In vitro selection of linezolid resistance in hypermutable and wild-type Staphylococcus aureus
Abstract number: 1134_02_173
North S.E., Ellington M.J., Johnson A.P., Livermore D.M., Woodford N.
Resistance to linezolid (lin) is rare, but can arise via mutations in the 23S rRNA genes. We hypothesised (a) that resistance would emerge preferentially in staphylococci with a hypermutable phenotype engineered by mutations in the mutS gene, and (b) that hypermutability might be co-selected with linezolid resistance. MutS is part of the DNA mismatch repair (MMR) system, which identifies and corrects genome alterations post-replication, thereby influencing the net mutation rate.
Linezolid-resistant (LinR) mutants of Staphylococcus aureus RN4220, its hypermutable mutS deletion mutant RN4220mutS, harbouring an erythromycin resistance marker gene (ermB), and a genetically-related pair of clinical isolates were selected by repeated passage with increasing concentrations of lin. Mutant parentage was confirmed by PFGE and susceptibility was tested by agar dilution. An amplified 694-bp fragment of 23S rRNA genes was studied by sequencing and by RFLP analysis. Frequencies at which LinR mutants yielded variants resistant to fusidic acid and rifampin were calculated in triplicate experiments.
Seventeen LinR mutants (MICs 864 mg/ml) were raised. Ten mutants had a G2447T mutation in their 23S rRNA genes; 4, all from RN4220mutS, had G2576T, typically seen in LinR gram-positive cocci from the clinic; 1 had T2504C; 2 had no identifiable mutations. Curiously 5 / 7 RN4220mutS mutants lost ermB during the course of the experiment, reverting to macrolide susceptible. LinR mutants had mutation frequencies for rifampicin and fusidic acid resistance comparable with those of their parents (106 to 109), implying no co-selection of stable hypermutability.
More LinR mutants were obtained from the hypermutable (RN4220mutS) strain than from the wild type strain, and G2576T mutants were only obtained from the hypermutable organism. These data suggest hypermutability facilitates the emergence of LinR, nevertheless, LinR mutants derived from wild-type parents did not have elevated mutation frequencies.
|Session name:||XXIst ISTH Congress|
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