Inés Martínez

Resistant Starches Types 2 and 4 Have Differential Effects on the Composition of the Fecal Microbiota in Human Subjects

Background To systematically develop dietary strategies based on resistant starch (RS) that modulate the human gut microbiome, detailed in vivo studies that evaluate the effects of different forms of RS on the community structure and population dynamics of the gut microbiota are necessary. The aim of the present study was to gain a community wide perspective of the effects of RS types 2 (RS2) and 4 (RS4) on the fecal microbiota in human individuals. Methods and Findings Ten human subjects consumed crackers for three weeks each containing either RS2, RS4, or native starch in a double-blind, crossover design. Multiplex sequencing of 16S rRNA tags revealed that both types of RS induced several significant compositional alterations in the fecal microbial populations, with differential effects on community structure. RS4 but not RS2 induced phylum-level changes, significantly increasing Actinobacteria and Bacteroidetes while decreasing Firmicutes. At the species level, the changes evoked by RS4 were increases in Bifidobacterium adolescentis and Parabacteroides distasonis, while RS2 significantly raised the proportions of Ruminococcus bromii and Eubacterium rectale when compared to RS4. The population shifts caused by RS4 were numerically substantial for several taxa, leading for example, to a ten-fold increase in bifidobacteria in three of the subjects, enriching them to 18–30% of the fecal microbial community. The responses to RS and their magnitudes varied between individuals, and they were reversible and tightly associated with the consumption of RS. Conclusion Our results demonstrate that RS2 and RS4 show functional differences in their effect on human fecal microbiota composition, indicating that the chemical structure of RS determines its accessibility by groups of colonic bacteria. The findings imply that specific bacterial populations could be selectively targeted by well designed functional carbohydrates, but the inter-subject variations in the response to RS indicates that such strategies might benefit from more personalized approaches.

Gut microbiome composition is linked to whole grain-induced immunological improvements.

The involvement of the gut microbiota in metabolic disorders, and the ability of whole grains to affect both host metabolism and gut microbial ecology, suggest that some benefits of whole grains are mediated through their effects on the gut microbiome. Nutritional studies that assess the effect of whole grains on both the gut microbiome and human physiology are needed. We conducted a randomized cross-over trial with four-week treatments in which 28 healthy humans consumed a daily dose of 60 g of whole-grain barley (WGB), brown rice (BR), or an equal mixture of the two (BR+WGB), and characterized their impact on fecal microbial ecology and blood markers of inflammation, glucose and lipid metabolism. All treatments increased microbial diversity, the Firmicutes/Bacteroidetes ratio, and the abundance of the genus Blautia in fecal samples. The inclusion of WGB enriched the genera Roseburia, Bifidobacterium and Dialister, and the species Eubacterium rectale, Roseburia faecis and Roseburia intestinalis. Whole grains, and especially the BR+WGB treatment, reduced plasma interleukin-6 (IL-6) and peak postprandial glucose. Shifts in the abundance of Eubacterium rectale were associated with changes in the glucose and insulin postprandial response. Interestingly, subjects with greater improvements in IL-6 levels harbored significantly higher proportions of Dialister and lower abundance of Coriobacteriaceae. In conclusion, this study revealed that a short-term intake of whole grains induced compositional alterations of the gut microbiota that coincided with improvements in host physiological measures related to metabolic dysfunctions in humans.

Barcoded Pyrosequencing Reveals That Consumption of Galactooligosaccharides Results in a Highly Specific Bifidogenic Response in Humans

Prebiotics are selectively fermented ingredients that allow specific changes in the gastrointestinal microbiota that confer health benefits to the host. However, the effects of prebiotics on the human gut microbiota are incomplete as most studies have relied on methods that fail to cover the breadth of the bacterial community. The goal of this research was to use high throughput multiplex community sequencing of 16S rDNA tags to gain a community wide perspective of the impact of prebiotic galactooligosaccharide (GOS) on the fecal microbiota of healthy human subjects. Fecal samples from eighteen healthy adults were previously obtained during a feeding trial in which each subject consumed a GOS-containing product for twelve weeks, with four increasing dosages (0, 2.5, 5, and 10 gram) of GOS. Multiplex sequencing of the 16S rDNA tags revealed that GOS induced significant compositional alterations in the fecal microbiota, principally by increasing the abundance of organisms within the Actinobacteria. Specifically, several distinct lineages of Bifidobacterium were enriched. Consumption of GOS led to five- to ten-fold increases in bifidobacteria in half of the subjects. Increases in Firmicutes were also observed, however, these changes were detectable in only a few individuals. The enrichment of bifidobacteria was generally at the expense of one group of bacteria, the Bacteroides. The responses to GOS and the magnitude of the response varied between individuals, were reversible, and were in accordance with dosage. The bifidobacteria were the only bacteria that were consistently and significantly enriched by GOS, although this substrate supported the growth of diverse colonic bacteria in mono-culture experiments. These results suggest that GOS can be used to enrich bifidobacteria in the human gastrointestinal tract with remarkable specificity, and that the bifidogenic properties of GOS that occur in vivo are caused by selective fermentation as well as by competitive interactions within the intestinal environment.

Depletion of luminal iron alters the gut microbiota and prevents Crohn's disease-like ileitis

Background Iron replacement therapy is a common treatment in patients with anaemia and Crohns disease, but oral iron supplements are less tolerated. The pathogenesis of Crohns disease is attributed to intestinal bacteria and environmental factors that trigger disease in a genetically predisposed host. The aim of this study was to characterise the interrelationship between luminal iron sulfate, systemic iron, the gut microbiota and the development of chronic ileitis in a murine model of Crohns disease. Methods Wild type (WT) and heterozygous TNFΔARE/WT mice were fed with an iron sulfate containing or iron sulfate free diet in combination with intraperitoneal control injections or iron injections for 11 weeks. Results TNFΔARE/WT mice develop severe inflammation of the distal ileum but remained completely healthy when transferred to an iron sulfate free diet, even if iron was systemically repleted. Absence of luminal iron sulfate reduced cellular markers of endoplasmic reticulum (ER) stress responses and pro-apoptotic mechanisms in the ileal epithelium. Phenotype or reactivity of major effector intraepithelial CD8αβ+ T cells were not altered in the absence of luminal iron. Interestingly, ER stress mechanisms sensitised the small intestinal epithelial cell (IEC) line Mode-K to cytotoxic function of effector T cells from TNF∆ARE/WT mice. Pyrosequencing of 16S rRNA tags of the caecal microbiota revealed that depletion of luminal iron sulfate induced significant compositional alterations, while total microbial diversity (Shannons diversity index) and number of total operational taxonomic units were not affected. Conclusion This study showed that an iron sulfate free diet in combination with systemic iron repletion prevents the development of chronic ileitis in a murine model of Crohns disease. Luminal iron may directly affect IEC function or generate a pathological milieu in the intestine that triggers epithelial cell stress-associated apoptosis through changes in microbial homeostasis. These results suggest that oral replacement therapy with iron sulfate may trigger inflammatory processes associated with progression of Crohns disease-like ileitis.

The Gut Microbiota of Rural Papua New Guineans: Composition, Diversity Patterns, and Ecological Processes

Although recent research revealed an impact of westernization on diversity and composition of the human gut microbiota, the exact consequences on metacommunity characteristics are insufficiently understood, and the underlying ecological mechanisms have not been elucidated. Here, we have compared the fecal microbiota of adults from two non-industrialized regions in Papua New Guinea (PNG) with that of United States (US) residents. Papua New Guineans harbor communities with greater bacterial diversity, lower inter-individual variation, vastly different abundance profiles, and bacterial lineages undetectable in US residents. A quantification of the ecological processes that govern community assembly identified bacterial dispersal as the dominant process that shapes the microbiome in PNG but not in the US. These findings suggest that the microbiome alterations detected in industrialized societies might arise from modern lifestyle factors limiting bacterial dispersal, which has implications for human health and the development of strategies aimed to redress the impact of westernization.

A dose dependent impact of prebiotic galactooligosaccharides on the intestinal microbiota of healthy adults.

The goal of this research was to determine the effect of different doses of galactooligosaccharide (GOS) on the fecal microbiota of healthy adults, with a focus on bifidobacteria. The study was designed as a single-blinded study, with eighteen subjects consuming GOS-containing chocolate chews at four increasing dosage levels; 0, 2.5, 5.0, and 10.0g. Subjects consumed each dose for 3 weeks, with a two-week baseline period preceding the study and a two-week washout period at the end. Fecal samples were collected weekly and analyzed by cultural and molecular methods. Cultural methods were used for bifidobacteria, Bacteroides, enterobacteria, enterococci, lactobacilli, and total anaerobes; culture-independent methods included denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR (qRT-PCR) using Bifidobacterium-specific primers. All three methods revealed an increase in bifidobacteria populations, as the GOS dosage increased to 5 or 10g. Enumeration of bifidobacteria by qRT-PCR showed a high inter-subject variation in bifidogenic effect and indicated a subset of 9 GOS responders among the eighteen subjects. There were no differences, however, in the initial levels of bifidobacteria between the responding individuals and the non-responding individuals. Collectively, this study showed that a high purity GOS, administered in a confection product at doses of 5g or higher, was bifidogenic, while a dose of 2.5g showed no significant effect. However, the results also showed that even when GOS was administered for many weeks and at high doses, there were still some individuals for which a bifidogenic response did not occur.

Long-Term Temporal Analysis of the Human Fecal Microbiota Revealed a Stable Core of Dominant Bacterial Species

Next-generation sequencing has greatly contributed to an improved ecological understanding of the human gut microbiota. Nevertheless, questions remain regarding the characteristics of this ecosystem and the ecological processes that shape it, and controversy has arisen regarding the stability of the bacterial populations and the existence of a temporal core. In this study, we have characterized the fecal microbial communities of three human individuals over a one-year period by 454 pyrosequencing of 16S rRNA tags in order to investigate the temporal characteristics of the bacterial communities. The findings revealed a temporal core of 33 to 40 species-level Operational Taxonomic Units (OTUs) within subjects. Although these OTUs accounted only for around 12% of the total OTUs detected, they added up to >75% of the total sequences obtained for each individual. In order to determine the capacity of the sequencing and bioinformatic approaches applied during this study to accurately determine the proportion of a core microbiota, we analyzed the fecal microbiota of nine mice with a defined three-member community. This experiment revealed that the sequencing approach inflated the amount of rare OTUs, which introduced a significant degree of artificial variation across samples, and hence reduced the apparent fraction of shared OTUs. However, when assessing the data quantitatively by focusing on dominant lineages, the sequencing approaches deliver an accurate representation of the community. In conclusion, this study revealed that the human fecal microbiota is dominated by around 40 species that maintain persistent populations over the duration of one year. The findings allow conclusions about the ecological factors that shape the community and support the concept of a homeostatic ecosystem controlled largely by deterministic processes. Our analysis of a three-member community revealed that methodological artifacts of OTU-based approaches complicate core calculations, and these limitations have to be considered in the interpretation of microbiome studies.

Characterization of the ileal microbiota in rejecting and nonrejecting recipients of small bowel transplants

Small bowel transplantation can be a life‐preserving procedure for patients with irreversible intestinal failure. Allograft rejection remains a major source of morbidity and mortality and its accurate diagnosis and treatment are critical. In this study, we used pyrosequencing of 16S ribosomal RNA gene tags to compare the composition of the ileal microbiota present during nonrejection, prerejection and active rejection states in small bowel transplant patients. During episodes of rejection, the proportions of phylum Firmicutes (p < 0.001) and the order Lactobacillales (p < 0.01) were significantly decreased, while those of the phylum Proteobacteria, especially the family Enterobacteriaceae, were significantly increased (p < 0.005). Receiver‐operating characteristic analysis revealed that relative proportions of several bacterial taxa in ileal effluents and especially Firmicutes, could be used to discriminate between nonrejection and active rejection. In conclusion, the findings obtained during this study suggest that small bowel transplant rejection is associated with changes in the microbial populations in ileal effluents and support microbiota profiling as a potential diagnostic biomarker of rejection. Future studies should investigate if the dysbiosis that we observed is a cause or a consequence of the rejection process.

Diet-Induced Alterations of Host Cholesterol Metabolism Are Likely To Affect the Gut Microbiota Composition in Hamsters

ABSTRACT The gastrointestinal microbiota affects the metabolism of the mammalian host and has consequences for health. However, the complexity of gut microbial communities and host metabolic pathways make functional connections difficult to unravel, especially in terms of causation. In this study, we have characterized the fecal microbiota of hamsters whose cholesterol metabolism was extensively modulated by the dietary addition of plant sterol esters (PSE). PSE intake induced dramatic shifts in the fecal microbiota, reducing several bacterial taxa within the families Coriobacteriaceae and Erysipelotrichaceae. The abundance of these taxa displayed remarkably high correlations with host cholesterol metabolites. Most importantly, the associations between several bacterial taxa with fecal and biliary cholesterol excretion showed an almost perfect fit to a sigmoidal nonlinear model of bacterial inhibition, suggesting that host cholesterol excretion can shape microbiota structure through the antibacterial action of cholesterol. In vitro experiments suggested a modest antibacterial effect of cholesterol, and especially of cholesteryl-linoleate, but not plant sterols when included in model bile micelles. The findings obtained in this study are relevant to our understanding of gut microbiota-host lipid metabolism interactions, as they provide the first evidence for a role of cholesterol excreted with the bile as a relevant host factor that modulates the gut microbiota. The findings further suggest that the connections between Coriobacteriaceae and Erysipelotrichaceae and host lipid metabolism, which have been observed in several studies, could be caused by a metabolic phenotype of the host (cholesterol excretion) affecting the gut microbiota.

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.