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Prevalence of plasmid-mediated resistance genes among multidrug-resistant uropathogens in Egypt

Background The emergence of multidrug-resistant (MDR) uropathogens has become a public health threat and current knowledge of the genotypic basis of bacterial resistance is essential for selecting appropriate treatment options. Objectives To determine the prevalence of antimicrobial resistance among MDR uropathogens and to elucidate the molecular bases of plasmid-mediated resistance. Methods Bacterial isolates were recovered from urine specimens of 150 out-patients with signs and symptoms of urinary tract infections (UTIs) at El-Demerdash Hospital, Cairo, Egypt. Standard methods were used for identification, antimicrobial susceptibility testing was performed according to CLSI guidelines. Results Among the recovered isolates, 22.7% and 77.3% were Gram-positive, and negative, respectively. Of which; 43.3% were MDR with 60% harboring plasmids. Extended spectrum β-lactamase (ESBL) genes blaCTX-M, blaSHV, and blaTEM were detected on plasmids of 89.7%, 41%, and 84.6% of the tested isolates, respectively. The aminoglycoside resistance gene aac6′-Ib/aac-6′-Ib-cr was found on plasmids of 92.3% of the tested isolates followed by qnrS (92.3%), qnrB (46.2%), and qnrA (7.7%). The most prevalent quinolone efflux pump gene was oqxB (38.5%), followed by oqxA (20.5%), then qepA (10.3%). Conclusion High levels of resistance to nitrofurans, β-lactam/β-lactamase inhibitor, cephalosporins, aminoglycosides, and fluoroquinolones were detected, and their use as empirical treatment for UTIs has become questionable.


Introduction
Urinary tract infection (UTI) is one of the most common infections worldwide which may be caused by Gram-negative or Gram-positive bacteria, as well as by some fungi. The most common causative organism is Escherichia E. coli 1 . Antimicrobial resistance (AMR) has become a ma-jor threat to public health in many countries. There has been a steady increase in AMR to the agents commonly used in treatment of UTIs 2,3 . In most UTI cases, empirical therapy is initiated before the results of urine culture and sensitivity are available, thus it is necessary to have AMR surveillance 4 . The emergence and spread of multidrug-resistant (MDR) organisms, which show resistance to three or more classes of antimicrobials 5 , is increasing over time; and UTI cases requiring intravenous therapy due to the lack of effective oral treatment has become a challenge for physicians, complicating a previously simple-to-treat infection 3 .
Extended Spectrum Beta Lactamases (ESBLs) have emerged as a chief mechanism of resistance among uropathogens 6 . These ESBLs are enzymes that trigger the resistance against β-lactam antibiotics by hydrolysis of the African Health Sciences Vol 20 Issue 1, March, 2020 190 β-lactam ring 7 . Regrettably, ESBL-producing organisms usually carry resistance determinants to other antimicrobial agents as well,such as aminoglycosides and fluoroquinolones, leaving a limited range of treatment options 6 . The aim of this study was to reveal the prevalence of antimicrobial resistance and the molecular bases of plasmid-mediated resistance among bacterial uropathogens in one of the major clinical settings in Cairo, Egypt.

Materials and methods Specimen collection
Starting October 2015 to May 2016, a total of 150 bacterial isolates were recovered from urine specimens of patients suffering signs and symptoms of UTIs at the outpatient clinics of El-Demerdash Hospital, Cairo, Egypt. All specimens were mid-stream urine and patients were instructed on how to collect specimens to avoid contamination. Patients included in the study were adults (ages ranging from 25 -45 years), symptomatic, with pyuria (Pus cells ≥20/HPF), and the bacterial count in urine was >10 5 cfu/ml. The study was approved by Faculty of Pharmacy, Ain Shams University Ethics Committee Nr. 212 and an informed consent was obtained from patients after explaining the study purpose.

Identification of the recovered bacterial isolates
Isolates were categorized based on their Gram reactions, followed by identification using standard methods. Identification to the species level was done for MDR isolates by using API ® 20E identification kit and API ® Staph identification kit (BioMérieux, France) for Gram-negative and Gram-positive isolates respectively.

Antimicrobial susceptibility testing
The Kirby-Bauer disk diffusion test was performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines 8,9 using commercially available antimicrobial disks (Oxoid, UK). The reference strains E. coli ATCC ® 25922 and Staphylococcus S. aureus ATCC ® 25923 were used for quality control. All MDR isolates were selected for further studying.

Determination of minimum inhibitory concentration of multidrug-resistant isolates
Minimum inhibitory concentration (MIC) values of MDR isolates were determined by broth microdilution method according to CLSI guidelines 9,10 using ceftriax-one, cefepime, meropenem, gentamicin, and ciprofloxacin. The reference strains E. coli ATCC ® 25922 and S. aureus ATCC ® 25923 were used for quality control.

Extraction of DNA plasmids from multidrug-resistant isolates
The extraction of DNA plasmids from MDR isolates was done using Zyppy™ Plasmid Miniprep Kit (Zymo Research, USA) according to the manufacturer's instructions. The extracted DNA plasmids were analyzed via agarose gel electrophoresis 11 and visualized by UV transilluminator.

Amplification of plasmid-encoded resistance genes
Amplification of antibiotic resistance genes was carried out by polymerase chain reaction (PCR) using the appropriate primers (Table 1); and the DNA plasmids of the MDR isolates as templates. Primers were manufactured by LGC Biosearch Technologies, USA. The amplified products were analyzed via agarose gel electrophoresis, and the expected DNA product size was determined by comparing to a 100 bp DNA ladder (New England Biolabs, UK). The antibiotic resistance genes amplified in this study included ESBL genes ( bla CTX-M, bla SHV, and bla TEM); the aac(6')-Ib gene conferring resistance to aminoglycosides, and its bifunctional variant aac(6')Ib-cr conferring resistance to both aminoglycosides and ciprofloxacin; low level resistance plasmid-mediated quinolone resistance (PMQR) genes (qnrA, qnrB, qnrS), and quinolone efflux pump genes (qepA, oqxA , oqxB).

Sequencing of selected PCR products
Some selected PCR products of amplified genes were sent for sequencing at GATC, Germany using ABI 3730 xl DNA Sequencer. The alignment and assembly of the obtained forward and reverse sequence files into the final consensus was done using BioEdit v7.2.5 software 16 .

Transformation
Plasmids extracted from the MDR isolates were used to transform competent E. coli DH5α prepared according to the modified Hanahan method 17 to test the phenotypic resistance of the transformants. Transformants were cultured on LB/ampicillin, LB/gentamicin, and LB/ciprofloxacin agar plates at concentrations of 100 µg/ml, 25 µg/ml, and 50 µg/ml, respectively.

Statistical analysis
Categorical variables were analyzed using the Chi-square test to determine statistical significance. Statistical analysis including descriptive statistics, frequency tables, and cross-tabulations was performed using Statistical Package for the Social Sciences software IBM ® SPSS ® version 20 18 . A value of P<0.05 was considered statistically significant, and significance was two-sided.

Antimicrobial susceptibility testing
The antimicrobial susceptibility patterns of the recovered isolates are shown in Table 2

Minimum inhibitory concentrations of MDR isolates
The obtained MIC results of MDR GNB and GPC iso-lates are shown in supplementary tables S2 and S3, respectively.   Extraction of DNA Plasmids from MDR isolates DNA plasmids were extracted from 39 (60%) of the 65 MDR isolates. The extracted plasmids were analyzed via agarose gel electrophoresis, and the band sizes were compared to a 1 kb DNA ladder (New England Biolabs, UK).

Amplification of some plasmid-encoded resistance genes
Results of PCR amplification of the ESBL genes ( bla C-TX-M, bla SHV, and bla TEM); the aac(6')-Ib gene, plasmid-mediated quinolone resistance (PMQR) genes (qnrA, qnrB, qnrS), and quinolone efflux pump genes (qepA, oqxA , oqxB) are depicted in figures S1, S2 and S3. The prevalence of amplified antibiotic resistance genes among MDR bacterial uropathogens is shown in Figure 3. Figure 3. Prevalence of some selected antibiotic resistance genes among MDR bacterial uropathogens. Prevalence was expressed as percent of isolates carrying the tested genes relative to total tested isolates (n=39)

Genotypes of MDR isolates
Among the 65 MDR isolates; 22 different genotypes were observed based on the PCR detection of antimicrobial resistance genes on the extracted DNA plasmids of the MDR isolates, as shown in Table 3.

Transformation
The results of transformation along with PCR amplification for the tested isolates are shown in Table S4. In case of plasmids harboring ESBL coding genes (n=39), successful transformation and gene expression was achieved with plasmids extracted from 28 isolates (71.8%) harboring such plasmids; demonstrated by the ability of transformants to grow on LB/ampicillin agar plates. Out of 36 MDR isolates that carried the aminoglycoside resistance gene aac(6')-Ib/aac(6')-Ib-cr, the plasmids extracted from 15 isolates (41.7%) were successfully transformed and resistance genes were expressed; which was demonstrated by the ability of transformants to grow on LB/ gentamicin agar plates. Isolates that carried any of the plasmid-mediated quinolone resistance (PMQR) genes (aac(6')-Ib-cr, qnrA, qnrB, qnrS, qepA, oqxA, and oqxB) were found to be 38 out of the 39 tested MDR isolates (97.4%); plasmids extracted from 10 of which (26.3%) were successfully transformed and resistance genes were expressed; demonstrated by the ability of transformants to grow on LB/ciprofloxacin agar plates.

Statistical analysis
Statistical analysis has shown that there is significant association between antimicrobial resistance and PCR detection of the respective genes on DNA plasmids. There is also significant co-existence of PCR-detected antibiotic resistance genes on DNA plasmids of the same isolate (P value <0.05). The statistical association and respective P values are shown in Table 3.

Discussion
As reported, UTIs are becoming more difficult to treat due to the emergence and prevalence of a wide range of antibiotic resistance mechanisms 3 . Accordingly, in this study we assessed both the phenotypic and genotypic bases of antimicrobial resistance of some MDR uropathogens against the most common antimicrobial agents used in treatment of UTIs. The antimicrobial susceptibility of the GPC collected in this study (n=34) showed that the lowest resistance was observed to doxycycline, vancomycin, and imipenem (5.9%, 8.8%, 10.3%). On the other hand, the highest resistance was observed to nitrofurantoin (50.0%); cefoxitin, ceftazidime (44.8% each); ampicillin/sulbactam and cefepime (41.4% each). The antimicrobial susceptibility of the GNB (n=116) showed that the lowest resistance was observed to doxycycline (11.0%) and imipenem (16.4%). The highest resistance was observed to ampicillin/sulbactam (52.3%); co-amoxiclav (49.5%); ceftazidime (42.2%); cefepime (41.4%); and ciprofloxacin (40.5%). These results limit the use of nitrofurans, cephalosporins, β-lactam/β-lactamase inhibitors, and fluoroquinolones as empirical treatment of UTIs, while tetracyclines and carbapenems still retain their efficacy in treating UTIs based on in vitro data. Some other studies deduced that imipenem showed highest efficacy and may be the drug of choice for empirical therapy of UTIs based on the in vitro data [19][20][21] .
In this study, PCR amplification was used to detect some plasmid-mediated antimicrobial resistance genes associated with the 39 MDR isolates harboring plasmids. Our results also showed that there is a significant association between the presence of bla CTX-M gene and aac6'-Ib/aac-6'-Ib-cr gene in the same isolate (P=0.001). Accordingly, isolates that produce ES-BLs also carry resistance genes to aminoglycosides and fluoroquinolones, thus reducing treatment options. This cross-resistance is more prominent in urinary isolates 3 . Therefore, carbapenems, which are less prone to hydrolysis by such enzymes, have become the preferred therapy for infections with ESBL-producing pathogens [24][25][26][27] . The most prevalent qnr gene in our study was qnrS gene (36/39; 92.3%), followed by qnrB (18/39; 46.2%), and qnrA (3/39; 7.7%). This prevalence pattern is in accordance to that obtained in a study conducted by several recent studies [28][29][30] . The quinolone efflux pump resistance genes were detected in 15 (38.5%) out of the 39 MDR isolates and their prevalence was highest for oqxB (15/39; 38.5%), followed by oqxA (8/39; 20.5%), and qepA (4/39; 10.3%). It should be noted that the acquisition of PMQR genes alone results in low levels of resistance to fluoroquinolones, and does not cause MICs to exceed the breakpoints of these agents 31 , but rather facilitates the selection of mutants of higher-level resistance 32 . This was evident in our study by the lack of statistically significant association between the presence of PMQR genes and resistance to fluoroquinolones, which means the presence of PMQR genes alone, did not confer resistance to fluoroquinolones.

Conclusion
High levels of resistance to antimicrobials commonly used for treatment of UTIs was detected among MDR uropathogens. The current efficacy of nitrofurans, β-lactam/β-lactamase inhibitor, cephalosporins, aminoglycosides, and fluoroquinolones has become questionable. Carbapenems, tetracyclines, and vancomycin have yet to retain their efficacy in treatment of UTIsbased on in vitro data. No significant correlation was observed between the presence of PMQR genes and fluoroquinolone resistance, indicating that PMQR genes alone do not grant phenotypic resistance to fluoroquinolones, however the resistance may have been due to co-existence of ESBL and/or aac6'-Ib/aac-6'-Ib-cr genes in the same isolate or even on the same plasmids.