Abstract
Herein, we present a new test, dubbed AntiBioVol, to be used for the quantitative evaluation of antibiofilm activity of volatile compounds in vitro. AntiBioVol is performed in two 24-well plates using a basic microbiological laboratory equipment. To demonstrate AntiBioVol usability, we have scrutinized the activity of volatilized eucalyptus, tea tree, thyme essential oils, and ethanol (used for method suitability testing) against biofilms of Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. We have also compared AntiBioVol with the standard disc volatilization method, placing a special stress on evaluating the impact of various technical parameters on the outcomes of the latter method. The obtained results indicate that AntiBioVol allows analyzing the antibiofilm activity of volatile compounds in a high number of repeats and provides semi-quantitative or quantitative results of high repeatability. In comparison to disc volatilization, AntiBioVol is a more space- and cost-effective method that allows analyzing various types of microbial aggregates. Moreover, we have indicated that the possible reasons for the discrepancies in the results obtained by means of the standard disc volatilization method may be related to various parameters of the testing dishes used (height, volume, diameter) and to various volumes of the agar medium applied. In turn, the application of a 24-well plate and a strictly defined AntiBioVol protocol provide a higher control of experimental conditions. Therefore, the application of AntiBioVol may enable an optimization of and introduction of volatile compounds to the fight against infective biofilms.
Highlights
Biofilm is a diversified and adaptive community of microbial cells that displays a high tolerance to conventional antimicrobials due to the presence of a protective extracellular matrix, diversification of metabolism within specific biofilm layers, and coordinated reactivity to stimuli [1]
The main aim of the current study was to improve the consistency of essential oils (EOs) analysis and to design a sensitive, cost-effective, and easy-to-perform test of antibiofilm activity of volatile compounds
To compare AntiBioVol with the methods previously developed for the assessment of antimicrobial activity of liquid EO fractions, we used the disc diffusion method [24], while to assess the activity of volatile fractions, we applied the inverted Petri plate method
Summary
Biofilm is a diversified and adaptive community of microbial cells that displays a high tolerance to conventional antimicrobials (antibiotics, antiseptics, disinfectants) due to the presence of a protective extracellular matrix, diversification of metabolism within specific biofilm layers, and coordinated reactivity to stimuli [1]. High tolerance to stressors and antibiotic resistance mechanisms displayed by microbial cells within the biofilm make the structure highly persistent and are the reasons why science has recognized the biofilm’s central role in the pathogenesis of infective diseases [2,3,4]. The introduction of novel antibiofilm countermeasures is one of the most pressing needs of contemporary medicine [7] The use of such antimicrobial substances of plant origin as essential oils (EOs) in biofilm eradication is considered a promising direction to follow [8]. Thanks to their high lipophilicity, EOs bind to and break the integrity of microbial cell walls and membrane structures, which results in cell lysis through a mechanism resembling the one displayed by antiseptics [9]. The above-mentioned features predispose EOs to be used either in monotherapy or as adjuvant substances for antibiotics [11]
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