864 Sudjana et al. The essential oil derived from the Australian native as previously described [13]with the addition of 0.001% plant Melaleuca alternifolia,tea tree oil (TTO),contains Tween 80 to enhance the solubility of the oil.Modal MICs predominantly monoterpenes and related alcohols and and MFCs were selected from a minimum of two indepen- composition is regulated by the International Standard dent replicates. 4730 for Oil of Melaleuca,terpinen-4-ol type(tea tree oil) [7].TTO has a broad antimicrobial activity spectrum [8] and exhibits in vitro fungicidal activity against C.albicans Mammalian cells at approximately 0.5-2%(v/v)oil [9,10].The oil acts Buccal epithelial cells(BECs)were harvested from healthy. largely by altering the properties and function of the cell adult,non-smoking female volunteers by gently swabbing membrane [10],which in turn leads to the loss of intracel- the inside of the cheeks with a cotton-tipped swab and lular components and eventually cell death.Previous stud- suspending cells in phosphate buffered saline(PBS),pH ies have demonstrated that formulations with TTO as the 7.4.BECs were pooled,washed twice with PBS and stan- active ingredient have clinical efficacy in AIDS patients dardized to 10>cells/ml.HeLa cells (human cervix epithe- with fluconazole-refractory oral candidiasis [11]and against lial adenocarcinoma)and A549 cells(human lung epithelial vaginal candidiasis in an animal model [12].While TTO adenocarcinoma)were cultured in Minimal Eagles medium may act in these instances by direct fungicidal activity,it containing 10%(v/v)foetal calf serum (FCS),2 mM glu- is likely that a range of concentration-dependent effects tamine,28 mM HEPES buffer and antibiotics (50 ug/ml occur,with more subtle antimicrobial actions occurring at vancomycin and 50 ug/ml streptomycin)(HGM-M).Main- sub-inhibitory TTO levels.For example,sub-inhibitory tenance medium (HMM-M)contained a lower concentra- concentrations of TTO inhibit the formation of germ tubes tion of 2%(v/v)FCS.For experiments,cells were grown by C.albicans [9,which is one of the early stages in the for 24 h in tissue culture flasks in HGM-M at 35C with development of invasive disease.Given that TTO shows 5%CO,to 80%confluency.Cells were washed with promise as a topical anti-candidal therapy,the aim of this Hank's buffered salt solution (HBSS)(for HeLa cells)or study was to determine whether TTO may act by inhibiting versene(for A549 cells),detached using trypsin(HeLa)or biofilm formation and the adhesion of C.albicans to trypsin-versene (A549)then collected in HMM-M for surfaces,and to investigate underlying mechanisms. cytotoxicity assays or double-strength RPMI 1640 for adhesion assays Materials and methods M.alternifolia essential oil Cytotoxicity assays M.alternifolia essential oil (batch 1216)was supplied by TTO cytotoxicity was evaluated for BECs using the trypan P.Guinane Pty.Ltd.,NSW,Australia.The major compo- blue exclusion method [14].BECs were exposed to solu- nents were terpinen-4-ol (42.4%),y-terpinene (20.1%), tions of TTO in PBS ranging from 0.25-0.008%(v/v)for o-terpinene (9.0%).1.8-cineole (3.7%).terpinolene (3.2%). 5 min then stained with trypan blue(0.4%w/v).The uptake p-cymene (3.1%)and a-terpineol (3.1%)which complies of dye was determined by light microscopy.A minimum with ISO 4730 [7].Fresh solutions of TTO were prepared of 100 BECs was examined for each TTO concentration volume/volume (v/v)prior to each experiment and were and the percentage viability was calculated. used within 2 h of preparation. For HeLa and A549 cells,the cytotoxicity of TTO was determined by the uptake of propidium iodide (PD)quanti- fied by flow cytometry.Briefly,24 h cultures of HeLa or Organisms A549 cells in 6-well tissue culture trays were exposed to The reference strains C.albicans ATCC 10231,ATCC doubling dilutions of TTO(0.5-0.001%v/v)in HMM-M for 90028 and ATCC 90029 and 25 clinical C.albicans iso- 90 min.The TTO was then removed and cells were washed lates were obtained from the Division of Microbiology and with HBSS or versene,removed by trypsinization,collected, Infectious Diseases at PathWest Laboratory Medicine washed again and resuspended in HMM-M to approximately WA.Nedlands.Western Australia.Routine culture was 1-5 X 10>cells/ml.PI (1 ul/ml)was then added for 15 min performed on Sabouraud dextrose agar (SDA)incubated before analyzing using a FACSCalibur flow cytometer(BD aerobically at 35C for 24-48 h. Biosciences,San Jose,CA).Cells were gated based on for- ward/side scatter and a total of 10,000 events were collected within the gate.PI was excited with a 488 nm laser and the In vitro susceptibility assays emission was collected with a 670 nm long pass filter.The Minimum inhibitory concentrations(MICs)and minimum percentage of live (PI negative)and dead(PI positive)cells fungicidal concentrations(MFCs)of TTO were determined in each sample was then calculated. @2012 ISHAM,Medical Mycology,50,863-870© 2012 ISHAM, Medical Mycology, 50, 863–870 864 Sudjana et al. The essential oil derived from the Australian native plant Melaleuca alternifolia , tea tree oil (TTO), contains predominantly monoterpenes and related alcohols and composition is regulated by the International Standard 4730 for Oil of Melaleuca , terpinen-4-ol type (tea tree oil) [7]. TTO has a broad antimicrobial activity spectrum [8] and exhibits in vitro fungicidal activity against C. albicans at approximately 0.5 – 2% (v/v) oil [9,10]. The oil acts largely by altering the properties and function of the cell membrane [10], which in turn leads to the loss of intracel￾lular components and eventually cell death. Previous stud￾ies have demonstrated that formulations with TTO as the active ingredient have clinical effi cacy in AIDS patients with fl uconazole-refractory oral candidiasis [11] and against vaginal candidiasis in an animal model [12]. While TTO may act in these instances by direct fungicidal activity, it is likely that a range of concentration-dependent effects occur, with more subtle antimicrobial actions occurring at sub-inhibitory TTO levels. For example, sub-inhibitory concentrations of TTO inhibit the formation of germ tubes by C. albicans [9], which is one of the early stages in the development of invasive disease. Given that TTO shows promise as a topical anti-candidal therapy, the aim of this study was to determine whether TTO may act by inhibiting biofi lm formation and the adhesion of C. albicans to surfaces, and to investigate underlying mechanisms. Materials and methods M. alternifolia essential oil M. alternifolia essential oil (batch 1216) was supplied by P. Guinane Pty. Ltd., NSW, Australia. The major compo￾nents were terpinen-4-ol (42.4%), γ -terpinene (20.1%), α -terpinene (9.0%), 1,8-cineole (3.7%), terpinolene (3.2%), ρ -cymene (3.1%) and α -terpineol (3.1%) which complies with ISO 4730 [7]. Fresh solutions of TTO were prepared volume/volume (v/v) prior to each experiment and were used within 2 h of preparation. Organisms The reference strains C. albicans ATCC 10231, ATCC 90028 and ATCC 90029 and 25 clinical C. albicans iso￾lates were obtained from the Division of Microbiology and Infectious Diseases at PathWest Laboratory Medicine WA, Nedlands, Western Australia. Routine culture was performed on Sabouraud dextrose agar (SDA) incubated aerobically at 35 ° C for 24 – 48 h. In vitro susceptibility assays Minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) of TTO were determined as previously described [13] with the addition of 0.001% Tween 80 to enhance the solubility of the oil. Modal MICs and MFCs were selected from a minimum of two indepen￾dent replicates. Mammalian cells Buccal epithelial cells (BECs) were harvested from healthy, adult, non-smoking female volunteers by gently swabbing the inside of the cheeks with a cotton-tipped swab and suspending cells in phosphate buffered saline (PBS), pH 7.4. BECs were pooled, washed twice with PBS and stan￾dardized to 10 5 cells/ml. HeLa cells (human cervix epithe￾lial adenocarcinoma) and A549 cells (human lung epithelial adenocarcinoma) were cultured in Minimal Eagles medium containing 10% (v/v) foetal calf serum (FCS), 2 mM glu￾tamine, 28 mM HEPES buffer and antibiotics (50 μ g/ml vancomycin and 50 μ g/ml streptomycin) (HGM-M). Main￾tenance medium (HMM-M) contained a lower concentra￾tion of 2% (v/v) FCS. For experiments, cells were grown for 24 h in tissue culture fl asks in HGM-M at 35 ° C with 5% CO 2 to 80% confl uency. Cells were washed with Hank ’ s buffered salt solution (HBSS) (for HeLa cells) or versene (for A549 cells), detached using trypsin (HeLa) or trypsin-versene (A549) then collected in HMM-M for cytotoxicity assays or double-strength RPMI 1640 for adhesion assays. Cytotoxicity assays TTO cytotoxicity was evaluated for BECs using the trypan blue exclusion method [14]. BECs were exposed to solu￾tions of TTO in PBS ranging from 0.25 – 0.008% (v/v) for 5 min then stained with trypan blue (0.4% w/v). The uptake of dye was determined by light microscopy. A minimum of 100 BECs was examined for each TTO concentration and the percentage viability was calculated. For HeLa and A549 cells, the cytotoxicity of TTO was determined by the uptake of propidium iodide (PI) quanti￾fi ed by fl ow cytometry. Briefl y, 24 h cultures of HeLa or A549 cells in 6-well tissue culture trays were exposed to doubling dilutions of TTO (0.5 – 0.001% v/v) in HMM-M for 90 min. The TTO was then removed and cells were washed with HBSS or versene, removed by trypsinization, collected, washed again and resuspended in HMM-M to approximately 1 – 5  10 5 cells/ml. PI (1 μ l/ml) was then added for 15 min before analyzing using a FACSCalibur fl ow cytometer (BD Biosciences, San Jose, CA). Cells were gated based on for￾ward/side scatter and a total of 10,000 events were collected within the gate. PI was excited with a 488 nm laser and the emission was collected with a 670 nm long pass fi lter. The percentage of live (PI negative) and dead (PI positive) cells in each sample was then calculated