Functional Characterization of a Multiple-Antibiotic Resistant Plasmid from Clinical Isolates of Methicillin-Resistant Staphylococcus aureus

Abstract

During surveillance done as part of the investigation of nosocominal infections due to methicillinresistantStaphylococcus aureus (MRSA), we have been aware of the close relationship between thepresence of certain plasmids and the characteristic patterns of antibiotic resistance. The hypothesisthat a mechanism for the rapid and widespread dissemination of resistance to multiple aminoglycosidesin clinical isolates of MRSA at our university hospital was the result of transfer of a singleplasmid among these strains was examined.
About 45% of the total isolates of MRSA (91/200 isolates) carried a 35.5 kb plasmid (designatedpCL4) and these isolates always showed resistance to gentamicin, tobramycin, kanamycin, amikacin, astromicin, and arbekacin. Mating experiments between a susceptible strain and resistant MRSA isolatescarrying pCL4 showed high frequency transfer of the plasmid. The aminoglycoside resistancepatterns 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 transformantsobtained by means of electroporation usually possessed the plasmid. The plasmid-encodedaminoglycoside-resistance determinant, which has been identified as the gene aacA/aphD that encodesthe bifunctional enzyme AAC (6') /APH (2''), either in the transconjugants and transformantscould be transposed to their chromosomes in the absence of whole-plasmid integration resulting froma recombination event. Southern hybridization analysis using an aacA /aphD specific probe demonstratedthat there are multiple sites of the insertions indistinguishable among the chromosomes ofplasmid-free transconjugants and transformants. These results indicate that rapid dissemination ofmultiple-aminoglycoside-resistance in nosocominal strains of MRSA resulted from transfer of a conjugativeplasmid and has been facilitated by translocations of the resistance gene to the chromosome.