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SummaryIt is known that changes in bacterial metabolism can contribute to the modulation of bacterial susceptibility to antibiotics. Understanding how bacterial metabolism is impacted by antibiotics may improve our understanding of the antibiotic mechanism of actions from a metabolic perspective. Here, we utilized a mass spectrometry‐based targeted metabolic profiling technique to characterize the metabolome of a pair of isogenic methicillin‐susceptible and resistant Staphylococcus aureus (MSSA and MRSA) strains RN450 and 450M treated with the sublethal dose of three antibiotics from different classes (β‐lactams, aminoglycosides and quinolones). These treatments induced a set of metabolic alterations after 6 h of co‐incubation with antibiotics. Similar and divergent metabolic perturbations were observed from different antibiotics to the tested strains. Different metabolic response from MSSA and MRSA to the same antibiotics was also detected in the study and indicated the potentially different stress response mechanism in MSSA and MRSA metabolism. This work has shown that a complex set of metabolic changes can be induced by a variety of antibiotics, and the comparative metabolomics strategy can provide a good understanding of this process from a metabolic perspective.

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