Macrolides-lincosamides-streptogramin B resistance in staphylococci can result from a gene, ermA, that comprises part of transposon Tn554. Tn554 is unusual in (i) its high specificity for a primary chromosomal attachment site, a1t554, and (ii) the variability of its 3'-terminal six or seven nucleotides, which appear to copy the six or seven chromosomal nucleotides 5' to the parent transposon during transposition. We characterized a novel Tn554 insert in the chromosomes of methicillin-resistant Staphylococcus aureus strains involved in a current outbreak. This insert was found to resemble an insert recently discovered in S. epidermidis in its junctional fragment restriction pattern. Sequence analysis of the junctional regions showed that the attachment site, att1SS, exhibited 78% similarity to att554 (39 of the 50 nucleotides flanking the insertion sites) for both S. aureus and S. epidermidis inserts and that the 3' hexanucleotide of the S. epidermidis transposon (GACATC) resembled the reverse complement (TACATC) of its commonly occurring S. aureus counterpart (GATGTA). Epidemiologic and molecular data indicated that attU55 is harbored by extra DNA characteristic of methicillin-resistant strains and absent from methicillin-susceptible ones. Further, Southern hybridization showed that, even in the absence of Tn554 inserts, some methicillin-resistant strains contain DNA related to attl55 and Tn554.Macrolides-lincosamides-streptogramin B (MLS) resistance was first described in Staphylococcus aureus (6) and is now common in this and other species of staphylococci (see, e.g., references 2, 12, and 43). Resistance is due to the action of an erm methylase which converts an adenosine residue of 23S RNA to m26A (38,42), thereby decreasing the affinity of the ribosome for all MLS antibiotics. In classical MLSr S. aureus, the resistance is inducible by erythromycin, the most widely used macrolide, but not by most other macrolides, the lincosamides, or the streptogramins (42).The erm methylase is encoded in S. aureus by three evolutionarily distinct classes of genes, so-called ermA, ermB, and ermC (summarized in reference 37). In a recent (1984) patterns resembling the classical 10 insert except for an apparent restriction site polymorphism and (ii) a 2°insert whose restriction pattern resembled one recently observed in S. epidermidis (37). The junctional sequences corresponding to this 20 insert were determined and found to be the same for S. aureus and S. epidermidis save for the extreme 3' six residues of the transposon. This difference can be explained, in general terms, on the basis of a peculiar pattern of information flow related to transposition of Tn554.Recent studies have indicated that Mcr determinant mec is part of ==35 kilobases (kb) of DNA not present in methicillinsusceptible (Mcs) strains (mec-associated DNA) (4,11,20,21). We present evidence that the DNA harboring the attachment site for the new S. aureus-S. epidermidis Tn554 insert (attiSS) is also absent from Mcs strains, suggesting the possibility that ...