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ОСОБЕННОСТИ
МИКРОМОРФОЛОГИИ
ASPERGILLUS TUBINGENSIS:
СКАНИРУЮЩАЯ
ЭЛЕКТРОННАЯ
МИКРОСКОПИЯ
Степанова А.А. (зав. лаб.)*, Васильева Н.В. (директор института, зав. кафедрой), 2Ямагучи M. (главный специалист), 2Чибана Х. (профессор), 1Чилина Г.А. (зав. лаб.), Богомолова Т.С. (зав. лаб.)
1НИИ медицинской микологии им. П.Н. Кашкина, СевероЗападный государственный медицинский университет им. И.И. Мечникова, Санкт-Петербург, Россия; 2Центр исследований по медицинской микологии, Университет г. Чиба, Япония
В сканирующем электронном микроскопе изучены особенности ультраструктуры клеток воздушного вегетативного мицелия и ко-нидиофоров у Aspergillus tubingensis (штамм PKnrF-104/BKMF-412). Клеточные стенки зрелых клеток гиф мицелия имели продольно-складчатую текстуру, что обусловлено наличием на их поверхности внеклеточного матрикса. B изученных колониях показано наличие двух типов конидиогенных аппаратов. Конидиеносцы зрелых конидиогенных аппаратов снабжены гладкими (мелких размеров ко-нидиофоры) и тонко-гранулярными (крупные конидиофоры) клеточными стенками. Фиалиды и конидии в конидиофорах A. tubingensis закладываются и формируются асинхронно. Зрелые конидии имели на своей поверхности густо расположенные шипики.
Ключевые слова: Aspergillus tubingensis, клетки гиф, конидиоген-ный аппарат, сканирующая электронная микроскопия, ультраструктура
THE PECULIARITY OF ASPERGILLUS TUBINGENSIS MICROMORPHOLOGY: SCANNING ELECTRON MICROSCOPY
1Stepanova A.A. (head of the laboratory), 1Vasilyeva N.V. (director of the institute, head of the department), 2Yamaguchi M (grand fellow), 2Chibana H. (associate professor), 1Chilina G.A. (head of the laboratory), 1Bogomolova T.C. (head of the laboratory)
1North-Western State Medical University named after I.I. Mechnikov, Kashkin Research Institute of Medical Mycology of, St. Petersburg, Russia; 2Medical Mycology Research Center, Chiba University, Chiba, Japan
Using scanning electronic microscope, the peculiarity of the ultrastructure of aerial hyphal cells and conidiogenous apparatus of the strain (PKnrF-104/BKMF-412) of Aspergillus tubingensis were studied. Cell walls of mature hyphae of mycelium in mature hyphal cells had longitudinally-folded texture that was caused by presence on their surface extracellular маМх. The existence of two types the conidiophores were demonstrated in investigated colonies. Stipes of the conidiogenous apparatus in all stages of development posses with smooth (small conidiophores) and fine-granular (large one) cell walls. It was shown thatphyalides and conidia in conidiophores of A. tubingensis developed asynchronously. Mature mnidia had densely located spinules on the surfaces.
Key words: Aspergillus tubingensis, conidiogenous apparatus, hyphal cells, scanning electron microscopy, ultrastructure
* Контактное лицо: Степанова Амалия Аркадьевна,
e-mail: [email protected]
INTRODUCTION
The A. niger complex includes several cryptic species. The most frequent one in the clinical setting was Aspergillus tubingensis Yauchi, Someya&Udagawa, which has been found with the same prevalence as A. niger [1]. A. tubingensis was the causive agent of the invasive aspergillosis [1], cutaneous infection keratitis [2], otomycosis [3] and osteomyelitis [4]. Previously using scanning electron microscope (SEM) the mature conidiophores [5, 7] and conidia [5, 6] of growing in vitro cultures A. tubingensis, which isolated from environmen were investigated.
This paper aimed to study the in vitro growing cultures of A. tubingensis and compared this data with previously investigated cryptic species A. niger.
МАТЕRIALS AND METHODS
We studied the strain (PKOTF-104/BKMF-412, Russian collection of pathogenic fungi) of A. tubingensis isolated from the patient. The fungal strain was identified as A. tubingensis on the basis of colony morphology, macroscopic, microscopic examination and by the DNA sequencing of partial ^-tubulin-coding gene.
The fungal cultures were cultivated on the Czapek's medium on 27 °C. After 10 and 20 days of cultivation the parts of fungal colony were fixed for electron microscopy in 3% glutaraldehyde (in cacodylate buffer pH 7.2), followed by 1% OsO 4, then dehydrated and embedded in mixed epon-araldit resin. Ultrathin sections were cut by Ultratome LKB V, stained with uranyl acetate and lead citrate and were observed on the SEM microscope JSM 35 (Jeol, Tokyo, Japan).
RESULTS AND DISCUSSION
Colony morphology. Colonies after 10 days of growing on the Czapek's medium on 27 °C showed the diameter of 8 cm, velvety, with different colored zones, regularly and radially distributes depression (Fig.1 a, b).
Aerial vegetative mycelium. Hyphae in the young marginal part of colony dispersed chaotic and loose (Fig. 1 c). In the mature part of colony their density of distribution was increased, they formed round gleam (Fig. 1 d, arrows). The surfaces of young hyphae were smooth (Fig. 1 e). In mature hyphal cells surfaces were longitudinally folded (Fig. 1 f) which was caused by existence on their surface so called "extracellular matrix". This layer were revealed on the surfaces of mature cells of aerial mycelium in another species from genus Aspergillus by using SEM [9, 10, etc.] and transmission electron microscopy (TEM) [11, 12].
Mature conidiophore. In direction from the central part of colony to its edge the frequency of mature conidiophores considerably decreased (Fig. 1. b, arrows). The specific peculiarities of this species were the presence in colony two types of conidiophores. In one type of which the stipe was long (5-6 mm) and narrow (15-20 (m), slightly wavy and with smooth cell walls in young conidiophores (Fig. 1 g-i) and fine-granular in mature (Fig. 1 j, arrows). Apical vesicles were roundish (40-60 (m), completely covered with biseriate metules (14-30 (m, Fig. 1 i, j) and phialides (7,010 x 2,5-3,1 (m, Fig. 1 i, j). Mature conidiophores posses with roundish apical head (Fig. 1 g-i). In comparison with another strains of A. niger [8], which also isolated from patients, the head contour in A. tubingensis conidiophores irregular (Fig. 1 g), which is caused by different height of phialides (Fig. 1 h).
Fig. 1. A. tubingensis: colonium which was cultivated 10 days in Czapek's medium (a), scanning electron microscopy (b-j). Explanation for this and another figures: Cn - conidia, Cr - conidiophore, Hd - head, Hp - hyphae, M - metule,
Ph - phialide(s), St - stipe.
Conidia developed asynchronously (Fig. 2 a) in the apex of phialides. Their formation start with origin the small swelling (conidial initial, fig. 2 a, arrows) which by isodiametrical grows led to formation the spherical in shape (from 3,0 to 3,3 (m) mature conidia (Fig. 1 h-j, 2 a, b). In the surfaces of mature conidia elements of external sculptures (Fig. 1 j, 2 a, b, 3 a, b) were in the form of spinules. For comparison the mature conidia of A. niger [9, 13, etc.] have the same surface micromorphology. When mature conidia in A. tubingensis undergo dehydration, their sizes became small, and elements of the sculpture - more expressed.
On the conidiophores of the second type the stipe not so long (3-4 mm), with width 10 (m (Fig. 2 c). The apical part of conidiophore ere inverse pear-shaped (5 x 6 (m) form (Fig. 1 c, d, arrows). On the apex of head present only phialides (Fig. 1 c-e). Mature conidia were spherical in shape (from 3,0 to 3,2 (m), formed in long sized chains (Fig. 2 b-e).
CONCLUSION
The ultrastructure of the mature hyphal cells walls in A. tubingensis was more regular in comparison with A. niger [9]. For last also was typical more variable structure
of stipe cells in conidiophores. In the colony of the investigated strain A. tubingensis we revealed two types of conidiophores which differ according its sizes, presence of metules, phyalides and the presence of conidial chains, which were not typical for A. niger [9, 13]. A. tubingensis the contour of mature conidiophore head was not strongly spherical as in A. niger [9, 13].
The mature conidia which developed in A. tubingensis showed two types of conidiophores which were simple according to the sizes and their cell walls architectonics and consist of from the densely located spinules (Fig. 3 a, b). For comparison mature conidia in A. niger posses with lowly sinuous folds (Fig. 1 c). For the conidiophores of the investigated strain of A. tubingensis were typical the asynchronously development of phialides and conidia, which what was typical also for A. niger [9, 13].
Thus our data demonstrated essentially differences in micromorphology of all types of cells between the two cryptic species A. tubingensis and A. niger. Actually to provide the study under TEM the ultrastructure of the septal pore apparatus in the hyphal cells of this two species for final resolving the question about the degree of filiation between this species.
LITERATURE
1. Alastruey-Izquierdo A., Mellado E., Pelaez T., et al. Population-based survey of filamentous fungi and an-tifungal resistance in Spain (FILPOP Study). Antimicrob. Agents Chemother. 2013; 57 (7): 3380-3387.
2. Arabatzis M., Kambouris M., Kyprianou M., et al. Polyphasic identification and susceptibility to seven antifungals of 102 Aspergillus isolates recovered from immunocompromised hosts in Greece. Antimicrob. Agents Chemother. 2011; 55: 3025-3030.
3. Kredics L., Varga J., Kocsube S., et al. Infectious keratitis caused by Aspergillus tubingensis. Cornea. 2009; 28: 951-954.
4. Szigeti G., Sedaghati E., Mahmoudabadi A.Z., et al. Species assignment and antifungal susceptibilities of black aspergilli recovered from otomycosis cases in Iran. Mycoses. 2012; 55: 333-338.
5. Bathoorn E., Escobar Salazar N., Sepehrkhouy S., et al. Involvement of the opportunistic pathogen Asper-gillus tubingensis in osteomyelitis of the maxillary bone: a case report BMC. Infect. Dis. 2013: 13-59.
6. Noman E.A., Al-Gheethi A.A., Norulaini N., et al. Assessment of relevant fungal species in clinical solid wastes. Environ. Sci. Pollut. Res. 2016.
7. Silva D.M., Batista L.R., Rezende E.F., et al. Identification of fungi of the genus Aspergillus section nigri using polyphasic taxonomy. Brasilian J. Microbiol. 2011; 42 (2).
8. Khan S., Nadir S., Shah Z.U., et al. Biodegradation of polyester polyurethane by Aspergillus tubingensis. Environmental pollution. 2017; 225: 489-480.
9. Степанова А.А., Васильева Н.В., Чилина Г.А. Сканирующая электронная микроскопия Aspergillus niger. Проблемы медицинской микологии. 2018; 20 (2): 40-44. [Stepanova A.A., Vasil'eva N.V., Chilina G.A. Skaniruyushchaya ehlektronnaya mikroskopiya Aspergillus niger. Problemy medicinskoj mikologii. 2018; 20 (2): 40-44 (In Russ)].
10. Степанова А.А., Чилина Г.А., Баракаева Ф.Р. Сканирующая электронная микроскопия Aspergillus fumigatus. Проблемы медицинской микологии. 2018; 20 (4): 39-42. [Stepanova A.A., Chilina G.A., Barakaeva F.R. Skaniruyushchaya ehlektronnaya mikroskopiya Aspergillus fumigatus. Problemy medicinskoj mikologii. 2018; 20 (4): 39-42 (In Russ)].
11. Степанова А.А., Синицкая И.А. Электронно-микроскопическое изучение клеток вегетативного ми-целия Aspergillus terreus Thom. Проблемы медицинской микологии. 2007; 9 (3): 26-33. [Stepanova A.A., Sinickaya I.A. Elektronno-mikroskopicheskoe izuchenie kletok vegetativnogo myceliya Aspergillus terreus Thom. Problemy medicinskoj mikologii. 2007; 9 (3): 26-33 (In Russ)].
12. Степанова А.А., Васильева Н.В., Чжан Ф., Тонг Д. Ультраструктурное исследование клеток вегетативного мицелия Aspergillus candidus Link, выращенных in vitro. Проблемы медицинской микологии. 2016; 18 (2): 23-27. [Stepanova A.A., Vasil'eva N.V., Chzhan F., Tong D. Ul'trastrukturnoe issledovanie kletok vegetativnogo miceliya Aspergillus candidus Link, vyrashchennyh in vitro. Problemy medicinskoj mikologii. 2016; 18 (2): 23-27 (in Russ)].
13. Степанова А.А., Синицкая И.А. Морфогенез конидиогенного аппарата Aspergillus niger по данным электронной микроскопии. Проблемы медицинской микологии. 2004; 6 (2): 37-48. [Stepanova A.A., Sinickaya I.A. Morfogenez konidiogennogo apparata Aspergillus nigerpo dannym ehlektronnoj mikroskopii. Problemy medicinskoj mikologii. 2004; 6 (2): 37-48 (In Russ)].
Поступила в редакцию журнала:11.03.2019 Рецензент: В.С. Митрофанов
