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。     

基于ERIC-PCR技术研究酪蛋白糖巨肽对小鼠肠道菌群结构的影响

目的了解酪蛋白糖巨肽(CGMP)对小鼠肠道中微生物群落结构及其动态变化的影响。方法采用ER IC-PCR技术分析鉴定在灌胃小鼠CGMP期间其肠道菌群结构的变化情况。在实验的第0、3、5、7、10、15和21天(灌胃停止后1周)分别提取对照组和CGMP组小鼠粪便总DNA,以此为模板进行PCR反应,获得肠道微生物群落的ER IC-PCR指纹图谱。结果小鼠个体在一段时间内微生物群落演替不明显,群落相似性较高。对照组小鼠肠道菌群多样性指数范围为1.75±0.06,CGMP组多样性指数范围为1.89±0.04,二者之间差异有统计学意义。结论小鼠肠道内的微生物群落非常丰富,普遍存在共有的优势菌群,且优势菌群的群落结构较为稳定;CGMP能够显著增加小鼠肠道菌群的多样性;聚类分析结果显示:对照组小鼠个体在不同时间的肠道菌群结构相似性较高,且ER IC-PCR指纹图谱没有明显的规律;CGMP组小鼠个体的ER IC-PCR指纹图谱被明显地分成2个亚族,说明小鼠灌胃CGMP 3~5 d后,其肠道菌群的群落结构开始发生明显变化。     

应用PCR与温度梯度凝胶电泳分析龈上菌斑微生物群落

目的应用PCR与温度梯度凝胶电泳(PCR—TGGE)分子技术对成年健康口腔的龈上菌斑中微生物群落组成进行分析。方法8例成年个体包括4男4女,年龄19～29岁,分别采取每例个体上下颌牙周龈上菌斑样品,共18份(个体Subl间隔10天采集2次样品)。提取菌斑DNA,PCR扩增16SrDNAV3可变区,产物经TGGE后进行相似系数分析。结果同一个体的上下颌微生物群落组成相似性系数为81％～95％,而不同个体的龈上菌斑微生物群落组成相似性系数,均在60％以下。结论不同个体具有其独特的牙周微生物群落,而且在一定时期内组成稳定。     

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。     

肠道微生物菌群分型的研究进展

人体肠道内存在着处于动态平衡中的复杂微生物群体,包含1000多种细菌和古生菌等共生微生物。它们广泛参与人体的营养、代谢和免疫等生理过程,是影响健康最重要的因素之一。同一个体不同胃肠道部位的微生物群落组成显著不同,而在不同个体的肠道微生物群落组成也存在很大差异。肠道微生物群落结构受到饮食习惯、药物干预以及生活环境等因素影响,形成了不同个体间菌群组成的差异。通过菌群测序分析和群体分型,可以将不同个体的肠道微生物群落组成分为拟杆菌、普氏菌和瘤胃球菌三种肠型。确定肠道微生物群落结构的分型,将复杂的肠道微生物系统模式化,有利于对大样本肠道微生物菌群进行分析,更好地指导相关疾病的诊断和治疗。本文综述了肠道微生物的分型和相关影响因素的进展。     

健康儿童与发育不佳儿童肠道菌群结构的比较研究

目的对健康儿童与发育不佳(FTT)儿童肠道中微生物区系的ERIC-PCR指纹图谱异同进行研究。方法根据美国疾病预防控制中心(CDC)对儿童生长发育的评价指标对某幼儿园200例4～6岁儿童进行评价,筛选出16例健康儿童和13例FTT儿童,每周1次连续3周跟踪取样,提取粪便样品中细菌总DNA,获得其ERIC-PCR指纹图谱,再将其中一个样品的ERIC-PCR产物作为混合探针通过杂交对指纹图谱上DNA条带序列的异同进一步比较。结果同一个体的肠道菌群结构在取样期间稳定性较好;虽然健康儿童间的肠道菌群结构也有一定差异,但它们却有着共同的结构特征;而健康儿童与FTT儿童的肠道菌群结构差异较大。结论儿童发育状况与肠道菌群结构有一定的关系。     

基于PCR-DGGE指纹图谱川纹笛鲷及圆白鲳消化道壁优势菌群结构比较分析

本文采用免培养的16S rDNA梯度凝胶电泳技术(DGGE)对集约化海水网箱养殖川纹笛鲷Lutjanus sebae及圆白鲳Ephippus orbis消化道壁优势菌群结构进行了比较分析。研究结果显示川纹笛鲷及圆白鲳消化道壁存在着大量细菌群落,对DGGE指纹图谱聚类分析表明两种鱼肠道壁及胃壁菌群组成相似度高于50%,其中二者肠道壁细菌组成相似性最高(67%),这些可能与两种鱼养殖在同一水域、摄食相同饵料相关,另外通过软件对DGGE指纹带谱相对丰度分析表明同种鱼肠道壁及胃壁具有相同最大优势菌群。同时,两种鱼消化道壁之间在细菌多样性及相对丰度上亦存在明显区别,圆白鲳消化道壁细菌多样性要高于川纹笛鲷,这可能归因于川纹笛鲷与圆白鲳在天然环境中栖息地的差异性。本研究通过首次建立不同海水鱼消化道壁16S rDNA-DGGE指纹图谱及比较分析,为澄清海水鱼消化道壁微生物区系奠定基础。     

用PCR指纹图技术分析焦化废水接触氧化池中悬浮污泥和生物膜的微生物种群组成

目的:应用分子生物学方法,以处理焦化工业废水(A2/O生物膜工艺)中的悬浮污泥和生物膜的微生物群落作为研究对象,分析不同环境微生物群落的组成差异.方法:首先提取群落的总DNA,获得ERIC-PCR和LP-RAPD指纹图谱并进行对比分析,然后结合群落探针杂交的技术,检查同样迁移率的条带的序列同源性,运用UVIBAND/MAP软件比较所得群落指纹图谱的相似性指数,从而可以得到群落差异的量化结果.结果:焦化废水接触氧化池中,悬浮污泥和生物膜的微生物群落组成存在相当大的差异.结论:通过这种差异的比较分析,有可能让我们更准确地了解氧化池中微生物的群落组成情况,有利于分析其与系统功能的关系.     

慢性牙周炎患者和牙周健康人唾液微生物多样性研究及临床意义

慢性牙周炎是一种常见的口腔疾病，其主要症状为牙周组织发炎和牙周袋，会导致牙齿松动甚至脱落。过去研究已经发现牙周炎与口腔微生物群落失衡密切相关。唾液是口腔中重要的微生物媒介，可以反映口腔微生物群落的特征。因此，研究慢性牙周炎患者与牙周健康人唾液微生物多样性的差异，对于深入了解慢性牙周炎的发病机制、个体化治疗及预防和促进口腔健康具有重要意义。文章针对慢性牙周炎患者与牙周健康人唾液微生物多样性的差异及其临床意义展开研究，以期为临床医生进行相关治疗提供参考。     

胜利油藏不同时间细菌群落结构的比较

利用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)和构建16S rRNA基因克隆文库2种方法,对孤岛油田两口井(注水井G和采油井L)在相距9个月的2个时间点(A和B)所采集样品的细菌群落结构进行了比较。DGGE图谱聚类分析表明注水井在2个时间点的微生物群落结构相似性为48.1%,而采油井的相似性只有28.7%。16S rRNA基因克隆文库结果表明,A时间点样品G中的优势菌群为Betaproteobacteria、Gammaproteobacteria,还有Deferribacteres、Firmicutes、Bacteroidetes等;而样品L中,Gammaproteobacteria中的Moraxellaceae含量达到97%。B时间点G中除了优势菌Betaproteobacteria之外,Deferribacteres的数量显著增加,成为优势菌;而L在B时间点优势菌除Gammaproteobacteria外,还有Betaproteobacteria和Firmicutes。采油井中的微生物群落结构随时间发生了显著改变,而注水井变化不显著。这一结果部分揭示了微生物采油过程中地层微生物群落的变化规律,有助于进一步阐明微生物驱油的机理。     

Extracellular Glycoside Hydrolase Activities in the Human Oral Cavity

Carbohydrate availability shifts when bacteria attach to a surface and form biofilm. When salivary planktonic bacteria form an oral biofilm, a variety of polysaccharides and glycoproteins are the primary carbon sources; however, simple sugar availabilities are limited due to low diffusion from saliva to biofilm. We hypothesized that bacterial glycoside hydrolase (GH) activities would be higher in a biofilm than in saliva in order to maintain metabolism in a low-sugar, high-glycoprotein environment. Salivary bacteria from 13 healthy individuals were used to grow in vitro biofilm using two separate media, one with sucrose and the other limiting carbon sources to a complex carbohydrate. All six GHs measured were higher in vitro when grown in the medium with complex carbohydrate as the sole carbon source. We then collected saliva and overnight dental plaque samples from the same individuals and measured ex vivo activities for the same six enzymes to determine how oral microbial utilization of glycoconjugates shifts between the planktonic phase in saliva and the biofilm phase in overnight dental plaque. Overall higher GH activities were observed in plaque samples, in agreement with in vitro observation. A similar pattern was observed in GH activity profiles between in vitro and ex vivo data. 16S rRNA gene analysis showed that plaque samples had a higher abundance of microorganisms with larger number of GH gene sequences. These results suggest differences in sugar catabolism between the oral bacteria located in the biofilm and those in saliva.     

Effects of sampling location and time,and host animal on assessment of bacterial diversity and fermentation parameters in the bovine rumen

Aims: To investigate, using culture‐independent methods, whether the ruminal bacterial structure, population and fermentation parameters differed between sampling locations and time. Methods and Results: The detectable bacteria and fermentation parameters in the digesta from five locations in the rumen of three cows at three time points were analysed. The PCR‐denaturing gradient gel electrophoresis (PCR‐DGGE) profiles were similar among digesta samples from five locations (95·4%) and three time points (93·4%) within cows; however, a lower similarity was observed for samples collected from different host animals (85·5%). Rumen pH and concentration of volatile fatty acids (VFA) were affected by time points of sampling relative to feeding. Conclusions: The detectable bacterial structure in the rumen is highly conserved among different locations and over time, while the quantity of individual bacterial species may change diurnally in response to the feeding. Significance and impact of the study: This study supplies the fundamental understanding of the microbial ecology in the rumen, which is essential for manipulation of ruminal microflora and subsequent improvement in animal production.     

Analysis of oral microbial community and Th17‐associated cytokines in saliva of patients with oral lichen planus

Oral lichen planus (OLP) is a chronic inflammatory disorder of oral mucosa of unknown cause. Microbial infection and dysimmunity appear to play important roles in its pathogenesis. In this study, differences in genetic profiling of salivary microbial communities in two subtypes of OLP and healthy controls were evaluated by means of PCR‐denaturing gradient gel electrophoresis (DGGE). Additionally, ELISA was used to investigate the possible role of Th17 in lesion formation by detecting two related cytokines IL‐17 and IL‐23 in the saliva of OLP patients. When the DGGE profiles were analyzed, the bacterial populations were found to be significantly less rich in subjects with reticular and erosive OLP than in healthy controls. There was significantly less microbial diversity, as denoted by the Shannon index, in saliva samples from subjects with erosive OLP than in those from healthy controls. Cluster analysis and principal component analysis showed that the DGGE profiles formed distinctly group‐specific clusters. Salivary concentrations of IL‐17 in subjects with erosive OLP group were significantly higher than in those with reticular OLP and healthy controls. What's more, significantly positive correlations were observed between salivary IL‐17 concentrations and disease clinical scores. Microbial richness and diversity was negatively correlated with salivary IL‐17 concentrations. These results suggest there is significantly less salivary bacterial diversity and complexity in subjects with OLP han in healthy controls and that the shifted community composition is closely related to an immune cytokine, IL‐17.     

Correlation network analysis applied to complex biofilm communities

The complexity of the human microbiome makes it difficult to reveal organizational principles of the community and even more challenging to generate testable hypotheses. It has been suggested that in the gut microbiome species such as Bacteroides thetaiotaomicron are keystone in maintaining the stability and functional adaptability of the microbial community. In this study, we investigate the interspecies associations in a complex microbial biofilm applying systems biology principles. Using correlation network analysis we identified bacterial modules that represent important microbial associations within the oral community. We used dental plaque as a model community because of its high diversity and the well known species-species interactions that are common in the oral biofilm. We analyzed samples from healthy individuals as well as from patients with periodontitis, a polymicrobial disease. Using results obtained by checkerboard hybridization on cultivable bacteria we identified modules that correlated well with microbial complexes previously described. Furthermore, we extended our analysis using the Human Oral Microbe Identification Microarray (HOMIM), which includes a large number of bacterial species, among them uncultivated organisms present in the mouth. Two distinct microbial communities appeared in healthy individuals while there was one major type in disease. Bacterial modules in all communities did not overlap, indicating that bacteria were able to effectively re-associate with new partners depending on the environmental conditions. We then identified hubs that could act as keystone species in the bacterial modules. Based on those results we then cultured a not-yet-cultivated microorganism, Tannerella sp. OT286 (clone BU063). After two rounds of enrichment by a selected helper (Prevotella oris OT311) we obtained colonies of Tannerella sp. OT286 growing on blood agar plates. This system-level approach would open the possibility of manipulating microbial communities in a targeted fashion as well as associating certain bacterial modules to clinical traits (e.g.: obesity, Crohn's disease, periodontal disease, etc).     

Molecular characterization of the bacteria adherent to human colorectal mucosa

AIMS: To study large intestinal mucosal bacterial communities by Denaturing Gradient Gel Electrophoresis (DGGE) profiling and sequencing of 16S rRNA gene polymerase chain reaction (PCR) products amplified from DNA extracted from colorectal biopsies taken from healthy individuals. The specific aims were to determine how similar the mucosa-associated bacterial communities are within and between individuals and also to characterize the phylogenetic origin of isolated DGGE bands. METHODS AND RESULTS: Human colorectal biopsies were taken at routine colonoscopy from 33 patients with normal looking mucosa. The DNA was extracted directly from single biopsies and the bacterial 16S rDNA PCR amplified. The PCR products were profiled using DGGE to generate a fingerprint of the dominant members of the bacterial community associated with the biopsy. The reproducibility of this method was high (>98%). Washed and unwashed biopsies gave similar DGGE banding patterns (Median Similarity Coefficient - MSC 96%, InterQuartile Range - IQR 3.0%, n = 5). Adjacent biopsies sampled from the same patient using different forceps gave similar DGGE profiles (MSC 94%, n = 2). Two colorectal biopsies sampled at locations 2-5 cm apart, from each of 18 patients, resulted in very similar profiles (MSC 100%, IQR 2.8%). Biopsies sampled from different locations within the large intestine of the same patient also gave similar DGGE profiles (MSC 98% IQR 3.3%n = 6). Although all patients (n = 33) gave different DGGE profiles, some similarity (c. 34%) was observed between profiles obtained from 15 patients arbitrarily selected. 35 DGGE bands were excised and sequenced. Many were found to be most closely related to uncultured bacterial sequence entries in the Genbank database. Others belonged to typical gut bacterial genera including Bacteroides, Ruminococcus, Faecalibacterium and Clostridium. CONCLUSIONS: Bacterial communities adherent to colorectal mucosa within a normal patient show little variation; in contrast, mucosal bacterial communities sampled from different patients with normal colorectal mucosa show a high degree of variation. SIGNIFICANCE AND IMPACT OF THE STUDY: This research demonstrates that DGGE profiling of 16S rRNA gene PCR products amplified from DNA extracted directly from mucosal samples offers fresh insight into the bacterial communities that are adherent to colorectal mucosa. These findings are important with respect to further studies on the gastrointestinal tract in health and disease.     

Identification of early microbial colonizers in human dental biofilm

AIMS: To elucidate the first colonizers within in vivo dental biofilm and to establish potential population shifts that occur during the early phases of biofilm formation. METHODS AND RESULTS: A 'checkerboard' DNA-DNA hybridization assay was employed to identify 40 different bacterial strains. Dental biofilm samples were collected from 15 healthy subjects, 0, 2, 4 and 6 h after tooth cleaning and the composition of these samples was compared with that of whole saliva collected from the same individuals. The bacterial distribution in biofilm samples was distinct from that in saliva, confirming the selectivity of the adhesion process. In the very early stages, the predominant tooth colonizers were found to be Actinomyces species. The relative proportion of streptococci, in particular Streptococcus mitis and S. oralis, increased at the expense of Actinomyces species between 2 and 6 h while the absolute level of Actinomyces remained unaltered. Periodontal pathogens such as Tannerella forsythensis(Bacteroides forsythus), Porphyromonas gingivalis and Treponema denticola as well as Actinobacillus actinomycetemcomitans were present in extremely low levels at all the examined time intervals in this healthy group of subjects. CONCLUSION: The data provide a detailed insight into the bacterial population shifts occurring within the first few hours of biofilm formation and show that the early colonizers of the tooth surface predominantly consist of beneficial micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: The early colonizers of dental plaque are of great importance in the succession stages of biofilm formation and its overall effect on the oral health of the host.     

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.     

Analysis of Oral Microbiota in Children with Dental Caries by PCR-DGGE and Barcoded Pyrosequencing

Oral microbiota plays a vital role in maintaining the homeostasis of oral cavity. Dental caries are among the most common oral diseases in children and pathogenic bacteria contribute to the development of the disease. However, the overall structure of bacterial communities in the oral cavity from children with dental caries has not been explored deeply heretofore. We used high-throughput barcoded pyrosequencing and PCR-denaturing gradient gel electrophoresis (DGGE) to examine bacterial diversity of oral microbiota in saliva and supragingival plaques from 60 children aged 3 to 6 years old with and without dental caries from China. The multiplex barcoded pyrosequencing was performed in a single run, with multiple samples tagged uniquely by multiplex identifiers. As PCR-DGGE analysis is a conventional molecular ecological approach, this analysis was also performed on the same samples and the results of both approaches were compared. A total of 186,787 high-quality sequences were obtained for evaluating bacterial diversity and 41,905 unique sequences represented all phylotypes. We found that the oral microbiota in children was far more diverse than previous studies reported, and more than 200 genera belonging to ten phyla were found in the oral cavity. The phylotypes in saliva and supragingival plaques were significantly different and could be divided into two distinct clusters (p < 0.05). The bacterial diversity in oral microbiome analyzed by PCR-DGGE and barcoded pyrosequencing was employed to cross validate the data sets. The genera of Streptococcus, Veillonella, Actinomyces, Granulicatella, Leptotrichia, and Thiomonas in plaques were significantly associated with dental caries (p < 0.05). The results showed that there was no one specific pathogen but rather pathogenic populations in plaque that significantly correlated with dental caries. The enormous diversity of oral microbiota allowed for a better understanding of oral microecosystem, and these pathogenic populations in plaque provide new insights into the etiology of dental caries and suggest new targets for interventions of the disease.     

Buccal Swabbing as a Noninvasive Method To Determine Bacterial,Archaeal, and Eukaryotic Microbial Community Structures in the Rumen

Analysis of rumen microbial community structure based on small-subunit rRNA marker genes in metagenomic DNA samples provides important insights into the dominant taxa present in the rumen and allows assessment of community differences between individuals or in response to treatments applied to ruminants. However, natural animal-to-animal variation in rumen microbial community composition can limit the power of a study considerably, especially when only subtle differences are expected between treatment groups. Thus, trials with large numbers of animals may be necessary to overcome this variation. Because ruminants pass large amounts of rumen material to their oral cavities when they chew their cud, oral samples may contain good representations of the rumen microbiota and be useful in lieu of rumen samples to study rumen microbial communities. We compared bacterial, archaeal, and eukaryotic community structures in DNAs extracted from buccal swabs to those in DNAs from samples collected directly from the rumen by use of a stomach tube for sheep on four different diets. After bioinformatic depletion of potential oral taxa from libraries of samples collected via buccal swabs, bacterial communities showed significant clustering by diet (R = 0.37; analysis of similarity [ANOSIM]) rather than by sampling method (R = 0.07). Archaeal, ciliate protozoal, and anaerobic fungal communities also showed significant clustering by diet rather than by sampling method, even without adjustment for potentially orally associated microorganisms. These findings indicate that buccal swabs may in future allow quick and noninvasive sampling for analysis of rumen microbial communities in large numbers of ruminants.