Michael J. Barratt

Persistent gut microbiota immaturity in malnourished Bangladeshi children

Therapeutic food interventions have reduced mortality in children with severe acute malnutrition (SAM), but incomplete restoration of healthy growth remains a major problem. The relationships between the type of nutritional intervention, the gut microbiota, and therapeutic responses are unclear. In the current study, bacterial species whose proportional representation define a healthy gut microbiota as it assembles during the first two postnatal years were identified by applying a machine-learning-based approach to 16S ribosomal RNA data sets generated from monthly faecal samples obtained from birth onwards in a cohort of children living in an urban slum of Dhaka, Bangladesh, who exhibited consistently healthy growth. These age-discriminatory bacterial species were incorporated into a model that computes a ‘relative microbiota maturity index’ and ‘microbiota-for-age Z-score’ that compare postnatal assembly (defined here as maturation) of a child’s faecal microbiota relative to healthy children of similar chronologic age. The model was applied to twins and triplets (to test for associations of these indices with genetic and environmental factors, including diarrhoea), children with SAM enrolled in a randomized trial of two food interventions, and children with moderate acute malnutrition. Our results indicate that SAM is associated with significant relative microbiota immaturity that is only partially ameliorated following two widely used nutritional interventions. Immaturity is also evident in less severe forms of malnutrition and correlates with anthropometric measurements. Microbiota maturity indices provide a microbial measure of human postnatal development, a way of classifying malnourished states, and a parameter for judging therapeutic efficacy. More prolonged interventions with existing or new therapeutic foods and/or addition of gut microbes may be needed to achieve enduring repair of gut microbiota immaturity in childhood malnutrition and improve clinical outcomes.

Gut bacteria that prevent growth impairments transmitted by microbiota from malnourished children

Microbiota and infant development Malnutrition in children is a persistent challenge that is not always remedied by improvements in nutrition. This is because a characteristic community of gut microbes seems to mediate some of the pathology. Human gut microbes can be transplanted effectively into germ-free mice to recapitulate their associated phenotypes. Using this model, Blanton et al. found that the microbiota of healthy children relieved the harmful effects on growth caused by the microbiota of malnourished children. In infant mammals, chronic undernutrition results in growth hormone resistance and stunting. In mice, Schwarzer et al. showed that strains of Lactobacillus plantarum in the gut microbiota sustained growth hormone activity via signaling pathways in the liver, thus overcoming growth hormone resistance. Together these studies reveal that specific beneficial microbes could potentially be exploited to resolve undernutrition syndromes. Science, this issue p. 10.1126/science.aad3311, p. 854 Microbes from healthy children protect mice from the detrimental effects of the microbiota of malnourished infants. INTRODUCTION As we come to appreciate how our microbial communities (microbiota) assemble following birth, there is an opportunity to determine how this facet of our developmental biology relates to the healthy or impaired growth of infants and children. Childhood undernutrition is a devastating global health problem whose long-term sequelae, including stunting, neurodevelopmental abnormalities, and immune dysfunction, remain largely refractory to current therapeutic interventions. RATIONALE To test the hypothesis that perturbations in the normal development of the gut microbiota are causally related to undernutrition, we first applied random forests (RF), a machine learning method, to bacterial 16S ribosomal RNA data sets generated from fecal samples that were collected serially from healthy Malawian infants and children during their first 3 postnatal years. Age-discriminatory bacterial taxa were identified with distinctive time-dependent changes in their relative abundances; they were used to construct a sparse RF-derived model describing a program of normal postnatal gut microbiota development that is shared across biologically unrelated individuals. A metric based on this model (microbiota-for-age Z-score) was used to define the state of development (maturation) of fecal microbiota from infants and children with varying degrees of undernutrition. Fecal samples obtained from 6- and 18-month-old children with healthy growth patterns or with varying degrees of undernutrition were transplanted into young germ-free mice that were fed a representative Malawian diet. The recipient animals’ rate of lean body mass gain was characterized by serial quantitative magnetic resonance, their metabolic phenotypes were determined by targeted mass spectrometry, and their femoral bone morphologic features were delineated by microcomputed tomography. RESULTS Undernourished children in the Malawian birth cohort that we studied have immature gut microbiota. Unlike microbiota from healthy children, immature microbiota transmit impaired growth, altered bone morphology, and metabolic abnormalities in the muscle, liver, and brain to recipient gnotobiotic mice. The representation of several age-discriminatory taxa in the transplanted microbiota harbored by recipient animals correlated with their growth rates. Microbiota from 6-month-old infants produced greater effects on growth than did microbiota from 18-month-old children, although in each age bin, the growth effects produced by a healthy donor’s community were greater than those produced by an undernourished donor’s community. Cohousing coprophagic mice shortly after they received microbiota from healthy or severely stunted and underweight 6-month-old infants resulted in the invasion of age- and growth-discriminatory taxa from the former into the latter microbiota in the recipient animals, with associated prevention of growth impairments. Introducing cultured members from this group of invasive species ameliorated growth and metabolic abnormalities in recipients of microbiota from undernourished donors. CONCLUSION These preclinical findings provide evidence that gut microbiota immaturity is causally related to childhood undernutrition. The age- and growth-discriminatory taxa that we identified should help direct studies of the effects of host and environmental factors on gut microbial community development, and they represent therapeutic targets for repairing or preventing gut microbiota immaturity. Preclinical evidence that gut microbiota immaturity is causally related to childhood undernutrition. (A) A model of normal gut microbial community development in Malawian infants and children, based on the relative abundances of 25 bacterial taxa that provide a microbial signature defining the “age,” or state of maturation, of an individual’s (fecal) microbiota. (Hierarchical clusterings of operational taxonomic units are indicated on the left.) (B) Fecal samples from healthy (H) or stunted and underweight (Un) infants and children were transplanted into separate groups of young germ-free mice that were fed a Malawian diet. The immature microbiota of Un donors transmitted impaired growth phenotypes to the mice

Sialylated Milk Oligosaccharides Promote Microbiota-Dependent Growth in Models of Infant Undernutrition

Identifying interventions that more effectively promote healthy growth of children with undernutrition is a pressing global health goal. Analysis of human milk oligosaccharides (HMOs) from 6-month-postpartum mothers in two Malawian birth cohorts revealed that sialylated HMOs are significantly less abundant in those with severely stunted infants. To explore this association, we colonized young germ-free mice with a consortium of bacterial strains cultured from the fecal microbiota of a 6-month-old stunted Malawian infant and fed recipient animals a prototypic Malawian diet with or without purified sialylated bovine milk oligosaccharides (S-BMO). S-BMO produced a microbiota-dependent augmentation of lean body mass gain, changed bone morphology, and altered liver, muscle, and brain metabolism in ways indicative of a greater ability to utilize nutrients for anabolism. These effects were also documented in gnotobiotic piglets using the same consortium and Malawian diet. These preclinical models indicate a causal, microbiota-dependent relationship between S-BMO and growth promotion.

Childhood undernutrition, the gut microbiota, and microbiota-directed therapeutics

Gut microbiota and undernutrition Poor nutrition during the early years of life can have severe consequences for subsequent skeletal, immunological, and intellectual development. Blanton et al. review the evidence showing that undernutrition is not caused by food insecurity alone. Other factors range from the length of the breastfeeding period and the availability of milk oligosaccharides, enteropathogen exposure, and enteric dysfunction marked by villus atrophy and loss of gut barrier function. Unfortunately, the current practice of nutritional restoration with or without antibiotic treatment may not be effective in the longer term. Differences in the succession of microbial establishment and maturity might contribute to family discordances in nutritional status. Thus, microbiota-directed therapeutics could be a promising route to nutritional restoration in these children. Science, this issue p. 1533 BACKGROUND Childhood undernutrition is a global health challenge. Undernutrition in early life is associated with a number of adverse outcomes, including persistent stunting, immune dysfunction, and neurocognitive deficits. Current approaches to treatment have only modest effects in correcting these long-term sequelae, suggesting that certain features of host biology are not being adequately repaired. This has led to the hypothesis that healthy growth is dependent, in part, on normal postnatal development of the gut microbiota and that perturbations in its development are causally related to undernutrition. Testing this hypothesis illustrates a number of the challenges that human microbial ecology research faces: (i) defining “normal,” both in terms of community structure and expressed functions; (ii) determining whether normal in one population generalizes to other populations; (iii) ascertaining whether deviations from normal correlate with disease and are a cause rather than an effect of pathology; (iv) determining whether abnormal microbial community configurations can be repaired in a sustained fashion, and what route and time course are optimal for such repair; (v) deciphering the short- and long-term effects and safety of repair; and (vi) proactively addressing the ethical, regulatory, and other societal implications of microbiota-directed food and/or microbial interventions designed to deliberately manipulate this facet of postnatal human development. ADVANCES Culture-independent studies of the gut microbiota of members of birth cohorts with healthy growth phenotypes have identified a program of community assembly (“maturation”) defined by the changing representation of a group of age-discriminatory bacterial taxa. Features of this program are shared across individuals living in several low-income countries. Applying metrics for defining deviations from this program (microbiota-for-age Z score) has disclosed that children with severe acute malnutrition have gut microbial communities that appear younger than would be expected on the basis of their chronological age. The resulting microbiota “immaturity” is not repaired by current therapeutic food interventions. Compared with healthy children, microbiota from undernourished children transmit impaired growth phenotypes to recipient gnotobiotic mice fed diets representative of those of the human donors; moreover, some of the transplanted age-discriminatory strains are growth-discriminatory. These findings provide early preclinical proof-of-concept that gut microbiota immaturity is causally related to a number of the manifestations of childhood undernutrition. OUTLOOK Gnotobiotic animal models can be used to test a number of concepts. Gut microbiota immaturity, increased enteropathogen burden, and gut barrier dysfunction are interrelated factors that affect disease risk and pathogenesis. Microbiota development is linked to maturation of the gut mucosal immune system, metabolic function in multiple host tissues, plus musculoskeletal and brain development. Age- and growth-discriminatory bacterial strains identified in the normally developing microbiota represent therapeutic targets in children with undernutrition. The representation of these strains provides a way not only for defining the efficacy of these therapeutic interventions but also for assessing the effects of various parameters postulated to contribute to disease risk and pathogenesis (such as maternal health status, breast milk composition, history and quality of complementary feeding, poor sanitation and enteropathogen burden, and antibiotic exposures). Microbiota-directed strategies for treating and ultimately preventing childhood undernutrition raise intriguing questions about the mechanisms that define human development. They also highlight the need to add a microbial dimension to our conceptualization of human biological immaturity and its associated adaptations and compensations, and to consider whether interventions that promote healthy microbiota development can spawn a form of preventative medicine that has lifelong benefits. The concept that impaired postnatal gut microbiota development (maturation) is causally related to childhood undernutrition. The representation of age-discriminatory bacterial taxa defines a program of normal gut microbiota development. Disrupting the coordinated functional codevelopment of microbiota and host affects multiple biological regulatory systems through largely unknown mechanisms. Developing effective strategies for sustained repair of microbiota immaturity though food or microbial interventions requires preclinical studies of these mechanisms and modeling of the effects of different rates and routes of repair. Childhood undernutrition is a major global health challenge. Although current therapeutic approaches have reduced mortality in individuals with severe disease, they have had limited efficacy in ameliorating long-term sequelae, notably stunting, immune dysfunction, and neurocognitive deficits. Recent work is providing insights about the role of impaired development of the human gut microbiota in disease pathogenesis, leading to new concepts for treatment and prevention. These findings raise intriguing basic questions about the mechanisms that direct normal gut microbial community assembly and functional maturation. Designing and implementing new microbiota-directed therapeutics for undernutrition highlights the need to simultaneously consider a variety of features of human biology as well as broader societal issues.

The effects of micronutrient deficiencies on bacterial species from the human gut microbiota

Mechanistic studies reveal pronounced effects of vitamin A deficiency on bacterial members of a defined human gut microbiota. A gut bacterial view of micronutrient deficiency Deficiencies in vitamins and minerals (micronutrients) are a global health challenge. In a new study, Hibberd et al. compare the effects of acute dietary deficiency of vitamin A, folate, iron, or zinc in gnotobiotic mice harboring bacterial strains common in the human gut. Vitamin A had the greatest effect on the structure of the bacterial community and gene expression. Bacteroides vulgatus, a bacterial species positively correlated with host growth in gnotobiotic mouse models of postnatal human microbiota development, had the biggest response to vitamin A deficiency, exhibiting an increase in its abundance. Genetic, multi-omic, and pharmacologic analyses indicated that retinol treatment affected B. vulgatus fitness through the activity of the bacterial AcrAB-TolC efflux system. These results suggest that micronutrient imbalances should be considered from the perspective of both the human host and the gut microbiota they possess. Vitamin and mineral (micronutrient) deficiencies afflict 2 billion people. Although the impact of these imbalances on host biology has been studied extensively, much less is known about their effects on the gut microbiota of developing or adult humans. Therefore, we established a community of cultured, sequenced human gut–derived bacterial species in gnotobiotic mice and fed the animals a defined micronutrient-sufficient diet, followed by a derivative diet devoid of vitamin A, folate, iron, or zinc, followed by return to the sufficient diet. Acute vitamin A deficiency had the largest effect on bacterial community structure and metatranscriptome, with Bacteroides vulgatus, a prominent responder, increasing its abundance in the absence of vitamin A. Applying retinol selection to a library of 30,300 B. vulgatus transposon mutants revealed that disruption of acrR abrogated retinol sensitivity. Genetic complementation studies, microbial RNA sequencing, and transcription factor–binding assays disclosed that AcrR is a repressor of an adjacent AcrAB-TolC efflux system. Retinol efflux measurements in wild-type and acrR-mutant strains plus treatment with a pharmacologic inhibitor of the efflux system revealed that AcrAB-TolC is a determinant of retinol and bile acid sensitivity in B. vulgatus. Acute vitamin A deficiency was associated with altered bile acid metabolism in vivo, raising the possibility that retinol, bile acid metabolites, and AcrAB-TolC interact to influence the fitness of B. vulgatus and perhaps other microbiota members. This type of preclinical model can help to develop mechanistic insights about the effects of, and more effective treatment strategies for micronutrient deficiencies.

The Gut Microbiota, Food Science, and Human Nutrition: A Timely Marriage

Analytic advances are enabling more precise definitions of the molecular composition of key food staples incorporated into contemporary diets and how the nutrient landscapes of these staples vary as a function of cultivar and food processing methods. This knowledge, combined with insights about the interrelationship between consumer microbiota configurations and biotransformation of food ingredients, should have a number of effects on agriculture, food production, and strategies for improving the nutritional value of foods and health status. These effects include decision-making about which cultivars of current or future food staples to incorporate into existing and future food systems, and which components of waste streams from current or future food manufacturing processes have nutritional value that is worth capturing. They can also guide which technologies should be applied, or need to be developed, to produce foods that support efficient microbial biotransformation of their ingredients into metabolic products that sustain health.

Food and microbiota in the FDA regulatory framework

How should microbiota-directed foods be regulated? New understanding of how our gut microbial communities (microbiota) transform dietary ingredients into metabolic products that affect human biology is altering our definitions of the nutritional value of foods (1, 2). We are coming to appreciate how much formation of the gut microbiota during early postnatal life and the traits encoded by its several million microbial genes (microbiome) are important determinants of healthy growth and of our metabolic, physiologic, immune, and perhaps neurologic phenotypes (3, 4). These advances are spawning efforts to develop foods that promote healthy microbiota development during early postnatal life, prevent the loss of microbial diversity associated with Western diets, and repair abnormalities associated with various disease states (5–8). But given the mechanisms by which such microbiota-directed foods (MDFs) may achieve their desired effects, the existing U.S. regulatory framework presents challenges in determining how MDFs should be classified. How these challenges are addressed will affect innovation incentives, product quality, consumer access, and public health. Although approaches to regulation vary among countries (9), we focus on the U.S. Food and Drug Administration (FDA) because of its global influence and because the products it regulates are often widely distributed.