Send Orders for Reprints to Reprints@benthamscience.ae Pmqr Determinants Expression in Gram-negative Uropathogens Clinically Isolated from Hospitalized Patients with Pyelonephritis in Kharkiv, Ukraine

Background:


INTRODUCTION
Urinary tract infections (UTIs) are thought to be the most common group of bacterial infections, worldwide.It is estimated that the proportion of UTIs remains at a high level reaching 150 million episodes per year worldwide and accounting for $6 billion in health care expenditures [1,2].In Ukraine, among the UTI, pyelonephritis is a leading cause of end-stage chronic kidney disease (CKD) [3].In the USA, more than 100,000 hospital admissions per year are the UTIs patients, among them, the patients with pyelonephritis are the most frequent.For the US outpatients, approximately 15% of antibiotics used are prescribed for UTIs [4].
Meanwhile, beta-lactams and fluoroquinolones are well-known as the main therapeutics effective to treat such infections [4].However, recent studies in Europe and the United States have demonstrated a steady increase in the rate of uropathogen resistance to commonly prescribed antibiotics, and this obviously will lead to a reduction in therapeutic possibilities of UTI [2,5].The most recent worldwidе estimates of glоbal antibiotic resistance, publishеd by the Wоrld Health Organization (WHO) in 2014, list Escherichia cоli, Klebsiella pneumoniae, and Staphylоcoccus aureus as the three agents of greatest concern, associated with both hospital -and cоmmunity acquirеd infections.In five of the six WHO rеgions, some countries reported E. coli rеsistance of more than 50 pеrcent to fluoroquinolones and thirdgeneration cephalоsporins [6].In the United States, CDC has estimated that more thаn 2 million infections and 23,000 dеaths are due to antibiоtic resistance each year [7].In Europe, an еstimated 25,000 deaths are аttributable to antibioticrеsistant infеctions [5].
The data about the prevalence of plasmid-mediated quinolone resistance genes in our country is quite limited.Moreover we did not find any publications regarding the prevalence of plasmid-mediated resistance genes to fluoroquinolones and its association with ESBL-prоducing plasmids in Ukraine as well as in contiguous countries, like Belarus, Moldova or Russia.
Therefore, the aim of our cross-sectional study was to determine the prevalence of plasmid-mediated quinolone resistance genes among urinary strains clinically isolated from hospitalized patients with pyelonephritis, treated in the Kharkiv City Clinical Emergency Hospital, Ukraine and to identify the presence of genes involved in the resistance, specifically -aac(6')-Ib-cr, qnrA and efflux pump QepA.

SUBJECTS AND METHODS
A cross-sectional study of 105 adult patients with pyelonephritis (the diagnosis pyelonephritis has been stated in accordance with the criteria established by European Association of Urologists), who were admitted in Kharkiv City Clinical Emergency Hospital, Ukraine, was carried out.The study has been carried out between April 2013 and February 2014.The patients were considered as those who have chronic kidney disease (CKD) G1-G4 stage, where the stage was evaluated using glomerular filtration rate (GFR) index calculated by formula CKD-EPI (KDIGO 2012) [16].
Our study is a prospective one.All the patients were discharged from the hospital; no mortality case was reported during hospitalization.

Bacterial Isolates
Midstream urine from the patients with pyelonephritis collected in a sterile container and processed in the medical biology department of the Kharkiv City Clinical Emergency Hospital within 2 h of collection.Urine samples were inoculated on blood agar or chromogenic media ChromID CPS (bioMerieux, France) then positive cases were incubated at 37°C for 24 hours, while negative cases -at 37°C for 48 hours.The samples were considered as significant if the number of colony forming units (CFU) was ≥10 5 CFU/ml of urine.Identification of Gram positive сatalase negative cocci (Streptococci, Enterococci and related genera) was performed using test systems ID 32 STREP production of bio Mérieux, France.Identification of Gram positive сatalase positive cocci (Staphylococcus, Micrococcus and related genera) was performed using test systems ID 32 STAPH production of bio Mérieux, France.Identification of Gram negative bacillus -Enterobacteriaceae and other non-fastidious Gram-negative Bacillus was performed using test systems ID 32 GN production of bio Mérieux, France.Commercial identification kits were used according to the manufacturer's instructions.

Detection of Plasmid-mediated Resistance Genes
The total DNA extraction was performed for all samples using the heat-shock technique.Screening for the presence of plasmid-mediated resistance genes was performed by polymerase chain reaction (PCR) sequencing assays.The following primers used for extended spectrum beta-lactamases and genes involved in the resistance to fluoroquinolones are shown in Table 1.PCR products were analyzed by agarose gel electrophoresis and stained with ethidium bromide.Plasmid DNA, used as a molecular weight marker, was hydrolyzed by the enzyme puc19 HpaII [18].

Statistical Analysis
Statistical analysis was performed using the statistical the Statistical Package for the Social Sciences (SPSS), version 20.Categorical data (sex, setting and susceptibility to antibiotics) were presented as the number and percentage.The percentages in different categories were compared using Chi square test.The statistical analysis was done by using the proportions of sensitive, resistant and intermediates.A difference was considered to be significant if the probability that chance would explain the results, was reduced to less than 5% (p ≤ 0.05).For each potential risk factor, odds ratios and 95% confidence intervals were calculated using multivariate analysis.

Ethics
Each patient was aware about the data collection, and written informed consent was obtained from each subject.The study protocol has been approved by the ethics committee at the Kharkiv Academy for Postgraduate Education, Kharkiv, Ukraine (No. 2, 22.02.2013).Anonymity was guaranteed during and after the study.The study is not invasive to be fully consistent with the Declaration of Helsinki as a statement of ethical principles for medical research involving human subjects approved by The World Medical Association.
Out of 105 patients, 20 (19%) and 9 (9%) had ESBLs and PMQR positive urine cultures, respectively.Two patients (2%) were infected with both ESBLs and PMQR producing urinary strains.Clinically, the vast majority of the patients with resistance genes had flank pain (92.6% vs. 85.2%P = 0.045), symptoms of lower urinary tract infection (59.8% vs. 45.6%P = 0.002), and costovertebral angle tenderness (72.2% vs. 64.9%P = 0.015).The mean body temperature in patients infected with resistance bacteria was 38.3±0.1 °C, whereas in patients without resistance gene was 37.9±0.3°C.In both the treatment groups, the prevalence of patients infected with ESBLs producing urinary strains was significantly higher, than the prevalence of patients, infected with PMQR producers.Treatment by fluoroquinolones demonstrated the best favorable clinical response in the patients infected with ESBL-producing organisms, whereas cephalosporins were the most effective in patients infected with fluoroquinolone resistance uropathogens.Of note, that 12 (44.4%) of the ESBL-producers were isolated on the fifth day after the beginning of antibiotic therapy.However, the hospitalization term was longer for the patients with plasmid-mediated genes compared to the patients without this gene (median 10.4 days vs. 8.4 days, P < 0.05).All the patients were discharged from the hospital; no mortality case was reported during hospitalization.In the treatment group 1, a favorable clinical response was seen in 6 of 13 patients (46.2%) infected with ESBL-producing organisms compared with 4 of 6 patients (66.7%) infected with PMQR-producing organisms (p<0.05).In the treatment group 2, a favorable clinical response was seen in 5 of 7 patients (85.7%) infected with ESBL-producing organisms compared with 1 of 3 patients (33.3%) infected with PMQRproducing organisms (p<0.05).In contrast, clinical efficacy of antibiotic therapy was higher in patients without plasmid-mediated genes existence and favorable clinical response was seen in 30 of 37 patients (81.1%) and 35 of 37 patients (94.6%) treated by cephalosporins and fluoroquinolones, respectively.
In this study, only meropenem exhibited a good enough activity against PMQR-producing urinary strains.Nitroxolinum and fosfomycin were also found to be highly effective drugs in vitro.The resistance rates of PMQRproduces were significantly higher compared to non-PMQR-producing urinary strains.Moreover, the resistance rates have been substantially growing up among all isolated uropathogens to commonly prescribed antibiotics, such as third generation cephalosporins and fluoroquinolones, which are the main therapeutics effective to treat such infections.Out of 105 patients, 31 (29.5%) were infected with resistance bacteria.Clinical efficacy of antibiotic therapy was lower in these patients.Moreover, the hospitalization term was longer for the patients with plasmid-mediated genes compared to the patients without this gene (median 10.4 days vs. 8.4 days, P < 0.05).Longhi C et al. suggest that the prevalence of PMQR genes among uropathogenic Escherichia coli was 11% in outpatients and 21% inpatients [24].
It has been clearly observed in other papers that therapeutic options are limited in patients infected with resistance bacteria.Moreover, antimicrobials activity in vitro and in vivo may be different in the case of such infections.According to Paterson and colleagues, who published a series that included 32 patients (age ≥ 16 years) with Klebsiella bacteremia where the organism was confirmed by phenotypic tests to produce an ESBL.Despite ESBL production, the MICs of all of these organisms were in the susceptible or intermediate range to the cephalosporins used for treatment.Out of 32 study patients, 19 (59%) experienced clinical failure despite the lack of laboratory-determined resistance to the drug used [25].Kim et al. examined patients with bloodstream infections due to E. coli or K. pneumoniae.Among patients treated with a cephalosporin to which the organism was susceptible, a favorable clinical response was seen in 9 out of 17 patients (52.9%) infected with ESBL-producing organisms compared with 47 out of 50 patients (94.0%) infected with non-ESBL-producing organisms (p<0.001)[26].In our study, treatment by fluoroquinolones demonstrated the best favorable clinical response in the patients infected with ESBL-producing organisms, whereas cephalosporins were the most effective in patients infected with fluoroquinolone resistance uropathogens.In the treatment group 1, a favorable clinical response was seen in 6 out of 13 patients (46.2%) infected with ESBL-producing organisms compared with 5 of 6 patients (83.3%) infected with PMQR-producing organisms (p<0.05).In the treatment group 2, a favorable clinical response was seen in 5 out of 7 patients (85.7%) infected with ESBL-producing organisms compared with 1 out of 3 patients (33.3%) infected with PMQR-producing organisms (p<0.05).Besides, 12 (44.4%) of the ESBL-producers were isolated on the fifth day after the beginning of antibiotic therapy.Thus, in vitro-in vivo differences in resistance bacteria became an established problem, and therefore, ESBL and PMQR producing оrgаnisms pose a major challenge for clinicians, limiting therapeutic options.
The known risk factors for quinolone resistance in uropathogenic E. coli are prior exposure to quinolones, previous hospitalization, recurrent UTIs, previous invasive procedures, the presence of complicated UTIs, chronic diseases including neurologic diseases, age over 50 years, and the presence of a urinary catheter in the past 6 months [27 -29].In our study, the main factors related to the appearance of plasmid-mediated resistance genes were Chronic Kidney Disease stage ІІІ (OR 2.03) and ІV (OR 1.1), hypertension (OR 2.57), age range above 55 years (OR 3.05), in-patient treatment history (OR 2.02), and the history of using antibiotics last year (OR 1.41).

CONCLUSION
The prevalence of PMQR among uropathogens, isolated from hospitalized patients with pyelonephritis was 12.2%.Efflux pump QepA was observed to be the most common isolated gene.Out of 105 patients, 31 (29.5%) were infected with resistance bacteria.Global resistance to fluoroquinolones was ≥ 20% among isolated gram-negative strains.In this study, 100% of the PMQR producing isolates were identified as meropenem susceptible.Fosfomycin, nitroxolinum and co-trimoxazole were also found to be highly effective drugs in vitro.Treatment by fluoroquinolones demonstrated the best favorable clinical response in the patients with ESBL-producing organisms, whereas cephalosporins were the most effective in patients infected with fluoroquinolone resistance uropathogens.The main risk factors related to the appearance of plasmid-mediated resistance genes were Chronic Kidney Disease stage ІІІ (OR 2.03) and ІV (OR 1.1), arterial hypertension (OR 2.57), age range above 55 years (OR 3.05), in-patient treatment history (OR 2.02), and the history of using antibiotics last year (OR 1.41).
Taking all these things into consideration, we can assume that isоlation and detеction of ESBLs and PMQRprоducing urinary strаins are еssential fоr the sеlection оf the mоst effеctive antibiоtic for the empiric trеatment.Since, the most of ESBLs and PMQR genes are cаrried by plаsmids, thеse gеnes could be eаsily trаnsferred amоng hоspitalized pаtients.This is a mаjor fаctor responsible for increаsing the sprеad of both ESBL and PMQR prоducеrs.Moreover, therapeutic alternatives for the treatment of UTI patients with resistant uropathogens, particularly in hospitalized patients, are limited.Further clinical studies are needed to establish the guideline for the management of patients with plasmid-mediated resistance.