title: Mutations in ribosomal protein L3 and 23S ribosomal RNA at the peptidyl transferase centre are associated with reduced susceptibility to tiamulin in Brachyspira spp. isolates.
abstract: The pleuromutilin antibiotic tiamulin binds to the ribosomal peptidyl transferase centre. Three groups of Brachyspira spp. isolates with reduced tiamulin susceptibility were analysed to define resistance mechanisms to the drug. Mutations were identified in genes encoding ribosomal protein L3 and 23S rRNA at positions proximal to the peptidyl transferase centre. In two groups of laboratory-selected mutants, mutations were found at nucleotide positions 2032, 2055, 2447, 2499, 2504 and 2572 of 23S rRNA (Escherichia coli numbering) and at amino acid positions 148 and 149 of ribosomal protein L3 (Brachyspira pilosicoli numbering). In a third group of clinical B. hyodysenteriae isolates, only a single mutation at amino acid 148 of ribosomal protein L3 was detected. Chemical footprinting experiments show a reduced binding of tiamulin to ribosomal subunits from mutants with decreased susceptibility to the drug. This reduction in drug binding is likely the resistance mechanism for these strains. Hence, the identified mutations located near the tiamulin binding site are predicted to be responsible for the resistance phenotype. The positions of the mutated residues relative to the bound drug advocate a model where the mutations affect tiamulin binding indirectly through perturbation of nucleotide U2504.
authors.affiliation: Department of Antibiotics, National Veterinary Institute, SE-75189 Uppsala, Sweden.
authors.forename: Märit
authors.initials: M
authors.lastname: Pringle
authors.forename: Jacob
authors.initials: J
authors.lastname: Poehlsgaard
authors.forename: Birte
authors.initials: B
authors.lastname: Vester
authors.forename: Katherine S
authors.initials: KS
authors.lastname: Long
mesh_terms: D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004224:Diterpenes / Q000378:metabolism / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012329:RNA, Bacterial / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000235:genetics*; D000090822:Ribosomal Protein L3; D012269:Ribosomal Proteins / Q000235:genetics*; D013144:Spirochaetales / Q000187:drug effects* / Q000235:genetics
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004224:Diterpenes; D011500:Protein Synthesis Inhibitors; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S; D000090822:Ribosomal Protein L3; D012269:Ribosomal Proteins; C014224:tiamulin