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Background and Importance Linezolid is an oxazolidinone antibiotic active against resistant Gram-positive bacteria. Although use beyond 28 days is not usually recommended by the Summary of Product Characteristics (SmPC) due to haematological toxicities, prolonged treatment may be necessary, requiring therapeutic drug monitoring to mitigate adverse effects and ensure therapeutic success.While linezolid does not affect human cytoplasmic ribosomes, it can impair mitochondrial protein synthesis, leading to side effects such as lactic acidosis and hypoglycaemia. Toxic levels affecting mitochondria are higher than those needed for antibacterial efficacy. A reduction in mitochondrial protein synthesis has been documented in peripheral blood mononuclear cells (PBMCs), mainly impacting complex IV of the respiratory chain.Aim and Objectives To evaluate mitochondrial function in PBMCs from patients treated with linezolid for different durations (1–7, 7–14, and >14 days) and to perform proteomic analysis of these cells.Material and Methods Blood samples were collected in EDTA tubes from healthy subjects (n=14) and linezolid-treated patients (n=40). PBMCs were isolated using the autoMACS Pro system and stored in liquid nitrogen until analysis.Mitochondrial respiration was assessed with Seahorse XF assays (n=8 controls, n=22 patients) using the Cell Mito Stress Kit. Two-way ANOVA corrected with Dunnett’s multiple comparisons test was performed. For proteomics, PBMCs (n=6 controls, n=18 patients) were processed with the PreOmics Kit and analysed by LC-MS/MS. Differential protein expression was evaluated in R, with Volcano plots and protein–protein interaction networks (STRING). Proteins with fold-change ≥1.5 and false discovery rate (FDR)-adjusted p<0.05 were considered significant.Results Mitochondrial function declined progressively with longer treatment duration, with maximal respiratory capacity as the most affected parameter (p<0.05*) ( figure 1). These findings suggest a dysfunction at the level of substrate oxidation and/or the electron transport chain. Proteomic profiling corroborated functional impairment, showing downregulation of proteins involved in electron transport–coupled proton transport, along with 27 of the 61 proteins associated with Complex I.Abstract 6ER-005 Figure 1Mitochondrial function as determined in our studyConclusion and Relevance Prolonged linezolid treatment is associated with progressive mitochondrial dysfunction in PBMCs, reflected at both the functional and proteomic levels. The concordance between respiration assays and protein expression patterns highlights mitochondrial involvement as a key mechanism underlying linezolid-related toxicity. These findings support the importance of therapeutic monitoring during extended treatments.Conflict of Interest No conflict of interest