Objectives: The study determined antibiotic resistance profiles of surgical wound’ multidrug-resistant (MDR) isolates belonging to the ESKAPE group and evaluated antibacterial and antibiofilm activities of geraniol (G), carvacrol (C), and eugenol (E), individually and in the selected binary mixtures against them. The cytotoxicity of monoterpenes and their combinations was also assessed. Methods: The antibacterial activity against E. faecium, S. aureus MRSA, K. pneumoniae, A. baumannii, P. aeruginosa and Enterobacter spp. was assessed in microdilution assay. Antibiofilm activity was tested in crystal violet assay. Cytotoxicity of the tested monoterpenes was estimated in XTT assay. Results: C proved the strongest antimicrobial activity (MIC 0.32±0.24 mg mL-1). Antibacterial checkerboard assay, used to study interactions, showed synergism between all monoterpenes. Significant biofilm inhibition (in crystal violet assay) was found for sole monoterpenes, with the most pronounced inhibition for G (38.78–84.72%, p< 0.05). The most significant biofilm eradication was demonstrated for C (25.21-61.34%, p< 0.05), while the G (28.83-55.26%) and E (21.74-52.83%) exhibited a weaker effect. G-C mixtures were further studied for the antibiofilm effect, showing notable inhibition of biofilm formation on all isolates except P. aeruginosa. However, the G-C mixtures exhibited biofilm eradication activity solely against P. aeruginosa. Cytotoxicity tested on normal human MRC-5 fibroblasts showed no cytotoxicity at tested doses of G, E and C, while G-C mixtures were less cytotoxic than povidone-iodine (p< 0.05), used as control. Conclusions: All monoterpenes and G-C mixture proved significant antibacterial and antibiofilm potential against ESKAPE isolates, and acceptable impact on cell viability.

Objectives: The study determined antimicrobial resistance profiles of surgical wound multidrug-resistant (MDR) isolates belonging to the ESKAPE group and evaluated antibacterial and antibiofilm activities of geraniol (G), carvacrol (C) and eugenol (E), individually and in the selected mixtures. The cytotoxicity of monoterpenes and combinations was also assessed. Methods: The antibacterial and antibiofilm activity against tested isolates of Enterococcus faecium, Staphylococcus aureus MRSA, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter sp. was assessed in microdilution and crystal violet assay, respectively, while cytotoxicity was estimated in XTT assay on human MRC-5 fibroblasts. Results: C showed the strongest antibacterial activity (MIC 0.32 ± 0.24 mg mL⁻¹). Monoterpenes induced synergism in certain combinations (FICI 0.09–0.31). All individual monoterpenes inhibited biofilm formation, but G was the most active (38.78–84.72%, p < 0.05). The most pronounced biofilm eradication was observed for C (25.21–61.34%, p < 0.05). G-C mixtures showed notable inhibition of biofilm formation in all isolates except P. aeruginosa, but, on the contrary, induced biofilm eradication against P. aeruginosa only. Cytotoxicity was not detected with the applied concentrations of monoterpenes, while the G-C mixtures exhibited lower cytotoxicity than povidone-iodine (p < 0.05) used as the control. Conclusions: All monoterpenes and G-C mixtures proved significant antibacterial and antibiofilm potential against ESKAPE isolates and acceptable impact on cell viability. Practical significance: The results highlight the potential of plant monoterpenes and their binary combinations as complementary agents for controlling MDR ESKAPE pathogens and biofilm-associated infections following surgical procedures. Further in vivo and safety studies are needed to confirm their applicability in clinical settings.