Macrolides-lincosamides-streptogramin B (MLS) resistance is commonly found in Staphylococcus aureus and coagulase-negative staphylococci (22 and 45%, respectively, among isolates from three New Jersey hospitals). We have examined representative subsets of 107 MLS-resistant isolates for the molecular nature of the resistance determinant, the erm gene, by dot blot and Southern hybridization analysis. All of 35 S. aureus isolates examined and 39 of 42 coagulase-negative isolates examined were found to harbor the ermA or ermC evolutionary variant. Genes of the ermC class occurred exclusively on a small plasmid similar to or indistinguishable from one (pNE131) previously described in S. epidermidis. Genes of the ermA class occurred exclusively in the chromosome, and restriction patterns indicated that they were part of a transposon, Tn554, characteristic of the classical S. aureus ermA strain. Unlike S. aureus ermA strains examined previously, which harbor TnS54 at a single specific (primary) site, four of our S. aureus isolates had second inserts at different chromosomal sites. The majority of our coagulase-negative isolates had two or more inserts, neither of which occurred at the classical primary site and many of which differed from one another in location (as inferred from restriction patterns). Coagulase-negative staphylococci constitute a large reservoir of the ermA and ermC class of determinants, with clear potential for interspecies spread.The MLS phenotype refers to cross-resistance to three groups of antibiotics: the macrolides (exemplified by erythromycin), the lincosamides (exemplified by clindamycin), and the streptogramins B (exemplified by vernamycin Ba). The classical phenotype was described for four Staphylococcus aureus isolates, in which resistance to the macrolide spiramycin was shown to be inducible by low levels of erythromycin (2). The phenotype has since been shown to occur in a variety of bacterial genera (5), but the S. aureus system has been subjected to the most intensive scrutiny and is understood in greatest detail (see references 26 and 36 for summaries). Neither lincosamides, streptogramins, nor most macrolides are effective inducers. The induction process entails activation of an mRNA that encodes a 23S RNA methylase, the erm methylase, which in turn renders newly synthesized ribosomes resistant to the MLS agents by methylating a specific adenosine residue (equivalent to Escherichia coli 23S RNA A2058 [33]) of the rRNA component of the peptidyl transferase center. The activation involves alteration in the secondary structure of the mRNA, resulting from a stall of an erythromycin-ribosome complex on the leader region(s) encoding short polypeptides. Constitutive variants readily arise via mutational changes in these leader regions.

A total of 332 staphylococcal and 263 streptococcal isolates from three hospital microbiology laboratories were tested with erythromycin, clindamycin, and vernamycin B. to determine the prevalence of macrolideslincosamides-streptogramin B resistance. Constitutive resistance was detected in 28 Staphylococcus aureus isolates (15.5%), 53 coagulase-negative staphylococci (35.1%), and 20 streptococci (7.6%). Inducible resistance was observed in 13 S. aureus isolates (7.2%), 25 coagulase-negative staphylococci (16.6%), and 2 streptococci (0.8%). Eleven coagulase-negative staphylococci (7.3%) exhibited a novel phenotype, namely inducible resistance to erythromycin and vernamycin Ba but not clindamycin. Among the staphylococci, two variants of the inducible phenotype detected with the agar disk diffusion assay correlated with the presence of classical ermA or ermC genes, respectively, by dot-blot analysis. The prevalences of the staphylococcal phenotypes were different in the hospitals surveyed, and there was an apparent inverse correlation between the resistance observed and the use of erythromycin in each hospital.Simultaneous resistance to macrolides, lincosamides, and the type-B streptogramins (MLS resistance) in clinical isolates is a form of acquired resistance due to several evolutionary variants of the resistance-conferring (erm) gene, which encodes a 23S rRNA methylase (34). The methylase, which renders affected ribosomes incapable of binding the MLS antibiotics, can be produced constitutively or inducibly (38), low levels of erythromycin being the most effective inducer (36).This pattern of resistance has been demonstrated in many bacterial species, and its prevalence has been monitored in Europe (10,11,15,16, 29, 30,39), Canada (7-9), and Japan (18,(20)(21)(22). In the United States, systematic studies of the epidemiology of MLS resistance are sparse. However, studies of bacterial susceptibilities to erythromycin and clindamycin suggest that MLS resistance is present in clinical isolates in the United States (2-4, 17, 28). To determine the prevalence of the MLS resistance phenomenon in our geographic area, we studied more than 800 clinical isolates, predominantly staphylococci and streptococci, from three hospitals affiliated with our institution. All isolates were tested for susceptibility to erythromycin, clindamycin, and vernamycin Ba. Resistant strains were tested further to determine whether resistance was constitutive or inducible. Finally, selected strains were characterized with respect to the evolutionary class (ermA, B, or C) of the resistance determinant.

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 ...