The increased incidence of mixed infections requires that the scientific community develop novel antimicrobial molecules. Essential oils and their bioactive pure compounds have been found to exhibit a wide range of remarkable biological activities and are attracting more and more attention. Therefore, the aim of this study was to evaluate myrtenol (MYR), one of the constituents commonly found in some essential oils, for its potential to inhibit biofilms alone and in combination with antimicrobial drugs against Candida auris/Klebsiella pneumoniae single and mixed biofilms. The antimicrobial activity of MYR was evaluated by determining bactericidal/fungicidal concentrations (MIC), and biofilm formation at sub-MICs was analyzed in a 96-well microtiter plate by crystal violet, XTT reduction assay, and CFU counts. The synergistic interaction between MYR and antimicrobial drugs was evaluated by the checkerboard method. The study found that MYR exhibited antimicrobial activity at high concentrations while showing efficient antibiofilm activity against single and dual biofilms. To understand the underlying mechanism by which MYR promotes single/mixed-species biofilm inhibition, we observed a significant downregulation in the expression of mrkA, FKS1, ERG11, and ALS5 genes, which are associated with bacterial motility, adhesion, and biofilm formation as well as increased ROS production, which can play an important role in the inhibition of biofilm formation. In addition, the checkerboard microdilution assay showed that MYR was strongly synergistic with both caspofungin (CAS) and meropenem (MEM) in inhibiting the growth of Candida auris/Klebsiella pneumoniae-mixed biofilms. Furthermore, the tested concentrations showed an absence of toxicity for both mammalian cells in the in vitro and in vivo Galleria mellonella models. Thus, MYR could be considered as a potential agent for the management of polymicrobial biofilms.

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