Antimicrobial Activity of Essential Oils

The strains were obtained during 72-h cultivation in the brain heart infusion agar (BHA, Becton Dickinson, Germany) + 7% horse blood in microaerophilic conditions (5% O₂, 15% CO₂ and 80% N₂). Cell concentration was determined using a densitometer (BioMerieux, Marcy l’Etoile, France). Bacterial suspensions with a density of three according to the McFarland scale, i.e., 3 × 10⁸ cells (CFU)/1 mL were used for the tests. Essential oils were screened for antibacterial activities by microdilution broth method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (www.eucast.org) using Mueller-Hinton broth with 5% lysed horse blood. Minimal Inhibitory Concentration (MIC) of the tested essential oils were evaluated for all H. pylori strains with method modification by addition after incubation of resazurin to visualize the growth of H. pylori. Appropriate DMSO control (at a final concentration of 10%), a positive control (containing inoculum without the tested essential oils) and negative control (containing the tested essential oils without inoculum) were included on each microplate.
Minimal bactericidal concentration (MBC) was determined by subculturing 5 μL of the microbial culture from each well that showed growth inhibition, from the last positive one and from the growth control onto the recommended agar plates. The plates were incubated at 35 °C for 72 h in microaerophilic conditions and the MBC was defined as the lowest concentration of the essential oil without growth of microorganism. Each experiment was triplicated. Representative data is presented.