Variants of the gentamicin and tobramycin resistance plasmid pRAY are widely distributed in Acinetobacter

To determine the cause of resistance to the aminoglycosides gentamicin and tobramycin in Acinetobacter isolates and the location of the resistance genes. Australian Acinetobacter baumannii isolates were screened for resistance to aminoglycosides. PCR followed by restriction digestion of amplicons was used to detect genes and plasmids. Plasmids were isolated and examined by restriction digestion. Plasmid DNA sequences were determined and bioinformatic analysis was used to identify features. The sequence of the bla(OXA-Ab) gene and multilocus sequence typing were used to determine strain types. Isolates that exhibited resistance to gentamicin, kanamycin and tobramycin were of diverse strain types. These isolates all carried the aadB gene cassette, and in all but one the cassette was in a 6 kb plasmid similar to pRAY. The three plasmid sequences determined revealed multiple frame-shift differences in the available pRAY sequence that altered the reading frames. In pRAY, mobA and mobC mobilization genes were identified, but no potential replication initiation protein was found. pRAY-v1 differed from pRAY by 66 single-base differences, and pRAY-v2 included two insertion sequences, ISAba22, located upstream of the aadB gene cassette, and IS18-like, within ISAba22. The plasmid pRAY and variants are widely distributed in Acinetobacter spp. and are the most common cause of resistance to gentamicin and tobramycin. Mobilization genes should assist in the dissemination of pRAY and its variants.