A revision of malbranchea-like fungi from clinical specimens in the United States of America reveals unexpected novelty

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 identified a representative number of clinical fungi belonging to that order from a reference laboratory in the USA. A total of 22 strains isolated from respiratory tract (40%) and human skin and nails (27.2%) showed a malbranchea-like morphology. Six genera were phenotypically and molecularly identified, 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 synonymized Auxarthron with Malbranchea, and erected two new genera: Pseudoarthropsis and Pseudomalbranchea. New species proposed are: Arachnomyces bostrychodes, A. graciliformis, Currahmyces sparsispora, Malbranchea gymnoascoides, M. multiseptata, M. stricta, Pseudoarthropsis crassispora, Pseudomalbranchea gemmata, and Spiromastigoides geomycoides, along with a new combination for Malbranchea gypsea. The echinocandins showed the highest in vitro antifungal activity against the studied isolates, followed by terbinafine and posaconazole; in contrast, amphotericin B, fluconazole, itraconazole and 5-fluorocytosine were less active or lacked in vitro activity against these fungi. Supplementary Information The online version contains supplementary material available at 10.1186/s43008-021-00075-x.


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(Currah , 1994Doveri et al. 2012;Dukik et al. 2017;Hubálek 2000;Hubka et al. 2013; Sharma and 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 and Carmichael 1976), Malbranchea pulchella has been suggested as a possible cause of sinusitis (Benda and Corey 1994), and M. cinnamomea was recovered from dystrophic nails in patients with underlying chronic illnesses (Lyskova 2007, Salar andAneja 2007). More recently, Malbranchea spp. have been proposed as one of the causative agents of Majocchi's granuloma (Govind et al. 2017;Durdu et al. 2019). In a study of 245 patients with fungal saprophytic infections of nails and skin, Malbranchea spp. were isolated in 1% of skin samples (Lyskova 2007). Other studies demonstrated the coexistence (0.3% of the cases) of Malbranchea spp. with the primary pathogen patients with tuberculosis (Benda and Corey 1994;Yahaya et al. 2015).
Malbranchea was erected by Saccardo in 1882 for a single species, Malbranchea pulchella. It is characterized by alternate arthroconidia originating in curved branches from the vegetative hyphae, which developed on the surface of wet cardboard collected by A. Malbranche in Normandy, France (Fig. 1). Cooney and Emerson reviewed the genus in 1964, providing an appropriated description for mesophilic (M. pulchella) and thermophilic (Malbranchea sulfurea) species. In a more recent revision by Sigler and Carmichael (1976) 12 species were accepted, while a close relationship with the genus Auxarthron (family Onygenaceae, order Onygenales) was reported, i.e. the species Auxarthron conjugatum forms a malbranchea-like asexual morph, and Malbranchea albolutea produces a sexual morph related to Auxarthron. Also, Sigler and co-workers (2002) connected Malbranchea filamentosa with Auxarthron based on molecular studies, and also reported the production of fertile ascomata after an in vitro mating of several sexually compatible strains of M. filamentosa. The genus Auxarthron produces reddish brown, appendaged gymnothecial ascomata with globose prototunicate 8spored asci, and globose or oblate, reticulate ascospores . Some species of this genus, such as Auxarthron ostraviense and A. umbrinum have been reported as producing onychomycosis in humans , and Auxarthron brunneum, A. compactum and A. zuffianum were also isolated from the lungs of kangaroo rats, A. conjugatum from lungs of rodents, and A. umbrinum from lung of dogs, bats and rodents Kuehn et al. 1964). Malbranchea-like asexual morphs are also present in other taxa of ascomycetes. The genus Arachnomyces (family Arachnomycetaceae, order Arachnomycetales; Malloch andCain 1970, Guarro et al. 1993), characterized by the production of brightly coloured cleistothecial ascomata bearing setae, and by the production of an onychocola-like (Sigler et al. 1994) or a malbranchea-like (Udagawa and Uchiyama 1999) asexual morph, have been also implicated in animal and human infections. Specifically, Arachnomyces nodosetosus and Arachnomyces kanei have been reported as causing nail and skin infections in humans (Sigler and Congly 1990;Sigler et al. 1994;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 A. 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 in 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.

Fungal strains
Twenty-two malbranchea-like fungal strains (19 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).

DNA extraction, amplification and sequencing
Total DNA was extracted as previously described (Valenzuela-Lopez et al. 2018), and the following phylogenetic markers were amplified: the internal transcribed spacers (ITS) (ITS5/ITS4 primers; White et al. 1990, and a fragment of the large subunit (LSU) gene (LR0R/LR5 primers; Vilgalys and Hester 1990;Rehner and Samuels 1994) of the nrDNA. 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 identification 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 identification. A maximum level of identity (MLI) ≥ 98% was used for specieslevel and < 98% for genus-level identification. 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 final matrices used for the phylogenetic analysis were deposited in TreeBASE (www.treebase.org; accession number: 25068).

Antifungal susceptibility testing
In vitro antifungal susceptibility testing was carried out following the broth microdilution method from the Clinical and Laboratory Standards Institute (CLSI) protocol M38 (CLSI 2017) with some modifications. The antifungal drugs tested were amphotericin B (AMB), fluconazole (FLC), voriconazole (VRC), itraconazole (ITC), posaconazole (PSC), anidulafungin (AFG), caspofungin (CFG), micafungin (MFG), terbinafine (TRB), and 5fluorocytosine (5-FC). Briefly, 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°C (without light or agitation) until the drug-free well displayed a visible fungal growth (minimum 48 h; maximum 10 days) for quantification 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 defined as the lowest concentration that completely inhibited the fungal growth. C. parapsilosis ATCC 22019 was used as the quality control strain in all experiments. Table 1 shows the identity of the 22 fungal strains studied. The highest number of strains corresponded to Auxarthron umbrinum (4), followed by A. alboluteum (2), A. conjugatum (2), and Malbranchea aurantiaca (2). Auxarthron zuffianum, Currahmyces indicus and M. flocciformis were represented by one strain each. Eight strains were only identified at genus-level (three belonging to Malbranchea, two to Spiromastigoides, two to Arachnomyces, and one to Arthropsis), one strain (FMR 17684) only at family-level (Onygenaceae).

Arachnomyces
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 described as new, here.

Currahmyces
Due to the strain FMR 17683 being placed into a terminal branch of Onygenaceae together with Currahmyces indicus (Sharma and Shouche 2019), and because they differ molecularly and phenotypically, we erect the new species Currahmyces sparsispora.
Taking into account that Auxarthron and Malbranchea are congeneric, as has been shown in previous studies Sarrocco et al. 2015) and here (Fig. 2), and that Malbranchea (Saccardo 1882) has historical priority (Turland et al. 2018) over Auxarthron  Because in a BLAST search using the ITS and LSU nucleotide sequences from the ex-type strains, Malbranchea circinata and M. flavorosea match with taxa in the family Myxotrichaceae, both those species are excluded to the genus.
After examination of the lectotype of Auxarthron indicum (Patil and Pawar 1987, as "indica"), 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 available we consider it as of uncertain application.
Haemolytic. 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 filamentosa but in a separate terminal branch, and because both differ morphologically and genotypically, the new species Malbranchea stricta is also described.
Diagnosis: Malbranchea stricta is phylogenetically close to M. filamentosa. Also, both species lack a sexual morph . However, M. filamentosa produces more regularly shaped conidia than M. stricta, and forms thick-walled brown setae, structures absent in M. stricta.
Diagnosis: Pseudoarthropsis crassispora is phylogenetically close to P. cirrhata. Nevertheless, the former produces holoarthric conidia, while they are enteroarthric in the latter. Also, the conidia of P. crassispora are ellipsoidal, globose or broadly barrel-shaped, while these are cylindrical to cuboid (often wider than they are long) in P. cirrhata (van Oorschot and de Hoog 1984). Moreover, the conidia are bigger in P. crassispora than in P. cirrhata (4.5-5.5 × 2.5-3.5 μm vs. 2.5-4.0 × 2.0-3.0 μm). Also, P. crassispora grows faster than P. cirrhata (on PYE at 25°C), and the maximum temperature of growth is at 37°C and 30°C, respectively.
Spiromastigoides Because strains FMR 17686 and FMR 17696 were placed together in a terminal branch close to the ex-type strain of M. gypsea in the Spiromastigaceae clade (Fig.  2), M. gypsea is combined into Spiromastigoides and these two strains are described as the new species S. geomycoides.
Description (adapted from the original description): Arthroconidia produced intercalary or terminally along straight primary hyphae, or on short or long lateral branches, separated each one by one or more alternate empty cells, or, rarely, formed immediately adjacent to each other. Arthroconidia unicellular, hyaline, smoothand thin-walled, cylindrical or slightly barrel-shaped, (2.5) 3-6 (9) × 2-2.5 μm, slightly broader than the interconnecting cells. No sexual morph obtained by matting. Colonies on PYE reaching 17-39 mm after three wk. at room temperature, chalky white to creamy white, downy to velvety, slightly raised, surface folded to convoluted, umbonated at centre, reverse buff. Optimum temperature of growth 25-30°C. Maximum temperature of growth 37°C (but strain depending).

IN VITRO ANTIFUNGAL SUSCEPTIBILITY TESTING
The results of the antifungal susceptibility test are summarized in Table 2. In general, the echinocandins (AFG, CFG and MFG) displayed the most potent in vitro antifungal activity, but TRB and PSC also demonstrated a good activity against these fungi. In contrast, limited to no inhibition of growth was observed with AMB, FLC, ITC and 5-FC. 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 wk. of incubation.

DISCUSSION
To our knowledge, this is the main study to be produced on malbranchea-like fungi from a clinical origin to date. 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 15 strains as belonging to the genus Malbranchea (syn. Auxarthron), of which three of them are described as new species. These results indicate a high diversity of onygenalean fungi in these sorts of substrates, which may be difficult to differentiate using only 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 and Trotter 1913;Cooney and Emerson 1964;Sigler and Carmichael 1976). All were able to grow at 30°C, and most of them at 35-37°C.
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 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 Malbranchea in the present study) reduced susceptibility to AMB, ITC and PSC was reported, but a high susceptibility to TRB was observed . Another study (Gupta and Kohli 2003) showed that strains of Arachnomyces nodosetosus (syn. Onychocola canadensis) where highly susceptible to ciclopirox 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.

CONCLUSIONS
From all malbranchea-like strains from clinical specimens (mostly human) in the USA that we studied, only 13 out of 22 could be identified at the species level, three of them belonging to the genus Malbranchea. With the exception of one strain initially identified as Currahmyces indicus, the others were identified as species of Auxarthron, a genus synonymized with Malbranchea during the course of the present work. Eight of the remaining strains have been assimilated to the genera Arachnomyces (2), Arthropsis (1), Malbranchea (3), and Spiromastigoides (2), the latter only located at family level (Onygenaceae). This is an extraordinary finding, because nearly half of the fungal strains presumed to belong to the genus Malbranchea resulted in becoming new taxa for science. Finally, despite the lack of histopathological data, which could have undoubtedly proven that these strains were the causative agents of the infections that led to the request for sample collection, we would highlight their poor sensitivity to first-line drugs such as AMB, FLC, and ITC, but better sensitivity to echinocandins and PSC.