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. Supplementary Information The online version contains supplementary material available at 10.1007/s11046-024-00851-4.


Introduction
The genus Trichosporon encompasses anamorphic basidiomycetous yeast-like fungi recognized for their remarkable adaptations to different hosts and habitats, including plants, soils, mammals, and human beings [1][2][3][4].Particularly in human hosts, these yeasts may be part of the commensal microbiota and some species can cause a wide range of superficial and invasive infections in susceptible patients [5][6][7][8].
Since its first description in 1865 by the German physician Hermann Beigel, the taxonomy of Trichosporon has undergone extensive revisions.Initially, the genus contained both ascomycetous and basidiomycetous yeasts that asexually reproduced with arthroconidia [4,9].For several decades, the systematics of this genus heavily relied on morphological, biochemical, and physiological characteristics, resulting in potential conflicts in genus and species identification [4,[10][11][12][13].In 2015, a phylogenetic classification framework was proposed for the families and genera within the Tremellomycetes, resulting in substantial steps toward understanding the taxonomy of Trichosporon, especially in terms of differentiating at genus and species levels [14,15].Consequently, most of the 51 species previously classified as Trichosporon species were reclassified or accommodated into proposed or amended genera within the Trichosporonaceae family, such as Cutaneotrichosporon, Apiotrichum, Haglerozyma, Effuseotrichosporon and Pascua [14][15][16][17].
In this study we describe a novel yeast species closely related to T. inkin, for which we proposed the name Trichosporon austroamericanum, a fungal pathogen cultured from a urine sample obtained by an ultrasound-guided percutaneous puncture performed in a Brazilian kidney transplant recipient with an abdominal collection secondary to urinary fistula.

Case Description
The yeast culture L9630 (= CBS 17435, ex-type) was initially isolated in 2013 from a urine sample collected intraoperatively during a surgery performed to correct a urinary fistula complicating the ureterovesical anastomosis of a 15-year-old female kidney transplant patient who was hospitalized at the Kidney and Hypertension Hospital in São Paulo, Brazil.
Fungal elements were abundantly observed in the urine sediment during a wet-mount examination, and the sample was subsequently cultured on routine microbiological media, including Sabouraud dextrose agar (Difco, BD, Franklin Lakes, NJ, USA).The culture media were incubated at 37 °C for 48 h, resulting in the growth of dry, creamy white colonies.The strain was determined as Trichosporon inkin by using the VITEK2 system (BioMérieux, Marcy l'Etoile, France).The strain was sent to Laboratório Especial de Micologia, UNIFESP, Brazil, for further analysis.Phylogenetic analysis of the intergenic spacer region (IGS1) of the ribosomal DNA placed the strain in a clade most closely related to T. inkin.The patient was successfully treated with fluconazole (400 mg/day) and was discharged from hospital 14 days after start of antifungal therapy.Additional Trichosporon Strains An additional set of strains was included in our analysis, which was composed of: (a) six clinical T. inkin isolates stored in our Yeast Collection at LEMI-UNIFESP.The strains were collected from six patients admitted to different Brazilian medical centers between 2013 and 2019.They were selected for analysis because they fell within the same IGS1clade as strain L9630 (= CBS 17435, ex-type); and (b) the type-strain of T. inkin CBS 5585 obtained from the CBS culture collection (Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands); finally (c) after finishing the initial molecular analyses we became aware of similar strains from France, hence we included a set of 27 clinical strains recovered between 2006 and 2023 from 18 French hospitals.These French strains were initially identified as Trichosporon sp., closely related to T. inkin, at the French National Reference Center for invasive Mycoses and Antifungals, Institut Pasteur.Next to their IGS1-sequence data we included the antifungal susceptibility testing (see below) results for the azoles.
The rDNA sequences were aligned via the online MAFFT platform and manually adjusted in MEGA v7.Additionally, an IGS1 sequence dataset for T. inkin was included in the analysis by collecting them from the NCBI GenBank database.Prior running the phylogenetic analysis, the best nucleotide substitution model was searched with MEGA v7 using the foreseen dataset as input, resulting in the Kimura-2-parameter with gamma distributed with invariant sites.These settings were used as options for the subsequent 1000× bootstrapped Maximum Likelihood (ML heuristic method = nearest-neighbor-interchange; gaps were treated as complete deletion) analysis in MEGA v7 [31][32][33][34].

Morphological and Physiological Characterization
Strain L9630 (= CBS 17435, ex-type) was further morphological and physiological characterized after growing it onto MEA, glucose yeast peptone agar (GYPA, Oxoid), and yeast morphology agar (YMoA, Difco).Plates were incubated at 25 °C for 7 days.Microscopic observations and measurements were carried out using an Axioskop 2 plus microscope (Carl Zeiss, Jena, Germany) fitted with a Nikon DS-Ri2 microscope camera (Nikon Instruments, Tokyo, Japan).
Physiological profiling was conducted using standard methods commonly employed in yeast taxonomy [37].Nitrogen and carbon compound utilization was assessed by auxanograms and liquid media, respectively.The growth profile at different temperatures was evaluated using GYPA plates for up to 1 week.
In addition, we assessed the identification of the strain by performing matrix-assisted laser desorption-ionization-time of flight mass spectrometry (MALDI-TOF MS) using the Biotyper database v.4.1 (Bruker Daltonics, Bremen, Germany).Main spectra (MSP) were created as previously published [38,39].

DNA-Based Characterization and Species Delineation
Based on IGS1-sequencing, strain L9630 (= CBS 17435, ex-type) and the six related Trichosporon isolates that were selected for the genetic analysis displayed a low identification score when compared by BLAST with data in the NCBI Nucleotide database, showing a low similarity score of 81% with T. inkin.The D1/D2 LSU sequences had > 99% similarity with that of T. inkin, T. dohaense, T. lactis, and T. ovoides.
Phylogenetic analysis using IGS1-sequences data from our seven clinical strains along with data from Institut Pasteur and the NCBI GenBank database encompassing all currently recognized species within the genus Trichosporon.In addition, we incorporated into our analyses all available IGS1-sequences of T. inkin deposited by several diagnostic reference laboratories.The IGS1 region analysis was chosen due to its superior capability in distinguishing phylogenetically related species when compared to the ITS sequence, as previously described [11].Our findings consistently demonstrated that DNA sequences of strain L9630 (= CBS 17435, ex-type) and its six Brazilian and 27 French siblings had identical IGS1sequences and yield a clearly defined and well-supported clade.This further supports our observation of a novel species, hereafter designated as Trichosporon austroamericanum (Fig. 1).As depicted by the 1,000× bootstrap Maximum Likelihood analysis, T. austroamericanum (L9630 = CBS 17435, ex-type) clustered most closely with T. inkin (CBS 5585 T ) followed distantly with T. ovoides and T. dohaense (Fig. 1).The recognition of T. austroamericanum as a distinct lineage within the genus Trichosporon was further substantiated through analysis of the commonly used ITS region (Table 1).Maximum likelihood phylogeny showed strong bootstrap support of 81%, respectively, although the species was found to be closely related to T. inkin (Supplementary Information 1) (Table 1).

Morphological and Physiological Characterization
The six isolates displayed the typical morphology of Trichosporon (Fig. 3).On MEA plates, colonies exhibited a cream to beige color, with a membranous, friable, and farinose texture.Colonies were raised, rough, featuring transversal/cerebriform marginal zones, along with a fringed appearance (Fig. 3a,  d).Likewise, on YMoA plates, the same mentioned characteristics were present, besides colonies showing a flat lateral zone with transversal lines (Fig. 3e).
Compared to T. inkin, T. austroamericanum exhibits distinctive characteristics, such as delayed but pronounced d-glucosamine and melibiose assimilation, weakly positive l-arabinose utilization, lack of growth on 50% glucose, resilience to 0.1% cycloheximide, and the ability to thrive at temperatures up to 45 °C (Table 3).Identification performed by MALDI-TOF      MALDI-TOF MS, as well as to CDC-hosted Micro-beNet (https:// www.cdc.gov/ micro benet/).The main growth requirements able to distinguish strain L9630 (= CBS 17435, ex-type) and their six relatives strains tested from T. inkin can be summarized as follows: capability to grow at 45 °C; utilization of l-sorbose, d-glucosamine, l-arabinose, d-arabinose, melibiose, inulin, methanol, and other sources detailed in Table 3, although growth may be weak or delayed for some; inability to use both succinate and citrate; and the species does not grow in media with elevated sugar concentration (50% glucose).Considering that physiological characterization can be susceptible to subjectivity in result interpretation, sequencing of the IGS1 region or application of MALDI-TOF MS provide a dependable tool for the precise identification of T. austroamericanum.
Here an integrated analysis, which incorporated a combination of morphology, physiology, and phylogeny, was employed to define our new species and formally describe it as T. austroamericanum below.

Discussion
The phylogenetic analysis and AFLP fingerprinting, that included the strain L9630 (= CBS 17435, extype) and an additional set of clinical Trichosporon strains obtained from our laboratory, showed that all these strains were closely related to T. inkin, but grouped together in a different clade.In addition, these strains displayed distinctive physiological characteristics when compared to T. inkin, The integration of morphological, physiological, and genetic analyses based on rDNA targets (ITS, IGS1) and AFLP-fingerprint provided a comprehensive characterization of the new species.The new clinically relevant yeast species Trichosporon austroamericanum (= CBS 17435, ex-type culture) was isolated from a urine sample collected from a kidney transplant recipient hospitalized at a Brazilian tertiary care center.The morphological observations on different growth media cosubstantiate the typical characteristics of Trichosporon species, without any morphological finding able to distinguish between the Trichosporon species.On the other hand, physiological profiling of strain L9630 (= CBS 17435, ex-type) could distinguish it from T. inkin as further detailed in Table 3 [9].Given the intrinsic resistances of basidiomycetous yeasts to echinocandins, this holds true for T. austroamericanum too.
The IGS1 region has been used in the characterization of Trichosporon species, emphasizing its significance in distinguishing potentially identical species that otherwise might appear genetically identical when using other rDNA regions, like ITS.The characterization of Trichosporon mycotoxinivorans was performed by phylogenetic analysis using different rDNA regions, the species exhibited high homology with T. loubieri for the ITS and D1/D2 loci.However, analysis of the commonly used IGS marker successfully differentiated both species, which were otherwise difficult to discriminate from each other using ITS and D1/D2 data.By IGS analysis these two species had only 66.7% similarity [40].Similarly, in the characterization of T. terricola, although D1/D2, ITS, and IGS sequences effectively placed the new species into a distinct clade, the nucleotide differences observed in the IGS1 region played a pivotal role in conclusively identifying the studied isolate as a species [41].We observed that the D1/D2 sequence of T. austroamericanum was > 99% similar to T. inkin, T. dohaense, T. lactis, and T. ovoides, showing that this part of the ribosomal DNA is not suitable to identify Trichosporon species, while IGS has a superior ability to do so [11].
Considering the occurrence of the novel species, BLAST searches and a subsequent phylogenetic analysis, showed that T. austroamericanum has been isolated from diverse clinical samples (Fig. 1), including clones obtained from invasive infections (MG872032, China), urine (ON051553, Thailand), blood (KX463498, Brazil), superficial mycosis (MZ269052, India), and others human sources (JX476301, Argentina; Fig. 1).Retrospectively, we identified the first clinical strains recovered in France in 2006 from an African immigrant.These findings underscore the apparent global distribution of this pathogen and the urgent requirement for further studies to characterize the burden, natural history, and potential therapeutic peculiarities of T. austroamericanum infections.
In conclusion, the identification and characterization of a novel species named T. austroamericanum contribute to the knowledge of the pathogenic species belonging to the basidiomycetous yeast genus Trichosporon.Nevertheless, additional investigations are warranted to fully uncover the extent of T.

Nutritional Data and Growth Temperatures
Trichosporon austroamericanum exhibits delayed but pronounced d-glucosamine and melibiose assimilation, weakly positive l-arabinose utilization, lack of growth on 50% glucose, resilience to 0.1% cycloheximide, and the ability to thrive at temperatures up to 45 °C.Urease-positive.The full dataset of physiological characteristics is provided in Table 3.

Ecology, and Occurrence
Trichosporon austroamericanum is often isolated from clinical specimens, but its environmental ecological niche is unknown.
The holotype CBS H-24937 was isolated from a urine sample obtained via an ultrasound-guided percutaneous puncture performed in a Brazilian kidney transplant recipient with an abdominal collection secondary to urinary fistula.Based on intergenic spacer (IGS1) ribosomal DNA locus available via the NCBI GenBank database, T. austroamericanum has been isolated from China, France, India, and Argentina, so far only from clinical sources.

Identification and Phenotypical Differences with Closely Related Species
Trichosporon austroamericanum can be distinguished from the other species belonging to the Trichosporon genus by sequencing the intergenic spacer (IGS1) ribosomal DNA locus.The expansion of MALDI-TOF MS databases has enabled the accurate identification of this species and has become a powerful tool capable of distinguishing T. austroamericanum from T. inkin.Furthermore, T. austroamericanum displays a broader range of physiological characteristics compared to T. inkin, including the ability to thrive at 45 °C.

Fig. 1
Fig.1 Phylogenetic tree of the here described novel species Trichosporon austroamericanum and currently recognized species within the Trichosporon genus based on IGS1sequences.The phylogenetic tree was drawn based on a 1000× bootstrap Maximum Likelihood analysis.The tree with the highest log likelihood (− 914.03) is shown.The percentage of trees in which the associated taxa clustered together is shown next to the branches.The tree is drawn to scale, with branch lengths measured in the number of nucleotide substitutions per site.All positions containing gaps and missing data were eliminated.There were a total of 113 positions in the final dataset.Data in bold indicates strains genotypically and phenotypically characterized in the current study.Strain numbers are followed by information about the country of origin, source and NCBI Genbank accession number

Table 3
Trichosporon austroamericanum physiological characteristics ues ranging from 0.00 to 1.69).The closest match generated by spectrometry was T. inkin with a score value of 1.68 for CBS 5585.Consequently, new MALDI-TOF MS main spectra (MSPs) were generated for the new species.The MSP was incorporated at the WI-KNAW in-house library, ensuring reliable identification through Bruker Daltonics

Table 3
Physiological data for reference strain CBS 5575 T were obtained from the CBS Culture Collection Database (https:// wi.knaw.nl/ page/ Colle ction).** CBS 17435 ex−type strain; Glucose was used to ensure the inability of Trichosporon species to ferment compounds.Trichosporon inkin displayed a restrictive physiology when compared to T. austroamericanum strains.Both species grew at a temperature ranging from 25 to 42 °C, and the T. austroamericanum was able to grow at 45 °C. *Vol:.(1234567890)