Exploration of Novel Antibacterial and Anti-adhesive Formulations from three Chemically Characterized Essential oils: Optimization using experimental design methodology

In this study, experimental design approach coupled with MIC assay were used to determine the combined treatment effect of three Moroccan essential oils (EOs) derived from Ammi visnaga (AVEO), Mentha suaveolens (MSEO) and Lavandula stoechas (LSEO), against S. aureus, E. coli and P. aeruginosa, as well as to predict the optimal formulations of these EOs to inhibit bacterial adhesion on the stainless steel (304L SS) surface. EOs were obtained via hydrodistillation and then, their volatile content was analyzed using Gas Chromatography-Mass Spectrometry (GC–MS). The antibacterial activity was evaluated using disc-diffusion, microdilution assays. Moreover, the anti-adhesive effect was assessed using two complementary methods, including contact angle measurements and scanning electron microscopy (SEM) analysis. The GC-MS analysis revealed the presence of various bioactive compounds in AVEO, MSEO and LSEO. The main identified components were piperitenone oxide (52.04%) and pulegone (19.78%) for MSEO; and camphor (23.97 %) and menthol (21.5%) for LSEO. Whereas, AVEO was mainly characterized by the presence of linalool (30.24 %). The antibacterial assays showed that all EOs are able to inhibit the bacterial growth at low concentration (MIC ranged from 0.015 to 4 v/v (%)). The optimization of mixtures antibacterial effect has demonstrated the synergistic effect between the three EOs. The optimal mixture predicted against E. coli, P. aeruginosa, and S. aureus corresponded to 30%/42%/28%, 50%/50%/0%, 43%/30%/27%, and AVEO, LSEO and MSEO, respectively. The contact angle assessment indicated that LSEO, MSEO, AVEO and their optimal mixtures remarkably inhibit bacterial adhesion by the alteration of the physico-chemical properties of 304L SS surface. These results are confirmed by SEM analysis, and proved that EOs formulation was able to inhibit more than 80% of bacterial adhesion to 304L SS surface. In light of these findings, EOs mixtures recommended here, can be applied as a promoting and eco-friendly agent for combating the formation of biofilms on stainless steel surface.