Candidosis management: antifungal, cytotoxic and immunomodulatory properties of tea tree oil and its derivative components

Milligan, Steven George (2010) Candidosis management: antifungal, cytotoxic and immunomodulatory properties of tea tree oil and its derivative components. MSc(R) thesis, University of Glasgow.

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Oropharyngeal candidosis (OPC) is a common opportunistic yeast infection in elderly and immuno-compromised populations, caused by Candida albicans and other Candida spp. forming biofilms on the oral epithelium or artificial denture surfaces. Oral thrush (pseudomembranous candidosis) is the most common type of OPC occurring when a biofilm grows on oral mucosal surfaces, while growth on dentures commonly causes denture stomatitis in denture-wearers. OPC causes significant morbidity with symptoms including inflammation, pain, burning, eating difficulties and alteration of taste sensation. Conventional antifungal treatments have limited success due to biofilm resistance mechanisms, with recurring infections promoting development of azole resistance. Other problems with current antifungal drugs include toxicity, drug interactions and unpleasant taste. Therefore, alternative methods for prophylactic or therapeutic management of Candida spp. biofilms are desirable. This study aimed firstly to evaluate the efficacy of tea tree oil (TTO) and its derivatives against biofilms formed by a clinically-diverse panel of C. albicans isolates; and secondly to assess the toxicological effects of TTO exposure using a clinically relevant oral cell line. Thirdly, this study aimed to further investigate previously reported anti-inflammatory effects of TTO. TTO is a complex mixture of essential oils; however, individual components of TTO are commercially available. TTO has broad spectrum antimicrobial activity and TTO oral products are currently available. However, evidence for antifungal efficacy is limited and there are concerns regarding safety of long-term use of TTO products. The data presented demonstrate TTO and its derivatives are effective antifungal agents. Minimal inhibitory concentrations (MIC) of TTO and seven components were determined for planktonic C. albicans cells (PMIC) using the standard CLSI dilution technique. The PMIC50 value for TTO was 0.5%, with lower values for two components - 0.25% for both terpinen-4-ol (T4-ol) and α-terpineol. Growth of all 100 strains was inhibited by 1% TTO, 0.5% terpinen-4-ol and 0.5% α-terpineol. A pilot study found no decrease in TTO sensitivity with multiple TTO exposure. Sessile susceptibilities (SMFC) were determined using a metabolic assay on C. albicans cells after 24 h treatment of pre-formed biofilms, to determine the most effective anti-biofilm components. T4-ol and α-terpineol were potent biofilm inhibitors, which could inhibit biofilm metabolism by 50% at PMIC50 concentrations (SMFC50 = 0.25%), exhibiting significantly greater anti-biofilm activity than TTO (SMFC50 = 1%). Strains isolated from different patient groups had similar biofilm susceptibilities. Other components tested had little effect on biofilm metabolism (SMFC50 of 2% to >4%). Shorter treatments modelling a ‘mouthwash’ exposure time produced moderate inhibition (50%) of pre-formed biofilm metabolism after 2 min in 1% α-terpineol, while longer exposures with 1% T4-ol (15 min) and 2% TTO (60 min) were required to give this level of inhibition. A time-dependent treatment effect for TTO and the single components was also seen at these concentrations, with longer exposures giving better inhibition of biofilm metabolism. Inhibition of biofilm formation and morphogenesis was also investigated to define effective components, concentrations and exposure times for prophylactic use. Presence of TTO, T4-ol or α-terpineol could prevent morphogenesis of C. albicans, and therefore block biofilm formation, if present within 2 hours of adherence of cells to a surface. One hour treatments with PMIC50 levels of TTO (0.5%) or the 2 components (0.25%) could effectively prevent biofilm formation. Pre-coating a plastic well with 1% TTO prior to inoculation resulted in strong inhibition (>50%) of biofilm formation. Cellular cytotoxicity studies demonstrated that antifungal concentrations of TTO and T4-ol were cytotoxic to human cells in vitro. Investigations using a human oral epithelial cell line (OKF6-TERT2) and primary oral fibroblasts indicated that 2 min exposures to TTO and T4-ol showed cytotoxic effects at 0.25%, comparable with 0.12% chlorhexidine, with 0.125% TTO / T4-ol being non-toxic. Previously reported immunomodulatory effects were investigated using non-toxic concentrations of TTO / T4-ol (0.125%). The cytokine response of oral epithelial cells following TTO / T4-ol treatment was monitored using quantitative PCR, protein arrays and an IL-8 ELISA. TTO did not exhibit any clear immunomodulatory effects, but T4-ol pre-treatment of zymosan-activated cells resulted in reduced IL-8 protein in ELISA assays, indicating a potential to reduce inflammation. Although inflammation is a major symptom of OPC infections, it is also an important part of the host response to control the yeast pathogen. An anti-inflammatory agent may help to control candidosis symptoms, but may cause problems in controlling the infection. These studies demonstrate that T4-ol could be suitable for use in prophylactic oral hygiene products such as mouthrinses and denture cleansers, and also as a novel treatment for established OPC infections. The use of T4-ol, a single component from TTO, has advantages over the complete essential oil in terms of product safety and consistency. Preclinical and clinical trials of mouthwashes or denture cleansers, containing the range of T4-ol concentrations (0.125 - 0.5%) investigated in these studies, would be required to validate the clinical use of such a product. In conclusion, TTO-derived mouthwashes and denture cleansers may offer both a suitable alternative to conventional azole treatment of OPC and also a safe prophylactic alternative for inhibiting microbial biofilms, as they exhibit potent antifungal activity.

Item Type: Thesis (MSc(R))
Qualification Level: Masters
Keywords: tea tree oil, terpinen-4-ol, oral candidosis, antifungal, cytotoxicity, immunomodulation, candida biofilm
Subjects: Q Science > QR Microbiology
R Medicine > RK Dentistry
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Medicine, Dentistry & Nursing > Dental School
Funder's Name: UNSPECIFIED
Supervisor's Name: Ramage, Dr. Gordon and Culshaw, Dr. Shauna, E
Date of Award: 2010
Depositing User: steven g milligan
Unique ID: glathesis:2010-2282
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 25 Nov 2010
Last Modified: 10 Dec 2012 13:53

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