Novel microplate sealing lid SLIM-V® for in vitro susceptibility testing of chemically characterized plant-derived volatile agents in vapour phase against pneumonia causing bacteria

This study describes a newly designed microplate sealing lid (SLIM-V®) for susceptibility testing of vapours of plant-derived volatiles against pneumonia causing bacteria. The in vitro antibacterial effect of volatile compounds (trans-anethole, 4-isopropyl-3-methylphenol, β-thujaplicin, thymoquinone) and essential oils (EOs) from aerial part of Thymus vulgaris, fruits of Hellwigia musifolia, leaves of Wurfbainia mindanaensis, and two forms of Kolowratia eruciformis with red and white calyces were evaluated in vapour and liquid phase using broh microdilution volatilization (BMV) assay and its modification employing SLIM-V. Thymoquinone was found to be the most active against all bacteria tested (MICs 16-64 μg/mL), followed by β-thujaplicin (32-1024 μg/mL). The consistent results of both methods support validity and reliability of SLIM-V. GC-MS/FID analysis using two detectors (mass spectrometer and flame ionization detector) and two columns of different polarity (HP-5MS/DB-HeavyWAX) showed that β-caryophyllene, α-pinene and p-cymene, thymol, and 1,8-cineol are predominant compounds of H. musifolia, K. eruciformis red and white forms, T. vulgaris, and W. mindanaensis EOs, respectively. More detailed analysis of K. eruciformis using GC×GC-TOF/MS confirmed variances in the chemical composition of EOs of both forms suggesting their chemotaxonomical distinction. Based on the results of antimicrobial assay, thymoquinone can be proposed for further research focused on development of inhalation therapies. In comparison with BMV assay, the experiments demonstrated that the use of SLIM-V represents a significant advancement in the methodologies assessing antimicrobial activity of volatiles. This innovative laboratory tool has potential to be safer, easier to handle, and provide more reproducible and sensitive results than other methods.