MATERIALS AND METHODS Bacterial strains and media. The source and properties of the staphylococci and their plasmids are listed in Table 1. We used the designations Cm, Tc, Sm, and Km for the phenotypes of resistance to chloramphenicol, tetracycline, streptomycin, and kanamycin. CY broth, 1%, a yeast extract-casein hydrolysate medium, was prepared according to Novick (24). Other media were commercial products. Plasmid elimination. For elimination of plasmids, two to three colonies of a culture grown on a brain heart infusion (BHI; Difco) agar plate containing 25 ig 9:84

Plasmid deoxyribonucleic acid was not detected by centrifugal analysis of lysates of penicillinase-negative strains of Staphylococcus aureus harboring a determinant of methicillin resistance derived from strain Villaluz. When these strains contained a penicillinase plasmid, the plasmid deoxyribonucleic acid of methicillin-resistant and methicillin-susceptible strains was indistinguishable by the methods employed. The results indicate that the genetic determinant for methicillin resistance in the strains examined was not associated with a circular plasmid comparable to those that have been shown to determine resistance to benzylpenicillin, tetracycline, and chloramphenicol in S. aureus .

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A circular covalently closed duplex deoxyribonucleic acid plasmid carrying genes for resistance to kanamycin/neomycin has been identified in Staphylococcus aureus E419. The plasmid has a molecular size of 9.2 x 106 daltons and can be transduced into or can transform competent susceptible strains of S. aureus to kanamycin/neomycin resistance.The determinant for kanamycin/neomycin (Km/Nm) resistance in Staphylococcus aureus has been found to be situated on extrachromosomal elements or plasmids that were irreversibly lost from the host cell by growth with acridine dyes (9, 16) or at elevated temperature (2). This determinant mediates resistance to these antibiotics (and to paromomycin) through the action of a phosphoryltransferase that catalyzes the phosphorylation of these antibiotics. Preparation of cell-free lysate. Cultures of the various strains were grown in 1% CY broth at 37 C to logarithmic phage (3-h culture) and then harvested by centrifugation at 10,000 rpm for 10 min at 4 C. The cells were suspended in buffer at pH 7.0 (10 mM MgCl2, 60 mM KCl, and 100 mM tris(hydroxymethyl)-aminomethane-chloride; 15) to which was added 50 Mg of lysostaphin per ml (Schwarz-Mann). The suspension was incubated at 37 C until it turned translucent (30 to 60 min). The lysate was then centrifuged at 16,000 rpm for 45 min at 4 C, and the supernatant fluid was saved.Inactivation of Km. A portion of the supernatant of a cell-free lysate of 8325-4, 8325-4(pSH2), or 8325(pSH2) was mixed with adenosine triphosphate to give a final concentration of 5 mM. Portions of this mixture were incubated ovemight at 37 C with varying concentrations of Km. A 0.02-ml sample from each reaction mixture was placed on a sterile paper disk, 6 mm in diameter, on a heart infusion agar plate freshly spread with a culture of Escherichia coli X (provided by R. Brubaker), a Km-susceptible indicator strain. The plates were incubated overnight at 37 C and the diameters of the zones of inhibited growth around each disk were measured.Density gradients. The S. aureus strains were labeled with [methyl-3H]thymidine (50 Ci/mmol; New England Nuclear) for at least two generations in 1% CY broth. The labeled cultures were lysed by the procedure of Novick and Bouanchaud (22) with minor modifications to be published elsewhere (P. W. Stiffler, H. M. Sweeney, and S. Cohen, J. Bacteriol., in press). Centrifugation of the cesium chlorideethidium bromide (CsCl-EtBr)-DNA solution was in a Spinco type 65 rotor at 43,000 rpm for 24 h at 20 C. Thirty 0.2-ml fractions (18 drops) were collected.Samples of 5 iliters were spotted on filter paper squares, washed, and counted for radioactivity (24