**2.2 Biofilm**

*Essential Oils - Oils of Nature*

language for most bacteria.

**2.1 QS in eukaryotes**

system common to both Gram-positive and Gram-negative bacteria, although its role as a true QSM has been doubted for some microorganisms [12–15]. This system might give rise to a family of molecules that are supposed to operate as a common

The production of the AHL involved in the QS mechanism was recently discovered also in several Gram-negative bacteria, such as *Roseobacter* sp. TB60 and *Psychrobacter* sp. TB67 associated with the Antarctic sponge, *Anoxycalyx joubini* [16], indicating this a certain "universality" of the system. Dong and Zhang [17] and McDougald and co-workers [18] demonstrated the existence of other two novel signaling pathways: hydroxyl-palmitic acid methyl ester and methyl-dodecanoic acid.

Some eukaryotic microorganisms monitor their population density through QS mechanisms [19, 20] too. This is not so surprising, taking into account that many bacteria and eukaryotic microorganisms inhabit in common ecological niches and often play similar challenges. In fungi, QS mechanisms are in charge to check and regulate processes such as sporulation and production of some molecules, such as secondary metabolites, as well as to those events giving rise to the morphological transition and enzyme secretion by the cells. Considering this and starting from the assumption that even this type of organisms is extremely varied, we can undoubtedly affirm that fungi exhibit different cell-cell communication mechanisms, using a wide variety of signal molecules [19]. Furthermore, fungi can communicate with bacteria and even with their plant or mammalian hosts. In yeasts and dimorphic fungi, aromatic alcohols originating from amino acids mediate the QS type of regulation [21]. Therefore, yeasts, through the production of tryptophol and phenylethyl alcohol, can manage the formation of pseudohyphae and biofilm [22] and probably trigger the virulence process toward some plants such as *Vitis vinifera* [23]. QSMs stimulate the exit from lag phase inducing germtube formation and hyphal development [24]. *Candida albicans* remains the most studied species from this point of view. It produces some QSMs, such as tyrosol, described also in other fungal species, such as *Saccharomyces cerevisiae* [25]. QSMs of *C. albicans* influence the formation and structure of biofilms [26, 27] as well as the dispersal of cells from a biofilm; hence, it, as well as other molecules, plays important roles in pathogenesis. E-farnesol, the other most known QSM produced by *C. albicans* [28], is an exogenous molecule that, on the contrary, inhibits biofilm formation when provided early during adherence; furthermore, it acts as an inhibitor of hyphal formation indeed [29]. Therefore, this organism can modulate its morphology (vegetative/hyphal) and, consequently, all events related, including the pathogenicity, through the modulating production mainly of these two QSMs. Dodecanol and γ-butyrolactone are other molecules identified as mediators of QS processes present in other eukaryotic organisms, such as the filamentous fungi, *Aspergillus* and *Penicillium* spp. Some species of *Penicillium*, such as *P. sclerotinum*, produce sclerotiorin, a secondary metabolite with antibiotic properties, and γ-butyrolactone-containing molecules such as multicolic acid, which act as QSMs [30]. Taking into account that Gram-negative bacteria produced lactones (AHLs) as QSMs and that filamentous fungi produce butyrolactone I [31], γ-heptalactone [32], and γ-butyrolactones [33], the discovery that γ-butyrolactones are produced also by the filamentous bacterium *Streptomyces* [11] suggests a convergent evolution or a horizontal gene transfer occurring during the evolution [30]. At the same time, different fungi, such as basidiomycete *Cryptococcus neoformans*, produce as QSMs some peptides, similar to how Gram-positive bacteria do. This means that fungi use a language "analogous" in some way to that exhibited by other phyla [10, 30].

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The term "biofilm" is referred to a structure, enough complex, formed by microbial cells, associated with each other that attach to a surface, which are in a certain sense kept isolated from the external environment (although they exert an important influence on this) through the formation of a sort of "dome" of polysaccharide nature [37]. Biofilm has generally a three-dimensional structure: it contains more or less channels and pores, used as a sort of intercellular communication channel and for the maintenance of the entire bacterial community [38, 39]. Biofilm is thus one of the subsequent mechanisms giving rise from the communication among bacteria, which precisely through the formation of biofilm and other microbial behaviors and social exchange (not only bioluminescence, conjugation, and virulence but also motility, sporulation, competence, etc.) form the social microbial system of interaction, the QS (**Figure 1**) [40, 41], that prokaryotes and some eukaryotes developed many millions of years before the actual human social media, which is certainly more organized and complex. The system is so organized and evolved that allows microorganisms to easily adapt to adverse environmental conditions and to use them even to switch to counterattack, with an action of growth, proliferation, and change in their metabolic pathways and morphology. Biofilms allow the survival of bacterial cells in a hostile environment; the extremely

**Figure 1.** *Mechanism of biofilm formation.*

complex structure and the metabolic and physiological heterogeneity that characterize them suggest an analogy between these communities and the tissues of higher organisms. Bacterial biofilms, not easily eradicated with the conventional antibiotic therapies, affect a large number of chronic bacterial infections. Biofilms represent a cohesive matrix of microorganisms and other cellular constituents that can be present in any natural environment; they are also characterized two pints by the ability to adhere to surfaces; by a structural heterogeneity; by a genetic diversity of the components; by complex interactions of communities, even mixed; and by an extracellular matrix of polymeric substances. At the end of the 1990s, it was ascertained that the so called planktonic growthis an artifact and that the type of growth prevalent in natural environments is sessile (fixed to a substrate). When nutrient intake is limited, biofilms tend to adhere to solid supports and remain stable at the solid/liquid interface, where nutrients are concentrated. Once adhered, the biofilms secrete exopolysaccharides that surround them, guaranteeing their cohesion to the support and between them. This creates biofilms, which in most cases are polymicrobial. Bacteria grow slowly inside the biofilm, forming microcolonies. The biofilms are mature when the growth reaches the point where most external cells come off, returning to the planktonic life and then starting the formation of new biofilms. The whole process takes from a few days to a few weeks. In mature biofilms, bacteria are present in different states, depending on the location: the innermost ones are metabolically less active, and the more external have metabolic characteristics similar to those of planktonic growth bacteria. At first, sessile growth attracted attention due to some negative effects of biofilm formation (corrosion of cables and submerged structures but also the dental plaque of many animals) and of the resistance of the bacteria included in the biofilm to the antimicrobials. Studies carried out with the confocal microscope have shown that the biofilm is highly organized within it, with channels through which the surrounding fluid circulates in the matrix carrying the nutrients and removing the toxic products. Maintaining a structure of this type requires complex mechanisms of cell-cell regulation and communication to prevent undifferentiated growth obstructing the canaliculi. The phenomenon of sessile growth has determined, in the last decade, the onset of new pathologies, linked to the colonization of prosthetic implants by bacterial biofilms [42]. Biofilms are present in the most diverse environments, e.g., in thermal springs or on the bottom of lakes and rivers, and can be used not only for the purification of water in an industrial environment but also for the removal of oil or other pollutants from contaminated marine areas. Moreover, it is now established that most bacterial species, when conditions allow it, modify their behavior to find true "microbial cities" in the form of biofilms. These include "fortification walls," consisting of a three-dimensional array of polymeric sugars, and "shipping channels" for the transport of nutrients and catabolites. Two main types of adhesion are involved in the formation of biofilm: *adhesion* of the bacterium to a solid substrate (supra inert) also by attacking host proteins and *intercellular adaption*, which determines the formation of multiple layers of the biofilm. In the biomedical field, biofilms are involved in a wide range of pathologies, involving cochlear, articular, orthopedic, etc. The sessile structures of which biofilms are endowed, the multi-species communities of which they are constituted, and the influence that the dynamics of fluid flows in which they are immersed exert on them are the factors that have contributed to considering biofilms as the core reefs of the microbial world. Obviously, the multicellularity of a biofilm translates into a better defense of microorganisms, contributing substantially to their survival. Nutrient depletion creates some areas of activity alteration; the outer cell layers of the biofilm contribute to the formation of a sort of barrier, capable to absorb external damage. The innermost microorganisms have the task, to some extent, to elaborate a response to

**171**

**3. Essential oils**

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

intrinsic stress. The biofilm bacteria are 10–10,000 times more resistant to antibiotic treatment than the planktonic phenotype. For this reason, biofilm infections show recurrent symptoms after cycles of antibiotic therapy. This persistence in the heart of the biofilm is linked to the presence of the so-called "sleeper" microorganisms, with low activity and which determine the phenomenon of persistence.

Biofilm adapts to environmental fluctuations, such as temperatures, pH variations, osmolarity, and nutrient availability through multiple gene expression; its resistance is not genotypic. Microbial cells contained within the biofilm are much more difficult to reach; moreover, they have the advantage, compared to the host organism, of being able to communicate outside the biofilm, through the previously indicated system of channels and pores. At the same time, it becomes more difficult for synthetic drugs to "break" the biofilm organization, just for how it is structured and how it is composed. After a certain threshold, bacteria change their life perspective, in the sense that they no longer act as a single cell, but as a component of a microbial team. Such community grows through the recruitment of other cells, which arrive there. In this manner, microbial colony can spread upward of the surface. At the beginning, this gives rise to the formation of small colonies and unripe biofilm. At the end of the process, the biofilm is ripe (**Figure 1**). The production of compounds such as exopolysaccharides determines the embedding of bacteria in a complex matter constituted also by nucleic acids, lipids, and proteins [43]. A so organized matrix supplies bacteria for several advantages: for instance, it can manage the flow of nutrients and protect bacteria against the action of antimicrobial substances and the host immune system, which encounter great difficulty in scratching the structure and organization of the biofilm matrix [44]. So, manipulation and inhibition of the QS system might open new scenarios and improve therapies for chronic bacterial diseases [45, 46], including even cancer [47, 48].

Several possible strategies could treat infections associated with biofilms: substances capable of destroying the biofilm matrix (e.g., dispersion B), substances capable of destroying resistant cells, quorum-quenching enzymes that interfere with the quorum sensing phenomenon, substances that cause self-destruction of the biofilm, and then, in particular, strategies to strengthen the action of antimicrobials. The treatment of biofilms with antibiotics often causes only partial killing, allowing the surviving bacteria, present in the depth of the biofilm, to act as a true nucleus of propulsion for the spread of the infection after the interruption of the antibiotic therapy. Antibiotics can be inactivated by the production of specific enzymes within the biofilm. In some extreme cases, even the sessile population must not be surgically removed from the body. Another aspect to take into consideration is the age of biofilm: the younger is the biofilm, the easier is its eradication. The need to identify substances/active ingredients able to replace synthetic drugs in the fight against pathogens, in particular against those more resistant to conventional treatments, also directed research toward (or better to say, to the rediscovery of) the "natural world," source of bioactive compounds used by traditional medicine since ancient times. Moreover, these substances have always exhibited a great spectrum of action that can be considered of great benefit, also due to the chemical structural differences of the active compounds. In such context, substances of vegetal origin, such as essential oils, have always been successfully used in traditional medicine and stimulated, practically always but particularly in recent decades, the scientific world to discover and identify substances, intended as a mixture or as single components that are able to fight pathogenic microorganisms. From this point of view, the study of

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

*Essential Oils - Oils of Nature*

complex structure and the metabolic and physiological heterogeneity that characterize them suggest an analogy between these communities and the tissues of higher organisms. Bacterial biofilms, not easily eradicated with the conventional antibiotic therapies, affect a large number of chronic bacterial infections. Biofilms represent a cohesive matrix of microorganisms and other cellular constituents that can be present in any natural environment; they are also characterized two pints by the ability to adhere to surfaces; by a structural heterogeneity; by a genetic diversity of the components; by complex interactions of communities, even mixed; and by an extracellular matrix of polymeric substances. At the end of the 1990s, it was ascertained that the so called planktonic growthis an artifact and that the type of growth prevalent in natural environments is sessile (fixed to a substrate). When nutrient intake is limited, biofilms tend to adhere to solid supports and remain stable at the solid/liquid interface, where nutrients are concentrated. Once adhered, the biofilms secrete exopolysaccharides that surround them, guaranteeing their cohesion to the support and between them. This creates biofilms, which in most cases are polymicrobial. Bacteria grow slowly inside the biofilm, forming microcolonies. The biofilms are mature when the growth reaches the point where most external cells come off, returning to the planktonic life and then starting the formation of new biofilms. The whole process takes from a few days to a few weeks. In mature biofilms, bacteria are present in different states, depending on the location: the innermost ones are metabolically less active, and the more external have metabolic characteristics similar to those of planktonic growth bacteria. At first, sessile growth attracted attention due to some negative effects of biofilm formation (corrosion of cables and submerged structures but also the dental plaque of many animals) and of the resistance of the bacteria included in the biofilm to the antimicrobials. Studies carried out with the confocal microscope have shown that the biofilm is highly organized within it, with channels through which the surrounding fluid circulates in the matrix carrying the nutrients and removing the toxic products. Maintaining a structure of this type requires complex mechanisms of cell-cell regulation and communication to prevent undifferentiated growth obstructing the canaliculi. The phenomenon of sessile growth has determined, in the last decade, the onset of new pathologies, linked to the colonization of prosthetic implants by bacterial biofilms [42]. Biofilms are present in the most diverse environments, e.g., in thermal springs or on the bottom of lakes and rivers, and can be used not only for the purification of water in an industrial environment but also for the removal of oil or other pollutants from contaminated marine areas. Moreover, it is now established that most bacterial species, when conditions allow it, modify their behavior to find true "microbial cities" in the form of biofilms. These include "fortification walls," consisting of a three-dimensional array of polymeric sugars, and "shipping channels" for the transport of nutrients and catabolites. Two main types of adhesion are involved in the formation of biofilm: *adhesion* of the bacterium to a solid substrate (supra inert) also by attacking host proteins and *intercellular adaption*, which determines the formation of multiple layers of the biofilm. In the biomedical field, biofilms are involved in a wide range of pathologies, involving cochlear, articular, orthopedic, etc. The sessile structures of which biofilms are endowed, the multi-species communities of which they are constituted, and the influence that the dynamics of fluid flows in which they are immersed exert on them are the factors that have contributed to considering biofilms as the core reefs of the microbial world. Obviously, the multicellularity of a biofilm translates into a better defense of microorganisms, contributing substantially to their survival. Nutrient depletion creates some areas of activity alteration; the outer cell layers of the biofilm contribute to the formation of a sort of barrier, capable to absorb external damage. The innermost microorganisms have the task, to some extent, to elaborate a response to

**170**

intrinsic stress. The biofilm bacteria are 10–10,000 times more resistant to antibiotic treatment than the planktonic phenotype. For this reason, biofilm infections show recurrent symptoms after cycles of antibiotic therapy. This persistence in the heart of the biofilm is linked to the presence of the so-called "sleeper" microorganisms, with low activity and which determine the phenomenon of persistence.

Biofilm adapts to environmental fluctuations, such as temperatures, pH variations, osmolarity, and nutrient availability through multiple gene expression; its resistance is not genotypic. Microbial cells contained within the biofilm are much more difficult to reach; moreover, they have the advantage, compared to the host organism, of being able to communicate outside the biofilm, through the previously indicated system of channels and pores. At the same time, it becomes more difficult for synthetic drugs to "break" the biofilm organization, just for how it is structured and how it is composed. After a certain threshold, bacteria change their life perspective, in the sense that they no longer act as a single cell, but as a component of a microbial team. Such community grows through the recruitment of other cells, which arrive there. In this manner, microbial colony can spread upward of the surface. At the beginning, this gives rise to the formation of small colonies and unripe biofilm. At the end of the process, the biofilm is ripe (**Figure 1**). The production of compounds such as exopolysaccharides determines the embedding of bacteria in a complex matter constituted also by nucleic acids, lipids, and proteins [43]. A so organized matrix supplies bacteria for several advantages: for instance, it can manage the flow of nutrients and protect bacteria against the action of antimicrobial substances and the host immune system, which encounter great difficulty in scratching the structure and organization of the biofilm matrix [44]. So, manipulation and inhibition of the QS system might open new scenarios and improve therapies for chronic bacterial diseases [45, 46], including even cancer [47, 48].
