Measurement of Accumulation of Antibiotics to Staphylococcus aureus in Phagosomes

Staphylococcus aureus ( S. aureus ) has evolved the ability to persist after uptake into host immune cells. This intracellular niche enables S. aureus to potentially escape host immune responses and survive the action of antibiotics. The elevated tolerance of S. aureus to small molecule antibiotics is likely to be multifactorial. We pose that there may be contributions related to permeation into phagosome, which would require translocation across two mammalian bilayers. To empirically test this, we adapted our recently developed permeability assay to determine the accumulation of FDA-approved antibiotics in phagocytic vacuoles. Bioorthogonal reactive handles were metabolically anchored within the surface of S. aureus , and complementary tags were chemically added to antibiotics. Following phagocytosis of labeled S. aureus cells, we were able to specifically analyze the accumulation levels of antibiotics within the phagosomes of infected macrophages. Our findings enabled the determination of differences between the permeability of antibiotics to extra- and intracellular S. aureus , thus providing a roadmap to dissect the contribution of antibiotic permeability to intracellular pathogens. ### Competing Interest Statement The authors have declared no competing interest.