Multiple pathways towards reduced membrane potential and concomitant reduction in aminoglycoside susceptibility in Staphylococcus aureus

Staphylococcus aureus is responsible for life-threatening and difficult-to-treat infections worldwide and antimicrobial resistance is an increasing concern. Whilst acquired resistance has been widely studied, little is known of the contributions from chromosomal determinants that upon inactivation may reduce the susceptibility of S. aureus to antibiotics. The aim of this study was to identify genetic determinants that upon inactivation reduce aminoglycoside susceptibility in S. aureus. The Nebraska Transposon Mutant Library of 1920 single-gene inactivations in S. aureus strain JE2 was screened for reduced susceptibility to gentamicin. Nine mutants were confirmed by Etest to display between 2- and 16-fold reduced susceptibility to this antibiotic. All of the identified genes were associated with the electron transport chain and energy metabolism. Four mutant strains (menD, hemB, aroC and SAUSA300_0355) conferred the largest decrease in gentamicin susceptibility and three exhibited a small colony variant phenotype, whereas the remaining mutants (qoxA, qoxB, qoxC, ndh and hemX) displayed colony morphology similar to the wild-type. All of the mutants, except hemX, displayed reduced membrane potential suggesting that reduced uptake of gentamicin is the predominant mechanism leading to reduced susceptibility. The results of this study demonstrate that S. aureus possesses multiple genes that upon inactivation by mutagenesis reduce the membrane potential and thereby reduce the lethal activity of gentamicin.