The Effect of Mutations in Type II Topoisomerases on Fluoroquinolone Resistance in Clinical Canine Urine Escherichia coli Isolates

Abstract

A series of experiments were preformed in order to validate a rapid FRET-PCR based assay for the detection of fluoroquinolone resistance in small animal Escherichia coli urinary tract infection (UTI) isolates. Three hundred and six canine UTI E. coli isolates from pure culture were subjected to the FRET-PCR assay. Fourty-three of 50 enrofloxacin resistant isolates were detected by FRET-PCR for a sensitivity of 86% and a specificity of 97%. Urine was then spiked with 7 isolates of varying minimum inhibitory concentration for enrofloxacin (MICEnro) to evaluate sensitivity of detection and resistant isolates were detected at concentrations as small as 103 CFUs. Lastly DNA extracted from 438 small animal urine samples was subjected to the FRET-PCR assay. Two hundred and seventy-eight were confirmed to contain E. coli, 18 of which were resistant to enrofloxacin based on susceptibility testing. The FRET assay positively identified 15 of 18 enrofloxacin resistant urine samples (sensitivity of 83.33%) and negatively identified 388 of 420 samples (specificity of 92.36%). When compared to FRET run on DNA extracted from isolates, isolates had better specificity and sensitivity than FRET run on DNA extracted from urine samples.