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Multiple mutations in the quinolone-determining resistant regions of topoisomerase enzymes are usually associated with high levels of fluoroquinolone resistance in E. coli. Gyr A gene encodes (gyrase II) enzyme that changes topology by introducing transient double-stranded breaks into DNA and passing a second double-stranded DNA segment through the break before resealing it. The current investigation aims to reveal the correlation between functional gyrase enzyme and non-functional gyrase with level MIC of quinolone resistance under study. Patients with various illnesses such as urinary tract infections (UTIs), gastro enteritis (diarrhea) and wound infections were involved in the investigation. A total of 200 clinical samples were obtained. There are 30 samples diagnostic as E. coli clinical strain after identification by biochemical test, VITEK-2 compact system, and by molecular method using 16SrDNA marker. Antimicrobial susceptibility testing and minimal inhibition concentration (MIC) for Nalidixic acid, Norfloxacin, ciprofloxacin, levofloxacin, and Gatifloxacin was performed by broth microdilution method. According to manufacturing company (Topogen-USA); cell lysate are produced;then Bacterial gyrase assay (Topogen-USA) has been used to show the activity of gyrase enzyme by observation of negative supercoiling of E. coli DNA. Gyr A gene is detectable in all tested E. coli (100%); but There are only 16/30 isolates have active topoisomerase type2 with percentage 53.33% only; while; (14) isolates with non-functional gyrase enzyme since they not produce negative supercoiling DNA bands after gel electrophoreses with ethidium bromide stain. non-functional gyrase enzyme are associated with high E. coli resistance level against quinolone antimicrobial under study. MIC value for two groups are show significantly different concerning Nalidixic acid, Norfloxacin, while no significant difference concerning Levofloxacin and Gatifloxacin antibiotics.As a result, this research representsto be an effective method for determining the primary target of a quinolone drug in E. coli clinical isolates, and for the purification and characterization of DNA Gyrase so. E. coli are highly isolated from vagina samples 40% and then from urine samples (32%) followed by (24%) from wounds infections and (21%) from stool .There are correlation between the reduced sensitivity of quinolone under study and bacterial topoisomerases activity in E. coli isolates.