A Molecular Enrichment Strategy Based on cpn60 for Detection of Epsilon-Proteobacteria in the Dog Fecal Microbiome
A Molecular Enrichment Strategy Based on cpn60 for Detection of Epsilon-Proteobacteria in the Dog Fecal Microbiome
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DOI：
10.1007/s00248-011-9931-7
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发表时间：
2012-02-01
影响因子：
3.6
通讯作者：
Hill, Janet E.
中科院分区：
文献类型：
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作者：
Chaban, Bonnie;Links, Matthew G.;Hill, Janet E.
稀有微生物群的成员可能是复杂微生物群落的重要组成部分。例如，宠物狗的主人是人类弯曲杆菌病的已知风险因素，并且通常通过有针对性的检测在狗的粪便中检测到弯曲杆菌。然而，这些生物还没有被元基因组学方法检测到。这项研究的目标是使用两种不同水平的分子检测来表征健康和腹泻宠物狗的粪便微生物区系。使用通用cpn60基因靶标的PCR扩增和焦磷酸测序来获得每只狗的微生物图谱。为了研究微生物组中相对罕见的epsilon-Protebacteria成分，我们使用了一种分子富集法，首先从epsilon-Producteria中扩增出cpn10-cpn60区域，然后进行通用的cpn60靶标扩增和焦磷酸测序。从非浓缩调查中，主要的发现是与腹泻狗相比，健康狗中类杆菌的比例明显更高，特别是普通拟杆菌。来自螺杆菌属和弯曲杆菌属的变形杆菌也在一些狗的非浓缩剖面中检测到低水平。分子浓缩增加了从每只狗身上检测到的epsilon蛋白细菌序列的比例，以及识别出在非浓缩图谱中未见的新的、推测为罕见的序列。丰富的图谱包含了Arcobacter，弯曲杆菌，Flexispira和螺旋杆菌的已知物种，并确定了两个可能的新物种。这些发现增加了我们对犬粪便微生物组的总体了解，特别是表型-变形杆菌组分，并为研究稀有微生物组的传统元基因组学方法提供了一种新的改进。
Members of the rare microbiome can be important components of complex microbial communities. For example, pet dog ownership is a known risk factor for human campylobacteriosis, and Campylobacter is commonly detected in dog feces by targeted assays. However, these organisms have not been detected by metagenomic methods. The goal of this study was to characterize fecal microbiota from healthy and diarrheic pet dogs using two different levels of molecular detection. PCR amplification and pyrosequencing of the universal cpn60 gene target was used to obtain microbial profiles from each dog. To investigate the relatively rare epsilon-proteobacteria component of the microbiome, a molecular enrichment was carried out using a PCR that first amplified the cpn10-cpn60 region from epsilon-proteobacteria, followed by universal cpn60 target amplification and pyrosequencing. From the non-enriched survey, the major finding was a significantly higher proportion of Bacteroidetes, notably Bacteroides vulgatus, in healthy dogs compared to diarrheic dogs. Epsilon-proteobacteria from the genera Helicobacter and Campylobacter were also detected at a low level in the non-enriched profiles of some dogs. Molecular enrichment increased the proportion of epsilon-proteobacteria sequences detected from each dog, as well as identified novel, presumably rare sequences not seen in the non-enriched profiles. Enriched profiles contained known species of Arcobacter, Campylobacter, Flexispira, and Helicobacter and identified two possibly novel species. These findings add to our understanding of the canine fecal microbiome in general, the epsilon-proteobacteria component specifically, and present a novel modification to traditional metagenomic approaches for study of the rare microbiome.