Multiple species of Trichosporon produce biofilms highly resistant to triazoles and amphotericin B

Abstract

Invasive infections caused by Trichosporon spp. have increased considerably in recent years, especially in neutropenic and critically ill patients using catheters and antibiotics. The genus presents limited sensitivity to different antifungal agents, but triazoles are the first choice for treatment. Here, we investigated the biofilm production and antifungal susceptibility to triazoles and amphotericin B of 54 Trichosporon spp. isolates obtained from blood samples (19), urine (20) and superficial mycosis (15). All isolates and 7 reference strains were identified by sequence analysis and phylogenetic inferences of the IGS1 region of the rDNA. Biofilms were grown on 96-well plates and quantitation was performed using crystal violet staining, complemented with Scanning Electron Microscopy (SEM). Susceptibility tests for fluconazole, itraconazole, voriconazole and amphotericin B were processed using the microdilution broth method (CLSI) for planktonic cells and XTT reduction assay for biofilm-forming cells. Our results showed that T. asahii was the most frequent species identified (66.7%), followed by T. faecale (11.1%), T. asteroides (9.3%), T. inkin (7.4%), T. dermatis (3.7%) and one T. coremiiforme (1.8%). We identified 4 genotypes within T. asahii isolates (G1, G3, G4 and G5) and 2 genotypes within T. faecale (G1 and G3). All species exhibited high adhesion and biofilm formation capabilities, mainly T. inkin, T. asteroides and T. faecale. Microscopy images of high biofilm-producing isolates showed that T. asahii presented mainly hyphae and arthroconidia, whereas T. asteroides exhibited mainly short arthroconidia and few filaments. Voriconazole exhibited the best in vitro activity against all species tested. Biofilm-forming cells of isolates and reference strains were highly resistant to all antifungals tested. We concluded that levels of biofilm formation by Trichosporon spp. were similar or even greater than those described for the Candida genus. Biofilm-forming cells were at least 1,000 times more resistant to antifungals than planktonic cells, especially to voriconazole.

Figure 1. Median-joining genotypes network of T. asahii, T. faecale and T. coremiiforme based on IGS1 rDNA sequences related to biofilm quantitation.

Dashed square groups the 12 different genotypes (G1 to G12) of T. asahii. Dashed circle groups the 3 different genotypes of T. faecale. Circumference sizes are proportional to the genotype frequencies. Black dots (mv = median vectors) are hypothetical missing intermediates.

Figure 2. Inter and intra species variation in the biofilm production of 54 Trichosporon spp. clinical isolates and 7 reference strains.

A- 19 isolates from blood identified as T. asahii, T. asteroides, T. coremiiforme and T. dermatis. B- 20 isolates from urine identified as T. asahii. C- 15 isolates from superficial mycosis/skin colonization identified as T. asahii, T. dermatis, T. faecale and T. inkin. D- 7 reference strains from CBS obtained from different sources.

Figure 3. Scanning electron microscopy of 4 Trichosporon spp. strains grown on catheter surfaces.

A and B: Low biofilm producer T. asahii 05-001 (CV-A₅₇₀  =  0.287); C and D: Medium biofilm producer T. asahii 18-001 (CV-A₅₇₀  =  1.557); E and F: High biofilm producer T. asahii 07-001A (CV-A₅₇₀  =  3.337); and G and H: High biofilm producer T. asteroides 13-001 (CV-A₅₇₀  =  2.755).

Figure 4. Comparative analysis of MIC values obtained against planktonic cells of T. asahii (36 isolates) versus non-T. asahii (18) isolates for all antifungals tested.

A- Fluconazole; B- Itraconazole; C- Voriconazole and D- Amphotericin B.