ANALYSIS OF CHEMICAL COMPOSITION OF THE MOST EFFICIENT ESSENTIAL OILS TOWARDS ENTEROCOCCUS FAECALIS REFERENT STRAIN ATCC 29212 AND CLINICAL ISOLATES

47.


INTRODUCTION
Allergy caused by antibiotics and antibiotic resistance have increased rapidly in recent years, causing a lot of concern in medical community [1]. Knowing the fact that nearly 60 % of all drugs introduced in therapy between 1981 and 2006, were first identified as natural products [2], it is easy to understand why researchers are seeking for alternatives in natural products, such as essential oils [3].
According to different authors, approximately 3000 plants species contain essential oils, among which only 300 are considered as commercially important [4,5,6]. Essential oils (EOs) are complex mixtures of volatile constituents, biosynthesized by plants [7]. They are frequently comprising 20 to 60 components at concentrations ranging from the fairly high (20-70 %) to the trace amounts [8].
The main group of constituents in the most EOs used to be terpenoids, which are, according to Maguna et al., [9], molecules capable of causing death of bacterial cells by following mechanisms; increasing the membrane permeability, affecting structural stability of the membrane or disrupting the lipid bilayer packing.
Enterococcus faecalis is a gram-positive, spherical bacterium, and one of the most resistant bacteria in infected human teeth root canals, whose presence is detected in teeth with periapical lesions [10], and particularly in persistent apical periodontitis [11,12]. Ability to invade dentinal tubules and survive harsh canal conditions, together with adaptability to lethal challenges, makes this pathogen very persistent to root canal treatments [13].
According to Stuart et al., [14], incidence of E. faecalis in infected root canals and in re-treatment cases of apical periodontitis ranges from 24 % to 77 %. Achieving the root canal treatment is highly related to the degree of reduction of the intracanal bacterial population. Although complete elimination of bacteria is challenging and might not always be successful, it generally might be achieved by chemo-mechanical root canal preparation with the aid of antimicrobial irrigation solutions and intracanal dressings between the treatment visits.
Since a standard endodontic procedure includes use of irrigation solutions, as a canal disinfectant, the most effective in removing endodontic biofilm, including E. faecalis, appears to be a 1-6 % sodium hypochlorite solution (NaOCl). However, if it is not used properly (with regard to concentration, pH and exposure time), NaOCl may easily create many problems outside the endodontic space, including inflammation, severe pain, extensive swelling, necrosis and cell destruction of exposed tissues, except the epithelium which is strongly keratinized [15].
Apart from its role in endodontic biofilm formation, the other difficulty with E. faecalis is its resistance to Ca(OH) 2 -based medicaments [13] used as an intersession remedy. In addition to this, a number of researchers also devoted their research to find out best sealer against this pathogen; Al Shwaimi et al., [16] in their systemic review, reported that there is no such a canal sealer that possesses satisfactory antimicrobial effect towards E. faecalis.
Knowing all disadvantages that follow E. faecalis, it's easy to understand why it is important to find something that will effectively control this pathogen, the same time being harmless to human tissues. As the standard antimicrobial agents in endodontic treatment seems to lack in efficacy toward E. faecalis [15,16,17], in addition to the fact that great efficiency of EOs towards many pathogenic oral microorganisms are already well-documented [1,4,18,19,20,21,22,23,24,25,26,27,28], we assumed the EOs might be a good source to search for efficient alternatives.
The aim of this review is to present EOs with the most significant activity against Enterococcus faecalis, to mark their common constituents and discuss their responsibility in the achieved antimicrobial effect.

MATERIAL AND METHODS
Original papers and /or reviews (521 selected bibliographic units) have been collected from the index base Web of Science (WoS) and screened according to pre-selected criteria in order to select appropriate results of their investigation and include them in this review. There were no time span limitations; all scientific manuscripts with pre-set criteria available from 1996 to 2016 were included.
The main key words used in searching procedure were Enterococcus faecalis and essential oil. Criteria for the final selection were as follows: Original papers presenting antimicrobial effect of EOs on E. faecalis with presented chemical composition of tested EOs the use of microdilution method for determination of MIC values the MIC values in accordance to our pre-set criterion: MIC ≤ 200 μL of EO / mL of growing medium (Müeller-Hinton Broth or Tryptone Soya Broth) -MIC values expressed only in μg or μL / mL -ATCC 29212 referent strain and /or clinical isolates of E. faecalis.
Screening also included results of previous investigation of the author of this rewiev [26].

Observation of EOs efficacy towards Enterococcus faecalis
As to the best of our knowledge, 21 EOs in available scientific literature documented their significant antimicrobial effect with regard to our pre-set criterion (MIC ≤ 200 μL / mL) towards E. faecalis (ATCC 29212 and or clinical isolates); the best effect MIC 0.4 μL / mL (approx. 0.26 μg / mL) achieved Satureja horvatii L. EO [18], while the EOs with the lowest antimicrobial efficacy were Rosmarinus officinalis L. and Achilea milefolium L., both with MIC 160.0 μg / mL [26].
During analysis of reported MIC values with desired efficacy and chemical composition of corresponding EOs, first we have observed the differences in susceptibility towards EOs between the reference strain ATCC 29212 and clinical isolates of E. faecalis, so we grouped them in accordance to this; the group that showed the most efficiant EOs towards ACTT 29212 included 11 EOs while the other one included 10 most efficient EOs towards clinical isolates of E. faecalis.
Analysis of the MIC values within the groups revealed that those for ATCC strain of E. faecalis are generally lower, ranging from 0.26 to 156 μg / mL , in comparison to those for clinical isolates, which ranged from 10 to 160 μg / mL . Similar observation reported Jaradat et al. [29] with Thymus bovei EO, which was tested towards clinicaly isolated MRSA Staphylococcus aureus and S. aureus ATCC 25923; the MIC value for the clinical isolate was two times higher. On the other hand, Subbiya at al. [30], reported difference in sensitivity between E. faecalis clinical isolate and the referent ATCC 29212; double concentration of the RC Solve (Prime Dental., Thane, Maharashtra, India) containing orange EO was required for ATCC 292121 in comparison to clinical isolates, implying that clinical isolates are more sensitive than the referent strain. In addition, Lysakowska at al., [31] investigated sensitivity of 21 clinical isolates of endodontic Enterococcus spp., and two referent E. faecalis strains (ATCC 29212 and ATCC 51299) to geranium EO; the MIC values for the referent strains were equal or lower then those for the most clinical isolates (only one proved to be more sensitive).
Such variability in results shows that complete understanding of the efficient MIC values of selected EOs towards E. faecalis should always include simultaneous testing on clinical isolates and referent strains with the use of the same EO of known chemic al composition, so that the outcome of investigation would be less confusing.

EOs constituents
The most common components contributing to the EOs with ≥ 1.0 %, range of their incidence in selected EOs, and a number of oils in which they were found, are presented in Tables 1 and 2.
While comparing presented components, we noticed following 12 that are common in EOs which MIC s were up to 200 μg / mL in testings towars both, the clinical and referent strains: Germacrene D, eugenol, cadinene, t-cadinol and δ-cadinene are components that are present in more than two EOs effective against ATCC, but not in EOs affective against clinical isolates, while camphor, citronellol, β-pinene, αterpineol, thymol, carvacrol, caryophyllene oxide, cis-caryophyllene, terpinene-4ol, bornyl acetate, β-elemene, citronellal and β-bisabolene were present in two or more EOs effective against clinical isolates but not in two or more EOs that are effective against ATCC 29212 strain.

Major constituents, common in the EOs
Comparison of EO constituents presented in Charts 1 and 2., reviled that only two of them, geraniol and 1,8-cineol, contributed with ≥ 10 % to more than one EO (MIC range 0.3-200 μg / mL) that showed efficiency against both, the ATCC and the clinical strains. Bearing in mind the fact that the EOs included in this rewiev proved highly efficient antimicrobial effect towards both, the clinical isolates and the referent strain, it cound be generally implied that all EOs containing geraniol and 1,8-cineol in amounts ≥ 10 % would probably achieve antimicrobial effect, on both E. faecalis.
Geraniol is an acyclic monoterpene alcohol [32]. According to previous reports geraniol exhibit high antimicrobial activities against various Gram-positive and Gram-negative bacteria and Candida spp. [25,33,34]. Deeper analysis of its content in EOs that we investigated, suggests that it is generally higher in EOs that are more effective against ATCC strains, with following content trend 79.9 % > 21,8 % > 7,2 %; it is maximal in the most effective oil that contains it (MIC 63 g / mL) and decreases as the EOs efficacy decrease (MIC 125 g / mL) [23], while in the case of EOs with efficiency against clinical isolates, the trend of geraniol content proved to be just the opposite, 3.3 % > 12.1 % > 19.2 %; content of geraniol was the lowest in most effective EO containing it (MIC 10 g / mL) and raised in less effective ones (MIC 130 g / mL) [26]. This may lead to conclusion that geraniol may play important role in antimicrobial activity towards both, clinical and referent E. faecalis strains, but it is obvious that the EOs containing this compound also contain some other compounds that interfere in the achieved activity, either enhancing it or retarding. It is already documented that eugenol /geraniol, geraniol / menthol [35] and geraniol / linalool [36], while in mixtures, interfere to each others activity; they acts synergisticaly towards B. cereus and S. aureus [35], and towards Candida strains [36].

Major constituents specific for the EOs
While comparing the content of EO constituents, we notice that there are several components whose percentage exceeded 10 %, also being specific for only one oil with efficacy towards E. faecalis ATCC or clinicaly isolated.
No correlation was observed between the content of major components presented in the Chart 3, and the MIC values of their corresponding EOs.
Also, no correlation was observed between the content of major components presented in the Chart 4, and the MIC values of their corresponding EOs.
It's quite known that major EO constituents are commonly charged for biological activities of complete EOs, such as intensity of antimicrobial activity [44]. Majority of compounds found in EOs presentd in Charts 3 and 4., belong to either phenoles or alcohols. Monoterpenes with phenolic structures, such as carvacrol, eugenol and thymol, are already known as highly active against many microorganisms [45,46,47], while the alcoholic monoterpenes (menthol, αeudesmol, citronellol, linalool, phenylethyl alcohol) are known to possess rather bactericidal than bacteriostatic activity [46]. Acording to Lakusic et al. [18], phenolic compounds are capable to increase cellular membrane permeability, which appear to be related to the loss of the cellular pH gradient, decreased ATP levels, and loss of the proton motive force, which finally causes cellular death.
Although phenols and alcohols are known by their antimicrobial activity, the other terpenes, for example, oxygenated monoterpenes, such as neral and geranial, may also achieve strong antibacterial effect; according to De Jesus et al., In inestigation of antimicrobial effect of aldehydes, it has been proposed that an aldehyde group conjugated to a carbon to carbon double bond is a highly electronegative arrangement, which may explain the achieved activity [50]; an increase in electronegativity could increase the antibacterial activity [51]. The aldehyde citronellal was tested against the 25 test microorganisms (16 Gramnegative bacteria and nine Gram-positive, among them E. faecalis) and was only active against few investigated bacteria B. subtilis, Clostridium sporogenes, Flavobacterium suaveolens, Micrococcus luteus and Pseudomonas aeruginosa [48]. In our results citronellal content is 36.7 % and it is one of three main componenets in EO Cymbopogon winterianus with MIC 125 μg / mL. Menthone is a ketone, and in our study its content was 11.3 %, in second the best EO with MIC 0.3 μL / mL (approx. 0.28 μg / mL). In the study of Dorman and Deans [48], menthone provided no inhibition growth of E. faecalis.
Pinene is a monoterpenes hydrocarbon, capable to destroy cellular integrity by inhibiting the respiration and ion transport processes, and may also increase the membrane permeability in isolated mitochondria [52]. α-Pinene in our sudy is a component with high content (31.2 % in EO towards clinical isolates of E. faecalis) (Chart 4), but is also a common component in six EOs effective towards clinicaly isolated E. faecalis in range of 1.1 -11.5 %, and two EOs effective agenst E. faecalis ATCC with range of 3.7 -31.2 % (Tables 1. and 2).
Similar situation is with p-cymene; its content, as a major component, is 20 %, in EOos effective against E. faecalis clinical isolates, but is also present in five EOs effective agenst E. faecalis in range 1.2 -20 %, clinicaly isolated and two E. faecalis ATCC in range 2.7 -4.5 % (Tables 1. and 2).

Major constituents, common in the most efficient EOs
Out of 21 highly efficient EOs selected in this study, six proved to be the most efficient (MIC ≤ 30 μg / mL ); three oils in control of E. faecalis ATCC strain (Satureja horvatii, Mentha pulegium and Rosmarinus officinalis) and other three in control of clinical isolates (Leptospermum petersonii, Thymus algeriensis and T. serpyllum). During analysis of their major constituents, certain regularity was noticed.
Thymol is a major component in three out of the six aforementioned most efficient EOs. Comparative observation of the MIC values for the oils that contain thymol (Table 3), reveals the highest thymol content (63 %) in EO with the smallest MIC (Satureja horvatii EO), while the EOs of T. algeriensis and T. serpyllum contained it in even lower amounts, 56 % and 38 %, respectively [18,26]. The observation that the lower thymol content correlates with the higher EO MIC value, points out that tymol might be used as a "marker constituent" for EOs with a solid potential against E. faecalis.
In addition, it is important to sress that thymol was the only component in selected EOs contributing to the oils with more then 30 %, the same time being common in even four out of thirteen EOs with MICs ≤ 100 μg / mL. None of other components contributed with such a high percentage (≥ 30 %) in more than one EO (Table 4).   [53] showed that only thymol, as a single component, showed antimicrobial activity almost 13 times stronger then those of Thymus glabrescens entire oil towards Enterococcus faecium ATCC 19433 (MIC of thymol was 195.2 μg / mL and MIC of Thymus glabrescens oil was 2508.4 μg / mL). However, this does not implicate that every single EO containing thymol in high percentage will be equally successful toward referent strain or clinically isolates of E. faecalis.
Even when it seems quite logical to attribute antibacterial effect of an EO to its one or few active components, it must not be forget that EOs are mixtures of so many different compounds [5,54,55,56,57] and their mutual interactions may be very variable; as the activity is dependent on the adequate number and the content of key bioactive compounds [19], the components may interact to either reduce or enforce it [45,58].
According our investigation, it could be outlined that the most efficiant combination towards E faecalis would probably combine three most common components that exideded 10 %, geraniol and 1,8-cineol, with the most common major oil component, thymol. However, mixtures might be quite tricky, particuarly in case of unexpected antagonism between them, as some of them are already proven in the available literature between thymol and geraniol [35] although in test on other bacterial species (B. cereus, S. aureus and E. coli). Researchers must always keep in mind the fact that the same mixture combination will not be equally efficiant in every bacterial species, and gold standard combination for S. aureus or E. coli must not nessery be efficiant towards E. faecalis [35]. Another question is, if clinical isolates and refferent strains will be equally susceptible to applied EOs or their mixtures, as it was not always the case, at least with oral flora microorganisms [27,28]; will the thymol / geraniol mixture be equally indiferent to both, the referent and the isolated strains of E. faecalis, stays unknown until results of a new study reviel it.

CONCLUSIONS
Complete understanding of the efficient MIC values of selected EOs towards E. faecalis should always include simultaneous testing on clinical isolates and referent strains with the use of the same EO of known chemic al composition, so that the outcome of investigation would be less confusing.
Major common constituents, geraniol and 1,8-cineol, again highlight differences in susceptibility between the referent and clinical strains of E. faecalis, thus confirming necessity to perform comparative testing of EOs on both.
1.8 cineol and geraniol can interfere with other components in EO.
No correlation between percentage of major components specific for one EO effective (MIC 0 -200 μg / mL) on E. faecalis clinical isolate / E. faecalis ATCC and MIC values could be noticed.
Tymol might be used as a "marker constituent" for EOs with a solid potential against E. faecalis.
According our investigation, it could be outlined that the most efficiant combination towards E. faecalis would probably combine two the most common components that exideded 10 %, geraniol and 1,8-cineol, with the most common major oil component, thymol.

AKNOWLEDGEMENTS
The authors are grateful to the Ministry of Education, Science and Technological Development of Serbia for financial support (Grants № 173032 and 41008).