|Rondeau, Gaelle -|
|Porwollik, Steffen -|
|Long, Fred -|
|Mcclelland, Michael -|
Submitted to: Microbial Drug Resistance
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 9, 2009
Publication Date: March 1, 2010
Citation: Frye, J.G., Lindsey, R.L., Rondeau, G., Porwollik, S., Long, F., Mcclelland, M., Jackson, C.R., Englen, M.D., Meinersmann, R.J., Berrang, M.E., Davis, J.A., Barrett, J.B., Turpin, J.B., Thitaram, S.N., Cray, P.J. 2010. Development of a DNA microarray to detect antimicrobial resistance genes identified in the national center for biotechnology information database. Microbial Drug Resistance. 16(1):9-19. Interpretive Summary: To study the spread of antimicrobial resistance in bacteria it is necessary to detect and characterize the genes responsible for resistance. Currently, each gene must be screened individually in order to identify the gene(s) responsible for the resistance expressed by a bacterium. The inability to rapidly identify these genes limits research progress. We have designed, constructed and validated a DNA microarray to simultaneously test for all identified sequenced resistance genes in one assay. The microarray can be used to detect 775 resistance genes in a variety of diverse bacteria including Salmonella, E. coli, Camplyobacter and Enterococcus. The assay will be used to identify the genes that are associated with antimicrobial resistant phenotypes, to study the epidemiology of these genes and to detect their dissemination throughout the ecosystem. This technological advance will accelerate scientific research and aid in the development of intervention strategies to prevent the spread of antimicrobial resistance.
Technical Abstract: High density genotyping techniques are needed for investigating antimicrobial resistance especially in the case of multi-drug resistant (MDR) isolates. To achieve this all antimicrobial resistance genes in the NCBI Genbank database were identified by key word searches of sequence annotations and then compiled into a non-redundant list of 775 genes. Unique 70mer oligonucleotide probes were designed to detect each of these genes and used to construct a DNA microarray. The microarray contains probes to detect 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 sequences encoding antimicrobial resistance associated genes, potential resistance transfer genes, and antimicrobial efflux pumps. The microarray was successfully tested with two fully sequenced control strains of Salmonella enterica: Typhimurium LT2 (sensitive) and Typhi CT18 (MDR). All resistance genes as well as all CT18 MDR plasmid pHCM1 genes with probes on the array were detected. The microarray was also tested with a variety of bacteria including MDR Salmonella enterica serovars, Escherichia coli, Campylobacter spp., Enterococcus spp., Staphylococcus (MRSA), Listeria spp., and Clostridium difficile. The genes detected correlated greater than 75% with phenotypes and alternative gene detection techniques. This demonstrates that virtually every known antimicrobial resistance gene can be detected in a variety of bacteria using this technique.