Alginate oligosaccharides inhibit fungal cell growth and potentiate the activity of antifungals against Candida and Aspergillus spp

Abstract

The oligosaccharide OligoG, an alginate derived from seaweed, has been shown to have anti-bacterial and anti-biofilm properties and potentiates the activity of selected antibiotics against multi-drug resistant bacteria. The ability of OligoG to perturb fungal growth and potentiate conventional antifungal agents was evaluated using a range of pathogenic fungal strains. Candida (n = 11) and Aspergillus (n = 3) spp. were tested using germ tube assays, LIVE/DEAD staining, scanning electron microscopy (SEM), atomic force microscopy (AFM) and high-throughput minimum inhibition concentration assays (MICs). In general, the strains tested showed a significant dose-dependent reduction in cell growth at ≥6% OligoG as measured by optical density (OD600; P<0.05). OligoG (>0.5%) also showed a significant inhibitory effect on hyphal growth in germ tube assays, although strain-dependent variations in efficacy were observed (P<0.05). SEM and AFM both showed that OligoG (≥2%) markedly disrupted fungal biofilm formation, both alone, and in combination with fluconazole. Cell surface roughness was also significantly increased by the combination treatment (P<0.001). High-throughput robotic MIC screening demonstrated the potentiating effects of OligoG (2, 6, 10%) with nystatin, amphotericin B, fluconazole, miconazole, voriconazole or terbinafine with the test strains. Potentiating effects were observed for the Aspergillus strains with all six antifungal agents, with an up to 16-fold (nystatin) reduction in MIC. Similarly, all the Candida spp. showed potentiation with nystatin (up to 16-fold) and fluconazole (up to 8-fold). These findings demonstrate the antifungal properties of OligoG and suggest a potential role in the management of fungal infections and possible reduction of antifungal toxicity.

Figure 1. Effect of increasing concentrations of OligoG (0, 2, 6, and 10%) on cell densities.

Cultivation in Mueller-Hinton broth for 48 h at 34°C for various Candida and Aspergillus species. Error bars represent standard deviation from the mean (n≥4). (*, data not significantly different from control results; P>0.05).

Figure 2. Germ tube assays.

(A) Light microscopy images of Candida albicans (CCUG 39343) cells grown with/without the presence of OligoG, (Scale bar is 100 µm). (B) Percentage of Candida cells producing hyphae for four different strains grown for 2 hours in the presence of OligoG (0, 0.2, 0.5, 2, 6 and 10%). Candida glabrata as a non-hyphae producer was the negative control. *indicates significantly different from the control, (P<0.05).

Figure 3. LIVE/DEAD fluorescence imaging of Candida tropicalis 519468.

Biofilms were grown for 24 h at 37°C showing vital live cells (green) versus dead cells (red). (A) Untreated control. (B) 2% OligoG.

Figure 4. Scanning electron microscopy of Candida tropicalis 519468 (x 1.20 K) treated with 2% OligoG with/without fluconazole (FLC).

FLC was used at concentrations of 0.5, 1 and 2 µg/mL (equivalent to ‘below’, ‘at’ and ‘above’ the MIC value respectively). Scale bar is 40 µm.

Figure 5. AFM imaging of Candida tropicalis 519468 grown on polystyrene with/without 2% OligoG and/or fluconazole (FLC).

FLC was used at 1 mg/L (equivalent to the MIC) with more rounded cells (post OligoG treatment), more flattened cells (post fluconazole treatment) and both flattened, and “wrinkled” cells (post combination treatment) apparent. Z scale of 7.5 µm. Scale bar is 15 µm.