曲霉生物膜的形成过程与结构特征

中国真菌学杂志　 2011, Vol. 6 　Issue (2): 93-97.

曲霉生物膜的形成过程与结构特征

李丽娟, 陈伟, 许辉, 万喆, 李若瑜, 刘伟

北京大学第一医院皮肤性病科真菌室, 北京大学真菌和真菌病研究中心, 北京, 100034

收稿日期:2010-09-17 出版日期:2011-04-28 发布日期:2011-04-28
通讯作者: 刘伟,E-mail:liuw90@yahoo.com E-mail:liuw90@yahoo.com
作者简介:李丽娟,女(汉族),硕士研究生在读.E-mail:lilj630@sina.com
基金资助:
国家自然科学基金(30970131);北京市自然科学基金(7102149)

Development and architecture characteristics of Aspergillus biofilm

LI Li-juan, CHEN Wei, XU Hui, WAN Zhe, LI Ruo-yu, LIU Wei

Department of Dermatovenerology, Peking University First Hospital, and Research Center for Medical Mycology, Peking University, Beijing, 100034, China

Received:2010-09-17 Online:2011-04-28 Published:2011-04-28

摘要/Abstract

摘要： 目的研究曲霉生物膜的形成过程和结构特征。方法我们利用一个曲霉生物膜体外模型研究其形成过程和结构特征。将200μL浓度为1×105孢子/mL的受试曲霉(烟曲霉AF293,黄曲霉BMU03940,土曲霉BMU00802,黑曲霉BMU04689)的孢子悬液加到24孔组织培养板中的无菌塑料细胞培养盖玻片上,37℃孵育不同时间(0、2、4、8、10、12、16、18、24、48、72h),加入25μmol/L的FUN-1室温避光染色后,用波长488nm激光激发,通过共聚焦激光扫描显微镜观察曲霉生物膜的形成过程;再用波长为488nm和633nm激光同时激发,将两个波长下的图像叠加后观察曲霉生物膜的活力;利用XYZ轴成像观察其结构特征。在上述不同的时间点用钙荧光白染色后,用波长为405nm的紫外光激发,观察曲霉生物膜细胞外基质的产生。结果烟曲霉AF293在第4h即开始有散在的孢子黏附于盖玻片上;8h时孢子开始萌芽,10-12h菌丝延长形成单细胞层;16-20h菌丝缠绕形成多层立体结构;24h形成一个具有复杂的三维立体结构特征的多细胞菌落,菌丝有序排列,细胞外基质弥散的分布在菌丝的周围;48-72h生物膜逐渐成熟。成熟的烟曲霉生物膜是由细胞外基质包裹的有序排列的菌丝形成的复杂立体结构。黄曲霉BMU03940、土曲霉BMU00802、黑曲霉BMU04689与烟曲霉AF293有类似的生物膜发育阶段,包括黏附、孢子萌芽、菌丝延长、菌丝有序排列形成三维立体结构。结论烟曲霉、黄曲霉、土曲霉和黑曲霉在体外都能形成典型的生物膜,它的形成过程和结构特征与其他真菌生物膜类似。

关键词: 曲霉, 生物膜, 结构特征

Abstract: Objective To investigate the development and architecture characteristics of Aspergillus biofilm.Methods In vitro biofilms of Aspergillus fumigatus(AF293),Aspergillus flavus(BMU03940),Aspergillus terreus(BMU00802) and Aspergillus niger(BMU04689) formed on the surface of sterile plastic cell culture coverslips in 24-well tissue culture plates by adding 200 μL of 1×105 spores/mL cell suspension to each well.After incubation at 37℃ for various time periods(2,4,8,10,12,16,18,24,48 and 72h),the biofilms were stained with FUN-1 in dark for 45 minites.The forming process and vitality were examined under confocal microscope by 488 or combination with 633 argon ion laser stimulation.Calcofluor White stain was applied to observe cytoplasm production with 405 argon ion laser triggering.Results A.fumigatus conidial adhesion occurred at 4h and conidia began to swell and germinate at 8h.Hyphae intertwined and amonolayer formed at 10 to 12h,followed by network structure forming at 16 to 20h.Then the complex three-dimensional architecture with extracellular matrix aroundhyphae was set up at 24h,with gradual matureation at 48 to 72h.Similar to A.fumigatus,the biofilms of A.flavus,A.terreus and A.nigerhad distinct developmental phases including adhesion,germination,filamentation,monolayer development,proliferation and maturation.Conclusions A.fumigatus,A.flavus,A.terreus and A.niger are able to form typical biofilm with distinct developmental phases and architecture characteristics.

Key words: Aspergillus spp, biofilm, architecture characteristic

中图分类号:

R379.6

李丽娟, 陈伟, 许辉, 万喆, 李若瑜, 刘伟. 曲霉生物膜的形成过程与结构特征[J]. 中国真菌学杂志, 2011, 6(2): 93-97.

LI Li-juan, CHEN Wei, XU Hui, WAN Zhe, LI Ruo-yu, LIU Wei. Development and architecture characteristics of Aspergillus biofilm[J]. Chinese Journal of Mycology, 2011, 6(2): 93-97.

参考文献

[1] Costerton JW,Lewandowski Z,Caldwell DE,Microbial biofilms[J] Annu Rev Microbiol,1995,49 (1):711-745.
[2] Donlan RM.Biofilms:microbial life on surfaces[J] Emerg Infect Dis,2002,8 (9):881-890.
[3] Chandra J,Patel JD,Li J,Modification of surface properties of biomaterials influences the ability of Candida albicans to form biofilms[J] Appl Environ Mierabiol,2005,71 (12):8795-8801.
[4] Emerson RJ,Camasano TA.Nanoscale investigation of pathogenic microbial adhesion to a biomaterial[J] Appl Environ Microbiol,2004,70 (10):6012-6022.
[5] Ramage G,Martinez JP,Lopez-Ribot JL.Candida biofilms on implanted biomaterials:a clinically significant problem[J] FEMS Yeast Res,2006,6 (7):979-986.
[6] Chandra J,Mukherjee PK,Leidich SD,Antifungal resistence of Candida biofilms formed on denture acrylic in vitro[J] J Dent Res,2001,80 (3):903-908.
[7] Denning DW.Invasive aspergillosis[J] Clin Infect Dis,1998,26 (4):781-803.
[8] Kontoyiannis DP,Bodey GP.Invasive aspergillasis in 2002:an update[J] Eur J Clin Microbiol Infect Dis,2002,21 (3):161-172.
[9] Beauvais A,Schmidt C,Guadagnini S,An extracellular matrix glues together the aerial-grewnhyphae of Aspergillus fumigatus[J] Cell Microbiol,2007,9 (6):1588-1600.
[10] Loussert C,Schmitt C,Prevost MC,In vivo biofilm composition of Aspergillus fumigatus[J] Cell Mierobiol,2010,12 (3):405-410.
[11] Ramage G,Vandewalle K,Wiekes BL,Characteristics of biofilm formation by Candida albicans[J] Rev Iberoam Micol,2001,18 (4):163-170.
[12] Ramage G,Saville SP,Thomas DP,Candida biofilms:an update[J] Eukaryot Cell,2005,4 (4):633-638.
[13] Mowat E,Butcher J,Lang S,Development of a simple model for studying the effects of antifungal agents on multicellular communities of Aspergillus fumigatus[J] J Med Microbiol,2007,56 (Pt9):1205-1212.
[14] Chandra J,Kuhn DM,Mukherjee PK,Biofilm formation by the fungal pathogen Candida albicans:development,architecture,and drug resistance[J] J Bacteriol,2001,183(18):5385-5394.
[15] A1-Fattani MA,Douglas LJ.Penetration of Candida biofilms by antifungal agents[J] Antimicrob Agents Chemother,2004,48(9):3291-3297.
[16] Baillie GS,Douglas LJ.Iron-limited biofilms of Candida albieans and their susceptibility to amphotericin B[J] Antimicrob Agents Chemother,1998,42(8):2146-2149.
[17] Garcia-Sanchez S,Aubert S,Iraqui I,Candida albicans biofilms:a developmental state associated with specific and stable gene expression patterns[J] Eukaryot Cell,2004,3 (2):536-545.
[18] Nikawa H,Jin C,Makihira S,Biofilm formation of Candida albieans on the surfaces of deteriorated soft denture lining materials caused by denture cleansers in vitro[J] J Oral Rehabil,2003,30(3):243-250.
[19] Blankenship JR,Mitchell AP.How to build a biofilm:a fungal perspective[J] Curt Opin Mierebiol,2006,9 (6):588-594.
[20] Villena GK,Gutierrez-Correa M.Morphological patterns of Aspergillus niger biofilms and pellets related to lignocellulolytic enzyme productivities[J] Lett Appl Micrebiol,2007,45(3):231-237.