**7. New opportunities from the use of essential oils**

In view of the importance of quorum sensing on the physiology regulation of microorganisms, research is also addressing toward the identification of new prospects for the use of essential oils in the blocking of cellular communication mechanisms. In recent years, the opportunity to associate essential oils or their main components with synthetic substances (drugs, enzymes, etc.) has been evaluated, to identify the associations that improve their performance in this sector. In particular, emerging resistance to last-resort antibiotics, such as carbapenems and polymixin B, led to theory of the so-called post-antibiotic era [99–101], and now research is moving to identify new compounds with stronger activity also against the most resistant pathogens. Different research groups are thus carrying a

**179**

*Essential Oils and Microbial Communication DOI: http://dx.doi.org/10.5772/intechopen.85638*

screening of "no-drug compounds," including the EOs, in association with nonsteroidal anti-inflammatory drugs (the so-called NSAIDs) for inhibition of quorum sensing and biofilm formation in pathogens. Some NSAIDs, such as Z-phytol and lonazolac, can block the QS system of *Salmonella enteritidis*, acting as antagonists with respect to the AHLs [102]. The association of EOs with azolic drugs gives interesting challenges in the treatment of fungal infections determined by fungi particularly difficult to be eradicated [103]. Thymol in combination with the azolic drug, fluconazole, is capable to act against several species of *Candida* isolated from clinical specimens [104]. Chitosan nanoparticles containing miconazole and farnesol can inhibit *Candida* proliferation. The presence of farnesol is also capable of decreasing the pathogenicity of infection, demonstrated through the absence of inflammation [105]. The combination between the EO of *Cinnamomum tamala*, or its main component cinnamaldehyde, and linalool, with a commercial DNase I and marine bacterial DNase, might offer an alternative strategy to fight the biofilm formation and quorum sensing-mediated virulence factors in the aquaculture pathogen *Vibrio parahaemolyticus*, acting as effective food-preserving agent too [106]. The synergistic association between some EOs and DNase can decrease the biofilm-forming ability of *S. aureus* [87]. The association between EOs and/or their main components with conventional antibiotics can allow to eradicate also some particularly resistant biofilm, decreasing concurrently their resistance threshold. This allows also to minimize the antibiotic concentration, decreasing also its potential accompanying toxic side effects [107]. Synergistic interactions between EOs and their components with antibiotics are recognized, including several instances of antibiotic re-sensitization in resistant isolates, in support of this strategy to control antibiotic resistance, although synergistic effects are not well explored outside a preliminary identification of antibacterial interactions and mechanism of action is seldom defined, despite many hypotheses and recommendations for future studies [108]. The possibility of using essential oils has also been evaluated in the prevention of biofilm development by microorganisms (*Klebsiella*, *S. aureus*, *Staphylococcus haemolyticus*, *E. coli*, *Enterococcus cloacae*, *Enterococcus faecalis*), and the EO of *Matricaria chamomilla* (Asteraceae) could be considered as a good candidate, encouraging research related to the application of essential oils to fight diabetic complications [109]. The nanomaterials including EOs, at subinhibitory concentration, can inhibit QS and prevent biofilm formation and virulence development in pathogens [110]. The capability of blocking the quorum sensing mechanisms and biofilm formation by EOs can be combined with conventional drugs for a better treatment efficacy, as well as to design new more effective drugs, capable of acting also against those particularly resistant bacteria and fungi. The possibility to use nanotechnology, through the production of nano-vesicles containing EO, can result useful in a variety of applications including medical and pharmaceutical recipients and in home products for treating or preventing microbial colonization, as well as avoiding biofilm development [111, 112], also in food technology [113, 114]. Nano-coating with different inorganic and organic materials also supported by EOs proves to be particularly useful in the treatment of chronic wounds, such as venous or arterial ulcers, diabetic foot ulcers, pressure sores, and non-healing surgical wounds, all scenarios associated with chronic mono- or polymicrobial biofilm infections, formed by different bacteria, such as *S. aureus* and *P. aeruginosa*, followed by various species of *Enterobacteriaceae* such as *E. coli*, *Klebsiella* spp., *Proteus* spp., *Enterobacter* spp., *Morganella morganii*, *Citrobacter freundii*, *Serratia marcescens*, *Providencia* spp., *Enterococcus* spp., *Streptococcus* spp., and rarely *Corynebacterium* spp. or *Acinetobacter baumannii* [115]. In agro-food industry, the increasing demand for eco-friendly material stimulated the study of the elaboration of complex bio-nanocomposite films containing also EOs, such as that of rosemary,

#### *Essential Oils and Microbial Communication DOI: http://dx.doi.org/10.5772/intechopen.85638*

*Essential Oils - Oils of Nature*

**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 disrup-

**178**

tion of cell membrane.

**7. New opportunities from the use of essential oils**

In view of the importance of quorum sensing on the physiology regulation of microorganisms, research is also addressing toward the identification of new prospects for the use of essential oils in the blocking of cellular communication mechanisms. In recent years, the opportunity to associate essential oils or their main components with synthetic substances (drugs, enzymes, etc.) has been evaluated, to identify the associations that improve their performance in this sector. In particular, emerging resistance to last-resort antibiotics, such as carbapenems and polymixin B, led to theory of the so-called post-antibiotic era [99–101], and now research is moving to identify new compounds with stronger activity also against the most resistant pathogens. Different research groups are thus carrying a

screening of "no-drug compounds," including the EOs, in association with nonsteroidal anti-inflammatory drugs (the so-called NSAIDs) for inhibition of quorum sensing and biofilm formation in pathogens. Some NSAIDs, such as Z-phytol and lonazolac, can block the QS system of *Salmonella enteritidis*, acting as antagonists with respect to the AHLs [102]. The association of EOs with azolic drugs gives interesting challenges in the treatment of fungal infections determined by fungi particularly difficult to be eradicated [103]. Thymol in combination with the azolic drug, fluconazole, is capable to act against several species of *Candida* isolated from clinical specimens [104]. Chitosan nanoparticles containing miconazole and farnesol can inhibit *Candida* proliferation. The presence of farnesol is also capable of decreasing the pathogenicity of infection, demonstrated through the absence of inflammation [105]. The combination between the EO of *Cinnamomum tamala*, or its main component cinnamaldehyde, and linalool, with a commercial DNase I and marine bacterial DNase, might offer an alternative strategy to fight the biofilm formation and quorum sensing-mediated virulence factors in the aquaculture pathogen *Vibrio parahaemolyticus*, acting as effective food-preserving agent too [106]. The synergistic association between some EOs and DNase can decrease the biofilm-forming ability of *S. aureus* [87]. The association between EOs and/or their main components with conventional antibiotics can allow to eradicate also some particularly resistant biofilm, decreasing concurrently their resistance threshold. This allows also to minimize the antibiotic concentration, decreasing also its potential accompanying toxic side effects [107]. Synergistic interactions between EOs and their components with antibiotics are recognized, including several instances of antibiotic re-sensitization in resistant isolates, in support of this strategy to control antibiotic resistance, although synergistic effects are not well explored outside a preliminary identification of antibacterial interactions and mechanism of action is seldom defined, despite many hypotheses and recommendations for future studies [108]. The possibility of using essential oils has also been evaluated in the prevention of biofilm development by microorganisms (*Klebsiella*, *S. aureus*, *Staphylococcus haemolyticus*, *E. coli*, *Enterococcus cloacae*, *Enterococcus faecalis*), and the EO of *Matricaria chamomilla* (Asteraceae) could be considered as a good candidate, encouraging research related to the application of essential oils to fight diabetic complications [109]. The nanomaterials including EOs, at subinhibitory concentration, can inhibit QS and prevent biofilm formation and virulence development in pathogens [110]. The capability of blocking the quorum sensing mechanisms and biofilm formation by EOs can be combined with conventional drugs for a better treatment efficacy, as well as to design new more effective drugs, capable of acting also against those particularly resistant bacteria and fungi. The possibility to use nanotechnology, through the production of nano-vesicles containing EO, can result useful in a variety of applications including medical and pharmaceutical recipients and in home products for treating or preventing microbial colonization, as well as avoiding biofilm development [111, 112], also in food technology [113, 114]. Nano-coating with different inorganic and organic materials also supported by EOs proves to be particularly useful in the treatment of chronic wounds, such as venous or arterial ulcers, diabetic foot ulcers, pressure sores, and non-healing surgical wounds, all scenarios associated with chronic mono- or polymicrobial biofilm infections, formed by different bacteria, such as *S. aureus* and *P. aeruginosa*, followed by various species of *Enterobacteriaceae* such as *E. coli*, *Klebsiella* spp., *Proteus* spp., *Enterobacter* spp., *Morganella morganii*, *Citrobacter freundii*, *Serratia marcescens*, *Providencia* spp., *Enterococcus* spp., *Streptococcus* spp., and rarely *Corynebacterium* spp. or *Acinetobacter baumannii* [115]. In agro-food industry, the increasing demand for eco-friendly material stimulated the study of the elaboration of complex bio-nanocomposite films containing also EOs, such as that of rosemary,

which can act as effective eco-friendly nanocomposite films in packaging industries [116]. Algae are beginning the new frontier for the supplement of new bioactive compounds with antimicrobial activity and represent a unique opportunity for the science. Some investigations are exploring the therapeutic potential of algal extracts and their chemical groups, including terpenes, capable to have antimicrobial activity and to block the mechanism of communication between microorganisms. The alga *Pithophora oedogonium* targets *Staphylococcus* and *Salmonella*. The algae *Rivularia bullata*, *Nostoc spongiaeforme*, *Codium fragile*, *Colpomenia peregrina*, *Cystoseira barbata*, and *Zanardinia typus* have already demonstrated activity against many Gram-negative and Gram-positive bacteria [117, 118]. Finally, innovative techniques, such as the optical technique of bio-speckle [119] and the biofilm electrostatic test (BET) [120], will support the research in the near future to have a very fast scenario of EO biological activity. Speckle decorrelation can lead us to visualize the effect of essential oil on the decrease of the usual self-propelling movement of microorganisms taking place when they interact with coherent light. BET is as a simple, rapid, and highly reproducible tool for evaluating *in vitro* the ability of bacteria to form biofilms through electrostatic interaction with a pyro-electrified carrier and for ascertaining the impeding effect of an EO on the microbial capability to form biofilm in just 3 h.
