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Keywords:

  • ESBL or AmpC-producing Escherichia coli;
  • Pediatrics;
  • PMQR;
  • quinolone resistance

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Three kinds of PMQR determinants (qnr genes, aac(6’)-Ib-cr, and qepA) have been discovered and shown to be widely distributed among clinical isolates. To characterize the prevalence of PMQR determinants in ESBL or AmpC-producing E. coli clinical isolates in Chinese children, a total of 292 ESBL or AmpC-producing E. coli clinical isolates collected from five children's hospitals in China from 2005 to 2006 were screened for PMQR determinants by PCR. Twenty (6.8%) of the 292 isolates were positive for PMQR determinants. A total of 12 (4.1%) isolates were positive for qnr genes, comprising three positive for qnrA (1.0%), three for qnrB (1.0%), and six for qnrS (2.1%). Twenty-four (8.2%) isolates were positive for aac(6’)-Ib, of which 10 (3.4% of 292) had the –cr variant. There was no qepA gene detected in the isolates. Conjugation revealed that qnr, aac(6’)-Ib-cr, and ESBL-encoding genes were transferred together.

List of Abbreviations: 
CLSI

Clinical and Laboratory Standards Institute

E. coli

Escherichia coli

ESBL

extended-spectrum beta-lactamase

K. pneumoniae

Klebsiella pneumoniae

LB

Luria-Bertani

MIC

minimum inhibitory concentration

PFGE

pulsed-field gel electrophoresis

PMQR

plasmid-mediated quinolone resistance

QRDR

quinolone resistance-determining region

Quinolones are an important group of antibiotics which are used broadly in adult patients because of their excellent bactericidal activity. Over the past few years however, resistance to these antibiotics has sharply risen in China due to their wide use (1). It is believed that quinolone resistance can only be acquired through a chromosomal mutation that includes the gyrA, gyrB, parC, and parE genes. The gyrA and parC genes are major factors for the mutation of the QRDR. Recently, three PMQR mechanisms have also been described. The first mechanism comprises qnr genes that encode target protection proteins of the pentapeptide repeat family (2, 3). The second mechanism is the aac(6’)-Ib-cr gene, which encodes a new variant of the common aminoglycoside acetyltransferase. Two single-nucleotide substitutions at codons 102 and 179 in the wild-type allele aac(6’)-Ib enable the gene product to be capable of acetylating and reducing the activity of some quinolones, including norfloxacin and ciprofloxacin (4). The third mechanism involves qepA, a new plasmid-mediated gene which encodes an efflux pump belonging to the major facilitator subfamily and is responsible for reduced fluoroquinolone susceptibility (5, 6).

Quinolones are restricted for use among children because they are associated with a variety of adverse side effects. However, our previous study showed there was both a high resistance rate against the quinolones and a high prevalence of PMQR determinants qnr gene among ESBL or AmpC beta-lactamase producing clinical K. pneumoniae isolates from Chinese pediatrics patients (7). The objective of this study was to screen for the presence of PMQR determinants in clinical isolates of ESBL or AmpC-producing E. coli from pediatric patients in China. We found a low carriage rate for qnr genes in those strains. However, we also found a close relationship between PMQR genes and beta-lactamase genes, as well as a high incidence of resistance rate to ciprofloxacin.

MATERIAL AND METHODS

  1. Top of page
  2. ABSTRACT
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Bacterial isolates

From 2005 to 2006 a total of 292 clinical isolates of phenotypic positive ESBL or AmpC beta-lactamase E. coli were collected at five children's hospitals located in China. Each isolate was obtained from a single patient. The isolates were obtained from patients admitted to the Department of Internal Medicine (240 strains, 82.2%), the Intensive Care Unit (20 strains, 6.8%), and the Department of Surgery (18 strains, 6.2%), and from patients seen by the Outpatient Service (14 strains, 4.8%). These isolates were obtained from the lower respiratory tract (60.6%), urine (21.9%), blood (3.4%), pus (3.1%), and other areas (10.9%). E. coli J53AzR, which is resistant to azide, was used as the recipient strain in the conjugation experiments (8).

Screening and confirmation of ESBL and AmpC

All isolates were screened for ESBL production using the double-disk synergy method (9), the results of which were confirmed by the CLSI ESBL disk confirmatory method (CLSI 2003). Insensitivity to cefoxitin was preliminarily regarded as AmpC positive, and a confirmatory experiment was further performed according to the 3-aminophenylboronic acid procedure (Sigma-Aldrich, Milwaukee, WI, USA) and double disk diffusion method (10) (Oxoid, Basingstoke, UK). Quality control was performed by testing E. coli ATCC25922 and K. pneumoniae ACTT700603.

Antibiotic sensitivity experiment

MIC of amikacin, ampicillin, ampicillin/clavulanic acid, cefepime, cefotaxime, ceftazidime, ciprofloxacin, gentamicin, and imipenem were determined by the agar dilution method according to the CLSI guidelines. Quality control was performed by testing E. coli ATCC25922. The cefotaxime sodium was from Sigma USA, while all the other antibiotics and the Mueller-Hinton medium were purchased from Oxoid, England.

Detection of PMQR determinants, gyrA, parC, gyrB, parE and beta-lactamase-encoding genes

The isolates were investigated for the presence of qnrA, qnrB, qnrS, aac(6’)-Ib, qepA, gyrA, parC, gyrB, parE, blaTEM, blaSHV, and blaCTX-M genes by PCR amplification using the primer sets described previously (5, 11–17). Because only a portion of the QRDR genes was sequenced, the sequenced range of the relevant nucleotide positions of gyrA, parC, gyrB, and parE genes were 193–536, 160–347, 1051–1424, and 1071–1496, respectively, compared with the DNA sequence of the QRDR genes of E. coli. The Genebank accession numbers are DQ447134.1, NC_011601.1, NC_012892.1, and NC_000913.2, respectively.

Amplification of AmpC beta-lactamase was performed according to Perez-Perez and Hanson (18). The colonies were transferred to water through an Eppendorf tube and boiled to prepare DNA templates for PCR. The results of the DNA sequences were compared with the BLAST online search engine from GenBank at the National Center for Biotechnology Information Web site (http://www.ncbi.nlm.nih.gov/BLAST/).

Conjugation experiments and plasmid detection

Conjugation experiments were carried out in an LB broth with E. coli J53AzR as the recipient as previously described (19). The PMQR-positive isolates were used for the conjugation experiments. Transconjugants were selected on trypticase soy agar plates containing sodium azide (150 μg/ml) for counterselection and sulfamethoxazole (300 μg/ml) to select for plasmid-encoded resistance. To determine if quinolone resistance was co-transferred, colonies were plated in replica onto trypticase soy agar plates with and without ciprofloxacin (0.06 μg/ml) .The MIC for the transconjugants were measured by an Epsilometer test, and the transconjugants carrying PMQR genes were then confirmed by PCR. Plasmid DNA of the donors and the transconjugants were extracted by alkaline lysis. E. coli V517 (plasmid sizes, 54, 5.6, 5.1, 3.9, 3.0, 2.7, and 2.1 kb) and E. coli J53 plac (plasmid size152 kb) were used as standards.

Pulsed-field gel electrophoresis

The genomic DNA of PMQR-producing isolates were analyzed by PFGE after digestion with XbaI (Sangon, Shanghai, China). The DNA fragments were separated by electrophoresis in 1% agaroseD-5 (TaKaRa, Shiga, Japan) in a 0.5 X tris-borate-EDTA buffer with the clamped homogenous electric fields apparatus (CHEF Mapper XA, Bio-Rad, Hercules, CA, USA) at 14°C, 6 volts/cm and with alternating pulses at a 120° angle in a 5–35 s pulse time gradient for 19 hr.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Prevalence of PMQR determinants and gyrA, parC, gyrB, and parE genes

Twenty (6.8%) of the 292 E. coli isolates were positive for PMQR determinants. A total of 12 (4.1%) of the 292 isolates were positive for qnr genes, comprising three positive for qnrA (1.0%), three for qnrB (1.0%), and six for qnrS (2.1%). A total of 24 (8.2%) isolates were positive for aac(6’)-Ib, of which 10 (3.4% of 292) had the –cr variant. There was no isolates positive for qepA. There was one isolate having both the qnrA and aac(6’)-Ib genes, one with both the qnrB and aac(6’)-Ib-cr genes, one carrying both the qnrS and aac(6’)-Ib-cr genes, and one carrying both the qnrS and aac(6’)-Ib genes.

QRDR PCR products were sequenced on both strands, and DNA sequences of gyrA, parC, gyrB, and parE genes for each of the PMQR-positive isolates were compared with the QRDR DNA sequences of E. coli. Among the PMQR-positive strains, gyrA mutations were observed in 5/20 (25%), of which three strains had both Asp87Asn and Ser83leu, one only Asp87Asn, while the third had only Ser83leu. In parC, the mutations showed up in four strains in the same Ser80Ile substitution. There were no mutations in gyrB and parE. (The Genebank accession numbers are DQ447134.1 for gyrA and NC_011601.1for parC.)

Detailed information on these PMQR determinant-positive isolates is presented in Table 1.

Table 1.  Characteristics of the PMQR-positive strains
Number of StrainsSpecimenDiagnosisAgePMQR determinantESBL or AmpC beta-lactamaseMIC-ciprofloxacinQRDR mutation in
DonorTransconjugantDonorTransconjugantDonorTransconjugantgyrApraC
  1. ALL, acute lymphoblastic leukemia; d, days; m, months; UTI, urinary tract infection; wt, wild type; y, years.

A26SputumPneumonia31daac(6’)-Ib-craac(6’)-Ib-crCTX-M-14-0.250.19wtwt
E38SputumPneumonia4mqnrB4qnrB4CTX-M-15, SHV-1, DHA-1CTX-M-1520.5wtwt
E47SputumPneumonia10maac(6’)-Ib-craac(6’)-Ib-crCTX-M-14CTX-M-14640.047wtwt
E52SputumPneumonia2yaac(6’)-Ib-cr-CTX-M-14-32-wtSer80Ile
g26UrineUTI7mqnrS1-CTX-M-14-1-wtwt
g39UrineUTI9mqnrA1qnrA1CTX-M-14CTX-M-141280.032wtwt
j6TrachealPneumonia4maac(6’)-Ib-craac(6’)-Ib-crCTX-M-14CTX-M-1440.047wtwt
m12SputumPneumonia1yaac(6’)-Ib-cr-CTX-M-14-256-wtwt
m33SputumPneumonia1yaac(6’)-Ib-craac(6’)-Ib-crCTX-M-14,SHV-12CTX-M-14,SHV-1240.25Ser83Leuwt
n16BloodALL3yqnrS1qnrS1CTX-M-24CTX-M-24162wtwt
n33SputumPneumonia12dqnrS1,aac(6’)-Ib-CTX-M-14-8-wtSer80Ile
n35SputumPneumonia7maac(6’)-Ib-craac(6’)-Ib-crCTX-M-14CTX-M-1440.38Asp87Asn,Ser83Leuwt
s21BloodALL1mqnrS1-CTX-M-14-8-Asp87Asn,Ser83Leuwt
s34SputumPneumonia6yqnrA1qnrA1CTX-M-14CTX-M-1420.38Asp87Asnwt
s46TrachealPneumonia2mqnrB4qnrB4CTX-M-14, SHV-1CTX-M-1480.38Asp87Asn,Ser83Leuwt
s47SputumPneumonia5mqnrS1qnrS1CTX-M-15CTX-M-1581.5wtwt
s48TrachealPneumonia11mqnrS1,aac(6’)-Ib-crqnrS1,aac(6’)-Ib-crCTX-M-15, SHV-1CTX-M-15, SHV-141.5wtwt
x17SputumPneumonia6yaac(6’)-Ib-craac(6’)-Ib-crCTX-M-14CTX-M-141280.047wtSer80Ile
x25SputumPneumonia9mqnrA1,aac(6’)-IbqnrA1CTX-M-15CTX-M-152560.032wtwt
x37UrineUTI12yqnrB4,aac(6’)-Ib-craac(6’)-Ib-crCTX-M-14,DHA-1CTX-M-141280.38wtSer80Ile
J53-------0.016-- 

Identification of ESBL and plasmid-mediated AmpC beta-lactamases

In total, 274 (93.8%) isolates were positive for ESBL BlaTEM and/or BlaSHV, and/or the BlaCTX-M-1 group and/or the BlaCTX-M-9 group. Five isolates were positive for AmpC genes, of which two were of the DHA-1 and three the CMY-2 type. In the 20 E. coli isolates with PMQR determinants, all had the BlaCTX-M and/or AmpC beta-lactamases. Among them, 15 isolates had the CTX-M-14, one the CTX-M-24, four the CTX-M-15, and two both the DHA-1 and CTX-M-14 or CTX-M-15 (Table 1).

Antibiotic susceptibility

Of the 292 ESBL or AmpC-producing E. coli isolates, the rate of resistance to ciprofloxacin was 55% and the intermediate rate was 5%. The rates of resistance to cefotaxime, cefepime, and ceftazidime were 65.4%, 47.6%, and 25.3%, respectively. The rates of resistance to gentamicin, amikacin, and ampicillin/clavulanic acid were 54.8%, 6.2%, and 22.3%, respectively. No isolates were resistant to imipenem. All the tested strains were resistant to ampicillin, and the MIC50 and MIC90 were all >512 μg/ml. The MIC50/MIC90 of cefepime, cefotaxime, ceftazidime, ciprofloxacin, gentamicin, amikacin, ampicillin/clavulanic acid, and imipenem were 32/256, 128/512, 4/64, 32/512, 16/256, 4/16, 16/32, and 0.125/0.25 μg/ml, respectively.

Conjugation experiments and plasmid detection

The PMQR genes could be transferred by conjugation in 15 of the 20 qnr and aac(6’)-Ib-cr-positive donors. Two isolates harboring more than one PMQR gene (x25 harbored qnrA1 and aac(6’)-Ib; x37 harbored qnrB4 and aac(6’)-Ib-cr) transferred only a single gene to the recipient (x25 transferred qnrA1 and x37 transferred aac(6’)-Ib-cr). The MIC of ciprofloxacin for 15 transconjugants were 0.032–2 μg/ml, representing a 2- to 125-fold increase compared with the recipient (MIC 0.016 μg/ml) (Table 1). Plasmid DNA was extracted from the 20 donors and the 15 transconjugants. Each of the positive donors contained one large plasmid (>152 kb) and one to four small plasmids (<54 kb). Moreover, the transconjugants carried the responding large plasmid.

Pulsed-field gel electrophoresis

The molecular characterization of PMQR-positive isolates by PFGE showed great genomic diversity amongst them, indicating that most of these strains were not clonal.

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

The rates of quinolone resistance in clinical isolates of E. coli and K. pneumoniae are unusually high in China; more than 50% of clinical strains of E. coli are resistant to ciprofloxacin (1), especially in strains producing extended-spectrum beta-lactamase (20, 21).However, the use of quinolones in children has been restricted. There are few data available on the prevalence of resistance of E. coli to quinolones in this population. Interestingly, our results show that even though there was a high rate of resistance to ciprofloxacin (55%) in isolates from Chinese pediatric patients, a low qnr genes carriage rate (4.1%) was found in those isolates producing ESBL or AmpC beta-lactamases. It should be noted that all the positive qnr isolates were distributed in pediatric patients: about 75% being isolated from children less than one year in age. Possibly the source of the qnr gene is not directly associated with selective pressure caused by the quinolones used in pediatric patients, but could be related to horizontal transmission from adults or other reservoirs. Recent findings have shown that these genes come from environmental Gram-negative bacterial species, such as Shewanella algae, the progenitor of the qnrA genes (22). In addition some studies have found qnr genes in bacteria from companion and food-producing animals (23) and in clinical isolates of E. coli from poultry and swine (16). This shows that a contaminated environment is an important reservoir for novel antibiotic resistance determinants.

To date, qnr genes have been widely detected in North and South America, in Europe, and in South and East Asia. A high prevalence of qnr among ESBL-producing enterobacterial species has been described. (24, 25). In this study, qnr genes were detected in 4.1% of the isolates from pediatric patients, which is more than recently reported in Europe (26) and Korea (27) but less than reported in the Zhejiang province of China (21). The prevalence of qnrA varied from less than 1% to greater than 20%. A recent European survey has suggested that qnrS may be more prevalent than qnrA in clinical Enterobacteriaceae (25). In our previous study, a low rate of qnrA was observed in ESBL or AmpC beta-lactamase of K. pneumoniae (2.4%), with qnrB and qnrS at 6.1% and 15.1%, respectively (7). Furthermore, in this study, the characteristic qnr gene distribution showed that, among ESBL or AmpC-producing E. coli isolates, qnrS was the most prevalent (2.1%), followed by qnrB (1.0%), and qnrA (1.0%). This result is similar to Jiang's report, in which the prevalence of qnrA, qnrB, and qnrS was 1.9%, 1.5%, and 1.9%, respectively (21), but different from that of Yang's (28). The current results indicate that the prevalence rates and distribution of plasmid-mediated qnr genes are different in isolates from different populations, and in species of Enterobacteriaceae in the same area. It has been reported that the qnr gene can co-exist with blaCTX-M and blaSHV alleles (21). In this study, all isolates in which qnr genes were detected also contained blaCTX-M-like genes.

Aac(6’)-Ib-cr, a novel PMQR protein, was first reported in 2003, but is now recognized to be widely disseminated. However, the presence of aac(6’)-Ib-cr in clinical isolates from Chinese children has not previously been evaluated. In this study, the prevalence rate of aac(6’)-Ibcr in ESBL or AmpC-producing E. coli isolates from Chinese children was about 3.4%, which was less than that in two recent reports from China (28) and Canada (29). The aac(6’)-Ib-cr genes have been reported to be co-associated with genes encoding ESBL or other beta-lactamases (24, 25) especially CTX-M-15 (29–31). However, in this study, nine out of ten isolates with aac(6’)-Ib-cr co-produced CTX-M-14, which is the prevalent type in China (20, 32). Only one of them co-produced CTX-M-15. Our conjugation experiments showed that the two genes can be transferred together, further suggesting that aac(6’)-Ib-cr may usually be co-associated with the predominant type of CTX-M in a certain region.

Furthermore, there are some isolates with no mutation in gyrA and parC which show a high MIC value against ciprofloxacin. This may suggest that, apart from chromosomal mutations in the genes encoding quinolone target enzymes, efflux pumps or porin channels could be mainly responsible for the high MIC value against ciprofloxacin (33).

In short, our study shows that, in the bacterial strains tested, resistance to quinolones is quite frequent despite their restricted use in children, and that PMQR in ESBL or AmpC-producing E. coli is prevalent in Chinese pediatric patients. These results suggest that the emergence of PMQR may have contributed to a rapid increase in bacterial resistance to quinolones in Chinese children.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

This study was sponsored by the Health Ministry of China (No. 2004BA720A09-01).We are grateful to George A. Jacoby (Lahey Clinic, Burlington, MA, USA) for kindly providing the E. coli J53AzR.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES
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