Imaging Staphylococcus aureus biofilms in vivo reveals neutrophil exclusion by PNAG exopolysaccharide

Staphylococcus aureus forms biofilms on biotic and abiotic surfaces, enabling persistent infections that evade immune clearance. Although many S. aureus strains can produce the biofilm-associated exopolysaccharide, poly-β-1,6-N-acetyl-D-glucosamine (PNAG), they often form protein-dominated biofilm matrices in vitro , leaving a role for PNAG unclear. Using intravital imaging in a foreign-body infection model, we found that PNAG in biofilms hinders neutrophil access and delays bacterial clearance. Neutrophil elastase was crucial for eventual biofilm clearance. In vivo PNAG labeling revealed that the exopolysaccharide forms a physical barrier that prevents neutrophils from reaching bacterial clusters. In contrast, PNAG-deficient strains permitted greater neutrophil infiltration and were cleared more rapidly than wild-type bacteria. Enzymatic degradation of PNAG with the glycoside hydrolases PgaB or dispersin B (DspB) disrupted the biofilm, restored neutrophil access, and enhanced bacterial clearance. Together, these findings identify PNAG as a key structural barrier protecting S. aureus from innate immunity and suggest that targeting PNAG with glycoside hydrolases may offer a promising therapeutic strategy for biofilm-associated S. aureus infections.