Sanger和Pyrosequencing测序法分析健康成人口腔菌群

目的对比Sanger和Pyrosequencing测序法分析健康人口腔菌群组成。方法收集6例健康成人唾液、舌背、黏膜、龈上及龈下菌斑并构建16SrRNA基因文库,分别用Sanger和Pyrosequencing测序法分析。结果 Sanger测序所得已知的序列有5,794条(占6,535总序列数88.7%)、75个属,396个序列划分操作分类单元(operational taxonomic units,OTUs,占总OTUs的61.4%)。Pyrosequencing测序所得已知的序列有10,771条(占11,103总序列数97.0%)、66个属,322个OTUs(占总OTUs的68.0%)。Sanger和Pyrosequencing测序法所得口腔菌群在门、属的水平分布趋势基本一致,但在种的水平分布差异显著。Sanger和Pyrosequencing测序法构建的口腔菌群文库均匀度值分别为0.016和0.007,说明Pyrosequencing分析口腔菌群物种数量分布比Sanger测序方法的文库均匀性稍差,但优势种更显著。结论 Pyrosequencing测序时所构建基因文库能代表口腔菌群的多样性且经济、省时,可以应用于口腔细菌物种的分析。     

松材线虫伴生细菌多样性的宏基因组分析

【目的】松材线虫是松材线虫病的病原,且与其伴生细菌之间存在互作关系,它们构成一个微生态系统。本研究旨在揭示松材线虫-伴生细菌群落细菌多样性。【方法】采用16S rRNA基因文库和454测序对伴生细菌群落的宏基因组进行初步分析。【结果】依据97%序列相似性划分OTU(Operational Taxonomic Unit),构建的16S rRNA文库包含25个OTU,分别属于Alphaproteobacteria、Betaproteobacteria、Gammaproteobacteria和Bacteroidetes,其中优势菌群为Gammaproteobacteria,特别是Stenotrophomonas maltophilia为优势细菌。在伴生细菌优势种群上,454测序结果与16SrRNA基因文库结果基本一致。【结论】松材线虫的伴生细菌多样性较高,这些细菌可能对松材线虫具有一定的生态意义。     

新疆断裂带含硫冷泉泉水细菌群落结构多样性

摘要：【目的】为了解新疆断裂带含硫冷泉泉水中细菌群落结构的组成和物种多样性。【方法】采用免培养法直接从冷泉水中提取环境总DNA,采用细菌通用引物对泉水中细菌的16S rRNA基因进行PCR扩增,构建16S rRNA基因克隆文库。使用限制性内切酶Hae Ⅲ对随机挑选的阳性克隆子进行限制性片段长度多态性分析(Restriction Fragment Length Polymorphism, RFLP),选出具有不同酶切图谱的序列进行测序、BLAST比对和构建16S rRNA基因系统发育树。【结果】共从细菌16S rRNA基因文库中筛选了228个阳性克隆,RFLP分型得到33个不同的操作分类单元 (Operational Taxonomic Unites, OTUs),覆盖度 (Coverage C) 为92%。BLAST比对、RDP归类及系统发育分析将这33个OTUs归为：变形菌门 (Proteobacteria)、拟杆菌门 (Bacteroidetes) 和厚壁菌门 (Firmicutes)。变形菌门为绝对优势类群,占整个细菌克隆文库的98%,,其中20%左右的类群与硫化物代谢相关的光合自养和化能自养类群纯培养菌具有高的相似性 (>97%)。此外,还发现大量类群 (总文库的64%,其中57%为军团菌属Legionella spp., 类群)与GenBank中已存细菌16S rRNA基因相似性小于96%。【结论】新疆断裂带含硫冷泉泉水中细菌类群的多样性较低,但可能存在大量潜在细菌新种和新分类。另外,该泉水可能是潜在的新军团菌病传播源,因而可能对下游人畜健康存在潜在威胁。     

豆腐废水UASB反应器中的原核生物多样性及主要功能菌群

通过构建16S rRNA基因文库,对豆腐废水UASB反应器中颗粒污泥的原核生物多样性进行了分析,并用MPN法对颗粒污泥中的互养产乙酸细菌和产甲烷菌进行了活菌数量测定。结果表明,33%的16S rRNA基因序列属于产甲烷菌,氢和乙酸盐营养型的产甲烷菌在颗粒污泥中数量最多,分别为1.1×10.9个/mL和4.5×10.8个/mL。低GC革兰氏阳性菌和δ-变形菌纲分支的细菌也是颗粒污泥中的主要菌群,它们的16S rRNA序列分别占22%和9%,其中互养产乙酸细菌在颗粒污泥中的数量可达4.5×10.7个/ml。绿色非硫细菌是另一类丰度很高的细菌,其16S rRNA序列占文库的12%。对各类微生物在颗粒污泥中可能的作用进行了讨论。通过研究不仅了解了特定环境中的微生物组成,还为从中分离特异类群的微生物提供了指导。     

基于培养组学的健康人口腔细菌分离鉴定初探CSCD

目的 通过对口腔的不同部位进行细菌分离,扩展对口腔微生物多样性的认识,以期为后续的口腔菌群研究提供参考。方法 通过培养组学方法分离口腔不同部位的细菌,采集2个健康人扁桃体、唾液、牙菌斑3个部位的样本,使用13种培养基并于需氧和厌氧条件下进行培养,采用16S rRNA基因全长测序分析。结果 总计获得144株菌,分属36个种,10个科,3个门。其中,链球菌属细菌种类最多。基于16S rRNA基因全长相似度98.65%的阈值判定,获得5种潜在全新细菌。3个部位中,扁桃体分离的细菌种类多样性最丰富。结论 13种培养基中,BHI培养基获得的口腔细菌多样性最好;BAB培养基与TSA培养基更适合口腔链球菌生长,链球菌的比例更高;LBB培养基上只有较单一的口腔细菌生长,不适于广谱分离口腔细菌。     

基于16S rRNA基因文库技术分析水性涂料菌群的多样性

采用未培养技术直接提取变质涂料微生物总DNA,使用通用引物进行PCR扩增、纯化、连接、克隆和测序,建立16S rRNA基因文库,分析变质水性涂料细菌的数量与种类。从96个克隆子中共检测到8种不同种属的细菌,假单胞菌属(Pseudomonas)为优势菌群,占总数的75%,软骨酸芽孢杆菌(Paenibacillus chondroitinus)占总数的15%,此外豚鼠气单胞菌(Aeromonas caviae)和粪产碱杆菌(Alcaligenes faecalis)分别占总数2%。结果表明16S rRNA基因文库是一种较好的研究水性涂料细菌群落多样性的方法。     

螺旋粉虱成虫体内细菌群落多样性的PCR-DGGE 和16S rRNA文库序列分析

为了解螺旋粉虱Aleurodicus dispersus体内细菌多样性和主要优势菌群结构, 用PCR-DGGE和16S rRNA文库对采自于海南省番石榴上螺旋粉虱雌、 雄成虫体内的细菌群落进行了分析。用PCR扩增体内细菌16S rRNA基因, 构建雌、 雄虫克隆文库; 再用限制性片段长度多态性（restriction fragment length polymorphism, RFLP）方法从文库中筛选不同16S rRNA基因图谱, 根据图谱对克隆子进行分型。从螺旋粉虱雌、 雄两个样品中共获得10 种分类操作单元(operational taxonomic unit, OTUs)。以16S rRNA基因为基础构建系统发育树, 系统发育分析表明, 螺旋粉虱雌、 雄成虫体内优势菌群主要为发酵菌属Zymobacter, 杀雄菌属Arsenophonus, 泛菌属Pantoea和假单胞菌属Pseudomonas。Candidatus Portiera aleyrodidarum和Arsenophonus sp.可能为其体内共生菌群, 在所有样品中均可稳定地检测到。这些微生物可能对螺旋粉虱生长发育、 繁殖和性比调控起到重要的协同作用。     

广西水牛瘤胃中的细菌多样性

[目的]了解广西水牛瘤胃中细菌的组成及其可能的降解纤维素细菌的主要类群。[方法]提取水牛瘤胃内容物和高效降解滤纸的水牛瘤胃内容物的富集培养物的宏基因组DNA,以宏基因组DNA为模板,扩增16S rRNA基因序列,构建该两种样品的细菌的16S rRNA基因文库。通过对16S rRNA基因序列的分析,了解这两种样品的细菌群体种类及数量。 [结果] 水牛瘤胃内容物与其富集培养物中均主要含有LGCGPB (low G+C Gram-Positive Bacteria)、CFB (Cytophaga-Flexibacter-Bacteroides)两大类菌群和少数的螺旋体菌（Spirochaetes）,且LGCGPB所占的比例都是最高的,LGCGPB在水牛瘤胃内容物细菌中的比例为56.66%,而在富集培养物细菌中的比例升高为73.33%。在水牛瘤胃内容物中,丝状杆菌（Fibrobacteres）占3.33%,但在富集培养物中未被检测到。而在富集培养物中占13.33%的变形杆菌（Proteobacteria）,在水牛瘤胃内容物中未被检测到。本研究还发现了分类地位尚未明确的一菌群（R46）。[结论]细菌类群LGCGPB、Proteobacteria可能在水牛瘤胃中的纤维素降解过程中起重要作用。此外,水牛瘤胃中的细菌组成和牦牛、牛、羊瘤胃中的细菌组成较相似但比例有所不同。     

沁水盆地寺河地区煤层水细菌多样性分析

[目的]了解沁水盆地寺河地区煤层水中细菌群落组成和物种多样性。[方法]采用免培养法提取煤层水中微生物总DNA,利用细菌通用引物构建16S r DNA基因克隆文库。采用HhaⅠ、MspⅠ限制性内切酶对克隆子进行RFLP分析,测序并构建16S r DNA基因系统发育树。[结果]从文库中筛选出234个阳性克隆,覆盖度为97.4%,聚类为28个操作分类单元。BLAST比对、RDP归类及系统发育分析将这234个克隆归为变形菌门、拟杆菌门、螺旋体门、疣微菌门、黏胶球形菌门。其中变形菌门为绝对优势类群,占整个细菌克隆文库的80.1%。变形菌门中的ε-变形菌纲,占整个基因文库的35%。[结论]应用16S r DNA克隆文库技术,分析沁水盆地寺河地区煤层水中细菌类群的多样性不高。     

应用16S rRNA基因文库技术分析土壤细菌群落的多样性

[目的]土壤微生物在菜田生态系统中具有重要的生态功能,通过16S rRNA基因克隆文库技术分析典型菜田土壤细菌群落结构的组成情况,为揭示典型的菜田土壤微生物的多样性以及土地利用变化与生态环境效应之间的关系奠定基础.[方法]采用未培养技术直接从北京和山东两地典型菜田土壤样品中提取微生物总的DNA,分别构建基于通用引物PCR扩增的土壤细菌16S rRNA基因克隆文库,通过Hinf Ⅰ和Hae Ⅲ限制性内切酶对两地土壤细菌16s rRNA基因文库中的克隆进行ARDRA(Amplified Ribosomal DNA Rstriction Analysis)分析,将所有阳性克隆分为若干个可操作分类单元(OTU).[目的]通过构建两地细菌克隆文库的系统发育树,并分析主要种群的组成表明:北京和山东菜田土壤细菌克隆文库的优势种群均为γ、β、α变形细菌亚群.两地的细菌种类组成分别包括124个OTUs和92个OTUs.[结论]北京地区和山东地区典型蔬菜地土壤细菌种群中优势种群均为变形细菌,但是土壤细菌多样性降低,这可能与典型菜田的多年连作,种植蔬菜种类单一直接相关.同时,也可能是造成菜田土壤病害普遍发生,土壤退化的一个重要原因.     

Investigation of the Effect of Type 2 Diabetes Mellitus on Subgingival Plaque Microbiota by High-Throughput 16S rDNA Pyrosequencing

Diabetes mellitus is a major risk factor for chronic periodontitis. We investigated the effects of type 2 diabetes on the subgingival plaque bacterial composition by applying culture-independent 16S rDNA sequencing to periodontal bacteria isolated from four groups of volunteers: non-diabetic subjects without periodontitis, non-diabetic subjects with periodontitis, type 2 diabetic patients without periodontitis, and type 2 diabetic patients with periodontitis. A total of 71,373 high-quality sequences were produced from the V1-V3 region of 16S rDNA genes by 454 pyrosequencing. Those 16S rDNA sequences were classified into 16 phyla, 27 classes, 48 orders, 85 families, 126 genera, and 1141 species-level OTUs. Comparing periodontally healthy samples with periodontitis samples identified 20 health-associated and 15 periodontitis-associated OTUs. In the subjects with healthy periodontium, the abundances of three genera (Prevotella, Pseudomonas, and Tannerella) and nine OTUs were significantly different between diabetic patients and their non-diabetic counterparts. In the subjects carrying periodontitis, the abundances of three phyla (Actinobacteria, Proteobacteria, and Bacteriodetes), two genera (Actinomyces and Aggregatibacter), and six OTUs were also significantly different between diabetics and non-diabetics. Our results show that type 2 diabetes mellitus could alter the bacterial composition in the subgingival plaque.     

Robustness of Gut Microbiota of Healthy Adults in Response to Probiotic Intervention Revealed by High-Throughput Pyrosequencing

Probiotics are live microorganisms that potentially confer beneficial outcomes to host by modulating gut microbiota in the intestine. The aim of this study was to comprehensively investigate effects of probiotics on human intestinal microbiota using 454 pyrosequencing of bacterial 16S ribosomal RNA genes with an improved quantitative accuracy for evaluation of the bacterial composition. We obtained 158 faecal samples from 18 healthy adult Japanese who were subjected to intervention with 6 commercially available probiotics containing either Bifidobacterium or Lactobacillus strains. We then analysed and compared bacterial composition of the faecal samples collected before, during, and after probiotic intervention by Operational taxonomic units (OTUs) and UniFrac distances. The results showed no significant changes in the overall structure of gut microbiota in the samples with and without probiotic administration regardless of groups and types of the probiotics used. We noticed that 32 OTUs (2.7% of all analysed OTUs) assigned to the indigenous species showed a significant increase or decrease of ≥10-fold or a quantity difference in >150 reads on probiotic administration. Such OTUs were found to be individual specific and tend to be unevenly distributed in the subjects. These data, thus, suggest robustness of the gut microbiota composition in healthy adults on probiotic administration.     

基于高通量测序技术研究水貂远端肠道细菌群落组成

【目的】研究水貂远端肠道微生物群落组成及其多样性。【方法】通过高通量测序技术研究水貂远端肠内容物细菌的组成和多样性。【结果】从10只健康水貂远端肠道内容物样品中得到146 287高质量序列代表17个菌门、167个细菌属。水貂肠道细菌以厚壁菌门(59.99%)、拟杆菌门(16.2%)、梭杆菌门(11.5%)、放线菌门(5.9%)和变形菌门(5.3%)为主,其中厚壁菌门最为丰富。厚壁菌门中的梭菌目是最丰富的目,而梭菌目中的链球菌占有50%以上的OTUs,是最大的细菌属。【结论】水貂肠道内存在复杂的微生物区系,这对进一步研究水貂对营养物质吸收利用提供了理论基础。     

16S rRNA Gene Sequence Analysis of Drinking Water Using RNA and DNA Extracts as Targets for Clone Library Development

The bacterial composition of chlorinated drinking water was analyzed using 16S rRNA gene clone libraries derived from DNA extracts of 12 samples and compared to clone libraries previously generated using RNA extracts from the same samples. Phylogenetic analysis of 761 DNA-based clone sequences showed that unclassified bacteria were the most abundant group, representing nearly 62% of all DNA sequences analyzed. Other phylogenetic groups identified included Proteobacteria (20%), Actinobacteria (9%), Cyanobacteria (4%), and Bacteroidetes (2%). The composition of RNA-based libraries (1122 sequences) was similar to the DNA-based libraries with a few notable exceptions: Proteobacteria were more dominant in the RNA clone libraries (i.e., 35% RNA; 20% DNA). Differences in the Proteobacteria composition were also observed; alpha-Proteobacteria was 22 times more abundant in the RNA-based clones while beta-Proteobacteria was eight times more abundant in the DNA libraries. Nearly twice as many DNA operational taxonomic units (OTUs) than RNA OTUs were observed at distance 0.03 (101 DNA; 53 RNA). Twenty-four OTUs were shared between all RNA- and DNA-based libraries (OTU_0.03) representing only 18% of the total OTUs, but 81% (1527/1883) of all sequences. Such differences between clone libraries demonstrate the necessity of generating both RNA- and DNA-derived clone libraries to compare these two different molecular approaches for community analyses.     

Comparison of the structure and composition of bacterial communities from temperate and tropical freshwater ecosystems

We used a partial 16S rRNA sequencing approach to compare the structure and composition of the bacterial communities in three large, deep subalpine lakes in France with those of communities in six shallow tropical reservoirs in Burkina Faso. Despite the very different characteristics of these ecosystems, we found that their bacterial communities share the same composition in regard to the relative proportions of the different phyla, suggesting that freshwater environmental conditions lead to convergence in this composition. In the same way, we found no significant difference in the richness and diversity of the bacterial communities in France and Burkina Faso. We defined core and satellite operational taxonomic units (OTUs) (sequences sharing at least 98% identity) on the basis of their abundance and their geographical distribution. The core OTUs were found either ubiquitously or only in temperate or tropical and subtropical areas, and they contained more than 70% of all the sequences retrieved in this study. In contrast, satellite OTUs were characterized by having a more restricted geographical distribution and by lower abundance. Finally, the bacterial community composition of these freshwater ecosystems in France and Burkina Faso was markedly different, showing that the history of these ecosystems and regional environmental parameters have a greater impact on the relative abundances of the different OTUs in each bacterial community than the local environmental conditions.     

PhyloChip hybridization uncovered an enormous bacterial diversity in the rhizosphere of different potato cultivars: many common and few cultivar-dependent taxa

The phylogenetic composition of bacterial communities in the rhizosphere of three potato cultivars grown at two distant field sites was analysed. Ribosomal gene fragments amplified from total community DNA were hybridized to PhyloChips. A total of 2432 operational taxonomic units (OTUs) were detected by the PhyloChips, of which 65% were found in the rhizosphere of all cultivars at both field sites. From all detected OTUs, 9% revealed a cultivar-dependent abundance at the one or the other field site and 4% at both sites. Differential abundance on the three cultivars was mainly observed for OTUs belonging to the Pseudomonadales, Actinomycetales and Enterobacteriales. More than 40% of OTUs belonging to Bradyrhizobiales, Sphingomonadales, Burkholderiales, Rhodocyclales, Xanthomonadales and Actinomycetales differed significantly in their abundance between the sites. A sequence analysis of six 16S rRNA gene clone libraries corresponded well with the taxonomic community structure evidenced by the PhyloChip hybridization. Most ribotypes matched OTUs detected by the PhyloChip. Those OTUs that responded to the potato cultivar at both field sites might be of interest in view of cultivar-specific effects on bacterial biocontrol strains and pathogens.     

Comparison of the Distal Gut Microbiota from People and Animals in Africa

The gut microbiota plays a key role in the maintenance of healthy gut function as well as many other aspects of health. High-throughput sequence analyses have revealed the composition of the gut microbiota, showing that there is a core signature to the human gut microbiota, as well as variation in its composition between people. The gut microbiota of animals is also being investigated. We are interested in the relationship between bacterial taxa of the human gut microbiota and those in the gut microbiota of domestic and semi-wild animals. While it is clear that some human gut bacterial pathogens come from animals (showing that human – animal transmission occurs), the extent to which the usually non-pathogenic commensal taxa are shared between humans and animals has not been explored. To investigate this we compared the distal gut microbiota of humans, cattle and semi-captive chimpanzees in communities that are geographically sympatric in Uganda. The gut microbiotas of these three host species could be distinguished by the different proportions of bacterial taxa present. We defined multiple operational taxonomic units (OTUs) by sequence similarity and found evidence that some OTUs were common between human, cattle and chimpanzees, with the largest number of shared OTUs occurring between chimpanzees and humans, as might be expected with their close physiological similarity. These results show the potential for the sharing of usually commensal bacterial taxa between humans and other animals. This suggests that further investigation of this phenomenon is needed to fully understand how it drives the composition of human and animal gut microbiotas.     

PCR指纹图谱技术分析人体口腔内微生物菌群结构

目的 应用PCR-DGGE指纹图谱技术对人体口腔微生物菌群结构进行系统性研究.方法 对1例健康人唾液周期性采集的样品和8例健康人个体的唾液与牙菌斑采集的样品,进行微生物群落总DNA的抽提.以此为模板扩增16S rRNA V3可变区,产物经DGGE指纹图谱分析其组成结构,并运用UVIBAND/MAP等软件比较所得群落指纹图谱的相似性指数.结果 同一健康人个体不同采样时间的唾液菌群结构相似性系数＞74%,通过对不同健康个体口腔样本的研究,发现同一个体的唾液与牙菌斑菌群结构存在差异（84%～95%）.结论 同一健康个体其唾液微生物菌群在一定时间内基本稳定,仅有微小的变化;唾液与同个体牙菌斑的微生物组成虽然存在差异,但这种差异要明显小于个体间的差异.     

Bacterial and archaeal communities inhabiting mussels,sediment and water in Indonesian anchialine lakes

Anchialine lakes are a globally rare and unique ecosystem consisting of saline lakes surrounded by land and isolated from the surrounding marine environment. These lakes host a unique flora and fauna including numerous endemic species. Relatively few studies have, however, studied the prokaryote communities present in these lakes and compared them with the surrounding ‘open water’ marine environment. In the present study, we used a 16S rRNA gene barcoded pyrosequencing approach to examine prokaryote (Bacteria and Archaea) composition in three distinct biotopes (sediment, water and the mussel Brachidontes sp.) inhabiting four habitats, namely, three marine lakes and the surrounding marine environment of Berau, Indonesia. Biotope and habitat proved significant predictors of variation in bacterial and archaeal composition and higher taxon abundance. Most bacterial sequences belonged to OTUs assigned to the Proteobacteria. Compared to sediment and water, mussels had relatively high abundances of the classes Mollicutes and Epsilonproteobacteria. Most archaeal sequences, in turn, belonged to OTUs assigned to the Crenarchaeota with the relative abundance of crenarchaeotes highest in mussel samples. For both Bacteria and Archaea, the main variation in composition was between water samples on the one hand and sediment and mussel samples on the other. Sediment and mussels also shared much more OTUs than either shared with water. Abundant bacterial OTUs in mussels were related to organisms previously obtained from corals, oysters and the deepsea mussel Bathymodiolus manusensis. Abundant archaeal OTUs in mussels, in contrast, were closely related to organisms previously obtained from sediment.