Betulinic Acid Eradicates Implant‐Associated Infections by Disrupting the <i>S. aureus</i> Biofilm Matrix and Potentiating Host Immune Clearance

Staphylococcus aureus biofilms are major contributors to chronic and recurrent infections due to their intrinsic tolerance to antibiotics and host immune clearance, highlighting the urgent need for safe and effective antibiofilm strategies. This study evaluated the inhibitory effects and underlying mechanisms of betulinic acid (BA), the principal active constituent of the traditional Chinese medicine Liquidambaris fructus, against S. aureus biofilms. In vitro assays demonstrated that the minimum biofilm inhibitory concentration (MBIC) of BA was 32 μg/mL, which was markedly lower than its minimum inhibitory concentration (MIC, 512 μg/mL), indicating preferential activity against biofilm formation. Serial passage experiments revealed no detectable induction of drug resistance. Mechanistic studies revealed that BA suppressed early biofilm adhesion and aggregation, downregulated the expression of adhesion-related genes (clfA, clfB, fnbpA and fnbpB), and reduced the production of extracellular polysaccharide (EPS) and extracellular DNA (eDNA). BA further disrupted mature biofilm architecture, promoted macrophage infiltration, enhanced bacterial clearance and attenuated the expression of immune evasion factors (scin, chip, lukE and nuc). In vivo, BA significantly alleviated implant-associated infections, mitigated local inflammatory responses and facilitated tissue repair. Collectively, these findings reveal that BA inhibits S. aureus biofilms through multiple coordinated mechanisms, with a low propensity for resistance development and favourable biosafety, supporting its potential as a promising lead compound for the development of novel antibiofilm therapeutics.