Commercial Essential Oils as Potential Antimicrobials to Treat Skin Diseases

Essential oils are one of the most notorious natural products used for medical purposes. Combined with their popular use in dermatology, their availability, and the development of antimicrobial resistance, commercial essential oils are often an option for therapy. At least 90 essential oils can be identified as being recommended for dermatological use, with at least 1500 combinations. This review explores the fundamental knowledge available on the antimicrobial properties against pathogens responsible for dermatological infections and compares the scientific evidence to what is recommended for use in common layman's literature. Also included is a review of combinations with other essential oils and antimicrobials. The minimum inhibitory concentration dilution method is the preferred means of determining antimicrobial activity. While dermatological skin pathogens such as Staphylococcus aureus have been well studied, other pathogens such as Streptococcus pyogenes, Propionibacterium acnes, Haemophilus influenzae, and Brevibacterium species have been sorely neglected. Combination studies incorporating oil blends, as well as interactions with conventional antimicrobials, have shown that mostly synergy is reported. Very few viral studies of relevance to the skin have been made. Encouragement is made for further research into essential oil combinations with other essential oils, antimicrobials, and carrier oils.


Introduction
The skin is the body's largest mechanical barrier against the external environment and invasion by microorganisms. It is responsible for numerous functions such as heat regulation and protecting the underlying organs and tissue [1,2]. The uppermost epidermal layer is covered by a protective keratinous surface which allows for the removal of microorganisms via sloughing off of keratinocytes and acidic sebaceous secretions. This produces a hostile environment for microorganisms. In addition to these defences, the skin also consists of natural microflora which offers additional protection by competitively inhibiting pathogenic bacterial growth by competing for nutrients and attachment sites and by producing metabolic products that inhibit microbial growth. The skin's natural microflora includes species of Corynebacterium, staphylococci, streptococci, Brevibacterium, and Candida as well as Propionibacterium [3][4][5][6][7][8].
In the event of skin trauma from injuries such as burns, skin thinning, ulcers, scratches, skin defects, trauma, or wounds, the skin's defence may be compromised, allowing for microbial invasion of the epidermis resulting in anything from mild to serious infections of the skin. Common skin infections caused by microorganisms include carbuncles, furuncles, cellulitis, impetigo, boils (Staphylococcus aureus), folliculitis (S. aureus, Pseudomonas aeruginosa), ringworm (Microsporum spp., Epidermophyton spp., and Trichophyton spp.), acne (P. acnes), and foot odour (Brevibacterium spp.) [3,[8][9][10][11]. Environmental exposure, for example, in hospitals where nosocomial infections are prominent and invasive procedures make the patient vulnerable, may also create an opportunity for microbial infection. For example, with the addition of intensive therapy and intravascular cannulae, S. epidermidis can enter the cannula and behave as a pathogen causing bloodborne infections. Noninfective skin diseases such as eczema can also result in pathogenic infections by 2 Evidence-Based Complementary and Alternative Medicine damaging the skin, thus increasing the risk of secondary infection by herpes simplex virus and/or S. aureus [5,8,12].
Skin infections constitute one of the five most common reasons for people to seek medical intervention and are considered the most frequently encountered of all infections. At least six million people worldwide are affected by chronic wounds and up to 17% of clinical visits are a result of bacterial skin infections and these wounds are a frequent diagnosis for hospitalised patients. These are experienced daily and every doctor will probably diagnose at least one case per patient. Furthermore, skin diseases are a major cause of death and morbidity [8,13,14]. The healing rate of chronic wounds is affected by bacterial infections (such as S. aureus, E. coli, and P. aeruginosa), pain, inflammation, and blood flow, and thus infection and inflammation control may assist in accelerating healing [15][16][17].
Topical skin infections typically require topical treatment; however, due to the ability of microbes to evolve and due to the overuse and incorrect prescribing of the current available conventional antimicrobials, there has been emergence of resistance in common skin pathogens such as S. aureus resulting as methicillin-resistant Staphylococcus aureus (MRSA) and other such strains. Treatment has therefore become a challenge and is often not successful [8,18,19]. In some regions of the world, infections are unresponsive to all known antibiotics [20]. This threat has become so severe that simple ulcers now require treatment with systemic antibiotics [21]. A simple cut on the finger or a simple removal of an appendix could result in death by infection. The World Health Organization (WHO) has warned that common infections may be left without a cure as we are headed for a future without antibiotics [22]. Therefore, one of the solutions available is to make use of one of the oldest forms of medicine, natural products, to treat skin infections and wounds [18,23].
Complementary and alternative medicines (CAMs) are used by 60-80% of developing countries as they are one of the most prevalent sources of medicine worldwide [24][25][26][27]. Essential oils are also one of the most popular natural products, with one of their main applications being for their use in dermatology [28][29][30]. In fact, of all CAMs, essential oils are the most popular choice for treating fungal skin infections [13,31]. Their use in dermatology, in the nursing profession, and in hospitals has been growing with great popularity worldwide, especially in the United States and the United Kingdom [1,27,[32][33][34][35]. Furthermore, the aromatherapeutic literature [1,2,26,32,[36][37][38][39][40][41][42][43] identifies numerous essential oils for dermatological use, the majority of which are recommended for infections. This brought forth the question as to the efficacy of commercial essential oils against the pathogens responsible for infections. The aim of this review was to collect and summarise the in vivo, in vitro, and clinical findings of commercial essential oils that have been tested against infectious skin diseases and their pathogens and, in doing so, offer aromatherapists and dermatologists valuable information regarding the effectiveness of essential oils for dermatological infections.
The readily available aromatherapeutic literature has reported over 90 (Table 1) commercial essential oils that may  be used for treating dermatological conditions [1,2,26,32,[36][37][38][39][40][41][42][43]. An overview of the skin related uses can be seen in Figure 1. Essential oils are mostly used for the treatment of infections caused by bacteria, fungi, or viruses (total 62%). This is followed by inflammatory skin conditions (20%) such as dermatitis, eczema, and lupus and then general skin maintenance (18%) such as wrinkles, scars, and scabs, which are the third most common use of essential oils. Other applications include anti-inflammatory and wound healing applications ( Figure 1). Of the 98 essential oils recommended for dermatological use, 88 are endorsed for treating skin infections. Of these, 73 are used for bacterial infections, 49 specifically for acne, 34 for fungal infections, and 16 for viral infections.

Searching Strategy/Selection of Papers.
The aim of the comparative review was to identify the acclaimed dermatological commercial essential oils according to the aromatherapeutic literature and then compare and analyse the available published literature. This will serve as a guideline in selecting appropriate essential oils in treating dermatological infections. The analysed papers were selected from three different electronic databases: PubMed, ScienceDirect, and Scopus, accessed during the period 2014-2016. The filters used included either "essential oils", "volatile oils", or "aromatherapy" or the scientific or common name for each individual essential oil listed in Table 1 and the additional filters "antimicrobial", "antibacterial", "skin", "infection", "dermatology", "acne", "combinations", "fungal infections", "dermatophytes", "Brevibacteria", "odour", "antiviral", "wounds", "dermatitis", "allergy", "toxicity", "sentitisation", or "phototoxicity".

Inclusion Criteria.
In order to effectively understand the possible implications and potential of essential oils, the inclusion criteria were broad, especially with this being the first review to collate this amount of scientific evidence with the aromatherapeutic literature. Inclusion criteria included the following: (i) Type of in vitro studies for bacterial and fungal pathogens by means of the microdilution assay, macrodilution assay, or the agar dilution assay (ii) In vivo studies (iii) Antiviral studies (iv) Case reports (v) Animal studies (vi) All clinical trials 2.3. Exclusion Criteria. Papers or pieces of information were excluded for the following reasons: (i) Lack of accessibility to the publication (ii) If the incorrect in vitro technique (diffusion assays) was employed (iii) Indigenous essential oils with no relevance to commercial oils (iv) If they were in a language not understood by the authors of the review (v) Pathogens studied not relevant to skin disease 2.4. Data Analysis. The two authors (Ané Orchard and Sandy van Vuuren) conducted their own data extraction independently, after which critical analysis was applied. Information was extrapolated and recorded and comments were made. Observations were made and new recommendations were made as to future studies.

Description of Studies.
After the initial database search, 1113 reports were screened. Duplicates were removed, which brought the article count down to 513, after which the abstracts were then read and additional reports removed based on not meeting the inclusion criteria. A final number of 349 articles were read and reviewed. Of these, 143 were in vitro bacterial and fungal studies (individual oil and 45 combinations), two in vivo studies, 15 antiviral studies, 19 clinical trials, and 32 toxicity studies. The process that was followed is summarised in Figure 2.

Experimental Approaches
3.2.1. Chemical Analysis. Essential oils are complex organic (carbon containing) chemical entities, which are generally made up of hundreds of organic chemical compounds in combination that are responsible for the essential oil's many characteristic properties. These characteristics may include medicinal properties, such as anti-inflammatory, healing, or antimicrobial activities, but may also be responsible for negative qualities such as photosensitivity and toxicity [37].
Even with the high quality grade that is strived for in the commercial sector of essential oil production, it must be noted that it is still possible for essential oil quality to display discrepancies, changes in composition, or degradation. The essential oil composition may even vary between the same species [1,44]. This may be due to a host of different factors Essential oils used in dermatology identified using layman's literature (books and information leaflets) available to the public (n = 13) Articles identified via database searching (n = 1113) Articles after duplicates were removed (n = 513) Abstracts read (n = 513) Articles included (n = 349) such as the environment or location that the plants are grown in, the harvest season, which part of the plant was used, the process of extracting the essential oil, light or oxygen exposure, the storage of the oil, and the temperature the oil was exposed to [45][46][47][48][49][50][51].
Gas chromatography in combination with mass spectrometry (GCMS) is the preferred technique for analysis of essential oils [52]. This is a qualitative and quantitative chemical analysis method which allows for the assurance of the essential oil quality through the identification of individual compounds that make up an essential oil [1,45,53]. It has clearly been demonstrated that there is a strong correlation between the chemical composition and antimicrobial activity [51,54,55]. Understanding the chemistry of essential oils is essential for monitoring essential oil composition, which then further allows for a better understanding of the biological properties of essential oils. It is recommended to always include the chemical composition in antimicrobial studies [56].

Antimicrobial Investigations.
Several methods exist that may be employed for antimicrobial analysis, with two of the most popular methods being the diffusion and the dilution methods [56][57][58][59].

Diffusion Method.
There are two types of diffusion assays. Due to the ease of application, the disc diffusion method is one of the most commonly used methods [60]. This is done by applying a known concentration of essential oil onto a sterile filter paper disc. This is then placed onto agar which has previously been inoculated with the microorganism to be tested, or it is spread on the surface. If necessary, the essential oil may also be dissolved in an appropriate solvent. The other diffusion method is the agar diffusion method, where, instead of discs being placed, wells are made in the agar into which the essential oil is instilled. After incubation, antimicrobial activity is then interpreted from the zone of inhibition (measured in millimetres) using the following criteria: weak activity (inhibition zone ≤ 12 mm), moderate activity (12 mm < inhibition zone < 20 mm), and strong activity (inhibition zone ≤ 20 mm) [24,[60][61][62].
Although this used to be a popular method, it is more suitable to antibiotics rather than essential oils as it does not account for the volatile nature of the essential oils. Essential oils also diffuse poorly through an aqueous medium as they are hydrophobic. Thus, the results are less reliable as they are influenced by the ability of the essential oil to diffuse through the agar medium, resulting in variable results, false negatives, or a reduction in antimicrobial activity [24,63]. The results have been found to vary significantly when tested this way and are also influenced by other factors such as disc size, amount of compound applied to the disc, type of agar, and the volume of agar [57,59,[64][65][66][67][68]. It has thus been recommended that results are only considered where the minimum inhibitory concentration (MIC) or cidal concentration values have been established [65].

Dilution
Methods. The dilution assays are reliable, widely accepted, and promising methods for determining an organism's susceptibility to inhibitors. The microdilution method is considered the "gold standard" [64,[68][69][70]. This is a quantitative method that makes it possible to calculate the MIC and allows one to understand the potency of the essential oil [68,71]. With one of the most problematic characteristics of essential oils being their volatility, the microdilution technique allows for an opportunity to work around this problem as it allows for less evaporation due to the essential oil being mixed into the broth [67].
This microdilution method makes use of a 96-well microtitre plate under aseptic conditions where the essential oils (diluted in a solvent to a known concentration) are serially diluted. Results are usually read visually with the aid of an indicator dye. The microdilution results can also be interpreted by reading the optical density [72,73]; however, the shortcoming of this method is that the coloured nature of some oils may interfere with accurate turbidimetric readings [74].
Activity is often classified differently according to the quantitative method followed. van Vuuren [56] recommended 2.00 mg/mL and less for essential oils to be considered as noteworthy, Agarwal et al. [75] regarded 1.00% and less, and Hadad et al. [76] recommended ≤250.00 g/mL. On considering the collection of data and frequency of certain MIC values, this review recommends MIC values of ≤1.00 mg/mL as noteworthy.
The macrodilution method employs a similar method to that of the microdilution method, except that, instead of a 96well microtitre plate being used, multiple individual test tubes are used. Although the results are still comparable, this is a time-consuming and a tedious method, whereas the 96-well microtitre plate allows for multiple samples to be tested per plate, allowing for speed, and it makes use of smaller volumes which adds to the ease of its application [77,78]. The agar dilution method is where the essential oil is serially diluted, using a solvent, into a known amount of sterile molten agar in bottles or tubes and mixed with the aid of a solvent. The inoculum is then added and then the agar is poured into plates for each dilution and then incubated. The absence of growth after incubation is taken as the MIC [79][80][81].

The Time-Kill Method.
The time-kill (or death kinetic) method is a labour intensive assay used to determine the relationship between the concentration of the antimicrobial and the bactericidal activity [82]. It allows for the presentation of a direct relationship in exposure of the pathogen to the antimicrobial and allows for the monitoring of a cidal effect over time [74]. The selected pathogen is exposed to the antimicrobial agent at selected time intervals and aliquots are then sampled and serially diluted. These dilutions are then plated out onto agar and incubated at the required incubation conditions for the pathogen. After incubation, the colony forming units (CFU) are counted. These results are interpreted from a logarithmic plot of the amount of remaining viable cells against time [74,82,83]. This is a timeconsuming method; however, it is very useful for deriving real-time exposure data.

Summary of Methods.
The variation in essential oil test methods makes it difficult to directly compare results [24,58]. Numerous studies were found to employ the use of a diffusion method due to its acclaimed "ease" and "time saving" ability of the application. Researchers tend to use this as a screening tool whereby results displaying interesting outcomes are further tested using the microdilution method [84][85][86][87]. The shortcoming of this method is that firstly, due to the discussed factors affecting the diffusion methods, certain essential oils demonstrate no inhibition against the pathogen, and thus further studies with the oils are overlooked. Secondly, the active oils are then investigated further using the microdilution method. Therefore, the researchers have now doubled the amount of time required to interpret the quantitative data. Thirdly, the method may be believed to be a faster method if one considers the application; however, if one considers the preparation of the agar plates and their risk of contamination as well as the overall process of this method, there is very little saving of time and effort.
It is recommended to follow the correct guidelines as set out by the Clinical and Laboratory Standards Institute M38-A (CLSI) protocol [88] and the standard method proposed by the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing (AFST-EUCAST) [89] for testing with bacteria and filamentous fungi.
Other factors that may affect results and thus make it difficult to compare published pharmacological results of essential oils are where data is not given on the chemical composition, the microbial strain number, temperature and length of incubation, inoculum size, and the solvent used. The use of appropriate solvents helps address the factor of poor solubility of essential oils. Examples include Tween, acetone, dimethylformamide (DMF), dimethylsulfoxide (DMSO), and ethanol. Tween, ethanol, and DMSO have, however, been shown to enhance antimicrobial activity of essential oils [24,53,90]. Soković et al. [91] tested antimicrobial activity with ethanol as the solvent and Tween. When the essential oils were diluted with Tween, it resulted in a greater antifungal activity; however, Tween itself does not display its own antimicrobial activity [92]. Eloff [93] identified acetone as the most favourable solvent for natural product antimicrobial studies.
The inoculum is a representative of the microorganisms present at the site of infection [94]. When comparing different articles, the bacterial inoculum load ranges from 5 × 10 2 to 5 × 10 8 CFU/mL. The antibacterial activity is affected by inoculum size [62,[95][96][97][98][99]. If this concentration is too weak, the effect of the essential oils strengthens; however, this does not allow for a good representation of the essential oil's activity. If the inoculum is too dense, the effect of the essential oil weakens and the inoculum becomes more prone to cross contamination [100]. Future studies should aim to keep the inoculum size at the recommended 5 × 10 6 CFU/mL [99].

Pathogenesis of Wounds and Skin Infections and the Use of Essential Oils
The pathogenesis of the different infections that are frequently encountered in wounds and skin infections is presented in Table 2. A more in-depth analysis of essential oils and their use against these dermatological pathogens follows.

Gram-Positive
Bacteria. The Gram-positive bacterial cell wall is comprised of a 90-95% peptidoglycan layer that allows for easy penetration of lipophilic molecules into the cells. This thick lipophilic cell wall also results in essential oils making direct contact with the phospholipid bilayer of the cell membrane which allows for a physiological response to occur on the cell wall and in the cytoplasm [183,184].

Staphylococcus aureus.
Staphylococcus aureus is a common Gram-positive bacterium that can cause anything from local skin infections to fatal deep tissue infections. The pathogen is also found colonising acne and burn wounds [185][186][187]. Methicillin-resistant S. aureus (MRSA) is one of the most well-known and widespread "superbugs" and is resistant to numerous antibiotics [158]. Methicillin-resistant S. aureus strains can be found to colonise the skin and wounds of over 63%-90% of patients and have been especially infamous as being the dreaded scourge of hospitals for several years [22,[188][189][190]. Staphylococcus aureus has developed resistance against erythromycin, quinolones, mupirocin, tetracycline, and vancomycin [190][191][192]. Table 3 shows some of the antimicrobial in vitro studies undertaken on commercial essential oils and additional subtypes against this most notorious infectious agent of wounds. Of the 98 available commercial essential oils documented from the aromatherapeutic literature for use for dermatological infections, only 54 oils have been tested against S. aureus and even fewer against the resistant S. aureus strain. This is troubling, especially if one considers the regularity of            When considering the antimicrobial activity of the tested essential oils, it can be noted how the main compounds influence overall antimicrobial activity. Melaleuca alternifolia (tea tree), rich in terpinen-4-ol, showed noteworthy activity, and Anthemis aciphylla var. discoidea (chamomile) containing -pinene and terpinen-4-ol displayed noteworthy activity (1.00 mg/mL) [114], whereas the essential oil predominantly containing terpinen-4-ol displayed an MIC value of 0.50 mg/mL. The Origanum spp. (Origanum scabrum and Origanum vulgare) were shown to display rather impressive antimicrobial activity, which appeared to predominantly be related to the amount of carvacrol [163]. Geraniol also appears to be a compound that influences antimicrobial activity against the staphylococci spp. as can be seen for Backhousia citriodora (lemon myrtle) and Cymbopogon martinii (palmarosa) (geraniol 61.6%) [125,126]. Cymbopogon martinii, with lower levels of geranial (44.80%), showed moderate antimicrobial activity [99]. Mentha piperita (peppermint) had higher antimicrobial activity for oils with higher concentrations of menthol [128,155]. Laurus nobilis (bay), Styrax benzoin, and Cinnamomum zeylanicum (cinnamon), each rich in eugenol, were found to have notable activity [99].
Some clinical studies included the evaluation of the effects of essential oils on malodorous necrotic ulcers of cancer patients. The use of an essential oil combination (mostly containing Eucalyptus globulus (eucalyptus)) resulted in a decrease in inflammation, reduction of the odour, and improved healing rates [193]. Edwards-Jones et al. [194] performed a clinical study with a wound dressing containing essential oils to decrease infection risk. Ames [195] found Melaleuca alternifolia (tea tree) to be effective in treating wounds; and Matricaria recutita (German chamomile) with L. angustifolia at a 50 : 50 ratio diluted in calendula oil was found to improve leg ulcers and pressure sores.
Methicillin-resistant S. aureus hinders the rate of wound healing, which may lead to chronic wounds [196]. Delayed wound healing has been proven to lead to psychological stress and social isolation [197,198]. A randomised controlled trial, consisting of 32 patients (16 in control group, 16 in placebo group) with stage II and above MRSA-colonised wounds that were not responding to treatment, was undertaken where the control group was treated with a 10% topical M. alternifolia preparation and was found to effectively decrease colonising MRSA in 87.5% of patients and result in a 100% healing rate within 28 days [196]. These studies lead to the high recommendation of the incorporation of this essential oil combination in palliative care.
Methicillin-resistant S. aureus may potentially be carried and propagated by hospital staff and patients, which is an acknowledged risk for hospital-acquired infections [147,189]. Therefore, successful decolonisation of MRSA from patients and good hygiene may improve the microbial load, number of reinfections, and ultimately therapeutic outcomes of patients [199]. A topical preparation containing M. alternifolia essential oil has been considered for assistance in eradicating MRSA in hospitals, due to its reported efficacy [200]. The largest randomised trial against MRSA colonisation included 224 patients where the control group was treated with 2% nasal mupirocin applied three times a day, 4% chlorhexidine gluconate soap used at least once a day, and 1% silver sulfadiazine cream applied to skin infections once a day. The study group was treated with 10% M. alternifolia oil nasal cream applied three times a day and 5% M. alternifolia oil body wash used at least once daily with a 10% M. alternifolia cream applied to skin infections. The results showed that 41% of patients in the study group were cleared as opposed to 49% of patients on the standard therapy [200]. A small threeday pilot study was designed by Caelli et al. [189] to observe whether daily washing with a 5% M. alternifolia oil would clear MRSA colonisation which may result in ICU patient outcome improvement [199]. The test group made use of 4% M. alternifolia nasal ointment and 5% M. alternifolia oil body wash and was compared to a conventional treatment consisting of 2% mupirocin nasal ointment and triclosan body wash. The test group overall was found to have more improvement at the infection site when compared to the control group. Although the pilot study was too small to be statistically significant, the researchers did find that the M. alternifolia oil performed better than the conventional treatment and was effective, nontoxic, and well tolerated [189]. Messager et al. [90] tested 5% M. alternifolia ex vivo in a formulation, where it again was proven to decrease the pathogenic bacteria on the skin. In another study, M. alternifolia oil was investigated to determine the influence on healing rates [201]. The patients were treated with water-miscible tea tree oil (3.30%) solution applied as part of the wound cleansing regimen. This study used this oil as a wash only three times a week which is not how this oil is prescribed and hence the results were not positive. A more accurate method of study was shown by Chin and Cordell [202], where M. alternifolia oil was used in a dressing for wound healing abilities. All patients, except for one, were found to have an accelerated healing rate of abscessed wounds and cellulitis. The concluding evidence shows that there is definitely potential for the use of M. alternifolia (tea tree) oil as an additional/alternative treatment to standard wound treatments [203].
The healing potential of Commiphora guidotti (myrrh) was investigated via excisions of rats. The authors could confidently report on an increased rate in wound contraction and candid wound healing activity that was attributed to the antimicrobial and anti-inflammatory effects of this oil [204]. Ocimum gratissimum (basil) was also found by Orafidiya et al. [205] to promote wound healing by eradicating the infectious pathogens and by inducing early epithelialisation Evidence-Based Complementary and Alternative Medicine 25 and moderate clotting formation, thereby accelerating scab formation, contraction, and granulation.
From these studies, clearly, M. alternifolia has shown great promise against S. aureus. However, considering the potential of essential oils in clinical practice and comparing them to essential oils with promising in vitro activity, other oils such as Cymbopogon citratus (lemongrass), Santalum album (sandalwood), and Vetiveria zizanioides/Andropogon muricatus (vetiver) should in the future be paid the same amount of attention.

Pathogens Involved in Acne.
Pathogens associated with acne include Propionibacterium acnes, Propionibacterium granulosum, and Staphylococcus epidermidis [206][207][208]. Methicillin-resistant S. epidermidis (MRSE) have become extensively problematic microorganisms in the recent years due to their antimicrobial resistance and P. acnes has developed resistance to tetracycline, erythromycin, and clindamycin. Both have also shown multidrug resistance, including against quinolones [158,188,206]. Table 4 displays the in vitro antimicrobial efficacies of commercial essential oils against bacteria involved in the pathogenesis of acne. When observing the number of commercial essential oils that are recommended for acne treatment, less than half of the commercial oils have actually focused on S. epidermidis, P. granulosum, and P. acnes. Overall, the acne pathogens have been sorely neglected in essential oil studies.
For Anthemis aciphylla var. discoidea (chamomile) 0.13-0.25 mg/mL, initially, it appeared that higher -pinene and lower terpinen-4-ol showed higher antimicrobial activity. However, the sample with terpinen-4-ol predominantly as its main component displayed the best activity at 0.06 mg/mL. This makes -pinene appear as an antimicrobial antagonist. Cinnamomum zeylanicum, Rosa centifolia (rose), L. angustifolia, and Syzygium aromaticum (clove) displayed noteworthy antimicrobial activity against both S. epidermidis and P. acnes. Only the latter two are, however, recommended in the aromatherapeutic literature for the treatment of acne. Leptospermum scoparium (manuka) showed noteworthy activity for both P. acnes and S. epidermidis; however, Tween 80 was used as a solvent, which may overexaggerate the antimicrobial activity. Another study also found L. scoparium to effectively inhibit P. acnes. As was seen against S. aureus, O. scabrum and O. vulgare also notably inhibited S. epidermidis. Unfortunately, these oils were not studied against P. acnes. Cymbopogon citratus was shown to effectively inhibit P. acnes; however, no data was available against S. epidermidis. Essential oils such as S. album, V. zizanioides, Viola odorata (violet), Citrus aurantium var. amara (petitgrain), and Citrus bergamia (bergamot) are a few that are recommended for the treatment of acne and other microbial infections [2,26,32,36,37,[40][41][42][43] in the aromatherapeutic literature that are yet to be investigated.
Some clinical studies have shown promising results. A four-week trial comparing O. gratissimum oil with 10% benzoyl peroxide and a placebo was conducted and was aimed at reducing acne lesions in students. The 2% and 5% O. gratissimum oils in the hydrophilic cetomacrogol base were found to reduce acne lesions faster than standard therapy, and they were well tolerated. The 5% preparation, despite being highly effective, caused skin irritation. Overall, O. gratissimum oil showed excellent potential in the management of acne as it was as effective as benzoyl peroxide, although it was less popular with patients due to the unpleasant odour [217].
Melaleuca alternifolia oil demonstrated in vitro antimicrobial and anti-inflammatory activity against P. acnes and S. epidermidis and is in fact the essential oil on which most clinical trials have been undertaken. Bassett et al. [218] performed one of the first rigorous single-blinded randomised (RCT) controlled trials consisting of 124 patients that assessed the efficacy of 5% M. alternifolia gel in comparison to 5% benzoyl peroxide lotion in the management of mild to moderate acne. Both treatments showed equal improvement in the acne lesions. Enshaieh et al. [219] evaluated the efficacy of 5% M. alternifolia on mild to moderate acne vulgaris. The 5% M. alternifolia oil was found to be effective in improving the number of papules in both inflammatory and noninflammatory acne lesions and was found to be more effective than the placebo. Proven efficacy has made M. alternifolia preparations popular in acne products.
Other oil studies included a gel formulation containing acetic acid, Citrus sinensis (orange), and Ocimum basilicum (sweet basil) essential oils, which was tested in acne patients. The combination of these antimicrobial essential oils and the keratolytic agent resulted in a 75% improvement in the rate of acne lesion healing [220].
If one examines the results displayed in Table 4, essential oils such as Anthemis aciphylla var. discoidea (chamomile), C. zeylanicum, Citrus aurantium (bitter orange), O. vulgare (oregano), and S. aromaticum displayed higher antimicrobial activity in vitro than M. alternifolia, yet these essential oils have to be investigated clinically.

Gram-Negative
Bacteria. The Gram-negative bacterial cell wall consists of a 2-3 nm thick peptidoglycan layer (thinner than Gram-positive bacteria), which means that the cell wall consists of a very small percentage of the bacteria. The cell wall is further surrounded by an outer membrane (OM) which is comprised of a double layer of phospholipids that are linked to an inner membrane by lipopolysaccharides (LPS). This OM protects the bacteria from lipophilic particles; however, it makes them more vulnerable to hydrophilic solutes due to the abundance of porin proteins that serve as hydrophilic transmembrane channels [184,221,222].
Gram-negative pathogens present a serious threat with regard to drug resistance, especially Escherichia coli and Pseudomonas aeruginosa [190,192]. These pathogens that are found to colonise wounds often cause multidrug resistance [166,223]. -Lactamase-positive E. coli is appearing frequently among nonhospital patients [224]. Pseudomonas aeruginosa is a regular cause of opportunistic nosocomial infections [187]. It is often involved in localised skin infections, green nail syndrome, and interdigital infection, colonises burn wounds, and may expand into a lifethreatening systemic illness [225].
A number of essential oils display antimicrobial activity against E. coli and P. aeruginosa with the predominant studies       (Table 5). The Gram-negative pathogens appear to be a lot more resistant to essential oil inhibition than the Gram-positive bacteria, but this a known fact. Aniba rosaeodora (rosewood) was found to inhibit E. coli at an MIC value of 0.40 mg/mL. No GC-MS data was given [85]. Anthemis aciphylla var. discoidea (chamomile) also displayed notable inhibition against E. coli and P. aeruginosa; however, the highest activity was seen for the essential oil containing high levels of -pinene (39.00%) and terpinen-4ol (32.10%) [114]. Cinnamomum zeylanicum, with the main compound cinnamaldehyde, was shown to have inhibited these two Gram-negative pathogens at noteworthy MIC values [80]. Noteworthy activity was also reported for Commiphora myrrha (myrrh) and Thymus numidicus (thyme) [99]. Syzygium aromaticum and S. album were reported to effectively inhibit P. aeruginosa [99]; and Thymus vulgaris (thyme) inhibits E. coli (including multidrug-resistant strains) [182].

Other Bacterial
Pathogens. Brevibacterium spp. form part of the Coryneform bacteria and are involved in foul body odour [3,103]. Insufficient quantitative studies have been conducted using commercial essential oils to treat problems caused by these microorganisms, even though there have been some earlier studies using the diffusion assays against B. linen [226][227][228]. One quantitative study reported on the activity of Ziziphora persica against B. agri (125 g/mL) and B. brevis (250 g/mL), in addition to Ziziphora clinopodioides against B. agri (31.25 g/mL) and B. brevis (125 g/mL) [229]. In another study, essential oils of Kunzea ericoides (Kānuka) and L. scoparium were able to inhibit three species of Brevibacterium (MIC: 0.06-1.00 mg/mL) [138]. Clearly, the lack of attention to this neglected group of microorganisms warrants further attention, especially considering that, to the best of our knowledge, not one essential oil recommended for odour has been investigated against relevant pathogens in vitro.
The -hemolytic Streptococcus (S. pyogenes) is a threatening pathogen that needs to be considered when investigating wound infections [166]. Group A Streptococcus (GAS) is usually involved in impetigo and necrotising fasciitis ("flesheating" disease). This pathogen has developed resistance to erythromycin, azithromycin, clarithromycin, clindamycin, and tetracycline [188,190]. Group B Streptococcus is also involved in skin infections and has developed resistance to clindamycin, erythromycin, azithromycin, and vancomycin [190]. Periorbital cellulitis is a common occurrence in children and is caused by Haemophilus influenzae [106], and Clostridium spp. (C. perfringens, C. septicum, C. tertium, C. oedematiens, and C. histolyticum) are involved in gas green/gangrene infections. Table 6 summarises the antimicrobial activity of essential oils that have been studied and shown to have some in vitro efficacy against these pathogens. The lack of studies against S. pyogenes, C. perfringens, and H. influenza highlights the need to investigate these sorely neglected dermatologically important pathogens, especially since the few available studies have shown these organisms to be highly susceptible to essential oil inhibition. These are also pathogens that cause deeper skin infections, so, with the enhanced penetration offered by essential oils, they may prove beneficial.

Fungal Infections: Yeasts.
Yeasts may act as opportunistic pathogens and can result in infection if presented with the opportunity, the most common pathogen being Candida albicans. Candida spp. can cause candidiasis at several different anatomical sites [230]. Candida has started developing resistance to first-line and second-line antifungal treatment agents such as fluconazole [190]. Essential oils demonstrating noteworthy activity against this organism are shown in Table 7. Candida albicans has been quite extensively investigated and most oils used in dermatology have been tested against this pathogen.
In an in vivo study, L. angustifolia was found to effectively inhibit growth of C. albicans isolated from 20 patients, which was comparative to the inhibition observed by clotrimazole [272].

Fungal Infections: Dermatophytes.
Infection with these organisms results in dermatophytosis, which affects the skin, nails, or hair [230,273,274]. There is a 10-20% risk of a person acquiring a dermatophyte infection [29], and although the symptoms do not necessarily pose a threat, the treatment is costly and onerous due to resistance and side effects [29]. Essential oils present an excellent option for treating superficial human fungal infections, especially when one is confronted with the effective antifungal results found in previous studies (Table 8). This is encouraging considering the difficulty and challenges faced in treating these infections.
The ability of topical formulations to penetrate the skin is crucial for the effective treatment of subcutaneous infections [108]. Melaleuca alternifolia oil has displayed in vitro activity against M. mycetomatis and M. furfur, proving its potential in treating eumycetoma, pityriasis, and seborrheic dermatitis, not only because of its antifungal activity, but also because of its ability to penetrate the skin due to its main compound (terpinen-4-ol) [108,109,275,276].
Onychomycosis is generally resilient to topical treatment of any kind; thus, there is a poor cure rate. It is usually treated systemically due to its infrequency in responding to topical treatments [277,278]. With onychomycosis being the most frequent cause of nail disease, Buck et al. [279] aimed to treat onychomycosis in clinical trials whereby 60% of patients were treated with M. alternifolia oil and 61% of patients were treated with 1% clotrimazole. There was only a 1% difference between the two study groups. What would be interesting for 32 Evidence-Based Complementary and Alternative Medicine

58
Evidence-Based Complementary and Alternative Medicine   [125,126] Evidence-Based Complementary and Alternative Medicine 59     M. sympodialis (10 clinical isolates)     future studies is to determine what the results would be when testing the same treatments against resistant strains. Tinea pedis is often treated topically, which presents an opportunity for essential oil use [280]. Melaleuca alternifolia oil was evaluated in two trials for treating tinea pedis. In the first trial by Tong et al. [281], the patients were treated with either a 10% M. alternifolia oil in sorbolene, 1% tolnaftate, or a placebo (sorbolene). The patients on M. alternifolia oil treatment had a mycological cure rate of 30%. Mycological cure rates of 21% were seen in the placebo group and of 85% in patients receiving tolnaftate, proving the essential oil to not be as effective. The second trial tested two solutions of 25% and 50% M. alternifolia oil in ethanol and polyethylene glycol. This was compared to a placebo containing only the vehicle in a double-blinded randomised controlled trial [282]. The placebo group showed a clinical response in 39% of patients. Melaleuca alternifolia oil test groups showed a 72% improvement. A higher concentration of the oil is thus required for treating this type of infection.
In spite of the dermatophytes showing susceptibility to essential oils, there are few studies dedicated to these pathogens. One would expect more essential oil treatments considering the difficulty in treating these infections which require expensive prolonged treatment. An essential oil with superior activity certainly warrants further investigation, particularly as essential oils work well on skin surfaces and are shown to display good penetration capabilities [283,284]. Madurella mycetomatis and Malassezia furfur are sorely neglected pathogens in research. Possibly their fastidious nature acts as a barrier for further research. As far as clinical studies are concerned, essential oils against fungal pathogens have also been neglected. Only M. alternifolia oil has been clinically studied extensively with investigations incorporating onychomycosis, tinea pedis, and dandruff [275,279,281,282,285]. It would be interesting to observe the antidermatophytic property of essential oils that have shown to be noteworthy in vitro antifungal activity such as for Apium nodiflorum (celery), Cedrus atlantica (cedar wood), C. citratus, Juniperus oxycedrus ssp. oxycedrus (cade), Pelargonium graveolens (geranium), S. aromaticum, and Thymus spp.

Essential Oil Combinations
Other than the use of oils within carrier oils, most essential oils are used in blends or combinations of two or more oils [32]. These blends are considered to be an art where the oils are carefully selected and combined with the intention of holistically healing the "whole" individual according to his/her symptoms. The goal of blending is to create a synergistic therapeutic effect where the combination of essential oils is greater than the sum of the individual oil [37,40,286]. The beneficial value of synergy has been notorious and used since antiquity [74]. Synergy can be achieved if the compounds in the oil are able to affect different target sites, or they may interact with one another to increase solubility thereby enhancing bioavailability [287][288][289]. Mechanisms that can lead to pharmacological synergy are (1) multitarget effect where multiple target sites of the bacterial cell are affected; (2) solubility and bioavailability enhancement; (3) the mechanism where the essential oil may inhibit the mutation mechanism of bacteria to the antimicrobial; or (4) the mechanism where the essential oil may inhibit the efflux pump of bacteria, thus allowing for the antimicrobial to accumulate inside the bacteria [11,288,290]. The goal is for a multitargeted treatment to decrease pathogen mutation and thus retard the development of resistance. The combined formulation also has the potential to decrease toxicity and adverse side effects by lowering the required dose [290][291][292]. This is not an infallible method, however, as even the combined penicillin with clavulanic acid has become prone to resistance [293,294].
When blends are created, the intention is to create therapeutic synergy [2,26,32]. The reasoning for the combinations is to produce a forceful blend that has more than one mode of action. For example, in the treatment of abscesses, C. bergamia and L. angustifolia may be used in combination. C. bergamia is used for its antiseptic properties and L. angustifolia for antiseptic and anti-inflammatory effects. Anthemis nobilis is also often used for anti-inflammatory effects [2,26,32,37]. The theory is sound and not too far off considering that numerous essential oils have been proven to possess additional pharmacological properties. For example, P. graveolens is known for antiseptic and antiinflammatory properties. It is often used for the ability to balance sebum secretions and clear oily and sluggish skin [295]. Eucalyptus globulus (eucalyptus) may be used for its proven antimicrobial and anti-inflammatory activity [296,297]. Often used on acne prone skin because of its antiseptic properties is L. angustifolia [298,299]. Anthemis nobilis is believed to ease inflammation and L. angustifolia assists with healing and regeneration [25]. Citrus aurantium (neroli) flower oil has displayed antioxidant activity [120], and the main component of M. alternifolia (terpinen-4-ol) has the ability to hinder tumour necrosis factor (TNF), interleukin-1, interleukin-8, and interleukin-10, and prostaglandin E 2 [300]. The anti-inflammatory activity of C. bergamia has been proven by several studies in vitro or on animal models [301,302]. This supports the theory behind therapeutic synergy; however, the mistaken belief that any essential oil blend will result in synergy is not fully accurate [33]. It is a complex area, because although a certain combination may have a synergistic therapeutic effect, it does not necessarily translate into antimicrobial synergy and this needs further investigation.
By reviewing the aromatherapeutic literature [1,2,26,32,[36][37][38][39][40][41][42][43], at least 1500 possible combinations (made up of two oils) could be identified for dermatology alone. This brings forth the question as to the antimicrobial effect of the overall combination. After all, if essential oils are to be investigated as options to curb antimicrobial resistance, the aim of combination therapy should be to broaden the spectrum of the antimicrobial activity and prevent development of additional resistance occurring [96]. The risk of resistance emerging against essential oils should not be disregarded because suboptimal doses of essential oils may impact these phenomena [303]. Sublethal concentration exposure to M. alternifolia has been proven to result in slightly lowered bacterial susceptibility to M. alternifolia and a larger decrease in susceptibility to conventional antimicrobials. The study concluded that essential oil products containing sublethal concentrations may result in stress-hardened (mutated) S. aureus isolates and possible treatment failure [146]. This highlights that although therapeutic synergy is strived for, these must still be verified in a controlled environment [288].
Studies have proven that essential oils, whether in combination with other essential oils [99] or in combination with conventional antimicrobials [304], can initiate a synergistic antimicrobial effect. This effect, however, is limited to the studied pathogen [290]. de Rapper et al. [99] demonstrated that even when essential oils displayed synergistic blends against one pathogen, the same could not be said against other pathogens. This highlights how the assumption should not be made that all synergistic blends are the same against all pathogens.
The fractional inhibitory concentration index (∑ FIC or FICI) is the commonly accepted mathematical method employed to interpret interactions in 1 : 1 combinations [74]. ∑ FIC is determined from the sum of all individual FICs of each of the test agents within the combination [305]. This then allows for the determination of their individual interactions in the combination [306]. The results are interpreted as synergistic (∑ FIC ≤ 0.5), additive (∑ FIC > 0.5-1.0), indifferent ∑ FIC (>1.0 ≤ 4.0), or antagonistic (∑ FIC > 4.0) [74]. Although using ∑ FIC calculations is an easy method, it is not without its limitation. When examining 1 : 1 ratios between two essential oils, it is assumed that half the concentration will only offer half the effect. This is not necessarily the case between agents, as two agents may not have the same dose response at the same concentrations [307]. An interactive assessment of the different ratio combinations is mostly carried out using the isobole method [308,309]. This method allows for more accurate valuation of the combination contribution made by each agent on a mathematical level line where all points are collected on a surface that lies at a specific value [288,305,310]. There are, however, other complex methods that can also be used [311,312].

Essential Oils in Combination with Other Essential Oils.
Although combinations are frequently mentioned in aromatherapy to treat skin ailments, only a handful of studies documenting essential oil combinations were found against skin pathogens ( Table 9). The combination studies are predominantly limited to S. aureus, P. aeruginosa, C. albicans, and, to a lesser extent, E. coli. Even fewer studies were found against the dermatophytes and acne pathogens. This is rather abysmal considering the amount of combinations and the regularity of their use. An interesting observation was made even in an early study [316], where it was shown that synergy found in the 1 : 1 combinations was apparent irrespective of the poor efficacy displayed by the individual oils. This indicates that essential oils do not necessarily have to be combined based purely on independent noteworthy antimicrobial activity.
One of the largest studies on combinations was done by de Rapper et al. [99], where 45 essential oils were combined with L. angustifolia, which is one of the most popular essential oils used in combination. What could be observed was that there was no predictive pattern as to what the combined FIC index would be. There were a few synergistic interactions, most of which against C. albicans and some antagonism; however, the majority of the combinations resulted in an indifferent or additive interaction. A study investigated the antimicrobial activity of the popular commercial product containing essential oils (Olbas). The individual essential oils were tested separately and then in the combined product [128]. The combination of the four oils showed no further enhancement in the antimicrobial. The combination of Syzygium aromaticum (clove) and Rosmarinus officinalis (rosemary) has also displayed synergy against C. albicans, at ratios of 1 : 5, 1 : 7, and 1 : 9 [169]. Synergy was observed with a combination of commercially popular L. angustifolia and M. alternifolia essential oils against dermatophytes T. rubrum and T. mentagrophytes var. interdigitale in various combinations [303]. Unfortunately, only a few essential oil combinations have been investigated in clinical settings.
Essential oil combinations have proven efficacy in clinical settings. L. angustifolia and Matricaria recutita (German chamomile) were investigated in a small trial involving eight patients with chronic leg ulcers. Five received a 6% mixture of the two essential oils mixed in Vitis vinifera (grape seed) carrier oil, and three received conventional wound care. It was noted that four of the five patients in the control group had complete healing of the wounds with the fifth patient making progress towards a recovery [317]. Another successful essential oil combination included L. angustifolia, Artemisia vulgaris (mugwort), and Salvia officinalis (sage) in treating chronic wounds such as venous ulcers, pressure sores, skin tears, and abrasions. It was speculated that the essential oils had increased circulation and vascular permeability resulting in accelerated angiogenesis [318]. An in vivo study by Mugnaini et al. [266] made use of a mixture composed of 5% O. vulgare, 5% R. officinalis, and 2% Thymus serpyllum (Breckland thyme), diluted in Prunus dulcis (sweet almond), and this was topically administered on M. canis lesions. A 71% success rate in treatment was observed.

Essential Oils in Combination with Conventional Antimicrobials.
In an effort to prevent resistance and increase antimicrobial efficacy against multidrug-resistant bacteria, the combination of essential oils with antibiotics has been investigated [182,[319][320][321]. Certain studies are based on the assumption that the antimicrobial and essential oils attack at different sites of the pathogen [304], while others believe this is due to the increase in chemical complexity, together with the added advantage of enhanced skin penetration by the essential oil components [322], or the hope that the essential oils will improve antibiotic diffusion across the bactericidal cell membranes and/or inhibit the Gram-negative efflux pump [323]. Conventional medication in combination with essential oils (bought over the counter or shelves) is also common among patients [183]; therefore, unknowingly, they may be causing enhancement or failure. Table 10 displays the studies validating the improvement of antimicrobial activity from the combined use of antimicrobials with essential oils. The majority of the studies have         shown essential oils to enhance antimicrobial activity of antibiotics and antifungals [81,346,347]. Origanum vulgare oil displayed synergy (FICs 0.4-0.5) when combined with doxycycline, florfenicol, or sarafloxacin against an ESBL producing E. coli [324]. This presents a possible solution for -lactamase antibiotic-resistant bacteria. Origanum vulgare essential oils were investigated and shown to improve the activity of -lactam antibiotics against both Grampositive and Gram-negative -lactamase-producing bacteria [77,324]. Helichrysum italicum (everlasting) (2.5%) reduced the multidrug resistance of Gram-negative bacteria, E. coli and P. aeruginosa, to chloramphenicol [320].
Four community-associated methicillin-resistant S. aureus (CA-MRSA) isolates were used to compare benzethonium chloride 0.2% with M. alternifolia and T. vulgaris combination with conventional antimicrobials (neomycin with polymyxin B sulphate and polymyxin B sulphate with gramicidin). The essential oil-antibiotic combination was found to be more effective than conventional medicines on their own [348]. In another study, however, where M. piperita, M. alternifolia, T. vulgaris, and R. officinalis were each individually combined with amphotericin B against C. albicans, antagonism was observed [304], indicating that there may still be risks present when combining essential oils with antimicrobials. Cinnamomum cassia showed potentiation of amphotericin B activity against C. albicans. The increased activity was attributed to the essential oil because synergy increased with an increase in essential oil concentration; however, antagonism was observed for combinations with a lower concentration of essential oil [238].
Although there have been some studies in vitro on essential oil combinations with antibiotics and antifungals, little attention has been paid to in vivo studies or clinical trials. Syed et al. [285] tested a 2% butenafine hydrochloride combination with a 5% M. alternifolia oil cream in a clinical trial, consisting of 60 patients, treating toenail onychomycosis. The control group showed an 80% cure rate compared to 0% by the placebo group containing M. alternifolia alone, allowing the study to conclude clinical effectiveness of butenafine hydrochloride and M. alternifolia in combination. However, in order to determine whether the same could be said for butenafine, a control group should have also been allowed for this product to allow for comparison.

Antiviral Studies
Viral infections are a worldwide threat, firstly due to the lack of effective treatments available and secondly due to resistance [333]. Essential oils are a potential source for novel medicines in this regard [30]. Certain essential oils have previously displayed antiviral activity [30,334], with the best viral inhibitors specifically acting on the steps involved in viral biosynthesis. These work by inhibiting viral replication, thereby limiting viral progeny production [30]. It is advantageous that the viral replication cycle consists of a complex sequence of different steps because it increases the chance of interference from antiviral agents [30].
Less than half of the essential oils recommended for skin infections have been studied for antiviral activity. Table 11 records the readily available studies. The most studied virus is the herpes simplex virus (HSV) and the most studied essential oil is M. alternifolia.
Antiviral studies encompass an extensive process where the cytotoxicity and antiviral activity need to be determined. Antiviral activity is usually tested via the plaque reduction assay on Vero (African green monkey kidney cells) cells infected with the virus. This assay determines the effective concentration inhibiting 50% of virus growth (IC 50 ). The selective indicator or selectivity index is calculated with the equation of CC 50 /IC 50 . An essential oil with a SI value greater than four is considered suitable as an antiviral agent [332,333]. Besides the criteria being made for the SI, no criteria for the IC 50 have been made. According to the results reviewed, an IC 50 value of less than 0.0010% or 1.00 g/mL should be considered as noteworthy.
Essential oils recommended in the aromatherapeutic literature, with supporting in vitro evidence, include Citrus limon (lemon), Lavandula latifolia (lavender), M. piperita, Santolina insularis (santolina), M. alternifolia, E. globulus, and S. officinalis. Of these oils, the latter three are not ideally suited for antiviral use against HSV-1, due firstly to the IC 50 values being weaker than what is recommended (less than 0.0010% or 1.00 g/mL) and due to their low selectivity index (below 4) [331,332,334,340,341]. Essential oils still to be studied according to the literature include C. zeylanicum, C. bergamia, Pelargonium odoratissimum (geranium), and Tagetes minuta (Mexican marigold).
In a small pilot study, consisting of 18 patients undergoing treatment of recurrent herpes labialis, a 6% M. alternifolia oil gel applied five times daily was compared to a placebo gel [349]. Reepithelialisation occurred after nine days for the test group compared to the placebo group where reepithelialisation occurred only after 12.5 days. Millar and Moore [350], undertook a case study of a patient with six reoccurring warts (human papillomavirus) after countless treatments with 12% w/w salicylic acid and lactic acid (4% w/w) for several weeks. Alternative treatment consisted of 100% topical M. alternifolia oil applied each evening straight after bathing and prior to bedtime. After five days, a significant reduction in wart size was observed, and, after an additional seven days, all warts were cleared, with complete reepithelialisation of the infected areas and no recurrence. The main shortfall of the two studies is the small sample size. It should also be recommended that any trial involving viral pathogens include a one-, two-, and six-month follow-up after the discontinuation of treatment, the reason being due to the tendency of viral pathogens remaining dormant for an extended period. It can then be observed how effective the essential oil is for longterm effects.
The nonenveloped (such as HPV) viruses have thus far been shown to be more resilient to essential oils [30] compared to the enveloped viruses (HSV) which are more susceptible to essential oils that could dissolve the lipid membrane [5]. Essential oil studies against viruses are clearly lacking. The most studied virus is HSV, which is one of the most prevalent viruses [351], and the most studied essential oil is M. alternifolia. Although numerous studies have proven efficacy of tea tree oil, the problem with a few of the studies is that these were compared to a placebo, which is expected to display poor activity. Although these studies demonstrate some antiviral activity, other viral pathogens (e.g., varicella zoster, herpes zoster, human papillomavirus, and Molluscum contagiosum) associated with skin infections have clearly been neglected and warrant further study.

Essential Oil Toxicity
Plants used for therapeutic purposes are normally assumed to be safe and free of toxicity. This misconception is mainly due to the long-term usage of medicinal plants for the treatment of diseases based on basic knowledge accumulated and shared from generation to generation over many centuries. However, scientific studies and reports have highlighted the toxic effects of essential oils used to treat skin ailments, which are known to produce adverse effects such as allergic contact dermatitis, skin irritation, or photosensitization [300]. Phenols and aldehyde containing oils may often cause irritation [352]. Furanocoumarin containing essential oils (such as C. bergamia) have been proven to induce phototoxicity [353][354][355]. The evidence based review on botanicals in dermatology by Reuter et al. [18] identifies certain medicinal plants which have been used for dermatological purposes, which have also reported toxic effects. These include C. bergamia and M. recutita. Mentha piperita oil has been reported to cause dermal irritation [356]. Prashar et al. [357] have shown in an in vitro study that L. angustifolia oil and linalool (one of the main compounds) are cytotoxic to human fibroblast and endothelial cells [357]. There have also been a few case reports on L. angustifolia use resulting in contact dermatitis [358][359][360].
Stonehouse and Studdiford [361] determined that nearly 5% of patients that use M. alternifolia oil will experience allergic contact dermatitis. Centred on a patch test study of 311 volunteers, it was determined that neat 5% tea tree oil can cause irritancy (mean irritancy score of 0.25) [362]. In contrast, however, the study of 217 patients from a dermatology clinic, subjected to a patch test with 10% M. alternifolia oil, showed no irritation [363]. Two additional studies tested the M. alternifolia in patch tests at concentrations of 5% and 10%; 0.15-1.8% of patients experienced allergic contact dermatitis [364,365]. However, considering that patch tests exaggerate real-world product use [366,367], they do not necessarily give a good indication of products containing the essential oils. This is evident in the discussed clinical trials using M. alternifolia oil where only mild reactions were observed [189,200,219,281,282,285]. Increasing the oil concentration to 25-100%, however, resulted in an increased risk of contact dermatitis in 2-8% of patients [275,279]. Several additional reports exist reporting contact dermatitis and one systemic hypersensitivity reaction, from the use of M. alternifolia [368][369][370][371].
As the prospective use of these essential oils may be for topical application, it is necessary to test toxicity against skin fibroblasts and human skin cell lines F1-73 [115]. Backhousia citriodora oil at a concentration of 1.00% showed low toxicity to human skin cells and skin fibroblasts [115], whereas neat B. citriodora oil and citral were shown to be toxic to human skin cells (F1-73) and skin fibroblasts [115]. Thymus quinquecostatus, when tested against fibroblast cells for cytotoxicity, showed low cytotoxicity at concentrations below 12.5 g/mL in fibroblast cells and thus may be suitable for topical treatment [207]. Mentha piperita is one of the most popularly used essential oils [372]; however, there have been reports that M. piperita oil can cause both dermal irritations [356]. A review by Reichling et al., containing more information regarding essential oil toxicity, is available [30].

Conclusion
Of all the skin pathogens studied, dermatophytes were found to be the most sensitive to essential oil inhibition, followed by the yeast C. albicans and then Gram-positive bacteria (anaerobes more than aerobes), with Gram-negative bacteria being the most resistant, especially P. aeruginosa [168,181]. The most frequently studied organisms are E. coli, P. aeruginosa, C. albicans, and S. aureus. However, less attention has been paid to pathogens such as S. epidermidis, H. influenzae, S. pyogenes, P. acnes, Clostridium spp., Brevibacterium spp., and the dermatophytes. The reason for this may be due to the difficulty in performing such studies on fastidious pathogens and the lack of a perceived threat. Furthermore, many of these pathogens are slow growing and, combined with the volatile nature of oils, may prove difficult in retaining the oil with the pathogen during the incubation period. Where possible, resistant strains should be included in essential oil studies, along with the reference strain [56,147]. Antiviral studies should extend to the neglected viruses. These should also report on which part of the cycle the inhibition occurred. The focus should be directed towards the aromatherapeutic recommendation of the essential oil and the responsible pathogens connected to the type of infection, together with the inclusion of the microorganism strain number, the solvent, essential oil composition, and the reason for testing. This is especially relevant for combination studies where it is ill advised to just randomly test different combinations.
Regardless of the frequency of the therapeutic claims made for essential oils and the proven in vitro activity, most evidence of the therapeutic efficacy of aromatherapy has been published in books about aromatherapy and not in peerreviewed journals. A few clinical trials have emerged, but their results are rarely confirmed completely to substantiate essential oil effectiveness. More rigorous clinical trials would establish confidence from the medical professionals [352].
Besides the antimicrobial activities, toxicity studies are also recommended using skin fibroblasts for sensitivity, as the use is topical. The toxicological effects of essential oils are important facets that need to be addressed. Discernment also needs to be applied as certain sensitivity studies may have been done on rabbit skin; however, human skin has been found to be more sensitive to irritants [115].
Further essential oil combinations need to be studied, along with the reason for the combination selection. Whether the interaction is synergistic, additive, indifferent, or antagonistic, each interaction is a valuable result. If antagonism is not reported, it will not be known to avoid those combinations, which in turn will result in their continuous use, which may eventually lead to resistance to the essential oils themselves. Including synergistic results will allow for these essential oil combinations to be used more frequently in practice. The inclusion of additive and indifferent interactions is also vital in order to report essential oil combinations already studied. This will prevent unnecessary duplication of combination research and confirm essential oil combinations that have useful antimicrobial activity. This research will provide an insight into the understanding of these combinations which could allow for newer directives for integrating essential oils into mainstream medicine. Although essential oil combinations with other essential oils and with antimicrobials have started gaining some attention, there is still a gap in the research with regard to carrier oils. Essential oils are seldom used directly on the skin because direct use onto the skin can cause irritation [26,38]. Therefore, essential oils are blended with carrier oils before they are applied to the skin. This raises the question as to whether or not the carrier oils influence the overall antimicrobial activity of the essential oils. Gemeda et al. [373] tested the antimicrobial activity of essential oils mixed in different hydrophilic and lipophilic bases. They found better effects in hydrophilic bases than in lipophilic bases. This study confirmed that the base may have an influence on the antimicrobial activity; however, carrier oils in combination have to the best of our knowledge not been studied further.
Essential oils, such as M. alternifolia, are often used in subinhibitory concentrations in commercial products such as shampoos, shower gels, and creams to enhance commercial selling point of a greener product or improve fragrance or desire for the product [202]. This in itself can cause resistance. Therefore, although essential oils are showing promise, the use of essential oils in subinhibitory concentrations in cosmetics and other dermatological formulations may weaken the efficacies of the essential oils as antiseptics, as was shown by Nelson [136]. This highlights the need to insure that there is sufficient evidence supporting aromatherapeutic combinations not only for therapeutics, but also in commercial products.
Resistant strains such as P. aeruginosa, MRSA, and methicillin-resistant S. epidermidis (MRSE) have become extensively problematic microorganisms in the recent years due to their antimicrobial resistance [158], and, as such, including these organisms in screening studies is becoming more and more important.
For viral studies, one needs to consider that genuine antiviral potential is seen for those essential oils that display activity after absorption into the host cell's nucleus because this is where viral DNA replicates by using viral DNA polymerase [30].
Clinical trial and ex vivo studies should consider regular essential oil dosing, instead of once daily, or every several days, application. According to the aromatherapeutic literature, essential oils are generally applied two to three times a day. The reason may be due to the volatile nature resulting in essential oil evaporation. Thus, in order to give credit to essential oil use, application studies should consider timed dosages.
Finally, M. alternifolia is the most studied of all commercial essential oils. However, many other oils have shown better antimicrobial activity. It is time essential oil researchers give just as much attention to oils such as C. zeylanicum, L. scoparium, O. vulgare, S. album, and S. aromaticum in the hope of increasing the global knowledge of essential oils used on the skin.