The Cfr rRNA methyltransferase confers resistance to Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A antibiotics

Abstract

A novel multidrug resistance phenotype mediated by the Cfr rRNA methyltransferase is observed in Staphylococcus aureus and Escherichia coli. The cfr gene has previously been identified as a phenicol and lincosamide resistance gene on plasmids isolated from Staphylococcus spp. of animal origin and recently shown to encode a methyltransferase that modifies 23S rRNA at A2503. Antimicrobial susceptibility testing shows that S. aureus and E. coli strains expressing the cfr gene exhibit elevated MICs to a number of chemically unrelated drugs. The phenotype is named PhLOPSA for resistance to the following drug classes: Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A antibiotics. Each of these five drug classes contains important antimicrobial agents that are currently used in human and/or veterinary medicine. We find that binding of the PhLOPSA drugs, which bind to overlapping sites at the peptidyl transferase center that abut nucleotide A2503, is perturbed upon Cfr-mediated methylation. Decreased drug binding to Cfr-methylated ribosomes has been confirmed by footprinting analysis. No other rRNA methyltransferase is known to confer resistance to five chemically distinct classes of antimicrobials. In addition, the findings described in this study represent the first report of a gene conferring transferable resistance to pleuromutilins and oxazolidinones.

FIG. 1.

Binding of the phenicol, lincosamide, pleuromutilin, and streptogramin A classes of antimicrobials to overlapping sites at the ribosomal peptidyl transferase center. (A) The structure of the bacterial 50S ribosomal subunit showing the slice plane used in panel B. (B) An expanded view showing the structures of four drugs bound at the peptidyl transferase center. The structural data can be found in reference and references therein. The names and chemical structures of the four antimicrobial agents are shown at the bottom on background colors that correspond to the bound structures (depicted in stick representation). The target of the Cfr methyltransferase, nucleotide A2503, is shown in red. The surrounding RNA is shown in light gray. (C) The Cfr-mediated resistance patterns with S. aureus for chloramphenicol, clindamycin, tiamulin, and virginiamycin M₁. The data are from Table 1. The MICs are depicted on a logarithmic scale with strains lacking Cfr shown in the left column of each pair of bars (marked −), whereas those of strains containing Cfr are shown in the right column of each pair of bars (marked +). The numbers above the +Cfr columns are the n-fold differences in MICs between −Cfr and +Cfr strains. Details on the visualization of the 50S ribosomal subunit and antibiotic-50S subunit complexes are provided in Materials and Methods.

FIG. 2.

Gel autoradiograms comparing antibiotic binding to E. coli 70S ribosomes isolated from cells lacking (−Cfr) or harboring (+Cfr) the Cfr methyltransferase. Footprints of the pleuromutilin drugs tiamulin and valnemulin (panel A) and the streptogramin A drug virginiamycin M₁ (panel B) are shown. Control lanes for each experiment contain unmodified 70S ribosomes. Lanes labeled CMCT denote 70S ribosomes modified with CMCT in the absence of drug. Wedges are used to indicate the increase in tiamulin (TIA), valnemulin (VAL), and virginiamycin M₁ (VIRM₁) concentration in reactions of 70S subunits modified with CMCT in the presence of 0.5, 2, or 10 μM tiamulin, 0.2, 0.5, or 2 μM valnemulin, or 0.5, 5, or 50 μM virginiamycin M₁. CMCT modifications are detected through primer extension analysis. The nucleotide positions in domain V of 23S rRNA exhibiting altered CMCT reactivity in the presence of the drugs are indicated. The Cfr modification at nucleotide A2503 is labeled (Cfr md.). Lanes marked G, A, U, and C denote dideoxy sequencing reactions.