**3. Essential oils**

### **3.1 General aspect**

Essential oils are compounds obtained from the secondary metabolism of the plants. They are characterized as complex mixtures of volatile compounds abundant in aromatic plants found in different parts of the plant, including leaves, flowers, stem, roots, seeds, and fruits [59]. There is a diversity of these substances described in the literature in commercial use, such as in perfumes, pharmaceuticals, cosmetics, insecticides, and food additives [60].

Generally, they are oily-looking liquids at room temperature of complex mixtures of volatile lipophilic substances, usually with pleasant scent. In water, EOs have a limited solubility, which allows their separation by steam or water distillation. Other methods to obtain EOs include cold-press extraction used for citrus peels, separation by solubility using organic solvents, and through supercritical fluid extraction [61]. They are usually unstable against environmental factors such as light, temperature, water activity, and salinity, affecting their constitution, contributing to the appearance of chemotypes with particular compositions. Depending on the technique used in the course of a separation, reactions such as ester hydrolysis, autoxidation, and rearrangements may occur, leading to the formation of artifacts and modifying their biological activity [62].

Compounds included in the EOs are produced in the cytoplasm and plastids of plant cells through the action of terpene synthase enzymes (TPSs), in which they use substrates from two pathways involved in the synthesis of terpenes: the mevalonate (MVA) and the methyl-eritritol phosphate (MEP) pathways [63]. They are localized and stored in complex secretory structures, such as glands, secretory cavities, hairs or trichomes, epidermal cells, internal secretory cells, and the secretory pockets [64]. Many of these substances are now known to be directly involved in the defense or attraction mechanisms of plants and often show interesting biological activities [65].

Despite containing two or three main components at a level of 20–70%, EOs are very complex mixtures of substances. In general, the majority components are formed by terpenes and phenylpropanoids [66]. In the very first definitions of EOs, these were frequently identified with terpenes, principally mono- and sesquiterpenes. Other substances have also been identified as alcohols, aldehydes, ketones, phenols, esters, ethers, oxides, peroxides, furans, organic acids, lactones, coumarins, sulfur compounds, anthraquinones, and alkaloids [61]. In the mixture, such compounds come in different concentrations. Usually, one of them is the majority compound, with others in lower grades and some in very low quantities (trace). EOs are composed of volatile hydrocarbons, and they may contain oxygen, sulfur, and halogens (rare) in their chemical structure [67]. In a reduced number of species, the predominant components are aromatic molecules, and these include thyme (thymol and carvacrol), peppermint (menthol), and anise (anethol) [68].

### **3.2 Antimicrobial and antibiofilm potential**

Humans have used EOs for thousands of years, not only as aromatic extracts and for beauty care and culinary uses, but also in folk medicine, due to their many different pharmacological activities, such as antiseptic, anti-inflammatory, and analgesic properties [65]. Some of the EOs, and their components, have demonstrated the relevant antimicrobial potential against a wide range of microbial pathogens [69]. Additionally, Gram-positive bacteria, such as *S. aureus*, seem to be much more susceptible to EOs than Gram-negative cells, probably due to cellular surface constitution. Gram-positive has only the inner membrane and a cell wall that allows hydrophobic molecules to easily penetrate into the cells. For instance, phenolic compounds have a dose-dependent effect, at low concentrations they interfere with enzymes involved in energy production, and at high amounts they can denature proteins [70, 71].

The broad-spectrum activity of EOs is related to the diverse chemical reactions of aldehydes, phenolic compounds, and terpenes, synthesized from secondary metabolism by different plant parts [10]. The EO action is attributed to the ability of their constituents to interact with the cell membrane and consequently disturb the microbial integrity, leading to cell death [72]. However, EO bioactive components can have several cellular targets, and they are mainly associated with cytoplasmic coagulation, inhibition of ATP-production enzymes, alteration in ion transport, cell-wall damage, and bacterial membrane destruction (**Figure 3**) [73].

The emergence of MRD pathogens has caused an interesting shift from the onerous development of novel classes of antibiotics to the more straightforward application of synergism or combinatory therapy in the hope of reviving the efficacy and effectiveness of existing antibiotics [74]. Several studies have demonstrated that there was synergetic effect when two or more EOs are mixed together. Moreover, there are also reports of synergistic activity of EOs when used in combination with well-known antibiotics. When blended with other antimicrobial agents, the

**169**

would be translated into antibiofilm drugs.

occur [77].

**Figure 3.**

motility [71].

*Essential Oils as an Innovative Approach against Biofilm of Multidrug-Resistant* Staphylococcus*…*

constituents of EOs can unlock the cell membrane channels, thus opening the pas-

The capacity of some EOs to inhibit biofilm formation has been less explored; however, some reports suggested their utilization as potent inhibitor of virulence factors and biofilm formation [76]. So far, a plethora of potential antibiofilm agents, mainly inspired by natural products, has been developed and shown great promise in either facilitating the dispersion of preformed biofilms or inhibiting the formation of new biofilms *in vitro* [77]. In contrast to conventional antibiotics, the recently developed antibiofilm molecules do not directly affect bacterial survival and thus the expectation is that resistance to these molecules will not readily

**Table 1** shows some studies based on the *S. aureus* antibiofilm activity of OEs extracted from several plant sources. The EO action on biofilm inhibition and dispersal can be related to reactivity, hydrophobicity, and the diffusion rate of the EO in the matrix, as well as the biofilm composition and structure [78]. Same studies correlated sublethal concentrations of EOs with their capability to inhibit the first steps of biofilm formation. The main constituents of OE can act by several ways to disturb the biofilm development, such as blockage of the quorum-sense system, inhibition of the flagellar gene transcription, or through interference with bacterial

Antibiofilm agents can have different therapeutic applications depending on their effects on the biofilm: compounds that interfere with biofilm formation could be exploited in the prophylaxis of implant surgery or for the coatings in medical devices, whereas agents able to disperse biofilm structure could be administered in combination with conventional antibiotics for the treatment of biofilm-associated infections [96]. Despite the growing number of new potent EO-based antibiofilm compounds described, there is still a great challenge in the development of antibiofilm drugs. Once the EO compounds, which has such activity, discovered so far need further optimizations to improve potency for it become one clinical candidate for such approach. Other EO features such as stability, volatility, encapsulation, and optimal dosage should be considered for the development of EO-based antibiofilm drugs. However, it is expected that in the coming years some of these compounds

sage of antimicrobial agents to reach their target sites [75].

*General antibacterial mechanisms of essential oils.*

*DOI: http://dx.doi.org/10.5772/intechopen.91833*

*Essential Oils as an Innovative Approach against Biofilm of Multidrug-Resistant* Staphylococcus*… DOI: http://dx.doi.org/10.5772/intechopen.91833*

### **Figure 3.**

*Bacterial Biofilms*

biological activities [65].

Compounds included in the EOs are produced in the cytoplasm and plastids of plant cells through the action of terpene synthase enzymes (TPSs), in which they use substrates from two pathways involved in the synthesis of terpenes: the mevalonate (MVA) and the methyl-eritritol phosphate (MEP) pathways [63]. They are localized and stored in complex secretory structures, such as glands, secretory cavities, hairs or trichomes, epidermal cells, internal secretory cells, and the secretory pockets [64]. Many of these substances are now known to be directly involved in the defense or attraction mechanisms of plants and often show interesting

Despite containing two or three main components at a level of 20–70%, EOs are very complex mixtures of substances. In general, the majority components are formed by terpenes and phenylpropanoids [66]. In the very first definitions of EOs, these were frequently identified with terpenes, principally mono- and sesquiterpenes. Other substances have also been identified as alcohols, aldehydes, ketones, phenols, esters, ethers, oxides, peroxides, furans, organic acids, lactones, coumarins, sulfur compounds, anthraquinones, and alkaloids [61]. In the mixture, such compounds come in different concentrations. Usually, one of them is the majority compound, with others in lower grades and some in very low quantities (trace). EOs are composed of volatile hydrocarbons, and they may contain oxygen, sulfur, and halogens (rare) in their chemical structure [67]. In a reduced number of species, the predominant components are aromatic molecules, and these include thyme (thymol

Humans have used EOs for thousands of years, not only as aromatic extracts and for beauty care and culinary uses, but also in folk medicine, due to their many different pharmacological activities, such as antiseptic, anti-inflammatory, and analgesic properties [65]. Some of the EOs, and their components, have demonstrated the relevant antimicrobial potential against a wide range of microbial pathogens [69]. Additionally, Gram-positive bacteria, such as *S. aureus*, seem to be much more susceptible to EOs than Gram-negative cells, probably due to cellular surface constitution. Gram-positive has only the inner membrane and a cell wall that allows hydrophobic molecules to easily penetrate into the cells. For instance, phenolic compounds have a dose-dependent effect, at low concentrations they interfere with enzymes involved in energy production, and at high amounts they

The broad-spectrum activity of EOs is related to the diverse chemical reactions of aldehydes, phenolic compounds, and terpenes, synthesized from secondary metabolism by different plant parts [10]. The EO action is attributed to the ability of their constituents to interact with the cell membrane and consequently disturb the microbial integrity, leading to cell death [72]. However, EO bioactive components can have several cellular targets, and they are mainly associated with cytoplasmic coagulation, inhibition of ATP-production enzymes, alteration in ion transport, cell-wall damage, and bacterial membrane destruction

The emergence of MRD pathogens has caused an interesting shift from the onerous development of novel classes of antibiotics to the more straightforward application of synergism or combinatory therapy in the hope of reviving the efficacy and effectiveness of existing antibiotics [74]. Several studies have demonstrated that there was synergetic effect when two or more EOs are mixed together. Moreover, there are also reports of synergistic activity of EOs when used in combination with well-known antibiotics. When blended with other antimicrobial agents, the

and carvacrol), peppermint (menthol), and anise (anethol) [68].

**3.2 Antimicrobial and antibiofilm potential**

can denature proteins [70, 71].

**168**

(**Figure 3**) [73].

*General antibacterial mechanisms of essential oils.*

constituents of EOs can unlock the cell membrane channels, thus opening the passage of antimicrobial agents to reach their target sites [75].

The capacity of some EOs to inhibit biofilm formation has been less explored; however, some reports suggested their utilization as potent inhibitor of virulence factors and biofilm formation [76]. So far, a plethora of potential antibiofilm agents, mainly inspired by natural products, has been developed and shown great promise in either facilitating the dispersion of preformed biofilms or inhibiting the formation of new biofilms *in vitro* [77]. In contrast to conventional antibiotics, the recently developed antibiofilm molecules do not directly affect bacterial survival and thus the expectation is that resistance to these molecules will not readily occur [77].

**Table 1** shows some studies based on the *S. aureus* antibiofilm activity of OEs extracted from several plant sources. The EO action on biofilm inhibition and dispersal can be related to reactivity, hydrophobicity, and the diffusion rate of the EO in the matrix, as well as the biofilm composition and structure [78]. Same studies correlated sublethal concentrations of EOs with their capability to inhibit the first steps of biofilm formation. The main constituents of OE can act by several ways to disturb the biofilm development, such as blockage of the quorum-sense system, inhibition of the flagellar gene transcription, or through interference with bacterial motility [71].

Antibiofilm agents can have different therapeutic applications depending on their effects on the biofilm: compounds that interfere with biofilm formation could be exploited in the prophylaxis of implant surgery or for the coatings in medical devices, whereas agents able to disperse biofilm structure could be administered in combination with conventional antibiotics for the treatment of biofilm-associated infections [96]. Despite the growing number of new potent EO-based antibiofilm compounds described, there is still a great challenge in the development of antibiofilm drugs. Once the EO compounds, which has such activity, discovered so far need further optimizations to improve potency for it become one clinical candidate for such approach. Other EO features such as stability, volatility, encapsulation, and optimal dosage should be considered for the development of EO-based antibiofilm drugs. However, it is expected that in the coming years some of these compounds would be translated into antibiofilm drugs.


#### **Table 1.**

*Summarized antibiofilm activity of EOs against MDR* S. aureus*.*
