Ribosomes from an oxazolidinone-resistant mutant confer resistance to eperezolid in a Staphylococcus aureus cell-free transcription-translation assay

Abstract

Oxazolidinone-resistant mutants of Staphylococcus aureus, isolated with a spiral plating technique, had a 16-fold higher MIC (2 versus 32 microg/ml) of eperezolid when compared to the parental sensitive strain. Eperezolid inhibited in vitro protein translation with 50% inhibitory concentrations of 30 microM for the oxazolidinone-sensitive S30 extract and 75 microM for the resistant extract. Experiments mixing various combinations of S100 and crude ribosome preparations from oxazolidinone-sensitive and -resistant S. aureus strains in a transcription-translation assay demonstrated that the resistant determinant resided within the ribosomal fraction. Ribosomes from the oxazolidinone-resistant strain bound less drug than ribosomes from the sensitive strain, indicating that the ribosome is the site of action for the oxazolidinones. These experiments demonstrate that an alteration of the ribosome is responsible for some or all of the oxazolidinone resistance observed in the S. aureus mutant.

FIG. 1

Effect of eperezolid on activity of S30 extracts from Oxa^s and Oxa^r strains in the S. aureus in vitro TT assay. Percent [³⁵S]methionine incorporation in TCA-precipitable material in the presence of increasing concentrations of eperezolid. •, SA31552 (Oxa^s); ▪, SA31593 (Oxa^r).

FIG. 2

Reconstitution of S. aureus TT in the presence of increasing concentrations of eperezolid (0 to 300 μM) with S100 and crude ribosome preparations from SA31552 and SA31593 in the S. aureus in vitro TT assay. Measuring TCA-precipitable [³⁵S]methionine incorporation. (A) S100 from the sensitive strain mixed with crude ribosomes from either the sensitive or the resistant strain. •, Oxa^s S100, Oxa^r ribosomes; □, Oxa^s S100, Oxa^s ribosomes. (B) S100 from the resistant strain mixed with crude ribosomes from either the sensitive or resistant strain. , Oxa^r S100, Oxa^r ribosomes; ◊, Oxa^r S100, Oxa^s ribosomes.

FIG. 3

Binding of [¹⁴C]eperezolid to increasing concentrations of purified, high-salt-washed Oxa^s and Oxa^r ribosomes. Specific binding is calculated as the total binding minus the nonspecific binding. Nonspecific binding represents the binding reaction in the presence of a 300-fold molar excess of cold drug competitor. Error bars indicate standard deviations (n = 3). □, total eperezolid binding; ▨, specific eperezolid binding.