Human serum promotes Candida albicans biofilm growth and virulence gene expression on silicone biomaterial.

OBJECTIVES: Systemic candidal infections are a common problem in hospitalized patients due to central venous catheters fabricated using silicone biomaterial (SB). We therefore evaluated the effect of human serum on C. albicans biofilm morphology, growth, and the expression of virulence-related genes on SB in vitro. METHODS: We cultivated C. albicans SC5314 (wild-type strain, WT) and its derivative HLC54 (hyphal mutant, HM) for 48 h in various conditions, including the presence or absence of SB discs, and human serum. The growth of planktonic and biofilm cells of both strains was monitored at three time points by a tetrazolium salt reduction assay and by scanning electron microscopy. We also analyzed by RT-PCR its expression of the virulence-related genes ALS3, HWP1, EAP1, ECE1, SAP1 - SAP10, PLB1, PLB2, PLC and PLD. RESULTS: At each time point, planktonic cells of WT strain cultured in yeast nitrogen base displayed a much higher expression of EAP1 and HWP1, and a moderately higher ALS3 expression, than HM cells. In planktonic cells, expression of the ten SAP genes was higher in the WT strain initially, but were highly expressed in the HM strain by 48 h. Biofilm growth of both strains on SB was promoted in the presence of human serum than in its absence. Significant upregulation of ALS3, HWP1, EAP1, ECE1, SAP1, SAP4, SAP6 - SAP10, PLB1, PLB2 and PLC was observed for WT biofilms grown on serum-treated SB discs for at least one time point, compared with biofilms on serum-free SB discs. CONCLUSIONS: Human serum stimulates C. albicans biofilm growth on SB discs and upregulates the expression of virulence genes, particularly adhesion genes ALS3 and HWP1, and hydrolase-encoding genes SAP, PLB1 and PLB2. This response is likely to promote the colonization of this versatile pathogen within the human host.

Genotypic, phenotypic, and proteomic characterization of Candida glabrata during sequential fluconazole exposure.

AIM: Candida glabrata is a major pathogen in humans known to be intrinsically resistant to fluconazole. However, genotypic, phenotypic, and proteomic changes associated with reduced susceptibility to fluconazole are not properly understood. The aim of this study was to observe specific phenotypic, chromosomal, and proteomic alterations in a Candida glabrata strain sequentially exposed to fluconazole. METHODS: Candida glabrata was exposed to increased concentrations of fluconazole in RPMI for 55 days. Phenotypic changes were evaluated using standard assays. Molecular/proteomic changes in C. glabrata were analyzed by contour-clamped homogeneous electric field electrophoresis, reverse transcription-polymerase chain reaction, and mass spectrometry. RESULTS: Candida glabrata demonstrated increased fluconazole resistance (>256 µg/mL), with extensive cross-resistance to ketoconazole (0.38-3.0 µg), itraconazole (8 to >32 µg), and voriconazole (0.125-1.5 µg). Morphologically dissimilar colonies on RPMI/fluconazole agar demonstrated variable chromosomal profiles compared with the control isolate. Stable chromosomal changes were associated with a significantly higher (P<0.05) mRNA level of the hemolysin gene compared with the control. Phenotypic switching on CuSO4 agar was associated with variable metallothionein mRNA transcription levels. The proteome analysis of a fluconazole-resistant offshoot demonstrated a total of 98 protein spots, 25 showing a twofold upregulation. CONCLUSION: Fluconazole exposure initiates the chance evolution of a new colonizing population with specific virulence traits.