Prevalence of Resistance to Quinolone and Fluoroquinolone Antibiotics and Screening of qnr Genes Among Escherichia coli Isolates From Urinary Tract Infection

Copyright © 2017 The Author(s); Published by Alborz University of Medical Sciences. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited. Background Urinary tract infection (UTI) is among the most prevalent bacterial infections.1 This disease can be caused by various bacterial agents but the most common agent is uropathogenic Escherichia coli (UPEC).2 Antibiotic therapy of UTI has encountered many problems; today, antibiotic resistance (AR) is an important challenge in prevention, treatment and control of infectious diseases (such as UTI) and is a public health threat for human.3 The most important causes of AR are incorrect usage of antibiotics by human and the excessive usage of antibiotics in food-producing animals.4 E. coli, the main member of the intestinal microbiota, has a primary role in acquisition and transmission of AR traits. E. coli may be resistant to various categories of antibiotics and employ different ways to transfer AR genes to other strains and bacteria through transposons, bacteriophages and plasmids.5 Of the commonest choices in antibiotic therapy of UTI originated from E. coli are quinolones and fluoroquinolones. These categories of antibiotics are effective against a wide range of bacteria and have oral absorption and renal excretion. They inhibit bacterial DNA synthesis by inhibition of DNA gyrase enzyme that causes the death of bacteria.6 These antibiotic families have four generations; for example, nalidixic acid, ciprofloxacin and levofloxacin belong to the first, second and third generations, respectively.6 Recently, resistance to these antibiotics has raised concerns; in the Netherlands, a yearly increase of resistance to fluoroquinolones in E. coli from UTI has been reported which may be associated with increased prescription of these antibiotics for UTI. Main mechanism of resistance to fluoroquinolones in Enterobacteriaceae is commonly associated with the mutation of chromosomal genes encoding DNA gyrase, topoisomerase IV, and regulatory efflux pumps.7 Moreover, plasmid-mediated quinolone resistance


Background
Urinary tract infection (UTI) is among the most prevalent bacterial infections. 1 This disease can be caused by various bacterial agents but the most common agent is uropathogenic Escherichia coli (UPEC). 2 Antibiotic therapy of UTI has encountered many problems; today, antibiotic resistance (AR) is an important challenge in prevention, treatment and control of infectious diseases (such as UTI) and is a public health threat for human. 3he most important causes of AR are incorrect usage of antibiotics by human and the excessive usage of antibiotics in food-producing animals. 4E. coli, the main member of the intestinal microbiota, has a primary role in acquisition and transmission of AR traits.E. coli may be resistant to various categories of antibiotics and employ different ways to transfer AR genes to other strains and bacteria through transposons, bacteriophages and plasmids. 5f the commonest choices in antibiotic therapy of UTI originated from E. coli are quinolones and fluoroquinolones.These categories of antibiotics are effective against a wide range of bacteria and have oral absorption and renal excretion.They inhibit bacterial DNA synthesis by inhibition of DNA gyrase enzyme that causes the death of bacteria. 6These antibiotic families have four generations; for example, nalidixic acid, ciprofloxacin and levofloxacin belong to the first, second and third generations, respectively. 6Recently, resistance to these antibiotics has raised concerns; in the Netherlands, a yearly increase of resistance to fluoroquinolones in E. coli from UTI has been reported which may be associated with increased prescription of these antibiotics for UTI.Main mechanism of resistance to fluoroquinolones in Enterobacteriaceae is commonly associated with the mutation of chromosomal genes encoding DNA gyrase, topoisomerase IV, and regulatory efflux pumps. 7oreover, plasmid-mediated quinolone resistance (PMQR) genes are alternative mechanisms for resistance to quinolones which enhance the resistance by the DNA gyrase protection proteins (via qnrA, qnrB and qnrS genes), an aminoglycoside-modifying enzyme [via aac(6')-Ib-cr genes] and efflux pumps (via oqxAB and qepA genes). 7In South Korea, the prevalence of PMQR genes in clinical isolates of Enterobacteriaceae have been reported over a 9-year period; their results showed that the increasing frequency of ciprofloxacin resistance in Enterobacteriaceae was related to an increasing prevalence of PMQR genes. 8scherichia coli can be classified into 4 major phylogenetic groups (A, B1, B2, and D) and 7 subgroups (A 0 , A 1 , B2 2 , B2 3 , B1, D 1 and D 2 ) according to the combination of the 3 genetic markers chuA, yjaA and TspE4.C2. 9 The distribution pattern of phylogenetic groups among clinical isolates of E. coli from UTI has been reported by Zhang et al; they stated B2 is the predominant phylogroup among E. coli strains isolated from UTI. 10 In this study, we wanted to know the prevalence of resistance to the 3 antibiotics including ciprofloxacin, levofloxacin and nalidixic acid among E. coli strains isolated from UTI cases.We were also interested to know the prevalence of qnrA, qnrB and qnrS genes in the strains, in order to identify the genes with more important role in development of resistance to quinolones and fluoroquinolones.

Urinary Sampling and Escherichia coli Isolation
The study was carried out using urine samples obtained from 105 patients with UTI in Bojnord city (northeast of Iran).In hospital laboratory, samples were cultured on MacConkey agar (Merck, Germany) and eosin methylene blue (EMB) agar (Merck, Germany) in aerobic condition at 37°C for 24-48 hours.MacConkey agar is useful for differentiation between lactose-positive and lactosenegative strains.In EMB agar, E. coli strains have green metallic appearance that helps to select the E. coli strains, more quickly and carefully.

Antibiogram for Identification of Escherichia coli Isolates Resistant to Quinolones and Fluoroquinolones
The AR to 3 antibiotics of ciprofloxacin (CP; 5 μg, Padtanteb, Iran), nalidixic acid (NA; 30 μg, Padtanteb, Iran) and levofloxacin (LOM; 5 μg, Padtanteb, Iran) was identified via Kirby Bauer disk diffusion method as described previously. 11Results were also analyzed on the basis of the Clinical and Laboratory Standards Institute (CLSI) guide. 11lymerase Chain Reaction for Screening of qnr Genes Escherichia coli isolates were subjected to phylogenotyping examinations.DNA was extracted by boiling method previously described. 12Two multiplex PCR assays were performed for detection of qnr resistance genes and phylogenetic grouping.
For screening of the resistance genes, we focused on DNA gyrase protection mechanism and studied the related genes including qnrA, qnrB and qnrS.

Prevalence of qnr Genes
In this study, the frequency of genes resistant to quinolone including qnrA, qnrB and qnrS was very low and only 4 strains were positive for these genes (Figure 1).One strain had qnrS gene and was resistant to ciprofloxacin and levofloxacin antibiotics.Two strains had qnrS gene but were susceptible to ciprofloxacin, levofloxacin and nalidixic acid antibiotics.Furthermore, 1 strain had qnrA gene and was susceptible to ciprofloxacin, levofloxacin and nalidixic acid.

Discussion
In this study, phenotypic AR to quinolone and fluoroquinolone antibiotics was found in approximately half of E. coli strains.Sedighi et al in Hamadan studied 120 E. coli isolates from UTIs in children; frequency of resistance to ciprofloxacin and nalidixic acid was 79.2% and 40.9%, respectively. 16Rezazadeh et al reported that prevalence of resistance to nalidixic acid, levofloxacin and ciprofloxacin was between 55% and 65%. 17     (20), D 2 ( 16), D 1 (10), B1(5), A 0 (5), A 1 (2), B2 2 (2) 60 prevalence of resistance to nalidixic acid was 54.16%; in their study resistance to ciprofloxacin was determined in 29.18% of UTI isolates. 18According to studies in Iran, resistance to ciprofloxacin varies between 45% and 55% 17,19 that is in correspondence with our findings.This resistance has been reported in European countries with high prevalence. 20Resistance to nalidixic acid in some countries such as India and Nigeria is very high 21,22 ; however nalidixic acid is not a common choice in UTI treatment.Prevalence of AR to levofloxacin in this study was very similar to the results of researches conducted in China 23 and Korea. 24The rate in developed countries such as Canada 25 and the United States 25 is very low and less than 10%.
It should be noted that resistance to quinolones and fluoroquinolones was almost close to 0, but it slowly increased from less than 1.2% in 1996 that indicates the current situation is alarming. 26Differences in frequency may be due to hygiene conditions and prescribing strategies in different countries. 27Treatment of patients with fluoroquinolones may select and enhance the fluoroquinolone-resistant commensal strains. 27Therefore, these antibiotics should only be employed with caution in UTI patients, if necessary.Consequently, environmental contamination and person-to-person transmission cause the resistance development.Moreover, purpose of each study can affect its results.In a research, 100% of extended-spectrum beta-lactamases (ESBL) producing E. coli strains were reported as fluoroquinolone-resistant isolates, explaining that only 11.8% of the isolates were  ESBL positive. 28n the current study, prevalence of the qnr-positive isolates was low.Frequency of qnrA in our research and majority of studies in Iran, 17,29 Kuwait 13 and Thailand 30 was 0. Prevalence of qnrS gene in our study was similar to prevalence of that in some studies in Iran 16,17 and other countries such as Kuwait 13 and Thailand. 30In Mashhad, 200 clinical isolates were studied by Harifi Mood et al and qnrA, qnrB, and qnrS genes were found in 31.5%,17% and 7% of the isolates, respectively. 31Vasilaki et al in Greece showed that 11 isolates (10%) carried qnrS gene and none of the isolates harbored qnrA and qnrB genes. 32The 2 recent studies are in contrast with our results.
Our results indicated that there was no significant relationship between existence of qnrA, qnrB and qnrS genes and resistance to ciprofloxacin, levofloxacin and nalidixic acid antibiotics.Because of low prevalence of the genes, it is possible that other types of qnr genes and/or other PMQR mechanisms are responsible for fluoroquinolones resistance in the present study.Minimum inhibitory concentration (MIC) can help understand the association between the presence of a PMQR gene and quantity of resistance to the 3 antibiotics; we suggest the performance of MIC for next similar researches.In qnrS-positive and sensitive strains to ciprofloxacin, levofloxacin and nalidixic acid antibiotics, it is possible that for any reason, qnrS gene was silent and not expressed; qnr genes enhance the quinolones resistance, commonly not to non-susceptibility, but help the selection of more resistant strains. 7n the present study, all isolates were classified into 7 phylogenetic subgroups and majority of isolates belonged to the group D and subgroup D 2 .Our results are in contrast with Baponi et al study that assigned E. coli isolates from hospitalized patients to the groups A (30%), B1 (12%), B2 (40%) and D (18%). 33Alizade et al reported that among 122 E. coli isolates from UTI patients in Bam city, 43.4% of the strains belonged to group D which is approximately in agreement with our results. 34The present study can also be comparable with Staji et al research that reported phylogenetic groups of E. coli isolates from 150 UTIs in women including: A (31%), D (31%), B2 (28%) and B1 (10%). 35revious studies indicate that in human, most of E. coli strains resistant to common antibiotics are related to A, B1 and D phylogenetic groups, and the members of phylogenetic group B2 are mostly susceptible to the antibiotics. 36Majority of commensal and non-virulent E. coli strains are associated with the groups A and B1. 37. coli strains belonging to group D and B2 often sustain their virulence traits. 38In our study, all qnrS-positives were assigned to A 0 , and the qnrA-positive isolate was placed in phylogroup D 2 .Moreover, resistant isolates were mostly classified into B2 and D, the groups with resistant and virulent nature.
To conclude, in the present study, only half of the E. coli isolates were susceptible to quinolones and fluoroquinolones.Prevalence of qnr genes was insignificant and majority of our isolates were classified into B2 and D phylogenetic groups.Prevalence of resistant isolates to ciprofloxacin, levofloxacin and nalidixic acid antibiotics was approximately similar to frequency of susceptible isolates; and this resistance rate may increase in the future.Correct strategies of antibiotic therapy and antibiogram tests before prescribing the drugs are critical in the management of UTIs.Quinolones and fluoroquinolones should be prescribed with caution even in patients.
In a study which was performed by Adib et al in Kerman, CGCGCCAACAAAGTATTACG a M = A or C; H = A or C or T; Y = C or T.

Table 1 .
Primers Used for Tracing of Antimicrobial Resistance Genes and Phylogenetic Sequences

Table 2 .
Antibiotic Resistance Patterns in Relation to Phylogenetic Groups

Table 3 .
Distribution of UTI Isolates in Phylogenetic Subgroups and Prevalence of Each Antimicrobial Resistance Phenotype Among Phylogroups