ERIC-PCR分子杂交技术分析大熊猫肠道菌群结构

目的了解大熊猫肠道微生物区系结构的相似性和稳定性,并找出大熊猫肠道微生物群落结构的变化与健康状况的关系。方法对上海动物园及上海野生动物园所饲养的3只大熊猫2次采集的粪便样品进行微生物群落总DNA的抽提,并以此为模板获得反映肠道微生物群落结构特征的ERIC—PCR和Southern杂交指纹图谱,比较各DNA样品指纹图谱的相似性指数。结果除国庆(大熊猫)的第1次采集的样品(当时处于腹泻状态),其他各DNA样品的ERIC-PCR及Southern杂交指纹图谱的相似性都达到85％～100％;佳斯及川川(大熊猫)2个个体2次采集的样品之间ERIC指纹图谱的相似性分别为93％和87％,而国庆腹泻时的样品与健康时的样品之间则为71％。结论大熊猫不同个体之间肠道微生物群落结构比较相似,而且同一个体在不同时期表现出比较高的稳定性,但当个体的健康出现问题时肠道优势菌菌群结构有一定波动。所采用的DNA提取方法、ERIC—PCR和Southern杂交指纹图谱的高度重复性证明了之一分子生态学技术在大熊猫肠道微生物区系动态监测中的可行性。     

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.     

健康儿童与轮状病毒感染儿童肠道菌群结构的比较研究

目的比较分析轮状病毒感染个体与健康个体肠道菌群结构的差异。方法采集11例轮状病毒感染个体及6例健康个体的粪便样品,提取粪便样品中细菌的混合DNA,先通过ERIC-PCR结合分子杂交的技术分析两组个体之间肠道微生物组成的相似性;再扩增粪便样品中菌群的16SrRNA基因,利用PCR—TGGE技术分析肠道菌群的组成情况。结果轮状病毒感染个体与健康个体相比,肠道菌群中GC含量较低的菌明显减少,同时肠道菌群有宿主专一性。结论轮状病毒感染会导致儿童肠道内菌群结构失调。     

应用TGGE技术分析人肠道中双歧杆菌的组成

用温度梯度凝胶电泳(TGGE)技术结合16SrDNA克隆、测序,对健康人肠道中双歧杆菌的组成进行了分析。10例健康人肠道双歧杆菌的TGGE分析显示:人肠道内双歧杆菌的组成具有宿主特异性,不同人肠道双歧杆菌种的多样性和种类不同。通过对一健康儿童肠道双歧杆菌属特异性PCR扩增产物的测序及TGGE电泳行为的分析发现,该个体双歧杆菌TGGE图谱中的条带分别代表Bifidobacteriumbifidum、B.infantis、B.longum、B.adolescentis、B.pseudocatenulatum等种和一新种,其中B.pseudocatenulatum(假小链双歧杆菌)是大多数个体共有且较优势的种类。用传统培养方法只检出B.pseudocatenulatum和B.longum两种。基于双歧杆菌属特异性PCR基础上的TGGE方法结合16SrDNA克隆文库分析可较灵敏、直观地反映人肠道中双歧杆菌的组成。     

DGGE/TGGE技术在土壤微生物分子生态学研究中的应用

传统的微生物生态学研究方法只限于环境样品中极少部分(0.1%￣1%)可培养的微生物类群,极大程度地限制了对土壤微生物群落结构的研究。综述了以16S rDNA为主要研究对象的DGGE/TGGE(Denaturing gradientgel electrophoresis,DGGE/Temperature gradient gel electrophoresis,TGGE)技术原理,以其为主要手段结合PCR扩增、克隆建库、序列测定以及种系分析对土壤微生物的群落结构和多样性研究的最新动态。DGGE/TGGE技术极大地推动了土壤微生物分子生态学的发展,同时也为实际问题的诊断、作物生长跟踪监测等提供了技术支撑,在土壤微生物分子生态学研究和生产实践中起着越来越重要的作用。     

TGGE分析焦化废水处理系统活性污泥细菌种群动态变化及多样性

应用TGGE指纹图谱技术对两个曝气池细菌种群的动态变化及多样性进行了研究。每3d进行1次,共8个监测时期中同一曝气池活性污泥的16SrDNAV3-PCRTGGE指纹图谱基本一致,图谱间的相似性系数(Cs)为100％。同一曝气池不同位点活性污泥的TGGE指纹图谱也完全一致。功能不同曝气池活性污泥TGGE指纹图谱存在差异,Cs为83．3％。对TJ1活性污泥TGGE图谱中9条主要条带回收、扩增、克隆建库,每个条带选4个转化子进行序列分析,结果显示TGGE条带是由序列不同的片段组成。32个序列在97％的相似性下分成16个分类操作单元(OTU),14个OTU与GenBank中已登录的细菌种群的同源性≥97％,2个OTU的同源性为95％和94％。与10个OTU同源性较高的细菌类群是在活性污泥或污染环境分离或发现的,与8个OTU相似的细菌类群目前尚无法分离培养。     

浓香型大曲原核微生物群落的PCR-SSCP解析

采用PCR-SSCP(单链构象多态性)技术对浓香型大曲发酵过程中原核微生物群落结构的变化情况进行了研究, 结果表明: (1)发酵各时期曲样的群落结构相似, 同时也具有多态性; (2)不同微生物群落具有协同和制约的复杂生态学效应; (3)各时期曲样微生物多样性指数均在1.69~2.01之间, 群落结构相对较稳定; (4)各曲样微生物群落相似性位于0.67~1.00之间, 邻近时期样品间的相似性程度较高。     

大港孔店油田水驱油藏微生物群落的分子分析

通过多聚酶链式反应温度梯度凝胶电泳（PCRTGGE）和构建16S rRNA基因克隆文库两种方法对比研究了大港油田孔二北断块注水井和采油井的微生物群落结构。16S rDNA V3区PCR扩增产物的TGGE图谱分析表明,这两个油井的微生物群落结构差异很大。注水井样品的TGGE图谱中有6条主要条带,而采油井样品中只有一个条带占绝对优势。同时,建立了两个样品的16S rRNA基因克隆文库,从中分别挑选了108和50个克隆进行限制性酶切片段长度多样性分析（ARDRA）。注水井样品有33个操作分类单元（OUT）,其中6个OUT是优势类型;而采油井样品只有8个OUT,有1个OUT在文库中占绝对优势。克隆文库和TGGE的研究结果一致,均表明注水井样品的微生物多样性比采油井丰富很多。每个OUT的代表克隆序列分析结果表明,注水井样品中的细菌主要属于α、β、γ变形菌纲和放线菌纲,尤其是红细菌亚纲（47%）。采油井样品的细菌主要属于α、β、γ变形菌纲,尤其是假单胞菌属（62%）。油藏微生物多样性的分子分析可为开展微生物采油技术研究奠定基础。     

T-RFLP技术分析油藏微生物多样性

应用T-RFLP(末端限制性片段长度多态性)技术分析和比较了胜利油田单12区块的一口注水井(S12-zhu)和三口采油井(S12-4、S12-5和S12-19)的油藏微生物多样性。基于T-RFLP图谱的多样性指数表明注入水样品具有更丰富的细菌和古菌多样性。相似性指数表明,样品间细菌群落结构的相似性介于22.4%～30.8%之间,古菌群落结构的相似性介于20.8%～34.5%之间。查询RDP数据库推测这4个油藏样品所共有的优势微生物可能为Pseudomonas属,Marinobacter属和产甲烷微生物。T-RFLP技术能方便快捷的分析微生物多样性,其在油藏微生物多样性研究上的应用可以为MEOR提供有用的信息。     

基于16S rRNA基因序列分析1例健康人龈上菌群

目的构建细菌16S rRNA基因文库分析健康人龈上菌群的组成。方法取1例健康成年女性龈上菌斑并构建细菌16S rRNA基因文库,分析其龈上菌群组成。结果 1例健康人龈上菌斑细菌的种水平分类有62种,其中可以培养的细菌有34种而尚无法培养的细菌有28种(45.1%);新发现的细菌物种有17种(27.4%);缓症链球菌是克隆子数最多的优势菌种;链球菌属、奈氏菌属和嗜血杆菌属占文库的60%,为主要菌群;构建的细菌16S rRNA基因文库的覆盖率为95%,文库的均匀度值为0.016。结论 1例健康人龈上菌群中45.1%的细菌尚无法分离培养、27.4%的物种尚不清楚,未培养菌及尚不清楚的菌种中可能藏匿着与口腔疾病密切相关的致病菌,全面地了解健康人龈上菌群的组成有助于研究龋病的发病机制。     

Oral cavity contains distinct niches with dynamic microbial communities

Microbes colonize human oral surfaces within hours after delivery. During postnatal development, physiological changes, such as the eruption of primary teeth and replacement of the primary dentition with permanent dentition, greatly alter the microbial habitats, which, in return, may lead to community composition shifts at different phases in people's lives. By profiling saliva, supragingival and mucosal plaque samples from healthy volunteers at different ages and dentition stages, we observed that the oral cavity is a highly heterogeneous ecological system containing distinct niches with significantly different microbial communities. More importantly, the phylogenetic microbial structure varies with ageing. In addition, only a few taxa were present across the whole populations, indicating a core oral microbiome should be defined based on age and oral niches.     

Supragingival Microbial Profiles of Permanent and Deciduous Teeth in Children with Mixed Dentition

Objectives

The present study was designed to investigate the microbial profiles of teeth in different locations in mixed-dentition-stage children, and to compare the microbiomes of permanent and deciduous teeth in the same healthy oral cavity.

Methods

Supragingival plaque samples of teeth in various locations—the first permanent molars, deciduous molars, deciduous canines and incisors and permanent incisors—were collected from 20 healthy mixed-dentition-stage children with 10–12 permanent teeth erupted. Plaque DNA was extracted, and the V3–V4 hypervariable region of the bacterial 16S rRNA gene was amplified and subjected to sequencing.

Results

On average, 18,051 high-quality sequences per sample were generated. Permanent tooth sites tended to host more diverse bacterial communities than those of deciduous tooth sites. A total of 12 phyla, 21 classes, 38 orders, 66 families, 74 genera were detected ultimately. Five predominant phyla (Proteobacteria, Firmicutes, Bacteroidetes, Fusobacteria and Actinobacteria) were highly variable among sites. Of 26 genera with a mean relative abundance of >0.1%, 16 showed significant differences in relative abundance among the groups. More than 20% of the total operational taxonomical units were detected only in permanent or deciduous teeth. The variation in the microbial community composition was due mainly to permanent teeth being enriched in Actinomyces and deciduous teeth in Treponema. The core microbiome of supragingival plaque in mixed dentition comprised 19 genera with complex correlationships.

Conclusion

Our results suggest differences in microbial diversity and composition between permanent and deciduous teeth sites in mixed dentition. Moreover, the core microbiome of these sites was determined. These findings enhance our understanding of the development of the native oral microbiota with age.     

Exploring the oral microflora of preschool children

The oral cavity is one of the most important and complicated habitats in our body and supports diverse microbial communities. In this study, we aimed to determine the bacterial diversity and composition of various oral micro-niches. Samples were collected from supragingival plaque, saliva, and tongue coating from 10 preschool children (30 samples total). 16S rRNA gene pyrosequencing dataset generated 314,639 clean reads with an average of 10,488 ± 2,787 reads per sample. The phyla Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, and Fusobacteria were predominant, accounting for more than 90% of the total sequences. We found the highest α diversity, microbial richness, and evenness in plaque, compared with saliva and tongue coating. Plaque was also distinguished from saliva and tongue coating by phylogenetic distances (weighted UniFrac). Taxa with different relative abundances were further identified, confirming the existence of microbial differences across the three niches. Core microbiomes were defined of each niche; however, only a small proportion of operational taxonomic units (8.07%) were shared by the three niches. Coaggregation between Actinomyces spp. and Streptococcus spp. and other correlations among periodontal pathogens, such as Prevotella, Fusobacteria, Capnocytophaga, and Tannerella, were shown by a co-occurrence network. In summary, our study provides a framework of oral microbial communities in the population of preschool children as a baseline for further studies of oral diseases related to microbes.     

健康青少年口腔正常菌群的初步研究

本文采用非选择性培养基对22名健康青少年的唾液、沟裂菌斑、龈上菌斑及龈下菌斑中的需氧菌、兼性厌氧菌及专性厌氧菌进行了分离培养,并计算其在不同标本中占可培养菌的百分比及检出率。结果共分离到包括18个菌属的35种细菌。其中,链球菌、放线菌、奈瑟氏球菌、二氧化碳噬纤维菌、类杆菌、梭杆菌,奴卡氏菌及棒状杆菌在口腔4个部位的检出率及所占比例均较高,是健康青少年口腔中的优势菌群.通过比较还发现,其中一些菌在口腔4个部位的分布存在一定差异.本文还采用刚果红负性染色涂片法,镜下观察龈上、龈下菌斑中的螺旋体,并计算其相对比例.结果龈下菌斑中螺旋体的相对比例明显高于龈上菌斑.     

Automated fluorescent in situ hybridization for the specific detection and quantification of oral streptococci in dental plaque

Our aim was to develop a rapid fluorescent in situ hybridization (FISH) assay for the identification of different oral groups of streptococci in dental plaque and to combine it with digital image analysis for the automated enumeration of target cells. Cy3-labeled oligonucleotide probes specific for 16S rRNA gene sequences of the anginosus, mitis, mutans, and salivarius groups of streptococci were hybridized under stringent conditions with bacterial cultures or supragingival plaque samples that had been permeabilized with lysozyme. Probe specificity was determined with strains from 30 different species, mainly of oral origin. Results showed that probes ANG541, MIT447, SSP001, and SAL090 with specificity for the anginosus, mitis, mutans, and salivarius groups, respectively, the pan-reactive streptococcal probe STR405, the S. mutans specific probe MUT590, and the S. sobrinus specific probe SOB174 were well-suited for the identification of cultured streptococci. Probes STR405, MIT447 and SSP001 were then successfully applied to enumerate automatically bacteria of the recognized taxa in 144 supragingival plaque samples. On the average, total streptococci accounted for 8.2%, streptococci of the mitis and mutans groups for 3.9 and 1.7%, respectively, of the plaques. The combined application of FISH and automated image analysis provides an objective time-saving alternative to culture or PCR for the enumeration of selected oral streptococci in dental plaque.     

Effects of lactoferrin and lactoperoxidase‐containing food on the oral microbiota of older individuals

The oral microbiota influences health and disease states. Some gram‐negative anaerobic bacteria play important roles in tissue destruction associated with periodontal disease. Lactoferrin (LF) and lactoperoxidase (LPO) are antimicrobial proteins found in saliva; however, their influence on the whole oral microbiota currently remains unknown. In this randomized, double‐blinded, placebo‐controlled study, the effects of long‐term ingestion of LF and LPO‐containing tablets on the microbiota of supragingival plaque and tongue coating were assessed. Forty‐six older individuals ingested placebo or test tablets after every meal for 8 weeks. The relative abundance of bacterial species was assessed by 16S rRNA gene high‐throughput sequencing. Most of the bacterial species in supragingival plaque and tongue coating that exhibited significant decreases in the test group were gram‐negative bacteria, including periodontal pathogens. Decreases in the total relative abundance of gram‐negative organisms in supragingival plaque and tongue coating correlated with improvements in assessed variables related to oral health, such as oral malodor and plaque accumulation. Furthermore, there was significantly less microbiota diversity in supragingival plaque at 8 weeks in the test group than in the placebo group and low microbiota diversity correlated with improvements in assessed variables related to oral health. These results suggest that LF and LPO‐containing tablets promote a shift from a highly diverse and gram‐negative‐dominated to a gram‐positive‐dominated community in the microbiota of supragingival plaque and tongue coating. This microbial shift may contribute to improvements in oral health, including oral malodor and state of the gingiva.

     

Experimental abscess formation caused by human dental plaque

Human dental plaque consists of a wide variety of microorganisms, some of which are believed to cause systemic infections, including abscesses, at various sites in the body. To confirm this hypothesis experimentally, we examined the abscess-forming ability of native dental plaque in mice, the microbial features of the infectious locus produced by the plaque, and the anti-phagocytic property of microbial isolates. Aliquots of a suspension of supragingival dental plaque containing 6 x 10(6) colony-forming unit of bacteria were injected subcutaneously into the dorsa of mice. Abscess formation was induced in 76 of 85 mice using ten different plaque samples. Thirteen microorganisms were isolated from pus samples aspirated from abscess lesions. The microbial composition of pus, examined in 17 of 76 abscesses, was very simple compared to that of the plaque sample that had induced the abscess. The majority of the isolates belonged to the Streptococcus anginosus group, normally a minor component of plaque samples. S. anginosus was the most frequently detected organism and the most prevalent in seven abscesses, and Streptococcus intermedius and Streptococcus constellatus were predominant in one and three abscess samples, respectively. Each isolate of S. anginosus group produced abscesses in mice, and heat-treated supragingival dental plaque influenced the abscess-forming ability of S. anginosus isolate. These isolates possessed a high antiphagocytic capacity against human polymorphonuclear leukocytes. Our results suggest that human supragingival dental plaque itself is a source of the infectious pathogens that cause abscess formation.     

The subgingival microbiome of clinically healthy current and never smokers

Dysbiotic oral bacterial communities have a critical role in the etiology and progression of periodontal diseases. The goal of this study was to investigate the extent to which smoking increases risk for disease by influencing the composition of the subgingival microbiome in states of clinical health. Subgingival plaque samples were collected from 200 systemically and periodontally healthy smokers and nonsmokers. 16S pyrotag sequencing was preformed generating 1 623 713 classifiable sequences, which were compared with a curated version of the Greengenes database using the quantitative insights into microbial ecology pipeline. The subgingival microbial profiles of smokers and never-smokers were different at all taxonomic levels, and principal coordinate analysis revealed distinct clustering of the microbial communities based on smoking status. Smokers demonstrated a highly diverse, pathogen-rich, commensal-poor, anaerobic microbiome that is more closely aligned with a disease-associated community in clinically healthy individuals, suggesting that it creates an at-risk-for-harm environment that is primed for a future ecological catastrophe.