The abundance of fecal Faecalibacterium prausnitzii in relation to obesity and gender in Chinese adults

The influence of gender and obesity on the abundance of human colonic Feacalibacterium prausnitzii is currently unclear. We collected fecal samples from 54 obese and 54 sex- and age-matched normal-weight Chinese adults and quantified the fecal F. prausnitzii as percentage of 16S rRNA gene copies of F. prausnitzii accounting to that of total gut bacteria with quantitative PCR. The fecal F. prausnitzii amount was not significantly different between obese and lean subjects. Men possessed significantly lower level of fecal F. prausnitzii than women, and the significant and positive correlation of fecal F. prausnitzii quantity with fasting glucose level was observed in men, not in women. Our results suggest that the gender effect, in addition to other factors including the geographic location, ethnicity, diet and gut transit times of study subjects, has to be considered when studying the relationship between gut F. prausnitzii and diseases.     

Structural segregation of gut microbiota between colorectal cancer patients and healthy volunteers

Despite a long-suspected role in the development of human colorectal cancer (CRC), the composition of gut microbiota in CRC patients has not been adequately described. In this study, fecal bacterial diversity in CRC patients (n=46) and healthy volunteers (n=56) were profiled by 454 pyrosequencing of the V3 region of the 16S ribosomal RNA gene. Both principal component analysis and UniFrac analysis showed structural segregation between the two populations. Forty-eight operational taxonomic units (OTUs) were identified by redundancy analysis as key variables significantly associated with the structural difference. One OTU closely related to Bacteroides fragilis was enriched in the gut microbiota of CRC patients, whereas three OTUs related to Bacteroides vulgatus and Bacteroides uniformis were enriched in that of healthy volunteers. A total of 11 OTUs belonging to the genera Enterococcus, Escherichia/Shigella, Klebsiella, Streptococcus and Peptostreptococcus were significantly more abundant in the gut microbiota of CRC patients, and 5 OTUs belonging to the genus Roseburia and other butyrate-producing bacteria of the family Lachnospiraceae were less abundant. Real-time quantitative PCR further validated the significant reduction of butyrate-producing bacteria in the gut microbiota of CRC patients by measuring the copy numbers of butyryl-coenzyme A CoA transferase genes (Mann–Whitney test, P<0.01). Reduction of butyrate producers and increase of opportunistic pathogens may constitute a major structural imbalance of gut microbiota in CRC patients.     

Faecalibacterium prausnitzii Inhibits Interleukin-17 to Ameliorate Colorectal Colitis in Rats

Background and Aims

It has been shown that Faecalibacterium prausnitzii (F. prausnitzii), one of the dominant intestinal bacterial flora, may protect colonic mucosa against the development of inflammation and subsequent inflammatory bowel disease (IBD), with the underlying mechanisms being unclear.

Methods

The impacts of F. prausnitzii and its metabolites on IL-23/Th17/IL-17 pathway markers were determined in human monocytes and a rat model of colitis induced by 2,4,6-trinitrobenzene sulfonic acid. F. prausnitzii and its culture medium (containing complete metabolites) were used to treat the rats in vivo, as well as rat splenocytes and human monocytes in vitro. Inflammatory cytokines were measured in colon tissue, plasma and cell culture medium.

Results

The culture supernatant of F. prausnitzii increased plasma anti-Th17 cytokines (IL-10 and IL-12)and suppressed IL-17 levels in both plasma and colonic mucosa, with ameliorated colonic colitis lesions. This inhibition of IL-17 release has also been observed in both rat splenocytes and human venous monocytes in vitro. The culture supernatant of F. prausnitzii also suppressed Th17 cell differentiation induced by cytokines (TGF-ß and IL-6) and bone marrow-derived dendritic cells (BMDCs) in vitro. The metabolites of F. prausnitzii in the culture supernatant exert a stronger anti-inflammatory effect than the bacterium itself. F. prausnitzii protected the colon mucosa against the development of IBD by its metabolites, suggesting a promising potential for the use of F. prausnitzii and its metabolic products in the treatment of IBD.     

Phylogenetic diversity of bacteria associated with the endemic freshwater sponge Lubomirskia baicalensis

In this study, for the first time the diversity of bacteria associated with the endemic freshwater sponge Lubomirskia baicalensis collected from the Sousern Basin of Lake Baikal was investigated employing cultivation-independent approaches. In total, 102 bacterial 16S rRNA clones were screened using restriction fragment length polymorphism (RFLP) and 30 were selected for sequencing. BLASTN and phylogenetic analysis based on near full length 16S rDNA sequences showed that 22 operational taxonomic units (OTUs) were clustered in six known phyla: Actinobacteria (8 OTUs), alpha-Proteobacteria (4 OTUs), beta-Proteobacteria (4 OTUs), Verrucomicrobia (4 OTUs), Nitrospiracea (1 OTU) and Bacteroidetes (1 OTU). Remarkably all phylotypes were affiliated to uncultured microorganisms, however, all alpha-Proteobacteria sequences were closely related to bacteria derived from the freshwater sponge Spongilla lacustris. Our results reveal a high diversity in the L. baicalensis bacterial community and provide an insight into microbial ecology and diversity within freshwater sponges inhabiting the ancient Lake Baikal ecosystem.     

Biodiversity, Community Structural Shifts, and Biogeography of Prokaryotes within Antarctic Continental Shelf Sediment

16S ribosomal DNA (rDNA) clone library analysis was conducted to assess prokaryotic diversity and community structural changes within a surficial sediment core obtained from an Antarctic continental shelf area (depth, 761 m) within the Mertz Glacier Polynya (MGP) region. Libraries were created from three separate horizons of the core (0- to 0.4-cm, 1.5- to 2.5-cm, and 20- to 21-cm depth positions). The results indicated that at the oxic sediment surface (depth, 0 to 0.4 cm) the microbial community appeared to be dominated by a small subset of potentially r-strategist (fast-growing, opportunistic) species, resulting in a lower-than-expected species richness of 442 operational taxonomic units (OTUs). At a depth of 1.5 to 2.5 cm, the species richness (1,128 OTUs) was much higher, with the community dominated by numerous gamma and delta proteobacterial phylotypes. At a depth of 20 to 21 cm, a clear decline in species richness (541 OTUs) occurred, accompanied by a larger number of more phylogenetically divergent phylotypes and a decline in the predominance of Proteobacteria. Based on rRNA and clonal abundance as well as sequence comparisons, syntrophic cycling of oxidized and reduced sulfur compounds appeared to be the dominant process in surficial MGP sediment, as phylotype groups putatively linked to these processes made up a large proportion of clones throughout the core. Between 18 and 65% of 16S rDNA phylotypes detected in a wide range of coastal and open ocean sediments possessed high levels of sequence similarity (>95%) with the MGP sediment phylotypes, indicating that many sediment prokaryote phylotype groups defined in this study are ubiquitous in marine sediment.     

Microbiota dysbiosis in inflammatory bowel diseases: <Emphasis Type="Italic">in silico</Emphasis> investigation of the oxygen hypothesis

Background

Inflammatory bowel diseases (IBD), which include ulcerative colitis and Crohn’s disease, cause chronic inflammation of the digestive tract in approximately 1.6 million Americans. A signature of IBD is dysbiosis of the gut microbiota marked by a significant reduction of obligate anaerobes and a sharp increase in facultative anaerobes. Numerous experimental studies have shown that IBD is strongly correlated with a decrease of Faecalibacterium prausnitzii and an increase of Escherichia coli. One hypothesis is that chronic inflammation induces increased oxygen levels in the gut, which in turn causes an imbalance between obligate and facultative anaerobes.

Results

To computationally investigate the oxygen hypothesis, we developed a multispecies biofilm model based on genome-scale metabolic reconstructions of F. prausnitzii, E. coli and the common gut anaerobe Bacteroides thetaiotaomicron. Application of low bulk oxygen concentrations at the biofilm boundary reproduced experimentally observed behavior characterized by a sharp decrease of F. prausnitzii and a large increase of E. coli, demonstrating that dysbiosis consistent with IBD disease progression could be qualitatively predicted solely based on metabolic differences between the species. A diet with balanced carbohydrate and protein content was predicted to represent a metabolic “sweet spot” that increased the oxygen range over which F. prausnitzii could remain competitive and IBD could be sublimated. Host-microbiota feedback incorporated via a simple linear feedback between the average F. prausnitzii concentration and the bulk oxygen concentration did not substantially change the range of oxygen concentrations where dysbiosis was predicted, but the transition from normal species abundances to severe dysbiosis was much more dramatic and occurred over a much longer timescale. Similar predictions were obtained with sustained antibiotic treatment replacing a sustained oxygen perturbation, demonstrating how IBD might progress over several years with few noticeable effects and then suddenly produce severe disease symptoms.

Conclusions

The multispecies biofilm metabolic model predicted that oxygen concentrations of ～1 micromolar within the gut could cause microbiota dysbiosis consistent with those observed experimentally for inflammatory bowel diseases. Our model predictions could be tested directly through the development of an appropriate in vitro system of the three species community and testing of microbiota-host interactions in gnotobiotic mice.

     

Phylogenetic analysis of 16S rRNA gene sequences reveals distal gut bacterial diversity in wild wolves (Canis lupus)

The aim of this study was to describe the microbial communities in the distal gut of wild wolves (Canis lupus). Fecal samples were collected from three healthy unrelated adult wolves captured at the nearby of Dalai Lake Nature Reserve in Inner Mongolia of China. The diversity of fecal bacteria was investigated by constructing PCR-amplified 16S rRNA gene clone libraries using the universal bacterial primers 27 F and 1493 R. A total of 307 non-chimeric near-full-length 16S rRNA gene sequences were analyzed and 65 non-redundant bacteria phylotypes (operational taxonomical units, OTUs) were identified. Seventeen OTUs (26%) showed less than 98% sequence similarity to 16S rRNA gene sequences were reported previously. Five different bacterial phyla were identified, with the majority of OTUs being classified within the phylum Firmicutes (60%), followed by Bacteroidetes (16.9%), Proteobacteria (9.2%), Fusobacteria (9.2%) and Actinobacteria (4.6%). The majority of clones fell within the order Clostridiales (53.8% of OTUs). It was predominantly affiliated with five families: Lachnospiraceae was the most diverse bacterial family in this order, followed by Ruminococcaceae, Clostridiaceae, Peptococcaceae and Peptostreptococcaceae.     

Fungal disease and temperature alter skin microbiome structure in an experimental salamander system

Pathogens compete with host microbiomes for space and resources. Their shared environment impacts pathogen–microbiome–host interactions, which can lead to variation in disease outcome. The skin microbiome of red‐backed salamanders (Plethodon cinereus) can reduce infection by the pathogen Batrachochytrium dendrobatidis (Bd) at moderate infection loads, with high species richness and high abundance of competitors as putative mechanisms. However, it is unclear if the skin microbiome can reduce epizootic Bd loads across temperatures. We conducted a laboratory experiment to quantify skin microbiome and host responses (P. cinereus: n = 87) to Bd at mimicked epizootic loads across temperatures (13, 17 and 21°C). We quantified skin microbiomes using 16S rRNA gene metabarcoding and identified operational taxonomic units (OTUs) taxonomically similar to culturable bacteria known to kill Bd (anti‐Bd OTUs). Prior to pathogen exposure, temperature changed the microbiome (OTU richness decreased by 12% and the abundance of anti‐Bd OTUs increased by 18% per degree increase in temperature), but these changes were not predictive of disease outcome. After exposure, Bd changed the microbiome (OTU richness decreased by 0.1% and the abundance of anti‐Bd OTUs increased by 0.2% per 1% increase in Bd load) and caused high host mortality across temperatures (35/45: 78%). Temperature indirectly impacted microbiome change and mortality through its direct effect on pathogen load. We did not find support for the microbiome impacting Bd load or host survival. Our research reveals complex host, pathogen, microbiome and environmental interactions to demonstrate that during epizootic events the microbiome will be unlikely to reduce pathogen invasion, even for putatively Bd‐resistant species.     

Among-Population Variation in Microbial Community Structure in the Floral Nectar of the Bee-Pollinated Forest Herb Pulmonaria officinalis L

Background

Microbial communities in floral nectar have been shown to be characterized by low levels of species diversity, yet little is known about among-plant population variation in microbial community composition.

Methodology/Principal Findings

We investigated the microbial community structure (yeasts and bacteria) in floral nectar of ten fragmented populations of the bee-pollinated forest herb Pulmonaria officinalis. We also explored possible relationships between plant population size and microbial diversity in nectar, and related microbial community composition to the distance separating plant populations. Culturable bacteria and yeasts occurring in the floral nectar of a total of 100 plant individuals were isolated and identified by partially sequencing the 16S rRNA gene and D1/D2 domains of the 26S rRNA gene, respectively. A total of 9 and 11 yeast and 28 and 39 bacterial OTUs was found, taking into account a 3% (OTU_0.03) and 1% sequence dissimilarity cut-off (OTU_0.01). OTU richness at the plant population level (i.e. the number of OTUs per population) was low for yeasts (mean: 1.7, range: 0–4 OTUs_0.01/0.03 per population), whereas on average 6.9 (range: 2–13) OTUs_0.03 and 7.9 (range 2–16) OTUs_0.01 per population were found for bacteria. Both for yeasts and bacteria, OTU richness was not significantly related to plant population size. Similarity in community composition among populations was low (average Jaccard index: 0.14), and did not decline with increasing distance between populations.

Conclusions/Significance

We found low similarity in microbial community structure among populations, suggesting that the assembly of nectar microbiota is to a large extent context-dependent. Although the precise factors that affect variation in microbial community structure in floral nectar require further study, our results indicate that both local and regional processes may contribute to among-population variation in microbial community structure in nectar.     

Altered microbiota associated with abnormal humoral immune responses to commensal organisms in enthesitis-related arthritis

Introduction

Prior studies have established altered microbiota and immunologic reactivity to enteric commensal organisms in inflammatory bowel disease (IBD). Since intestinal inflammation is present in a subset of patients with both pediatric and adult spondyloarthritis (SpA), we hypothesized that SpA patients may also have altered microbiota and immune responsiveness to enteric organisms.

Methods

Stool and blood specimens were collected from children with enthesitis-related arthritis (ERA) and non-inflammatory controls. DNA purified from stool was subject to PCR amplification and sequencing of the variable IV region from the 16S rDNA gene. IgA and IgG Enzyme-linked Immunosorbent Assays (ELISAs) were performed on select species of bacteria in most subjects.

Results

Twenty-five children with ERA and 13 controls were included. The ERA patients had less Faecalibacterium prausnitzii (3.8% versus 10%, P = 0.008) and lachnospiraceae family (12 versus 7.0%, P = 0.020), a statistically significant increase in bifidobacterium (1.8% versus 0%, P = 0.032) and a non-statistically significant increase in Bacteroides (21% versus 11%, P = 0.150). Akkermansia muciniphila was abundant (>2%) in 7/27 ERA patients but none of the controls (P = 0.072.) Cluster analysis revealed two clusters of ERA patients: Cluster one (n = 8) was characterized by high levels of Bacteroides genus, while a second (n = 15) cluster had similar levels as the controls. Seven of 17 (41%) of the ERA subjects in Cluster 2 compared to 0/8 of the subjects in Cluster 1 had abundant Akkermansia muciniphila (P = 0.057). Serum IgA and IgG antibody levels against F. prausnitzii and B. fragilis were similar between patients and controls, whereas the two groups showed divergent responses when the fecal relative abundances of F. prausnitzii and Bacteroides were compared individually against IgA antibody levels recognizing F. prausnitzii and B. fragilis, respectively.

Conclusion

The abundance of F. prausnitzii in the stool among patients with ERA is reduced compared to controls, and Bacteroides and A. muciniphila are identified as associative agents in subsets of ERA patients. Differences in the humoral responses to these bacteria may contribute to disease.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0486-0) contains supplementary material, which is available to authorized users.     

Divergence thresholds and divergent biodiversity estimates: can metabarcoding reliably describe zooplankton communities?

DNA metabarcoding is a promising method for describing communities and estimating biodiversity. This approach uses high‐throughput sequencing of targeted markers to identify species in a complex sample. By convention, sequences are clustered at a predefined sequence divergence threshold (often 3%) into operational taxonomic units (OTUs) that serve as a proxy for species. However, variable levels of interspecific marker variation across taxonomic groups make clustering sequences from a phylogenetically diverse dataset into OTUs at a uniform threshold problematic. In this study, we use mock zooplankton communities to evaluate the accuracy of species richness estimates when following conventional protocols to cluster hypervariable sequences of the V4 region of the small subunit ribosomal RNA gene (18S) into OTUs. By including individually tagged single specimens and “populations” of various species in our communities, we examine the impact of intra‐ and interspecific diversity on OTU clustering. Communities consisting of single individuals per species generated a correspondence of 59–84% between OTU number and species richness at a 3% divergence threshold. However, when multiple individuals per species were included, the correspondence between OTU number and species richness dropped to 31–63%. Our results suggest that intraspecific variation in this marker can often exceed 3%, such that a single species does not always correspond to one OTU. We advocate the need to apply group‐specific divergence thresholds when analyzing complex and taxonomically diverse communities, but also encourage the development of additional filtering steps that allow identification of artifactual rRNA gene sequences or pseudogenes that may generate spurious OTUs.     

Biochar,Bentonite and Zeolite Supplemented Feeding of Layer Chickens Alters Intestinal Microbiota and Reduces Campylobacter Load

A range of feed supplements, including antibiotics, have been commonly used in poultry production to improve health and productivity. Alternative methods are needed to suppress pathogen loads and maintain productivity. As an alternative to antibiotics use, we investigated the ability of biochar, bentonite and zeolite as separate 4% feed additives, to selectively remove pathogens without reducing microbial richness and diversity in the gut. Neither biochar, bentonite nor zeolite made any significant alterations to the overall richness and diversity of intestinal bacterial community. However, reduction of some bacterial species, including some potential pathogens was detected. The microbiota of bentonite fed animals were lacking all members of the order Campylobacterales. Specifically, the following operational taxonomic units (OTUs) were absent: an OTU 100% identical to Campylobacter jejuni; an OTU 99% identical to Helicobacter pullorum; multiple Gallibacterium anatis (>97%) related OTUs; Bacteroides dorei (99%) and Clostridium aldenense (95%) related OTUs. Biochar and zeolite treatments had similar but milder effects compared to bentonite. Zeolite amended feed was also associated with significant reduction in the phylum Proteobacteria. All three additives showed potential for the control of major poultry zoonotic pathogens.     

Analysis of the Rumen Bacterial Diversity under two Different Diet Conditions using Denaturing Gradient Gel Electrophoresis,Random Sequencing,and Statistical Ecology Approaches

Bacterial community structure and diversity in the rumen of steers in conditions of hay and corn diets was assessed by in vitro retrieval and analysis of the variable region (V3) of 16S rDNA. Two types of libraries were generated in this study: DGGE libraries, which further were analysed by excising, reamplification, and sequencing, and random shotgun sequence libraries. Phylogenetic and sequence similarity analyses of the resultant 68 clone sequences in DGGE libraries revealed the presence of 42 operational taxonomic units (OTUs) or phylotypes defined as having more than 97% of sequence similarity. One hundred and thirty four clone sequences in shotgun libraries were clustered into 72 phylotypes. The phylotype similarity, diversity, richness, and evenness in these libraries were estimated using a variety of diversity indices. In relation to diet, the corn-fed animals displayed more diverse and rich bacterial populations, which were mostly contributed by CFB-related phylotypes. Proteobacteria were also numerically prevalent on this diet (27%) but were represented by a few phylotypes thus diminishing the overall diversity and species richness values. On hay diet, the principal contributors to general diversity and species richness appeared to be low-G + C gram-positives. Although the ruminal Treponemaes were encountered only in hay-fed animals, their impact on species diversity on hay diet was low because of the limited number of phylotypes.     

Faecalibacterium prausnitzii Strain HTF-F and Its Extracellular Polymeric Matrix Attenuate Clinical Parameters in DSS-Induced Colitis

A decrease in the abundance and biodiversity of intestinal bacteria within the Firmicutes phylum has been associated with inflammatory bowel disease (IBD). In particular, the anti-inflammatory bacterium Faecalibacterium prausnitzii, member of the Firmicutes phylum and one of the most abundant species in healthy human colon, is underrepresented in the microbiota of IBD patients. The aim of this study was to investigate the immunomodulatory properties of F. prausnitzii strain A2-165, the biofilm forming strain HTF-F and the extracellular polymeric matrix (EPM) isolated from strain HTF-F. For this purpose, the immunomodulatory properties of the F. prausnitzii strains and the EPM were studied in vitro using human monocyte-derived dendritic cells. Then, the capacity of the F. prausnitzii strains and the EPM of HTF-F to suppress inflammation was assessed in vivo in the mouse dextran sodium sulphate (DSS) colitis model. The F. prausnitzii strains and the EPM had anti-inflammatory effects on the clinical parameters measured in the DSS model but with different efficacy. The immunomodulatory effects of the EPM were mediated through the TLR2-dependent modulation of IL-12 and IL-10 cytokine production in antigen presenting cells, suggesting that it contributes to the anti-inflammatory potency of F. prausnitzii HTF-F. The results show that F. prausnitzii HTF-F and its EPM may have a therapeutic use in IBD.     

Microbial Contaminants of Cord Blood Units Identified by 16S rRNA Sequencing and by API Test System,and Antibiotic Sensitivity Profiling

Over a period of ten months a total of 5618 cord blood units (CBU) were screened for microbial contamination under routine conditions. The antibiotic resistance profile for all isolates was also examined using ATB strips. The detection rate for culture positive units was 7.5%, corresponding to 422 samples.16S rRNA sequence analysis and identification with API test system were used to identify the culturable aerobic, microaerophilic and anaerobic bacteria from CBUs. From these samples we recovered 485 isolates (84 operational taxonomic units, OTUs) assigned to the classes Bacteroidia, Actinobacteria, Clostridia, Bacilli, Betaproteobacteria and primarily to the Gammaproteobacteria. Sixty-nine OTUs, corresponding to 447 isolates, showed 16S rRNA sequence similarities above 99.0% with known cultured bacteria. However, 14 OTUs had 16S rRNA sequence similarities between 95 and 99% in support of genus level identification and one OTU with 16S rRNA sequence similarity of 90.3% supporting a family level identification only. The phenotypic identification formed 29 OTUs that could be identified to the species level and 9 OTUs that could be identified to the genus level by API test system. We failed to obtain identification for 14 OTUs, while 32 OTUs comprised organisms producing mixed identifications. Forty-two OTUs covered species not included in the API system databases. The API test system Rapid ID 32 Strep and Rapid ID 32 E showed the highest proportion of identifications to the species level, the lowest ratio of unidentified results and the highest agreement to the results of 16S rRNA assignments. Isolates affiliated to the Bacilli and Bacteroidia showed the highest antibiotic multi-resistance indices and microorganisms of the Clostridia displayed the most antibiotic sensitive phenotypes.     

Statistical approaches for estimating actinobacterial diversity in marine sediments

Bacterial diversity in a deep-sea sediment was investigated by constructing actinobacterium-specific 16S ribosomal DNA (rDNA) clone libraries from sediment sections taken 5 to 12, 15 to 18, and 43 to 46 cm below the sea floor at a depth of 3,814 m. Clones were placed into operational taxonomic unit (OTU) groups with >/= 99% 16S rDNA sequence similarity; the cutoff value for an OTU was derived by comparing 16S rRNA homology with DNA-DNA reassociation values for members of the class Actinobacteria. Diversity statistics were used to determine how the level of dominance, species richness, and genetic diversity varied with sediment depth. The reciprocal of Simpson's index (1/D) indicated that the pattern of diversity shifted toward dominance from uniformity with increasing sediment depth. Nonparametric estimation of the species richness in the 5- to 12-, 15- to 18-, and 43- to 46-cm sediment sections revealed a trend of decreasing species number with depth, 1,406, 308, and 212 OTUs, respectively. Application of the LIBSHUFF program indicated that the 5- to 12-cm clone library was composed of OTUs significantly (P = 0.001) different from those of the 15- to 18- and 43- to 46-cm libraries. F(ST) and phylogenetic grouping of taxa (P tests) were both significant (P < 0.00001 and P < 0.001, respectively), indicating that genetic diversity decreased with sediment depth and that each sediment community harbored unique phylogenetic lineages. It was also shown that even nonconservative OTU definitions result in severe underestimation of species richness; unique phylogenetic clades detected in one OTU group suggest that OTUs do not correspond to real ecological groups sensu Palys (T. Palys, L. K. Nakamura, and F. M. Cohan, Int. J. Syst. Bacteriol. 47:1145-1156, 1997). Mechanisms responsible for diversity and their implications are discussed.     

CD4CD8αα Lymphocytes,A Novel Human Regulatory T Cell Subset Induced by Colonic Bacteria and Deficient in Patients with Inflammatory Bowel Disease

How the microbiota affects health and disease is a crucial question. In mice, gut Clostridium bacteria are potent inducers of colonic interleukin (IL)-10-producing Foxp3 regulatory T cells (Treg), which play key roles in the prevention of colitis and in systemic immunity. In humans, although gut microbiota dysbiosis is associated with immune disorders, the underlying mechanism remains unknown. In contrast with mice, the contribution of Foxp3 Treg in colitis prevention has been questioned, suggesting that other compensatory regulatory cells or mechanisms may exist. Here we addressed the regulatory role of the CD4CD8 T cells whose presence had been reported in the intestinal mucosa and blood. Using colonic lamina propria lymphocytes (LPL) and peripheral blood lymphocytes (PBL) from healthy individuals, and those with colon cancer and irritable bowel disease (IBD), we demonstrated that CD4CD8αα (DP8α) T lymphocytes expressed most of the regulatory markers and functions of Foxp3 Treg and secreted IL-10. Strikingly, DP8α LPL and PBL exhibited a highly skewed repertoire toward the recognition of Faecalibacterium prausnitzii, a major Clostridium species of the human gut microbiota, which is decreased in patients with IBD. Furthermore, the frequencies of DP8α PBL and colonic LPL were lower in patients with IBD than in healthy donors and in the healthy mucosa of patients with colon cancer, respectively. Moreover, PBL and LPL from most patients with active IBD failed to respond to F. prausnitzii in contrast to PBL and LPL from patients in remission and/or healthy donors. These data (i) uncover a Clostridium-specific IL-10-secreting Treg subset present in the human colonic LP and blood, (ii) identify F. prausnitzii as a major inducer of these Treg, (iii) argue that these cells contribute to the control or prevention of colitis, opening new diagnostic and therapeutic strategies for IBD, and (iv) provide new tools to address the systemic impact of both these Treg and the intestinal microbiota on the human immune homeostasis.     

Dysbiosis Signature of Fecal Microbiota in Colorectal Cancer Patients

The human gut microbiota is a complex system that is essential to the health of the host. Increasing evidence suggests that the gut microbiota may play an important role in the pathogenesis of colorectal cancer (CRC). In this study, we used pyrosequencing of the 16S rRNA gene V3 region to characterize the fecal microbiota of 19 patients with CRC and 20 healthy control subjects. The results revealed striking differences in fecal microbial population patterns between these two groups. Partial least-squares discriminant analysis showed that 17 phylotypes closely related to Bacteroides were enriched in the gut microbiota of CRC patients, whereas nine operational taxonomic units, represented by the butyrate-producing genera Faecalibacterium and Roseburia, were significantly less abundant. A positive correlation was observed between the abundance of Bacteroides species and CRC disease status (R?=?0.462, P?=?0.046?<?0.5). In addition, 16 genera were significantly more abundant in CRC samples than in controls, including potentially pathogenic Fusobacterium and Campylobacter species at genus level. The dysbiosis of fecal microbiota, characterized by the enrichment of potential pathogens and the decrease in butyrate-producing members, may therefore represent a specific microbial signature of CRC. A greater understanding of the dynamics of the fecal microbiota may assist in the development of novel fecal microbiome-related diagnostic tools for CRC.     

Next Generation Sequencing to Define Prokaryotic and Fungal Diversity in the Bovine Rumen

A combination of Sanger and 454 sequences of small subunit rRNA loci were used to interrogate microbial diversity in the bovine rumen of 12 cows consuming a forage diet. Observed bacterial species richness, based on the V1–V3 region of the 16S rRNA gene, was between 1,903 to 2,432 species-level operational taxonomic units (OTUs) when 5,520 reads were sampled per animal. Eighty percent of species-level OTUs were dominated by members of the order Clostridiales, Bacteroidales, Erysipelotrichales and unclassified TM7. Abundance of Prevotella species varied widely among the 12 animals. Archaeal species richness, also based on 16S rRNA, was between 8 and 13 OTUs, representing 5 genera. The majority of archaeal OTUs (84%) found in this study were previously observed in public databases with only two new OTUs discovered. Observed rumen fungal species richness, based on the 18S rRNA gene, was between 21 and 40 OTUs with 98.4–99.9% of OTUs represented by more than one read, using Good’s coverage. Examination of the fungal community identified numerous novel groups. Prevotella and Tannerella were overrepresented in the liquid fraction of the rumen while Butyrivibrio and Blautia were significantly overrepresented in the solid fraction of the rumen. No statistical difference was observed between the liquid and solid fractions in biodiversity of archaea and fungi. The survey of microbial communities and analysis of cross-domain correlations suggested there is a far greater extent of microbial diversity in the bovine rumen than previously appreciated, and that next generation sequencing technologies promise to reveal novel species, interactions and pathways that can be studied further in order to better understand how rumen microbial community structure and function affects ruminant feed efficiency, biofuel production, and environmental impact.     

The 16S rRNA and mcrA gene based comparative diversity of methanogens in cattle fed on high fibre based diet

In the present study, the diversity of rumen methanogens in crossbred Karan Fries cattle was determined by constructing 16S rRNA and mcrA (methyl coenzyme-M reductase α subunit) gene libraries using specific primers. All thirteen OTUs or phylotypes from 16S rRNA library clustered with order Methanobacteriales, twelve of which aligned with Methanobrevibacter spp., whereas one OTU resemble with Methanosphaera stadtmanae. Out of eighteen OTUs identified from mcrA gene library, fifteen clustered with order Methanobacteriales, two resemble with Methanomicrobiales and remaining one grouped with Methanosarcinales. These results revealed that Methanobrevibacter phylotype was predominantly present in Karan Fries crossbred cattle fed on high fibrous diet containing wheat straw. Compared to 16S rRNA gene, mcrA gene OTUs clustered in three orders providing better insights of rumen methanogens diversity in cattle.