Development of a DNA Probe for the Structural Gene of the 2''-O-Adenyltransferase Aminoglycoside-Modifying Enzyme

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Abstract
Analysis of aminoglycoside-resistant Enterobacteriaceae isolated from patients at the Seattle Veterans Administration Medical Center indicated that a single 68-kilobase R factor was responsible for the epidemic spread of low-level resistance to gentamicin, kanamycin, and tobramycin. An examination, by means of the phosphocellulose paper binding assay, of resistant strains carrying this R factor resulted in the identification of a 2″-O-adenyltransferase [ANT(2″)]-modifying enzyme. This enzyme was later detected in strains containing 150-kilobase plasmids. For more convenient monitoring of the dissemination of the ANT(2″) gene among clinical isolates at the medical center, a DNA probe was developed by cloning of the ANT(2″) structural gene from the 68-kilobase R factor into pBR322. A 310-base pair Ava I restriction fragment isolated from the interior of the cloned ANT(2″) gene was radiolabeled and used in Southern hybridization gels as a probe for plasm ids isolated from aminoglycoside-resistant organisms. The probe proved to be highly specific and was more sensitive than enzymologic techniques for detection of the ANT(2″) gene in clinical isolates with complex aminoglycoside resistance phenotypes.