Evaluation of Biofilm Formation in Candida tropicalis Using a Silicone-Based Platform with Synthetic Urine Medium

Abstract

Molecular mechanisms of biofilm formation in Candida tropicalis and current methods for biofilm analyses in this fungal pathogen are limited. (2) Methods: Biofilm biomass and crystal violet staining of the wild-type and each gene mutant strain of C. tropicalis were evaluated on silicone under synthetic urine culture conditions. (3) Results: Seven media were tested to compare the effects on biofilm growth with or without silicone. Results showed that biofilm cells of C. tropicalis were unable to form firm biofilms on the bottom of 12-well polystyrene plates. However, on a silicone-based platform, Roswell Park Memorial Institute 1640 (RPMI 1640), yeast nitrogen base (YNB) + 1% glucose, and synthetic urine media were able to induce strong biofilm growth. In particular, replacement of Spider medium with synthetic urine in the adherence step and the developmental stage is necessary to gain remarkably increased biofilms. Interestingly, unlike Candida albicans, the C. tropicalis ROB1 deletion strain but not the other five biofilm-associated mutants did not cause a significant reduction in biofilm formation, suggesting that the biofilm regulatory circuits of the two species are divergent. (4) Conclusions: This system for C. tropicalis biofilm analyses will become a useful tool to unveil the biofilm regulatory network in C. tropicalis.

Keywords: Candida tropicalis; ROB1; biofilms; synthetic urine.

Figure 1

Effects of different culture conditions on C. tropicalis biofilm formation on the bottom of 12-well polystyrene plates: (a) Representative images of biofilm formation of the C. tropicalis wild-type strain (MYA3404) on the bottom of 12-well polystyrene plates. Quantitative results showed that no medium induced cell adhesion and biofilm formation significantly compared to that of the Spider medium on the bottom of 12-well polystyrene plates. Values are the mean ± SD from six replicates. (b) C. tropicalis exhibited a significant increase in the slime index in synthetic urine medium. Values are the mean ± SD from six replicates. ** p < 0.01.

Figure 2

RPMI, YNB + 1% glucose, and synthetic urine (SU) media as adhesion conditions exhibited increased biofilm dry weights. The C. tropicalis wild-type strain MYA3404 was cultured under different culture conditions for biofilm analysis in 12-well tissue culture plates with silicone. (a) Representative images of biofilm formation of the C. tropicalis wild-type strain on silicone. (b) Quantitative analysis of the C. tropicalis wild-type strain in a biofilm assay on silicone squares: Values are the mean ± SD from five replicates. * p < 0.05; ** p < 0.01, and *** p < 0.001.

Figure 3

Quantitation of biofilms of C. albicans and C. tropicalis wild-type (WT) and efg1Δ strains in the Spider and SU biofilm formation conditions: (a) The use of SU medium resulted in increased C. tropicalis biofilm formation, whereas C. albicans showed a similar biomass when cultured with Spider medium and SU. Representative images of biofilm formation were shown below. (b) The extent of C. albicans and C. tropicalis biofilms were measured using crystal violet and resulted in similar conclusions in Figure 3a. Values are the mean ± SD from four replicates. ** p < 0.01. The C. albicans efg1Δ and C. tropicalis efg1Δ strains were used as negative controls.

Figure 4

Effects of the ingredients of SU on biofilm formation in C. tropicalis: (a) Depletion of MgCl₂ or KH₂PO₄ in SU significantly inhibited biofilm formation. (b) The addition of exogenous MgCl₂ in Spider medium caused very only slight effects on biofilm dry weights in both C. albicans and C. tropicalis. Values are the mean ± SD from five replicates. * p < 0.05; *** p < 0.001. (c) Growth curves of C. tropicalis strains at 30 °C in SU with or without additional MgCl₂ showed the requirement of magnesium for C. tropicalis proliferation. Growth rates were monitored every 2 h using a Biowave density meter.

Figure 5

Except the rob1Δ mutant strain, the lack of each gene in C. tropicalis caused a significant reduction in biofilms. Biofilms were determined by (a) biomass dry weight and (b) the OD₆₀₀ at the wavelength for crystal violet absorbance. Each experiment was repeated independently at least three times. (c) Reintroduction of each functional gene in each respective mutant strain except the ROB1 complementary strain was able to recover biofilm formation. Values are the mean ± SD from five replicates. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 6

Heterogeneous expression of C. tropicalis ROB1 in the C. albicans rob1Δ could not recover biofilm growth. Values are the mean ± SD from three replicates. Reintroduction of CtBCR1 and CtEFG1 into C. albicans bcr1Δ and efg1Δ strains, respectively, restored biofilm development in C. albicans, whereas Carob1Δ::CtROB1 exhibited few biofilms. *** p < 0.001.