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.

Hemagglutination ability and hemolytic activity of Trichosporon asahii.

Trichosporon asahii is a human fungal pathogen that causes deep-seated infections in immunocompromised patients. While the pathogenic mechanisms of T. asahii remain unknown, our previous studies indicate that adherent colony morphologies were generated from parent strains, which may contribute to their pathogenicity. In the present study, we analyzed the hemolytic and hemagglutination activities of T. asahii. We report that T. asahii cells demonstrate hemagglutination and hemolytic activities, and that cell surface molecules play a role in the hemagglutination activity of adherent strains. These observations suggest that hemagglutination and hemolysis may be one of the pathogenic mechanisms of T. asahii.

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.

Trichosporon jirovecii infection of red swamp crayfish (Procambarus clarkii).

One hundred and twenty-nine isolates of Trichosporon jirovecii were isolated from the melanized exoskeleton as well as eyestalks, gills, muscle and haemolymph of red swamp crayfish (Procambarus clarkii) collected from the River Nile, during summer 2015. Isolates were similar morphologically, biochemically and genetically. Also, random amplified polymorphic DNA (RAPD) analysis exhibited no polymorphism among the tested isolates. Virulence factors such as chitinase, protease, lipase activities and biofilm formation were examined. Challenge test, using a representative isolate (Tj_ASU8), proved its pathogenicity against crayfish. Magnesium oxide nanoparticles had a good antifungal activity with a minimum fungicidal concentration of 8 mg/ml. To the best of our knowledge, this is the first report for isolation of T. jirovecii from red swamp crayfish, showing melanization, from the River Nile. We assume that infected crayfish may act as a vector for this fungus and can disseminate infection to all susceptible hosts in the vicinity.

The effects of TORC signal interference on lipogenesis in the oleaginous yeast Trichosporon oleaginosus.

BACKGROUND: Oleaginous organisms are a promising, renewable source of single cell oil. Lipid accumulation is mainly induced by limitation of nutrients such as nitrogen, phosphorus or sulfur. The oleaginous yeast Trichosporon oleaginosus accumulates up to 70% w/w lipid under nitrogen stress, while cultivation in non-limiting media only yields 9% w/w lipid. Uncoupling growth from lipid accumulation is key for the industrial process applicability of oleaginous yeasts. This study evaluates the effects of rapamycin on TOR specific signaling pathways associated with lipogenesis in Trichosporon oleaginosus for the first time. RESULTS: Supplementation of rapamycin to nutrient rich cultivation medium led to an increase in lipid yield of up to 38% g/L. This effect plateaued at 40 µM rapamycin. Interestingly, the fatty acid spectrum resembled that observed with cultivation under nitrogen limitation. Significant changes in growth characteristics included a 19% increase in maximum cell density and a 12% higher maximum growth rate. T. oleaginosus only has one Tor gene much like the oleaginous yeast Rhodosporidium toruloides. Consequently, we analyzed the effect of rapamycin on T. oleaginosus specific TORC signaling using bioinformatic methodologies. CONCLUSIONS: We confirm, that target of rapamycin complex 1 (TORC1) is involved in control of lipid production and cell proliferation in T. oleaginosus and present a homology based signaling network. Signaling of lipid induction by TORC1 and response to carbon depletion to this complex appear to be conserved, whereas response to nitrogen limitation and autophagy are not. This work serves as a basis for further investigation regarding the control and induction of lipid accumulation in oil yeasts.

Cell surface hydrophobicity and colony morphology of Trichosporon asahii clinical isolates.

Trichosporon asahii is a pathogenic basidiomycetous yeast. Individual strains of T. asahii have different colony morphologies. However, it is not clear whether cell surface phenotypes differ among the colony morphologies. Here we characterized the cell surface hydrophobicity and analysed the carbohydrate contents of the cell surface polysaccharides in T. asahii clinical isolates with various colony morphologies. Among the three distinctive colony morphologies obtained from one clinical isolate, the white-type morphology exhibited higher hydrophobicity. The hydrophobicity of heat-killed T. asahii cells was greatly reduced after periodate oxidation of the cell surface carbohydrates. Furthermore, the cell wall and extracellular polysaccharide components differed among the morphologies. Our results suggest that T. asahii cell surface hydrophobicity is affected by cell surface carbohydrate composition. Copyright © 2016 John Wiley & Sons, Ltd.

In Vitro Activity of Berberine Alone and in Combination with Antifungal Drugs Against Planktonic Forms and Biofilms of Trichosporon Asahii.

Trichosporon asahii (T. asahii) is an opportunistic pathogen that can cause life-threatening infections in immunocompromised patients, with high mortality rates up to 80% despite treated with antifungal drugs. The biofilms-forming ability of T. asahii on indwelling medical devices may account for the resistance to antifungal drugs. Berberine (BBR) has been demonstrated to have antifungal activity and synergistic effects in combination with antifungal drugs against pathogenic fungi. In the present study, the in vitro activities of BBR alone or combined with fluconazole (FLC), itraconazole (ITC), voriconazole (VRC), caspofungin (CAS) and amphotericin B (AMB) against planktonic forms and biofilms of 21 clinical T. asahii isolates were evaluated using checkerboard microdilution method and XTT reduction assay, respectively. The fractional inhibitory concentration index (FICI) was used to interpret drug interactions. BBR alone did not exhibit significant antifungal activities against both T. asahii planktonic cells (MICs, 32 â†’ 128 µg/ml) and T. asahii biofilms (SMICs, >128 µg/ml). However, BBR exhibited synergistic effects against T. asahii planktonic cells in combination with AMB, FLC and CAS (FICI ≤ 0.5) and exhibited synergistic effects against T. asahii biofilms in combination with AMB and CAS (FICI ≤ 0.5). BBR/ITC and BBR/VRC combinations yielded mainly indifferent interactions against T. asahii planktonic cells. BBR/FLC, BBR/ITC and BBR/VRC combinations also yielded indifferent interactions against T. asahii biofilms. Our study highlights the therapeutic potential of BBR to be used as an antifungal synergist in combination with antifungal drugs against T. asahii infections, especially BBR/AMB combination. Further in vivo studies are needed to validate our findings.

Lumping kinetics of ABE fermentation wastewater treatment by oleaginous yeast Trichosporon cutaneum.

Lumping kinetics models were built for the biological treatment of acetone-butanol-ethanol (ABE) fermentation wastewater by oleaginous yeast Trichosporon cutaneum with different fermentation temperatures. Compared with high temperature (33°C, 306 K) and low temperature (23°C, 296 K), medium temperature (28°C, 301 K) was beneficial for the cell growth and chemical oxygen demand (COD) degradation during the early stage of fermentation but the final yeast biomass and COD removal were influenced little. By lumping method, the materials in the bioconversion network were divided into five lumps (COD, lipid, polysaccharide, other intracellular products, other extracellular products), and the nine rate constants (k1-k9) for the models can well explain the bioconversion laws. The Gibbs free energy (G) for this bioconversion was positive, showing that it cannot happen spontaneous, but the existence of yeast can after the chemical equilibrium and make the bioconversion to be possible. Overall, the possibility of using lumping kinetics for elucidating the laws of materials conversion in the biological treatment of ABE fermentation wastewater by T. cutaneum has been initially proved and this method has great potential for further application.

Switching of colony morphology and adhesion activity of Trichosporon asahii clinical isolates.

Trichosporon asahii is a pathogenic yeast that causes trichosporonosis, a deep-seated infection, in immunocompromised hosts. Pathogenic factors involved in this infection have not been investigated in detail, but morphological phenotype switching is thought to be important for T. asahii pathogenesis. Therefore, we analyzed adhesion, which may be a key early step in T. asahii infection, after morphological phenotype switching. T. asahii clinical isolates show several colony morphologies. In this study, colonies showing white-farinose (W), off-white-smooth (O), off-white-rugose (OR), smooth (S), and yellowish-white (Y) morphologies were obtained from three isolates and compared in an adhesion assay performed in cell culture dishes. At least one type of colony morphology from each clinical isolate adhered strongly to the culture dish surface, although the colony type that displayed strong adherence varied among the strains. Thus, morphological phenotype switching altered the adhesion of T. asahii strains.

Identification of three yeast species using the conventional and internal transcribed spacer region sequencing methods as first or second global record from human superficial infections.

During the mycological analysis of skin and nail samples taken from patients with onychomycosis and tineas in Assiut city, it is interesting to report that yeast fungi were the main causal agents being cultured from 45.79% of total cases. In general, 21 species of yeast were isolated. Some of these are reported for the first time from clinical specimens. From the literature available up-to-date around the world, this study reports for the first time Saccharomycopsis fibuligera as the causal agent of four clinical cases: two onychomycoses, one tinea capitis and one tinea amiantacea. Also, it is reported here the second record for Trichosporon dohaense from a case of onychomycosis of a 40-year-old woman (after its original description in 2009 by Taj-Aldeen et al. J Clin Microbiol 47: 1791). Candida galli was also reported for the first time from clinical specimen (tinea unguium) in 2014 by Galán-Sánchez et al. Mycopathol 178: 303, and this study reports the second case of onychomycosis by C. galli. These strains were identified on the basis of their phenotypic, biochemical, physiological and genotypic features. Strains and internal transcribed spacer (ITS) gene sequences of these species are deposited at Assiut University Mycological Center Culture Collection (AUMC) and National Center for Biotechnological Information (NCBI) respectively.

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.

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.

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.

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.

Biostimulation by methanol enables the methylotrophic yeasts Hansenula polymorpha and Trichosporon sp. to reveal high formaldehyde biodegradation potential as well as to adapt to this toxic pollutant.

The methylotrophic yeasts Hansenula polymorpha and Trichosporon sp. revealed enhanced biodegradation capability of exogenously applied formaldehyde (Fd) upon biostimulation achieved by the presence of methanol, as compared to glucose. Upon growth on either of the above substrates, the strains proved to produce the activity of glutathione-dependent formaldehyde dehydrogenase-the enzyme known to control the biooxidative step of Fd detoxification. However, in the absence of methanol, the yeasts' tolerance to Fd was decreased, and the elevated sensitivity was especially pronounced for Trichosporon sp. Both strains responded to the methanol and/or Fd treatment by increasing their unsaturation index (UI) at xenobiotic levels below minimal inhibitory concentrations. This indicated that the UI changes effected from the de novo synthesis of (poly) unsaturated fatty acids carried out by viable cells. It is concluded that the yeast cell response to Fd intoxication involves stress reaction at the level of membranes. Fluidization of the lipid bilayer as promoted by methanol is suggested as a significant adaptive mechanism increasing the overall fitness enabling to cope with the formaldehyde xenobiotic via biodegradative pathway of C1-compound metabolism.

Trichosporon vanderwaltii sp. nov., an asexual basidiomycetous yeast isolated from soil and beetles.

During a survey of unidentified yeast isolates deposited in the UNESCO-MIRCEN Biotechnological Yeast Culture Collection housed at the Department of Microbial, Biochemical and Food Biotechnology of the University of the Free State, one isolate obtained from soil in South Africa showed 100 % identity in D1/D2 rDNA sequence with undescribed basidiomycetous yeasts isolated from the gut of beetles from the United States of America and forest soil from Taiwan in the NCBI sequence database. Phylogenetic analyses using sequences of the D1/D2 rDNA and ITS regions indicated that all these isolates form a well-supported sub-clade that is the sister clade to the Brassicae plus Porosum clades of Trichosporon in the order Trichosporonales. Subsequent phenotypic tests revealed that asexual reproduction by budding is rare but dominated by arthroconidia resulting from segmentation of hyphae and that fusiform giant cells are characterized by budding from a broad base. These findings further suggest that these isolates belong to a single tremellomycetous yeast species for which the name Trichosporon vanderwaltii CBS 12124(T) (=NRRL Y-48732(T), =UOFS Y-1920(T)) is proposed.

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.

Antifungal Activity and Potential Action Mechanism of Allicin against Trichosporon asahii.

Trichosporon asahii is an emerging opportunistic pathogen that causes potentially fatal disseminated trichosporonosis. The global prevalence of coronavirus disease 2019 (COVID-19) poses an increasing fungal infection burden caused by T. asahii. Allicin is the main biologically active component with broad-spectrum antimicrobial activity in garlic. In this study, we performed an in-depth analysis of the antifungal characteristics of allicin against T. asahii based on physiological, cytological, and transcriptomic assessments. In vitro, allicin inhibited the growth of T. asahii planktonic cells and biofilm cells significantly. In vivo, allicin improved the mean survival time of mice with systemic trichosporonosis and reduced tissue fungal burden. Electron microscopy observations clearly demonstrated damage to T. asahii cell morphology and ultrastructure caused by allicin. Furthermore, allicin increased intracellular reactive oxygen species (ROS) accumulation, leading to oxidative stress damage in T. asahii cells. Transcriptome analysis showed that allicin treatment disturbed the biosynthesis of cell membrane and cell wall, glucose catabolism, and oxidative stress. The overexpression of multiple antioxidant enzymes and transporters may also place an additional burden on cells, causing them to collapse. Our findings shed new light on the potential of allicin as an alternative treatment strategy for trichosporonosis. IMPORTANCE Systemic infection caused by T. asahii has recently been recognized as an important cause of mortality in hospitalized COVID-19 patients. Invasive trichosporonosis remains a significant challenge for clinicians, due to the limited therapeutic options. The present work suggests that allicin holds great potential as a therapeutic candidate for T. asahii infection. Allicin demonstrated potent in vitro antifungal activity and potential in vivo protective effects. In addition, transcriptome sequencing provided valuable insights into the antifungal effects of allicin.

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.