868 Sudjana et al. Table 3 Proportions of mammalian cells with adherent Candida receptor-ligand interactions and the previously mentioned albicans after incubation with tea tree oil. inhibition of germ tube formation may be involved.Numer- Treatment (TTO v/v) ous C.albicans adhesins have been identified [23]and at least one adhesin,Eaplp,has been shown to play a role in Isolate Cell type Control 0.016 0.031 0.062 adhesion to both mammalian cells and polystyrene [24]. ATCC BEC 1.0±0.00.56±0.21* Although TTO significantly reduced adhesion to poly- 10231 styrene in all C.albicans isolates,C.albicans ATCC 10231 HeLa 1.0±0.01.16±0.090.95±0.03t0.27±0.14* was the only strain with a significant reduction at the low- A549 1.0±0.01.00±0.000.99±0.020.99±0.02 4047 BEC est TTO concentration of 0.008%.This may indicate dif- 1.0±0.00.75±0.06* 137548LBEC 1.0±0.00.71±0.53 ferences between this strain and the remaining isolates in terms of adhesion mechanisms.such as adhesins and other *Values are significant (Student's t-test,2-tailed,assuming unequal cell surface glycoproteins [23,25].Furthermore,C.albicans variance).Significant by the one-tailed test only. Discussion (A)1.6 The ability of C.albicans to adhere and develop biofilm is 1.4 ■Control important in the progression of infection.This study dem- ☐0.062 onstrates that subinhibitory concentrations of TTO decrease 1.2 00.125 biofilm formation,decrease adhesion to both polystyrene and epithelial cells and reduce CSH.These findings sup- 1.0 port the hypothesis that inhibition of biofilm formation and adhesion may be factors contributing to the clinical effi- 0.8 cacy of TTO.Similarly,previous studies have demonstrated 0.6 that TTO affects biofilms formed by Staphylococcus aureus [19]and coagulase-negative staphylococci [20]and the 0.4 viability of mature C.albicans biofilm is decreased by treatment with monoterpenoids similar to those in TTO 0.2 such as carvacrol and geraniol [21],and thymol [21,22]. 0.0 Interestingly,this study showed that biofilm formation 1 was inhibited at concentrations well below the TTO MIC. 2 suggesting that a specific process necessary for biofilm Time (h) formation is being inhibited,rather than a non-specific effect such as a global reduction in growth.The key steps (B)1.6 in the development of fungal biofilm include adhesion, Control proliferation (which includes germ tube formation and 14 口0.062 hyphal development),maturation and the production of 1.2 口0.125 extracellular matrix and finally dispersal [21.Previous stud- ies have demonstrated that monoterpenes and monoter- 1.0 pene-rich essential oils are capable of inhibiting the transition from yeast to hypha (germ tube formation) 0.8 [9,22],indicating that this is one of the stages of biofilm formation that is likely to be adversely affected by TTO. 0.6 Adhesion is also likely to be a stage of biofilm formation 0.4 adversely affected by TTO,based on the current findings. Adhesion is mediated by many different factors which may 0.2 be classed as non-specific,such as cell surface hydropho- bicity (CSH)and van der Waals forces,or specific,such as 0.0 receptor-ligand interactions [5].Data from the current 4 study indicate that TTO may affect CSH,but only at rela- Time (h) tively high TTO concentrations and adhesion inhibition occurred at concentrations lower than those altering CSH. Fig.1 Relative cell surface hydrophobicity of (A)Candida albicans ATCC 10231.and (B)C.albicans 137548L treated with TTO (v/v) This suggests that CSH changes play only a minor role in measured by flow cytometry.For statistical significance.*denotes adhesion reduction and that additional factors,such as P<0.05 and **denotes P<0.01 2012 ISHAM,Medical Mycology,50,863-870© 2012 ISHAM, Medical Mycology, 50, 863–870 868 Sudjana et al. Discussion The ability of C. albicans to adhere and develop biofi lm is important in the progression of infection. This study dem￾onstrates that subinhibitory concentrations of TTO decrease biofi lm formation, decrease adhesion to both polystyrene and epithelial cells and reduce CSH. These fi ndings sup￾port the hypothesis that inhibition of biofi lm formation and adhesion may be factors contributing to the clinical effi - cacy of TTO. Similarly, previous studies have demonstrated that TTO affects biofi lms formed by Staphylococcus aureus [19] and coagulase-negative staphylococci [20] and the viability of mature C. albicans biofi lm is decreased by treatment with monoterpenoids similar to those in TTO such as carvacrol and geraniol [21], and thymol [21,22]. Interestingly, this study showed that biofi lm formation was inhibited at concentrations well below the TTO MIC, suggesting that a specifi c process necessary for biofi lm formation is being inhibited, rather than a non-specifi c effect such as a global reduction in growth. The key steps in the development of fungal biofi lm include adhesion, proliferation (which includes germ tube formation and hyphal development), maturation and the production of extracellular matrix and fi nally dispersal [2]. Previous stud￾ies have demonstrated that monoterpenes and monoter￾pene-rich essential oils are capable of inhibiting the transition from yeast to hypha (germ tube formation) [9,22], indicating that this is one of the stages of biofi lm formation that is likely to be adversely affected by TTO. Adhesion is also likely to be a stage of biofi lm formation adversely affected by TTO, based on the current fi ndings. Adhesion is mediated by many different factors which may be classed as non-specifi c, such as cell surface hydropho￾bicity (CSH) and van der Waals forces, or specifi c, such as receptor-ligand interactions [5]. Data from the current study indicate that TTO may affect CSH, but only at rela￾tively high TTO concentrations and adhesion inhibition occurred at concentrations lower than those altering CSH. This suggests that CSH changes play only a minor role in adhesion reduction and that additional factors, such as receptor-ligand interactions and the previously mentioned inhibition of germ tube formation may be involved. Numer￾ous C. albicans adhesins have been identifi ed [23] and at least one adhesin, Eap1p, has been shown to play a role in adhesion to both mammalian cells and polystyrene [24]. Although TTO signifi cantly reduced adhesion to poly￾styrene in all C. albicans isolates, C. albicans ATCC 10231 was the only strain with a signifi cant reduction at the low￾est TTO concentration of 0.008%. This may indicate dif￾ferences between this strain and the remaining isolates in terms of adhesion mechanisms, such as adhesins and other cell surface glycoproteins [23,25]. Furthermore, C. albicans 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 124 Time (h) Relative hydrophobicity Control 0.062 0.125 ∗ ∗∗ (A) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1 2 4 Time (h) Relative hydrophobicity Control 0.062 0.125 ∗∗ ∗∗ (B) Fig. 1 Relative cell surface hydrophobicity of (A) Candida albicans ATCC 10231, and (B) C. albicans 137548L treated with TTO (% v/v) measured by fl ow cytometry. For statistical signifi cance, * denotes P  0.05 and * * denotes P  0.01. Table 3 Proportions of mammalian cells with adherent Candida albicans after incubation with tea tree oil. Treatment (TTO % v/v) Isolate Cell type Control 0.016 0.031 0.062 ATCC 10231 BEC 1.0  0.0 0.56  0.21 * HeLa 1.0  0.0 1.16  0.09 0.95  0.03 † 0.27  0.14 * A549 1.0  0.0 1.00  0.00 0.99  0.02 0.99  0.02 4047 BEC 1.0  0.0 0.75  0.06 * 137548L BEC 1.0  0.0 0.71  0.53 * Values are signifi cant (Student ’ s t -test, 2-tailed, assuming unequal variance). † Signifi cant by the one-tailed test only