinolones and fluoroquinolones are broad-spectrum antimicrobial agents used extensively in humans, and the rates of resistance to these agents have been increasing worldwide over the past few years.¹ A well-established mechanism of resistance to quinolones is chromosomal mutation in the quinolone resistance-determining region (QRDR) containing the DNA gyrase (gyrA, gyrB) and topoisomerase IV (parC, parE) encoding genes.² Since 1998, a plasmid-mediated quinolone resistance (PMQR) mechanism has been reported and three types of PMQR mechanisms have been described that include the topoisomerase protection proteins (qnrA, qnrB, qnrS),³ the acetylation enzyme variant (aac(6′)-Ib-cr),⁴ and the multidrug resistance efflux pump (qepA).⁵ The function of the qnr proteins that confer quinolone resistance is to protect the gyrase proteins; the aac(6′)-Ib-cr protein is an acetyltransferase that modifies the quinolones, and the qepA protein is an active efflux pump.⁶ The PMQR mechanisms usually mediate lower level resistance below the susceptible minimal inhibitory concentration (MIC) breakpoints and can be transferred horizontally.⁷

In China, a recent study reported that the ciprofloxacin resistance rate of community-onset Escherichia coli in 30 county hospitals was 51.2%, and at least one PMQR genes was carried by 37.3% isolates.⁸ As a result of their side effects, quinolones are restricted for use in children. However, there have also been a few reports on PMQR strains isolated from pediatric patients.^9–11 Although the PMQR is not capable of conferring resistance clinically, the prevalence of this genetic region is of great concern because PMQR genes are reportedly located on the same plasmid as the extended-spectrum β-lactamase (ESBL) genes.¹¹ β-lactam is the main antibiotic used in the treatment of bacterial infections, so there is concern that the horizontal transmission of PMQR will concurrently lead to more ESBL resistance. Therefore, the objective of this study was to characterize quinolones and fluoroquinolone resistance (PMQR genes and QRDR region) in Enterobacteriaceae from pediatric isolates in China. In addition, the identification of ESBL genes was carried out, as well as the transfer of quinolone resistance genes.

Materials and Methods

Ethics statement

This study was performed in compliance with the Helsinki Declaration (Ethical Principles for Medical Research Involving Human Subjects) and was approved by the research board of the Ethics Committee of the Capital Institute of Pediatrics, Beijing, China. All specimens used in this study are part of routine patient management without any additional collection, and all patient data were anonymously reported. Based on the guidelines of the Ethics Committee of the Capital Institute of Pediatrics, there is no consent given in this study.

Bacterial strains

In total, 131 Klebsiella pneumoniae and 112 E. coli nonduplicate clinical isolates recovered from sputum (n = 183), feces (n = 10), urine (n = 42), blood cultures (n = 3), and cerebrospinal fluid (n = 5) were collected from the Affiliated Children's Hospital of the Capital Institute of Pediatrics in Beijing from January 2010 to December 2012. Among these, 143 (68 K. pneumoniae and 75 E. coli) were ESBL producers and 100 (63 K. pneumoniae and 37 E. coli) were non-ESBL producers. All isolates were identified using the Gram-negative card with the automated Vitek Compact system (BioMérieux) and K. pneumoniae species were verified by polymerase chain reaction (PCR).¹² Clinical and epidemiological data were collected.

Antimicrobial susceptibility testing

The MIC of quinolones (nalidixic acid) and fluoroquinolones (ciprofloxacin, Levofloxacin) were determined using the broth microdilution method according to the guidelines issued by the CLSI. Susceptibility to other antimicrobial agents (imipenem, amikacin, gentamicin, amoxicillin/clavulanic acid, ceftazidime, cefotaxime, cefepime, and aztreonam) was tested using the AST GN13 card for the Vitek Compact system (BioMérieux). E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality control strains. All of the tests were performed according to the CLSI 2013.¹³

Detection of resistance genes

Total DNA was extracted by boiling. All isolates collected were screened for the presence of the PMQR genes, including qnrA, qnrB, qnrS, qepA, and aac(6′)-Ib-cr, and point mutations in the QRDR genes gyrA and parC from the isolates displaying ciprofloxacin resistance were determined by PCR and DNA sequencing as described previously.¹⁴ ESBL genes were determined by PCR using previously reported primers, including blaTEM, blaSHV, and blaCTX-M.^15,16 All PCR products were directly sequenced and the sequences were compared with those in the GenBank nucleotide database (www.ncbi.nlm.nih.gov/blast/).

Conjugation experiments

Conjugation experiments were carried out in LB broth using E. coli strain J53AzR as the recipient as previously described.¹⁷ All PMQR-positive isolates were used for the conjugation experiments. Transconjugants were selected on trypticase soy agar plates containing sodium azide (150 μg/ml) and ciprofloxacin (0.03 μg/ml) for selection of plasmid-mediated quinolone resistance. The MIC of quinolones for the transconjugants were measured by the broth microdilution method, and the PMQR genes in the transconjugants were detected by PCR.

Strain typing by Enterobacterial repetitive intergenic consensus sequence PCR analysis

All PMQR determinant-positive isolates were analyzed by Enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) using the ERIC1R (5′-ATGTAAGCTCCTGGGGATTCA-3′) and ERIC2 (5′-AAGTAAGTGACTGGGGTGAGCG-3′) primers as previously described.¹⁸ The samples were amplified as follows: 95°C for 7 min to denature the template; then 40 cycles of 95°C for 1 min, 52°C for 1 min, and 72°C for 4 min; and finally, 72°C for 10 min.

Results

Prevalence of PMQR genes

Among the 243 clinical isolates, 55 (22.63%), including 45 K. pneumoniae isolates and 10 E. coli isolates, were positive for PMQR. Of these, 51 (92.73%, 51/55) isolates produced ESBLs (41 K. pneumoniae and 10 E. coli). The PMQR detection rate among ESBL isolates was 35.66% (51/143), the other four isolates (4%, 4/100) were non-ESBL expressing isolates (χ² = 33.7, p < 0.05). QnrB, qnrS, and aac(6′)-Ib-cr were detected alone or in combination in 9 (3.7%, 9/243), 32 (13.2%, 32/243), and 15 (6.2%, 15/243) isolates, respectively. The genes qnrA and qepA were not detected. Seven isolates were positive for two genes: two isolates carried qnrB and aac(6′)-Ib-cr, one isolate carried qnrB and aac(6′)-Ib, two isolates carried qnrS and aac(6′)-Ib-cr, and two isolates carried qnrB and qnrS (Table 1).

Table 1.

Main Features of PMQR-Positive Klebsiella pneumoniae and Escherichia coli Isolate

								Minimal inhibitory concentration
												QRDR
Isolates	Service	Sample	Species	Diagnosis	Age	PMQR determinant	β-lactamases	Resistance phenotype (IMP, AMK, GEN, AMC, CAZ, CTX, FEP, ATM)	MIC-NAL	MIC-CIP	MIC-LEX	gyrA	parC
N1044	Surgery	Urine	K. pneumoniae	UTI	1m6d	qnrS1	SSSSSRRR	CTX-M-9G. SHV-1	6	0.125	0.25	wt	wt
N1047	Surgery	Urine	K. pneumoniae	UTI	1m4d	qnrS1	SSSSSRRS	CTX-M-1G, SHV-1	4	0.25	0.5	wt	wt
T4617	Neonatology	Sputum	K. pneumoniae	Pneumonia	1d	qnrS1	SSRSSRSR	CTX-M-1G, CTX-M-9G, TEM-1, SHV-1	12	0.125	0.25	wt	wt
T4611	Neonatology	Sputum	K. pneumoniae	Pneumonia	3m18d	qnrS1	SSRSSRSS	CTX-M-9G	8	0.25	0.5	wt	wt
N1054	Hematology	Urine	K. pneumoniae	UTI	1y10m	qnrS1, aac(6′)-Ib-cr	SSRRRRRR	CTX-M-1G, TEM-1	4	0.25	0.5	wt	wt
N1061	Neonatology	Urine	K. pneumoniae	UTI	1m29d	qnrS1	SSRRRRRR	CTX-M- 1G, CTX-M-9G, SHV-1	6	0.25	0.25	wt	wt
T4263	Neonatology	Sputum	K. pneumoniae	Pneumonia	2m29d	qnrS1	SSRRRRRR	CTX-M-1G, TEM-1, SHV-11	3	0.25	0.25	wt	wt
T4338	ICU	Sputum	K. pneumoniae	Pneumonia	2d	qnrS1	SSRRRRRR	CTX-M-1G, TEM-1, SHV-1	12	0.25	0.5	wt	wt
T4359	ICU	Sputum	K. pneumoniae	Pneumonia	8d	qnrS1	SSRRRRRR	CTX-M-1G, CTX-M-9G, TEM-1, SHV-1	8	0.25	0.5	wt	wt
N1007	ICU	Sputum	K. pneumoniae	Pneumonia	2m10d	qnrS1	SSRRRRRS	CTX-M-1G, CTX-M-9G, TEM-1, SHV-2	4	0.25	0.5	wt	wt
T3811	ICU	Sputum	K. pneumoniae	Pneumonia	1m1d	qnrS1	SSSSSRRS	CTX-M-9G, SHV-1	6	1	0.5	wt	wt
T4577	Neonatology	Sputum	K. pneumoniae	Pneumonia	3m15d	qnrB4	SSSRRRRS	CTX-M-1G, TEM-1, SHV-11	4	0.25	0.25	wt	wt
T4550	Rheumatism immunity	Sputum	K. pneumoniae	Pneumonia	4m3d	qnrS1	SSRSSRSS	CTX-M-9G, SHV-1	8	0.125	0.5	wt	wt
N1010	ICU	Sputum	K. pneumoniae	Pneumonia	6y0m8d	qnrS1, aac(6′)-Ib-cr	SSRRRRRS	CTX-M-9G, TEM-1	4	0.03	0.06	wt	wt
T4488	Neonatology	Sputum	K. pneumoniae	Pneumonia	1m1d	aac(6′)-Ib	SSSSSRRR	CTX-M-9G	3	0.06	0.125	wt	wt
T4339	Neonatology	Sputum	K. pneumoniae	Pneumonia	20d	aac(6′)-Ib	SSSSSRRR	CTX-M-9G	16	0.03	0.125	wt	wt
T4413	ICU	Sputum	K. pneumoniae	Pneumonia	12d	qnrS1	SSRRRRRR	CTX-M-9G, TEM-1, SHV-1	12	0.25	0.5	wt	wt
T3540	Neonatology	Sputum	K. pneumoniae	Pneumonia	23d	qnrS1	SSRRRRRR	CTX-M-1G, TEM-1, SHV-11	12	0.25	0.5	wt	wt
T3521	Respiratory	Sputum	K. pneumoniae	Pneumonia	3m3d	aac(6′)-Ib	SSRRRRRS	CTX-M-1G, TEM-1	8	0.125	0.125	wt	wt
T4415	ICU	Sputum	K. pneumoniae	Pneumonia	13d	qnrS1	SSRRRRRR	CTX-M-1G, TEM-1, SHV-1	12	0.25	0.5	wt	wt
N994	Surgery	Urine	K. pneumoniae	UTI	14y1m	aac(6′)-Ib	SSSRRRSR	CTX-M-1G, CTX-M-9G	8	0.016	0.06	wt	wt
N1030	Surgery	Urine	K. pneumoniae	UTI	6m13d	qnrS1	SSSSSSSR	ND	4	0.016	0.03	wt	wt
T3635	Neonatology	Sputum	K. pneumoniae	Pneumonia	2m11d	qnrB4	SRRRRRSR	TEM-1, SHV-12	24	0.25	0.5	wt	wt
T3950	ICU	Sputum	K. pneumoniae	Pneumonia	1m2d	qnrS1	SSSRRRSS	CTX-M-1G, TEM-1	8	0.25	0.5	wt	wt
T4044	ICU	Sputum	K. pneumoniae	Pneumonia	1m9d	qnrB4, aac(6′)-Ib-cr	SSRRRRRR	CTX-M-1G, TEM-1	12	0.25	0.5	wt	wt
T4097	ICU	Sputum	K. pneumoniae	Pneumonia	1m17d	qnrS1	SSSSSRRS	CTX-M-9G, SHV-2	32	0.25	0.25	wt	wt
T3798	ICU	Sputum	K. pneumoniae	Pneumonia	3m8d	aac(6′)-Ib-cr	SSSSSSSS	ND	16	1	0.5	wt	wt
T4414	ICU	Sputum	K. pneumoniae	Pneumonia	3m10d	qnrS1	SSSSSSSS	ND	8	0.25	0.5	wt	wt
T20891	Neonatology	Sputum	K. pneumoniae	Pneumonia	19d	qnrS1	SSRRRRRR	CTX-M-1G, TEM-1, SHV-1	8	0.25	0.5	wt	wt
T20595	ICU	Sputum	K. pneumoniae	Capillary bronchitis	2m1d	qnrS1	SSRSRRRR	CTX-M-1G, TEM-1, SHV-28	8	0.25	0.5	wt	wt
T14250	Respiratory	Sputum	K. pneumoniae	Capillary bronchitis	2y10m	qnrB1, aac(6′)-Ib-cr	SSRRRRRR	TEM-1	4	0.016	0.06	wt	wt
T13828	ICU	Sputum	K. pneumoniae	Pneumonia	1m9d	qnrS1	SSRSSRRS	CTX-M-9G, SHV-11	12	0.125	0.5	wt	wt
T20500	ICU	Sputum	K. pneumoniae	Capillary bronchitis	11m15d	aac(6′)-Ib	SSRRRRRR	TEM-1, SHV-36	4	0.06	0.06	wt	wt
T20400	Neonatology	Sputum	K. pneumoniae	Capillary bronchitis	1m12d	qnrS1	SSRSSRRS	CTX-M-9G, SHV-1	4	0.25	0.5	wt	wt
T20502	Neonatology	Sputum	K. pneumoniae	Pneumonia	10d	aac(6′)-Ib-cr	SSSRRRSS	TEM-1	4	0.03	0.06	wt	wt
T20096	ICU	Sputum	K. pneumoniae	Capillary bronchitis	2m9d	qnrB4, qnrS1	SSRRSRRS	CTX-M-9G, SHV-1	16	0.25	0.5	wt	wt
T20844	ICU	Sputum	K. pneumoniae	Capillary bronchitis	21d	qnrS1	SSRSRRRR	CTX-M-9G, TEM-1, SHV-28	128	0.25	0.5	wt	wt
T14058	Neonatology	Sputum	K. pneumoniae	Pneumonia	1m28d	qnrS1	SSSSSSSS	ND	8	0.25	0.5	wt	wt
T14305	Hematology	Sputum	K. pneumoniae	Septicemia	3y1m	qnrB4	SSSRRRRR	CTX-M-1G, TEM-1, SHV-36	8	0.25	0.25	wt	wt
T14210	Hematology	Sputum	K. pneumoniae	Pneumonia	1m1d	qnrB4	SSSRRRRR	CTX-M-1G, TEM-1, SHV-36	>256	64	32	wt	Ser80Ile
T13041	Respiratory	Sputum	K. pneumoniae	Pneumonia	1y8m	qnrS1	SSRRSRRR	CTX-M-1G, TEM-1, SHV-1	4	0.125	0.5	wt	wt
N10583	ICU	Urine	K. pneumoniae	Pneumonia	2m3d	qnrB4, qnrS1	SSRRRRRS	CTX-M-1G, TEM-1, SHV-12	16	0.5	1	wt	wt
N13068	ICU	Urine	K. pneumoniae	Pneumonia	1y6m	aac(6′)-Ib-cr	SSSSRRRR	CTX-M-1G, TEM-1, SHV-1	>256	32	32	Ser83Leu, Asp87Ala	wt
T13115	ICU	Sputum	K. pneumoniae	Pneumonia	2m24d	qnrB4, aac(6)-Ib	SSSSSRRS	CTX-M-1G, SHV-12	8	0.016	0.03	wt	wt
T13014	ICU	Sputum	K. pneumoniae	Pneumonia	1m13d	qnrS1	SSSRRRRS	CTX-M-1G, TEM-1	8	0.06	0.06	wt	wt
T3470	Respiratory	Sputum	E. coli	Meningitis	3m	aac(6′)-Ib-cr	SSRSRRRR	CTX-M-1G	>256	>64	>64	Ser83Leu, Asp87Asn	wt
N949	Respiratory	Urine	E. coli	UTI	1y3m	aac(6′)-Ib-cr	SSRRRRRR	CTX-M-1G, CTX-M-9G	>256	>64	>64	Ser83Leu, Asp87Asn	wt
N1024	Hematology	Urine	E. coli	UTI	6y0m24d	aac(6′)-Ib-cr	SSRRRRRR	CTX-M-1G	>256	>64	32	Ser83Leu, Asp87Asn	wt
T3931	Neonatology	Sputum	E. coli	Pneumonia	1m4d	qnrS1	SSRSRRRR	CTX-M-1G, TEM-1	>256	0.25	0.25	wt	wt
N952	Respiratory	Urine	E. coli	UTI	1y3m	aac(6′)-Ib-cr	SSRRRRRR	CTX-M-1G	>256	>64	16	Ser83Leu, Asp87Asn	wt
N981	Hematology	Urine	E. coli	UTI	2y1m	aac(6′)-Ib-cr	SSRRRRRR	CTX-M-1G	>256	>64	>64	Ser83Leu, Asp87Asn	wt
T14073	Neonatology	Sputum	E. coli	Pneumonia	20d	aac(6′)-Ib-cr	SSRSSRRR	CTX-M-1G	64	0.125	0.125	Ser83Leu	wt
N2022	ICU	Urine	E. coli	Urethritis	1m8d	aac(6′)-Ib-cr	SSSSRRRR	CTX-M-1G, TEM-1	>256	>64	>64	Ser83Leu, Asp87Asn	wt
NJ2055	Neonatology	CSF	E. coli	Septicemia	7d	aac(6′)-Ib-cr	SSSSSRSS	CTX-M-1G	128	0.06	0.25	wt	wt
T14229	ICU	Sputum	E. coli	Capillary bronchitis	3m16d	qnrS1	SSRSSRRS	CTX-M-1G, TEM-1	6	0.125	0.25	wt	wt

AMC, amoxicillin/clavulanic acid; AMK, amikacin; ATM, aztreonam; CAZ, ceftazidime; CIP, ciprofloxacin; CSF, cerebrospinal fluid; CTX-M-1G, CTX-M-1group; CTX-M-9G, CTX-M-9 group; CTX, cefotaxime; d, days; FEP, cefepime; GEN, gentamicin; IMP, imipenem; LEX, Levofloxacin; m, month; MIC, minimal inhibitory concentration; ND, not detected; PMQR, plasmid-mediated quinolone resistance; QRDR, quinolone resistance-determining region; UTI, urinary tract infection; y, year.

Mutations in the gyrA and parC genes in the QRDR

QRDR PCR products were sequenced on both strands along with the DNA sequences of the gyrA and parC genes for each of the PMQR-positive isolates, and the sequences were compared with the QRDR DNA sequences of E. coli and K. pneumoniae present in the GenBank database. Among the PMQR-positive strains, gyrA mutations were observed in 8/55 (14.55%) isolates, of which six isolates displayed both Ser83leu and Asp87Asn, one displayed Ser83leu and Asp87Ala, and one displayed Ser83leu. For the parC gene, only one mutation (1.82%, 1/55) was detected in one isolate, a Ser80Ile substitution. Detailed information on these PMQR-positive isolates is provided in Table 1.

Detailed analysis of the ESBL genotypes in PMQR-positive isolates

Among the 51 ESBL-positive strains, 47 isolates were positive for CTX-M gene (28 isolates belonged to the CTX-M-1 group, 13 belonged to the CTX-M-9 group, and 6 isolates belonged to both CTX-M-1 and CTX-M-9 group). Twenty-nine isolates were positive for SHV gene (15 for SHV-1 gene, 4 for SHV-11, 3 for SHV-36, 2 for SHV-28, 3 for SHV-12, and 2 for SHV-2). Thirty isolates were positive for TEM-1 gene (Table 1).

Antibiotic susceptibility

Among the 243 isolates, 51 (20.99%, 51/243) were resistant to nalidixic acid or ciprofloxacin or levofloxacin using the broth microdilution method. Of these, 13 (9.92%, 13/131) isolates were K. pneumoniae and 38 (33.93%, 38/112) were E. coli. Of the 55 PMQR-positive isolates, 12 (21.82%, 12/55) isolates were resistant to quinolones, 3 of which were K. pneumoniae and the other 9 were E. coli.

Among the 55 PMQR-positive isolates, most were resistant to cephalosporin, aztreonam, and aminoglycosides. The rates of resistance to ceftazidime, cefotaxime, and cefepime were 61.82% (34/55), 92.73% (51/55), and 78.18% (43/55), respectively. The rate of resistance to aztreonam was 60% (33/55). The rates of resistance to gentamicin and amoxicillin/clavulanic acid were 61.82% (34/55) and 54.55% (30/55), respectively. All of the strains were susceptible to imipenem, and only one isolate was resistant to amikacin (Table 1).

Conjugation and antimicrobial susceptibility

Of the 55 PMQR-positive isolates, 46 were used in a conjugation test (the other nine were excluded because they were naturally resistant to sodium azide at 150 μg/ml). The results showed that quinolone resistance could be transferred by conjugation in 28 of the 46 (60.87%) PMQR-positive donors. Five qnrB-positive, 19 qnrS-positive, and four aac(6′)-Ib-cr-positive transconjugants were obtained. PCR experiments confirmed that the transconjugants harbored the same PMQR genes as their donors. Antibiotic susceptibilities for the 28 transconjugants showed that the nalidixic acid or ciprofloxacin resistance MIC value increased 2- and 32-fold, respectively (Table 2).

Table 2.

The Ciprofloxacin Minimal Inhibitory Concentration Results of Transconjugants

		PMQR determinant		MIC-NAL		MIC-CIP
Isolates	Species	Donors	Conjugants	Donors	Conjugants	Donors	Conjugants
N1044	K. pneumoniae	qnrS1	qnrS1	6	6	0.125	0.03
N1047	K. pneumoniae	qnrS1	qnrS1	4	8	0.25	0.064
T4617	K. pneumoniae	qnrS1	qnrS1	12	12	0.125	0.032
N1061	K. pneumoniae	qnrS1	qnrS1	6	6	0.25	0.064
T4338	K. pneumoniae	qnrS1	qnrS1	12	16	0.25	0.064
T4359	K. pneumoniae	qnrS1	qnrS1	8	16	0.25	0.032
N1007	K. pneumoniae	qnrS1	qnrS1	4	4	0.25	0.064
T3811	K. pneumoniae	qnrS1	qnrS1	6	24	1	0.12
T4577	K. pneumoniae	qnrB4	qnrB4	4	8	0.25	0.032
T4550	K. pneumoniae	qnrS1	qnrS1	8	8	0.125	0.064
T4413	K. pneumoniae	qnrS1	qnrS1	12	6	0.25	0.032
T3540	K. pneumoniae	qnrS1	qnrS1	12	8	0.25	0.032
T3635	K. pneumoniae	qnrB4	qnrB4	24	12	0.25	0.016
T3950	K. pneumoniae	qnrS1	qnrS1	8	8	0.25	0.12
T4044	K. pneumoniae	qnrB4,aac(6)-Ib-cr	qnrB4	12	8	0.25	0.25
T20891	K. pneumoniae	qnrS1	qnrS1	8	16	0.25	0.016
T14250	K. pneumoniae	qnrB1,aac(6)-Ib-cr	qnrB1	4	8	0.016	0.016
T13828	K. pneumoniae	qnrS1	qnrS1	12	32	0.125	0.032
T20400	K. pneumoniae	qnrS1	qnrS1	4	4	0.25	0.016
T20096	K. pneumoniae	qnrB4,qnrS1	qnrS1	16	8	0.25	0.032
T14305	K. pneumoniae	qnrB4	qnrB4	8	8	0.25	0.032
T13041	K. pneumoniae	qnrS1	qnrS1	4	8	0.125	0.032
T13014	K. pneumoniae	qnrS1	qnrS1	8	4	0.06	0.032
T3470	E. coli	aac(6)-Ib-cr	aac(6)-Ib-cr	>256	16	>64	0.5
N1024	E. coli	aac(6)-Ib-cr	aac(6)-Ib-cr	>256	24	>64	0.25
N952	E. coli	aac(6)-Ib-cr	aac(6)-Ib-cr	>256	8	>64	0.125
N981	E. coli	aac(6)-Ib-cr	aac(6)-Ib-cr	>256	24	>64	0.5
T14229	E. coli	qnrS1	qnrS1	6	4	0.125	0.032
J53AzR	E. coli			2		0.008

CIP, ciprofloxacin; NAL, nalidixic acid.

Strain typing by ERIC-PCR

ERIC-PCR analysis indicated that most PMQR determinant-positive isolates of E. coli and K. pneumoniae showed different DNA banding patterns, indicating that they were not clonally related.

Discussion

As broad spectrum antibiotics, quinolones and fluoroquinolones are widely used in adults. The risk of side effects, however, limits the use of these antibiotics in children, and hospitals are not advised to administer them to children. In China, quinolones are not used on children younger than 17 years of age; however, resistant isolates have been detected in pediatric patients. In this study, the rate of resistance to quinolones in pediatric isolates was 20.99%, of which E. coli isolates showed a higher rate of resistance than K. pneumoniae (33.93% vs. 9.92%). This finding was consistent with a previous study showing that K. pneumoniae presented a lower ciprofloxacin resistance rate than E. coli (59.3% vs. 91.9%).¹⁹ In addition, only a few PMQR-positive isolates were quinolone resistant (21.82%, 12/55), whereas most were ESBL producing strains (92.73%, 51/55). This finding demonstrated that the presence of PMQR did not correlate with a quinolone resistance phenotype; it was more related to ESBL production. This was consistent with a study by Wang and colleagues that showed that all of the 21 qnr-positive isolates were quinolone susceptible.²⁰ The PMQR genes might not, therefore, be directly associated with the selective pressure caused by the quinolones used in pediatric patients, and it could be inferred that the presence of PMQR is more closely linked to cephalosporin use than quinolone use, suggesting that other resistance genes may have a greater effect on PMQR prevalence than the low-level quinolone resistance that PMQR itself provides.²¹

Over the past few years, PMQR in Enterobacteriaceae has been widely reported in North and South America and European countries,^22,23 but only a few studies have been reported on pediatric isolates. In China, a previous study in ESBL-positive E. coli pediatric isolates collected from 2005 to 2006 showed that the prevalence rate of PMQR was 6.8%.⁹ Another study showed that the PMQR detection rate in Typhimurium isolates from hospitalized pediatric patients with diarrhea was 37.1%.²⁴ No other reports about PMQR in pediatric patients have been reported. In the current study, the PMQR detection rate was 22.63% among the 243 clinical isolates and 35.66% (51/143) among ESBL-positive isolates. This rate was higher than a previous study in China,⁹ and it was also higher compared with pediatric isolates collected from other countries, like the prevalence of PMQR genes among Enterobacteriaceae pediatric isolates in Mexico was 32.1% (36/112),²⁵ in Uruguay was 25% (5/20),¹¹ while in a study in Tunisia, the detection rate was 14.4%,¹⁰ and in a Korean study, the PMQR detection rate was 9.7% and this rate had increased over time.²⁶

In this study, the most frequently detected PMQR gene was qnrS, followed by aac(6′)-Ib-cr and qnrB in all the isolates, while aac(6′)-Ib-cr was the most prevalent gene in PMQR-positive E. coli isolates. This result was consistent with a previous study in Japan, in which 150 ceftazidime or cefotaxime-resistant clinical isolates of K. pneumoniae and E. coli sampled between 2008 and 2011 were found to possess the qnrB, aac(6′)-Ib-cr, and qnrS genes in high prevalence, with the qnrA and qepA genes being less frequently detected in K. pneumoniae (66.7%) and to a lesser extent in E. coli (0.8%).²⁷ In another study in France analyzing 47 isolates, the qnrS gene was detected in two K. pneumoniae isolates and 11 E. coli isolates, but only one K. pneumoniae isolate harbored the aac(6′)-Ib-cr gene, whereas no qnrA or qnrB genes were detected.²⁸ The same trend was not observed in Mexico, where among 44 E. coli isolates qnr-determinants were detected in 13.6%, while the aac(6′)-Ib-cr gene was not detected.²⁵

Conjugation experiments showed that 60.87% (28/46) of isolates were able to transfer the PMQR genes to transconjugants. This rate of transferability confirmed previous findings that not all PMQR-positive strains were able to transfer quinolone resistance.⁸ Transconjugants showed increased MIC values to nalidixic acid or ciprofloxacin of 2- to 32-fold, respectively, suggesting the horizontal transmission of this gene. The results of ERIC-PCR showed that the positive PMQR strains had different DNA banding patterns, thereby suggesting that the PMQR in clinical isolates was not caused by the spread of identical strains.

Conclusion

Our study showed that PMQR detection rates in Chinese pediatric isolates were high and increased compared with the previous study in China. PMQR genes were more frequently detected in ESBL-producing isolates and ciprofloxacin susceptible. Qnr genes were mainly found in K. pneumoniae, while the aac(6′)-Ib-cr gene was more commonly detected in E. coli. Conjugation experiments showed that these genes could be transferred horizontally. This is an alarming situation with the high prevalence of PMQR in pediatrics in China.

Footnotes

Acknowledgments

The authors thank Professor Minggui Wang (Huashan Hospital, Fudan University, Shanghai, China) for kindly providing the recipient strain E. coli J53AzR. This work was supported by the Natural Science Foundation of China (81401678) and the Foundation of Capital Institute of Pediatrics.

Disclosure Statement

No competing financial interests exist.

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