Distribution of dfrA1 and cat1 antibiotic resistance genes in uropathogenic Escherichia coli isolated from teens pregnant women in Iraq

The present study aims to detect the distribution of dfrA1 and cat1 antibiotic resistance genes among uropathogenic Escherichia coli (UPEC) in pregnant teens women and determine their susceptibility to common antibiotic uses. We collected urine (116) samples from patients in hospitals in Baghdad, Iraq. Isolation and identification of bacteria (culturing, biochemical test, and genetically by 16S rRNA gene), antibiotic susceptibility tests (eight antibiotics), and detection of the dfrA1 and cat1 resistance genes, and used SPSS program for statistically analyzing the results. The distributed UPEC in patients most than another causative agent in percentage (50%). It was highly resistant to Trimethoprim (82%) and Cefotaxime (82%) antibiotics. And they highly distributed frequency for dfrA1 -gene (Trimethoprim resistance gene) (74%) than cat1 -gene (Chloramphenicol resistance gene) (38%).

The urine samples from patients who were admitted with UTIs were collected from Al-Alwaiya Maternity educational Hospital, and Abu-Ghraib hospital in sterile containers, the containers transfer immediately to the laboratory for microbiological analysis. Through During the period from 1st December 2019 to 1st March 2020, samples were collected from pregnant women in an aged less than 18 years old (teens). Isolation and identification of bacterial isolates The urine samples were cultured on different media Blood agar and MacConkey agar and incubated at 37 ˚C for overnight. The E. coli isolates was identified using conventional methods and molecular method by detection detecting the 16R rRNA gene at the microbiology research laboratory / Department of the Biotechnology/ College of Science/ University of Baghdad. Antibiotic susceptibility tests The E. coli isolates were tested to for the susceptibility of antibiotics by disk diffusion (Kirby-Bauer) method, by cultured culturing the bacterial colony on Muller-Hinton (MH) agar. Eight antibiotics (Mastdisc, U.K.) used in these studies: Aztreonam (ATM, 30µg), Chloramphenicol (C, 30µg), Cefotaxime (CTX, 30µg), Gentamicin (GM, 10µg), Imipenem (IMI, 10µg), Nitrofurantoin (NI, 300µg), Norfloxacin (NOR, 10µg), and Trimethoprim (TM, 5µg), the diameter of inhibition zone was measured and compared to the chart provided by National Committee for Clinical Laboratory Standard Institute (CLSI, 2017). Calculated the Multidrug resistance (MDR) when isolate which showed resistance to 3 classes of antibiotics or more. Molecular Assay The E coli Genomic was extracted using Geneaid™ DNA Isolation Kit. The extracted DNA is used as a templet template for polymerase chain reaction (PCR) amplification. PCR analysis was performed for the detection gene associated with the identification of E. coli (16S rRNA), Trimethoprim resistant (dfrA1) gene, and Chloramphenicol resistant (cat1) gene, sequence of primers and size of the product are described in Table 1.   * Statistically significant at p-value ≤ 0.05, First-trimester: (1-3pregnant month), Secondtrimester: (4-6pregnant month), Third-trimester: (7-9pregnant month). During pregnancy, UTIs with a high frequency are commonly agreeable because a the human body undergoes physiological changes in a pregnant situation [21]. The risk factors for UTIs during pregnancy beside that it could be explained to by the presence of unique structures in gram-negative bacteria which help attachment to the uroepithelial cells and prevent bacteria from urinary lavage, allowed allowing multiplication and tissue invasion, resulting in invasive infection and pyelonephritis in pregnancy as a complicated state [1]. The E. coli isolates showed at age 17 years were higher than other (15, and 16) years, and in the second pregnant were higher than other (first, and third) pregnant frequency, also in the second trimester of pregnancy was higher than a first or third trimester. E. coli has a high frequency with of UTIs in this study and other studies [22,23]. An increase in the lactose and amino acid levels during pregnancy promotes E. coli growth. There is a diversity of the pathogens which are responsible for the urinary tract infection and that would be due to the differences in host susceptibility to pathogens as a result of both biological and environmental factors which encourage biodiversity in host, pathogens, vectors, and social factors such as people's efforts in controlling disease [24]. Many virulence factors that enable infection with UTIs either secreted or surface virulence factors explain a high distribution incidence of E. coli isolates. The secreted virulence factors such as α-haemolysin (the most important secreted virulence factor) is a pore forming toxin gain enhance access to host nutrient and iron stores, and damage effector immune cells [25]. Surface virulence factors such as fimbria (adhesive molecules), these types of organelles different ways to in virulence like promoting bacterial invasion, directly triggering host and bacterial cell signaling pathways, and facilitating the delivery of other bacterial products [26,27].

Multidrug resistance
Grouping isolates to obtain a pattern of resistance is essential for clearing the view of their infectivity behavior. Accordingly, the results shown in Figure 3 indicate that forty E. coli isolates (80%) showed multiple resistance to various types of antibiotics used in this study.
The results of antibiotics show that most isolates of E. coli are resistance frequency to four and five antibiotics that are more than others antibiotic antibiotics. Lately used trimethoprim_sulfamethoxazole as the criterion antibiotic for UTIs therapy and because of UPEC strains increasing resistance to this antibiotics class. Therefore, using broadspectrum antibiotic agents fluoroquinolones with rising recurrence in complicated and uncomplicated UTIs [28], but emerged resistant to fluoroquinolones after a short time [29]. In contrast, the results showed the nitrofurantoin is a choice drug due to its lowest antibiotic resistance 16% in UPEC strains. The previous study reported that 8% to 15% of UPEC strains were resistant to nitrofurantoin [30]. Our results showed that resistance to chloramphenicol in percentage 12%, but this is a forbidden antibiotic because of having a high degree of mutation that increases bacterial resistance to this antibiotic. The Momtaz et al., 2013 study indicates that the irregular and unauthorized use in drug therapy [31]. The resistance of E. coli against the B-lactam group is the highest to cefotaxime (3rd generation cephalosporins) at 82%, and the aztreonam (monobactam antibiotic) resistance at 78%. The resistance of E. coli against β-lactam group is because of many reasons the most important ability of E. coli is the producing production of β-lactamases enzyme that lysis the β-lactam ring [32][33][34]. Resistance of bacteria to Trimethoprim often results from overproduction of the enzyme (dihydrofolate reductase, DHFR) targeted promoter for the antibiotic by promoter mutation [35]. The resistance of E. coli to Norfloxacin (fluoroquinolone) due to mutations in the enzyme DNA gyrase (decrease the binding ability of the antibiotic), the target for this antibiotic and in the topoisomerase IV the target for Norfloxacin, can render bacterial cells resistant to those antibiotics [36]. Gram-negative bacteria have developed resistance to numerous antibiotics, posing a therapeutic challenge in hospitals and communities [37]. Therefore, it is essential to raise awareness about the non-use of antibiotics only after the made of tests that reveal the sensitivity of antibiotics and describe at being by doctors in this specialty. The antibiotic resistance of bacteria could be due to transferable plasmids carrying resistant genes that are transferred among pathogenic bacteria such as pBS13, pBS12, pB2, and pMS4 plasmids [38,39]. In addition to that, a particular mutation could occur due to random use and overuse of antibiotics [40]. In recent years, the recurrence of antibiotic resistances among human E. coli clinical isolates rose substantially, posing a severe challenge in treating these illnesses. This high rate of antibiotic resistance might be attributed to the overuse of antibiotics, which has resulted in a selection of novel antibiotic-resistant bacterium strains. In the case of UPEC strains, this event is apparent, antibiotics readily available over the counter without a prescription of a registered medical practitioner, inadequate doses of antibiotics intake, lack of dependency on laboratory guidance, comparatively cheaper antibiotics intake, and selfprescription policy. The treatment of an infections that are causing caused by E. coli has become more challenging, and it can even rising a morbidity or mortality of a UTIs simple [41]. The preponderance of Gram-negative bacteria, generally Enterobacteriaceae, and notably E. coli, has been shown in several studies to restrict the regional diversity of pathogen incidence in UTIs. The resistance patterns of these organisms might differ substantially between hospitals, countries, and continents in different parts of the world [42]. Because of rising resistance to common antibiotic uses, clinicians have very few antibiotic choices for the UTIs treatment [43]. A transmission phenomenon of antibiotic resistance from bacteria to others spreads worldwide, and the results are dangerous. The selection of optimum antibiotics for treatment is essential to limit this phenomenon and form economic loss and human health. Clearly from the previous, that the choice of suitable antibiotic for treatment is not random. Still, it depends on the antibiotic suitability tests against the microbial isolate to find the appropriate antibiotic to eliminate them. To stop the emergence of resistance and obtain high efficiency in the treatment, choosing an effective antibiotic against the infectious microbial and determining its value and dose [44].

Distribution of UPEC Antibiotic Resistance Gene
The distribution of E. coli resistance gene of patients with UTI for 50 DNA extraction of isolates that identification as E. coli in this study. The results of electrophoresis appearance in 1.5% agarose gel at 70 volts for 80 mins shown in Figure 4 and Figure 5, were dfrA1 (Trimethoprim resistance) gene detected in 37 isolates (74%) of E. coli, cat1 (Chloramphenicol resistance) gene detected in 19 isolates (38%). In order to confirm the result of the uniplex presence of dfrA1 and cat1 genes in E. coli isolates, several attempts were made to standardize the reaction conditions for genes under study for Diplex PCR. Results of gel electrophoresis showed in Figure 6.   Cross-tabulation between antibiotic resistance gene and antibiotic susceptibility results of UPEC isolates for pregnant women with UTIs, and Chi-square test. The relationship between Chloramphenicol antibiotic with cat1-Gene (Chloramphenicol resistance gene) is shown in Table 4 appearance the result of the Chi-square test non-significance between them due to a present gene in 17 isolates (34%) in sensitive E. coli isolates. Table 5 showing shows the relationship between Trimethoprim antibiotics with dfrA1-Gene (Trimethoprim resistance gene). The Chi-square test result is significant between them (P<0.05).   Table 6 shows cross-tabulation between a cat1 gene with pregnant months, pregnancy frequency, and sample age. In contrast, Table 7 shows cross-tabulation between the dfrA1 gene with pregnant months, pregnancy frequency, and sample age.  The results from Table 6 and Table 7 show that these genes are more distributed in the second trimester, second pregnancy, and at 17 years for both genes.  [22] found the percentage was 15.44% and 11.3%, respectively less than for this study and in Siasi et al., 2017 [49] was 92.1% most more than for this study.
Using Diplex PCR in this study aims to reduce time and cost by detecting more than one gene in one reaction compared to the Uniplex PCR. This method was used by researcher Al-Alak, 2012 [50] in a molecular study of the adhesion factors in UPEC and its relationship to fluoroquinolones resistance. Kareem also used this method, 2013 [51] researcher to detect the S.T. and L.T. genes in an E. coli bacteria isolated from urine and stool samples by using multiplex PCR technique. Also used this method by Baqer (2013) [52] investigate method comparative study of E. coli O157:H7 isolates from patients in Baghdad, as well as used by Mohammed and Rasheed (2015) [53] for the detection of fimh, sfa, pap, and afa genes in E. coli bacteria isolated from urine samples used multiplex PCR technique.

Conclusion
The genetic detection for of microbial is more efficient than other methods. The E. coli is the most causative agent causing UTIs in patients in our study. The appearance shows variable resistances to various antibiotics, and the dfrA1 gene for Trimethoprim resistance is more than the cat1 gene for Chloramphenicol resistance.