Environmental enteric dysfunction (EED), a small intestinal disorder prevalent in undernourished children with stunted growth and their undernourished mothers, is associated with gut mucosal barrier disruption and decreased absorptive capacity. Here we provide preclinical evidence that intergenerational transmission of a perturbed small intestinal microbiota contributes to pathogenesis. One of two bacterial consortia cultured from duodenal aspirates obtained from Bangladeshi children with EED induced local and systemic inflammation in female gnotobiotic mice, resulting in impaired prenatal and postnatal growth in their offspring. Immunologic changes in pups phenocopied features of EED in children, and dam-to-pup transmission of this consortium altered signalling pathways related to intestinal epithelial cell renewal, barrier integrity and immune function. Screening of co-housed mice harbouring the inflammatory or non-inflammatory consortia identified Campylobacter concisus as an inducer of pro-inflammatory cytokine signalling in a host nitric oxide synthase-dependent manner. This preclinical model could facilitate small intestinal microbiota-targeted therapeutics for intergenerational undernutrition.

Studies have implicated perturbations in the postnatal development of the gut microbiome as a contributing factor to childhood undernutrition. Compared to a standard ready-to-use supplementary food, a microbiome-directed complementary food (MDCF-2) designed to repair these perturbations produced superior improvements in ponderal and linear growth in clinical trials of Bangladeshi children with moderate acute malnutrition. Here, "reverse translation" experiments are performed where intact fecal microbiomes collected from trial participants before and at the end of treatment are introduced into female gnotobiotic mice just after delivery of their pups. Pups received diets designed to resemble those consumed by children in the trials to recreate "unrepaired" and "repaired" gut ecosystems. Analyses of the abundances of bacterial strains (metagenome-assembled genomes), their expressed genes, and metabolic products, combined with assessments of ponderal growth and intestinal epithelial lineage transcriptomes (single-nucleus RNA-Seq with follow-up immunocytochemistry) disclosed effects of MDCF-2 associated microbiome repair that cannot be determined, in part because "no treatment" control arms cannot be ethically incorporated into these trials. Specifically, microbiome repair in these mice produced significant increases in ponderal growth, changes in microbial gene expression consistent with a less virulent gut ecosystem and alterations in expression of i) components of cell junctions in the enterocytic and goblet cell lineages, ii) pathways for synthesis and secretion of eicosanoid immune effectors in chemosensory tuft cells, and iii) goblet cell pathways involved in glycosylation and secretion of mucin. Experiments of the type described can help formulate and test hypotheses about how microbiome repair affects host biology.

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.

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.

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.

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.

Preclinical and clinical studies are providing evidence that the healthy growth of infants and children reflects, in part, healthy development of their gut microbiomes1-5. This process of microbial community assembly and functional maturation is perturbed in children with acute malnutrition. Gnotobiotic animals, colonized with microbial communities from children with severe and moderate acute malnutrition, have been used to develop microbiome-directed complementary food (MDCF) formulations for repairing the microbiomes of these children during the weaning period5. Bangladeshi children with moderate acute malnutrition (MAM) participating in a previously reported 3-month-long randomized controlled clinical study of one such formulation, MDCF-2, exhibited significantly improved weight gain compared to a commonly used nutritional intervention despite the lower caloric density of the MDCF6. Characterizing the 'metagenome assembled genomes' (MAGs) of bacterial strains present in the microbiomes of study participants revealed a significant correlation between accelerated ponderal growth and the expression by two Prevotella copri MAGs of metabolic pathways involved in processing of MDCF-2 glycans1. To provide a direct test of these relationships, we have now performed 'reverse translation' experiments using a gnotobiotic mouse model of mother-to-offspring microbiome transmission. Mice were colonized with defined consortia of age- and ponderal growth-associated gut bacterial strains cultured from Bangladeshi infants/children in the study population, with or without P. copri isolates resembling the MAGs. By combining analyses of microbial community assembly, gene expression and processing of glycan constituents of MDCF-2 with single nucleus RNA-Seq and mass spectrometric analyses of the intestine, we establish a principal role for P. copri in mediating metabolism of MDCF-2 glycans, characterize its interactions with other consortium members including Bifidobacterium longum subsp. infantis, and demonstrate the effects of P. copri-containing consortia in mediating weight gain and modulating the activities of metabolic pathways involved in lipid, amino acid, carbohydrate plus other facets of energy metabolism within epithelial cells positioned at different locations in intestinal crypts and villi. Together, the results provide insights into structure/function relationships between MDCF-2 and members of the gut communities of malnourished children; they also have implications for developing future prebiotic, probiotic and/or synbiotic therapeutics for microbiome restoration in children with already manifest malnutrition, or who are at risk for this pervasive health challenge.

Evidence is accumulating that perturbed postnatal development of the gut microbiome contributes to childhood malnutrition1-4. Designing effective microbiome-directed therapeutic foods to repair these perturbations requires knowledge about how food components interact with the microbiome to alter its expressed functions. Here we use biospecimens from a randomized, controlled trial of a microbiome-directed complementary food prototype (MDCF-2) that produced superior rates of weight gain compared to a conventional ready-to-use supplementary food (RUSF) in 12-18-month-old Bangladeshi children with moderate acute malnutrition (MAM)4. We reconstructed 1000 bacterial genomes (metagenome-assembled genomes, MAGs) present in their fecal microbiomes, identified 75 whose abundances were positively associated with weight gain (change in weight-for-length Z score, WLZ), characterized gene expression changes in these MAGs as a function of treatment type and WLZ response, and used mass spectrometry to quantify carbohydrate structures in MDCF-2 and feces. The results reveal treatment-induced changes in expression of carbohydrate metabolic pathways in WLZ-associated MAGs. Comparing participants consuming MDCF-2 versus RUSF, and MDCF-2-treated children in the upper versus lower quartiles of WLZ responses revealed that two Prevotella copri MAGs positively associated with WLZ were principal contributors to MDCF-2-induced expression of metabolic pathways involved in utilization of its component glycans. Moreover, the predicted specificities of carbohydrate active enzymes expressed by polysaccharide utilization loci (PULs) in these two MAGs correlate with the (i) in vitro growth of Bangladeshi P. copri strains, possessing differing degrees of PUL and overall genomic content similarity to these MAGs, cultured in defined medium containing different purified glycans representative of those in MDCF-2, and (ii) levels of carbohydrate structures identified in feces from clinical trial participants. In the accompanying paper5, we use a gnotobiotic mouse model colonized with age- and WLZ-associated bacterial taxa cultured from this study population, and fed diets resembling those consumed by study participants, to directly test the relationship between P. copri, MDCF-2 glycan metabolism, host ponderal growth responses, and intestinal gene expression and metabolism. The ability to identify bioactive glycan structures in MDCFs that are metabolized by growth-associated bacterial taxa will help guide recommendations about use of this MDCF for children with acute malnutrition representing different geographic locales and ages, as well as enable development of bioequivalent, or more efficacious, formulations composed of culturally acceptable and affordable ingredients.

Conotruncal heart defects (CTDs) are heart malformations that affect the cardiac outflow tract and typically cause significant morbidity and mortality. Evidence from epidemiological studies suggests that maternal folate intake is associated with a reduced risk of heart defects, including CTD. However, it is unclear if folate-related gene variants and maternal folate intake have an interactive effect on the risk of CTDs. In this study, we performed targeted sequencing of folate-related genes on DNA from 436 case families with CTDs who are enrolled in the National Birth Defects Prevention Study and then tested for common and rare variants associated with CTD. We identified risk alleles in maternal MTHFS (ORmeta = 1.34; 95% CI 1.07 to 1.67), maternal NOS2 (ORmeta = 1.34; 95% CI 1.05 to 1.72), fetal MTHFS (ORmeta = 1.35; 95% CI 1.09 to 1.66), and fetal TCN2 (ORmeta = 1.38; 95% CI 1.12 to 1.70) that are associated with an increased risk of CTD among cases without folic acid supplementation. We detected putative de novo mutations in genes from the folate, homocysteine, and transsulfuration pathways and identified a significant association between rare variants in MGST1 and CTD risk. Results suggest that periconceptional folic acid supplementation is associated with decreased risk of CTD among individuals with susceptible genotypes.

mass spectrometry to directly characterize the metabolic profiles of discrete gut cell populations

Disk diffusion is a slow but reliable standard method for measuring the antimicrobial susceptibility of microorganisms. Our objective was to improve the turnaround time for this method by reducing the time that cultures are incubated before setting up disk diffusion testing. For initial method development, clinical isolates (n = 13) and quality control strains (n = 8) of bacteria were inoculated on blood agar and were incubated at 35 degrees C for either 6, 10, or 24 h before performing disk diffusion testing, in triplicate, using a panel of clinically appropriate antimicrobial agents. Disk diffusion zone sizes were interpreted using Clinical and Laboratory Standards Institute (CLSI) guidelines. Compared to standard 24 h of incubation, early 6-h growth had 1.3% major errors (MEs) and 1.9% very major errors (VMEs), whereas 10-h growth yielded 0.7% MEs and no VMEs. Categorical agreement with standard incubation was similar for both 6 h (96.7%) and 10 h (96.7%) growth. Inhibitory zone size from 6 h (r(2) = 0.98) and 10 h (r(2) = 0.99) growth correlated well with results from standard conditions. Based on these results, we performed disk diffusion under optimized conditions (6 h growth), using 100 additional clinical isolates, demonstrating a high level of categorical agreement (917 of 950 measurements [96.5%]; 95% confidence interval [CI], 95.2 to 97.5%), as well as no VMEs or MEs. Using early growth for disk diffusion testing is a simple and accurate method for susceptibility testing that can reduce time to results by as much as 18 h, compared to standard incubation, with no additional supply costs or equipment/instrumentation.

Plant fibers in byproduct streams produced by non-harsh food processing methods represent biorepositories of diverse, naturally occurring, and physiologically active biomolecules. To demonstrate one approach for their characterization, mass spectrometry of intestinal contents from gnotobiotic mice, plus in vitro studies, revealed liberation of N-methylserotonin from orange fibers by human gut microbiota members including Bacteroides ovatus. Functional genomic analyses of B. ovatus strains grown under permissive and non-permissive N-methylserotonin "mining" conditions revealed polysaccharide utilization loci that target pectins whose expression correlate with strain-specific liberation of this compound. N-methylserotonin, orally administered to germ-free mice, reduced adiposity, altered liver glycogenesis, shortened gut transit time, and changed expression of genes that regulate circadian rhythm in the liver and colon. In human studies, dose-dependent, orange-fiber-specific fecal accumulation of N-methylserotonin positively correlated with levels of microbiome genes encoding enzymes that digest pectic glycans. Identifying this type of microbial mining activity has potential therapeutic implications.

ABSTRACTEthanol-water extraction of phenolics from cowpeas was modeled and optimized by response surface methodology (RSM). The ethanol concentration and extraction temperature were shown to have a significant effect on phenolic extraction and antioxidant capacity. Modeling predicted that extraction of phenolics from cowpea flour for 42.8 minutes at 58.6°C with 58.4% ethanol would maximize the radical scavenging capacity of solutes. Extraction of phenolics under these optimized conditions yielded 11.05 ± 0.10 mg chlorogenic acid equivalents (CAE)/g cowpea flour. These extracts contained 10.41 % ± 0.11% phenolics by weight and had an antioxidant capacity of 0.45 ± 0.02, closely approximating the predicted phenolic content of 10.11% ± 0.44% and antioxidant capacity of 0.42 ± 0.04. Extracted material was characterized by HPLC, and the predominant phenolic compounds detected were epicatechin and ferulic acid. Cowpea’s low cost, ease of storage, and high antioxidant capacity reflect their potential for use as a naturally-derived antioxidant additive in foods.

Programs for treating malnutrition in children should consider how food formulations affect postnatal gut microbiome development.

Our objective was to evaluate the diagnostic yield and accuracy of the BioFire FilmArray pneumonia panel (BFPP) for identification of pathogens in lower respiratory tract specimens ( n = 200) from emergency department (ED) and intensive care unit (ICU) patients at a tertiary care academic medical center. Specimens were collected between January and November 2018, from patients ≥18 years of age, and culture was performed as part of standard-of-care testing.

Abstract Background Heparin-induced thrombocytopenia (HIT) is a life-threatening complication of exposure to heparin that is caused by autoantibodies against heparin-PF4 complexes. We recently changed our in-house HIT screening platform from a manual, daily batched ELISA (Stago-Asserochrom HPIA Immunoassay) to an automated, on-demand latex immunoturbidimetric assay (LIA, HemosIL HIT-Ab) and have also implemented a reflex from a positive LIA result to the confirmatory serotonin release assay (SRA). We compared the two methods in terms of utilization, test performance, and turnaround time. Methods Data were collected retrospectively from a 7-month period before (June-December 2017) and after (June-December 2018) implementation of the HemosIL LIA in the clinical laboratory at a large academic institution. This study includes consecutive test results from adults (median age: 64 years, range: 19-98 years) seen at our 1,300-bed main hospital. Test utilization, turnaround time (sample receipt to verification), and test performance characteristics were compared between the two methods. Repeat testing was excluded from the analysis. Samples with a positive result on the HemosIL LIA were reflexed to a serotonin release assay (SRA), performed at a large reference laboratory, whereas samples tested with the earlier ELISA assay were referred for SRA testing based upon clinical judgment. When performed, SRA was considered the gold standard for diagnosis of HIT. Results During the 7 months before and after switching methods, there were 109 of 594 (18.4%) positive ELISA results and 45 of 523 (8.6%) positive LIA results. Only 90 of 109 (82%) of the positive results from the ELISA HIT Ab test were sent out by clinicians for SRA testing, whereas 45 of 45 (100%) of the positive results from LIA testing were reflexed to SRA per protocol. Although fewer LIA tests were sent out for SRA testing, there were an equal number of SRA-confirmed cases of HIT with the ELISA (PPV: 16/90 [17.8%]) and LIA methods (PPV: 16/45 [35.6%]), resulting in a high positive predictive value (PPV) with the newly implemented method. Not only was the PPV higher with the LIA test, but it had a significantly shorter mean turnaround time of 96 minutes compared to the ELISA TAT of 1,234 minutes (P < .0001). Conclusions With the new testing protocol, patients received results faster (average 96-minute TAT) and had fewer false-positive results (74/594 pre vs 29/523 post), with no apparent reduction in detection of true-positive cases of HIT (16/594 pre vs 16/523 post).

BACKGROUND: Pneumatic tube systems (PTSs) provide rapid transport of patient blood samples, but physical stress of PTS transport can damage blood cells and alter test results. Despite this knowledge, there is limited information on how to validate a hospital PTS. METHODS: We compared 2 accelerometers and evaluated multiple PTS routes. Variabilities in PTS forces over the same routes were assessed. Response curves that demonstrate the relationship between the number and magnitude of accelerations on plasma lactate dehydrogenase (LD), hemolysis index, and potassium in PTS-transported blood from volunteers were generated. Extrapolations from these relationships were used to predict PTS routes that may be prone to false laboratory results. Historical data and prospective patient studies were compared with predicted effects. RESULTS: The maximum recorded g-force was 10g for the smartphone and 22g for the data logger. There was considerable day-to-day variation in the magnitude of accelerations (CV, 4%-39%) within a single route. The linear relationship between LD and accelerations within the PTS revealed 2 PTS routes predicted to increase LD by >= 20%. The predicted increase in LD was similar to that observed in patient results when using that PTS route. CONCLUSIONS: Hospital PTSs can be validated by documenting the relationship between the concentrations of analytes in plasma, such as LD, with PTS forces recorded by 3-axis accelerometers. Implementation of this method for PTS validation is relatively inexpensive, simple, and robust. (c) 2019 American Association for Clinical Chemistry

Background The development of conotruncal heart defects (CTDs) involves a complex relationship among genetic variants and maternal lifestyle factors. In this article, we focused on the interactions between 13 candidate genes within folate, homocysteine, and transsulfuration pathways for potential association with CTD risk. Methods Targeted sequencing was used for 328 case-parental triads enrolled in the National Birth Defects Prevention Study (NBDPS). To evaluate the interaction of two genes, we applied a conditional logistic regression model for all possible SNP pairs within two respective genes by contrasting the affected infants with their pseudo-controls. The findings were replicated in an independent sample of 86 NBDPS case-parental triads genotyped by DNA microarrays. The results of two studies were further integrated by a fixed-effect meta-analysis. Results One SNP pair (i.e., rs4764267 and rs6556883) located in gene MGST1 and GLRX, respectively, was found to be associated with CTD risk after multiple testing adjustment using simpleM, a modified Bonferroni correction approach (nominal p-value of 4.62e-06; adjusted p-value of .04). Another SNP pair (i.e., rs11892646 and rs56219526) located in gene DNMT3A and MTRR, respectively, achieved marginal significance after multiple testing adjustment (adjusted p-value of .06). Conclusion Further studies with larger sample sizes are needed to confirm and elucidate these potential interactions.