Synergistic Effect of Calcineurin Inhibitors and Fluconazole against Candida albicans Biofilms

C. albicans strains. C. albicans strains used in this study were SC5314, K1, and DAY185, C. albicans calcineurin B homozygous mutant cnb1/cnb1 (DAY364) and its complemented strain cnb1/cnb1+CNB1 (MCC85), C. albicans zinc finger transcription factor mutant crz1/crz1 (OCC1.1) and its complemented strain crz1/crz1+CRZ1 (OCC7), the FK506-resistant calcineurin mutant CNB1-1/CNB1 (JRB61), and the rbp1/rbp1 C. albicans strain lacking FKBP12 (YAG171). All strains except K1 are congenic with the SC5314 strain background. The strains were routinely maintained and grown in YPD liquid medium (20 g of peptone, 10 g of yeast extract, 20 g of glucose per liter) at 30°C. All strains had normal and comparable growth rates.

Biofilm growth conditions.Two in vitro models were used to assess biofilm growth and drug susceptibility. The first model utilized a 96-well polystyrene plate as a substrate, as previously described (22, 28). Briefly, cells were grown in YPD overnight at 37°C and resuspended in RPMI buffered with HEPES at a concentration of 10⁶ cells/ml based on hemocytometer calculations. An inoculum (100 μl) was added to each well of a 96-well flat-bottom plate. After a 24-h incubation at 37°C, the wells were washed with phosphate-buffered saline (PBS) three times to remove any nonadherent cells.

In the second model, biofilms were also grown on silicon elastomer (SE) surfaces as described previously (31). Briefly, strains were grown overnight in YPD medium at 30°C and diluted to an optical density at 600 nm of 0.5 in Spider medium. The suspension was added to a sterile 12-well or a 48-well plate containing bovine serum (B-9433; Sigma)-treated SE squares (cardiovascular instrument silicone sheets; PR72034-06N). The inoculated plates were incubated at 37°C for 90 min at 150 rpm agitation for the initial adhesion of cells. The squares were washed with PBS, transferred to fresh plates containing fresh Spider medium, and incubated at 37°C for 48 h at 150 rpm agitation to allow biofilm formation.

Quantitative measurement of biofilms.Biofilms were quantified by both dry weight measurements and colorimetrically by a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT; Sigma) reduction assay. For dry weight measurements, the SE squares were weighed prior to biofilm development. After 48 h of biofilm formation, the biofilms were washed gently with sterile PBS and air dried at 37°C for 24 h. The total biomass of each biofilm was calculated by subtracting the weight of the silicone prior to biofilm growth from the weight of the silicone after biofilm growth.

XTT solution (1 mg/ml) was prepared in PBS; menadione solution (0.4 mM; Sigma) was prepared in acetone. The two solutions were added to the wells containing PBS-washed biofilms at a ratio of 5:1 (vol/vol), and the plates were incubated for 3 h at 37°C in the dark. After incubation, the liquid was removed from each well and clarified by centrifugation, and XTT formazan production was measured by determining the absorbance at 492 nm.

In vitro biofilm drug susceptibility.Biofilms were formed in 96-well plates as described above. After 24 h, fresh medium (RPMI/HEPES) and drugs were added to wells. Dilutions of fluconazole (0.25 to 1,000 μg/ml), FK506 (0.005 to 300 μg/ml), and cyclosporine (1.2 to 300 μg/ml) were examined alone and in combination in a checkerboard format. The ranges of concentrations of the drugs used included the standard doses given to humans. After 24 h of incubation at 37°C, biofilms were washed twice with PBS. Measurement of biofilm cell metabolic activity using the XTT reduction assay was performed as previously described (22, 28). Briefly, 90 μl of XTT (1 mg/ml) and 10 μl phenazine methosulfate (320 μg/ml) were added to each well, and the plate was incubated at 37°C for 2 h. Absorbance at 492 nm was measured using an automated plate reader. Assays were performed in triplicate. We determined the drug concentration associated with a 50% reduction in optical density compared to the no-drug control wells (EC₅₀). The fractional inhibitory concentration (FIC) was then calculated as follows: [(EC₅₀ of drug A in combination)/(EC₅₀ of drug A alone)] + [(EC₅₀ of drug B in combination)/(EC₅₀ of drug B alone)]. Values of ≤0.5 revealed synergy, those of >0.5 but <2 indicated no interaction, and those of >2 were antagonistic (22).

Under many conditions, drug susceptibility testing was also performed for cells in biofilms grown on SE. Mature biofilms were washed gently with sterile PBS and transferred to fresh 12-well plates containing Spider medium supplemented with the appropriate concentration(s) of a single drug or a combination of two drugs. The biofilms were further incubated for 48 h, and metabolic activities of biofilms were measured by the XTT reduction assay. The MIC was defined as the antifungal concentration that caused a 50% reduction in metabolic activity of a C. albicans biofilm compared with the control (incubated in the absence of drug). The drugs used were fluconazole (Sigma, St. Louis, MO), FK506 (Prograf; Astellas Pharma US, Inc., Deerfield, IL), and CsA (Alexis Corporation, San Diego, CA).

In vivo biofilm drug susceptibility.A rat central venous catheter infection model was selected for in vivo biofilm studies (1, 21). Specific-pathogen-free Sprague-Dawley rats weighing ∼400 g were used (Harlan Sprague-Dawley, Indianapolis, IN). A heparinized (100 U/ml) polyethylene catheter was surgically inserted into the external jugular vein and advanced to a site above the right atrium (2-cm length). The catheter was secured to the vein, and the proximal end was tunneled subcutaneously to the midscapular space and externalized through the skin. The catheters were implanted 24 h prior to inoculation. Infection was achieved by intraluminal instillation of 500 μl C. albicans strain K1 (10⁶ cells/ml). After 4 h, the catheters were flushed and maintained with heparinized 0.15 M NaCl for 24 h to allow for biofilm formation. Catheters were then treated with fluconazole (125 μg/ml), FK506 (20 μg/ml), or a combination for 16 h. These concentrations were picked based on their efficacies in the in vitro drug combination experiments. For controls, animals were treated with saline. Two animals were used per time and treatment point. At the end of the observation period, the catheters were removed and processed for electron microscopy, as described below.

Confocal microscopy.Biofilms were stained with 25 μg/ml concanavalin A-Alexa Fluor 594 conjugate (C-11253; Molecular Probes, Eugene, OR) for 1 h in the dark at 37°C. Confocal scanning laser microscopy was performed with a Zeiss LSM 510 upright confocal microscope using a Zeiss Achroplan 40×, 0.8-W objective. Concanavalin A conjugate staining was observed using a HeNe1 laser with an excitation wavelength of 543 nm. Images were assembled into side and depth views using the Zeiss LSM Image Browser v4.2 software. Artificially colored depth view images indicate cell depth using a color gradient, where cells closest to the SE are represented in blue and the cells farthest away are represented in red.

Scanning electron microscopy.Silicon elastomer squares containing biofilms were introduced without fixation into the environmental scanning electron microscope (Philips XL30 ESEM TMP; FEI Company, Hillsboro, OR; NSF award DBI-0098534). The ESEM allows examination of any specimen, wet or dry, in situ and close to its natural state. Samples were examined at 10 kV, 5 torr pressure, and at a temperature of 3°C. The samples remained hydrated throughout the process. Catheter segments were processed for scanning electron microscopy as previously described (1). After overnight fixation (4% formaldehyde, 1% glutaraldehyde in PBS), catheter segments were washed for 5 min in PBS and placed in 1% osmium tetroxide for 30 min. Drying was accomplished using a series of alcohol washes followed by critical point drying. Catheter segments were mounted and gold coated. Images were obtained with a scanning electron microscope (JEOL JSM-6100) in the high-vacuum mode at 10 kV. The images were assembled using Adobe Photoshop 7.0.1.

Calcineurin is not required for C. albicans biofilm formation in vitro or in vivo.Calcineurin governs several important physiological properties in C. albicans, including C. albicans survival in serum, virulence in a murine model of systemic candidiasis, and resistance to azole antifungals (7, 9, 11, 23, 32). Azole tolerance is mediated in part via the calcineurin target Crz1 (15, 25, 33). Although calcineurin does not control C. albicans properties of adhesion or filamentation (3, 4, 9), two properties important for biofilm formation, given its broad physiological roles we hypothesized that calcineurin might be involved in C. albicans biofilm formation. To test this, C. albicans cnb1/cnb1 (lacking calcineurin B) and crz1/crz1 (lacking a transcription factor controlled by calcineurin) mutants were first used to develop biofilms in vitro on silicone elastomer squares. After 48 h of development at 37°C, biofilms were quantitated by two independent methods, dry weight measurements and XTT assays. No significant difference (P > 0.3) was observed between the biofilms formed by the C. albicans wild-type strain and the C. albicans calcineurin or crz1 mutant strains (Fig. 1). Also, as expected, the complemented strains of the C. albicans calcineurin and the crz1 mutants developed biofilms as robustly as the C. albicans wild-type strain (Fig. 1).

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FIG. 1.

Calcineurin is not required for C. albicans biofilm formation. Biofilms were grown as described in Materials and Methods, metabolic activity was measured by XTT assay (A), and biomass was measured based on dry weight (B). Assays were performed in triplicate. Biofilms were not significantly different from each other (P > 0.3, analysis of variance).

The ability of the C. albicans cnb1/cnb1 mutant to form a biofilm in vivo was examined using the venous catheter animal model (Fig. 2). Similar to the wild-type strain, cnb1/cnb1 formed a mature biofilm 24 h after inoculation. Scanning electron microscopy imaging demonstrated similar biofilm architecture between the wild-type and mutant strains. Both biofilms were of comparable thickness and consisted of a combination of adherent yeast and hyphae surrounded by matrix and host components.

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FIG. 2.

The C. albicans calcineurin mutant forms a biofilm in vivo. C. albicans wild type and cnb1/cnb1 were instilled in rat venous catheters. The ability to form a biofilm and biofilm architecture were evaluated by scanning election microscopy imaging at 24 h. The top row represents a 50× magnification (bar, 600 μm), and the bottom row represents 1,000× magnification (bar, 30 μm). Experiments were performed in duplicate. Biofilm thickness and architecture were similar between the two strains.

Calcineurin is involved in C. albicans biofilm resistance to fluconazole. C. albicans cnb1/cnb1 and crz1/crz1 mutant strains are known to be hypersensitive to fluconazole compared to wild-type cells under planktonic growth conditions (23, 25, 30, 33). The fact that these mutants formed morphologically wild-type biofilms allowed us to test if these strains were also azole hypersensitive under biofilm conditions. Mature biofilms were treated for 48 h with a range of fluconazole concentrations (0.25 μg/ml to 1,024 μg/ml). The MIC₅₀ of fluconazole for biofilm cells was calculated based on results from the XTT assay. C. albicans cnb1/cnb1 biofilm cells were at least 15-fold more sensitive to fluconazole than wild-type biofilms, and those formed by the C. albicans crz1/crz1 mutant were at least 8-fold more sensitive (Table 1). Complementation of the cnb1 and crz1 mutations with the wild-type gene restored wild-type levels of azole resistance in biofilms (Table 1). These results are complementary to earlier reports that, under planktonic conditions, crz1/crz1 mutants are less fluconazole hypersensitive than cnb1/cnb1 mutants (but both strains are significantly more sensitive than wild-type C. albicans cells) (15, 25).

C. albicans biofilms are hypersensitive to calcineurin inhibitor-fluconazole combinations.The increased fluconazole susceptibility of the cnb1/cnb1 mutant biofilms indicated that calcineurin may be responsible for the resistance of C. albicans biofilms to fluconazole. This hypothesis was further tested by exposing wild-type C. albicans biofilms to a combination of calcineurin inhibitors and fluconazole. Previous studies demonstrated that C. albicans planktonic cells are hypersensitive to combinations of calcineurin inhibitor (either FK506 or cyclosporine A) and fluconazole (11, 17, 18). If calcineurin were indeed responsible for mediating fluconazole resistance, then biofilms cells might also exhibit sensitivity to the drug combination.

Measurement of metabolic activity of biofilm cells in an XTT assay revealed that biofilm cells were resistant to CsA (1 to 300 μg/ml), FK506 (0.005 to 300 μg/ml), or fluconazole (0.25 to 1,024 μg/ml) delivered individually (Table 2). However, biofilm cells were found to be exquisitely sensitive to either an FK506-fluconazole or CsA-fluconazole combination, with FIC indexes as low as 0.08 and 0.13 for the respective drug combinations, indicating a synergistic interaction (Table 2).

In addition to the XTT assays, we performed confocal scanning laser microscopy (CSLM) to examine the architecture of biofilms treated with the FK506-fluconazole combination compared to individual drug treatment. Side and depth biofilm analyses revealed no significant difference between the biofilms formed by the wild-type untreated, fluconazole-treated, or FK506-treated cells (∼300 μm) (Fig. 3). However, the biofilms treated with the FK506-fluconazole combination were significantly reduced in thickness by ∼5-fold to 20% the depth produced by cells under the other conditions (Fig. 3).

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FIG. 3.

C. albicans biofilms are hypersensitive to the tacrolimus (FK506)-fluconazole combination. C. albicans wild-type biofilms (untreated) (A) or treated with fluconazole (B), FK506 (C), or a combination of fluconazole and FK506 (D) were stained with concanavalin A conjugate for CSLM visualization, and image reconstructions were created to provide side views (A to D). CSLM depth views for the wild-type biofilm (E) and biofilm treated with the two-drug combination (G) were artificially colored: blue represents cells closest to the silicone, and red represents cells farthest from the silicone. SEM images of the latter two conditions were also obtained (F and H). Biofilms treated with only fluconazole or only FK506 showed depth view and SEM patterns similar to the wild type and are not included in the figure. Bars in SEM images, 100 μm.

To examine the impact of the FK506-fluconazole combination in vivo, we used a rat venous catheter biofilm model and imaged treated and control biofilms by scanning electron microscopy (Fig. 4). Addition of fluconazole alone (125 μg/ml) or FK506 alone (20 μg/ml) had minimal impact on mature wild-type C. albicans biofilms, compared to a saline-treated control. However, imaging of the two-drug combination demonstrated the complete inhibition of the biofilm.

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FIG. 4.

Impact of fluconazole and FK506 alone and in combination as lock therapy against C. albicans biofilm cells in an in vivo catheter model. The biofilms were grown for 24 h followed by intraluminal drug treatment for 16 h. Following compound exposure, the catheters were removed for SEM processing. For each of the four panels, the top row represents a 50× magnification (bar, 600 μm) and the bottom row represents 1,000× magnification (bar, 30 μm). Panel columns: no treatment, images from a control biofilm treated with saline; fluconazole, images after a 125-μg/ml fluconazole exposure; FK506, images from catheters exposed to FK506 at 20 μg/ml; fluconazole + FK506, images from a combination of fluconazole and FK506 at 125 μg/ml and 20 μg/ml, respectively.

To test if calcineurin is involved in the toxic effect of the FK506-fluconazole combination, two FK506-resistant strains were analyzed: first, a strain lacking the RBP1 gene, whose product is required to bind FK506 and inhibit calcineurin (10, 11), and second, CNB1-1, a mutant strain that expresses a dominant drug-resistant form of calcineurin B that has reduced affinity for FKBP12-FK506 but wild-type sensitivity to cyclophilin A-CsA (11, 14). If calcineurin is required for the observed synergistic interaction between FK506 and fluconazole in C. albicans, then both the rbp1/rbp1 and CNB1-1 mutations should confer resistance to the drug combination while continuing to be sensitive to the CsA-fluconazole combination. Indeed, both the rbp1/rbp1 and CNB1-1 strains were completely resistant to the FK506-fluconazole combination (FIC, 2.0) but remained sensitive to the CsA-fluconazole combination (FIC, 0.31 to 0.37), confirming that calcineurin is directly involved in biofilm resistance to fluconazole (Tables 1 and 2).