Biofilm Formed by Candida haemulonii Species Complex: Structural Analysis and Extracellular Matrix Composition

Abstract

Candida haemulonii species complex (C. haemulonii, C. duobushaemulonii, and C. haemulonii var. vulnera) has emerged as opportunistic, multidrug-resistant yeasts able to cause fungemia. Previously, we showed that C. haemulonii complex formed biofilm on polystyrene. Biofilm is a well-known virulence attribute of Candida spp. directly associated with drug resistance. In the present study, the architecture and the main extracellular matrix (ECM) components forming the biofilm over polystyrene were investigated in clinical isolates of the C. haemulonii complex. We also evaluated the ability of these fungi to form biofilm on catheters used in medical arena. The results revealed that all fungi formed biofilms on polystyrene after 48 h at 37 °C. Microscopic analyses demonstrated a dense network of yeasts forming the biofilm structure, with water channels and ECM. Regarding ECM, proteins and carbohydrates were the main components, followed by nucleic acids and sterols. Mature biofilms were also detected on late bladder (siliconized latex), nasoenteric (polyurethane), and nasogastric (polyvinyl chloride) catheters, with the biomasses being significantly greater than on polystyrene. Collectively, our results demonstrated the ability of the C. haemulonii species complex to form biofilm on different types of inert surfaces, which is an incontestable virulence attribute associated with devices-related candidemia in hospitalized individuals.

Susceptibility of the Candida haemulonii Complex to Echinocandins: Focus on Both Planktonic and Biofilm Life Styles and a Literature Review

“…The viability of the fungal cells forming the biofilm was determined using a colorimetric assay that measures the metabolic reduction of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT; Sigma-Aldrich) to a water-soluble brown formazan product [ 20 , 21 ]. A XTT/menadione solution was prepared as follows: 2 mg of XTT was dissolved in 10 mL of pre-warmed PBS solution supplemented with 100 μL of a menadione stock solution (made by dissolving 55 mg of menadione in 100 mL of acetone).…”

“…A XTT/menadione solution was prepared as follows: 2 mg of XTT was dissolved in 10 mL of pre-warmed PBS solution supplemented with 100 μL of a menadione stock solution (made by dissolving 55 mg of menadione in 100 mL of acetone). The XTT/menadione solution (200 μL) was added to all wells containing the biofilms (see Section 2.4 above) and incubated in the dark at 37 °C for 3 h. One hundred microliters of the supernatant from each well were then transferred to a new microplate and the colorimetric readings were measured at 492 nm using a microplate reader (SpectraMax M3; Molecular Devices, Sunnyvale, CA, USA) [ 21 ].…”

“…After discarding the crystal violet solution, the wells were washed once with PBS to remove excess stain and the biomass in each well was then decolorized by adding 200 μL of 33% acetic acid for 5 min. One hundred microliters of the acetic acid solution were transferred to a new 96-well plate and the absorbance measured at 590 nm using a microplate reader (SpectraMax M3; Molecular Devices) [ 21 ].…”

“…Biofilms were formed on a polystyrene surface and treated as described above with different concentrations of micafungin (0.5–2.0 mg/L). Then, the biofilms were stained with Calcofluor white (Sigma-Aldrich) solution (5 µg/mL) for 1 h at room temperature and protected from the light [ 21 , 22 , 23 ]. Subsequently, the biofilms were washed twice with PBS and covered with n -propyl-gallate for observation using a confocal microscope (Leica TCS SP5 with OBS, Berlin, Germany).…”

“…Subsequently, the biofilms were washed twice with PBS and covered with n -propyl-gallate for observation using a confocal microscope (Leica TCS SP5 with OBS, Berlin, Germany). Fiji ImageJ2 software (UW-Madison LOCI, Madison, WI, USA), was used to obtain three-dimensional (3-D) reconstitutions of the biofilms [ 21 , 24 ]. In this way, image analysis was performed using z -series image stacks from five randomly chosen spots on each biofilm [ 21 ].…”