Molecular Pro(cid:2510)ile of Aminoglycoside, Fluoroquinolone, and Class 1 Integron Genes among Gentamicin-Resistant Escherichia coli in Najaf City, Iraq

Escherichia coli has a major cause of women urinary tract infection, which it harbours various kinds of drug resistance-associated genes. So, the current study examined the prevalence and frequency of genes. These genes are responsible for the resistance of aminoglycoside and (cid:977)luoroquinolone drugs in uropathogenic gentamicin-resistant E. coli isolated from urinary tract infections among women. Six hundred urine specimens were tested. The data revealed 348 (58%) and 70 (11.66%) had gram-positive and gram-negative, respectively. The other 182 (30.33%) were found without any growth. A total of 600 clinical specimens were 167(27.833%) identi(cid:977)ied as E.coli isolate according to biochemical tests and Vitek-2 System. The phenotypic gentamicin-resistant screening (MIC and disk diffusion) revealed out of 167(27.833%) E.coli isolates were 25(4.166%) gentamicin-resistance. Antibacterial agents susceptibility of 25 gentamicin-resistant E.coli isolates showed concern level of resistance among different categories of antibacterial agents, ranged from high resistance 25(100%) for nalidixic acid to less rate of resistance 4/25(16%) by imipenem drug. Molecular data have demonstrated the prevalence of associated resistance genes for both aminoglycosides and (cid:977)luoroquinolones. Among 25 gentamicin-resistant E.coli isolates 24/25(96%) were harbours for the genes gyr-B, aac(6’)-Ib-cr, strA/B, and 23/25(92%) of isolates were harbouring for the genes gyrA, qnrS, and aacC-2. In con-trast, qnr-B, aac(6 ′ )/aph(2 ′ ), and aph(3)lla were identi(cid:977)ied in 20/25(80%), 11/25(44%) and 8/25(32%) respectively. At the same respect, aacC-1, qnrA, and qnrC genes were no detect in the current study. However, 24/25(96%) of isolates were carrying the class 1integron (intel-1) gene.


INTRODUCTION
Urinary tract infection (UTIs) is one of the infectious diseases that have a high frequency and prevalence among different groups of society. Uropathogenic Escherichia coli have a signi icant role in most hospital-acquired infections as well as It is the principal cause of various clinical specimens, especially urine (Abad et al., 2019).
Antimicrobial resistance has been a cause of concern and alarm over the period time worldwide, especially in developed and developing countries (Rather et al., 2017). Aminoglycosides drugs are signi icant antimicrobial agents for the treating of several infections in humans. At the same respect, gentamicin has shown marked as perfect curative options for UTIs treatment (Goodlet et al., 2018).
Quinolones drugs are broadly applied to heal UTIs generated via E. coli. This expanded application of quinolones becomes led to enhanced resistance in E. coli. Target alteration and modi ications in the permeability of the cell membrane can give quinolone resistance. Moreover,plasmid-mediated quinolones genes such as qnrA,qnrB, and qnrS can promote resistance to these drugs (Düzgün et al., 2019).
Recently, traditional drug resistances and increase of multi-drug resistance (MDR) organisms in UTIs are related to higher averages of unsuitable empirical medication due to weakening drug covering. E. coli, like other gram-negative bacteria, the aminoglycosides resistance usually are mediated mainly through enzyme production; it changes or modiies antibiotics either by adenylation, acetylation or phosphorylation and maybe by ef lux pump mechanism. (Reygaert, 2018).

Specimens collection
The contemporary study was associated with 600 non-duplicate women patients undergoing UTIs of both Medical Al-Sader City and Al-Hakim General Hospitals as well as leading clinical laboratories in Najaf City, from January to June of 2019. A clean catching midstream urine specimens collected in a sterile urine container and carried directly to the Advance Medical Bacteria Laboratory, Department of Biology, Faculty of Science, University of Kufa, Iraq.
All specimens were cultured and streaking on different media included blood agar (Oxoid, England), MacConkey agar (Oxoid, England), chromagar orientation (CHROMagar TM , France) till reached a single colony. The petri-dish were incubated under aerobic conditions overnight at 37 • C. Moreover, a negative growth incubated for two days. The present investigation depended on specimens that concentration bacteria at least 10 5 colony-forming unit (cfu) per ml. Isolates of E.coli were diagnosis based on the characters of microscopic, the colour of Chromagar Orientation media., IMViC tests, motility and oxidase tests (MacFaddin, 2000). Vitek-2 system (bioMèrieux France) used to con irm the diagnosis.

Screening of gentamicin-resistant E.coli isolates using minimal inhibitory concentration (MIC) strip and disk diffusion
Phenotypically, all isolates of uropathogenic E.coli procedure were investigated and screened. These were to detect gentamycin-resistant E.coli. For detecting these E.coli isolates, both gentamicin MIC strip (Lio ilchem ® , MTS, Italy), covering of 0.016-266 µg/mL and gentamicin disk (10µg) (Bioanalyse, Turkey) were used. It was employed on sterile media of Mueller Hinton agar (England) The suspension of all tested isolates were achieved based on 0.5 McFarland standard. The MIC is recorded at the point where the edge of the inhibition ellipse touches with the Strip further the results were described via the instructions guide of the Clinical Laboratory Standards Institute (CLSI, 2018). At the same time, all these achieved at the same moment beside the procedure of disk diffusion; furthermore, all plates were incubated at the same conditions. The strain of E.coli ATCC 25922 employed as the negative control.

Antibacterial Agents susceptibility
The present study covered testing susceptibility pro ile of various commercial classes of antibacterial agents (Bioanalyse, Turkey) against all 25 isolates of gentamycin-resistant E.coli according to Kirby-Bauer procedure using sterile media of Mueller Hinton agar (Oxoid, England), (Bauer et al., 1966). The resistance, intermediate and sensitive of isolates were expressed according to guide instructions of the clinical and Laboratory Standards Institute (CLSI, 2018). Disks of antibacterial agents and their concentration marked in the Table 5.

Total DNA extraction
The entire genomic DNA of 25 isolates of gentamycin-resistant E.coli was extracted after 24 hours of liquid growth for these pathogens using a kit of the total genomic DNA extraction (iNtRoN, Biotech. Inc., Korea), wherever the extraction was completed according to the instructions of manufacture company. The DNA was saved below -20 o C situation employing deep freezing apparatus, till executed PCR to the investigation of gyr-A, gyr-B, aac(6')-Ib -cr, qnrA, qnrB, qnrC, qnrS, aacC-1, aacC-2, strA/B, aac(6 ′ )/aph(2 ′ ), aph(3)lla and Int-1 genes by speci ic primers and requirements listed in Table 1 and Table 2. The system of gel document (Cleaver, United Kindom), applied to examine and separate the migration of PCR products using 1% agarose (iNtRoN, Biotech. Inc., Korea), after staining the gel with 0.5 µg/ml ethidium bromide.

Specimens collection and E.coli identi ication
The present study was involved 600 no duplicate urine specimens taken from woman patients suffering from UTI, who attended to main hospitals and clinical laboratories in Najaf city-Iraq, through the period the time ive months (from January to June of 2019). Results in Table 3 were demonstrated that the percentage of the bacterial growth was 348 (58%) and 70(11.66%) to both Gram-negative and gram-positive bacteria, while no bacterial growth was 182(30.33%).
According to microscopic features, culture growth on MacConkey Agar, Chromagar Orientation, biochemical tests and inally all suspected E. coli isolates were con irmed using the Vitek-2 system, the data showed the number and percentage of this pathogen reached to 167(27. 833%) isolates.

Antibacterial agents susceptibility of gentamicin-resistant E.coli isolates
Depending on the results of 20 antibacterial agents susceptibility in the Table 5, most gentamicinresistant E.coli isolates were high resistance against    most various antibiotics categories used in the current study. At same respect, data of antibacterial agents susceptibility showed that uropathogenic gentamicin-resistant E.coli isolates regarded as multidrug resistance (MDR) through all isolates were resisted to three classes of antibacterial agents and above. However, these pathogens revealed a high effect on beta-lactam drugs (except imipenem) where resistance rates were 24(96%), 23(92%), 22(88%) and 20(80%) to Ampicillin, ceftriaxone, cefoxitin and aztreonam respectively. While imipenem drug was the best in inhibition the bacterial growth, the resistance rate was 4(16%) and this lowest rate among all antibacterial agents which used in current work.
Although all 25 E.coli isolates were resistant to gentamicin, the data in the Table 5 proved different resistance rates among aminoglycosides drugs. They were high resistance reached to 24(96%) in both amikacin and streptomycin, 23(92%) in tobramycin, while they were low resistance reached to 10(40%) in both o loxacin and nor loxacin. At the same time, moderate resistance to netilmicin reached to17(68%).
This study also focused on investigation and distribution of genes which were responsible for the resistance of both quinolones and luoroquinolones drugs among 25 isolates of gentamicin-resistant E.coli which isolated from the urine of non-duplicate women. The data in Figure 5and Figure 6 revealed that 24/25(96%) were harbours for the genes gyr-B, and aac(6')-Ib-cr, as well as 23/25(92%) of isolates, were harbours for the genes gyr-A, and qnrS, (Figure 7 and Figure 8), while qnrB, was identi ied in 20/25(80%), (Figure 9). At the same respect, qnrA and qnrC genes were no detect in the current study. However, 24/25(96%) of isolates were carrying the intel-1 gene ( Figure 10).

Specimens collection and E.coli identi ication
UTIs are the common persistent infection in women usually generated by bacteria. The current study appeared E. coli isolates remained the most prevalent species isolated from UTIs. Data of this study was following the indings of numerous articles from various sections of the world (Kulkarni et al., 2017).
Domestically, several studies have been conducted on the bacterial causes of urinary tract infections, all of which showed that the frequency of E. coli is the highest between gram-negative and positive bacteria. These results were near to a study carries out in Iraq (Erbil) found the rate of E.coli isolates was 44.6 % and it is highest amongst other bacteria causes which isolated from a pregnant woman suffering from UTIs (Mohammad et al., 2018).
Antibacterial agents susceptibility of gentamicin-resistant E.coli isolates MDR E. coli becomes expanded in the recent years reasonably because of the increasing and wrong application of antimicrobials agents (Kulkarni et al.,2017). The susceptibility results are shown in Table 5 revealed high resistance to E. coli isolates for different groups of antibiotics, especially those drugs used in UTIs (aminoglycosides, Fluoroquinolones, and sulfonamides drugs), which constitute a source of reduced human health, especially the elderly or those with weak immunity. Therefore, a realistic plan and solutions must be put in place to limit its spread. However, the results of this study are closely related and somewhat compatible with previous local studies (Almamoori et al., 2019). The study also showed the sensitivity of bacteria to imipenem. Despite its high ef icacy, it is used only in critical illness cases which reduces the exposure of bacteria to this antagonist and thus reduces the chance of a mutation.

Detection of aminoglycoside, luoroquinolones and Integron class 1 genes
Aminoglycoside resistance is expanding year after year. It is signi icant and dangerous rate; this is so, not because of their capability to create chronic diseases but also because they are capable of building resistance to conventional drugs. globally and locally, Found an abundance of articles about resistance to aminoglycosides in E.coli isolates. (Chaudhary and Payasi, 2014;Fasugba et al., 2015;Tawfeeq et al., 2017).
Previous articles have done through Ho et al. (2010) observes the appearance of aacC-2 gene were a rate of 84.1% and 75.5% from E.coli isolated from human and animal, respectively. A further article by Dias-Goncalves et al. (2015) manifested about 80% of the gentamicin-resistant E. coli isolates were carried out the aacC-2 gene. However, this gene was found and detected in a study that done in Iraq on E.coli isolates and other Enterobacteriaceae (Tawfeeq et al., 2017). Sunde and Norstrom (2005) conducted the study and found the strA/B gene was high frequency among streptomycin-resistant E.coli strain. Locally, several reports achieved in Najaf City, Iraq that indicated the frequency of strA/B gene among gramnegative isolated from clinical specimens Locally, many reports delivered in Najaf City, Iraq stated the frequency of aacC-2 gene among gram-negative isolated from clinical and environmental samples (Hayder and Aljanaby, 2019; Tuwaij et al., 2019).
The horizontal transfer of genes by genetic elements like integron or plasmid among microorganisms or chromosomal mutation possesses an essential role in the acquisition of new genes to contribute drug resistance (Düzgün et al., 2019). This may be one of the reasons that explain the vast spreading of luoroquinolones genes among E. coli isolates in the current study.
An earlier article by Abbasi and Ranjbar (2018), data of PCR revealed that among 100 clinical uropathogenic E.coli isolates obtained 0%, 25% and 36% for qnrA, qnrB, and qnrS respectively. A study achieved by Ranjbar et al. (2018) in Iran found a high frequency of qnrS 92/95 (96.84%)among clinical quinolone-resistant E.coli isolates. Data of current work about the qnrA gene similar to previ-ous articles, which showed none of this contained qnrA gene (Vaz-Moreira et al., 2016;Conte et al., 2017). However, Among 25 gentamicin-resistant E.coli isolates 24/25(96%) were harbours for the genes aac(6')-Ib-cr, and this result was congruence with previous local article done by Almamoori et al. (2019) showed that 98.3% of clinical uropathogenic E.coli isolates contain aac(6')-Ib-cr gene. The primary goal for destroying quinolones drug in isolates of E.coli usually by produce DNA gyrase that constituted from two subunits encoding through gyrA and gyrB (Jaktaji and Mohiti, 2010). The results of PCR of current work revealed a high rate of gyrA and gyrB among gentamicin-resistance E.coli isolates, however, these data trend to accordance with earlier surveillance (Bhatnagar and Wong, 2019;Hassan et al., 2019).
Integrons can be found inside plasmid or transposons and transport along with them, as well as it promotes the diffusion of antibacterial resistance genes among microorganisms causing dangerous public health effects (Khoramrooz et al., 2016). A high prevalence of intl1in current study may be due to all isolates were clinical and isolated from the patient. A study achieved by Oliveira-Pinto et al. (2017) pointed that molecular examination of integrase gene (intl-1) exhibited a higher frequency of class 1 integrons in isolates of uropathogenic E. coli reach to 65 % compared with 11.9 % in commensal isolates. However, numerous researches are examining the predominance of integrons in uropathogenic E. coli isolates have recorded a notable relationship between antibacterial resistance and integrin. (Khoramrooz et al., 2016).

CONCLUSION
The current research proved that gentamicinresistant E.coli isolated from women infected with UTIs was a signi icant rate, as well as these isolates, were resistant to multiple commercial antimicrobials agents. In the same respect, imipenem drug was an effect on bacterial growth. A high frequency of aminoglycoside and luroquinolone genes is a concern in the country.