**3. Antibacterial activity of essential oils and plant extracts against** *S. aureus*

Essential oils (EO) and their components are becoming increasingly popular as antimicrobial agents. They belong to the group of secondary metabolites that are enriched in compounds based on an isoprene structure and are called terpenes. They occur as di-, tri-, tetra-, hemi- and sesquiterpenes. The compounds that contain additional elements, usually oxygen, are termed terpenoids. Terpenes, terpenoids, as well as essential oils containing these substances exhibit antibacterial activity against broad spectrum of microorganisms including staphylococci [66].

One of the best characterized EO, which effectively inhibits growth of *S. aureus*, including MRSA isolates, is tea tree oil (TTO) derived mainly from the Australian native plant *Melaleuca alternifolia*. The antimicrobial activity of TTO is attributed mainly to its major component terpinen-4-ol, and α-terpineol which is present in a lower concentration [67]. Several groups of researchers have evaluated the activity of TTO against MRSA. Carson et al. examined 64 MRSA isolates from Australia and the United Kingdom and showed that the MIC and MBC for the Australian isolates were 0.25 and 0.5% (v/v), respectively, while for the United Kingdom isolates were 0.312 and 0.625%, respectively [68]. The TTO has been also evaluated as an alternative decolonization agent for MRSA. The ointment and body wash containing 4 and 5% of TTO, respectively, were found as more effective in eradication of MRSA carriers than classical therapies with using 2% mupirocin nasal ointment and triclosan body wash [69]. Carson and coworkers revealed that TTO and its main components compromise the cytoplasmic membrane of *S. aureus* [70]. The most important consequences of the damages of lipid bilayer are as follows: leakage of important cytoplasmic components, inhibition of respiration (leakage of potassium ions), loss of sodium chloride tolerance and some changes in cell morphology [70, 71]. TTO, terpinen-4-ol and α-terpineol showed strong activity against biofilm formed by *S. aureus* on biomaterials. Partial destruction of 24-h-old biofilms was achieved in the concentration 4–8 times greater than MIC after 1 h, whereas 2–4 × MIC was adequate to obtain 90% reduction metabolic activity of biofilm after 4 h of treatment [72]. The research of some authors showed transient decreases in antibiotic susceptibility in several bacteria that had been exposed to TTO. It raises concerns that TTO may hinder the effectiveness of conventional antibiotics and influence the development of resistance [73–75]. However, recently Hammer et al. showed that the presence of TTO or terpinen-4-ol resulted in only minor changes in antibiotic susceptibility of *S. aureus* isolates that were serially subcultured with sub-inhibitory TTO or terpinen-4-ol [76].

a bacteriocin which strongly inhibits the growth of *Bacillus cereus* [65]. Thus, it was shown that antimicrobial activity of honey is attributed not only to hydrogen peroxide (formed by glucose oxidase originating from honeybees), antioxidant components or dimethyloglyoxal (in the case of Manuka honey), but also to microorganisms which are present in this product. However, results of some successful investigations have been published the trials of isolation of bacteriocinogenic bacterial strains from honey and other than honeybee products were rarely carried out to date. In our opinion, especially, promising source of bacteria producing interesting bacteriocins could be fermented pollen—it is pollen which is collected by bees for the winter and early spring. The high antimicrobial activity of fermented pollen is the consequence of lactic acid bacteria (LAB) presence in this product, and the products of LAB

**3. Antibacterial activity of essential oils and plant extracts against** *S. aureus*

Essential oils (EO) and their components are becoming increasingly popular as antimicrobial agents. They belong to the group of secondary metabolites that are enriched in compounds based on an isoprene structure and are called terpenes. They occur as di-, tri-, tetra-, hemi- and sesquiterpenes. The compounds that contain additional elements, usually oxygen, are termed terpenoids. Terpenes, terpenoids, as well as essential oils containing these substances exhibit antibacterial activity against broad spectrum of microorganisms including staphylococci [66]. One of the best characterized EO, which effectively inhibits growth of *S. aureus*, including MRSA isolates, is tea tree oil (TTO) derived mainly from the Australian native plant *Melaleuca alternifolia*. The antimicrobial activity of TTO is attributed mainly to its major component terpinen-4-ol, and α-terpineol which is present in a lower concentration [67]. Several groups of researchers have evaluated the activity of TTO against MRSA. Carson et al. examined 64 MRSA isolates from Australia and the United Kingdom and showed that the MIC and MBC for the Australian isolates were 0.25 and 0.5% (v/v), respectively, while for the United Kingdom isolates were 0.312 and 0.625%, respectively [68]. The TTO has been also evaluated as an alternative decolonization agent for MRSA. The ointment and body wash containing 4 and 5% of TTO, respectively, were found as more effective in eradication of MRSA carriers than classical therapies with using 2% mupirocin nasal ointment and triclosan body wash [69]. Carson and coworkers revealed that TTO and its main components compromise the cytoplasmic membrane of *S. aureus* [70]. The most important consequences of the damages of lipid bilayer are as follows: leakage of important cytoplasmic components, inhibition of respiration (leakage of potassium ions), loss of sodium chloride tolerance and some changes in cell morphology [70, 71]. TTO, terpinen-4-ol and α-terpineol showed strong activity against biofilm formed by *S. aureus* on biomaterials. Partial destruction of 24-h-old biofilms was achieved in the concentration 4–8 times greater than MIC after 1 h, whereas 2–4 × MIC was adequate to obtain 90% reduction metabolic activity of biofilm after 4 h of treatment [72]. The research of some authors showed transient decreases in antibiotic susceptibility in several bacteria that had been exposed to TTO. It raises concerns that TTO may hinder the effectiveness of conventional antibiotics and influence the development of resistance [73–75].

metabolism—lactic acid, as well as bacteriocins.

212 Frontiers in Frontiers in Staphylococcus Aureus *Staphylococcus aureus*

Promising results were also obtained in the investigation of antimicrobial potential of lavender oil (LO). The oil obtained from *Lavandula angustifolia* demonstrated *in vitro* activity against MRSA at concentration of <1% [77]. Several chemically characterized lavender oils were assessed for their antibacterial activity using the disc diffusion method. All tested lavender oils inhibited growth of both MSSA and MRSA with inhibition zones ranged from 8 to 30 mm in diameter at oil doses ranging from 1 to 20 μL, respectively [78]. Some significant differences in the chemical composition and antibacterial activity of LO, which mainly depend on the origin of the lavender samples, were observed in research carried out by different authors. For example, it was demonstrated that oil from lavender of Bulgarian origin, which contains 51.1% linalool and 9.5% linalyl acetate as main components, was more effective against bacteria than oil originated from lavender sample of French origin containing 29.1% linalool and 43.2% linalyl acetate. It is believed, however, that lavender oil may be useful, first and foremost, as a prophylactic or topical application for surface infection [79].

Another interesting, from the point of view of its antimicrobial properties, is Thymus essential oils (TOs). Its main chemical components are α-thujone, α-pinene, camphene, β-pinene, p-cymene, α-terpinene, linalool, borneol, β-caryophyllene, thymol and carvacrol [80]. Different chemotypes of the essential oil from the genus *Thymus* were distinguished based on the presence of chemical components [81]. The antimicrobial properties of TOs are related to their high content of carvacrol and thymol, which were identified as the most efficient against bacteria [80]. Using an agar dilution method, the MIC values for MSSA and MRSA were reported for carvacrol (0.015–0.03%, v/v) followed by thymol (0.03–0.06%, v/v) [82]. The *Thymus* essential oils blended, in which the principal components were thymol, linalool, terpinen-4-ol and α-terpinene, exhibited significant inhibitory and bactericidal effects against strains of epidemic MRSA. The mean MIC and MBC values for the oil blend was 0.3 and 0.6%, respectively, whereas for the linalool chemotype thyme oil the MIC and MBC values were 0.4 and 0.8%, respectively. In the disc diffusion assay, the essential oils blended resulted in the formation of mean zone of inhibition size of 34.8 mm, while linalool chemotype produced a mean zone of 20.7 mm [83].

Recently research of many authors showed also the antimicrobial activity of geranium oil (GO) against MRSA [84, 85]. Among 67 components of geranium oil from *Pelargonium graveolens* Ait, citronellol, geraniol, nerol, citronellyl formate, isomenthone and linalool are the main constituents responsible for its biological activity. The research based on agar dilution method showed that the geranium oil had very strong activity against the clinical *S. aureus* strains, including MRSA strains, exhibiting MIC values of 0.25–2.50 μL/mL [85]. Moreover, Rosato et al. showed the occurrence of a synergism between geranium oil and norfloxacin against reference *S. aureus* strains [86].

The antibacterial activity of essential oils from oregano (*Origanum vulgare*) against multiresistant bacteria, including MRSA, was analysed by Costa et al. [87]. The MIC values were determined by the microdilution method. MRSA were inhibited by the essential oil at the concentration of 0.125%. Nostro et al. investigated activity of essential oils from oregano against biofilm-grown *S. aureus* and the effects of the oil on biofilm formation. The biofilm inhibitory concentrations (0.125–0.500%, v/v) and biofilm eradication concentrations (0.25–1.0%, v/v) were twofold or fourfold greater than the concentration inhibitory planktonic growth. Sub-inhibitory concentrations of the oils from oregano prevented biofilm formation by *S. aureus* strains [82].

*Nigella sativa* is a herbaceous plant cultivated in many countries in the world [88]. Crude extract and seed essential oil possess antibacterial activity against several bacteria [89]. The antibacterial effect may be due to the presence of the two important active compounds of *N. sativa*, thymoquinone and melanin [90]. The activity of *N. sativa* extract against clinical isolates MRSA was investigated by Hannan et al. [91]. They showed that all MRSA isolates were sensitive to *N. sativa* extract at a concentration of 4 mg/disc and MIC was in the range of 0.2–0.5 mg/mL. On the other hand, the multidrug resistant *S. aureus* strains isolated from nasal and milk samples of cows and buffalo were completely inhibited by *N. sativa* extract at concentration of 40 μg/mL on disc and MIC values were between 0.3 and 2.5 mg/mL [92].

Essential oil of cinnamon and cinnamaldehyde, which is main chemical constituent of this oil, also showed activity against MRSA. Essential oil from *Cinnamomum osmophloeum* (clone B) had an excellent inhibitory effect with the MIC of the essential oil and cinnamaldehyde against MRSA from human stand at 250 μg/mL [93]. The antimicrobial activity of cinnamon essential oil and *trans*-cinnamaldehyde against *Staphylococcus* spp. from clinical mastitis of cattle and goats was not dependent on the antibacterial susceptibility profile. However, the best antimicrobial activity was showed with *trans*-cinnamaldehyde and this compound could be used in the treatment of mastitis [94].

The number of research on antibacterial properties of extracts from medicinal plants against MRSA increased in recent years. These researches are conducted in different countries and show that extracts of plants are rich source of unique phytochemicals with activity against MRSA. Among recently investigated plant was *Schinus areira* L., which grows naturally in Argentina, Peru, Bolivia and Northern Chile. The essential oil from leaves and fruits of two specimens of *S. areira* differ in chemical profile. The limonene-rich oil isolated from the leaves and fruits had potent antibacterial effect on MRSA. When using 3.2 and 15 μL/mL (MICs value) of essential oil from leaves and fruits, respectively, the complete inhibition of MRSA growth was observed. Leaves and fruits oils showed bactericidal action after incubation for 24 h with 20 and 40 μL/mL, respectively. On the other hand, the α-phellandrene-rich fruit oil, having a lower content of limonene, was inactive against MRSA [95].

According to research of Endo and Dias Filho [96], MRSA is also sensitive to berberine (plant alkaloid) which is used in Chinese medicine. MICs values of berberine ranged from 62.5 to 250 μg/mL and MBC values were the same or twofold above the MIC. Highly potent anti-MRSA activity with MIC values in range of 25–50 mg/mL was detected among Libyan medicinal plants such as *Cistus salviifolius, Salvia officinalis, Pistacia atlantica, Arbutus pavarii* and *Myrtus communis* [97]. Significant anti-MRSA activity was documented in many studies on extracts of plants used in traditional medicine in Brazil. A mixture of hydrolyzable tannins from *Punica granatum* and the naphthoquinones α-lapachone I and α-xyloidone II from *Tabebuia avellanedae* showed antibacterial activity against all *S. aureus* strains tested, including MRSA isolates [98]. *Turnera ulmifolia* L. occurs in the north and northeast Brazilian regions and ethanol extract from this plant showed synergistic effect on gentamicin and kanamycin against MRSA strains. Coutinho et al. [99] found that the presence of ethanol extract of *T. ulmifolia* in growth medium at concentration of 32 μg/mL causes a significant reduction in the MIC for these antibiotics. The other studies conducted in India reported that ethanol, methanol and acetone extracts of *Moringa oleifera, Elettaria cardamomum* and *Tamarindus indica* seeds from India showed antibacterial activities against multidrug resistant MRSA isolates from wound infection [100].
