High-throughput sequencing has revolutionized microbial ecology, but read quality remains a significant barrier to accurate taxonomy assignment and alpha diversity assessment for microbial communities. We demonstrate that high-quality read length and abundance are the primary factors differentiating correct from erroneous reads produced by Illumina GAIIx, HiSeq, and MiSeq instruments. We present guidelines for user-defined quality-filtering strategies, enabling efficient extraction of high-quality data from, and facilitating interpretation of Illumina sequencing results.

A low-error 16S rRNA amplicon sequencing method (LEA-Seq) plus whole genome sequencing of >500 cultured isolates were used to characterize bacterial strain composition in the fecal microbiota of 37 USA adults sampled for up to five years. Microbiota stability follows a power law function which, when extrapolated, suggests that most strains in an individual are residents for decades. Shared strains were recovered from family members, but not from unrelated individuals. Sampling individuals for up to 32 weeks while consuming a monotonous liquid diet indicated that changes in weight are more predictive of changes in strain composition than sampling interval. This combination of stability and responsiveness to physiologic change confirms the potential of the gut microbiota as a diagnostic tool and therapeutic target.

Therapeutic food interventions have reduced mortality in children with severe acute malnutrition (SAM) but incomplete restoration of healthy growth remains a major problem1,2. 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 rRNA datasets generated from monthly fecal samples obtained from a birth-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 development (defined here as maturation) of a child’s fecal 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 diarrhea), 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.

Undernourished children exhibit impaired gut microbiota development. Transplanting microbiota from 6- and 18-month old healthy or undernourished Malawian donors into young germ-free mice fed a Malawian diet revealed that immature microbiota from undernourished infants/children transmit impaired growth phenotypes. The representation of several age-discriminatory taxa in recipient animals correlated with lean body mass gain, liver, muscle, and brain metabolism, plus bone morphology. Co-housing mice shortly after receiving microbiota from healthy (H) or severely stunted/underweight (Un) infants demonstrated that invasion of age-/growth-discriminatory taxa from H to Un cagemates’ microbiota ameliorates growth faltering. Adding two invasive species, Ruminococcus gnavus and Clostridium symbiosum, to the Un microbiota also ameliorated growth and metabolic abnormalities. These results provide evidence that microbiota immaturity is causally related to undernutrition, and reveal potential therapeutic targets and agents.

To examine the contributions of impaired gut microbial community development to childhood undernutrition, we combined metabolomic and proteomic analyses of plasma samples with metagenomic analyses of fecal samples to characterize the biological state of Bangladeshi children with severe acute malnutrition (SAM) as they transitioned, after standard treatment, to moderate acute malnutrition (MAM) with persistent microbiota immaturity. Host and microbial effects of microbiota-directed complementary food (MDCF) prototypes targeting weaning-phase bacterial taxa underrepresented in SAM and MAM microbiota were characterized in gnotobiotic mice and gnotobiotic piglets colonized with age- and growth-discriminatory bacteria. A randomized, double-blind controlled feeding study identified a lead MDCF that changes the abundances of targeted bacteria and increases plasma biomarkers and mediators of growth, bone formation, neurodevelopment, and immune function in children with MAM.

Given the global burden of diarrheal diseases1, it is important to understand how members of the gut microbiota affect the risk for, course of, and recovery from disease in children and adults. The acute, voluminous diarrhea caused by Vibrio cholerae represents a dramatic example of enteropathogen invasion and gut microbial community disruption. We have conducted a detailed time-series metagenomic study of fecal microbiota collected during the acute diarrheal and recovery phases of cholera in a cohort of Bangladeshi adults living in an area with a high burden of disease2. We find that recovery is characterized by a pattern of accumulation of bacterial taxa that shows similarities to the pattern of assembly/maturation of the gut microbiota in healthy Bangladeshi children3. To define underlying mechanisms, we introduced into gnotobiotic mice an artificial community that was composed of human gut bacterial species that directly correlate with recovery from cholera in adults and are indicative of normal microbiota maturation in healthy Bangladeshi children3. One of the species, Ruminococcus obeum, exhibited consistent increases in its relative abundance upon V. cholerae infection of the mice. Follow-up analyses, including mono- and co-colonization studies, established that R. obeum restricts V. cholerae colonization, that R. obeum luxS [autoinducer-2 (AI-2) synthase] expression and AI-2 production increase significantly with V. cholerae invasion, and that R. obeum AI-2 causes quorum-sensing mediated repression of several V. cholerae colonization factors. Co-colonization with V. cholerae mutants disclosed that R. obeum AI-2 reduces Vibrio colonization/pathogenicity through a novel pathway that does not depend on the V. cholerae AI-2 sensor, LuxP. The approach described can be used to mine the gut microbiota of Bangladeshi or other populations for members that use autoinducers and/or other mechanisms to limit colonization with V. cholerae, or conceivably other enteropathogens.

Identifying the factors that influence the outcome of host-microbial interactions is critical to protecting biodiversity, minimizing agricultural losses, and improving human health. A few genes that determine symbiosis or resistance to infectious disease have been identified in model species, but a comprehensive examination of how a host's genotype influences the structure of its microbial community is lacking. Here we report the results of a field experiment with the model plant Arabidopsis thaliana to identify the fungi and bacteria that colonize its leaves and the host loci that influence the microbes’ numbers. The composition of this community differs among accessions of A. thaliana. Genome-wide association studies (GWAS) suggest that plant loci responsible for defense and cell wall integrity affect variation in this community. Furthermore, species richness in the bacterial community is shaped by host genetic variation, notably at loci that also influence the reproduction of viruses, trichome branching and morphogenesis.

Characterizing the organization of the human gut microbiota is a formidable challenge given the number of possible interactions between its components. Using a statistical approach initially applied to financial markets, we measured temporally conserved covariance among bacterial taxa in the microbiota of healthy members of a Bangladeshi birth cohort sampled from 1 to 60 months of age. The results revealed an “ecogroup” of 15 covarying bacterial taxa that provide a concise description of microbiota development in healthy children from this and other low-income countries, and a means for monitoring community repair in undernourished children treated with therapeutic foods. Features of ecogroup population dynamics were recapitulated in gnotobiotic piglets as they transitioned from exclusive milk feeding to a fully weaned state consuming a representative Bangladeshi diet.