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Antimicrobial Agents and Chemotherapy, October 2003, p. 3290-3295, Vol. 47, No. 10
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.10.3290-3295.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Identifying Antimicrobial Resistance Genes with DNA Microarrays

Douglas R. Call,1* Marlene K. Bakko,1 Melissa J. Krug,1 and Marilyn C. Roberts2

Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, Washington 99164,1 Department of Pathobiology, University of Washington, Seattle, Washington 981952

Received 24 April 2003/ Returned for modification 6 June 2003/ Accepted 2 July 2003

We developed and tested a glass-based microarray suitable for detecting multiple tetracycline (tet) resistance genes. Microarray probes for 17 tet genes, the ß-lactamase blaTEM-1 gene, and a 16S ribosomal DNA gene (Escherichia coli) were generated from known controls by PCR. The resulting products (ca. 550 bp) were applied as spots onto epoxy-silane-derivatized, Teflon-masked slides by using a robotic spotter. DNA was extracted from test strains, biotinylated, hybridized overnight to individual microarrays at 65°C, and detected with Tyramide Signal Amplification, Alexa Fluor 546, and a microarray scanner. Using a detection threshold of 3x the standard deviation, we correctly identified tet genes carried by 39 test strains. Nine additional strains were not known to harbor any genes represented on the microarray, and these strains were negative for all 17 tet probes as expected. We verified that R741a, which was originally thought to carry a novel tet gene, tet(I), actually harbored a tet(G) gene. Microarray technology has the potential for screening a large number of different antibiotic resistance genes by the relatively low-cost methods outlined in this paper.

* Corresponding author. Mailing address: Department of Veterinary Microbiology and Pathology, Washington State University, 402 Bustad Hall, Pullman, WA 99164-7040. Phone: (509) 335-6313. Fax: (509) 335-8529. E-mail: drcall{at}wsu.edu.

Antimicrobial Agents and Chemotherapy, October 2003, p. 3290-3295, Vol. 47, No. 10
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.10.3290-3295.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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