Metal resistance inCandidabio¢lmsJoe J. Harrison1,2, Maryam Rabiei1, Raymond J. Turner1, Erin A. Badry1, Kimberley M. Sproule1 &Howard Ceri1,2

1Department of Biological Sciences, University of Calgary, Calgary, Canada; and 2Biofilm Research Group, University of Calgary, Calgary, Canada

Correspondence: Raymond J. Turner,

Department of Biological Sciences, University

of Calgary, 2500 University Drive NW,

Calgary, AB, Canada T2N 1N4. Tel.: 11403

220 4308; fax: 11403 289 9311;

e-mail: turnerr@ucalgary.ca

Received 27 July 2005; revised 8 September

2005; accepted 8 September 2005.

First published online 5 January 2006.

doi:10.1111/j.1574-6941.2005.00045.x

Editor: Gary King

Keywords

Candida tropicalis ; biofilm; metal tolerance;

antifungal resistance; metalloid; extracellular

polymeric matrix.

Abstract

Yeasts are often successful in metal-polluted environments; therefore, the ability of

biofilm and planktonic cell Candida tropicalis to endure metal toxicity was

investigated. Fifteen water-soluble metal ions, chosen to represent groups 6A to

6B of the periodic table, were tested against this organism. With in vitro exposures

as long as 24 h, biofilms were up to 65 times more tolerant to killing by metals than

corresponding planktonic cultures. Of the most toxic heavy metals tested, only

very high concentrations of Hg21, CrO42� or Cu21 killed surface-adherent

Candida. Metal-chelator precipitates could be formed in biofilms following

exposure to the heavy metals Cu21 and Ni21. This suggests that Candida biofilms

may adsorb metal cations from their surroundings and that sequestration in the

extracellular matrix may contribute to resistance. We concluded that biofilm

formation may be a strategy for metal resistance and/or tolerance in yeasts.

Introduction

In ecological studies of yeast populations, Candida repre-

sents an abundant fungal genus found in rich soil and

aquatic habitats (Slavikova & Vadkertiova, 1997; Ahearn,

1998; Lopez-Archilla et al., 2004). Toxic metals may be

naturally spread into these places by volcanic activity or by

the weathering of minerals (Brown et al., 2003). Urbaniza-

tion and industrialization have also disseminated water-

soluble metal ions into our environment, including into

forests, rivers and even protected regions that may otherwise

be considered pristine (De Vries et al., 2002; Hernandez

et al., 2003; Schparyk & Parpan, 2004). As examples, Zn21,

Ni21 and Mn21 have been reported at concentrations of 19,

6.5 and 39 mM, respectively, in the mine-polluted Huelva

estuary, Spain (Morillo et al., 2005). In the severely con-

taminated River Avoca in southeast Ireland, the heavy

metals Cu21 and Pb21 are present in the subsurface sedi-

ment at 14.2 and 2.2 mmol g� 1, respectively (Gaynor &

Gray, 2004). Yeasts often become the predominant microbial

species in aqueous locales contaminated by heavy metal

pollutants (Hagler & Mendocs-Hageler, 1981).

Candida spp. have been isolated from metal-contami-

nated pulp-mill wastewater and acid mine runoff (Suihko &

Hoekstra, 1999; Lopez-Archilla et al., 2004). Candida albi-

cans and Candida tropicalis are known for high levels of

resistance to the water-soluble ions Hg21, Pb21, Cd21,

arsenate (AsO43� ) and selenite (SeO3

2� ) (Berdicevsky

et al., 1993). Many Candida spp. also have the capacity to

adsorb and/or accumulate metals from their surroundings

(Podgorskii et al., 2004). Metalloadsorption by nonpatho-

genic and cell-surface engineered microbes is a promising

technology for the bioremediation of metal-polluted sites

(Costley & Wallis, 2001; Lee et al., 2003). Our work is

focused on the ability of C. tropicalis biofilms to survive

exposure to metals and adsorb them from an aqueous

growth medium.

The majority of microorganisms found in medical, in-

dustrial and natural environments grow in multicellular,

surface-adherent assemblages termed biofilms. Approxi-

mately 10% of Candida spp., including C. tropicalis, are

opportunistic human pathogens that are normal inhabitants

of human skin, and vaginal, gastrointestinal and urinary

tracts (Ahearn, 1998). The majority of studies of Candida

biofilms have been centred on medical applications. Candi-

diasis is most often associated with biofilm formation on

indwelling medical devices (Nyguyen et al., 1995; Chandra

et al., 2001a; Kojic & Darouiche, 2004). Candida spp. are

frequently recovered from hard surfaces in hospitals as well

as from sites of fecal contamination (Eggimann et al., 2003).

Within these settings, Candida biofilms are infamous for

FEMS Microbiol Ecol 55 (2006) 479–491 c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

high levels of resistance to disinfectants and antifungal

agents (Lamfon et al., 2004; Ramage et al., 2005).

Although the tolerance of bacterial biofilms to metal ions

has been documented in a number of studies (Teitzel &

Parsek, 2003; Harrison et al., 2004a, b, 2005b, c), no study to

date has specifically examined the susceptibility of yeast

biofilms to metals. This study employed a high-throughput

method previously developed by our research group to

examine the susceptibility of bacterial biofilms to antibio-

tics, disinfectants and metals (Ceri et al., 1999; Harrison

et al., 2004a). Here we modified this technique to examine

the ability of C. tropicalis biofilms to survive exposure to a

diverse array of toxic metal ions. Candida tropicalis biofilms

survived (and in some instances grew) in high concentra-

tions of these antimicrobials, whereas corresponding plank-

tonic cells died in a time-dependent fashion. Furthermore,

the ability of yeast biofilms to adsorb metals was investi-

gated. When treating exposed biofilms with the organic

chelator diethyldithiocarbamate, highly coloured metal pre-

cipitates rapidly became visible in these surface-adherent

microbial communities. We suggest that biofilm formation

may function as a mechanism of metal resistance and/or

tolerance for Candida.

Materials andmethods

Strainsandgrowthmedia

Candida tropicalis 99916 (an isolate from the Foothills

Hospital in Calgary, AB, Canada) was stored at � 70 1C in

a MicrobankTM (ProLab Diagnostics, Toronto, Canada)

according to the manufacturer’s directions. This yeast was

grown in tryptic soy broth (TSB, EMD Chemicals Inc.,

Gibbstown, NJ) for all metal susceptibility assays. Subcul-

tures, fungicidal assays and viable cell counts were carried

out by plating on tryptic soy agar (TSA) (EMD Chemicals

Inc.), and incubating at 30 1C for 48 h. Serial dilutions were

performed using 0.9% saline.

Biofilm cultivation

Biofilms were grown in the MBECTM Physiology and

Genetics assay (MBEC BioProducts Inc., Edmonton, Canada;

http://www.mbec.ca). This method was previously described

by Ceri et al. (1999, 2001), and uses a plastic lid with 96 pegs

that fits inside a standard 96-well microplate. This device

and the cultivation methods were modified to facilitate the

growth of yeast biofilms. The peg lid was immersed into a

sterile solution of 1% L-lysine in double-distilled water

(ddH2O) and incubated at room temperature for 16 h. The

lids were then dried upside down in a laminar-flow hood for

30 min. Coating the pegs with a positively charged amino

acid enhanced the ability of C. tropicalis 99916 to form

biofilms on this polystyrene surface.

Starting from cryogenic stocks, C. tropicalis was streaked

out twice on TSA. An inoculum was prepared by suspending

colonies from the second agar subculture in TSB to match a

1.0 McFarland standard. This standard inoculum was diluted

30-fold in TSB to attain a starting viable cell count of roughly

1.0� 105 CFU mL� 1. One hundred and fifty microliters mL

of this inoculum was transferred into each well of a 96-well

microtitre plate. The dried, L-lysine-coated peg lids were then

inserted into the microplates containing this inoculum. These

devices were placed on a gyrorotary shaker at 125 rpm for

48 h in an incubator at 35 1C and 95% relative humidity.

Biofilms were rinsed once with 0.9% saline (by placing

the lid in a microplate containing 200 mL of saline in each

well) to remove loosely adherent planktonic cells. Biofilm

formation was evaluated by breaking four pegs from each

device after it had been rinsed. Biofilms were disrupted into

200 mL of 0.9% saline using a water table sonicator on the

setting ‘high’ for a period of 5 min (Aquasonic model 250

HT; VWR Scientific, Mississauga, Canada). Alternatively,

microbial growth was evaluated for statistical equivalence by

disrupting all biofilms at the same time from the lid into a

microplate with 200mL of saline in each well. The disrupted

biofilms were serially diluted and plated onto agar for viable

cell counting.

Stock solutionsofmetal compounds

Sodium arsenite (NaAsO2), cadmium sulphate (CdSO4 � 8/3

H2O), lead nitrate [Pb(NO3)2], manganese chloride

(MnCl2 � 6H2O), nickel sulphate (NiSO4 � 6H2O), sodium

selenite (Na2SeO3), silver nitrate (AgNO3), and potassium

tellurite (K2TeO3) were obtained from Sigma Chemical Com-

pany (St Louis, MO). Aluminum sulphate [Al2(SO4)3 � 18

H2O], sodium hydrogen arsenate (Na2HAsO4), cupric sul-

phate (CuSO4 � 5H2O), potassium dichromate (K2Cr2O7),

mercuric chloride (HgCl2), and zinc sulphate (ZnSO4 � 7H2O)

were obtained from Fischer Scientific (Ottawa, Canada).

Cobalt chloride was purchased from British Drug Houses

Limited (Poole, England). All metal compounds were dis-

solved in ddH2O to obtain a stock solution of the metal

counterion at a concentration five times the maximum used

in susceptibility assays. These solutions were passed through

0.22mm syringe filters into sterile glass vials and stored at

room temperature until used. Working solutions of metals

were prepared in TSB from stock metal cation or oxyanion

solutions not more than 60 min prior to exposure.

Metal susceptibility testingof biofilms

The rinsed peg lids were transferred to 96-well microtitre

plates containing serial twofold dilutions of metal com-

pounds in TSB. The first and last well of every row served as

a sterility and growth control, respectively. These plates were

incubated at 35 1C and 95% relative humidity for either 5 or

FEMS Microbiol Ecol 55 (2006) 479–491c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

480 J.J. Harrison et al.

24 h. Biofilm metal susceptibility testing was performed

according to the method of Harrison et al. (2004a). All

metal susceptibility assays used a neutralizing step as pre-

viously described (Harrison et al., 2004a, b, 2005b). Co21,

Zn21, Cd21, Pb21 and all metal oxyanions were neutralized

using 10 mM reduced glutathione (Harrison et al., 2004a,

b). Cu21 and Ni21 were chelated with 0.5 mM sodium

diethyldithiocarbamate (DDTC) (Harrison et al., 2004a,

2005c), a maximum concentration dictated by its toxicity

to yeast cells. Minimum lethal concentrations for planktonic

cells (MLCP) and biofilms (MLCB) were redundantly deter-

mined without the use of this agent. Silver was complexed

with 10 mM sodium citrate, Al31 and Mn21 with�1–2 mM

acetylsalicylic acid, and Hg21 with 10 mM L-cysteine (Har-

rison et al., 2004a). All neutralizing agents were purchased

from Sigma Chemical Company.

Following exposure, peg lids were rinsed twice with 0.9%

saline, and the biofilms were disrupted into fresh recovery

media (which contained the neutralizing agents). Twenty-

microlitre aliquots of the recovery cultures (containing

the disrupted biofilms) were plated onto TSA. These plates

were incubated for 48 h and were scored for growth to obtain

MLCB values. Alternatively, log-killing was evaluated by serial

dilution of the recovery cultures and by plating onto TSA.

Planktonic cell susceptibility testing

To evaluate planktonic minimal inhibitory concentration

(MIC) and MLCP values we used a modified protocol

for yeast susceptibility testing suggested by the American

Society for Microbiology (Fothergill & McGough, 1995).

Challenge plates of metals were prepared as described above.

A 1.0 McFarland standard was prepared in a manner

identical to that used for biofilm cultivation. This standard

was diluted twofold in TSB, and 5 mL of this was added

to each well of the challenge plates. Forty-microlitre aliquots

of the planktonic cultures were transferred to 96-well

microtitre plates (which contained neutralizing agents).

Twenty-microlitre aliquots of the neutralized planktonic

cultures were plated onto TSA. These plates were incubated

for 48 h and scored for growth to obtain MLCP values.

Alternatively, log-killing was evaluated by serial dilution

of neutralized cultures and plating onto TSA. MIC values

were obtained after 48 h incubation at 35 1C by reading

the optical density at 650 nm (OD650) of the challenge

plates using a THERMOmax microplate reader with

SOFTmax Pro data analysis software (Molecular Devices

Corporation, Sunnyvale, CA).

Confocal laser scanningmicroscopy (CLSM)

Pegs were broken from the lid of the MBECTM assay. A

portion of these pegs were immersed in 200mg mL� 1 tetra-

methylrhodamine conjugated concanavalin A (TRITC) (Con

A, Molecular Probes, Burlington, Canada) and incubated at

30 1C for 90 min. Con A is a lectin with high specificity for

mannose sugars present in the cell walls and biofilm matrix

of Candida albicans (Jin et al., 2005). These pegs were

subsequently treated with the nucleic acid stain Syto 9

(Molecular Probes) at the dilution of the commercially

available stock suggested by the manufacturer. Biofilms were

incubated in Syto 9 at 30 1C for 20 min. The remaining pegs

were stained by immersion into 200mL of 0.05% weight in

volume (w/v) acridine orange (Sigma Chemical Co.) for

5 min at room temperature. Acridine orange is a nucleic acid

stain that interchelates dsDNA and binds ssDNA through

dye-base stacking (Bernas et al., 2005).

Cell viability staining of yeast using the Live/Dead

BacLightTM Kit (Molecular Probes) was carried out accord-

ing to the method of Jin et al. (2005). Biofilms exposed to

metals were rinsed twice with 0.9% saline. Subsequently,

biofilm pegs were stained concomitantly with Syto 9 and

propidium iodide at 30 1C for 30 min. Syto 9 (green emis-

sion) stains all cells in the microbial population regardless of

viability. This technique relies on the impermeability of the

yeast membrane to propidium iodide (PI, red emission),

which only penetrates and stains cells with compromised

membrane integrity. Thus live cells stain green, and dead

cells stain orange-red. This has been previously shown to

correlate well with viable cell counts of a calibrated suspen-

sion of C. albicans cells (Jin et al., 2005).

Fluorescently labelled biofilm pegs were placed in 2 drops

of phosphate-buffered saline (pH 7.2) on the top of a glass

coverslip. For Live/Dead staining, 0.9% saline was used

instead. These pegs were examined using a Leica DM IRE2

spectral confocal and multiphoton microscope with a Leica

TCS SP2 acoustic optical beam splitter (AOBS) (Leica

Microsystems, Richmond Hill, Canada). To eliminate arti-

facts associated with single-wavelength excitation, samples

were sequentially scanned, frame by frame, first at 488 nm

and then at 543 nm. Fluorescence emission was then sequen-

tially collected in the green and red regions of the spectrum,

respectively, for the dye pairs Syto 91PI, and Syto

91TRITC-Con A. A 63�water immersion objective was

used in all imaging experiments. Image capture and three-

dimensional reconstruction of z-stacks were performed

using Leica Confocal Software Lite (LCS Lite, available free

of charge from Leica Microsystems at http://www.leica-

microsystems.com/website/lms.nsf).

Precipitationof heavymetal cations inCandida biofilms

The organic chelator sodium diethyldithiocarbamate

(DDTC) causes the rapid precipitation of some transition

metals as well as tellurite in vitro. In particular, DDTC causes

FEMS Microbiol Ecol 55 (2006) 479–491 c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

481Metal resistance in Candida biofilms

Cu21, Ni21 and TeO32� to form brown, light-yellow, and

dark-yellow precipitates, respectively (Cheng et al., 1982;

Turner et al., 1992). Our research group has previously

exploited this to show that metal cations may be retained

inside bacterial biofilms (Harrison et al., 2005b). To test if

this also occurred for biofilms of yeast, Candida tropicalis

was grown on the pegs of the MBECTM device and exposed

to Cu21, Ni21 or TeO32� for 5 h. Next, the exposed biofilms

were rinsed twice with 0.9% saline for 10 min each time. The

peg lids were then inserted into a 96-well microtitre plate

with 200 mL of 37.5 mM DDTC in each well. Planktonic

cultures in metal challenge media were also exposed to

DDTC as a positive control. In this case, 12.5mL aliquots

from wells of the challenge plate (following biofilm expo-

sure) were mixed with 37.5 mL of 150 mM DDTC in the wells

of a microtitre plate.

Statistical tests anddataanalysis

One-way analysis of variances (ANOVA) was used to analyse

log10-transformed raw data with MINITABs Release 14

(Minitab Inc., State College, PA). Mean and standard

deviation calculations of MIC, MLCP, and MLCB values

were performed using Microsofts Excel XP (Microsoft

Corporation, Redmond, WA).

Results

Definitionsofmeasurements

The minimum lethal concentration (MLC) has been defined

as the concentration of an antimicrobial required to kill

99.95% of the fungal population (Fothergill & McGough,

1995). This definition will be retained for planktonic cell

susceptibility assays and will be referred to here as MLCP.

The minimum lethal concentration for biofilms (MLCB) will

be similarly defined here as the concentration of an anti-

microbial required to kill 99.95% of the fungal biofilm

population. In terms of our assays, this implies that any

plate with 1 or no colonies (from a 20mL test spot) was

scored negative for growth. Here we will define the fold

tolerance as the ratio of MLCB:MLCP. This measurement is

quantitative and also represents a logical argument. If the

MLCB is greater than the maximum concentration exam-

ined and the MLCp is known, then the fold tolerance is

greater than or equal to twice the ratio of the highest

concentration examined over the MLCP. This takes into

account the sensitivity of the assays on a log2 scale. Strictly

speaking, resistance is defined as the ability of a microorgan-

ism to continue growing in the presence of an antimicrobial

compound. Tolerance is defined as the ability of a micro-

organism to survive exposure to, but not grow in the

presence of, an antimicrobial.

Biofilmformationand inocula for susceptibilityassays

For each MBECTM assay, four pegs were broken from the peg

lid and the biofilm growth was evaluated by viable cell

counting (this gave a total of 168 control measurements).

Candida tropicalis grew to a mean cell density (with stan-

dard deviation) of 4.3� 0.5 log10 CFU peg� 1. To ensure that

biofilm growth was even across the rows of pegs in the

MBECTM device, all of the biofilms from a single peg lid

were plated for viable cell counting. Growth of C. tropicalis

biofilms was statistically equivalent between the different

rows of pegs by means of one-way ANOVA (P = 0.402). These

data are presented in Fig. S1 (available as supplementary

material). For planktonic susceptibility assays, each well of a

separate set of challenge plates was inoculated with

4.5� 0.1 log10 CFU of planktonic C. tropicalis (a starting

number derived from six viable-cell-counting assays of the

inoculum). This starting cell number was calibrated to

imitate the initial biofilm cell density.

Candida tropicalis biofilms were examined in situ using

confocal laser-scanning microscopy (CLSM). Three different,

complementary staining techniques were used. In one set of

experiments, acridine orange was used to stain C. tropicalis

on the pegs of the MBECTM device (green, Figs 1a and d).

Biofilms were approximately 7–30mm in thickness, forming

layers and ridges of cells that covered the peg heteroge-

neously. In a separate set of experiments, Syto 9 (green, Fig.

1b) and TRITC–Con A (red, Fig. 1c) were concomitantly

used to label biofilms. Con A is a glucose- and mannose-

specific lectin that has been used to label the extracellular

polymeric matrix and cell walls of Candida (Chandra et al.,

2001a; Jin et al., 2005). Correlative with previous reports, we

see that Con A highlighted cell walls and stained EPS to a

lesser extent (Chandra et al., 2001a). An overlay of these

pictures shows cells in physical contact with their neigh-

bours, joined either by their cell walls or a thin layer of EPS

(Fig. 1e). Although the majority of the fungal biomass was

composed of yeast cells, some sparsely distributed pseudo-

hyphae and hyphal structures could be observed near the

air–liquid-surface interface of the pegs in some instances.

SusceptibilityofCandida tropicalis biofilms tometals

Means and standard deviations (SD) for metal cation and

oxyanion MIC, MLCP and MLCB values observed for

Candida tropicalis planktonic and biofilm cultures are sum-

marized in Table 1. Frequently, the same value was obtained

from every trial for the same compound (i.e. SD = 0). Values

reported represent four to eight independent trials each.

Planktonic C. tropicalis was not killed by exposure to

three of the metals tested: Ni21, Zn21 and Al31 (corre-

sponding to maximum concentrations of 280, 490 and

FEMS Microbiol Ecol 55 (2006) 479–491c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

482 J.J. Harrison et al.

590 mM, respectively). However, planktonic yeast cells were

killed in a time-dependent fashion by other metals. In the

cases of CrO42� , Mn21, Co21, Cd21, AsO4

3� and AsO2� ,

there was a minimum fold decrease in the MLCP of 8.3, 4.0,

33, 7.7, 4.1 and 2.6, respectively (between 5 and 24 h

exposure). These data indicate that planktonic C. tropicalis

exhibits time-dependent tolerance to high concentrations of

metals. Mercury (Hg21) was the most toxic heavy metal

tested, inhibiting yeast growth and killing both planktonic

cells and biofilms over a narrow concentration range of

0.5–2.0 mM. As the sole exception, biofilms were no more

tolerant to Hg21 than planktonic cells.

Biofilm C. tropicalis was highly tolerant to metal toxicity,

surviving in vitro concentrations of more than 120, 140 and

500 mM of the heavy metal(loid)s AsO43� , Co21 and

SeO32� , respectively. As another example, cationic silver

(Ag1) was highly toxic to planktonic cells, inhibiting growth

and killing this form of Candida at concentrations of

approximately 0.5–1.0 mM and 20–25 mM, respectively. In

contrast, biofilms were highly tolerant to Ag1 and were not

killed at the highest concentration added in vitro (150 mM).

Given the sensitivity of this assay on a log2 scale, this implied

that C. tropicalis biofilms were minimally 17–26 times more

tolerant to cationic silver than corresponding planktonic

cells. Notably (in comparison with planktonic cells), MLCB

values did not decrease with increasing exposure time to

metals. Because there was no time-dependent eradication of

these surface-adherent microbes, logically, biofilms may

have been resistant and/or tolerant to these compounds.

KillingofCandida tropicalis byCo21andSeO32�

To differentiate between tolerance and resistance in fungal

biofilms, the lethality of one metal cation and one metalloid

oxyanion was evaluated using viable cell counting and Live/

Dead staining in conjunction with CLSM. Co21 was selected

as a representative heavy metal cation, because Candida

biofilms had the greatest fold tolerance (Z65) to this

compound. SeO32� was chosen as a representative oxya-

nion, because relative to Co21 (as well as to other metals)

planktonic cells were not killed time-dependently. Biofilms

were at least 21 times more tolerant to SeO32� than

planktonic cells at both 5 and 24 h exposure (Table 1).

Mean viable cell counts and log-killing of C. tropicalis

biofilm and planktonic cells by Co21 are presented in Fig. 2.

At concentrations above the planktonic MIC (4.3 mM),

both planktonic and biofilm cells were killed by Co21. In

the case of planktonic cells, every cell in the population had

died by 24 h exposure to 8.6 mM of this heavy metal. In

contrast, although a portion of the biofilm population died

on exposure to high concentrations (35 or 140 mM) of

Co21, there were many survivors even after an in vitro

exposure of 1 day (Fig. 2d–h). The size of yeast cells exposed

to Co21 for 24 h was larger than that in growth controls

(compare Fig. 2c or f–h). There was some differentiation of

Fig. 1. Confocal laser scanning microscopy (CLSM) images of Candida

tropicalis biofilms grown for 48 h in tryptic soy broth on the pegs of the

MBECTM P&G assay. Candida tropicalis formed uneven layers, cell

clusters, ridges and ripples across the surface of the plastic pegs. (a) A

three-dimensional (3D) model of a C. tropicalis biofilm stained with

acridine orange. Biofilms were up to 30 mm in thickness. (b) A two-

dimensional (2D) average of an image z-stack of yeast cells stained with

the nucleic acid stain Syto-9. (c) The biofilms in panel B were concomi-

tantly stained with tetramethylrhodamine isothioncyanate (TRITC) con-

jugated concanavalin A (Con A). This lectin is specific for the

carbohydrate-rich biofilm extracellular polymeric substance. (d) The 2D

average of the image z-stack used to construct the 3D model in panel A.

(e) An overlay of the Syto 9 and TRITC–Con A stained images from panels

B and C, respectively. Yeast cells in the biofilm were surrounded by thick

cell walls and some EPS that physically joined individuals to their

neighbours. Each CLSM panel represents a 2D average of a 3D z-stack

with a length and width of 238.1 mm taken at a magnification of� 630.

FEMS Microbiol Ecol 55 (2006) 479–491 c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

483Metal resistance in Candida biofilms

Candida into hyphae and pseudohyphae in these controls

(Fig. 2f). Furthermore, yeast cells from control samples were

approximately 2.8–4.2 mm in diameter. After 24 h exposure

to Co21, the majority of cells were 5.2–7.0 mm in diameter.

Many of these large, metal-exposed cells were dead, and this

was reflected in a marked decrease in mean viable cell

counts. These data indicate that C. tropicalis biofilms were

highly tolerant to Co21.

A different scenario existed for SeO32� . Mean viable cell

counts and log-killing of C. tropicalis biofilm and planktonic

cells by this heavy metalloid oxyanion are presented in Fig. 3.

At concentrations above the MIC (56 mM), planktonic C.

tropicalis was rapidly killed (which correlated closely with the

data in Table 1). Strikingly, at an in vitro concentration of

125 mM SeO32� , mean viable cell counts for biofilms in-

creased during the first 5 h of exposure. CLSM of Live/Dead

stained biofilms exposed to SeO32� (Fig. 3d) showed a

corresponding increase in the number of live cells in the

biofilms, which generally gave greater surface coverage of the

peg. By 24 h exposure to 125 mM SeO32� (Fig. 3g), mean

viable cell counts of biofilms were similar to the starting

number of cells in biofilms. In contrast, 500 mM SeO32�

resulted in rapid yeast-cell death in biofilms (Fig. 3e and h). By

24 h, many of the dead cells had been lost from the peg, and the

biofilm covered a smaller surface area than before exposure.

These data indicate that, under certain conditions, biofilm C.

tropicalis was more resistant to SeO32� than planktonic cells.

Retentionofdivalent heavymetal cationsinyeast biofilms

The ability of bacterial biofilms to trap metal cations has

been previously reported (Harrison et al., 2005b). We thus

Table 1. Susceptibility of biofilm and planktonic Candida tropicalis to metals

Periodic

group

Metal

ion n

Exposure

time (h)

Broth microdilution assay MBECTM assayFold

tolerance�MIC (mM) MLCP (mM) MLCB (mM)

6B CrO42� 4 5 4.4� 0 44� 17 (1.0� 0.4)�102w

2.3

24 5.3� 4.1 4 71 Z26

7B Mn21 4 5 nd 4 5.8� 102 4 5.8�102 na

24 (3.0� 0)� 102 4 5.8�102 3.8

8B Ni21 4 5 70� 0 4 2.8� 102 4 2.7�102 na

24 4 2.8� 102 4 2.8�102 na

Co21 4 5 4.3� 0 4 1.4� 102 4 1.4�102 na

24 4.3� 0 4 1.4�102Z65

1B Cu21 4 5 64� 0 64� 0 64�0 1.0

24 64� 0 4 1.3�102Z4.1

Ag1 5 5 1.2� 0 23� 9 4 1.5�102Z26

24 19� 18 4 1.6�102Z17

2B Zn21 4 5 39� 16 4 4.9� 102 4 4.9�102 na

24 4 4.9� 102 4 4.9�102 na

Cd21 5 5 2.8� 1.1 64� 18 4 1.4�102Z4.4

24 8.3� 7.4 4 1.4�102Z34

Hg21 6 5 1.3� 0 1.3� 0 1.0� 0.3 0.8

24 1.3� 0 1.9� 0.7 1.5

3A Al31 3 5 5.9� 102 4 5.9� 102 4 5.9�102 na

24 4 5.9� 102 4 5.9�102 na

4A Pb21 6 5 39� 0 79� 0 4 77 Z2.0

24 (1.2� 0.5)� 102 4 77 na

5A AsO43� 4 5 59� 0 4 1.2� 102 4 1.2�102 na

24 29� 20 4 1.2�102Z8.2

AsO2� 4 5 19� 0 4 3.0� 102 4 3.0�102 na

24 (2.3� 1.4)� 102 4 3.0�102Z2.6

6A SeO32� 4 5 56� 16 48� 19 4 5.0�102

Z21

24 48� 19 4 5.0�102Z21

TeO32� 5 5 4 11 4 11 4 11 na

24 4 11 4 11 na

n denotes the principal quantum number of the element.

na, not applicable.

nd indicates an MIC that could not be determined owing to metal precipitation or to production of a coloured reduction product in the well of the

planktonic cells.�The fold tolerance, given the sensitivity of the assay on a log2 scale, is equal to the ratio of the means of MLCB : MLCP (see Section 3.1).wDenotes that the culture was killed at the threshold of the highest concentration of the metal used in these assays.

FEMS Microbiol Ecol 55 (2006) 479–491c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

484 J.J. Harrison et al.

Fig. 2. Candida tropicalis 99916 biofilms are highly tolerant to the heavy metal Co21. Biofilms were grown for 48 h on the peg lid of the MBECTM-high

throughput device before exposure for 5 or 24 h to Co21. In a separate series of assays, an equivalent number of planktonic cells were added to metal

challenge media. Biofilm and planktonic cells were recovered in saline, serially diluted 10-fold, and then plated onto agar for viable cell counts. Each

data point is the mean of three or four independent replicates, and error bars denote standard deviation. An asterisk denotes complete killing of the

fungal culture at the reported concentration. Confocal laser scanning microscopy (CLSM) and Live/Dead staining were used to complement cell-

counting assays of biofilms on the pegs. (a) Mean viable cell counts of C. tropicalis with respect to concentration of Co21. (b) Log-killing of biofilm and

planktonic cells. (c) Biofilm before exposure to Co21. The vast majority of biomass was yeast cells and the vast majority were alive. (d and e) Biofilm after

5 h exposure to 35 and 140 mM Co21, respectively. The number of cells that were dead increased with the concentration of heavy metal cations in the

growth medium. (f) Biofilm after an additional 24 h incubation, but not exposed to metals. There was some differentiation of yeast into pseudohyphae

and hyphae. (g and h) Biofilm after 24 h exposure to 35 and 140 mM Co21, respectively. There were fewer cells and less biofilm coverage of pegs in

samples exposed to Co21 for 24 h. Yeast cells were notably larger at 24 h exposure to Co21 than at 5 h. Planktonic cell populations were killed

(completely) in a time-dependent fashion by Co21. Many biofilm cells were killed by prolonged exposure to metals. However, some cells in the biofilm

population survived exposure to this highly toxic metal even with 1 day of exposure in vitro. Each CLSM panel represents a 2-dimensional average of a

3-dimensional z-stack with a length and width of 238.1 mm taken at a magnification of�630.

FEMS Microbiol Ecol 55 (2006) 479–491 c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

485Metal resistance in Candida biofilms

investigated whether yeast biofilms may similarly sequester

metal cations or oxyanions from their aqueous surround-

ings. Biofilms exposed to the metals Cu21, Ni21, and TeO32�

were rinsed and then treated with the chelator diethyldithio-

carbamate (DDTC). This is pictured in Fig. 4. In Cu21 and

Ni21 exposed biofilms, DDTC caused the rapid formation

of brown and yellow metal-chelates, respectively. However,

biofilms exposed to the metalloid oxyanion tellurite did not

Fig. 3. Candida tropicalis 99916 biofilms are highly resistant to the heavy metalloid oxyanion SeO32� . Experimental conditions were similar to those

described in the legend of Fig. 2. (a) Mean viable cell counts of C. tropicalis with respect to concentration of SeO32� . (b) Log-killing of biofilm and

planktonic cells. (c) Biofilm before exposure to SeO32� . (d and e) Biofilm after 5 h exposure to 125 and 500 mM SeO3

2� , respectively. Candida

tropicalis biofilms continued to grow in 125-mM SeO32� , a concentration that was two to four fold larger than the planktonic minimal inhibitory

concentration. In contrast, 500 mM SeO32� killed a large portion of biofilm cell populations. (f) Biofilm after an additional 24 h incubation, but not

exposed to metals. (g and h) Biofilm after 24 h exposure to 125 and 500 mM SeO32� , respectively. Viable cell counts as well as the number of cells in

biofilms were generally lower at longer exposure times. Yeast cells were notably larger at 24 h exposure to SeO32� than at 5 h. Each confocal laser

scanning microscopy panel represents a 2-dimensional average of a 3-dimensional z-stack with a length and width of 238.1 mm taken at a magnification

of�630.

FEMS Microbiol Ecol 55 (2006) 479–491c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

486 J.J. Harrison et al.

change colour when immersed in DDTC. This suggests that

C. tropicalis biofilms may adsorb positively charged metal

ions. Conversely, negatively charged metal ions may not be

sequestered in the extracellular matrix of Candida biofilms.

Discussion

Species of the dimorphic yeast Candida are prevalent in soil

and aquatic environments. In these niches, microorganisms

are frequently exposed to highly toxic, water-soluble metal

ions. This study focused on a high-throughput in vitro

system for examining the susceptibility of yeast biofilms to

metals. Two complementary but different approaches were

used to examine and compare planktonic cells of Candida

tropicalis with biofilms. The first system was the MBECTM

assay, and biofilm susceptibility to metals could be discerned

using this technique. This model reflects the integrated

lifestyle of yeasts in the environment, where planktonic and

biofilm forms may grow together in the same locale. How-

ever, a limitation to this technique is that the number of cells

shed from biofilms is unknown. Thus, the second approach

used a well-defined inoculum with an initial number of cells

per well of challenge medium equivalent to the biofilm cell

density per peg (each of which was placed in a single well of

a microtitre plate). These experiments suggested that, rela-

tive to planktonic cells, C. tropicalis biofilms were less

susceptible to metal toxicity.

Candida tropicalis 99916 was fastidious with regards to

biofilm growth in vitro. This microorganism required 2 days

of incubation in a rich growth medium (tryptic soy broth)

as well as a positively charged substratum. For this latter

requirement, the plastic pegs of the MBECTM device were

coated with L-lysine prior to biofilm cultivation. Biofilms of

C. tropicalis could not be grown using Sabaroud dextrose

broth, a standard growth medium for yeast (J.J. Harrison,

R.J. Turner, and H. Ceri, unpublished data). Although

minimal medium formulations designed to reduce inorgan-

ic precipitation are desirable for metal susceptibility testing,

these could not be used here owing to the laboratory

cultivation requirements of C. tropicalis.

There are several in vitro models that have been used to

examine biofilm formation by C. albicans. Candida albicans

grows on surfaces in a series of organized, developmental

stages (for a review, see Mukherjee & Chandra, 2004). When

grown on polyvinylchloride or elastomer surfaces, C. albi-

cans forms an adherent layer of cells that is later covered by

hyphal elements and embedded in a matrix of extracellular

polymeric substance (EPS) (Chandra et al., 2001a; Kuhn

et al., 2002). On denture acrylic, mature C. albicans biofilms

form EPS-encased ridges of high cell density (Chandra et al.,

2001a). On the polystyrene surface of the MBECTM device,

C. tropicalis biofilms formed crests of densely packed cells

and/or single-cell layers that later developed into dome-

shaped microcolonies. Microcolony formation was

Fig. 4. Metal cations were sequestered in the extracellular polymeric matrix of Candida tropicalis biofilms. Biofilms grown in the MBECTM physiology &

genetics assay were exposed to log2 concentration gradients of the heavy metals copper (Cu21), nickel (Ni21) and tellurite (TeO32� ) for 5 h (these

gradients are illustrated in the bottom panel of each column). Planktonic cultures exposed to these metals are pictured in panels a, b and c, respectively.

The addition of the organic chelator sodium diethyldithiocarbamate (DDTC) to aliquots of these planktonic cultures produced brown, light-yellow and

dark-yellow precipitates, respectively (panels d, e and f). The biofilms from the corresponding wells illustrated in panels a, b and c were removed from

the metals and rinsed twice with 0.9% saline. These pegs were subsequently treated with DDTC. Brown and light-yellow precipitates formed in biofilms

exposed to Cu21 and Ni21 (panels g and h, respectively). There was no colour change in biofilms exposed to TeO32� (panel I). This suggests that biofilms

of Candida tropicalis have the ability to trap and sequester positively charged metal ions. Each panel represents four independent assays.

FEMS Microbiol Ecol 55 (2006) 479–491 c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

487Metal resistance in Candida biofilms

particularly evident by 64 h in our studies (data not shown).

Furthermore, there was some limited differentiation of yeast

cells into hyphal structures near the air–liquid-surface inter-

face of the biofilm under certain test conditions. Candida

tropicalis biofilms were also encased in a layer of EPS, which

is best observed using scanning electron microscopy rather

than CLSM (H. Ceri, D. Fogg, and M. Olson, unpublished

data). The pattern of C. tropicalis 99916 biofilm growth is

thus similar to that reported for C. parapsilosis, which forms

patches of mushroom-shaped communities rather than the

biphasic arrangement of layers seen in C. albicans (Kuhn

et al., 2002).

An advantage to a high-throughput method for metal

susceptibility testing of biofilms is the potential to compare a

number of compounds for microbiological toxicity. Correla-

tive with a previous report examining bacterial biofilm

tolerance to metals (Harrison et al., 2004a), the toxicity of

metal ions belonging to a single periodic group generally

increased with principal quantum number (n). For example

(as shown in Table 2), in group 2B, Hg21 (n = 6, MIC = 1.3�0 mM) was more toxic than Cd21 (n = 5, MIC = 2.8�1.1 mM), which was much more toxic than Zn21 (n = 4,

MIC = 39� 0 mM). A similar trend was observed for group

1B, in which Cu21 (n = 4, MIC = 64� 0) was less toxic than

Ag1 (n = 5, MIC = 1.2� 0). In this latter case, this trend was

not true of MLCB values, as Cu21 killed C. tropicalis biofilms

whereas Ag1 did not. However, the lower solubility of Ag1 in

TSB relative to Cu21 is a likely explanation for this exception.

No correlation between MIC, MLCP and MLCB values and

oxidation state or standard reduction potential could be

distinguished. Similar to the case for bacteria (Pseudomonas

aeruginosa, Escherichia coli, and Staphylococcus aureus), Hg21

and Ag1 were the two of the most biologically toxic

compounds to C. tropicalis (Harrison et al., 2004a).

Bacterial biofilm tolerance to antimicrobials, including

metals, is currently regarded as a multifactorial phenomenon

(for reviews, see Lewis, 2001; Harrison et al., 2005c). In this

model, there are at least four independent mechanisms at

work: (1) metabolic heterogeneity arising from the restricted

penetration of oxygen and nutrients into the biofilm extra-

cellular polymeric matrix (Walters et al., 2003; Borriello

et al., 2004); (2) a distinct biofilm physiology resulting from

cell-to-cell signalling in the biofilm (Harrison et al., 2004b;

Bjarnsholt et al., 2005); (3) restricted diffusion and/or

penetration of antimicrobials containing charged moieties

into the biofilm matrix (Hall-Stoodley et al., 2004; Harrison

et al., 2005b); and (4) a small subpopulation of persister cells

(Spoering & Lewis, 2001; Keren et al., 2004; Harrison et al.,

2005a,b). The factors contributing to antimicrobial tolerance

in Candida biofilms are largely unknown.

Unlike the case for bacterial biofilms, metabolic stratifica-

tion does not appear to exist in Candida biofilms, because

basal layers of cells still possess high metabolic activity

(Chandra et al., 2001a, b). Neither is there evidence in the

literature that Candida produces specialized persister cells.

However, quorum-sensing in C. albicans biofilms is

mediated by the small molecule farnesol (Hornby et al.,

2001) as well as by the contact-activated kinase Mkc1p,

which signals biofilm development in this dimorphic yeast

(Kumamoto, 2005). Within 1 h of attachment to a substra-

tum, C. albicans has undergone a change in physiological

state that gives elevated resistance to antifungals (Ramage

et al., 2002; Mukherjee & Chandra, 2004). For instance, C.

albicans biofilms have been observed to continue to grow in

high concentrations of fluconazaole that were otherwise

inhibitory to planktonic cell growth (Ramage et al., 2002).

The data in this report suggest that a similar phenomenon

may exist to enable C. tropicalis biofilms to resist metal

toxicity. In particular, planktonic cells were killed in a time-

dependent manner by toxic metal cations and oxyanions. In

our assays, this was observed as a decreasing MLCP with

increasing exposure times in broth microdilution assays

performed using a defined inoculum. However (and dissim-

ilar to the case for bacteria), MLCP values discerned using

the MBECTM assay were comparable between 5 and 24 h

exposure times (J.J. Harrison, H. Ceri, and R.J. Turner,

unpublished data). A potential explanation for these ob-

servations is that the biofilms on the pegs may be continuing

to grow, shedding cells into the growth medium in the

presence of metals. In this way, dead and dying planktonic

cells would be continually replenished by the biofilm,

leading to increased MLCP values at prolonged exposures.

A similar trend was observed in our studies for nystatin (J.J.

Harrison, H. Ceri, and R.J. Turner, unpublished data). These

observations were the rationale for the examination of Co21

and SeO32� in greater detail. The data presented in Fig. 3

indicate that C. tropicalis biofilms continued to grow in

concentrations of SeO32� (125 mM) that were approxi-

mately twice the concentration that was lethal to planktonic

cells (approximately 50 mM).

This trend is conspicuously different from that in bacter-

ial biofilms. For Pseudomonas aeruginosa and Escherichia

coli, a majority of the population dies at relatively low

concentrations of antibiotics or toxic metals, and a small

‘persister’ cell population survives to high concentrations of

these compounds (Harrison et al., 2005a, b). Strictly speak-

ing, persister cells are not resistant, as they do not grow in

the presence of bactericidal agents. Rather, persisters do not

die and thus exhibit multidrug tolerance (Keren et al., 2004).

Similarly, planktonic cells of Candida exhibit time-depen-

dent tolerance to metals. However, biofilms of C. tropicalis

may continue to grow in the presence of high concentrations

of some antifungals and metals, and thus may be considered

multidrug- and metal-resistant.

A contributing factor to metal resistance in C. tropicalis

biofilms may be sequestration of cations in the extracellular

FEMS Microbiol Ecol 55 (2006) 479–491c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

488 J.J. Harrison et al.

matrix. A previous report has shown that the diffusion of

fluconazole and 5-fluorocytosine through Candida biofilms

is slowed, requiring 3 to 6 h to equilibrate across these

surface-adherent communities (Al-Fattani & Douglas,

2004). However, Candida biofilms exposed for this period

of time continued to grow in the presence of these agents,

indicating that poor penetration cannot solely account for

drug resistance. Similarly, the formation of visible precipi-

tates of metal cations in biofilms suggests that sequestration

and limited penetration may be a contributing factor to

resistance. However, metal oxyanions could not be precipi-

tated in a similar fashion, indicating that these negatively

charged compounds may diffuse across the biofilm matrix.

Furthermore, although CrO42� , AsO2

� and AsO43� killed

planktonic C. tropicalis time-dependently, biofilms survived

high concentrations of these antimicrobials at the longest

exposure time assayed. Together, these data suggest that

restricted penetration of metals into the biofilm matrix of

C. tropicalis is only one contributing factor for biofilm

resistance and/or tolerance.

In this study, we have used C. tropicalis as a model

organism for yeasts growing in the environment. The high-

est concentrations of many metals examined in vitro were

not lethal to fungal biofilms. We note that the concentra-

tions at the lower end of these in vitro gradients may be

similar to those found in some polluted environments

(Lopez-Archilla & Amils, 2001; De Vries et al., 2002;

Hernandez et al., 2003; Schparyk & Parpan, 2004). These

same concentrations of metal cations are lethal to some soil

bacteria (for example Pseudomonas aeruginosa) (Harrison

et al., 2005a, b). This is consistent with reports that yeasts

may overwhelm natural systems contaminated with heavy

metals (Hagler & Mendocs-Hageler, 1981). Candida spp.

may survive bactericidal concentrations of these compounds

and may continue growth as biofilms.

Overall, the data suggest that, similar to the case for

bacterial biofilms, Candida biofilm resistance is a complex

process that relies on more than one mechanism. In our

model system, biofilms of C. tropicalis were highly resistant

and/or tolerant to a wide range of metal compounds. This

suggests that biofilm formation may be an innate means for

yeasts to survive metal toxicity in the environment.

Supplementarymaterial

The following supplementary material is available for this

article online:

Acknowledgements

This work was supported through operating grants from the

Natural Sciences and Engineering Research Council

(NSERC) of Canada to Drs Raymond J. Turner and Howard

Ceri. NSERC also provided an Industrial Postgraduate

Scholarship (IPS1) to Joe J. Harrison. Corporate funding

was provided by MBEC BioProducts Inc. NSERC addition-

ally provided a summer studentship for Erin A. Badry.

Kimberley M. Sproule was supported by a summer student-

ship from the Alberta Heritage Foundation for Medial

Research (AHFMR). Maryam Rabiei was funded by a Post-

Doctoral Fellowship from the Iranian Ministry of Health

and Medical Education through the Faculty of Pharmacy at

Shahid Beheshti University of Medical Sciences. Confocal

laser-scanning microscopy was made possible through a

Canadian Foundation for Innovation (CFI) Bone and Joint

Disease Network grant to Dr Howard Ceri. Lastly, thanks to

Nicole J. Roper, Darrin Fogg, Daniel Joo and Carol A.

Stremick for their hard work, valuable input and/or techni-

cal advice. JJH and MR contributed equally to this work and

should be considered equal first authors.

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