Novel Insights of Gentamicin Activity and Resistance in Staphylococcus aureus

Abstract

The desKR-like two-component system (TCS) of Staphylococcus aureus was identified previously to be activated by stimuli of low temperature, sapienic acid and subinhibitory concentration of the aminoglycoside antibiotic gentamicin. The desKR TCS regulates expression of an ABC transporter and might sense changes in the cell membrane and act to efflux antibiotics to maintain membrane and cellular function. The Nebraska transposon mutant library (NTML) was screened for growth phenotypes at 1⁄4 MIC gentamicin to identify genetic loci that contribute to the cell response to gentamicin that would allow testing of their effects on the S. aureus. The NTML screen revealed 11 mutants associated with electron transport pathways (aroC, menD, ispA, cyoE, qoxA, and qoxC), lipid metabolism (bfmBAB and fakA), DNA repair (xerC) and potassium uptake/regulation (ktrA and ktrD). Mutations were transduced from S. aureus USA300 to SH1000 and revealed most NTML mutations in S. aureus SH1000 did not confer preserved phenotypes, except for three gene mutants: qoxC, fakA, and ktrD, which were tested for their effect on desKR-like TCS gene expression. Each mutant had reduced desKR gene expression after exposure to 1⁄4 MIC gentamicin compared to pre- treatment conditions for each strain with reference to WT. Allelic replacement mutants were constructed for qoxC and ktrD genes in S. aureus SH1000 to compare with the their NTML mutants to investigate potential effects from the transposon. The constructed mutants confirmed the gentamicin phenotype. Subsequently, small colony variant (SCV) phenotype frequencies were explored after exposure to 1⁄4 MIC of gentamicin and showed greater SCV numbers compared to WT. Gentamicin at 1⁄4 MIC activates the desKR-like TCS regulon and it also selects for SCV growth. Specifically, SCV selection data demonstrated that strains lacking either desK or 01312 genes had reduced formation of SCV after gentamicin exposure, whereas a S. aureus desK-SNP (H001) strain with constitutive regulon expression had a higher emergence of SCVs than both inactivation mutant strains. The desK::tet and Δ01312 complemented mutants demonstrated similar SCV formation to WT. Collectively, these data are supportive evidence that the TCS operon contributes to antibiotic resistance. Whole genome sequence of selected SCV clones revealed mutations in menadione biosynthesis as the primary metabolic pathway disrupted (menA, menB and menE_2). One stable SCV of desK::tet identified aroA2 as an unreported gene mutation linked to the SCV phenotype. SNP analysis of both normal and small colony phenotype clones identified gtf1, ebh, pbuE, mutM, and comEC mutations that were selected in desKR operon mutants, which might collectively contribute to improved survival/fitness. While the roles of these genes in this context remain unknown they provide future targets for exploration of both the impact of gentamicin antibiotic on the cell and the role of the desKR-like TCS in S. aureus.