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.

Effect of resveratrol and Regrapex-R-forte on Trichosporon cutaneum biofilm.

Microorganisms that cause chronic infections exist predominantly as surface-attached stable communities known as biofilms. Microbial cells in biofilms are highly resistant to conventional antibiotics and other forms of antimicrobial treatment; therefore, modern medicine tries to develop new drugs that exhibit anti-biofilm activity. We investigated the influence of a plant polyphenolic compound resveratrol (representative of the stilbene family) on the opportunistic pathogen Trichosporon cutaneum. Besides the influence on the planktonic cells of T. cutaneum, the ability to inhibit biofilm formation and to eradicate mature biofilm was studied. We have tested resveratrol as pure compound, as well as resveratrol in complex plant extract-the commercially available dietary supplement Regrapex-R-forte, which contains the extract of Vitis vinifera grape and extract of Polygonum cuspidatum root. Regrapex-R-forte is rich in stilbenes and other biologically active substances. Light microscopy imaging, confocal microscopy, and crystal violet staining were used to quantify and visualize the biofilm. The metabolic activity of biofilm-forming cells was studied by the tetrazolium salt assay. Amphotericin B had higher activity against planktonic cells; however, resveratrol and Regrapex-R-forte showed anti-biofilm effects, both in inhibition of biofilm formation and in the eradication of mature biofilm. The minimum biofilm eradicating concentration (MBEC80) for Regrapex-R-forte was found to be 2222 mg/L (in which resveratrol concentration is 200 mg/L). These methods demonstrated that Regrapex-R-forte can be employed as an anti-biofilm agent, as it has similar effect as amphotericin B (MBEC80 = 700 mg/L), which is routinely used in clinical practice.

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 asahii secretes a 30-kDa aspartic peptidase.

Trichosporon asahii is a fungal opportunistic pathogen that causes superficial and deep-seated infections presenting high mortality. Very little is known about the virulence attributes produced by this fungus. Herein, aspartic peptidase production was identified in Brazilian clinical isolates of T. asahii by different methodologies. Initially, T. asahii strain 250 (from skin lesion) was inoculated in both liquid and solid culture media containing bovine serum albumin (BSA) as the sole nitrogenous source. A translucent halo around the fungal colony was observed from the 5th day of culture. The cell-free culture supernatant revealed that soluble BSA was hydrolyzed along the growth, generating low molecular mass polypeptides as observed by electrophoresis. Subsequently, the secretions from four clinical strains of T. asahii were analyzed by BSA-SDS-PAGE and a single proteolytic band of 30-kDa was detected under acidic pH at 37°C. The secreted aspartic peptidase of T. asahii efficiently cleaved the cathepsin D peptide substrate, but not the substrates with specificity to HIV-1 peptidase and rennin. The capability to cleave either cathepsin D substrate in a fluorogenic assay or BSA immobilized within a gel matrix varied according to the T. asahii isolate. T. asahii extracellular peptidase activity was strongly inhibited by pepstatin A and HIV peptidase inhibitors, classifying it as an aspartic-type peptidase. Human serum albumin, mucin, non-immune immunoglobulin G and gelatin induced, in different levels, the secretion of this aspartic peptidase. With these results, T. asahii must be included in the list of many human fungal opportunistic pathogens able to secrete an aspartic-type peptidase.

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.

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.

Inhibitory effect of essential oils against Trichosporon ovoides causing Piedra Hair Infection

Piedra, is an asymptomatic fungal infection of the hair shaft, resulting in the formation of nodules of different hardness on the infected hair. The infection also known as Trichomycosis nodularis is a superficial fungal infection arising from the pathogen being restricted to the stratum corneum with little or no tissue reaction. The nodules are a concretion of hyphae and fruiting bodies of the fungus. Two varieties of Piedra may be seen, Black Piedra and White Piedra. The fungus Trichosporon ovoides is involved in the occurrence of both types of Piedras. The purpose of this study was to examine the effectiveness of selected essential oils for the control of growth of the fungus and to determine whether the antifungal effect was due to the major compounds of the oils. Two screening methods viz. Agar well diffusion assay and Minimum Inhibitory Concentration were adopted for the study. MIC and MFC were determined by tube dilution method. Essential oils from Eucalyptus, Ocimum basilicum, Mentha piperita, Cymbopogon flexuosus, Cymbopogon winterians, Trachyspermum ammi, Zingiber officinalis, Citrus limon, Cinnamomon zeylanicum, Salvia sclarea, Citrus aurantifolia, Melaleuca alternifolia, Citrus aurantium, Citrus bergamia, Pogostemon pathchouli, Cedrus atlantica, Jasminum officinale, Juniperus communis, Abelmoschus moschatus, Cyperus scariosus, Palargonium graveolens, Boswellia carterii, Rosa damascene, Veteveria zizanoides and Commiphora myrrha were evaluated. The essential oils of Cymbopogon winterians, Mentha piperita, Cinnamomum zeylanicum, Melaleuca alternifolia and Eucalyptus globulus were proved to be most effective against the fungus Trichosporon ovoides.

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.

[Microbial oil production by Trichosporon cutaneum B3 using cassava starch].

Microbial oil, as raw material for biodiesel, can be produced by Trichosporon cutaneum B3 using cassava starch hydrolysate. Batch cultures demonstrated that there was little inhibitory effect with the concentration of cassava starch hydrolysate up to 90 g/L. The favorable initial pH, C/N molar ratio, nitrogen source and its concentration were 6.0, 116, yeast extract and 3.0 g/L, respectively. Under the optimized conditions, dry biomass reached 15.2 g/L and lipid content reached 40.9% after culture for 144 h in flask. Batch cultures in a 2 L stirred-tank fermenter were run for 44 h and resulted in dry biomass, lipid content and lipid yield of 28.7 g/L, 42.8% and 12.27 g/L, respectively. The chemical compositions of biodiesel prepared from lipids of T cutaneum B3 mainly included palmitic acid methyl ester, stearic acid methyl ester, oleic acid methyl ester and linoleic acid methyl ester etc., and its main physicochemical properties were in compliance with relevant national diesel standards. Therefore, the biodiesel prepared from lipids of T cutaneum B3 can serve as a potential fossil fuel alternatives.

Antifungal susceptibility profile of trichosporon isolates: correlation between CLSI and etest methodologies

The aim of the present study was to evaluate the antifungal susceptibility profile of Trichosporon species isolated from different sources employing the Clinical and Laboratory Standards Institute (CLSI) method and E-test method. Thirty-four isolates of Trichosporon spp. and six CBS reference samples were tested for their susceptibility to Amphotericin B, 5-flucytosine, Fluconazole, Itraconazole, Voriconazole and Terbinafine. All species showed high Minimun Inhibitory Concentrations (MIC) for Itraconazole and susceptibility to Fluconazole, The comparison among the results obtained by the CLSI method and E-test revealed larger discrepancies among 5-flucytosine and Itraconazole. The present work provides epidemiological data that could influence therapeutic choices. Furthermore, the comparison between different methodologies could help to analyze results obtained by different laboratories.

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.

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.

Glucose exerts a negative effect over a peroxidase from Trichosporon asahii, with carotenoid cleaving activity.

Tobacco aroma compounds were generated via lutein cleavage by the combined action of a yeast and a bacterium identified as Trichosporon asahii and Paenibacillus amylolyticus, respectively. In this study, an inverse relationship between glucose concentration and the generation of three compounds, present in the tobacco aroma profile, was observed in mixed cultures. In order to identify the organism sensitive to the sugar effect, both were grown separately. The presence of glucose suppressed beta-ionone production by T. asahii grown with lutein. However, the biotransformation of the ionone into its reduced derivatives (7,8-dihydro-beta-ionone and 7,8-dihydro-beta-ionol) by P. amylolyticus was not affected by the sugar. This pointed to the cleavage of lutein, a step within the process necessary for the synthesis of beta-ionone, as the target of the glucose effect. In vitro studies with crude extracts and concentrated cell-free medium derived from T. asahii cultures showed that the carotenoid breakdown activity was located extracellularly and only detected in supernatants from yeast cells grown in the absence of the sugar. Rather than an inhibition or a mechanism affecting the enzyme secretion, the glucose effect on lutein degradation comprised another regulatory level. Further experiments showed that the enzyme responsible for lutein breakdown and susceptible to the sugar effect exhibited a high degree of identity to fungal peroxidases, studied as well, for their involvement in carotenoid cleavage.

Bioconversion of lutein using a microbial mixture--maximizing the production of tobacco aroma compounds by manipulation of culture medium.

The generation of aroma compounds by carotenoid cleavage in the 9-10 position was studied, due to the importance of these compounds in the flavor and fragrance industry. The bioconversion of the carotenoid lutein to C(13) norisoprenoids utilizing a microbial mixture composed of Trichosporon asahii and Paenibacillus amylolyticus was carried out by a fermentation process. Applying an experimental design methodology, the effects of nutritional factors on the production of aroma compounds present in the tobacco profile were studied. After an assessment of the significance of each nutritional factor, the levels of the variables yielding the maximum response were calculated. Glucose, tryptone, and yeast extract exerted a strong negative effect over the objective function, with glucose being the strongest. Lutein possessed a positive effect over the tobacco aroma production, while sodium chloride and trace elements showed no influence over the process. The yield attained after culture medium manipulation was almost ten-fold higher, compared with the base medium; and the aroma mixture was characterized as: 7,8-dihydro-beta-ionol (95.2%), 7,8-dihydro-beta-ionone (3.7%), and beta-ionone (1.1%).

Disseminated trichosporonosis in China.

A 20-year-old female patient presented with erythematous plaques on the nose which were progressively spreading to the trunk and the extremities, sometimes with erosions and scars. The patient was misdiagnosed as having seborrhoeic dermatitis and subacute cutaneous lupus erythematosus. The histopathological biopsy revealed mycotic infectious granuloma. Samples taken from skin lesions and other locations grew Trichosporon asahii in cultures. The identification was confirmed by molecular biological methods. The patient was treated successfully with liposomal amphotericin B in combination with fluconazole orally.

Experimental model of progressive disseminated trichosporonosis in mice with latent trichosporonemia.

Trichosporon asahii and Trichosporon mucoides are the most common strains of fungi that cause disseminated trichosporonosis, a severe opportunistic infection in immunocompromised hosts. We have previously established a nested PCR assay using serum samples for detection of both strains. Here we describe a new experimental animal model for investigating the underlying mechanisms of disseminated trichosporonosis. T. asahii (OMU239, a clinical isolate from a patient with acute myelogenous leukemia) and 8-week-old ICR male mice were used in all experiments. A suspension of T. asahii (3 x 10(6) CFU/animal) was injected into the caudal vein of each mouse after immunosuppression with cyclophosphamide (200 mg/kg of body weight/day for 2 days) and prednisolone (30 mg/kg/day for 1 day). Mice were then divided into four subgroups (R0, R1, R2, and R3) based on the time of reimmunosuppression. The latter was performed using the same drugs 1 week (group R1), 2 weeks (group R2), and 3 weeks (group R3) after fungal infection. Reimmunosuppression was not performed in group R0. The 5-week-survival rates of mice after T. asahii infection were 0% for group R1, 50% for group R2, 80% for group R3, and 80% for group R0. There was a significant difference in the survival rates between group R1 and either group R0 or R3 (P < 0.05). Fungal clearance in peripheral blood and various organs of group R1 and R2 was delayed relative to that of group R0 but was similar to the control in group R3 in spite of reimmunosuppression. Our results suggest that the critical period for the development of disseminated trichosporonosis in our model is shorter than 3 weeks after T. asahii infection. We concluded that mice during this critical period were in a state of latent trichosporonemia. Comparison of the survival rates suggests that the nested PCR assay was more useful than blood culture and glucuronoxylomannan antigen assay in the detection of this latent trichosporonemia.

Fluconazole therapy in a rhesus monkey (Macaca mulatta) with epidural Trichosporon beigelii in a cephalic recording cylinder.

An adult female rhesus monkey with a cephalic recording cylinder surgically implanted over a craniotomy site developed cloudy cylinder fluid and a white gelatinous plaque on the epidural capsule surface. Results of baseline hematologic, serum biochemical, and cerebrospinal fluid analysis and simian retrovirus panel were unremarkable. Aerobic culture of the cylinder fluid yielded a pure culture of Trichosporon beigelii. This organism is a ubiquitous saprophytic fungus that is a potential pathogen, especially in the immunocompromised host. Factors that could have contributed to the infection included a microenvironment in the cylinder that was favorable to fungal growth, and presence of a dural pseudocapsule of collagen and granulation tissue in the implant which could have inhibited cellular defense mechanisms. An intravenous formulation of fluconazole was selected for direct application into the recording cylinder on the basis of safety and efficacy. Fluconazole is a highly water-soluble, metabolically stable bis-triazole antifungal with excellent cerebrospinal fluid penetration and low toxicity. A 4-week course of treatment eliminated Trichosporon organisms from the cylinder. Change to oral administration of fluconazole was made at that time to allow use of cephalic cylinder antibiotics that are incompatible with fluconazole. Further treatment with fluconazole was continued orally for 3 more months to prevent fungal recrudescence. Culture of cylinder fluid was performed periodically for 6 months after resolution, and results remained negative for T. beigelii. This case is believed to be the first reported T. beigelii infection in a non-human primate. Fluconazole was effective in eliminating the infection from the cylinder and preventing its recurrence.

Trichosporon on humans: a practical account.

Six human pathogenic Trichosporon species are described with respect to criteria for routine identification, epidemiology and clinical origin: T. ovoides, T. inkin, T. asahii, T. asteroides (Fissuricella filamenta), T. cutaneum, and T. mucoides. These species are causative agents of white piedra and cutaneous infections and are involved in systemic, localized or disseminated mycoses, particularly in patients with underlying haematological malignancy. Data on in vitro sensitivity to antifungal drugs are provided.

Disseminated trichosporonosis in a neutropenic murine model.

Life-threatening disseminated infection with Trichosporon beigelii (trichosporonosis) is a rare mycosis most commonly seen in patients with hematologic malignancies made neutropenic by cytotoxic therapy. This infection is usually resistant to conventional antifungal therapies. Poor correlation between therapeutic outcome of trichosporonosis and in vitro susceptibility of clinical isolates of T. beigelii to antifungal agents is often reported. To obtain a better understanding of its pathogenesis, and to aid in the future study of the therapy of this disease, a murine model of trichosporonosis was developed. The in vitro growth of clinical isolates of T. beigelii was first studied. Subsequently, mice made neutropenic with cyclophosphamide were inoculated intravenously with the fungus to produce the disease model. Inoculum size which produced 100% mortality, yet allowed an apparent therapeutic window (6 x 10(6)) was determined. Tissue distribution and burden of organism during the course of infection was examined by viability and histopathologic studies. T. beigelii disseminated rapidly in this model, involving numerous organs including the heart, brain, kidneys, lungs, and liver. The heart and kidneys of the infected animals showed evidence of infection as early as 6 hours following inoculation. Further understanding of the pathogenesis of trichosporonosis in the neutropenic host was imparted by this study. This will aid in the future study of antibiotic treatment of this disease and its untreated progression.