Isolation of an antibiotic multiresistance plasmid from Pseudomonas aeruginosa

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Abstract
We have described a Pseudomonas aeruginosa plasmid, an isolate from pus, which carries resistance to several antibiotics [carbenicillin (Cb), streptomycin (Sm), spectinomycin (Sp), neomycin (Nm), paromomycin (Pm), lividomycin (Lv), ribostamycin (Rm), kanamycin (Km), gentamicin (Gm), sissomicin (Ss), chloramphenicol (Cm), tetracycline (Tc), sulphonamide (Su) and mercury]. This plasmid, named pYMBI, is transferable to Pseudomonas aeruginosa with a transfer frequency between 10−5 to 10−7 (recipient strains PA01670, PA08) and also to Escherichia coli (J5 strain). This last strain when stored for several weeks at +4°C, lost Sm, Sp, Gm and Ss resistance. Cure of the recipient was obtained with sub-inhibition concentrations of ethidium bromide, resistance to carbenicillin and most aminoglycosides being lost. Incompatibility studies with RPI Cb3 plasmid show that this plasmid belongs to the Pl group. The β-lactamase enzyme, conferring carbenicillin resistance had a substrate profile (Vmax penicillin 100, carbenicillin 3, ampicillin 105, cephaloridine 180) and an isoelectric point (electrofocusing in polyacrylamide gel 5%, ampholine 24%) similar to that of RPI-β-lactamase (TEM 2 enzyme). The plasmid was isolated, having obtained a clear lysate, by denaturation of DNA in an alkaline medium and subsequent chromatography on a Sepharose 4B-nitrocellulose column. Electron microscopy after AU/Pd shadowing showed a circular DNA molecule of 25 (±0·5) μm length (molecular weight approximately 52 × 106 daltons). In conclusion, our isolation of a multi-resistance plasmid and demonstration of its stability in different strains of Pseudomonas aeruginosa, shows the extent of diffusion of these types of polyresistance plasmids within this species and their relative instability in a different genus (E. coil). The presence of Pl plasmids (Pseudomonas aeruginosa, i.e. P plasmids E. coli) seems frequent in human Pseudomonas aeruginosa. As well it is known that a gene for a broadly spreading TEM 2 β-lactamase in Enterobacteriaceae may be acquired by transposition.