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 (Fig. 2). However, none of the three yeasts were able to grow at 4 and 45°C. Colonies of the three isolates were initially white to beige, both on the surface and 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 mycelium growth.
Microscopic characterisation
The microscopic observation of the three strains was 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, 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 side and most often in clusters. Collarettes were only found in both Coniochaeta massiliensis and C. mutabilis. No chlamydospore was observed (Fig. 3).
Antifungal susceptibility testing (AFST)
The minimum inhibitory concentration (MIC) values of the three species for all nine antifungal drugs are shown in (Table 3). The MIC endpoints for all strains were determined as the lowest concentration inhibiting the growth of 90% of the strains and were determined as described in Perdomo et al. (2011), since there are no validated AFST guidelines for this genus. The MICs of AMB, 5-FC, ITC, POS and VOR were low for the three isolates. The new species of Coniochaeta displayed low echinocandins (AND and CAS) MICs and a high FL MIC, while C. hoffmannii and C. mutabilis demonstrated the opposite for these four antifungal drugs.
Table 3. Results of in vitro antifungal susceptibility testing.
MIC* (mg/L) read at 48h
|
|
|
AMB
|
AND
|
CAS
|
5-FC
|
FL
|
ITC
|
MIF
|
POS
|
VOR**
|
|
Coniochaeta massiliensis PMML0158
|
0,25
|
0,5
|
1
|
2
|
8
|
0,25
|
1
|
0,25
|
0,12
|
|
Coniochaeta hoffmannii DSM 2693
|
0,12
|
4
|
4
|
1
|
4
|
0,06
|
>32
|
0,12
|
0,12
|
|
|
Coniochaeta mutabilis DSM 10716
|
0,12
|
4
|
8
|
1
|
2
|
0,015
|
>32
|
0,03
|
0,03
|
|
|
*MIC, minimum inhibitory concentration
** AMB, amphotericin B; AND, anidulafungin; CAS, caspofungin; 5-FC, 5-fluorocytosine; FL, fluconazole; ITC, itraconazole; MIF, micafungin; POS, posaconazole; and VOR, voriconazole.
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. The score value generated was 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, were collected and have been incremented in the MALDI-TOF MS database.
Physiological analysis
1. EDX (Energy-Dispersive X-ray Spectroscopy)
The weight 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 (Fig. 4), each Coniochaeta species was very distant from the others, demonstrating highly heterogeneous chemical profiles.
2. Biolog™ system
The Biolog™ advanced phenotypic technology was very useful for the phenotypic characterisation. The oxidation and assimilation test results are illustrated using heat maps (Fig. 5, 6). 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 analysis
We built a phylogenetic tree using MEGA 11 software based on the assembled and concatenated sequences of ITS, Beta-tubulin2 and D1/D2 genetic regions for one clinical isolate, two reference strains and sequences of eight strains collected from GenBank (Table 2).
The multilocus analysis of the tree strains (Fig. 1) revealed the presence of 2 main clades; the first one is divided into five subclades. The second one includes the newly isolated strain of Coniochaeta PMML0158, which appears distinct from all other Coniochaeta species.
Taxonomy
Coniochaeta massiliensis Kabtani J. & Ranque S. sp. nov.
MycoBank: MB843839
(Fig. 3. A-L)
Etymology: Named in honour of Marseille (France), the city where it was isolated.
Diagnosis: Closely similar to the other two Coniochaeta species examined by 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 by lacking 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. At this point, C. massiliensis is closer to C. mutabilis, due to the decisive presence of the collarette.
Type: France: Marseille. Human body (abscess of the hand), 15 July 2020. (Holotype IHEM 28559). GenBank: OM366153 (ITS), ON000097 (Btub2), OM640093 (TEF-1a), OM366268 (D1/D2).
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 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 mycelium 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.
The Biolog™ carbon sources assimilation profile showed that C. massiliensis PMML0158 can assimilate different carbon substrates, such as 2 -keto-D-gluconic acid, D-gluconic acid, D-ribose, D-xylose, D-glucosamine, D-cellobiose, D-melibiose, Palatinose, Turanose, L-sorbose, and β-methyl-D-glucoside. Based on this phenotypic analysis, C. massiliensis PMMFL0158 appears similar to C. hoffmannii DSM 2693.
Host: Human
Additional specimen examined: C. hoffmannii: Type: Country of origin unknown. Human body, before 08 July 1983. (Holotype DSM 2693 - ATCC 34158 – SP33–4). GenBank: OM366155 (ITS), ON000099 (Btub2), OM640095 (TEF-1a), OM366270 (D1/D2).
Additional specimen examined: C. mutabilis: Type: Sweden. Human body, before 14 June 1996. (Holotype DSM 10716 - EMPA 573, S24 E). GenBank: OM366154 (ITS), ON000098 (Btub2), OM640094 (TEF-1a), OM366269 (D1/D2).