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Hospital and agriculture wastewater is mostly responsible for causing environmental pollution by spreading un-metabolized antibiotics and resistant bacteria, especially in Bangladesh. Here, we studied the influence of the most frequently prescribed antibiotic, fluoroquinolone (~72%), on the development of antibiotic resistance in Escherichia coli. Out of 300, 24 ciprofloxacin resistant E. coli isolates were selected for the study that showed the MBC higher than expected (600 μg/mL). Here, we profiled plasmid, sequenced gyr genes, screened mutations and analyzed the effect of mutation on drug-protein interaction through molecular docking approach. We found that (1) out of 10, most of them (n = 7) had large plasmid(s); (2) all ciprofloxacin-resistant isolates had gyrA double mutations (S83L and D87Y); (3) no isolate had qnr gene; and (4) docking of ciprofloxacin with DNA gyrase A subunit suggests that acquisition of double mutation leads to alteration of the ciprofloxacin binding pocket. [ABSTRACT FROM AUTHOR]