Detection of plasmid-mediated qnr genes among the quinolone-resistant Escherichia coli isolates in Iran

Introduction: Plasmid-mediated quinolone resistance, which complicates treatment, has been increasingly identified in Escherichia coli isolates worldwide. The purpose of this study was to identify the plasmid-mediated qnrA and qnrB genes among the quinolone-resistant Escherichia coli isolated from urinary tract infections in Iran. Methodology: A total of 140 Escherichia coli isolates were collected between March and October 2012 from urinary tract infections in Khorram Abad, Iran. All isolates were tested for quinoloe resistance using the disk diffusion method. Also, all quinolone-resistant isolates were screened for the presence of the qnrA and qnrB genes by polymerase chain reaction. Minimum inhibitory concentrations (MICs) of ciprofloxacin for the qnr-positive isolates were determined. Results: One hundred sixteen (82.8%) of 140 Escherichia coli isolates were nalidixic acid-resistant; among them, 14 (12.1%) and 9 (7.8%) were qnrA and qnrB-positive, respectively. Two quinolone-resistant isolates harbored both qnrA and qnrB. Among 63 ciprofloxacin-resistant isolates, 14 (22.2%) and 9 (14.3%) were found to carry qnrA and qnrB genes, respectively. The ciprofloxacin MIC range was 0.25–512 μg/mL for 23 qnr-positive Escherichia coli isolates, 18 of which had MICs values of 4–512 μg/mL. Conclusion: Our study shows that the frequency of plasmid-mediated quinolone resistance genes among E. coli isolates in Iran is high.


Introduction
Escherichia coli (E.coli) is the major cause of urinary tract infections (UTIs), including cystitis and pyelonephritis [1].Fluoroquinolones are the drugs of choice for the treatment of UTI infections in many countries due to widespread resistance to other antibiotics [1][2][3].Based on its availability in oral and intravenous formulations, ciprofloxacin is preferred as the initial antibiotic for empiric therapy of UTIs in Iran [4].Fluoroquinolone-resistant E. coli isolates have been increasingly reported worldwide, thus limiting therapeutic options [2,5].
Resistance to quinolones in E. coli isolates is usually associated with mutations in gyrA and parC genes [6,7].In addition, efflux pumps and changes in the outer membrane proteins also contribute to chromosomal-mediated quinolone resistance [8].Moreover, plasmid-mediated resistance genes play an important role in the development of low-level resistance to fluoroquinolones [9][10][11].These genes include the qnr genes.The qnrA was the first plasmidmediated gene to be detected, followed by qnrB, qnrS, aac(6')-Ib, and qepA genes that increase the MICs of quinolones four to eight times [12,13].The qnrB is another plasmid-mediated quinolone resistant gene that has been identified worldwide and shares 43% amino acid identity with qnrA [13].The horizontal transfer of plasmids carrying quinolone resistance determinants and the accumulation of chromosomal mutations play an important role in increasing rates of resistance [14,15].
Little is known about the frequency of qnr genes in E. coli isolates recovered from clinical specimens in Iran.Therefore, the aim of this study was to investigate the presence of plasmid-mediated qnrA and qnrB genes among the quinolone-resistant E. coli isolated from urinary tract infections of patients admitted to the two hospitals in Khorram Abad, Iran.

Bacterial isolates and antimicrobial susceptibility testing
One hundred forty non-duplicate Escherichia coli isolates were collected from cases of urinary tract infections admitted to Shahid Madani and Shohaday-e-Ashayer hospitals in Khorram Abad, west Iran, between March and October 2012.All isolates were screened for quinolone and fluoroquinolone resistance using the disk diffusion method according to Clinical and Laboratory Standards Institute (CLSI) guidelines [16].The following antibiotic disks were used for antimicrobial susceptibility testing: nalidixic acid (30 µg), norfloxacin (10 µg), levofloxacin (5 µg), ciprofloxacin (5 µg), and ofloxacin (5 µg).The reference strain E. coli ATCC 25922 was used as a control.Results were interpreted as susceptible or resistant according to the criteria recommended by the CLSI and the manufacturer protocols (Mast Group, Bootle, UK).
MICs of ciprofloxacin for E. coli isolates that showed resistance to ciprofloxacin by the disk diffusion method, carrying qnrA or qnrB genes, were determined by the microbroth dilution method according to CLSI guidelines [16].The quality control organisms were E. coli ATCC 25922 and P. aeroginosa ATCC 27853.

DNA extraction and qnr gene amplification
The DNA of 116 E. coli isolates found to be resistant to at least one antibiotic in the disk diffusion method were extracted using a DNA extraction kit (Bioneer, Daejeon, South Korea).Before DNA extraction, the antibiotic-resistant E. coli strains were cultured in Luria-Bertani broth at 37°C for 18 hours.
A polymerase chain reaction (PCR) assay was used to evaluate the presence of qnrA and qnrB resistance genes in quinolone-and fluoroquinoloneresistant strains of E. coli.The primers used for amplification of qnrA and qnrB genes were as follows: 5′-ATTTCTCACGCCAGGATTTG-3′ and 5′-GATCGGCAAAGGTTAGGTCA-3′ for qnrA determinant to detect a 516-bp amplicon, and 5'-GATCGTGAAAGCCAGAAAGG-3' and 5′ -ACGATGCCTGGTAGTTGTCC-3′ for qnrB determinant to detect a 469-bp amplicon [13].The amplification conditions were 5 minutes at 95ºC and 35 cycles consisting of 94°C for 45 seconds, 51°C for 45 seconds and 72°C for 45 seconds, and 72°C for 7 minutes in the final extension.The electrophoresis of PCR products was performed on 1.2% agarose gel, and then the gels were stained in ethidium bromide for 15 minutes and visualized in a gel document system (Bio-Rad, Hemel Hempsted, UK).

Discussion
Plasmid-mediated quinolone resistance mechanisms play an important role in the development of quinolone and fluoroquinolone resistance [14,15].Urinary tract infections caused by such resistant isolates can be difficult to treat [2,6].In this study, the resistance rates to nalidixic acid and ciprofloxacin were 82.8% and 45%, respectively, indicating a high rate of resistance to quinolone and fluoroquinolone by E. coli isolates associated with UTIs in Khorram Abad.Since the development of nalidixic acid in 1962, the drug has been used for the treatment of UTIs for more than five decades [17].The resistance rate for nalidixic acid, therefore, is expected to be higher than for fluoroquinolones.Ciprofloxacin is frequently used for treatment of UTIs in Iran [9].The high ciprofloxacin resistance rate (45%) found in this study could be due to the inappropriate use of ciprofloxacin in Iran.
Other studies in most parts of the world have indicated that fluoroquinolone resistance in E. coli isolates is increasing [1,2,5,8,14].In Pakistan, the frequencies of nalidixic acid and ciprofloxacin resistance among the E. coli isolated from UTIs were 84.2% and 36.5%,respectively, which is in agreement with our findings [18].In addition, a study in China showed the frequency of ciprofloxacin resistance to have increased to 59.4% [19].
On the other hand, our findings are in contrast to studies conducted in the United States showing that   21% and 12% of uropathogenic E. coli were resistant to quinolones and fluoroquinolones, respectively [20].There are many reasons for this discrepancy, including the situation of drug use in Iran where people take antimicrobial drugs without a prescription, differences in animal husbandry, and over-the-counter use of quinolones in veterinary medicine, as well as environmental conditions.
A significant relationship between fluoroquinolone use and resistance to these antibiotics has been documented [5].The frequency of qnr genes in the ciprofloxacin-resistant E. coli isolates in the present study was higher than that found in China, where the rate of qnr genes among ciprofloxacin-resistant isolates of E. coli was 7.5% [19].The results of a survey in Japan showed that qnrA was detected in 6.5% of E. coli clinical isolates and that the qnrB and qnrS genes were not found [21].QnrA determinants were found in up to 32% of multidrug-resistant Enterobacteriaceae from blood cultures in Liverpool, United Kingdom [12].In a similar study, approximately 30% of the quinolone-resistant E. coli isolates harbored the qnr genes [13].Rates of 48% in Thailand and 86% in Vietnam have been reported [22,23].In Greece, 10% of ciprofloxacin-resistant E. coli clinical isolates were qnr-positive [24], which is similar to the findings of our study.
In the current study, the findings that qnrA compared to qnrB was found in the majority of resistant isolates is similar to results from previous studies in Jamaica [14], the United Kingdom [12], and Spain [25], while in contrast to results of other studies [26,27].For instance, in Korea, qnrB was the predominant qnr determinant identified among E. coli isolates [28].Similarly, a remarkable dissemination of qnrB determinant in commensal enterobacteria isolates from healthy children was reported in Peru and Bolivia [29].The results of a study in China revealed that qnrA was more common than the other three qnr gene groups [19], similar to our results.A high frequency of qnr determinants was identified in ciprofloxacinresistant isolates compared to the nalidixic acidresistant isolates, indicating that other mechanisms may contribute to nalidixic acid resistance.
The finding of our study that some qnrA and qnrBnegative isolates were also resistant to ciprofloxacin signifies that other qnr genes or resistance mechanisms, such as mutations in the target enzyme (e.g., DNA gyrase and topoisomerase IV) and/or activation of efflux pumps, may be involved.
According to Jeong et al. (2005), the presence of a qnr determinant increases resistance to nalidixic acid and fluoroquinolones four to eight times [30].In our study, the majority of ciprofloxacin-resistant isolates carrying qnr genes showed complete resistance to ciprofloxacin (MICs > 32 μg/mL).The high MIC values for qnr-positive ciprofloxacin-resistant isolates shows the extent of the therapeutic challenge for such resistant isolates.
In conclusion, our finding showed high frequencies of plasmid-mediated quinolone-resistant genes in urinary tract infection isolates of E. coli in Iran.Appropriate use of these antibiotics may be useful to limit the potential spread of resistant genes and reserve their application for therapeutic uses.

Table 1 .
Quinolone and fluoroquinolone-resistant isolates by disk diffusion method