Validation of Qualitative Broth Volatilization Checkerboard Method for Testing of Essential Oils: Dual-Column GC-FID/MS Analysis and In Vitro Combinatory Antimicrobial Effect of Origanum vulgare and Thymus vulgaris against Staphylococcus aureus in Liquid and Vapor Phases.

Combinatory action of antimicrobial agents such as essential oils (EOs) show to be an effective strategy to overcome the problem with increasing antibiotic resistance of microorganisms, including Staphylococcus aureus. The objective of this study was to evaluate in vitro antimicrobial interactions between Origanum vulgare and Thymus vulgaris EOs against various S.aureus strains in both liquid and vapor phases using the broth volatilization checkerboard method. Fractional inhibitory concentrations (FICs) were determined for both liquid and vapor phases, and the composition of EOs was analyzed by gas chromatography-mass spectrometry using dual-column/dual-detector gas chromatograph. Results of oregano and thyme EOs combination showed additive effects against all S. aureus strains in both phases. In several cases, sums of FICs were lower than 0.6, which can be considered a strong additive interaction. The lowest FICs obtained were 0.53 in the liquid phase and 0.59 in the gaseous phase. Chemical analysis showed that both EOs were composed of many compounds, including carvacrol, thymol, γ-terpinene, and p-cymene. This is the first report on oregano and thyme EOs interactions against S. aureus in the vapor phase. It also confirms the accuracy of the broth volatilization checkerboard method for the evaluation of combinatory antimicrobial effects of EOs in the vapor phase.

Evaluation of in vitro growth-inhibitory effect of carvacrol and thymol combination against Staphylococcus aureus in liquid and vapour phase using new broth volatilization chequerboard method.

Carvacrol and thymol, both plant-derived volatile compounds, have extensively been studied individually as well as in combination with other agents for their antimicrobial activity in liquid phase. However, in contrast to well-established assays for testing of antimicrobial combinatory effects in liquid media, there are no standardized methods for evaluation of interactions between volatile compounds in vapour phase. The objective of this study was to verify new broth volatilization chequerboard method by testing the combination of carvacrol and thymol and to determine in vitro inhibitory effect of these compounds in liquid and vapour phase against twelve Staphylococcus aureus strains. The new method, based on combination of standard microdilution chequerboard and new broth volatilization tests allowing calculation of fractional inhibitory concentrations (FICs), was used. Combination of carvacrol and thymol produced the additive antimicrobial effect against all strains tested. In several cases, they reached ΣFIC values lower than 0.6, which can be considered as a strong additive interaction. The best result was found in vapour phase against one standard strain at combination of 128 µg/mL of carvacrol and 16-256 µg/mL of thymol (ΣFIC = 0.51) and in liquid phase against one clinical isolate at combination of 256 µg/mL of carvacrol and 256 µg/mL of thymol (ΣFIC = 0.53). The study verified that the new technique is suitable for simple and rapid high-throughput combinatory antimicrobial screening of volatile compounds simultaneously in vapour and liquid phase and that it allows determination and comparison of MIC and FIC values in both, liquid and solid media.