Unveiling Trichosporon austroamericanum sp. nov.: A Novel Emerging Opportunistic Basidiomycetous Yeast Species.

During an epidemiological survey, a potential novel species within the basidiomycetous yeast genus Trichosporon was observed. The clinical strain was obtained from a urine sample taken from a Brazilian kidney transplant recipient. The strain was molecularly identified using the intergenic spacer (IGS1) ribosomal DNA locus and a subsequent phylogenetic analysis showed that multiple strains that were previously reported by other studies shared an identical IGS1-genotype most closely related to that of Trichosporon inkin. However, none of these studies provided an in-depth characterization of the involved strains to describe it as a new taxon. Here, we present the novel clinically relevant yeast for which we propose the name Trichosporon austroamericanum sp. nov. (holotype CBS H-24937). T. austroamericanum can be distinguished from other siblings in the genus Trichosporon using morphological, physiological, and phylogenetic characters.

Identification of heparin-binding proteins expressed on Trichosporon asahii cell surface.

Trichosporon asahii is a pathogenic yeast that cause trichosporonosis. T. asahii exhibits several colony morphologies, such as white (W)- or off-white (O)-type, which may affect virulence. In this study, we compared the expression pattern of heparin-binding proteins in various colony morphologies and identified heparin-binding protein in T. asahii. Surface plasmon resonance analysis revealed that cell surface molecules attached more strongly to heparin in W- than O-type cells. We purified and identified a heparin-binding protein strongly expressed in W-type cells using heparin-Sepharose beads, named it heparin-binding protein 1 (HepBP1), and expressed Flag-tagged HepBP1 in mammalian cells. The heparin-binding ability of Flag-tagged HepBP1 was confirmed by pulldown assay using heparin-Sepharose beads. Thus, HepBP1 is a heparin-binding protein on T. asahii cell surface. These results suggest that several T. asahii cell surface proteins interact with glycosaminoglycans; therefore, they could contribute to infection.

Skin Manifestations of Micafungin Breakthrough Disseminated Trichosporonosis in Acute Megakaryoblastic Leukemia.

Disseminated trichosporonosis is a rare fungal infection whose risk factors are hematological malignancies and neutropenia. Recently, breakthrough Trichosporon infections after administration of micafungin, the first-line systemic antifungal agent in compromised hosts, have been widely recognized. A man in his seventies about 1 month into chemotherapy for acute megakaryoblastic leukemia presented with a worsening fever and dyspnea. The patient was being administered with empirical micafungin therapy for suspected candidiasis. As the symptoms progressed, scattered erythema appeared on the trunk, some with a dark red vesicle at the center. Blood cultures identified Trichosporon asahii, as did the specimen of the skin biopsy. On the basis also of the presence of pneumonia on chest computed tomography, we confirmed the diagnosis of disseminated trichosporonosis and changed the antifungal agent from micafungin to voriconazole. Blood culture turned out to be negative 1 month after administrating voriconazole. However, the patient died of the leukemia. Our review of previous reports on cutaneous manifestations of disseminated trichosporonosis revealed that despite their morphological diversity, erythema with a red papule or vesicle at the center, implying necrosis, was also observed in previous cases. Our case report suggests that dermatologists should be aware of skin manifestations of disseminated trichosporonosis after micafungin administration, especially in cases of hematological malignancies.

Genetic analysis of emerging fungal pathogens: Trichosporon asahii.

Trichosporon asahii is an emerging opportunistic fungus that mainly causes fatal disseminated trichosporonosis, especially in immunocompromised patients. T. asahii infection has been reported in Thailand, but few studies of this fungus have been published. Therefore, this study investigated the genetic diversity of 51 clinical strains of T. asahii from urine samples in Thailand. We sequenced and characterized the beta-1-tubulin (TUB1), copper-exporting ATPase (ATP), phosphate carrier protein (PHCP), and topoisomerase-1 (TOP1) genes. In addition, intergenic spacer 1 (IGS1) sequences from our previous studies were investigated. The numbers of haplotypes were 3, 3, 2, 2, and 2 for IGS1, TUB1, ATP, PHCP, and TOP1, respectively. The results suggested a relatively low level of genetic diversity among the strains. The findings illustrated that IGS1, TUB1, ATP, PHCP, and TOP1 can be collectively used as an alternative molecular typing tool for investigating the population diversity and structure of T. asahii.

Nasal trichosporonosis in 2 mixed-breed ewes.

Two adult mixed-breed ewes were presented with a 2-wk history of upper respiratory disease. Both animals were depressed, with bilateral serosanguineous nasal discharge and harsh bronchovesicular sounds accompanied by crackles and wheezes on auscultation. One animal was recumbent and was euthanized at presentation. The other animal with similar signs, as well as exophthalmos, was euthanized because of a mass in the nasal passages. On autopsy, severe pyogranulomatous and necrotizing ethmoidal rhinitis with focal pyogranulomatous pneumonia was diagnosed in both animals. An intralesional fungal organism was identified in the nares and lungs of both animals. The organism could not be isolated via fungal culture but was identified as Trichosporon sp. by a PCR assay. Trichosporon spp. are rarely associated with disease in veterinary medicine. This ubiquitous fungus might cause disease following trauma to the nasal passages or secondary to immunocompromise.

Interaction of Host Proteins with Cell Surface Molecules of the Pathogenic Yeast Trichosporon asahii.

Trichosporon asahii is an invasive pathogenic yeast that infects immunocompromised hosts. Several virulence factors contribute to the fungal infection; however, the factors that contribute to the occurrence of T. asahii infections remain unclear. Since adhesins are typical virulence factors reported for pathogenic fungi, we looked for host proteins that interact with the T. asahii cell surface. T. asahii and Candida albicans were used for screening using a pull-down assay with fetal bovine serum. Serum albumin and elongation factor 2 were identified as the yeast-binding serum proteins. Additionally, we investigated the interactions of the cell surface-associated molecules (CSM) of T. asahii with vitronectin (VTN), fibronectin, fetuin-A, and alpha-1antitrypsin (AAT). The surface plasmon resonance (SPR) method was used to examine the interaction between CSM and human proteins. On the other hand, the pull-down assay was used to examine the interaction between human proteins and the T. asahii cell surface. Serum albumin, AAT, and VTN were found to interact with T. asahii in both SPR and pull-down assays. This study identified several proteins that interact with T. asahii, suggesting that these proteins play a role in infection mechanisms.

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.

Identification of Clinical Trichosporon asteroides Strains by MALDI-TOF Mass Spectrometry: Evaluation of the Bruker Daltonics Commercial System and an In-House Developed Library.

Trichosporon asteroides is an emerging yeast-like pathogen commonly misidentified by commercial biochemical identification systems. We evaluated the performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the identification of 21 clinical T. asteroides strains using the Bruker Daltonics database (BDAL) and an in-house developed library. Mass spectra were obtained by the FlexControl system v.3.4, and characterizations were performed in the Biotyper BDAL database v.4.1 and the developed in-house library. Species identification for T. asteroides failed as all 21 strains were misidentified as T. japonicum (log-scores 1.89-2.19). Extending the existing database was crucial to achieving 100% correct species-level identification and accurate distinction between species. Our results indicate that the commercial BDAL database has no discriminatory power to distinguish between T. japonicum and T. asteroides. Whereas improvement of the current BDAL database is pending, we strongly advise system users not to exclude the possibility of the failure to report T. asteroides.

Hyphal Growth in Trichosporon asahii Is Accelerated by the Addition of Magnesium.

Fungal dimorphism involves two morphologies: a unicellular yeast cell and a multicellular hyphal form. Invasion of hyphae into human cells causes severe opportunistic infections. The transition between yeast and hyphal forms is associated with the virulence of fungi; however, the mechanism is poorly understood. Therefore, we aimed to identify factors that induce hyphal growth of Trichosporon asahii, a dimorphic basidiomycete that causes trichosporonosis. T. asahii showed poor growth and formed small cells containing large lipid droplets and fragmented mitochondria when cultivated for 16 h in a nutrient-deficient liquid medium. However, these phenotypes were suppressed via the addition of yeast nitrogen base. When T. asahii cells were cultivated in the presence of different compounds present in the yeast nitrogen base, we found that magnesium sulfate was a key factor for inducing cell elongation, and its addition dramatically restored hyphal growth in T. asahii. In T. asahii hyphae, vacuoles were enlarged, the size of lipid droplets was decreased, and mitochondria were distributed throughout the cell cytoplasm and adjacent to the cell walls. Additionally, hyphal growth was disrupted due to treatment with an actin inhibitor. The actin inhibitor latrunculin A disrupted the mitochondrial distribution even in hyphal cells. Furthermore, magnesium sulfate treatment accelerated hyphal growth in T. asahii for 72 h when the cells were cultivated in a nutrient-deficient liquid medium. Collectively, our results suggest that an increase in magnesium levels triggers the transition from the yeast to hyphal form in T. asahii. These findings will support studies on the pathogenesis of fungi and aid in developing treatments. IMPORTANCE Understanding the mechanism underlying fungal dimorphism is crucial to discern its invasion into human cells. Invasion is caused by the hyphal form rather than the yeast form; therefore, it is important to understand the mechanism of transition from the yeast to hyphal form. To study the transition mechanism, we utilized Trichosporon asahii, a dimorphic basidiomycete that causes severe trichosporonosis since there are fewer studies on T. asahii than on ascomycetes. This study suggests that an increase in Mg2+, the most abundant mineral in living cells, triggers growth of filamentous hyphae and increases the distribution of mitochondria throughout the cell cytoplasm and adjacent to the cell walls in T. asahii. Understanding the mechanism of hyphal growth triggered by Mg2+ increase will provide a model system to explore fungal pathogenicity in the future.

Aspects related to biofilm production and antifungal susceptibility of clinically relevant yeasts of the genus Trichosporon.

Trichosporonosis corresponds to a systemic fungal disease that leads to high mortality rates and is frequently associated with medical devices. It affects immunosuppressed patients in particular and is strongly linked to acquired human immunodeficiency, organ and tissue transplants, and malignant hematologic diseases such as leukemia and lymphomas. Trichosporon infections have been increasingly reported worldwide; however, little information is available either about their characteristics or the causative microorganism. Thus, the aims of the present study were: to investigate 59 yeasts of the genus Trichosporon by verifying the biofilm formation capacity of isolates; to analyze the susceptibility patterns of planktonic cells against the antifungals fluconazole, itraconazole, amphotericin-B, voriconazole, and caspofungin by comparing European Committee for Antimicrobial Susceptibility Testing (EUCAST) broth microdilution technique with the commercial method Etest; and to assess the susceptibility patterns of biofilm cells (sessile) against the same antifungals through broth microdilution. The ability to form biofilm on the surface of polystyrene plates was noted for all isolates, and 54.3% of samples were considered strong producers. Comparison between the antifungal susceptibility techniques evidenced that Etest showed higher and discordant minimum inhibitory concentrations (MICs) from those obtained by the microdilution method, especially for fluconazole, itraconazole, and caspofungin. Considering the susceptibility of biofilms, most species had high MIC50 and MIC90 against the tested antifungals, showing 4-to-66-fold higher concentrations for amphotericin B and 2-to-33-fold greater concentrations for caspofungin. These results highlight the importance of further studies with Trichosporon spp. for comparison between laboratory findings and in vivo response, considering both the susceptibility tests and the behavior of biofilm cells against drugs.

This study investigated 59 isolates of the medically important yeast Trichosporon in relation to their ability to form biofilms and the susceptibility of biofilms to antifungal agents. All isolates were able to produce biofilms and biofilms showed lower antifungal susceptibility.

Genotyping, antifungal susceptibility testing, and biofilm formation of Trichosporon spp. isolated from urine samples in a University Hospital in Bangkok, Thailand.

The basidiomycetes yeast Trichosporon is widespread in the natural environment, but can cause disease, mainly in immunocompromised patients. However, there have been only few studies about this infection in Thailand. In this study, we characterized 53 Trichosporon spp. isolated from urine samples from patients admitted to a single hospital in Bangkok, Thailand over a one-year period from 2019 to 2020. The strains were identified using colony morphology, microscopy, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and nucleotide sequence analysis of intergenic spacer 1 (IGS1). Fifty-one isolates were Trichosporon asahii, and the remaining isolates were Trichosporon inkin and other Trichosporon species. Three genotypes of IGS1-1, 3, and 7 were observed among T. asahii. The sensitivity of the yeasts to the antifungal drugs amphotericin B, fluconazole, and voriconazole ranged from 0.25 to >16 µg ml-1, 0.5-8 µg ml-1, and 0.01-0.25 µg ml-1, respectively. We investigated biofilm formation by the isolates, and no biofilm production was found in one isolate, low biofilm production in forty-four isolates, and medium biofilm production in six isolates. T. inkin produced biofilms at low levels, and Trichosporon spp. produced biofilms at medium levels. This research increases our understanding of the molecular epidemiology of Trichosporon spp. isolated from one university hospital in Bangkok, Thailand, and reveals their genetic diversity, antifungal susceptibility profiles, and capacity for in vitro biofilm production.

Possible Trichosporon asahii urinary tract infection in a critically ill COVID-19 patient.

BACKGROUND: Trichosporon asahii, an emerging fungal pathogen, has been frequently associated with invasive infections in critically ill patients. CASE REPORT: A 74-year-old male patient diagnosed with COVID-19 was admitted to an Intensive Care Unit (ICU). During hospitalization, the patient displayed episodes of bacteremia by Staphylococcus haemolyticus and a possible urinary tract infection by T. asahii. While the bacterial infection was successfully treated using broad-spectrum antibiotics, the fungal infection in the urinary tract was unsuccessfully treated with anidulafungin and persisted until the patient died. CONCLUSIONS: With the evolving COVID-19 pandemic, invasive fungal infections have been increasingly reported, mainly after taking immunosuppressant drugs associated with long-term broad-spectrum antibiotic therapy. Although Candida and Aspergillus are still the most prevalent invasive fungi, T. asahii and other agents have emerged in critically ill patients. Therefore, a proper surveillance and diagnosing any fungal infection are paramount, particularly in COVID-19 immunocompromised populations.

Trichosporon asahii causing subcutaneous mycoses in an immunocompetent patient: case report and a minireview.

Trichosporon spp. are a constituent of the normal flora of humans that can cause both superficial and invasive infections, mainly in immunocompromised and immunocompetent hosts, respectively. Herein, we a report of Trichosporon asahii causing subcutaneous fungal infection (SFI) in an immunocompetent patient after carpal tunnel surgery. Although susceptible to fluconazole, the treatment of SFI failed even using high doses of this azole. The skin lesion improved following the administration of voriconazole. We conducted a literature minireview searching reports on SFI in immunocompetent patients to check for epidemiological, diagnostic, therapeutic, and outcome characteristics. A total of 32 cases were reported. Despite being uncommon, the clinical suspicion and early diagnosis of SFI in immunocompetent patients undergoing previous surgery are important. Our study indicated that the azoles are the most active antifungal agents against Trichosporon spp., except for fluconazole, and voriconazole can be considered the first therapeutic option.

JMM Profile: Trichosporon yeasts: from superficial pathogen to threat for haematological-neutropenic patients.

Trichosporon yeasts are classical agents of superficial mycoses, and they are ranked as the first to second predominant basidiomycetous yeast able to cause invasive infections. The clinical presentation of Trichosporon infections varies with the affected anatomical site, with fungaemia present in the majority of invasive trichosporonosis cases. Only a limited number of antifungal compounds can be used to treat Trichosporon infections. Azoles are the first choice due to their intrinsic resistance to echinocandins. Better laboratory methods and up-to-date databases of commercial platforms are required to improve identification, susceptibility testing and surveillance of this potentially threating infection.

Development of an efficient gene-targeting system for elucidating infection mechanisms of the fungal pathogen Trichosporon asahii.

Trichosporon asahii is a pathogenic fungus that causes severe, deep-seated fungal infections in neutropenic patients. Elucidating the infection mechanisms of T. asahii based on genetic studies requires a specific gene-targeting system. Here, we established an efficient gene-targeting system in a highly pathogenic T. asahii strain identified using the silkworm infection model. By comparing the pathogenicity of T. asahii clinical isolates in a silkworm infection model, T. asahii MPU129 was identified as a highly pathogenic strain. Using an Agrobacterium tumefaciens-mediated gene transfer system, we obtained a T. asahii MPU129 mutant lacking the ku70 gene, which encodes the Ku70 protein involved in the non-homologous end-joining repair of DNA double-strand breaks. The ku70 gene-deficient mutant showed higher gene-targeting efficiency than the wild-type strain for constructing a mutant lacking the cnb1 gene, which encodes the beta-subunit of calcineurin. The cnb1 gene-deficient mutant showed reduced pathogenicity against silkworms compared with the parental strain. These results suggest that an efficient gene-targeting system in a highly pathogenic T. asahii strain is a useful tool for elucidating the molecular mechanisms of T. asahii infection.

MALDI-TOF MS characterisation, genetic diversity and antifungal susceptibility of Trichosporon species from Iranian clinical samples.

BACKGROUND: Trichosporonosis is an emerging fungal infection caused by Trichosporon species, a genus of yeast-like fungi, which are frequently encountered in human infections ranging from mild cutaneous lesions to fungemia in immunocompromised patients. The incidence of trichosporonosis has increased in recent years, owing to higher numbers of individuals at risk for this infection. Although amphotericin B, posaconazole and isavuconazole are generally effective against Trichosporon species, some isolates may have variable susceptibility to these antifungals. OBJECTIVES: Herein, we evaluated the species distribution, genetic diversity and antifungal susceptibility profiles of Trichosporon isolates in Iran. METHODS: The yeasts were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Phylogenetic analysis was performed based on amplified fragment length polymorphism (AFLP). The in vitro susceptibilities of eight antifungal agents were analysed using the Clinical and Laboratory Standards Institute broth microdilution methods. RESULTS: The isolates belonged to the species T asahii (n = 20), T japonicum (n = 4) and T faecale (n = 3). A dendrogram of the AFLP analysis demonstrated that T asahii and non-asahii Trichosporon strains (T japonicum and T faecale) are phylogenetically distinct. While voriconazole was the most active agent (GM MIC = 0.075 µg/ml), high fluconazole MICs (8 µg/ml) were observed for a quarter of Trichosporon isolates. The GM MIC value of amphotericin B for T asahii and non-asahii Trichosporon species was 0.9 µg/ml. CONCLUSIONS: The distribution and antifungal susceptibility patterns of the identified Trichosporon species could inform therapeutic choices for treating these emerging life-threatening fungi.

Implication of efflux pumps and ERG11 genes in resistance of clinical Trichosporon asahii isolates to fluconazole.

Introduction. Trichosporon asahii has been recognized as an opportunistic agent having a limited sensitivity to antifungal treatment.Hypothesis/Gap Statement. Molecular mechanisms of azole resistance have been rarely reported for Trichosproron asahii. Similar to other fungi, we hypothesized that both ERG11 gene mutation and efflux pumps genes hyper-expression were implicated.Aim. The current work aimed to study the sensitivity of clinical T. asahii isolates to different antifungal agents and to explore their resistance mechanisms by molecular methods including real-time PCR and gene sequencing.Methods. The sensitivity of T. asahii isolates to fluconazole, amphotericin B and voriconazole was estimated by the Etest method. Real-time PCR was used to measure the relative expression of Pdr11, Mdr and ERG11 genes via the ACT1 housekeeping gene. Three pairs of primers were also chosen to sequence the ERG11 gene. This exploration was followed by statistical study including the receiver operating characteristic (ROC) curve analysis to identify a relationship between gene mean expression and the sensitivity of isolates.Results. In 31 clinical isolates, the resistance frequencies were 87, 16.1 and 3.2 %, respectively, for amphotericin B, fluconazole and voriconazole. Quantitative real-time PCR demonstrated that only Mdr over-expression was significantly associated with FCZ resistance confirmed by univariate statistical study and the ROC curve analysis (P <0.05). The ERG11 sequencing revealed two mutations H380G and S381A in TN325U11 (MIC FCZ=8 µg ml-1) and H437R in TN114U09 (MIC FCZ=256 µg ml-1) in highly conserved regions (close to the haem-binding domain) but their involvement in the resistance mechanism has not yet been assigned.Conclusion. T. asahii FCZ resistance mechanisms are proven to be much more complex and gene alteration sequence and/or expression can be involved. Only Mdr gene over-expression was significantly associated with FCZ resistance and no good correlation was observed between FCZ and VCZ MIC values and relative gene expression. ERG11 sequence alteration seems to play a major role in T. asahii FCZ resistance mechanism but their involvement needs further confirmation.

Specific microRNA/mRNA expression profiles and novel immune regulation mechanisms are induced in THP-1 macrophages by in vitro exposure to Trichosporon asahii.

BACKGROUND: Trichosporon asahii is considered the most prominent species associated with invasive trichosporonosis, but little is known about the pathogenesis of T. asahii infection in the host. MicroRNAs (miRNAs) are a class of noncoding endogenous small RNAs that play vital roles by manipulating immune responses against pathogenic microorganisms. Nevertheless, the exact functions of miRNAs in T. asahii infection are still unknown. OBJECTIVE: To investigate the interactions involved in the miRNA immune response in THP-1 macrophages following in vitro exposure to T. asahii. METHODS: We utilized next-generation sequencing to detect differentially expressed (DE) miRNAs and mRNAs in THP-1 cells after 24 h of in vitro exposure to T. asahii. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to verify the sequencing results. The miRNA-mRNA regulatory network was constructed with the DE miRNAs and DE mRNAs. We performed Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis of the predicted targeting mRNAs in the miRNA-mRNA network. A dual-luciferase reporter assay and enzyme-linked immunosorbent assay (ELISA) were utilized to demonstrate the reliability of the miR-342-3p/Dectin-1 pair. RESULTS: A total of 120 DE miRNAs and 588 DE mRNAs were identified after 24 h of in vitro exposure to T. asahii. The miRNA-mRNA regulatory network was constructed with 39 DE miRNAs and 228 DE mRNAs. KEGG pathway analysis revealed that the up-regulated DE mRNAs in the complex interaction network were mainly involved in immune-related pathways. In addition, we verified the target relationship between miR-342-3p and Dectin-1 and found that miR-342-3p could promote the expression of TNF-α and IL-6 by negatively regulating Dectin-1. CONCLUSIONS: This study evaluated the expression profiles of miRNA/mRNA and revealed the immunological consequences of THP-1 macrophages in response to T. asahii exposure. Moreover, our data suggest that miR-342-3p can indirectly promote inflammatory responses and may be a potential therapeutic target against trichosporonosis.