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.

Efficient xylose utilization leads to highest lipid productivity in Candida tropicalis SY005 among six yeast strains grown in mixed sugar medium.

Six local isolates of yeasts were screened for cell mass and lipid production in mixed glucose and xylose medium. Candida tropicalis SY005 and Trichosporon (Apiotrichum) loubieri SY006 showed significant lipid accumulation of 24.6% and 32% (dry cell weight), respectively when grown in medium containing equal mass of both the sugars. SY005 produced relatively higher cell mass of 9.66 gL-1 due to higher rate of sugar consumption, which raised the lipid productivity of the organism to 0.792 gL-1day-1 as compared to 0.446 gL-1day-1 in SY006. When grown with each sugar separately, the xylose consumption rate of SY005 was found to be 0.55 gL-1 h-1 after 4 days as compared to 0.52 gL-1 h-1 for SY006. Transcript expression of the high affinity xylose transporter (Cthaxt), xylose reductase (Ctxyl1), and xylitol dehydrogenase (Ctxyl2) of SY005 was monitored to unravel such high rate of sugar consumption. Expression of all the three genes was observed to vary in mixed sugars with Cthaxt exhibiting the highest expression in presence of only xylose. Expression levels of both Ctxyl1 and Ctxyl2, involved in xylose catabolism, were maximum during 24-48 h of growth, indicating that xylose utilization started in the presence of glucose, which was depleted in the medium after 96 h. Together, the present study documents that C. tropicalis SY005 consumes xylose concomitant to glucose during early period of growth, and it is a promising yeast strain for viable production of storage lipid or other high-value oleochemicals utilizing lignocellulose hydrolysate.

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.

Farnesol inhibits planktonic cells and antifungal-tolerant biofilms of Trichosporon asahii and Trichosporon inkin.

Trichosporon species have been considered important agents of opportunistic systemic infections, mainly among immunocompromised patients. Infections by Trichosporon spp. are generally associated with biofilm formation in invasive medical devices. These communities are resistant to therapeutic antifungals, and therefore the search for anti-biofilm molecules is necessary. This study evaluated the inhibitory effect of farnesol against planktonic and sessile cells of clinical Trichosporon asahii (n = 3) andTrichosporon inkin (n = 7) strains. Biofilms were evaluated during adhesion, development stages and after maturation for metabolic activity, biomass and protease activity, as well as regarding morphology and ultrastructure by optical microscopy, confocal laser scanning microscopy, and scanning electron microscopy. Farnesol inhibited Trichosporon planktonic growth by 80% at concentrations ranging from 600 to 1200 µM for T. asahii and from 75 to 600 µM for T. inkin. Farnesol was able to reduce cell adhesion by 80% at 300 µM for T. asahii and T. inkin at 600 µM, while biofilm development of both species was inhibited by 80% at concentration of 150 µM, altering their structure. After biofilm maturation, farnesol decreased T. asahii biofilm formation by 50% at 600 µM concentration and T. inkin formation at 300 µM. Farnesol inhibited gradual filamentation in a concentration range between 600 and 1200 µM. Farnesol caused reduction of filament structures of Trichosporon spp. at every stage of biofilm development analyzed. These data show the potential of farnesol as an anti-biofilm molecule.

In vivo pathogenicity of Trichosporon asahii isolates with different in vitro enzymatic profiles in an immunocompetent murine model of systemic trichosporonosis.

Trichosporon asahii is an opportunistic yeastlike fungus that colonizes the gastrointestinal and respiratory tracts and human skin. Although it is an important cause of disseminated infections by non-Candida species, there are a few reports related to its virulence factors and their possible role in in vivo pathogenicity. We developed a murine model of disseminated trichosporonosis in immunocompetent mice for the evaluation of the in vivo pathogenicity of 6 T. asahii isolates with different in vitro virulence factor profiles. Tissue fungal burden was determined on days 1, 3, 7, 15, and 25 post-challenge. Overall, the largest fungal load was detected in the kidney on the 5 experimental days, while brain, spleen, and liver displayed a comparatively low fungal count. We observed a fungal burden decrease in most experimental groups from day 15. Histological analysis showed the presence of T. asahii in tissue and a generalized inflammatory infiltrate of polymorphonuclear cells in the kidney, liver, red pulp of the spleen, and the hippocampus. Even though our isolates showed different in vitro virulence factors profiles, we did not detect relevant differences when assayed in vivo, except for a higher persistence of a protease- and biofilm-producing strain in kidney, liver, and brain.

Biophysical and biological properties of small linear peptides derived from crotamine, a cationic antimicrobial/antitumoral toxin with cell penetrating and cargo delivery abilities.

Crotamine is a natural polypeptide from snake venom which delivers nucleic acid molecules into cells, besides having pronounced affinity for negatively charged membranes and antifungal activity. We previously demonstrated that crotamine derived short linear peptides were not very effective as antifungal, although the non-structured recombinant crotamine was overridingly more potent compared to the native structured crotamine. Aiming to identify the features necessary for the antifungal activity of crotamine, two linear short peptides, each comprising half of the total positively charged amino acid residues of the full-length crotamine were evaluated here to show that these linear peptides keep the ability to interact with lipid membrane model systems with different phospholipid compositions, even after forming complexes with DNA. Interestingly, the presence of cysteine residues in the structure of these linear peptides highly influenced the antifungal activity, which was not associated to the lipid membrane lytic activity. In addition to the importance of the positive charges, the crucial role of cysteine residues was noticed for these linear analogs of crotamine, although the tridimensional structure and lipid membrane lytic activity observed only for native crotamine was not essential for the antifungal activity. As these peptides still keep the ability to form complexes with DNA molecules with no prejudice to their ability to bind to lipid membranes, they may be potentially advantageous as membrane translocation vector, as they do not show lipid membrane lytic activity and may harbor or not antifungal activity, by keeping or not the semi-essential amino acid cysteine in their sequence.

Trichosporon: another yeast-like organism responsible for immune reconstitution inflammatory syndrome in patients with hematological malignancy.

Trichosporon has recently emerged as a life-threatening opportunistic fungal pathogen, notably in patients with hematological malignancy. Fungemia, sometimes associated with cutaneous lesions and/or pneumonitis, is the major clinical form. Here, we report two cases of patients suffering from acute leukaemia who developed hepatic and/or splenic lesions apart from Trichosporon positive blood cultures. The appearance of hepatic and splenic lesions following the recovery from neutropenia is highly suggestive of a chronic disseminated infection, now considered as an immune reconstitution inflammatory syndrome. Treatment with corticosteroid therapy led to clinical improvement in both cases. Copyright © 2016 John Wiley & Sons, Ltd.

Growth Inhibition of an Opportunistic Yeast Pathogen Trichosporon asahii by Staphylococcus epidermidis.

In the co-culture of Staphylococcus epidermidis and Trichosporon asahii, a fungal pathogen, it was observed that live S. epidermidis inhibited the growth of T. asahii. Soluble active anti-T. asahii substances were speculated to be produced by S. epidermidis in culture medium. Using 1H- and 13C-NMR spectra and electron ionization-high resolution mass spectrometry (HR-negative-FAB-MS), we separated the active molecule and identified it as lactic acid. Commercially available L-lactic acid and D-lactic acid inhibited the growth of T. asahii. These results show that metabolites from bacterial populations are involved in the interactions of pathogenic fungi. The use of antibacterial agents to treat primary diseases could lead to the disruption of normal microbial communities and could cause opportunistic infections such as trichosporonosis.

The HIV aspartyl protease inhibitor ritonavir impairs planktonic growth, biofilm formation and proteolytic activity in Trichosporon spp.

This study evaluated the effect of the protease inhibitor ritonavir (RIT) on Trichosporon asahii and Trichosporon inkin. Susceptibility to RIT was assessed by the broth microdilution assay and the effect of RIT on protease activity was evaluated using azoalbumin as substrate. RIT was tested for its anti-biofilm properties and RIT-treated biofilms were assessed regarding protease activity, ultrastructure and matrix composition. In addition, antifungal susceptibility, surface hydrophobicity and biofilm formation were evaluated after pre-incubation of planktonic cells with RIT for 15 days. RIT (200 µg ml-1) inhibited Trichosporon growth. RIT (100 µg ml-1) also reduced protease activity of planktonic and biofilm cells, decreased cell adhesion and biofilm formation, and altered the structure of the biofilm and the protein composition of the biofilm matrix. Pre-incubation with RIT (100 µg ml-1) increased the susceptibility to amphotericin B, and reduced surface hydrophobicity and cell adhesion. These results highlight the importance of proteases as promising therapeutic targets and reinforce the antifungal potential of protease inhibitors.

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.

The Yeast Fungus Trichosporon lactis Found as an Epizoic Colonizer of Dung Beetle Exoskeletons.

The study on the biology and biodiversity of coprophagous Scarabaeoidea carried out in the Polish Carpathians revealed the occurrence of unusual epizoic excrescences on various dung beetles species of the genus Onthophagus. The excrescences occur on the elytra, prothorax, and head of the studied beetles. Detailed research on this phenomenon determined that the fungus grew in the form of multicellular thalli. The ITS-based identification of fungal material collected from beetles' exoskeletons resulted in a 100 % match with Trichosporon lactis. Until now, only a yeast lifestyle/stage was known for this basidiomycete species. Therefore, in this paper, we describe a new substrate for growth of T. lactis and its unknown and intriguing relationship with dung beetles. The results obtained in this study open up numerous research possibilities on the new role of dung beetles in terrestrial ecosystems, as well as on using the physiological properties of T. lactis to restore soils.

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.

Beneficial effect of corncob acid hydrolysate on the lipid production by oleaginous yeast Trichosporon dermatis.

In this work, corncob acid hydrolysate and its simulated medium whose sugar composition was the same as the corncob acid hydrolysate were used as fermentation substrate for lipid production by oleaginous yeast Trichosporon dermatis. On the corncob acid hydrolysate, after 7 days of fermentation, the biomass, lipid content, lipid yield, and lipid coefficient of T. dermatis were 17.3 g/L, 40.2%, 7.0 g/L, and 16.5%, respectively. Interestingly, during the lipid fermentation on the corncob acid hydrolysate, glucose, xylose, arabinose, and even acetic acid could be well utilized as carbon sources by T. dermatis. Surprisingly, the lipid yield (7.0 g/L) of T. dermatis on the corncob acid hydrolysate was much higher than that (3.8 g/L) on the simulated medium, in spite of the fact that the lipid coefficient (17.4%) on the simulated medium was a little higher. This phenomenon further showed that lignocellulosic acid hydrolysate was a suitable substrate for lipid fermentation by T. dermatis. This work would help the comprehensive utilization of lignocellulosic biomass for lipid production.

Antimicrobial lipopeptaibol trichogin GA IV: role of the three Aib residues on conformation and bioactivity.

The lipopeptaibol trichogin GA IV is a natural, non-ribosomally synthesized, antimicrobial peptide remarkably resistant to the action of hydrolytic enzymes. This feature may be connected to the multiple presence in its sequence of the non-coded residue α-aminoisobutyric acid (Aib), which is known to be responsible for the adoption of particularly stable helical structures already at the level of short peptides. To investigate the role of Aib residues on the 3D-structure and bioactivity of trichogin GA IV, we synthesized and fully characterized four analogs where one or two Aib residues are replaced by L-Leu. Our extensive conformational studies (including an X-ray diffraction analysis) and biological assays performed on these analogs showed that the Aib to L-Leu replacements do not affect the resistance to proteolysis, but modulate the bioactivity of trichogin GA IV in a 3D-structure related manner.

Microbial oil production from corncob acid hydrolysate by Trichosporon cutaneum.

Corncob was treated by dilute H(2)SO(4). The hydrolysate contained 45.7 g sugar/l. Without concentration or adding other nutrients, the hydrolysate, after being detoxified by overliming and adsorption with activated charcoal, was used for oil production using Trichosporon cutaneum. After 8 days' growth in shake-flasks, the biomass was 22.1 g/l with a lipid content of 36%. The lipid yield per mass of sugar was 17.4% (w/w). Corncob thus is a promising raw material for microbial oil production by this yeast.

Galectin-9 expands immunosuppressive macrophages to ameliorate T-cell-mediated lung inflammation.

Galectin-9 (Gal-9) plays pivotal roles in the modulation of innate and adaptive immunity to suppress T-cell-mediated autoimmune models. However, it remains unclear if Gal-9 plays a suppressive role for T-cell function in non-autoimmune disease models. We assessed the effects of Gal-9 on experimental hypersensitivity pneumonitis induced by Trichosporon asahii. When Gal-9 was given subcutaneously to C57BL/6 mice at the time of challenge with T. asahii, it significantly suppressed T. asahii-induced lung inflammation, as the levels of IL-1, IL-6, IFN-gamma, and IL-17 were significantly reduced in the BALF of Gal-9-treated mice. Moreover, co-culture of anti-CD3-stimulated CD4 T cells with BALF cells harvested from Gal-9-treated mice on day 1 resulted in diminished CD4 T-cell proliferation and decreased levels of IFN-gamma and IL-17. CD11b(+)Ly-6C(high)F4/80(+) BALF Mphi expanded by Gal-9 were responsible for the suppression. We further found in vitro that Gal-9, only in the presence of T. asahii, expands CD11b(+)Ly-6C(high)F4/80(+) cells from BM cells, and the cells suppress T-cell proliferation and IFN-gamma and IL-17 production. The present results indicate that Gal-9 expands immunosuppressive CD11b(+)Ly-6C(high) Mphi to ameliorate Th1/Th17 cell-mediated hypersensitivity pneumonitis.

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.

Proteinase and phospholipase activities and development at different temperatures of yeasts isolated from bovine milk.

The presence of yeasts in milk may cause physical and chemical changes limiting the durability and compromising the quality of the product. Moreover, milk and dairy products contaminated by yeasts may be a potential means of transmission of these microorganisms to man and animals causing several kinds of infections. This study aimed to determine whether different species of yeasts isolated from bovine raw milk had the ability to develop at 37°C and/or under refrigeration temperature. Proteinase and phospholipase activities resulting from these yeasts were also monitored at different temperatures. Five genera of yeasts (Aureobasidium sp., Candida spp., Geotrichum spp., Trichosporon spp. and Rhodotorula spp.) isolated from bovine raw milk samples were evaluated. All strains showed one or a combination of characteristics: growth at 37°C (99·09% of the strains), psychrotrophic behaviour (50·9%), proteinase production (16·81% of the strains at 37°C and 4·09% under refrigeration) and phospholipase production (36·36% of the isolates at 37°C and 10·9% under refrigeration), and all these factors may compromise the quality of the product. Proteinase production was similar for strains incubated at 37°C (16·81% of the isolates) and room temperature (17·27%) but there was less amount of phospholipase-producing strains at room temperature (15·45% of the isolates were positive) when compared with incubation at 37°C (36·36%). Enzymes production at 37°C by yeasts isolated from milk confirmed their pathogenic potential. The refrigeration temperature was found to be most efficient to inhibit enzymes production and consequently ensure better quality of milk. The viability of yeasts and the activity of their enzymes at different temperatures are worrying because this can compromise the quality of dairy products at all stages of production and/or storage, and represent a risk to the consumer.

Fungal F8-Culture Filtrate Induces Tomato Resistance against Tomato Yellow Leaf Curl Thailand Virus.

Tomato (Solanum lycopersicum) is an important economic crop worldwide. However, tomato production is jeopardized by the devastating tomato yellow leaf curl disease caused by whitefly-transmitted begomoviruses (WTBs). In this study, we evaluated the efficacy of our previously developed plant antiviral immunity inducer, fungal F8-culture filtrate, on tomato to combat tomato yellow leaf curl Thailand virus (TYLCTHV), the predominant WTB in Taiwan. Our results indicated that F8-culture filtrate treatment induced strong resistance, did not reduce the growth of tomato, and induced prominent resistance against TYLCTHV both in the greenhouse and in the field. Among TYLCTHV-inoculated Yu-Nu tomato grown in the greenhouse, a greater percentage of plants treated with F8-culture filtrate (43-100%) were healthy-looking compared to the H2O control (0-14%). We found that TYLCTHV cannot move systemically only on the F8-culture filtrate pretreated healthy-looking plants. Tracking the expression of phytohormone-mediated immune maker genes revealed that F8-culture filtrate mainly induced salicylic acid-mediated plant immunity. Furthermore, callose depositions and the expression of the pathogen-induced callose synthase gene, POWDERY MILDEW RESISTANT 4 were only strongly induced by TYLCTHV on tomato pretreated with F8-culture filtrate. This study provides an effective way to induce tomato resistance against TYLCTHV.