Abstract Children of undernourished women have impaired pre-and postnatal growth. Undernourished women and children have a high incidence of environmental enteric dysfunction (EED), an enteropathy characterized by gut barrier dysfunction and systemic inflammation. Here, we employ gnotobiotic mice to compare the effects of bacterial consortia cultured from the duodenal microbiota of Bangladeshi women with EED and their healthy counterparts. Female mice harboring the EED-derived consortium exhibited fetal and placental growth restriction. Transcriptomic and proteomic analyses disclosed pronounced effects of the EED-derived consortium on the decidual component of the maternal-fetal interface involving tissue-resident uterine natural killer (uNK) cells and disruption of TGF-β signaling between uNK and decidual stromal cells. Co-housing mice with EED and healthy consortia ameliorated these effects, disclosing bacterial targets to improve prenatal development.

Bifidobacteria are among the earliest colonizers of the human gut and are widely used as probiotics for their health-promoting properties. However, individual responses to probiotic supplementation may vary with strain type(s), microbiota composition, diet, or lifestyle conditions, highlighting the need for strain-level insights into the bifidobacterial metabolism of dietary and host glycans. Here, we systematically reconstructed 68 pathways involved in the utilization of mono-, di-, oligo-, and polysaccharides by analyzing the distribution of 589 curated metabolic functional roles (catabolic enzymes, transporters, transcriptional regulators) in 3083 non-redundant cultured Bifidobacterium isolates and metagenome-assembled genomes (MAGs) of human origin. Our analysis uncovered extensive inter- and intraspecies heterogeneity, including a distinct clade within the Bifidobacterium longum species capable of metabolizing α-glucans. We also identified isolates of Bangladeshi origin that harbor unique gene clusters implicated in the breakdown of xyloglucan and human milk oligosaccharides. Thirty-eight predicted carbohydrate utilization phenotypes were experimentally validated in 30 geographically diverse Bifidobacterium isolates in vitro. Our large-scale genomic compendium expands the knowledge of bifidobacterial carbohydrate metabolism and can inform the rational design of probiotic and synbiotic formulations tailored to strain-specific nutrient preferences.

Environmental enteric dysfunction (EED), a small intestinal disorder found at a high prevalence in stunted children, is associated with gut mucosal barrier disruption and decreased absorptive capacity 1-4. To test the hypothesis that intergenerational transmission of a perturbed small intestinal microbiota contributes to undernutrition by inducing EED 5, we characterized two consortia of bacterial strains cultured from duodenal aspirates from stunted Bangladeshi children with EED - one of which induced local and systemic inflammation in female gnotobiotic mice. Offspring of dams colonized with the inflammatory consortium exhibited impaired prenatal and postnatal growth, as well as immunologic changes phenocopying features of EED in children. Dam-to-pup transmission of the inflammatory consortium produced, in recently weaned offspring, alterations in inter-cellular signaling pathways related to intestinal epithelial cell renewal, barrier integrity and immune function. Cohousing of mice harboring the inflammatory or non-inflammatory consortia and subsequent screening of candidate disease-promoting bacterial isolates identified Campylobacter concisus, an organism typically found in the oral microbiota, as a contributor to enteropathy. The C. concisus strain induced, in a host nitric oxide synthase (NOS)-dependent manner, pro-inflammatory cytokine signaling. Moreover, host-derived nutrients generated by NOS augmented C. concisus growth. This preclinical model should facilitate identification of small intestinal microbiota-targeted therapeutics for (intergenerational) undernutrition.

Generating metagenome-assembled genomes from DNA shotgun sequencing datasets can demand considerable computational resources. Here, we describe a sequential co-assembly method that reduces the assembly of duplicate reads through successive application of single-node computing tools for read assembly and mapping. Using a simulated mouse microbiome DNA shotgun sequencing dataset, we demonstrated that this approach shortens assembly time, uses less memory than traditional co-assembly, and produces significantly fewer assembly errors. Applying sequential co-assembly to shotgun sequencing reads from (1) a longitudinal study of gut microbiomes from undernourished Bangladeshi children and (2) a 2.3-terabyte dataset generated from gnotobiotic mice colonized with pooled microbiomes from these children that was too large to be handled by a traditional co-assembly approach also demonstrated significant reductions in assembly time and memory requirements. These results suggest that this approach should be useful in resource-constrained settings, including in low- and middle-income countries.

Despite established links between the infant gut microbiome and health, how complementary feeding shapes colonization remains unclear. Using FoodSeq, a DNA-based dietary assessment technique, we surveyed food intake across 729 children (0-3 y) from North and Central America, Africa, and Asia. We detected 199 unique plant food sequences, with only eight staples shared across all countries. Despite this global variation, early-life diets followed a common trajectory: weaning stage emerged as the dominant dietary signature across populations. Crucially, dietary diversity did not correlate with gut microbial diversity. Instead, dietary diversity and weaning stage specifically predicted the abundance of adult-associated bacteria. Our findings support a two-stage maturation model: an initial milk-dominated phase, followed by a diet-responsive phase that yields adult-like communities. Monitoring and promoting plant dietary diversity may thus support timely microbiome maturation worldwide.

The contribution of placental immune responses to congenital Zika virus (ZIKV) syndrome remains poorly understood. Here, we leveraged a mouse model of ZIKV infection to identify mechanisms of innate immune restriction exclusively in the fetal compartment of the placenta. ZIKV principally infected mononuclear trophoblasts in the junctional zone, which was limited by mitochondrial antiviral-signaling protein (MAVS) and type I interferon (IFN) signaling mechanisms. Single nuclear RNA sequencing revealed MAVS-dependent expression of IFN-stimulated genes (ISGs) in spongiotrophoblasts but not in other placental cells that use alternate pathways to induce ISGs. ZIKV infection of Ifnar1-/- or Mavs-/- placentas was associated with greater infection of the adjacent immunocompetent decidua, and heterozygous Mavs+/- or Ifnar1+/- dams carrying immunodeficient fetuses sustained greater maternal viremia and tissue infection than dams carrying wild-type fetuses. Thus, MAVS-IFN signaling in the fetus restricts ZIKV infection in junctional zone trophoblasts, which modulates dissemination and outcome for both the fetus and the pregnant mother.

Undernutrition in Bangladeshi children is associated with disruption of postnatal gut microbiota assembly; compared with standard therapy, a microbiota-directed complementary food (MDCF) substantially improved their ponderal and linear growth. Here, we characterize a fatty acid amide hydrolase (FAAH) from a growth-associated intestinal strain of Faecalibacterium prausnitzii cultured from these children. This enzyme, expressed and purified from Escherichia coli, hydrolyzes a variety of N -acylamides, including oleoylethanolamide (OEA), neurotransmitters, and quorum sensing N -acyl homoserine lactones; it also synthesizes a range of N -acylamides, notably N -acyl amino acids. Treating germ-free mice with N -oleoylarginine and N -oleolyhistidine, major products of FAAH OEA metabolism, markedly affected expression of intestinal immune function pathways. Administering MDCF to Bangladeshi children considerably reduced fecal OEA, a satiety factor whose levels were negatively correlated with abundance and expression of their F. prausnitzii FAAH. This enzyme, structurally and catalytically distinct from mammalian FAAH, is positioned to regulate levels of a variety of bioactive molecules.

Severe acute malnutrition (SAM), defined anthropometrically as a weight-for-length z-score more than 3 standard deviations below the mean (WLZ<-3), affects 19 million children under 5-years-old worldwide. Complete anthropometric recovery after standard inventions is rare with children often left with moderate acute malnutrition (MAM; WLZ -2 to -3). Here we conduct a randomized controlled trial (RCT), involving 12-18-month-old Bangladeshi children from urban and rural sites, who after hospital-based treatment for SAM received a 3-month intervention with a microbiota-directed complementary food (MDCF-2) or a ready-to-use supplementary food (RUSF) as they transitioned to MAM. The rate of WLZ improvement was significantly greater with MDCF-2 than the more calorically-dense RUSF, as we observed in a previous RCT of Bangladeshi children with MAM without antecedent SAM. A correlated meta-analysis of aptamer-based measurements of 4,520 plasma proteins in this and the prior RCT revealed 215 proteins positively-associated with WLZ (prominently those involved in musculoskeletal and CNS development) and 44 negatively-associated proteins (related to immune activation), with a significant enrichment in levels of the positively WLZ-associated proteins in the MDCF-2 arm. Characterizing changes in 754 bacterial metagenome-assembled genomes in serially collected fecal samples disclosed the effects of acute rehabilitation for SAM on the microbiome, its transition as each child achieves a state of MAM, and how specific strains of Prevotella copri function at the intersection between MDCF-2 glycan metabolism and the rescue of growth faltering. These results provide a rationale for further testing the generalizability of the efficacy of MDCF and identify biomarkers for defining treatment responses.

ABSTRACT Environmental enteric dysfunction (EED) is a subclinical syndrome of altered small intestinal function postulated to be an important contributor to childhood undernutrition. The role of small intestinal bacterial communities in the pathophysiology of EED is poorly defined due to a paucity of studies where there has been a direct collection of small intestinal samples from undernourished children. Sixty-three members of a Pakistani cohort identified as being acutely malnourished between 3 and 6 months of age and whose wasting (weight-for-length Z -score [WLZ]) failed to improve after a 2-month nutritional intervention underwent esophagogastroduodenoscopy (EGD). Paired duodenal luminal aspirates and duodenal mucosal biopsies were obtained from 43 children. Duodenal microbiota composition was characterized by sequencing bacterial 16S rRNA gene amplicons. Levels of bacterial taxa (amplicon sequence variants [ASVs]) were referenced to anthropometric indices, histopathologic severity in biopsies, expression of selected genes in the duodenal mucosa, and fecal levels of an immunoinflammatory biomarker (lipocalin-2). A “core” group of eight bacterial ASVs was present in the duodenal samples of 69% of participants. Streptococcus anginosus was the most prevalent, followed by Streptococcus sp., Gemella haemolysans , Streptococcus australis , Granulicatella elegans , Granulicatella adiacens, and Abiotrophia defectiva . At the time of EGD, none of the core taxa were significantly correlated with WLZ. Statistically significant correlations were documented between the abundances of Granulicatella elegans and Granulicatella adiacens and the expression of duodenal mucosal genes involved in immune responses (dual oxidase maturation factor 2, serum amyloid A, and granzyme H). These results suggest that a potential role for members of the oral microbiota in pathogenesis, notably Streptococcus , Gemella, and Granulicatella species, warrants further investigation. IMPORTANCE Undernutrition among women and children is a pressing global health problem. Environmental enteric dysfunction (EED) is a disease of the small intestine (SI) associated with impaired gut mucosal barrier function and reduced capacity for nutrient absorption. The cause of EED is ill-defined. One emerging hypothesis is that alterations in the SI microbiota contribute to EED. We performed a culture-independent analysis of the SI microbiota of a cohort of Pakistani children with undernutrition who had failed a standard nutritional intervention, underwent upper gastrointestinal tract endoscopy, and had histologic evidence of EED in their duodenal mucosal biopsies. The results revealed a shared group of bacterial taxa in their duodenums whose absolute abundances were correlated with levels of the expression of genes in the duodenal mucosa that are involved in inflammatory responses. A number of these bacterial taxa are more typically found in the oral microbiota, a finding that has potential physiologic and therapeutic implications.

Interleukin 22 (IL-22) promotes intestinal barrier integrity, stimulating epithelial cells to enact defense mechanisms against enteric infections, including the production of antimicrobial peptides. IL-22 binding protein (IL-22BP) is a soluble decoy encoded by the Il22ra2 gene that decreases IL-22 bioavailability, attenuating IL-22 signaling. The impact of IL-22BP on gut microbiota composition and functioning is poorly understood. We found that Il22ra2-/- mice are better protected against Clostridioides difficile and Citrobacter rodentium infections. This protection relied on IL-22-induced antimicrobial mechanisms before the infection occurred, rather than during the infection itself. Indeed, the gut microbiota of Il22ra2-/- mice mitigated infection of wild-type (WT) mice when transferred via cohousing or by cecal microbiota transplantation. Indicator species analysis of WT and Il22ra2-/- mice with and without cohousing disclosed that IL22BP deficiency yields a gut bacterial composition distinct from that of WT mice. Manipulation of dietary fiber content, measurements of intestinal short-chain fatty acids and oral treatment with acetate disclosed that resistance to C. difficile infection is related to increased production of acetate by Il22ra2-/--associated microbiota. Together, these findings suggest that IL-22BP represents a potential therapeutic target for those at risk for or with already manifest infection with this and perhaps other enteropathogens.

Microbiota-directed complementary food (MDCF) formulations have been designed to repair the gut communities of malnourished children. A randomized controlled trial demonstrated that one formulation, MDCF-2, improved weight gain in malnourished Bangladeshi children compared to a more calorically dense standard nutritional intervention. Metagenome-assembled genomes from study participants revealed a correlation between ponderal growth and expression of MDCF-2 glycan utilization pathways by Prevotella copri strains. To test this correlation, here we use gnotobiotic mice colonized with defined consortia of age- and ponderal growth-associated gut bacterial strains, with or without P. copri isolates closely matching the metagenome-assembled genomes. Combining gut metagenomics and metatranscriptomics with host single-nucleus RNA sequencing and gut metabolomic analyses, we identify a key role of P. copri in metabolizing MDCF-2 glycans and uncover its interactions with other microbes including Bifidobacterium infantis. P. copri-containing consortia mediated weight gain and modulated energy metabolism within intestinal epithelial cells. Our results reveal structure-function relationships between MDCF-2 and members of the gut microbiota of malnourished children with potential implications for future therapies.

Synthetic glycans (SGs) containing glycosidic linkages and structures not identified in nature offer a means for deliberately altering microbial community properties. Here pools of SG oligosaccharides were generated via polymerization of monosaccharides and screened for their ability to increase saccharolytic Bacteroides in ex vivo cultures of human fecal samples. A lead SG preparation was orally administered to gnotobiotic mice harboring a consortium of 56 cultured, phylogenetically diverse human gut bacteria and fed a Western diet. The abundances of 3 of 15 Bacteroides strains increased, most prominently B. intestinalis. Underlying mechanisms were characterized by analyzing in vivo expression of the carbohydrate utilization machinery, using retrievable microscopic paramagnetic particles with bound SG oligosaccharides and assaying SG degradation by individual purified B. intestinalis glycoside hydrolases. The results reveal that SGs can selectively co-opt carbohydrate utilization machinery in different human gut Bacteroides and demonstrate a means for identifying artificial carbohydrate structures for targeted bacterial manipulation.

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. 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.

Studies of the microbes living on and in our bodies are conducted mainly in a few rich countries, squandering opportunities to improve the health of people globally.

BACKGROUND:A randomized controlled trial in Bangladeshi children aged 12 to 18 months with moderate acute malnutrition found that dietary supplementation with the microbiota-directed complementary food (MDCF-2) significantly improved weight gain and repaired gut microbiota compared to the ready-to-use supplementary food. However, the MDCF-2 formulation was made daily from locally available ingredients and the need for a packaged, nutritionally compliant, and organoleptically acceptable MDCF-2 prototype was essential for future large-scale clinical studies. OBJECTIVE:The study aimed to develop and assess the acceptability of 3 alternative foil-packaged formulations of MDCF-2 in comparison to current MDCF-2. METHODS:Of the 3 packaged formulations, the Jumpstart version was provided in 2 sachets, the other 2 formulations were provided in a retort-stable foil pouch extended by sterilization, and microbiological growth was monitored over 10 months. The acceptability study included 40 children aged 8 to 12 months living in an urban slum in Dhaka, and the organoleptic properties were assessed using a 7-point hedonic scale. RESULTS:In the 100 g distributed over the 2 sessions, children consumed 82.5 ± 7.84 g (mean ± SD) of kitchen-prepared MDCF-2, 85.4 ± 7.15 g of the "Jumpstart" MDCF-2 formulation, 85.4 ± 8.70 g of the MDCF-2 with green banana powder, and 86.2 ± 4.26 g of the MDCF-2 with sweet potato formulation. The "Jumpstart" MDCF-2 and MDCF-2 with sweet potato achieved the highest overall acceptability scores on the hedonic scale; although none of the shelf-stable formulations were significantly different from the kitchen-prepared MDCF-2. CONCLUSIONS:Packaged, shelf-stable prototypes of MDCF-2 exhibited comparable acceptability among Bangladeshi children aged 8 to 12 months to the original freshly prepared formulation. PLAIN LANGUAGE TITLE:Development and Acceptability of Shelf-Stable Microbiota-Directed Complementary Foods.

Routine coffee consumption is associated with a gut microbiome signature characterized by elevated levels of Lawsonibacter asaccharolyticus. The growth of this bacterium is stimulated by coffee in vitro and its activities may influence the biotransformation of coffee-associated metabolites.

Background Globally, stunting affects similar to 150 million children under five, while wasting affects nearly 50 million. Current interventions have had limited effectiveness in ameliorating long-term sequelae of undernutrition including stunting, cognitive deficits and immune dysfunction. Disrupted development of the gut microbiota has been linked to the pathogenesis of undernutrition, providing potentially new treatment approaches. Methods 124 Bangladeshi children with moderate acute malnutrition (MAM) enrolled (at 12 - 18 months) in a previously reported 3-month RCT of a microbiota-directed complementary food (MDCF-2) were followed for two years. Weight and length were monitored by anthropometry, the abundances of bacterial strains were assessed by quantifying metagenome-assembled genomes (MAGs) in serially collected fecal samples and levels of growthassociated proteins were measured in plasma. Findings Children who had received MDCF-2 were significantly less stunted during follow-up than those who received a standard ready-to-use supplementary food (RUSF) [linear mixed-effects model, beta treatment group x study week (95% CI) = 0.002 (0.001, 0.003); P = 0.004]. They also had elevated fecal abundances of Agathobacter faecis, Blautia massiliensis, Lachnospira and Dialister, plus increased levels of a group of 37 plasma proteins (linear model; FDR-adjusted P < 0.1), including IGF-1, neurotrophin receptor NTRK2 and multiple proteins linked to musculoskeletal and CNS development, that persisted for 6-months post-intervention. Interpretation MDCF-2 treatment of Bangladeshi children with MAM, which produced significant improvements in wasting during intervention, also reduced stunting during follow-up. These results suggest that the effectiveness of supplementary foods for undernutrition may be improved by including ingredients that sponsor healthy microbiotahost co-development. Copyright (c) 2024 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

The gut microbiota and tumor-associated macrophages (TAMs) affect tumor responses to anti–programmed cell death protein 1 (PD-1) immune checkpoint blockade. Reprogramming TAM by either blocking or deleting the macrophage receptor triggering receptor on myeloid cells 2 (TREM2) attenuates tumor growth, and lack of functional TREM2 enhances tumor elimination by anti–PD-1. Here, we found that anti–PD-1 treatment combined with TREM2 deficiency in mice induces proinflammatory programs in intestinal macrophages and a concomitant expansion of Ruminococcus gnavus in the gut microbiota. Gavage of wild-type mice with R. gnavus enhanced anti–PD-1–mediated tumor elimination, recapitulating the effect occurring in the absence of TREM2. A proinflammatory intestinal environment coincided with expansion, increased circulation, and migration of TNF-producing CD4 + T cells to the tumor bed. Thus, TREM2 remotely controls anti–PD-1 immune checkpoint blockade through modulation of the intestinal immune environment and microbiota, with R. gnavus emerging as a potential probiotic agent for increasing responsiveness to anti-PD-1.