#119 In-silico assessment of gyrA obtained from fluoroquinolone-resistant urinary isolates of E. coli

Abstract

Urinary Tract Infection (UTI) is one of the most common infections affecting the human race. Although it is prevalent in the female gender, it also affects men. Escherichia coli are one of the most common culprits causing UTI in 90% of the cases. Ciprofloxacin and other fluoroquinolones have been successfully used to treat UTI caused by E. coli. However, this bacteria has obtained Multiple Drug Resistance (MDR) against more than one fluoroquinolone class by horizontal transfer of genes. Most of this MDR occurs by alteration of the gyrA genes. This study examines the mutation of gyrA within the quinolone-resistance determining region (QRDR) of the resistant E. coli isolated from the urine of the UTI patients. This study also involves the molecular docking of ligands on the fluoroquinolones to examine their binding efficacy and effect on the resistant strains. Understanding the correlation between genetic determinants of pathogenicity and phenotypic antibiotic resistance is key to preventing the spread of resistant bacterial strains, especially in specific population groups based on hospital antibiograms and analysis of prevalent resistant strains. To validate this, 50 urinary isolates of E. coli were analyzed for resistance towards fluoroquinolone antibiotics. The resistant strains were chosen based on their definite resistance to at least two fluoroquinolones (Ciprofloxacin, Norfloxacin, Ofloxacin, Levofloxacin). Genomic DNA extraction was carried out after strain characterization, and isolates showing a 260/280 ratio within 1.6-1.9 were labeled pure. Primers were designed to target a mutation hotspot, Quinolone resistance determining region (QRDR), in the gyrA gene. As confirmed by AGE, the synthesized oligonucleotides were used to run a PCR assay to amplify a gene target spanning ~350bp. The PCR products were purified using a spin-column format, and the purified amplicons were subject to single direction reads of Sanger’s sequencing technique. 25% of the samples were sequenced. The data suggested that mutations in amino acid residues 83 and 87 were prevalent in the chosen population and parallel studies of similar scope.