Host-Defense Extracellular Vesicle Protein Changes in Antibiotic- and Staphylococcus aureus–Treated Blood

Sepsis accounts for nearly 20% of global mortality, with antibiotic resistance worsening clinical outcomes. Rapid antibiotic administration and accurate pathogen identification remain crucial. It is well now known that extracellular vesicles (EVs) from human cells and bacterial membrane vesicles (bMVs) play a central role in the interaction between host and pathogen and represent promising biomarkers for early infections. This study investigated how antibiotic exposure alters EV responses in Staphylococcus aureus ( SA )–spiked blood and compared these findings with EV proteome profiles from bacteremia patients. Whole blood from healthy donors was spiked with SA (Multiplicity of infection: 0.001) and treated with piperacillin–tazobactam (Pip-Tazo), vancomycin, or moxifloxacin at clinically relevant concentrations. EVs were isolated using the Miltenyi Pan EV Kit, and bMVs were captured with magnetic beads conjugated to anti-OmpA and anti-GroEL. EVs and bMVs were analyzed using bead-based flow cytometry. Proteomics of total plasma, EVs and bMVs high-resolution LC–MS/MS. Patients’ serum EVs from 6 healthy controls and 12 bacteremia patients (6 blood culture–positive, 6 culture-negative) were processed using the same workflow to assess both host and bacterial proteins. Flow cytometry revealed that levels of CellMask Orange⁺ (CMO⁺) CD45⁺ PanEV⁺ SA⁺ extracellular vesicles increased in blood samples exposed to low concentrations of Pip–Tazo and high concentrations of vancomycin, despite minimal changes in vesicle size distribution and total particle counts. Proteomic analysis identified notable alterations in EV-associated proteins, including strong elevation of the ribosomal protein bL21 in SA-spiked samples and those treated with vancomycin. Gene ontology analysis indicated enrichment of innate immune and exosome-related pathways. In patient samples, EVs were enriched with acute-phase proteins such as PLSCR1, haptoglobin, CRP, and SAA1–4, along with canonical EV markers CD81 and MFGE8, irrespective of culture positivity. Antibiotic treatment leads to significant remodeling of the EV proteome, characterized by enhanced presence of immune and bacterial response proteins, even when vesicle numbers remain constant. These protein shifts appeared in both culture-positive and culture-negative patient samples, supporting the idea that EV-associated proteins could serve as early, host-derived indicators of bloodstream infection.