Antifungal susceptibility test.

Caspofungin (GoldBio, St. Louis, MO) was dissolved in sterile Milli-Q water to a concentration of 1 mg/ml. The caspofungin stock solution was stored at −80°C in individual aliquots for one-time use. The CLSI M27-A2 (73) testing standard was slightly modified and used to test Candida planktonic caspofungin susceptibility. The cells were diluted to a concentration of 5.0 × 10² to 2.5 × 10³ cells/ml in YNB medium (50 mM glucose, pH 7). The cultures were allowed to grow for 48 h in test tubes at 37°C without agitation. The optical density at 600 nm (OD₆₀₀) of planktonic cultures was measured after 48 h. To test biofilm susceptibility, 96-well microtiter plates were coated with HI FBS and placed at 37°C overnight. The wells were washed once with PBS before inoculating with prepared cell cultures as mentioned above. The final volume in the wells was 100 μl. Biofilms were allowed to grow for 24 h at 37°C. After 24 h, the biofilms were washed twice with 200 μl PBS, and fresh YNB medium or YNB medium with caspofungin was added to the wells. The plates were placed back at 37°C for 24 h. After 48 h of total growth, the biofilms were washed twice with 200 μl PBS and processed with either the XTT assay or CLSM for biofilm cell counts.

The quantitation of Candida biofilms was performed as described previously (74, 75) using both a biochemical assay, the XTT reduction assay, and CFU measurements via confocal microscopy. XTT is reduced by mitochondrial dehydrogenase into a water-soluble formazan product that is measured spectrophotometrically. Briefly, XTT (Sigma Chemical Co. [St. Louis, MO] or Amresco) was dissolved in PBS to a final concentration of 0.5 mg/ml and filter sterilized. Menadione (Spectrum, NJ) was dissolved in acetone to a final concentration of 10 mM. XTT and menadione aliquots were stored at −80°C for future use. The menadione solution was added to the XTT solution for a final concentration of 1 μM. One hundred microliters of the XTT-menadione solution was added to the wells, and the plates were wrapped in foil and incubated at 37°C for 3 h. Eighty microliters of the supernatant was added to a new 96-well plate and measured at 490 nm (Tecan Infinite M200). The percent viability was calculated using the following equation: percent viability = 100 × (A₄₉₀ treated − A₄₉₀ background)/(A₄₉₀ untreated − A₄₉₀ background).

To count biofilm cells from the antifungal susceptibility test, cells were removed from the 96-well plates by the addition of 100 μl PBS and scraped with a pipette tip. A total of 3 technical replicates were collected and combined for one sample in a microcentrifuge tube. The samples were vortexed vigorously and 10 μl of cell suspension was added to a coverslip and imaged. Images were taken using a Leica TCS SP5 (Leica, Germany) using an HCX Plan-Apo 40×/0.85 dry objective with 488 nm and 543 nm laser lines with a 1.7× zoom. Ten images were taken per sample, and cell counts from a total of 3 biological replicates were counted using Image J software (70).