Development of a DNA Microarray to Detect Antimicrobial Resistance Genes Identified in the National Center for Biotechnology Information Database

To understand the mechanisms and epidemiology of antimicrobial resistance (AR), the genetic elements responsible must be identified. Due to the myriad of possible genes, a high-density genotyping technique is needed for initial screening. To achieve this, AR genes in the National Center for Biotechnology Information GenBank database were identified by their annotations and compiled into a nonredundant list of 775 genes. A DNA microarray was constructed of 70mer oligonucelotide probes designed to detect these genes encoding resistances to aminoglycosides, beta-lactams, chloramphenicols, glycopeptides, heavy metals, lincosamides, macrolides, metronidazoles, polyketides, quaternary ammonium compounds, streptogramins, sulfonamides, tetracyclines, and trimethoprims as well as resistance transfer genes. The microarray was validated with two fully sequenced control strains of Salmonella enterica: Typhimurium LT2 (sensitive) and Typhi CT18 (multidrug resistance [MDR]). All resistance genes encoded on the MDR plasmid, pHCM1, harbored by CT18 were detected in that strain, whereas no resistance genes were detected in LT2. The microarray was also tested with a variety of bacteria, including MDR Salmonella enterica serovars, Escherichia coli, Campylobacter spp., Enterococcus spp., methicillin-resistant Staphylococcus aureus, Listeria spp., and Clostridium difficile. The results presented here demonstrate that a microarray can be designed to detect virtually all AR genes found in the National Center for Biotechnology Information database, thus reducing the subsequent assays necessary to identify specific resistance gene alleles.