As indicated in Figure 1 the mitochondrial metabolism of biofilms of C. albicans and C. dubliniensis, assessed by XTT assay, was significantly inhibited by 1 mM 18:4 n-3, 20:5 n-3 and 22:5 n-3. In addition, biofilms of C. albicans were also sensitive to 20:4 n-6, while biofilms of C. dubliniensis were not significantly inhibited. Interestingly, 22:6 n-3 did not significantly inhibit mitochondrial metabolism of C. albicans or C. dubliniensis biofilms. As a result 20:4 n-6 and 22:6 n-3 were not used in further experiments.

Although several authors use the XTT reduction assay as an indicator of biofilm biomass, Kuhn and co-workers [19] has cautioned against this approach. Therefore, the effect of the marine PUFAs on biofilm biomass production was determined by dry weight. As indicated in Figure 2, biofilm biomass production by C. dubliniensis was susceptible to the three tested PUFAs, with 18:4 n-3 and 20:5 n-3 resulting in a reduction of circa 82% and 71%, respectively. C. dubliniensis biofilm biomass was less susceptible to 22:5 n-3, which produced an inhibition of circa 19%. Similar results were obtained between the two species for the XTT reduction assay, however, the biofilm biomass of C. albicans was generally less susceptible to the tested PUFAs and a reduction of only circa 25% and 22% was seen for 18:4 n-3 and 22:5 n-3, respectively. Although a circa 16% reduction in C. albicans biofilm biomass was observed with 20:5 n-3, this was not statistically significant. These results may indicate the increased ability of the C. albicans strain to obtain energy through pathways that do not require mitochondrial metabolism.

Figure 3 depicts biofilms of C. albicans grown in the presence and absence of 1 mM 18:4 n-3, 20:5 n-3 and 22:5 n-3. In the absence of the PUFAs, the cell surface appeared smooth (Figure 3a) and when grown in the presence of PUFAs, cells had a rough appearance with protuberances (Figure 3b, c, d). Similar results were also observed for biofilms of C. dubliniensis when grown in the absence (Figure 4a), and the presence (Figure 4b, c, d), of the PUFAs with protuberances and fibrillar structures visible on the cell surface. Similar rough cell surfaces were observed when C. albicans was exposed to miconazole [20], which is known to cause an increase in reactive oxygen species in Candida biofilm cells [21]. Lemar et al. [22] also found that C. albicans cells were not smooth in the presence of alyl alcohol, which increases oxidative stress. Furthermore, in a study by Leeuw et al. [23] on the yeast Cryptococcus curvatus grown on oxidised lipids, protuberances were observed on the cell surface. We therefore speculate that the changes in cell surface in the presence of PUFAs might be due to increased lipid peroxidation and resultant oxidative stress. This will be studied in future.

Candida albicans CBS 562T and Candida dubliniensis NRRL Y-17841T were used in this study and were maintained on yeast malt extract (YM) agar plates (10 g/L glucose, 3 g/L yeast extract, 3 g/L malt extract, 5 g/L peptone, 16 g/L agar) at room temperature. The strains were stored on agar slants at 4 °C.

Cells of C. albicans and C. dubliniensis were grown separately on YM agar plates and incubated at 30 °C for 24 hours. After incubation, a loop-full of the cells was inoculated into 20 mL of yeast nitrogen base (YNB) glucose medium (10 g/L glucose, 6.7 g/L YNB) and incubated for 48 hours at 30 °C. Cells were washed three times with phosphate buffered saline (PBS) and diluted in RPMI-1640 medium (Sigma-Aldrich, UK) to an initial cell concentration of 1 × 10⁶ cells/mL. A volume of 100 μL of the standardized cell suspension was dispensed into a 96-well microtiter plate (Corning Incorporated, Costar^®, U.S.) and incubated for 1 hour at 37 °C to allow adherence of cells to the surface [24]. Wells were washed twice with PBS to remove non-adherent cells. Mature biofilms were formed at 37 °C for 47 hours in the presence and absence of 1 mM of the marine fatty acids (18:4 n-3, 20:4 n-3, 20:5 n-3, 22:5 n-3, 22:6 n-3) (Sigma-Aldrich, UK). The reduction of (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) (XTT) (Sigma Aldrich) was used to examine the yeast viability by measuring the mitochondrial metabolic activity of the biofilms, according to the method of Kuhn et al. [19]. XTT is converted to the diffusible, water soluble formazan that is colored and is easily measured in cellular supernatants in terms of optical density at 492 nm. This experiment was done in duplicate on two different days and four values obtained for each repetition (n = 8). The average and standard deviations were calculated and the student’s t-test was used to determine the significance of data sets.

Twenty milliliters of a standardized cell suspension (1 × 10⁶ cells/mL), prepared as before, was inoculated into Petri dishes containing RPMI-1640 medium and allowed to adhere to the surface for 1 hour at 37 °C. Non-adherent cells were removed with PBS and mature biofilms were formed in the presence of 1 mM of the marine fatty acids (18:4 n-3, 20:5 n-3, 22:5 n-3) for 47 hours at 37 °C. Untreated biofilms served as controls. Mature biofilms were washed to remove non-adherent cells, scraped off and resuspended in PBS. Cells were filtered on pre-weighed 0.2 μm cellulose acetate filters (Lasec, SA). The filters were dried to constant weight for 48 hours at 37 °C and the biomass determined. This experiment was done in duplicate and the mean and range calculated.

The standardized cell suspension (1 × 10⁶ cells/mL), prepared as before, was added to chamber slides (Lab-Tek^® Chamber Slide™ System 177372) containing silicone rubber disks (diam 5.5 mm) and 4 mL RPMI-1640 medium. Cells were allowed to adhere for 1 hour at 37 °C. Non-adherent cells were removed with PBS and mature biofilms were formed in the presence of 1 mM of the marine fatty acids (18:4 n-3, 20:5 n-3, 22:5 n-3) for 47 hours at 37 °C, with appropriate controls. The silicone rubber disks were removed and fixed for 2 hours using 3% (v/v; 1.0 M) sodium phosphate buffered glutardialdehyde, followed by fixing for 1 hour with a similarly buffered solution of osmium tetroxide (1% m/v). The disks were dehydrated in a graded series of ethanol solutions (50%, 70% and 95%) for 20 minutes and absolute ethanol for 1 hour. They were then critical-point dried, mounted and coated with gold to make them electrically conductive and finally visualized on a Shimadzu SSX550 SEM (Japan) microscope according to the method of van Wyk and Wingfield [25].