Effect of Marine Polyunsaturated Fatty Acids on Biofilm Formation of Candida albicans and Candida dubliniensis

The effect of marine polyunsaturated fatty acids on biofilm formation by the human pathogens Candida albicans and Candida dubliniensis was investigated. It was found that stearidonic acid (18:4 n-3), eicosapentaenoic acid (20:5 n-3), docosapentaenoic acid (22:5 n-3) and docosahexaenoic acid (22:6 n-3) have an inhibitory effect on mitochondrial metabolism of both C. albicans and C. dubliniensis and that the production of biofilm biomass by C. dubliniensis was more susceptible to these fatty acids than C. albicans. Ultrastructural differences, which may be due to increased oxidative stress, were observed between treated and untreated cells of C. albicans and C. dubliniensis with formation of rough cell walls by both species and fibrillar structures in C. dubliniensis. These results indicate that marine polyunsaturated fatty acids may be useful in the treatment and/or prevention of biofilms formed by these pathogenic yeasts.


Introduction
Candida albicans and Candida dubliniensis are dimorphic yeasts, able to grow both as yeasts and mycelia. Several members of the genus Candida exist as commensals of the human gastrointestinal and OPEN ACCESS genitourinary tract in healthy individuals [1][2][3]. However, in individuals whose immune system is compromised, such as those that are HIV positive, C. albicans can cause diseases ranging from superficial infections to deep seated mycoses [4,5]. Candida dubliniensis is a species closely related to C. albicans and a causative agent of oropharyngeal candidiasis in immunocompromised humans [5,6]. Biofilm formation is a major virulence factor in the pathogenicity of Candida species, partly due to their increased resistance to antifungal treatment [7,8]. Since biofilm associated infections have many clinical and economic consequences, recent research into the pathogenicity of Candida species has focused on the prevention and management of these biofilms. Fatty acids have been known to have antibacterial [9] and antifungal properties and especially capric acid (10:0) and lauric acid (12:0) are known to have anti-Candida effects by inhibiting growth of planktonic cells [10] and butyric acid (4:0) was shown to inhibit hyphal formation by C. albicans [11].

Inhibition of biomass production
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 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.

Strains used
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.

XTT assay of biofilms
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 × 10 6 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.

Biomass determination
Twenty milliliters of a standardized cell suspension (1 × 10 6 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.

Morphological examination
The standardized cell suspension (1 × 10 6 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].

Conclusions
Certain marine PUFAs, especially 18:4 n-3, 20:5 n-3 and 22:5 n-3, have an inhibitory effect on the mitochondrial activity of both C. albicans and C. dubliniensis biofilms and significantly inhibited biofilm biomass of C. dubliniensis. These marine PUFAs also affected cellular morphology of biofilms of both species. This may be due to increased oxidative stress as a result of incorporation of PUFAs into the cellular lipids. These findings suggest that marine PUFAs may be useful in the treatment and/or prevention of Candida biofilms, which are known to have increased antifungal resistance compared to free-living cells.