Transcriptional Profiling of Phagocytic Leukocytes and Microglia Reveals a Critical Role for Reactive Oxygen Species in Biofilm Containment During Staphylococcus aureus Craniotomy Infection

Introduction: Craniotomies are performed to treat a variety of intracranial pathology. Surgical site infection remains a complication of craniotomy despite the use of prophylactic antibiotics and universal sterile precautions. Infections occur in 1-3% of procedures, with approximately half caused by Staphylococcus aureus (S. aureus) that forms a biofilm on the bone flap and is recalcitrant to systemic antibiotic therapy.

Methods: We utilized a S. aureus-dsRed construct to compare the phagocytic capacity of leukocytes and microglia in vitro and in vivo using a mouse model of craniotomy infection. In addition, single cell RNA-sequencing (scRNA-seq) was applied to determine whether a transcriptional signature could be identified for phagocytic vs. non-phagocytic cells in vivo.

Results: S. aureus was phagocytosed to equivalent extents in microglia, macrophages, neutrophils, and granulocytic myeloid-derived suppressor cells in vitro; however, microglial uptake of S. aureus was limited in vivo, whereas the other leukocyte populations exhibited phagocytic activity. scRNA-seq comparing the transcriptional signatures of phagocytic (S. aureus-dsRed+) vs. non-phagocytic (S. aureus-dsRed-) leukocytes identified classical pathways enriched in phagocytic cells (i.e., reactive oxygen/nitrogen species (ROS/RNS), lysosome, iron uptake and transport) whereas non-phagocytic populations had increased ribosomal, interferon, and hypoxia signatures. scRNA-seq also revealed a robust ROS profile, which led to the exploration of craniotomy infection in NADPH oxidase (NOX2) knockout (KO) mice. S. aureus burden, leukocyte recruitment, and intracellular bacterial load were significantly increased in NOX2 KO compared to wild-type animals.

Conclusion : Collectively, these results highlight the importance of ROS generation in phagocytes for S. aureus biofilm containment, but not clearance, during craniotomy infection.