Staphylococcus aureus is a leading cause of musculoskeletal infection with frequent recurrence despite surgery and antibiotic therapy. Its persistence in human tissues under antibiotic pressure remains unclear. We performed longitudinal in situ analyses of matched tissue samples before and during therapy from patients undergoing repeated surgery using advanced imaging and tissue-based metagenomics. S. aureus remained detectable in all samples - including culture-negative specimens - and whole-genome sequencing confirmed persistence of the infecting strain. Antibiotic initiation reduced bacterial burden by 1.05log10 bacteria/mm^3 but residual loads stabilized thereafter. Quantitative imaging identified a culture detection threshold (~10^3bacteria/mm^3), indicating that culture negativity reflects diagnostic sensitivity limits rather than true bacterial clearance. Persistence under antibiotic therapy was associated with high baseline burden and intracellular sequestration, whereas long-term persistence was characterized by predominantly extracellular bacteria capable of driving relapse. These findings reveal sustained tissue persistence despite apparent microbiological clearance and guide precision strategies to target resilient bacterial reservoirs.
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