Diutina (Candida) rugosa complex: The biofilm ultrastructure, extracellular matrix, cell wall component and antifungal susceptibility to amphotericin B, caspofungin, fluconazole and voriconazole

Abstract

The genus Candida is the most common etiological factor of opportunistic fungal infections in humans. The virulence of Candida species is due to a wide repertoire of factors, specifically, the ability to form biofilms. Medical devices such as intravenous catheters, prosthetic heart valves and surgical interventions provide pathogenic microorganisms with a surface to adhere to form biofilm. The objectives of this study were to investigate the biofilm ultrastructure of Diutina (Candida) rugosa (D. rugosa) at different developmental phases using Confocal scanning laser microscopy (CSLM) and scanning electron microscopy (SEM), quantify {beta}-glucan, total carbohydrate and total protein in the extracellular matrix (ECM) using enzymatic {beta}-glucan kit, phenol-sulfuric acid method and Bradfords method, respectively, and to identify Sessile Minimum Inhibition Concentrations (SMICs) of amphotericin B, caspofungin, fluconazole, and voriconazole using serial doubling dilution. From the SEM micrographs, D. rugosa biofilms were composed of adherent yeast cells and blastospores with hyphal elements. The ultrastructure of the yeast cells was collapsed and disfigured upon exposure to amphotericin B, fluconazole and voriconazole and the biofilms presented with punctured yeast morphology upon exposure to caspofungin at their respective SMICs. The matrix thickness of embedded yeast cells from CLSM micrographs was 3.9{micro}m at 48h. However, there was reduction in the thickness of the biofilms upon antifungal exposure. The antifungal exposed biofilms exhibit bright, diffuse, green-yellow fluorescence that were not seen in the control. D. rugosa biofilm matrices revealed 172.57{micro}g/mL of carbohydrate, and 27.11{micro}g/mL of protein content. The {beta}-glucan yield in D. rugosa complex planktonic cells were in the range of 2.5 to 4.38%, on the contrary, {beta}-glucan was not detected in the ECM. The SMICs of Diutina biofilm for amphotericin B is 1024g/mL, caspofungin is 512 g/mL, whereas fluconazole and voriconazole is 2048 g/mL, respectively.