A subpopulation of Candida albicans and ... - Wiley Online Library

A subpopulation of Candida albicans and Candida tropicalis bio¢lm cells are highly tolerant to chelating agents Joe J. Harrison1,2, Raymond J. Turner1 & Howard Ceri1,2 1

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

Correspondence: Howard Ceri, Department of Biological Sciences, University of Calgary, 2500 University Drive N.W., Calgary, Alberta, Canada T2N 1N4. Tel.: 1403 220 6960; fax: 1403 289 9311; e-mail: [email protected] Received 19 January 2007; revised 3 April 2007; accepted 4 April 2007. First published online 8 May 2007. DOI:10.1111/j.1574-6968.2007.00745.x Editor: Jeff Cole Keywords Candida tropicalis ; Candida albicans ; biofilm; multidrug resistance; sodium diethyldithiocarbamate; tetrasodium ethylenediaminetetraacetic acid.

Abstract Many Candida spp. produce surface-adherent biofilm populations that are resistant to antifungal compounds and other environmental stresses. Recently, certain chelating agents have been recognized as having strong antimicrobial activity against biofilms of Candida species. This study investigated and characterized the concentration- and time-dependent killing of Candida biofilms by the chelators tetrasodium EDTA and sodium diethyldithiocarbamate. Here, Candida albicans and Candida tropicalis biofilms were cultivated in the Calgary Biofilm Device and then exposed to gradient arrays of these agents. Population survival was evaluated by viable cell counting and by confocal laser scanning microscopy (CLSM) in conjunction with fluorescent viability staining. At concentrations of Z2 mM, both EDTA and diethyldithiocarbamate killed c. 90–99.5% of the biofilm cell populations. Notably, a small fraction (c. 0.5–10%) of biofilm cells were able to withstand the highest concentrations of these antifungals that were tested (16 and 32 mM for EDTA and diethyldithiocarbamate, respectively). Interestingly, CLSM revealed that these surviving cells were irregularly distributed throughout the biofilm community. These data suggest that the use of chelating agents against biofilms of Candida spp. may be limited by the refractory nature of a variant cell subpopulation in the surface-adherent community.

Introduction Biofilms are cell–cell and solid surface-attached assemblages of microorganisms that are encased in a self-produced matrix of extracellular polymers. Biofilm formation is part of the ecological cycle for many microorganisms in the environment and in disease, and this includes fungi from the genus Candida (Hall-Stoodley et al., 2004). Although only one in 10 Candida species are human opportunistic pathogens, these polymorphic yeasts represent a widespread cause of device-associated biofilm infection from which systemic Candidemia frequently occurs (Ramage et al., 2006). In most cases, Candida albicans is the implicated etiological agent; however, Candida tropicalis may represent up to 25% of these infections (Douglas, 2003). In comparison with planktonic suspensions of cells, Candida biofilms have innate resistance and/or tolerance to hard surface disinfectants, metal ions and antifungal drugs (Chandra et al., 2001; Harrison et al., 2006b). One potential contributor to biofilm antimicrobial tolerance is a subpopulation of persister cells (Lewis, 2007). 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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Originally described in bacterial biofilm models, persisters are metabolically quiescent cells that neither grow nor die in the presence of antimicrobials (Balaban et al., 2004; Keren et al., 2004). Persisters may withstand a diverse range of structurally and chemically distinct bactericidal agents that include antibiotics (Brooun et al., 2000), peroxides and acids (Spoering & Lewis, 2001), as well as metal cations and oxyanions (Harrison et al., 2005a, 2005c). Candida albicans biofilms produce an analogous subpopulation of metabolically slowed cells that are highly tolerant to azole drugs and amphotericin B (Lafleur et al., 2006). Similarly, Lamfon et al. (2005) have identified that 0.1–10% of Candida spp. in mixed species microcosm denture plaque biofilms persist after pulsed exposure to miconazole and chlorhexidine. Several recent publications have indicated that metal chelators may be effective antibiofilm agents. For instance, tetrasodium EDTA is known to have high activity against biofilms of Gram-positive and Gram-negative bacteria, as well as C. albicans (Percival et al., 2005). EDTA has been used as an irrigant of root canals and in comparison with other antifungals, has high activity against clinical oral FEMS Microbiol Lett 272 (2007) 172–181

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Chelator tolerance in Candida biofilms

isolates of C. albicans (Sen et al., 2000). Similarly, it has recently been identified that the chelator sodium diethyldithiocarbamate possesses high antifungal activity against C. tropicalis biofilms (J.J. Harrison, R.J. Turner and H. Ceri, unpublished data). Diethyldithiocarbamate has a history of therapeutic use as a broad-spectrum antifungal agent and has been reported to inhibit planktonic growth of clinical C. albicans isolates and other Candida spp. at concentrations of o 1.2 mg mL 1 (Allerberger et al., 1989; Shah et al., 1997). Here, the in vitro concentration- and time-dependent killing of C. tropicalis and C. albicans biofilms by the chelating agents diethyldithiocarbamate and EDTA was examined. Analogous to the well-characterized killing kinetics of bacterial biofilm populations by antimicrobial agents, it was discovered that a small subpopulation of C. albicans and C. tropicalis biofilm cells (representing 10% or less of the total biofilm population) were not killed by high concentrations of these agents. Furthermore, survivors from EDTA exposure that were propagated in vitro and gave rise to biofilms with unchanged susceptibility to this agent. These data suggest that a subpopulation of C. albicans and C. tropicalis biofilm cells are innately recalcitrant to the fungicidal action of organic metal ion chelators.

Materials and methods Strains and growth media Candida albicans 3153A (Lafleur et al., 2006) and C. tropicalis 99916 (Harrison et al., 2006b) were stored at 70 1C in MicobankTM vials as described by the manufacturer (ProLab Diagnostics, Toronto, ON). Tryptic soy agar (TSA) was used to grow all subcultures and to perform viable cell counts (EMD Chemicals Inc., Gibbstown, NJ). Inoculated TSA plates were incubated at 35 1C for 48 h. Tryptic soy broth (TSB) or Roswell Park Memorial Institute medium (RPMI) 1640 with 5 mM L-glutamine (SigmaAldrich, Oakville, ON, Canada) supplemented with 0.165 M 3-N-morpholinopropanesulfonic acid and 2.0 g L 1 sodium bicarbonate (RQMB) were used to grow these Candida spp. as indicated throughout this manuscript. All growth media were adjusted to a pH of 7.2 0.1. Serial dilutions were performed using 0.9% saline.

Biofilm cultivation Biofilms were grown in the Calgary Biofilm Device (CBD, Innovotech, Edmonton, AB, Canada) as described previously (Ceri et al., 1999). This device consists of a polystyrene lid with 96 pegs that may be fitted inside a standard 96-well microtiter plate. The CBD and manufacturer’s protocols were modified in different ways to facilitate C. albicans and C. tropicalis biofilm formation. FEMS Microbiol Lett 272 (2007) 172–181

In the case of C. albicans, the CBD pegs were coated with a solution of 100% w/v trichloroacetic acid (TCA), dried upside down in a laminar flow hood and then treated with ethylene oxide gas (Anprolenes, Anderson Products Inc., Oyster Bay, NY). Candida albicans cells were streaked out twice on TSA and colonies from the second agar subcultures were suspended in RQMB so as to match a 1.0 McFarland optical standard. Aliquots (150 mL) of this standard were placed into the wells of a microtiter plate and the chemically modified peg lid was inserted into this. The inoculated devices were incubated under static conditions at 35 1C and 95% relative humidity for 3 h, after which the peg lid was transferred into a microtiter plate containing 150 mL of fresh RQMB in each well. These devices were then placed on a gyrorotary shaker for 48 h at 75 r.p.m. in an incubator at 35 1C and 95% relative humidity. In the case of C. tropicalis, the pegs were coated with a sterile solution of 1% L-lysine and then dried upside-down in a laminar flow hood as described previously (Harrison et al., 2006a). Following surface modification, the peg lids were inserted into microtiter plates containing 150 mL of a standardized inoculum that contained 1.0 105 CFU mL 1 (corresponding to a one in 30 dilution of a 1.0 McFarland standard). Biofilms of C. tropicalis were formed by incubating the inoculated devices on a gyrorotary shaker at 125 r.p.m., 35 1C and 95% relative humidity for 48 h. Candida tropicalis biofilms were cultivated in TSB or in RQMB medium. Following this initial period of growth, biofilms were rinsed once with 0.9% saline by placing the peg lid in a microtiter plate containing 200 mL of saline in each well. Biofilm formation was evaluated by breaking three or four pegs from each device after it had been rinsed. Biofilms from these pegs were disrupted into 200 mL of 0.9% saline using a sonicator (Aquasonic model 250HT; VWR Scientific, Mississauga, ON, Canada) as described previously (Ceri et al., 1999; Harrison et al., 2006b). These growth controls were serially diluted and plated for viable cell counting.

Stock solutions of antifungals and chelators All antifungals and chelating agents were purchased from Sigma-Aldrich. Amphotericin B desoxycholate was made up to 5120 mg mL 1 in sterile double-distilled water (ddH2O). The chelating agents tetrasodium EDTA and sodium diethyldithiocarbamate were prepared in sterile ddH2O at a concentration of 256 mM. All of the stock antifungal and chelator solutions were stored at 20 1C. Working solutions, corresponding to the maximum antifungal concentration used in susceptibility assays, were prepared by making a minimum fivefold dilution of the stock agent in TSB or RQMB. 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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Biofilm susceptibility testing High-throughput antimicrobial susceptibility testing of microbial biofilms using the CBD has been previously described by the authors’ research group (Ceri et al., 1999; Harrison et al., 2005b) and is succinctly summarized here. Serial two- or fourfold dilutions of antimicrobial working solutions were prepared in 96-well microtiter plates with sterility and growth controls situated in the first and last well of each row, respectively. Peg lids with C. albicans or C. tropicalis biofilms were inserted into these antimicrobial challenge plates for the desired exposure time. Following exposure, the peg lids were removed from the challenge plates and then inserted into microtiter plates containing 200 mL of recovery medium in each well (the recovery plates). Recovery medium consisted of TSB or RQMB that had been enriched with 1 mM CaCl2 (Sigma-Aldrich) and 10 mM MgSO4 (Fisher Scientific, Ottawa, ON, Canada) as neutralizing agents for chelators. The biofilms were then disrupted into the recovery medium using a sonicator (as described above), and following this, the recovered cells were serially diluted and then plated for viable cell counting.

J.J. Harrison et al.

membrane to the nucleic acid intercalators Syto-9 (green emission, membrane permeant) and propidium iodide (PI, red emission, membrane impermeant); cells with compromised membrane integrity are presumably dead and stain orange-red, whereas live cells stain green. Alternatively, biofilms were fixed with 5% glutaraldehyde and stained with Syto-9 and tetramethylrhodamine isothiocyanate-conjugated concanavalin A (TRITC-ConA) as described previously (Harrison et al., 2006a). ConA is a lectin that specifically binds to glucose, glucosamine and mannose sugars, all of which are present in the extracellular matrix of C. albicans and C. tropicalis (Al-Fattani & Douglas, 2006). In this latter set of experiments, biofilms were also examined using Syto-24 (Molecular Probes), a DNA-specific intercalator (green emission, membrane permeant), in lieu of Syto-9. CBD biofilms were examined using a Leica DM IRE2 spectral confocal and multiphoton microscope with a Leica TCS SP2 acoustic optical beam splitter (Leica Microsystems, Richmond Hill, ON, Canada) as described previously (Harrison et al., 2006a). Image capture and three-dimensional reconstruction of z-stacks were performed using LEICA CONFOCAL software (Leica Microsystems).

Planktonic cell susceptibility testing A broth microdilution assay was used to test the susceptibility of planktonic C. albicans and C. tropicalis to antifungals (Fothergill & McGough, 1995). Antifungal challenge plates were prepared as described above and then 5 mL of a 1.0 McFarland standard solution (c. 1.0 104 CFU mL 1) was added to each well, except for sterility controls. During this exposure, challenge plates were incubated at 35 1C and 95% relative humidity. A neutralizing regime was used for planktonic cultures by transferring 40 mL aliquots from challenge plates into neutralizing plates that contained (in each well) 10 mL of TSB or RQMB with 5 mM CaCl2 and 50 mM MgSO4 added. Thus, the final concentration of neutralizing agent used was identical for biofilm and planktonic cells. The recovery medium that contained the recovered planktonic cells was serially diluted and then plated for viable cell counting. Minimum inhibitory concentration (MIC) values were evaluated by determining the OD at 650 nm (OD650 nm) of challenge plates after a 24-h incubation at 35 1C using a THERMOmax microplate reader with SOFTMAX PRO data analysis software (Molecular Devices Corporation, Sunnyvale, CA).

Confocal laser scanning microscopy (CLSM) In one set of experiments, CBD biofilms were stained with the Live/Deads BacLightTM Kit (Molecular Probes, Burlington, ON, Canada) as described previously (Harrison et al., 2006a). In principle, this fluorescent viability stain exploits the differential permeability of the yeast cytoplasmic 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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Digital photography Digital pictures were captured using a Kodak EasyShare C340 5.0 MegaPixel digital camera. Captured images were contrast and brightness enhanced using ADOBEs s PHOTOSHOP 7.0.

Results and discussion Biofilm formation by C. albicans and C. tropicalis In this study, two different clinical isolates of two different Candida spp. were examined: fluconazole-resistant C. tropicalis 99916 and fluconazole-sensitive C. albicans 3153A. These two strains have been previously characterized as robust biofilm formers that give rise to surface-adherent populations with decreased susceptibility to various antifungal agents relative to the corresponding planktonic cell populations (Harrison et al., 2006b; Lafleur et al., 2006). When cultivated in TSB or RQMB media, C. tropicalis 99916 formed biofilms in the CBD with a mean cell density and an SD of 4.5 0.3 log10 CFU peg 1 (21 replicates) and 4.4 0.3 log10 CFU peg 1 (four replicates), respectively. When cultivated in RQMB medium, C. albicans 3153A formed biofilms with a mean cell density and an SD of 2.9 0.5 log10 CFU peg 1 (four replicates). To characterize the present model system for susceptibility testing, biofilm formation was also examined using CLSM. FEMS Microbiol Lett 272 (2007) 172–181

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Candida tropicalis biofilms grown in TSB were treated with Syto-9 and PI (the Live/Deads viability stain, Fig. 1a and b). These biofilms were composed primarily of a layer of budding yeast cells that were up to 20 mm thick at the air–liquid–surface interface. The vast majority of these cells were alive (i.e. green). Candida tropicalis and C. albicans biofilms grown in RQMB medium were also stained using Syto-9 or Syto-24 in conjunction with TRITC-ConA (Fig. 1c and d). This latter approach was used to stain cellular nucleic acids as well as cell walls and extracellular polysaccharides, respectively. Both Candida spp. formed biofilms

where yeast and hyphal cells were in contact with their neighbors via the cell wall and/or were surrounded by a thin layer of extracellular polysaccharides. In contrast to C. tropicalis, C. albicans formed biofilms with a greater number of hyphae among the surface-adherent population.

A subpopulation of C. tropicalis biofilm cells is highly tolerant to chelating agents Lafleur et al. (2006) have recently reported that a subpopulation of C. albicans cells are tolerant to the polyene

Fig. 1. CLSM of Candida albicans 3153A and Candida tropicalis 99916 biofilms grown on surface-modified pegs of the CBD. Each imaging experiment was performed in triplicate and representative examples are shown here. (a) A C. tropicalis biofilm grown on an L-lysine coated CBD using TSB as the growth medium. This sample was stained using a Syto-9 and PI; therefore, live cells are green and dead cells are red. (b) A three-dimensional visualization of the biofilm pictured in (a). (c) A C. tropicalis biofilm grown on an L-lysine coated CBD using RQMB as the growth medium. This sample was stained with Syto-9 and TRITC-ConA; thus, all cells are stained green and the cell walls as well as the extracellular matrix are stained red. (d) A C. albicans biofilm on a CBD that was chemically modified with TCA and EtO and cultivated using RQMB as the growth medium. This sample was stained with Syto-24 and TRITC-ConA; thus, cell nuclei are stained green and the cell walls as well as the extracellular matrix are stained red. Each panel represents an area of c. 238 238 mm.

FEMS Microbiol Lett 272 (2007) 172–181

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antifungal amphotericin B. The altered susceptibility of this group of cells to antimicrobial agents resulted in biphasic killing kinetics of C. albicans biofilm populations that were both concentration- and time-dependent. Here, the killing kinetics of C. tropicalis 99916 biofilms by diethyldithiocarbamate and EDTA at 5 and 24 h was examined and this was compared (concomitantly) with amphotericin B (Fig. 2).


It was observed that relatively low concentrations of both diethyldithiocarbamate and EDTA (1 and 2 mM, respectively) killed Z90% of the C. tropicalis biofilm cell populations, a value that was comparable with biofilm treatment with amphotericin B (Z16 mg mL 1). Notably, a small fraction (c. 0.5–10%) of biofilm cells were able to withstand the highest concentrations of all three of these antifungals

Fig. 2. A subpopulation of Candida tropicalis 99916 biofilms was highly tolerant to the microbicidal action of organic chelators. Here, C. tropicalis biofilms were cultivated on L-lysine coated pegs in the CBD using TSB as the growth medium. After 48 h of growth, biofilms were exposed to the indicated antifungal agent for either 5 h (’ with solid lines) or 24 h (m with dotted lines). Biofilms were treated with a neutralizing agent, recovered from the CBD and then plated for viable cell counting. Each data point indicates the mean and SD of four independent replicates. (a and b) Mean viable cell counts and log-killing of biofilms by diethyldithiocarbamate (DDTC), respectively. (c and d) Mean viable cell counts and log-killing of biofilms by EDTA, respectively. (e and f) Mean viable cell counts and log-killing of biofilms by amphotericin B, respectively.


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that were tested in vitro (8, 16 mM and 512 mg mmL 1 for diethyldithiocarbamate, EDTA and amphotericin B, respectively). In the case of EDTA treatment, the size of the surviving subpopulation decreased with time (10% with 5 h exposure vs. 0.1% at 24 h exposure). The spatial localization of the surviving cells in the biofilms was investigated by staining the exposed samples with Syto-9 and PI (Fig. 3). Under the conditions of antimicrobial exposure, growth controls showed an increase in the mean viable cell count (Fig. 2b, d and f). This corresponded to an increase in biofilm thickness that was paralleled by hyphal formation at the biofilm air– liquid–surface interface (Fig. 3a and e). In contrast, the addition of millimolar concentrations of diethyldithiocarbamate and EDTA killed the vast majority of cells in these biofilm populations (Fig. 3b–d and f–H), with the surviving cells (coloured green) sporadically distributed across the peg surface. This pattern of C. tropicalis biofilm killing was similar to that produced by amphotericin B at concentrations in excess of 16 mg mL 1 (Fig. 3d and h, as well as data not shown). In summary, these data suggest that C. tropicalis 99916 biofilms have a subpopulation of cells that exhibit altered susceptibility to killing by organic chelators. Furthermore,

the biphasic pattern of population killing was similar to that described previously for bacterial and fungal persister cells (Lewis, 2007).

Comparison of planktonic and biofilm susceptibility to organic chelators Although this study initially focused on a C. tropicalis biofilm model system using TSB as the growth medium, antifungal susceptibility testing routinely uses C. albicans in a defined medium that is based on RPMI 1640. Therefore, biofilm and planktonic cell susceptibility testing was additionally performed for C. tropicalis 99916 and C. albicans 3153A in RQMB medium, a buffered medium derived from RPMI 1640. The MIC, as well as the minimum lethal concentration for planktonic cells (MLCP) and biofilms (MLCB) for diethyldithiocarbamate, EDTA and amphotericin B is summarized in Table 1. Here, the MLC was defined as the concentration of the antimicrobial required to kill 99.95% of the fungal population. Each value is expressed as the median and range (where applicable) of four independent replicates. Using this method, C. tropicalis biofilms were at least 4 , 8 and 1024 more tolerant to killing by diethyldithiocarbamate, EDTA and amphotericin B,

Fig. 3. CLSM of Candida tropicalis 99916 biofilms exposed to diethyldithiocarbamate, EDTA and amphotericin B. Biofilms were cultivated on L-lysinecoated pegs in the CBD using TSB as the growth medium. Samples were stained using a Syto-9 and PI; thus, live cells are green and dead cells are red. Each imaging experiment was performed in triplicate and representative examples are shown here. (a and e) Candida tropicalis biofilms that were not treated with an antifungal grew during the 5- and 24-h exposure period, respectively. In many cases, some cells in the outermost edge of the biofilm underwent hyphal cell differentiation. (b, c and d) After a 5-h exposure to diethyldithiocarbamate, EDTA or amphotericin B, respectively, the vast majority of biofilm cells were dead, with a few survivors intermingled in the population. (f, g, and h) Similarly, after 24-h exposure to diethyldithiocarbamate, EDTA or amphotericin B, respectively, only a few C. tropicalis biofilm cells remained intact. Each panel represents an area of c. 238 238 mm.



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Table 1. Susceptibility of Candida tropicalis 99916 and Candida albicans 3153A to diethyldithiocarbamate, EDTA and amphotericin B in minimal medium Strain

C. tropicalis 99916 Broth microdilution assay

C. albicans 3153A

Method Antifungal

MIC

MLCP

CBD assay MLCB

Diethyldithiocarbamate (mM) EDTA (mM) Amphotericin B (mg mL 1)

8.0 (8.0–16) 1.0 0.50

8.0 (8.0–16) 2.0 (1.0–2.0) 0.50

32 (32 to 4 32) 4 16 512 (2 to 4 512)

Broth microdilution assay MIC

MLCP

CBD assay MLCB

0.06 0.06 (0.06–0.12) 0.50

0.06 (0.06–0.50) 2.0 (0.06 to 4 16) 0.50

4 32 (32 to 4 32) 4 16 2.0 (0.50–8.0)

Fig. 4. Biofilms (m with solid lines) of Candida tropicalis 99916 and Candida albicans 3153A were more tolerant to organic chelators than the corresponding planktonic cell populations (’ with dotted lines). Furthermore, this tolerance was mediated by a subpopulation of cells representing c. 0.5–10% of the starting population. Here, C. tropicalis and C. albicans biofilms were cultivated in RQMB medium on CBD pegs that were coated with L-lysine or chemically modified with TCA and EtO, respectively. After 48 h of growth, biofilms were exposed to the indicated antifungal agent for 24 h, treated with a neutralizing agent, recovered from the CBD and then plated for viable cell counting. Planktonic cell susceptibility testing was performed using a broth microdilution assay with a number of planktonic cells in the starting inoculum that was equivalent to the biofilm cell density per peg. Each data point indicates the mean and SD of three or four independent replicates. (a, c and e) The susceptibility of C. tropicalis to diethyldithiocarbamate (DDTC), EDTA and amphotericin B, respectively. (b, d, f) The susceptibility of C. albicans to diethyldithiocarbamate EDTA and amphotericin B, respectively. 2007 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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respectively, than the corresponding planktonic cells. For C. albicans, biofilms were at least 533 , 8 and 4 more tolerant to killing by these agents, respectively, than the corresponding planktonic cultures. The concentration-dependent killing of biofilm and planktonic C. tropicalis and C. albicans were also examined in this defined medium by diethyldithiocarbamate, EDTA and amphotericin B (Fig. 4). In every case, biofilm tolerance to these compounds was mediated by a small portion ( 10%) of the starting population. Using this assay method, no subpopulation of planktonic cells that were tolerant to amphotericin B could be detected (Fig. 4e and f), which corresponds to the previous report by Lafleur et al. (2006). However, a subpopulation of diethyldithiocarbamate- and EDTA-tolerant planktonic cells could be detected for C. tropicalis and C. albicans, although the size of this population was much smaller (10 2–10 1) than that of biofilms (Fig. 4a–d). It is also worth noting that these planktonic cell survivors expired at

much lower concentrations of diethyldithiocarbamate and EDTA than the corresponding biofilms.

Survivors of EDTA exposure give rise to biofilms with normal susceptibility to chelators Bacterial and fungal persisters give rise to cultures with unchanged susceptibility to antimicrobial agents (Spoering & Lewis, 2001; Harrison et al., 2005c; Lafleur et al., 2006). Here, four single C. tropicalis 99916 colonies were isolated after exposure of biofilms to 8, 4, 4 and 2 mM EDTA, and these were denoted persister strains A, B, C and D, respectively. These strains were cultivated on agar plates and produced colonies with a morphology identical to that of the original inoculating strain (Fig. 5b–f). Furthermore, relative to the inoculating C. tropicalis 99916 strain, these persister strains produced biofilms of similar cell density and displayed similar concentration-dependent killing

Fig. 5. Cells surviving EDTA exposure gave rise to biofilms with susceptibility similar to the inoculating Candida tropicalis 99916 strain. Here, C. tropicalis biofilms were cultivated on L-lysine-coated pegs in the CBD using TSB as the growth medium. After 48 h of growth, biofilms were exposed to the indicated concentrations of EDTA for 24 h. Biofilms were treated with a neutralizing agent, recovered from the CBD and then plated for viable cell counting. Each data point indicates the mean and SD of four independent replicates. (a) The concentration-dependent killing of C. tropicalis biofilms by EDTA. The inoculating strain and persister strains A–D are indicated by ’ with a black line, m with an orange line, . with a green line, ^ with a blue line and with a red line, respectively. Persister strains were derived from colonies recovered on agar plates following separate EDTA exposures (see the text for details). (b–f) The inoculating strain and persister strains had a similar colony morphology when streaked out on TSA plates. Each panel represents an area of c. 2 2 cm. Images were contrast and brightness enhanced using ADOBEs PHOTOSHOPs 7.0.



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kinetics when subsequently exposed to EDTA for 24 h (Fig. 5a). In broth microdilution assays, all of these persister strains had an MIC of 1.0 mM EDTA, which was identical to that of the inoculating strain (Table 1). These results indicate that chelator exposure did not select for genetic variants in the C. tropicalis biofilm; rather, these data suggest that the chelator-tolerant subpopulation reflected intrinsic cell-to-cell heterogeneity in the biofilm population.

Conclusions Candida spp. form exopolysaccharide-entrenched infectious biofilms that are highly resistant and/or tolerant to most of the commonly used antifungal agents (Chandra et al., 2001). Amphotericin B as well as the metal chelators EDTA and diethyldithiocarbamate are known to have potent antibiofilm activity (unpublished data as well as Ramage et al., 2002; Percival et al., 2005). Although these antifungals rapidly kill a large portion of C. albicans and C. tropicalis biofilm populations, the data in this study show that a small subpopulation of biofilm cells may withstand these fungicidal agents even at very high concentrations. This gave rise to chelator tolerance in the form of biphasic population killing that was similar in many regards to that previously reported for bacterial and fungal persister cells (Lewis, 2007). By using this information to re-evaluate the antifungal efficacy of diethyldithiocarbamate and EDTA, C. albicans and C. tropicalis biofilms were 4 to 533 more tolerant to these organic chelators than the corresponding planktonic cell populations. These data suggest that, similar to other antimicrobials, the use of organic chelators as agents against biofilms of Candida spp. may be limited by the refractory nature of a variant cell subpopulation in the surfaceadherent community.

Acknowledgements This work was supported through discovery grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada to R.J.T. and H.C. NSERC has provided a Canada Graduate Scholarship Doctoral (CGSD2) to J.J.H., who was additionally supported through a Ph.D. Studentship from the Alberta Heritage Foundation for Medical Research (AHFMR). The Calgary Biofilm Devices used in this study were gifts kindly provided by Innovotech. CLSM was made possible through a Canadian Foundation for Innovation (CFI) Bone and Joint Disease Network grant to H.C.

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