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- MATERIAL AND METHODS
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.
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.
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- MATERIAL AND METHODS
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’)-Ib–cr 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.