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The intestinal microbiota actively converts dietary flavanols into phenolic acids, some of which are bioavailable in vivo and may promote resilience to select neurological disorders by interfering with key pathologic mechanisms. Since every person harbors a unique set of gut bacteria, we investigated the influence of the gut microbiota's interpersonal heterogeneity on the production and bioavailability of flavonoid metabolites that may interfere with the misfolding of alpha (α)-synuclein, a process that plays a central role in Parkinson's disease and other α-synucleinopathies. We generated two experimental groups of humanized gnotobiotic mice with compositionally diverse gut bacteria and orally treated the mice with a flavanol-rich preparation (FRP). The two gnotobiotic mouse groups exhibited distinct differences in the generation and bioavailability of FRP-derived microbial phenolic acid metabolites that have bioactivity towards interfering with α-synuclein misfolding or inflammation. We also demonstrated that these bioactive phenolic acids are effective in modulating the development and progression of motor dysfunction in a Drosophila model of α-synucleinopathy. Lastly, through in vitro bacterial fermentation studies, we identified select bacteria that are capable of supporting the generation of these bioavailable and bioactive phenolic acids. Outcomes from our studies provide a better understanding of how interpersonal heterogeneity in the gut microbiota differentially modulates the efficacy of dietary flavanols to protect against select pathologic mechanisms. Collectively, our findings provide the basis for future developments of probiotic, prebiotic, or synbiotic approaches for modulating the onset and/or progression of α-synucleinopathies and other neurological disorders involving protein misfolding and/or inflammation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Microbiota are thought to influence the development and progression of inflammatory bowel disease (IBD), but determining generalizable effects of microbiota on IBD etiology requires larger-scale functional analyses. We colonized germ-free mice with intestinal microbiotas from 30 healthy and IBD donors and determined the homeostatic intestinal T cell response to each microbiota. Compared to microbiotas from healthy donors, transfer of IBD microbiotas into germ-free mice increased numbers of intestinal Th17 cells and Th2 cells and decreased numbers of RORγt + Treg cells. Colonization with IBD microbiotas exacerbated disease in a model where colitis is induced upon transfer of naive T cells into Rag1 -/- mice. The proportions of Th17 and RORγt + Treg cells induced by each microbiota were predictive of human disease status and accounted for disease severity in the Rag1 -/- colitis model. Thus, an impact on intestinal Th17 and RORγt + Treg cell compartments emerges as a unifying feature of IBD microbiotas, suggesting a general mechanism for microbial contribution to IBD pathogenesis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Accumulating evidence indicates that the health impact of dietary phenolic compounds, including the principal grape-derived polyphenols, (+)‑catechin and (-)‑epicatechin, is exerted by not only the parent compounds but also their phenolic metabolites generated by the gut microbiota. In this work, a new high-throughput, sensitive and reproducible analytical method was developed employing ultra-high performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS) for the simultaneous analysis of 16 microbial-generated phenolic acid metabolites (PAMs) along with their precursors, catechin and epicatechin. Following optimizing the solvent system, LC conditions and MS parameters, method validation was carried out to evaluate the sensitivity, selectivity, accuracy and precision of the proposed method, and to ensure promising recovery of all analytes extracted from the matrix prior to bioanalysis. Results showed that the optimized analytical method allowed successful confirmation and quantitation of all analytes under dynamic multiple reaction monitoring mode using trans‑cinnamic acid‑d 7 as an internal standard (I.S.). Excellent sensitivity and linearity were obtained for all analytes, with lower limits of detection (LLODs) and lower limits of quantification (LLOQs) in the ranges of 0.225-2.053 ng/mL and 0.698-8.116 ng/mL, respectively. By examining blank matrix spiked with standard mixture at different concentration levels, promising recoveries at two spiking levels (low level, 91.2-115%; high level 90.2-121%), and excellent precision (RSD < 10%) were obtained. This method was then successfully applied to an in vitro study where catechin/epicatechin-enriched broth samples were anaerobically fermented with gut microbes procured from healthy human donors. All sources of bacteria employed showed remarkable activity in metabolizing grape polyphenols and distinct variations in the production of PAMs. The successful application of this method in the in vitro fermentation assays demonstrates its suitability for high-throughput analysis of polyphenol metabolites, particularly catechin/epicatechin-derived PAMs, in biological studies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background & Aims: Several studies have shown that signaling via the interleukin 23 (IL23) receptor is required for development of colitis. We studied the roles of IL23, dietary factors, alterations to the microbiota, and T cells in the development and progression of colitis in mice., Methods: All mice were maintained on laboratory diet 5053, unless otherwise noted. We generated mice that express IL23 in CX3CR1-positive myeloid cells (R23FR mice) upon cyclic administration of tamoxifen dissolved in diet 2019. Diets 2019 and 5053 have minor differences in the overall composition of protein, fat, fiber, minerals, and vitamins. CX3CR1 CreER mice (FR mice) were used as controls. Some mice were given antibiotics, and others were raised in a germ-free environment. Intestinal tissues were collected and analyzed by histology and flow cytometry. Feces were collected and analyzed by 16S rDNA sequencing. Feces from C57/Bl6, R23FR, or FR mice were fed to FR and R23FR germ-free mice in microbiota transplant experiments. We also performed studies with R23FR/Rag -/- , R23FR/Mu -/- , and R23FR/Tcrd -/- mice. R23FR mice were given injections of antibodies against CD4 or CD8 to deplete T cells. Mesenteric lymph nodes and large intestine CD4 + cells from R23FR or FR mice in remission from colitis were transferred into Rag -/- mice. CD4 + cells were isolated from donor R23FR mice and recipient Rag -/- mice, and T-cell receptor sequences were determined., Results: Expression of IL23 led to development of a relapsing-remitting colitis that was dependent on the microbiota and CD4 + T cells. The relapses were caused by switching from the conventional diet used in our facility (diet 5053) to the diet 2019 and were not dependent on tamoxifen after the first cycle. The switch in the diet modified the microbiota but did not alter levels of IL23 in intestinal tissues compared with mice that remained on the conventional diet. Mesenteric lymph nodes and large intestine CD4 + cells from R23FR mice in remission, but not from FR mice, induced colitis after transfer into Rag -/- mice, but only when these mice were placed on the diet 2019. The CD4 + T-cell receptor repertoire of Rag -/- mice with colitis (fed the 2019 diet) was less diverse than that from donor mice and Rag -/- mice without colitis (fed the 5053 diet) because of expansion of dominant T-cell clones., Conclusions: We developed mice that express IL23 in CX3CR1-positive myeloid cells (R23FR mice) and found that they are more susceptible to diet-induced colitis than mice that do not express IL23. The R23FR mice have a population of CD4 + T cells that becomes activated in response to dietary changes and alterations to the intestinal microbiota. The results indicate that alterations in the diet, intestinal microbiota, and IL23 signaling can contribute to pathogenesis of inflammatory bowel disease., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The development of a given botanical preparation for eventual clinical application requires extensive, detailed characterizations of the chemical composition, as well as the biological availability, biological activity, and safety profiles of the botanical. These issues are typically addressed using diverse experimental protocols and model systems. Based on this consideration, in this study we established a comprehensive database and analysis framework for the collection, collation, and integrative analysis of diverse, multiscale data sets. Using this framework, we conducted an integrative analysis of heterogeneous data from in vivo and in vitro investigation of a complex bioactive dietary polyphenol-rich preparation (BDPP) and built an integrated network linking data sets generated from this multitude of diverse experimental paradigms. We established a comprehensive database and analysis framework as well as a systematic and logical means to catalogue and collate the diverse array of information gathered, which is securely stored and added to in a standardized manner to enable fast query. We demonstrated the utility of the database in (1) a statistical ranking scheme to prioritize response to treatments and (2) in depth reconstruction of functionality studies. By examination of these data sets, the system allows analytical querying of heterogeneous data and the access of information related to interactions, mechanism of actions, functions, etc., which ultimately provide a global overview of complex biological responses. Collectively, we present an integrative analysis framework that leads to novel insights on the biological activities of a complex botanical such as BDPP that is based on data-driven characterizations of interactions between BDPP-derived phenolic metabolites and their mechanisms of action, as well as synergism and/or potential cancellation of biological functions. Out integrative analytical approach provides novel means for a systematic integrative analysis of heterogeneous data types in the development of complex botanicals such as polyphenols for eventual clinical and translational applications.

Background & Aims: It is not clear how the complex interactions between diet and the intestinal microbiota affect development of mucosal inflammation or inflammatory bowel disease. We investigated interactions between dietary ingredients, nutrients, and the microbiota in specific pathogen-free (SPF) and germ-free (GF) mice given more than 40 unique diets; we quantified individual and synergistic effects of dietary macronutrients and the microbiota on intestinal health and development of colitis., Methods: C56BL/6J SPF and GF mice were placed on custom diets containing different concentrations and sources of protein, fat, digestible carbohydrates, and indigestible carbohydrates (fiber). After 1 week, SPF and GF mice were given dextran sulfate sodium (DSS) to induce colitis. Disease severity was determined based on the percent weight change from baseline, and modeled as a function of the concentration of each macronutrient in the diet. In unchallenged mice, we measured intestinal permeability by feeding mice labeled dextran and measuring levels in blood. Feces were collected and microbiota were analyzed by 16S rDNA sequencing. We collected colons from mice and performed transcriptome analyses., Results: Fecal microbiota varied with diet; the concentration of protein and fiber had the strongest effect on colitis development. Among 9 fiber sources tested, psyllium, pectin, and cellulose fiber reduced the severity of colitis in SPF mice, whereas methylcellulose increased severity. Increasing dietary protein increased the density of the fecal microbiota and the severity of colitis in SPF mice, but not in GF mice or mice given antibiotics. Psyllium fiber reduced the severity of colitis through microbiota-dependent and microbiota-independent mechanisms. Combinatorial perturbations to dietary casein protein and psyllium fiber in parallel accounted for most variation in gut microbial density and intestinal permeability in unchallenged mice, as well as the severity of DSS-induced colitis; changes in 1 ingredient could be offset by changes in another., Conclusions: In an analysis of the effects of different dietary components and the gut microbiota on mice with and without DSS-induced colitis, we found complex mixtures of nutrients affect intestinal permeability, gut microbial density, and development of intestinal inflammation., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)

A major challenge in inflammatory bowel disease (IBD) is the integration of diverse IBD data sets to construct predictive models of IBD. We present a predictive model of the immune component of IBD that informs causal relationships among loci previously linked to IBD through genome-wide association studies (GWAS) using functional and regulatory annotations that relate to the cells, tissues, and pathophysiology of IBD. Our model consists of individual networks constructed using molecular data generated from intestinal samples isolated from three populations of patients with IBD at different stages of disease. We performed key driver analysis to identify genes predicted to modulate network regulatory states associated with IBD, prioritizing and prospectively validating 12 of the top key drivers experimentally. This validated key driver set not only introduces new regulators of processes central to IBD but also provides the integrated circuits of genetic, molecular, and clinical traits that can be directly queried to interrogate and refine the regulatory framework defining IBD.

The development of fecal microbiota transplantation and defined live biotherapeutic products for the treatment of human disease has been an empirically driven process yielding a notable success of approved drugs for the treatment of recurrent Clostridioides difficile infection. Assessing the potential of this therapeutic modality in other indications with mixed clinical results would benefit from consistent quantitative frameworks to characterize drug potency and composition and to assess the impact of dose and composition on the frequency and duration of strain engraftment. Monitoring these drug properties and engraftment outcomes would help identify minimally sufficient sets of microbial strains to treat disease and provide insights into the intersection between microbial function and host physiology. Broad and correct usage of strain detection methods is essential to this advancement. This article describes strain detection approaches, where they are best applied, what data they require, and clinical trial designs that are best suited to their application.

It has been 35 y since Carl Woese reported in PNAS how sequencing ribosomal RNA genes could be used to distinguish the three domains of life on Earth. During the past decade, 16S rDNA sequencing has enabled the now frequent enumeration of bacterial communities that populate the bodies of humans representing different ages, cultural traditions, and health states. A challenge going forward is to quantify the contributions of community members to wellness, disease risk, and disease pathogenesis. Here, we explore a theoretical framework for studies of the inheritance of bacterial strains and discuss the advantages and disadvantages of various study designs for assessing the contribution of strains to complex diseases.

The gut microbiota codevelops with the immune system beginning at birth. Mining the microbiota for bacterial strains responsible for shaping the structure and dynamic operations of the innate and adaptive arms of the immune system represents a formidable combinatorial problem but one that needs to be overcome to advance mechanistic understanding of microbial community and immune system coregulation and to develop new diagnostic and therapeutic approaches that promote health. Here, we discuss a scalable, less biased approach for identifying effector strains in complex microbial communities that impact immune function. The approach begins by identifying uncultured human fecal microbiota samples that transmit immune phenotypes to germ-free mice. Clonally arrayed sequenced collections of bacterial strains are constructed from representative donor microbiota. If the collection transmits phenotypes, effector strains are identified by testing randomly generated subsets with overlapping membership in individually housed germ-free animals. Detailed mechanistic studies of effector strain-host interactions can then be performed., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The role of specific gut microbes in shaping body composition remains unclear. We transplanted fecal microbiota from adult female twin pairs discordant for obesity into germ-free mice fed low-fat mouse chow, as well as diets representing different levels of saturated fat and fruit and vegetable consumption typical of the U.S. diet. Increased total body and fat mass, as well as obesity-associated metabolic phenotypes, were transmissible with uncultured fecal communities and with their corresponding fecal bacterial culture collections. Cohousing mice harboring an obese twin's microbiota (Ob) with mice containing the lean co-twin's microbiota (Ln) prevented the development of increased body mass and obesity-associated metabolic phenotypes in Ob cage mates. Rescue correlated with invasion of specific members of Bacteroidetes from the Ln microbiota into Ob microbiota and was diet-dependent. These findings reveal transmissible, rapid, and modifiable effects of diet-by-microbiota interactions.

A low-error 16S ribosomal RNA amplicon sequencing method, in combination with whole-genome sequencing of >500 cultured isolates, was used to characterize bacterial strain composition in the fecal microbiota of 37 U.S. adults sampled for up to 5 years. Microbiota stability followed a power-law function, which when extrapolated suggests that most strains in an individual are residents for decades. Shared strains were recovered from family members but not from unrelated individuals. Sampling of individuals who consumed a monotonous liquid diet for up to 32 weeks indicated that changes in strain composition were better predicted by changes in weight than by differences in sampling interval. This combination of stability and responsiveness to physiologic change confirms the potential of the gut microbiota as a diagnostic tool and therapeutic target.

High-throughput sequencing has revolutionized microbial ecology, but read quality remains a considerable barrier to accurate taxonomy assignment and α-diversity assessment for microbial communities. We demonstrate that high-quality read length and abundance are the primary factors differentiating correct from erroneous reads produced by Illumina GAIIx, HiSeq and MiSeq instruments. We present guidelines for user-defined quality-filtering strategies, enabling efficient extraction of high-quality data and facilitating interpretation of Illumina sequencing results.

The interrelationships between our diets and the structure and operations of our gut microbial communities are poorly understood. A model community of 10 sequenced human gut bacteria was introduced into gnotobiotic mice, and changes in species abundance and microbial gene expression were measured in response to randomized perturbations of four defined ingredients in the host diet. From the responses, we developed a statistical model that predicted over 60% of the variation in species abundance evoked by diet perturbations, and we were able to identify which factors in the diet best explained changes seen for each community member. The approach is generally applicable, as shown by a follow-up study involving diets containing various mixtures of pureed human baby foods.

The proportion of the human gut bacterial community that is recalcitrant to culture remains poorly defined. In this report, we combine high-throughput anaerobic culturing techniques with gnotobiotic animal husbandry and metagenomics to show that the human fecal microbiota consists largely of taxa and predicted functions that are represented in its readily cultured members. When transplanted into gnotobiotic mice, complete and cultured communities exhibit similar colonization dynamics, biogeographical distribution, and responses to dietary perturbations. Moreover, gnotobiotic mice can be used to shape these personalized culture collections to enrich for taxa suited to specific diets. We also demonstrate that thousands of isolates from a single donor can be clonally archived and taxonomically mapped in multiwell format to create personalized microbiota collections. Retrieving components of a microbiota that have coexisted in single donors who have physiologic or disease phenotypes of interest and reuniting them in various combinations in gnotobiotic mice should facilitate preclinical studies designed to determine the degree to which tractable bacterial taxa are able to transmit donor traits or influence host biology.