Adhesion and biofilm formation by two clinical isolates of Trichosporon Cutaneum in various environmental conditions.

Trichosporon spp. is an emerging opportunistic pathogen and a common cause of both superficial and invasive infections. Although Trichosporon asahii is the most frequently isolated species, Trichosporon cutaneum is also widely observed, as it is the predominant agent in cases of white Piedra and onychomycosis. Trichosporon spp. is a known to produce biofilms, which serve as one of its virulence mechanisms, however, there is limited data available on biofilms formed by T. cutaneum. Thus, the aim of this study was to assess the adhesion and biofilm formation of two clinical isolates of T. cutaneum under various environmental conditions (including temperature, nutrient availability, and carbon source), as well as their tolerance to fluconazole. Adhesion was tested on common abiotic substrates (such as silicone, glass, and stainless steel), revealing that T. cutaneum readily adhered to all surfaces tested. CV staining was applied for the evaluation of the environment influence on biofilm efficiency and it was proved that the nutrient availability has a major impact. Additionaly, fluorescent staining was employed to visualize the morphology of T. cutaneum biofilm and its survival in the presence of fluconazole. Hyphae production was shown to play a role in elevated biofilm production in minimal medium and increased tolerance to fluconazole.

Vancomycin enhances growth and virulence of Trichosporon spp. planktonic cells and biofilms.

Invasive fungal infections (IFIs) are important worldwide health problem, affecting the growing population of immunocompromised patients. Although the majority of IFIs are caused by Candida spp., other fungal species have been increasingly recognized as relevant opportunistic pathogens. Trichosporon spp. are members of skin and gut human microbiota. Since 1980's, invasive trichosporonosis has been considered a significant cause of fungemia in patients with hematological malignancies. As prolonged antibiotic therapy is an important risk factor for IFIs, the present study investigated if vancomycin enhances growth and virulence of Trichosporon. Vancomycin was tested against T. inkin (n = 6) and T. asahii (n = 6) clinical strains. Planktonic cells were evaluated for their metabolic activity and virulence against Caenorhabditis elegans. Biofilms were evaluated for metabolic activity, biomass production, amphotericin B tolerance, induction of persister cells, and ultrastructure. Vancomycin stimulated planktonic growth of Trichosporon spp., increased tolerance to AMB, and potentiates virulence against C. elegans. Vancomycin stimulated growth (metabolic activity and biomass) of Trichosporon spp. biofilms during all stages of development. The antibiotic increased the number of persister cells inside Trichosporon biofilms. These cells showed higher tolerance to AMB than persister cells from VAN-free biofilms. Microscopic analysis showed that VAN increased production of extracellular matrix and cells in T. inkin and T. asahii biofilms. These results suggest that antibiotic exposure may have a direct impact on the pathophysiology of opportunistic trichosporonosis in patients at risk. LAY ABSTRACT: This study showed that the vancomycin stimulated Trichosporon growth, induced morphological and physiological changes on their biofilms, and also enhanced their in vivo virulence. Although speculative, the stimulatory effect of vancomycin on fungal cells should be considered in a clinical scenario.

Inhibitory effect of a lipopeptide biosurfactant produced by Bacillus subtilis on planktonic and sessile cells of Trichosporon spp.

The present study aimed to investigate the inhibitory effect of a bacterial biosurfactant (TIM96) on clinical strains of Trichosporon. Additionally, the effect of TIM96 on the ergosterol content, cell membrane integrity, and the hydrophobicity of planktonic cells was assessed. The inhibitory activity of TIM96 against Trichosporon biofilms was evaluated by analyzing metabolic activity, biomass and morphology. MIC values ranged from 78.125 to 312.5 µg ml-1 for TIM96; time-kill curves revealed that the decline in the number of fungal cells started after incubation for 6 h with TIM96 at both MIC and 2×MIC. The biosurfactant reduced the cellular ergosterol content and altered the membrane permeability and the surface hydrophobicity of planktonic cells. Incubation at 10×MIC TIM96 reduced cell adhesion by up to 96.89%, thus interfering with biofilm formation. This concentration also caused up to a 99.2% reduction in the metabolic activity of mature biofilms. The results indicate potential perspectives for the development of new antifungal strategies.

Biofilm formation and antifungal susceptibility of Trichosporon asahii isolates from Mexican patients.

BACKGROUND: Trichosporon asahii is a yeast-like fungus that has recently gained importance as a cause of opportunistic systemic infections. The pathogenicity and virulence factors of T. asahii remain largely unknown. Because of the association between invasive infections and the use of catheters and related devices, the ability of the microorganism to adhere and form biofilms may play an important role in the pathogenicity during a trichosporonosis. AIMS: The aim of this study is to identify an association between biofilm formation by T. asahii isolates and their genotype and/or clinical source. METHODS: The biofilm production of 49 T. asahii strains isolated from Mexican patients was measured using the crystal violet stain method, and a comparison made with different adhesion phase incubation times. Antifungal susceptibility testing was performed using a modified CLSI protocol coupled with the quantification of the viable cells with the XTT reduction method. RESULTS: All the T. asahii isolates assayed were able to produce biofilm in vitro, with an intraspecific variability being observed. Overall, increased biofilm production was found when extending the adhesion phase incubation time from 2 to 4h. No association could be established between the biofilm-producing phenotype and either the genotype or clinical source. Higher antifungal resistance to amphotericin B and fluconazole was linked to increased biofilm production by T. asahii. CONCLUSIONS: All clinical isolates tested were able to produce biofilm. No association could be established between biofilm formation and genotype or clinical source.

Genotype, antifungal susceptibility, and biofilm formation of Trichosporon asahii isolated from the urine of hospitalized patients.

In this study, the culture analysis of urine samples from patients hospitalized in the Central-West region of Brazil was performed, and the isolated microorganisms were phylogenetically identified as Trichosporon asahii. Maximum parsimony analysis of the IGS1 sequences revealed three novel genotypes that have not been described. The minimum inhibitory concentrations of the nine isolates identified were in the range of 0.06-1 µg/ml for amphotericin B, 0.25-4 µg/ml for fluconazole, and 0.03-0.06 µg/ml for itraconazole. Approximately 6/9 of the T. asahii isolates could form biofilms on the surface of polystyrene microplates. This study reports that the microorganisms isolated here as T. asahii are agents of nosocomial urinary tract infections. Furthermore, the IGS1 region can be considered a new epidemiological tool for genotyping T. asahii isolates. The least common genotypes reported in this study can be related to local epidemiological trends.

Yeast microbiota of natural cavities of manatees (Trichechus inunguis and Trichechus manatus) in Brazil and its relevance for animal health and management in captivity.

The aim of this study was to characterize the yeast microbiota of natural cavities of manatees kept in captivity in Brazil. Sterile swabs from the oral cavity, nostrils, genital opening, and rectum of 50 Trichechus inunguis and 26 Trichechus manatus were collected. The samples were plated on Sabouraud agar with chloramphenicol and incubated at 25 °C for 5 days. The yeasts isolated were phenotypically identified by biochemical and micromorphological tests. Overall, 141 strains were isolated, of which 112 were from T. inunguis (Candida albicans, Candida parapsilosis sensu stricto, Candida orthopsilosis, Candida metapsilosis, Candida guilliermondii, Candida pelliculosa, Candida tropicalis, Candida glabrata, Candida famata, Candida krusei, Candida norvegensis, Candida ciferri, Trichosporon sp., Rhodotorula sp., Cryptococcus laurentii) and 29 were from T. manatus (C. albicans, C. tropicalis, C. famata, C. guilliermondii, C. krusei, Rhodotorula sp., Rhodotorula mucilaginosa, Rhodotorula minuta, Trichosporon sp.). This was the first systematic study to investigate the importance of yeasts as components of the microbiota of sirenians, demonstrating the presence of potentially pathogenic species, which highlights the importance of maintaining adequate artificial conditions for the health of captive manatees.

Trichosporon inkin biofilms produce extracellular proteases and exhibit resistance to antifungals.

The aim of this study was to determine experimental conditions for in vitro biofilm formation of clinical isolates of Trichosporon inkin, an important opportunistic pathogen in immunocompromised patients. Biofilms were formed in microtitre plates in three different media (RPMI, Sabouraud and CLED), with inocula of 104, 105 or 106 cells ml- 1, at pH 5.5 and 7.0, and at 35 and 28 °C, under static and shaking conditions for 72 h. Growth kinetics of biofilms were evaluated at 6, 24, 48 and 72 h. Biofilm milieu analysis were assessed by counting viable cells and quantification of nucleic acids released into biofilm supernatants. Biofilms were also analysed for proteolytic activity and antifungal resistance against amphotericin B, caspofungin, fluconazole, itraconazole and voriconazole. Finally, ultrastructural characterization of biofilms formed in microtitre plates and catheter disks was performed by scanning electron microscopy. Greater biofilm formation was observed with a starter inoculum of 106 cells ml- 1, at pH 7.0 at 35 °C and 80 r.p.m., in both RPMI and Sabouraud media. Growth kinetics showed an increase in both viable cells and biomass with increasing incubation time, with maximum production at 48 h. Biofilms were able to disperse viable cells and nucleic acids into the supernatant throughout the developmental cycle. T. inkin biofilms produced more protease than planktonic cells and showed high tolerance to amphotericin B, caspofungin and azole derivatives. Mature biofilms were formed by different morphotypes, such as blastoconidia, arthroconidia and hyphae, in a strain-specific manner. The present article details the multicellular lifestyle of T. inkin and provides perspectives for further research.

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

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.

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).

The biofilms formed by opportunistic yeasts serve as a persistent reservoir of infection and impair the treatment of fungal diseases. The aim of this study was to evaluate photodynamic inactivation (PDI) of biofilms formed by Candida spp. and the emerging pathogens Trichosporon mucoides and Kodamaea ohmeri by a cationic nanoemulsion of zinc 2,9,16,23-tetrakis(phenylthio)-29H,31H-phthalocyanine (ZnPc). Biofilms formed by yeasts after 48 h in the bottom of 96-well microtiter plates were treated with the photosensitizer (ZnPc) and a GaAlAs laser (26.3 J cm(-2)). The biofilm cells were scraped off the well wall, homogenized, and seeded onto Sabouraud dextrose agar plates that were then incubated at 37°C for 48 h. Efficient PDI of biofilms was verified by counting colony-forming units (CFU/ml), and the data were submitted to analysis of variance and the Tukey test (p < 0.05). All biofilms studied were susceptible to PDI with statistically significant differences. The strains of Candida genus were more resistant to PDI than emerging pathogens T. mucoides and K. ohmeri. A mean reduction of 0.45 log was achieved for Candida spp. biofilms, and a reduction of 0.85 and 0.84, were achieved for biofilms formed by T. mucoides and K. ohmeri, respectively. Therefore, PDI by treatment with nanostructured formulations cationic zinc 2,9,16,23- tetrakis (phenylthio)- 29H, 31H- phthalocyanine (ZnPc) and a laser reduced the number of cells in the biofilms formed by strains of C. albicans and non-Candida albicans as well the emerging pathogens T. mucoides and K. ohmeri.

Current knowledge of Trichosporon spp. and Trichosporonosis.

Trichosporon spp. are basidiomycetous yeast-like fungi found widely in nature. Clinical isolates are generally related to superficial infections. However, this fungus has been recognized as an opportunistic agent of invasive infections, mostly in cancer patients and those exposed to invasive medical procedures. It is possible that the ability of Trichosporon strains to form biofilms on implanted devices, the presence of glucuronoxylomannan in their cell walls, and the ability to produce proteases and lipases are all factors likely related to the virulence of this genus and therefore may account for the progress of invasive trichosporonosis. Disseminated trichosporonosis has been increasingly reported worldwide and represents a challenge for both diagnosis and species identification. Phenotypic identification methods are useful for Trichosporon sp. screening, but only molecular methods, such as IGS region sequencing, allow the complete identification of Trichosporon isolates at the species level. Methods for the diagnosis of invasive trichosporonosis include PCR-based methods, Luminex xMAP technology, and, more recently, proteomics. Treating patients with trichosporonosis remains a challenge because of limited data on the in vitro and in vivo activities of antifungal drugs against clinically relevant species of the genus. Despite the mentioned limitations, the use of antifungal regimens containing triazoles appears to be the best therapeutic approach.

Determinación de zearalenona por cromatografía líquida de alta resolución: normalización de un nuevo método para raciones especiales para cerdos / Zearalenone determination in high performance liquid chromatography: a new standardization method for commercial swine rations

The aim of this work was to offer a new method of high performance liquid chronomatography (HPLC) to evaluate commercial swine rations (CSR) contaminated by zearalenone (ZEA). After ZEA extraction and purification from CSR, the samples were eluted with acetonitrile, methanol and water solvent system. The results indicated that the proposed method showed to be rapid and efficient for the detection and quantification of ZEA in CSR, since its recovery was 102.62 percent, it offered excellent precision with a coefficient of variation of 0.9992. Furthermore, it is also proposed a as a biocontrol assay for micotoxigenic fungi isolated and maintained in the laboratory. The test was performed with the killer yeast Trichosporum insectorum CBS 10422 against Fusarium sp and Aspergillus flavus, which demonstrated to be effective against the latter.

El propósito de este articulo es ofrecer un nuevo método de cromatografía líquida de alta resolución (CLAR) para evaluar las raciones especiales para cerdos (REC) contaminado con zearalenona (ZEA). Después de la extracción y purificación de ZEA, las muestras se eluyeron con acetonitrilo, metanol y agua del sistema disolvente. Los resultados indican que el método propuesto demostró ser rápido y eficaz para la detección y cuantificación de ZEA en REC, ya que sus indicadores se presentan capaces de recuperación de 102,62 por ciento, además de ofrece una excelente precision, con un çõefiCiênte de variación de 0,9992. Por otra parte, también se propone una prueba de control biológico de hongos micotoxige-nic aislados y mantenidos en el laboratorio. La prueba se realizó con la levadura killer Trichosporum insectorum CBS 10422 contra Fusarium sp y Aspergillus flavus, mostrando eficaces sólo contra Aspergillus.

Trichosporon insectorum sp. nov., a new anamorphic basidiomycetous killer yeast.

Three killer yeasts, isolated from the gut of insects in Panama and artisanal cheese in Brazil, were shown to be related to the Ovoides clade of the genus Trichosporon. Sequencing of the D1/D2 region of the LSU rDNA and physiological characterization revealed a distinct taxonomic position in relation to known species of the genus. Conspecificity of the three killer isolates was reinforced by similar M13 fingerprinting and killer profiles. We propose a new species in this genus: Trichosporon insectorum. The type strain is CBS 10422(T) (syn. NRRL Y-48120). This anamorphic species produces arthroconidia but not appressoria, and its killer character seems to be associated with dsRNA.

Susceptibility patterns and molecular identification of Trichosporon species.

The physiological patterns, the sequence polymorphisms of the internal transcriber spacer (ITS), and intergenic spacer regions (IGS) of the rRNA genes and the antifungal susceptibility profile were evaluated for their ability to identify Trichosporon spp. and their specificity for the identification of 49 clinical isolates of Trichosporon spp. Morphological and biochemical methodologies were unable to differentiate among the Trichosporon species. ITS sequencing was also unable to differentiate several species. However, IGS1 sequencing unambiguously identified all Trichosporon isolates. Following the results of DNA-based identification, Trichosporon asahii was the species most frequently isolated from deep sites (15 of 25 strains; 60%). In the main, other Trichosporon species were recovered from cutaneous samples. The majority of T. asahii, T. faecale, and T. coremiiforme clinical isolates exhibited resistance in vitro to amphotericin B, with geometric mean (GM) MICs >4 mug/ml. The other species of Trichosporon did not show high MICs of amphotericin B, and GM MICs were <1 mug/ml. Azole agents were active in vitro against the majority of clinical strains. The most potent compound in vitro was voriconazole, with a GM MIC