Development and validation of a diagnostic DNA microarray to detect quinolone-resistant Escherichia coli among clinical isolates

Abstract

The incidence of resistance against fluoroquinolones among pathogenic bacteria has been increasing in accordance with the worldwide use of this drug. Escherichia coli is one of the most relevant species for quinolone resistance. In this study, a diagnostic microarray for single-base-mutation detection was developed, which can readily identify the most prevalent E. coli genotypes leading to quinolone resistance. Based on genomic sequence analysis using public databases and our own DNA sequencing results, two amino acid positions (83 and 87) on the A subunit of the DNA gyrase, encoded by the gyrA gene, have been identified as mutation hot spots and were selected for DNA microarray detection. Oligonucleotide probes directed against these two positions were designed so that they could cover the most important resistance-causing and silent mutations. The performance of the array was validated with 30 clinical isolates of E. coli from four different hospitals in Germany. The microarray results were confirmed by standard DNA sequencing and were in full agreement with phenotypic antimicrobial susceptibility testing.

FIG. 1.

Frequency of reported cases of mutation in the literature according to amino acid position of the A subunit of the E. coli gyrase (gyrA product) (literature data analysis from January 1985 to June 2003 through PubMed of NCBI). Positions with silent mutations are indicated with an asterisk.

FIG. 2.

Diagnostic microarray results for three clinical E. coli isolates. (A) Partial microarray layout (position 83); (B to D) microarray images of E. coli isolates 1, 5, and 8, respectively; (E to G) quantitative fluorescent signal intensity analysis for E. coli isolates 1, 5, and 8, respectively.