Genetic Environment and Stability of cfr in Methicillin-Resistant Staphylococcus aureus CM05

Abstract

The Cfr methyltransferase confers resistance to many 50S ribosomal subunit-targeted antibiotics, including linezolid (LZD), via methylation of the 23S rRNA base A2503 in the peptidyl transferase center. Methicillin-resistant Staphylococcus aureus strain CM05 is the first clinical isolate documented to carry cfr. While cfr is typically plasmid borne, in CM05 it is located on the chromosome and is coexpressed with ermB as part of the mlr operon. Here we evaluated the chromosomal locus, association with mobile genetic elements, and stability of the cfr insertion region in CM05. The cfr-containing mlr operon is located within a 15.5-kb plasmid-like insertion into 23S rRNA allele 4. The region surrounding the cfr gene has a high degree of sequence similarity to the broad-host-range toxin/antitoxin multidrug resistance plasmid pSM19035, including a second ermB gene downstream of the mlr locus and istAS-istBS. Analysis of several individual CM05 colonies revealed two distinct populations for which LZD MICs were either 8 or 2 g/ml. In the LZD s colonies (designated CM05⌬), a recombination event involving the two ermB genes had occurred, resulting in the deletion of cfr and the 3= flanking region (cfr-istAS-istBS-ermB). The fitness advantage of CM05⌬ over CM05 (though not likely due to the cfr deletion itself) results in the predominance of CM05⌬ in the absence of selective pressure. Minicircles resulting from the ermB recombination event and the novel association of cfr with the pSM19035 plasmid system support the potential for the continued dissemination of cfr. R esistance to linezolid (LZD) was reported shortly after its introduction into the clinic and included both staphylococcal and enterococcal strains carrying G2576T mutations in one or more alleles of the domain V-encoding region of 23S rRNA genes (21, 57). These mutations perturb the conformation of the peptidyl transferase center, which is the target of oxazolidinones in the 50S ribosomal subunit (30,52,59). More recently, LZD resistance has been linked to additional mutations identified in the genes encoding 50S ribosomal proteins L3 and L4 (34, 58). Most problematic has been the identification of cfr, a horizontally transferable gene encoding a ribosomal methyltransferase that confers resistance to multiple classes of antibacterial agents targeting the peptidyl transferase center by posttranscriptional modification of the 23S rRNA (29). Recent studies have shown that Cfr methylates carbon 8 of nucleotide A2503 (20), thereby conferring a PhLOPS A phenotype characterized by decreased binding of phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A class antibiotics (29,37,56), as well as of 16-membered ring macrolides (55), to Cfr-methylated ribosomes. Of further concern is the low fitness cost associated with Cfr methylation (31) and the documentation of clinical cfr strains possessing co-occurring mutations in 23S rRNA (14) and ribosomal proteins L3 (36) and L4 (5). The need to maintain potency against strains with cfr and/or other LZ...

Antimicrobial Resistance and Virulence: a Successful or Deleterious Association in the Bacterial World?

“…However, in some clinical isolates of S. aureus, cfr coexpressed with the erm gene, which encodes a methyltransferase with another targeting 23S rRNA residue, A2058. The dimethylation of A2058 by Erm increases the fitness cost associated with Cfr-mediated methylation of A2503 (173,174).…”

First Report of the Multidrug Resistance Gene cfr in Enterococcus faecalis of Animal Origin

“…Since its initial identification on the multiresistance plasmid pSCFS1 from Staphylococcus sciuri in 2000 (21), several studies have reported the cfr gene in staphylococcal isolates of animal and human origin. In most reports, the cfr gene was located on different plasmids (9,10,16), while in a few cases, it was also detected in the chromosomal DNA (9,14,27). Recently, the first outbreak case of linezolid-resistant, cfr-carrying Staphylococcus aureus was reported in Spain (17), and the cfr gene has also been identified in Panton-Valentine leukocidin-positive sequence type 8 (ST8) methicillin-resistant S. aureus IVa (USA300) (23).…”

The Genetic Environment of the cfr Gene and the Presence of Other Mechanisms Account for the Very High Linezolid Resistance of Staphylococcus epidermidis Isolate 426-3147L

“…In this S. aureus isolate, designated CM05, the cfr gene was located in the chromosome, and its genetic environment differed greatly from that seen in the plasmids (19,26,27). In the CM05 isolate, cfr is located immediately downstream of the Tn917-derived gene erm(B), which encodes a methyltransferase that modifies A2058 in 23S rRNA and confers resistance to macrolides, lincosamides, and streptogramin B.…”

“…The 1.5-kb cfr-containing region is inserted in the 23S rRNA gene of the rrn4 allele. Active transcription of the rRNA operon might contribute to cfr expression in the CM05 isolate (27).…”