- Email: [email protected]
Susceptibility to Melaleuca alternifolia (tea tree) oil of yeasts isolated from the mouths of patients with advanced cancer
Oral Oncology (2006) 42, 487–492
http://intl.elsevierhealth.com/journals/oron/
Susceptibility to Melaleuca alternifolia (tea tree) oil of yeasts isolated from the mouths of patients with advanced cancer Jeremy Bagg a,*, Margaret S. Jackson a, M. Petrina Sweeney a, Gordon Ramage b, Andrew N. Davies c a
University of Glasgow Dental School, 378 Sauchiehall Street, Glasgow G2 3JZ, United Kingdom Glasgow Caledonian University, Cowcaddens Road, Glasgow G4 0BA, United Kingdom c Department of Palliative Medicine, Royal Marsden Hospital, Downs Road, Sutton, Surrey SM2 5PT, United Kingdom b
Received 22 September 2005; accepted 3 October 2005
KEYWORDS
Summary Yeasts that are resistant to azole antifungal drugs are increasingly isolated from the mouths of cancer patients suffering from oral fungal infections. Tea tree oil is an agent possessing antimicrobial properties that may prove useful in the prevention and management of infections caused by these organisms. In this study, 301 yeasts isolated from the mouths of 199 patients suffering from advanced cancer were examined by an in vitro agar dilution assay for susceptibility to tea tree oil. All of the isolates tested were susceptible, including 41 that were known to be resistant to both fluconazole and itraconazole. Clinical studies of tea tree oil as an agent for the prevention and treatment of oral fungal infections in immunocompromised patients merit consideration. c 2005 Elsevier Ltd. All rights reserved.
Yeasts; Oral infection; Cancer; Tea tree oil
Introduction
Oral fungal infections, caused predominantly by Candida species, are common in patients suffering from advanced cancer.1–3 Evidence from recently
* Corresponding author. Tel.: +44 141 211 9701; fax: +44 141 353 1593. E-mail address: [email protected] (J. Bagg).
published studies involving this patient group suggests an increasing prevalence of infections caused by yeasts other than Candida albicans, so-called non-albicans yeasts.4,5 It is notable that some of these non-albicans species, for example Candida glabrata, often demonstrate reduced susceptibility, or resistance, to fluconazole and itraconazole,5 the commonly used triazole antifungal drugs. Furthermore, some of these resistant isolates show cross-resistance with the new triazole
1368-8375/$ - see front matter c 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2005.10.002
488 agent voriconazole.6 There is a need to identify new methods of preventing and treating oral candidosis in this highly compromised group of patients. Institution of regular oral hygiene procedures and use of agents to reduce oral dryness are helpful in this regard,7 but the applicability of novel antifungal agents also merits investigation. One agent which may be considered is tea tree oil (TTO).8 Tea tree oil is produced as a distillate of leaves of the Melaleuca alternifolia shrub, which grows in New South Wales, Australia. It comprises a complex mixture of approximately 100 components, most of which are monoterpenes, sesquiterpenes and their related alcohols.9 Tea tree oil has been shown to possess a number of therapeutic properties, including anti-inflammatory activities.10,11 However, it is best known for its antimicrobial activity against a wide spectrum of microorganisms, for example Staphylococcus aureus (including MRSA),12–14 a range of oral bacteria,15 and certain viruses, including herpes simplex virus.16,17 Tea tree oil also has potent activity against many fungi.18–20 In view of the relatively high prevalence of non-albicans yeasts, together with the increasing problem of azole resistance alluded to earlier, it is of interest that many azole-resistant types of yeast apparently remain susceptible to TTO.21–23 This raises the possibility of using TTO preparations for the treatment of established oral candidal infections. Furthermore, incorporation of TTO into preparations used for routine mouth care in palliative medicine may have a role in preventing fungal infections and reducing resultant inflammation. Many oral care products are now available containing TTO, and a recent study of a topically applied TTO-containing gel demonstrated significant reductions in the Gingival Index and Papillary Bleeding Index, although there were no reductions in plaque scores.24 The role of TTO-containing mouthwashes in treatment of oral candidosis among AIDS patients has been reported,21,22 but to the authors’ knowledge there have been no published clinical trials of TTO-containing oral care products in other groups of compromised patients, including those with advanced cancer. In order to inform the preparations for a clinical trial of a TTO-containing oral care product in a hospice setting, the present study has examined the in vitro susceptibility to TTO of a collection of well-characterised yeasts, including azole-resistant strains, isolated from the mouths of patients with advanced cancer.
J. Bagg et al.
Materials and methods Patients Yeast isolates were available from the mouths of 199 patients who were receiving palliative care for advanced cancer. These isolates had been collected and characterised during two previous studies of fungal carriage among randomly selected patients receiving palliative care in the UK.4,5 All of these patients had received the standard forms of treatment appropriate to their particular malignancies, for example surgery, radiotherapy and chemotherapy, but such modalities were no longer being pursued with a curative intent. The patients were under the care of one of three hospices, either as inpatients or day-stay patients, and all were suffering from very advanced disease with limited life expectancies. The most common diagnoses were carcinomas of the breast, bronchus, prostate and large bowel. This large group of patients was, therefore, demographically representative of those receiving treatment in a palliative medicine setting.
Yeast isolates A collection of 301 clinical isolates of oral yeasts was available. These yeasts had been isolated from oral swabs and oral rinses of the 199 patients described above. The yeast isolates were identified by germ tube tests and API 20C AUX and API ID 32C (bio-Me ´rieux S A, Marcy-l’Etoile, France) profiles. Isolates with indeterminate results were further investigated using species-specific PCR and 26S rDNA gene sequencing. All germ tube-positive isolates were screened by PCR using C. dubliniensis-specific and fungal universal primers.25 Candida albicans ATCC 90028 and C. glabrata ATCC 90030 were set up as control strains in each run of the tea tree oil susceptibility assay.
Tea tree oil susceptibility assay The susceptibility of the yeast isolates to tea tree oil was determined by an agar dilution method based on NCCLS methodology.26 To prepare the plates, aliquots (60 ml) of Mueller Hinton Agar (Oxoid, Basingstoke, England) were melted by steaming and kept molten in a 50 C water-bath. Tween 80 (300 lL) was added to each aliquot to give a final concentration of 0.5% Tween 80. Pharmaceutical grade 100% tea tree oil (Thursday Plantation; supplied by Wellbeing, Birmingham, UK), which complied with the International
Tea tree oil and oral yeasts
489
Standard ISO 4730,27 was used. This standard27 contains a chromatographic profile that stipulates minimum and maximum percentage composition values for 14 components. Serial doubling dilutions of the tea tree oil were made from 20% to 1.25% in Mueller Hinton broth. An aliquot (3 mL) of each dilution of tea tree oil was added to the molten agar samples, mixed thoroughly and each poured into a 140 mm diameter Petri dish. This gave a range of tea tree oil concentrations from 25% to 0.0625% in agar plates of a depth of 3–4 mm. A growth control plate containing no tea tree oil was included in each test run. The plates were used immediately or stored at 5 C for up to 5 days. To prepare the yeast inocula, test and control organisms were cultured at 37 C for 18–24 h on Sabouraud’s agar (Lab M, Bury, Lancashire, England). A direct suspension of six isolated colonies was prepared from a fresh plate, in Mueller Hinton broth. This suspension was adjusted to a turbidity of 0.5 against a MacFarland standard, equating to 1–2 · 108 CFU/mL. A further 1 in 10 dilution of this suspension was prepared in Mueller Hinton broth to yield a cell concentration of approximately 1 · 107 CFU/mL, and this was used as the inoculum. The prepared yeast cell suspensions were inoculated onto the agar plates within 15 min. The plates were inoculated with the yeast cell suspensions using a multipoint inoculator (WR Ltd, Leicestershire, England). This applied 1–2 lL (approximately 104 CFU) of each inoculum onto the respective plates. A growth control plate, containing no tea tree oil, was inoculated first then the remaining plates were inoculated, starting with the plate containing the lowest concentration of tea tree oil and working up to the most concentrated. Finally, a second growth control plate was inoculated to ensure that no carry-over of tea tree Table 1
oil had occurred on the inoculator tips. The plates were allowed to stand for 15 min after inoculation, to allow the inocula an opportunity to localise on the TTO-containing agar, then incubated at 30 C for 48 h. The lowest concentration of TTO to inhibit visible growth of the yeast was recorded as the MIC. If any inconsistencies were noted on the control plates, then results from that assay run were disregarded.
Results The MIC values for the control strains were 0.125– 1% for C. albicans ATCC 90028 and 0.125–0.5% for C. glabrata ATCC 90030. The range of MIC’s of tea tree oil for the yeasts tested is shown in Table 1. The MIC50 values were 0.5% for C. albicans and C. dubliniensis and 0.25% for C. glabrata, C. tropicalis and S. cerevisiae. The MIC90 values for C. albicans, C. glabrata and C. dubliniensis were 1%, whilst for C. tropicalis and S. cerevisiae the MIC90 was 0.5%. It was not possible to determine accurate MIC50 and MIC90 values for C. parapsilosis, C. krusei, C. guilliermondii, C. lusitaniae and C. famata, because there were insufficient isolates of each of these species. Table 2 summarises the MIC’s of tea tree oil for the 41 isolates that were dually resistant to both fluconazole and itraconazole. Ten of the C. glabrata isolates had MIC’s to voriconazole of P2 lg/mL, suggesting that this subset of isolates was also voriconazole resistant.28 The MIC50 values of TTO were 0.5% for C. albicans and 0.25% for C. glabrata. The MIC90 values for both C. albicans and C. glabrata were 1%. There is no evidence to suggest that the azole-resistant yeasts had reduced susceptibility to TTO.
The minimum inhibitory concentrations of tea tree oil for the 301 yeast isolates tested
Species
Minimum inhibitory concentration of tea tree oil (% v/v) 0.25%
Total
1%
0.5%
0.125%
C. albicans C. glabrata S. cerevisiae C. tropicalis C. dubliniensis C. parapsilosis C. krusei C. guilliermondii C. lusitaniae C. famata
32 19 2 1 3 – – – – –
58 15 9 8 5 1 1 1 1 –
68 34 6 11 4 3 2 2 2 1
1 3 7 – 1 – – – – –
159 71 24 20 13 4 3 3 3 1
Total
57
99
133
12
301
490
J. Bagg et al.
Table 2 The minimum inhibitory concentrations of tea tree oil for the 41 yeast isolates which were resistant to fluconazole and itraconazole Species
Minimum inhibitory concentration of tea tree oil (% v/v)
Total
1%
0.5%
0.25%
0.125%
C. albicans C. glabrata S. cerevisiae C. tropicalis C. parapsilosis
4 1 – – –
2 8 4 – –
5 13 – 1 1
– 2 – – –
11 24 4 1 1
Total
5
14
20
2
41
Discussion Oral candidosis is a common opportunistic infection among patients with advanced cancer1–3 and its effective management is therefore an important element of palliative medicine.7 The availability of effective systemic antifungal drugs such as fluconazole has been a significant clinical advance, but the recurrent nature of oral candidosis in these patients and use of repeated courses of antifungal agents has raised fears of epidemiological shifts in fungal populations together with associated antifungal drug resistance.4–6 This is not just an issue in palliative medicine, but also in other branches of oncology29,30 and in a range of medical and surgical specialties.31 Novel approaches to prevention and treatment of mucosal fungal infections are, therefore, applicable in many clinical fields. New antifungal drugs, for example caspofungin,32 show promise, but use of a topical agent such as TTO merits consideration, since resistance is less likely to become a problem.23 The results of this in vitro susceptibility study suggest that TTO should be considered as a potential preventive or therapeutic agent in the management of oral candidosis among patients with advanced cancer. It could be used as a mouthwash or, for severely debilitated patients, used as an adjunctive measure during regular oral lavage. The broad spectrum of activity of TTO would also allow it to play a role in reducing colonisation with Staphylococcus aureus12–14 and coliforms,33 other potential pathogens commonly isolated from the mouths of cancer patients.34 The activity of TTO against herpes simplex virus16,17 may also be helpful in the prevention of severe HSV reactivations, another important clinical problem for the terminally ill.7 It is important that mouthwash preparations for this group of patients are water-based rather than alcohol-based. Tea tree oil is not readily watersoluble, but both alcohol-based and water-based mouthwashes are now available. There is some evi-
dence that both are clinically effective in HIVinfected patients with oral candidosis,22 but additional clinical trial data based on studies with larger numbers of participants and other patient groups are necessary. Another important issue to consider is the effectiveness of antifungal agents against yeasts growing within the oral cavity as complex biofilm structures. In vitro antifungal drug susceptibility testing is generally carried out on planktonic organisms, but it is now clear that fungi in nature typically exist in a biofilm lifestyle.35 Many antifungal agents, including azoles, are significantly less effective against yeasts which are growing in biofilms.35 Consequently, the potential for alternative therapies that are active against oral biofilms is an exciting prospect. The effectiveness of TTO against yeasts in biofilms remains to be determined and is the subject of ongoing work by our group. One of the most important findings of this study and of other recently published papers21–23 is the effectiveness of TTO against azole-resistant yeasts. All of the isolates tested in the present study, including the fluconazole- and itraconazole-resistant subset, were susceptible to TTO at concentrations that are present in commercially available oral care products. These findings support the establishment of further clinical trials to examine the role of TTO-containing oral care products. Two additional factors that need to be examined carefully are patient compliance and the potential toxicity of TTO. Tea tree oil has a strong taste and the maximum concentration that can be tolerated is 1%, though flavourings can be added to help to mask the taste. There have also been isolated case reports of cutaneous sensitivity to tea tree oil, though the basis of the sensitivity and its incidence rate are not known.36 However, such reactions are uncommon in response to application of pharmaceutical grade tea tree oil. Adverse effects, including a burning sensation,21,22,37 stinging22 and unpleasant taste,37 have all been reported
Tea tree oil and oral yeasts by patients using TTO mouthwash. However, two of these studies used alcohol-based preparations, which may explain the burning. Furthermore, the burning was often noted during the early stages of therapy and reduced as the oral lesions responded to treatment. This would be less of a problem if the TTO preparation were used as a prophylactic measure. Clearly, product formulation will be critical and further research in this area is required. In summary, this study has shown that a large collection of yeasts isolated from the mouths of patients receiving palliative care for advanced cancer in three UK hospices were all susceptible in vitro to TTO. The potential role of water-based TTO preparations for the prevention and treatment of oral candidosis in this patient population should now be examined in controlled clinical trials.
Acknowledgement Professor David Coleman, University of Dublin, is thanked for his help in confirming the identifications of isolates of C. dubliniensis.
References 1. Aldred MJ, Addy M, Bagg J, Finlay I. Oral health in the terminally ill: a cross-sectional pilot survey. Spec Care Dentist 1991;11(2):59–62. 2. Clarke JGM, Wilson J, Von Haacke NP, Milne LJR. Oral candidiasis in terminal illness. Health Bull (Edin) 1987;45(5):268–71. 3. Finlay IG. Oral symptoms and candida in the terminally ill. Brit Med J 1986;292:592–3. 4. Davies AN, Brailsford S, Broadley K, Beighton D. Oral yeast carriage in patients with advanced cancer. Oral Microbiol Immunol 2002;17(2):79–84. 5. Bagg J, Sweeney MP, Lewis MAO, et al. High prevalence of non-albicans yeasts and detection of anti-fungal resistance in the oral flora of patients with advanced cancer. Palliative Med 2003;17(6):477–81. 6. Bagg J, Sweeney MP, Davies AN, Jackson MS, Brailsford S. Voriconazole susceptibility of yeasts isolated from the mouths of patients with advanced cancer. J Med Microbiol 2005;54(10):959–64. 7. Davies A, Finlay I. Oral care in advanced disease. Oxford: Oxford University Press; 2005. 8. Carson CF, Riley TV. Antimicrobial activity of the essential oil of Melaleuca alternifolia. Lett Appl Microbiol 1993;16(1):49–55. 9. Brophy JJ, Davies NW, Southwell IA, Stiff IA, Williams IR. Gas chromatographic quality control for oil of Melaleuca terpinen-4-ol (Australian tea tree). J Agric Food Chem 1989;37(5):1330–5. 10. Brand C, Grimbaldeston MA, Gamble JR, Drew J, FinlayJones JJ, Hart PH. Tea tree oil reduces the swelling associated with the efferent phase of a contact hypersensitivity response. Inflamm Res 2002;51(5):236–44.
491 11. Koh KJ, Pearce AL, Marshman G, Finlay-Jones JJ, Hart PH. Tea tree oil reduces histamine-induced skin inflammation. Brit J Dermatol 2002;147(6):1212–7. 12. Chan CH, Loudon KW. Activity of tea tree oil on methicillinresistant Staphylococcus aureus (MRSA). J Hosp Infect 1998;39(3):244–5. 13. Caelli M, Porteous J, Carson CF, Heller R, Riley TV. Tea tree oil as an alternative topical decolonization agent for methicillin-resistant Staphylococcus aureus. J Hosp Infect 2000;46(3):236–7. 14. Dryden MS, Dailly S, Crouch M. A randomized, controlled trial of tea tree topical preparations versus a standard topical regimen for the clearance of MRSA colonization. J Hosp Infect 2004;56(4):283–6. 15. Hammer KA, Dry L, Johnson M, Michalak EM, Carson CF, Riley TV. Susceptibility of oral bacteria to Melaleuca alternifolia (tea tree) oil in vitro. Oral Microbiol Immunol 2003;18(6):389–92. 16. Schnitzler P, Scho ¨n K, Reichling J. Antiviral activity of Australian tea tree oil against herpes simplex virus in cell culture. Pharmazie 2001;56(4):343–7. 17. Carson CF, Ashton L, Dry L, Smith DW, Riley TV. Melaleuca alternifolia (tea tree) oil gel (6%) for the treatment of recurrent herpes labialis. J Antimicrob Chemother 2001;48(3):450–1. 18. Hammer KA, Carson CF, Riley TV. Antifungal activity of the components of Melaleuca alternifolia (tea tree) oil. J Appl Microbiol 2003;95(4):853–60. 19. Oliva B, Piccirilli E, Ceddia T, Pontieri E, Aureli P, Ferrini AM. Antimycotic activity of Melaleuca alternifolia essential oil and its major components. Lett Appl Microbiol 2003;37(2):185–7. 20. Hammer KA, Carson CF, Riley TV. Antifungal effects of Melaleuca alternifolia (tea tree) oil and its components on Candida albicans, Candida glabrata and Saccharomyces cerevisiae. J Antimicrob Chemother 2004;53(6):1081–5. 21. Jandourek A, Vaishampayan JK, Vazquez JA. Efficacy of melaleuca oral solution for the treatment of fluconazole refractory oral candidiasis in AIDS patients. AIDS 1998;12(9):1033–7. 22. Vazquez JA, Zawawi AA. Efficacy of alcohol-based and alcohol-free melaleuca oral solution for the treatment of fluconazole-refractory oral candidiasis in patients with AIDS. HIV Clin Trials 2002;3(5):379–85. 23. Mondello F, De Bernardis F, Girolamo A, Salvatore G, Cassone A. In vitro and in vivo activity of tea tree oil against azole-susceptible and—resistant human pathogenic yeasts. J Antimicrob Chemother 2003;51(5):1223–9. 24. Soukoulis S, Hirsch R. The effects of a tea tree oilcontaining gel on plaque and chronic gingivitis. Aust Dent J 2004;49(2):78–83. 25. Donnelly SM, Sullivan DJ, Shanley DB, Coleman DC. Phylogenetic analysis and rapid identification of Candida dubliniensis based on analysis of ACT1 intron and exon sequences. Microbiology 1999;145(8):1871–82. 26. NCCLS. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 4th ed. Approved Standard, January 1997, M7-A4, vol. 17(2). 27. International Organization for Standardization. ISO 4730 Oil of Melaleuca, Terpinen-4-ol type (Tea Tree Oil), 1996. Geneva: International Organization for Standardization. 28. Pelletier R, Loranger L, Marcotte H, de Carolis E. Voriconazole and fluconazole suceptibility of Candida isolates. J Med Microbiol 2002;51(6):479–83. 29. Redding SW, Dahiya MC, Kirkpatrick WR, Coco BJ, Patterson TF, Fothergill AW, et al. Candida glabrata is an emerging cause of oropharyngeal candidiasis in patients
492
30.
31.
32.
33.
receiving radiation for head and neck cancer. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;97(1):47–52. Chryssanthou E, Cherif H, Petrini B, Kalin M, Bjorkholm M. Surveillance of triazole susceptibility of colonizing yeasts in patients with haematological malignancies. Scand J Infect Dis 2004;36(11–12):855–9. Krcmery V, Barnes AJ. Non-albicans Candida spp causing fungaemia: pathogenicity and antifungal resistance. J Hosp Infect 2002;50(4):243–60. Groll AH, Walsh TJ. Caspofungin: pharmacology, safety and therapeutic potential in superficial and invasive fungal infections. Expert Opin Invest Drugs 2001;10(8):1545–58. Banes-Marshall L, Cawley P, Phillips CA. In vitro activity of Melaleuca alternifolia (tea tree) oil against bacterial and Candida spp. isolates from clinical specimens. Br J Biomed Sci 2001;58(3):139–45.
J. Bagg et al. 34. Jobbins J, Bagg J, Parsons K, Finlay I, Addy M, Newcombe RG. Oral carriage of yeasts, coliforms and staphylococci in patients with advanced malignant disease. J Oral Pathol Med 1992;21(7):305–8. 35. Ramage G, Lo ´pez-Ribot JL. Fungal biofilms and drug resistance. In: San Blas G, Calderone RA, editors. Pathogenic fungi: host interactions and emerging strategies for control. Norwich: Horizon Scientific Press; 2004, Chapter 11. 36. Carson CF, Riley TV, Cookson BD. Efficacy and safety of tea tree oil as a topical antimicrobial agent. J Hosp Infect 1998;40(3):175–8. 37. Groppo FC, Ramacciato JC, Simoes RP, Florio FM, Sartoratto A. Antimicrobial activity of garlic, tea tree oil, and chlorhexidine against oral microorganisms. Int Dent J 2002;52(6):433–7.
http://intl.elsevierhealth.com/journals/oron/
Susceptibility to Melaleuca alternifolia (tea tree) oil of yeasts isolated from the mouths of patients with advanced cancer Jeremy Bagg a,*, Margaret S. Jackson a, M. Petrina Sweeney a, Gordon Ramage b, Andrew N. Davies c a
University of Glasgow Dental School, 378 Sauchiehall Street, Glasgow G2 3JZ, United Kingdom Glasgow Caledonian University, Cowcaddens Road, Glasgow G4 0BA, United Kingdom c Department of Palliative Medicine, Royal Marsden Hospital, Downs Road, Sutton, Surrey SM2 5PT, United Kingdom b
Received 22 September 2005; accepted 3 October 2005
KEYWORDS
Summary Yeasts that are resistant to azole antifungal drugs are increasingly isolated from the mouths of cancer patients suffering from oral fungal infections. Tea tree oil is an agent possessing antimicrobial properties that may prove useful in the prevention and management of infections caused by these organisms. In this study, 301 yeasts isolated from the mouths of 199 patients suffering from advanced cancer were examined by an in vitro agar dilution assay for susceptibility to tea tree oil. All of the isolates tested were susceptible, including 41 that were known to be resistant to both fluconazole and itraconazole. Clinical studies of tea tree oil as an agent for the prevention and treatment of oral fungal infections in immunocompromised patients merit consideration. c 2005 Elsevier Ltd. All rights reserved.
Yeasts; Oral infection; Cancer; Tea tree oil
Introduction
Oral fungal infections, caused predominantly by Candida species, are common in patients suffering from advanced cancer.1–3 Evidence from recently
* Corresponding author. Tel.: +44 141 211 9701; fax: +44 141 353 1593. E-mail address: [email protected] (J. Bagg).
published studies involving this patient group suggests an increasing prevalence of infections caused by yeasts other than Candida albicans, so-called non-albicans yeasts.4,5 It is notable that some of these non-albicans species, for example Candida glabrata, often demonstrate reduced susceptibility, or resistance, to fluconazole and itraconazole,5 the commonly used triazole antifungal drugs. Furthermore, some of these resistant isolates show cross-resistance with the new triazole
1368-8375/$ - see front matter c 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2005.10.002
488 agent voriconazole.6 There is a need to identify new methods of preventing and treating oral candidosis in this highly compromised group of patients. Institution of regular oral hygiene procedures and use of agents to reduce oral dryness are helpful in this regard,7 but the applicability of novel antifungal agents also merits investigation. One agent which may be considered is tea tree oil (TTO).8 Tea tree oil is produced as a distillate of leaves of the Melaleuca alternifolia shrub, which grows in New South Wales, Australia. It comprises a complex mixture of approximately 100 components, most of which are monoterpenes, sesquiterpenes and their related alcohols.9 Tea tree oil has been shown to possess a number of therapeutic properties, including anti-inflammatory activities.10,11 However, it is best known for its antimicrobial activity against a wide spectrum of microorganisms, for example Staphylococcus aureus (including MRSA),12–14 a range of oral bacteria,15 and certain viruses, including herpes simplex virus.16,17 Tea tree oil also has potent activity against many fungi.18–20 In view of the relatively high prevalence of non-albicans yeasts, together with the increasing problem of azole resistance alluded to earlier, it is of interest that many azole-resistant types of yeast apparently remain susceptible to TTO.21–23 This raises the possibility of using TTO preparations for the treatment of established oral candidal infections. Furthermore, incorporation of TTO into preparations used for routine mouth care in palliative medicine may have a role in preventing fungal infections and reducing resultant inflammation. Many oral care products are now available containing TTO, and a recent study of a topically applied TTO-containing gel demonstrated significant reductions in the Gingival Index and Papillary Bleeding Index, although there were no reductions in plaque scores.24 The role of TTO-containing mouthwashes in treatment of oral candidosis among AIDS patients has been reported,21,22 but to the authors’ knowledge there have been no published clinical trials of TTO-containing oral care products in other groups of compromised patients, including those with advanced cancer. In order to inform the preparations for a clinical trial of a TTO-containing oral care product in a hospice setting, the present study has examined the in vitro susceptibility to TTO of a collection of well-characterised yeasts, including azole-resistant strains, isolated from the mouths of patients with advanced cancer.
J. Bagg et al.
Materials and methods Patients Yeast isolates were available from the mouths of 199 patients who were receiving palliative care for advanced cancer. These isolates had been collected and characterised during two previous studies of fungal carriage among randomly selected patients receiving palliative care in the UK.4,5 All of these patients had received the standard forms of treatment appropriate to their particular malignancies, for example surgery, radiotherapy and chemotherapy, but such modalities were no longer being pursued with a curative intent. The patients were under the care of one of three hospices, either as inpatients or day-stay patients, and all were suffering from very advanced disease with limited life expectancies. The most common diagnoses were carcinomas of the breast, bronchus, prostate and large bowel. This large group of patients was, therefore, demographically representative of those receiving treatment in a palliative medicine setting.
Yeast isolates A collection of 301 clinical isolates of oral yeasts was available. These yeasts had been isolated from oral swabs and oral rinses of the 199 patients described above. The yeast isolates were identified by germ tube tests and API 20C AUX and API ID 32C (bio-Me ´rieux S A, Marcy-l’Etoile, France) profiles. Isolates with indeterminate results were further investigated using species-specific PCR and 26S rDNA gene sequencing. All germ tube-positive isolates were screened by PCR using C. dubliniensis-specific and fungal universal primers.25 Candida albicans ATCC 90028 and C. glabrata ATCC 90030 were set up as control strains in each run of the tea tree oil susceptibility assay.
Tea tree oil susceptibility assay The susceptibility of the yeast isolates to tea tree oil was determined by an agar dilution method based on NCCLS methodology.26 To prepare the plates, aliquots (60 ml) of Mueller Hinton Agar (Oxoid, Basingstoke, England) were melted by steaming and kept molten in a 50 C water-bath. Tween 80 (300 lL) was added to each aliquot to give a final concentration of 0.5% Tween 80. Pharmaceutical grade 100% tea tree oil (Thursday Plantation; supplied by Wellbeing, Birmingham, UK), which complied with the International
Tea tree oil and oral yeasts
489
Standard ISO 4730,27 was used. This standard27 contains a chromatographic profile that stipulates minimum and maximum percentage composition values for 14 components. Serial doubling dilutions of the tea tree oil were made from 20% to 1.25% in Mueller Hinton broth. An aliquot (3 mL) of each dilution of tea tree oil was added to the molten agar samples, mixed thoroughly and each poured into a 140 mm diameter Petri dish. This gave a range of tea tree oil concentrations from 25% to 0.0625% in agar plates of a depth of 3–4 mm. A growth control plate containing no tea tree oil was included in each test run. The plates were used immediately or stored at 5 C for up to 5 days. To prepare the yeast inocula, test and control organisms were cultured at 37 C for 18–24 h on Sabouraud’s agar (Lab M, Bury, Lancashire, England). A direct suspension of six isolated colonies was prepared from a fresh plate, in Mueller Hinton broth. This suspension was adjusted to a turbidity of 0.5 against a MacFarland standard, equating to 1–2 · 108 CFU/mL. A further 1 in 10 dilution of this suspension was prepared in Mueller Hinton broth to yield a cell concentration of approximately 1 · 107 CFU/mL, and this was used as the inoculum. The prepared yeast cell suspensions were inoculated onto the agar plates within 15 min. The plates were inoculated with the yeast cell suspensions using a multipoint inoculator (WR Ltd, Leicestershire, England). This applied 1–2 lL (approximately 104 CFU) of each inoculum onto the respective plates. A growth control plate, containing no tea tree oil, was inoculated first then the remaining plates were inoculated, starting with the plate containing the lowest concentration of tea tree oil and working up to the most concentrated. Finally, a second growth control plate was inoculated to ensure that no carry-over of tea tree Table 1
oil had occurred on the inoculator tips. The plates were allowed to stand for 15 min after inoculation, to allow the inocula an opportunity to localise on the TTO-containing agar, then incubated at 30 C for 48 h. The lowest concentration of TTO to inhibit visible growth of the yeast was recorded as the MIC. If any inconsistencies were noted on the control plates, then results from that assay run were disregarded.
Results The MIC values for the control strains were 0.125– 1% for C. albicans ATCC 90028 and 0.125–0.5% for C. glabrata ATCC 90030. The range of MIC’s of tea tree oil for the yeasts tested is shown in Table 1. The MIC50 values were 0.5% for C. albicans and C. dubliniensis and 0.25% for C. glabrata, C. tropicalis and S. cerevisiae. The MIC90 values for C. albicans, C. glabrata and C. dubliniensis were 1%, whilst for C. tropicalis and S. cerevisiae the MIC90 was 0.5%. It was not possible to determine accurate MIC50 and MIC90 values for C. parapsilosis, C. krusei, C. guilliermondii, C. lusitaniae and C. famata, because there were insufficient isolates of each of these species. Table 2 summarises the MIC’s of tea tree oil for the 41 isolates that were dually resistant to both fluconazole and itraconazole. Ten of the C. glabrata isolates had MIC’s to voriconazole of P2 lg/mL, suggesting that this subset of isolates was also voriconazole resistant.28 The MIC50 values of TTO were 0.5% for C. albicans and 0.25% for C. glabrata. The MIC90 values for both C. albicans and C. glabrata were 1%. There is no evidence to suggest that the azole-resistant yeasts had reduced susceptibility to TTO.
The minimum inhibitory concentrations of tea tree oil for the 301 yeast isolates tested
Species
Minimum inhibitory concentration of tea tree oil (% v/v) 0.25%
Total
1%
0.5%
0.125%
C. albicans C. glabrata S. cerevisiae C. tropicalis C. dubliniensis C. parapsilosis C. krusei C. guilliermondii C. lusitaniae C. famata
32 19 2 1 3 – – – – –
58 15 9 8 5 1 1 1 1 –
68 34 6 11 4 3 2 2 2 1
1 3 7 – 1 – – – – –
159 71 24 20 13 4 3 3 3 1
Total
57
99
133
12
301
490
J. Bagg et al.
Table 2 The minimum inhibitory concentrations of tea tree oil for the 41 yeast isolates which were resistant to fluconazole and itraconazole Species
Minimum inhibitory concentration of tea tree oil (% v/v)
Total
1%
0.5%
0.25%
0.125%
C. albicans C. glabrata S. cerevisiae C. tropicalis C. parapsilosis
4 1 – – –
2 8 4 – –
5 13 – 1 1
– 2 – – –
11 24 4 1 1
Total
5
14
20
2
41
Discussion Oral candidosis is a common opportunistic infection among patients with advanced cancer1–3 and its effective management is therefore an important element of palliative medicine.7 The availability of effective systemic antifungal drugs such as fluconazole has been a significant clinical advance, but the recurrent nature of oral candidosis in these patients and use of repeated courses of antifungal agents has raised fears of epidemiological shifts in fungal populations together with associated antifungal drug resistance.4–6 This is not just an issue in palliative medicine, but also in other branches of oncology29,30 and in a range of medical and surgical specialties.31 Novel approaches to prevention and treatment of mucosal fungal infections are, therefore, applicable in many clinical fields. New antifungal drugs, for example caspofungin,32 show promise, but use of a topical agent such as TTO merits consideration, since resistance is less likely to become a problem.23 The results of this in vitro susceptibility study suggest that TTO should be considered as a potential preventive or therapeutic agent in the management of oral candidosis among patients with advanced cancer. It could be used as a mouthwash or, for severely debilitated patients, used as an adjunctive measure during regular oral lavage. The broad spectrum of activity of TTO would also allow it to play a role in reducing colonisation with Staphylococcus aureus12–14 and coliforms,33 other potential pathogens commonly isolated from the mouths of cancer patients.34 The activity of TTO against herpes simplex virus16,17 may also be helpful in the prevention of severe HSV reactivations, another important clinical problem for the terminally ill.7 It is important that mouthwash preparations for this group of patients are water-based rather than alcohol-based. Tea tree oil is not readily watersoluble, but both alcohol-based and water-based mouthwashes are now available. There is some evi-
dence that both are clinically effective in HIVinfected patients with oral candidosis,22 but additional clinical trial data based on studies with larger numbers of participants and other patient groups are necessary. Another important issue to consider is the effectiveness of antifungal agents against yeasts growing within the oral cavity as complex biofilm structures. In vitro antifungal drug susceptibility testing is generally carried out on planktonic organisms, but it is now clear that fungi in nature typically exist in a biofilm lifestyle.35 Many antifungal agents, including azoles, are significantly less effective against yeasts which are growing in biofilms.35 Consequently, the potential for alternative therapies that are active against oral biofilms is an exciting prospect. The effectiveness of TTO against yeasts in biofilms remains to be determined and is the subject of ongoing work by our group. One of the most important findings of this study and of other recently published papers21–23 is the effectiveness of TTO against azole-resistant yeasts. All of the isolates tested in the present study, including the fluconazole- and itraconazole-resistant subset, were susceptible to TTO at concentrations that are present in commercially available oral care products. These findings support the establishment of further clinical trials to examine the role of TTO-containing oral care products. Two additional factors that need to be examined carefully are patient compliance and the potential toxicity of TTO. Tea tree oil has a strong taste and the maximum concentration that can be tolerated is 1%, though flavourings can be added to help to mask the taste. There have also been isolated case reports of cutaneous sensitivity to tea tree oil, though the basis of the sensitivity and its incidence rate are not known.36 However, such reactions are uncommon in response to application of pharmaceutical grade tea tree oil. Adverse effects, including a burning sensation,21,22,37 stinging22 and unpleasant taste,37 have all been reported
Tea tree oil and oral yeasts by patients using TTO mouthwash. However, two of these studies used alcohol-based preparations, which may explain the burning. Furthermore, the burning was often noted during the early stages of therapy and reduced as the oral lesions responded to treatment. This would be less of a problem if the TTO preparation were used as a prophylactic measure. Clearly, product formulation will be critical and further research in this area is required. In summary, this study has shown that a large collection of yeasts isolated from the mouths of patients receiving palliative care for advanced cancer in three UK hospices were all susceptible in vitro to TTO. The potential role of water-based TTO preparations for the prevention and treatment of oral candidosis in this patient population should now be examined in controlled clinical trials.
Acknowledgement Professor David Coleman, University of Dublin, is thanked for his help in confirming the identifications of isolates of C. dubliniensis.
References 1. Aldred MJ, Addy M, Bagg J, Finlay I. Oral health in the terminally ill: a cross-sectional pilot survey. Spec Care Dentist 1991;11(2):59–62. 2. Clarke JGM, Wilson J, Von Haacke NP, Milne LJR. Oral candidiasis in terminal illness. Health Bull (Edin) 1987;45(5):268–71. 3. Finlay IG. Oral symptoms and candida in the terminally ill. Brit Med J 1986;292:592–3. 4. Davies AN, Brailsford S, Broadley K, Beighton D. Oral yeast carriage in patients with advanced cancer. Oral Microbiol Immunol 2002;17(2):79–84. 5. Bagg J, Sweeney MP, Lewis MAO, et al. High prevalence of non-albicans yeasts and detection of anti-fungal resistance in the oral flora of patients with advanced cancer. Palliative Med 2003;17(6):477–81. 6. Bagg J, Sweeney MP, Davies AN, Jackson MS, Brailsford S. Voriconazole susceptibility of yeasts isolated from the mouths of patients with advanced cancer. J Med Microbiol 2005;54(10):959–64. 7. Davies A, Finlay I. Oral care in advanced disease. Oxford: Oxford University Press; 2005. 8. Carson CF, Riley TV. Antimicrobial activity of the essential oil of Melaleuca alternifolia. Lett Appl Microbiol 1993;16(1):49–55. 9. Brophy JJ, Davies NW, Southwell IA, Stiff IA, Williams IR. Gas chromatographic quality control for oil of Melaleuca terpinen-4-ol (Australian tea tree). J Agric Food Chem 1989;37(5):1330–5. 10. Brand C, Grimbaldeston MA, Gamble JR, Drew J, FinlayJones JJ, Hart PH. Tea tree oil reduces the swelling associated with the efferent phase of a contact hypersensitivity response. Inflamm Res 2002;51(5):236–44.
491 11. Koh KJ, Pearce AL, Marshman G, Finlay-Jones JJ, Hart PH. Tea tree oil reduces histamine-induced skin inflammation. Brit J Dermatol 2002;147(6):1212–7. 12. Chan CH, Loudon KW. Activity of tea tree oil on methicillinresistant Staphylococcus aureus (MRSA). J Hosp Infect 1998;39(3):244–5. 13. Caelli M, Porteous J, Carson CF, Heller R, Riley TV. Tea tree oil as an alternative topical decolonization agent for methicillin-resistant Staphylococcus aureus. J Hosp Infect 2000;46(3):236–7. 14. Dryden MS, Dailly S, Crouch M. A randomized, controlled trial of tea tree topical preparations versus a standard topical regimen for the clearance of MRSA colonization. J Hosp Infect 2004;56(4):283–6. 15. Hammer KA, Dry L, Johnson M, Michalak EM, Carson CF, Riley TV. Susceptibility of oral bacteria to Melaleuca alternifolia (tea tree) oil in vitro. Oral Microbiol Immunol 2003;18(6):389–92. 16. Schnitzler P, Scho ¨n K, Reichling J. Antiviral activity of Australian tea tree oil against herpes simplex virus in cell culture. Pharmazie 2001;56(4):343–7. 17. Carson CF, Ashton L, Dry L, Smith DW, Riley TV. Melaleuca alternifolia (tea tree) oil gel (6%) for the treatment of recurrent herpes labialis. J Antimicrob Chemother 2001;48(3):450–1. 18. Hammer KA, Carson CF, Riley TV. Antifungal activity of the components of Melaleuca alternifolia (tea tree) oil. J Appl Microbiol 2003;95(4):853–60. 19. Oliva B, Piccirilli E, Ceddia T, Pontieri E, Aureli P, Ferrini AM. Antimycotic activity of Melaleuca alternifolia essential oil and its major components. Lett Appl Microbiol 2003;37(2):185–7. 20. Hammer KA, Carson CF, Riley TV. Antifungal effects of Melaleuca alternifolia (tea tree) oil and its components on Candida albicans, Candida glabrata and Saccharomyces cerevisiae. J Antimicrob Chemother 2004;53(6):1081–5. 21. Jandourek A, Vaishampayan JK, Vazquez JA. Efficacy of melaleuca oral solution for the treatment of fluconazole refractory oral candidiasis in AIDS patients. AIDS 1998;12(9):1033–7. 22. Vazquez JA, Zawawi AA. Efficacy of alcohol-based and alcohol-free melaleuca oral solution for the treatment of fluconazole-refractory oral candidiasis in patients with AIDS. HIV Clin Trials 2002;3(5):379–85. 23. Mondello F, De Bernardis F, Girolamo A, Salvatore G, Cassone A. In vitro and in vivo activity of tea tree oil against azole-susceptible and—resistant human pathogenic yeasts. J Antimicrob Chemother 2003;51(5):1223–9. 24. Soukoulis S, Hirsch R. The effects of a tea tree oilcontaining gel on plaque and chronic gingivitis. Aust Dent J 2004;49(2):78–83. 25. Donnelly SM, Sullivan DJ, Shanley DB, Coleman DC. Phylogenetic analysis and rapid identification of Candida dubliniensis based on analysis of ACT1 intron and exon sequences. Microbiology 1999;145(8):1871–82. 26. NCCLS. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 4th ed. Approved Standard, January 1997, M7-A4, vol. 17(2). 27. International Organization for Standardization. ISO 4730 Oil of Melaleuca, Terpinen-4-ol type (Tea Tree Oil), 1996. Geneva: International Organization for Standardization. 28. Pelletier R, Loranger L, Marcotte H, de Carolis E. Voriconazole and fluconazole suceptibility of Candida isolates. J Med Microbiol 2002;51(6):479–83. 29. Redding SW, Dahiya MC, Kirkpatrick WR, Coco BJ, Patterson TF, Fothergill AW, et al. Candida glabrata is an emerging cause of oropharyngeal candidiasis in patients
492
30.
31.
32.
33.
receiving radiation for head and neck cancer. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;97(1):47–52. Chryssanthou E, Cherif H, Petrini B, Kalin M, Bjorkholm M. Surveillance of triazole susceptibility of colonizing yeasts in patients with haematological malignancies. Scand J Infect Dis 2004;36(11–12):855–9. Krcmery V, Barnes AJ. Non-albicans Candida spp causing fungaemia: pathogenicity and antifungal resistance. J Hosp Infect 2002;50(4):243–60. Groll AH, Walsh TJ. Caspofungin: pharmacology, safety and therapeutic potential in superficial and invasive fungal infections. Expert Opin Invest Drugs 2001;10(8):1545–58. Banes-Marshall L, Cawley P, Phillips CA. In vitro activity of Melaleuca alternifolia (tea tree) oil against bacterial and Candida spp. isolates from clinical specimens. Br J Biomed Sci 2001;58(3):139–45.
J. Bagg et al. 34. Jobbins J, Bagg J, Parsons K, Finlay I, Addy M, Newcombe RG. Oral carriage of yeasts, coliforms and staphylococci in patients with advanced malignant disease. J Oral Pathol Med 1992;21(7):305–8. 35. Ramage G, Lo ´pez-Ribot JL. Fungal biofilms and drug resistance. In: San Blas G, Calderone RA, editors. Pathogenic fungi: host interactions and emerging strategies for control. Norwich: Horizon Scientific Press; 2004, Chapter 11. 36. Carson CF, Riley TV, Cookson BD. Efficacy and safety of tea tree oil as a topical antimicrobial agent. J Hosp Infect 1998;40(3):175–8. 37. Groppo FC, Ramacciato JC, Simoes RP, Florio FM, Sartoratto A. Antimicrobial activity of garlic, tea tree oil, and chlorhexidine against oral microorganisms. Int Dent J 2002;52(6):433–7.