Candida albicans adhesion to human epithelial cells and polystyrene and formation of biofi lm is reduced by sub-inhibitory Melaleuca alternifolia（tea tree）essential oil

informa Medical Mycology November 2012,50,863-870 healthcare Candida albicans adhesion to human epithelial cells and polystyrene and formation of biofilm is reduced by sub-inhibitory Melaleuca alternifolia (tea tree)essential oil AURELIA N.SUDJANA*,CHRISTINE F.CARSON*,KERRY C.CARSONt,THOMAS V.RILEY*& KATHERINE A.HAMMER* *Discipline of Microbiology and Immunology(M502),School of Biomedical,Biomolecular and Chemical Sciences,The University ofWestern Australia,Crawley,Western Australia,Australia,and Division of Microbiology and Infectious Diseases,PathWest Laboratory Medicine WA,Nedlands,Western Australia,Australia This study investigated the effects of the volatile terpene-rich oil from Melaleuca alterni- folia (tea tree oil)on the formation of biofilms and the adhesion of C.albicans cells to both biotic and abiotic surfaces.Biofilm formation on polystyrene was significantly inhibited for 70%of the isolates at the lowest test concentration of 0.016%of tea tree oil (TTO)when quantified by XTT and 40%of isolates when measured by crystal violet staining.Adhesion to polystyrene,quantified by crystal violet staining,was significantly reduced for 3 isolates at 0.031%,6 isolates at 0.062%and 0.125%and for all 7 isolates at 0.25%TTO.Reductions in adhesion were not due to loss of viability (at concentra- tions of0.125%)or interactions between the TTO and polystyrene.Similarly,adhe- sion to buccal epithelial and HeLa cells was also significantly reduced in the presence of 0.016-0.062%TTO.Treatment with 0.125%TTO,but not 0.062%,decreased the cell surface hydrophobicity of C.albicans,indicating one potential mechanism by which adhesion may be reduced.These data demonstrate that sub-inhibitory TTO reduces the adhesion of C.albicans to both human cells and polystyrene,inhibits biofilm formation and decreases cell surface hydrophobicity. Keywords candidiasis,virulence,alternative therapies,monoterpenes Introduction Biofilms,which are complex structured microbial com- munities,are increasingly being recognized as having an Candida albicans is a commensal of the human orophar- important role in infection and disease.Microbial biofilms are ynx,as well as the urogenital and gastrointestinal tracts. Under certain conditions C.albicans is capable of causing typically difficult to eradiate once established as the organ- superficial infections such as oral or vaginal candidiasis or isms in the biofilms are less susceptible to antimicrobial agents and immune defences than their planktonic counter- disseminated,systemic diseases including bloodstream parts [2].Recent studies have demonstrated for the first time infections.It has been estimated that the prevalence of vulvo-vaginal candidiasis is 5-15%amongst adult women, that biofilms are formed on mucosa in vivo [3,4].confirming the importance of biofilm in the disease process.Adhesion, with approximately 75%of all women suffering from at which is the attachment of microbial cells to biological or least one episode during their lifetime [1]. non-biological substrates.has a critical role in the initial stages of biofilm formation and is also a major virulence fac- Received 13 December 2011;Received in final revised form 28 February tor of C.albicans [5].Since biofilm and adhesion both play 2012;Accepted 2 April 2012 important roles in oral and vaginal candidiasis,therapies for Correspondence:K.A.Hammer,Discipline of Microbiology and treating such infections may need to target these virulence Immunology (M502),School of Biomedical,Biomolecular and Chemical Sciences,The University of Western Australia,35 Stirling Hwy, mechanisms in addition,or as an adjunct,to causing cell Crawley,6009,Western Australia,Australia.Tel:+61 8 9346 1986. death.This approach of targeting virulence has been proposed Fax:+61 8 9346 2912.E-mail:katherine.hammer@uwa.edu.au. as a new tactic for the treatment of infectious diseases [6]. ©20I2 ISHAM D○10.3109/13693786.2012.683540

Biofi lms, which are complex structured microbial com￾munities, are increasingly being recognized as having an important role in infection and disease. Microbial biofi lms are typically diffi cult to eradiate once established as the organ￾isms in the biofi lms are less susceptible to antimicrobial agents and immune defences than their planktonic counter￾parts [2]. Recent studies have demonstrated for the fi rst time that biofi lms are formed on mucosa in vivo [3,4], confi rming the importance of biofi lm in the disease process. Adhesion, which is the attachment of microbial cells to biological or non-biological substrates, has a critical role in the initial stages of biofi lm formation and is also a major virulence fac￾tor of C. albicans [5]. Since biofi lm and adhesion both play important roles in oral and vaginal candidiasis, therapies for treating such infections may need to target these virulence mechanisms in addition, or as an adjunct, to causing cell death. This approach of targeting virulence has been proposed as a new tactic for the treatment of infectious diseases [6]. Received 13 December 2011 ; Received in fi nal revised form 28 February 2012; Accepted 2 April 2012 Correspondence: K. A. Hammer, Discipline of Microbiology and Immunology (M502), School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, 35 Stirling Hwy, Crawley, 6009, Western Australia, Australia. Tel: 61 8 9346 1986. Fax: 61 8 9346 2912. E-mail: katherine.hammer@uwa.edu.au. Candida albicans adhesion to human epithelial cells and polystyrene and formation of biofi lm is reduced by sub-inhibitory Melaleuca alternifolia (tea tree) essential oil AURELIA N. SUDJANA *,CHRISTINE F. CARSON *, KERRY C. CARSON †, THOMAS V. RILEY * , † & KATHERINE A. HAMMER * *Discipline of Microbiology and Immunology (M502), School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, Crawley, Western Australia, Australia,and †Division of Microbiology and Infectious Diseases, PathWest Laboratory Medicine WA, Nedlands, Western Australia, Australia This study investigated the effects of the volatile terpene-rich oil from Melaleuca alterni￾folia (tea tree oil) on the formation of biofi lms and the adhesion of C. albicans cells to both biotic and abiotic surfaces. Biofi lm formation on polystyrene was signifi cantly inhibited for 70% of the isolates at the lowest test concentration of 0.016% of tea tree oil (TTO) when quantifi ed by XTT and 40% of isolates when measured by crystal violet staining. Adhesion to polystyrene, quantifi ed by crystal violet staining, was signifi cantly reduced for 3 isolates at 0.031%, 6 isolates at 0.062% and 0.125% and for all 7 isolates at 0.25% TTO. Reductions in adhesion were not due to loss of viability (at concentra￾tions of 0.125%) or interactions between the TTO and polystyrene. Similarly, adhe￾sion to buccal epithelial and HeLa cells was also signifi cantly reduced in the presence of 0.016 – 0.062% TTO. Treatment with 0.125% TTO, but not 0.062%, decreased the cell surface hydrophobicity of C. albicans , indicating one potential mechanism by which adhesion may be reduced. These data demonstrate that sub-inhibitory TTO reduces the adhesion of C. albicans to both human cells and polystyrene, inhibits biofi lm formation and decreases cell surface hydrophobicity. Keywords candidiasis , virulence , alternative therapies , monoterpenes Introduction Candida albicans is a commensal of the human orophar￾ynx, as well as the urogenital and gastrointestinal tracts. Under certain conditions C. albicans is capable of causing superfi cial infections such as oral or vaginal candidiasis or disseminated, systemic diseases including bloodstream infections. It has been estimated that the prevalence of vulvo-vaginal candidiasis is 5 – 15% amongst adult women, with approximately 75% of all women suffering from at least one episode during their lifetime [1]. © 2012 ISHAM DOI: 10.3109/13693786.2012.683540 Medical Mycology November 2012, 50, 863–870

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

Tea tree oil reduces Candido adhesion and biofilm 365 Biofilm prevention assay tray containing serial dilutions of TTO but no microorgan- isms was incubated for 90 min at 35C.TTO was then Ten isolates including reference strains C.albicans ATCC removed and wells were washed twice with PBS with 10231.90028 and 90029 were examined for biofilm for- mation in the presence of TTO.Inocula were prepared by 0.001%Tween 80.After air drying.100 ul of a standardized collecting cells from overnight cultures in Sabouraud dex- suspension of C.albicans ATCC 10231 or C.albicans trose broth (SDB).washing.then resuspending in 0.85% 137548L was aliquoted into rows of the tray which was saline to~2X 10 cfu/ml.Doubling dilutions of TTO were then incubated for 90 min with shaking(75 rpm)at 35C. Well contents were removed.wells were washed and stained prepared in double-strength RPMI 1640 medium in 100 ul volumes in a 96-well flat-bottomed polystyrene microtitre with CV and optical densities determined as described tray (Nunc,Roskilde,Denmark).A final concentration of above.To assess whether TTO exposure alone reduced 0.001%Tween 80 was included throughout to aid oil sol- viability,suspensions of C.albicans ATCC 10231,137548L and 4047 prepared as described above were inoculated into ubility.Wells with no TTO served as positive growth con- solutions of TTO in PBS at final concentrations of 0.0.062 trols.Two dilution series were inoculated with 100 ul and 0.125%(v/v).Treatments were incubated with shaking volumes of inoculum per isolate to result in final concen- trations of~1X 106 cfu/ml and 0.25-0.016%(v/v)TTO. at 35C for 90 min and then viable counts were performed Corresponding TTO control wells each contained only using the Miles-Misra drop count technique. TTO solution and sterile growth medium.Microtitre trays were then incubated at 35C for 24 h,after which the trays Adhesion to mammalian cells were inverted to remove well contents and wells were Flow cytometry with FUN-1 stain was used to identify washed three times with PBS to remove any residual planktonic cells.Biofilm was then quantified by XTT(2.3- C.albicans bound to mammalian cells.Inocula were pre- bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5- pared by collecting cells of C.albicans ATCC 10231,4047 and 137548L from overnight cultures in SDB,washing,then carboxanilide)reduction or crystal violet (CV)staining. XTT stain was prepared as described previously [15]. resuspending them in HEPES buffer(10 mM HEPES with with the exception that the XTT was prepared at 1 mg/ml 2%glucose)to a concentration of 10?cfu/ml.One ml vol- in PBS and menadione was prepared at 0.4 mM in ethanol umes of suspensions of each C.albicans strain,BEC,HeLa or A549 cells (10 cells/ml in 2X RPMI 1640).TTO solution (final concentration of 1 uM).Volumes of 200 uL of XTT/ and FUN-1 stain(2-chloro-4-(2.3-dihydro-3-methyl-(benzo- menadione were added to each well and trays were incu- 1,3-thiazol-2-yl)-methylidene)-1-phenylquinoliniumiodide, bated at 35C for 2 h in the dark.Then.100 ul volumes 0.5 uM,Invitrogen)were combined and incubated in the were transferred to new 96-well trays and the absorbance was determined at 490 nm using a microplate reader dark for 90 min at 35C with shaking at 50 rpm.Final TTO (Molecular Devices,Sunnydale,CA).Averages of the concentrations were 0,0.016.0.031 and 0.062%(v/v). Adhesion was then assessed by flow cytometry using a duplicate rows were calculated and the appropriate TTO blank value was subtracted. FACSCaliburTM flow cytometer.Mammalian cells (BEC, HeLa or A549)were gated based on forward/side scatter and For CV staining,biofilms were air dried and fixed with a total of 10,000 events were collected within the gate. methanol and then stained for 5 min with 1%(w/v)CV FUN-1 was excited with a 488 nm laser and the emission (225 ul per microtitre tray well).Excess stain was removed by rinsing under running tap water.After air drying,CV was collected with a 670 nm long pass filter(red live cells). was eluted by adding 225 ul of 33%(v/v)glacial acetic acid Mammalian cells positive for FUN-1 stain were indicative of binding of C.albicans.The following controls were to each well and leaving for 15 min.The absorbance of each included:(i)mammalian cells only.(ii)unstained C.albicans well was then determined at 540 nm as described above. and (iii)stained C.albicans.The percentage of mammalian cells with adherent C.albicans was calculated for each sample using CellQuest software (BD Biosciences,San Jose. Adhesion to polystyrene CA).Proportions of mammalian cells with adherent C.albi- Seven isolates including C.albicans ATCC 10231 were cans were calculated by dividing all test and control values examined for adhesion to polystyrene.The assay was per- by the relevant control.To examine the effects of TTO on formed as described above for the biofilm prevention assay pre-adhered C.albicans,equal volumes of C.albicans and with the exception that incubation was for 90 min at 35C HeLa cells prepared as described above were combined in a with shaking at 75 rpm [16]and final TTO concentrations glass McCartney bottle and incubated for 1 h at 35C with ranged from 0.25-0.008%(v/v).To eliminate the possibil- shaking at 50 rpm.TTO was then added to final concentra- ity that reductions in adhesion were an artefact of interac- tions of 0,0.031 or 0.062%and cells were incubated for a tions between TTO and the polystyrene tray,a microtitre further 90 min under the same conditions.Cells were then 2012 ISHAM,Medical Mycology,50,863-870

© 2012 ISHAM, Medical Mycology, 50, 863–870 Tea tree oil reduces Candida adhesion and biofi lm 865 tray containing serial dilutions of TTO but no microorgan￾isms was incubated for 90 min at 35 ° C. TTO was then removed and wells were washed twice with PBS with 0.001% Tween 80. After air drying, 100 μ l of a standardized suspension of C. albicans ATCC 10231 or C. albicans 137548L was aliquoted into rows of the tray which was then incubated for 90 min with shaking (75 rpm) at 35 ° C. Well contents were removed, wells were washed and stained with CV and optical densities determined as described above. To assess whether TTO exposure alone reduced viability, suspensions of C. albicans ATCC 10231, 137548L and 4047 prepared as described above were inoculated into solutions of TTO in PBS at fi nal concentrations of 0, 0.062 and 0.125% (v/v). Treatments were incubated with shaking at 35 ° C for 90 min and then viable counts were performed using the Miles-Misra drop count technique. Adhesion to mammalian cells Flow cytometry with FUN-1 stain was used to identify C. albicans bound to mammalian cells. Inocula were pre￾pared by collecting cells of C. albicans ATCC 10231, 4047 and 137548L from overnight cultures in SDB, washing, then resuspending them in HEPES buffer (10 mM HEPES with 2% glucose) to a concentration of 10 7 cfu/ml. One ml vol￾umes of suspensions of each C. albicans strain, BEC, HeLa or A549 cells (10 6 cells/ml in 2  RPMI 1640), TTO solution and FUN-1 stain (2-chloro-4-(2,3- dihydro-3-methyl-(benzo- 1,3-thiazol-2-yl)- methylidene)-1- phenylquinolinium iodide, 0.5 μ M, Invitrogen) were combined and incubated in the dark for 90 min at 35 ° C with shaking at 50 rpm. Final TTO concentrations were 0, 0.016, 0.031 and 0.062% (v/v). Adhesion was then assessed by fl ow cytometry using a FACSCalibur ™ fl ow cytometer. Mammalian cells (BEC, HeLa or A549) were gated based on forward/side scatter and a total of 10,000 events were collected within the gate. FUN-1 was excited with a 488 nm laser and the emission was collected with a 670 nm long pass fi lter (red live cells). Mammalian cells positive for FUN-1 stain were indicative of binding of C. albicans . The following controls were included; (i) mammalian cells only, (ii) unstained C. albicans and (iii) stained C. albicans . The percentage of mammalian cells with adherent C. albicans was calculated for each sample using CellQuest software (BD Biosciences, San Jose, CA). Proportions of mammalian cells with adherent C. albi￾cans were calculated by dividing all test and control values by the relevant control. To examine the effects of TTO on pre-adhered C. albicans , equal volumes of C. albicans and HeLa cells prepared as described above were combined in a glass McCartney bottle and incubated for 1 h at 35 ° C with shaking at 50 rpm. TTO was then added to fi nal concentra￾tions of 0, 0.031 or 0.062% and cells were incubated for a further 90 min under the same conditions. Cells were then Biofi lm prevention assay Ten isolates including reference strains C. albicans ATCC 10231, 90028 and 90029 were examined for biofi lm for￾mation in the presence of TTO. Inocula were prepared by collecting cells from overnight cultures in Sabouraud dex￾trose broth (SDB), washing, then resuspending in 0.85% saline to ∼ 2  10 6 cfu/ml. Doubling dilutions of TTO were prepared in double-strength RPMI 1640 medium in 100 μ l volumes in a 96-well fl at-bottomed polystyrene microtitre tray (Nunc, Roskilde, Denmark). A fi nal concentration of 0.001% Tween 80 was included throughout to aid oil sol￾ubility. Wells with no TTO served as positive growth con￾trols. Two dilution series were inoculated with 100 μ l volumes of inoculum per isolate to result in fi nal concen￾trations of ∼ 1  10 6 cfu/ml and 0.25 – 0.016% (v/v) TTO. Corresponding TTO control wells each contained only TTO solution and sterile growth medium. Microtitre trays were then incubated at 35 ° C for 24 h, after which the trays were inverted to remove well contents and wells were washed three times with PBS to remove any residual planktonic cells. Biofi lm was then quantifi ed by XTT (2,3- bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5- carboxanilide) reduction or crystal violet (CV) staining. XTT stain was prepared as described previously [15], with the exception that the XTT was prepared at 1 mg/ml in PBS and menadione was prepared at 0.4 mM in ethanol (fi nal concentration of 1 μ M). Volumes of 200 μ L of XTT/ menadione were added to each well and trays were incu￾bated at 35 ° C for 2 h in the dark. Then, 100 μ l volumes were transferred to new 96-well trays and the absorbance was determined at 490 nm using a microplate reader (Molecular Devices, Sunnydale, CA). Averages of the duplicate rows were calculated and the appropriate TTO blank value was subtracted. For CV staining, biofi lms were air dried and fi xed with methanol and then stained for 5 min with 1% (w/v) CV (225 μ l per microtitre tray well). Excess stain was removed by rinsing under running tap water. After air drying, CV was eluted by adding 225 μ l of 33% (v/v) glacial acetic acid to each well and leaving for 15 min. The absorbance of each well was then determined at 540 nm as described above. Adhesion to polystyrene Seven isolates including C. albicans ATCC 10231 were examined for adhesion to polystyrene. The assay was per￾formed as described above for the biofi lm prevention assay with the exception that incubation was for 90 min at 35 ° C with shaking at 75 rpm [16] and fi nal TTO concentrations ranged from 0.25 – 0.008% (v/v). To eliminate the possibil￾ity that reductions in adhesion were an artefact of interac￾tions between TTO and the polystyrene tray, a microtitre

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

Tea tree oil reduces Candido adhesion and biofilm 86 Table 1 Relative biofilm formed (mean standard deviation)by Candida albicans in the presence of TTO(%v/v)quantified by XTT metabolism and crystal violet staining. Relative biofilm formed in the presence of TTO (v/v)at: Isolate Stain 0 0.016 0.031 0.062 0.125 0.25 ATCC 10231 XTT 1.00±0.00 0.47±0.20* 0.39±0.16* 0.22±0.07* 0.06±0.04 0.03±0.04* CV 1.00±0.00 0.45±0.09 0.60±0.34 0.64±0.97 0.27±0.43 -0.04±0.26t ATCC 90028 XTT 1.00±0.00 0.58±0.27* 0.56±0.20* 0.30±0.10* 0.10±0.07* 0.04±0.02* CV 1.00±0.00 0.63±0.29 0.36±0.14 0.35±0.37 0.44±0.78 0.03±0.10* ATCC 90029 XTT 1.00±0.00 0.58±0.13* 0.47±0.14* 0.26±0.13* 0.05±0.03* 0.01±0.02* CV 1.00±0.00 0.60±0.16 0.49±026 0.31±0.10t 0.04±0.11 -0.04±0.18* 137442J XTT 1.00±0.00 0.50±0.22* 0.23±0.10* 0.08±0.05* 0.03±0.02* 0.09±0.08* CV 1.00±0.00 0.27±0.13* 0.09±0.04* 0.03±0.01* 0.01±0.01* 0.03±0.02* 137645E XTT 1.00±0.00 0.87±0.04 0.71±0.16 0.58±0.18* 0.31±0.05* 0.14±0.14* CV 1.00±0.00 0.89±0.22 0.48±0.15 0.45±0.22 0.96±0.97 0.25±0.22 142457X XTT 1.00±0.00 0.94±0.17 0.78±0.18 0.55±0.11* 0.20±0.08* 0.12±0.05* CV 1.00±0.00 0.75±0.43 039±0.09 0.41±0.19 0.18±0.07* 0.17±0.29* 147168E XTT 1.00±0.00 0.62±0.23t 0.60±0.24t 0.37±0.15* 0.12±0.06* 0.05±0.04* CV 1.00±0.00 0.54±0.27 0.47±0.24t 0.35±0.13* 0.11±0.08* 0.17±0.33* FF225 XTT 1.00±0.00 0.53±0.29t 0.32±031* 0.15±0.15* 0.05±0.05◆ 0.14±0.19* CV 1.00±0.00 0.21±0.10* 0.09±0.09* 0.04±0.04* 0.02±0.01* 0.06±0.08* KE216 XTT 1.00±0.00 0.63±0.23t 0.48±0.33↑ 0.35±0.28* 0.13±0.13* 0.08±0.13* CV 1.00±0.00 0.34±0.20* 0.14±0.09* 0.05±0.02* 0.02±0.01* 0.00±0.01* LL031 XTT 1.00±0.00 0.67±0.21 0.63±0.25↑ 0.52±0.29 0.21±0.22* 0.05±0.07* CV 1.00±0.00 0.64±0.40 0.54±0.18 0.40±0.08* 0.15±0.09* 0.02±0.05* Relative biofilm production was determined by dividing each test OD value by the control (no TTO).Values that differ significantly from the control are shown with a dagger ()(P<0.05)or with an asterisk ()(P<0.001). (not significant).The number of C.albicans ATCC 10231 cells adhered in the presence and absence of TTO would cells pre-adhered to HeLa cells was significantly less than be overstated. the control after exposure to 0.062%TTO (P=0.032) whereas no significant change was seen after exposure to 0.031%TTO (P=0.11).Numbers of C.albicans cells per Hydrophobicity HeLa cell were3.18±0.74.2.53±0.16and1.74±0.09 Both isolates were significantly less hydrophobic after 4 h for the control,0.031%treatment and 0.062%treatment, treatment with 0.125%TTO (Fig.1).C.albicans ATCC respectively.However,since non-adherent C.albicans 10231 was also significantly less hydrophobic after 1 h cells were not removed after the initial adhesion phase it with 0.125%TTO and isolate 137548L was significantly is possible that further adhesion occurred during the next less hydrophobic after 2 h with 0.125%TTO.No signifi- experimental phase,which was TTO treatment.If this cant changes were evident after treatment with 0.062% occurred.the differences between numbers of C.albicans TTO at any time point,including 18 h. Table 2 Relative adhesion"(mean standard deviation)of Candida albicans to polystyrene in the presence of TTO(%v/v)quantified by crystal violet staining. Relative adhesion in the presence of TTO (v/v)at: Isolate 0 0.008 0.016 0.031 0.062 0.125 0.25 ATCC 10231 1.00±0.00 0.66±0.22 0.67±0.09片 0.54±0.16* 0.46±0.20* 0.33±0.14* 0.28±0.11* 4047 1.00±0.00 0.79±0.25 0.78±0.28 0.82±0.29 0.67±0.28 0.56±0.32 0.43±0.271 137548L 1.00±0.00 0.83±0.23 0.72±0.26 0.54±0.231 0.35±0.12* 024±0.10* 0.27±0.15◆ 142455R 1.00±0.00 0.95±0.02 0.85±0.21 0.75±0.12 0.61±0.16 0.34±0.09* 0.29±0.14 142629J 1.00±0.00 0.92±0.34 0.79±0.30 0.67±0.26 0.43±0.23 039±0.20片 0.41±0.331 142636J 1.00±0.00 0.95±0.26 0.86±0.30 0.61±0.30 0.41±0.24÷ 0.27±0.20* 0.39±0.30* 147131G 1.00±0.00 1.02±0.28 0.89±0.36 0.55±0.17+ 0.51±0.16 0.41±0.14 0.46±0.22÷ Relative adhesion was determined by dividing each test OD value by the control (no TTO).Values that differ significantly from the control are shown with a dagger ()(P<0.05)or with an asterisk ()(P<0.001). 2012 ISHAM,Medical Mycology,50,863-870

© 2012 ISHAM, Medical Mycology, 50, 863–870 Tea tree oil reduces Candida adhesion and biofi lm 867 (not signifi cant). The number of C. albicans ATCC 10231 cells pre-adhered to HeLa cells was signifi cantly less than the control after exposure to 0.062% TTO ( P  0.032) whereas no signifi cant change was seen after exposure to 0.031% TTO ( P  0.11). Numbers of C. albicans cells per HeLa cell were 3.18  0.74, 2.53  0.16 and 1.74  0.09 for the control, 0.031% treatment and 0.062% treatment, respectively. However, since non-adherent C. albicans cells were not removed after the initial adhesion phase it is possible that further adhesion occurred during the next experimental phase, which was TTO treatment. If this occurred, the differences between numbers of C. albicans cells adhered in the presence and absence of TTO would be overstated. Hydrophobicity Both isolates were signifi cantly less hydrophobic after 4 h treatment with 0.125% TTO (Fig. 1). C. albicans ATCC 10231 was also signifi cantly less hydrophobic after 1 h with 0.125% TTO and isolate 137548L was signifi cantly less hydrophobic after 2 h with 0.125% TTO. No signifi - cant changes were evident after treatment with 0.062% TTO at any time point, including 18 h. Table 1 Relative biofi lm a formed (mean  standard deviation) by Candida albicans in the presence of TTO (% v/v) quantifi ed by XTT metabolism and crystal violet staining. Relative biofi lm formed in the presence of TTO (% v/v) at: Isolate Stain 0 0.016 0.031 0.062 0.125 0.25 ATCC 10231 XTT 1.00  0.00 0.47  0.20 * 0.39  0.16 * 0.22  0.07 * 0.06  0.04 * 0.03  0.04 * CV 1.00  0.00 0.45  0.09 0.60  0.34 0.64  0.97 0.27  0.43  0.04  0.26 † ATCC 90028 XTT 1.00  0.00 0.58  0.27 * 0.56  0.20 * 0.30  0.10 * 0.10  0.07 * 0.04  0.02 * CV 1.00  0.00 0.63  0.29 0.36  0.14 0.35  0.37 0.44  0.78 0.03  0.10 * ATCC 90029 XTT 1.00  0.00 0.58  0.13 * 0.47  0.14 * 0.26  0.13 * 0.05  0.03 * 0.01  0.02 * CV 1.00  0.00 0.60  0.16 0.49  0.26 0.31  0.10 † 0.04  0.11 *  0.04  0.18 * 137442J XTT 1.00  0.00 0.50  0.22 * 0.23  0.10 * 0.08  0.05 * 0.03  0.02 * 0.09  0.08 * CV 1.00  0.00 0.27  0.13 * 0.09  0.04 * 0.03  0.01 * 0.01  0.01 * 0.03  0.02 * 137645E XTT 1.00  0.00 0.87  0.04 0.71  0.16 † 0.58  0.18 * 0.31  0.05 * 0.14  0.14 * CV 1.00  0.00 0.89  0.22 0.48  0.15 0.45  0.22 0.96  0.97 0.25  0.22 142457X XTT 1.00  0.00 0.94  0.17 0.78  0.18 † 0.55  0.11 * 0.20  0.08 * 0.12  0.05 * CV 1.00  0.00 0.75  0.43 0.39  0.09 † 0.41  0.19 † 0.18  0.07 * 0.17  0.29 * 147168E XTT 1.00  0.00 0.62  0.23 † 0.60  0.24 † 0.37  0.15 * 0.12  0.06 * 0.05  0.04 * CV 1.00  0.00 0.54  0.27 † 0.47  0.24 † 0.35  0.13 * 0.11  0.08 * 0.17  0.33 * FF225 XTT 1.00  0.00 0.53  0.29 † 0.32  0.31 * 0.15  0.15 * 0.05  0.05 * 0.14  0.19 * CV 1.00  0.00 0.21  0.10 * 0.09  0.09 * 0.04  0.04 * 0.02  0.01 * 0.06  0.08 * KE216 XTT 1.00  0.00 0.63  0.23 † 0.48  0.33 † 0.35  0.28 * 0.13  0.13 * 0.08  0.13 * CV 1.00  0.00 0.34  0.20 * 0.14  0.09 * 0.05  0.02 * 0.02  0.01 * 0.00  0.01 * LL031 XTT 1.00  0.00 0.67  0.21 0.63  0.25 † 0.52  0.29 † 0.21  0.22 * 0.05  0.07 * CV 1.00  0.00 0.64  0.40 † 0.54  0.18 † 0.40  0.08 * 0.15  0.09 * 0.02  0.05* a Relative biofi lm production was determined by dividing each test OD value by the control (no TTO). Values that differ signifi cantly from the control are shown with a dagger ( † ) ( P  0.05) or with an asterisk ( * ) ( P  0.001). Table 2 Relative adhesion a (mean  standard deviation) of Candida albicans to polystyrene in the presence of TTO (% v/v) quantifi ed by crystal violet staining. Relative adhesion a in the presence of TTO (% v/v) at: Isolate 0 0.008 0.016 0.031 0.062 0.125 0.25 ATCC 10231 1.00  0.00 0.66  0.22 † 0.67  0.09 † 0.54  0.16 * 0.46  0.20 * 0.33  0.14 * 0.28  0.11 * 4047 1.00  0.00 0.79  0.25 0.78  0.28 0.82  0.29 0.67  0.28 0.56  0.32 0.43  0.27 † 137548L 1.00  0.00 0.83  0.23 0.72  0.26 0.54  0.23 † 0.35  0.12 * 0.24  0.10 * 0.27  0.15 * 142455R 1.00  0.00 0.95  0.02 0.85  0.21 0.75  0.12 0.61  0.16 † 0.34  0.09 * 0.29  0.14 * 142629J 1.00  0.00 0.92  0.34 0.79  0.30 0.67  0.26 0.43  0.23 † 0.39  0.20 † 0.41  0.33 † 142636J 1.00  0.00 0.95  0.26 0.86  0.30 0.61  0.30 0.41  0.24 † 0.27  0.20 * 0.39  0.30 * 147131G 1.00  0.00 1.02  0.28 0.89  0.36 0.55  0.17 † 0.51  0.16 † 0.41  0.14 † 0.46  0.22 † a Relative adhesion was determined by dividing each test OD value by the control (no TTO). Values that differ signifi cantly from the control are shown with a dagger ( † ) ( P  0.05) or with an asterisk ( * ) ( P  0.001)

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

Tea tree oil reduces Candido adhesion and biofilm 869 ATCC 10231 is atypical as it produces the quorum sensing 3 Dongari-Bagtzoglou A,Kashleva H,Dwivedi P,Diaz P,Vasilakos J. molecule farnesoic acid [26]but not farnesol [27].Quorum Characterization of mucosal Candida albicans biofilms.PLoS One sensing is important in biofilm formation and since farne- 2009:4:e7967.doi:10.1371/joural.pone.0007967. 4 Harriott MM,Lilly EA,Rodriguez TE,Fidel PL,Jr.,Noverr MC. soic acid has approximately 3%of the quorum sensing Candida albicans forms biofilms on the vaginal mucosa.Microbiol- activity of farnesol [27],this may have significant implica- 0gy2010:156:3635-3644. tions.It is recommended that studies investigating adhe- 5 Cotter G,Kavanagh K.Adherence mechanisms of Candida albicans. sion and/or biofilm formation use typical and well Br J Biomed Sci 2000:57:241-249. characterized strains,such as C.albicans ATCC 90028,so 6 Clatworthy AE,Pierson E,Hung DT.Targeting virulence:a new paradigm for antimicrobial therapy.Nat Chem Biol 2007:3: that results are more representative. 541-548. Diseases of the mucous membranes caused by Candida 7 International Organisation on Standardisation.ISO 4730:2004.Oil of spp.,including oral and vaginal candidiasis,are not life- Melaleuca,terpinen-4-ol type (tea tree oil).Geneva,Switzerland: threatening but may cause significant discomfort to suffer- International Organisation for Standardisation,2004. ers and episodes of disease can be recurring or chronic 8 Carson CF.Hammer KA,Riley TV.Melaleuca alternifolia (tea tree) oil:a review of antimicrobial and other medicinal properties.Clin While oral and vaginal candidiasis can be treated systemi- Microbiol Rev 2006:19:50-62. cally with oral antifungal agents,evidence from clinical 9 Hammer KA,Carson CF,Riley TV.Melaleuca alternifolia (tea tree) and animal studies indicate that there may be a role for oil inhibits germ tube formation by Candida albicans.Med Mycol topically applied TTO or individual essential oil compo- 2000:38:355-362. nents in the treatment of both infections [11,12,28.The 10 Hammer KA,Carson CF,Riley TV.Antifungal effects of Melaleuca alternifolia (tea tree)oil and its components on Candida albicans. therapeutic efficacy of terpenoid essential oils such as TTO Candida glabrata and Saccharomyces cerevisiae.J Antimicrob may be due,in part,to their ability to inhibit biofilm forma- Chemother2004:53:1081-1085. tion and reduce adhesion.However,many additional ther- 11 Vazquez JA,Zawawi AA.Efficacy of alcohol-based and alcohol-free apeutic factors such as essential oil formulation,frequency melaleuca oral solution for the treatment of fluconazole-refractory and duration of therapy need to be optimized to ensure the oropharyngeal candidiasis in patients with AlDS.HIV Clin Trials 2002:3:379-385 best possible clinical outcomes. 12 Mondello F,De Bernardis F,Girolamo A,Salvatore G.Cassone A.In In conclusion,the present study showed that TTO vitro and in vivo activity of tea tree oil against azole-susceptible and decreases biofilm formation and the adhesion of C.albi- -resistant human pathogenic yeasts.J Antimicrob Chemother 2003; cans to both biotic and abiotic surfaces.These novel data. 51:1223-1229. in addition to previously published information describing 13 Clinical and Laboratory Standards Institute.Reference Method for the effects of TTO against Candida,provide further sup- Broth Dilution Antifungal Susceptibility Testing of Yeasts,approved standard,3rd ed,document M27-A3.Wayne,PA:Clinical and Labo- port for the potential therapeutic use of TTO to treat super- ratory Standards Institute,2008. ficial Candida infections. 14 Altman S,Randers L,Rao G.Comparison of trypan blue dye exclu- sion and fluorometric assays for mammalian cell viability determina- tions.Biotechnol Prog 1993:9:671-674. Acknowledgments 15 Ramage G,Vande Walle K,Wickes BL,Lopez-Ribot JL.Stan- dardized method for in vitro antifungal susceptibility testing of The authors acknowledge the facilities,scientific and tech- Candida albicans biofilms.Antimicrob Agents Chemother 2001;45: nical assistance of the Australian Microscopy Micro- 2475-2479. analysis Research Facility at the Centre for Microscopy, 16 Jin Y,Samaranayake L,Samaranayake Y,Yip H.Biofilm formation Characterisation Analysis,The University of Western of Candida albicans is variably affected by saliva and dietary sugars. Arch Oral Biol 2004;49:789-798. Australia,a facility funded by the University,State and 17 Pizzo G.Giuliana G.Milici M.D'Angelo M.Effect of antimicrobial Commonwealth Governments.This work was supported mouthrinses on the in vitro adhesion of Candida albicans to human by Rural Industries Research and Development Corpora- buccal epithelial cells.Clin Oral Invest 2001;5:172-176. tion grants PRJ-5 and UWA-90A. 18 Colling L.Carter R.Essman M.Larsen B.Evaluation of relative yeast cell surface hydrophobicity measured by flow cytometry.Infect Dis Obstet Gynecol 2000;13:43-48. Declaration of interest:The authors report no conflicts of 19 Kwiecinski J,Eick S,Wojcik K.Effects of tea tree (Melaleuca alterni- interest.The authors alone are responsible for the content folia)oil on Staphylococcus aureus in biofilms and stationary growth phase.Int J Antimicrob Agents 2009;33:343-347. and writing of the manuscript. 20 Brady A,Loughlin R.Gilpin D,Kearney P,Tunney M.In vitro activity of tea-tree oil against clinical skin isolates of meticillin-resistant and References -sensitive Staphylococcus aureus and coagulase-negative staphylo- cocci growing planktonically and as biofilms.Med Microbiol 2006: 1 Sobel JD.Vulvovaginal candidosis.Lancet 2007;369:1961-1971. 55:1375-1380. 2 Ramage G,Mowat E,Jones B,Williams C,Lopez-Ribot J.Our cur- 21 Dalleau S.Cateau E,Berges T,Berjeaud JM,Imbert C.In vitro rent understanding of fungal biofilms.Crit Rev Microbiol 2009:35: activity of terpenes against Candida biofilms.Int J Antimicrob Agents 340-355. 2008:31:572-576. 2012 ISHAM,Medical Mycology,50,863-870

© 2012 ISHAM, Medical Mycology, 50, 863–870 Tea tree oil reduces Candida adhesion and biofi lm 869 ATCC 10231 is atypical as it produces the quorum sensing molecule farnesoic acid [26] but not farnesol [27]. Quorum sensing is important in biofi lm formation and since farne￾soic acid has approximately 3% of the quorum sensing activity of farnesol [27], this may have signifi cant implica￾tions. It is recommended that studies investigating adhe￾sion and/or biofi lm formation use typical and well characterized strains, such as C. albicans ATCC 90028, so that results are more representative. Diseases of the mucous membranes caused by Candida spp., including oral and vaginal candidiasis, are not life￾threatening but may cause signifi cant discomfort to suffer￾ers and episodes of disease can be recurring or chronic. While oral and vaginal candidiasis can be treated systemi￾cally with oral antifungal agents, evidence from clinical and animal studies indicate that there may be a role for topically applied TTO or individual essential oil compo￾nents in the treatment of both infections [11,12,28]. The therapeutic effi cacy of terpenoid essential oils such as TTO may be due, in part, to their ability to inhibit biofi lm forma￾tion and reduce adhesion. However, many additional ther￾apeutic factors such as essential oil formulation, frequency and duration of therapy need to be optimized to ensure the best possible clinical outcomes. In conclusion, the present study showed that TTO decreases biofi lm formation and the adhesion of C. albi￾cans to both biotic and abiotic surfaces. These novel data, in addition to previously published information describing the effects of TTO against Candida , provide further sup￾port for the potential therapeutic use of TTO to treat super￾fi cial Candida infections. Acknowledgments The authors acknowledge the facilities, scientifi c and tech￾nical assistance of the Australian Microscopy & Micro￾analysis Research Facility at the Centre for Microscopy, Characterisation & Analysis, The University of Western Australia, a facility funded by the University, State and Commonwealth Governments. This work was supported by Rural Industries Research and Development Corpora￾tion grants PRJ-5 and UWA-90A. Declaration of interest: The authors report no confl icts of interest. The authors alone are responsible for the content and writing of the manuscript. References 1 Sobel JD. Vulvovaginal candidosis. Lancet 2007; 369 : 1961 – 1971. 2 Ramage G, Mowat E, Jones B, Williams C, Lopez-Ribot J. Our cur￾rent understanding of fungal biofi lms. Crit Rev Microbiol 2009; 35 : 340 – 355. 3 Dongari-Bagtzoglou A, Kashleva H, Dwivedi P, Diaz P, Vasilakos J. Characterization of mucosal Candida albicans biofi lms. PLoS One 2009; 4 : e7967. doi:10.1371/journal.pone.0007967. 4 Harriott MM, Lilly EA, Rodriguez TE, Fidel PL, Jr., Noverr MC. Candida albicans forms biofi lms on the vaginal mucosa. Microbiol￾ogy 2010; 156 : 3635 – 3644. 5 Cotter G, Kavanagh K. Adherence mechanisms of Candida albicans . Br J Biomed Sci 2000; 57 : 241 – 249. 6 Clatworthy AE, Pierson E, Hung DT. Targeting virulence: a new paradigm for antimicrobial therapy. Nat Chem Biol 2007; 3 : 541 – 548. 7 International Organisation on Standardisation. ISO 4730:2004, Oil of Melaleuca, terpinen-4-ol type (tea tree oil) . Geneva, Switzerland: International Organisation for Standardisation, 2004. 8 Carson CF, Hammer KA, Riley TV. Melaleuca alternifolia (tea tree) oil: a review of antimicrobial and other medicinal properties. Clin Microbiol Rev 2006; 19 : 50 – 62. 9 Hammer KA, Carson CF, Riley TV. Melaleuca alternifolia (tea tree) oil inhibits germ tube formation by Candida albicans . 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Comparison of trypan blue dye exclu￾sion and fl uorometric assays for mammalian cell viability determina￾tions. Biotechnol Prog 1993; 9 : 671 – 674. 15 Ramage G, Vande Walle K, Wickes BL, Lopez-Ribot JL. Stan￾dardized method for in vitro antifungal susceptibility testing of Candida albicans biofi lms. Antimicrob Agents Chemother 2001; 45 : 2475 – 2479. 16 Jin Y, Samaranayake L, Samaranayake Y, Yip H. Biofi lm formation of Candida albicans is variably affected by saliva and dietary sugars. Arch Oral Biol 2004; 49 : 789 – 798. 17 Pizzo G, Giuliana G, Milici M, D ’ Angelo M. Effect of antimicrobial mouthrinses on the in vitro adhesion of Candida albicans to human buccal epithelial cells. Clin Oral Invest 2001; 5 : 172 – 176. 18 Colling L, Carter R, Essman M, Larsen B. Evaluation of relative yeast cell surface hydrophobicity measured by fl ow cytometry. Infect Dis Obstet Gynecol 2000; 13 : 43 – 48. 19 Kwiecinski J, Eick S, Wojcik K. 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870 Sudjana et al. 22 Braga PC,Culici M.Alfieri M.Dal Sasso M.Thymol inhibits Can- 26 Oh KB,Miyazawa H.Naito T.Matsuoka H.Purification and char- dida albicans biofilm formation and mature biofilm.Int J Antimicrob acterization of an autoregulatory substance capable of regulating the Agents2008:31:472-477. morphological transition in Candida albicans.Proc Nat Acad Sci 23 Grubb SE.Murdoch C.Sudbery PE,et al.Candida albicans-endothelial 2001:98:46644668. cell interactions:a key step in the pathogenesis of systemic candidi- 27 Hornby JM.Nickerson KW.Enhanced production of farnesol asis.nfect Immun2008;76:4370-4377. by Candida albicans treated with four azoles.Antimicrob Agents 24 Li F.Palecek SP.EAP1,a Candida albicans gene involved in binding Chemother2004;48:2305-2307. human epithelial cells.Eukaryor Cell 2003;2:1266-1273. 28 Maruyama N.Takizawa T.Ishibashi H,et al.Protective activity of 25 ten Cate JM,Klis FM,Pereira-Cenci T,Crielaard W.de Groot PW. geranium oil and its component,geraniol,in combination with vaginal Molecular and cellular mechanisms that lead to Candida biofilm for- washing against vaginal candidiasis in mice.Biol Pharm Bull 2008; mation.Dent Res 2009;88:105-115. 31:1501-1506. This paper was first published online on Early Online on 15 May 2012. 2012 ISHAM,Medical Mycology,50,863-870

© 2012 ISHAM, Medical Mycology, 50, 863–870 870 Sudjana et al. 22 Braga PC, Culici M, Alfi eri M, Dal Sasso M. Thymol inhibits Can￾dida albicans biofi lm formation and mature biofi lm. Int J Antimicrob Agents 2008; 31 : 472 – 477. 23 Grubb SE, Murdoch C, Sudbery PE, et al . Candida albicans -endothelial cell interactions: a key step in the pathogenesis of systemic candidi￾asis. Infect Immun 2008; 76 : 4370 – 4377. 24 Li F, Palecek SP. EAP1, a Candida albicans gene involved in binding human epithelial cells. Eukaryot Cell 2003; 2 : 1266 – 1273. 25 ten Cate JM, Klis FM, Pereira-Cenci T, Crielaard W, de Groot PW. Molecular and cellular mechanisms that lead to Candida biofi lm for￾mation. J Dent Res 2009; 88 : 105 – 115. 26 Oh KB, Miyazawa H, Naito T, Matsuoka H. Purifi cation and char￾acterization of an autoregulatory substance capable of regulating the morphological transition in Candida albicans . Proc Nat Acad Sci 2001; 98 : 4664 – 4668. 27 Hornby JM, Nickerson KW. Enhanced production of farnesol by Candida albicans treated with four azoles. Antimicrob Agents Chemother 2004; 48 : 2305 – 2307. 28 Maruyama N, Takizawa T, Ishibashi H, et al . Protective activity of geranium oil and its component, geraniol, in combination with vaginal washing against vaginal candidiasis in mice. Biol Pharm Bull 2008; 31 : 1501 – 1506. This paper was fi rst published online on Early Online on 15 May 2012