Proteomic Alterations in Bacterial and Human Vesicles Under Various Antibiotic Treatments in Blood Spiked with Staphylococcus Aureus
Background
Sepsis remains a significant global health challenge, contributing to 20% of annual deaths worldwide, with 71% of these attributed to antibiotic-resistant infections. Effective management hinges on timely antibiotic administration and accurate bacterial detection in blood samples. Bacterial membrane vesicles (bMVs), ranging from 20 to 400 nm and loaded with diverse biomolecules, play pivotal roles in inter-bacterial communication, modulation of host immune responses, and potential organ failure. These bMVs have been identified in both blood and tissues, prompting investigations into their protein cargo, along with the proteome of human extracellular vesicles (EVs), under conditions mimicking bacterial infection and antibiotic exposure.
Methods
To simulate sepsis conditions, 5 ml of healthy donor EDTA-blood (MOI: 0.0001) was spiked with Staphylococcus aureus (SA), either alone or in the presence of antibiotics commonly used in German hospitals: Piperacillin-Tazobactam (Pip-Tazo: 25, 50, and 100 µg/ml), Vancomycin (6.25, 12.5, and 25 µg/ml), and Moxifloxacin (0.18, 0.73, and 2.9 µg/ml). Human EVs were isolated using the Miltenyi Biotec Pan EV Isolation Kit, followed by incubation of unbound plasma with in-house coupled 1 µm magnetic beads with anti-OmpA/GroEL antibodies. Subsequently, human EVs and the bMV-BacBead complex were stained with CellMask™ Deep Red (CMD), anti-PanEV, anti-SA antibodies, and analyzed using flow cytometry. Ongoing proteomic analysis aims to thoroughly characterize the protein profiles of plasma, human EVs, and bMVs across mock control, SA-only infection, and the highest concentration of three antibiotic groups.
Results
Initial flow cytometry results indicated elevated levels of CMD+ PanEV+/- SA+ on human EVs and bMVs at the highest concentrations of Pip-Tazo and Vancomycin compared to SA-only infection, though not with Moxifloxacin. Our preliminary proteomics investigations enable us to gain a deeper insight into bacterial-host interactions and inflammation.
Conclusion
In conclusion, our extensive proteomic profiles can help us understand the dysregulation of inflammation caused by bMVs in patients with sepsis.
Keywords
Bacterial membrane vesicle, Human extracellular vesicle, Staphylococcus aureus, Antibiotics, Proteomics
Funding/Acknowledgments
Zentrales Innovationsprogramm Mittelstand (ZIM)
Authors
Dapi Menglin Chiang1,2 (Corresponding Author: dapi.chiang[at]mytum[dot]de), Michael W. Pfaffl1, Christina Ludwig3, Susanne I Wudy3, Agnes Meidert4, Florian Brandes4, Gustav Schelling4, Benedikt Kirchner1,2, Mia S.C. Yu1, Christian Zenner5, Laurent Muller6,7, and Marlene Reithmair2