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Cell Density and Cell Aging as Factors Modulating Antifungal Resistance of Candida albicans Biofilms

Cell Density and Cell Aging as Factors Modulating Antifungal Resistance of Candida albicans Biofilms

C. J. Seneviratne, L. J. Jin, [...], and L. P. Samaranayake

ABSTRACT
Biofilm for m a t ion is a m a jor v ir u len ce a t t r ibu t e of Candida pa t h og en icit y w h ich con t r ibu t es t o h ig h er a n t ifu n g a l r esist a n ce. W e in v est ig a t ed t h e r oles of cell den sit y a n d cellu la r a g in g on t h e r ela t iv e a n t ifu n g a l su scept ibilit y of pla n k t on ic, biofilm , a n d biofilm -der iv ed pla n k t on ic m odes of Candida . A r efer en ce a n d a w ild-t y pe st r a in of Candida albicans w er e u sed t o ev a lu a t e t h e MICs of ca spofu n g in (CA S), a m ph ot er icin B (A MB), n y st a t in (NY T ), k et ocon a zole (KT C), a n d flu cy t osin e (5 FC). St a n da r d, NCCLS, a n d Eu r opea n Com m it t ee on A n t ibiot ic Su scept ibilit y T est in g m et h ods w er e u sed for pla n k t on ic MIC det er m in a t ion . Candida biofilm s w er e t h en dev eloped on poly st y r en e w ells, a n d MICs w er e det er m in ed w it h a st a n da r d 2 ,3 -bis(2 -m et h ox y -4 -n it r o-5 -su lfoph en y l)-5 [(ph en y la m in o)ca r bon y l]-2 H-t et r a zoliu m h y dr ox ide a ssa y . Su bsequ en t ly , a n t ifu n g a l su scept ibilit y t est in g w a s per for m ed for g r ea t er in ocu lu m con cen t r a t ion s a n d 2 4 - a n d 4 8 -h -old cu lt u r es of pla n k t on ic Candida . Fu r t h er m or e, Candida biofilm -der iv ed pla n k t on ic cells (BDPC) w er e a lso su bject ed t o a n t ifu n g a l su scept ibilit y t est in g . T h e MICs for bot h C. albicans st r a in s in t h e pla n k t on ic m ode w er e low , a lt h ou g h on in cr ea sin g t h e in ocu lu m con cen t r a t ion (u p t o 1 1 0 8 cells/m l), a v a r ia ble MIC w a s n ot ed. On t h e con t r a r y , for Candida biofilm s, t h e MICs of a n t ifu n g a ls w er e 1 5 - t o >1 ,0 0 0 -fold h ig h er . In t er est in g ly , t h e MICs for BDPC w er e low er a n d w er e sim ila r t o t h ose for pla n k t on ic-m ode cells, pa r t icu la r ly t h ose of CA S a n d A MB. Ou r da t a in dica t e t h a t h ig h er a n t ifu n g a l r esist a n ce of Candida biofilm s is a n in t r in sic fea t u r e possibly r ela t ed t o t h e biofilm a r ch it ect u r e r a t h er t h a n cellu la r den sit y or cellu la r a g in g . Candida is by fa r t h e m ost im por t a n t fu n g a l pa t h og en of h u m a n s, ca u sin g disea ses v a r y in g fr om su per ficia l m u cosa l in fect ion s t o life-t h r ea t en in g sy st em ic disor der s (3 4 ). Candida in fect ion s h a v e been docu m en t ed a s a lea din g ca u se of n osocom ia l in fect ion , w it h r epor t ed m or t a lit y r a t es a s h ig h a s 4 0 % (6 , 4 1 , 4 2 ). Fu r t h er , pr ev a ilin g in m a n y g u ises, Candida ca u ses sig n ifica n t m or t a lit y a n d m or bidit y in com pr om ised pa t ien t popu la t ion s su ch a s h u m a n im m u n odeficien cy v ir u s/A IDS pa t ien t s a n d or g a n t r a n spla n t r ecipien t s on im m u n osu ppr essiv e t h er a py (3 3 ). Candida in fect ion s pr im a r ily beg in w it h a dh er en ce a n d colon iza t ion of a n a r t ificia l or a biot ic h ost su r fa ce, lea din g t o t h e for m a t ion of su r fa ce-a t t a ch ed com m u n it ies k n ow n a s biofilm s. T h ese st r u ct u r ed com m u n it ies, en ca sed in a m a t r ix of ex opoly m er ic su bst a n ces, displa y u n iqu e ch a r a ct er ist ics t h a t con fer su r v iv a l a dv a n t a g es ov er t h eir pla n k t on ic cou n t er pa r t s (1 2 , 1 4 ). In deed, it h a s been docu m en t ed t h a t a t lea st 6 5 % of a ll m icr obia l in fect ion s a r e r ela t ed t o biofilm s (2 9 ). Candida ca n dev elop biofilm s on a lm ost a ll m edica l dev ices in cu r r en t u se, in clu din g st en t s, sh u n t s, pr ost h eses (v oice box , h ea r t v a lv es, den t u r es, et c.), im pla n t s (len s, br ea st , et c.), a n d v a r iou s t y pes of ca t h et er s (3 1 ). T h e m a jor clin ica l r elev a n ce of Candida biofilm s, a n d in fa ct of a ll m icr obia l biofilm s, is t h eir h ig h r esist a n ce t o a n t im icr obia ls. Candida biofilm s a r e k n ow n t o ex h ibit elev a t ed a n t ifu n g a l r esist a n ce com pa r ed t o t h eir pla n k t on ic cou n t er pa r t s in liqu id cu lt u r e for a n u m ber of a n t ifu n g a l a g en t s in clu din g a m ph ot er icin B (A MB), flu con a zole, it r a con a zole, a n d k et ocon a zole (KT C) (3 , 1 1 , 1 6 ). Som e st u dies, h ow ev er , su g g est t h a t n ew er a n t ifu n g a l for m u la t ion s su ch a s ech in oca n din s a n d liposom a l for m u la t ion s of A MB a r e m u ch m or e a ct iv e a g a in st Candida biofilm s (2 , 2 1 ).

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A lt h ou g h biofilm -for m in g a bilit y is a m a jor pa t h og en ic a t t r ibu t e of t h is u biqu it ou s fu n g u s, it s pr oper t ies in clu din g t h e m ech a n ism s of r esist a n ce t o a n t ifu n g a ls h a v e y et t o be defin ed. Pr ev iou s w or k er s h a v e post u la t ed fa ct or s su ch a s low er m et a bolic a ct iv it y , con t a ct -in du ced g en e ex pr ession , t h e pr esen ce of a n ex t r a cellu la r m a t r ix , a n d per sist er cells a s possible r ea son s for biofilm -a ssocia t ed a n t ifu n g a l r esist a n ce (for a r ecen t r ev iew , see r efer en ce 3 5 ). In t h e la t t er st u dies, t h e NCCLS m icr odilu t ion a ssa y h a s been com m on ly u sed for MIC det er m in a t ion for pla n k t on ic-m ode Candida w h ile a lt er n a t iv e color im et r ic a ssa y s su ch a s t h e 3 -(4 -5 -dim et h y lt h ia zol-2 -y l)2 ,5 -diph en y lt et r a zoliu m br om ide a n d 2 ,3 -bis(2 -m et h ox y -4 -n it r o-5 -su lfoph en y l)-5 -[(ph en y la m in o)ca r bon y l]-2 H-t et r a zoliu m h y dr ox ide (X T T ) r edu ct ion a ssa y s h a v e been u sed by a few for t h e sa m e pu r pose (1 1 , 1 6 ). On t h e con t r a r y , color im et r ic a ssa y s h a v e been t h e cor n er st on e of MIC det er m in a t ion for t h e biofilm m ode of Candida . A ccor din g t o t h e cu r r en t m et h odolog y , t h e pla n k t on ic MIC is com pa r ed w it h t h a t for 2 4 - or 4 8 -h biofilm s (1 0 , 1 6 , 2 3 , 3 2 ). It is n ot ew or t h y , h ow ev er , t h a t t h e NCCLS M-2 7 A m icr odilu t ion a ssa y u sed con v en t ion a lly for MIC det er m in a t ion for Candida w a s or ig in a lly desig n ed for a st a n da r d con cen t r a t ion of t h e pla n k t on ic m ode of Candida (0 .5 1 0 3 cells/m l) (2 7 ). Nev er t h eless, t h e Candida biofilm cell den sit y a ft er 2 4 or 4 8 h is fa r g r ea t er t h a n 0 .5 1 0 3 cells/m l a n d it is t em pt in g t o specu la t e t h a t t h e r epor t ed a n t ifu n g a l r esist a n ce of biofilm s m a y be du e t o t h e in cr ea sed cell m a ss of biofilm s. In deed, in a r ecen t a r t icle, a cla im h a s been m a de t h a t cell den sit y does a ffect t h e a n t ifu n g a l r esist a n ce of Candida (2 8 ). Fu r t h er m or e, t h er e is a lso a n a n om a ly w it h r eg a r d t o t h e a g e of t h e cells su bject ed t o a n t ifu n g a l su scept ibilit y t est in g ; i.e., NCCLS MIC det er m in a t ion for pla n k t on ic-m ode cells is per for m ed w it h fr esh cu lt u r es w h er ea s a 2 4 - or 4 8 -h -old cell popu la t ion is u sed for t h e biofilm m ode. Hen ce, it cou ld be su r m ised t h a t cellu la r a g in g cou ld con t r ibu t e t o a cqu ir ed h ig h er dr u g r esist a n ce in Candida biofilm s. T h er efor e, t h e a im of t h is st u dy w a s t o in v est ig a t e t h e r oles of cell den sit y a n d cellu la r a g in g on t h e com pa r a t iv e a n t ifu n g a l su scept ibilit ies of pla n k t on ic, biofilm , a n d biofilm -der iv ed pla n k t on ic cells (BDPC) of Candida . Fu r t h er m or e, w e a lso in v est ig a t ed t h e effect s of g r ow t h m ediu m a n d in cu ba t ion t im e w it h dr u g s on t h e r ela t iv e MICs for t h e pla n k t on ic v er su s t h e biofilm m ode of Candida .

MATERIALS AND METHODS

Organisms and growth conditions. T w o st r a in s of Candida albicans , n a m ely , a r efer en ce st r a in , A T CC 9 0 0 2 8 , obt a in ed fr om t h e a r ch iv a l collect ion of t h e Or a l Bioscien ces la bor a t or y of t h e Fa cu lt y of Den t ist r y , T h e Un iv er sit y of Hon g Kon g , a n d a w ild-t y pe (W T ) clin ica l st r a in , BF-1 , u sed pr ev iou sly for biofilm st u dies in ou r la bor a t or y , w er e u sed for t h is st u dy (1 8 , 1 9 ). T h e iden t it ies of t h e y ea st isola t es w er e con fir m ed w it h t h e com m er cia lly a v a ila ble A PI 3 2 C iden t ifica t ion sy st em (BioMr ieu x , Ma r cy l'Et oile, Fr a n ce). Bot h Candida st r a in s w er e su bcu lt u r ed on Sa bou r a u d dex t r ose a g a r (SDA ; Gibco Lt d., Pa isley , Un it ed Kin g dom ) a n d m a in t a in ed a t 4 C du r in g t h e ex per im en t a l per iod. Cu lt u r e pu r it y w a s con fir m ed per iodica lly by Gr a m st a in v isu a liza t ion a n d t h e g er m t u be t est . Antifungal agents. Fiv e a n t ifu n g a ls com m on ly u sed t o t r ea t or oph a r y n g ea l a n d sy st em ic ca n didia sis w er e select ed for t h is st u dy , v iz., ca spofu n g in (CA S) fr om Mer ck a n d n y st a t in (NY T ), A MB, KT C, a n d flu cy t osin e (5 FC) fr om Sig m a . A n t ifu n g a l a g en t s w er e pr epa r ed a s pr ev iou sly descr ibed (1 ). Determination of MICs for planktonic Candida cells. Fir st w e det er m in ed t h e MICs of t h e a n t ifu n g a ls CA S, NY T , A MB, KT C, a n d 5 FC for t w o Candida st r a in s w it h t h e st a n da r d NCCLS (n ow k n ow n a s t h e Clin ica l a n d La bor a t or y St a n da r ds In st it u t e [CLSI]) cr it er ia (2 7 ). Br iefly , in ocu la fr om 2 4 -h y ea st cu lt u r es on SDA w er e st a n da r dized t o a t u r bidit y equ iv a len t t o a 0 .5 McFa r la n d st a n da r d a t 5 2 0 n m w it h a spect r oph ot om et er . T h e su spen sion s w er e fu r t h er dilu t ed in Rosw ell Pa r k Mem or ia l In st it u t e (RPMI) 1 6 4 0 m ediu m (Life T ech n olog ies, New Y or k , NY ) t o y ield a n in ocu lu m con cen t r a t ion of a ppr ox im a t ely
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0 .5 1 0 3 t o 2 .5 1 0 3 cells/m l. T h e MIC a ssa y w a s per for m ed w it h 9 6 -w ell pla t es (Iw a k i, T ok y o, Ja pa n ), a n d ea ch of t h e Candida species w a s ex posed t o a dou ble dilu t ion of t h e a for em en t ion ed a n t ifu n g a ls. T h e pla t es w er e in cu ba t ed a t 3 5 C for 4 8 h t o ev a lu a t e MICs. Fu r t h er m or e, MICs for pla n k t on ic Candida cells w er e a lso det er m in ed a ccor din g t o t h e A n t ifu n g a l Su scept ibilit y T est in g Su bcom m it t ee of t h e Eu r opea n Com m it t ee on A n t ibiot ic Su scept ibilit y T est in g (A FST -EUCA ST ) m et h od (1 3 ). T h e EUCA ST m et h od u ses RPMI 1 6 4 0 m ediu m su pplem en t ed w it h 2 % g lu cose a s t h e t est m ediu m a n d a n in ocu lu m con cen t r a t ion of 0 .5 1 0 5 t o 2 .5 1 0 5 cells/m l. Ea ch of t h ese ex per im en t s w a s per for m ed on t h r ee differ en t occa sion s. Preparation of standard yeast cell suspensions for biofilm studies. Candida cells w er e g r ow n in SDA m ediu m a t 3 7 C for 1 8 h . A loopfu l of t h e y ea st w a s t h en in ocu la t ed in t o y ea st n it r og en ba se (Y NB; Difco) m ediu m su pplem en t ed w it h 5 0 m M g lu cose in a r ot a r y sh a k er a t 7 5 r pm . A ft er ov er n ig h t br ot h cu lt u r e, t h e y ea st cells w er e h a r v est ed in t h e la t e ex pon en t ia l g r ow t h ph a se a n d w a sh ed t w ice w it h 2 0 m l of ph osph a t e-bu ffer ed sa lin e (PBS; pH 7 .2 , 0 .1 M) pr ior t o u se in t h e biofilm st u dies. Biofilm formation. Candida biofilm s w er e dev eloped a ccor din g t o a pr ev iou sly pu blish ed pr ot ocol (1 8 , 1 9 ). In br ief, w a sh ed y ea st cells w er e r esu spen ded in Y NB m ediu m su pplem en t ed w it h 1 0 0 m M g lu cose a n d a dju st ed t o a n opt ica l den sit y a t 5 2 0 n m of 0 .3 8 (1 1 0 7 cells/m l). T h e st a n da r d cell su spen sion w a s u sed im m edia t ely t o dev elop biofilm s on com m er cia lly a v a ila ble pr est er ilized 9 6 -w ell poly st y r en e pla t es (Iw a k i, T ok y o, Ja pa n ). Fir st , 1 0 0 l of a st a n da r dized cell su spen sion (1 1 0 7 cells/m l) w a s pipet t ed in t o ea ch w ell of a m icr ot it er pla t e a n d in cu ba t ed for 1 .5 h a t 3 7 C in a sh a k er a t 7 5 r pm t o per m it y ea st a dh er en ce t o t h e w ell su r fa ce (a dh er en ce ph a se). For con t r ols, a w ell of ea ch m icr ot it er pla t e w a s h a n dled in a n iden t ica l fa sh ion ex cept t h a t n o Candida su spen sion w a s a dded. Follow in g t h e a dh esion ph a se, t h e cell su spen sion s w er e a spir a t ed a n d ea ch w ell w a s w a sh ed w it h 1 0 0 l of PBS t o r em ov e loosely a dh er en t cells. A 2 0 0 - l v olu m e of Y NB m ediu m w it h 1 0 0 m M g lu cose w a s t h en pipet t ed in t o ea ch of t h e w a sh ed w ells, a n d t h e pla t es w er e in cu ba t ed a t 3 7 C in a sh a k er a t 7 5 r pm for 4 8 h . A ft er t h e 4 8 -h g r ow t h ph a se, m icr oscopic ex a m in a t ion of t h e cu lt u r es w a s per for m ed t o r u le ou t con t a m in a t ion . Antifungal susceptibility test (AST) for Candida biofilms. A ST s of Candida biofilm s w er e ca r r ied ou t a s pr ev iou sly descr ibed (3 2 ). A ft er 4 8 h , t h e su spen din g m ediu m w a s a spir a t ed a n d biofilm s w er e w a sh ed w it h 1 0 0 l PBS t o r em ov e t h e n on a dh er en t cells. T h e st ock solu t ion s w er e dilu t ed t w ofold w it h RPMI 1 6 4 0 m ediu m su pplem en t ed w it h 2 % g lu cose t o obt a in dr u g con cen t r a t ion s of 1 0 0 t o 0 .1 g /m l for CA S, 2 4 0 t o 0 .2 2 5 g /m l for A MB, 6 4 t o 0 .1 2 5 g /m l for NY T a n d KT C, a n d 4 2 0 t o 0 .4 g /m l for 5 FC. A t ot a l of 1 0 0 l of t h e dr u g solu t ion w a s a dded t o t h e m icr ot it er pla t e con t a in in g Candida biofilm s. Biofilm s w er e t h en in cu ba t ed a t 3 7 C for 2 4 or 4 8 h w it h t h e a n t ifu n g a ls, a n d a ft er w a r ds t h e m et a bolic a ct iv it y of fu n g a l cells w a s det er m in ed by t h e X T T r edu ct ion a ssa y w it h m in or m odifica t ion s (1 8 , 3 2 ). XTT reduction assay. T h e X T T r edu ct ion a ssa y u sed w a s a m odifica t ion of t h e m et h ods descr ibed pr ev iou sly (1 8 , 1 9 , 3 2 ). Br iefly , a n X T T (Sig m a , St . Lou is, MO) solu t ion (1 m g /m l in PBS) w a s pr epa r ed, filt er st er ilized t h r ou g h a 0 .2 2 - m -por e-size filt er , a n d st or ed a t 7 0 C. A m en a dion e (Sig m a ) solu t ion (0 .4 m M) w a s pr epa r ed a n d filt er st er ilized im m edia t ely befor e ea ch a ssa y . Pr ior t o ea ch a ssa y , t h e X T T solu t ion w a s t h a w ed a n d m ix ed w it h t h e m en a dion e solu t ion a t a r a t io of 5 t o 1 by v olu m e. T h e biofilm s w er e fir st w a sh ed t h r ee t im es w it h 2 0 0 l of PBS, a n d t h en 2 0 0 l of PBS-X T T -m en a dion e solu t ion w a s a dded t o ea ch of t h e pr ew a sh ed w ells a n d t h e con t r ol w ells. T h e m icr ot it er pla t e w a s t h en in cu ba t ed in t h e da r k for 3 h a t 3 7 C. Follow in g in cu ba t ion , 1 0 0 l of solu t ion w a s t r a n sfer r ed t o n ew w ells a n d t h e color ch a n g e in t h e solu t ion w a s m ea su r ed w it h a m icr ot it er pla t e r ea der (Spect r a MA X 3 4 0 T u n a ble Micr opla t e Rea der ; Molecu la r Dev ices Lt d., Su n n y v a le, CA ) a t 4 9 0 n m . T h e a bsor ba n ce v a lu es for a ll n ew w ells w er e r ea d a t 4 9 0 n m . T h e a bsor ba n ce v a lu es for t h e con t r ols w er e t h en
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su bt r a ct ed fr om t h e v a lu es for t h e t est w ells t o elim in a t e spu r iou s r esu lt s du e t o ba ck g r ou n d in t er fer en ce. T h e MIC 50 for biofilm Candida cells w a s defin ed a s t h e low est dr u g con cen t r a t ion w it h a 5 0 % r edu ct ion in opa cit y com pa r ed w it h t h e dr u g -fr ee con t r ol. T o ex clu de t h e effect of t h e g r ow t h m ediu m on t h e MIC en dpoin t for t h e biofilm s, w e per for m ed t h e a n t ifu n g a l a ssa y s w it h t w o differ en t m edia , n a m ely , RPMI 1 6 4 0 m ediu m su pplem en t ed w it h 2 % g lu cose a n d Y NB m ediu m su pplem en t ed w it h 1 0 0 m M g lu cose. Ea ch ex per im en t w a s per for m ed t h r ee t im es w it h fou r r eplica t es on sepa r a t e occa sion s. Determination of cell density in Candida biofilm and age-matched planktonic cultures. Sin ce t h e MICs of a ll of t h e a n t ifu n g a ls t est ed w er e h ig h er for t h e biofilm m ode of Candida com pa r ed t o t h e pla n k t on ic m ode, w e qu er ied w h et h er t h is in cr ea sed r esist a n ce w a s a n a r t ifa ct du e t o t h e h ig h er cell den sit y of t h e biofilm s. T h er efor e, t h e cell den sit ies of t h e dev elopin g biofilm s w er e a ssessed by CFU cou n t in g a n d spect r om et r ic a n a ly sis a s descr ibed pr ev iou sly (3 9 ). Candida biofilm s w er e dev eloped a s descr ibed a bov e, a n d cell den sit ies w er e det er m in ed for t h e 2 4 - a n d 4 8 -h t im e poin t s. Cell den sit ies w er e a lso det er m in ed in pa r a llel for a g e-m a t ch ed pla n k t on ic cu lt u r es t h a t w er e pr opa g a t ed u n der iden t ica l con dit ion s, a n d iden t ica l in it ia l in ocu la (1 0 7 cells/m l) w er e u sed for t h e biofilm ex per im en t s. Drug susceptibility testing with higher-density planktonic suspensions. Sin ce t h e a for em en t ion ed ex per im en t r ev ea led t h a t t h e cell den sit ies of t h e 2 4 - a n d 4 8 -h biofilm s w er e h ig h er t h a n t h e st a n da r d in ocu lu m con cen t r a t ion u sed for t h e det er m in a t ion of MICs for pla n k t on ic Candida by eit h er t h e NCCLS or t h e EUCA ST m et h od, w e su r m ised t h a t t h e r epor t ed in cr ea se in a n t ifu n g a l r esist a n ce of biofilm m ode cells w a s du e t o t h e in cr ea sed cell den sit y . T o fu r t h er in v est ig a t e t h is possibilit y , w e in cr ea sed t h e cell den sit y of t h e pla n k t on ic cu lt u r es fr om 1 1 0 5 t o 1 1 0 8/m l a n d con du ct ed A ST s w it h t h e br ot h m icr odilu t ion a ssa y a s descr ibed ea r lier . Cla ssica l g r ow t h cu r v es cou ld be obser v ed w it h a cell con cen t r a t ion of 1 1 0 7/m l for MIC det er m in a t ion . How ev er , on ce t h e cell con cen t r a t ion in cr ea sed bey on d 1 1 0 8/m l, Candida su spen sion g r ow t h cu r v es w er e n ot clea r ly discer n ible du e t o t h e v er y h ig h opt ica l den sit y of t h e su spen sion s. T h er efor e, w e r epea t ed t h e A ST s of h ig h er -in ocu lu m Candida su spen sion s (1 1 0 7 a n d 1 1 0 8 cells/m l) w it h X T T a s descr ibed a bov e. Dr u g s w er e ser ia lly dou ble dilu t ed in 9 6 -w ell m icr ot it er pla t es, a n d h ig h cell den sit y pla n k t on ic su spen sion s w er e a dded a s descr ibed for t h e NCCLS m et h od. A ll of t h e a ssa y s w er e per for m ed in pa r a llel w it h bot h RPMI 1 6 4 0 m ediu m su pplem en t ed w it h 2 % g lu cose a n d Y NB m ediu m su pplem en t ed w it h 1 0 0 m M g lu cose. Antifungal susceptibility of C . albicans SC5314. T h e fir st set of ex per im en t s descr ibed a bov e in dica t ed t h a t , in t h e biofilm m ode, bot h t h e r efer en ce a n d W T C. albicans st r a in s w er e r esist a n t t o CA S a n d A MB a lt h ou g h t h e h ig h -den sit y pla n k t on ic cu lt u r es w er e su scept ible t o bot h dr u g s. Sin ce a cla im h a s been m a de in a r ecen t r epor t (2 8 ) t h a t , in pla n k t on ic m ode, C. albicans SC5 3 1 4 dr u g r esist a n ce depen ds on cell den sit y , w e r epea t ed t h e a bov e-descr ibed ex per im en t s w it h t h e sa m e st r a in , C. albicans SC5 3 1 4 , u sed by t h e pr ev iou s w or k er s. Hen ce, for t h e A ST of pla n k t on ic-m ode C. albicans SC5 3 1 4 , t h e NCCLS m et h od a n d h ig h -den sit y cu lt u r es (1 1 0 8 cells/m l) w er e u sed. C. albicans SC5 3 1 4 biofilm s w er e for m ed a s descr ibed a bov e for 2 4 h , a n d cell den sit y w a s det er m in ed by CFU cou n t in g a n d spect r om et r ic a n a ly sis. MICs for C. albicans SC5 3 1 4 biofilm s w er e det er m in ed w it h t h e X T T r edu ct ion a ssa y a s descr ibed a bov e. Antifungal susceptibility testing of age-matched planktonic cultures. Nex t , w e a sk ed w h a t ot h er fa ct or s, a pa r t fr om cell den sit y , cou ld con t r ibu t e t o t h e h ig h er a n t ifu n g a l r esist a n ce seen in Candida biofilm s com pa r ed t o pla n k t on ic cu lt u r es. Sin ce biofilm s a r e pr ein cu ba t ed for 2 4 or 4 8 h befor e t h e a ddit ion of dr u g s, cell a g in g m a y con t r ibu t e t o t h eir a n t ifu n g a l r esist a n ce. Fu r t h er m or e, it h a s been r epor t ed t h a t a n t ifu n g a l-r esist a n t cell popu la t ion s dev elop w it h t h e m a t u r a t ion of Candida biofilm s (1 0 ). T h er efor e, t o ex plor e t h is possibilit y , 1 1 0 7-cell/m l st a n da r d su spen sion s w er e pr epa r ed a s descr ibed a bov e for biofilm ex per im en t s. T h ese su spen sion s w er e div ided in t o t w o a liqu ot s a n d pr opa g a t ed in t h e pla n k t on ic
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a n d biofilm m odes u n der iden t ica l con dit ion s. A lon g w it h t h e biofilm , a g e-m a t ch ed pla n k t on ic cu lt u r es, for 2 4 - a n d 4 8 -h per iods, w er e ev a lu a t ed by A ST w it h t h e a for em en t ion ed X T T r edu ct ion a ssa y . Antifungal susceptibility of BDPC. Nex t , w e ev a lu a t ed w h et h er t h e h ig h a n t ifu n g a l r esist a n ce of Candida cells in t h e biofilm m ode of g r ow t h is du e t o a ph en ot y pic or a g en ot y pic a lt er a t ion of cells. W e post u la t ed t h a t if t h e dr u g r esist a n ce is a g en et ica lly a cqu ir ed t r a it , t h en on ce biofilm cells a r e r esu spen ded in t h e pla n k t on ic m ode, t h ey sh ou ld m a in t a in t h e a n t ifu n g a l r esist a n ce a cqu ir ed in t h e biofilm m ode. T o ev a lu a t e t h is h y pot h esis, w e g r ew Candida biofilm s a s m en t ion ed a bov e. A ft er 2 4 a n d 4 8 h , t h e biofilm s w er e w a sh ed g en t ly a n d scr a ped a n d pipet t ed in t o a n ew 9 6 -w ell m icr ot it er pla t e con t a in in g ser ia lly dou ble-dilu t ed dr u g s a s descr ibed ea r lier . T h ese BDPC w er e in cu ba t ed w it h a ll of t h e a n t ifu n g a ls t est ed for eit h er 2 4 or 4 8 h , a n d MICs w er e det er m in ed by X T T r edu ct ion a ssa y a s descr ibed ea r lier .

RESULTS AND DISCUSSION

Biofilm for m a t ion is a m a jor v ir u len ce a t t r ibu t e of Candida . Sin ce t h e pion eer in g w or k of Ha w ser a n d Dou g la s in 1 9 9 4 (1 6 ), h ig h er a n t ifu n g a l r esist a n ce of Candida biofilm s h a s been sh ow n by a n u m ber of ot h er in v est ig a t or s (1 0 , 2 3 , 3 2 ). How ev er , in t h ese st u dies, t h e MICs of a n t ifu n g a ls for t h e biofilm m ode of Candida der iv ed fr om color im et r ic a ssa y s su ch a s t h e X T T r edu ct ion a ssa y w er e com pa r ed w it h t h ose of pla n k t on ic-m ode Candida cells by u sin g MICs der iv ed by t h e NCCLS m icr odilu t ion m et h od. T h e la t t er m et h od u ses pla n k t on ic-m ode Candida su spen sion s a t con cen t r a t ion s of 1 1 0 3 t o 1 1 0 4 cells/m l for t h e a n t ifu n g a l a ssa y . In con t r a st , t h e biofilm m ode Candida popu la t ion u sed in t h e X T T r edu ct ion a ssa y h a s a m u ch h ig h er den sit y . T h is ca st s dou bt on t h e possibilit y t h a t t h e h ig h er cell den sit y in t h e biofilm m ode is a con t r ibu t or y fa ct or in t h e h ig h er MICs for t h e Candida biofilm m ode, a s r ecen t ly cla im ed by Per u m a l et a l. (2 8 ). Antifungal susceptibility of planktonic and biofilm mode Candida by standard methodology. In t h e fir st set of ex per im en t s in t h e pr esen t st u dy , st a n da r d A ST s, i.e., t h e NCCLS a n d EUCA ST m et h ods, w er e u sed t o ev a lu a t e t h e MICs for t h e C. albicans A T CC a n d W T st r a in s. T h e MICs of a ll fiv e a n t ifu n g a ls (v iz., CA S, NY T , A MB, KT C, a n d 5 FC) w er e low er for bot h t h e A T CC a n d W T pla n k t on ic-ph a se C. albicans st r a in s com pa r ed w it h biofilm ph a se cells w h en ev a lu a t ed by eit h er t h e NCCLS or t h e EUCA ST m et h od (T a ble 1 ). T h u s, ou r r esu lt s a r e in a g r eem en t w it h pr ev iou s r epor t s t h a t biofilm s a r e m or e r esist a n t t o a n t ifu n g a ls. How ev er , CA S, w h ich h a s been r epor t ed t o be effect iv e a g a in st t h e Candida biofilm m ode (2 , 2 1 , 3 8 ), w a s n ot pa r t icu la r ly a ct iv e a g a in st Candida biofilm s in ou r st u dy , con fir m in g t h e obser v a t ion s t h a t t h e su scept ibilit y of Candida biofilm s t o CA S is v a r ia ble, possibly du e t o r esist a n t st r a in s (1 7 , 3 7 ).
TA BLE 1 . MICs of anti f ungal agents f or the pl ank toni c v er sus b i of i l m modes of Candida

Antifungal susceptibility of high-density Candida planktonic suspensions. On ce Candida cell den sit ies w er e det er m in ed a ft er t h e a dh esion ph a se (i.e., 1 .5 h ), it w a s fou n d t h a t a ppr ox im a t ely 1 0 5 cells/m l r em a in ed in ea ch w ell of a 9 6 -w ell pla t e fr om a n in it ia l st a n da r d in ocu lu m of 1 1 0 7 cells/m l. Su bsequ en t ly , biofilm cell den sit ies r ea ch ed a ppr ox im a t ely 1 1 0 7 a n d 1 1 0 8/m l a ft er 2 4 a n d 4 8 h , r espect iv ely (Fig . 1 ). W h en w e r epea t ed t h e A ST of pla n k t on ic-m ode cells w it h a g r ea t er in ocu lu m con cen t r a t ion of 1 1 0 8 cells/m l, u p t o 8 -, 2 5 6 -, a n d >1 ,0 0 0 -fold in cr ea sed MICs of NY T , KT C, a n d 5 FC, r espect iv ely , w er e n ot ed (T a ble 1 ). How ev er , t h e MICs of CA S a n d A MB did n ot ch a n g e sig n ifica n t ly , ev en w it h t h e h ig h er in ocu lu m con cen t r a t ion of pla n k t on ic Candida . T h u s, t h e MICs of t h e la t t er t w o dr u g s w er e eit h er sim ila r t o or
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on e- t o t w ofold g r ea t er t h a n t h e MICs a t t h e low er in ocu lu m con cen t r a t ion . For in st a n ce, t h e MICs of CA S for t h e C. albicans W T st r a in a t 1 1 0 8 a n d 1 1 0 5 cells/m l w er e 0 .4 a n d 0 .2 g /m l, r espect iv ely (T a ble 1 ). Hen ce, it a ppea r ed t h a t t h e r esist a n ce of pla n k t on ic-m ode Candida m a y depen d on t h e cell den sit y of t h e su spen sion s u n der som e cir cu m st a n ces, in pa r t icu la r , for fu n g ist a t ic dr u g s su ch a s KT C a n d 5 FC.

FIG. 1 . Hi stogr am i l l ustr ati ng the r el ati v e v ar i ati ons i n c el l densi ty of Candida b i of i l ms dur i ng matur ati on and of age-matc hed pl ank toni c c ul tur es. N ote the r educ ti on i n the i ni ti al i noc ul um c onc entr ati on of 1 1 0 7 to 1 1 0 5 c el l s/ml i n the ...

How ev er , a s sh ow n a bov e, a lt h ou g h t h e h ig h er in ocu lu m con cen t r a t ion s of pla n k t on ic Candida w er e su scept ible t o fu n g icida l dr u g s su ch a s CA S a n d A MB, t h eir biofilm cou n t er pa r t s h a v in g a n equ iv a len t cell den sit y w er e r esist a n t t o t h e la t t er dr u g s. T h er efor e, w e cou ld con clu de t h a t t h e h ig h er a n t ifu n g a l r esist a n ce seen in t h e Candida biofilm m ode com pa r ed t o t h e pla n k t on ic m ode is n ot solely du e t o t h e h ig h er cell den sit y . It t h er efor e a ppea r s t h a t t h e n a t u r e of t h e a n t ifu n g a l pla y s a cr it ica l r ole in det er m in in g t h e r ela t iv e r esist a n ce of t h e biofilm v er su s pla n k t on ic-m ode Candida . Ou r da t a con fir m r esu lt s of pr ev iou s st u dies w h ich sh ow ed t h a t h ig h -den sit y (1 0 7 cells/m l) pla n k t on icm ode Candida cells w er e su scept ible t o a n t ifu n g a ls w h er ea s biofilm m ode cells w er e r esist a n t (4 , 1 1 ). In con t r a st , Per u m a l et a l. (2 8 ) r ecen t ly cla im ed t h a t cell den sit y is a m a jor fa ct or con t r ibu t in g t o t h e h ig h er a n t ifu n g a l r esist a n ce of Candida biofilm s. T h e la t t er w or k er s obser v ed t h a t h ig h -den sit y pla n k t on ic cu lt u r es of C. albicans SC5 3 1 4 (equ a l t o biofilm cell con cen t r a t ion s) sh ow ed a r esist a n ce t o CA S a n d A MB sim ila r t o t h a t of biofilm m ode cells. T h er efor e, t o ex plor e t h e possibilit y of a st r a in v a r ia t ion effect on a n t ifu n g a l su scept ibilit y , w e u sed t h e sa m e C. albicans st r a in , SC5 3 1 4 , for t h e n ex t set of A ST s (2 4 ). W h en w e com pa r ed t h e a n t ifu n g a l MICs obt a in ed by t h e NCCLS m et h od for t h e pla n k t on ic m ode w it h t h ose obt a in ed for t h e biofilm m ode, h ig h er MICs of a ll of t h e a n t ifu n g a l t est ed, i.e., CA S, A MB, KT C, a n d 5 FC, w er e obser v ed (T a ble 2 ). How ev er , on ce t h e pla n k t on ic-m ode C. albicans SC5 3 1 4 con cen t r a t ion w a s in cr ea sed t o 1 1 0 8 cells/m l, it w a s obser v ed t h a t t h e cells w er e r esist a n t t o KT C a n d 5 FC bu t n ot t o CA S a n d A MB (T a ble 2 ; Fig . 2 a n d 3 ). Hen ce, ou r da t a on CA S a n d A MB a r e in lin e w it h pr ev iou s st u dies (4 , 1 1 ) bu t con t r a dict t h e obser v a t ion s r epor t ed by Per u m a l et a l. (2 8 ). Nev er t h eless, ou r da t a on KT C a r e in a g r eem en t w it h t h e obser v a t ion of Per u m a l et a l. (2 8 ) t h a t h ig h -den sit y pla n k t on ic-m ode C. albicans cells a r e t oler a n t of h ig h a zole dr u g con cen t r a t ion s. T h ese discr epa n cies in r esu lt s cou ld be du e t o t h e m odified ex per im en t a l pr ot ocols u sed in t h e st u dies. T h er efor e, it is im per a t iv e t h a t st a n da r d pr ot ocols be u sed by fu t u r e w or k er s in or der t o u n der st a n d t h e en ig m a of t h e a n t ifu n g a l r esist a n ce of Candida biofilm s. Con sider in g t h e da t a fr om t h e pr esen t a n d ot h er , r ela t ed, st u dies, w e cou ld con clu de t h a t a lt h ou g h t h e h ig h er cell den sit y seen in t h e biofilm m ode m a y pa r t ia lly con t r ibu t e t o a n t ifu n g a l r esist a n ce, t h er e a r e ot h er , a s-y et -u n discov er ed, fa ct or s w h ich m odu la t e t h is ph en om en on . T h ese fa ct or s m a y in clu de t h e n a t u r e of t h e a n t ifu n g a l, a s w ell a s con t a ct -in du ced g en e ex pr ession or differ en t ia lly ex pr essed pr ot ein biom a r k er s r ela t ed t o t h e su r fa ce-a t t a ch ed biofilm m ode (2 6 , 3 5 , 3 6 ).

FIG. 2. XTT r educ ti on assay of r esi stanc e of hi gh-densi ty (1 0 8 c el l s/ml ) pl ank toni c c ul tur es of C. albic ans SC531 4 and W T BF-1 to CA S (l i nes 1 to 4) and A MB (l i nes 5 to 8). N ote the par adox i c al ef f ec t of C. albic ans str ai n r esi stanc e to CA S at hi gher c onc entr ati ons ...

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FIG. 3. XTT r educ ti on assay of r esi stanc e of hi gh-densi ty (1 0 8 c el l s/ml ) pl ank toni c c ul tur es of C. albic ans SC531 4 to CA S (l i nes 1 and 2), A MB (l i nes 3 and 4), KTC (l i nes 4 to 6), and 5FC (l i nes 7 and 8).

TA BLE 2. MICs deter mi ned b y the N CCLS method and A ST of pl ank toni c -mode and 24-h b i of i l m C. albic ans SC531 4 c el l s

A n ot h er in t er est in g obser v a t ion of t h e pr esen t st u dy is t h e pa r a dox ica l effect sh ow n by C. albicans st r a in s for CA S (Fig . 2 ). T h u s, g r ow t h of C. albicans cou ld be obser v ed a t dr u g con cen t r a t ion s h ig h er t h a n t h e MIC, i.e., 1 2 .5 t o 5 0 g /m l. T h is obser v a t ion is in a g r eem en t w it h pr ev iou s r epor t s on t h e pa r a dox ica l g r ow t h of C. albicans in t h e pr esen ce of ech in oca n din dr u g s (9 , 2 5 ). A lt h ou g h sev er a l h y pot h eses h a v e been pr oposed t o ex pla in t h is ph en om en on , t h e ex a ct m ech a n ism (s) h a s y et t o be fou n d. Antifungal susceptibility of age-matched planktonic cells and biofilms. T o ev a lu a t e t h e effect of cell a g in g on MICs, w e per for m ed r ela t iv e a n t ifu n g a l su scept ibilit y t est in g w it h a g e-m a t ch ed biofilm a n d pla n k t on ic-m ode Candida cells k ept u n der iden t ica l con dit ion s for u p t o 2 4 a n d 4 8 h (a g e m a t ch ed r efer s t o t h e in cu ba t ion per iod for w h ich t w o g r ow t h m odes of Candida w er e pr opa g a t ed; h ow ev er , it sh ou ld be n ot ed t h a t , du e t o t h e dispa r a t e g r ow t h r a t es of t h e biofilm a n d pla n k t on ic-m ode cells, t h e qu a lit y of t h e r esu lt a n t cell popu la t ion s a t 2 4 or 4 8 h is u n lik ely t o be t h e sa m e). W h en a n A ST of t h e a for em en t ion ed a g e-m a t ch ed pla n k t on ic cu lt u r es a t 2 4 or 4 8 h w a s per for m ed, w e fou n d a MIC pr ofile v er y sim ila r t o t h a t of h ig h -den sit y pla n k t on ic cu lt u r es (T a ble 3 ). For in st a n ce, h ig h er MICs of t h e fu n g ist a t ic dr u g s KT C a n d 5 FC a n d low er MICs of CA S a n d A MB w er e n ot ed. A s t h e cell den sit y of pla n k t on ic cu lt u r es in cr ea ses a ft er 2 4 or 4 8 h , t h is m a y h a v e been a con t r ibu t or y fa ct or in t h e low er su scept ibilit y t o fu n g ist a t ic dr u g s. In con t r a st , t h e MICs of CA S a n d A MB for r espect iv e a g e-m a t ch ed biofilm m ode cells of Candida w er e r ela t iv ely h ig h er (T a ble 3 ). It is k n ow n t h a t a g in g Candida cells or cu lt u r es en t er in t o a st a t ion a r y ph a se a n d, in doin g so, a cqu ir e a deg r ee of r esist a n ce t o a n t ifu n g a ls (7 , 8 , 4 0 ). How ev er , ou r da t a in dica t e t h a t t h e a n t ifu n g a l su scept ibilit y of a g ed cu lt u r es v a r ies, depen din g on t h e a n t ifu n g a ls a s w ell a s t h e g r ow t h m ode (T a ble 3 ). T a k en t og et h er , it cou ld be su r m ised t h a t fa ct or s ot h er t h a n cellu la r a g in g pla y a r ole in t h e h ig h er a n t ifu n g a l r esist a n ce of Candida cells in t h e biofilm m ode.
TA BLE 3. Compar ati v e MICs of anti f ungal agents f or 24- and 48-h pl ank toni c , b i of i l m, and b i of i l m-der i v ed pl ank toni c Candida c el l s

Effect of culture medium and incubation period on antifungal susceptibility. In or der t o det er m in e w h et h er t h e differ en ce in t h e m ediu m or in cu ba t ion t im e u sed cou ld a ffect t h e r ela t iv e MICs of dr u g s for pla n k t on ic v er su s biofilm m ode Candida cells, w e u sed t w o differ en t m edia a n d in cu ba t ion t im es for ou r A ST . W e n ot ed t h a t for pla n k t on ic a n d biofilm cells, in cu ba t ion for eit h er 2 4 or 4 8 h w it h t h e a n t ifu n g a ls t est ed y ielded a lm ost iden t ica l r esu lt s (da t a n ot sh ow n ). Fu r t h er m or e, in cu ba t ion in RPMI 1 6 4 0 m ediu m w it h
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2 % g lu cose or Y NB m ediu m w it h 1 0 0 m M g lu cose h a d n o effect on t h e MICs for eit h er pla n k t on ic or biofilm cells (da t a n ot sh ow n ). It sh ou ld be n ot ed t h a t , on occa sion , t h e cu lt u r e m ediu m a n d in cu ba t ion per iod r esu lt ed in a dev ia t ion of t h e MIC by 1 dilu t ion . Hen ce, w e cou ld ex clu de t h e qu a lit y of t h e cu lt u r e m ediu m u sed or t h e in cu ba t ion per iod a s a possible con t r ibu t or y fa ct or in t h e h ig h er MICs seen in ou r A ST of Candida biofilm s. Antifungal susceptibility of BDPC. Nex t , t o ev a lu a t e t h e possibilit y t h a t t h e h ig h r esist a n ce seen in Candida biofilm s is a ph en ot y pic or a g en ot y pic fea t u r e, w e per for m ed a n A ST on BDPC. W e collect ed biofilm cells a ft er 2 4 a n d4 8 h a n d t r a n sfer r ed t h e cells in t o w ells of a n ew 9 6 -w ell pla t e, ch a n g in g t h e cells t o t h e pla n k t on ic m ode. T h e a n t ifu n g a l su scept ibilit y of t h ese BDPC w a s a lm ost iden t ica l t o t h a t of 4 8 -h pla n k t on ic Candida su spen sion s, ex cept for A MB, t o w h ich cells w er e m or e r esist a n t (T a ble 3 ). T h u s, t h e MIC of A MB for BDPC of C. albicans A T CC 9 0 0 2 8 w a s 1 .8 7 g /m l, com pa r ed w it h 0 .4 6 g /m l for t h e cor r espon din g 4 8 -h pla n k t on ic cu lt u r es. On ly a few st u dies t h u s fa r h a v e com pa r ed t h e a n t ifu n g a l su scept ibilit ies of biofilm cells a n d BDPC (4 , 5 , 2 0 , 2 8 , 3 0 ). A lt h ou g h it is difficu lt t o dr a w fir m con clu sion s du e t o differ en ces in t h e m et h odolog ies, a n t ifu n g a l a g en t s, a n d st r a in s em ploy ed in t h ese st u dies, t h e g en er a l con sen su s is t h a t a n t ifu n g a l r esist a n ce is a ph en ot y pic fea t u r e r a t h er t h a n a g en ot y pic a lt er a t ion of biofilm cells. How ev er , it is n ot ew or t h y t h a t som e w or k er s h a v e sh ow n t h a t t h er e is a sm a ll su bpopu la t ion of biofilm cells k n ow n a s per sist er s w it h h ig h er a n t ifu n g a l r esist a n ce t h a n t h e r est of t h e biofilm popu la t ion (1 5 , 2 0 , 2 2 ). Hen ce, t h e m a r g in a l in cr ea se in t h e r esist a n ce of BDPC t o a n t ifu n g a ls obser v ed in ou r st u dy m a y be du e t o t h e pr esen ce of t h ese per sist er s or a n ot h er t r a it a cqu ir ed t h r ou g h t h e biofilm life-st y le. In con clu sion , w e h a v e ca r r ied ou t a com pr eh en siv e st u dy of com pa r a t iv e t est in g of t h e a n t ifu n g a l su scept ibilit y of pla n k t on ic cells, biofilm cells, a n d BDPC t o fiv e a n t ifu n g a ls in clin ica l u se. Ou r st u dy r ev ea led t h a t NCCLS or EUCA ST det er m in a t ion s a lon e a r e in su fficien t t o obt a in a h olist ic pict u r e of a n t ifu n g a l su scept ibilit y , especia lly w h en t h e or g a n ism s a r e in t h e biofilm m ode. T h er efor e, it is im per a t iv e t o det er m in e t h e MICs of a n t ifu n g a ls a t a h ig h er cell den sit y of t h e pla n k t on ic a n d biofilm m odes. Da t a fr om su ch a pr ot ocol w ill per h a ps be m or e u sefu l pa r t icu la r ly in clin ica l set t in g s t o select a n a ppr opr ia t e a n t ifu n g a l. Fu r t h er m or e, it cou ld be con clu ded t h a t a lt h ou g h ph y sica l fa ct or s su ch a s cell den sit y , cellu la r a g in g , a n d g r ow t h m ediu m m a y pa r t ia lly con t r ibu t e t o t h e h ig h er a n t ifu n g a l r esist a n ce seen in Candida biofilm s, t h er e a r e ot h er fa ct or s r ela t ed t o t h e biofilm m ode w h ich det er m in e it s h ig h er a n t ifu n g a l r esist a n ce. Cu r r en t st u dies w it h n ew er m olecu la r biolog y t ools a n d g en om ic a n d pr ot eom ic a ppr oa ch es sh ou ld elu cida t e t h e in t r ig u in g a n d a s-y et -u n discov er ed a spect s of t h e a n t ifu n g a l r esist a n ce of ca n dida l biofilm s.

ACKNOWLEDGMENTS
T h is w or k w a s su ppor t ed by t h e Hon g Kon g Resea r ch Gr a n t s Cou n cil, RGC n o. HKU 7 6 2 4 /0 6 M.

FOOTNOTES
Published ahead of print on 14 July 2008.

ARTICLE INFORMATION
Antimicrob Agents Chemother. 2008 September; 52(9): 32593266. Published online 2008 July 14. doi: 10.1128/AAC.00541-08 PMCID: PMC2533466 C. J. Seneviratne,* L. J. Jin, Y. H. Samaranayake, and L. P. Samaranayake

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* Corresponding

Cell Density and Cell Aging as Factors Modulating Antifungal Resistance of Candida albicans Biofilms

Oral Biosciences, Faculty of Dentistry, The University of Hong Kong, Hong Kong author. Mailing address: Oral Biosciences, Faculty of Dentistry, The University of Hong Kong, Prince Philip Dental Hospital, 34 Hospital Road, Hong Kong. Phone: (852) 28590480. Fax: (852) 25476133. E-mail: lakshman/at/hku.hk Received April 28, 2008; Revised May 17, 2008; Accepted July 6, 2008. Copyright 2008, American Society for Microbiology This article has been cited by other articles in PMC. Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)

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