Mechanism of action of Melaleuca armillaris (Sol. Ex Gaertu) Sm. essential oil on six LAB strains as assessed by multiparametric flow cytometry and automated microtiter-based assay

Abstract Little is known concerning the effects of essential oils (EOs) against lactic acid bacteria (LAB), either of the human gastrointestinal microflora or those involved in industrial processes. GC and GC/MS analysis of Melaleuca armillaris EO resulted in the identification of 68 compounds comprising 99.6% of the oil. Eucalyptol (1,8-cineole) was the major compound (68.9%) and the composition was largely dominated by the oxygenated monoterpenes fraction (77.3%). The anti-LAB activities of the EO were first checked by the disc diffusion assay. In a second phase, time-survival kinetics of each strain incubated with increasing concentrations of the EO (0.25, 2.5, 5 and 25 μg/ml) were established using an automated microtiter assay (Bioscreen C). Bacteriostatic or bactericidal effects were noticed depending on the studied strain and on the applied concentration of the EO. The mathematical modelling of the kinetics showed that in presence of increasing concentrations of M. armillaris EO, the lag phases of growth were extended (0.69%–97.5%) and both the growth rate and final cell density were reduced. Variations depending on the strain were noticed. Live/dead assays of the multiparametric flow cytometry technique (combining carboxyfluorescein diacetate (cFDA) and propidium iodide (PI) fluorescent probes) were performed by dual staining of each sample culture to differentiate viable, dead and stressed cells. The behaviour of each strain, in presence of increasing concentrations of M. armillaris EO, was evaluated by quantifying the relative percentages of each subpopulation throughout 3 days of culture. Results displayed disparate patterns of subpopulations which revealed dynamic changes in cell behaviour. This is probably due to disparate influences of the EO components on cellular physiological properties throughout the incubation period. This study proved that multiparametric flow cytometry was a convenient and rapid tool to evaluate the viability of LAB, and was well correlated with plate count results. Such study could be useful to understand how to fully take advantage of LAB as probiotics or as potential candidates to improve food hygiene and to assure food quality; namely when they are associated with natural preservatives such as EOs.