Bartholomeus van den Bogert

The human small intestinal microbiota is driven by rapid uptake and conversion of simple carbohydrates

The human gastrointestinal tract (GI tract) harbors a complex community of microbes. The microbiota composition varies between different locations in the GI tract, but most studies focus on the fecal microbiota, and that inhabiting the colonic mucosa. Consequently, little is known about the microbiota at other parts of the GI tract, which is especially true for the small intestine because of its limited accessibility. Here we deduce an ecological model of the microbiota composition and function in the small intestine, using complementing culture-independent approaches. Phylogenetic microarray analyses demonstrated that microbiota compositions that are typically found in effluent samples from ileostomists (subjects without a colon) can also be encountered in the small intestine of healthy individuals. Phylogenetic mapping of small intestinal metagenome of three different ileostomy effluent samples from a single individual indicated that Streptococcus sp., Escherichia coli, Clostridium sp. and high G+C organisms are most abundant in the small intestine. The compositions of these populations fluctuated in time and correlated to the short-chain fatty acids profiles that were determined in parallel. Comparative functional analysis with fecal metagenomes identified functions that are overrepresented in the small intestine, including simple carbohydrate transport phosphotransferase systems (PTS), central metabolism and biotin production. Moreover, metatranscriptome analysis supported high level in-situ expression of PTS and carbohydrate metabolic genes, especially those belonging to Streptococcus sp. Overall, our findings suggest that rapid uptake and fermentation of available carbohydrates contribute to maintaining the microbiota in the human small intestine.

Microarray Analysis and Barcoded Pyrosequencing Provide Consistent Microbial Profiles Depending on the Source of Human Intestinal Samples

ABSTRACT Large-scale and in-depth characterization of the intestinal microbiota necessitates application of high-throughput 16S rRNA gene-based technologies, such as barcoded pyrosequencing and phylogenetic microarray analysis. In this study, the two techniques were compared and contrasted for analysis of the bacterial composition in three fecal and three small intestinal samples from human individuals. As PCR remains a crucial step in sample preparation for both techniques, different forward primers were used for amplification to assess their impact on microbial profiling results. An average of 7,944 pyrosequences, spanning the V1 and V2 region of 16S rRNA genes, was obtained per sample. Although primer choice in barcoded pyrosequencing did not affect species richness and diversity estimates, detection of Actinobacteria strongly depended on the selected primer. Microbial profiles obtained by pyrosequencing and phylogenetic microarray analysis (HITChip) correlated strongly for fecal and ileal lumen samples but were less concordant for ileostomy effluent. Quantitative PCR was employed to investigate the deviations in profiling between pyrosequencing and HITChip analysis. Since cloning and sequencing of random 16S rRNA genes from ileostomy effluent confirmed the presence of novel intestinal phylotypes detected by pyrosequencing, especially those belonging to the Veillonella group, the divergence between pyrosequencing and the HITChip is likely due to the relatively low number of available 16S rRNA gene sequences of small intestinal origin in the DNA databases that were used for HITChip probe design. Overall, this study demonstrated that equivalent biological conclusions are obtained by high-throughput profiling of microbial communities, independent of technology or primer choice.

A comprehensive metatranscriptome analysis pipeline and its validation using human small intestine microbiota datasets

BackgroundNext generation sequencing (NGS) technologies can be applied in complex microbial ecosystems for metatranscriptome analysis by employing direct cDNA sequencing, which is known as RNA sequencing (RNA-seq). RNA-seq generates large datasets of great complexity, the comprehensive interpretation of which requires a reliable bioinformatic pipeline. In this study, we focus on the development of such a metatranscriptome pipeline, which we validate using Illumina RNA-seq datasets derived from the small intestine microbiota of two individuals with an ileostomy.ResultsThe metatranscriptome pipeline developed here enabled effective removal of rRNA derived sequences, followed by confident assignment of the predicted function and taxonomic origin of the mRNA reads. Phylogenetic analysis of the small intestine metatranscriptome datasets revealed a strong similarity with the community composition profiles obtained from 16S rDNA and rRNA pyrosequencing, indicating considerable congruency between community composition (rDNA), and the taxonomic distribution of overall (rRNA) and specific (mRNA) activity among its microbial members. Reproducibility of the metatranscriptome sequencing approach was established by independent duplicate experiments. In addition, comparison of metatranscriptome analysis employing single- or paired-end sequencing methods indicated that the latter approach does not provide improved functional or phylogenetic insights. Metatranscriptome functional-mapping allowed the analysis of global, and genus specific activity of the microbiota, and illustrated the potential of these approaches to unravel syntrophic interactions in microbial ecosystems.ConclusionsA reliable pipeline for metatransciptome data analysis was developed and evaluated using RNA-seq datasets obtained for the human small intestine microbiota. The set-up of the pipeline is very generic and can be applied for (bacterial) metatranscriptome analysis in any chosen niche.

Diversity of human small intestinal Streptococcus and Veillonella populations

Molecular and cultivation approaches were employed to study the phylogenetic richness and temporal dynamics of Streptococcus and Veillonella populations in the small intestine. Microbial profiling of human small intestinal samples collected from four ileostomy subjects at four time points displayed abundant populations of Streptococcus spp. most affiliated with S. salivarius, S. thermophilus, and S. parasanguinis, as well as Veillonella spp. affiliated with V. atypica, V. parvula, V. dispar, and V. rogosae. Relative abundances varied per subject and time of sampling. Streptococcus and Veillonella isolates were cultured using selective media from ileostoma effluent samples collected at two time points from a single subject. The richness of the Streptococcus and Veillonella isolates was assessed at species and strain level by 16S rRNA gene sequencing and genetic fingerprinting, respectively. A total of 160 Streptococcus and 37 Veillonella isolates were obtained. Genetic fingerprinting differentiated seven Streptococcus lineages from ileostoma effluent, illustrating the strain richness within this ecosystem. The Veillonella isolates were represented by a single phylotype. Our study demonstrated that the small intestinal Streptococcus populations displayed considerable changes over time at the genetic lineage level because only representative strains of a single Streptococcus lineage could be cultivated from ileostoma effluent at both time points.

The small intestine microbiota, nutritional modulation and relevance for health.

The intestinal microbiota plays a profound role in human health and extensive research has been dedicated to identify microbiota aberrations that are associated with disease. Most of this work has been targeting the large intestine and fecal microbiota, while the small intestine microbiota may also have a profound impact on various aspects of the hosts physiology, including immune, metabolic and endocrine functions. This review highlights the recent advances made in the study of the human small intestine microbiota. In addition, it describes recent human and animal studies that underpin the importance of this part of the intestine for health of the host organism.

Dietary Heme Alters Microbiota and Mucosa of Mouse Colon without Functional Changes in Host-Microbe Cross-Talk

Colon cancer is a major cause of cancer deaths in Western countries and is associated with diets high in red meat. Heme, the iron-porphyrin pigment of red meat, induces cytotoxicity of gut contents which injures surface cells leading to compensatory hyperproliferation of crypt cells. This hyperproliferation results in epithelial hyperplasia which increases the risk of colon cancer. In humans, a high red-meat diet increases Bacteroides spp in feces. Therefore, we simultaneously investigated the effects of dietary heme on colonic microbiota and on the host mucosa of mice. Whole genome microarrays showed that heme injured the colonic surface epithelium and induced hyperproliferation by changing the surface to crypt signaling. Using 16S rRNA phylogenetic microarrays, we investigated whether bacteria play a role in this changed signaling. Heme increased Bacteroidetes and decreased Firmicutes in colonic contents. This shift was most likely caused by a selective susceptibility of Gram-positive bacteria to heme cytotoxic fecal water, which is not observed for Gram-negative bacteria, allowing expansion of the Gram-negative community. The increased amount of Gram-negative bacteria most probably increased LPS exposure to colonocytes, however, there is no appreciable immune response detected in the heme-fed mice. There was no functional change in the sensing of the bacteria by the mucosa, as changes in inflammation pathways and Toll- like receptor signaling were not detected. This unaltered host-microbe cross-talk indicates that the changes in microbiota did not play a causal role in the observed hyperproliferation and hyperplasia.

Comparative Genomics Analysis of Streptococcus Isolates from the Human Small Intestine Reveals their Adaptation to a Highly Dynamic Ecosystem

The human small-intestinal microbiota is characterised by relatively large and dynamic Streptococcus populations. In this study, genome sequences of small-intestinal streptococci from S. mitis, S. bovis, and S. salivarius species-groups were determined and compared with those from 58 Streptococcus strains in public databases. The Streptococcus pangenome consists of 12,403 orthologous groups of which 574 are shared among all sequenced streptococci and are defined as the Streptococcus core genome. Genome mining of the small-intestinal streptococci focused on functions playing an important role in the interaction of these streptococci in the small-intestinal ecosystem, including natural competence and nutrient-transport and metabolism. Analysis of the small-intestinal Streptococcus genomes predicts a high capacity to synthesize amino acids and various vitamins as well as substantial divergence in their carbohydrate transport and metabolic capacities, which is in agreement with observed physiological differences between these Streptococcus strains. Gene-specific PCR-strategies enabled evaluation of conservation of Streptococcus populations in intestinal samples from different human individuals, revealing that the S. salivarius strains were frequently detected in the small-intestine microbiota, supporting the representative value of the genomes provided in this study. Finally, the Streptococcus genomes allow prediction of the effect of dietary substances on Streptococcus population dynamics in the human small-intestine.

Effects of pectin supplementation on the fermentation patterns of different structural carbohydrates in rats

SCOPE We aimed to investigate and compare the effects of four types of pectins on dietary fiber (DF) fermentation, microbiota composition, and short chain fatty acid (SCFA) production throughout the large intestine in rats. METHODS AND RESULTS Male Wistar rats were given diets supplemented with or without 3% structurally different pectins for 7 weeks. Different fermentation patterns of pectins and different location of fermentation of pectin and diet arabinoxylans (AXs) in the large intestine were observed. During cecal fermentation, sugar beet pectin significantly stimulated Lactobacillus (p < 0.01) and Lachnospiraceae (p < 0.05). The stimulating effects of sugar beet pectin on these two groups of microbes are stronger than both other pectins. In the cecum, low-methyl esterified citrus pectin and complex soy pectin increased (p < 0.05) the production of total SCFAs, propionate and butyrate, whereas high-methyl esterified pectin and sugar beet pectin did not. The fermentation patterns of cereal AXs in the cecum were significantly different upon supplementation of different pectins. These differences, however, became smaller in the colon due to an enhanced fermentation of the remaining DFs. CONCLUSION Dietary supplementation of pectin is a potential strategy to modulate the location of fermentation of DFs, and consequently microbiota composition and SCFA production for health-promoting effects.

Immunomodulatory properties of Streptococcus and Veillonella isolates from the human small intestine microbiota.

The human small intestine is a key site for interactions between the intestinal microbiota and the mucosal immune system. Here we investigated the immunomodulatory properties of representative species of commonly dominant small-intestinal microbial communities, including six streptococcal strains (four Streptococcus salivarius, one S. equinus, one S. parasanguinis) one Veillonella parvula strain, one Enterococcus gallinarum strain, and Lactobacillus plantarum WCFS1 as a bench mark strain on human monocyte-derived dendritic cells. The different streptococci induced varying levels of the cytokines IL-8, TNF-α, and IL-12p70, while the V. parvula strain showed a strong capacity to induce IL-6. E. gallinarum strain was a potent inducer of cytokines and TLR2/6 signalling. As Streptococcus and Veillonella can potentially interact metabolically and frequently co-occur in ecosystems, immunomodulation by pair-wise combinations of strains were also tested for their combined immunomodulatory properties. Strain combinations induced cytokine responses in dendritic cells that differed from what might be expected on the basis of the results obtained with the individual strains. A combination of (some) streptococci with Veillonella appeared to negate IL-12p70 production, while augmenting IL-8, IL-6, IL-10, and TNF-α responses. This suggests that immunomodulation data obtained in vitro with individual strains are unlikely to adequately represent immune responses to mixtures of gut microbiota communities in vivo. Nevertheless, analysing the immune responses of strains representing the dominant species in the intestine may help to identify immunomodulatory mechanisms that influence immune homeostasis.

T lymphocytes control microbial composition by regulating the abundance of Vibrio in the zebrafish gut

Dysbiosis of the intestinal microbial community is considered a risk factor for development of chronic intestinal inflammation as well as other diseases such as diabetes, obesity and even cancer. Study of the innate and adaptive immune pathways controlling bacterial colonization has however proven difficult in rodents, considering the extensive cross-talk between bacteria and innate and adaptive immunity. Here, we used the zebrafish to study innate and adaptive immune processes controlling the microbial community. Zebrafish lack a functional adaptive immune system in the first weeks of life, enabling study of the innate immune system in the absence of adaptive immunity. We show that in wild type zebrafish, the initial lack of adaptive immunity associates with overgrowth of Vibrio species (a group encompassing fish and human pathogens), which is overcome upon adaptive immune development. In Rag1-deficient zebrafish (lacking adaptive immunity) Vibrio abundance remains high, suggesting that adaptive immune processes indeed control Vibrio species. Using cell transfer experiments, we confirm that adoptive transfer of T lymphocytes, but not B lymphocytes into Rag1-deficient recipients suppresses outgrowth of Vibrio. In addition, ex vivo exposure of intestinal T lymphocytes to Rag1-deficient microbiota results in increased interferon-gamma expression by these T lymphocytes, compared to exposure to wild type microbiota. In conclusion, we show that T lymphocytes control microbial composition by effectively suppressing the outgrowth of Vibrio species in the zebrafish intestine.