Rapid and sensitive detection of fluoroquinolone-resistant Escherichia coli from urine samples using a genotyping DNA...

Urinary tract infections (UTI) are among the most common bacterial infections in humans, with Escherichia coli being the major cause of infection. Fluoroquinolone resistance of uropathogenic E. coli has increased significantly over the last decade. In this study a microarray-based assay was developed and applied, which provides a rapid, sensitive and specific detection of fluoroquinolone-resistant E. coli in urine. The capture probes were designed against previously identified and described hotspots for quinolone resistance (codons 83 and 87 of gyrA). The key goals of this development were to reduce assay time while increasing the sensitivity and specificity as compared with a pilot version of a gyrA genotyping DNA microarray. The performance of the assay was demonstrated with pure cultures of 30 E. coli isolates as well as with urine samples spiked with 6 E. coli isolates. The microarray results were confirmed by standard DNA sequencing and were in full agreement with the phenotypic antimicrobial susceptibility testing using standard methods. The DNA microarray test displayed an assay time of 3.5h, a sensitivity of 100CFU/ml, and the ability to detect fluoroquinolone-resistant E. coli in the presence of a 10-fold excess of fluoroquinolone-susceptible E. coli cells. As a consequence, we believe that this microarray-based determination of antibiotics resistance has a true potential for the application in clinical routine laboratories in the future.