**6. Action of EOs on biofilm of eukaryotes**

More recently, the role of EOs and their components was studied for their potential capability to block the formation of biofilms in eukaryotes [64]. Terpenes are capable to inhibit the formation of biofilm through different mechanisms of action. Thymol, for instance, can affect the envelope of the planktonic form of *C. albicans*: it reaches to alter its membrane permeability [92] by infiltrating between the fatty acyl chains of the membrane lipid bilayers, with subsequent disruption of the lipid organization and damage to membrane fluidity. These events led to important alterations of yeast cell and can also reduce its adherence capability, which represents a major step in biofilm formation. Eugenol acts as a potent agent in blocking the biofilms associated with polystyrene too. Also in the case of *C. albicans*, the action of terpenes on the biofilm formation depends on the concentration of the compound used. Thus, a decrease of approximatively 50% of the metabolic pathway linked to biofilm is observed with 0.016% of carvacrol, geraniol, or thymol; however, a higher concentration is requested when we want to use citral, and even a percentage > or equal to 0.25% is necessary to decrease at 50% the biofilm of *C. albicans* if we want use 1,8-cineole, eugenol, farnesol, linalool, menthol, and α-terpinene [93]. Also using the same terpenes at the same concentration, the capability to inhibit the biofilm formation is related to the strain within the same species of the yeast [94], to the different species belonging to the same genus [95] or even to the genera considered [96]. It should not be overlooked the fact that much often, due to the different mechanisms of communication between bacteria, between fungi and among different bacteria and fungi, the own nature led to the formation of complex biofilm containing mixed population. Even, we can find such very frequent situation. In this case, it could be also more difficult to eradicate a biofilm, being the strength of more than a unique type of microorganisms against whom to combat. For instance, *C. albicans* may be associated with mixed infections of *Streptococcus mutans* to form plaque biofilms [97]. The chemical interaction between these two pathogens results in mixed biofilm development, mainly at oral level; therefore, there are no effective treatments in preventing or inhibiting the formation of mixed biofilms or in preventing inter-microbial communication. Eugenol, at sub-MICs, inhibits single and mixed biofilms of *C. albicans* and *S. mutans*. Also in this case, such capability cannot be expanded to every strain of *C. albicans* neither to all strains of *S. mutans*. In fact, Jafri and co-workers [98] ascertained that eugenol is effective against the biofilm formed by two of more than ten strains of *C. albicans* (in particular, the strain CAJ-01) and *S. mutans* (strain MTCC497), studied singularly on in mixed form, with a concurrent reduction of cell viability and a disruption of cell membrane.
