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The placenta is the principal organ nurturing the fetus during pregnancy and was traditionally considered to be sterile. Recent work has suggested that the placenta harbours microbial communities, however the location and possible function of these microbes remain to be confirmed and elucidated. Here, we employed genomic DNA sequencing of multiple variable (V) regions of the bacterial 16S ribosomal gene, to interrogate microbial profiles in term pregnancies, from the basal plate, which is in direct contact with maternal uterine, endothelial, and immune cells; placental villi, which are bathed in maternal blood, and fetal membranes, which encapsulate the amniotic cavity. QIIME, R package “Phyloseq” analysis was used to assess alpha and beta diversity and absolute abundance of the 16S rRNA gene per location. We demonstrate that (1) microbiota exhibit spatially distinct profiles depending on the location within the placenta and (2) “semi-composite” 16S profiles using multiple V regions validated by quantitative PCR analysis confirmed that distinct bacterial taxa dominate in different placental niches. Finally, profiles are not altered by mode of delivery. Together these findings suggest that there is niche-specificity to the placental microbiota and placental microbiome studies should consider regional differences, which may affect maternal, fetal, and/or neonatal health and physiology.

Significance Dietary fibers contain complex mixtures of biomolecules, making it difficult to develop/test hypotheses about how different fiber-types impact different components of the human gut microbiome and how microbiome changes that they produce are linked to human physiology. Here, we analyze microbiome and plasma proteome responses to consumption of two fiber-enriched snacks in two human studies. We use a variety of computational methods to correlate their effects on gut microbiome genes encoding enzymes that degrade complex fiber-associated polysaccharides, the microbial products of polysaccharide degradation, and plasma proteins representing diverse physiological processes. This approach can be used to guide the design of fiber-containing snacks that more precisely manipulate microbiome features in ways that improve nutritional and health status.

Changing food preferences brought about by westernization that have deleterious health effects 1 , 2 —combined with myriad forces that are contributing to increased food insecurity—are catalysing efforts to identify more nutritious and affordable foods 3 . Consumption of dietary fibre can help to prevent cardiovascular disease, type 2 diabetes and obesity 4 – 6 . A substantial number of reports have explored the effects of dietary fibre on the gut microbial community 7 – 9 . However, the microbiome is complex, dynamic and exhibits considerable intra- and interpersonal variation in its composition and functions. The large number of potential interactions between the components of the microbiome makes it challenging to define the mechanisms by which food ingredients affect community properties. Here we address the question of how foods containing different fibre preparations can be designed to alter functions associated with specific components of the microbiome. Because a marked increase in snack consumption is associated with westernization, we formulated snack prototypes using plant fibres from different sustainable sources that targeted distinct features of the gut microbiomes of individuals with obesity when transplanted into gnotobiotic mice. We used these snacks to supplement controlled diets that were consumed by adult individuals with obesity or who were overweight. Fibre-specific changes in their microbiomes were linked to changes in their plasma proteomes indicative of an altered physiological state. Fibre snacks that target distinct features of the microbiomes of donors with obesity transplanted into gnotobiotic mice also lead to fibre-specific changes in the microbiome and physiology when used in controlled-diet human studies.

Carbohydrates comprise the largest fraction of most diets and exert a profound impact on health. Components such as simple sugars and starch supply energy, while indigestible components, deemed dietary fiber, reach the colon to provide food for the tens of trillions of microbes that make up the gut microbiota. The interactions between dietary carbohydrates, our gastrointestinal tracts, the gut microbiome and host health are dictated by their structures. However, current methods for analysis of food glycans lack the sensitivity, specificity and throughput needed to quantify and elucidate these myriad structures. This protocol describes a multi-glycomic approach to food carbohydrate analysis in which the analyte might be any food item or biological material such as fecal and cecal samples. The carbohydrates are extracted by ethanol precipitation, and the resulting samples are subjected to rapid-throughput liquid chromatography (LC)-tandem mass spectrometry (LC-MS/MS) methods. Quantitative analyses of monosaccharides, glycosidic linkages, polysaccharides and alcohol-soluble carbohydrates are performed in 96-well plates at the milligram scale to reduce the biomass of sample required and enhance throughput. Detailed stepwise processes for sample preparation, LC-MS/MS and data analysis are provided. We illustrate the application of the protocol to a diverse set of foods as well as different apple cultivars and various fermented foods. Furthermore, we show the utility of these methods in elucidating glycan–microbe interactions in germ-free and colonized mice. These methods provide a framework for elucidating relationships between dietary fiber, the gut microbiome and human physiology. These structures will further guide nutritional and clinical feeding studies that enhance our understanding of the role of diet in nutrition and health. Key points It is important to understand how carbohydrates are digested—both by human enzymes and by microorganisms present in the gut. This protocol is designed to characterize and quantify food and fecal polysaccharides at the monosaccharide, linkage and polysaccharide level. Analysis is performed by LC-MS/MS. Higher-throughput sample preparation in 96-well plates is possible by using a custom-made clamp to hold the plate lids closed during heating. Complex carbohydrates that are not broken down by human enzymes are food sources for gut microbiota. Toward understanding this process, this protocol describes the quantitative analysis of carbohydrates in food and fecal samples by using LC-MS/MS.

Our H. sapiens genome does not encode enzymes required to degrade most of the complex plant-derived polysaccharides present in our diets (e.g., hemicelluloses, xylans, resistant starches, pectins). Our gut microbiomes, which contain ~100 times more genes than our human genome, encode a vast repertoire of carbohydrate-active enzymes that catalyze the metabolism of these dietary glycans to products that benefit members of the microbiota and their human host. Western diets are characterized by a paucity of fruits and vegetables and by extension, dietary fiber. Low-fiber diets, in turn, have been associated with a loss of diversity in the microbiota. This loss of diversity is a characteristic of the microbial communities of obese individuals.Members of the lab have conducted a screen of 34 food-grade fibers in gnotobiotic mice harboring a defined human gut microbial community composed of cultured sequenced bacterial strains and fed a representative USA diet high in saturated fats and low in fruits and vegetables (HiSF-LoFV). This screen, which involved testing the effects of almost 144 systematically manipulated diets on the fitness of community members, was designed to identify components of fibers that targeted ‘beneficial’ saccharolytic Bacteroides species underrepresented in the gut communities of obese (Ob) co-twin members of obesity-discordant twin pairs. Bioactive components of fibers that selectively increase the representation of these taxa, and expression of their beneficial metabolic features, are candidates for incorporation into microbiota-directed food prototypes designed to ameliorate the obesity and metabolic dysfunction phenotypes that are transmitted by Ob co-twin microbiota to recipient germ-free mice and abrogated by the introduction of the targeted Bacteroides.My thesis represents a partnership between food science and microbiome science. It focuses on an analysis of the mechanisms by bioactive components of lead plant fibers emerging from this screen incorporated into food prototypes that affect the structural and functional properties of microbial communities from obese co-twins transplanted into gnotobiotic mice and whether results from these mouse models translate to humans.I colonized groups of germ-free mice with intact uncultured fecal microbiota from obese co-twins in obesity concordant or discordant pairs. I subjected mice colonized with a given donor microbiota to a diet oscillation. The diet oscillation consisted of feeding mice a HiSF-LoFV diet followed by the same diet + one lead fiber, then by a return to the unsupplemented diet, followed by HiSF-LoFV + a second lead fiber, then by a return to the unsupplemented diet, and finished by supplementation with a third lead fiber. The effects of fiber consumption on (i) body weight and composition (the latter defined by quantitative magnetic resonance), (ii) microbiota/ microbiome configuration (sequencing V4-bacterial 16S rDNA amplicons and whole community DNA), and (iii) community metabolism (short-chain fatty acids, monosaccharide, and linkage-analysis of carbohydrates) were defined. I found that supplementation with lead fibers (i) significantly reduced body weight gain, (ii) significantly increase the abundance of targeted Bacteroides plus CAZymes and mcSEED metabolic pathways/modules involved in the utilization of bioactive components of the lead fibers. Higher-order singular value decomposition (HOSVD) analysis of microbiome datasets revealed temporal projections associated with fiber consumption and identified robust responsive, and hypo-responsive donor microbiota and the microbiome features driving the variance.I subsequently tested the effects of one of the lead fibers in two separate pilot clinical studies, one involving female twins discordant or concordant for obesity, including those whose microbiota had been selected for the preclinical gnotobiotic mouse experiments, and the other involving overweight and obese singleton adults from the St. Louis metropolitan region. The study with twins was done supplementing their regular diets with a snack food prototype enriched with the lead-fiber. The other study involved controlled feeding with a diet high in saturated fats and low in fruits and vegetables, and supplemented with the same fiber snacks. HOSVD plus linear models disclosed significant relationships between fiber-snack consumption, changes in microbiota/microbiome structural and functional configurations, and changes in the plasma proteome. Understanding the transformation capacity of the bioactive components of fibers by the gut microbiota and their effects on host health will be critical in the development of microbiota-directed foods (MDFs).

Carbohydrate-active enzymes (CAZymes) are crucial for digesting glycans, but bioinformatics tools for CAZyme profiling and interpretation of substrate preferences in microbial community data are lacking. To address this, we developed a CAZyme profiler (Cayman) and a hierarchical substrate annotation scheme. Leveraging these, we genomically survey CAZymes in human gut microbes (n=107,683 genomes), which suggests novel mucin-foraging species. In a subsequent meta-analysis of CAZyme repertoires in Western versus non-Western gut metagenomes (n=4,281) we find that non-Western metagenomes are richer in fibre-degrading CAZymes despite lower overall CAZyme richness. We additionally pinpoint the taxonomic drivers underlying these CAZyme community shifts. A second meta-analysis comparing colorectal cancer patients (CRC) to controls (n=1,998) shows that CRC metagenomes are deprived of dietary fibre-targeting, but enriched in glycosaminoglycan-targeting CAZymes. A genomic analysis of co-localizing CAZyme domains predicts novel substrates for CRC-enriched CAZymes. Cayman is broadly applicable across microbial communities and freely available from https://github.com/zellerlab/cayman.Competing Interest StatementHT and ODB are employees of Societe des Produits Nestle, Switzerland, however Nestle was not involved in funding the study. HT and ODB contributed as experts on the topic.