title: Novel Pathways for Ameliorating the Fitness Cost of Gentamicin Resistant Small Colony Variants.
abstract: Small colony variants (SCVs) of the human pathogenStaphylococcus aureusare associated with persistent infections. Phenotypically, SCVs are characterized by slow growth and they can arise upon interruption of the electron transport chain that consequently reduce membrane potential and thereby limit uptake of aminoglycosides (e.g., gentamicin). In this study, we have examined the pathways by which the fitness cost of SCVs can be ameliorated. Five gentamicin resistant SCVs derived fromS. aureusJE2 were independently selected on agar plates supplemented with gentamicin. The SCVs carried mutations in the menaquinone and hemin biosynthesis pathways, which caused a significant reduction in exponential growth rates relative to wild type (WT; 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 of gentamicin). For SCVs evolved in the absence of gentamicin, 12 out of 15 lineages derived from SCVs with point mutations acquired intra-codonic suppressor mutations restoring membrane potential, growth rate, gentamicin susceptibility and colony size to WT levels. For the SCVs carrying deletions, all lineages enhanced fitness independent of membrane potential restoration without alterations in gentamicin resistance levels. By whole genome sequencing, we identified compensatory mutations in genes related to the σBstress response (7 out of 10 lineages). Inactivation ofrpoFthat encode for the alternative sigma factor SigB (σB) partially restored fitness of SCVs. For all lineages passaged in the presence of gentamicin, fitness compensation via membrane potential restoration was suppressed, however, selected for secondary mutations infusAandSAUSA300_0749. This study is the first to describe fitness compensatory events in SCVs with deletion mutations and adaptation of SCVs to continued exposure to gentamicin.
authors.affiliation: Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen Frederiksberg, Denmark.
authors.forename: Martin
authors.initials: M
authors.lastname: Vestergaard
authors.affiliation: Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen Frederiksberg, Denmark.
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Paulander
authors.affiliation: Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen Frederiksberg, Denmark.
authors.forename: Bingfeng
authors.initials: B
authors.lastname: Leng
authors.affiliation: MRSA Knowledge Center, Department of Clinical Microbiology, Hvidovre Hospital Hvidovre, Denmark.
authors.forename: Jesper B
authors.initials: JB
authors.lastname: Nielsen
authors.affiliation: MRSA Knowledge Center, Department of Clinical Microbiology, Hvidovre Hospital Hvidovre, Denmark.
authors.forename: Henrik T
authors.initials: HT
authors.lastname: Westh
authors.affiliation: Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen Frederiksberg, Denmark.
authors.forename: Hanne
authors.initials: H
authors.lastname: Ingmer
keywords: Staphylococcus aureus; electron transport chain; evolution; gentamicin; resistance; small colony variants