Effect of Myrtenol and Its Synergistic Interactions with Antimicrobial Drugs in the Inhibition of Single and Mixed Biofilms of Candida auris and Klebsiella pneumoniae

The increased incidence of mixed infections requires that the scientific community developnovel antimicrobial molecules. Essential oils and their bioactive pure compounds have been found toexhibit 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 commonlyfound in some essential oils, for its potential to inhibit biofilms alone and in combination withantimicrobial drugs against Candida auris/Klebsiella pneumoniae single and mixed biofilms. Theantimicrobial 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 antimicrobialdrugs was evaluated by the checkerboard method. The study found that MYR exhibited antimicrobialactivity at high concentrations while showing efficient antibiofilm activity against single and dualbiofilms. To understand the underlying mechanism by which MYR promotes single/mixed-speciesbiofilm 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 wellas 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 withboth caspofungin (CAS) and meropenem (MEM) in inhibiting the growth of Candida auris/Klebsiellapneumoniae-mixed biofilms. Furthermore, the tested concentrations showed an absence of toxicityfor both mammalian cells in the in vitro and in vivo Galleria mellonella models. Thus, MYR could beconsidered as a potential agent for the management of polymicrobial biofilms.