Fecal microbiota of three bactrian camels(Camelus ferus and Camelus bactrianus) in China by high throughput sequencing of the V3-V4 region of the 16S rRNA gene

This study aimed to reveal the microbial diversity in the fecal samples of bactrian camels using the 16 S r RNA sequencing analysis on the Illumina Mi Seq platform. Three fecal samples were collected from two geographical regions in China. Operational taxonomic unit（OTU） clustering was performed by identifying an OTU at 97% sequence identity. The alpha and beta diversities were applied to estimate the differences in microbial diversity among the three fecal samples. Totally, 4409, 3151 and 4075 OTUs in the fecal samples were identified in the Lop Nor wild camel（Camelus ferus）, the domestic camel（C. bactrianus） and Dunhuang wild camel（C. ferus）, respectively. The majority of bactreria were affiliated with phylum Firmicutes and Bacteroidetes in the three samples. The wild camels had higher gastrointestinal tract microbial diversity than the domestic one, while the microbial composition of the Lop Nor wild camel shared higher similarity with domestic camel at the genus and family levels than that of the Dunhuang wild camel did. Our results may provide a theoretical basis for assessing their health conditions and may thus be useful for protecting the critically endangered species of C. ferus.

Diversity of Microflora in Colonic Mucus from Severe Ulcerative Colitis Patients Analyzed by Terminal Restriction Fragment Length Polymorphism and Clone Libraries of Bacterial 16S rRNA Gene Sequences

Although the gut microflora is thought to be an essential factor in the development of ulcerative colitis (UC), the entire gut microflora occurring in UC remains unknown. Most studies use feces to represent the microflora distribution;however, here we analyzed the bacterial diversity in colonic mucus from UC patients receiving colectomy surgery and control patients. The diversity of microflora was investigated using a combination of terminal restriction fragment length polymorphism (T-RFLP) and clone library analyses of the 16S rRNA gene sequences. In the T-RFLP analysis, the number of terminal restriction fragments (T-RFs) decreased significantly in UC patients when compared to control samples. Also in the clone library analysis, the number of operational taxonomic units (OTU) and the Shannon diversity index were reduced significantly in UC patients. These molecular analyses reveal an overall dysbiosis in UC patients. No specific pathogen was found, and a strong negative correlation in relative abundance of bacterial populations was observed between the phyla Bacteroidetes and Firmicutes in the UC patients. This is the first report showing a significant correlation between these two phyla, which may be important characteristics in the pathogenesis of UC.

Identification of Bacterial Fish Pathogens in Brazil by Direct Colony PCR and 16S rRNA Gene Sequencing

Intensive fish farming systems in Brazil have increased the disease incidence, mainly of bacterial origin, due to higher stocking density, high organic matter levels and poor quality of the aquatic environment that causes high mortality rates during outbreaks. The identification of pathogenic species using a fast and reliable method of diagnosis is essential for successful epidemiological studies and disease control. The present study evaluated the use of direct colony PCR in combination with 16S rRNA gene sequencing to diagnose fish bacterial diseases, with the goal of reducing the costs and time necessary for bacterial identification. The method was successful for all 178 isolates tested and produced bands with the same intensity as the standard PCR performed using pure DNA. In conclusion, the genetics methods allowed detecting the most common and important pathogens in Aquaculture, including 12 species of occurrence in Brazilian fish farms. The results of the present study constitute an advance in the available diagnostic methods for bacterial pathogens in fish farms.

基于PacBio 16S rRNA基因全长测序调查某校园水体微生物群落结构及多样性研究

水体是校园环境的重要部分,了解水体特征对于校园水体治理和完善校园生态环境研究具有重要意义。采用PacBio 16S rRNA基因全长测序技术,对某校园水体微生物群落的结构及多样性进行分析。结果显示各处校园水体的群落丰度与多样性呈现出差异,源湖、南一楼西湖和东湖的微生物群落丰度和多样性要高于东九湖、醉晚亭西湖和东湖。具体表现为水体的菌门和菌属的组成与丰度有所不同,呈现出复杂的变化情况。Beta多样性分析结果显示空间距离小的水体的微生物组成更为相似。水体微生物群落多样性受到NH_(3)-N、TP、DO等多种环境因子的共同作用。

16S rRNA Gene-Based Metagenomic Analysis of Soil Bacterial Diversity in Brazzaville, Republic of the Congo

Soil contains a great diversity of microorganisms, among which are bacteria. This study aimed to explore bacterial diversity in soil samples in Brazzaville in the Republic of the Congo. Environmental DNA was extracted. The illumina MiSeq sequencing was held and the diversity indices have been computed. Illumina MiSeq sequencing revealed 21 Phyla, four of which were abundant: Proteobacteria, Acidobacteria, Actinobacteria and Bacteroidetes. Soil microbial communities in the studied samples were phylogenetically diverse but with a stable community structure. 17 classes are represented with relative abundances of Rihzobiales, Bacillales, Actinomycetales and Acidobacteriales. 40 families, the Alphaproteobacteria, the Bacilli and the 12 Actinobacteria. 83 orders among which the Rhizobiales are the most abundant followed by Bacillales and the least abundant followed by the Flavobacteriaceae. Of the 28 genera listed, the Bradyrhizobium is the most dominant in Mw3 and Mw4. 25 listed species, Bradyrhizobium, Bacillus, Actinoplanes, and Candidatu coribacter Acidobacterium are the most abundant species. The Shannon indices of Mw3 and Mw4 are equal, the H’max of Mw4 is greater than the H’max of Mw3. The Simpson index of Mw4 is equal to the Simpson index of Mw3, and the Pielou index (J) of Mw4 is less than the R of Mw3, but very close. This study opens interesting perspectives on the knowledge and exploitation of telluric bacteria in several areas of life.

High-throughput sequencing of 16S r RNA amplicons characterizes gut microbiota shift of juvenile sea cucumber Apostichopus japonicus feeding with three antibiotics

Sea cucumber Apostichopus japonicus is an important marine economic species in Asian countries due to its profound nutritional and medicinal value. So far, with the rapid development of intensifi ed artifi cial aquaculture of sea cucumbers, the use of antibiotics is still an inexpensive and dispensable way to treat pathogenic infections, especially during the nursery phase. However, there is little information on the eff ects of antibiotics on the intestinal microbiota of sea cucumber. Therefore an Illumina based sequencing method was used to examine the intestinal bacterial composition of juvenile A . japonicas following diets with three typical antibiotics (tetracycline, erythromycin, and norfl oxacin) under 15, 30, and 45 d. The fi ndings reveal that diff erent antibiotics have distinct eff ects on the growth performance of juvenile sea cucumbers. However, the richness and diversity of microbiota were barely aff ected by antibiotics but the community composition alterations indicated that the three antibiotics exhibited their respective patterns of reshaping the intestinal bacteria of juvenile sea cucumbers. In common, the abundance of some sensitive genera with helpful functions, such as Thalassotalea , Shewanella , Sulfi tobacter , and Halomonas decreased signifi cantly with exposure to antibiotics and the abundance of multiple potential pathogenic- and suspected antibiotic-resistant microorganisms like Arcobacter , Leucothrix , and Clostridium_sensu_stricto_1 was found increased signifi cantly in the antibiotic groups. These results suggest that low doses of antibiotics could aff ect the composition of the intestinal microbiota of sea cucumbers and might increase the risk of infection of the hosts. This study could help us to explore how antibacterial compounds modify the gut microbiota of sea cucumbers and provide theoretical guidance in hatchery management by scientifi c antibiotic use in sea cucumber mariculture.

Evaluation of Real-Time 16S rDNA PCR and Pyrosequencing for Routine Identification of Bacteria in Joint Fluid and Tissue Specimens

16S rDNA PCR and sequencing are powerful tools for bacterial detection and identification, although their routine use is not currently widespread in the field of clinical microbiology. The availability of pyrosequencing now makes 16S rDNA assays more accessible to routine diagnostic laboratories, but this approach has had limited evaluation in general diagnostic practice. In this study we evaluated a real-time 16S rDNA PCR and pyrosequencing assay for use in a routine microbiology laboratory, by retrospectively testing joint fluid and joint tissue specimens received for conventional culture. We found that use of the real-time 16S rDNA assay was clinically valuable in this specimen type because it enabled us to identify a small number of culture-negative infections. Although faster and less labour-intensive, we found that the utility of pyrosequencing for pathogen identification is still hampered by shorter read lengths compared to conventional (Sanger) sequencing. Combining results from both molecular and conventional culture methods, bacteria were only detected in 11.8% specimens in this study. However, the detection rate was increased to 18.6% if specimens were only included from patients with a documented clinical suspicion of infection. In conclusion, while pyrosequencing had significant advantages in speed and ease-of-use over conventional sequencing, multiple reactions will be required to deliver comparable species-level identification, thus negating many of the benefits of using the technique. We found that 16S rDNA PCR and sequencing should be rationally targeted on the basis of good clinical information in the routine diagnostic setting, and not used as a general screening test for the exclusion of bacterial infection in joint specimens.

中国对虾16SrRNA基因序列多态性的研究

利用PCR技术扩增获得中国对虾 (Penaeuschinensis)线粒体DNA的 16SrRNA基因片段 ,通过测定该基因片段的序列 ,分析了取自我国烟台、长岛、青岛近海和宁波养殖的 17只中国对虾遗传多态性。结果发现 ,不同地理种群存在丰富的DNA序列多态性 ,17只个体具有 17种基因型 ,在扩增的长为 5 2 3bp的基因片段中 ,共检测到 3 7个多态性核苷酸位点 ( 7 0 7% )。UPGMA法构建的分子系统树表明不同地理种群中国对虾存在一定程度的遗传分化 ,长岛群体与烟台群体遗传关系较近 ,宁波群体次之 ,青岛群体为相对独立的一支。

福建华溪蟹线粒体DNA COI和16S rRNA基因序列的遗传多样性

目的探讨福建华溪蟹(Sinopotamon fukienense)的遗传多样性。方法采用PCR结合DNA测序技术,测定S.fukienense的线粒体COI和16SrRNA基因序列的组成。经比对获得639bp长度的COI基因序列和526bp的16SrRNA基因序列,以对比分析S.fukienense的遗传多样性。结果 S.fukienense基于COI基因核苷酸多样性(Pi)为0.048 4,高于其基于16SrRNA基因核苷酸多样性(Pi)为0.021 6。同时,S.fukienense基于COI基因单倍型间的平均遗传距离(P)为0.048,大于其基于16SrRNA基因单倍型间的平均遗传距离(P)0.026。结论 COI序列在分析S.fukienense遗传异变时的作用更优于16SrRNA基因序列。

5种经济海胆线粒体16S rRNA基因片段的序列分析

对光棘球海胆、中间球海胆、马粪海胆,海刺猬和紫海胆线粒体DNA 16SrRNA基因片段进行了PCR扩增和测序,得到395 bp的可信片段。通过ClustalX 1.83和Mega 3.0软件对所得线粒体16SrRNA片段序列进行比较,共检测到118个碱基变异,其中简约信息位点为33个。应用Mega 3.0计算各种间的相对遗传距离,以糙海参为外类群,得到NJ系统树和MP系统树,系统树各分支的置信度由“Bootstrap”1000次循环检验。结果表明,马粪海胆与光棘球海胆的亲缘关系较近,其次为中间球海胆,而紫海胆、海刺猬与这3种海胆的关系相对较远。

两种大眼蟹线粒体16S rRNA基因序列分析

测定了宽身大眼蟹和日本大眼蟹线粒体16SrRNA基因部分片段的序列,其长度均为517bp。二者的核苷酸序列A,T,G,C的含量相似:宽身大眼蟹分别为33.3%,35.6%,19.9%,11.2%;日本大眼蟹分别为35.2%,35.4%,18.4%,11.0%。不包括6处插入/缺失位点,两序列间有63个变异位点,核苷酸差异率为12.26%,其中转换32个、颠换31个,转换与颠换比约为1.0。对国内外大眼蟹的24条长度为415bp的16SrRNA基因同源序列进行分析,A+T的平均含量为71.7%,明显高于G+C的平均含量,且存有变异位点171个,简约信息位点137个。中国和日本的日本大眼蟹之间的核苷酸差异率为4.42%,表明二者已有明显的遗传分化;中国的日本大眼蟹与日本的万岁大眼蟹之间的核苷酸差异率仅为0.21%,表明二者有可能为同一物种。上述结果得到了系统发生树拓扑结构的支持。

基于16S rRNA测序分析冰鲜鸡腐败过程中微生物变化

为分析冰鲜鸡腐败过程中微生物变化,筛选冰鲜鸡腐败相关菌群,研究利用16 S rRNA测序技术分析了保存1、3、5和7 d冰鲜鸡样本微生物组成和多样性,利用正交偏最小二乘法回归和逐步多元回归分析筛选与冰鲜鸡腐败相关的菌属。结果显示:冰鲜鸡腐败过程中微生物丰富度和均匀度均显著下降,保存5 d和7 d样本与保存1 d和3 d样本微生物组成存在显著差异,4 d可能为冰鲜鸡腐败变质的临界点;筛选到27个冰鲜鸡腐败相关的候选菌属,其中肉食杆菌属、沙雷氏菌属和发光杆菌属可能为引起冰鲜鸡腐败变质的优势菌属。研究结果有助于加深对冰鲜鸡腐败变质机理的理解。

16S rRNA序列分析技术对临床标本中疑难细菌的鉴定

目的应用16S rRNA序列分析技术鉴定临床少见或疑难细菌,提高细菌鉴定的准确性。方法收集临床少见或疑难细菌44株,提取DNA,采用通用引物进行PCR扩增及目的片段基因测序,并将测序结果在核酸数据库中比对分析以确定菌种。结果 44株临床少见或疑难细菌均扩增到16S rRNA目的基因片段并成功测序,40株(90.9%)鉴定到"种",4株(9.1%)鉴定到"属";其中常规方法与测序方法在"属"水平一致的有34株(77.3%),不一致的有10株(22.7%)。结论 16S rRNA序列分析技术可以准确、快速地鉴定临床少见或疑难细菌,可以作为临床细菌鉴定的重要方法之一。

黄芪根瘤菌CA8561和JL84的部分16SrRNA基因序列的测定

报导了黄芪根瘤菌ＣＡ８５６１和ＪＬ８４的部分１６ｓｒＲＮＡ基因序列，测算了它们与已知根瘤菌、茎瘤菌各属模式种之间的遗传距离．结果表明，ＣＡ８５６１和ＪＬ８４均具有独立的进化路线，充分显示了黄芪根瘤菌的遗传多样性．

Relationship between hyperlipidemia and the gut microbiome of rats, characterized using high-throughput sequencing

Objective:To determine the effects of a high-fat diet(HFD)on the gut microbiome in rats,to explore the relationship between the intestinal flora and blood lipid profile.Methods:SpragueeDawley rats were fed an HFD for four weeks to induce hyperlipidemia,then 16S rRNA sequencing was used to compare the intestinal flora between hyperlipidemic and control diet-fed rats.Results:The microbiome of rats fed an HFD for four weeks differed from that of control diet-fed rats.Bacterial species that were less abundant were most affected by HFD feeding,among which were many pathogenic species,which became significantly more abundant.Eighteen genera were present in significantly different numbers in hyperlipidemic and control rats,more than half of which have been linked to infection and inflammation,or energy intake and obesity.The results indicated a type of stress response of the flora to a high-fat environment.In addition,the age of the rats tended to influence the gut microbial composition.Conclusion:These findings suggest that HFD may induce hyperlipidemia by affecting the gut microbial composition.Changes in the abundance of pro-inflammatory and pathogenic bacteria,and those that influence energy intake and obesity,may be important mediators of this.

中华绒螯蟹颤抖病螺原体分布及16 S-23 S rRNA基因间隔区序列的分析

为了探究中华绒螯蟹颤抖病螺原体(Spiroplasma eriocheiris)在江苏省的分布,以2003年至2009年从江苏省养殖池塘收集的发病中华绒螯蟹(Eriocheir sinensis)为研究对象,利用螺原体体外培养、光电镜检测和16 S-23 S rRNA基因间隔区序列比较等方法进行了系统分析。结果表明,S.eriocheiris是一种广泛分布于江苏省养殖中华绒螯蟹体内的病原微生物,它们不仅在7月底8月初的高温季节引起中华绒螯蟹发病和暴发性死亡,还能在3月和11月等低温季节引起部分中华绒螯蟹的发病和死亡,其严重危害中国江苏省中华绒螯蟹养殖业的健康发展。

16S rRNA Cloning and Identification of a Strain Isolated from Intestinal Tract of Rex Rabbit

This study was conducted to identify bacterial strain HeNan-001 isolated from intestinal tract of healthy rex rabbit. The strain was identified by gram staining, and OD600 values at different culture time were measured to develop a bacterial growth curve. The metabolic pathways of the bacterium using sugar in fermentation was identified with biochemical tubes. Total RNA of the strain was extracted, and total eDNA was obtained by Oligo （dT） method. Primers were designed using Primer 5.0 hip-software according to the 16S rRNA sequence published in GenBank, through cloning, ligation to vector PMD18T, construction of PMD18T/16S rRNA vector and transformation into DHSα, plasmid was extracted and sequenced, and the sequencing result was compared with sequences in NCBI followed by drawing of phylogenetic tree. The strain was verified to be double-stranded gram-positive cocci, which could ferment glucose, mannose, maltose and sorbitol, producing acids production, but failed to ferment sucrose, serum inulin and β-galactosidase. The phylogenetie tree analysis showed that the isolated bacterium shared the highest homology with an India isolate, and belongs to diplostreptococcus. This study provides significance experimental data and theoretical guidance for further development and utilization of the bacterial strain.

Enterococcus faecalis provides protection during scavenging in carrion crow (Corvus corone)

The gut microbiota significantly influences host physiology and provides essential ecosystem services.While diet can affect the composition of the gut microbiota,the gut microbiota can also help the host adapt to specific dietary habits.The carrion crow(Corvus corone),an urban facultative scavenger bird,hosts an abundance of pathogens due to its scavenging behavior.Despite this,carrion crows infrequently exhibit illness,a phenomenon related to their unique physiological adaptability.At present,however,the role of the gut microbiota remains incompletely understood.In this study,we performed a comparative analysis using 16S rRNA amplicon sequencing technology to assess colonic content in carrion crows and 16 other bird species with different diets in Beijing,China.Our findings revealed that the dominant gut microbiota in carrion crows was primarily composed of Proteobacteria(75.51%)and Firmicutes(22.37%).Significant differences were observed in the relative abundance of Enterococcus faecalis among groups,highlighting its potential as a biomarker of facultative scavenging behavior in carrion crows.Subsequently,E.faecalis isolated from carrion crows was transplanted into model mice to explore the protective effects of this bacterial community against Salmonella enterica infection.Results showed that E.faecalis down-regulated the expression of pro-inflammatory cytokines tumor necrosis factor alpha(TNF-α),interferon gamma(IFN-γ),and interleukin 6(IL-6),prevented S.enterica colonization,and regulated the composition of gut microbiota in mice,thereby modulating the host’s immune regulatory capacity.Therefore,E.faecalis exerts immunoregulatory and anti-pathogenic functions in carrion crows engaged in scavenging behavior,offering a representative case of how the gut microbiota contributes to the protection of hosts with specialized diets.

Next-generation sequencing revealed specific microbial symbionts in Porites lutea with pigment abnormalities in North Sulawesi, Indonesia

Bacterial diseases affecting corals pose an enormous threat to the health of coral reefs. The relationship between certain bacterial species and coral diseases remain largely unknown. Pigment abnormalities are common in Porites lutea. Here we used Illumina 16S rRNA gene sequencing to analyze the bacterial communities associated with healthy P. lutea and P. lutea with pigment abnormalities. We observed an increase of alpha diversity of the bacterial community of P. lutea with pigment abnormalities, relative to healthy corals. We then identified changes in the abundance of individual operational taxonomic units(OTUs) between pigmented and healthy corals. We were able to identify eight OTUs associated with pigment abnormalities, which are possibly the causative agents of pigment abnormalities.

Alterations in gut microbiota are related to metabolite profiles in spinal cord injury

Studies have shown that gut microbiota metabolites can enter the central nervous system via the blood-spinal cord barrier and cause neuroinflammation, thus constituting secondary injury after spinal cord injury. To investigate the correlation between gut microbiota and metabolites and the possible mechanism underlying the effects of gut microbiota on secondary injury after spinal cord injury, in this study, we established mouse models of T8–T10 traumatic spinal cord injury. We used 16 S rRNA gene amplicon sequencing and metabolomics to reveal the changes in gut microbiota and metabolites in fecal samples from the mouse model. Results showed a severe gut microbiota disturbance after spinal cord injury, which included marked increases in pro-inflammatory bacteria, such as Shigella, Bacteroides, Rikenella, Staphylococcus, and Mucispirillum and decreases in anti-inflammatory bacteria, such as Lactobacillus, Allobaculum, and Sutterella. Meanwhile, we identified 27 metabolites that decreased and 320 metabolites that increased in the injured spinal cord. Combined with pathway enrichment analysis, five markedly differential amino acids(L-leucine, L-methionine, L-phenylalanine, L-isoleucine and L-valine) were screened out, which play a pivotal role in activating oxidative stress and inflammatory responses following spinal cord injury. Integrated correlation analysis indicated that the alteration of gut microbiota was related to the differences in amino acids, which suggests that disturbances in gut microbiota might participate in the secondary injury through the accumulation of partial metabolites that activate oxidative stress and inflammatory responses. Findings from this study provide a new theoretical basis for improving the secondary injury after spinal cord injury through fecal microbial transplantation.