Abstract

Although biofilm-based fungal infections are an important cause of morbidity and mortality in patients, there is no standardized method for the in vitro evaluation of the drug susceptibility of biofilms. We investigated a high-throughput method for determining the susceptibility of Candida albicans biofilms that uses the oxidation reduction indicator Alamar blue (AB). Biofilms from the tested Candida albicans strains were markedly resistant to amphotericin B (AMB), nystatin (NYT), fluconazole (FLC) and 5-fluorouracil (5FC), but susceptible to Conflikt disinfectant. The latter was used in comparative studies of AB reduction with two other methods for assessing in vitro drug susceptibility i.e., 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction and enumeration of viable colony counts (CFU/ml). AB results correlated well with XTT (r=0.88–0.93) and CFU/ml (r=0.93–0.99) for all four C. albicans test strains. This simple, reproducible method for determining in vitro drug susceptibility should facilitate discovery of antifungals active against Candida biofilms.

Introduction

Biofilm formation is important in both superficial and systemic candidiasis. Currently, there is no Clinical Laboratory Standards Institute (CLSI) method for determining the drug susceptibility of Candida biofilms. Recent studies evaluating the drug susceptibility of Candida biofilms have used a variety of techniques including dry weight and/or wet weight measurements 1–3, crystal violet stain results 6–11.

XTT has been the method of choice because it depends on metabolic mitochondrial succinoxidases and cytochrome P450 systems for formazan production 12. However, there are drawbacks with the use of XTT such as our unpublished observations which suggest that the concentration of XTT greatly affects the correlation of formazan production with CFU/ml. Although the formazan product of XTT reduction is allegedly water soluble, we and others 13 have observed an orange residue on biofilms. Additionally, the electron coupling agent (menadione) used in XTT preparation is toxic to humans.

The use of Alamar blue (AB; Trek Diagnostic Systems), in comparison with these other methods of susceptibility testing, may have many potential advantages. For example, AB results can be read visually, spectrofluorometrically or spectrophotometrically, its reduction is dependent on active metabolism, it is amenable to high-throughput, its use is cost effective and AB lacks toxicity to humans and microorganisms. The AB assay was recently standardized for biofilms from multiple strains of Staphylococcus epidermidis, and had good to excellent correlation with XTT reduction and CFU/ml 14. In the current study, one Candida albicans ATCC isolate, one National Collection of Yeast Cultures isolate and two clinical isolates were used to determine the suitability of the AB assay for susceptibility testing of C. albicans biofilms.

Materials and methods

Candida albicans ATCC 14116, GDH 2346 (National Collection of Yeast Cultures) and clinical isolates Y1.914 and Y1.815 were maintained on Yeast Mold (YM) agar (Difco) at 35°C. To confirm biofilm formation and the presence of matrix material, biofilms from these isolates were prepared as in the AB biofilm susceptibility assay described below. Concanavalin A (ConA) was used to stain biofilm matrix as it binds to α-mannopyranosyl and α-glucopyranosyl residues 15 on cell surfaces and in the biofilm matrix. Wells were stained with ConA (25 µg/ml) and viewed with a confocal laser scanning microscope. Matrix materials was present for all strains (data not shown) and staining was conducted in duplicate on separate days.

The US Food and Drug Administration-approved antifungals amphotericin B (AMB) (Sigma), fluconazole (FLC) (ICN Biomedicals), 5-fluorouracil (5FC) (Aldrich), and nystatin (NYT) (Sigma) were hydrated according to the manufacturers’ instructions. AB was stored according to the manufacturers’ instructions. Prior to use, AB was thawed to room temperature and vortexed thoroughly. Due to the heat and light sensitivity of AB, exposure to these factors was minimized.

Planktonic assays were performed using the standard CLSI protocol 16, with results read at 48 h. After planktonic minimum inhibitory concentrations (CSLI MICs) were recorded, 5 µl AB was added to each well, the plates shaken gently for 10 sec in a Wallac 1420 Victor3 multi-label counter (PerkinElmer Systems), and incubated for 3 h at 35°C. The plates were then again shaken in the plate reader, and the lowest drug concentration with a blue well (AB MIC) recorded. Experiments were done in duplicate on separate days and the values reported represent the range of values observed.

The endpoint of AB reduction for C. albicans biofilms was determined as previously described 14, with modification. Briefly, 48 h biofilms were mock drug-treated with saline for 48 h, AB was then added and absorbance was recorded hourly for 12 h and then at 24 h and 48 h. Well color and absorbances remained stable from 3–48 h.

Modification of a previously published AB protocol was used for the biofilm susceptibility assays 14. Candida albicans colonies from 48 h YM plates were used to prepare inocula suspensions. Assays were performed in 96-well, flat-bottom, polystyrene, non-tissue-culture-treated plates (Falcon). In each well, 50 µl of Roswell Park Memorial Institute (RPMI) media was combined with 50 µl of 106 cells/ml saline inoculum solution. Plates were incubated at 35°C without shaking. Drug dilutions were prepared separately in 96-well 1 ml DeepWell microplates (VWR). After 48 h, all liquid was aspirated from biofilm wells and 50 µl of fresh RPMI and 50 µl of drug dilutions were added to each well. Biofilms were exposed to drugs for 48 h at 35°C without shaking. After the 48 h incubation, 5 µl of AB was added to each of the wells. Plates were shaken gently for 10 sec in a plate reader and incubated for 3 h at 35°C. Plates were then returned to the microplate reader, shaken gently for 10 sec and absorbances at 570 nm and 600 nm were obtained. Plates were kept in the incubator at 35°C between time points. There is no standard definition for minimum biofilm inhibitory concentration (MBIC). In our study, the AB MBIC was defined as the lowest drug concentration resulting in ≤50% reduction and a blue well 3 h after the addition of AB. Biofilm susceptibility experiments were performed in triplicate on separate days. Controls included media plus AB plus drug dilution and cells plus media plus AB.

The manufacturers’ formula for calculating percent reduction of AB includes the implicit assumption that all AB in the control is in the oxidized form. Because of this assumption, percent reduction is artificially inflated to values that may be significantly larger than 100%. This can be corrected by subtracting the background concentration measured in an uninoculated control: 

formula
% reduction is then given by, 
formula
 
formula
 
formula
Where

AB = Alamar blue, ɛox=molar extinction coefficient of Alamar Blue oxidized form (blue), ɛred=molar extinction coefficient of Alamar Blue reduced form (red), λ1=570 nm, λ2=600 nm, A = absorbance of test well, A1=absorbance of negative control (media + Alamar blue + experimental concentration of drug; no cells) 14, A0=absorbance of positive control (media + Alamar blue + cells; no drug).

The range of AB MBICs is shown (Table 1). We were unable to acquire lipid-based amphotericin B and echinocandins for this study. As all other drugs available to our laboratory failed to inhibit the biofilms, an alternate positive control, Conflikt (Decon) detergent, a quaternary ammonium based disinfectant that exhibits antibacterial, antiviral and antifungal activities, was used for correlation studies 17.

Table 1

Alamar blue MICs and MBICs of approved drugs and Conflikt® against planktonic and biofilm-grown Candida albicans strains.

 AMB 5FC FLC NYT CONFLIKT® 
C. albicans strain AB MIC* (µg/ml) AB MBIC** (µg/ml) AB MIC (µg/ml) AB MBIC (µg/ml) AB MIC (µg/ml) AB MBIC (µg/ml) AB MIC (µg/ml) AB MBIC (µg/ml) AB MIC (µg/ml) AB MBIC (µg/ml) 
ATCC 10231 <0.5 >512 <0.5 >512 64 >1024 >512 <0.02 0.625 
Y1.914 >512 <0.5 >512 >64 >1024 >512 <0.02 0.625 
Y1.815 <0.5 >512 >512 >64 >1024 >512 <0.02 0.625 
GDH 2346 >512 <0.5 >512 64 >1024 >512 <0.02 0.625 
 AMB 5FC FLC NYT CONFLIKT® 
C. albicans strain AB MIC* (µg/ml) AB MBIC** (µg/ml) AB MIC (µg/ml) AB MBIC (µg/ml) AB MIC (µg/ml) AB MBIC (µg/ml) AB MIC (µg/ml) AB MBIC (µg/ml) AB MIC (µg/ml) AB MBIC (µg/ml) 
ATCC 10231 <0.5 >512 <0.5 >512 64 >1024 >512 <0.02 0.625 
Y1.914 >512 <0.5 >512 >64 >1024 >512 <0.02 0.625 
Y1.815 <0.5 >512 >512 >64 >1024 >512 <0.02 0.625 
GDH 2346 >512 <0.5 >512 64 >1024 >512 <0.02 0.625 

AMB, Amphotericin B; 5FC, 5-fluorouracil; FLC, Fluconazole; NYT, Nystatin; *AB MIC, Alamar blue minimum inhibitory concentration; **AB MBIC, Alamar blue minimum biofilm inhibitory concentration.

The XTT susceptibility assay, with modification, was employed as previously described 2–4. Biofilms were grown and treated with Conflikt as in the AB susceptibility assay. To each well, 50 µl XTT solution (1 mg/ml in phosphate-buffered saline [PBS]) and 4 µl of menadione solution (1 mM in acetone) were added and gently shaken in a plate reader. Plates were incubated at 35°C for 3 h. Controls included media alone, media plus XTT/menadione, media plus XTT/menadione plus a drug concentration equal to each experimental well (negative control), and cells plus media plus XTT/menadione (positive control) 14. Percent formazan produced was calculated as previously described 14.

The biofilm CFU/ml assay was a modification of that described by Pettit et al. 14. Briefly, wells containing biofilms from the AB susceptibility assay were thoroughly scraped, cells were washed 3 times, diluted in saline blanks and inoculated onto warm YM plates. Colonies were counted after 48 h of incubation at 35°C. In controls, exposure to AB alone for 48 h caused no alteration in CFU/ml counts (data not shown). Growth control wells at 48 h were washed and the cells plated in the same manner as the drug-treated biofilms. The 48 h CFU/ml untreated growth control counts were similar to the CFU/ml counts from the lowest Conflikt concentration wells (Fig. 1). In control experiments, the presence of hyphae in biofilms did not noticeably impact CFU/ml determination relative to planktonic-grown cells.

Fig. 1

Percent reduction of Alamar blue, percent formazan production, and CFU/ml (right y axis) for biofilms from Candida albicans strains ATCC 10231, Y1.914, Y1.815 and GDH 2346 (A–D) treated for 48 h with twofold dilutions of Conflikt®. Arrow indicates biofilm CFU/ml at 48 h (prior to drug treatment).

Fig. 1

Percent reduction of Alamar blue, percent formazan production, and CFU/ml (right y axis) for biofilms from Candida albicans strains ATCC 10231, Y1.914, Y1.815 and GDH 2346 (A–D) treated for 48 h with twofold dilutions of Conflikt®. Arrow indicates biofilm CFU/ml at 48 h (prior to drug treatment).

Correlation of AB reduction to XTT reduction and CFU/ml was completed with a Pearson correlation. Pearson's two-tailed correlations were calculated with Prism 4 software using averaged data from the entire range of drug concentrations.

Results

Planktonic AB MICs and AB MBICs were obtained for four strains of C. albicans (Table 1). Results confirmed that Candida biofilms are highly resistant to antifungal drugs. AB MICs were either identical or within one 2-fold dilution of the CLSI MIC (data not shown). AB MBICs increased dramatically relative to AB MICs (Table 1). Susceptibility testing was not performed at doses higher than 1,024 µg/ml due to drug precipitation.

To determine if a correlation existed between results from the AB assay, the XTT assay and CFU/ml counts, the methods were performed in parallel. Conflikt was used since no approved antifungals available to our lab visibly inhibited the Candida biofilms. Conflikt was found to be effective at low concentrations against Candida biofilms (Table 1). The AB MBIC for strain ATCC 10231, for example, was ≥1,024 µg/ml for FLC, but was only 0.625 µg/ml for Conflikt (Table 1).

Preformed C. albicans biofilms were treated with Conflikt for 48 h at 35°C. AB or XTT solution were then added, and after 3 h absorbances were recorded. AB wells were then scraped and cell dilutions plated to determine CFU/ml counts (Fig. 1). There appeared to be a correlation between the results obtained with AB and XTT, as well as between AB and CFU/ml data. These correlations were confirmed by Pearson's two-tailed correlation coefficients as calculated from the data in Fig. 1. For strains ATCC 10231, Y1.914, Y1.815 and GDH 2346, correlation of the AB biofilm assay with that of the XTT biofilm assay was good to excellent, with r=0.93, 0.98, 0.88 and 0.91, respectively. For strains ATCC 10231, Y1.914, Y1.815 and GDH 2346, correlation of the AB biofilm assay as compared to CFU/ml was also good to excellent, with r=0.99, 0.98, 0.93 and 0.98, respectively.

Discussion

The increased drug resistance of biofilm vs. planktonic C. albicans described here is consistent with previous studies 18–20. We found that the AB susceptibility assay is reproducible and correlates well with both XTT and CFU/ml results for biofilms of multiple C. albicans isolates. While AB and XTT reduction both depend on metabolically active cells, AB poses no known safety issues. The AB method is considerably easier and less costly than CFU/ml assays, and is very amenable to high-throughput. The disadvantages of AB are light and heat sensitivity, but these are very minor problems under normal laboratory conditions. An additional benefit of AB is that results can be measured with fluorescence for increased sensitivity. AB has been evaluated for susceptibility testing of S. epidermidis and C. albicans biofilms, and should now be applied to other medically relevant microorganisms. A rapid, standardized drug susceptibility assay for fungal biofilms is critical to the discovery of biofilm-specific agents.

Acknowledgements

We thank K. Hazen (Department of Pathology, University of Virginia Health System) for supplying all clinical isolates. This research was supported by the Arizona Disease Control Research Commission, the Robert B. Dalton Endowment Fund and Dr Alec Keith.

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