Evaluation of a fluorescence resonance energy transfer quantitative polymerase chain reaction assay for identification of gyrA mutations conferring enrofloxacin resistance in canine urinary Escherichia coli isolates and canine urine specimens

OBJECTIVE To evaluate a fluorescence resonance energy transfer quantitative PCR (FRET-qPCR) assay for detection of gyrA mutations conferring fluoroquinolone resistance in canine urinary Escherichia coli isolates and canine urine specimens. SAMPLE 264 canine urinary E coli isolates and 283 clinical canine urine specimens. PROCEDURES The E coli isolates were used to validate the FRET-qPCR assay. Urine specimens were evaluated by bacterial culture and identification, isolate enrofloxacin susceptibility testing, and FRET-qPCR assay. Sensitivity and specificity of the FRET-qPCR assay for detection of gyrA mutations in urine specimens and in E coli isolated from urine specimens were computed, with results of enrofloxacin susceptibility testing used as the reference standard. RESULTS The validated FRET-qPCR assay discriminated between enrofloxacin-resistant and enrofloxacin-susceptible E coli isolates with an area under the receiver operating characteristic curve of 0.92. The assay accurately identified 25 of 40 urine specimens as containing enrofloxacin-resistant isolates (sensitivity, 62.5%) and 226 of 243 urine specimens as containing enrofloxacin-susceptible isolates (specificity, 93.0%). When the same assay was performed on E coli isolates recovered from these specimens, sensitivity (77.8%) and specificity (94.8%) increased. Moderate agreement was achieved between results of the FRET-qPCR assay and enrofloxacin susceptibility testing for E coli isolates recovered from urine specimens. CONCLUSIONS AND CLINICAL RELEVANCE The FRET-qPCR assay was able to rapidly distinguish between enrofloxacin-resistant and enrofloxacin-susceptible E coli in canine clinical urine specimens through detection of gyrA mutations. Therefore, the assay may be useful in clinical settings to screen such specimens for enrofloxacin-resistant E coli to avoid inappropriate use of enrofloxacin and contributing to antimicrobial resistance.