Staphylococcus aureus and wounds: A review of tea tree oil as a promising antimicrobial

Staphylococcus aureus and wounds: A review of tea tree oil as a promising antimicrobial Linda Halco´n, PhD, MPH, RN,a and Kelly Milkus, BAb Minneapolis, Minnesota

Antibiotic-resistant bacteria continue to be a major health concern worldwide. In particular, Staphylococcus aureus, both methicillin-resistant and -sensitive, are of concern in their ability to cause difficult skin and underlying tissue infections. Melaleuca alternifolia oil (tea tree oil), an essential oil, has demonstrated promising efficacy in treating these infections. Tea tree oil has been used for centuries as a botanical medicine, and has only in recent decades surfaced in the scientific literature as a promising adjunctive wound treatment. Tea tree oil is antimicrobial, anti-inflammatory, and has demonstrated ability to activate monocytes. There are few apparent side effects to using tea tree oil topically in low concentrations, with contact dermatitis being the most common. Tea tree oil has been effective as an adjunctive therapy in treating osteomyelitis and infected chronic wounds in case studies and small clinical trials. There is a need for larger clinical trials to further examine efficacy of tea tree oil as an adjunctive wound therapy, as well as improved guidelines for developing plant-based medicines. (Am J Infect Control 2004;32:402-8.)

In recent decades there has been a marked increase in difficult-to-treat skin and underlying tissue infections associated with gram-positive bacteria, notably methicillin-resistant and -sensitive Staphylococcus aureus (MRSA and MSSA).1-3 Patients with skin lesions are particularly susceptible to long-term MRSA carriage. MRSA carriers are more likely to have chronic skin lesions and subsequently be readmitted for care, potentially exposing other vulnerable patients.4-6 Many long-term care facilities will not admit patients with MRSA-infected wounds, compromising patients’ ability to access appropriate health care. In addition to the epidemiologic implications of MSSA and MRSA infection, persistent wounds have a profound effect on individual patients’ sense of well-being, comfort level, and quality of life, and they are costly in terms of medication costs, health care providers’ time and increased length of hospitalization.7,8 New strategies are needed to treat wounds infected with S aureus (MSSA and MRSA).2,9-11 Many pharma-

From the School of Nursing, University of Minnesotaa and Minnesota Program in CAM Clinical Research, Berman Center for Outcomes and Clinical Research, Minneapolis Medical Research Foundation.b Reprint requests: Linda Halco´n, PhD, MPH, RN, University of Minnesota School of Nursing, 6-101 Weaver-Densford Hall, 308 Harvard Street SE, Minneapolis, MN 55455. During preparation of this manuscript, Dr Halco´n was supported in part by the A. Marilyn Sime Faculty Research Fellowship, grant no. R25AT00556 (P.I. Kreitzer) from NIH-NCCAM, and T80-MC00021 (P.I. Bearinger) from the Maternal Child Health Bureau (Title V, Social Security ACT), HRSA, DHHS. 0196-6553/$30.00 Copyright ª 2004 by the Association for Professionals in Infection Control and Epidemiology, Inc. doi:10.1016/j.ajic.2003.12.008

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cologic agents have been used with varying efficacy; however, treatment side effects and microbial resistance continue to be problematic.9,12-14 Even in wounds infected with MSSA isolates, there is a need for more convenient and less costly treatment options, as standard treatment consists of parenteral antibiotics or very expensive oral antibiotics. There is considerable and growing international literature on the use of plant essential oils against pathogenic microorganisms.15-24 In vitro studies suggest that some essential oils and their components have strong bactericidal action.24-28 Over the past 30 years there have been recurring reports of the efficacy of Melaleuca alternifolia (tea tree oil) essential oil against bacterial pathogens.20,21,29-35 Many S aureus isolates (MSSA and MRSA) have been found to be susceptible to M alternifolia (tea tree) essential oil.10,25,36-40

HISTORY OF USE Tea tree oil has been used as a botanical medicine in various forms over the centuries, and over 70 years for medicinal use as an essential oil. Australian aboriginal people used tea tree oil for a variety of medicinal purposes. Tea tree was used in the early 20th century as a medicinal antiseptic in Australia.41 Tea tree oil has a long history of clinical use in the treatment of foot problems such as tinea pedis and toenail onychomycosis.31,42-45 Other dermatologic studies have been conducted with tea tree oil in the treatment of acne, dandruff, head lice, and recurrent herpes labialis, in which effects were found to be either similar or better than traditional treatment, and often with fewer side effects.35,46-48 A few published studies report the successful use of tea tree oil in treating mucous membrane infections, including Trichomonas vaginalis,49 and against oral bacteria and oropharyngeal candidiasis.50,51

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CHEMISTRY M alternifolia essential oil is known by many names, including tea tree essential oil, tea tree oil, Melaleuca oil, and TTO. There are many varieties of plants commonly referred to as tea tree; thus, it is important to clearly identify genus and species. Tea tree essential oil is steam distilled from the leaves and terminal branchlets of M alternifolia. It is a clear, mobile liquid with no visible trace of water and has a distinct odor.52 The exact constituency of tea tree oil varies. According to the Australian and International Standards Organizations, the substance known as tea tree oil from M alternifolia has a chromatographic profile within given ranges (Table 1).53

ACTIVITY AND MECHANISM OF ACTION The pharmacology of M alternifolia essential oil has been identified in part through bacteriologic and animal studies. Several published reports have addressed minimum inhibitory and bactericidal concentrations of tea tree oil against clinical isolates of S aureus. A study of 105 clinical isolates of S aureus using a broth microdilution method found the MIC90 of tea tree oil to be 0.5%.25 A later study of 100 clinical isolates of MRSA found the MIC90 of tea tree oil to be 0.32%.54 Susceptibility of normal skin flora and disease isolates to tea tree oil has been examined, although S aureus has been the focus of most research. A study of 15 clinical isolates of S pyogenes found the MIC90 of tea tree oil to be 0.12%.55 A study of 4 clinical isolates of coagulase-negative staphylococci found the MIC90 of tea tree oil to be 2% to 4% by broth microdilution and 1% with agar dilution.56 Another study examining several species of normal skin flora reported MIC90 for 6 microorganisms as follows: 10 clinical isolates of Corynebacteria species, 0.25%; 15 isolates of S epidermidis, 1%; 10 isolates of S capitis, 1%; 10 isolates of S hominis, 0.5%; 4 isolates of S saprophyticus, 0.25% to 1%; and 5 isolates of Micrococcus species, 0.06% to 5%. These values are, in general, above the MIC90 for S aureus, suggesting that it is possible to treat S aureus infections while somewhat preserving the population of normal flora.22 The response of other disease-causing bacteria to tea tree oil has also been addressed in the literature. A study of 10 clinical isolates of b-hemolytic streptococci found a MIC90 of 1% by broth microdilution, and 0.5% by agar dilution. The same study found the MIC90 of tea tree oil for 3 clinical isolates of Pseudomonas aeruginosa to be 1% to 5% with broth microdilution and less than 2% with agar dilution.56 Another study found the MIC90 of tea tree oil to 10 clinical isolates of P aeruginosa to be 3%.22

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Table 1. Chemistry and selected properties of Melaleuca alternifolia essential oil (Australian Standard AS27821997: oil of melaleuca, terpinen-4-ol type [tea tree oil]) Components

Minimum %

a-Pinene Sabinene a-Terpinene Limonene r-Cymene 1,8-Cineole g-Terpinene Terpinolene Terpinen-4-ol a-Terpineol Aromadendrene d-Cadinene Globulol Viridiflorol

1 Traces 5 0.5 0.5 — 10 1.5 30 1.5 Traces Traces Traces Traces

Maximum % 6 3.5 13 4 12 15 28 5 — 8 7 8 3 1.5

Relative density at 208C/208C: 0.885 to 0.906; refractive index at 208C: 1.4750 to 1.4820; optical rotation at 208C: +58 to +158; miscibility at 208C: 1 part soluble in 2 parts 85% v/v ethanol. Source: Council of Standards Australia.53

Based on results of laboratory and animal studies, there are several likely mechanisms by which a topical, tea tree oil preparation may facilitate healing in chronic Staphylococcus-infected wounds. Preliminary studies suggest both reduction in microbial load and changes in immune function related to tea tree oil applications. Terpenen-4-ol, linalool, and alpha-terpineol are the most studied active antibacterial components of tea tree essential oil.15,55,57-60 These components, primarily terpinen-4-ol, have been shown to affect the bacterial cell wall, demonstrated by the loss of 260-nm nuclear material, K+, salt tolerance, and the presence of mesosome-like structures by electron microscopy after in vitro treatment of S aureus with tea tree oil. Inhibition of glucose-dependent respiration has also been demonstrated.20,61-63 Studies of the whole essential oil and several of its components (1,8 cineole, terpinen-4-ol, and a-terpineol) suggest that tea tree oil compromises the cytoplasmic membrane of S aureus, giving it a bacteriostatic and bactericidal effect.40,61,62 Tea tree oil also has been shown to increase monocytic differentiation in vitro.64 The anti-inflammatory properties of tea tree oil are also likely to assist in healing chronic wounds. The main component of tea tree oil, terpinen-4-ol, has been shown to suppress inflammatory mediator production by monocytes activated in vitro.65,66 Anti-inflammatory properties are also suggested by studies showing that the application of topical tea tree oil regulated edema associated with the contact hypersensitivity response in mice.66-68 More specifically, terpinen-4-ol showed potential for selective regulation of cell types during inflammatory processes.69 A study of 27 human

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volunteers demonstrated that tea tree oil can reduce histamine-induced skin inflammation.70 There is little known to date as to whether bacterial resistance may be an issue with tea tree oil. Nelson38 induced low-level resistance to tea tree oil in subpopulations of 3 clinical isolates and 2 laboratory strains of S aureus that had no previous exposure to tea tree oil. The MIC90 for these resistant subpopulations ranged from 0.5% to 16%. Gustafson et al.71 found that after induction of the mar operon in Escherichia coli, leading to expression of the MAR (multiple antibiotic resistance) phenotype, there was an increased tolerance of, but not resistance to tea tree oil. This increased tolerance has been hypothesized to be caused by the same mechanisms that lead to the MAR, namely a membrane efflux pump and a decrease in the membrane binding of organic solvents.72-74 P aeruginosa has also shown more tolerance to tea tree oil than other bacteria. This increased tolerance is thought to be due to qualities of the outer membrane of P aeruginosa, as using polymyxin B nonapeptide to permeabilize the membrane decreases the tolerance of P aeruginosa to tea tree oil.75 Research on resistance of P aeruginosa to other essential oils has demonstrated the existence of a plasmid thought to be involved in the increased resistance.76 These mechanisms have not been demonstrated, but may also be involved in staphylococcal resistance to tea tree oil.

TOXICOLOGY The general toxicology profile of M alternifolia essential oil suggests that severe reactions would be extremely rare in the absence of ingestion. d

d

d d

d

Oral LD50 in rats: 1.9 to 2.6 mL/kg of body weight.41,77 Acute dermal LD50 in rabbits: 5.0 gm/kg77 pure (100%) tea tree oil applied to the skin of albino rabbits and maintained at 2 gm/kg for 24 hours resulted in no signs of toxicity.41 Average lethal dose for a 3-year-old child: 26 mL78 Irritation: Draize skin irritancy index 5.0, based on application of 100% tea tree oil to intact and abraded skin of albino rabbits, signifying that tea tree oil could cause dermatitis in some users.41,52 A 30-day dermal irritation test in rabbits using 25% tea tree oil in paraffin on shaved skin did not result in apparent irritation.41 Sensitization: Nil skin sensitization in the guinea pig.41,52

Hayes et al.79 tested tea tree oil and components of tea tree oil on several human cell lines in vitro. Cytotoxicity with 100% tea tree oil ranged from 0.02 to 2.8 gm/L, with epithelial-like cells being the most

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robust, and liver-derived cells being the most susceptible. Cytotoxicity for the components of tea tree oil was as follows: 1,8-cineole, from 0.14 to 4.2 gm/L; terpinen-4-ol, from 0.06 to 2.7 gm/L; and a-terpineol, 0.02 to 1.1 gm/L. It is important to note that this study does not take into account the body’s metabolism of tea tree oil. This study supports the use of tea tree oil as a topical treatment, as epithelial cells seem to be the most resilient to tea tree oil. There have been case reports of dermal sensitivity, contact dermatitis, and oral toxicity related to tea tree essential oil.81-84 Varma et al.85 reported a case of vaginal application of tea tree oil and lavender oil in a patient with concurrent severe eczema. Bhushan and Beck86 reported a case of blistering dermatitis where a wart paint containing tea tree oil had been used for a period of 4 months. The man had a positive patch test to 1% tea tree oil, while 50 controls were negative on testing with 1% and 5% aqueous tea tree solutions. The case patient was treated with topical corticosteroids and recovered with no known sequelae. Del Beccaro87 and Jacobs and Hornfeldt88 reported treating toddlers showing signs of toxicity after accidental ingestion of small amounts of 100% tea tree oil. Drowsiness, ataxia, and disorientation were the major signs of toxicity. Several other reports of oral toxicity can be found in the literature.89,90 Tea tree oil was found to produce ototoxicity when applied in the ears of guinea pigs at 100% concentration, but no ototoxicity was found for 2% solutions.91 De Groot and Weyland80 reported a case of contact dermatitis related to extended use of full-strength tea tree oil topically, followed by oral ingestion of tea tree oil. In this report, 1,8 cineole or eucalyptol was found to be the offending substance. Other cases of allergic contact dermatitis related to eucalyptol have been reported92; however, results are conflicting on this point.93 More recent case reports have revealed additional, possibly isolated, adverse events with topical use of tea tree oil. Perrett et al.94 reported a case in which an 18-year-old woman developed linear IgA disease precipitated by contact dermatitis after 3 weeks of selftreatment with tea tree oil. Her condition improved after topical steroid treatment. Dharmagunawardena et al.95 used gas chromatography to determine common constituents of several essential oils in 2 aroma therapists who developed contact dermatitis to several essential oils, including tea tree oil. Alpha-terpinene was confirmed as the common allergen in both cases. This is a major constituent in tea tree oil and may be one of the components responsible for tea tree oil sensitivity. In addition to case studies, there have been experiments on dermal sensitivity, but for the most part these have been limited to small studies. One study of 13 participants who underwent patch testing with 5% tea

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tree oil found no cases of sensitivity.96 An earlier test of 1% tea tree oil among 22 subjects also found no irritation or sensitivity.77 In another study of 28 subjects exposed multiple times to a 25% tea tree solution, 25 had no reaction and the 3 reactions were shown to be sensitivity rather than irritation.97,98 Fritz et al.99 patch tested patients with various products, including several containing M alternifolia as an ingredient, and various concentrations of M alternifolia. Fourteen of 1216 subjects reported that they had developed contact dermatitis previously with use of tea tree oil products, and 7 of those 14 demonstrated sensitivity and an eczematous reaction upon patch testing. Sherry et al.100 reported only 5 cases of localized sensitivity among 510 orthopedic surgical wounds treated with products containing tea tree oil. Because essential oils are not regulated as pharmaceutical products, there is concern about the sterility or purity of these substances. Two studies have addressed this issue. The microbiological safety of essential oils, including tea tree for patient use, was examined in a study using products obtained from retail outlets. The authors concluded that all of the oils were sterile, antimicrobial against 7 bacterial pathogens and Candida albicans, and thus considered safe for human use. Potential contamination while in use by patients cannot be precluded, however.24 A study of the effects of conifer resin acids and tea tree oil on human fibroblasts and epithelial cells found that tea tree oil exhibited a different and less potent pattern of cytotoxicity than the other substances tested, in that cytotoxicity towards normal cells was lower than against pathogenic microorganisms.101 Proper storage and handling of essential oils, including tea tree oil, is important in reducing the likelihood of sensitization or irritation. In one study, fresh and deteriorated tea tree oil, both the whole oil and 15 components, were applied topically to guinea pigs. Oil that had been exposed to heat and light was 3 times more likely to elicit sensitivity responses.102 Other studies suggest that when allergic dermatitis follows the use of formulated products containing tea tree oil, it is important to determine whether the sensitizing agent is the essential oil or other constituents.103 Intact human skin has been shown to be less permeable to essential oils than rat, rabbit, or pig skin, with pig skin closest in permeability to human skin; thus, animal toxicology studies may not reflect the true penetration potential or systemic effects of essential oils topically applied in humans.104 A study of essential oil penetration in porcine mucosal tissue found high permeability of monoterpenols and enhanced penetration with the addition of a hydrogel carrier.105 Thus, good penetration might be expected using gel forma-

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tions of essential oils on wounds with a dilute solution unlikely to be associated with serious adverse events.

HUMAN STUDIES Findings from published case reports and uncontrolled studies in humans with chronic wounds support further research in this area; however, these studies employed combinations of essential oils; thus, it is difficult to attribute the effect to a single oil. Sherry et al.106 reported using a mixture of tea tree oil and other essential oils for percutaneous treatment of 2 patients with osteomyelitis that had not resolved with surgery or multiple courses of antibiotics over months to years. The essential oil mixture was applied daily with noted healing after a few days. After a few weeks, the wounds had healed, cultures were clear, and symptoms had resolved. In a study of 25 patients with MRSA-infected wounds,100 a similar tea tree mixture was applied following wound debridement. Twentytwo of the wounds resolved with (n = 3) or without (n = 19) concurrent antibiotic therapy. These same authors recently published an account of using a waterbased Melaleuca solution as a successful adjunctive therapy for gangrenous leg wounds in diabetic patients.107 In an uncontrolled study among 100 longterm care residents with chronic wounds, an essential oil mixture containing tea tree oil was used as an adjunctive topical therapy. Most of the wounds reportedly healed in less time than expected with rapid reduction in inflammation, pain and wound odor.108 The efficacy of using essential oils, although not tea tree, in wound care was investigated in another small uncontrolled study among patients in a long-term care setting.109 A review article summarizing controlled trials of herbal medicines for treating bacterial infections critiqued 2 tea tree oil studies. The authors concluded that there is a need for rigorous clinical trials to evaluate efficacy.110 Tea tree oil has been evaluated as an alternative topical decolonization agent for MRSA. Tea tree oil 4% in a nasal ointment and 5% in a body wash were compared with standard treatment of 2% mupirocin nasal ointment and triclosan body wash in patients with MRSA skin colonization. Although the difference was not statistically significant, tea tree oil appeared more efficacious than the standard treatment and was better tolerated by patients.111 In this study, tea tree oil appeared to produce similar results to a standard treatment, and the authors concluded that tea tree oil may be beneficial as an alternative treatment in cases of antibiotic resistance.

DISCUSSION Both in vitro studies and case studies suggest evidence for the adjunctive use of tea tree oil in wound care. The in

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vitro studies on tea tree oil have shown good S aureus microbicidal activity at fairly low levels. Beginning research suggests possible mechanisms of action, although more work is needed in this area. In addition, tea tree oil has produced promising results in case studies and small clinical trials. The case studies included complicated disease processes that had not responded to traditional treatments. Combined, these studies present compelling evidence that tea tree could be a possible therapy for difficult to treat S aureus infections. Plant-based products and therapies, such as essential oils, are gaining recognition as promising therapeutic products. However, development of these products as therapeutic substances may not fit the traditional, pharmaceutical drug development model. First, the pharmaceutical model does not include a mechanism for recognizing natural products’ long history of human use. Second, the chemical constituents of plant-based products often vary within established ranges; thus, exact standardization may not be possible without altering the nature of the products. Third, interest in botanicals such as tea tree oil by major drug companies may be hindered by inability to obtain patent control. Finally, laboratory standards for testing products may not fit natural products, making it difficult to meet Food and Drug Administration requirements for testing. An example of the difficulty in using the classical drug development model for tea tree oil products involves antimicrobial susceptibility testing. National Committee for Clinical Laboratory Standards (NCCLS) for disc diffusion and broth microdilution susceptibility testing were created for use with watersoluble experimental drugs. Essential oils are lipophilic, and therefore cannot be solubilized in the watersoluble broths and agars that meet the NCCLS standards. Different broths or solubilizing agents need to be used to test essential oils in this manner, but these methods have not been validated according to the NCCLS standards, making it difficult to prove in vitro efficacy.25 Schuster112 outlined the drug development process and contrasted it with the World Health Organization’s Guidelines for the Assessment of Herbal Medicines. The WHO guidelines preserve most of the pharmaceutical drug development process, but also take into account the history of human use as it applies to safety and efficacy. Clinical research on tea tree oil is limited. Although there are many in vitro studies on tea tree oil and S aureus, there have been few published animal or human studies in the English-language scientific literature. Those that have been published seem to demonstrate efficacy, but have been case studies, employ small numbers, or are uncontrolled. There is little known about the pharmacology of tea tree oil in the human body. In addition, toxicology information is

limited to in vitro studies on cell lines or clinical case reports. There is also little information on mechanisms of toxicity. There is a need for more research in this area to further demonstrate efficacy and patient tolerance of tea tree oil as a topical treatment. There are more than 100 components in tea tree oil, and only about 10 have been identified as major components and active substances. More work is needed to identify active components, as well as to identify synergy or antagonism between the components. There is also a need for randomized, controlled clinical trials to demonstrate efficacy and toxicity. In order for this to occur, mechanisms need to be established to study natural products using procedures and methods that take into account their history of use and unique chemical makeup. References 1. Witte W. Antibiotic resistance in gram-positive bacteria: epidemiological aspects. J Antimicrob Chemother 1999;44(Suppl A):1-9. 2. Mulligan ME, Murray-Leisure KA, Ribner BS, et al. Methicillin-resistant Staphylococcus aureus: a consensus review of the microbiology, pathogenesis, and epidemiology with implications for prevention and management. Am J Med 1993;94:313-28. 3. Gorak EJ, Yamada SM, Brown JD. Community-acquired methicillin-resistant Staphylococcus aureus in hospitalized adults and children without known risk factors. Clin Infect Dis 1999;29:797-800. 4. Blok HE, Vriens MR, Weersink AJ, et al. Carriage of methicillinresistant Staphylococcus aureus (MRSA) after discharge from hospital: follow-up for how long? A Dutch multi-centre study. J Hosp Infect 2001;48:325-7. 5. MacKinnon MM, Allen KD. Long-term MRSA carriage in hospital patients. J Hosp Infect 2000;46:216-21. 6. Thomas MJ. Overview of resistance in the 1990’s. Chest 1999;115: 3S-8S. 7. Theaker C, Ormond-Walshe S, Azadian B, et al. MRSA in the critically ill. J Hosp Infect 2001;48:98-102. 8. Tonge H. Special focus: tissue viability. The management of infected wounds. Nurs Standard 1997;12(12):49-53. 9. Wright JB, Lam K, Burrell RE. Wound management in an era of increasing bacterial antibiotic resistance: a role for topical silver treatment. Am J Infect Control 1998;26:572-7. 10. Chan CH, Loudon KW. Activity of tea tree oil on methicillin-resistant Staphylococcus aureus (MRSA). J Hosp Infect 1998;39:244-5. 11. Arnold MS, Dempsey JM, Fishman M, et al. The best hospital practices for controlling methicillin-resistant Staphylococcus aureus: on the cutting edge. Infect Control Hosp Epidemiol 2002;23:69-76. 12. Maple PA, Hamilton-Miller JM, Brumfitt W. Comparison of the invitro activities of the topical antimicrobials azelaic acid, nitrofurazone, silver sulphadiazine and mupirocin against methicillin-resistant Staphylococcus aureus. J Antimicrob Chemother 1992;29:661-8. 13. Riley TV, Carson CF, Bowman RA, et al. Mupirocin-resistant methicillin-resistant Staphylococcus aureus in Western Australia. Med J Aust 1994;161:397-8. 14. Irizarry L, Merlin T, Rupp J, et al. Reduced susceptibility of methicillin-resistant Staphylococcus aureus to cetylpyridinium chloride and chlorhexidine. Chemotherapy 1996;42:248-52. 15. May J, Chan CH, King A, et al. Time-kill studies of tea tree oils on clinical isolates. J Antimicrob Chemother 2000;45:639-43.

Halco´n and Milkus 16. Hayashi K, Kamiya M, Hayashi T. Virucidal effects of the steam distillate from Houttuynia cordata and its components on HSV-1, influenza virus, and HIV. Planta Med 1995;61:237-41. 17. Larrondo JV, Agut M, Calvo-Torras MA. Antimicrobial activity of essences from labiates. Microbios 1995;82:171-2. 18. Tirillini B, Velasquez ER, Pellegrino R. Chemical composition and antimicrobial activity of essential oil of Piper angustifolium. Planta Med 1996;62:372-3. 19. Mikus J, Harkenthal M, Steverding D, et al. In vitro effect of essential oils and isolated mono- and sesquiterpenes on Leishmania major and Trypanosoma brucei. Planta Med 2000;66:366-8. 20. Gustafson JE, Liew YC, Chew S, et al. Effects of tea tree oil on Escherichia coli. Lett Appl Microbiol 1998;26:194-8. 21. Shapiro S, Meier A, Guggenheim B. The antimicrobial activity of essential oils and essential oil components towards oral bacteria. Oral Microbiol Immunol 1994;9:202-8. 22. Carson CF, Riley TV. Antimicrobial activity of tea tree oil. Nedlands, Western Australia: Rural Industries Research and Development Corporation; July 1998. 23. Serkedjieva J, Hay AJ. In vitro anti-influenza virus activity of a plant preparation from Geranium sanguineum L. Antiviral Res 1998;37:121-30. 24. Maudsley F, Kerr KG. Microbiological safety of essential oils used in complementary therapies and the activity of these compounds against bacterial and fungal pathogens. Support Care Cancer 1999;7:100-2. 25. Carson CF, Hammer KA, Riley TV. Broth micro-dilution method for determining the susceptibility of Escherichia coli and Staphylococcus aureus to the essential oil of Melaleuca alternifolia (tea tree oil). Microbios 1995;82:181-5. 26. Harkenthal M, Reichling J, Geiss HK, et al. Comparative study on the in vitro antibacterial activity of Australian tea tree oil, cajuput oil, niaouli oil, manuka oil, kanuka oil, and eucalyptus oil. Pharmazie 1999;54:460-3. 27. Pattnaik S, Subramanyam VR, Bapaji M, et al. Antibacterial and antifungal activity of aromatic constituents of essential oils. Microbios 1997;89:39-46. 28. Peana AT, Moretti MD, Juliano C. Chemical composition and antimicrobial action of the essential oils of Salvia desoleana and S. sclarea. Planta Med 1999;65:752-4. 29. Feinblatt HM. Cajeput-type oil for the treatment of furunculosis. J Natl Med Assoc 1960;52:32-4. 30. Hammer KA, Carson CF, Riley TV. In vitro susceptibilities of lactobacilli and organisms associated with bacterial vaginosis to Melaleuca alternifolia (tea tree) oil. Antimicrob Agents Chemother 1999;43:196. 31. Walker M. Clinical investigation of Australian Melaleuca Alternifolia oil for a variety of common foot problems. Curr Podiatry 1972;43:28-34. 32. Hammer KA, Carson CF, Riley TV. Susceptibility of transient and commensal skin flora to the essential oil of Melaleuca alternifolia (tea tree oil). Am J Infect Control 1996;24:186-9. 33. Faoagali J, George N, Leditschke JF. Does tea tree oil have a place in the topical treatment of burns? Burns 1997;23:349-51. 34. Blackwell AL. Tea tree oil and anaerobic (bacterial) vaginosis. Lancet 1991;337:300. 35. Bassett IB, Pannowitz DL, Barnetson RS. A comparative study of tea-tree oil versus benzoylperoxide in the treatment of acne. Med J Aust 1990;153:455-8. 36. Carson CF, Cookson BD, Farrelly HD, et al. Susceptibility of methicillin-resistant Staphylococcus aureus to the essential oil of Melaleuca alternifolia. J Antimicrob Chemother 1995;35:421-4. 37. Nelson RR. In-vitro activities of five plant essential oils against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. J Antimicrob Chemother 1997;40:305-6. 38. Nelson RR. Selection of resistance to the essential oil of Melaleuca alternifolia in Staphylococcus aureus. J Antimicrob Chemother 2000; 45:549-50. 39. Carson CF, Riley TV. The antimicrobial activity of tea tree oil. Med J Aust 1994;160:236.

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