Department of Cariology, Endodontology and Periodontology, University of Leipzig, Liebigstraße 12, 04103 Leipzig, Germany.
Department of Environmental Microbiology, Research Group Flow Cytometry, Helmholtz Centre for Environmental Research - UFZ, Permoserstr. 15, 04318 Leipzig, Germany.
Methods. 2018 Feb 1;134-135:67-79. doi: 10.1016/j.ymeth.2017.08.009. Epub 2017 Aug 24.
Microbial flow cytometry is an established fast and economic technique for complex ecosystem studies and enables visualization of rapidly changing community structures by measuring characteristics of single microbial cells. Cytometric evaluation routines are available such as flowCyBar which are useful for automatic data processing. Here, a cytometric workflow was established which allows to routinely analyze salivary microbiomes on the example of ten oral healthy subjects. First, saliva was collected within a 3-month period, cytometrically analyzed and the evolution of the microbiomes followed as well as the calculation of their intra- and inter-subject similarity. Second, the respective microbiomes were stressed by exposition to high sugar or acid concentrations and immediate changes were recorded. Third, bactericide solutions were tested on their impact on the microbiomes. In all three set ups huge intra-individual variations in cytometric community structures were found to be largely absent, even under stress, while inter-individual diversity was obvious. The bacterial cell counts of saliva samples were found to vary between 3.0×10 and 6.2×10 cells per sample and subject in undisturbed environments. The application of the two bactericides did not cause noteworthy diversity changes but the loss in cell numbers by about 50% was high after treatment. Illumina® sequencing of whole microbiomes or sorted sub-microbiomes revealed typical phyla such as Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes and Fusobacteria. This approach is useful for fast monitoring of individual salivary microbiomes and automatic calculation of intra- and inter-individual dynamic changes and variability and opens insight into ecological principles leading to their sustainment in their individual environment.
微生物流式细胞术是一种成熟的快速且经济的技术，适用于复杂生态系统的研究，能够通过测量单个微生物细胞的特征来可视化快速变化的群落结构。现在已经有了诸如 flowCyBar 等流式细胞术评估程序，可用于自动数据处理。本研究建立了一个流式细胞术工作流程，该流程可用于以十个口腔健康受试者为例，常规分析唾液微生物组。首先，在三个月的时间内收集唾液，进行流式细胞术分析，然后跟踪微生物组的演变，并计算其个体内和个体间的相似性。其次，通过暴露于高糖或高酸浓度来对各自的微生物组进行应激处理，并记录立即发生的变化。第三，测试杀菌剂对微生物组的影响。在所有这三个设置中，发现即使在应激条件下，个体间的微生物组结构的个体内巨大差异也很大程度上不存在，而个体间的多样性则很明显。在未受干扰的环境中，唾液样本的细菌细胞计数在 3.0×10 和 6.2×10 个细胞/样本和个体之间变化。两种杀菌剂的应用并未导致明显的多样性变化，但处理后细胞数量减少约 50%。对整个微生物组或分选的亚微生物组进行 Illumina®测序揭示了典型的门，例如厚壁菌门、变形菌门、放线菌门、拟杆菌门和梭杆菌门。这种方法可用于快速监测个体唾液微生物组，并自动计算个体内和个体间的动态变化和可变性，并深入了解导致其在个体环境中维持的生态原则。
