PCR amplification plays an integral role in the measurement of mixed microbial communities via high-throughput DNA sequencing of the 16S ribosomal RNA (rRNA) gene. Yet PCR is also known to introduce multiple forms of bias in 16S rRNA studies. Here we present a paired modeling and experimental approach to characterize and mitigate PCR NPM-bias (PCR bias from non-primer-mismatch sources) in microbiota surveys. We use experimental data from mock bacterial communities to validate our approach and human gut microbiota samples to characterize PCR NPM-bias under real-world conditions. Our results suggest that PCR NPM-bias can skew estimates of microbial relative abundances by a factor of 4 or more, but that this bias can be mitigated using log-ratio linear models.

Pediatric obesity remains a public health burden and continues to increase in prevalence. The gut microbiota plays a causal role in obesity and is a promising therapeutic target. Specifically, the microbial production of short-chain fatty acids (SCFA) from the fermentation of otherwise indigestible dietary carbohydrates may protect against pediatric obesity and metabolic syndrome. Still, it has not been demonstrated that therapies involving microbiota-targeting carbohydrates, known as prebiotics, will enhance gut bacterial SCFA production in children and adolescents with obesity (age, 10 to 18 years old). Here, we used an in vitro system to examine the SCFA production by fecal microbiota from 17 children with obesity when exposed to five different commercially available over-the-counter (OTC) prebiotic supplements. We found microbiota from all 17 patients actively metabolized most prebiotics. Still, supplements varied in their acidogenic potential. Significant interdonor variation also existed in SCFA production, which 16S rRNA sequencing supported as being associated with differences in the host microbiota composition. Last, we found that neither fecal SCFA concentration, microbiota SCFA production capacity, nor markers of obesity positively correlated with one another. Together, these in vitro findings suggest the hypothesis that OTC prebiotic supplements may be unequal in their ability to stimulate SCFA production in children and adolescents with obesity and that the most acidogenic prebiotic may differ across individuals. IMPORTANCE Pediatric obesity remains a major public health problem in the United States, where 17% of children and adolescents are obese, and rates of pediatric “severe obesity” are increasing. Children and adolescents with obesity face higher health risks, and noninvasive therapies for pediatric obesity often have limited success. The human gut microbiome has been implicated in adult obesity, and microbiota-directed therapies can aid weight loss in adults with obesity. However, less is known about the microbiome in pediatric obesity, and microbiota-directed therapies are understudied in children and adolescents. Our research has two important findings: (i) dietary prebiotics (fiber) result in the microbiota from adolescents with obesity producing more SCFA, and (ii) the effectiveness of each prebiotic is donor dependent. Together, these findings suggest that prebiotic supplements could help children and adolescents with obesity, but that these therapies may not be “one size fits all.”

The mucophilic anaerobic bacterium Akkermansia muciniphila is a prominent member of the gastrointestinal (GI) microbiota and the only known species of the Verrucomicrobia phylum in the mammalian gut. A high prevalence of A. muciniphila in adult humans is associated with leanness and a lower risk for the development of obesity and diabetes. Four distinct A. muciniphila phylogenetic groups have been described, but little is known about their relative abundance in humans or how they impact human metabolic health. In this study, we isolated and characterized 71 new A. muciniphila strains from a cohort of children and adolescents undergoing treatment for obesity. Based on genomic and phenotypic analysis of these strains, we found several phylogroup-specific phenotypes that may impact the colonization of the GI tract or modulate host functions, such as oxygen tolerance, adherence to epithelial cells, iron and sulfur metabolism, and bacterial aggregation. In antibiotic-treated mice, phylogroups AmIV and AmII outcompeted AmI strains. In children and adolescents, AmI strains were most prominent, but we observed high variance in A. muciniphila abundance and single phylogroup dominance, with phylogroup switching occurring in a small subset of patients. Overall, these results highlight that the ecological principles determining which A. muciniphila phylogroup predominates in humans are complex and that A. muciniphila strain genetic and phenotypic diversity may represent an important variable that should be taken into account when making inferences as to this microbe’s impact on its host’s health. IMPORTANCE The abundance of Akkermansia muciniphila in the gastrointestinal (GI) tract is linked to multiple positive health outcomes. There are four known A. muciniphila phylogroups, yet the prevalence of these phylogroups and how they vary in their ability to influence human health is largely unknown. In this study, we performed a genomic and phenotypic analysis of 71 A. muciniphila strains and identified phylogroup-specific traits such as oxygen tolerance, adherence, and sulfur acquisition that likely influence colonization of the GI tract and differentially impact metabolic and immunological health. In humans, we observed that single Akkermansia phylogroups predominate at a given time but that the phylotype can switch in an individual. This collection of strains provides the foundation for the functional characterization of A. muciniphila phylogroup-specific effects on the multitude of host outcomes associated with Akkermansia colonization, including protection from obesity, diabetes, colitis, and neurological diseases, as well as enhanced responses to cancer immunotherapies.

Culture and screening of gut bacteria enable testing of microbial function and therapeutic potential. However, the diversity of human gut microbial communities (microbiota) impedes comprehensive experimental studies of individual bacterial taxa. Here, we combine advances in droplet microfluidics and high-throughput DNA sequencing to develop a platform for separating and assaying growth of microbiota members in picoliter droplets (MicDrop). MicDrop enabled us to cultivate 2.8 times more bacterial taxa than typical batch culture methods. We then used MicDrop to test whether individuals possess similar abundances of carbohydrate-degrading gut bacteria, using an approach which had previously not been possible due to throughput limitations of traditional bacterial culture techniques. Single MicDrop experiments allowed us to characterize carbohydrate utilization among dozens of gut bacterial taxa from distinct human stool samples. Our aggregate data across nine healthy stool donors revealed that all of the individuals harbored gut bacterial species capable of degrading common dietary polysaccharides. However, the levels of richness and abundance of polysaccharide-degrading species relative to monosaccharide-consuming taxa differed by up to 2.6-fold and 24.7-fold, respectively. Additionally, our unique dataset suggested that gut bacterial taxa may be broadly categorized by whether they can grow on single or multiple polysaccharides, and we found that this lifestyle trait is correlated with how broadly bacterial taxa can be found across individuals. This demonstration shows that it is feasible to measure the function of hundreds of bacterial taxa across multiple fecal samples from different people, which should in turn enable future efforts to design microbiota-directed therapies and yield new insights into microbiota ecology and evolution. IMPORTANCE Bacterial culture and assay are components of basic microbiological research, drug development, and diagnostic screening. However, community diversity can make it challenging to comprehensively perform experiments involving individual microbiota members. Here, we present a new microfluidic culture platform that makes it feasible to measure the growth and function of microbiota constituents in a single set of experiments. As a proof of concept, we demonstrate how the platform can be used to measure how hundreds of gut bacterial taxa drawn from different people metabolize dietary carbohydrates. Going forward, we expect this microfluidic technique to be adaptable to a range of other microbial assay needs.

Background Short-chain fatty acids (SCFAs) derived from gut bacteria are associated with protective roles in diseases ranging from obesity to colorectal cancers. Intake of microbially accessible dietary fibers (prebiotics) lead to varying effects on SCFA production in human studies, and gut microbial responses to nutritional interventions vary by individual. It is therefore possible that prebiotic therapies will require customizing to individuals. Results Here, we explored prebiotic personalization by conducting a three-way crossover study of three prebiotic treatments in healthy adults. We found that within individuals, metabolic responses were correlated across the three prebiotics. Individual identity, rather than prebiotic choice, was also the major determinant of SCFA response. Across individuals, prebiotic response was inversely related to basal fecal SCFA concentration, which, in turn, was associated with habitual fiber intake. Experimental measures of gut microbial SCFA production for each participant also negatively correlated with fiber consumption, supporting a model in which individuals’ gut microbiota are limited in their overall capacity to produce fecal SCFAs from fiber. Conclusions Our findings support developing personalized prebiotic regimens that focus on selecting individuals who stand to benefit, and that such individuals are likely to be deficient in fiber intake.

Many ecosystems retain an ecological memory of past conditions that affects responses to future stimuli. However, it remains unknown what mechanisms and dynamics may govern such a memory in microbial communities. Here, in both a human dietary intervention cohort and an artificial gut, we show that the human gut microbiome encodes a memory of past carbohydrate exposures. Changes in the relative abundance of primary degraders were sufficient to enhance metabolism, and these changes were mediated by transcriptional changes within hours of initial exposure. We further found that ecological memory of one carbohydrate species impacted metabolism of others. These findings demonstrate that the human gut microbiome's .

Eating a varied diet is a central tenet of good nutrition. Here, we develop a molecular tool to quantify human dietary plant diversity by applying DNA metabarcoding with the chloroplast trnL -P6 marker to 1,029 fecal samples from 324 participants across two interventional feeding studies and three observational cohorts. The number of plant taxa per sample (plant metabarcoding richness or pMR) correlated with recorded intakes in interventional diets and with indices calculated from a food frequency questionnaire in typical diets (ρ = 0.40 to 0.63). In adolescents unable to collect validated dietary survey data, trnL metabarcoding detected 111 plant taxa, with 86 consumed by more than one individual and four (wheat, chocolate, corn, and potato family) consumed by >70% of individuals. Adolescent pMR was associated with age and household income, replicating prior epidemiologic findings. Overall, trnL metabarcoding promises an objective and accurate measure of the number and types of plants consumed that is applicable to diverse human populations.

23Pediatric obesity remains a public health burden and continues to increase in 24 prevalence. The gut microbiota plays a causal role in obesity and is a promising 25 therapeutic target. Specifically, the microbial production of short-chain fatty acids 26 (SCFA) from the fermentation of otherwise indigestible dietary carbohydrates may 27 protect against pediatric obesity and metabolic syndrome. Still, it has not been 28 demonstrated that therapies involving microbiota-targeting carbohydrates, known as 29 prebiotics, will enhance gut bacterial SCFA production in children and adolescents with 30 obesity (age 10-18). Here, we used an in vitro system to examine the SCFA production 31 by fecal microbiota from 17 children with obesity when exposed to five different 32 commercially available over-the-counter (OTC) prebiotic supplements. We found 33 microbiota from all 17 patients actively metabolized most prebiotics. Still, supplements 34 varied in their acidogenic potential. Significant inter-donor variation also existed in SCFA 35 production, which 16S rRNA sequencing supported as being associated with differences 36 in the host microbiota composition. Last, we found that neither fecal SCFA 37 concentration, microbiota SCFA production capacity, nor markers of obesity positively 38 correlated with one another. Together, these in vitro findings suggest the hypothesis that 39 OTC prebiotic supplements may be unequal in their ability to stimulate SCFA production 40 in children and adolescents with obesity, and that the most acidogenic prebiotic may 41 differ across individuals. 42 43 44 IMPORTANCE Pediatric obesity remains a major public health problem in the US, where 45 17% of children and adolescents are obese, and rates of pediatric 'severe obesity' are 46 increasing. Children and adolescents with obesity face higher health risks, and non-47 invasive therapies for pediatric obesity often have limited success. The human gut 48 microbiome has been implicated in adult obesity, and microbiota-directed therapies can 49 aid weight loss in adults with obesity. However, less is known about the microbiome in 50 pediatric obesity, and microbiota-directed therapies are understudied in children and 51 adolescents. Our research has two important findings: 1) dietary prebiotics (fiber) cause 52 the microbiota from adolescents with obesity to produce more SCFA, and 2) the 53 effectiveness of each prebiotic is donor-dependent. Together, these findings suggest 54 that prebiotic supplements could help children and adolescents with obesity, but that 55 these therapies may not be one-size-fits-all. 56 developed long-term complications, we pursued three specific lines of inquiry: 1) whether 132 different types of prebiotics lead to differences in SCFA production by gut microbiota from 133 adolescents with obesity; 2) whether the effects of prebiotics are shaped by inter-134 individual differences in gut microbiota structure; and, 3) whether fecal SCFA production 135 is associated with protection from obesity. 136