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8УДК 57.012.4: 576.3:582.282
СРАВНИТЕЛЬНЫЙ УЛЬТРАСТРУКТУРНЫЙ АНАЛИЗ IN VITRO РАСТУЩИХ КЛЕТОК ГИФ SCEDOSPORIUM AURANTIACUM
''Степанова А.А. (зав. лаб.)*, 2Ямагучи M. (главный специалист), 2Чибана Х. (профессор), ''Васильева Н.В. (директор института, зав. кафедрой), 1Шульгина М.В. (зам. директора по научной работе)
1 НИИ медицинской микологии им. П.Н. Кашкина, СевероЗападный государственный медицинский университет им. И.И. Мечникова, Санкт-Петербург, Россия; 2 Центр исследований по медицинской микологии, Университет г. Чиба, Япония
©Коллектив авторов, 2017
При сравнительном ультраструктурном анализе особенностей морфогенеза клеток гиф Scedosporium aurantiacum выявили значительные различия между штаммом CBS 136.046 и тремя другими (CBS 116910, CBS 136047, CBS 136049). Показаны различия в характере споруляции изученных культур, размерах интерфазных ядер, морфологии оболочки ядра, присутствии/отсутствии митохондри-ального ретикулума, а также микротелец, компонентов эндомем-бранной системы, типе аккумулируемых запасных веществ и внеклеточных вторичных метаболитов. Клетки вегетативного мицелия колоний всех изученных штаммов содержали одно интерфазное ядро с низким уровнем конденсированного хроматина. Выявленные различия в морфогенезе клеток гиф коррелируют с данными по строению стенки конидий и септального порового аппарата изученных штаммов. Впервые склероции обнаружены в культурах разных штаммов S. aurantiacum, а также отмечена важность использования данных ультраструктуры клеток гиф, выращенных in vitro, для таксономических исследований.
Ключевые слова: сравнительный анализ, клетки гиф, морфогенез, Scedosporium aurantiacum, склероций, in vitro, ультраструктура
COMPARATIVE ULTRASTRUCTURAL ANALYSIS OF THE IN VITRO GROWING HYPHAL CELLS OF SCEDOSPORIUM AURANTIACUM
1Stepanova A.A. (head of the laboratory), 2Yamaguchi M.M. (grand-fellow), 2Chibana H. (associated professor), Vasilyeva N.V. (director of the institute, head of the chair), 1Shulgina M.V. (deputy director for scientific work) 1Kashkin Research Institute of Medical Mycology, Northwestern State Medical University named after I.I. Mechnikov, St. Petersburg, Russia; 2Medical Mycology Research Center, Chiba University, Chiba, Japan
© Collective of authors, 2017
Comparative ultrastructural analyses of the peculiarities of Scedosporium aurantiacum hyphal cells show significant differences between the strain CBS 136.046 and another three (CBS 116910, CBS 136047, CBS 136049). Differences in the pattern of sporulation within cultures, interphase nucleus size, morphology of nuclear envelope, presence/ absence of the mitochondrial reticulum, and also microbodies, components of endomembrane system, types of accumulated storage substances and extracellular secondary metabolites were revealed. The cells of vegetative mycelium in colony of all studied strains contained one interphase nucleus with lower level of condensed chromatin. The morphological differences in the hyphal cells correlated with the data about the structure of conidial wall and septal pore apparatus of studied strains. For the first time the sclerotia were revealed in cultures of different strains of S. aurantiacum; and also importance of the in vitro growing hyphal cell ultrastructural data for taxonomical investigations was shown.
Key words: comparative analysis, hyphal cells, morphogenesis, Scedosporium aurantiacum, sderotium, in vitro, ultrastructure
Контактное лицо: Степанова Амалия Аркадьевна, тел.: (812) 303-51-40
Scedosporium aurantiacum Gilgado, Cano, Gene & Guarro - opportunistic pathogenic fungi, was found in soil, sewage and polluted waters [1]. This fungus cause malignant otitis externa, osteomyelitis, invasive sinusitis, keratitis, brain infection, cystic fibrosis and pneumonia [2-5]. The infection developed also after solid organ transplantation [6]. Previously we demonstrated distinct differences in the conidial wall and septal pore apparatus structure of the hyphal cells of S. aurantiacum strains and type species of genus Scedosporium - S. apiospermum [7]. Ultrastructural study of the in vitro growing hyphal cell morphogenesis of S. aurantiacum has not been performed. Thus, it may be interesting to study peculiarity in pattern of morphogenesis of vegetative mycelium cells on different S. aurantiacum strains and compare these data with the strains of S. apiospermum [8].
MATERIALS AND METHODS
In present work we investigate the cells of four strains of S. aurantiacum Gilgado, Cano, Gene & Guarro (CBS 116910, CBS 136046, CBS 136047, CBS 136049, Centraalbureau voor Schimmelcultures Fungal Biodiversity Centre), which were cultured on potato dextrose agar at 28 °C and fixed after 7 and 20 days of culture. The stains were verified by rDNA ITS sequencing. The strain CBS 116910 was isolated from human wound exudate, CBS 136046 - from human lung and CBS 136047, CBS 136049 - from soil).
For scanning electron microscopy the part of fungal colonies with nutrient medium after 7 and 20 days of sowing was transported in 3% glutaraldehyde (on 0,1 M cacodylate buffer) for 3 hour, post-fixed overnight in 1% osmium tetroxide in same buffer, dehydrated by ethanol series (30°^70°), critical-point dried (HCP-2) for 15 min, coated with gold and observed in JSM 35 (JEOL, Tokyo, Japan).
For transmission electron microscopy (TEM) the pieces of medium with different part of fungal colonies were fixed for 3 h in 3% glutaraldehyde and post-fixed in 1% osmium tetroxide for 10 h. Then samples were dehydrated through series of ethanol and acetone and embedded in epon-araldite epoxy resin.
We carried out the light microscopic investigations of the semi-thin epoxy sections (3-5 ^m), which were cut on the Pyramitome 1180 (LKB, Bromma, Sweden) using of glass knives and then colored with toluidine blue. After investigations of the semi-thin sections under light microscope Leica DM 4000, we revealed area, which necessary for subsequent target trimming (on aerial or submerged mycelium). Ultrathin sections were cut on Ultratome 2088 (LKB, Bromma, Sweden), stained with uranyl acetate and lead citrate and then examined under trans-mission electron microscope JEM-100 CX II (JEOL, Tokyo, Japan).
RESULTS AND DISCUSSION
Longitudinal semi-thin epoxy sections of the 20-days part of strains colonies demonstrated the presence of three distinct layers (Fig. 1 a-c, layers 1-3). The arrows on fig. 1 a-c demonstrated the nutrient medium surfaces. The 1st outer layer was thicker in culture of strain CBS 136049 (Fig. 1 a, layer 1). Contrary in the culture CBS 136046 this layer was very thin (Fig. 1 c, layer 1) and in CBS 116910 and CBS 136047 (Fig. 1 b, layer 1) - with intermediate thickness. In this layer we found dead or on different stage of senescence hyphal cells and mature conidia; there were no intact hyphae
as a rule. The mature conidia of all investigated strains were concentrated in upper part of submerged mycelium (Fig. 1 a-c, layer 2). Concentration on conidia was higher in the semi-thin sections of fungal colony of strains CBS 136049 (Fig. 1 a, layer 2) and CBS 136046 (Fig. 1 c, layer 2). The 3-rd layer in submerged mycelium was composed of practically only with dead hyphae and in different stages of senescence and destruction.
Fig. 1. Light microscopy of longitudinal semi-thin epoxy sections of the mature part of colony S. aurantiacum. Sc -sclerotia. f - demonstrated nutrient medium surface; a - CBS 136049, b - CBS 116910, CBS 136047, c - CBS 136.046. Scale: a-c - 100 |m.
During «maturation» of the hyphal cells of aerial mycelium in all strains they produce thin-fibrillar matrix (Fig. 1, layer 1). The upper border of this layer was visible under light (Fig. 1 a-b), TEM (Fig. 2 d, arrows) and SEM (Fig. 4 g, arrow) microscopes. In the mature cultures of strain CBS 136.046 we did not find this border under TEM and the cells of aerial mycelium which produce lipophilic substance (Fig. 3 c, arrows).
The diameter of the mature hyphal cells of aerial and submerged mycelium in cultures of S. aurantiacum varied from 3,5 to 4,0 ^m. The cells of aerial and submerged mycelium contain one ellipsoidal (Table 1) interphase nucleus (Fig. 2 a, h, 3 h, i) with regular (CBS 116910, CBS 136047, CBS 136049) or irregular (CBS 136.046) envelope. The cells of vegetative mycelium of the strain CBS 136.046 considerably differ from another one by the presence of large interphase nuclei (Table). The interphase nucleus situated in the central part of cells occupied all its lumen and contain moderate level of randomly distributed condensed chromatin. Nucleolus localized near nuclear envelope, is spherical (0,5 ^m), and contain granular and fibrillar components, but the former was dominated (Fig. 3 h). The topography, number, sizes and level of chromatization of interphase nuclei in the cells of 4th analyzed strains on this and in another stages was similar.
Fig. 2. Ultrastructure of the aerial (a, c, d, f-i) and submerged (b, j) cells of S. aurantiacum. a, d - CBS 117410, b, h - 136049, c, e, f, g, i - CBS 136.046, j - CBS 116910. Abbreviations used on this and another figures: C - conidium; CW - cell wall; G - glycogen; GER - granular endoplasmic reticulum; H - hypha(e); LI - lipid inclusion(s); Ls - lomasomes; M - mitochondrium(a); Mt - microtubule; N - nucleus; Nu - nucleolus; S - septum; SER - smooth endoplasmic reticulum; V - vacuole(s), Vs - vesicles. Scale: a, b, e, g, h - 1
|m; c, d, i - 0,5 |m, f, j - 4 |m.
Fig. 3. Ultrastructure of the aerial (a-e, g) and submerged (f, h, i) cells of S. aurantiacum. f - on c show extra-cellular matrix, on f -discrete dark external deposition, on g - protein globule in vacuole. a, b, e - CBS 136049, c, d, f, g, i - CBS 136.046, h - CBS 301.79.
Scale: a, c, d - 3 |m, b, e-h - 1 |m, i - 2 |m.
Fig. 4. Ultrastructure of the sclerotiums in cultures of S. aurantiacum. f - on a, b, c, h - demonstrated sclerotiums, on g - the outer surfaces of the thin-fibrillar matrix. a, b - CBS 116910, c, d - CBS 136047, e, f - CBS 136049; g, h, i - CBS 136.046. Scale: a, c, e, h -
100 |m; b - 30 |m; d, f, i - 5 |m.
Fig. 5. Schematic drawing of S. aurantiacum young actively growing (a), mature (b, c) and in early stage senescence hyphal cells
(d). a, b, d - CBS 116910, 136047, 136049; a, c, d - CBS 136046.
Table
The main cytological characteristics of in vitro growing S. aurantiacum strains
Strains (CBS) The sizes of mature Sclerotium, |jm The number of interphase nucleus, |jm Diameter ofthe inter-phase nucleus, |jm The level of chroma-tization The contour of nuclear envelope The presence of mitochondrial reticulum in aerial and submerged mycelium The types of storage substances in mature cells The presence ofGER and SER The presence of micro-bodies The presence of extra-cellular lipophylic substance The presence of extracellular slime and dark external substance
116910 100-120 1 1,78x1,42 Low Reqular -/- LI, a +- - - +/-
136047 80-110 1 1,80x1,6 Low Reqular -/- LI, a +- - - +/-
136049 90-100 1 1,70x1,4 Low Reqular -/- LI, a +- - - +/-
136.046 40 x 60 -70 x 80 1 2,5x2,0 Low Irregular +/+ LI, LIV, a, GV ++ + + +/+
Notice: a - alpha glycogen; GER - granular endoplasmic reticulum; GV - protein granule in vacuole; LI - lipid inclusions in cytosole; LIV - lipophilic substances in vacuole; SER - smooth endoplasmic reticulum.
Under TEM the morphogenesis of the cells of aerial and submerged mycelium in all analyzed strains pass on three main stages: 1) growth, 2) maturation and 3) senescence. The 1st (Fig. 5 a) and 3rd (Fig. 5 d) stages in all strains pass regarding the one «scenario», but in 2nd stage the significant differences were found between the strain CBS 116910, 1360476 136049 (Fig. 5 b) and CBS 136046 (Fig. 5 c).
In the young active growing cells of mycelium, vacuoles were uniformly localized in cytosol. They were a few in number, small in size and show the light content or filled thin-fibrillar material (Fig. 2 a). Mitochondria as a rule situated near lateral cell wall (Fig. 2 a-e). The number on median section of cell varied from 5 to 8. They were single or in small groups, spherical (0,4-0,5 ^m) or ellipsoidal (0,5 x 0,6 ^m) in form. They have more electron dense matrix in comparison with the cytosol.
The growing hyphal cells differ according the type of endoplasmic reticulum (ER, Table). The long slightly curved cisterns of rough ER (Fig. 2 d) were found in the hyphal cells of all studied strains. The aggregations of the curved and tightly oriented tubular smooth ER were detected in the cells of aerial mycelium in only one strain (CBS 136.046, fig. 2 e). They formed the large (0,8-12,0 ^m) single irregular in form aggregations near cell wall (Fig. 2 e). Small number (from 3 to 7 on median cell section) of single (Fig. 2 c) secretory vesicles (60-70 nm) with thin-fibrillar content was distributed near cell wall. We found such types of vesicles in the hyphal cells of aerial mycelium in cultures of all investigated strains. In the hyphal cells of S. apiospermum [8] we also observed secretory vesicles with the same morphology. Only in the content of cells of aerial mycelium of one strain (CBS 136.046) we find large (90-110 nm) vesicles (Fig. 2 c) with more dark tightly oriented microfibrills. We did not find the possible sources of secretory vesicles - single Golgi cisterns, in the cells of mycelium of all investigated strains. For comparison, we observed the rare single horseshoe-shaped Golgi cisterns in the in vitro growing cells of S. apiospermum [8].
In the cytosol of investigated hyphal cells we did not find microbodies (Table). Contrary, in the hyphal cells of in vitro growing S. apiospermum [8] we observed them. Several single short straight microtubules were found deep in the cytosol (Fig. 2 e). Cytosol had moderate electron density and posses with numerous free ribosomes.
Plasma membrane was three-layered, closely contact with the cell wall. The cell wall was thin (0,05 ^m, Fig. 2 b) and two-layered. For the cell walls ofsubmerged hyphae were typical the presence the outer dark so-called «extracellular matrix» (Fig. 2 a, i), which consisted of tightly localized dark microfibrills. The thickness of extracellular matrix
was identical in all investigated strains cells and in 6 times thicker than the cell walls [7]. On the surfaces of living and senescent cells of aerial hyphal cells of all studied strains we found the discrete accumulations of thin-fibrillar slime (Fig. 2 c, e, 3 b, e, arrows). Another type of external fungal metabolite, which is typical for cells of aerial mycelium, was the discrete dark small practically homogenous deposition (Fig. 2 f, arrow). Very often the large amount of external slime and dark substances was also found between adjacent cells. In a whole, the amount of slime was predominated. Slime was also found on the surfaces of hyphal cells of aerial mycelium in S. apiospermum [8]. For this species the dark external substances was not typical. We suppose that in analyzed strains colonies S. aurantiacum the secretory vesicles with thin-fibrillar content participate in synthesis and secretion of external slime and the same with more dark content - the external dark substances.
During the maturation of the hyphal cells of S. aurantiacum the synthesis of storage substances (lipid inclusions, rosettes of a-glycogen, protein globules and lipophilic substances in vacuoles, Table, Fig. 5 b, c) were start. Lipid inclusions uniformly distributed in the hyphal cell cytosol and variable in size (0,4-0,7 ^m, Fig. 2 h, j, 3 g) and with median electron density. Initially in some cells the synthesis of lipid inclusions (Fig. 2 h) was start, but in another - lipid inclusions and rosettes of a-glycogen (Fig. 2 j). As a rule, in last type of mature cells lipid inclusions oriented in its central part and closely one to another. They were large sizes (1,0-1,6 x 0,5-0,8 ^m) and surrounded with aggregation of light rosettes of a-glycogen. The light color of last, perhaps, may be result of its elution during sample preparation. Only in vacuolar content of the mature hyphal cells of strain (CBS 136.046) we found the large (0,8-1,0 ^m) dark protein globule in vacuolar content (Fig. 2 g, arrows, 5 c, arrow) which localized near tonoplast and lipophilic substances (Fig. 3 g). In mature cells of vegetative mycelium of all analyzed strains in depending from its type (aerial, submerged) the amount of storage substances were identical.
In all analyzed strains cultures the number of mitochondria were increased (before 11-18 on median section) during the growth of the cells of aerial and submerged mycelium. They were polymorphic (0,2-0,4 ^m), straight or slightly curved and situated in near cell wall and in the space between the nucleus and cell wall. Only on serial sections of aerial and submerged cells of mycelium of the strain CBS 136.046 we revealed long (3,0-4,0 ^m) profiles of the one giant organelle, so called «mitochondrial reticulum» (Fig. 2 i, Table). In S. apiospermum [8] giant mitochondrion was developed also in all cells of mycelium of the two compared strains.
The cells of aerial mycelium in mature cultures (strain CBS 136.046), contrary with submerged, produced the third type of secondary lipophilic substance metabolite -homogenous median electron density external (outside the cell walls) and often in extracellular space (Fig. 3 c, arrows). It was interesting that in vacuolar content of mature aerial hyphae (Fig. 3 a) we also found this lipophilic substance. Previously we found the same type of external metabolite in the mature cultures of 2-wo strains of S. apiospermum [8]. But in mature cultures of last species this layer external occupied the lower part of aerial mycelium and so that not visible under SEM. Also we observed in the cells of aerial mycelium of this strains special type of tubular smooth endoplasmic reticulum which in our opinion may participate in the synthesis of lipophilic substances. Previously we suppose that in S. apiospermum [8] this lipophilic substance contain pigment, which presence of in extracellular space of aerial mycelium determinate its yellow color [8].
In the final stage of hyphal cells morphogenesis -senescence, large central vacuole was developed (Fig. 3 i, 5 d). The volume of cytosol, the amount of the storage substances and cell organelles significantly decrease (Fig.
3 f, i, 5 d). The volume of the protein globules (Fig. 3 g, h, arrows) and lipophilic substances in vacuoles gradually decreased and they finally disappeared. Numerous lomasomes (Fig. 3 i) were present near cell wall. In the whole, process of senescence pass according the way, which was described for another species of filamentous fungi [9-10; Stepanova A.A., Vasilyev A.E., 1994], including S. apiospermum [8].
Under the light (Fig. 1 c) and SEM (Fig. 4 a-i) microscopes in the surfaces of central mature parts of 20 days cultures of all investigated S. aurantiacum strains we found the single small or variable in sizes clusters of bodies (Fig. 4 e, h, arrows). In cultures of the strains CBS 116910, CBS 136047 and CBS 136049 they were spherical in size (100-135 ^m, Fig. 4 a-f, Table). The specific peculiarities of these bodies in culture of strain CBS 136047 was the presence in its apical part spherical flat smooth areas (Fig.
4 c, arrows). In culture of strain CBS 136.046 such type of bodies ellipsoidal in form (Fig. 4 h) and smaller in sizes (40-60 x 70-80 ^m). For wall surfaces of this morphological formation was typical uniformly good pronounced external crystalline texture (Fig. 4 b, d, f, i) and epidermoidea type of surfaces, which were typical for Pseudallescheria [2]. Only in strain CBS 136049 this bodies were covered with rare randomly distributed hyphae, which typical for ascomata of Pseudallescheria species [2]. The teleomorph stage for S. aurantiacum was unknown [2]. According the morphological charac-teristics it may be sclerotia. For comparison, in previously studied strains cultures of type species from genus Scedosporium - S. apiospermum we did not find the similar morphological formations [8].
Comparative analyses of the peculiarities of the morphogenesis of analyzed S. aurantiacum strains show significant differences between the strain CBS 136.046 and remains ones. Differences in the amount of formed conidia under light microscope were revealed - more numerous conidia we observed in the 20-days cultures of strains CBS 136049 and CBS 136.046.
The cells of the vegetative mycelium in cultures of all investigated strains contain one interphase nuclei with lower level of condensed chromatin, what was typical for
previously investigated strains S. apiospermum [7]. For the hyphal cells of strain CBS 136.046, in contrast to another one, was typical presence the large interphase nucleus with irregular outer envelope. For growing hyphal cells of this strain also were typical presence of the mitochondrial reticulum which demonstrated the evidence of its higher level of functional activity in comparison of another studied strains. The mature cells in cultures of strain CBS 136.046 accumulated not only lipid inclusions and a-glycogen, as other ones, but also large protein granule and lipophilic substances in vacuolar content. This data indicate about the differences in their physiology during growth on the same medium. The specific ultrastructural feature of the cells of this strain was the absence of microbodies and presence of the tubular smooth endoplasmic reticulum, which presence demonstrated the ability of this strain to synthesize the external lipophilic substances by the cells of aerial part of mycelium, what was also typical for growing in vitro S. apiospermum strains [8].
All cells of aerial mycelium of investigated strains synthesized and secreted the external secondary metabolites - slime and only hyphal cells of strain CBS 136.046 -discrete dark small droplets and homogenous median electron density lipophilic substance. It was interesting, that according the date Frisvad J.C. and Nielsen K.F. [11] strains of the S. aurantiacum, which we investigated in present work, divided on two groups according its possibility to produce the secondary metabolites: 1) CBS 116910, CBS 136046 and 2) CBS 136047, CBS 136049. As decided this authors, this distinction demonstrated its functional (physiological) relationship, rather than a taxonomical. Contrary, previously discovered differences in the ultrastructure of conidial sporoderm and septal pore apparatus between, at first time, S. apiospermum and S. auratiacum, and, at second time, different strains of last species [7] correlated with ultra-structural distinction in its hyphal cells in vitro morphogenesis [S. apiospermum: 8; S. aurantiacum: data of the present work]. For the first time we observed in the colony of investigated strains S. auratiacum presence of sclerotia, what significantly distinguished this species from type species - S. apiospermum for which typical only large cleistothecia, but sclerotia - not [2]. From the members of the genus Scedosporium sclerotia were described for S. boydii [2]. For the strain CBS 136.046, contrary with another one, was typical ellipsoidal sclerotia. According the data of several authors [12; Chang P.-K., Bennett J. W., Cotty P.J., 2001] in filamentous fungi were present the correlation between the secondary metabolism and production of sclerotia. The opinion of Calvo A.M. and Cary J. W. [12] that «they represent the principal source of primary inoculum for some phytopathogenic fungi» actually also for investigated in present work human pathogenic fungus and give them additional «card blanche» in confrontation with environmental condition, perhaps including the host tissues.
There were few studies on differences in pattern of morphogenesis between the strains of one filamentous fungal species in vitro [8]. We found that the main pattern of morphogenesis of three analyzed S. aurantiacum strains (CBS 116910, CBS 136047, CBS 136049) were identical. They were common also according to mature conidia sporoderm and septal pore apparatus ultrastructure [7]. These strains differ concordant the volume of accumulated external slime in layer 1: the cells of strains culture CBS
136049 was more active.
For comparison the similarity in ultrastructural aspects of the morphogenesis was typical for two strains previously investigated another species from the genus Scedosporium - S. apiospermum [8]. In last cases differences only in the area of sporulation and level of accumulated lipophilic substances were revealed what demonstrated the mediate influence of the secondary metabolites on the level of sporulation and inter strains differences.
Thus, the investigated S. aurantiacum strain CBS 136.046 significantly differ in comparisonоn with the another one according the presence of ellipsoidal sclerotia, differences
in size and contour of nuclear envelope, presence of mitochondrial reticulum, diversity of accumulated storage substances and secreted secondary metabolites. Before we revealed considerable differences of the strain CBS 136.046 from another investigated in respect to sporoderm and septal pore apparatus ultrastructure [7]. Presented data, for the first time, demonstrated importance of TEM morphogenetic data for the hyphal cell ultrastructure for taxonomical investigations.
ACKNOWLEDGEMENT
We are sincerely thanks to Prof. G. Sybren de Hoog for providing the Scedosporium auraticum cultures.
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Поступила в редакцию журнала:16.10.2017
Рецензент: В.Г. Корнишева
