From Chemistry to Bioactivity: HS-SPME-GC-MS Profiling and Bacterial Growth Inhibition of Three Different Propolis Samples from Romania, Australia, and Uruguay

Propolis is a valuable natural product whose chemical composition and bioactivity are strongly dependent on its geographical and botanical origin. This study presents a comprehensive comparative analysis of the volatile profiles and antibacterial properties of propolis from Romania, Australia, and Uruguay, benchmarked against previously published data from samples from Poland and Turkey. Volatile compounds were profiled using headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry. The resulting data were interrogated using multivariate chemometric analyses (HCA, PCA), and antibacterial activity was assessed via the disk diffusion method against five bacterial strains. Chemometric analysis revealed a clear demarcation into two primary chemotypes: a European type (Poland, Romania, Turkey) dominated by aromatic compounds such as benzoic acid, and a non-European type (Australia, Uruguay) characterized by a high abundance of terpenes. The Australian propolis exhibited a complex terpene profile rich in α-copaene and pinenes, while the Uruguayan sample was distinguished by an exceptionally high concentration of α-pinene. All active extracts showed selective, concentration-dependent inhibition against Gram-positive <i>Staphylococcus aureus</i> and <i>Streptococcus mutans</i>. The terpene-rich Australian propolis displayed the highest antibacterial potency, particularly against <i>S. mutans</i>. Crucially, Pearson correlation analysis revealed a counter-intuitive relationship: the most abundant terpenes in the non-European samples (e.g., α-pinene, verbenone) were significantly negatively correlated with antibacterial activity (r ≈ −0.99). Conversely, less abundant compounds, including linalool and acetic acid, were identified as strong positive predictors of inhibition (r > 0.90). These findings underscore a complex geography-chemotype-bioactivity relationship, where the overall synergistic effect of a mixed chemical profile, rather than the dominance of a single compound, determines antibacterial potency. The initially proposed markers provide a basis for origin-based standardization and highlight Australian propolis as a promising source of natural antibacterial agents.