Coniochaeta massiliensis sp. nov. Isolated from a Clinical Sampl28

The genus Coniochaeta belongs to the class Ascomycota and the family Coniochaetaceae. Some of the Coniochaeta species are plant and animal pathogens, while others are known to be primarily involved in human diseases. In the last few decades, case reports of human infections with Coniochaeta have increased, mainly in immunocompromised hosts. We have described and characterised a new species in the genus Coniochaeta, here named Coniochaeta massiliensis (PMML0158), which was isolated from a clinical sample. Species identification and thorough description were based on apposite and reliable phylogenetic and phenotypic approaches. The phylogenetic methods included multilocus phylogenetic analyses of four genomic regions: ITS (rRNA Internal Transcribed Spacers 1 and 2), TEF-1α (Translation Elongation Factor-1alpha), B-tub2 (β-tubulin2), and D1/D2 domains (28S large subunit rRNA). The phenotypic characterisation consisted, first, of a physiological analysis using both EDX (energy-dispersive X-ray spectroscopy) and BiologTM advanced phenotypic technology for fixing the chemical mapping and carbon-source oxidation/assimilation profiles. Afterwards, morphological characteristics were highlighted by optical microscopy and scanning electron microscopy. The in vitro antifungal susceptibility profile was characterised using the E-testTM exponential gradient method. The molecular analysis revealed the genetic distance between the novel species Coniochaeta massiliensis (PMML0158) and other known taxa, and the phenotypic analysis confirmed its unique chemical and physiological profile when compared with all other species of this genus.


Introduction
Coniochaeta spp. are pleomorphic ascomycetous fungi belonging to the family Coniochaetaceae [1]. Some of these yeasts are also classified within dematiaceous fungi due to the presence of melanin in cell walls, known for emitting dark pigments in culture, which is perceived as a virulence factor [2]. Coniochaeta species are ubiquitously distributed in the environment. They have been isolated from several natural substrates, including soil [3,4], wood [5], plants [6], water [7], and food [8]. The previously given name Coniochaeta has been retained for the Lecythophora genus, following the "one fungus, one name" nomenclature change in January 2013. Therefore, all six species of the Lecythophora genus (L. decumbens, L. fasciculata, L. hoffmannii, L. lignicola, L. luteoviridis, and L. mutabilis) were transferred to the Coniochaeta genus [9,10]. C. hoffmannii and C. mutabilis are considered the most frequent species of the Coniochaeta genus found in clinical samples. These yeasts are known to be human pathogens, causing invasive fungal infections, occasionally with fatal outcomes, particularly in immunocompromised patients [11]. C. hoffmannii has been described as a plant pathogen [12]. It has also been implicated in emerging human fungal infections, including subcutaneous abscess [12], keratitis [13], and sinusitis [14], and in canine osteomyelitis in a dog [15]. However, Coniochaeta mutabilis [12] is known to be more frequently involved in severe infections. It has been involved in human peritonitis [16], endocarditis [17,18], septic shock [19], endophthalmitis [20,21], and keratomycosis [22]. The present study aimed to properly describe and characterise, genetically and phenotypically, a new species of Coniochaeta isolated from a human abscess.

Fungal Strains
The phenotypic features of Coniochaeta massiliensis PMML0158 were compared with those of two reference strains, C. mutabilis DSM 10716 and C. hoffmannii DSM 2693. The genotypic characteristics of these three strains were also compared with those of seven other reference strains in the genus Coniochaeta, including C. fasciculate CBS 205.38, C. lignicola CBS 267.33, C. luteoviridis CBS 206.38, C. hoffmannii CBS 245.38, C. mutabilis CBS 157.44, C. lignaria DWS9m2/SMH2569/95.605, and C. cateniformis UTHSC 01-1644. Phialemonium obovatum CBS 279.76 was used as an outgroup in the phylogenetic analyses.

Macroscopic Characterisation
The temperature growth profiles and macroscopic features, including colony time of growth, aspect, and surface/reverse colour, for all strains were determined with fiveday-old colonies cultivated on Sabouraud Dextrose Agar (SDA) plates supplemented with Gentamycin and Chloramphenicol (GC). Colonies were inoculated on other, new plates of SDA GC at 4, 25, 30, 33, 37, 40, and 45 • C.

Microscopic Characterisation
Microscopic features were first analysed with optical microscopy. Microscopic slides were prepared using the cellophane adhesive-tape method with lactophenol cotton blue (LCB). Photographs were taken with the DM 2500 (LEICA CAMERA SARL, Paris, France) camera. Further observation was performed using scanning electron microscopy. A fragment of fungal colonies was fixed on a microscope slide with 400 µL of glutaraldehyde 2.5% in 0.1 M sodium cacodylate buffer and stored at 30 • C for at least 30 min. Photographs were taken with the TM4000 Plus microscope (Hitachi High-Technologies, Tokyo, Japan) adjusted to 15 KeV lens mode 4 with a back-scattered electron detector (BSE).

MALDI-TOF MS Identification
The fungi were incubated on SDA GC at 30 • C for five to eight days. Once grown, colonies were identified with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), using the procedure described in Cassagne et al. (2016) [23]. The Microflex LT TM instrument and the MALDI Biotyper TM system (Bruker Daltonics GmbH, Bremen, Germany) were used, along with the manufacturer's and inhouse reference spectra databases, as described in Normand et al. (2017) [24].

Antifungal Susceptibility Testing (AFST)
The in vitro sensitivity of nine antifungal drugs was tested against the three Coniochaeta strains using the Sensititre YeastOne TM (Thermo Fisher Scientific, Dardilly, France) microdilution system, following the supplier's recommendations. Briefly, isolates were grown on SDA GC until maturity, the inoculum was suspended in 2 mL of saline, and turbidity was adjusted to 0.5 McFarland, to obtain an inoculum of~1.5 × 10 8 CFU/mL. Next, 20 µL of this solution was added to 10 mL of YeastBroth TM (Thermo Fisher Scientific, Illkirch, France) before 100 µL of this final solution was transferred into each Sensititre YeastOne TM YO09 (Thermo Fisher Scientific) plate well. MICs were read after 48 h incubation at 35 • C.
2.6. Physiological Analyses 2.6.1. Energy-Dispersive X-ray Spectroscopy (EDX) Analysis Fresh cultures of the three strains were fixed in 1 mL of 2.5% Glutaraldehyde in 0.1 M Sodium Cacodylate buffer for at least 1 h. A cytospin was performed with 200 µL of the fixed fungal solution and centrifugation at 800 rpm for eight minutes. EDX was achieved with an INCA X-Stream-2 detector (Oxford Instruments) associated with TM4000 Plus scanning electron microscopy and AztecOne software (Oxford Instruments, Pasadena, CA, USA). The chemical mapping was performed blindly and took into consideration all chemical elements. The results for the weights and atomic percentages of chemical elements for each strain were subjected to principal component analysis with the XLSTAT (Addinsoft, Paris, France) software.

Biolog TM Phenotypic Analysis
Biolog's advanced phenotypic technology was used for the phenotypic analysis. YT MicroPlates TM (Gen III) (Biolog catalogue no. 1005) were used for oxidation tests and carbon-source assimilation. The selected carbon sources can discriminate between the different profiles of fungal phenotypes [25]. The 96-well YT MicroPlate TM contains a patented Redox tetrazolium dye that changes colour when cellular respiration occurs, conferring a metabolic fingerprint. All strains were cultivated on Biolog Universal Yeast TM (BUY) Agar medium (Biolog catalogue no. 70005). Colonies must be fresh and welldeveloped. Incubation time depends on the genus. For Coniochaeta spp., maximal growth was observed after four to six days of incubation. A fungal suspension was prepared by swabbing some conidia into YT Inoculating Fluid TM (Biolog catalogue no. 72501) adjusted to a 47% transmittance level with the Biolog Turbidimeter TM (Biolog catalogue no. 3587). Then, 100 µL of this suspension was pipetted into each YT MicroPlates TM well. The plates were incubated at 26 • C for one week. The results are shown in the form of a heat map generated by XLSTAT.

DNA Extraction and Sequencing
After five days of incubation on SDA GC at 30 • C, DNA was extracted from the fungal colonies with the Qiagen TM Tissue kit after mechanical lysis using the FastPrep TM -24 instrument in bead tubes with G2 lysis buffer (provided with the Qiagen TM Tissue kit). The extraction was performed with the EZ1 Advanced XL TM instrument, following the manufacturer's instructions.
Four genomic regions were amplified: the rRNA internal transcribed spacers 1 and 2 (ITS1-2), a fragment of the β-tubulin gene (B-tub2), a fragment of the translation elongation factor 1-alpha gene (TEF-1-α), and the D1/D2 domains of the rRNA large subunit (LSU) ( Table 1). PCR mixes included 5 µL of DNA extract to 20 µL of mix (12.5 µL ATG (Ampli Taq Gold TM 360 Master Mix, Applied Biosystems), 6 µL sterile water (DNase/RNase-free), 0.75 µL forward/reverse primer) to a total volume of 25 µL/well. The PCR program for all gene amplifications was as follows: an initial denaturation step at 95 • C for 15 min, followed by 39 cycles of: 1 min denaturation at 95 • C, 30 s annealing at 56 • C, 1 min extension at 72 • C, and a final 5 min extension at 72 • C. The amplicons were visualized on 2% agarose gel with Sybr Safe DNA TM gel stain (Invitrogen, Waltham, MA, USA) using the Safe Imager 2.0 Blue-Light Transilluminator TM (Invitrogen). Sequencing was performed on a 3500 Genetic Analyser TM (Applied Biosystems, Inc.). The sequences were assembled and corrected using Chromas Pro 2.0 and analysed using the BLASTn tool with the reference data available from the GenBank database of the National Center for Biotechnology Information (NCBI).

Phylogenetic Analyses
Five phylogenetic trees were built. The first one was a multilocus tree constructed after concatenating the ITS, B-tub2, and D1/D2 nucleotide sequences of the clinical isolate, two reference strains, and sequences of other Coniochaeta species obtained from the GenBank database. The four other trees were each built with only one locus and included several Coniochaeta species also collected from GenBank database (accession numbers are detailed in Table 2). All sequences were aligned with Muscle (a tool available in MEGA 11 software). Phialemonium obovatum CBS 279.76 was used as the outgroup. The maximum-parsimony phylogenetic trees were constructed with the default settings and branch-robustness estimation was tested using 1000 bootstrap replications with the molecular evolutionary genetics analysis (MEGA) software version 11.

Macroscopic Characterisation
Macroscopic features confirmed the rapid growth time of Coniochaeta species on SDA GC medium at an optimal temperature of 25 • C for all species (Figure 1). However, none of the three yeasts was able to grow at 4 and 45 • C. Colonies of the three isolates were initially white to beige, both on the surface and on the reverse. After four to five days of incubation, Coniochaeta massiliensis turned light orange to salmon. All colonies were flat and moist. Coniochaeta hoffmannii DSM 2693 and the newly isolated yeast (PMML0158) presented a glabrous aspect, while Coniochaeta mutabilis DSM 10716 was typified by an aerial mycelial growth.

Microscopic Characterisation
The microscopic observation of the three strains revealed wide septate hyphae, numerous cylindrical adelophialides (short phialides without septum), discrete phialides with conical tips exhibiting ellipsoidal to cylindrical and rarely curved conidia, and nonseptate with thin and smooth conidial walls (2 to 3 by 6 to 10 µm). Several conidia were observed aggregating on the hyphae's sides and most often in clusters. Collarettes were only found in both Coniochaeta massiliensis and C. mutabilis. No chlamydospore was observed ( Figure 2).

Antifungal Susceptibility Testing (AFST)
The minimum inhibitory concentration (MIC) values for the three species for all nine antifungal drugs are shown in (Table 3). The MIC endpoints for all strains were determined as the lowest concentrations inhibiting the growth of 90% of the strains and were determined as described in Perdomo

MALDI-TOF MS Identification
The MALDI-TOF MS identification score (log score < 1.90) was below the limit required for obtaining a good identification. The isolate was identified at the genus level as Coniochaeta sp. identification score values were generated from MALDI-TOF MS spectra of the new isolate and the two other reference strains from the DSMZ collection, Coniochaeta mutabilis DSM 10716 and Coniochaeta hoffmannii DSM 2693, which were collected and have been entered in the MALDI-TOF MS database.

EDX (Energy-Dispersive X-ray Spectroscopy)
The weights and atomic percentages of chemical elements resulting from the chemical mapping performed on the three species of the Coniochaeta genus displayed three distinct profiles. In the principal component analysis (Figure 3), each Coniochaeta species was very distant from the others, demonstrating highly heterogeneous chemical profiles.

Biolog TM System
Biolog TM advanced phenotypic technology was very useful for the phenotypic characterisation. The oxidation and assimilation test results were illustrated using heat maps (Figures 4 and 5). The majority of the substrates were not oxidized/assimilated. Each heat map was quite heterogeneous, and both demonstrated similar findings: Coniochaeta mutabilis DSM 10716 appeared more divergent from Coniochaeta massiliensis (PMML0158) than from Coniochaeta hoffmannii DSM 2693. However, Coniochaeta massiliensis (PMML0158) appeared to be closely related to Coniochaeta hoffmannii DSM 2693.

Phylogenetic Analyses
We built phylogenetic trees using MEGA 11 software. The multilocus analysis of the first tree ( Figure 6) based on the assembled sequences of the ITS, B-tub2, and D1/D2 genetic regions revealed that the new isolate Coniochaeta massiliensis (PMML0158) clustered apart and seemed quite distinct from all other Coniochaeta species. Then, we constructed four other trees (Figure 7) with only one locus each, including supplementary Coniochaeta and Colletotrichum species. Within both ITS and TEF-1a trees, Coniochaeta massiliensis PMML0158 clustered with other Coniochaeta species. However, the D1/D2 and B-tub2 trees could not clearly position the new species PMML0158 in the Coniochaeta or Colletotrichum genus. However, the TEF-1a tree ( Figure 7D) clearly positioned Coniochaeta massiliensis (PMML0158) as a distinct species within the Coniochaeta genus, which was unambiguously separated from the Colletotrichum genus.

Taxonomy
Coniochaeta massiliensis Kabtani J. & Ranque S. sp. nov. MycoBank: MB843839 (Figure 2A-L). Etymology: Named in honour of Marseille (France), the city where it was isolated. Diagnosis: Closely similar to the other two Coniochaeta species examined, displaying the same flat and moist colonies aspect, as well as the absence of the dematiaceous appearance. However, relying on macroscopic features, it differs from C. mutabilis in that it lacks aerial growth. On the other hand, C. massiliensis presented the same microscopic structures as other species, such as the presence of several adelophialides, discrete phialides, and cylindrical or curved conidia. In this respect, C. massiliensis is closer to C. mutabilis, due to the decisive presence of the collarette.
Description: the macroscopic features were characterised by a rapid growth time on SDA GC medium at an optimal temperature of 25 • C. However, Coniochaeta massiliensis was not able to grow at 4 and 45 • C. Colonies were first white to beige, both on the surface and on the reverse after four to five days of incubation, then turned light orange to salmon. Colonies were flat and moist, with a glabrous aspect. There was no aerial mycelial growth. The microscopic features were characterised by the presence of wide septate hyphae, numerous cylindrical adelophialides (short phialides without septum), discrete phialides with conical tips, exhibiting ellipsoidal to cylindrical and rarely curved conidia, nonseptate with thin and smooth conidial walls (2 to 3 by 6 to 10 µm). Several conidia were observed aggregating on the side of the hyphae and most often in clusters. Collarettes were present, but chlamydospores formation was not observed.

Discussion
Coniochaeta mutabilis and Coniochaeta hoffmannii are the most familiar human pathogens of the Coniochaeta genus and the most widespread and commonly encountered in human samples and severe infections. The MALDI-TOF MS identifications for these two species were relevant, with log scores >2.0. However, this tool was not able to perform a species identification for the new isolate, which led to a molecular analysis targeting four relevant genes: the internal transcribed spacer (ITS1/ITS2) in the RNA ribosomal small subunit (SSU), a fragment of the translation elongation factor 1-alpha gene (TEF-1-α), a fragment of the β-tubulin gene (B-tub2), and the D1/D2 domains of the ribosomal DNA large subunit (LSU).
The multilocus phylogenetic tree ( Figure 6) constructed with the concatenated sequences of ITS, B-tub2, and D1D2 showed that the type strains C. mutabilis CBS 157.44 and C. hoffmannii CBS 245.38 clustered within distinct clades and that both were distant from Coniochaeta massiliensis (PMML0158). Indeed, this newly isolated yeast was divergent from all other Coniochaeta species present in the tree. However, in the single-locus trees for ITS and B-tub2, Coniochaeta massiliensis (PMML0158) seemed closer to Coniochaeta species than in other single-locus trees for D1/D2 and TEF-1a, where it appeared much more distant from Coniochaeta species than the genus Colletotrichum.
Based on phylogeny, the new isolate seems divergent from the group's other known taxa of Coniochaeta. de Hoog et al. (2000) described C. lignaria as being genetically closely related to C. hoffmannii. However, in our phylogenetic tree, these yeasts clustered into two different clades, indicating a large distance between the two species. These findings are in line with those of both Perdomo et al. (2011) and Weber and Begerow (2002), who reported significant differences between these species.
Antifungal susceptibility testing showed relatively low MICs for AMB, 5-FC, ITC, POS, and VOR in all strains. These results are in line with those of Perdomo et al. (2011) but contrast with those of other authors [9,13,20] who reported relatively high MICs for AMB and ITC against C. hoffmannii and C. mutabilis.
We macroscopically observed a fast-growing phenotype in Coniochaeta species. This finding contrasts with those of Ahmad et al. (1985), who reported slow colony growth. All species displayed the same flat and moist colonies. Only C. mutabilis additionally displayed low aerial growth, as reported by Drees et al. (2007). The primary colony colour remained unchanged (white/beige to orange/salmon). None of the colonies turned brown/dark in culture even after one month of incubation. This aspect has been well described in C. hoffmanii, a species that lacks the dematiaceous aspect, whereas C. mutabilis is classified within dematiaceous fungi, which are particularly typified by the presence of melanin in hyphae and conidia cell walls, which is responsible for dark pigment emission. Occasionally, certain C. mutabilis species lack the melanin property, as mentioned in Khan Perdomo et al. (2011). This was noted for the C. mutabilis DSM 10716 strain. Furthermore, in this study, the three species lacked the dematiaceous aspect.
The most remarkable morphological characteristics of the Coniochaeta species that have been described were the presence of several adelophialides, discrete phialides, and cylindrical or curved conidia, in addition to the presence or absence of collarettes and chlamydospores, depending on the species [9,14,18,29]. Most of these specific characteristics were observed in the newly described species (PMML0158). Based on this microscopic analysis, we infer that Coniochaeta massiliensis is closer to C. mutabilis, due to the decisive presence of a collarette.
Morphological characterisation helped for species differentiation, and physiological analysis was more convincing in distinguishing the three species, with the aim of describing the new one as thoroughly as possible. EDX (energy-dispersive X-ray spectroscopy) and Biolog TM phenotypic technology revealed divergent chemical mapping and carbon-source oxidation/assimilation profiles between the new isolate and the two main species of Coniochaeta genus. Moreover, the Biolog TM system findings were more relevant, as they revealed that the physiological profile of Coniochaeta massiliensis was closer to C. hoffmannii DSM 2693 than to C. mutabilis DSM 10716.
In conclusion, the clinical yeast isolate PMML0158 is herein described as Coniochaeta massiliensis, a new species that can be easily discriminated from the other species in the Coniochaeta genus owing to distinct genomic sequences and chemical and physiological profiles.

Data Availability Statement:
The Coniochaeta massiliensis holotype is available in the IHU MI (no. PMML0158) and IHEM (no. 28559) strain collections. The nucleotide sequences are available in GenBank (accession nos. OM366153, ON000097, OM640093, and OM366268). The datasets analysed in the current study are available from the corresponding author upon reasonable request.

Acknowledgments:
The authors gratefully acknowledge wholehearted and fruitful discussions with Pierre Pontarotti concerning phylogenetic analysis and the technical support of Anthony Fontanini for electron microscopy investigations. We also sincerely thank Takashi Irie, Kyoko Imai, Shigeki Matsubara, Taku Sakazume, Toshihide Agemura, and the Hitachi team in Japan for the collaborative study conducted together with IHU Méditerranée Infection and for the installation of TM4000 Plus microscopes at the IHU Méditerranée Infection facility.

Conflicts of Interest:
The authors have no conflicts of interest to declare regarding this study. Partial translation elongation factor 1-alpha gene B-tub2