[Functional characterization of a multiple-antibiotic resistant plasmid from clinical isolates of...

During surveillance done as part of the investigation of nosocominal infections due to methicillin-resistant Staphylococcus aureus (MRSA), we have been aware of the close relationship between the presence of certain plasmids and the characteristic patterns of antibiotic resistance. The hypothesis that a mechanism for the rapid and widespread dissemination of resistance to multiple aminoglycosides in clinical isolates of MRSA at our university hospital was the result of transfer of a single plasmid among these strains was examined. About 45% of the total isolates of MRSA (91/200 isolates) carried a 35.5 kb plasmid (designated pCL4) and these isolates always showed resistance to gentamicin, tobramycin, kanamycin, amikacin, astromicin, and arbekacin. Mating experiments between a susceptible strain and resistant MRSA isolates carrying pCL4 showed high frequency transfer of the plasmid. The aminoglycoside resistance patterns of all the transconjugants obtained corresponded well with those of parental strains. However, the plasmid could not necessarily be detected in the transconjugants, whereas the transformants obtained by means of electroporation usually possessed the plasmid. The plasmid-encoded aminoglycoside-resistance determinant, which has been identified as the gene aacA/aphD that encodes the bifunctional enzyme AAC (6')/APH(2"), either in the transconjugants and transformants could be transposed to their chromosomes in the absence of whole-plasmid integration resulting from a recombination event. Southern hybridization analysis using an aacA/aphD specific probe demonstrated that there are multiple sites of the insertions indistinguishable among the chromosomes of plasmid-free transconjugants and transformants. These results indicate that rapid dissemination of multiple-aminoglycoside-resistance in nosocominal strains of MRSA resulted from transfer of a conjugative plasmid and has been facilitated by translocations of the resistance gene to the chromosome.