The emergence of antibiotic resistance greatly increases the difficulty of treating bacterial infections. In order to develop effective treatments, the underlying mechanisms of antibiotic resistance must be understood. In this study, Staphylococcus aureus ATCC6538 strain was passaged in medium with and without gentamicin and obtained lab-evolved gentamicin-resistant S. aureus (R_GEN) and gentamicin-sensitive S. aureus (S_GEN) strains, respectively. Data-Independent Acquisition (DIA)-based proteomics approach was applied to compare the two strains. A total of 1426 proteins were identified, of which 462 were significantly different: 126 were upregulated and 336 were downregulated in R_GEN compared to S_GEN. Further analysis found that reduced protein biosynthesis was a characteristic feature in R_GEN, related to metabolic suppression. The most differentially expressed proteins were involved in metabolic pathways. In R_GEN, central carbon metabolism was dysregulated and energy metabolism decreased. After verification, it was found that the levels of NADH, ATP, and reactive oxygen species (ROS) decreased, and superoxide dismutase and catalase activities increased. These findings suggest that inhibition of central carbon and energy metabolic pathways may play an important role in the resistance of S. aureus to gentamicin, and that gentamicin resistance is associated with oxidative stress. Significance: The overuse and misuse of antibiotics have led to bacterial antibiotic resistance, which is a serious threat to human health. Understanding the mechanisms of antibiotic resistance will help better control these antibiotic-resistant pathogens in the future. The present study characterized the differential proteome of gentamicin-resistant Staphylococcus aureus using the most advanced DIA-based proteomics technology. Many of the differential expressed proteins were related to metabolism, specifically, reduced central carbon and energy metabolism. Lower levels of NADH, ROS, and ATP were detected as a consequence of the reduced metabolism. These results reveal that downregulation of protein expression affecting central carbon and energy metabolisms may play an important role in the resistance of S. aureus to gentamicin.

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