Construction of a linezolid-resistant strain of methicillin-susceptible Staphylococcus aureus and its multi-omics based mechanism study

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Abstract
Background: The emergence of linezolid−resistant methicillin−susceptible Staphylococcus aureus (MSSA) is a growing clinical threat, but the underlying mechanisms are unclear. Here, we generated stable resistant derivatives and used a multi−omics strategy to investigate the transcriptional and metabolic regulation underlying linezolid resistance in MSSA. Methods: A clinical isolate of linezolid-susceptible S. aureus (strain S. aureus _97) was used as the parental strain. A stable linezolid-resistant derivative (designated LZR) was generated through stepwise induction with subinhibitory concentrations of linezolid. The genetic stability of the resistant phenotype was subsequently confirmed via phenotypic stability assays. To elucidate the mechanisms underlying resistance, comparative transcriptomic (RNA-seq) and untargeted metabolomic (LC-MS/MS) analyses were performed to identify differential gene expression and metabolic profiles between the resistant (LZR) and susceptible (LSS) strains. Results: The successfully induced linezolid-resistant derivative (LZR) exhibited a 16-fold increase in minimum inhibitory concentration (MIC) against linezolid (32 μg/mL) compared with the parental strain (2 μg/mL), and this resistant phenotype remained stable after 50 serial passages in antibiotic-free medium. CCCP was observed to reduce the MIC of LZR in a concentration-dependent manner. Transcriptomic and metabolomic analyses revealed widespread transcriptional reprogramming and metabolic perturbations in the resistant strain. Differentially expressed genes were mainly enriched in pathways associated with oxidative phosphorylation, ribosome function, and glycerolipid metabolism, while differential metabolites were primarily involved in the biosynthesis of secondary metabolites and amino acid metabolism. Integrated analysis identified key co-enriched pathways with distinct regulatory patterns, which collectively remodeled bacterial material and energy metabolism and were closely associated with the emergence of linezolid resistance. Conclusion: A stable linezolid-resistant MSSA strain was developed in this work, revealing an intricate network of regulatory interactions that drive resistance at both the transcriptional and metabolic levels. These findings provide an experimental foundation and theoretical basis for further understanding the mechanisms of linezolid resistance in MSSA and for developing strategies to combat resistance.

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