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

Iturrieta-González IA, Padovan AC, Bizerra FC, Hahn RC, Colombo AL - PLoS ONE (2014)

Bottom Line: Voriconazole exhibited the best in vitro activity against all species 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratório Especial de Micologia, Disciplina de Infectologia, Universidade Federal de São Paulo, São Paulo, SP, Brazil.

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.

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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.
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pone-0109553-g001: 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.

Mentions: Clinical isolates identified by IGS1 rDNA sequencing as T. asahii (36) and T. faecale (6) were subjected to phylogenetic analysis to confirm their identification and characterize their genotypes (Figure 1 and Figure S1). The network analysis (Figure 1) showed that 36 clinical isolates identified as T. asahii grouped into 4 different genotypes: 1, 3, 4 and 5. Trichosporon asahii G1 comprised 30 clinical isolates, the 2 reference strains CBS 2479 and CBS 7631, the G1 reference sequence (GenBank number: AB066386) and the supposedly G11 reference sequence (GenBank number: EU441160); T. asahii G3 consisted of 3 clinical isolates and the G3 reference sequence (GenBank number: AB066397); T. asahii G4 grouped 1 clinical isolate and the G4 reference sequence (GenBank number: AB180191); and T. asahii G5 included 2 clinical isolates and the G5 reference sequence (GenBank number: AB071387). The other reference sequences obtained from GenBank that did not grouped with our clinical isolates were: G2: AB072606, G6: AB180192, G7: AB180194, G8: AB439002, G9: AB439003, G10: EU441158, and G12: JF412789. Four of 6 T. faecale clinical isolates and the T. faecale CBS 4828 were classified as genotype 1 (G1 reference sequence number: AB066413). The isolates EB087B and EB108A had their molecular identification confirmed as T. faecale genotype 3 (G3 reference sequence number: AB439006). Trichosporon faecale G2 reference sequence (GenBank number: AB439004) did not group with any of the clinical isolates.


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

Iturrieta-González IA, Padovan AC, Bizerra FC, Hahn RC, Colombo AL - PLoS ONE (2014)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215839&req=5

pone-0109553-g001: 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.
Mentions: Clinical isolates identified by IGS1 rDNA sequencing as T. asahii (36) and T. faecale (6) were subjected to phylogenetic analysis to confirm their identification and characterize their genotypes (Figure 1 and Figure S1). The network analysis (Figure 1) showed that 36 clinical isolates identified as T. asahii grouped into 4 different genotypes: 1, 3, 4 and 5. Trichosporon asahii G1 comprised 30 clinical isolates, the 2 reference strains CBS 2479 and CBS 7631, the G1 reference sequence (GenBank number: AB066386) and the supposedly G11 reference sequence (GenBank number: EU441160); T. asahii G3 consisted of 3 clinical isolates and the G3 reference sequence (GenBank number: AB066397); T. asahii G4 grouped 1 clinical isolate and the G4 reference sequence (GenBank number: AB180191); and T. asahii G5 included 2 clinical isolates and the G5 reference sequence (GenBank number: AB071387). The other reference sequences obtained from GenBank that did not grouped with our clinical isolates were: G2: AB072606, G6: AB180192, G7: AB180194, G8: AB439002, G9: AB439003, G10: EU441158, and G12: JF412789. Four of 6 T. faecale clinical isolates and the T. faecale CBS 4828 were classified as genotype 1 (G1 reference sequence number: AB066413). The isolates EB087B and EB108A had their molecular identification confirmed as T. faecale genotype 3 (G3 reference sequence number: AB439006). Trichosporon faecale G2 reference sequence (GenBank number: AB439004) did not group with any of the clinical isolates.

Bottom Line: Voriconazole exhibited the best in vitro activity against all species 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratório Especial de Micologia, Disciplina de Infectologia, Universidade Federal de São Paulo, São Paulo, SP, Brazil.

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.

Show MeSH
Related in: MedlinePlus