Additional DNA was shown to be present in methicillin-resistant Staphylococcus aureus by one-and two-dimensional restriction endonuclease analyses of the chromosomal DNA. A 3.5-kilobase BglII fragment, which was present in methicillin-resistant strains but not in the isogenic methicillin-sensitive parental strain, was cloned into newly constructed plasmid pWDB1 in Escherichia coli. Hybridization of this 3.5-kilobase BglII fragment with different methicillin-Sensitive and methicillin-resistant S. aureus clinical isolates indicated that the fragment represents part of the methicillin resistance determinant (mec). In addition, the fragment carries a sequence that is present in some large staphylococcal plasmids, as well as in penicillinase plasmid p1524.Methicillin is the first 1-lactamase-resistant semisynthetic penicillin. Methicillin resistance in Staphylococcus aureus is due to intrinsic resistance to semisynthetic penicillins and cephalosporins (7). Methicillih resistance is not due to drug inactivation (8). The exact resistance mechanism is still not known. The methicillin resistance determinant (mec) is chromosomally located and is linked to the purA-nov-his cluster (10).Although methicillin sensitivity and purA+ are cotransducible at a frequency of 15%, mec cannot be cotransduced with purA+. This behavior supports the hypothesis that mec resides on an inserted DNA sequence in S. aureus and that there is no allelic equivalent in methicillin-sensitive strains (17). The methicillin resistance determinant might be integrated into a transposable element (4).Efforts to clone the methicillin resistance determinant by using molecular cloning methods have so far been unsuccessful, perhaps due to the rather large size or the unusual sequence of the methicillin resistance determinant. We attempted to identify and clone a methicillin-specific DNA fragment in order to obtain a hybridization probe for further investigations and for cloning the total methicillin resistance determinant. MATERIALS AND METHODSBacterial strains, plasmids, and cultivation conditions. The bacteria and plasmids used in this study are listed in Table 1.The strains were grown in Luria-Bertani broth (12) at 37°C. Solid medium contained 1.5% agar (Difco Laboratories). Ampicillin (Bayer Leverkusen) was used at a concentration of 50 ,ug/ml for selection after transformation.Isolation and cloning of DNA. Cellular DNA was prepared by phenol extracting a staphylococcal cell lysate and spooling out the DNA on a glass rod as described previously (3), except that the preparation was precipitated with spermine (6) before restriction nuclease analysis. Small-scale plasmid preparations of Escherichia coli were nmade by heat and alkali denaturation of a lysate and neutralization by using acidic phenol, as described by Kieser (9). For large-scale plasmid preparations the plasmids were amplified with chloramphenicol (12) before lysis and were purified by CsCI density gradient centrifugation. Ligation of DNA with T4 DNA ligase (Anglian Biotechnology Ltd.) was carr...

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