Genetic diversity of viable, injured, and dead fecal bacteria assessed by fluorescence-activated cell sorting and 16S rRNA gene analysis
Genetic diversity of viable, injured, and dead fecal bacteria assessed by fluorescence-activated cell sorting and 16S rRNA gene analysis
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DOI：
10.1128/aem.71.8.4679-4689.2005
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发表时间：
2005-08-01
影响因子：
4.4
通讯作者：
de Vos, WM
中科院分区：
文献类型：
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作者：
Ben-Amor, K;Heilig, H;de Vos, WM
采用流式细胞仪原位活性分析与16S rRNA基因分析相结合的方法，研究粪便微生物区系多样性与活性的关系。碘化丙啶(PI)和Syto BC同时染色可清晰区分完整细胞(49%)、损伤或受损细胞(19%)和死亡细胞(32%)。用变性梯度凝胶电泳法(DGGE)对总的和14个双歧杆菌群落的16S rRNA基因扩增产物进行了分类和鉴定。这一分析表明，每个个体不仅具有总群落的特征，而且具有不同的亚种群特征。DGGE图谱优势条带的克隆和测序表明，从活的、受伤的和死亡的馏分中回收的克隆大多属于球状梭状芽孢杆菌、淡色梭状芽胞杆菌和类杆菌。我们发现，在采样时，一些与丁酸产生相关的细菌，如真细菌和哈利氏真细菌，显然是活的。然而，特别是从死亡人群中获得了类杆菌和反式瘤胃球菌、类双形肠杆菌以及交叉丁状弧菌的附属扩增产物。此外，从所有分选的组分中回收了一些细菌克隆，这对轻质梭状芽胞杆菌集群尤其明显。鉴定出的双歧杆菌系统类型分别为青春型双歧杆菌、长双歧杆菌、婴儿双歧杆菌、拟卡氏双歧杆菌和双歧双歧杆菌。对活细胞、死亡细胞和损伤细胞的系统发育分析表明，这些细菌种群内存在显著的生理异质性；从所有分选的组分中检索到长双胞菌和婴儿双胞菌，而从分选的死组分中主要回收青春型双歧杆菌。
A novel approach combining a flow cytometric in situ viability assay with 16S rRNA gene analysis was used to study the relationship between diversity and activity of the fecal microbiota. Simultaneous staining with propidium iodide (PI) and SYTO BC provided clear discrimination between intact cells (49%), injured or damaged cells (19%), and dead cells (32%). The three subpopulations were sorted and characterized by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene amplicons obtained from the total and 14 bifidobacterial communities. This analysis revealed that not only the total community but also the distinct subpopulations are characteristic for each individual. Cloning and sequencing of the dominant bands of the DGGE patterns showed that most of clones retrieved from the live, injured, and dead fractions belonged to Clostridium coccoides, Clostridium leptum, and Bacteroides. We found that some of the butyrate-producing related bacteria, such as Eubacterium rectale and Eubacterium hallii, were obviously viable at the time of sampling. However, amplicons affiliated with Bacteroides and Ruminococcus obeum- and Eubacterium biforme-like bacteria, as well as Butyrivibrio crossotus, were obtained especially from the dead population. Furthermore, some bacterial clones were recovered from all sorted fractions, and this was especially noticeable for the Clostridium leptum cluster. The bifidobacterial phylotypes identified in total samples and sorted fractions were assigned to Bifidobacterium adolescentis, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium pseudocatenulatum, and Bifidobacterium bifidum. Phylogenetic analysis of the live, dead, and injured cells revealed a remarkable physiological heterogeneity within these bacterial populations; B. longum and B. infantis were retrieved from all sorted fractions, while B. adolescentis was recovered mostly from the sorted dead fraction.