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Orthopaedic Proceedings
Vol. 105-B, Issue SUPP_17 | Pages 20 - 20
24 Nov 2023
THE ROLE OF MACROPHAGES AND INTRACELLULAR SURVIVAL OF STAPHYLOCOCCUS AUREUS: NEW INSIGHTS FROM DIRECT EVALUATION OF HUMAN TISSUE SAMPLES
Morin B Tripathi V Iizuka A Clauss M Morgenstern M Baumhoer D Jantarug K Fuentes PR Kuehl R Bumann D Khanna N
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Aim

Staphylococcus aureus (SA) can cause various infections and is associated with high morbidity and mortality rates of up to 40%. Antibiotic treatment often fails to eradicate SA infections even if the causative strain has been tested susceptible in vitro. The mechanisms leading to this persistence is still largely unknown. In our work, we to reveal SA interactions with host cells that allow SA to persist at the site of infection.

Method

We established a sampling workflow to receive tissue samples from patients requiring surgical debridement due to SA bone-and joint or soft-tissue infections. We developed a multiplex immunofluorescent staining protocol which allowed us to stain for SA, leukocytes, neutrophils, macrophages, B-cells, T-cells, DAPI and cytoplasmatic marker on the same sample slide. Further, distance of SA to cell nuclei was measured. Interaction of immune cells and SA on a single cell level was investigated with high-resolution 3D microscopy. We then validated our findings applying fluorescence-activated cell sorting (FACS) on digested patient samples. Finally, we aimed to reproduce our ex vivo patient results in an in vitro co-culture model of primary macrophages and clinical SA strains, where we used live cell microscopy and high-resolution microscopy to visualize SA-immune cell interactions and a gentamicin protection assay to assess viability of SA.