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item Frye, Jonathan
item Jesse, Troy
item Long, Fred
item Rondeau, Gaelle
item Porwollik, Steffen
item Mcclelland, Michael
item Jackson, Charlene
item Englen, Mark
item Cray, Paula

Submitted to: International Journal of Antimicrobial Agents
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/17/2005
Publication Date: 1/19/2006
Citation: Frye, J.G., Jesse, T.W., Long, F., Rondeau, G., Porwollik, S., McClelland, M., Jackson, C.R., Englen, M.D., Cray, P.J. 2006. DNA microarray detection of antimicrobial resistance genes in diverse bacteria. International Journal of Antimicrobial Agents. 27(2):138-151.

Interpretive Summary: The lack of a method for rapid identification of the genes responsible for multi drug resistance in bacteria hinders research into this important problem. Currently, it may be necessary to individually assay for dozens of possible genes to identify the genetic element responsible for each observed resistance. We have designed, constructed and validated a DNA microarray to simultaneous test for more than 90 genes per assay. The micro array detected resistance genes in a variety of diverse bacteria including Salmonella, E. coli, Camplyobacter and Enterococcus and yielded results similar to the current standard techniques of PCR and Southern blotting. The microarray enables the identification of genes responsible for multi drug resistance in a single assay and can be expanded to cover all known resistance genes. Scientist will now be able to rapidly study the development and dissemination of multi drug resistance in bacteria in order to develop intervention strategies.

Technical Abstract: Investigation into the molecular mechanisms of antimicrobial resistance has been confounded by the emergence of multi drug resistant bacteria. Characterization of these strains can require hundreds of assays to identify the genes conferring antibiotic resistance. We have designed a microarray for the rapid detection of antibiotic resistance genes found in diverse bacteria. The array was designed to detect over 90 genes commonly found in a variety of bacteria which confer resistance to an assortment of antimicrobial drug classes. Oligonucleotide probes were designed, synthesized, robotically arrayed onto glass slides which were processed, hybridized, scanned and scored following standard microarray techniques. Preliminary tests were conducted with diverse collections of Gram negative and Gram positive pathogens and commensal bacteria. The probes demonstrated high specificity, low background and low cross reactivity. Test strains were found to harbor antimicrobial resistance genes consistent with their antibiograms. Several genes were confirmed by alternate methods including PCR and Southern blotting. Additionally, conjugation experiments established that the microarray could be used to trace the horizontal transfer of multiple antibiotic resistance genes. The results demonstrate that these techniques can be used to quickly design a microarray for detecting horizontally exchanged DNA elements in virtually any bacteria.