Essential Oils as Antimicrobial Agents—Myth or Real Alternative?

Herbs and the essential oils derived from them have been used from the beginning of human history for different purposes. Their beneficial properties have been applied to mask unpleasant odors, attract the attention of other people, add flavor and aroma properties to prepared dishes, perfumes, and cosmetics, etc. Herbs and essential oils (EOs) have also been used in medicine because of their biological properties, such as larvicidal action, analgesic and anti-inflammatory properties, antioxidant, fungicide, and antitumor activities, and many more. Many EOs exhibit antimicrobial properties, which is extremely important in fields of science and industry, such as medicine, agriculture, or cosmetology. Among the 250 EOs which are commercially available, about a dozen possess high antimicrobial potential. According to available papers and patents, EOs seem to be a potential alternative to synthetic compounds, especially because of the resistance that has been increasingly developed by pathogenic microorganisms. In this review we summarize the latest research studies about the most-active EOs that are known and used because of their antimicrobial properties. Finally, it is noteworthy that the antimicrobial activities of EOs are not preeminent for all strains. Further investigations should, thus, focus on targeting EOs and microorganisms.


Introduction
Essential oils (EOs) are defined as volatile secondary metabolites of plants that give the plant a distinctive smell, taste, or both. EOs are produced by more than 17,500 species of plants from many angiosperm families, e.g., Lamiaceae, Rutaceae, Myrtaceae, Zingiberaceae, and Asteraceae, but only about 300 of them are commercialized [1]. Compounds included in the EOs are synthesized in the cytoplasm and plastids of plant cells through the pathways of malonic acid, mevalonic acid, and methyl-d-erythritol-4-phosphate (MEP). They are produced and stored in complex secretory structures, such as glands, secretory cavities, and resin conduits, and are present as drops of liquid in the leaves, stems, flowers and fruits, bark, and roots of plants. Despite containing two or three main components at a level of 20-70%, EOs are very complex mixtures of mainly terpenes, terpenoids, and phenylpropanoids. They may also contain many other compounds, such as fatty acids, oxides, and sulfur derivatives [2]. al. [12]. The three species most commonly grown types are: L. angustifolia Mill. (narrow-leaved lavender, usually medical), L. stoechas (French lavender), L. latifolia, and their hybrids. L. angustifolia Mill. (formerly synonymous with L. officinalis L. or L. vera DC), is a species with the most significant industrial importance because of the EO derived from it. EO of lavender has unique biological activity, not only antimicrobial properties [13]. World trade in lavender EO is estimated at 50 million dollars [14].

Antimicrobial Properties of Lavender EO
Lavender EO showed antiviral activity against Herpes simplex virus type 1 [17]; it has shown antibacterial activity to a much greater extent. In dermatology, lavender EO can be used to treat ulcers, burns, and scarring that are difficult to cure. An extensive review of the properties of many different oils, including lavender EO, in relation to the pathogens responsible for dermatological diseases is detailed in Orchard et al. [18]. It is worth noting that the EO of L. angustifolia Mill. has a strong antiseptic effect against antibiotic-resistant strains, e.g., Staphylococcus aureus (MRSA) resistant to methicillin or vancomycin-resistant strains of Enterococcus sp. (VRE) [19,20]. Lavender EO is also active against the strain of E. coli J53 R1 resistant to piperacillin. Essential oil caused the strain's sensitivity to this antibiotic by altering the permeability of the outer membrane, as demonstrated by decreased bioluminescence. The activity of lavender EO against 24 strains of L. monocytogenes was also tested (minimal inhibitory concentration (MIC) 2.5-5.0 µL/mL). In the case of L. monocytogenes L120 resistant to chloramphenicol, the MIC was 1.3 µL/mL [21].
The activity of lavender EO against Shigella flexneri, which causes dysentery, has also been demonstrated [22]. Researchers have found that antimicrobial activity of this EO is a result of the synergistic action of major and minor components of the essential oil. In turn, using commercial L. angustifolia Mill. EO (Pollena Aroma, Warsaw), it was shown that the non-sporulating Gram-negative bacteria from the Enterobacteriaceae family (MBC = 1.25 and 2.5 mg/mL) were more sensitive to essential oil than Gram-positive S. aureus bacteria (MBC = 5 mg/mL) [23].
Lavender EO also has antibacterial activity against clinical strains of bacteria, which were isolated from patients with respiratory tract infections [24]. It is used to treat infections of the mouth, throat, upper respiratory tract, and lungs [25]. In addition, antibacterial activity may be accompanied by an immunostimulatory effect. After inhalation, no sputum bacteria were found in the essential oil, while the symptoms and increased chemiluminescence of granulocytes were alleviated and the incidence of infections was reduced in patients with bacterial respiratory infections [19]. Haemophilus influenzae may be responsible for such infections and lavender EO inhibited the growth of strains that were resistant to erythromycin or amoxycycline [25].

The Sources of Thyme EO
The genus Thymus, consisting of over 400 species, belongs to the Lamiaceae family. Thyme grows as a shrub with a stumpy, raised stem, on which small, longitudinal leaves and two-lobed pink-violet, light-violet, or white flowers in pairs glow. Oblong, brown splines are obstructions This plant has a height of 10-40 cm. EO is found in glandular hairs on leaves and flowers [15,16]. Plants of this type have been used in folk medicine for thousands of years, especially in the countries of the Mediterranean region. Antimicrobial activity is the most extensively studied in the case of Thymus vulgaris L. (common thyme, German thyme) [30]. The high intraspecific polymorphism of T. vulgaris L. in the production of terpenes is noteworthy, since 6 chemotypes of this species were found [31,32]. Pharmacopeia refers to thymol chemotype. Thyme EO (Latin thymi aetheroleum) is obtained by steam distillation of fresh aerial parts of T. vulgaris L.; T. zygis Loefl (Spanish thyme, white thyme), or mixtures of both species. The highest level of EO production usually occurs during the flowering period of the plant [30].

Antimicrobial Properties of Thyme EO
Thyme EO showed antiviral activity, as it was active against Herpes simplex (HSV1, DNA virus) with IC 50 values of 11 µg/mL [34]. This EO also displayed 100% inhibitory activity in the liquid phase at 3.1 µL/mL concentration against influenza virus A1/Denver/1/57 (H1N1) with 30 min exposure [35]. Nevertheless, the most important effect of thyme EO relates to bacteria. Particularly high bacteriostatic activity against the majority of Gram-positive and Gram-negative bacteria is demonstrated by the thymol chemotype of T. vulgaris L. EO, which has been described in many reviews [30,[36][37][38][39]. This activity is due to the high content of thymol and carvacrol, which due to the acidic nature of the hydroxyl group, can form hydrogen bonds with the active site of many enzymes. In 2017, Schött [40] described the activity of thyme EOs against S. mutans. This bacteria is responsible for the development of dental caries but may also cause infective endocarditis. The EO from T. zygis was more active than EO from T. vulgaris. Only thymol chemotype was active.
The activity of thyme EO against strains responsible for acute bacterial pharyngitis and throat inflammation was also examined. This infection is caused by β-haemolytic Streptococci strains, e.g., S. pyogenes. T. vulgaris EO was active against the S. pyogenes strains isolated from the throat of patients [41]. The activity of thyme EO and carvacrol was examined against Malassezia furfur-an opportunistic yeast that can develop on the face, forearms, and scalp, especially during adolescence. The MIC was in the range of 450-900 µg/mL [42].
Thyme EO was active against S. aureus ATCC 25923 and K. pneumoniae ATCC 13882. It also inhibited the growth of Brachyspira hyodysenteriae, which causes swine dysentery [30].
On the other hand, thyme EO rich in γ-terpinene (68.415%) and p-thymol (24.721%) completely inhibited the growth of Fusarium graminearum Fg 06-17 at a concentration of 0.06% [43]. However, again the antifungal activity of thyme EO is mainly attributed to the presence of thymol, which may, due to its low polarity, interfere with the lipid bilayer of the cell membrane, changing its fluidity and permeability. Other mechanisms of action are based on cell wall damage and changes in fungal morphology. Thyme EO inhibited aflatoxin production, caused the cytoplasmic spill of A. flavus, and was responsible for degenerative changes in its hyphae, which even led to complete disappearance of conidia [44]. It also influenced the morphology and growth of A. niger [45]. Thyme EO showed a synergistic effect in combination with cinnamon and rosemary EOs on Botrytis cinerea ATCC12481 and Penicillium expansum. The mean lesion diameter of the pears treated with the cinnamon-rosemary-thyme EOs combination (78, 1250, 39 µg/mL, respectively) was 6 mm and 8 mm against B. cinerea and P. expansum, respectively, after 10 days at 25 • C [46].

The Sources of Peppermint EO
The genus Mentha is part of the family of Lamiaceae (Lamiaceae) and includes about 30 species grown in temperate climate zones around the world, especially in Europe, North America, North Africa (Syria, Ethiopia), and northern parts of Iran. Due to the high variability of the species and great ease of crossing species, the chemical composition of the EOs obtained from them is very diverse [2]. One of the species-peppermint-is a natural hybrid of two species: Mentha spicata L. and Mentha aquatic L. Peppermint is a perennial with a height of 30-90 cm with numerous underground and aboveground runners and a purple stem. The leaves are oblong and dark green. Purple flowers are collected in capitate inflorescences. This plant requires fertile and permeable soil. EO from peppermint is obtained after distillation of dried leaves with water vapor. As a result of this process, a light yellow or greenish liquid with an intense mint aroma is obtained [15,16,47].

Antimicrobial Properties of Peppermint EO
The broad spectrum of biological activity of plants of the genus Mentha was discussed recently in a review article [2]. Peppermint essential oil exhibited high levels of virucidal activity against HSV-1 and HSV-2 in viral suspension tests. Both kinds of viruses were significantly inhibited when Herpes simplex virus was pre-treated with the EO prior to adsorption. Peppermint EO affected the virus before adsorption, but not after penetration into the host cell [48].
Peppermint EO has a weak antibacterial activity, hence it is usually included in complex preparations. Its widespread use is due more to a pleasant mint flavor and the feeling of coolness than to its antimicrobial properties. However, it is believed that the higher menthol content in peppermint EO has more antimicrobial activity [18]. In the diffusion test, peppermint EO (20 µL) inhibited the growth of bacterial strains, such as E. coli WDCM 00013, L. monocytogenes WDCM 00020, P. aeruginosa WDCM 00024, S. enterica WDCM 00030, and S. aureus WDCM 00032. The inhibition zone was from 12 mm for P. aeruginosa up to 37.66 mm for. S. aureus [49].
Recently, the effect of peppermint EO on the development of yeast has been investigated. These microorganisms may be responsible for the spoiling of cashew, guava, mango, and pineapple juices. In preliminary studies, the MIC was 1.875 µL/mL against C. albicans, C. tropicalis, Pichia anomala, and S. cerevisiae. However, when used in mango and pineapple juices, even at higher concentrations of EO (7.5 µL/mL), no significant reduction in yeast was observed. In the case of S. cerevisiae, the addition of 1.875 µL/mL of mint EO to the cashew and guava juices strongly weakened the membrane permeability, membrane potential, and activity of the efflux pump in the yeast cells. It is true that mint EO did not affect the appearance, smell, and viscosity of fruit juices, but negatively influenced their taste [50]. In turn, Benzaid et al. [7] determined that mint EO in the volatile form inhibits the development of C. albicans comparable to amphotericin-B, influencing the expression of various genes, such as secreted aspartyl proteinases (SAP 1, 2, 3, 9, 10), and being associated with the process of adhesion of hyphal wall protein 1 (HWP1).
It is worth noting that although the mint EO alone has a weak antibacterial activity, it may have a synergistic effect with other EOs or substances. For example, it increased the activity of Pongamia pinnata EO and additionally increased over 30 times the sensitivity of bacteria to gentamicin, E. coli pMG309 harboring and plasmid encoding β-lactamase, KPC-3 on meropenem, and caused a strong anti-candidal effect with azole antibiotics, such as Fluconazole and Ketoconazole, and a weaker synergistic effect with Clotrimazole and Itraconazole. The research was carried out using Candida strains isolated from patients affected by skin diseases and urine samples [51].
One of the popular preparations used to treat headaches, colds, coughs, mild spinal gastrointestinal complaints, and to relieve local muscle pain is Olbas ® Tropfen (Olbas). It contains EOs such as peppermint EO (5.3g), eucalyptus (2.1g), cajuput, and a smaller amount of juniper EO (0.3g). Olbas ® has shown antibacterial activity against many strains, including methilicin-resistant S. aureus (MRSA) and vancomycin-resistant Enterococcus. Hence, Olbas ® could be used to treat uncomplicated skin and respiratory infections [52]. This activity may result from the high content of monoterpenes, especially menthol, which due to their hydrophobicity, affect the fluidity and permeability of the cell membrane. Monoterpenes also affect the conformation of proteins embedded in the membrane, thus inhibiting the process of cellular respiration and disrupting the transport of ions through cell membranes, which can lead to cell death [53,54].
The possibility of using peppermint EO introduced into a chitosan nanogel was investigated in the protection of plaque against S. mutans causing caries. The maximum release of peppermint EO from the nanogel was about 50% after 360 h in an aqueous-alcoholic solvent at ambient temperature. The adhesion of bacterial cells was highly sensitive to nanoformulation of mint EO, as compared to the unloaded chitosan nanogel. Inhibition of biosynthesis of S. mutans occurred at a concentration of 50 µg/mL, compared to 400 µg/mL for a nanogel without EO. In addition, it was found that chitosan nanogel containing mint EO inhibited the activity of glycosyltransferase genes (gtf B, gtf C, and gtf D) involved in the formation of extracellular polymers [55]. Moreover, peppermint EO also inhibited the growth of fungi strains, such as Alternaria alternata, Aspergillus flavus, Aspergillus niger, Colletotrichum gloeosporioides, Fusarium solani, and Macrophomina phaseol [56].

Source
Cajuput oil is obtained from leaves and small branches of the cajuput tree (Melaleuca leucadendron L.; syn of M. cajuputi Powell, syn of M. minor Smith), which belongs to the Myrtaceae family and is native to Southeast Asia and North East Australia [57,58]. The cajuput tree reaches a height of 25 m, and has a characteristic white and twisted trunk, similar to birch trees. Furthermore, the bark of the cajuput tree has a thick flaky texture that detaches itself from the trunk and hangs downwards [59].
The branches are arranged irregularly, covered with white bark, and in the case of young branches, slightly silvery. The leaves are thick, lanceolate, about 10 cm long, and 1.5 cm wide. Oil reservoirs are visible on the leaf surface, making the leaves highly aromatic. Their smell is associated with camphor, rosemary, and cardamom. The small, white, individual flowers are collected in spiky inflorescences. Flowers bloom in early summer or spring and developed fruits are green-brown sacks [60].
The cajuput EO is obtained both from wild plants and those cultivated on plantations. It is obtained as a result of distillation with steam from fresh twigs. The cajuput EO content ranges from 1.5-3.0% [61]. The cajuput EO is yellow-green with an intense, herbal scent reminiscent of eucalyptus EO.

Source
Cinnamon is a natural plant that grows in Sri Lanka and tropical Asian countries. It is an important spice plant widely used throughout the world. Over 250 species of the genus Cinnamomum are known [66].
Cinnamon oil, known for its healing properties, is a product of the distillation with water vapor of three raw materials: (i) leaves and young twigs of Chinese cinnamon (Cinnamomoum cassia [syn aromaticum] Blume); (ii) Ceylon cinnamon leaves (Cinnamomum zeylanicum Blume [syn C. verum]; and (iii) Ceylon cinnabar bark [33].
These types of cinnamon vary in appearance, taste, and place of cultivation. C. cassia is grown mainly in China and is characterized by a dark reddish-brown color and a sharp and bitter taste, while C. zeylanicum grown in Sri Lanka is characterized by a bright to medium-dark reddish-brown color and sweet taste [67]. Moreover, these are differences in the classification regarding raw material used in medicine. In India and Sri Lanka, C. cassia is considered a substitute for C. zeylanicum. In turn, Chinese, Korean, Taiwanese, and Japanese pharmacopoeias allow usage of only the first one [68].

Antimicrobial Activity of Cinnamon EO
In numerous studies of antimicrobial activity, cinnamon EOs of various origins are used. EO of Cinnamomum zeylanicum displayed 100% inhibitory activity at 3.1 µL/mL concentration against influenza virus A1/Denver/1/57 (H1N1) with 30 min exposure. Eugenol, the major component of Cinnamomum zeylanicum EO, possessed the most potent anti-influenza activity in both liquid and vapour phases [35]. Brochot et al. investigated the antiviral activity of a blend that was composed of equal parts (3.52% each) of Eucalyptus globulus CT cineol (leaf) and Cinnamomum zeylanicum CT cinnamaldehyde (bark), 3.00% Rosmarinus officinalis CT cineol (leaf), 1.04% Daucus carota CT carotol (seed), and 88.90% Camelina sativa oil (seed). This blend significantly reduced viral units of H1N1 and HSV1. For both viruses, a reduction greater than 99% was observed with 1% blend and 60-min contact time [71]. Intorasoot et al. [72] and Feng et al. [73] carried out studies involving EO from the bark of C. zeylanicum, which is rich in cinnamic aldehyde. They showed that the EO has a better effect compared to other EOs used (including clove EO, lemongrass, tea tree, ginger, basil), with very good activity against S. aureus, E. coli, A. baumannii, and P. aeruginosa [74], and even better activity than the antibiotic against B. burgdorferi [73]. Cinnamon EO can cause destruction of bacterial cell membranes [74]. Furthermore, the antimicrobial activity of cinnamaldehyde against S. aureus has been previously confirmed by Ferro [75]. In a study carried out by Aumeerudda-Elalfi et al. [76], C. zeylanicum EO from leaves was used. The experiment indicated better antimicrobial activity than antibiotics (ampicillin, chloramphenicol, tetracycline) against E. faecalis, E. coli, P. auerginosa, and S. aureus. However, it did not show better antimicrobial activity than other EOs examined during the study.
Cinnamon oil obtained from the leaves and bark of C. zeylanicum inhibited the growth of Salmonella typhimurium and Listeria monocytogenes (MIC = 0.5% (v/v)), which constitute a serious public health problem. Listeria is responsible for about 1600 cases of food poisoning in the US and 260 deaths annually, while Salmonella caused about 19,000 hospitalizations and 380 deaths annually in the US. Both Salmonella and Listeria can contaminate food, such as fruits, vegetables, seafood, and dairy products [6]. Furthermore, Brnawi et al. [6] investigated whether the cinnamon EO would inhibit the growth of bacteria growing on celery stored at 4 • C for 7 days. The EO at a concentration of 0.5% significantly reduced (p < 0.05) the growth of S. typhimurium (4-week-old chicken heart and liver) and L. monocytogenes (V7 serotype 1/2a, isolated from raw milk).
Cinnamon EO has recently been used to modify the zein film used for food packaging, which contains an additional 4% chitosan nanoparticles (CNP). The results showed that the combination of EO with nanoparticles not only gave antimicrobial properties by inhibiting the growth of Escherichia coli (PTCC 1163) and Staphylococcus aureus PTTC 25923, but also improved the tensile strength and decreased the elongation of the composite zein film [77].
A cinnamon-rich bark EO from Cinnamomum cassia (L.) J. Presl, rich in cinnamic aldehyde, was used to prepare a herbal toothpaste (91.8%), which inhibited the growth of Streptococcus mutans, which causes the development of dental caries. The toothpaste Jack N' Jill, which initially showed no antibacterial effect, showed a 39 mm inhibition zone of S. mutans ATCC 35668 after adding cinnamon EO. The results of this study indicate that herbal toothpaste shows statistically higher antimicrobial activity against S. mutans ATCC 35668 (p < 0.05) after adding EOs than their initial forms without EO [78].
In another study carried out by Oliveira et al. [79], it was pointed out that the cinnamon leaf EO counteracts the development of Candida yeast, which can settle in the mouth. Ultimately, the antimicrobial activity of cinnamon EO against S. aureus, E. coli, and C. albicans was confirmed by Herman's research [80], in which the author does not indicate the raw material from which the EO was obtained. Similarly, without giving the raw material from which the EO was obtained, Sumalan et al. [81] reported activity indicating the inhibition of the growth of filamentous fungi of the genera Aspergillus, Cladosporium, and Fusarium. Overall, the antimicrobial activity of cinnamon EO is mainly associated with the content of cinnamaldehyde, for which the MIC was in the range of 0.5 to 1000 µg/mL in relation to 20 strains of P. aeruginosa [66].

Source
Clove EO is obtained from spicy clove (Eugenia caryophyllata Thunb, son E. aromatica Baill., E. caryophyllus L.; Syzygium aromaticum Merr. Et Perry). The genus Eugenia (Syzygium) belongs to the Myrtaceae family. The homeland of cloves is islands in Indonesia-the Moluckie islands. Nowadays, cloves are it is grown on large plantations in many regions, mainly on the islands of Zanzibar, Pemba, Penang, Amboine, Java, Ceylon, Madagascar, Réunion, and in Africa and South America [82]. Cloves are 15 m high trees with a dense pyramidal crown. Leaves are placed opposite each other and have an elliptic-lanceolate shape. They are leathery, dark green, and shiny on top with translucent oil reservoirs. The flowers are concentrated for 5-20 in baldachogrona. and consist of four-colored red chalices, a four-petal pink crown, and many stamens and pistils. The fruit is a dark red single-or double-edged berry. Nevertheless, the raw material from which the EO is obtained is undeveloped flower buds of Eugenia caryophyllata Thunb, which are subjected to steam distillation for 9-24 h after crushing. The EO yield is high and amounts to 15-20%. The EO itself is heavier than water, and colorless or light yellow. It has a durable, intense, spicy, sweet fragrance [83].

Chemical Composition of Clove EO
Clove EO was already obtainable in the fifteenth century, and in the nineteenth century, eugenol was identified in it. At present, about 100 compounds that are components of this EO have been identified. The dominant component of clove EO is eugenol, the content of which varies from 30-95%, with the lowest content of this compound (28%) in EO isolated from growing leaves [84]. There are also eugenol acetate, β-caryophyllene, α-ilangene, δ-cadinene, as well as compounds with an aromatic structure, i.e., methyl eugenol, anetol, chavikol, vanillin, benzyl alcohol, cinnamic aldehyde, benzyl salicylate, and calamenene.
The chemical composition of the oils depends on the origin of the oil and the degree of development of the leaves or pitch. Leaf oil differs from the bud oil, with low content of eugenyl acetate.

Antimicrobial Activity of Clove EO
The in vitro antiviral activity of eugenol-the main component of clove EO-has been tested against HSV-1 and HSV-2 viruses [85]. The replication of these viruses was inhibited with IC 50 values of 25.6 µg/mL and 16.2 µg/mL against HSV-1 and HSV-2, respectively. Additional investigations revealed synergistic interactions with a combination of eugenol and acyclovir, a known antiviral drug. Studies have shown that application of eugenol delayed the development of herpes virus-induced keratitis in a mouse model [34]. Eugenol was evaluated for its anti-HSV properties on the plaque reduction assay. Only two isolates were inhibited by eugenol, but the inhibition against these isolates was greater than for the extract obtained from the flower buds of E. caryophyllata [86].
The antimicrobial activity of clove EO was compared with cinnamon EO, oregano, and bay leaves against medically important bacterial strains, such as Bacillus cereus, Escherichia coli, Listeria innocua, and Salmonella typhimurium. The diffusion test showed that clove EO was the most active, with MIC ranging from 1.25% (v/v) (B. cereus) to 2.50% (v/v) (S. typhimurium and E. coli). In addition, a synergistic effect of the clove EO with 1.8% NaCl (g/v) was observed, which can be used to improve the organoleptic properties of food [10,87]. Moreover, cloves are commonly used in the food industry as a natural additive or antiseptic to extend the shelf life due to the effective antibacterial action against certain food-borne pathogens [10].
Due to the similarity of the composition of EO, tests of the antimicrobial activity of clove EO and cinnamon leaf EO are often carried out in parallel. Both EOs showed strong bacteriostatic activity at concentrations of 0.03-0.05% and bactericidal activity at concentrations of 0.04-0.1% in relation to S. aureus, L. monocytogenes, S. enteritidis, C. jejuni, and E. coli. The activity of both EOs was also compared in the case of Yersinia enterocolitica and 5 strains of Salmonella enteritidis isolated in the hospital. Clove EO was slightly weaker than cinnamon EO in relation to strains of bacteria attacking the human respiratory system, such as K. pneumoniae, Haemophilus influenzae, Streptococcus agalactiae, and S. pyogenes. The antimicrobial properties of clove EO with cardamon EO, cinnamon EO, and 10% phenol solution were also compared. The research was carried out using the diffusion method with 9 strains of Gram-positive bacteria, 4 Gram-negative bacteria, and 7 strains of molds. Clove EO was the most active. Schmidt et al. [89] conducted a study in which eugenol-rich clove EO was active against 38 strains of Candida albicans isolated from the oropharynx, damaged skin, and vagina. Interestingly, eugenol itself showed weaker activity than the clove EO isolated from leaves.
It has been proven that compounds forming clove EO can destroy the wall and cellular membrane of microorganisms, pass through cytoplasmic membranes or enter cells, and then inhibit the proper synthesis of DNA and proteins. In the case of L. monocytogenes treated with clove EO, the DNA content decreased from 15.004 µg/mL to 5,587 µg/mL, thus by 62.76%. In addition, the bioluminescence assay showed 81.7% reduction in intracellular ATPase, which means that clove oil can damage the cell membrane and cause ATP outflow from the cell. This oil also inhibits the action of β-galactosidase, which by hydrolyzing lactose to galactose and glucose determines the use of this sugar as a source of carbon and energy. It also inhibits bacterial respiratory metabolism by affecting the tricarboxylic acid cycle pathway [90]. In another study, it was found that eugenol, the main component of the EO, can inhibit the production of α-amylase, protease, and subtilisin by Bacillus subtilis, with the ability to destroy the cell wall and cause lysis of cells [91].

Source
Eucalyptus oil is obtained from the leaves of eucalyptus globose (Eucalyptus globulus Labill) [33], which belongs to the Myrtaceae family and is the dominant plant in the forests of Australia [92]. Eucalypti are evergreen trees or shrubs that reach a height of up to 100 m. Most species drop their barks every year. Leaves of E. globulus, from which oil is obtained, should be greyish-green, up to 25 cm long and up to 5 cm wide, thick, and sickle-shaped. The flowers are usually white, but in some species yellow, pink, or red.

Antimicrobial Activity of Eucalyptus EO
Eucalyptus EO has antimicrobial activity against viruses, bacteria, yeasts, and filamentous fungi. Eucalyptus EO, rich in 1,8-cineole (88%), was active against HSV-1 in vitro. EO was able to suppress viral multiplication by >96%. This EO exhibits an anti-HSV1 activity by directly inactivating free-virus particles and might also interfere with virion envelope structures required for entry into host cells [34]. In turn, the antiviral activity of Eucalyptus species grown in Tunisia against Coxsakievirus B3 Nancy strain was studied using Confluent Vero cell cultures that were treated with non-cytotoxic concentrations of the EOs, both during and after viral infections; activity depended on Eucalyptus species. EOs from E. sideroxylon, E. lehmannii, E. leucoxylon, and E. odorata had no antiviral activity, while those from E. bicostata (IC 50 = 0.7-4.8 mg/mL), E. astringens, and E. maidennii (IC 50 = 136.5-233.5 mg/mL) were the most active against viral infections [96,97]. Eucalyptus globulus EO was mildly active against mumps virus isolated from clinical specimens of patients with respiratory tract infection. The antiviral activity was assessed by plaque reduction assay [98]. This EO also displayed 100% inhibitory activity at 3.1 µL/mL concentration against influenza virus A1/Denver/1/57 (H1N1) after 10 min exposure [35].
Antibacterial activity has been determined to a much greater extent. It is active, for example, against bacteria such as P. gingivalis and S. mutant, which cause periodontitis and other dental ailments. Therefore, its main component 1,8-cineneol has been used in products for oral hygiene, such as Listerine Cleanmint (Pfizer), Listerine Freshmint (Pgizer), and Walgreens Fresh Breath Antiseptic Mouth Rinse (Walgreen Co.) [99]. The EO was also active against E. tarda, V. ichthoenteri, V. harveyi, P. damselae, S. iniae, S. parauberis, and L. garviae, considered to be pathogens of fish [100]. Furthermore, it exhibits toxic effects against S. aureus and E. coli [101]. Eucalyptus EO is more active against Gram-negative than Gram-positive bacteria, which is attributed to the presence of components such as 1.8-cineole, p-cymene, cis-geraniol, and terpinolene, which can increase the permeability of the cytoplasmic membrane through functional impairment. The most sensitive bacterium is Acinetobacter baumannii [99].
In the case of filamentous fungi, Martins et al. [102] have shown that eucalyptus EO is toxic for M. hiemalis Wehmer, Alternaria alternata (Fr.) Keissl, Penicillium glabrum (Wehmer), and Fusarium roseum (Link). In turn, Tyagi et al. [96] had shown activity of the oil in relation to yeasts S. cerevisiae, Z. bailli, A. pullulans, C. diversa, P. fermentans, P. kluyveri, P. anomala, and H. polymorpha, and indicated the possibility of using eucalyptus EO in the food industry as a preservative in beverages. Eucalyptus EO toxicity studies performed in vitro and in animals have shown that it is low in toxicity. In addition, studies conducted with the participation of human volunteers indicated its low allergenicity [99].

Source
The Salvia genus is probably the largest genus in the Lamiaceae family, consisting of almost 1000 species [9]. Salvia was used in medicine as early as in ancient times and the Latin name of the plant comes from the words salvare, to cure, and officina, a pharmacy. Sage EO is obtained from flowering plants or only sage leaves (Salvia officinalis L. syn S. graniflor Ten.). Sage comes from the Middle East, but grows throughout Europe, as well as in Asia Minor and North America [103]. It is a small shrub (20-70 cm) with silvery-grey hairy leaves. White or purple flowers appear in May or June. Salvia prefers environments with good sunlight and fertile soil that is rich in calcium [15,16]. Sage EO is extracted by distillation with superheated steam from partly dried raw material. The duration of the process is about 1 h and the efficiency varies between 1 and 2%.

Antimicrobial Activity of Sage Oil
The use of sage EO in phytopharmacy has recently been described in two review articles [9,103]. Both the plant raw material as well as the extracts and EO obtained from sage are used as herbal medicines with mainly sanitization and antiseptic effects. EO of Salvia officinalis was active against severe acute respiratory coronavirus SARS-CoV (RNA virus), which was obtained from the sputum of a patient hospitalized with a diagnosis of SARS (severe acute respiratory syndrome) in Frankfurt University Hospital. It is worth noting that the overriding clinical feature of SARS is the rapidity with which many patients develop symptoms of acute respiratory distress syndrome (ARDS). Essential oil was weakly active at IC 50 = 870 mg/ mL [112].
Moreover, plants of the genus Salvia are used as a component of herbal teas and as flavoring for food. At the same time, the number of microorganisms developing tolerance to existing preservative techniques is constantly growing, and the unwillingness to use synthetic preservatives and antibiotics is prevalent. Therefore, the interest for food processors and consumers in antibacterial preservatives of plant origin, including the possibility of using EOs, is increasing [9]. For example, sage EO at a concentration of 2.0% has a bacteriostatic effect on the strains of Salmonella anatum [118] and Salmonella enteritidis growing in minced meat. Unfortunately, such a high concentration of essential oil had a negative effect on taste. This problem was solved by using a lower concentration of the sage EO and the addition of sodium chloride, and the products were stored at a low temperature. However, with other meat products, there may be a problem with the formulation of the finished product, because both the NaCl concentration and the sage EO can negatively affect the taste sensation [119].

Antimicrobial Activity of Tea Tree EO
So far, the most promising tea tree EO in terms of antimicrobial properties is derived from a chemotype characterized by 30-40% terpinen-4-ol content [8]. EO was active against Herpes simplex virus type 1 in vitro. IC 50 of this EO was determined as 2.0 µg/mL. EO was able to suppress viral multiplication by >96% [8,34]. Additionally, a randomized, placebo-controlled, investigator-blinded protocol was used to evaluate the efficacy of tea tree essential oil (6% tea tree EO gel) in the treatment of recurrent herpes labialis. The median time of reepithelization after treatment with this EO was 9 days compared to 12.5 days after placebo, indicating some benefit from tea tree EO treatment [129]. Meanwhile, the antibacterial activity has been determined to a much greater extent. The tea tree EO inhibited the growth of S. aureus and E. coli at a concentration of 0.78% and inhibited the adhesion of S. mutans [130] and the development of Listeria monocytogenes ATCC 7644 (MIC = 0.10 µL/g) in ground beef [131].
Tea tree EO, thanks to its antimicrobial properties, has been used in products for oral hygiene and dermatological applications [129,132]. Furthermore, Graziano et al. [126] determined that tea tree EO reduces the growth of bacteria responsible for halitosis-Porphyromonas gingivalis (MIC and MBC = 0.007%) and Porphyromonas endodontalis (MIC = 0.007% and MBC = 0.5%). The most important element of the study was the finding that tea tree EO not only inhibits the growth of bacteria responsible for halitosis leading to personal and social discomfort, but also limits the production of volatile sulfur compounds-H 2 S by P. gingivalis and CH 3 SH by P. endodontalis. Moreover, tea tree EO has shown an inhibitory effect against Candida albicans. For good therapeutic activity, the tea tree EO must contain terpineol at a minimum of 30% and a maximum of 15% cineol to simultaneously achieve very low skin irritation [129]. The aim of the study conducted by Mertas et al. [131] was the reduction of yeast resistance to the drug fluconazole due to the simultaneous use of tea tree EO or its main component (terpinen-4-ol). The authors indicated that as many as 62.5% of the 32 resistant strains became susceptible to the drug after 24 h exposure to the essential oil or its component. The minimum fluconazole concentration inhibiting fungal growth also decreased significantly (from 244.0 µg/mL to 38.46 µg/mL), along with its minimum fungicidal concentration (from 254.47 µg/mL to 66.62 µg/mL).
Recently [8], the antimicrobial properties of commercially available tea tree EOs were investigated. Of the 10 EOs, 5 were active. Identified components of the tea tree EO reduced the survival of the bacteria in the Pseudomonas aeruginosa biofilm and caused oxidative damage in Candida glabrata. On the other hand, EO of Melaleuca alternifolia) had only marginal antifungal activity against Aspergillus niger (MIC = 625 µg/mL), attributed to its active components terpinen-4-ol and α-terpineol [133]. Another example is Penicillium expansum, which is a cosmopolitan pathogen that can cause damage to fruit crops. Germination of this fungus was completely inhibited at the concentration of tea tree EO 250 µg/mL. Tea tree EO caused damage to the plasma membrane, leading to DNA, protein, lipid, and glucose leakage [134]. In turn, the effects of tea tree EO on mitochondrial morphology and function in a culture of Botrytis cinerea was investigated. This EO at a concentration of 2 mL/L severely damaged mitochondria, resulting in matrix loss and increased mitochondrial irregularity. The next consequence of mitochondrial damage was the disruption of the tricarboxylic acid cycle and the accumulation of reactive oxygen species [135].

Conclusions
Contrary to common opinion, limited EOs possess demonstrated potential as antimicrobial agents. It should be emphasized that although the antimicrobial activity is well established, the real effect is significantly weaker compared to synthetic compounds (including antibiotics). Gram-positive bacteria seem to be much more susceptible to essential oil than Gram-negative organisms. According to available data, the activity is usually correlated with phenolic, aromatic, or alcohol groups. Due to their high volatility, the effective time of action is limited, and features such as encapsulation could be changed. On the other hand, low toxicity level, as well as their natural origin, makes them an attractive alternative in both the food as well as in cosmetic industries. Several practical application could be implemented in these industries. In summary, it should be underlined that the use of EO in microbial stabilization is possible, but all cases must be individually examined.
Supplementary Materials: The following are available online. Figure S1. The chromatogram of lavender oil; Figure S2. The chromatogram of thyme oil; Figure S3. The chromatogram of peppermint oil; Figure S4. The chromatogram of cajeput oil; Figure S5. The chromatogram of cinnamon oil; Figure S6. The chromatogram of eucalyptus oil; Figure S7. The chromatogram of clove oil; Figure S8. The chromatogram of sage oil; Figure S9. The chromatogram of tea tree oil; Table S1. Chemical composition of Eos; Table S2: The antimicrobiological activity of EOs, Materials and Methods.