Comparison of three DNA hybridization methods for detection of the aminoglycoside 2"-O-adenylyltransferase gene in clinical bacterial isolates

Abstract

Two rapid DNA hybridization methods in which whole-cell lysates fixed to nitrocellulose were used were compared with Southern hybridization of purified plasmid or chromosomal DNA for the ability to identify the 2"-O-adenylyltransferase [ANT(2")] gene in 42 enzymatically defined isolates of gram-negative bacilli. A DNA restriction fragment isolated from an ANT(2") gene cloned into pBR322 and radiolabeled with 32P was used as the probe in all three procedures. Under conditions of high stringency, agreement was obtained between the Southern hybridization method and detection of the ANT(2") enzyme by the phosphocellulose paper binding assay or resistance phenotype in 39 of the 42 strains tested. By using these characterized strains, colony hybridization was shown to be unsatisfactory as a rapid technique for detecting the ANT(2") gene, due to the high number of false-positive and -negative signals obtained. Compared with Southern hybridization, however, spot hybridization (SPH) proved highly reliable for detecting the ANT(2") gene in both members of Enterobacteriaceae and Pseudomonas aeruginosa harboring R factors ranging in size from 23 to 150 kilobases. The relatively low copy number of the 150-kilobase plasmids decreased the sensitivity of SPH, necessitating a minimum cell density of 5 X 10(6) cells per spot. SPH proved to be a very useful method for rapidly screening large numbers of clinical isolates for this resistance determinant.