Towards the human intestinal microbiota phylogenetic core

The paradox of a host specificity of the human faecal microbiota otherwise acknowledged as characterized by global functionalities conserved between humans led us to explore the existence of a phylogenetic core. We investigated the presence of a set of bacterial molecular species that would be altogether dominant and prevalent within the faecal microbiota of healthy humans. A total of 10 456 non-chimeric bacterial 16S rRNA sequences were obtained after cloning of PCR-amplified rDNA from 17 human faecal DNA samples. Using alignment or tetranucleotide frequency-based methods, 3180 operational taxonomic units (OTUs) were detected. The 16S rRNA sequences mainly belonged to the phyla Firmicutes (79.4%), Bacteroidetes (16.9%), Actinobacteria (2.5%), Proteobacteria (1%) and Verrumicrobia (0.1%). Interestingly, while most of OTUs appeared individual-specific, 2.1% were present in more than 50% of the samples and accounted for 35.8% of the total sequences. These 66 dominant and prevalent OTUs included members of the genera Faecalibacterium , Ruminococcus , Eubacterium , Dorea , Bacteroides , Alistipes and Bifidobacterium . Furthermore, 24 OTUs had cultured type strains representatives which should be subjected to genome sequence with a high degree of priority. Strikingly, 52 of these 66 OTUs were detected in at least three out of four recently published human faecal microbiota data sets, obtained with very different experimental procedures. A statistical model confirmed these OTUs prevalence. Despite the species richness and a high individual specificity, a limited number of OTUs is shared among individuals and might represent the phylogenetic core of the human intestinal microbiota. Its role in human health deserves further study.     

Comparative assessment of human and farm animal faecal microbiota using real-time quantitative PCR

Pollution of the environment by human and animal faecal pollution affects the safety of shellfish, drinking water and recreational beaches. To pinpoint the origin of contaminations, it is essential to define the differences between human microbiota and that of farm animals. A strategy based on real-time quantitative PCR (qPCR) assays was therefore developed and applied to compare the composition of intestinal microbiota of these two groups. Primers were designed to quantify the 16S rRNA gene from dominant and subdominant bacterial groups. TaqMan^® probes were defined for the qPCR technique used for dominant microbiota. Human faecal microbiota was compared with that of farm animals using faecal samples collected from rabbits, goats, horses, pigs, sheep and cows. Three dominant bacterial groups ( Bacteroides/Prevotella, Clostridium coccoides and Bifidobacterium ) of the human microbiota showed differential population levels in animal species. The Clostridium leptum group showed the lowest differences among human and farm animal species. Human subdominant bacterial groups were highly variable in animal species. Partial least squares regression indicated that the human microbiota could be distinguished from all farm animals studied. This culture-independent comparative assessment of the faecal microbiota between humans and farm animals will prove useful in identifying biomarkers of human and animal faecal contaminations that can be applied to microbial source tracking methods.     

PCR/DGGE and 16S rRNA gene library analysis of the colonic microbiota of HLA-B27/beta2-microglobulin transgenic rats

AIMS: To determine the phylogenetic composition of the colonic microbiota of transgenic (TG) HLA-B27 rats using 16S ribosomal RNA (rRNA) gene sequences obtained from denaturing gradient gel electrophoresis (DGGE) gels and sequences from a 16S rRNA gene library. METHODS AND RESULTS: Colonic microbiota of TG and nontransgenic (NT) rats harboured by 10-week-old and 6-month-old animals was screened using PCR/DGGE. Six months old TG rats had marked inflammation of the colon compared with 10-week-old TG and NT rats. The DGGE profiles of rats with inflamed colon were similar from rat to rat (Dice's Similarity Coefficient proximal colon 73%, distal colon 83%) whereas profiles from animals without inflammation were dissimilar (52-64%). Identifications of bacterial origins of 16S rRNA gene sequences obtained from DGGE gels (200 bp) and from 16S rRNA clones (450 bp) of the colonic microbiota of diseased rats gave sequences most closely phylogenetically affiliated with uncultured or unknown bacteria. CONCLUSIONS: PCR/DGGE was shown to be an effective method to compare the colonic microbiota composition of TG and NT rats relative to the progression of inflammatory disease. Sequencing of 16S rRNA gene fragments from DGGE gels or 16S rRNA gene clones from a random library showed that uncultured or unknown bacteria were most commonly detected by both methods. It can be concluded that it would be better in future studies to search for the antigens produced by the gut microbiota against which the dysfunctional immune system reacts rather than seek phylogenetic associations. SIGNIFICANCE AND IMPACT OF THE STUDY: PCR/DGGE can be used as a rapid initial screening method to compare the composition of bacterial communities of initially unknown composition that are associated with the development of intestinal disease.     

基于性别二分类的青海天峻藏民肠道菌群差异分析

目的 对青海省天峻县的藏族牧民健康志愿者肠道菌群进行研究,探讨青海藏民肠道菌群结构及性别对肠道菌群组成的影响。方法 以28例藏族牧民志愿者的粪便样品作为研究对象,应用基于16S rDNA V3+V4可变区的高通量测序技术测定肠道菌群组成及核心菌群;通过db-RDA和菌群多样性分析,探索性别因素对肠道菌群结构的影响。结果 在门水平上,厚壁菌门和拟杆菌门是青海藏族志愿者的优势菌群。在属水平上,优势菌属是普氏菌属、Lachnospiracea_incertae_sedis、Faecalibacterium和拟杆菌属;特有优势菌属是Lachnospiracea_incertae_sedis。db-RDA结果显示性别因素对肠道微生物群结构有一定分离趋势,多样性结果进一步证实性别差异显著影响菌群结构。男性和女性存在12种差异菌群,包括普氏菌属、Acidaminobacter属等。结论 性别差异对青海藏民肠道菌群影响显著。     

Dominant bacterial communities in the rumen of Gayals (Bos frontalis), Yaks (Bos grunniens) and Yunnan Yellow Cattle (Bos taurs) revealed by denaturing gradient gel electrophoresis

The dominant rumen bacteria in Gayals, Yaks and Yunnan Yellow Cattle were investigated using PCR-DGGE approach. The analysis of DGGE profiles, identification of dominant bands and phylogenetic analysis 16S rDNA sequences in DGGE profiles were combined to reveal the dominant bacterial communities and compared the differences between those cattle species. DGGE profiles revealed that Gayals had the most abundant dominant bacteria and the lowest similarity of intraspecies between individuals than other two cattle species. A total of 45 sequences were examined and sequence similarity analysis revealed that Gayals had the most sequences appeared to uncultured bacteria, accounting for 85.0% of the total sequences, Yaks and Yunnan Yellow Cattle had 44.4 and 68.8% uncultured bacterial sequences, respectively. According to phylogenetic analysis, the rumen dominant bacteria of Gayals were mainly phylogenetically placed within phyla firmicutes and bacteroidetes, and the known bacteria were mainly belonged to the genera Lachnospiraceae bacterium, Ruminococcus flavefaciens and Clostridium celerecrescens. Moreover, the dominant bacteria of Yaks were also mainly belonged to phyla firmicutes and bacteroidetes, and the known dominant bacteria were including Ruminococcus flavefaciens, Butyrivibrio fibrisolvens, Pseudobutyrivibrio ruminis, Schwartzia succinivorans and Clostridiales bacterium, most of them are common rumen bacteria. In addition, the dominant bacteria in Yunnan Yellow Cattle were belonged to phyla firmicutes, bacteroidetes and Actinobacteria, and the known dominant bacteria containing Prevotella sp., Staphylococci lentus, Staphylococcus xylosus and Corynebacterium casei. Present study first detected Staphylococcus lentus and Staphylococcus xylosus in the rumen of cattle.     

Denaturing gradient gel electrophoresis (DGGE)-based monitoring of intestinal lactobacilli and bifidobacteria of pigs during a feeding trial

The aim of this study was to determine the influence of different feeding strategies on the gut microbiota of organic growing-finishing pigs. A total of 76 pigs were allocated to four different dietary treatments (control, probiotics, maize silage and grass silage). Effects of the applied probiotic preparation on the composition of the intestinal and faecal microbiota were monitored. By using a DGGE (denaturing gradient gel electrophoresis)-based methodology, fingerprints of the intestinal microbiota were obtained. The total microbial DNA was isolated from faecal and colon samples and amplified with PCR using different primer sets to detect bifidobacteria and lactobacilli. PCR products were separated using DGGE and the resulting profiles were compared with the findings of the other dietary treatments. Bands were excised from the gels and sequenced for further identification. Particularly two different DGGE profiles of bifidobacteria were observed, while lactobacilli showed larger variety within the dietary treatments.     

圈养食叶猴的肠道菌群特征

充分认识圈养食叶猴的肠道菌群组成特征, 为饲养管理方法的改进提供参考依据。

通过MiSeq高通量测序平台对3个物种的19个个体[黑叶猴(n=5)、川金丝猴(n=9)和西非黑白疣猴(n=5)]的肠道菌群16S rRNA V4-V5区进行测序和分析。

食叶猴肠道菌群以厚壁菌门和拟杆菌门为优势菌门, 瘤胃菌科、普氏菌科、理研菌科和毛螺菌科为优势菌科, 普氏菌属、密螺旋体属和瘤胃球菌属为优势菌属; 食叶猴物种间肠道菌群组成存在显著差异, 肠道菌群按照宿主物种聚类, 而不受环境因素的影响。

宿主物种是决定肠道微生物组成的重要因素, 食叶猴肠道菌群特征反映了宿主对其食性的适应。

     

Development of Faecalibacterium 16S rRNA gene marker for identification of human faeces

Aims:  The focus of this study was to identify a bacterial 16S rRNA gene sequence, unique to microbiota in the human gut, for use in development of a dependable PCR assay to detect human faecal pollution in water.
Methods and Results:  Suppression subtractive hybridization (SSH) and bioinformatics were used to identify a genetic marker, within the 16S rRNA gene of Faecalibacterium , for the detection of human faeces. DNA sequencing analysis demonstrated that a majority (16) of 74 clones of the SSH library contained insertion sequences identified as Faecalibacterium 16S rRNA genes . Human faeces-specific sequences were derived and six PCR primer sets designed and tested against faecal DNA samples from human and nonhuman sources. One PCR primer set, HFB-F3 and HFB-R5, was exclusively associated with human faeces. These primers generated a human faeces-specific amplicon of 399 bp from 60·2% of human faecal samples and 100% of sewage samples.
Conclusions:  The subject Faecalibacterium marker is specific for sewage.
Significance and Impact of the Study:  This study represents the initial report of a Faecalibacterium marker for human faeces, which may prove useful for microbial source tracking.     

16S rRNA gene-based analysis of mucosa-associated bacterial community and phylogeny in the chicken gastrointestinal tracts: from crops to ceca

Mucosa-associated microbiota from different regions of the gastrointestinal (GI) tract of adult broilers was studied by analysis of 16S rRNA gene sequences. The microbiota mainly comprised Gram-positive bacteria along the GI tract. Fifty-one operational taxonomic units (OTUs) (from 98 clones) were detected in the ceca, as compared with 13 OTUs (from 49 clones) in the crops, 11 OTUs (from 51 clones) in the gizzard, 14 OTUs (from 52 clones) in the duodenum, 12 OTUs (from 50 clones) in the jejunum and nine OTUs (from 50 clones) in the ileum. Ceca were dominantly occupied by clostridia-related sequences (40%) with other abundant sequences being related to Faecalibacterium prausnitzii (14%), Escherichia coli (11%), lactobacilli (7%) and Ruminococcus (6%). Lactobacilli were predominant in the upper GI tract and had the highest diversity in the crop. Both Lactobacillus aviarius and Lactobacillus salivarius were the predominant species among lactobacilli. Candidatus division Arthromitus was also abundant in the jejunum and ileum.     

Differences in faecal bacteria populations and faecal bacteria metabolism in healthy adults and celiac disease patients

Differences in the intestinal microbiota between children and adults with celiac disease (CD) have been reported; however, differences between healthy adults and adults with CD have not been clearly demonstrated. The aim of this study was to evaluate the differences in the intestinal microbiota between adults with CD and healthy individuals. Microbial communities in faecal samples were evaluated by PCR-denaturing gradient gel electrophoresis (DGGE) and gas-liquid chromatography of short chain fatty acids (SCFAs). The study group included 10 untreated CD patients, 11 treated CD patients and 11 healthy adults (in normal gluten diet and in GFD). UPGMA clustered the dominant microbial communities of healthy individuals together and separated them from the dominant microbial communities of the untreated CD patients. Most of the dominant microbial communities of the treated CD patients clustered together with those of healthy adults. The treated CD patients showed a reduction in the diversity of Lactobacillus and Bifidobacterium species. The presence of Bifidobacterium bifidum was significantly higher in untreated CD patients than healthy adults. There was a significant difference between untreated CD patients and healthy adults, as well as between treated CD patients and healthy adults, regarding acetic acid, propionic acid, butyric acid, and total SCFAs. In conclusion: healthy adults have a different faecal microbiota from that of untreated CD patients. A portion of the treated CD patients displayed a restored "normal" microbiota. The treated CD patients significantly reduce the Lactobacillus and Bifidobacterium diversity. Healthy adults have a different faecal SCFAs content from that of CD patients.     

What do we mean when we refer to Bacteroidetes populations in the human gastrointestinal microbiota?

Recent large-scale cloning studies have shown that the ratio of Bacteroidetes to Firmicutes may be important in the obesity-associated gut microbiota, but the species these phyla represent in this ecosystem has not been examined. The Bacteroidetes data from the recent Turnbaugh study were examined to determine those members of the phylum detected in human faecal samples. In addition, FISH analysis was performed on faecal samples from 17 healthy, nonobese donors using probe Bac303, routinely used by gut microbiologists to enumerate Bacteroides – Prevotella populations in faecal samples, and another probe (CFB286) whose target range has some overlap with that of Bac303. Sequence analysis of the Turnbaugh data showed that 23/519 clones were chimeras or erroneous sequences; all good sequences were related to species of the order Bacteroidales , but no one species was present in all donors. FISH analysis demonstrated that approximately one-quarter of the healthy, nonobese donors harboured high numbers of Bacteroidales not detected by probe Bac303. It is clear that Bacteroidales populations in human faecal samples have been underestimated in FISH-based studies. New probes and complementary primer sets should be designed to examine numerical and compositional changes in the Bacteroidales during dietary interventions and in studies of the obesity-associated microbiota in humans and animal model systems.     

维吾尔族与汉族儿童肠道细菌群落分析

【背景】肠道菌群是人体的重要组成部分,在人体的多种生命活动中发挥着重要作用。【目的】探究维吾尔族和汉族儿童肠道细菌群落特征,为儿童营养健康状况监测和营养改善工作提供更有效精准的营养干预策略。【方法】从新疆维吾尔自治区泽普县维吾尔族和河南省民权县汉族人群中分别随机选取10?12岁学龄期儿童各20名,同一时间段收集其新鲜粪便,提取细菌总DNA,通过高通量测序和生物信息学分析,研究两地区健康维吾尔族儿童与汉族儿童肠道细菌群落的差异。【结果】获得测序序列2 007 100条,归类于994个OTU;所有样本共含15个细菌门139属。α多样性和β多样性分析表明,调查地区的2个民族儿童肠道细菌的丰富度和多样性均有统计学意义上的差异,维吾尔族儿童肠道细菌群落丰富度高于汉族儿童,而物种多样性不如汉族儿童。其中,维吾尔族儿童肠道细菌丰度相对占优势的门和属及其丰度值为：拟杆菌门(Bacteroidetes,63%)、厚壁菌门(Firmicutes,22%)、普氏菌属(Prevotella,61%)、琥珀酸弧菌属(Succinivibrio,9%)和粪杆菌属(Faecalibacterium,5%);汉族儿童肠道细菌丰度占优势的门和属及其丰度值为：厚壁菌门(57%)、拟杆菌门(23%)、粪杆菌属(16%)、普氏菌属(11%)和拟杆菌属(Bacteroides,11%)。【结论】调查地区维吾尔族与汉族儿童肠道细菌群落差异较大,这为进一步研究肠道菌群与膳食因素及人体营养健康状况之间的关系提供了依据。     

Distal gut microbiota of adolescent children is different from that of adults

Human intestinal microbiota plays a number of important roles in human health and is also implicated in several gastrointestinal disorders. Although the diversity of human gut microbiota in adults and in young children has been examined, few reports of microbiota composition are available for adolescents. In this work, we used Microbiota Array for high-throughput analysis of distal gut microbiota in adolescent children 11-18 years of age. Samples obtained from healthy adults were used for comparison. Adolescent and adult groups could be separated in the principal components analysis space based on the relative species abundance of their distal gut microbiota. All samples were dominated by class Clostridia. A core microbiome of 46 species that were detected in all examined samples was established; members of genera Ruminococcus, Faecalibacterium, and Roseburia were well represented among core species. Comparison of intestinal microbiota composition between adolescents and adults revealed a statistically significantly higher abundance of genera Bifidobacterium and Clostridium among adolescent samples. The number of detected species was similar between sample groups, indicating that it was the relative abundances of the genera and not the presence or absence of a specific genus that differentiated adolescent and adult samples. In summary, contrary to the current belief, this study suggests that the gut microbiome of adolescent children is different from that of adults.     

血液恶性肿瘤患者肠道菌群分析

目的 将血液恶性肿瘤患者肠道菌群与健康个体进行比较,观察血液肿瘤患者肠道菌群结构的变化,探讨肠道菌群与血液恶性肿瘤发生发展的联系。方法 收集血液恶性肿瘤患者与健康志愿者粪便样品,提取样品中菌群总DNA,然后通过变性凝胶梯度电泳（DGGE）技术分析肠道菌群多样性和差异性。结果 血液恶性肿瘤组与健康组肠道菌群的DGGE指纹图谱有明显差异。与正常组相比,患者组肠道大肠埃希菌呈现过度增长趋势,有益菌柔嫩梭菌减少或缺失,某些患者肠道内一些细菌呈现特异性增长,如粪肠球菌、硫磺肠球菌、约氏不动杆菌等。结论 与健康对照组相比,血液恶性肿瘤患者肠道菌群结构与多样性发生改变,这可能为血液恶性肿瘤早期抗感染提供实验依据。     

Molecularly assessed shifts of Bifidobacterium ssp. and less diverse microbial communities are characteristic of 5-year-old allergic children

The composition of intestinal microbiota and the Bifidobacterium group community in 20 allergic and 20 nonallergic 5-year-old children was visualized by PCR-denaturing gradient gel electrophoresis (DGGE). The number of dominant bands in the DGGE profiles was smaller in allergic children than in nonallergic children (P<0.001). Allergic children mainly formed a single group upon cluster analysis, whereas nonallergic children were divided between four different groups. In allergic children the Bifidobacterium adolescentis species prevailed, and in nonallergic children the Bifidobacterium catenulatum/pseudocatenulatum prevailed (P=0.01 and P=0.01, respectively). The less diverse composition of intestinal microbiota and prevalence of particular species of Bifidobacterium were characteristic of allergic children even at the age of 5 years.     

Alterations in the gut microbiota of patients with acquired immune deficiency syndrome

Acquired immune deficiency syndrome (AIDS), caused by infection with human immunodeficiency virus (HIV), is associated with gastrointestinal disease, systemic immune activation and changes in the gut microbiota. Here, we aim to investigate the gut microbiota patterns of HIV‐infected individuals and HIV‐uninfected individuals in populations from South China. We enrolled 33 patients with HIV (14 participants treated with highly active antiretroviral therapy [HAART] for more than 3 months; the remaining 19 individuals had not received treatment) and 35 healthy controls (HC) for a cross‐sectional comparison of gut microbiota using stool samples. Gut microbial communities were profiled by sequencing the bacterial 16S rRNA genes. Dysbiosis was more common among patients with AIDS compared with healthy individuals. Dysbiosis was characterized by decreased α‐diversity, low mean counts of Bacteroidetes, Faecalibacterium, Prevotella, Bacteroides vulgatus, Dialister and Roseburia inulnivorans, and high mean counts of Proteobacteria, Enterococcus, Streptococcus, Lactobacillus, Lachnociostridium, Ruminococcus gnavus and Streptococcus vestibularis. Increased abundance of Bacilli was observed in homosexual patients. Proteobacteria were higher among heterosexual patients with HIV infections. Tenericutes were higher among patients with history of intravenous drug abuse. Restoration of gut microbiota diversity and a significant increase in abundance of Faecalibacterium, Blautia and Bacteroides were found in patients receiving HAART compared to those who did not receive. HIV infection‐associated dysbiosis is characterized by decreased levels of α‐diversity and Bacteroidetes, increased levels of Proteobacteria and the alterations of gut microbiota correlate with the route of HIV transmission. The imbalanced faecal microbiota of HIV infection is partially restored after therapy.     

The Development and Stability of the Genus <Emphasis Type="Italic">Bacteriodes</Emphasis> from Human Gut Microbiota in HFA Mice Model

Human flora-associated (HFA) mice are frequently applied in studying the ecology and metabolism of human gut microbiota. However, the development and stability of the genus Bacteriodes, a prominent bacteria group of human gut microbiota, in HFA mice have not yet fully been examined. In this study, PCR-denaturing gradient gel electrophoresis (DGGE) analysis was employed to monitor the Bacteriodes community in the fecal microbiota of six HFA Kunming mice during a period of 3 weeks. Based on the DGGE banding patterns, the majority of prominent bands in the HFA mice DGGE profile were also typical bands in the human DGGE profile, despite the absence of three bands (corresponding to two different B. thetaiotaomicron strains and one B. intestinalis strain) from the human DGGE profile. The Dice coefficient of similarity for the fecal microbiota of HFA mice in comparison to the human donor sample ranged between 74 ± 6% and 81 ± 7%. The phylogeny of bands in the DGGE profile showed that the dominant Bacteriodes species in the fecal microbiota of HFA mice were B. thetaiotaomicron, representing 66.7% of all bands. Our results indicate that the genus Bacteriodes in the fecal microbiota of HFA mice was selected from the human donor and could remain relatively stable over time.     

1 067例不同年龄及地域人群肠道菌群特征

目的 探索不同人群在不同年龄阶段的菌群结构特点,了解环境因素对肠道菌群组成及发展的影响。方法 收集65例中国健康儿童粪便样本并进行16S rRNA检测,同时从已发表文献中获取来自亚洲、非洲、欧洲、北美洲及南美洲国家的1 002例健康人群肠道菌群数据,分析来自不同年龄阶段健康人群肠道菌群的特征。结果 3岁以下全球各地儿童肠道菌群的种类较为一致：拟杆菌属和双歧杆菌属在儿童肠道菌群中占据主导地位,且它们在免疫调节上有重要作用。3岁以上健康人群的肠道菌群则展示出了地域性特征：亚洲健康人群的肠道代表菌属为考拉杆菌属和巨单胞菌属,非洲健康人群的肠道代表菌属为粪杆菌属,欧洲健康人群的肠道代表菌属也是粪杆菌属,北美洲健康人群的肠道代表菌属为拟杆菌属,南美洲健康人群的肠道代表菌属为普氏菌属。另外,经济较发达的欧洲及北美洲健康人群的肠道菌群多样性显著高于其他经济较落后地区。结论 本研究加深了对不同地域人群肠道菌群特点以及它们随年龄增长而发展变化的认识。