866 Sudjana et al. fixed with methanol for 10 min and the number of adhered Cytotoxicity C.albicans cells per HeLa cell(minimum of 50)was counted The highest TTO concentrations resulting in less than 10% using a Nikon inverted light microscope at 20 X magnifica- cell death were 0.031%for BECs with 93.5%+3.1% tion.Adherent C.albicans cells were quantified according to previously published criteria [17]. (mean+standard deviation)of cells viable and 0.062%for both HeLa (94.0%+2.8%viability)and A549 (90.9% 3.4%)cells Assessment of hydrophobicity by flow cytometry Hydrophobicity was assessed based on a microsphere Biofilm development adhesion method [18].Inocula of C.albicans ATCC 10231 The presence of TTO resulted in significantly less biofilm and clinical isolate 137548L were prepared by collecting cells from overnight cultures in SDB,washing,then resus- being formed by all isolates at one or more concentrations. pending to 107 cfu/ml in PBS.Cells were added to glass When quantified by XTT reduction,biofilm formation was conical flasks containing PBS with 0.001%Tween 80 and significantly reduced at the lowest test concentration of TTO at final concentrations of 0.125,0.06 and 0%(con- 0.016%TTO for 7(70%)of isolates and at the next high- est concentration of 0.031%for all isolates (Table 1).Using trol).Cells with TTO were incubated at 35C with shaking CV staining,biofilm was significantly reduced at 0.016% and at 1.2 and 4 h cells were removed,washed twice and resuspended in PBS.The 18 h treatment (0.062%TTO) for 50%of isolates and at 0.25%TTO.90%of isolates showed significantly inhibited biofilm formation.In addi- was conducted identically except that cells were incubated in RPMI 1640 growth medium instead of PBS.To assess tion,at 0.25%TTO,biofilm values(relative to the control) hydrophobicity,1 ml volumes of TTO-treated cells were ranged from 0.03-0.31 when quantified by XTT metabo- incubated with 10 ul of Fluoresbrite YellowGreen 0.8 um lism and from 0.01-0.97 for CV.Comparison of XTT and CV results demonstrated that the lowest concentration to polystyrene latex microspheres (Polysciences Inc.)for 60 significantly inhibit biofilm formation was identical or dif- min at room temperature then analyzed by flow cytometry using a FACSCalibur flow cytometer.Cells were gated fered only by one dilution for 60%of isolates.The data based on forward/side scatter and a total of 10,000 events obtained using CV staining were more variable than those obtained with XTT,meaning that less significant differ- were collected within the gate.The microspheres were ences were evident. excited with a 488 nm laser and the emission was collected with a 530/30 band pass filter.The geometric mean of fluorescence intensity was obtained for control and TTO- Adhesion to polystyrene treated cells as a function of the relative number of micro- Adhesion was significantly reduced at 0.25%for all iso- spheres adhered per cell.Cells with greater hydrophobicity lates and for six of the seven isolates at 0.062 and 0.125% will bind a higher number of microspheres. (Table 2).For C.albicans ATCC 10231 adhesion was also significantly reduced at 0.008%and 0.016%TTO.Pre- Statistical analyses conditioning of 96-well tray wells with TTO did not sig- Data were normalized by dividing all data points by the nificantly alter subsequent adhesion (data not shown) relevant control (no TTO).All data are the mean (stan- indicating that any physical interactions between TTO and dard deviation)of at least three independent experiments. the polystyrene of the 96-well tray had negligible effects Results were statistically analyzed using One way ANOVA on adhesion.In addition,viability assays showed no followed by Dunnett's multiple comparison test(P<0.05) significant alterations in viability after the exposure of (GraphPad Prism Version 3.03).The exceptions were data C.albicans to 0.062 or 0.125%TTO for 90 min when for the adhesion of C.albicans to BECs and the removal compared to the control.However,viability after exposure of pre-adhered C.albicans from HeLa cells.which were to 0.25%TTO was not determined and,in fact,reductions analyzed by paired Student's t-tests (1-tailed,P<0.05). in adhesion at this concentration may be a result of decreased cell viability Results Adhesion to mammalian cells In vitro susceptibility TTO significantly reduced the adhesion of C.albicans Tea tree oil MICs ranged from 0.25-0.5%with an MIC9o of ATCC 10231 to HeLa cells and BECs (Table 3)but not 0.5%.MFCs ranged from 0.5-1%(v/v)and the MFC was A549 cells.The relative adhesion of clinical isolates 4047 1%.MICs and MFC were equivalent for 31%of isolates and 137548L to BECs in the presence of 0.016%TTO and differed by one doubling dilution for the remainder. was0.75±0.06(P<0.001)and0.71±0.53，respectively @2012 ISHAM,Medical Mycology,50,863-870© 2012 ISHAM, Medical Mycology, 50, 863–870 866 Sudjana et al. fi xed with methanol for 10 min and the number of adhered C. albicans cells per HeLa cell (minimum of 50) was counted using a Nikon inverted light microscope at 20  magnifi ca￾tion. Adherent C. albicans cells were quantifi ed according to previously published criteria [17]. Assessment of hydrophobicity by fl ow cytometry Hydrophobicity was assessed based on a microsphere adhesion method [18]. Inocula of C. albicans ATCC 10231 and clinical isolate 137548L were prepared by collecting cells from overnight cultures in SDB, washing, then resus￾pending to 10 7 cfu/ml in PBS. Cells were added to glass conical fl asks containing PBS with 0.001% Tween 80 and TTO at fi nal concentrations of 0.125, 0.06 and 0% (con￾trol). Cells with TTO were incubated at 35 ° C with shaking and at 1, 2 and 4 h cells were removed, washed twice and resuspended in PBS. The 18 h treatment (0.062% TTO) was conducted identically except that cells were incubated in RPMI 1640 growth medium instead of PBS. To assess hydrophobicity, 1 ml volumes of TTO-treated cells were incubated with 10 μ l of Fluoresbrite YellowGreen 0.8 μ m polystyrene latex microspheres (Polysciences Inc.) for 60 min at room temperature then analyzed by fl ow cytometry using a FACSCalibur fl ow cytometer. Cells were gated based on forward/side scatter and a total of 10,000 events were collected within the gate. The microspheres were excited with a 488 nm laser and the emission was collected with a 530/30 band pass fi lter. The geometric mean of fl uorescence intensity was obtained for control and TTO￾treated cells as a function of the relative number of micro￾spheres adhered per cell. Cells with greater hydrophobicity will bind a higher number of microspheres. Statistical analyses Data were normalized by dividing all data points by the relevant control (no TTO). All data are the mean (  stan￾dard deviation) of at least three independent experiments. Results were statistically analyzed using One way ANOVA followed by Dunnett ’ s multiple comparison test ( P  0.05) (GraphPad Prism ® Version 3.03). The exceptions were data for the adhesion of C. albicans to BECs and the removal of pre-adhered C. albicans from HeLa cells, which were analyzed by paired Student ’ s t -tests (1-tailed, P  0.05). Results In vitro susceptibility Tea tree oil MICs ranged from 0.25 – 0.5% with an MIC 90 of 0.5%. MFCs ranged from 0.5 – 1% (v/v) and the MFC 90 was 1%. MICs and MFC were equivalent for 31% of isolates and differed by one doubling dilution for the remainder. Cytotoxicity The highest TTO concentrations resulting in less than 10% cell death were 0.031% for BECs with 93.5%  3.1% (mean  standard deviation) of cells viable and 0.062% for both HeLa (94.0%  2.8% viability) and A549 (90.9%  3.4%) cells. Biofi lm development The presence of TTO resulted in signifi cantly less biofi lm being formed by all isolates at one or more concentrations. When quantifi ed by XTT reduction, biofi lm formation was signifi cantly reduced at the lowest test concentration of 0.016% TTO for 7 (70%) of isolates and at the next high￾est concentration of 0.031% for all isolates (Table 1). Using CV staining, biofi lm was signifi cantly reduced at 0.016% for 50% of isolates and at 0.25% TTO, 90% of isolates showed signifi cantly inhibited biofi lm formation. In addi￾tion, at 0.25% TTO, biofi lm values (relative to the control) ranged from 0.03 – 0.31 when quantifi ed by XTT metabo￾lism and from 0.01 – 0.97 for CV. Comparison of XTT and CV results demonstrated that the lowest concentration to signifi cantly inhibit biofi lm formation was identical or dif￾fered only by one dilution for 60% of isolates. The data obtained using CV staining were more variable than those obtained with XTT, meaning that less signifi cant differ￾ences were evident. Adhesion to polystyrene Adhesion was signifi cantly reduced at 0.25% for all iso￾lates and for six of the seven isolates at 0.062 and 0.125% (Table 2). For C. albicans ATCC 10231 adhesion was also signifi cantly reduced at 0.008% and 0.016% TTO. Pre￾conditioning of 96-well tray wells with TTO did not sig￾nifi cantly alter subsequent adhesion (data not shown) indicating that any physical interactions between TTO and the polystyrene of the 96-well tray had negligible effects on adhesion. In addition, viability assays showed no signifi cant alterations in viability after the exposure of C. albicans to 0.062 or 0.125% TTO for 90 min when compared to the control. However, viability after exposure to 0.25% TTO was not determined and, in fact, reductions in adhesion at this concentration may be a result of decreased cell viability. Adhesion to mammalian cells TTO signifi cantly reduced the adhesion of C. albicans ATCC 10231 to HeLa cells and BECs (Table 3) but not A549 cells. The relative adhesion of clinical isolates 4047 and 137548L to BECs in the presence of 0.016% TTO was 0.75  0.06 ( P  0.001) and 0.71  0.53, respectively