Antibacterial and antibiofilm activity of a triterpenoid-rich Eucalyptus globulus leaf extract against gram-positive bacteria

Eucalyptus globulus leaf extract (EGL) is a phytocomplex associated with antimicrobial and anti-inflammatory effects, but a comprehensive characterization of its chemical composition and its relationship with biological activity remains limited. In this study, we combined untargeted metabolomic profiling with functional antibacterial and antibiofilm assays to characterize EGL and to investigate the relationship between its chemical composition and biological activity. Untargeted UHPLC–TWIMS–QTOF analysis revealed a complex profile dominated by polyphenols and pentacyclic triterpenoids. Targeted quantification identified asiatic acid and ursolic acid as major triterpenoid components, present at concentrations of 47.29 ± 0.13 µg/mL and 16.12 ± 0.32 µg/mL, respectively. The antibacterial and antibiofilm activities of EGL were evaluated against clinically relevant Gram-positive pathogens, including Streptococcus pyogenes, Staphylococcus aureus, methicillin-resistant S. aureus and Streptococcus mutans. The two major triterpenoids identified in the extract, asiatic acid and ursolic acid, were tested separately as reference compounds to explore their individual antibacterial and antibiofilm activity in comparison with the whole extract. EGL inhibited bacterial growth with MIC values ranging from 62.5 µg/mL to 437.5 µg/mL, while the triterpenoids showed stronger activity, with MIC values of 2 µg/mL for ursolic acid and 4 µg/mL for asiatic acid against Streptococcus pyogenes. In antibiofilm assays, asiatic acid and ursolic acid showed MBIC values between 2 and 32 µg/mL, whereas the whole extract displayed partial inhibitory activity. Pre-exposure of bacterial cultures to EGL or its major triterpenoids enhanced antibiofilm efficacy, reducing MBIC values by up to fourfold. Overall, these results indicate that EGL is a chemically characterized multicomponent system with measurable antibacterial and antibiofilm activity against Gram-positive pathogens. The study highlights the importance of integrating chemical profiling with functional assays to better understand the activity of complex phytocomplexes and supports further investigation of phytochemical-based strategies targeting early stages of biofilm formation.