Floor Hugenholtz

The Mouse Intestinal Bacterial Collection (miBC) provides host-specific insight into cultured diversity and functional potential of the gut microbiota

Intestinal bacteria influence mammalian physiology, but many types of bacteria are still uncharacterized. Moreover, reference strains of mouse gut bacteria are not easily available, although mouse models are extensively used in medical research. These are major limitations for the investigation of intestinal microbiomes and their interactions with diet and host. It is thus important to study in detail the diversity and functions of gut microbiota members, including those colonizing the mouse intestine. To address these issues, we aimed at establishing the Mouse Intestinal Bacterial Collection (miBC), a public repository of bacterial strains and associated genomes from the mouse gut, and studied host-specificity of colonization and sequence-based relevance of the resource. The collection includes several strains representing novel species, genera and even one family. Genomic analyses showed that certain species are specific to the mouse intestine and that a minimal consortium of 18 strains covered 50-75% of the known functional potential of metagenomes. The present work will sustain future research on microbiota-host interactions in health and disease, as it will facilitate targeted colonization and molecular studies. The resource is available at www.dsmz.de/miBC.Intestinal bacteria influence mammalian physiology, but many types of bacteria are still uncharacterized. Moreover, reference strains of mouse gut bacteria are not easily available, although mouse models are extensively used in medical research. These are major limitations for the investigation of intestinal microbiomes and their interactions with diet and host. It is thus important to study in detail the diversity and functions of gut microbiota members, including those colonizing the mouse intestine. To address these issues, we aimed at establishing the Mouse Intestinal Bacterial Collection (miBC), a public repository of bacterial strains and associated genomes from the mouse gut, and studied host-specificity of colonization and sequence-based relevance of the resource. The collection includes several strains representing novel species, genera and even one family. Genomic analyses showed that certain species are specific to the mouse intestine and that a minimal consortium of 18 strains covered 50–75% of the known functional potential of metagenomes. The present work will sustain future research on microbiota–host interactions in health and disease, as it will facilitate targeted colonization and molecular studies. The resource is available at www.dsmz.de/miBC.

Metaproteome Analysis and Molecular Genetics of Rat Intestinal Microbiota Reveals Section and Localization Resolved Species Distribution and Enzymatic Functionalities

The digestion of food ingredients depends on the action of the gut microbiota and has a significant influence on the health, especially in the case of metabolic diseases, of the host organism. Despite the relevance of the structure and functionalities in the microbiota for the metabolism of the host, the spatial resolution of microbial consortia and the functionalities in the different gut sections of the rat are mostly unknown. Since there are suitable rat models for human metabolic diseases, the microbiota of the rat is of special interest. Samples along the intestinal tract of rats were investigated using metaproteomics and 16S rRNA gene pyrosequencing. The procedures for harvesting bacteria from the mucus and the content of the gut sections and feces were optimized leading to 2802 nonredundant bacterial protein groups in total that were assigned to spectra measured by liquid chromatography-tandem mass spectrometry. The majority of 16S rRNA genes and protein groups belonged to members of Firmicutes, Bacteroidetes and Proteobacteria. The functionalities in the enzyme repertoire were compared between the mucus and the content of the large intestine sections and the feces samples. This spatial resolution allowed pinpointing changes in the community to specific metabolic capacities like carbohydrate transport and energy conservation. The results showed that the mere analysis of feces samples reflects the functions of the gut microbiota only to a minor extent and sheds light on the metabolic interchange between the microbiota and the host organism.

IL-22-STAT3 Pathway Plays a Key Role in the Maintenance of Ileal Homeostasis in Mice Lacking Secreted Mucus Barrier

Background:Muc2-deficient mice show no signs of ileal pathology but the mechanisms remained unknown. Methods:Wild-type (WT), Muc2+/−, and Muc2−/− mice were killed at 2, 4, and 8 weeks of age. Total RNA from ileum was used for full genome transcriptome analysis and qPCR. Microbiota composition was determined using a mouse intestinal chip (MITChip). Morphological and immunohistological studies were performed on segments of ileum. Results:The ileum was colonized by more diverse microbiota in young (week 4) WT than in Muc2−/− mice, and composition was influenced by genotype. Weaning was associated with major changes in the transcriptome of all mice, and the highest number of differentially expressed genes compared with adults, reflecting temporal changes in microbiota. Although the spatial compartmentalization of bacteria was compromised in Muc2−/− mice, gene set enrichment analysis revealed a downregulation of Toll-like receptor, immune, and chemokine signaling pathways compared to WT mice. The predicted effects of enhanced IL-22 signaling were identified in the Muc2−/− transcriptome as the upregulation of epithelial cell proliferation altered expression of mitosis and cell-cycle control pathways. This is consistent with increased villus length and number of Ki67+ epithelial cells in Muc2−/− mice. Additionally, expression of the network of IL-22 regulated defense genes, including Fut2, Reg3&bgr;, Reg3&ggr;, Relmb, and the Defensin Defb46 were increased in Muc2−/− mice. Conclusions:These findings highlight a role for the IL-22-STAT3 pathway in maintaining ileal homeostasis when the mucus barrier is compromised and its potential as a target for novel therapeutic strategies in inflammatory bowel disease.

Intestinal microbiome landscaping : Insight in community assemblage and implications for microbial modulation strategies

Abstract High individuality, large complexity and limited understanding of the mechanisms underlying human intestinal microbiome function remain the major challenges for designing beneficial modulation strategies. Exemplified by the analysis of intestinal bacteria in a thousand Western adults, we discuss key concepts of the human intestinal microbiome landscape, i.e. the compositional and functional ‘core’, the presence of community types and the existence of alternative stable states. Genomic investigation of core taxa revealed functional redundancy, which is expected to stabilize the ecosystem, as well as taxa with specialized functions that have the potential to shape the microbiome landscape. The contrast between Prevotella- and Bacteroides-dominated systems has been well described. However, less known is the effect of not so abundant bacteria, for example, Dialister spp. that have been proposed to exhibit distinct bistable dynamics. Studies employing time-series analysis have highlighted the dynamical variation in the microbiome landscape with and without the effect of defined perturbations, such as the use of antibiotics or dietary changes. We incorporate ecosystem-level observations of the human intestinal microbiota and its keystone species to suggest avenues for designing microbiome modulation strategies to improve host health.

A Multi-Platform Flow Device for Microbial (Co-) Cultivation and Microscopic Analysis

Novel microbial cultivation platforms are of increasing interest to researchers in academia and industry. The development of materials with specialized chemical and geometric properties has opened up new possibilities in the study of previously unculturable microorganisms and has facilitated the design of elegant, high-throughput experimental set-ups. Within the context of the international Genetically Engineered Machine (iGEM) competition, we set out to design, manufacture, and implement a flow device that can accommodate multiple growth platforms, that is, a silicon nitride based microsieve and a porous aluminium oxide based microdish. It provides control over (co-)culturing conditions similar to a chemostat, while allowing organisms to be observed microscopically. The device was designed to be affordable, reusable, and above all, versatile. To test its functionality and general utility, we performed multiple experiments with Escherichia coli cells harboring synthetic gene circuits and were able to quantitatively study emerging expression dynamics in real-time via fluorescence microscopy. Furthermore, we demonstrated that the device provides a unique environment for the cultivation of nematodes, suggesting that the device could also prove useful in microscopy studies of multicellular microorganisms.

Mining microbial metatranscriptomes for expression of antibiotic resistance genes under natural conditions

Antibiotic resistance genes are found in a broad range of ecological niches associated with complex microbiota. Here we investigated if resistance genes are not only present, but also transcribed under natural conditions. Furthermore, we examined the potential for antibiotic production by assessing the expression of associated secondary metabolite biosynthesis gene clusters. Metatranscriptome datasets from intestinal microbiota of four human adults, one human infant, 15 mice and six pigs, of which only the latter have received antibiotics prior to the study, as well as from sea bacterioplankton, a marine sponge, forest soil and sub-seafloor sediment, were investigated. We found that resistance genes are expressed in all studied ecological niches, albeit with niche-specific differences in relative expression levels and diversity of transcripts. For example, in mice and human infant microbiota predominantly tetracycline resistance genes were expressed while in human adult microbiota the spectrum of expressed genes was more diverse, and also included β-lactam, aminoglycoside and macrolide resistance genes. Resistance gene expression could result from the presence of natural antibiotics in the environment, although we could not link it to expression of corresponding secondary metabolites biosynthesis clusters. Alternatively, resistance gene expression could be constitutive, or these genes serve alternative roles besides antibiotic resistance.

The Impact of Gut Microbiota on Gender-Specific Differences in Immunity

Males and females are known to have gender-specific differences in their immune system and gut microbiota composition. Whether these differences in gut microbiota composition are a cause or consequence of differences in the immune system is not known. To investigate this issue, gut microbiota from conventional males or females was transferred to germ-free (GF) animals of the same or opposing gender. We demonstrate that microbiota-independent gender differences in immunity are already present in GF mice. In particular, type I interferon signaling was enhanced in the intestine of GF females. Presumably, due to these immune differences bacterial groups, such as Alistipes, Rikenella, and Porphyromonadaceae, known to expand in the absence of innate immune defense mechanism were overrepresented in the male microbiota. The presence of these bacterial groups was associated with induction of weight loss, inflammation, and DNA damage upon transfer of the male microbiota to female GF recipients. In summary, our data suggest that microbiota-independent gender differences in the immune system select a gender-specific gut microbiota composition, which in turn further contributes to gender differences in the immune system.

High-fat feeding rather than obesity drives taxonomical and functional changes in the gut microbiota in mice

BackgroundIt is well known that the microbiota of high-fat (HF) diet-induced obese mice differs from that of lean mice, but to what extent, this difference reflects the obese state or the diet is unclear. To dissociate changes in the gut microbiota associated with high HF feeding from those associated with obesity, we took advantage of the different susceptibility of C57BL/6JBomTac (BL6) and 129S6/SvEvTac (Sv129) mice to diet-induced obesity and of their different responses to inhibition of cyclooxygenase (COX) activity, where inhibition of COX activity in BL6 mice prevents HF diet-induced obesity, but in Sv129 mice accentuates obesity.ResultsUsing HiSeq-based whole genome sequencing, we identified taxonomic and functional differences in the gut microbiota of the two mouse strains fed regular low-fat or HF diets with or without supplementation with the COX-inhibitor, indomethacin. HF feeding rather than obesity development led to distinct changes in the gut microbiota. We observed a robust increase in alpha diversity, gene count, abundance of genera known to be butyrate producers, and abundance of genes involved in butyrate production in Sv129 mice compared to BL6 mice fed either a LF or a HF diet. Conversely, the abundance of genes involved in propionate metabolism, associated with increased energy harvest, was higher in BL6 mice than Sv129 mice.ConclusionsThe changes in the composition of the gut microbiota were predominantly driven by high-fat feeding rather than reflecting the obese state of the mice. Differences in the abundance of butyrate and propionate producing bacteria in the gut may at least in part contribute to the observed differences in obesity propensity in Sv129 and BL6 mice.

Comparison of the effects of five dietary fibers on mucosal transcriptional profiles, and luminal microbiota composition and SCFA concentrations in murine colon

SCOPE The aim of our study was to investigate and compare the effects of five fibers on the mucosal transcriptome, together with alterations in the luminal microbiota composition and SCFA concentrations in the colon. METHODS AND RESULTS Mice were fed fibers that differed in carbohydrate composition or a control diet for 10 days. Colonic gene expression profiles and luminal microbiota composition were determined by microarray techniques, and integrated using multivariate statistics. Our data showed a distinct reaction of the host and microbiota to resistant starch, a fiber that was not completely fermented in the colon, whereas the other fibers induced similar responses on gene expression and microbiota. Consistent associations were revealed between fiber-induced enrichment of Clostridium cluster IV and XIVa representatives, and changes in mucosal expression of genes related to energy metabolism. The nuclear receptor PPAR-γ was predicted to be an important regulator of the mucosal responses. CONCLUSION Results of this exploratory study suggest that despite different sources and composition, fermentable fibers induce a highly similar mucosal response that may at least be partially governed by PPAR-γ.

Effect of a long-term high-protein diet on survival, obesity development, and gut microbiota in mice

Female C57BL/6J mice were fed a regular low-fat diet or high-fat diets combined with either high or low protein-to-sucrose ratios during their entire lifespan to examine the long-term effects on obesity development, gut microbiota, and survival. Intake of a high-fat diet with a low protein/sucrose ratio precipitated obesity and reduced survival relative to mice fed a low-fat diet. By contrast, intake of a high-fat diet with a high protein/sucrose ratio attenuated lifelong weight gain and adipose tissue expansion, and survival was not significantly altered relative to low-fat-fed mice. Our findings support the notion that reduced survival in response to high-fat/high-sucrose feeding is linked to obesity development. Digital gene expression analyses, further validated by qPCR, demonstrated that the protein/sucrose ratio modulated global gene expression over time in liver and adipose tissue, affecting pathways related to metabolism and inflammation. Analysis of fecal bacterial DNA using the Mouse Intestinal Tract Chip revealed significant changes in the composition of the gut microbiota in relation to host age and dietary fat content, but not the protein/sucrose ratio. Accordingly, dietary fat rather than the protein/sucrose ratio or adiposity is a major driver shaping the gut microbiota, whereas the effect of a high-fat diet on survival is dependent on the protein/sucrose ratio.