ADAPTATION OF PSEUDOMONAS-AERUGINOSA HARBORING THE MULTI-RESISTANCE PLASMID RIP64 TO HIGH-LEVELS OF MERCURY-CHLORIDE, CARBENICILLIN AND GENTAMICIN

In order to study phenotypic and genotypic alterations in response to selected conditions of stress, Pseudomonas aeruginosa PU21, harboring the multiresistance plasmid, Rip64 (142.5kb), was adapted by serial su bcultivation on selective agar plates, amended with either Hg2+, carbe nicillin, or gentamicin. Concentrations of the selective agents, to wh ich P. aeruginosa PU21 Rip64 carried plasmid-encoded resistance genes, were close to or slightly above the respective minimal inhibitory con centration. Three different substrains were obtained: R64-M was adapte d to Hg2+, R64-C to carbenicillin, and R64-G to gentamicin. All three substrains showed elevated levels of Hg2+ and carbenicillin resistance . Resistance to other antibiotics was either increased, decreased or u naffected. Increased Hg2+ resistance in all adapted substrains was acc ompanied by higher activity and less stringent induction of mercury re ductase, as determined by Hg-volatilization assays with resting cells. No plasmid alterations compared to the nonadapted strain were detecte d in R64-C, but in R64-M and R64-G plasmid sizes increased by approx. 23 kb, as detected by pulsed field gel electrophoresis and the occurre nce of additional DNA fragments after digestions with several restrict ion endonucleases. The plasmid localized gene rearrangements were acco mpanied by additional DNA fragments hybridizing with a merA gene probe .