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Background The chemical composition and biological activity of Eucalyptus essential oils have been studied extensively (EOs). A few of them were tested for antibacterial effectiveness against otitis strains. The chemical composition and antibacterial activity of the EOs of eight Tunisian Eucalyptus species were assessed in the present study. Methods Hydrodistillation was used to extract EOs from the dried leaves of eight Eucalyptus species: Eucalyptus accedens, Eucalyptus punctata, Eucalyptus robusta, Eucalyptus bosistoana, Eucalyptus cladocalyx, Eucalyptus lesouefii, Eucalyptus melliodora and Eucalyptus wandoo. They are assessed by GC/MS and GC/FID and evaluated for antibacterial activity using agar diffusion and broth microdilution techniques against three bacterial isolates (Haemophilus influenzae, Haemophilus parainfluenzae, Klebsiella pneumoniae) and three reference bacteria strains (Pseudomonas aeruginosa, ATTC 9027; Staphylococcus aureus, ATCC 6538; and Escherichia coli, ATCC 8739). Furthermore, the selected twenty-one major compounds and all values of the inhibition zone diameters were subjected to further statistical analysis using PCA and HCA. Results The EO yields of the studied Eucalyptus species range from 1.4 ± 0.4% to 5.2 ± 0.3%. Among all the species studied, E. lesouefii had the greatest mean percentage of EOs. The identification of 128 components by GC (RI) and GC/MS allowed for 93.6% – 97.7% of the total oil to be identified. 1,8-cineole was the most abundant component found, followed by α-pinene, p-cymene, and globulol. The chemical components of the eight EOs, extracted from the leaves of Eucalyptus species, were clustered into seven groups using PCA and HCA analyses, with each group forming a chemotype. The PCA and HCA analyses of antibacterial activity, on the other hand, identified five groups. Conclusion The oils of E. melliodora, E. bosistoana, and E. robusta show promise as antibiotic alternatives in the treatment of otitis media.

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