Antifungal Susceptibility of Candida Biofilms: Unique Efficacy of Amphotericin B Lipid Formulations and Echinocandins

Organisms.

The C. albicans and C. parapsilosis isolates used in this study were obtained by subculturing clinical specimens from the microbiology laboratory at the University Hospitals of Cleveland (29). Speciation was performed using routine germ tube tests and API20C-AUX methods. C. albicans strain M61 was obtained at our institution from an intravascular line culture, C. parapsilosis strain P/A71 was from a sputum culture, and C. parapsilosis strain P92 was from a blood culture. C. albicans strain GDH 2346 (GDH) was obtained from a patient with documented denture stomatitis (obtained from L. Julia Douglas, University of Glasgow, Glasgow, United Kingdom) and has been previously shown to produce biofilm (13, 14).

Medium and growth conditions.

All Candida strains were grown in yeast nitrogen base (YNB) medium (Difco Laboratories, Detroit, Mich.) supplemented with 50 mM glucose (14). Fifty milliliters of medium (in 250-ml Erlenmeyer flasks) was inoculated with Candida cells from fresh Sabouraud dextrose agar plates (Difco) and incubated for 24 h at 37°C in a water bath orbital shaker at 60 rpm. Cells were harvested and washed twice with 0.15 M phosphate-buffered saline (PBS; pH 7.2; Ca²⁺ and Mg²⁺ free). Yeast cells were resuspended in 10 ml of PBS, counted after serial dilution using a hemocytometer, standardized to 10⁷ cells/ml, and used immediately.

Substrate material.

Silicone elastomer (SE) sheets were obtained from Cardiovascular Instrument Corp. (Wakefield, Mass.). As per the manufacturer's instructions, the material was cleaned by washing in hand soap and water, rinsed with distilled water, and autoclaved. Flat circular disks, 1.5 cm in diameter, were obtained by cutting with a cork borer (29).

Biofilm formation.

SE disks were placed in 12-well tissue culture plates (Becton Dickinson, Franklin Lakes, N.J.) and incubated in fetal bovine serum for 24 h at 37°C on a rocker table (14, 29). To ensure uniform biofilm formation, the SE disks were immersed in a Candida cell suspension. Three milliliters of standardized cell suspension, containing 10⁷ blastospores/ml, was added to the wells and the disks were incubated for 90 min at 37°C on a rocker table (“adhesion phase”). Disks were gently agitated and transferred to new plates to ensure removal of nonadherent cells. Disks were then immersed in YNB medium with 50 mM glucose and incubated for 48 h at 37°C on a rocker table (“biofilm formation phase”). For controls, disks were handled in an identical fashion except that no Candida cells were added. All assays were carried out in quadruplicate and on different days.

Quantitation of biofilm.

Quantitation of Candida biofilms was performed as described previously (14) using both a biochemical assay, the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay (22), and dry weight (DW) measurements. XTT (Sigma Chemical Co., St. Louis, Mo.) is reduced by mitochondrial dehydrogenase into a water-soluble formazan product that is measured spectrophotometrically. Following the biofilm formation phase, SE disks containing C. albicans or C. parapsilosis biofilms were transferred to a new 12-well tissue culture plate containing 3 ml of PBS per well. For C. albicans strains, 50 μl of XTT salt solution (1 mg/ml in PBS) and 4 μl of menadione solution (1 mM in acetone; Sigma) were added to each well. Due to the lower XTT metabolism of the C. parapsilosis P/A71 and P92 strains (29), when these isolates were assayed we used 50 μl of a 5-mg/ml XTT solution and 12 μl of menadione. Plates were incubated at 37°C for 5 h and then the medium was removed and centrifuged for 5 min at 6,000 × g to pellet any suspended cells or debris. XTT-formazan in the supernatant was measured at 492 nm by using a spectrophotometer (Genesys 5; Spectronic Instruments, Rochester, N.Y.). DW measures total biofilm mass, including fungal cells and extracellular matrix (ECM) (for details of this technique see references 14, 20, and 29). Briefly, biofilms were scraped off the surface of the disks by using a cell scraper (Becton Dickinson), and both disks and scrapers were rinsed with PBS to remove residual biofilm. The material collected was filtered using a 0.45-μm-pore-size Millipore filter, air dried at 37°C for 48 h, and weighed (29).

Antifungal susceptibility.

Fluconazole (FLC) and voriconazole (VRC) were obtained from Pfizer Pharmaceuticals Group (New York, N.Y.); ravuconazole (Ravu; BMS 207-147) was from Bristol Meyers Squibb (Wallingford, Conn.); chlorhexidine (Chlor), amphotericin B (AMB), and nystatin (NYT) were from Sigma; terbinafine (TRB) was from the Novartis Research Institute (Vienna, Austria); caspofungin (Casp) was from Merck (Rahway, N.J.); micafungin (Mica; FK 463) was from Fujisawa (Deerfield, Ill.); AMB lipid complex (ABLC) was from Elan Pharmaceuticals/The Liposome Co. (Princeton, N.J.); liposomal AMB was from Fujisawa Healthcare, Inc. and Gilead Sciences (San Dimas, Calif.); and lipid complex-NYT was from Aronex Pharmaceuticals, Inc. (The Woodlands, Tex.). Drugs were solubilized in sterile saline (FLU, ABLC, Casp, Mica), sterile water (liposomal AMB), or dimethyl sulfoxide (AMB, NYT, TRB, Chlor, VRC, Ravu).

We used our published method to determine antifungal susceptibilities of biofilm-grown Candida isolates (14, 22, 29). Briefly, following biofilm formation, disks were gently agitated and transferred to new culture plates to remove non-biofilm-associated cells. YNB (3 ml) containing different concentrations of the antifungal agent was added to each well to produce final concentrations from 0.063 to 256 μg/ml. Biofilm activity at 48 h was determined using the XTT assay. The antifungal concentration which caused a 50% reduction in metabolic activity (50% RMA) of biofilm compared with control (incubated in the absence of drug) was then determined (22). As shown previously in planktonic cells, in this assay the 50% RMA is equivalent to the MIC₅₀ (minimum drug concentration at which there is 50% growth inhibition compared to control), as determined by the NCCLS M27-A method (14, 22, 35). Antifungal susceptibility of planktonic cells against the different antifungals was determined using the M27-A technique, as reported previously (13, 29, 35), and the MIC₅₀ was noted as the relevant endpoint (46). Resistance breakpoints were defined as follows (in micrograms per milliliter): FLC, >32; AMB, >0.5 (35, 46). For the other antifungal agents (VRC, Ravu, Chlor, NYT, TRB, Casp, Mica, ABLC, liposomal AMB, and lipid complex-NYT), clear breakpoints have not been established. As such, we defined resistance as a >5-fold increase in the MIC₅₀ since, conventionally, MICs within three dilutions are considered equivalent.

For CSLM of antifungal effects upon planktonic cells, C. albicans strain M61 was grown overnight as described above, washed with PBS, and then resuspended in 50 ml of fresh YNB medium (in 250-ml Erlenmeyer flasks) containing no drug (control), VRC (16 μg/ml), Casp (4 μg/ml), or ABLC (4 μg/ml), with a final concentration of 10⁷ cells/ml. Planktonic cells were incubated in the presence of the agents for 24 to 48 h in a water bath orbital shaker at 60 rpm. Antifungal effects on biofilms were examined in a similar fashion. At 24 to 48 h of formation, biofilms were transferred into new cell culture plates containing 3 ml of medium alone (control), VRC (16 μg/ml), Casp (4 μg/ml), or ABLC (4 μg/ml). Plates were then incubated at 37°C on a rocker for 24 to 48 h. After exposure to antifungals, planktonic cells and biofilms were prepared for CSLM imaging as below.

Subinhibitory concentration experiments were conducted using the technique of Ghannoum et al. (19) Briefly, one-fourth concentrations of the MIC₅₀s for individual drugs against planktonic C. albicans (VRC, 0.125 μg/ml; NYT, 0.5 μg/ml; Chlor, 2 μg/ml) were added to the initial culture medium of organisms (10⁷ blastospores/ml) and 50 ml of medium. After overnight incubation, cells were washed, standardized, and used to form biofilms as described above.

Confocal microscopy.

Biofilm staining and CSLM were performed as described previously (13, 29). FUN-1 (Molecular Probes, Eugene, Oreg.) is a fluorescent dye taken up by fungal cells; in the presence of metabolic viability it is converted from a diffuse yellow cytoplasmic stain to red, rod-like dye aggregates. Concanavalin A-alexa fluor 488 conjugate (CAAF; Molecular Probes) selectively binds to polysaccharides, including α-mannopyranosyl and α-glucopyranosyl residues, and gives a green fluorescence. Following biofilm formation and antifungal exposure, disks were removed and transferred to a new 12-well plate. Four microliters of FUN-1 (from a 10 mM stock) and 15 μl of CAAF (from a 5-mg/ml stock) were mixed into 3 ml of PBS to give final concentrations of 10 μM and 25 μg/ml, respectively. This mixture was added to wells containing biofilm disks. The plate was then incubated for 45 min at 37°C on a rocker table. Disks were removed from wells, placed in 35-mm glass-bottom microwell dishes (MatTek Corp., Ashland, Mass.), inverted, and observed using a Zeiss Axiovert 100 M confocal scanning laser microscope (using a rhodamine-fluorescein isothiocyanate protocol with excitation at 543 nm [HeNe laser] and 488 nm [argon laser], beam-splitting at 488/543 nm, and emission at 560 and 505 nm for FUN-1 [red] and CAAF [green], respectively). Lenses used included Zeiss Achroplan 20×/0.5 and C-Apochromat 40×/1.2 water immersion objectives. Images were captured and processed using Zeiss LSM510 v2.8 and Adobe Photoshop v5.5 (Adobe Systems, Inc., San Jose, Calif.) software.

For imaging of planktonic cells, a modified procedure was used. Cell suspensions were centrifuged as above and resuspended in 10 to 50 ml of medium with FUN-1 and CAAF in concentrations equivalent to those mentioned above. Several drops of stained cell suspension were placed in a cytospin apparatus and centrifuged for 8 min at 200 rpm in a Cytospin 3 centrifuge (Shandon Inc., Pittsburgh, Pa.) (15). Relevant fields on the prepared slides were then processed with Permount histological mounting medium (Fischer Scientific, Pittsburgh, Pa.) and glass coverslips were applied. Cells were imaged using CSLM as described above.

Statistical analysis.

Each experiment was performed in quadruplicate on at least two separate days; the data shown below in the Fig. 1 and 2 are from one representative experiment. Comparative results among different isolates were normalized to those for the C. albicans M61 strain, which by definition was considered to have 100% activity (29). Statistical analysis, including analysis of variance post hoc analysis using the Bonferroni-Dunn calculation (4), was performed using StatView v5.0.1 software (SAS Institute, Cary, N.C.). Using the Bonferroni-Dunn calculation, P values of <0.0083 were considered significant in Fig. 2, below (29).

Activities of conventional antifungals against C. albicans and C. parapsilosis biofilms.

As shown by our lab and others, mature Candida biofilms exhibit profound resistance to many antifungal agents (13, 21, 29). Antifungal susceptibilities of two strains each of C. albicans and C. parapsilosis were examined initially. As shown in Table 1, testing of conventional agents, including FLC, AMB, NYT, Chlor, and TRB, revealed resistance by all four isolates when grown as biofilms, compared to planktonic forms. While the two new triazoles (VRC and Ravu) were effective against planktonically growing C. albicans and C. parapsilosis, they failed to inhibit the same Candida strains after they were grown as biofilms (Table 1). Despite repeated attempts, we were unable to determine reproducible susceptibilities for TRB and Ravu against C. parapsilosis strain P/A71 biofilms, perhaps due to this strain's scant, disruptable biofilms (29).

Inhibitory activity of lipid formulations of polyene antifungals against Candida biofilms.

Lipid formulations of polyene antifungals have either recently come to market (liposomal AMB and ABLC) or are under development (lipid complex-NYT). Given issues of lipid complex size and the fact that standard formulations fail to demonstrate antibiofilm efficacy, lipid agents might not be expected to be effective in treating biofilm-associated Candida. However, as Table 1 shows, both liposomal AMB and ABLC exhibited inhibitory activities against C. albicans biofilms, with MICs similar to those seen for planktonic cells. C. parapsilosis strain P/A71 biofilms were susceptible to ABLC but had a slightly higher MIC₅₀ (1 rather than 0.25 μg/ml) for liposomal AMB. We were unable to determine reproducible MICs for biofilms formed by C. parapsilosis strain P92, perhaps due to this strain's heterogeneous biofilm formation (29). Although lipid complex-NYT showed potent activity against planktonically grown Candida, this antifungal failed to inhibit biofilms (Table 1). Lipid complex-NYT also thus acts as an internal control, indicating that lipid formulations per se are not the active antifungal agent (the lipid agents used in ABLC and lipid complex-NYT are the same, dimyristoyl phosphatidylcholine and -glycerol).

Inhibitory activity of echinocandins against Candida biofilms.

The new class of antifungals, echinocandins, is represented by Casp and Mica in this study. The class has demonstrated effectiveness against resistant Candida infections (9, 17), although none of the agents are yet approved by the Food and Drug Administration for such treatment. These agents have a novel mechanism of action, blocking the production of 1,3-β-d-glucan (3), a fundamental component of the fungal cell wall. As we had shown earlier, fungal cell wall polysaccharides are components of the Candida biofilm ECM (13, 29). Thus, we hypothesized that the echinocandins might have activity against Candida biofilms. As shown in Table 1, both Casp and Mica were, in fact, active against biofilm-associated C. albicans (both strains). Interestingly, both echinocandins were able to inhibit C. parapsilosis strain PA/71 while exhibiting high MICs for strain P92.

Figure 1 shows representative curves for the inhibition of biofilm-grown C. albicans (strain M61) in the presence of different concentrations of FLC, AMB, Casp, and ABLC. As can be seen, there is a dramatic difference in the susceptibility plots (as measured by the XTT method) between conventional agents (FLC and AMB) and the novel drugs (Casp and ABLC). Mica and liposomal AMB produced similar curves to the latter agents; effects were similar for agents effective with strains GDH, P/A71, and P92 (data not shown).

Effects of C. albicans preincubation with sub-MIC levels of antifungals on subsequent biofilm formation.

In an effort to determine if antifungal agents could affect the formation of mature biofilms, we exposed biofilm-precursor planktonic cells (C. albicans strain M61) to subinhibitory levels of antifungal agents. Planktonic cultures were grown in the presence of one-fourth MICs of antifungal agents and then used to form biofilms in the standard fashion. Cells grown in subinhibitory concentrations of AMB and Casp failed to form an adequate mass of planktonic cells to conduct subsequent experiments (data not shown). The results for VRC, NYT, and Chlor are shown in Fig. 2. Results have been normalized to the control, an untreated M61 strain (29). As can be seen, pretreatment of C. albicans with sub-MIC levels of any of the three antifungals caused a marked decrease in the ability of cells to form a biofilm, as measured by DW (P < 0.0005 for comparison of VRC and Chlor with untreated control cells). This effect was maximal with VRC. At the same time, there was an increase in relative XTT activity for all three treatment groups, although the difference was not statistically significant. These findings confirm our prior results of frequent XTT-DW discordance (29).

Confocal microscopic examination of antifungal effects on planktonic compared to biofilm-associated C. albicans cells.

In an effort to correlate our MIC results with Candida cellular and biofilm changes, we used CSLM to examine the effects of representative antifungal agents on both planktonic and biofilm-associated cells. Figure 3 shows the effects on planktonic C. albicans (strain M61) of Casp (Fig. 3B), ABLC (Fig. 3C), and VRC (Fig. 3D) compared with untreated control cells (Fig. 3A). Caspofungin-treated cells exhibit grossly distorted cell walls, with minimal cytoplasmic staining and no evidence of viability. ABLC-treated cells were small with diffusely yellow-staining cytoplasm, indicating lack of viability, and large vacuoles (the dark areas in the blastospores). VRC-treated cells also lacked viability. Cell wall architecture in these latter cells was distorted, but to a lesser degree than with Casp. Planktonic cells were exposed to antifungals for 24 and 48 h. Effects were the same for both groups (data not shown).

Figure 4 shows the effects of the same antifungals on C. albicans biofilms, specifically the basal blastospore layer (13, 29). As expected, a large number of control blastospores were still viable (Fig. 4A) (29). The most striking result was for the Casp-treated cells (Fig. 4B). Despite greatly reduced viability compared with controls (as expected from the MIC data above), there were minimal apparent distortions of cellular architecture. There were rare red FUN-1 aggregates noted. In concordance with the MIC results, ABLC-treated cells (Fig. 4C) displayed uniform nonviability, with shrunken cells and large vacuoles similar to planktonically treated cells. Also in agreement with the MIC results, VRC-treated biofilm cells (Fig. 4D) appeared minimally affected, although grossly there was an increase in the number of nonviable cells (by FUN-1 staining) and mild distortion in cell wall architecture with some apparent enlargement of blastospores compared to controls. Biofilms were grown for both 24 and 48 h, and both were exposed to antifungals for 24 and 48 h. Effects were the same for all four groups (data not shown). Due to destabilization of older (e.g., 72 to 92 h) biofilms by the CSLM dyes, we were unable to adequately visualize the entire biofilm matrix, including the hyphae that usually pervade this matrix.