Malnutrition and Dietary Repair
Undernourishment of childhood is accompanied by the growth muscle and immaturity of the intestinal microbial. Even after therapeutic intervention with common commercial supplemental foods, children may fail to thrive. Gehrig [etal and Raman et al. monitored metabolic parameters in healthy Bangladeshi children and those recovering from severe acute malnutrition. The authors examined the interactions between therapeutic diet, microbiota development and growth recovery. Diets were then designed using pig and mouse models to tie the microbiote to a mature post-weaning condition that can be expected to support the baby's growth. These were first tested in mice inoculated with age-characteristic intestinal microbiota. The designed diets led to the maturation of the children's microbiota and put their metabolic and growth profiles on a healthier pathway.
Science this issue p. Eaau4732, p. eaau4735
Repair of microbial obsolescence and determination of the degree to which such repair restores healthy growth requires the identification of microbial targets which are not only biomarkers of community assembly but also mediators of various aspects of growth. Identification of ingredients in complementary foods consumed during the transition from exclusive milk feeding to a fully weaned state, which increases the representation and expressed beneficial functions of growth-promoting bacterial taxa in the developing microbiote can provide an effective, affordable, culturally acceptable and sustainable methodomic and proteomic assay of Serially collected plasma samples were combined with metagenomic assays of serially collected faecal samples from severe acute malnutrition (SAM) children treated with standard treatment. The results provided a reading of their biological properties which they switched from SAM to a state of persistent moderate acute malnutrition (MAM) with consequent persistent microbiota abnormality. Significant correlations were identified between the levels of plasma proteins, anthropometry, plasma metabolites and the representation of bacteria in their microbiota. Gnotobiotic mice were then colonized with a defined consortium of bacterial strains representing different phases of microbiota development in healthy children from Bangladesh. Administration of various combinations of Bangladeshis complementary food ingredients to colonized mice and bacterial-free controls revealed diet-dependent increases in abundance and changes in the metabolic activities of targeted weaning phase strains as well as diet and colon-dependent increased growth of growth-promoting host signaling pathways. Host and microbial effects of prototypes with microbial complementary food (MDCF) were later investigated in gnotobiotic mice colonized with immature microbiots from children with post-SAM MAM and in gnotobiotic piglets colonized with a defined consortium of targeted age and growth discriminatory tariffs. A randomized double-blind study of standard therapy versus various MDCF prototypes derived from these preclinical models, conducted in Bangladeshi children with MAM, identified a leading MDCF as increasing levels of biomarkers and mediators of growth, bone formation, neurodevelopment and immune function against a similar condition. healthy children. Using an approach inspired by statistical methods applied to the financial markets, we show in the attached document by Raman et al .
These findings show the translatability of results obtained from preclinical gnotobiotic animal models to humans, directly supports the hypothesis that healthy microbiota development is a causal link to healthy growth, illustrating a way to treat childhood malnutrition, and capable of deliberately reconfiguring immature microbiota , propose a means to decipher how parts of gut microbial communities work to regulate various host systems involved in healthy growth.
To investigate contributions from impaired gut microbial community development to childhood and nutrition, we combined metabolomic and proteomic analyzes of plasma samples with metagenomic assays of fecal samples to characterize the biological condition of Bangladeshi children with severe acute malnutrition (SAM) then after standard treatment they switched to moderate acute malnutrition (MAM) with persistent microbiota immaturity. Host and microbial effects of prototypes with microbiotic complementary food (MDCF) targeting weaning bacterial taxa underrepresented in SAM and MAM microbiotes were characterized by gnotobiotic mice and gnotobiotic piglets colonized with age and growth discriminating bacteria. A randomized, double-blind controlled feed study identified a lead MDCF that alters the diversity of targeted bacteria and increases the plasma biotomorphs and mediators of growth, bone formation, neurodevelopment and immune function in children with MAM.
Evidence accumulates the disruption of "normal" gut community (microbiota) development can contribute to the pathogenesis of malnutrition. Using culture-independent studies, bacterial memberships have been defined in fecal samples collected each month during the first two postnatal years of healthy members of a birth cohort living in an urban slum (Mirpur) in Dhaka, Bangladesh ( 1 2 ) . Application of Machine Learning [Random Forests (RF)] to the Resulting 16 [ribosomalDNARatio(rDNA)gavea"sparse"modelconsistingofthemostage-discriminatingbacterialstrains;changesintherelativeabundanceoftheseorganismsdescribedaprogramfornormalmicrobioticdevelopment( 2 ). This RF-derived model was then used to characterize fecal samples collected from children from Bangladesh with severe acute malnutrition [SAM;definite-weight-for-height z -score (WHZ)> 3 standard deviations under the median for a health society for the World Health Organization. (WHO) 3 )]. The results revealed gut communities that resembled the healthy children who were chronologically younger. This microbiota "immaturity" was more pronounced in children with SAM compared to those with moderate acute malnutrition (MAM, WHZ scores between -2 and -3) and was not repaired in a clinical trial that tested the effects of two therapeutic foods (). 2 ).
Weakened microbiota development has also been documented in underground Malawian children ( 4 ). To investigate the functional significance of this deterioration, microbial communities were transplanted from healthy and diseased or underweight 6- and 18-month-old Malawian children to groups of newly-awaited sex-free mice who received a diet representative of that consumed by the human donor population. The results showed that compared to mice colonized with normal maturing microbiota from the healthy donors, animals such as outmoded microbiota exhibited reduced rates of lean body mass enhancement, changes in bone growth and metabolic abnormalities ( 4 ). These studies provided preclinical evidence of causality between microbiota immaturity and malnutrition; They also revealed that a subset of the age-discriminating tribes is growth discriminatory. In addition, a cultured consortium of these age- and growth-discriminating tariffs improved the weakened growth phenotype transmitted to recipient gnotobiotic mice by an immature microbiota ( 4 ).
An issue arising from these observations is how do we design optimal foods that control a microbiota in an age-appropriate and healthy state? Breastfeeding plays an important role in reducing the childcare industry. As such, WHO and UNICEF recommend exclusive breastfeeding during the first 6 months of postnatal life and continued breastfeeding after the introduction of supplemental foods up to 24 months ( 5 ). Suboptimal complementary feeding is important contributors to malnutrition in children under 2 years of age ( 6 ). The current supplemental feeding instructions, however, are not based on knowledge of how food affects the developmental biology of the intestinal microbiota during the weaning process. Together, these observations raise the issue: Do some complementary food ingredients or combinations of ingredients allow to selectively increase the representation and express beneficial functions of age and growth discriminating strains lacking SAM or MAM associated microbiota? If the answer is yes, the prescribed feeding of these ingredients can help "repair" or prevent the development of microbiota immaturity in children, with potentially persistent health promoting effects.
Here we describe a process for identifying microbiota-directed complementary foods (MDCF) designed to treat children with acute malnutrition. We first characterized the gut microbial community and host responses for 12 months in Bangladeshi children who were treated for SAM with one of three conventional therapeutic foods. Measurement of the levels of 1305 human plasma proteins including regulators and effectors of physiological, metabolic and immune functions combined with mass spectrometric profiling of plasma metabolites and culture-independent assays of serially collected fecal samples gave a "reading" of the biological properties of these children as they evolved from SAM to a state of incomplete recovery (post-SAM MAM) with persistent microbiota obscurity. This reading contained correlations between plasma proteins, anthropometry, plasma base metrics and representation of age-discriminating members in their microbiota. We then screened complementary foods in gnotobiotic mice colonized with a consortium of bacterial strains grown from children living in Mirpur to identify ingredients that promote the representation of constituent age-discriminating strains that are under-represented in the determination of acute malnutrition. Subsequently, a representative microbiota was transplanted from a child with post-SAM MAM to gnotobiotic mice. Receiving animals were fed a diet similar to that consumed by children in Mirpur but supplemented with ingredients identified on the screen to determine if one or more of these MDCF formulations could repair a microbiota from an individual who had already received conventional therapy. Lead formulations were then tested on gnotobiotic piglets colonized with a defined consortium of age and growth discriminating strains to test their biological effects in a host species that is physiologically and metabolically more similar to humans than mice. Lastly, three MDCF prototypes were administered to children with MAM and their effects on microbiotic and host biological conditions were determined.
Effects of Conventional Therapeutic Foods on Children's Biological Condition with SAM
A total of 343 children in Bangladesh aged 6 to 36 months with SAM enrolled in a multi-center randomized double-blind "noninferiority" study comparing two locally produced therapeutic foods ( complementary materials, materials and methods) with a commercially available, ready-treated therapeutic food (RUTF) () (study design provided in Figure 1A and the compositions of these therapeutic foods are available in Table S1A). Children received standard care for SAM during the acute stabilization phase of hospital treatment, including a brief course of antibiotics. Eligible children were then randomized to one of the three therapeutic arms (~ 200 kcal / kg / day, mean 16.1 ± 10.3 days) (Table S1B). Children were released according to meeting criteria described in additional materials, materials and methods. In a subset of 54 children, fecal samples were collected at enrollment [age 15.2 ± 5.1 months (mean ± SD)] prior to randomization, twice during treatment with a therapeutic food, and at regular intervals up to 12 months after discharge (Figure 1A; clinical metadata is provided in Table S1B). Blood samples (plasma) were also obtained during enrollment, discharge and 6 months after discharge for targeted mass spectrometry (MS) -based metabolic profiling; A sufficient amount of blood was obtained from eight children at all three times for aptamer-based proteomic analysis ( 8 – ) ). Of these children, 44% had MAM at 12 months of follow-up. None of the therapeutic foods had a significant effect on their severe stunt score (HAZ)] (Figure 1B and Table S1B).
Significant correlations between levels of plasma proteins, anthropometric indices, plasma metabolites and host signal pathways that regulate important growth factors are described in the supplemental text, the results (Table S3, A and B) -example growth hormone (GH) -IGF components. , including soluble growth hormone receptor (also known as growth hormone binding protein), multiple IGF binding proteins (IGFBP) and regulators of IGFBP turnover (metalloprotease cardiac lysine-1 and its inhibitor stanniocalcin-1). Intestinal Microbioma [sparseRF-derivedmodelofnormalgutmicrobiotadevelopmentinvolvingbacterialtaxa[operational taxonomic units (OTUs)] and obtained from healthy members of a birth cohort living in Mirpur (Table S4, A to C) was applied to bacterial V4. R16 dataset generated from serial fecal samples is retrieved from the children in the SAM study ( n = 539 samples). This model allowed us to define microbiota for age scores (MAZ) as a function of treatment arm and time [9.3 ± 3.7 samples/child (mean ± SD)]. The MAZ value measures the deviation in the development of a child's microbiota from that of chronologically age-related reference healthy children based on the representation of the ensemble of age-discriminating strains found in the RF-derived model ( 2 ]). Significant microbiota omotide was evident in the SAM and post SAM MAM groups (Fig. 1C and Table S5A). In addition, MAZ scores in this SAM cohort were significantly correlated with WHZ, HAZ and WAZ [Pearsoncorrelationcoefficient( r ) = 0.16, P = 0.0004; R [0.145] = [0.005]; and = 0.10, P = 0.02, respectively]. The MAZ score was no different at discharge but improved 1 month later ( P = 0.0051 for entry, Mann-Whitney test). This improvement may reflect increased dietary diversity, reduced use of antibiotics (Table S1B) and / or other factors associated with returning to the home environment. MAZ scores did not change significantly thereafter (Figure 1B).
A number of the age-discriminating strains were significantly correlated with anthropometric indices as well as with plasma proteins involved in biological processes such as media growth. We also identified significant negative correlations between these rates and mediators of systemic inflammation and anorexia / cachexia. Bifidobacterium longum (OTU 559527) had the largest number of significant correlations (114) [table S3C; further discussion is available in supplementary text, results].
The effects of the therapeutic food interventions on the representation of metabolic pathways in the intestinal microbiome were defined by shotgun sequencing of 331 fecal DNA samples obtained from 30 members of the Mirpur birth cohort with consistent healthy anthropometry and 15 of the 54 children enrolled in the SAM study ( Table S5B); These 15 children were selected by their age (12-18 months) and that we had corresponding plasma metabolomic and proteomic data sets for at least two of the three times sampled. The amounts of microbial genes that map pathways in the microbial communities of the SEED (mcSEED) database ( 12 ) – related to the metabolism of amino acids, carbohydrates, fermentation products and B vitamins and related cofactors were first defined in healthy children sampled. monthly from birth to 2 years. A set of age-discriminating metabolic pathways (mcSEED "subsystems" or path modules) were identified. The resulting sparse RF-derived model (Figs. S1, A and B, and materials and methods) allowed us to assign a developmental state (functional age or "maturity") to the fecal microbiomas of the 15 children treated for SAM. Relative functional maturity was significantly correlated with MAZ, WHZ and WAZ scores during the experiment (Pearson r and P values are MAZ, r = 0, P = 0.0011; P = [0.23] P P P P P P = [0.013]. When enrolling and just before administering therapeutic foods, children with SAM had more immature microbiomas [onewayanalysisvariance(ANOVA) P = 0.0002; Dunnett's multiple comparison test for healthy compared to SAM adjusted P values at the two time points, 0.027 and 0.0001, respectively. There was a statistically significant improvement in functional maturity from the initiation of therapeutic food treatment for delivery and at 1 and 6 months after discharge (Tukey's multiple comparison test, adjusted P = 19399, 0.0028 and 0.025, respectively). ). However, this improvement did not occur at later times (Fig. S1D). Comparing the relative excesses of the 30 most discriminatory pathways at six times revealed that the SAM microbiome had significantly reduced representation of (i) amino acid metabolic pathways, including those involved in isoleucine, leucine, valine biosynthesis and uptake; (ii) multiple carbohydrate utilization pathways (arabinose and arabinosides, rhamnose and rhamnogalacturonan and sialic acid); and (iii) multiple pathways involved in vitamin B metabolism, including "niacin / NADP (nicotinamide adenine dinucleotide phosphate) biosynthesis" (Figs. S1E and Table S5C). The observed under-representation of age-discriminatory OTUs and metabolic pathways in the gut communities of children with post-SAM MAM provided the basis for developing a pipeline to test complementary food ingredients for their ability to repair this inhumanity.
Screening complementary food ingredients  Nine age-discriminating bacterial strains were grown from the fecal microbiote of three healthy children aged 6 to 23 months living in Mirpur, and genomes of these isolates were sequenced (Table S6, A and C). Seven of the nine isolates had V4-16 rDNA sequences corresponding to age-discriminating OTUs, the representation of which is associated with the period of complementary food consumption ("weaning phase" OTU) (Fig. S2A), while two, Bifidobacterium longum subsp. Infantis and Bifidobacterium letters are most prominent during the period of exclusive, predominant milk feeding (Fig. S2A) ( 13 ). OTUs representing seven of the nine cultured strains were significantly depleted in the fecal microbiota of Bangladeshi children with SAM prior to treatment (Table S7 and Fig. S3). Seven additional age-discriminating strains were grown from the immature fecal microbiote of a 24-month-old SAM child enrolled in the same study as the subcohort shown in Figure 1 (Table S6, A and C). Together, the consortium of 16 strains represented OTUs that directly matched 65.6 ± 22.8% (mean ± SD) of V4-16 rDNA sequencing generated from 1039 fecal samples collected from 53 healthy members of Mirpur. birth cohort during the first 2 postnatal years and 74.2 ± 25.2% of the readers made from fecal samples obtained from 38 children with SAM (Table S7).
In order to identify complementary foods that selectively increase the representation of the age-discriminatory strain of the rejection phase deficient in immature SAM-associated microbiota, we colonized 5-week-old, bacterial-free C57B1 / 6J mice with the consortium of cultured, sequenced bacterial strains. After colonization, an 8-week period of diet "oscillations" was initiated (Fig. S2B). We incorporated 12 complementary food ingredients commonly consumed in Mirpur ( 6) into 14 different diets using a random sampling strategy (Table S8, A to E, and materials and methods). The composition of these complementary food combinations (CFCs) and their order of administration to mice was based on considerations described in the legend of Figs. S2, B and C. Spearman's rank correlation coefficients were calculated between the relative excesses of the 14 bacterial strains as colonized mice and levels. of complementary food ingredients in the 14 CFCs tested (Fig. S2D and Table S9A). Chickpeas and bananas had strong positive correlations with the largest number of strains representing weaning phase with age discrimination. Tilapia had a narrower number of significant positive effects (Fig. S2D). Chickpeas, bananas and tilapia also had significant negative correlations with the levels of the prophylactic milk-boron B. longum subsp.  infantis isolate. A sobering observation was that a number of complementary food ingredients typically represented in diets consumed by 18-month-old children living in Mirpur had significant negative correlations with six dilution phase with age discrimination, including rice, milk powder, potato, spinach and sweet pumpkin (Fig. S2D). ). Rice grains with milk are the most common first complementary food given to children from Bangladesh ( 14 ). Furthermore, eggs contained in a number of regimens for nutritional rehabilitation of children with acute malnutrition (19459007) are negatively correlated with the abundance of two weaning phase strains, [Dor. ] Blautia luti .
Testing an Initial MDCF Prototype
Khichuri-halwa (KH) is a therapeutic food administered jointly with milk sugary (MS) to Mirpur children with SAM. A previous study documented the inability of this procedure to repair the stomach microbiota inability ( 2 ). We prepared a diet that mimicked MS and KH (MS / KH) (Table S8, D and E); 7 of its 16 ingredients are commonly consumed complementary foods that had little, if any, effect on the representation of the weaning phase's age-discriminating strains (rice, red lentils, potatoes, pumpkin, spinach, whole meal flour, and powdered milk) (Fig. S2D). The effects of MS / KH on the members of the 14-member consortium and values were compared to those prepared by an initial MDCF prototype containing chickpeas, bananas and tilapia (Table S9B). Five-week-free C57B1 / 6J mice colonized with the consortium were fed monotonically with either of the two ad libitum diets for 25 days.
Microbial community responses
Community profiling using short read shotgun sequencing (COPRO-seq) of cecal DNA revealed that compared to MS / KH, consumption of the MDCF prototype resulted in significantly higher relative redundancies of a number of phase-out age discriminating rates, including Faecalibacterium prausnitzii Dorea longicatena and B. luti ( P <0.01; Mann-Whitney test) (Figure 3A and Table S9B). This prototype did not promote the suitability of SAM donor-derived strains, with the exception of Escherichia Fergusonii .
Vi använde riktade MS för att kvantifiera cecalnivåer av kolhydrater, kortkedjiga fettsyror, plus amino acids and their catabolites (table S10, A to D). Germ-free animals served as reference controls to define levels of cecal nutrients that, by inference, would be available for bacterial utilization in the different diet contexts. There were several noteworthy findings: (i) Levels of butyrate and succinate were significantly higher in colonized animals consuming MDCF compared with MS/KH (Fig. 3B and table S10B). (ii) There were no statistically significant diet-associated differences in levels of any of the amino acids measured in germ-free animals, but when compared with their colonized MS/KH–fed counterparts, colonized MDCF-consuming animals had significantly elevated cecal levels of six amino acids classified as essential in humans (the three branched-chain amino acids leucine, isoleucine, and valine plus phenylalanine and tryptophan) (Fig. 3C and table S10C). And (iii) two tryptophan-derived microbial metabolites that play important roles in suppressing inflammation and are neuroprotective, 3-hydroxyanthranillic acid (3-HAA) and indole-3-lactic acid (16–21), were significantly elevated in colonized animals fed MDCF compared with their MS/KH–treated counterparts (table S10D).
Findings from RNA-sequencing (RNA-seq) analysis of the transcriptional responses of community members to the two diets based on Kyoto Encyclopedia of Genes and Genomes (KEGG)– and mcSEED-derived annotations of the 40,735 predicted protein-coding genes present in consortium members are described in tables S9C and S11, A to C, and supplementary text, results, and in silico predictions of their ability to produce, use, and/or share nutrients are provided in table S6, D and E. For example, community-level analysis revealed specific members manifested MDCF-associated increases in expression of genes involved in biosynthe sis of the essential amino acids, including branched-chain amino acids (Ruminococcus obeum and Ruminococcus torques), and generation of aromatic amino acid metabolites (R. obeumR. torquesand F. prausnitzii) (table S11C, ii).
Serum levels of IGF-1 were significantly higher in colonized mice that consumed the initial MDCF prototype compared with those that consumed MS/KH. This effect was diet- and colonization-dependent, with germ-free animals exhibiting significantly lower levels of IGF-1 in both diet contexts (Fig. 3D). Serum insulin levels were also higher in colonized animals that consumed MDCF compared with MS/KH [8007±3029ng/mL(mean±SD)versus5187±1351ng/mLrespectively;P = 0.06; unpaired t test].
IGF-1 binding to its receptor tyrosine kinase, IGF-1R, affects a variety of signal transduction pathways, including one involving the serine/threonine kinase Akt/PKB, phosphatidylinositol-3 kinase (PI3K), and the mammalian target of rapamycin (mTOR). Absorption of several amino acids from the gut—notably, branched-chain amino acids and tryptophan—leads to activation of mTOR (22). Colonized animals fed MDCF had significantly higher levels of hepatic phosphoSer473-Akt, which is consistent with activation of Akt by IGF-1 signaling through the PI-3K pathway (Fig. 3E). Levels of phospho–AMPK (5′ adenosine monophosphate-activated protein kinase) were not significantly affected by diet (Fig. 3E), suggesting that Akt phosphorylation is not caused indirectly by altered hepatic energy status. Phosphorylation of hepatic Jak 2 (Tyr1007/1008) and mTOR (Ser2448), which are involved in IGF-1 production, was significantly increased in colonized mice consuming MDCF (Fig. 3E), whereas phosphorylation of STAT5, also implicated in IGF-1 production, was not significantly altered.
Previous studies of adult germ-free mice reported increases in serum IGF-1 after their colonization with gut microbiota from conventionally raised mice; increased IGF-1 levels were also associated with increased bone formation (2324). Micro-computed tomography (μCT) of mouse femurs revealed a significant increase in femoral cortical bone area in MDCF-fed animals; the effect was both diet- and microbiota-dependent (Fig. 3F).
We used targeted MS to quantify levels of amino acids, acyl–coenzyme As (acylCoAs), acylcarnitines, and organic acids in serum, liver, and gastrocnemius muscle (table S12). Products of nonoxidative metabolism of glucose and pyruvate (lactate from glycolysis and alanine from transamination of pyruvate, respectively) were significantly lower in mice fed MDCF compared with mice fed MS/KH; this was true for alanine in serum, skeletal muscle, and liver and for lactate in liver (table S12, A to C and H). Oxidative metabolism of glucose is associated with nutritionally replete, anabolic conditions. These findings are consistent with the observed elevations of the anabolic hormone IGF-1 in MDCF-fed compared with MS/KH–fed mice. MDCF-fed mice had significantly higher circulating levels of branched-chain amino acids than those of their MS/KH–fed counterparts (Fig. 3G and table S12, A to C). Skeletal muscle C5 carnitine and the closely related metabolite C5-OH/C3 carnitine were significantly higher in animals consuming MDCF (Fig. 3G and table S12F). In liver, C3 and C5 acylcarnitines were significantly lower in MDCF-treated mice (Fig. 3G and table S12E), suggesting that the more nutritionally replete state associated with MDCF may act to limit branched-chain amino acid oxidation in this tissue.
Testing additional MDCF prototypes in gnotobiotic mice
Incorporating tilapia into MDCF prototypes poses several problems: Its organoleptic properties are not desirable, and its cost is higher than commonly consumed plant-based sources of protein. To identify alternatives to tilapia, we selected an additional 16 plant-derived complementary food ingredients with varied levels and quality of protein (25) that are culturally acceptable, affordable, and readily available in Bangladesh (fig. S4A and table S13, A and B). Their effects were tested in gnotobiotic mice colonized with a defined, expanded consortium of 18 age- and growth-discriminatory bacterial strains (table S6A). We generated 48 mouse diets by supplementing a prototypic base diet representative of that consumed by 18-month-old children living in Mirpur (Mirpur-18), with each of the individual ingredients incorporated at three different concentrations (fig. S4A and table S13A). The results revealed that in this defined community context, peanut flour had the greatest effect on the largest number of targeted weaning-phase age-discriminatory taxa, followed by chickpea flour (fig. S4B and table S13C). Soy flour, which promoted the representation of two of these taxa, had the second-highest percentage protein after peanut flour (fig. S4A), and its protein quality was among the highest of the ingredients tested (table S13B). On the basis of these observations, we chose soy and peanut flours as replacements for tilapia in subsequent MDCF formulations.
We reasoned that by transplanting a representative immature intact microbiota into young, germ-free mice, we could investigate whether gut health (defined by relative abundances of community members, expression of microbial genes in mcSEED metabolic pathways, and biomarkers and mediators of gut barrier function) was improved by supplementing the Mirpur-18 diet with one or more complementary food ingredients that target weaning-phase age-discriminatory taxa. Fifteen fecal samples from 12 different children, obtained during or after treatment for SAM, were screened in gnotobiotic mice to identify samples containing the greatest number of transmissible weaning-phase age-discriminatory taxa and to assess their response to supplementation of Mirpur-18 (table S14A). We selected a sample obtained from a donor (PS.064) who had post-SAM MAM; in addition to the successful transmission of targeted taxa, 88.7 ± 1.3% (mean ± SD) of the recipient animals’ gut communities consisted of OTUs that were detected at >0.1% relative abundance in the donor sample (table S14B). Three groups of mice were colonized with this microbiota and monotonously fed one of three diets: unsupplemented Mirpur-18, Mirpur-18 supplemented with peanut flour [Mirpur(P)]or Mirpur-18 supplemented with four of the lead ingredients [Mirpur(PCSB), with peanut flour, chickpea flour, soy flour, and banana] (Fig. 4A and table S15A). Three control groups were maintained as germ-free; each group was fed one of the three diets.
We characterized the effects of diet supplementation on cecal and serum levels of metabolites as well as on expression of genes in various microbial metabolic pathways (tables S15, B, D, and E, and S16 and supplementary text, results). Eighteen mcSEED pathway modules involved in amino acid metabolism were significantly up-regulated in the cecal microbiomes of mice consuming Mirpur(PCSB) or Mirpur(P) compared with those consuming Mirpur-18, with the most up-regulated being “isoleucine, leucine, valine biosynthesis” [other age-discriminatory mcSEED pathway modules that showed significantly lower abundances in the fecal microbiomes of Bangladeshi children with SAM and whose expression was increased by Mirpur(PCSB) or Mirpur(P) in gnotobiotic mice are provided in Fig. 4B and fig. S1E]. Serum levels of a product of branched-chain amino acid metabolism, C5:1-acylcarnitine, were significantly higher in mice consuming Mirpur(PCSB) compared with unsupplemented Mirpur-18 (0.148 ± 0.015 versus 0.086 ± 0.0098 μM, respectively; P = 0.014, unpaired t test). Findings from mass spectrometric analysis of cecal contents, isolation, and comparative genomic analysis of an F. prausnitzii strain prominently represented in the transplanted community, and characterization of the in vivo transcriptional responses of this strain to the different diets, are described in table S15F and supplementary text results.
Gut mucosal barrier function
Epithelium and overlying mucus from the proximal, middle, and distal thirds of the small intestine were recovered with laser capture microdissection (LCM) (Fig. 4D). Listed in table S15C are the 30 most abundant OTUs identified by means of V4-16S rDNA analysis of LCM mucosal DNA obtained from the different small intestinal segments within a given diet group and between similarly positioned segments across the different diet treatments. For example, Mirpur(PCSB) produced a statistically significant increase in the relative abundance of F. prausnitzii in the proximal two-thirds of the small intestine, without significantly affecting the proportional representation of a milk-associated age-discriminatory Bifidobacteria OTU (Fig. 4, C and D).
Gene expression was characterized in the jejunal mucosa (Fig. 4D, SI-2 segment) recovered by LCM from mice belonging to all six treatment groups. Significant differences in expression were categorized based on enriched Gene Ontology (GO) terms for “Molecular Function.” In colonized mice, Mirpur(P) and Mirpur(PCSB) significantly up-regulated genes assigned to “cadherin binding” (GO: 0045296) and “cell adhesion molecule binding” (GO: 0050839) compared with Mirpur-18 (table S17A). The diet effect was colonization-dependent; there were no significant differences in expression of these genes or these GO categories in germ-free mice consuming supplemented versus unsupplemented diets (table S17). (Further discussion is available in the supplementary text, results, and histochemical and immunohistochemical analyses of tissue sections prepared along the length of the small intestines of these mice are provided in fig. S6). On the basis of its effects on microbial community organismal composition, gene expression, and gut barrier function, we deemed Mipur-18 supplemented with the four lead complementary foods [Mirpur(PCSB)] superior to that supplemented with just peanut flour [Mirpur(P)].
Characterizing MDCF prototypes in gnotobiotic piglets
We examined the effects of MDCF prototypes in a second host species whose physiology and metabolism are more similar to that of humans. Gnotobiotic piglets provide an attractive model for these purposes; piglets manifest rapid growth rates in the weeks after birth (26), and methods for conducting experiments with gnotobiotic piglets have been described (27).
On the basis of the results from the gnotobiotic mouse studies, we designed two MDCF prototypes. One prototype was formulated to be analogous to Mirpur-18, which contains milk powder; this prototype was supplemented with peanut flour, chickpea flour, soy flour, and banana [MDCF(PCSB)]. The other diet lacked milk powder and was supplemented with just chickpea flour and soy flour [MDCF(CS)]. The two MDCFs were isocaloric, matched in lipid levels and total protein content (with equivalent representation of amino acids), and also met current ready-to-use therapeutic food guidelines for children with respect to macro- and micronutrient content (table S18A) (28).
Four-day-old germ-free piglets fed a sow milk–based formula were colonized with a 14-member consortium of bacterial strains that consisted of the same nine Bangladeshi age-discriminatory strains used for the diet oscillation experiments described in fig. S2, plus five weaning-phase age-discriminatory strains cultured from Malawian children (table S6B). In an earlier study, several members of this consortium (B. longumF. prausnitziiClostridiumRuminococcus gnavusand D. formicigenerans) had been classified as growth-discriminatory by means of a RF-based analysis of their representation in gnotobiotic mouse recipients of healthy and undernourished donor microbiota and the animals’ weight and lean body mass gain phenotypes (4). After gavage, the two groups of piglets were weaned over the course of 10 days (supplementary materials, materials and methods) onto one or the other irradiated MDCF prototypes, which they consumed ad libitum for the remainder of the experiment (n = 4 piglets/treatment arm) (Fig. 5A). Animals were euthanized on day 31 after a 6-hour fast.
Piglets fed MDCF(PCSB) exhibited significantly greater weight gain than those receiving MDCF(CS) (Fig. 5B). Micro-computed tomography of their femurs revealed that they also had significantly greater cortical bone volume (Fig. 5C). COPRO-seq analysis disclosed that piglets fed MDCF(PCSB) had significantly higher relative abundances of C. symbiosumR. gnavusD. formicigeneransR. torquesand Bacteroides fragilis in their cecum and distal colon compared with those of piglets consuming MDCF(CS) (Fig. 5D and table S18B); all are weaning-phase age-discriminatory strains, and the former three were, as noted above, also defined as growth-discriminatory. Conversely, the relative abundances of three members of Bifidobacteria (including two milk-associated age-discriminatory strains, B. breve and B. longum subsp. infantis) were significantly higher in the ceca and distal colons of piglets fed MDCF(CS) (table S18B). These findings led us to conclude that MDCF(PCSB) promoted a more weaning-phase–like (mature) community configuration than MDCF(CS). (genome annotations, microbial RNA-seq, and targeted MS analyses of cecal metabolites are provided in tables S18, C and D, and S19A and supplementary text, results).
The effects of the two MDCF prototypes on host biology were defined by means of MS-based serum metabolomic and proteomic analyses (tables S19 and S20). Notable findings included significant increases in levels of tryptophan, methionine, and C3-acylcarnitine with MDCF(PCSB) as well as changes produced in the serum proteome that are shared with children in the SAM trial (Fig. 5, E and F, and supplementary text, results).
Testing MDCFs in Bangladeshi children with MAM
To assess the degree to which results obtained from the gnotobiotic mouse and piglet models translate to humans, we performed a pilot randomized, double-blind controlled feeding study of the effects of three MDCF formulations. The formulations (MDCF-1, -2, and -3) were designed to be similar in protein energy ratio and fat energy ratio and provide 250 kcal/day (divided over two servings). MDCF-2 contained all four lead ingredients (chickpea flour, soy flour, peanut flour, and banana) at higher concentrations than in MDCF-1. MDCF-3 contained two lead ingredients (chickpea flour and soy flour). A rice- and lentil-based ready-to-use supplementary food (RUSF), included as a control arm, lacked all four ingredients but was otherwise similar in energy density, protein energy ratio, fat energy ratio, and macro- and micronutrient content to those of the MDCFs (Fig. 6A). Milk powder was included in MDCF-1 and RUSF. All formulations were supplemented with a micronutrient mixture designed to provide 70% of the recommended daily allowances for 12- to 18-month-old children. The formulations were produced locally and tested for organoleptic acceptability before initiating the trial (table S21A).
Children from Mirpur with MAM and no prior history of SAM were enrolled (mean age at enrollment, 15.2 ± 2.1 months, mean WHZ –2.3 ± 0.3). Participants were randomized into one of the four treatment groups (14 to 17 children per group) and received 4 weeks of twice-daily feeding under supervision at the study center, preceded and followed by 2 weeks of observation and sample collection. Mothers were encouraged to continue their normal breastfeeding pactices throughout the study (Fig. 6A). There were no significant differences in the mean daily amount of each MDCF or RUSF consumed per child or in the mean incidence of morbidity across the four treatment groups (table S21, B to D). All three MDCFs and the RUSF control improved WHZ scores [–19±05(mean±SD)atthecompletionofinterventioncomparedwith–22±04atthestartofintervention;n = 63 children, P = 2.06 × 10−11 for all groups combined, paired t test]. There were no statistically significant differences between the four interventions in the change in WHZ (P = 0.31, one-way ANOVA). Despite the small group size and the short length of the study, there were significant differences in treatment effects on another anthropometric indicator, with MDCF-2 producing a significantly greater increase in mid-upper arm circumference (MUAC) than MDCF-3 (one-way ANOVA, P = 0.022; with Tukey’s multiple comparisons test, P = 0.017) (table S21E).
Effects on biological state
To contextualize the biological effects of the dietary interventions, we performed quantitative proteomics (SOMAscan) on plasma collected from 21 12- to 24-month-old Mirpur children with healthy growth phenotypes (mean age, 19.2 ± 5.1 months; WHZ, 0.08 ± 0.58; HAZ, –0.41 ± 0.56, WAZ, –0.12 ± 0.60) and 30 children with SAM before treatment (Fig. 1A, B1 sample; WHZ < –3; mean age 15.2 ± 5.1 months) [metadata associated with the healthy, SAM, and MAM (MDCF trial) cohorts are provided in table S22]. We rank-ordered all detected proteins according to fold differences in their abundances in plasma collected from healthy children compared with children with untreated SAM. The top 50 most differentially abundant proteins (P < 10−7; R package “limma”) that were significantly higher in healthy children were designated “healthy growth-discriminatory,” whereas the top 50 differentially abundant proteins that were significantly higher in children with SAM were designated “SAM-discriminatory” (table S23A). We next compared the mean difference for each protein in the pre- versus post-intervention plasma samples for all children in each MDCF/RUSF treatment group. Proteins were then ranked according to the fold differences of the pre- versus post-treatment levels in each of the four groups (table S23B), and these treatment effects were mapped onto the 50 most healthy growth-discriminatory and 50 most SAM-discriminatory proteins. Strikingly, MDCF-2 elicited a biological response characterized by a marked shift in the plasma proteome toward that of healthy children and away from that of children with SAM; MDCF-2 increased the abundance of proteins that are higher in plasma from healthy children and reduced the levels of proteins elevated in SAM plasma samples (Fig. 6B).
Aggregating proteomic datasets from the combined cohort of 113 children with SAM, MAM, and healthy growth phenotypes for whom plasma samples were available, we identified a total of 27 plasma proteins that were significantly positively correlated with HAZ and 57 plasma proteins that were significantly negatively correlated with HAZ [absolutevalueofPearsoncorrelation>025falsediscoveryrate(FDR)–correctedP value < 0.05]. Among the treatments, MDCF-2 was distinctive in its ability to increase the abundances of a broad range of proteins positively correlated with HAZ, including the major IGF-1 binding protein IGFBP-3, growth hormone receptor (GHR), and leptin (LEP) (Fig. 6C). Growth differentiation factor 15 (GDF15) was reduced after 4 weeks of dietary supplementation with MDCF-2 (Fig. 6C). This transforming growth factor–β superfamily member, which was negatively correlated with HAZ, has been implicated in the anorexia and muscle wasting associated with cancer and with chronic heart failure in children; it was elevated in children with SAM and positively correlated with their lipolytic biomarkers NEFA and ketones (supplementary text, results). Peptide YY, an enteroendocrine cell product elevated in SAM plasma that reduces appetite and negatively correlated with HAZ, was also decreased by MDCF-2.
We identified GO terms that were enriched among the group of treatment-responsive proteins and ranked them according to the P value of their enrichment (table S23C). Proteins belonging to GO terms significantly higher in healthy compared with SAM plasma samples were deemed “healthy growth-discriminatory,” whereas those that were significantly higher in SAM were deemed “SAM-discriminatory” (fold-difference >30%; FDR-adjusted P value <0.05). This analysis revealed multiple healthy growth-discriminatory proteins associated with GO processes “osteoblast differentiation” and “ossification” that were increased by supplementation with MDCF-2 (Fig. 7A and table S23C). Examples include key markers or mediators of osteoblast differentiation [osteopontin (SPP1), bone sialoprotein 2 (IBSP), and bone morphogenetic protein 7 (BMP7)] as well as matrix metalloproteases (MMP-2 and MMP-13) involved in terminal differentiation of osteoblasts into osteocytes and bone mineralization.
A number of plasma proteins categorized under the GO process “CNS development,” including those involved in axon guidance and neuronal differentiation, were also affected by MDCF-2 supplementation. Levels of the SAM-discriminatory semaphorin SEMA3A, a potent inhibitor of axonal growth, decreased with this treatment, whereas healthy growth-discriminatory semaphorins (SEMA5A, SEMA6A, and SEMA6B) increased (Fig. 7B). Other healthy growth-discriminatory proteins whose abundances increased with MDCF-2 included receptors for neurotrophin (NTRK2 and NTRK3) plus various axonal guidance proteins [netrin (UNC5D), ephrin A5 (EFNA5), roundabout homolog 2 (ROBO2), and SLIT and NTRK-like protein 5 (SLITRK5)] (Fig. 7B). Expression of a number of neurotrophic proteins belonging to these families has been reported to be influenced by nutrient availability in primates (29).
Compared with healthy children, the plasma proteome of children with SAM was characterized by elevated levels of acute phase proteins [such as C-reactive protein (CRP) or interleukin-6 (IL-6)] and inflammatory mediators, including several agonists and components of the nuclear factor–κB (NF-κB) signaling pathway (Fig. 7C). These components include the pro-inflammatory cytokines IL-1β, tumor necrosis factor–α (TNF-α), and CD40L, plus ubiquitin-conjugating enzyme E2 N (UBE2N), which is involved in induction of NF-κB– and mitogen-activated protein kinase (MAPK)–responsive inflammatory genes (30). MDCF-2 supplementation was associated with reductions in the levels of all of these SAM-associated proteins (Fig. 7C).
Effects on the microbiota
Our analysis of fecal microbiota samples revealed no significant change in the representation of enteropathogens within and across the four treatment groups (fig. S7A and table S21F). MDCF-2–induced changes in biological state were accompanied by increases in the relative abundances of several weaning-phase taxa, including OTUs assigned to F. prausnitzii (OTU 851865) and a Clostridiales sp. (OTU 338992) that are closely related to taxa ranked first and second in feature importance in the sparse Bangladeshi RF-derived model of gut microbiota maturation (fig. S7, B and C). MDCF-2 supplementation was associated with a significant decrease in B. longum (OTU 559527) (fig. S7B), which is ranked third in feature importance in the RF-derived model and discriminatory for a young, milk-oriented microbiota. None of the other members of the 30 OTU model showed significant changes. By contrast, MDCF-1 did not produce significant increases in any of the taxa in the model. The other two formulations were each associated with a significant change in just one member [anincreaseintherelativeabundanceofanearlyage-discriminatoryOTU(Streptococcus; ranked 30th) with MDCF-3 supplementation, and a decrease in another OTU (Enterococcus faecalis; ranked 29th) with RUSF supplementation] (table S4B).
MAZ scores were not significantly different between groups at enrollment, nor were they significantly improved by any of the formulations. Interpretation of this finding was confounded by unexpectedly high baseline microbiota maturity scores in this group of children with MAM [MAZ, –0.01 ± 1.12 (mean ± SD)] (table S22) compared with a small, previously characterized Mirpur cohort with untreated MAM (2). Hence, we developed an additional measure of microbiota repair (31). This involved a statistical analysis of covariance among bacterial taxa in the fecal microbiota of anthropometrically healthy members of a Mirpur birth cohort who had been sampled monthly over a 5-year period. Using approaches developed in the fields of econophysics and protein evolution to characterize the underlying organization of interacting systems with seemingly intractable complexity, such as financial markets, we found that the gut community in healthy children could be decomposed into a sparse unit of 15 covarying bacterial taxa termed an “ecogroup” (31). These ecogroup taxa include a number of age-discriminatory strains in the Bangladeshi RF-derived model (such as B. longumF. prausnitziiand Prevotella copri). We used the ecogroup to show that in addition to its effects on host biological state, MDCF-2 was also the most effective of the four treatments in reconfiguring the gut bacterial community to a mature state similar to that characteristic of healthy Bangladeshi children.
We have integrated preclinical gnotobiotic animal models with human studies to understand the contributions of perturbed gut microbiota development to childhood undernutrition and to identify new microbiota-directed therapeutic approaches. We identified a set of proteins that distinguish the plasma proteomes of healthy children from those with SAM. Using these data, we have developed a supplemental food prototype, MDCF-2, that shifted the plasma proteome of children with MAM toward that of healthy individuals, including proteins involved in linear growth, bone development, neurodevelopment, and immune function. MDCF-2 is a tool for investigating, in larger studies across different populations with varying degrees of undernutrition, how repair of gut microbiota immaturity affects various facets of child growth.
Overview of methods
Children aged 6 to 59 months with SAM (n = 343 participants) were enrolled in a study entitled “Development and field testing of ready-to-use therapeutic foods (RUTF) made of local ingredients in Bangladesh for the treatment of children with severe acute malnutrition.” The study was approved by the Ethical Review Committee at icddr,b (ClinicalTrials.gov identifier NCT01889329). Written informed consent was obtained from their parent or guardian. A subset of 54 children were included in a substudy that involved regular fecal sampling and three blood draws for up to 1 year after discharge. Children aged 12 to 18 months with MAM who were no longer exclusively breastfed (n = 63 participants) were enrolled in a double-blind, randomized, four-group, parallel assignment interventional trial study (ClinicalTrials.gov identifier NCT03084731) approved by the Ethical Review Committee at icddr,b. Fecal and plasma samples were collected as described in the supplementary materials, materials and methods, and stored at –80°C. Samples were shipped to Washington University with associated clinical metadata and maintained in a dedicated biospecimen repository with approval from the Washington University Human Research Protection Office.
Analysis of plasma samples
Methods for targeted MS-based metabolomics are described in the supplementary materials. The SOMAscan 1.3K Proteomic Assay plasma/serum kit (SomaLogic, Boulder, Colorado,) was used to measure 1305 proteins. The R package “limma” (Bioconductor) was used to analyze differential protein abundances (32). Spearman correlation analyses were performed between measured proteins and anthropometric scores, plasma metabolites, and the abundances of bacterial taxa in fecal samples. Plasma proteins in children with healthy growth phenotypes or SAM (before treatment) were rank-ordered according to the fold-difference in their levels between these two groups. The top 50 most differentially abundant proteins in healthy compared with SAM were designated as healthy growth-discriminatory proteins, and the top 50 most differentially abundant in SAM compared with healthy were designated as SAM-discriminatory proteins. The average fold-change for these healthy growth- and SAM-discriminatory proteins was then calculated for each treatment arm in the MDCF trial (before versus after MDCF/RUSF treatment) and normalized to the mean fold-change across all four arms. Limma was used to calculate statistical significance. All 1305 proteins were mapped to all GO “Biological Processes” in the GO database (www.geneontology.org). SetRank, a gene set enrichment analysis (GSEA) algorithm (33), was used to identify GO Biological Processes that were significantly enriched for proteins that exhibited changes in abundance from before to after treatment with MDCF/RUSF. Enrichment was calculated by using the setRankAnalysis function in the SetRank R library (parameters were use.ranks = TRUE; setPCutoff = 0.01; and fdrCutoff = 0.05). The average fold-change for each protein in the statistically significant Biological Process category was calculated for each treatment arm and normalized to the mean fold-change across all four arms. We defined proteins within the GO Biological Process as “healthy growth-discriminatory” if they were at least 30% more abundant in healthy individuals compared with those with SAM and “SAM-discriminatory” if they were at least 30% more abundant in children with SAM compared with those who were classified as healthy.
Characterizing human fecal microbial communities
Methods for V4-16S rRNA gene sequencing and data analysis, calculation of MAZ scores and functional microbiome maturity, and quantification of enteropathogen burden by means of multiplex quantitative polymerase chain reaction (qPCR) are described in the supplementary materials.
All mouse experiments were performed by using protocols approved by the Washington University Animal Studies Committee. Mice were housed in plastic flexible film gnotobiotic isolators under a 12-hour light cycle. Male germ-free C57BL/6 mice were initially weaned onto an autoclaved, low-fat, high-plant polysaccharide chow that was administered ad libitum (diet 2018S, Envigo). Animals were maintained on this diet until 3 days before the beginning of experiments involving tests of the effects of complementary food ingredients. Defined consortia of sequenced bacterial strains cultured from Bangladeshi children, or intact uncultured microbiota from donors with post-SAM MAM, were introduced by means of gavage into recipient mice at 5 weeks of age. Methods for identifying and characterizing the effects MDCF prototypes—including (i) design and preparation of diets; (ii) culturing of age-discriminatory and SAM-associated bacterial strains; (iii) shotgun sequencing of DNA isolated from serially collected fecal samples; (iv) microbial RNA-seq of cecal contents; (v) targeted MS of cecal contents, liver, gastrocnemius muscle, and serum samples for measurement of amino acids, acylcarnitines, organic acids, and acylCoAs; (vi) Western blot analysis of IGF-1 pathway components in liver; (vii) μCT of bone; and (viii) characterizing the effects of a transplanted fecal microbiome from a donor with post-SAM MAM in recipient gnotobiotic mice as a function of diet treatment by histochemical and immunohistochemical analysis of their intestinal segments, LCM of their small intestinal epithelium, and RNA-seq analysis of gene expression in LCM mucosa—are all described in the supplementary materials.
Experiments were performed under the supervision of a veterinarian by using protocols approved by the Washington University Animal Studies Committee and that followed American Veterinary Medical Association guidelines for euthanasia. The protocol for generating germ-free piglets; preparing diets, feeding, colonization, and husbandry of piglets; measurement of weight gain; μCT of femurs; and liquid chromatography–MS (LC-MS)/MS–based serum proteomics are all described in the supplementary materials.