Striking Essential Oil: Novel tools and new insights to study the biological activities of essential oils (EOs) and their components (EOCs)

The increasing level of antimicrobial resistance poses the threat that no suitable antimicrobials will be available for systematic treatment of common diseases. More and more Candida infections are caused by intrinsically and multi-resistant Candida isolates. There is clearly renewed scientific interest in discovering drugs from natural sources, although natural product-based drug discovery is considered intrinsically complex and requires a highly integrated interdisciplinary approach. Medicinal plants that are used to treat infectious diseases appear to be an abundant source of new bioactive secondary metabolites. One such class of plant-based molecules are essential oils which have been used therapeutically for millennia as they have a broad range of biological activities. Reanalyzing the data of a large antimicrobial screening (2012) of essential oils in microtiter plates systematically showed false-positive hits. We discovered that these false-positives were mediated by the vapor-phase of some essential oils in adjacent wells. The challenge was to circumvent this problem before we could start screening our latest essential oil (component) collection against two potentially disease-causing fungi i.e. C. albicans and C. glabrata. We developed an alternative plate set-up for the new screening in a way that it intrinsically does not modify the standardized protocol, while controlling for false positive results caused by the vapor-phase-mediated antimicrobial activity of the tested essential oils and their components. However, the alternative plate set-up and its derivatives are not limited to the testing of essential oils and their components but can be used with any volatile compound; nor is the set-up limited to testing antimicrobial activity but can essentially be extended to any biological activity tested in a microtiter plate. Therefore, we recommend these adaptations as good laboratory practices when working with volatiles in microtiter plates. An in vitro assay to test the vapor-phase-mediated antimicrobial activity of volatiles did not exist. From a drug discovery point such an assay is interesting as a model for testing compounds for e.g. the treatment of tract-related infections. We hypothesized that if we can detect false-positives in microtiter plates caused by the vapor-phase of volatiles, it should be possible to develop a microtiter plate-based assay. We developped the qualitative vapor-phase-mediated patch assay, a relatively simple test to