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 g/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.Candida species are the most common cause of disseminated nosocomial fungal infections (34). Invasive infections by rarer opportunistic fungal pathogens, however, have recently emerged as a significant problem in treatment of immunocompromised patients (8, 34).In particular, disseminated life-threatening Trichosporon infection is becoming increasingly common in patients with underlying hematological malignancies, extensive burns, solid tumors, and AIDS, accounting for approximately 10% of all confirmed cases of disseminated fungal infections (8,12,14,(32)(33)(34). Likewise, nonimmunosuppressed patients have suffered from Trichosporon infections associated with ophthalmologic surgery, infections of prosthetic devices, intravenous drug abuse, and peritoneal dialysis (1, 17, 21).Trichosporon beigelii was formerly considered the causative agent in trichosporonosis, but recent taxonomic findings based on partial sequences of large-subunit rRNA and DNA relatedness (9, 29, 30) revealed that T. beigelii actually consists of six distinct pathogenic Trichosporon species.In particular, Trichosporon asahii is the major cause of disseminated or deep-seated trichosporonosis (12,30). Although most of the reported cases of hematogenous T. asahii infections occurred in patie...

Trichosporon asahii causing nosocomial urinary tract infections in intensive care unit patients: genotypes, virulence factors and antifungal susceptibility testing

“…Biofilm formation was assayed colorimetrically based on sodium 39-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) reduction as previously reported (Di Bonaventura et al, 2006). Briefly, T. asahii isolates grown overnight on Sabouraud dextrose agar plates at 35 uC were harvested, washed once with sterile PBS and then resuspended in RPMI 1640 (Sigma Aldrich; R8758) adjusted to pH 7.0 with 0.165 M MOPS (Sigma Aldrich; 1132-61-2) to a concentration of 10 5 c.f.u.…”

The development of animal infection models and antifungal efficacy assays against clinical isolates ofTrichosporon asahii,T. asteroidesandT. inkin

“…[13][14][15][16][17][18] In spite of the fact that Trichosporon spp are probably the second or third most common non-Candida yeast infections causing invasive disease in patients with hematological cancer, there are few reports related to virulence factors of this genus. 2,[19][20][21] The main causes of infection by invasive Trichosporon spp. are central and vesical venous catheters, and peritoneal catheter-related devices.…”

Photodynamic inactivation of biofilms formed by Candida spp., Trichosporon mucoides, and Kodamaea ohmeri by cationic nanoemulsion of zinc 2,9,16,23-tetrakis(phenylthio)-29H, 31H-phthalocyanine (ZnPc)

“…Once established, yeasts biofilms serve as persistent reservoirs of infection and, in addition, confer greater resistance to antifungal agents and protection against host immune defenses [7,14,34,35]. In this study, we evaluated the effects of photodynamic inactivation mediated by cationic nanoemulsion of zinc 2,9,16,23-tetrakis(phenylthio)-29H,31H-phthalocyanine on biofilms formed by Candida spp., T. mucoides, and K. ohmeri.…”

“…In the literature, there have been reports of reduced susceptibility to amphotericin B and antifungal activity of fluconazole; however, in vitro observations of fluconazole action do not have a strong correlation with good clinical outcomes [10,13]. Furthermore, Trichosporon is capable of forming complex biofilms similar to Candida with intrinsic resistance to the antifungals amphotericin B, caspofungin, fluconazole, and voriconazole [14].…”