Development of a DNA probe for the structural gene of the 2"-O-adenyltransferase aminoglycoside-modifying enzyme

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 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 plasmids 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.