Multiplex FISH analysis of a six-species bacterial biofilm
Multiplex FISH analysis of a six-species bacterial biofilm
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DOI：
10.1016/j.mimet.2003.09.003
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发表时间：
2004-01-01
影响因子：
2.2
通讯作者：
Guggenheim, B
中科院分区：
文献类型：
--
作者：
Thurnheer, T;Gmür, R;Guggenheim, B
已建立的程序使用不同的和看似不相容的实验方案，用于革兰氏阴性和革兰氏阳性细菌的荧光原位杂交（FISH）。本研究的目的是开发一种基于FISH和共聚焦激光扫描显微镜（CLSM）的程序，用于分析含有革兰氏阴性和革兰氏阳性口腔细菌的体外生物膜的空间组织。在唾液预处理的羟基磷灰石圆盘上，37℃厌氧培养64.5 h，由6种口腔细菌组成的生物膜分别为纳氏放线菌、白色念珠菌、核梭杆菌、口腔链球菌、sobrin链球菌和细粒微孔菌。通过系统地改变固定和暴露于溶菌酶，寻求革兰氏阴性菌和革兰氏阳性菌同时原位杂交的条件。固定和渗透后的生物膜用FISH标记16S rrna靶向寡核苷酸探针ANA103（用于检测纳斯lundii）、EUK116（白色假单胞菌）、FUS664（核仁假单胞菌）、MIT447和MIT588 （S. oralis）、SOB174 （S. sobrinus）和VEI217 （V. dispar）。探针作为6-FAM、Cy3或Cy5的偶联物，使靶细胞分别发出绿色、橙红色或深红色荧光。因此，用三个携带不同荧光标记的探针进行两次独立的三重杂交，可以看到所有六个物种。结果表明，用FISH同时研究具有不同革兰氏染色特性的多种细菌组成的复杂生物膜是可能的。结合CLSM的光学切片特性，该技术在分析生物膜组成的空间变化以应对环境挑战方面具有很大的前景。(C) 2003 Elsevier B.V.版权所有
Established procedures use different and seemingly incompatible experimental protocols for fluorescent in situ hybridization (FISH) with Gram-negative and Gram-positive bacteria. The aim of this study was to develop a procedure, based on FISH and confocal laser scanning microscopy (CLSM), for the analysis of the spatial organization of in vitro biofilms containing both Gram-negative and Gram-positive oral bacteria. Biofilms composed of the six oral species Actinomyces naeslundii, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus sobrinus, and Veillonella dispar were grown anaerobically for 64.5 h at 37 degreesC on hydroxyapatite disks preconditioned with saliva. Conditions for the simultaneous in situ hybridization of both Gram-negative and Gram-positive bacteria were sought by systematic variation of fixation and exposure to lysozyme. After fixation and permeabilization biofilms were labeled by FISH with 16S rRNA-targeted oligonucleotide probes ANA103 (for the detection of A. naeslundii), EUK116 (C. albicans), FUS664 (F. nucleatum), MIT447 and MIT588 (S. oralis), SOB174 (S. sobrinus), and VEI217 (V. dispar). Probes were used as 6-FAM, Cy3 or Cy5 conjugates, resulting in green, orange-red or deep-red fluorescence of target cells, respectively. Thus, with two independent triple-hybridizations with three probes carrying different fluorescence-tags, all six species could be visualized. Results show that the simultaneous investigation by FISH of complex biofilms composed of multiple bacterial species with differential Gram-staining properties is possible. In combination with the optical sectioning properties of CLSM the technique holds great promise for the analysis of spatial alterations in biofilm composition in response to environmental challenges. (C) 2003 Elsevier B.V. All rights reserved.