Rare malbranchea-like fungal isolates from clinical specimens in United States of America.

The fungi of the order Onygenales can cause important human infections; however, their taxonomy and worldwide occurrence is still little known. We have studied and identied a representative number of clinical fungi belonging to that order from a reference laboratory in the USA. A total of twenty-two strains isolated from respiratory tract (40 %) and human skin and nails (27.2 %) showed a malbranchea-like morphology. Six genera were phenotypically and molecularly identied, i.e. Auxarthron/Malbranchea (68.2 %), Arachnomyces (9.1 %), Spiromastigoides (9.1 %), and Currahmyces (4.5 %), and two newly proposed genera (4.5 % each). Based on the results of the phylogenetic study, we synonymysed Auxarthron to Malbranchea, and erected two new genera: Pseudoarthropsis and Pseudomalbranchea. New species are proposed: Arachnomyces bostrychodes, A. graciliformis, Currahmyces sparsispora, Malbranchea gymnoascoidea, M. multiseptata, M. stricta, Pseudoarthropsis crassispora, Pseudomalbranchea gemmata and Spiromastigoides geomyces, along with a new combination for Malbranchea gypsea. The echinocandins showed the highest in vitro antifungal activity against the studied isolates, followed by terbinane and posaconazole; in contrast, amphotericin B, uconazole, itraconazole and 5-uorocytosine were less active or lacked in vitro activity against these fungi. μm wide, forming randomly intercalary and terminally arthroconidia. Conidia enteroarthric, hyaline, one-celled, smooth-walled, cylindrical, barrel-shaped, and nger-like-shaped when terminal, 4.0–8.0 × 1.0–2.0 μm, mostly curved and truncated at one or (mostly) both ends, separated from the fertile hyphae by rhexolysis. Chlamydospores, racquet hyphae, setae, and sexual morph not observed. Culture characteristics: Colonies on PYE reaching 19–20 mm diam. after 2 weeks at 25 °C, elevated, cottony, margins regular, white (5A1), sporulation absent; reverse light orange (5A4). Colonies on PDA reaching 11–12 mm diam. after 2 weeks at 25 °C, elevated, velvety with occose patches, margins regular, yellowish white (4A2), sporulation abundant; reverse greyish yellow (4B6). Colonies on PDA reaching 13–14 mm diam. after 2 weeks at 30 ºC, slightly elevated, velvety to occose, regular margins, white (4A1), sporulation sparse; reverse, greyish yellow (4B6). Colonies on OA researching 13–14 mm diam. after 2 weeks at 25 °C, attened, smooth and granulose, irregular margins, yellowish white (2A2) at centre and light yellow (2A5) at edge, sporulation abundant. Exudate and diffusible pigment absent. and diffusible pigment absent, Minimum, optimal and maximum temperature of and respectively. Non-haemolytic. Casein hydrolyzed without pH change. Not inhibited by cycloheximide. Urease and esterase tests positive. The fungus grows up to NaCl 10 % w/w, but not at 20 % w/w. slightly elevated, margins sporulation sparse. Exudate and diffusible absent all tested. Minimum, optimal and maximum temperature of growth on Haemolytic. Casein not hydrolysed. Not by cycloheximide. Urease and esterase tests Description: Vegetative hyphae septate, hyaline, smooth- and thin-walled, mostly straight, rarely branched, 1.5–2.5 μm wide. Asexual morph consisting in undifferentiated fertile hyphae which form randomly intercalary and terminally arthroconidia. Conidia enteroarthric, unicellular, hyaline, smooth- and thin-walled, mostly barrel-shaped, sometimes cylindrical or irregularly-shaped, 6.0–10.0 × 1.5–2.0 μm, detached by rhexolysis. Ascomata gymnothecial, solitary or in clusters, hyaline at rst, becoming orange brown with the age, globose or nearly so, 130–250 μm diam. excluding the appendages, which cover entirely the surface. Peridial hyphae septate, orange brown, branching and anastomosing to form a reticulate network, asperulate, very thick-walled, 3.5–5.5 μm wide, fragmenting by the septa when ageing, with lateral appendages. Appendages 0–1-septate, orange brown, asperulate, thick-walled, progressively tapering towards the apex, apex sinuous, 250–400 μm long, connected by basal knuckle joints. Asci 8-spored, globose or nearly so, 4–7 μm diam., soon deliquescent. elevated, occose, white sporulation abundant; reverse yellowish brown at and greyish yellow at margins. 16–17 mm weeks 25 °C, sporulation sparse.


Introduction
The order Onygenales includes medically important fungi, such as the dermatophytes and the thermally dimorphic systemic pathogens (Histoplasma, Coccidioides and related fungi), which are naturally present in keratinous substrates, in soil, and in freshwater sediments (Currah 1985, Doveri et al. 2012, Hubálek 2000, Hubka et al. 2013, Sharma & Shouche 2019. The genus Malbranchea, which is characterized by the production of alternate arthroconidia in branches from the vegetative hyphae, is one of the genus-form of this order; however, it´s pathogenic role in human infections is little known. Only a few cases of fungal infections by species of this genus have been described: Malbranchea dendritica has been recovered from lungs, spleen and liver of mice (Sigler & Carmichael 1976), Malbranchea pulchella has been suggested as a possible cause of sinusitis (Benda & Corey 1994), and Malbranchea cinnamomea was recovered from dystrophic nails in patients with underlying  or a malbranchea-like (Udagawa & Uchiyama 1999) asexual morph, have been also implicated in animal and human infections. Speci cally, Arachnomyces nodosetosus and Arachnomyces kanei have been reported as causing nail and skin infections in humans (Sigler & Congly 1990, Campbell et al. 1997, Contet-Audonneau et al. 1997, Kane et al. 1997, Koenig et al. 1997, Gupta et al. 1998, Erbagci et al. 2002, Gibas et al. 2002, Llovo et al. 2002, O'Donoghue et al. 2003, Gibas et al. 2004, Stuchlík et al. 2011, Järv 2015, Gupta et al. 2016. More recently, Arachnomyces peruvianus has been reported to cause cutaneous infection (Brasch et al. 2017) and Arachnomyces glareosus was isolated from nail and skin samples (Gibas et al. 2004;Sun et al. 2019).
Due to the limited knowledge of Malbranchea and their relatives on human infections, we have studied phenotypically and molecularly a set of malbranchea-like fungal strains from clinical specimens received in a fungal reference centre in the USA. Phylogenetic study and an antifungal susceptibility testing were also carried out.

Materials And Methods
Fungal strains Twenty-two malbranchea-like fungal strains (nineteen from human specimens and three from animals) from different locations in USA were included in this study. The strain number, anatomical source, and geographic origin of the specimens are listed in Table 1. They were provided by the Fungus Testing Laboratory of the University of Texas Health Science Centre at San Antonio (UTHSC; San Antonio, Texas, USA).
Amplicons were sequenced at Macrogen Europe (Macrogen Inc., Madrid, Spain) using the same pair of primers. Consensus sequences were obtained by SeqMan software v. 7 (DNAStar Lasergene, Madison, WI, USA). Sequences generated in this work were deposited in GenBank (Table 1).

Phylogenetic analysis
A preliminary molecular identi cation of the isolates was carried out with ITS and LSU nucleotide sequences using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi), and only the sequences of ex-type or reference strains from GenBank were included for identi cation. A maximum level of identity (MLI) ≥ 98% was used for species-level and < 98% for genus-level identi cation. A maximum-likelihood (ML) and Bayesian-inference (BI) phylogenetic analyses of the concatenated ITS-LSU sequences were performed in order to determine the phylogenetic placement of our clinical strains. Species of the order Arachnomycetales were used as outgroup. The sequence alignments and ML / BI analyses were performed according to Valenzuela-Lopez et al. (2018). The nal matrices used for the phylogenetic analysis were deposited in TreeBASE (www.treebase.org; accession number: 25068).
Brie y, incubation media, temperature and time were set to the sporulation requirements of every strain, and conidia suspensions were inoculated into the microdilution trays after being adjusted by haemocytometer counts. Incubation was set at 35 ºC (without light or agitation) until the drug-free well displayed a visible fungal growth (minimum 48 h; maximum 10 days) for quanti cation of the Minimal Effective Concentrations (MEC) for the echinocandins and the Minimal Inhibitory Concentrations (MIC) for the other tested antifungals. The MEC value was stablished as the lowest drug concentration at which short, stubby and highly branched hyphae were observed, while the MIC value was de ned as the lowest concentration that completely inhibited the fungal growth. Candida parapsilosis ATCC 22019 was used as the quality control strain in all experiments.

Molecular phylogeny
Our phylogenetic study included 92 sequences corresponding to 75 species with a total of 1,213 characters (700 ITS and 513 LSU) including gaps, of which 579 were parsimony informative (402 ITS and 177 LSU). The ML analysis was congruent with that obtained in the BI analysis, both displaying trees with similar topologies. The datasets did not show con ict with the tree topologies for the 70% reciprocal bootstrap trees, which allowed the two genes to be combined for the multi-locus analysis.

Taxonomy
Since the strains FMR 17685 and FMR 17691 represented two species of Arachnomyces that were different from the other species of the genus, they are proposed as new, i.e. Arachnomyces bostrychodes and Arachnomyces graciliformis, respectively.
Due to the strain FMR 17683 being placed into a terminal branch of the Onygenaceae together with Currahmyces indicus (Sharma & Shouche 2019), and because they differ molecularly and phenotypically, we propose the erection of the new species Currahmyces sparsispora.
Etymology: From Latin sparsa-, splashed, -sporarum, spore, due to the disposition of the conidia along the hyphae.
Because in a Blast search using the ITS and LSU nucleotide sequences from the ex-type strains, Malbranchea circinata and Malbranchea avorosea match with taxa into the family Myxotrichaceae, both species are excluded to the genus.
After examination of the lectotype of Auxarthron indicum (Patil & Pawar 1987), we concluded that this fungus must be excluded from Malbranchea because its sexual morph differs mainly from all species described for the former genus. Whereas Auxarthron indicum produces smooth-walled ellipsoidal ascospores and gymnothecial ascomata lacking of true appendages, in Malbranchea spp. the ascospores are globose and mostly ornamented, and the ascomata have appendages. Based on the fact that there is no type strain of this species, we consider it as invalid.
Consequently, an emended description of the genus Malbranchea is provided as follows: Malbranchea Sacc. MycoBank MB 8833.
Vegetative hyphae septate, hyaline, smooth-and thin-walled, straight or branched. Asexual morph consisting in undifferentiated fertile hyphae, and/or well-differentiated lateral branches, curved or not, which form randomly or basipetally terminal and intercalary arthroconidia. Conidia enteroarthric, rarely holoarthric, unicellular, hyaline, smooth-and thin-walled, mostly cylindrical, barrel-shaped, or irregularly shaped, sometimes cylindrical, detached from the fertile hyphae by rhexolysis. Sexual morph (when present) consisting in ascomata formed by of an anastomosing network of orange to brown, ornamented or not thick-walled hyphae (gymnothecia), bearing elongate appendages and/or spine projections, within there are small, evanescent, in ated asci which forms eight globose to oblate ascospores, whose cell wall is ornamented with a (coarse or thin) reticulate pattern. Species homothallic or heterothallic, thermotolerant or thermophilic, keratinolytic, chitinolytic or cellulolytic.
Despite the strain FMR 17695 being phylogenetically close to Malbranchea longispora, it differs phylogenetically and morphologically from it. Consequently, we propose the erection of the new species Malbranchea multiseptata.
Casein hydrolyzed without pH change. Not inhibited by cycloheximide. Urease positive. Growth occurs at NaCl 3 % w/w, but not at 10 %w/w. Neither grow on TOTM.
Because the strain FMR 17680 was placed phylogenetically close to Malbranchea lamentosa but in a separate terminal branch, and because both differ morphologically and genotypically, the new species Malbranchea stricta is proposed.
Etymology: Latin stricta, strict, due to the production of the typical reproductive structures of the genus.Diagnosis: Malbranchea stricta is phylogenetically close to M. lamentosa. Also, both species lack of a sexual morph  Description: Vegetative hyphae hyaline, smooth-and thin-walled, straight to sinuous, sparsely branched, 1.5-2.0 μm wide. Fertile hyphae well-developed, arising as lateral branches from the vegetative hyphae, mostly unbranched, right or slightly sinuous, contorted or arcuate at the end, up to 25 μm long, 1.5-2.0 μm wide, or developing at the extremes of the vegetative hyphae, in both cases forming arthroconidia randomly intercalary and terminally. Arthroconidia enteroarthric, hyaline, becoming yellowish with the age, barrel-shaped, "T"-shaped, "Y"-shaped, ngershaped or irregularly-shaped 2.0-6.0 × 1.0-2.0 μm, with rhexolytic secession. Chlamydospores, racquet hyphae, and sexual morph not observed. Minimum, optimum and maximum temperature of growth on PDA: 10 °C, 30 °C, and 37 °C, respectively. Colonies haemolytic (on BA), and casein hydrolyzed without pH changes at 25 °C (on BCP-MS-G). Not inhibited by cycloheximide. Urease and esterase tests positive. Growth occurs at NaCl 10 % w/w, but not at 20 % w/w.
1a Etymology: Because the morphological similarity with Malbranchea.
Antifungal activity was evaluated against all strains with the exception of FMR 17691, due to the scarce production of conidia and because this strain does not grow in RPMI medium, even after two weeks of incubation.

Discussion
To our knowledge, this is the main study on malbranchea-like fungi from a clinical origin. We have shown that several of these fungi have not been reported previously from human specimens, and although the pathologic role remains uncertain, their diversity is of interest since some represent new species.
Morphological and physiological characterization and phylogenetic analysis has allowed us to identify fteen strains as belonging to the genus Malbranchea (syn. Auxarthron), of which three of them are proposed as new species. These results indicate a high diversity of onygenalean fungi in these sorts of substrates, which may be di cult to differentiate only by using phenotypic characteristics.
All strains belonging to Malbranchea displayed thermotolerance, suggesting the potential pathogenicity of this genus in animals, including humans, as has been previously noted by others (Saccardo 1908, Saccardo & Trotter 1913, Cooney & Emerson 1964, Sigler & Carmichael 1976. In fact, all them were able to grow at 30 ºC, and most of them at 35-37 ºC. Malbranchea-like fungi were most commonly isolated from the respiratory tract (40 %) followed by nails and skin (27.2 %). Currahmyces sparsispora, Malbranchea albolutea, M. conjugata, M. gymnoascoides, M. multiseptata, Pseudoarthropsis crassispora and Pseudomalbranchea gemmata were all recovered from respiratory tract specimens (mostly obtained by bronchial-alveolar washing), while those of M. umbrina were isolated from the widest variety of anatomical sites. The rest of the taxa isolated were mostly from skin and annexes.
Regarding to the antifungal susceptibility of malbranchea-like fungi, limited data are available. However, in a previous study on onychomycosis-causing strains of Auxarthron ostraviense and Auxarthron umbrinum (transferred to the genus Malbranchea in the present study) reduced susceptibility to AMB, ITC and PSC was reported, but a high susceptibility to TRB was observed (Hubka et al. 2013). Another study (Gupta & Kohli 2003) showed that strains of Arachnomyces nodosetosus (syn. Onychocola canadensis) where highly susceptible to cicciclopirox and TRB. Our results are consistent with such previous studies, but we also demonstrated the enhanced susceptibility of the malbranchea-like fungi to the echinocandins.

Adherence to national and international regulations
The authors con rm that this manuscript respects the Nagoya Protocol to the Convention on Biological Diversity.

Consent for publication
Not applicable.

Availability of data and material
All data generated or analysed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.

Funding
The authors are indebted to the Instituto de Ciencia, Tecnología e Innovación (Mexico) and the Consejo Nacional de Ciencia y Tecnología (Mexico) for the scholarship 440135 with scholar 277137. This work was supported by the Spanish Ministerio de Economía y Competitividad, grant CGL2017-88094-P. APC is the recipient of a FI fellowship from Generalitat de Catalunya (Spain).

Authors´ contributions
ER-A performed all the experimental work, performing their phenotypic characterization, as well as the DNA extraction and puri cation, gene sequencing and data processing for phylogenetic analysis, being one of the major contributors of this manuscript. PC-A, performed and supervised with ER-A all the Antifungal susceptibility testing, reviewed the draft and writing part of "Materials and methods". AMS, because their experience on fungi belonging to Onygenales, supervised all steps of the experimental work by ER-A, collaborating in the description of the novel Page 23/35 fungi and in the writing of chapters "Introduction" and "Discussion", reviewing of the draft several times. WN, carried out the collection and morphological identi cation of the analysed strains, and reviewing the draft. JG contributed actively in the identi cation and taxonomy of the fungal strains, and reviewed the draft several times. JFC-L supervised the nucleotide sequence alignment and phylogenetic reconstruction, took the pictures that appear in the gures, contributed actively in the identi cation and taxonomy of the fungal strains, gave useful suggestions to write the manuscript and reviewed several times the draft. All authors read and approved the nal manuscript.