Abstract
BackgroundWe previously reported the case of a 64-year-old man with mediastinitis caused by Staphylococcus aureus in which the infecting bacterium acquired linezolid resistance after only 14 days treatment with linezolid. We therefore investigated relevant clinical isolates for possible mechanisms of this rapid acquisition of linezolid resistance.MethodsUsing clinical S. aureus isolates, we assessed the in vitro mutation rate and performed stepwise selection for linezolid resistance. To investigate homologous recombination, sequences were determined for each of the 23S ribosomal RNA (23S rRNA) loci; analyzed sequences spanned the entirety of each 23S rRNA gene, including domain V, as well as the 16S-23S intergenic spacer regions. We additionally performed next-generation sequencing on clinical strains to identify single-nucleotide polymorphisms compared to the N315 genome.ResultsStrains isolated from the patient prior to linezolid exposure (M5-M7) showed higher-level linezolid resistance than N315, and the pre-exposure strain (M2) exhibited more rapid acquisition of linezolid resistance than did N315. However, the mutation rates of these and contemporaneous clinical isolates were similar to those of N315, and the isolates did not exhibit any mutations in hypermutation-related genes. Sequences of the 23S rRNA genes and 16S-23S intergenic spacer regions were identical among the pre- and post-exposure clinical strains. Notably, all of the pre-exposure isolates harbored a recQ missense mutation (Glu69Asp) with respect to N315; such a lesion may have affected short sequence recombination (facilitating, for example, recombination among rrn loci). We hypothesize that this mechanism contributed to rapid acquisition of linezolid resistance.ConclusionsHypermutation and homologous recombination of the ribosomal RNA genes, including 23S rRNA genes, appear not to have been sources of the accelerated acquisition of linezolid resistance observed in our clinical case. Increased frequency of short sequence recombination may have resulted from a recQ variant in the infecting organism.
Highlights
Linezolid (LZD) is the first approved antibiotic of the oxazolidinone class
We previously reported the case of a 64-year-old man with mediastinitis caused by Staphylococcus aureus in which the infecting bacterium acquired linezolid resistance after only 14 days treatment with linezolid
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of Strains isolated from the patient prior to linezolid exposure (M5-M7) showed higher-level linezolid resistance than N315, and the pre-exposure strain (M2) exhibited more rapid acquisition of linezolid resistance than did N315
Summary
Linezolid (LZD) is the first approved antibiotic of the oxazolidinone class This agent is effective for the treatment of infectious diseases caused by Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). The clinical occurrence of LZD resistance is rare, and has been observed only following prolonged exposure to the drug [5] This observation is consistent with the low frequency of in vitro mutation, reported as less than 8 × 10−11 [6]. The minimum inhibitory concentration (MIC) of LZD following exposure was 32 μg/ml, compared to an MIC of 4 μg/ml in the parent strain These LRSA strains harbored a G2576T mutation in multiple 23S rRNA loci. We investigated relevant clinical isolates for possible mechanisms of this rapid acquisition of linezolid resistance.
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