Syncephalastrum massiliense sp. nov. and Syncephalastrum timoneanum sp. nov. Isolated from Clinical Samples

Abstract

Mucormycosis is known to be a rare opportunistic infection caused by fungal organisms belonging to the Mucorales order, which includes the Syncephalastrum species. These moulds are rarely involved in clinical diseases and are generally seen as contaminants in clinical laboratories. However, in recent years, case reports of human infections due to Syncephalastrum have increased, especially in immunocompromised hosts. In this study, we described two new Syncephalastrum species, which were isolated from human nails and sputum samples from two different patients. We used several methods for genomic and phenotypic characterisation. The phenotypic analysis relied on the morphological features, analysed both by optical and scanning electron microscopy. We used matrix-assisted laser desorption-ionization time-of-flight mass spectrometry, energy-dispersive X-ray spectroscopy, and BiologTM technology to characterise the proteomic, chemical mapping, and carbon source assimilation profiles, respectively. The genomic analysis relied on a multilocus DNA sequence analysis of the rRNA internal transcribed spacers and D1/D2 large subunit domains, fragments of the translation elongation factor-1 alpha, and the β-tubulin genes. The two novel species in the genus Syncephalastrum, namely S. massiliense PMMF0073 and S. timoneanum PMMF0107, presented a similar morphology: irregular branched and aseptate hyphae with ribbon-like aspects and terminal vesicles at the apices all surrounded by cylindrical merosporangia. However, each species displayed distinct phenotypic and genotypic features. For example, S. timoneanum PMMF0107 was able to assimilate more carbon sources than S. massiliense PMMF0073, such as adonitol, α-methyl-D-glucoside, trehalose, turanose, succinic acid mono-methyl ester, and alaninamide. The polyphasic approach, combining the results of complementary phenotypic and genomic assays, was instrumental for describing and characterising these two new Syncephalastrum species.

Keywords: Mucorales; Syncephalastrum; genotype; mucormycosis; one new taxon; phenotype.

Figure 7

Lactophenol cotton blue mount of Syncephalastrum massiliense PMMF0073. (A) Sporangiophore with apical vesicles and merosporangial sacks enclosing merospores. (B) Columella and hyphae ribbon-like aspect. (C) Merospores. Optical microscopy (magnification ×1000). Scale bars: 50 μm.

Figure 8

Lactophenol cotton blue mount of Syncephalastrum timoneanum PMMF0107. (A) Sporangiophore with apical vesicles and merosporangial sacks enclosing merospores. (B) Columella and hyphae ribbon-like aspect. (C) Merospores. Optical microscopy (magnification ×1000). Scale bars: 50 μm.

Figure 9

Morphology of Syncephalastrum spp. (A) Sporangiophore with apical vesicles and merosporangial sacks of Syncephalastrum racemosum DSM 859. (B–D) Sporangiophore with apical vesicles and merosporangia of S. monosporum CBS 567.91, S. monosporum CBS 568.91, and S. monosporum CBS 569.91. (E,F) Columella of S. racemosum DSM 859 and S. monosporum CBS 567.91. Optical microscopy (magnification ×1000). Scale bars: 50 μm.

Figure 10

Vesicles, sporangiophores, merosporangia, and merospores of Syncephalastrum massiliense PMMF0073. Scanning electron microscopy TM 4000Plus (15 KeV lens mode 4). Scale bars: (A) = 50 μm, (B) = 30 μm, (C) = 200 μm, and (D) = 40 μm.

Figure 11

Vesicles, sporangiophores, merosporangia, and merospores of Syncephalastrum timoneanum PMMF0107. Scanning electron microscopy TM 4000Plus (15 KeV lens mode 4). Scale bars: (A) = 50 μm, (B) = 30 μm, (C) = 50 μm, and (D) = 20 μm.

Figure 12

Morphology of Syncephalastrum spp. (A) Sporangiophore with apical vesicles and merosporangial sacks of S. racemosum DSM 859. (B–D) Sporangiophore with apical vesicles and merosporangia of S. monosporum CBS 567.91, S. monosporum CBS 568.91, and CBS 569.91, respectively. Scanning electron microscopy TM 4000Plus (15 KeV lens mode 4). Scale bars: (A) = 40 μm, (B,C) = 30 μm, and (D) = 50 μm.

Figure 13

Principal component analysis (PCA) of the different structure measurements (hyphae, columella, sporangiola, merosporangium, and sporangiospores number within the merosporangial sack) using the TM4000 Plus microscope (SEM) for the four reference strains and two new species of Syncephalastrum. In this analysis, computed using the XLSTAT software V.2022.4.1, the principal components F1 and F2 explained 90.3% of the fungi structure variance.

Figure 1

MALDI-TOF MS dendrogram based on the protein expression intensity profile for the four reference strains and two new species of Syncephalastrum generated using the MALDI-TOF Biotyper Compass Explorer software V.4.1 (Bruker Daltonics).

Figure 2

Maximum parsimony dendrogram based on the ITS sequences, including Syncephalastrum massiliense PMMF0073, Syncephalastrum timoneanum PMMF0107, and 20 other Syncephalastrum spp. reference strains. Rhizopus microsporus ATCC 52813 was used as the outgroup. The tree was constructed via the maximum parsimony method using MEGA 11 software. The bootstrap values were estimated at 1000 replications.

Figure 3

Bayesian phylogenetic tree based on the ITS sequences of Syncephalastrum massiliense PMMF0073, S. timoneanum PMMF0107, and 20 Syncephalastrum spp. reference strains. Rhizopus microsporus ATCC 52813 was used as the outgroup. The tree was constructed using the MrBayes software (3.2.7a) and Figtree (V.1.4.4).

Figure 4

Maximum parsimony dendrogram based on the concatenated ITS, TUB2, TEF1, and D1/D2 sequences of Syncephalastrum massiliense PMMF0073, S. timoneanum PMMF0107, and four Syncephalastrum spp. type strains. Rhizopus microsporus ATCC 52813 was used as the outgroup. The tree was constructed via the maximum parsimony method using the MEGA 11 software. The bootstrap values were estimated at 1000 replications.

Figure 5

Bayesian phylogenetic tree based on the concatenated ITS, TUB2, TEF1, and D1/D2 sequences of Syncephalastrum massiliense PMMF0073, S. timoneanum PMMF0107, and four Syncephalastrum spp. type strains. Rhizopus microsporus ATCC 52813 was used as the outgroup. The tree was constructed using the MrBayes software (3.2.7a) and Figtree (V.1.4.4).

Figure 6

Culture growth on the SDA GC medium at 25 °C. (A) Syncephalastrum massiliense PMMF0073. (B) Syncephalastrum timoneanum PMMF0107. (C) S. racemosum DSM 859. (D) S. monosporum CBS 567.91. (E) S. monosporum CBS 568.91. (F) S. monosporum CBS 569.91.

Figure 14

Principal component analysis of the EDX (energy-dispersive X-ray spectroscopy) chemical mapping profile for the four reference strains and the two novel Syncephalastrum species. In this analysis, computed using the XLSTAT software V.2022.4.1, the principal components F1 and F2 explained 41.7% of the EDX data variance.

Figure 15

Heat map of the carbon sources assimilation assessed using the Biolog™ system for the four reference strains and two species of Syncephalastrum, computed using the XLSTAT software V.2022.4.1. Colour gradient interpretation: the most assimilated substrates are in red, and the least assimilated substrates are in yellow.