Systems-level analysis of microbial community organization through combinatorial labeling and spectral imaging
Systems-level analysis of microbial community organization through combinatorial labeling and spectral imaging
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DOI：
10.1073/pnas.1101134108
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发表时间：
2011-03-08
影响因子：
11.1
通讯作者：
Borisy, Gary G.
中科院分区：
文献类型：
--
作者：
Valm, Alex M.;Welch, Jessica L. Mark;Borisy, Gary G.
自然界中的微生物经常作为复杂的多分类群的成员发挥作用，但由于标记和成像技术的限制，这些社区在微米水平上的结构组织知之甚少。我们在这里报告了一种结合光谱图像采集和分析的组合标记策略，该策略大大增加了单个图像中可区分的荧光特征的数量。作为成像原理的证明，我们首先展示了通过荧光原位杂交（FISH）标记的大肠杆菌的可视化，该荧光原位杂交具有8种荧光团的28种不同的二元组合。作为原理的生物学证明，我们然后应用这种组合标记和光谱成像FISH（CLASI-FISH）策略，使用属和家族特异性探针同时可视化并区分实验室培养微生物的人工混合物中的15种不同的细菌类型。然后，我们说明了我们的方法的效用的天然微生物群落的结构分析，即，人类牙菌斑，微生物生物膜。我们证明，15个分类群的斑块社区可以同时成像和分析，这个社会是占主导地位的早期殖民者，包括链球菌，普氏菌，放线菌，韦荣氏球菌。邻近分析被用来确定的频率间和内轴突细胞与细胞的协会，揭示统计学显着的分类群间配对。普雷沃氏菌属和放线菌属的细胞表现出最多的种间关联，这表明这些属在建立和维持生物膜复杂性中起着核心作用。结果提供了一个初步的系统级结构分析的生物膜组织。
Microbes in nature frequently function as members of complex multitaxon communities, but the structural organization of these communities at the micrometer level is poorly understood because of limitations in labeling and imaging technology. We report here a combinatorial labeling strategy coupled with spectral image acquisition and analysis that greatly expands the number of fluorescent signatures distinguishable in a single image. As an imaging proof of principle, we first demonstrated visualization of Escherichia coli labeled by fluorescence in situ hybridization (FISH) with 28 different binary combinations of eight fluorophores. As a biological proof of principle, we then applied this Combinatorial Labeling and Spectral Imaging FISH (CLASI-FISH) strategy using genus-and family-specific probes to visualize simultaneously and differentiate 15 different phylotypes in an artificial mixture of laboratory-grown microbes. We then illustrated the utility of our method for the structural analysis of a natural microbial community, namely, human dental plaque, a microbial biofilm. We demonstrate that 15 taxa in the plaque community can be imaged simultaneously and analyzed and that this community was dominated by early colonizers, including species of Streptococcus, Prevotella, Actinomyces, and Veillonella. Proximity analysis was used to determine the frequency of inter- and intrataxon cell-to-cell associations which revealed statistically significant intertaxon pairings. Cells of the genera Prevotella and Actinomyces showed the most interspecies associations, suggesting a central role for these genera in establishing and maintaining biofilm complexity. The results provide an initial systems-level structural analysis of biofilm organization.