Folia Microbiol (2015) 60:45–51 DOI 10.1007/s12223-014-0338-y

The impact of growth conditions on biofilm formation and the cell surface hydrophobicity in fluconazole susceptible and tolerant Candida albicans Anna Kolecka & Dušan Chorvát Jr. & Helena Bujdáková

Received: 24 October 2013 / Accepted: 16 July 2014 / Published online: 7 August 2014 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2014

Abstract The influence of acidic (5.6) and neutral (7.0) pH and glucose concentrations (0.9 and 2 %) was determined in in vitro biofilm formation and the cell surface hydrophobicity (CSH) in fluconazole (FLC) susceptible and tolerant yeasts of Candida albicans. The determination of biofilm viability using tetrazolium salt XTT showed that both FLC-tolerant C. albicans 1173 and FLC-sensitive C. albicans SC 5314 formed more robust biofilm in the YNB medium at pH 7.0 in the absence of FLC than that at acidic pH. Tested glucose concentrations did not show any direct effect on formation of biofilm under all conditions. However, determination of biofilm dry mass that contains also extracellular matrix suggested some effect of 2 % D-glucose. An increase in CSH (for about 10 %) was estimated in C. albicans SC 5314 in the presence of FLC, while the FLC-tolerant isolate proved a weak increase of CSH only in the YNB media containing 2 % D-glucose. Additionally, strain C. albicans SC 5314 strongly flocculated at neutral pH in the absence of FLC, but this phenomenon was not observed in the presence of FLC. Subinhibitory concentration of FLC influenced biofilm cells and CSH, but FLC susceptibility versus tolerance of C. albicans tested strains did not directly affect biofilm formation and/or CSH. A. Kolecka : H. Bujdáková (*) Department of Microbiology and Virology, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynska dolina B-2, 842 15 Bratislava, Slovakia e-mail: [email protected] D. Chorvát Jr. Department of Biophotonics, International Laser Centre, Ilkovicova 3, 81219 Bratislava, Slovak Republic Present Address: A. Kolecka Department of Yeast and Basidiomycete Research, CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands

Introduction Candida albicans is a leading opportunistic human fungal pathogen able to adapt to various external environmental conditions affecting its persistence as well as pathogenesis (Desai et al. 2011; Leach et al. 2012). C. albicans developed different strategies to stay protected from stress conditions (Ramsdale et al. 2008; Desai et al. 2011). This adaptation has already been extensively studied in vitro, mainly on planktonic cells of clinical isolates or specific mutants (Enjalbert et al. 2003; Yu et al. 2012). The interest was focused on pH changes (Konno et al. 2006), temperature shift (Leach et al. 2012), nutrient limitation (nitrogen, amino acids, glucose starvation; Yin et al. 2004; Tsao et al. 2009; Ene et al. 2012), response to limited aeration (Rosa et al. 2008), and different salt tolerance (Kolecka et al. 2009). Additionally, an exposure to antifungal drugs was postulated as a factor evoking endogenous stress response as well (Cannon et al. 2007; Singh et al. 2012). In nature, many microorganisms prefer to survive in ubiquitous form—biofilm rather than free planktonic cells (Brablcová et al. 2013). Moreover, during the last decade, biofilm-associated infections have become a serious therapeutic problem because of resistance phenomenon (Ramage et al. 2006; Berila et al. 2011; Tumbarello et al. 2012). Both in vitro and in vivo biofilm formations are a process during which cultivation conditions (optimal or nonoptimal) have a direct impact on its development. For instance, Garcia-Sanchez et al. (2004) proved that increased extracellular matrix production was observed in biofilm formed under flow conditions when compared with a static growth model. On the other hand, depletion of oxygen observed in the later biofilm stadiums can lead to an alteration in sterol composition of fungal cells (Garcia-Sanchez et al. 2004; Borecká-Melkusová et al. 2009; Stichternoth and Ernst 2009). Additionally, Biswas and Chaffin (2005) and Kucharíková et al. (2011) confirmed that

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biofilm growth is pH dependent, while Hawser and Douglas (1994) described that the type and concentration of carbon sources (galactose or glucose) may directly affect biofilm formation resulting in a stimulatory effect on the yeast adhesion to solid surface and on the extracellular polymeric material production. The very important factor promoting adhesion and biofilm formation is the cell surface hydrophobicity (CSH; Yoshijima et al. 2010). The hydrophobic properties related to the yeast surface physicochemical characteristics are involved in the process of cell aggregation that promotes cell attachment during flocculation but also contributes to the mating between yeasts and finally facilitates biofilm development (Magee et al. 2002; Verstrepen et al. 2004; Verstrepen and Klis 2006; Li et al. 2012). It is of interest that flocculation is also observed at limited access to nutrients or under other stress conditions (Verstrepen and Klis 2006; Linder and Gustafsson 2008). This work investigated the influence of acidic (5.6) and neutral (7.0) pH, and two different glucose concentrations (0.9 and 2 %) on in vitro formed biofilm and CSH in the yeast C. albicans. Additionally, it was studied whether fluconazole (FLC) susceptibility versus tolerance can be of concern with tested conditions to both biofilm and CSH.

Materials and methods Strains For this research, clinical isolate of C. albicans 1173 with decreased susceptibility to FLC (Melkusová et al. 2004) and reference standard strain C. albicans SC 5314 was used (Gillum et al. 1984). Before use, strains were cultivated at 28 °C on YPD agar plate (2 % yeast extract, 2 % mycological peptone, 2 % glucose, 2 % agar, w/v; Biomark Laboratories, India) for 24 h. Isolates were maintained in glycerol/YPD at −80 °C until used. Susceptibility profiles determined as minimal inhibitory concentration (MIC)80 and MIC95 already published by Melkusová et al. (2004) were repeated and confirmed using a broth microdilution method according to the National Committee for Clinical Laboratory Standards (2008) protocol. According to determined MIC95 for both strains, subinhibitory concentrations of FLC (0.5× MIC95; 32 μg/mL for C. albicans 1173 and 0.5 μg/mL for C. albicans SC 5314) were used for proceeding with the majority of experiments. Determination of biofilm by XTT reduction assay and dry mass measurement The loop of cells cultivated on Sabouraud dextrose agar (Biomark Laboratories, India) at 28 °C for 24 h was

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transferred into yeast nitrogen base medium with amino acids (YNB broth, Difco, USA). Media were supplemented with Dglucose (Applichem, Germany) to a concentration of 0.9 or 2 % (w/v). The pH of nonadjusted YNB medium was 5.6 while that with pH 7.0 was buffered by addition of 0.05 mol/L Na2HPO4 and 0.05 mol/L KH2PO4. Additionally, cells were growing at the presence of subinhibitory concentration of FLC (0.5 μg/mL for SC 5314 and 32 μg/mL for 1173). After overnight cultivation at 37 °C in YNB medium (0.9 or 2 % D-glucose, with pH 5.6 or 7.0, with or without FLC), cells were harvested (4,470 g for 5 min) and washed twice with 0.5 mL of sterile 1× PBS. Then, cells were resuspended in fresh YNB medium and adjusted to A580 =1.0 (corresponding to approximately 5×107 cells/mL, MRX Microtiter plate absorbance reader, Dynex Technologies, USA). Biofilm was developed in polystyrene 96-well microtiter plates (flat bottom; Sarstedt, Germany) according to protocol of Li et al. (2003) with minor modifications. Briefly, 100 μL of the cell suspension with A580 = 1.0 was transferred into wells and cultivated for 90 min to allow cell attachment to surface. Afterward, medium containing nonadherent cells was removed, and adherent cells were gently washed twice with 150 μL of sterile 1× PBS. Next, 100 μL of fresh YNB medium was added to each well. Plates were covered and incubated at 37 °C. After 48 h, medium containing dispersal cells was removed, and wells with mature biofilm were washed with 200 μL of sterile 1× PBS for three times. Biofilm was quantified by the colorimetric assay using the XTT ([2,3-bis(2m e t ho x y - 4 - ni t r o- 5 - s u l p h en y l ) - 2H - t et r a z o l i u m - 5 carboxanilide] sodium salt, Sigma-Aldrich, USA) at A490 on a microplate reader after 3-h incubation at 37 °C in the dark. Experiment was performed with three parallels in three independent biological replicas for each strain. YNB medium containing no inoculum was used as a negative control. For biofilm dry mass determination, standard method according to Li et al. (2003) protocol was modified. For each strain, 4×20 mL of the cell suspension (A580 =1.0) was used to inoculate four polystyrene Petri dishes (Sarstedt, Germany). The plates were incubated statically for 90 min at 37 °C. Nonadherent cells were removed, and adherent cells were gently washed twice with 20 mL of sterile 1× PBS buffer. For mature biofilm, 20 mL of fresh YNB medium (supplemented to 0.9 or 2 % (w/v) with Dglucose, pH 5.6 or 7.0, in the presence or absence of FLC) was added to each Petri dish. Samples were statically cultivated at 37 °C. After 48 h, mature biofilm was washed twice with 20 mL of sterile 1× PBS, scratched from the Petri dish surface using a cell scraper, and collected to preweighted Eppendorf tubes by centrifugation. Pellets were deep frozen in liquid nitrogen and lyophilized (Lyovac GT2, Leybold-Heraeus, Germany) at

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room temperature to constant mass and then weighted again. The experiment was handled with two parallel samples collected from two different biological experiments. The standard deviations (SD) were calculated for each experiment to establish reproducibility and select the average values for the mean and new SD calculation. The final dry mass was calculated according to formula [(weight of dried Eppendorf tube − mass of empty Eppendorf tube)×1,000]/20 and presented as mg of dry mass per mL. Determination of CSH Inoculum for experiment was prepared like it was already described in previous paragraph. The CSH was estimated by the two-phase separation method, as described previously (Klotz et al. 1985). From 1.5 mL of cell suspension (A580 =1.0), 1.2 mL was placed in a clean glass tube and later extracted to the 300 μL overlay of n-octane (Merck, Germany) by vortexing for 3 min (Vortex Mixer VM300P, Taiwan). After separation of phases, the 3 × 100 μL of aqueous phase was collected to measure A580. Relative percentage of CSH was calculated as published by Borecká-Melkusová and Bujdáková (2008). Values of CSH were averages of triplicate independent measurement determinations±SD. Confocal scanning laser microscopy (CSLM) Biofilms for CSLM were formed on sterile, highly adherent, polystyrene cover slips (25 mm, Sarstedt, Germany). The YNB medium with glucose concentration of 0.9 %, but at different pH levels and in the presence/absence of FLC, was selected for microscopic examination. For each strain, two cover slips were placed in one polystyrene Petri dish (Sarstedt, Germany), inoculated with 30 mL of the cell suspension (A580 =1.0) and incubated at 37 °C for 90 min. Cell suspension was prepared, as it was mentioned previously for standard in vitro biofilm. After the period of adhesion (90 min), medium with nonadherent cells was removed, and cells attached to cover slip surface were washed twice with 20 mL of sterile 1× PBS. Next, each Petri dish with cover slips was overlaid again with 30 mL of fresh YNB medium and incubated at 37 °C for 48 h. Later, cover slips were gently washed with 1× PBS and transferred to another Petri dish with aluminum foil. Mature biofilms were stained with 20 μL of tetramethylrhodamine methyl ester (TMRM) and 20 μL tetramethylrhodamine ethyl ester (TMRE) (Invitrogen, USA; exCitation wavelength 549 nm, emission wavelength 573 nm), diluted in distilled deionized water to a final 5 μmol/L. Stained biofilms were observed with LSM 510 META confocal scanning laser microscope

Fig. 1 Variations between C. albicans SC 5314 (a) and C. albicans 1173 (b) in in vitro formed biofilm estimated by XTT reduction assay

mounted on Axiovert 200 M inverted microscope (both Carl Zeiss, Germany) as it was already described (Bujdáková et al. 2008). Biofilms were documented in the 3D reconstructed images used to determine biofilm thickness, and 2D images were employed for the biofilm architecture and morphological diversity. The thickness

Fig. 2 The quantification of C. albicans SC 5314 (a) and C. albicans 1173 (b) biofilm formation by dry mass in regard to different growth conditions

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was estimated from outer edges of the area, where signal gain intensity above half of its maximum.

Results and discussion The yeasts as many other microorganisms regulate their adaptation in order to survive in environment (Mitchell et al. 2009). C. albicans is the dimorphic yeast responding to extracellular pH and glucose level through the yeast to mycelial transition (Mitchell 1998; Sabina and Brown 2009). Additionally, environmental sensing of diverse glucose concentration has a significant impact on the entire yeast metabolism and physiology (Rodaki et al. 2009). Therefore, glucose concentration and pH are predicted to be very important factors not only for the yeasts growing as planktonic cells, but also for those organized in biofilm. Yeasts in their normal habitat conditions prefer acidic pH (pH64 μg/mL). The standard strain C. albicans SC 5314 showed FLC susceptibility (MIC80 and MIC95 =1 μg/mL). An ability of subpopulation of C. albicans cells to survive in FLC concentrations higher than 64 μg/mL (break point for resistance according to CLSI M27-A3 protocol) is characteristic for trailing isolates (Agrawal et al. 2007). The trailing phenomenon associated with the low-high MIC phenotype (Alp et al. 2010) was also confirmed in tolerant C. albicans 1173. Both C. albicans formed strong biofilm in the YNB medium with neutral pH in the absence of FLC (Fig. 1a, b; whitehatched columns); this feature seemed to be glucose

(a, e); pH 5.6, presence of FLC (b, f); pH 7.0, absence of FLC (c, g); pH 7.0, presence of FLC (d, h). Scale bar is 20 μm

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concentration independent. The above observation could be associated with the stimulating effect of neutral pH on hyphae formation (Biswas et al. 2007) or it can relate to higher expression of genes coding for proteins participating in the increase of adherence during biofilm formation (for example, the ALS3 gene; Kucharíková et al. 2011). The presence of subinhibitory concentration of FLC reduced ability to form biofilm in both strains under all tested conditions (Fig. 1a, b; gray bars and gray-hatched columns), with exception for SC 5314 biofilm grown in the YNB medium with 2 % D-glucose, pH 7.0 (Fig. 1a, the second gray-hatched column). The reduction of viability of biofilm cells in the presence of subinhibitory concentration of FLC in the tolerant strain C. albicans 1173 (despite tolerance of planktonic cells to this drug) determined by XTT reduction assay is possible to explain through an existence of the mix population of tolerant and susceptible cells in C. albicans culture that is frequently associated with the low-high MIC phenotype in trailing clinical isolates (Agrawal et al. 2007; Alp et al. 2010). Such mixed population expresses increased MIC to different drugs, but it is not usually able to form well-built biofilm. This observation is in agreement with previous results of Bruzual et al. (2007) or BoreckáMelkusová and Bujdáková (2008) who published that FLC can reduce C. albicans biofilm formed by FLC susceptible or resistant/tolerant strains. While XTT reduction assay did not prove any influence of glucose concentration on biofilm, determination of biofilm dry mass suggested some effect of 2 % D-glucose in the

Fig. 4 The influence of cultivation conditions and subinhibitory concentration of FLC on the cell surface hydrophobicity of C. albicans SC 5314 (a) and C. albicans 1173 (b)

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Fig. 5 The yeast suspensions of C. albicans SC 5314 before n-octane overlay in YNB medium without FLC pH 5.6 (a) and pH 7.0 (b) and supplemented with the subinhibitory concentration of FLC at pH 5.6 (c) and pH 7.0 (d)

absence of FLC to both tested strains, but up to different extent (Fig. 2a, b; the second white and the second white-hatched columns). This effect can be expected due to formation of the extracellular matrix, production of which is strain dependent, and it is involved in the total dry mass estimation. Contrary, it is not possible to evaluate using the XTT reduction assay that concerns only viable cells. The presence of FLC markedly reduced biofilm formation in both strains with stronger impact in media with pH 5.6 (Fig. 2a, b; gray and gray-hatched columns). At neutral pH, this effect was partially reduced probably because of higher vitality of biofilm.

Fig. 6 The C. albicans SC 5314 flocculation phenomenon observed in YNB medium containing 0.9 % D-glucose with pH 7.0 in the absence (a) and the presence (b) of FLC

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CLSM (Fig. 3) showed that biofilm of the strain C. albicans SC5314 consisted mainly of hyphae (Fig. 3a, c, d), while biofilm of C. albicans 1173 was formed by the dense mass of cells attached to each other (Fig. 3e–h). Media with neutral pH in the absence of FLC stimulated biofilm formation of both strains (Fig. 3c, g). However, the presence of FLC in media with pH 5.6 affected biofilm formation, and the strains developed only monolayer of attached cells (Fig. 3b, f). On the other hand, neutral pH stimulated biofilm dense despite FLC presence (Fig. 3d, h). The FLC did not affect only integrity of biofilm but also its thickness. The basal layer of biofilm developed by the standard strain C. albicans SC 5314 in medium with pH 5.6 was grown up to 10 μm and increased double, up to 20 μm, in medium with pH 7.0. However, in the presence of FLC, biofilm was reduced to 5 μm at pH 5.6 and to 10 μm at neutral pH. The isolate C. albicans 1173 showed better growth in the absence of FLC at pH 5.6 (16 μm) than at pH 7 (11 μm). In the presence of FLC and pH 5.6, it was not able to form biofilm (basal layer was on the detection limit, ≤4 μm), and at pH 7.0, it was reduced to 8 μm. The FLC presence proved that an effect on CSH C. albicans SC 5314 showed an increase in CSH for about 10 %, in the presence of FLC in YNB medium under all tested conditions (Fig. 4a, gray and gray-hatched columns). Additionally, while the changes in CSH showed to be glucose concentration independent, CSH was slightly increased in media with pH 7 (Fig. 4a all hatched columns). The FLC-tolerant C. albicans 1173 strain exhibited the other behavior under tested conditions (Fig. 4b). In medium containing 2 % D-glucose, and without FLC, cells converted from hydrophobic to more hydrophilic (Fig. 4b, the second white and the second white-hatched columns). On the other hand, in the presence of FLC, the clinical isolate showed a weak increasing of hydrophobic cells only in YNB medium containing 2 % D-glucose at both pH (Fig. 4b, the second gray and the second gray-hatched colums). Rodaki et al. (2009) published that upregulation of genes participating in resistance to osmotic stress, oxidative stress, and azole drugs in C. albicans is glucose concentrationdependent process. The changes in CSH can also be associated with a reorganization of fibrillar layer in the cell wall upon FLC treatment resulted in reducing the surface density of the fibrils (Hazen et al. 2000). The increase of CSH in FLC presence was also described in some C. albicans strains by BoreckáMelkusová and Bujdáková (2008), but the same authors concluded that this effect was proven to be strain dependent. It is of interest that C. albicans SC 5314, when cultivated in media with pH 7.0 in the absence of FLC, strongly flocculated (Fig. 5c). The flocs consisted mainly of the yeast cells, firmly attached to filamentous forms (Fig. 6a). Flocculation 3 was evidently reduced when cells were incubated in the FLC presence (Fig. 5) and microscopy did not reveal the true hyphae present in culture that contained only budding yeasts

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and pseudohyphal forms (Fig. 6b). This phenomenon was not observed in acidic pH (Fig. 5a, b). Flocculation development is quite well prescribed in model organism Saccharomyces cerevisiae. During this process, neighboring cells start cross-binding that leads to the cell aggregation. Additionally, external proteins present in the yeast cell wall participating in flocculation may influence CSH (Verstrepen and Klis 2006; de Groot and Klis 2008). Contrary to S. cerevisiae, C. albicans is a dimorphic organism, and filamentation is supposed to be an important factor involved in flocculation. Bauer and Wendland (2007) confirmed that flocs of C. albicans are composed of the yeasts and the hyphae. Taken together, some effect of glucose concentration, but mainly, influence of neutral pH was observed on biofilm formation and CSH status in tested C. albicans strains. FLC susceptibility versus tolerance of C. albicans tested strains did not directly affect biofilm formation and/or CSH. Acknowledgments We would like to thank Mgr Eva Sodomova for helpful assistance with the photographic examination of the flocculation phenomenon. This research was supported by EU grant Marie Curie Research Training Network MRTN-CT-2004-512481 CanTrain, by the Slovak Research and Development Agency under the contract No. APVV-0291-11, and by the grant VEGA 1/0966/12 supported by the Slovak Ministry of Education, Research, Science and Sport. The authors report no conflicts of interest; AK performed all experiments and participated in writing, DCh prepared the confocal scanning laser microscopy images, HB designed experimental research and participated in writing of manuscript.

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