Mitigation of Fe(0) nanoparticles toxicity to Trichosporon cutaneum by humic substances.

Zero-valent iron nanoparticles (nZVI) are a relatively new option for the treatment of contaminated soil and groundwater. However, because of their apparent toxicity, nZVI in high concentrations are known to interfere with many autochthonous microorganisms and, thus, impact their participation in the remediation process. The effect of two commercially available nZVI products, Nanofer 25 (non-stabilized) and Nanofer 25S (stabilized), was examined. Considerable toxicity to the soil yeast Trichosporon cutaneum was observed. Two chemically different humic substances (HSs) were studied as a possible protection agent that mitigates nZVI toxicity: oxidized oxyhumolite X6 and humic acid X3A. The effect of addition of HSs was studied in different phases of the experiment to establish the effect on cells and nZVI. SEM and TEM images revealed an ability of both types of nZVI and HSs to adsorb on surface of the cells. Changes in cell surface properties were also observed by zeta potential measurements. Our results indicate that HSs can act as an electrosteric barrier, which hinders mutual interaction between nZVI and treated cell. Thus, the application of HS seems to be a promising solution to mitigating the toxic action of nZVI.

The antifungal effect of silver nanoparticles on Trichosporon asahii.

BACKGROUND/PURPOSE: Silver nanoparticles are receiving increasing attention in biomedical applications. This study aims at evaluating the antifungal properties of silver nanoparticles against the pathogenic fungus Trichosporon asahii. METHODS: The growth of T. asahii on potato dextrose agar medium containing different concentrations of silver nanoparticles was examined and the antifungal effect was evaluated using minimum inhibitory concentration. Scanning and transmission electron microscopy were also used to investigate the antifungal effect of silver nanoparticles on T. asahii. RESULTS: Silver nanoparticles had a significant inhibitory effect on the growth of T. asahii. The minimum inhibitory concentration of silver nanoparticles against T. asahii was 0.5 µg/mL, which was lower than amphotericin B, 5-flucytosine, caspofungin, terbinafine, fluconazole, and itraconazole and higher than voriconazole. Silver nanoparticles obviously damaged the cell wall, cell membrane, mitochondria, chromatin, and ribosome. CONCLUSION: Our results demonstrate that silver nanoparticles have good antifungal activity against T. asahii. Based on our electron microscopy observations, silver nanoparticles may inhibit the growth of T. asahii by permeating the fungal cell and damaging the cell wall and cellular components.

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

The present study developed Galleria mellonella and murine infection models for the study of Trichosporon infections. The utility of the developed animal models was demonstrated through the assessment of virulence and antifungal efficacy for 7 clinical isolates of Trichosporon asahii, T. asteroides and T. inkin. The susceptibility of the Trichosporon isolates to several common antifungal drugs was tested in vitro using the broth microdilution and the E-test methods. The E-test method depicted a lower minimal inhibitory concentration (MIC) for amphotericin and a slightly higher MIC for caspofungin, while MICs observed for the azoles were different but comparable between both methods. All three Trichosporon species established infection in both the G. mellonella and immunosuppressed murine models. Species and strain dependent differences were observed in both the G. mellonella and murine models. T. asahii was demonstrated to be more virulent than the other 2 species in both animal hosts. Significant differences in virulence were observed between strains for T. asteroides in the murine model. In both animal models, fluconazole and voriconazole were able to improve the survival of the animals compared to the untreated control groups infected with any of the 3 Trichosporon species. In G. mellonella, amphotericin was not able to reduce mortality in any of the 3 species. In contrast, amphotericin was able to reduce murine mortality in the T. asahii or T. inkin models, respectively. Hence, the developed animal infection models can be directly applicable to the future deeper investigation of the molecular determinants of Trichosporon virulence and antifungal resistance.

Sweetpotato vines hydrolysate promotes single cell oils production of Trichosporon fermentans in high-density molasses fermentation.

This study investigated the co-fermentation of molasses and sweetpotato vine hydrolysate (SVH) by Trichosporon fermentans. T. fermentans showed low lipid accumulation on pure molasses; however, its lipid content increased by 35% when 10% SVH was added. The strong influence of SVH on lipid production was further demonstrated by the result of sensitivity analysis on effects of factors based on an artificial neural network model because the relative importance value of SVH dosage for lipid production was only lower than that of fermentation time. Scanning electron microscope observation and flow cytometry of yeast cells grown in culture with and without SVH showed that less deformation cells were involved in the culture with SVH. The activity of malic enzyme, which plays a key role in fatty acid synthesis, increased from 2.4U/mg to 3.7U/mg after SVH added. All results indicated SVH is a good supplement for lipid fermentation on molasses.

Differential diagnosis of trichosporonosis using conventional histopathological stains and electron microscopy.

AIMS: Although Trichosporon is a causative pathogen of white piedra and summer-type hypersensitivity pneumonitis, fatal disseminated trichosporonosis cases have recently been increasing. However, Trichosporon is often confused with other fungi, especially Candida, in pathological specimens. The aim was to determine the utility of histopathological stains and electron microscopy for diagnosing trichosporonosis. METHODS AND RESULTS: Autopsy cases of trichosporonosis, candidiasis, aspergillosis and cryptococcosis were investigated using histopathological stains and electron microscopy. Using Grocott's method, Trichosporon was weakly detected compared with other fungi. In contrast, diluted periodic acid methenamine silver (PAM) stain clearly enhanced the intensity of staining of Trichosporon compared with Candida. Furthermore, Alcian blue and colloidal iron stains predominantly detected Trichosporon. Electron microscopy after staining with diluted PAM demonstrated that Trichosporon has a variety of hyphal sizes and laminar deposition of rough silver granules, whereas Candida has uniform pseudohyphae and fine granules. The average diameter and population area of the granules were significantly higher in Trichosporon compared with Candida (P<0.01). Meanwhile, the laminar structure was preserved in the cell walls of Trichosporon without silver stains, whereas a low-density structure was observed in Candida. CONCLUSIONS: Histopathological staining patterns and electron microscopic findings can facilitate the diagnosis of trichosporonosis.

Uncommon characteristics of the structure and development of Trichosporon asahii.

BACKGROUND: Trichosporon asahii (T. asahii) is one of the most important pathogenic fungus in the genus of trichosporon. Although the species identification of T. asahii was based upon the complicated results of morphologic, biochemical and biologic examination, the morphology characteristic is still the first clue to the species. Some common structures of T. asahii had been described such as arthrofilaments and arthroconidia, but other important structures of T. asahii were unclear. METHODS: Six strains of T. asahii were incubated on the slant and micro culture of Sabouraud's dextrose agar at 30 degrees C for 7 days. Samples were fixed using 2% paraformaldehyde and 2.5% glutaraldehyde. T. asahii was observed under scanning electron microscope and transmission electron microscope. RESULTS: The detailed characteristics of the diverse sites of germination, as well as some uncommon structures such as giant cell, sarcinate, and club-shaped macroconidia, were presented. The pseudohyphae of T. asahii were noted to produce true hyphae, either along the longitude axis or on the flank. T. asahii was noted to have blastic and thallic conidiation. Digitated branches, trichoid structures and septa inside the spores were detected. CONCLUSION: These results may add our knowledge to the structure and development of T. asahii.

An outbreak of scalp white piedra in a Brazilian children day care.

White piedra is a superficial mycosis caused by Trichosporon spp. that affects the hair shaft of any part of the body. It is presented an outbreak of scalp white piedra seen in 5.8% of the children frequenting a day care in Northeastern of São Paulo State, Brazil. Mycological exam and culture identified T. cutaneum in all five cases, and scanning electron microscopy of nodules around hair shaft infected by Trichosporon spp. is demonstrated comparing them with those of black piedra and with nits of Pediculous capitis.

An outbreak of scalp white piedra in a Brazilian children day care / Surto de piedra branca afetando os cabelos do couro cabeludo de crianças em creche brasileira

White piedra is a superficial mycosis caused by Trichosporon spp. that affects the hair shaft of any part of the body. It is presented an outbreak of scalp white piedra seen in 5.8 percent of the children frequenting a day care in Northeastern of São Paulo State, Brazil. Mycological exam and culture identified T. cutaneum in all five cases, and scanning electron microscopy of nodules around hair shaft infected by Trichosporon spp. is demonstrated comparing them with those of black piedra and with nits of Pediculous capitis.

Piedra branca caracteriza-se por ser micose superficial, causada por Trichosporon spp., que compromete a haste dos pelos de qualquer região do corpo. Um surto de piedra branca, afetando os cabelos do couro cabeludo, foi registrado em 5,8 por cento das crianças que freqüentavam uma creche na região nordeste do estado de São Paulo. Exame micológico direto e cultura identificaram T. cutaneum nas cinco crianças afetadas. Enfatiza-se a utilização da microscopia eletrônica de varredura, que mostrou nódulos circundando a haste dos cabelos infectada por Trichosporon spp., comparando-os com nódulos de Piedra nigra e com lêndeas de Pediculus capitis.

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

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.

Microbiological characteristics of medically important Trichosporon species.

Trichosporon species are opportunistic pathogens associated with a high mortality rate in immunocompromised patients. Disseminated trichosporonosis is uncommon but reports are increasing. In this study, using 16 stock clinical isolates of suspected Trichosporon species and 4 known Trichosporon strains, we investigated the morphology, physio-biochemistry, molecular biology and antifungal susceptibility characteristics of these Trichosporon spp. and discovered that ITS sequence-based identification is a rapid and accurate identification alternative to most phenotypic or physio- biochemical methods. In vitro antifungal susceptibility tests showed high amphotericin B, itraconazole and terbinafine MIC value in these Trichosporon strains.

Fungicidal effect of indolicidin and its interaction with phospholipid membranes.

The fungicidal effect and mechanism of a tryptophan-rich 13-mer peptide, indolicidin derived from granules of bovine neutrophils, were investigated. Indolicidin displayed a strong fungicidal activity against various fungi. In order to understand the fungicidal mechanism(s) of indolicidin, we examined the interaction of indolicidin with the pathogenic fungus Trichosporon beigelii. Fluorescence confocal microscopy and flow cytometry analysis revealed that indolicidin acted rapidly on the plasma membrane of the fungal cells in an energy-independent manner. This interaction is also dependent on the ionic environment. Furthermore, indolicidin caused significant morphological changes when tested for the membrane disrupting activity using liposomes (phosphatidylcholine/cholesterol; 10:1, w/w). The results suggest that indolicidin may exert its fungicidal activity by disrupting the structure of cell membranes, via direct interaction with the lipid bilayers, in a salt-dependent and energy-independent manner.

Structural differences between two types of basidiomycete septal pore caps.

The septal pore cap (SPC) of Trichosporon sporotrichoides CBS 8245 is vesicular-tubular, connected with flat-tubular endoplasmic reticulum (ER), and stains densely with zinc/iodine/osmium tetroxide, as does the ER. The SPC of Schizophyllum commune CBS 340.81 is more complex, about 600 nm in diameter, with perforations of 80-120 nm diameter, and stains less densely with zinc/iodine/osmium tetroxide than the ER. In high-pressure frozen and freeze-substituted hyphae of T. sporotrichoides the ER is present parallel to the dolipore septa, and electron-dense material occurs opposite the septal pore channel; the SPC rarely showed smooth vesicular-tubular membranes, suggesting that this is an ephemeral function of the SPC. The SPC of S. commune has a smooth outer and inner membrane, which enclose a matrix with a palisade-like substructure. A thin layer of electron-dense material covers the inner surface of the SPC of S. commune, from which beaded filamentous structures connect the SPC and the pore-occluding material. These filamentous structures may maintain the intracellular position of the SPC and possibly play a role in plugging the septal pore channel. The septal pore swellings of T. sporotrichoides contain more 1,6-beta-glucan than the septum, and intracellular glucans are also present near the septal pore channel. This cytosolic 1,6-beta-glucan in T. sporotrichoides may serve as a matrix to keep the tubular membranous structures of the SPC together. In contrast, 1,6-beta-glucan is not observed in the SPC and in the pore-occluding material of S. commune, and hyphal septa of this species show less labelling of 1,6-beta-glucan than the septal swelling. The evolutionary transition from simple to more complex types of SPCs may have resulted in a requirement for different components to maintain the morphological integrity and cell biological function.

[Fungi of the Trichosporon genus. Identification, epidemiology and significance of dermatologic disease pictures]. / Hefen der Gattung Trichosporon. Identifizierung, Epidemiologie und Bedeutung der dermatologischen Krankheitsbilder.

Based on molecular data, the genus Trichosporon was recently reclassified. Six human pathogenic species, which are closely related to specific types of infections can be differentiated. By means of commercial test systems and simple key criteria according to Gueho et al. [15], 44 isolates that had been identified as Tr. cutaneum between 7/92 and 1/96, were reclassified. The evaluation of clinical data also included 66 isolates that had not been preserved, and 27 strains of Tr. inkin. Trichosporon species were found in 4.8% of all yeast isolates (3.8% Tr. cutaneum, 1% Tr. inkin). Nearly every other isolate of Tr. cutaneum was cultivated from nail material, while Tr. inkin was mainly isolated from skin in the anogenital region. In 38 cases, reclassification revealed Tr. mucoides; Tr. ovoides and Tr. asteroides were identified in 3 cases each, while Tr. asahii, an especially remarkable pathogen in systemic mycoses, was not found. Clinically, isolates of Tr. mucoides were predominant in toenail mycoses which might be considered in the therapy of onychomycosis. Furthermore, isolates from the skin should be evaluated with respect to the increasing incidence of systemic Trichopsoron mycoses.

The cell wall of the oleaginous yeast Trichosporon cutaneum.

The cell wall of Trichosporon cutaneum consists of 11% protein, 63% neutral carbohydrate, 9% glucosamine and 13% glucuronic acid. The sugars include glucose (32%), mannose (6%) and traces of xylose and galactose. The cell wall was fractionated with alkali to yield a mixture of alkali-soluble matrix components, and an alkali-insoluble glucan associated with chitin. The alkali-insoluble glucan contained a mixture of (1-3) and (1-6) glycosidic linkages. It was only partly susceptible to digestion by the beta(1-3) glucanase, Zymolyase. The alkali-soluble fraction contained glucan, mannan and acidic polymers. The glucan was (1-3)-linked with no (1-6) linkages and only trace amounts of (1-3-6)-linked glucose. It was resistant to digestion by Zymolyase. Extensive hydrolysis of this fraction with trifluoroacetic acid released a high-molecular-mass glucuronan which had 1H- and 13C-NMR profiles matching those of the beta(1-4) glucuronan, mucoric acid. Xylomannan was purified from isolated cell walls and from whole cells. It contained glucose, mannose, xylose, and D-glucuronic acid. It was very similar in composition and structure to the capsular polysaccharides of Cryptococcus neoformans, and to an extracellular polysaccharide produced by another yeast described as T. cutaneum. Electron microscopy showed that the cell wall of T. cutaneum has a lamellar structure characteristic of a basidiomycetous yeast rather than the electron-dense 'fuzzy coat' seen in Candida albicans.

Experimental Trichosporon infection in persistently granulocytopenic rabbits: implications for pathogenesis, diagnosis, and treatment of an emerging opportunistic mycosis.

Disseminated Trichosporon infection, an uncommon but emerging opportunistic mycosis due to Trichosporon beigelii, is frequently difficult to diagnose, refractory to treatment, and associated with a high attributable mortality. Models of disseminated and gastrointestinal Trichosporon infection were developed in persistently granulocytopenic rabbits. The patterns of infection resembled those of clinical disease, including cutaneous lesions, chorioretinitis, renal infection, pulmonary infection, and antigenemia cross-reactive with cryptococcal capsular polysaccharide. Antigenemia, an early manifestation of disseminated Trichosporon infection, originated in vivo from a fibrillar extracellular matrix. Trichosporon organisms disseminated from the gastrointestinal tract to visceral tissue in colonized immunosuppressed rabbits, whereas there was no dissemination from the gastrointestinal tract of otherwise normal rabbits. The antifungal triazoles, fluconazole and SCH 39304, were most active; maximum tolerated doses of amphotericin B and liposomal amphotericin B were ineffective. Trichosporon antigenemia declined in response to antifungal therapy. These findings contribute to improved understanding of the pathogenesis, diagnosis, and treatment of disseminated Trichosporon infection.

Contributions to a revision of the genus Trichosporon.

The genus Trichosporon was revised using characters of morphology, ultrastructure, physiology, ubiquinone systems, mol% G + C of DNA, DNA/DNA reassociations and 26S ribosomal RNA partial sequences. A total of 101 strains was used, including all available type and authentic cultures of previously described taxa. Nineteen taxa could be distinguished, 15 of which having Q-9 coenzyme systems and 4 having Q-10. Sixteen previously described names were reduced to synonymy. One new species was described. The genus is characterized by the presence of arthroconidia. Few species possess further diagnostic morphological characters, such as the presence of appressoria, macroconidia or meristematic conidiation. The septa of two species were found to be non-perforate, while those of the remaining species contained dolipores at variable degrees of differentiation, with or without vesicular or tubular parenthesomes. All species were able to assimilate a large number of carbon compounds; visible CO2 production was absent. The genus was found to be fairly homogeneous on the basis of a phylogenetic analysis of partial 26S rRNA sequences, with the exception of T. pullulans which proved to be unrelated. Most taxa were found to occupy well-defined ecological niches. Within the group of taxa isolated from humans, a distinction could be made between those involved in systemic mycoses and those which mainly caused pubic or non-pubic white piedras, respectively. One species was consistently associated with animals, while others came mainly from soil or water. One species was mesophilic and another psychrophilic.

Demonstration of a cell wall antigen cross-reacting with cryptococcal polysaccharide in experimental disseminated trichosporonosis.

Patients with disseminated infections caused by Trichosporon beigelii have a circulating antigen that cross-reacts with the polysaccharide capsule of Cryptococcus neoformans. We studied the localization of this antigen by immunoelectron microscopy in a rabbit model of experimental disseminated trichosporonosis. Deparaffinized lung sections were examined by using a murine monoclonal anti-cryptococcal polysaccharide antibody and colloidal gold particles coated with goat antibody to murine immunoglobulin G. Antigen that cross-reacted with the monoclonal antibody was observed in the T. beigelii cell wall and in a fibrillar matrix extending from the cell wall.

White piedra: evidence for a synergistic infection.

To determine the relative roles of coryneform bacteria and Trichosporon beigelii in the pathogenesis of genital white piedra, scrotal hair from 10 subjects was studied. Hairs were examined by light microscopy to determine the relative proportions of each organism, and were also cultured for coryneforms and yeast. Histologically, hair nodules from five out of nine cases showed a mixture of yeasts and bacteria, four had bacteria alone, and none showed yeast alone. Five strains of T. beigelii were cultured, two strains of Saccharomyces cerevisiae and 22 strains of coryneforms. The isolates were tested for synergism by a plate-overlay method. Growth of coryneforms occurred over and around sections of the plate inoculated with T. beigelii but not around the control yeast, S. cerevisiae. There were strain differences in the stimulatory response of both T. beigelii and coryneform strains. In reverse experiments coryneforms did not enhance growth of T. beigelii. It was concluded that white piedra is a mixed infection caused by the synergistic action between T. beigelii and a specific coryneform bacteria resulting in invasion of the hair cuticle and cortex.

White piedra: ultrastructure and a new microecological aspect.

White piedra or trichosporosis is a superficial mycosis of the hair shaft, caused by the yeast Trichosporon beigelii; it has been found in all continents and may involve the hair of any part of the body. We report a case of white piedra on the hairs of the inguinal fold with ultrastructural studies. Transmission electron microscopy showed that the nodules have the same morphological aspects as the fungus in culture (hyphae and arthrospores) except for the presence of a cementant substance. By scanning electron microscopy the elimination of spores was seen on the nodule surface. Interestingly similar nodules were found on cotton fibres of the patient's underwear, which were also studied by scanning electron microscopy. This finding can explain therapeutic failure and demands special hygienic conditions related to clothes.