Identifying antimicrobial resistance genes with DNA microarrays

Abstract

We developed and tested a glass-based microarray suitable for detecting multiple tetracycline (tet) resistance genes. Microarray probes for 17 tet genes, the beta-lactamase bla(TEM-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 degrees 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.

FIG. 1.

Hybridization results illustrating specificity of tet gene probes. Probes were printed in quadruplicate in two columns. From top to bottom of each array, the left column included tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(H), tet(L), tet(M), and tet(O). The right column included tetP(A), tetP(B), tet(S), tet(W), tet(Z), otr(B), tet(30), 16S rDNA, bla_TEM-1, and a biotin marker.

FIG. 2.

Example of quantified signal intensity for positive control hybridizations tet(A) (upper panel) and tet(S) (lower panel). The horizontal line represents a detection threshold equivalent to the average median intensity plus 3× SD for all tet probes.