الكشف الجزيئي لبكتريا Escherichia coli المعزولة من اصابات المسالك البولية والمقاومة للمضادات الحيوية المثبطة لبناء الحمض النووي الرايبوزي منقوص الأوكسجين

Molecular Detection of Escherichia coli isolated from Urinary tract infections and resistant to DNA synthesis inhibitors antibiotics.

Summary
Urinary tract infection is one of the most common bacterial diseases, and women are usually more susceptible to it than men. E.coli bacteria are often the major cause of urinary tract infections. In this research, mutations in E.coli bacteria resistant to antibiotics (quinolones) in Karbala Governorate and changes in amino acids were studied, the three-dimensional shape of the gyrB gene for the DNAgyrase enzyme was studied, and the possibility of proposing compounds that can bind to the active sites of the enzyme and inhibit its work.
Two hundred and twenty-three (223) urine samples were collected from patients with urinary tract infections (males and females) aged between 6-77 years from those attending government and private hospitals and private clinics in the holy Karbala Governorate (Imam Hussein Medical City Teaching Hospital, Imam Hassan Al-Mujtaba Hospital, Obstetrics and Gynecology Teaching Hospital, Karbala Children’s Teaching Hospital, and Imam Al-Hujjah Hospital) under the supervision of specialist doctors for the period from 3/8/2023 to 25/11/2023, and were directly cultured on culture media and incubated at 37°C for 24 hours .
It 60/223 (27%) Escherichia coli isolates were isolated and diagnosed from the total urine samples. The isolates were diagnosed based on morphological, microscopic and biochemical tests and using the API-20E system to confirm the diagnosis.
The susceptibility test of E.coli isolates showed that they were multi-resistant, and their resistance was varying, and their resistance was high to Ampicillin (AMP) 100%, Co-trimoxazole (COT) 73.33%, Tetracycline (TE) 68.33%, and Ceftriaxone (CTR) 65%, while to a lesser extent to Nalidixic acid (NA) 46.66%, Ciprofloxacin (CIP) and Levofloxacin (LEV) 36.66%, Gatifloxacin (GAT) 33.33%, and Imipenem (IMP) 31.66%, and they were least resistant to Amikacin (AK) 6.66% .
All E. coli isolates showed multiple resistance to a variety of antibiotics, as one isolate was resistant to seven types of antibiotics (E31), two isolates were resistant to five types of antibiotics (E19 , E44), two isolates were resistant to eight types of antibiotics (E45 , E57), three isolates were resistant to two types of antibiotics (E7 , E34 , E37), three isolates were resistant to all antibiotics (E22 , E50 , E60), which were ten antibiotics used in this study, while four other isolates showed resistance to three types of antibiotics (E1 , E2 , E8 , E14), four isolates were moderately resistant to one type of antibiotic (E8 , E21 , E31 , E45), and 14 isolates showed resistance to one type of antibiotic. (E4 , E9 , E13 , E15 , E20 , E23 , E25 , E28 , E30 , E33 ,E35 , E38 , E39 , E41), and 15 isolates resistant to four types of antibiotics (E10 , E16 , E17 , E18 , E21, E29 , E36 , E42 , E43 , E46 , E47 , E48 , E49 , E52 , E53), while 16 isolates showed resistance to nine types of antibiotics (E3 , E5 , E6 , E11, E12 , E24 , E26 , E27 , E32 , E40 , E51 , E54 , E55 , E56 , E58 , E59).
The gyrB gene of 40 E.coli isolates(from 60 isolated of E.coli) was doubled by conventional polymerase chain reaction, and the isolates showed that the percentage of the presence of the gyrB gene was 100%. DNA sequencing tests was performed for two parts of the gyrB gene for 40 bacterial isolates (80 segments of DNA), the first part was 998 base pairs long and the second part was 971 base pairs, and the results of the DNA sequencing tests showed no mutations related to resistance to the quinolones antibiotics used in this experiment .
The alignment sequences were assigned to all samples by using the MEGA11 program and using the ClustalW selection, and the phylogenetic tree analysis of the gyrB gene, thus including the samples of local clinical isolates with corresponding global sequences belonging to different isolates, and most of the results for the local isolates showed a 100% match with the global isolates. While some of them showed a 99% match.
The three-dimensional shape was studied and the ATP binding pocket in the gyrB gene was identified, and the results showed that there are some proposed chemical compounds that have the ability to bind to the amino acids present in the binding pocket and inhibit their action. Some of the proposed chemical compounds with high binding affinity to the gene were subjected to Lipinski’s rule of five to determine the possibility of taking the drug orally.