Ribosomal and non‐ribosomal resistance to oxazolidinones: species‐specific idiosyncrasy of ribosomal alterations

Summary A derivative of Mycobacterium smegmatis, which carries only one functional rRNA (rrn) operon, was used to isolate mutants resistant to the ribosome‐targeted antibiotic linezolid. Isolation and characterization of linezolid‐resistant clones revealed two classes of mutants. Ribosomes from class I mutants are resistant to oxazolidinones in an in vitro peptidyl transferase assay, indicating that resistance maps to the ribosome component. In contrast, ribosomes from class II mutants show wild‐type susceptibility to a linezolid derivative in vitro, pointing to a non‐ribosomal mechanism of resistance. Introduction of a wild‐type ribosomal RNA operon into linezolid‐resistant strains restored linezolid sensitivity in class I mutants, indicating that resistance (i) maps to the rRNA and (ii) is recessive. Sequencing of the entire rrn operon identified a single nucleotide alteration in 23S rRNA of class I mutant strains, 2447G → T (Escherichia coli numbering). Introduction of mutant rrl2447T into M. smegmatis rrn– resulted in a linezolid‐resistant phenotype, demonstrating a cause–effect relationship of the 2447G → T alteration. The 2447G → T mutation, which renders M. smegmatis linezolid resistant, confers lethality in E. coli. This finding is strong evidence of structural and pos‐sibly functional differences between the ribosomes of Gram‐positive and Gram‐negative bacteria. In agreement with the results of the in vitro assay, class II mutants show a wild‐type sequence of the complete rRNA operon. The lack of cross‐resistance of the class II mutants to other antibiotics suggests a resistance mechanism other than activation of a broad‐spectrum multidrug transporter.