Antifungal Activity of the Essential Oil of Melaleuca alternifolia (Tea Tree Oil) against Pathogenic Fungi in vitro

Transcription

1 Original Research Article Skin Pharmacol I 996;9: P. Nenoff' U.-F Haustein.a W. Brandtb Department of Dermatology, University of Leipzig, and b Dr. K. Hollborn & Sohne GmbH & Co. KG, Leipzig, Germany Antifungal Activity of the Essential Oil of Melaleuca alternifolia (Tea Tree Oil) against Pathogenic Fungi in vitro Key Words Tea tree oil Me/aleuca a/ternifo/ia Miconazole Dermatophytes Yeasts Malasseziafurfur In vitro susceptibility Minimun inhibitory concentration Abstract The in vitro antifungal activity of tea oil, the essential oil of Melaleuca alternifolia, has been evaluated against 26 strains of various dermatophyte species, 54 yeasts, among them 32 strains of Candida albicans and other Candida sp. as well as 22 different Ma/assezia furfur strains. Minimum inhibitory concentrations (MIC) of tea tree oil were measured by agar dilution technique. Tea tree oil was found to be able to inhibit growth of all clinical fungal isolates. For the investigated dermatophytes MIC values from I, to 4,450.0 µg/ml with a geometric mean of 1,431.5 µg/ml were demonstrated. Both C. a/bicans strains and the other strains belonging to the genus Candida and Trichosporon appeared to be slightly less susceptible to tea tree oil in vitro. However, their MIC values, which varied from 2,225.0 to 4,450.0 tg/ml (geometric mean 4,080 µg/ml), indicated moderate susceptibility to the essential oil of M. alternifo/ia. The lipophilic yeast M. furfur seemed to be most susceptible to tea tree oil. MIC values between and 4,450.0 µg/ml (geometric mean 1,261.5 µg/ml) were found against the tested M. furfur strains. However, when calculated as percentage tea tree oil of the agar, the above-mentioned concentrations correspond to % tea tree oil content. These values are far below the usual relatively high therapeutic concentrations of the agent; approximately 5-10% solution or even the concentrated essential oil are used for external treatment. Jn comparison with tea tree oil, in vitro susceptibility against miconazole, an established topical antifungal, was tested. As expected, very low MIC values for miconazole were found for dermatophytes (geometric mean 0.2 µg/ml), yeasts (geometric mean 1.0 µg/ml), and M. fifffur (geometric mean 2.34 µg/ml). It is suggested that the in vivo effect of tea tree oil ointment in the therapy of fungal infections of the skin and mucous membranes as well as in the treatment of dandruff, a mild form of scborrheic dermatitis, may be at least partly due to an antifungal activity of tea tree oil. KARGER &Mail karger@karger.eh Fax S. Karger AG. Basel Dr. Pietro NenofT I $ I 0.00/0 Department of Dennatology University of Leipzig Licbigstrassc 21 D Leipzig (Germany) Received: February Accepted: August

2 Introduction The essential oil of Me/a/euca a/ternifolia, or tea tree oil, has been used medicinally for almost 70 years [ 1 ]. It is becoming increasingly popular as a naturally occurring antimicrobial agent not only in Australia, where the myrtle tree itself grows, but more and more in Central Europe, particularly in Germany. Tea tree oil represents a very complex mixture of hydrocarbons and terpenes consisting of approximately 100 components. For commercial purposes, levels of two of the components have been stipulated by an Australian standard (AS ): a maximum of 15% 1,8- cineole and a minimum of 30% 1-terpinen-4- ol [2, 3). Previous examinations of selected microbial reference strains revealed a growth-inhibiting effect of tea tree oil against different species of bacteria [ 4-6). It has been suggested that an antifungal mode of action of tea tree oil may be responsible, at least in part, for the therapeutic efficacy in mycotic infections of the skin and mucous membranes. Previously, the antimycotic activity of tea tree oil was estimated against single reference strains of fungi. Among the tested fungi the yeast Candida albicans and some dermatopbytes showed susceptibility to Melaleuca oil [7]. The aim of the present study was to determine the minimal inhibitory concentrations (MIC) of tea tree oil and to evaluate a possible in vitro antifungal activity of this agent against a multitude of dermatophyte and yeast strains, among the latter 31 strains of the lipophilic yeast Malasseziafurfur. In comparison, in vitro susceptibility of the fungi against miconazole was investigated. Materials and Methods Fungal Strains a11d C11/111re Conditio11s Clinical isolates of dermatophytes from 25 patients suffering from tinea unguium, tinea pcdis, and tinca corporis were tested. Investigated species were Tricl10- phy1011 rubrum. (n = 17), Trichophy e111agrophytes (n = 8), and Microsporum ca11is (n = 1 ). The dermatophyte isolates were differentiated according to the regulations of the Deutsche Gesellschaft fiir Hygiene und Mikrobiologie (8), which include identification based on the macroscopic and microscopic features of the fungi when grown in culture and some additional tests. These are the ability to produce red pigment in maize. glucose agar and the urease test, which was positive within 3 days for T. 111e111agrophy1es and M. can is. Sixty-three clinical yeast isolates were collected for testing. Among these yeasts were 31 strains belonging to the genus Candida and one Trichosporon c111ane11m strain. The collection included 8 isolates of C. albicans and 23 of Candida sp., among them Candida parapsilosis (n = 9), Candida glabrata (n = 4), Candida tropicalis (n = 1 ), Candida kefyr (n = 1 ), Candida krusei (n = 3), Candida g11illier111011dii (n = 3), Ca11didc1 s1ea1olytica (n = 1), and Candida lusi1a11iae (n = 1 ). These isolates were all recent clinical isolates from individual outpatients and inpatients of the department of Dermatology. The samples were cultured on Sabouraud 4%-dextrose agar plates (S!Fl, Berlin), identified on rice agar and biotypcd by ID 32 C (API system, bio Merieux, Marcy l'etoile, France). Altogether 31 different M. f11rf11r strains were isolated from different patients suffering from dandruff ( n = 23), seborrheic dermatitis (n = 3), and pityriasis versicolor (n = 5). A fungal isolate was considered to be J\f. furfur if it was lipophilic and if the microscopic morphology conformed with descriptions of Yarrow and Ahearn (9]. M. fwfur isolates were cultivated and further maintained on Sabouraud 4%-dextrose agar (ph 5. 7) containing 2% olive oil and 0.2% Tween 80 at 37 C. M. alternifolia oil Tea tree oil or M. altemifo!ia oil (batch ) was obtained from Mineral & Chemical Traders, Cremorne, New South Wales, Australia, for use in the investigation. This sample fulfilled the criteria of the Australian Standard with a terpinen-4-ol level of 40.1 % and a 1,8-cineole level of 5.1 % as determined by gas liquid chromatographical analysis. In vitro S11scep1ibili1y Testing In vitro susceptibility testing was carried out as previously described (10-12]. Agar dilution test for MIC Antifungal Activity of Tea Tree Oil Skin Pharmacol 1996:9:

3 Table 1. MIC levels of tea tree oil for 26 dematophyte and 32 yeast strains (cell suspensions of I 06 cfu/ml) Species 1,112 µg/ml 2,225 µg/ml 4,450 µg/ml (0.11 %) (0.22%) (0.44%) Trichophyton rubrum Trichophyton mentagrophytes Microsporum canis (n= l 7) 14 (n = 8) 2 (n = I) Candida a/bicans Candida sp. Trichosporon cutaneum (n = 8) (n = 23) (n = I) The proportional tea tree oil content of agar is indicated in parentheses. investigation of tea tree oil was carried out using DST agar (Unipath Ltd., Oxoid, Bassingstoke, Hampshire, England; ph adjusted to 5.7). Both tea tree oil and miconazole (Dr. K. Hollborn & Sohne KG, Leipzig, Germany) were suspended in 96% ethanol and further diluted in sterile distilled water. Serial twofold dilutions of the essential oil of M. a/tern((olia ranging from 2 to 8,900 µg/ml and of miconazole ranging from to 100 µg/ml were prepared in DST agar. For susceptibility testing of the M. furfur strains this agar contained additionally 2% olive oil and 0.2% Tween 80 to allow growth of this lipophilic yeast. For susceptibility testing 2-day-old cultures of yeasts and 7-day-old cultures of dermatophytes on Sabouraud 4% dextrose agar were used. Suspensions of yeast blastospores and dermatophyte conidia were prepared in sterile physiological NaCl solution ( 106 cfu/ ml). These suspensions were inoculated onto tea tree oil-containing media using a multipoint inoculator which delivered 3-4 µl per spot, resulting in a final concentration of 3-4 x 103 cfu. The plates were read after 48 h incubation at 37 C for yeasts and after 5 days incubation at 26 C for dermatophytes. M. fa.fur cell aliquots were prepared as suspensions with olive oil, sterile distilled water and Tween 80 (3:5:2). These suspensions were vortexed twice for 20 s. Plates were inoculated with a multipoint inoculator. The yeast cell suspension (approximately l os cfu/ ml) was inoculated onto coal tar gel-containing media using a multipoint inoculator which delivered 3-4 µl per spot, resulting in a final concentration of 3-4 x 105 cfu. Results were recorded after incubation at 37 C for 96 h. The lowest drug concentration at which a strain showed no growth was considered to be the MIC. To rule out any inhibitory effect of DST medium on the tested substances growth controls were carried out on the medium without the active agent. Results No growth inhibition of the fungi was achieved at concentrations of tea tree oil which are normally used for the determination of in vitro susceptibility ( µg/ ml). However, all investigated dermatophytes, yeasts, and M. fwjur strains were susceptible to tea tree oil at higher concentrations of the agent. The data of the in vitro experiments are given in table 1 and 2. Control cultures of all yeast and dermatophyte strains developed normally. The MIC determined in vitro showed susceptibility of the various fungal species to tea tree oil. In the dermatophytic group (n = 26) MIC values were in the range from 1,112.5 to 4,450.0 µg/ml with a geometric mean of 1,531.5 µg/ml. When calculat- 390 Skin Pharmacol 1996:9: NenofTIHaustcin/Brandt

4 Table 2. MIC levels of tea tree oil for 22 M.furfurstrains M. furfur isolated from patients with 556 µg/ml 1,112 µg/ml 2,225 µg/ml 4,450 µg/ml (0.05%) (0.11%) (0.22%) (0.44%) Dandruff (n = 15) Seborrheic dermatitis (n = 3) 2 1 Pityriasis versicolor (n = 4) 2 The proportion al tea tree oil content in the agar is indicated in parentheses. Table 3. MIC levels of miconazole for 25 dermatophytc and 24 yeast strains (cell suspensions of 106 cfu/ml) Species Trichophyron rubr11111 (n = 17) 16 Trichophy1011 meragrophyres (n = 7) 4 2 J\licrosporwn canis (n = I) µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml Candida a/bicans (n = 8) I 4 2 Candidasp. (n = 16) Table 4. MIC levels of miconazole for 31 M. furfur strains M. fur fur isolated from patients with µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml µg/ml Dandruff (n = 23) 4 Seborrheic dermatitis (n = 3) Pityriasis vcrsicolor (n = 5) ed as percentage tea tree oil of the agar the above-mentioned concentrations correspond to % tea tree oil content. The tested yeasts seemed to be somewhat less susceptible to tea tree oil. Growth of the investigated yeast strains was inhibited at a MIC between 2,225.0 and 4,450.0 µg/ml with a geometric mean of 4,080.0 µg/ml. Tea tree oil showed inhibition of all investigated M. furfur strains at concentrations of ,450.0 µg/ml with a geometric mean of 1,261.5 µg/ml. Anti fungal Activity of Tea Tree Oil Skin Pharmacol 1996:9:

5 The in vitro susceptibilities of the fungi to miconazole are outlined in tables 3 and 4. For this classic topically used azole antifungal very low MIC values could be demonstrated. For dermatophytes MIC values were in the range from 0.1 to µg/ml (geometric mean 0.2 µg/ml), for yeasts from 0.1 to 6.25 µg/ml (geometric mean 1.0 µg/ml), and for M. fwjur between 0.05 and 50 µg/ml (geometric mean 2.34 µg/ml). Discussion In the early part of this century, an essential oil extracted from the Australian native tree M. alternifolia and commonly known as tea tree oil was widely used as a germicidal agent [I]. In recent years there has been a revival of interest in 'natural' medicinal procedures. Particularly tea tree oil is currently experiencing a renaissance in popularity and a broad and diverse range of tea tree oil products is widely available [6, 13]. Tea tree oil was used for a wide variety of complaints, e.g., wound infections and bacterial, viral, and fungal infections of the skin and mucous membranes of the mouth and genital tract. Reports on the clinical efficacy of tea tree oil in the above-mentioned skin diseases are largely anecdotal [ 14-16]. There are only few clinically controlled studies confirming the effect of tea tree oil. Bassett et al. [7] performed a single-blind, randomized clinical trial on 124 patients to evaluate the efficacy and skin tolerance of 5 % tea tree oil gel in the treatment of mild to moderate acne vulgaris when compared with 5% benzoyl peroxide lotion. The results of this study showed that both 5% tea tree oil and 5% benzoyl peroxide had a significant effect in ameliorating the patient's acne by reducing the number of inflamed and noninflamed lesions, although the onset of action in the case of tea tree oil was slower. The efficacy of tea tree oil formulations in the treatment of tinea pedis, anaerobic vaginosis, and other cutaneous infections was evaluated in recent studies (14-16]. The in vitro antimicrobial activity of tea tree oil has been demonstrated against several bacterial species, e.g., Staphylococcus aureus, Escherichia coli, and Propionibacterium acnes. In addition, the antimycotic activity of tea tree oil has previously been determined against selected reference fungal strains [7]. MIC90 (MIC to inhibit 90% of tested organisms) values between 0.25 and 0.750/ocould be demonstrated for C. albicans, T. rubrum, and T. mentagrophytes. These results are in accordance with the present investigation of the in vitro susceptibility testing of clinical isolates of dermatophytes and yeasts, including the lipophilic yeast M. fwfur, against tea tree oil. At MIC values ranging from to 4,450.0 µg/ml complete growth suppression of the tested fungal isolates was found. The results of the present study concerning the in vitro antif ungal activity of tea tree oil are in accordance with a recent report by Carson and Riley [5], who evaluated the antimicrobial activity of the major components of the essential oil of M alternifolia. For one investigated C. albicans strain (A TCC I 0231) they could demonstrate that the component terpinen-4-ol of tea tree oil exhibits a fungistatic effect in vitro at a MIC value of 0.25%, and 1.0% for 1,8-cineole. Considering the fact that the tea tree oil that was used contains only 40.1 % terpinen-4-ol and 5.1 % 1,8-cineole, much higher MIC values of the essential oil could be expected. Thus, the achieved MIC values of the complex mixture were similar to the values of the single antimicrobially active components of tea tree oil. Presumably, a summation eltect of the different components can be suggested, however, the role of synergistic or antagonistic interactions needs to be evaluated. 392 Skin Phannacol 1996:9: Nc1101T/Haustcin/Bra11d1

6 The usual therapeutic concentration of the agent is approximately 5-10%, but even concentrated tea tree oil is used for external treatment. These concentrations exceed the experimentally determined MICs for tea tree oil markedly. In order to compare the in vitro sensitivity of the investigated fungal strains against a given established locally applied antifungal agent, MIC values to miconazole were determined as well. It is not surprising that miconazole was a more effective agent for growth inhibition of the pathogenic fungi in vitro. The activity of miconazole found in vitro was comparable to previously demonstrated data on the in vitro suceptibility of dermatophytes [ 17]. It is suggested that such a true antimycotic preparation is a much more effective drug in human disease. Nevertheless, despite a generally good tolerability of tea tree oil and minor side effects [7] the occurrence of contact allergy to the essential oil has been reported in 3 patients [ 18, 19] and 1 patient with systemic contact allergy for which eucalyptol (1,8-cineole), the common ingredient in essential oils, was proved as the actual allergen [20]. The results obtained from the present in vitro susceptibility testing indicate that this antifungal mode of action may contribute to the therapeutic in vivo effect of tea tree oil in several fungal infections of the skin and mucous membrane by dermatophytes and yeasts, respectively. In addition, it is concluded that the tested tea tree oil itself, which is increasingly used for therapy of dandruff, a mild form of seborrheic dermatitis, exerts its efficacy, at least in part, through inhibition of M. fwfur. These investigations of the antifungal activity of Me/aleuca oil may have significant implications for the use of tea tree oil as an antimicrobial, particularly antimycotic agent in the future. However, the benefit-risk ratio in terms of the development of contact eczema has to be taken into consideration, the more so as potent antimicrobial agents are available on the market. References I Penfold AR, Grant R: The germicidal values of some Australian essen- 1 ial oils and their pure constituents: Together with those for some essential oil isolates. and synthetics. Part III. 1 Proc R Soc N SW 1925;59: Swordos G. Hunter GLK: Composition of Australian tea tree oil (Melaleuca altemifolia). J Agric Food Chem I 978;26: Brophy JJ. Davies NW. Southwell IA, Stiff IA, Williams LR: Gas chromatographic quality control for oil of Mclalcuca terpinen-4-ol type (Australian tea-tree). 1 Agric Food Chem 1989;37: Carson CF, Riley TV: Susceptibility of Propio11ibacteri11111 acnes to essential oil of Melaleuca altemifolia. Lett Appl Microbiol 1994; 19: Carson CF, Riley TV: Antimicrobial activity of the major components of the essential oil of Melaeuca alternijolia. J Appl Bacteriol I 995;78: Carson CF. Hammer KA. Riley TV: Broth microdilution method for determining the susceptibility of Escherichia coli and Staphylococcus 011re11s 10 the essential oil of J\llelaleuca altem(folia (lea tree oil). Microbios 1995;82: Bassell IB, Pannowitz DL, Barnctson RSC: A comparative study of tea-tree oil versus benzoylperoxide in the treatment of acne. Med J Aust 1990: 153: MeinhofW: lsolierung und Identifizierung von Dermatophyten. Zentralbl Bakteriol 1990:273: Yarrow D. Ahearn DG: Genus 7: Malassezia; in Kregger van Rij NJW (ed): The Yeasts: A Taxonomic Study, ed 2. Amsterdam, Elsevier, 1988, pp IO NenofT P. Haustein U-F: In vitro susceptibility testing of Pit.rrosporum ova/e against anti fungal. antiscborrheic and antipsoriatic agents. J Eur Acad Dermatol Venereal 1994: 3: Antifungal Activity of Tea Tree Oil Skin Pharmacol 1996:9:

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