Use of a DNA Microarray for Simultaneous Detection of Antibiotic Resistance Genes among Staphylococcal Clinical Isolates

We developed a multiplex asymmetric PCR (MAPCR)-based DNA microarray assay for characterization of the clinically relevant antibiotic resistance genes leading to penicillin, methicillin, aminoglycoside, macrolide, lincosamide, and streptogramin B (MLSB ) resistance in staphylococci. The DNA-based assay involves detection of specific conserved regions of themecA ,blaZ (methicillin and penicillin resistance),aac (6′)-Ie-aph (2‴) (aminoglycoside resistance),ermA andermC genes (MLSB resistance), and themsrA gene (macrolide and streptogramin B resistance). The microarray uses a variable sequence region of the 16S rRNA gene to broadly differentiate betweenStaphylococcus aureus and other coagulase-negative staphylococci (CoNS). The performance of the microarray was validated with a total of 178 clinically importantS. aureus and 237 CoNS isolates, with correlations of 100% forS. aureus to CoNS discrimination and more than 90% for antibiotic resistance between the genotypic analysis determined by the microarray and the phenotype determined by standard methods of species identification and susceptibility testing. The major discrepant results were 17mecA -positive CoNS and 60aac (6′)-Ie-aph (2‴)-positive CoNS isolates measured by microarray that were susceptible to the corresponding antibiotics based on disk diffusion assay. Overall, this microarray-based assay offers a simultaneous, fast (≤5 h), and accurate identification of antibiotic resistance genes from a single colony, as well as species classification. Our extensive validation of the microarray suggests that it may be a useful tool to complement phenotypic susceptibility testing in clinical laboratories and to survey the spread of antibiotic resistance determinants in epidemiological studies.