In vitro susceptibility of Madurella mycetomatis, prime agent of Madura foot, to tea tree oil and artemisinin
1 Department of Medical Microbiology & Infectious Diseases, Erasmus MC, University Medical Center Rotterdam, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands 2 Mycetoma Research Centre, University of Khartoum, Khartoum, Sudan 3 Microbiology and Immunology, The University of Western Australia, Perth, Australia
* Corresponding author. Tel: +31-10-4632176; Fax: +31-10-4633875; E-mail: w.vandesande{at}erasmusmc.nl
Received 2 October 2006; returned 24 November 2006; revised 27 November 2006; accepted 3 December 2006
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Abstract |
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Objectives: Eumycetoma caused by Madurella mycetomatis is treated with surgery and high doses of itraconazole and ketoconazole. These agents are toxic, and new therapies are required.
Methods: MICs were determined for artemisinin and tea tree oil, two natural herbal compounds.
Results: Artemisinin was not active against M. mycetomatis, but tea tree oil did inhibit its growth. Since tea tree oil's prime component easily penetrates the skin, tea tree oil could be a useful agent in the treatment of eumycetoma.
Conclusions: Tea tree oil is active in vitro against M. mycetomatis.
Keywords: MICs , essential oils , mycetoma , itraconazole , ketoconazole
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Introduction |
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Eumycetoma is a subcutaneous disease caused by a variety of microorganisms, both bacteria and fungi. The most common fungal causative agent is Madurella mycetomatis. Eumycetoma is usually treated for extended periods of time with high doses of either itraconazole or ketoconazole. Prolonged treatment with high doses of azoles can result in hepatic toxicity. In order to identify alternative antifungal therapies, the susceptibility of M. mycetomatis to other frequently used antifungal agents (amphotericin B, 5-flucytosine and voriconazole) has been determined previously. M. mycetomatis remained most susceptible towards the azoles; no activity was seen with 5-flucytosine.1 In the search for new antifungal agents, research is shifting to more traditional anti-infective folk medicines. An example of such a natural compound is artemisinin, which is isolated from the plant Artemisia annua and used in traditional Chinese medicine. In the early 1970s, it was established that artemisinin was the compound in this plant extract with antiparasitic activities against Plasmodium falciparum. A. annua was also resistant to common plant pathogenic fungi.2 This resistance was due to substances produced by endophytes present on A. annua, but artemisinin has antifungal activities also.2 Cryptococcus neoformans and Saccharomyces cerevisiae were inhibited by artemsinin, but Candia albicans was not.3 Another example of a natural compound that appears to be successful in curing infections is tea tree oil, extracted from Melaleuca alternifolia. This oil was used by the Australian Bundjalung Aborigines of New South Wales for its anti-inflammatory and antimicrobial properties.4,5
In vitro MICs have been determined for a wide variety of microorganisms, and clinical studies indicated that treatment with tea tree oil could improve the course of acne, dandruff, onychomycosis, oral candidiasis and tinea pedis.4 Since both artemisinin and tea tree oil appear to be successful in clearing certain fungal infections, the in vitro susceptibilities of a collection of strains of M. mycetomatis to both agents and itraconazole, the agent most widely used in eumycetoma treatment, were compared.
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Materials and Methods |
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In this study, MICs of itraconazole (Janssen Pharmaceutical Products, Beerse, Belgium), artemisinin (Sigma-Aldrich, Zwijndrecht, The Netherlands) and tea tree oil (Novasel, Skarup, Denmark) were determined for a total of 34 clinical isolates of M. mycetomatis (32 obtained from the Mycetoma Research Centre, University of Khartoum, Sudan, and two obtained from Mali). The strains were isolated from biopsies and maintained on Sabouraud dextrose agar (Difco Laboratories, Paris). The strains were previously identified on the basis of morphology and PCR-RFLP.6,7 MICs were determined independently in triplicate using the previously reported 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assay.1,8 In short, in this assay a hyphal inoculum of 70% transmission was prepared in RPMI medium. After 7 days of incubation at 37°C, XTT was administered, and MICs were determined. The MIC end-points for each antifungal agent were defined as the first concentration where spectrophotometrically 80% or more reduction was measured. Drug concentrations used ranged from 0.002 to 16 mg/L for itraconazole and artemisinin, and from 0.002 to 1% (v/v) for tea tree oil. All dilutions were prepared in DMSO; the final concentration of DMSO per inoculum was as described by the CLSI criteria.9
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Results |
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In concordance with previously published MICs for M. mycetomatis, all strains were strongly inhibited by itraconazole, the drug of choice to treat eumycetoma in Sudan (Figure 1). MICs of itraconazole were evenly distributed in concentrations ranging from <0.002 to 0.06 mg/L (Figure 1). A concentration of 0.03 mg/L was needed to inhibit the growth of 90% of the isolates (Table 1).
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Artemisinin was not active against the isolates. Only two isolates were inhibited in growth by artemisinin, with MICs of 0.03 mg/L and 0.5 mg/L, respectively. The other 32 isolates were not inhibited, not even by 16 mg/L artemisinin. In 19 of the 34 strains, there was a marginal inhibition in growth visible. Inhibition ranged from 20% to 50%. In the other 15 strains, no inhibition and sometimes even stimulation of growth were noted, resulting in growth percentages sometimes even as high as 150–200%.
In contrast to artemisinin, tea tree oil did inhibit M. mycetomatis growth. As shown in Figure 1, MICs ranged from 0.008% (v/v) to 0.25% (v/v). A concentration of 0.06% (v/v) was needed to inhibit 50% of the isolates, and a concentration of 0.25% (v/v) was needed to inhibit 90% of the isolates (Table 1).
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Discussion |
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The most common antifungal agents used in the treatment of mycetoma caused by M. mycetomatis are ketoconazole and itraconazole.10 Success rates with these drugs are variable, though, and toxic side effects occur. Therefore, the antifungal susceptibility of M. mycetomatis to a variety of other antifungal agents should be analysed. In vitro antifungal susceptibilities to amphotericin B, itraconazole, ketoconazole, fluconazole, voriconazole and 5-flucytosine have already been described.1 In the present study, less common antifungal agents were explored, namely tea tree oil and artemisinin, both representatives of traditional folk medicines. For artemisinin, some antifungal effects against C. neoformans and S. cerevisiae, but not against C. albicans, were noted previously.3,11 M. mycetomatis was not inhibited by this drug. Interestingly, very high MICs for S. cerevisiae were obtained when this species was cultured in medium with a fermentable carbon source such as glucose, while low MICs were found when non-fermentable carbon sources, such as glycerol or ethanol, were used.3,11 In S. cerevisiae, the inhibitory effect of artemisinin was shown to be caused by disrupting the depolarization of the mitochondrial membrane potential.3,11 Another striking feature of artemisinin is its binding to the translationally controlled tumour protein (TCTP). Generally, the higher the TCTP expression, the more susceptible the cells were to artemisinin.12 Although M. mycetomatis expresses TCTP both in vivo and in vitro, this did not result in growth inhibition by artemisinin.13
More interestingly, M. mycetomatis was found to be highly susceptible to tea tree oil. Concentrations below 0.25% (v/v) resulted in full inhibition of fungal growth. This is comparable to the susceptibilities found for the yeasts Candida spp. and Malassezia spp. and the filamentous fungi Alternaria spp., Cladosporium spp., Fusarium spp. and Penicillium spp.4 The susceptibilities of Aspergillus spp. were variable, with Vazquez reporting high MICs exceeding 2% (v/v), while Hammer et al. reported MICs in the range 0.12% (v/v).4 Aspergillus niger required concentrations as high as 8% (v/v) to be inhibited.4 Tea tree oil has also been used in clinical studies in the treatment of onychomycosis, refractory oral candidiasis and tinea pedis with varying success.4 In order for a topical treatment with tea tree oil to be effective in the treatment of subcutaneous mycetoma, the antifungal components of the agent must penetrate the skin. Nielsen and Nielsen showed that the least lipophilic constituents of tea tree oil could penetrate the skin.14 Among these less lipophilic agents were eucalyptol, 
-terpineol and terpinene-4-ol.14 Terpinen-4-ol, -terpineol, -pinene, aromadendrene and ß-pinene all had antifungal activities.5 The antifungal activities against C. albicans of the first four components were comparable to the antifungal activities found with tea tree oil itself, but much lower MICs of ß-pinene were found.5 Tea tree oil consists of 40% terpinen-4-ol, which is an active antifungal agent and able to penetrate the skin. Considering that the growth of M. mycetomatis is inhibited by tea tree oil, this would suggest that tea tree oil might be useful as a topical agent in the treatment of eumycetoma.
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None to declare.
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References |
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1 van de Sande WW, Luijendijk A, Ahmed AO, et al. (2005) Testing of the in vitro susceptibilities of Madurella mycetomatis to six antifungal agents by using the Sensititre system in comparison with a viability-based 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assay and a modified NCCLS method. Antimicrob Agents Chemother 49:1364–8.
2 Liu CH, Zou WX, Lu H, et al. (2001) Antifungal activity of Artemisia annua endophyte cultures against phytopathogenic fungi. J Biotechnol 88:277–82.[CrossRef][Web of Science][Medline]
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Carson CF, Hammer KA, Riley TV. (2006) Melaleuca alternifolia (tea tree) oil: a review of antimicrobial and other medicinal properties. Clin Microbiol Rev 19:50–62.
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Ahmed AO, Mukhtar MM, Kools-Sijmons M, et al. (1999) Development of a species-specific PCR-restriction fragment length polymorphism analysis procedure for identification of Madurella mycetomatis. J Clin Microbiol 37:3175–8.
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van de Sande WW, Gorkink R, Simons G, et al. (2005) Genotyping of Madurella mycetomatis by selective amplification of restriction fragments (amplified fragment length polymorphism) and subtype correlation with geographical origin and lesion size. J Clin Microbiol 43:4349–56.
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Ahmed AO, van de Sande WW, van Vianen W, et al. (2004) In vitro susceptibilities of Madurella mycetomatis to itraconazole and amphotericin B assessed by a modified NCCLS method and a viability-based 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assay. Antimicrob Agents Chemother 48:2742–6.
9 National Committee for Clinical Laboratory Standards. NCCLS2002Wayne, PA, USAReference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi: Approved Standard M38-A.
10 Fahal AH. (2004) Mycetoma: a thorn in the flesh. Trans R Soc Trop Med Hyg 98:3–11.[CrossRef][Web of Science][Medline]
11 Li W, Mo W, Shen D, et al. (2005) Yeast model uncovers dual roles of mitochondria in action of artemisinin. PLoS Genet 1:e36.[CrossRef][Medline]
12 Efferth T. (2005) Mechanistic perspectives for 1,2,4-trioxanes in anti-cancer therapy. Drug Resist Updat 8:85–97.[CrossRef][Web of Science][Medline]
13
van de Sande WW, Janse DJ, Hira V, et al. (2006) Translationally controlled tumor protein from Madurella mycetomatis, a marker for tumorous mycetoma progression. J Immunol 177:1997–2005.
14 Nielsen JB and Nielsen F. (2006) Topical use of tea tree oil reduces the dermal absorption of benzoic acid and methiocarb. Arch Dermatol Res 297:395–402.[CrossRef][Web of Science][Medline]
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