Metaproteomics and DNA metabarcoding as tools to assess dietary intake in humans.

Objective biomarkers of food intake are a sought-after goal in nutrition research. Most biomarker development to date has focused on metabolites detected in blood, urine, skin or hair, but detection of consumed foods in stool has also been shown to be possible via DNA sequencing. An additional food macromolecule in stool that harbors sequence information is protein. However, the use of protein as an intake biomarker has only been explored to a very limited extent. Here, we evaluate and compare measurement of residual food-derived DNA and protein in stool as potential biomarkers of intake. We performed a pilot study of DNA sequencing-based metabarcoding (FoodSeq) and mass spectrometry-based metaproteomics in five individuals' stool sampled in short, longitudinal bursts accompanied by detailed diet records (n=27 total samples). Dietary data provided by stool DNA, stool protein, and written diet record independently identified a strong within-person dietary signature, identified similar food taxa, and had significantly similar global structure in two of the three pairwise comparisons between measurement techniques (DNA-to-protein and DNA-to-diet record). Metaproteomics identified proteins including myosin, ovalbumin, and beta-lactoglobulin that differentiated food tissue types like beef from dairy and chicken from egg, distinctions that were not possible by DNA alone. Overall, our results lay the groundwork for development of targeted metaproteomic assays for dietary assessment and demonstrate that diverse molecular components of food can be leveraged to study food intake using stool samples.

The Binding Properties of Antibodies to Z-DNA in the Sera of Normal Healthy Subjects.

Antibodies to DNA are a diverse set of antibodies that bind sites on DNA, a polymeric macromolecule that displays various conformations. In a previous study, we showed that sera of normal healthy subjects (NHS) contain IgG antibodies to Z-DNA, a left-handed helix with a zig-zig backbone. Recent studies have demonstrated the presence of Z-DNA in bacterial biofilms, suggesting a source of this conformation to induce responses. To characterize further antibodies to Z-DNA, we used an ELISA assay with brominated poly(dGdC) as a source of Z-DNA and determined the isotype of these antibodies and their binding properties. Results of these studies indicate that NHS sera contain IgM and IgA as well as IgG anti-Z-DNA antibodies. As shown by the effects of ionic strength in association and dissociation assays, the anti-Z-DNA antibodies bind primarily by electrostatic interactions; this type of binding differs from that of induced anti-Z-DNA antibodies from immunized animals which bind by non-ionic interactions. Furthermore, urea caused dissociation of NHS anti-Z-DNA at molar concentrations much lower than those for the induced antibodies. These studies also showed IgA anti-Z-DNA antibodies in fecal water. Together, these studies demonstrate that antibodies to Z-DNA occur commonly in normal immunity and may arise as a response to Z-DNA of bacterial origin.

Ultra-processed food intake, gut microbiome, and glucose homeostasis in mid-life adults: Background, design, and methods of a controlled feeding trial.

BACKGROUND: Aging is associated with gut dysbiosis, low-grade inflammation, and increased risk of type 2 diabetes (T2D). Prediabetes, which increases T2D and cardiovascular disease risk, is present in 45-50% of mid-life adults. The gut microbiota may link ultra-processed food (UPF) with inflammation and T2D risk. METHODS: Following a 2-week standardized lead-in diet (59% UPF), adults aged 40-65 years will be randomly assigned to a 6-week diet emphasizing either UPF (81% total energy) or non-UPF (0% total energy). Measurements of insulin sensitivity, 24-h and postprandial glycemic control, gut microbiota composition/function, fecal short chain fatty acids, intestinal inflammation, inflammatory cytokines, and vascular function will be made before and following the 6-week intervention period. Prior to recruitment, menus were developed in order to match UPF and non-UPF conditions based upon relevant dietary factors. Menus were evaluated for palatability and costs, and the commercial additive content of study diets was quantified to explore potential links with outcomes. RESULTS: Overall diet palatability ratings were similar (UPF = 7.6 ± 1.0; Non-UPF = 6.8 ± 1.5; Like Moderately = 7, Like Very Much = 8). Cost analysis (food + labor) of the 2000 kcal menu (7-d average) revealed lower costs for UPF compared to non-UPF diets ($20.97/d and $40.23/d, respectively). Additive exposure assessment of the 2000 kcal UPF diet indicated that soy lecithin (16×/week), citric acid (13×/week), sorbic acid (13×/week), and sodium citrate (12×/week) were the most frequently consumed additives. CONCLUSIONS: Whether UPF consumption impairs glucose homeostasis in mid-life adults is unknown. Findings will address this research gap and contribute information on how UPF consumption may influence T2D development.

A pilot study of fecal pH and redox as functional markers in the premature infant gut microbiome.

The infant gut microbiome is a crucial factor in health and development. In preterm infants, altered gut microbiome composition and function have been linked to serious neonatal complications such as necrotizing enterocolitis and sepsis, which can lead to long-term disability. Although many studies have described links between microbiome composition and disease risk, there is a need for biomarkers to identify infants at risk of these complications in practice. In this pilot study, we obtained stool samples from preterm infant participants longitudinally during the first postnatal months, and measured pH and redox, as well as SCFA content and microbiome composition by 16S rRNA gene amplicon sequencing. These outcomes were compared to clinical data to better understand the role of pH and redox in infant gut microbiome development and overall health, and to assess the potential utility of pH and redox as biomarkers. We found that infants born earlier or exposed to antibiotics exhibited increased fecal pH, and that redox potential increased with postnatal age. These differences may be linked to changes in SCFA content, which was correlated with pH and increased with age. Microbiome composition was also related to birth weight, age, pH, and redox. Our findings suggest that pH and redox may serve as biomarkers of metabolic state in the preterm infant gut.

Fecal pH and redox as functional markers in the premature infant gut microbiome.

The infant gut microbiome is a crucial factor in health and development. In preterm infants, altered gut microbiome composition and function have been linked to serious neonatal complications such as necrotizing enterocolitis and sepsis, which can lead to long-term disability. Although many studies have described links between microbiome composition and disease risk, there is a need for biomarkers to identify infants at risk of these complications in practice. In this study, we obtained stool samples from preterm infant participants longitudinally during the first postnatal months, and measured pH and redox, as well as SCFA content and microbiome composition by 16S rRNA gene amplicon sequencing. These outcomes were compared to clinical data to better understand the role of pH and redox in infant gut microbiome development and overall health, and to assess the potential utility of pH and redox as biomarkers. We found that infants born earlier or exposed to antibiotics exhibited increased fecal pH, and that redox potential increased with postnatal age. These differences may be linked to changes in SCFA content, which was correlated with pH and increased with age. Microbiome composition was also related to birth weight, age, pH, and redox. Our findings suggest that pH and redox may serve as biomarkers of metabolic state in the preterm infant gut.

Diversity of plant DNA in stool is linked to dietary quality, age, and household income.

Eating a varied diet is a central tenet of good nutrition. Here, we develop a molecular tool to quantify human dietary plant diversity by applying DNA metabarcoding with the chloroplast trnL-P6 marker to 1,029 fecal samples from 324 participants across two interventional feeding studies and three observational cohorts. The number of plant taxa per sample (plant metabarcoding richness or pMR) correlated with recorded intakes in interventional diets and with indices calculated from a food frequency questionnaire in typical diets (ρ = 0.40 to 0.63). In adolescents unable to collect validated dietary survey data, trnL metabarcoding detected 111 plant taxa, with 86 consumed by more than one individual and four (wheat, chocolate, corn, and potato family) consumed by >70% of individuals. Adolescent pMR was associated with age and household income, replicating prior epidemiologic findings. Overall, trnL metabarcoding promises an objective and accurate measure of the number and types of plants consumed that is applicable to diverse human populations.

A genetic system for Akkermansia muciniphila reveals a role for mucin foraging in gut colonization and host sterol biosynthesis gene expression.

Akkermansia muciniphila, a mucophilic member of the gut microbiota, protects its host against metabolic disorders. Because it is genetically intractable, the mechanisms underlying mucin metabolism, gut colonization and its impact on host physiology are not well understood. Here we developed and applied transposon mutagenesis to identify genes important for intestinal colonization and for the use of mucin. An analysis of transposon mutants indicated that de novo biosynthesis of amino acids was required for A. muciniphila growth on mucin medium and that many glycoside hydrolases are redundant. We observed that mucin degradation products accumulate in internal compartments within bacteria in a process that requires genes encoding pili and a periplasmic protein complex, which we term mucin utilization locus (MUL) genes. We determined that MUL genes were required for intestinal colonization in mice but only when competing with other microbes. In germ-free mice, MUL genes were required for A. muciniphila to repress genes important for cholesterol biosynthesis in the colon. Our genetic system for A. muciniphila provides an important tool with which to uncover molecular links between the metabolism of mucins, regulation of lipid homeostasis and potential probiotic activities.

Horizontal gene transfer enables programmable gene stability in synthetic microbiota.

The functions of many microbial communities exhibit remarkable stability despite fluctuations in the compositions of these communities. To date, a mechanistic understanding of this function-composition decoupling is lacking. Statistical mechanisms have been commonly hypothesized to explain such decoupling. Here, we proposed that dynamic mechanisms, mediated by horizontal gene transfer (HGT), also enable the independence of functions from the compositions of microbial communities. We combined theoretical analysis with numerical simulations to illustrate that HGT rates can determine the stability of gene abundance in microbial communities. We further validated these predictions using engineered microbial consortia of different complexities transferring one or more than a dozen clinically isolated plasmids, as well as through the reanalysis of data from the literature. Our results demonstrate a generalizable strategy to program the gene stability of microbial communities.

Microbiota responses to different prebiotics are conserved within individuals and associated with habitual fiber intake.

BACKGROUND: Short-chain fatty acids (SCFAs) derived from gut bacteria are associated with protective roles in diseases ranging from obesity to colorectal cancers. Intake of microbially accessible dietary fibers (prebiotics) lead to varying effects on SCFA production in human studies, and gut microbial responses to nutritional interventions vary by individual. It is therefore possible that prebiotic therapies will require customizing to individuals. RESULTS: Here, we explored prebiotic personalization by conducting a three-way crossover study of three prebiotic treatments in healthy adults. We found that within individuals, metabolic responses were correlated across the three prebiotics. Individual identity, rather than prebiotic choice, was also the major determinant of SCFA response. Across individuals, prebiotic response was inversely related to basal fecal SCFA concentration, which, in turn, was associated with habitual fiber intake. Experimental measures of gut microbial SCFA production for each participant also negatively correlated with fiber consumption, supporting a model in which individuals' gut microbiota are limited in their overall capacity to produce fecal SCFAs from fiber. CONCLUSIONS: Our findings support developing personalized prebiotic regimens that focus on selecting individuals who stand to benefit, and that such individuals are likely to be deficient in fiber intake. Video Abstract.

Ecological memory of prior nutrient exposure in the human gut microbiome.

Many ecosystems have been shown to retain a memory of past conditions, which in turn affects how they respond to future stimuli. In microbial ecosystems, community disturbance has been associated with lasting impacts on microbiome structure. However, whether microbial communities alter their response to repeated stimulus remains incompletely understood. Using the human gut microbiome as a model, we show that bacterial communities retain an "ecological memory" of past carbohydrate exposures. Memory of the prebiotic inulin was encoded within a day of supplementation among a cohort of human study participants. Using in vitro gut microbial models, we demonstrated that the strength of ecological memory scales with nutrient dose and persists for days. We found evidence that memory is seeded by transcriptional changes among primary degraders of inulin within hours of nutrient exposure, and that subsequent changes in the activity and abundance of these taxa are sufficient to enhance overall community nutrient metabolism. We also observed that ecological memory of one carbohydrate species impacts microbiome response to other carbohydrates, and that an individual's habitual exposure to dietary fiber was associated with their gut microbiome's efficiency at digesting inulin. Together, these findings suggest that the human gut microbiome's metabolic potential reflects dietary exposures over preceding days and changes within hours of exposure to a novel nutrient. The dynamics of this ecological memory also highlight the potential for intra-individual microbiome variation to affect the design and interpretation of interventions involving the gut microbiome.

Modulation of microbial community dynamics by spatial partitioning.

Microbial communities inhabit spatial architectures that divide a global environment into isolated or semi-isolated local environments, which leads to the partitioning of a microbial community into a collection of local communities. Despite its ubiquity and great interest in related processes, how and to what extent spatial partitioning affects the structures and dynamics of microbial communities are poorly understood. Using modeling and quantitative experiments with simple and complex microbial communities, we demonstrate that spatial partitioning modulates the community dynamics by altering the local interaction types and global interaction strength. Partitioning promotes the persistence of populations with negative interactions but suppresses those with positive interactions. For a community consisting of populations with both positive and negative interactions, an intermediate level of partitioning maximizes the overall diversity of the community. Our results reveal a general mechanism underlying the maintenance of microbial diversity and have implications for natural and engineered communities.

Tracking defined microbial communities by multicolor flow cytometry reveals tradeoffs between productivity and diversity.

Cross feeding between microbes is ubiquitous, but its impact on the diversity and productivity of microbial communities is incompletely understood. A reductionist approach using simple microbial communities has the potential to detect cross feeding interactions and their impact on ecosystem properties. However, quantifying abundance of more than two microbes in a community in a high throughput fashion requires rapid, inexpensive assays. Here, we show that multicolor flow cytometry combined with a machine learning-based classifier can rapidly quantify species abundances in simple, synthetic microbial communities. Our approach measures community structure over time and detects the exchange of metabolites in a four-member community of fluorescent Bacteroides species. Notably, we quantified species abundances in co-cultures and detected evidence of cooperation in polysaccharide processing and competition for monosaccharide utilization. We also observed that co-culturing on simple sugars, but not complex sugars, reduced microbial productivity, although less productive communities maintained higher community diversity. In summary, our multicolor flow cytometric approach presents an economical, tractable model system for microbial ecology using well-studied human bacteria. It can be extended to include additional species, evaluate more complex environments, and assay response of communities to a variety of disturbances.

Measuring and mitigating PCR bias in microbiota datasets.

PCR amplification plays an integral role in the measurement of mixed microbial communities via high-throughput DNA sequencing of the 16S ribosomal RNA (rRNA) gene. Yet PCR is also known to introduce multiple forms of bias in 16S rRNA studies. Here we present a paired modeling and experimental approach to characterize and mitigate PCR NPM-bias (PCR bias from non-primer-mismatch sources) in microbiota surveys. We use experimental data from mock bacterial communities to validate our approach and human gut microbiota samples to characterize PCR NPM-bias under real-world conditions. Our results suggest that PCR NPM-bias can skew estimates of microbial relative abundances by a factor of 4 or more, but that this bias can be mitigated using log-ratio linear models.

Genotypic and Phenotypic Diversity among Human Isolates of Akkermansia muciniphila.

The mucophilic anaerobic bacterium Akkermansia muciniphila is a prominent member of the gastrointestinal (GI) microbiota and the only known species of the Verrucomicrobia phylum in the mammalian gut. A high prevalence of A. muciniphila in adult humans is associated with leanness and a lower risk for the development of obesity and diabetes. Four distinct A. muciniphila phylogenetic groups have been described, but little is known about their relative abundance in humans or how they impact human metabolic health. In this study, we isolated and characterized 71 new A. muciniphila strains from a cohort of children and adolescents undergoing treatment for obesity. Based on genomic and phenotypic analysis of these strains, we found several phylogroup-specific phenotypes that may impact the colonization of the GI tract or modulate host functions, such as oxygen tolerance, adherence to epithelial cells, iron and sulfur metabolism, and bacterial aggregation. In antibiotic-treated mice, phylogroups AmIV and AmII outcompeted AmI strains. In children and adolescents, AmI strains were most prominent, but we observed high variance in A. muciniphila abundance and single phylogroup dominance, with phylogroup switching occurring in a small subset of patients. Overall, these results highlight that the ecological principles determining which A. muciniphila phylogroup predominates in humans are complex and that A. muciniphila strain genetic and phenotypic diversity may represent an important variable that should be taken into account when making inferences as to this microbe's impact on its host's health.IMPORTANCE The abundance of Akkermansia muciniphila in the gastrointestinal (GI) tract is linked to multiple positive health outcomes. There are four known A. muciniphila phylogroups, yet the prevalence of these phylogroups and how they vary in their ability to influence human health is largely unknown. In this study, we performed a genomic and phenotypic analysis of 71 A. muciniphila strains and identified phylogroup-specific traits such as oxygen tolerance, adherence, and sulfur acquisition that likely influence colonization of the GI tract and differentially impact metabolic and immunological health. In humans, we observed that single Akkermansia phylogroups predominate at a given time but that the phylotype can switch in an individual. This collection of strains provides the foundation for the functional characterization of A. muciniphila phylogroup-specific effects on the multitude of host outcomes associated with Akkermansia colonization, including protection from obesity, diabetes, colitis, and neurological diseases, as well as enhanced responses to cancer immunotherapies.

The Pediatric Obesity Microbiome and Metabolism Study (POMMS): Methods, Baseline Data, and Early Insights.

OBJECTIVE: The purpose of this study was to establish a biorepository of clinical, metabolomic, and microbiome samples from adolescents with obesity as they undergo lifestyle modification. METHODS: A total of 223 adolescents aged 10 to 18 years with BMI ≥95th percentile were enrolled, along with 71 healthy weight participants. Clinical data, fasting serum, and fecal samples were collected at repeated intervals over 6 months. Herein, the study design, data collection methods, and interim analysis-including targeted serum metabolite measurements and fecal 16S ribosomal RNA gene amplicon sequencing among adolescents with obesity (n = 27) and healthy weight controls (n = 27)-are presented. RESULTS: Adolescents with obesity have higher serum alanine aminotransferase, C-reactive protein, and glycated hemoglobin, and they have lower high-density lipoprotein cholesterol when compared with healthy weight controls. Metabolomics revealed differences in branched-chain amino acid-related metabolites. Also observed was a differential abundance of specific microbial taxa and lower species diversity among adolescents with obesity when compared with the healthy weight group. CONCLUSIONS: The Pediatric Metabolism and Microbiome Study (POMMS) biorepository is available as a shared resource. Early findings suggest evidence of a metabolic signature of obesity unique to adolescents, along with confirmation of previously reported findings that describe metabolic and microbiome markers of obesity.

Predicting Vibrio cholerae Infection and Disease Severity Using Metagenomics in a Prospective Cohort Study.

BACKGROUND: Susceptibility to Vibrio cholerae infection is affected by blood group, age, and preexisting immunity, but these factors only partially explain who becomes infected. A recent study used 16S ribosomal RNA amplicon sequencing to quantify the composition of the gut microbiome and identify predictive biomarkers of infection with limited taxonomic resolution. METHODS: To achieve increased resolution of gut microbial factors associated with V. cholerae susceptibility and identify predictors of symptomatic disease, we applied deep shotgun metagenomic sequencing to a cohort of household contacts of patients with cholera. RESULTS: Using machine learning, we resolved species, strains, gene families, and cellular pathways in the microbiome at the time of exposure to V. cholerae to identify markers that predict infection and symptoms. Use of metagenomic features improved the precision and accuracy of prediction relative to 16S sequencing. We also predicted disease severity, although with greater uncertainty than our infection prediction. Species within the genera Prevotella and Bifidobacterium predicted protection from infection, and genes involved in iron metabolism were also correlated with protection. CONCLUSION: Our results highlight the power of metagenomics to predict disease outcomes and suggest specific species and genes for experimental testing to investigate mechanisms of microbiome-related protection from cholera.

Naught all zeros in sequence count data are the same.

Genomic studies feature multivariate count data from high-throughput DNA sequencing experiments, which often contain many zero values. These zeros can cause artifacts for statistical analyses and multiple modeling approaches have been developed in response. Here, we apply different zero-handling models to gene-expression and microbiome datasets and show models can disagree substantially in terms of identifying the most differentially expressed sequences. Next, to rationally examine how different zero handling models behave, we developed a conceptual framework outlining four types of processes that may give rise to zero values in sequence count data. Last, we performed simulations to test how zero handling models behave in the presence of these different zero generating processes. Our simulations showed that simple count models are sufficient across multiple processes, even when the true underlying process is unknown. On the other hand, a common zero handling technique known as "zero-inflation" was only suitable under a zero generating process associated with an unlikely set of biological and experimental conditions. In concert, our work here suggests several specific guidelines for developing and choosing state-of-the-art models for analyzing sparse sequence count data.

Conserved anti-inflammatory effects and sensing of butyrate in zebrafish.

Short-chain fatty acids (SCFAs) are produced by microbial fermentation of dietary fiber in the gut. Butyrate is a particularly important SCFA with anti-inflammatory properties and is generally present at lower levels in inflammatory diseases associated with gut microbiota dysbiosis in mammals. We aimed to determine if SCFAs are produced by the zebrafish microbiome and if SCFAs exert conserved effects on zebrafish immunity as an example of the non-mammalian vertebrate immune system. We demonstrate that bacterial communities from adult zebrafish intestines synthesize all three main SCFA in vitro, although SCFA were below our detectable limits in zebrafish intestines in vivo. Immersion in butyrate, but not acetate or propionate, reduced the recruitment of neutrophils and M1-type pro-inflammatory macrophages to wounds. We found conservation of butyrate sensing by neutrophils via orthologs of the hydroxycarboxylic acid receptor 1 (hcar1) gene. Neutrophils from Hcar1-depleted embryos were no longer responsive to the anti-inflammatory effects of butyrate, while macrophage sensitivity to butyrate was independent of Hcar1. Our data demonstrate conservation of anti-inflammatory butyrate effects and identify the presence of a conserved molecular receptor in fish.

Short-Chain Fatty Acid Production by Gut Microbiota from Children with Obesity Differs According to Prebiotic Choice and Bacterial Community Composition.

Pediatric obesity remains a public health burden and continues to increase in prevalence. The gut microbiota plays a causal role in obesity and is a promising therapeutic target. Specifically, the microbial production of short-chain fatty acids (SCFA) from the fermentation of otherwise indigestible dietary carbohydrates may protect against pediatric obesity and metabolic syndrome. Still, it has not been demonstrated that therapies involving microbiota-targeting carbohydrates, known as prebiotics, will enhance gut bacterial SCFA production in children and adolescents with obesity (age, 10 to 18 years old). Here, we used an in vitro system to examine the SCFA production by fecal microbiota from 17 children with obesity when exposed to five different commercially available over-the-counter (OTC) prebiotic supplements. We found microbiota from all 17 patients actively metabolized most prebiotics. Still, supplements varied in their acidogenic potential. Significant interdonor variation also existed in SCFA production, which 16S rRNA sequencing supported as being associated with differences in the host microbiota composition. Last, we found that neither fecal SCFA concentration, microbiota SCFA production capacity, nor markers of obesity positively correlated with one another. Together, these in vitro findings suggest the hypothesis that OTC prebiotic supplements may be unequal in their ability to stimulate SCFA production in children and adolescents with obesity and that the most acidogenic prebiotic may differ across individuals.IMPORTANCE Pediatric obesity remains a major public health problem in the United States, where 17% of children and adolescents are obese, and rates of pediatric "severe obesity" are increasing. Children and adolescents with obesity face higher health risks, and noninvasive therapies for pediatric obesity often have limited success. The human gut microbiome has been implicated in adult obesity, and microbiota-directed therapies can aid weight loss in adults with obesity. However, less is known about the microbiome in pediatric obesity, and microbiota-directed therapies are understudied in children and adolescents. Our research has two important findings: (i) dietary prebiotics (fiber) result in the microbiota from adolescents with obesity producing more SCFA, and (ii) the effectiveness of each prebiotic is donor dependent. Together, these findings suggest that prebiotic supplements could help children and adolescents with obesity, but that these therapies may not be "one size fits all."