Biofilm formation by the emerging fungal pathogen Trichosporon asahii: development, architecture, and antifungal resistance

Abstract

Trichosporon asahii is the most common cause of fatal disseminated trichosporonosis, frequently associated with indwelling medical devices. Despite the use of antifungal drugs to treat trichosporonosis, infection is often persistent and is associated with high mortality. This drove our interest in evaluating the capability of T. asahii to form a biofilm on biomaterial-representative polystyrene surfaces through the development and optimization of a reproducible T. asahii-associated biofilm model. Time course analyses of viable counts and a formazan salt reduction assay, as well as microscopy studies, revealed that biofilm formation by T. asahii occurred in an organized fashion through four distinct developmental phases: initial adherence of yeast cells (0 to 2 h), germination and microcolony formation (2 to 4 h), filamentation (4 to 6 h), and proliferation and maturation (24 to 72 h). Scanning electron microscopy and confocal scanning laser microscopy revealed that mature T. asahii biofilms (72-h) displayed a complex, heterogeneous three-dimensional structure, consisting of a dense network of metabolically active yeast cells and hyphal elements completely embedded within exopolymeric material. Antifungal susceptibility testing demonstrated a remarkable rise in the MICs of sessile T. asahii cells against clinically used amphotericin B, caspofungin, voriconazole, and fluconazole compared to their planktonic counterparts. In particular, T. asahii biofilms were up to 16,000 times more resistant to voriconazole, the most active agent against planktonic cells (MIC, 0.06 microg/ml). Our results suggest that the ability of T. asahii to form a biofilm may be a major factor in determining persistence of the infection in spite of in vitro susceptibility of clinical isolates.

FIG.1.

Optimization of Trichosporon asahii biofilm growth on polystyrene surface. Effect of adhesion time (30 [▪], 60 [▴], or 120 [▾] min), incubation time (24, 48, or 72 h), and inoculum concentration (A, B: 10⁴ CFU/ml; C, D: 10⁵ CFU/ml; E, F: 10⁶ CFU/ml) on T. asahii ATCC 201110 biofilm formation assessed by viable count (A, C, and E) and XTT (B, D, and F) assays. Values were plotted as means ± standard deviations (bars).

FIG. 2.

Kinetic of biofilm formation by T. asahii ATCC 201110 as determined by both XTT (optical density at 492 nm/optical density at 620 nm [OD_492/620]) and viable-count (CFU/plate) assays. Values were plotted as means ± standard deviations (bars). Linear regression analysis revealed a regression coefficient of 0.936 (P < 0.0001) between viable-count and XTT assay results.

FIG. 3.

(A to F) SEM images of T. asahii ATCC 201110 biofilm formed on a polystyrene surface after 30 min or 4, 8, 24, 48, or 72 h, respectively. (G) Magnification of F. Magnification, ×100 (A to F) or ×500 (G).

FIG. 4.

SEM images of mature (72-h-old) T. asahii TA309 biofilms on polystyrene. Biofilm cells stained without (A) or with (B) 0.1% alcian blue. Fixation for 22 h in aldehyde containing 0.1% alcian blue visualizes an extensive network of EPS filaments surrounding cells and bridging cell surfaces. Magnification, ×7,000.

FIG. 5.

CSLM examination of T. asahii TA311 biofilm with both FUN-1 and ConA stains after 72 h of development. (A) Orthogonal images of the basal layer show that mature biofilm consisted of mostly metabolically active (red, FUN1-stained) cells embedded in the polysaccharide extracellular material (green, ConA stained). (B to D) Three-dimensional representations of T. asahii biofilm: (B) top view; (C) basal layer, lateral view; (D) upper layer, lateral view. Image capture was set for simultaneous visualization of both green and red fluorescence (A, C, D) or for visualization of red fluorescence only (B). Magnification, ×100.