Detection of resistance genes (gyrA,qepA,drf1,drf17) for E.coli in Iraqi aquatic environment

The control of water represents the safe key for fair and optimal use to protect water resources due to human activities, including untreated wastewater, which is considered a carrier of a large number of antibiotic-resistant bacterial species. This study aimed to investigate the prevalence of antibiotic-resistance to E. coli in Tigris River by the presence of resistance genes for aminoglycoside( qepA ( ,quinolone ( gyrA ), and sulfa drugs( dfr1 ,dfr17 ) due to the frequent use of antibiotics and their release into wastewater of hospitals. Samples were collected from three sites on Tigris River: S1( station wastewater in Adhamiya), S2 (station wastewater in Baghdad Medical city hospital), S3 (station wastewater in Abu Nuwas) from February-July 2021. Out of 67 isolates of bacteria, only 40 isolates of E. coli were detected by Vitek2. The antibiotic-resistance was estimated by the disk diffusion method. All E.coli isolates were tested against 6 antibiotics. The results showed the high resistance antibiotic of E. coli against Ceftazidime 70%, with intermediate resistance to Cefotaxime 47.5%, and low resistance to the sulfa drugs as Trimethoprim 27.5% and quinolones antibiotics as ciprofloxacin 17.5%, aminoglycosides as Amikacin and Gentamycin 5% and 7.5%. Moreover, the results revealed that gyrA gene was detected in 4 isolates (10%) while drf1 and drf17 genes were in 2 isolates of each gene (5%).Whereas qepA gene has not appeared in isolates. In conclusion, the isolates of E.coli from the Tigris River showed low resistance to sulfa drugs and quinolones , aminoglycosides. The resistance genes ( gyrA,drf1,drf17 ) were detected in a few isolates which may be explained by the horizontal transfer of plasmids that carried genes and their distribution among the family Enterobacteriaceae.


Introduction
Aquatic environment pollution remains a foremost public health hazards, and symbolizes an important reservoir of releasing antibiotic-resistance bacteria due to human activities, including untreated wastewater, which is considered a carrier of a large number of antibiotic-resistance bacterial species 1 . The excessive use of quinolones and sulfa drugs, in different proportions, according to the health institutions that use antibiotics, and the geographic region, has led to the emergence of new types of resistance to antibiotics. Therefore, studying the genes responsible for resistance to antibiotics against bacteria is important, including the genes responsible for resistance to quinolone and sulfa drugs. E. coli is one of the intestinal microbiomes for more than 90% of people 2 , E. coli represents commensal bacteria in the intestine as microflora of animals and humans, some E. coli strains can cause disease in humans,

Abstract
The control of water represents the safe key for fair and optimal use to protect water resources due to human activities, including untreated wastewater, which is considered a carrier of a large number of antibiotic-resistant bacterial species. This study aimed to investigate the prevalence of antibioticresistance to E. coli in Tigris River by the presence of resistance genes for aminoglycoside(qepA( ,quinolone (gyrA), and sulfa drugs( dfr1 ,dfr17) due to the frequent use of antibiotics and their release into wastewater of hospitals. Samples were collected from three sites on Tigris River: S1( station wastewater in Adhamiya), S2 (station wastewater in Baghdad Medical city hospital), S3 (station wastewater in Abu Nuwas) from February-July 2021. Out of 67 isolates of bacteria, only 40 isolates of E. coli were detected by Vitek2. The antibiotic-resistance was estimated by the disk diffusion method. All E.coli isolates were tested against 6 antibiotics. The results showed the high resistance antibiotic of E. coli against Ceftazidime 70%, with intermediate resistance to Cefotaxime 47.5%, and low resistance to the sulfa drugs as Trimethoprim 27.5% and quinolones antibiotics as ciprofloxacin 17.5%, aminoglycosides as Amikacin and Gentamycin 5% and 7.5%. Moreover, the results revealed that gyrA gene was detected in 4 isolates (10%) while drf1 and drf17 genes were in 2 isolates of each gene (5%).Whereas qepA gene has not appeared in isolates. In conclusion, the isolates of E.coli from the Tigris River showed low resistance to sulfa drugs and quinolones , aminoglycosides. The resistance genes (gyrA,drf1,drf17) were detected in a few isolates which may be explained by the horizontal transfer of plasmids that carried genes and their distribution among the family Enterobacteriaceae.
Keywords: aquatic environment, drf1 gene ,drf17 gene, E.coli, gyrA gene.  4 . They are highly adaptable bacteria that can survive and grow in outdoor environments, involving the river 5 . Quinolones are a class of synthetic and broad-spectrum antibacterial agents that interfere with bacterial DNA gyrase (bacterial topoisomerase II) and topoisomerase IV, preventing the supercoiling of DNA, and ultimately promoting DNA strand breakage 6,7 .Resistance to fluoroquinolone (ciprofloxacin(, which is of the hydrophilic type, appears by plasmid efflux controlled by the QepA pump, Trimethoprim resistance in gram-negative bacteria occurs through the horizontal transfer of dfr gene that is responsible for coding for folic acid reductase 8,9 . Poirel et al 10 showed in their study the dfr , dfrA7, dfrA12 and dfrA17 genes were found in 15 isolates of nonpathogenic and resistant to E. coli that were found in food and animals and of human origin. Also, Alwash and Al-Rafyai. 11 appeared in their study that the spread of antibiotic resistance of E. coli isolates was associated with the proximity of Marjan Hospital for Internal Medicine and Cardiology to the river , where there was a direct discharge of wastewater in Hilla. The Hilla River showed a high prevalence of antibiotic-resistance to E. coli with MDR rates of 80.3%.
The study aimed to investigate the prevalence of antibiotic-resistance to E. coli in Tigris River by the presence of resistant genes for aminoglycoside (qepA( quinolone (gyrA( and sulfa drug( dfr1 ,dfr17( , due to the frequent use of antibiotics and their release into wastewater of hospitals.

Samples collection
The samples were collected from three sites of the station including S1 (station wastewater in Adhamiya), S2 (station wastewater in Baghdad Medical city hospital) which was thrown into the river and S3 (station wastewater in Abu Nuwas). A volume of (1000ml) was collected samples to estimate the rate of river pollution by medical wastewater. The period collection was from February -July 2021 with three times for each month so the total samples were 21 samples.

Identification of E. coli
E.coli were isolated by serial dilution method in normal saline, 1 ml from each dilution was dispensed into Petri-dishes; then poured Nutrient agar, MacConkey agar, and EMB agar. Plates were incubated for 48 hours at 37°C. E.coli colonies were initially identified by macroscopic and microscopic methods (gram stain(.Further biochemical tests ( oxidase, catalase, methyl red, indole test, Voges-Proskauer, and Citrate utilization) were used followed by VITEK2 Compact system. The isolates were subcultured and were stored at 4° C for further analysis 12 .

Genetic analysis for genes from E. coli isolates
The whole genome DNA of bacteria isolates was extracted using Bacterial DNA MiniPrep™ Zymo Inc. Catalog No. D6005. Primers for the genes from IDT (Integrated DNA Technologies company, Canada).Moreover, the concentration and purity of DNA estimated by nanodrop spectrophotometer (Nabi /Korea), concentration was 77-295 ng/μL and purity was 1.8-2. Primers for genes were chosen according to 15

Results and Discussion
Identification of E. coli It was diagnosed 40 isolates of E.coli out of 67 bacteria species by biochemical tests that showed positive for Methyl red, Catalase, Indole and negative for Citrate utilization, Voges-Proskauer,and Oxidase. The Vitek 2Compact outcomes appeared with a probability of 95% for isolates of E.coli. The results of antibiotics resistance showed different resistance for the forty isolates. The results of antibiotics sensitivity for E .coli appeared its resistant to one or ≥3 antibiotics categories,it was represented MDR (Multidrug-resistant ) which was represented in 7 isolates that were resistant to four antibiotics .The highest resistance to all antibiotics appeared in two isolates representing XDR (Extensive drug-resistant) and the rest of E. coli isolates were less resistant to antibiotics (Fig.1(.

Figure 1. Antibiotics resistant profile of E. coli isolates (Blue color represent sensitivity of antibiotics ,Red color represent intermediate resistance for 3 antibiotics and Green color represent strong resistance for more than 3 antibiotics
The number of isolates in each station was shown in Table 3, in which the high percentage was in station wastewater of Baghdad Medical City Hospital (S2) at a percentage of 50% with significant differences among the stations of wastewater P<0.05.

Genetic analysis for genes from E. coli isolates
Concerning the outcomes of resistance genes, four isolates have gyrA genes and two isolates for each of drf1 and drf17 genes (Fig. 3 ,4,5). The distribution percentage for gyrA gene was 10% (4/40 isolates), and 5% (2/40 isolates) for each of drf1 and drf17.While qepA gene has not appeared in isolates.

Discussion
Wastewater considers the reservoir and environmental source of antibiotic-resistance. It has been suggested to be a point for horizontal transmission of genes, enabling the diffusion of antibiotic-resistant genes among different bacterial spp 17 . In the current study, one or more than 3 of E .coli isolates were resistant to antibiotics , it was shown that seven isolates resistant to four antibiotics represented MDR. While two isolates were highly resistant to all antibiotics representing XDR.
So this study agreed with other study found that most E.coli isolates were MDR and XDR respectively 18 .In one study conducted to know the resistance of E. coli isolated from urine, it appeared high resistance with a percentage 80.56 % of multidrug E. coli isolates also showed high resistance against -lactamase 19 .
Besides, in the current study appeared E .coli isolates in all stations due to the waste excreted from the Baghdad hospitals in the Tigris river. Where another study conducted in Iraq, E.coli was shown to be the majority as MDR at 78.5% and XDR at 22.5% 11 .
The results of the current study were similar to another study which observed that E.coli isolates a high resistance against Ceftazidime and a little resistance to Amikacin and Gentamycin 20 .
Meanwhile, one study in Iraq reported spreading resistance of E. coli to antibiotics involving: cephalosporins, penicillins, imipenem, fosfomycin, and aminoglycosides 11 . Also, other studies showed the main antibiotic resistance types of E. coli noticed in the current study were prevalent in aquatic environments 21,22 . This resistance paradigm is real because, due to a lack of awareness and education, antibiotics can be purchased without a prescription in Iraq. Furthermore, these antibiotics are used to promote growth in animal farming and agricultural applications. Thus, wastewater discharges and compost effluents may contain antibiotics that pollute the aquatic environment 11 . Antibiotic resistance is activated by several mechanisms, either through genetic resistance, including the efflux mechanism or through acquired mechanisms and mutations in the genetic material and transferring by plasmids 23 . E. coli isolates appear resistant to quinolone (Ciprofloxacin).This can occur by either chromosomal mutations in DNA gyrase genes or the acquisition of transferable plasmidmediated quinolone resistance (PMQR) genes 24 . Generally, plasmid-mediated resistance is a rising concern and can be transferred among various bacterial species and stimulated for transferring into other pathogenic species through horizontal gene transfer (HGT) 25 . It was considered that quinolone resistance was only chromosomally regulated by nucleotide substitution in the quinolone resistance-determination location of DNA gyrase (gyrA and B) and also topoisomerase IV, which are the major target molecules for quinolones. Besides, the PMQR gene represents qnrA, which encodes a protein that conserves topoisomerase type II from quinolone 30 . The reason for the resistance due to a mutation in their target proteins; DNA gyrase and topoisomerase IV ,and changes in the permeability of the cell membrane 31,32 A sulfa drug such as trimethoprim appeared to have a low resistance to E. coli, caused by modifications in the target enzyme dihydrofolate reductase (dfr) encoded by dfr-genes 33 and can bacteria use folic acid that they get from the environment to undergo changes in their metabolism, allowing them to acquire resistance to trimethoprim and sulfonamides 34 Moreover, the E. coli resistance to aminoglycosides, which include Amikacin and Gentamycin, was low. The reason to penetrate the inner cytoplasmic membrane of E. coli and bind the 30S subunit of the bacterial ribosome inhibiting 35 Also, one mechanism of PMQR was the covalent modulation of particular quinolones such as ciprofloxacin by a plasmid-encoding various of aminoglycoside acetyltransferase. another mechanism of PMQR is the quinolones efflux via the efflux pump-encoded genes such as qepA and oqxAB 30 . Furthermore, the presence of determinants PMQR on mobile genetic factors may result in their distribution within the family Enterobacteriaceae 15 .
Numerous factors might have been involved in the release of resistance in the environment. Insufficient sanitation facilities 36 Indiscriminate use of antibiotics, and lack of well-managed sewerage systems are important contributing factors 37,38 .

Conclusion
It was found that wastewater from Baghdad Medical City Hospital had more E. coli number, where seven isolates of E .coli were MDR and two isolates were XDR. The gyrA genes were found in four isolates. While two isolates had drf1, and drf17 genes.