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.

Abstract

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
• 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
• URI: https://theses.gla.ac.uk/id/eprint/2282