Sodium butyrate inhibits planktonic cells and biofilms of Trichosporon spp.

Trichosporon spp. have been increasingly recognized as an important pathogen of invasive and disseminated infections in immunocompromised patients. These species are prone to form biofilms in medical devices such as catheters and prosthesis, which are associated with antifungal resistance and therapeutic failure. Therefore, new antifungals with a broader anti-biofilm activity need to be discovered. In the present study we evaluate the inhibitory potential of sodium butyrate (NaBut) - a histone deacetylase inhibitor that can alter chromatin conformation - against planktonic and sessile cells of T. asahii and T. inkin. Minimum inhibitory concentration (MIC) of NaBut against planktonic cells was evaluated by microdilution and morphological changes were analyzed by optical microscopy on malt agar supplemented with NaBut. Biofilms were evaluated during adhesion, development and after maturation for metabolic activity and biomass, as well as regarding ultrastructure by scanning electron microscopy and confocal laser scanning microscopy. NaBut inhibited the growth of planktonic cells by 50% at 60 mM or 120 mM (p < 0.05) and also reduced filamentation of Trichosporon spp. NaBut reduced adhesion of Trichosporon cells by 45% (10xMIC) on average (p < 0.05). During biofilm development, NatBut (10xMIC) reduced metabolic activity and biomass up to 63% and 81%, respectively (p < 0.05). Mature biofilms were affected by NaBut (10xMIC), showing reduction of metabolic activity and biomass of approximately 48% and 77%, respectively (p < 0.05). Ultrastructure analysis showed that NaBut (MIC and 10xMIC) was able to disassemble mature biofilms. The present study describes the antifungal and anti-biofilm potential of NaBut against these opportunist emerging fungi.

A Polysaccharide Derived from a Trichosporon sp. Culture Strongly Primes Plant Resistance to Viruses.

Plant viruses cause devastating diseases in plants, yet no effective viricide is available for agricultural application. We screened cultured filtrates derived from various soil microorganisms cultured in vegetable broth that enhanced plant viral resistance. A cultured filtrate, designated F8 culture filtrate, derived from a fungus belonging to the genus Trichosporon, induced strong resistance to various viruses on different plants. Our inoculation assay found the infection rate of Tobacco mosaic virus (TMV)-inoculated Nicotiana benthamiana with F8 culture filtrate pretreatment may decrease to 0%, whereas salicylic acid (SA)-pretreated N. benthamiana attenuated TMV-caused symptoms but remained 100% infected. Tracking Tobacco mosaic virus tagged with green fluorescence protein in plants revealed pretreatment with F8 culture filtrate affected the initial establishment of the virus infection. From F8 culture filtrate, we identified a previously unknown polysaccharide composed of D-mannose, D-galactose, and D-glucose in the ratio 1.0:1.2:10.0 with a α-D-1,4-glucan linkage to be responsible for the induction of plant resistance against viruses through priming of SA-governed immune-responsive genes. Notably, F8 culture filtrate only triggered local defense but was much more effective than conventional SA-mediated systematic acquired resistance. Our finding revealed that microbial cultured metabolites provided a rich source for identification of potent elicitors in plant defense.

The dynamic structure of Spitzenkörpers of Trichosporon asahii examined by the fluorescent probe FM4-64

Abstract The Spitzenkörper is a dynamic and specialized multicomponent cell complex present in the tips of hyphal cells. The amphiphilic styryl dye FM4-64 was found to be ideal for imaging the dynamic changes of the apical vesicle cluster within growing hyphal tips. It is widely used as a marker of endocytosis and to visualize vacuolar membranes. Here we performed uptake experiments using FM4-64 to study the dynamic of the Spitzenkörper in Trichosporon asahii. We observed that Spitzenkörpers were present at the tip of the budding site of the spore, blastospore, and the germ tube of T. asahii. We also found that Spitzenkörpers were present at the tip of the hyphae as well as the subapical regions. Cytochalasin D, an inhibitor of actin polymerization, leads to abnormal Spitzenkörper formation and loss of cell polarity.

The dynamic structure of Spitzenkörpers of Trichosporon asahii examined by the fluorescent probe FM4-64.

The Spitzenkörper is a dynamic and specialized multicomponent cell complex present in the tips of hyphal cells. The amphiphilic styryl dye FM4-64 was found to be ideal for imaging the dynamic changes of the apical vesicle cluster within growing hyphal tips. It is widely used as a marker of endocytosis and to visualize vacuolar membranes. Here we performed uptake experiments using FM4-64 to study the dynamic of the Spitzenkörper in Trichosporon asahii. We observed that Spitzenkörpers were present at the tip of the budding site of the spore, blastospore, and the germ tube of T. asahii. We also found that Spitzenkörpers were present at the tip of the hyphae as well as the subapical regions. Cytochalasin D, an inhibitor of actin polymerization, leads to abnormal Spitzenkörper formation and loss of cell polarity.

Strategies of preserving lipids in microorganism after fermentation.

Microbial oil is accumulated by microorganisms as stored energy during cultivation, and will be degraded to generate energy when they are not able to obtain external energy source. As the lipid is the desired production, the in situ degradation of oil by microorganisms after fermentation or during downstream processing has to be prevented. This study investigated the effect of pH, thermal, salinity, bead milling, ultrasonication, and microwave treatment on the viability of Trichosporon oleaginosus when it was used for lipid production. The cells in broth were completely inactivated with the treatment of pH 1 and 2 for 1h, temperature 80 °C for 10 min, ultrasonication for 8 min, and microwave for 6 min, respectively. It was observed that these treatments had no impact on final product (biodiesel) composition and were considered as safe and efficient methods to preserve lipid in cells.

Efficacy of Ethanol against Trichosporon asahii Biofilm in vitro.

Trichosporon asahii (T. asahii) can cause invasive infections, particularly catheter-related bloodstream infections (CR-BSIs). T. asahii biofilm, which is resistant to the most common clinical antifungal agents, may play an important role in these life-threatening infections. This study focused on the effects of ethanol on the different phases of T. asahii biofilm formation. At the concentrations clinically used, ethanol killed T. asahii planktonic cells (MIC90 = 15% and m-MIC90 = 15%) and biofilm (SMIC90 = 50%), and exposure to 25% ethanol for 12 h or to 50% ethanol for 8 h completely inhibited biofilm development and eradicated mature T. asahii biofilm. Thus, our results showed that ethanol effectively inhibited the main phases of T. asahii biofilm formation. This study reveals a new potential strategy to prevent and treat T. asahii biofilm-related CR-BSIs.

Sweetpotato vines hydrolysate induces glycerol to be an effective substrate for lipid production of Trichosporon fermentans.

By co-fermented with sweetpotato vines hydrolysate (SVH), glycerol can be used as an effective substrate for Trichosporon fermentans to produce lipids. Submerged fermentation results showed that T. fermentans exhibited a maximum lipid yield of 7.45 g l(-1) with a biomass of 17.95 g l(-1) on 10% SVH added glycerol mineral medium (Glycerol MM-10% SVH), which was 4.34-fold higher than that on glycerol mineral medium. Lipids produced on glycerol based media exhibited a significantly different fatty acid composition profile from that produced on sugar based media accompanying by a sharp increase in polyunsaturated fatty acids content. Biochemical behaviors characterization further demonstrated that SVH has a remarkable promoting effect on the biomass formation and glycerol uptake. The extremely high lipid yield on Glycerol MM-10% SVH was mainly attributed to the enhancement of SVH on biomass, although SVH is an excellent nutrient supplier for lipid accumulation due to its low nitrogen availability.

Improvement of FISH-FCM enumeration performance in filamentous yeast species in activated sludge by snailase partial digestion.

This paper developed a novel strategy to improve the fluorescence in situ hybridization-flow cytometry (FISH-FCM) enumeration performance in filamentous yeast species in activated sludge by snailase partial digestion to fully disaggregate filamentous yeast chains into single cells. A 2 h 2% snailase partial digestion liberated more rod-shaped yeast single cells from intertwined filamentous yeast samples than did sonication disaggregation, based on an optical microscopic observation and the forward-light-scatter frequency histogram of FCM analysis. However, adding snailase resulted in a fluorescence-quenching phenomenon of the hybridized filamentous yeast cells, which was minimized by lowering the snailase concentration. An approximately 3 h 0.5% snailase partial digestion conducted between sonication and hybridization significantly improved the FISH-FCM enumeration performance for filamentous yeast species by 37%. The results presented here will facilitate the rapid detection, identification and exact enumeration of specific filamentous fungal species in environmental samples.

Artificial construction and characterization of a fungal consortium that produces cellulolytic enzyme system with strong wheat straw saccharification.

A consortium-APcT2 composed of 92% of Trichoderma sp. T-1, 6.7% of P. chrysosporium and 1.3% of A. oryzae A-4 that produces cellulolytic enzyme system with strong wheat straw saccharification was constructed using Taguchi design combined with variance analysis (ANOVA). Among 32 consortia constructed from 6 fungi using Taguchi design, consortium 20 with the strongest composition was selected. The inhibitive fungal constituent in consortium 20 was subsequently removed according to the ANOVA results. The finally optimized consortium-APcT2 yielded 805.12 mg gds(-1) sugars, 26.98% higher than the pure Trichoderma sp. T-1. Protein profile analysis of the cellulolytic enzyme systems, sugar composition analysis of the hydrolysates and compatibility evaluation of the fungal constituents showed that the enhanced straw saccharification of the consortium-APcT2 could be mainly attributed to the enhancement of the co-cultivation for enzyme production and the synergistic action of different types of enzymes in the hydrolysis process.

Extensive white piedra of the scalp caused by Trichosporon inkin: A case report and review of literature.

Trichosporon species cause wide varieties of fungal infections. White piedra, a superficial fungal infection on the hair shaft, is caused by Trichosporon species. We report here a case of white piedra over the scalp due to T. inkin, a rarely isolated pathogen from such a lesion. The identification of the fungus was confirmed on the basis of morphology, carbohydrate assimilation tests, and sequencing of the ITS region of rDNA. The available literature on infections due to T. inkin is also reviewed.

Characterization of oleaginous yeasts revealed two novel species: Trichosporon cacaoliposimilis sp. nov. and Trichosporon oleaginosus sp. nov.

Two new species in the anamorphic basidiomycetous genus Trichosporon (Tremellomycetes, Agaricomycotina) were uncovered in a DNA sequence-based molecular analysis of oleaginous yeasts maintained in the ATCC Mycology Collection. One yeast is named as Trichosporon cacaoliposimilis sp. nov. for its capability of synthesizing and accumulating a large amount of lipids having a composition equivalent to that of natural cacao butter. The type strain is ATCC 20505(T), originally deposited as Trichosporon sp. The other can use food industry wastes and agricultural byproducts as the substrate for growth and accumulation of a high level of oil and accordingly is named Trichosporon oleaginosus sp. nov. The type strain is ATCC 20509(T), previously identified as Cryptococcus curvatus. Molecular phylogenetic analyses indicate that T. cacaoliposimilis is a novel taxon in the Gracile clade of the genus, close to T. gracile and T. dulcitum, and that T. oleaginosus belongs to the Cutaneum clade, with T. jirovecii as the closest sister taxon. Other oleaginous yeasts were identified as new strains of known taxa, T. insectorum, Candida orthopsilosis and C. palmioleophila.

Microbial oil production from rice straw hydrolysate by Trichosporon fermentans.

Microbial oil production from sulphuric acid treated rice straw hydrolysate (SARSH) by Trichosporon fermentans was performed for the first time. Fermentation of SARSH without detoxification gave a poor lipid yield of 1.7 g/l, which was much lower than the result with glucose or xylose as the single carbon source (13.6 g/l or 9.9 g/l). The detoxification pretreatment, including overliming, concentration, and adsorption by Amberlite XAD-4 improved the fermentability of SARSH significantly by removing the inhibitors in SARSH. A total biomass of 28.6 g/l with a lipid content of 40.1% (corresponding to a lipid yield of 11.5 g/l) could be achieved after cultivation of T. fermentans on the detoxified SARSH for 8 days. Moreover, besides SARSH, T. fermentans could also utilize mannose, galactose, or cellobiose, in hydrolysates of other natural lignocellulosic materials as the single carbon source to grow and accumulate lipid with a high yield (at least 10.4 g/l). Hence, it is a promising strain for microbial oil production and thus biodiesel preparation from agro-industrial residues, especially lignocellulosic materials.

Morphological and biochemical characterization of the aetiological agents of white piedra

The Trichosporon genus is constituted by many species, of which Trichosporon ovoides and Trichosporon inkin are the causative agents of white piedra. They can cause nodules in genital hair or on the scalp. At present, Brazilian laboratory routines generally do not include the identification of the species of Trichosporon genus, which, although morphologically and physiologically distinct, present many similarities, making the identification difficult. The aim of this study was to identify the aetiological agents at the species level of white piedra from clinical specimens. Therefore, both the macro and micro morphology were studied, and physiological tests were performed. Trichosporon spp. was isolated from 10 clinical samples; T. ovoides was predominant, as it was found in seven samples, while T. inkin was identified just in two samples. One isolate could not be identified at the species level. T. inkin was identified for the first time as a white piedra agent in the hair shaft on child under the age of 10.

First isolation of the anamorphic basidiomycetous yeast Trichosporon faecale in Germany, from the skin of a patient with tinea pedis.

Mycoses due to yeasts belonging to other genera than Candida have become common in the last years especially in immuno-compromised patients. Species of the anamorphic basidiomycetous yeast genus Trichosporon are such opportunistic human pathogenic yeasts which cause several diseases. In this study, Trichosporon faecale is reported in Germany for the first time. The isolate was taken from a human foot, where it was associated with a tinea pedis. The fungal isolate was identified by investigating the morphology, physiology by a commercial API 32 C-set and molecular data of SSU and LSU rDNA as well as the ITS region.

Morphological and biochemical characterization of the aetiological agents of white piedra.

The Trichosporon genus is constituted by many species, of which Trichosporon ovoides and Trichosporon inkin are the causative agents of white piedra. They can cause nodules in genital hair or on the scalp. At present, Brazilian laboratory routines generally do not include the identification of the species of Trichosporon genus, which, although morphologically and physiologically distinct, present many similarities, making the identification difficult. The aim of this study was to identify the aetiological agents at the species level of white piedra from clinical specimens. Therefore, both the macro and micro morphology were studied, and physiological tests were performed. Trichosporon spp. was isolated from 10 clinical samples; T. ovoides was predominant, as it was found in seven samples, while T. inkin was identified just in two samples. One isolate could not be identified at the species level. T. inkin was identified for the first time as a white piedra agent in the hair shaft on child under the age of 10.

Mineralization of chlorpyrifos by co-culture of Serratia and Trichosporon spp.

A bacterial strain (Serratia sp.) that could transform chlorpyrifos to 3,5,6-trichloro-2-pyridinol (TCP) and a TCP-mineralizing fungal strain (Trichosporon sp.) were isolated from activated sludge by enrichment culture technique. The fungus could also degrade 50 mg chlorpyrifos l(-1) within 7 days. Co-cultures completely mineralized 50 mg chlorpyrifos l(-1) within 18 h at 30 degrees C and pH 8 using a total inocula of 0.15 g biomass l(-1).

Trichosporon mycotoxinivorans sp. nov., a new yeast species useful in biological detoxification of various mycotoxins.

A yeast strain isolated from the hindgut of the lower termite Mastotermes darwiniensis (Mastotermitidae) was found to represent a new member of the genus Trichosporon. Trichosporon mycotoxinivorans is closely related to T. loubieri on the basis of the phylogenetic trees based on the D1/D2 region of 26S rDNA, an approx. 600 bp fragment of the 18S rDNA and both ITS regions. However, the two species differ at nine positions in the D1/D2 region of 26S rDNA. The IGS1 region of T. mycotoxinivorans is 401 bp long. T. mycotoxinivorans is distinguished from T. loubieri by its ability to assimilate inulin and galactitol, and its inability to grow at 40 degrees C. The name of this newly isolated strain refers to an important characteristics of T. mycotoxinivorans to detoxify mycotoxins such as ochratoxin A and zearalenone. Therefore this strain can be used for the deactivation of the respective mycotoxins in animal feeds.

Disseminated trichosporonosis in a murine model of chronic granulomatous disease.

Over a period of ten months, five mice submitted to our service (the Pathology Section of the Veterinary Resources Program, Office of Research Services at the National Institutes of Health, Bethesda, Md.) were diagnosed with disseminated trichosporonosis. These mice had pyogranulomatous inflammation in multiple organs, including lung, liver, lymph nodes, salivary gland, and skin. Fungal elements in many of the lesions were identified, using special histochemical stains, and Trichosporon beigelii was obtained by use of culture of specimens at affected sites. This saprophytic fungus has caused disseminated disease in immunosuppressed humans. However, despite widespread use of immunosuppressed rodents in research, to the authors' knowledge, this organism had not previously been reported to cause spontaneous disseminated disease in laboratory mice. All affected mice had a genetically engineered defect in p47(phox), a critical component of the nicotinamide dinucleotide phosphate (NADPH) oxidase, the enzyme responsible for generating the phagocyte oxidative burst. These animals are used as a murine model of human chronic granulomatous disease. We discuss the lesions, differential diagnosis, identification of the organism, and the role of NADPH oxidase in protecting against disseminated trichosporonosis.

Increased release of glucuronoxylomannan antigen and induced phenotypic changes in Trichosporon asahii by repeated passage in mice.

Clinically important fungi such as Candida albicans and Cryptococcus neoformans are known to undergo phenotypic changes after repeated subculture or passages in vivo. However, there are no reports describing this phenomenon in Trichosporon species. This study investigated whether in-vivo passages of environmental isolates of Trichosporon asahii in mice changes their phenotype; three environmental isolates and 14 clinical isolates (from deep-seated infections) were used. The shape of the colony and cell type were observed, and the titre of glucuronoxylomannan (GXM) antigen and concentration of (1-->3)-beta-D-glucan were measured for each isolate. Changes in these features were also examined after three passages of the environmental isolates in mice. The shape of colonies and cell types were clearly different in environmental and clinical isolates. Furthermore, the clinical isolates released significantly higher levels of GXM antigen than environmental isolates (titre: log2 9.4 SD 0.7 versus log2 5.4 SD 1.4). The phenotype of passaged isolates was significantly different from the original environmental isolates with respect to the morphology of colonies and cell type and GXM release (titre: log2 10.0 SD 0.7 versus log2 5.4 SD 1.4). These results suggest that the phenotypic changes in T. asahii occur as a result of in-vivo passages. This process may allow a proportion of the fungal population to escape eradication by the host immune system, as GXM antigen is considered to protect the fungi against phagocytosis by polymorphonuclear leucocytes and monocytes in vivo.