| description |
Small colony variants (SCVs) of the human pathogen Staphylococcus aureus are associated with persistent and recurrent infections. They are characterized by slow growth and can arise due to interrupted electron transport that consequently reduce membrane potential and thereby limit uptake of aminoglycosides (e.g. gentamicin). Here we have examined by which pathways the fitness cost of SCVs can be ameliorated. Five gentamicin resistant SCVs were independently selected on agar plates supplemented with gentamicin. The SCVs were found to carry mutations in the menaquinone and hemin biosynthesis pathways, which caused a significant reduction in exponential growth rates relative to wild type (0.59-0.72) and reduced membrane potentials. Fifty independent lineages of the low-fitness, resistant mutants were serially passaged for up to 500 generations with or without sub-lethal concentrations of gentamicin. Amelioration of the fitness cost followed three evolutionary trajectories and was dependent on the initial mutation type (point mutation vs. deletion) and the passage condition (absence or presence gentamicin). For SCVs evolved in the absence of gentamicin, 12 out of 15 lineages derived from SCVs with point mutations acquired intragenic suppressor mutations restoring membrane potential, growth rate, gentamicin susceptibility and colony size to wild type levels. For the SCVs carrying deletion mutations all lineages enhanced fitness independent of membrane potential restoration without alterations in gentamicin resistance levels and by whole genome sequencing, we identified compensatory mutations in genes related to the σB stress response (7 out of 10 lineages). Inactivation of rpoF that encode for the alternative sigma factor SigB (σB) partially restored fitness of SCVs. In the presence of gentamicin, fitness was improved by acquisition of additional resistance mutations while membrane potential remained unaltered for all lineages. This study is the first to describe fitness compensatory events in SCVs with deletion mutations and adaptation of SCVs to continued exposure to gentamicin. |