Growth Inhibition Studies

The effects of the presence of Lactobacillus strains on the growth of C. albicans were determined in vitroon agar plates and in liquid cultures. The intestinal Lactobacillus johnsonii strain PV016 and the non-lactic acid bacterium Staphylococcus aureus ATCC 25923 were used as controls. Plate assays with deferred inoculation of C. albicans were performed on MRS or MRS-MOPS (MRS medium buffered with 0.165 M 3-morpholinopropane-1-sulfonic acid to pH 7.0) media. For the plate overlay assays, overnight cultures of the bacteria were diluted to an OD_{600 nm} of 1 and 4 μL of the dilution were spotted on MRS or MRS-MOPS agar plates. After incubation for 2 days at 37°C in a CO₂ incubator, plates were overlayed with a C. albicans suspension (OD_{600 nm} = 0.01) in MRS soft agar (0.7% agar) that was prepared after cooling the autoclaved agar medium to 45°C. Following solidification of the soft agar overlay, plates were incubated for one day at 37°C in normal aerobic atmosphere. Overlay plates were inspected for clear zones with no C. albicans growth surrounding bacterial colonies capable of inhibiting growth of the fungi. Disk diffusion assays were used to detect inhibition of C. albicans growth by lactic acid. Briefly, 20 μL of lactic acid dilutions were pipetted onto antibiotic assay disks (diameter 6 mm, Whatman, GE Health Care, Piscataway, NJ, USA) and the disks were placed on MRS or MRS-MOPS agar plates inoculated for dense growth of C. albicans. Clear zones around the disks after 24 hr incubation at 37°C indicated fungal growth inhibition by lactic acid.

2.3. Candida albicans Viability Assay

Viability of C. albicans cells during coculture with lactobacilli was qualitatively assessed using the FUN 1 viability indicator (LIVE/DEAD Yeast Viability Kit; Invitrogen, Carlsbad, CA, USA) and fluorescence microscopy. After a 24 hr incubation of C. albicans cells in MRS at 37°C in presence or absence (control) of lactobacilli, cells were washed with GH buffer (10 mM Na-HEPES buffer pH 7.2 containing 2% glucose). FUN 1 cell stain was diluted in the same buffer to a concentration of 5 μM and the solution was added to the cells. Following incubation for 30 min in the dark, a Biorad MRC-1024 confocal laser scanning microscope was used to determine the metabolic activity of the yeast cells. Excitation and emission filter sets for green and red fluorescence of FUN 1 were used according to the manufacturer's directions. Intact metabolically active C. albicans cells converted the green-fluorescent intracellular FUN 1 dye to red fluorescent intravacuolar structures. Cells with little or no metabolic activity but still intact membranes just show diffuse cytoplasmic green fluorescence and no red intravacuolar bodies. In contrast, diffuse and extremely bright green-yellow fluorescence indicates dead cells. For semiquantitative assessment of fungal viability, the FUN 1 viability assay was adapted for fluorometric measurement of metabolic activity according to the manufacturer's recommendations. Following overnight incubation ofC. albicans cells (starting OD_{600 nm} 0.1) in MRS with bacteria (starting OD_{600 nm} 0.05) or in the presence of lactic acid, the viability of C. albicans cells was determined by kinetic readings of red (excitation filter 485/20, emission filter 590/35) and green (excitation filter 485/20, emission filter 528/20) fluorescence in a BioTek Synergy 2 multimode microplate reader (BioTek, Winooski, VT, USA). For this purpose, aliquots from cultured cells were washed in GH buffer, adjusted to OD_{600 nm} 0.5 in 200 μL GH + 5 μM FUN 1 and subsequently incubated for 1 hr in the microplate reader at 30°C. Red and green fluorescence readings were taken every 5 minutes and red/green ratios were determined to monitor the viability of fungal cells. An increase in the red/green ratio over time indicated the presence of the metabolic activity required for formation of the red fluorescent intravacuolar structures. Bacteria did not convert the dye (data not shown).

Date: 2016-01-03; view: 742