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.
With aim to develop effective proof-of-concept approach which can be used in a development of new preparations for the inhalation therapy, we designed a new screening method for simple and rapid simultaneous determination of antibacterial potential of plant volatiles in the liquid and the vapour phase at different concentrations. In addition, EVA (ethylene vinyl acetate) capmat™ as vapour barrier cover was used as reliable modification of thiazolyl blue tetrazolium bromide (MTT) assay for cytotoxicity testing of volatiles on microtiter plates. Antibacterial activity of carvacrol, cinnamaldehyde, eugenol, 8-hydroxyquinoline, thymol and thymoquinone was determined against Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae using new broth microdilution volatilization method. The cytotoxicity of these compounds was evaluated using MTT test in lung fibroblast cells MRC-5. The most effective antibacterial agents were 8-hydroxyquinoline and thymoquinone with the lowest minimum inhibitory concentrations (MICs) ranging from 2 to 128μg/mL, but they also possessed the highest toxicity in lung cell lines with half maximal inhibitory concentration (IC50) values 0.86-2.95μg/mL. The lowest cytotoxicity effect was identified for eugenol with IC50 295.71μg/mL, however this compound produced only weak antibacterial potency with MICs 512-1024μg/mL. The results demonstrate validity of our novel broth microdilution volatilization method, which allows cost and labour effective high-throughput antimicrobial screening of volatile agents without need of special apparatus. In our opinion, this assay can also potentially be used for development of various medicinal, agricultural, and food applications that are based on volatile antimicrobials.
- MeSH
- Acrolein analogs & derivatives chemistry MeSH
- Anti-Bacterial Agents chemistry MeSH
- Benzoquinones chemistry MeSH
- Cell Line MeSH
- Eugenol chemistry MeSH
- Phytochemicals chemistry MeSH
- Haemophilus influenzae drug effects MeSH
- Humans MeSH
- Microbial Sensitivity Tests methods MeSH
- Monoterpenes chemistry MeSH
- Oxyquinoline chemistry MeSH
- Staphylococcus aureus drug effects MeSH
- Streptococcus pneumoniae drug effects MeSH
- Volatile Organic Compounds chemistry MeSH
- Tetrazolium Salts MeSH
- Thiazoles MeSH
- Thymol chemistry MeSH
- Volatilization * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
