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UNLABELLED: Large-scale studies are essential to answer questions about complex microbial communities that can be extremely dynamic across hosts, environments, and time points. However, managing acquisition, processing, and analysis of large numbers of samples poses many challenges, with cross-contamination being the biggest obstacle. Contamination complicates analysis and results in sample loss, leading to higher costs and constraints on mixed sample type study designs. While many researchers opt for 96-well plates for their workflows, these plates present a significant issue: the shared seal and weak separation between wells leads to well-to-well contamination. To address this concern, we propose an innovative high-throughput approach, termed as the Matrix method, which employs barcoded Matrix Tubes for sample acquisition. This method is complemented by a paired nucleic acid and metabolite extraction, utilizing 95% (vol/vol) ethanol to stabilize microbial communities and as a solvent for extracting metabolites. Comparative analysis between conventional 96-well plate extractions and the Matrix method, measuring 16S rRNA gene levels via quantitative polymerase chain reaction, demonstrates a notable decrease in well-to-well contamination with the Matrix method. Metagenomics, 16S rRNA gene amplicon sequencing (16S), and untargeted metabolomics analysis via liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed that the Matrix method recovers reproducible microbial and metabolite compositions that can distinguish between subjects. This advancement is critical for large-scale study design as it minimizes well-to-well contamination and technical variation, shortens processing times, and integrates with automated infrastructure for enhancing sample randomization and metadata generation. IMPORTANCE: Understanding dynamic microbial communities typically requires large-scale studies. However, handling large numbers of samples introduces many challenges, with cross-contamination being a major issue. It not only complicates analysis but also leads to sample loss and increased costs and restricts diverse study designs. The prevalent use of 96-well plates for nucleic acid and metabolite extractions exacerbates this problem due to their wells having little separation and being connected by a single plate seal. To address this, we propose a new strategy using barcoded Matrix Tubes, showing a significant reduction in cross-contamination compared to conventional plate-based approaches. Additionally, this method facilitates the extraction of both nucleic acids and metabolites from a single tubed sample, eliminating the need to collect separate aliquots for each extraction. This innovation improves large-scale study design by shortening processing times, simplifying analysis, facilitating metadata curation, and producing more reliable results.

High levels of social connectivity among group-living animals have been hypothesized to benefit individuals by creating opportunities to rapidly reseed the microbiome and maintain stability against disruption. We tested this hypothesis by perturbing the microbiome of a wild population of Grants gazelles with an antibiotic and asking whether microbiome recovery differs between individuals with high versus low levels of social connectivity. We found that after treatment, individuals with high social connectivity experienced a faster increase in microbiome richness than less socially connected individuals. Unexpectedly, the rapid increase in microbiome richness of highly connected individuals that received treatment led to their microbiomes becoming more distinct relative to the background population. Our results suggest that the microbiome of individuals with high social connectivity can be rapidly recolonized after a perturbation event, but this leads to a microbiome that is more distinct from, rather than more similar to the unperturbed state. This work provides new insight into the role of social interactions in shaping the microbiome.

Studies using 16S rRNA and shotgun metagenomics typically yield different results, usually attributed to PCR amplification biases. We introduce Greengenes2, a reference tree that unifies genomic and 16S rRNA databases in a consistent, integrated resource. By inserting sequences into a whole-genome phylogeny, we show that 16S rRNA and shotgun metagenomic data generated from the same samples agree in principal coordinates space, taxonomy and phenotype effect size when analyzed with the same tree.

In 2020, we identified cancer-specific microbial signals in The Cancer Genome Atlas (TCGA) [1]. Multiple peer-reviewed papers independently verified or extended our findings [2-12]. Given this impact, we carefully considered concerns by Gihawi et al. [13] that batch correction and database contamination with host sequences artificially created the appearance of cancer type-specific microbiomes. (1) We tested batch correction by comparing raw and Voom-SNM-corrected data per-batch, finding predictive equivalence and significantly similar features. We found consistent results with a modern microbiome-specific method (ConQuR [14]), and when restricting to taxa found in an independent, highly-decontaminated cohort. (2) Using Conterminator [15], we found low levels of human contamination in our original databases (~1% of genomes). We demonstrated that the increased detection of human reads in Gihawi et al. [13] was due to using a newer human genome reference. (3) We developed Exhaustive, a method twice as sensitive as Conterminator, to clean RefSeq. We comprehensively host-deplete TCGA with many human (pan)genome references. We repeated all analyses with this and the Gihawi et al. [13] pipeline, and found cancer type-specific microbiomes. These extensive re-analyses and updated methods validate our original conclusion that cancer type-specific microbial signatures exist in TCGA, and show they are robust to methodology.

The composition of the human vaginal microbiome has been linked to a variety of medical conditions including yeast infection, bacterial vaginosis, and sexually transmitted infection. The vaginal microbiome is becoming increasingly acknowledged as a key factor in personal health, and it is essential to establish methods to collect and process accurate samples with self-collection techniques to allow large, population-based studies. In this study, we investigate if using AssayAssure Genelock, a nucleic acid preservative, introduces microbial biases in self-collected vaginal samples. To our knowledge, we also contribute some of the first evidence regarding the impacts of multiple swabs taken at one time point. Vaginal samples have relatively low biomass, so the ability to collect multiple swabs from a unique participant at a single time would greatly improve the replicability and data available for future studies. This will hopefully lay the groundwork to gain a more complete and accurate understanding of the vaginal microbiome.

The gut microbiome is important for human health, yet modulation requires more insight into inter-individual variation. Here, we explored latent structures of the human gut microbiome across the human lifespan, applying partitioning, pseudotime, and ordination approaches to >35,000 samples. Specifically, three major gut microbiome branches were identified, within which multiple partitions were observed in adulthood, with differential abundances of species along branches. Different compositions and metabolic functions characterized the branches' tips, reflecting ecological differences. An unsupervised network analysis from longitudinal data from 745 individuals showed that partitions exhibited connected gut microbiome states rather than over-partitioning. Stability in the Bacteroides-enriched branch was associated with specific ratios of Faecalibacterium:Bacteroides. We also showed that associations with factors (intrinsic and extrinsic) could be generic, branch- or partition-specific. Our ecological framework for cross-sectional and longitudinal data allows a better understanding of overall variation in the human gut microbiome and disentangles factors associated with specific configurations.

Introduction: During adulthood, the skin microbiota can be relatively stable if environmental conditions are also stable, yet physiological changes of the skin with age may affect the skin microbiome and its function. The microbiome is an important factor to consider in aging since it constitutes most of the genes that are expressed on the human body. However, severity of specific aging signs (one of the parameters used to measure apparent age) and skin surface quality (e.g., texture, hydration, pH, sebum, etc.) may not be indicative of chronological age. For example, older individuals can have young looking skin (young apparent age) and young individuals can be of older apparent age. Methods: Here we aim to identify microbial taxa of interest associated to skin quality/aging signs using a multi-study analysis of 13 microbiome datasets consisting of 16S rRNA amplicon sequence data and paired skin clinical data from the face. Results: We show that there is a negative relationship between microbiome diversity and transepidermal water loss, and a positive association between microbiome diversity and age. Aligned with a tight link between age and wrinkles, we report a global positive association between microbiome diversity and Crows feet wrinkles, but with this relationship varying significantly by sub-study. Finally, we identify taxa potentially associated with wrinkles, TEWL and corneometer measures. Discussion: These findings represent a key step towards understanding the implication of the skin microbiota in skin aging signs.

Despite advances in sequencing, lack of standardization makes comparisons across studies challenging and hampers insights into the structure and function of microbial communities across multiple habitats on a planetary scale. Here we present a multi-omics analysis of a diverse set of 880 microbial community samples collected for the Earth Microbiome Project. We include amplicon (16S, 18S, ITS) and shotgun metagenomic sequence data, and untargeted metabolomics data (liquid chromatography-tandem mass spectrometry and gas chromatography mass spectrometry). We used standardized protocols and analytical methods to characterize microbial communities, focusing on relationships and co-occurrences of microbially related metabolites and microbial taxa across environments, thus allowing us to explore diversity at extraordinary scale. In addition to a reference database for metagenomic and metabolomic data, we provide a framework for incorporating additional studies, enabling the expansion of existing knowledge in the form of an evolving community resource. We demonstrate the utility of this database by testing the hypothesis that every microbe and metabolite is everywhere but the environment selects. Our results show that metabolite diversity exhibits turnover and nestedness related to both microbial communities and the environment, whereas the relative abundances of microbially related metabolites vary and co-occur with specific microbial consortia in a habitat-specific manner. We additionally show the power of certain chemistry, in particular terpenoids, in distinguishing Earth's environments (for example, terrestrial plant surfaces and soils, freshwater and marine animal stool), as well as that of certain microbes including Conexibacter woesei (terrestrial soils), Haloquadratum walsbyi (marine deposits) and Pantoea dispersa (terrestrial plant detritus). This Resource provides insight into the taxa and metabolites within microbial communities from diverse habitats across Earth, informing both microbial and chemical ecology, and provides a foundation and methods for multi-omics microbiome studies of hosts and the environment.

BackgroundIndividual diet components and specific dietary regimens have been shown to impact the gut microbiome.ObjectivesHere, we explored the contribution of long-term diet by searching for dietary patterns that would best associate with the gut microbiome in a population-based cohort.MethodsUsing a priori and a posteriori approaches, we constructed dietary patterns from an FFQ completed by 1800 adults in the American Gut Project. Dietary patterns were defined as groups of participants or combinations of food variables (factors) driven by criteria ranging from individual nutrients to overall diet. We associated these patterns with 16S ribosomal RNA-based gut microbiome data for a subset of 744 participants.ResultsCompared to individual features (e.g., fiber and protein), or to factors representing a reduced number of dietary features, 5 a posteriori dietary patterns based on food groups were best associated with gut microbiome beta diversity (P ≤ 0.0002). Two patterns followed Prudent-like diets-Plant-Based and Flexitarian-and exhibited the highest Healthy Eating Index 2010 (HEI-2010) scores. Two other patterns presented Western-like diets with a gradient in HEI-2010 scores. A fifth pattern consisted mostly of participants following an Exclusion diet (e.g., low carbohydrate). Notably, gut microbiome alpha diversity was significantly lower in the most Western pattern compared to the Flexitarian pattern (P ≤ 0.009), and the Exclusion diet pattern was associated with low relative abundance of Bifidobacterium (P ≤ 1.2 × 10-7), which was better explained by diet than health status.ConclusionsWe demonstrated that global-diet a posteriori patterns were more associated with gut microbiome variations than individual dietary features among adults in the United States. These results confirm that evaluating diet as a whole is important when studying the gut microbiome. It will also facilitate the design of more personalized dietary strategies in general populations.

Preterm infants are at a greater risk for the development of asthma and atopic disease, which can lead to lifelong negative health consequences. This may be due, in part, to alterations that occur in the gut microbiome and metabolome during their stay in the Neonatal Intensive Care Unit (NICU). To explore the differential roles of family history (i.e., predisposition due to maternal asthma diagnosis) and hospital-related environmental and clinical factors that alter microbial exposures early in life, we considered a unique cohort of preterm infants born ≤ 34 weeks gestational age from two local level III NICUs, as part of the MAP (Microbiome, Atopic disease, and Prematurity) Study. From MAP participants, we chose a sub-cohort of infants whose mothers had a history of asthma and matched gestational age and sex to infants of mothers without a history of asthma diagnosis (control). We performed a prospective, paired metagenomic and metabolomic analysis of stool and milk feed samples collected at birth, 2 weeks, and 6 weeks postnatal age. Although there were clinical factors associated with shifts in the diversity and composition of stool-associated bacterial communities, maternal asthma diagnosis did not play an observable role in shaping the infant gut microbiome during the study period. There were significant differences, however, in the metabolite profile between the maternal asthma and control groups at 6 weeks postnatal age. The most notable changes occurred in the linoleic acid spectral network, which plays a role in inflammatory and immune pathways, suggesting early metabolomic changes in the gut of preterm infants born to mothers with a history of asthma. Our pilot study suggests that a history of maternal asthma alters a preterm infants' metabolomic pathways in the gut, as early as the first 6 weeks of life.

BackgroundEarly microbiota perturbations are associated with disorders that involve immunological underpinnings. Cesarean section (CS)-born babies show altered microbiota development in relation to babies born vaginally. Here we present the first statistically powered longitudinal study to determine the effect of restoring exposure to maternal vaginal fluids after CS birth.MethodsUsing 16S rRNA gene sequencing, we followed the microbial trajectories of multiple body sites in 177 babies over the first year of life; 98 were born vaginally, and 79 were born by CS, of whom 30 were swabbed with a maternal vaginal gauze right after birth.FindingsCompositional tensor factorization analysis confirmed that microbiota trajectories of exposed CS-born babies aligned more closely with that of vaginally born babies. Interestingly, the majority of amplicon sequence variants from maternal vaginal microbiomes on the day of birth were shared with other maternal sites, in contrast to non-pregnant women from the Human Microbiome Project (HMP) study.ConclusionsThe results of this observational study prompt urgent randomized clinical trials to test whether microbial restoration reduces the increased disease risk associated with CS birth and the underlying mechanisms. It also provides evidence of the pluripotential nature of maternal vaginal fluids to provide pioneer bacterial colonizers for the newborn body sites. This is the first study showing long-term naturalization of the microbiota of CS-born infants by restoring microbial exposure at birth.FundingC&D, Emch Fund, CIFAR, Chilean CONICYT and SOCHIPE, Norwegian Institute of Public Health, Emerald Foundation, NIH, National Institute of Justice, Janssen.

Changes in the gut microbiota during pathogen infection are often predicted to influence disease outcomes. However, studies exploring whether pathogens induce microbiota shifts have yielded inconsistent results. This suggests that variation in infection, rather than the presence of infection alone, might shape pathogen-microbiota relationships. For example, most hosts are coinfected with multiple pathogens simultaneously, and hosts vary in how long they are infected, which may amplify or diminish microbial shifts expected in response to a focal pathogen. We used a longitudinal anthelmintic treatment study of free-ranging African buffalo (Syncerus caffer) to examine whether (i) coinfection with bovine tuberculosis (Mycobacterium bovis, TB) and gastrointestinal nematodes, and (ii) the duration of TB infection, modified effects of single pathogens on the gut microbiota. By accounting for the interaction between TB and nematodes, we found that coinfection affected changes in microbial abundance associated with single infections. Furthermore, the duration of TB infection predicted more microbiota variation than the presence of TB. Importantly, coinfection and infection duration had nearly as much influence on microbial patterns as demographic and environmental factors commonly examined in microbiota research. These findings demonstrate that acknowledging infection heterogeneities may be crucial to understanding relationships between pathogens and the gut microbiota.

As the number of human microbiome studies expand, it is increasingly important to identify cost-effective, practical preservatives that allow for room temperature sample storage. Here, we reanalyzed 16S rRNA gene amplicon sequencing data from a large sample storage study published in 2016 and performed shotgun metagenomic sequencing on remnant DNA from this experiment. Both results support the initial findings that 95% ethanol, a nontoxic, cost-effective preservative, is effective at preserving samples at room temperature for weeks. We expanded on this analysis by collecting a new set of fecal, saliva, and skin samples to determine the optimal ratio of 95% ethanol to sample. We identified optimal collection protocols for fecal samples (storing a fecal swab in 95% ethanol) and saliva samples (storing unstimulated saliva in 95% ethanol at a ratio of 1:2). Storing skin swabs in 95% ethanol reduced microbial biomass and disrupted community composition, highlighting the difficulties of low biomass sample preservation. The results from this study identify practical solutions for large-scale analyses of fecal and oral microbial communities.IMPORTANCE Expanding our knowledge of microbial communities across diverse environments includes collecting samples in places far from the laboratory. Identifying cost-effective preservatives that will enable room temperature storage of microbial communities for sequencing analysis is crucial to enabling microbiome analyses across diverse populations. Here, we validate findings that 95% ethanol efficiently preserves microbial composition at room temperature for weeks. We also identified the optimal ratio of 95% ethanol to sample for stool and saliva to preserve both microbial load and composition. These results provide rationale for an accessible, nontoxic, cost-effective solution that will enable crowdsourcing microbiome studies, such as The Microsetta Initiative, and lower the barrier for collecting diverse samples.

Standard workflows for analyzing microbiomes often include the creation and curation of phylogenetic trees. Here we present EMPress, an interactive web tool for visualizing trees in the context of microbiome, metabolome, and other community data scalable to trees with well over 500,000 nodes. EMPress provides novel functionality-including ordination integration and animations-alongside many standard tree visualization features and thus simplifies exploratory analyses of many forms of 'omic data.IMPORTANCE Phylogenetic trees are integral data structures for the analysis of microbial communities. Recent work has also shown the utility of trees constructed from certain metabolomic data sets, further highlighting their importance in microbiome research. The ever-growing scale of modern microbiome surveys has led to numerous challenges in visualizing these data. In this paper we used five diverse data sets to showcase the versatility and scalability of EMPress, an interactive web visualization tool. EMPress addresses the growing need for exploratory analysis tools that can accommodate large, complex multi-omic data sets.

BackgroundImbalances in the gut microbial community (dysbiosis) of vertebrates have been associated with several gastrointestinal and autoimmune diseases. However, it is unclear which taxa are associated with gut dysbiosis, and if particular gut regions or specific time periods during ontogeny are more susceptible. We also know very little of this process in non-model organisms, despite an increasing realization of the general importance of gut microbiota for health.MethodsHere, we examine the changes that occur in the microbiome during dysbiosis in different parts of the gastrointestinal tract in a long-lived bird with high juvenile mortality, the ostrich (Struthio camelus). We evaluated the 16S rRNA gene composition of the ileum, cecum, and colon of 68 individuals that died of suspected enterocolitis during the first 3 months of life (diseased individuals), and of 50 healthy individuals that were euthanized as age-matched controls. We combined these data with longitudinal environmental and fecal sampling to identify potential sources of pathogenic bacteria and to unravel at which stage of development dysbiosis-associated bacteria emerge.ResultsDiseased individuals had drastically lower microbial alpha diversity and differed substantially in their microbial beta diversity from control individuals in all three regions of the gastrointestinal tract. The clear relationship between low diversity and disease was consistent across all ages in the ileum, but decreased with age in the cecum and colon. Several taxa were associated with mortality (Enterobacteriaceae, Peptostreptococcaceae, Porphyromonadaceae, Clostridium), while others were associated with health (Lachnospiraceae, Ruminococcaceae, Erysipelotrichaceae, Turicibacter, Roseburia). Environmental samples showed no evidence of dysbiosis-associated bacteria being present in either the food, water, or soil substrate. Instead, the repeated fecal sampling showed that pathobionts were already present shortly after hatching and proliferated in individuals with low microbial diversity, resulting in high mortality several weeks later.ConclusionsIdentifying the origins of pathobionts in neonates and the factors that subsequently influence the establishment of diverse gut microbiota may be key to understanding dysbiosis and host development. Video Abstract.

Systematic characterization of the cancer microbiome provides the opportunity to develop techniques that exploit non-human, microorganism-derived molecules in the diagnosis of a major human disease. Following recent demonstrations that some types of cancer show substantial microbial contributions1-10, we re-examined whole-genome and whole-transcriptome sequencing studies in The Cancer Genome Atlas11 (TCGA) of 33 types of cancer from treatment-naive patients (a total of 18,116 samples) for microbial reads, and found unique microbial signatures in tissue and blood within and between most major types of cancer. These TCGA blood signatures remained predictive when applied to patients with stage Ia-IIc cancer and cancers lacking any genomic alterations currently measured on two commercial-grade cell-free tumour DNA platforms, despite the use of very stringent decontamination analyses that discarded up to 92.3% of total sequence data. In addition, we could discriminate among samples from healthy, cancer-free individuals (n = 69) and those from patients with multiple types of cancer (prostate, lung, and melanoma; 100 samples in total) solely using plasma-derived, cell-free microbial nucleic acids. This potential microbiome-based oncology diagnostic tool warrants further exploration.

Lifestyle factors, such as diet, strongly influence the structure, diversity, and composition of the microbiome. While we have witnessed over the last several years a resurgence of interest in fermented foods, no study has specifically explored the effects of their consumption on gut microbiota in large cohorts. To assess whether the consumption of fermented foods is associated with a systematic signal in the gut microbiome and metabolome, we used a multi-omic approach (16S rRNA amplicon sequencing, metagenomic sequencing, and untargeted mass spectrometry) to analyze stool samples from 6,811 individuals from the American Gut Project, including 115 individuals specifically recruited for their frequency of fermented food consumption for a targeted 4-week longitudinal study. We observed subtle but statistically significant differences between consumers and nonconsumers in beta diversity as well as differential taxa between the two groups. We found that the metabolome of fermented food consumers was enriched with conjugated linoleic acid (CLA), a putatively health-promoting molecule. Cross-omic analyses between metagenomic sequencing and mass spectrometry suggest that CLA may be driven by taxa associated with fermented food consumers. Collectively, we found modest yet persistent signatures associated with fermented food consumption that appear present in multiple -omic types which motivate further investigation of how different types of fermented food impact the gut microbiome and overall health.IMPORTANCE Public interest in the effects of fermented food on the human gut microbiome is high, but limited studies have explored the association between fermented food consumption and the gut microbiome in large cohorts. Here, we used a combination of omics-based analyses to study the relationship between the microbiome and fermented food consumption in thousands of people using both cross-sectional and longitudinal data. We found that fermented food consumers have subtle differences in their gut microbiota structure, which is enriched in conjugated linoleic acid, thought to be beneficial. The results suggest that further studies of specific kinds of fermented food and their impacts on the microbiome and health will be useful.

Diet and host phylogeny drive the taxonomic and functional contents of the gut microbiome in mammals, yet it is unknown whether these patterns hold across all vertebrate lineages. Here, we assessed gut microbiomes from ∼900 vertebrate species, including 315 mammals and 491 birds, assessing contributions of diet, phylogeny, and physiology to structuring gut microbiomes. In most nonflying mammals, strong correlations exist between microbial community similarity, host diet, and host phylogenetic distance up to the host order level. In birds, by contrast, gut microbiomes are only very weakly correlated to diet or host phylogeny. Furthermore, while most microbes resident in mammalian guts are present in only a restricted taxonomic range of hosts, most microbes recovered from birds show little evidence of host specificity. Notably, among the mammals, bats host especially bird-like gut microbiomes, with little evidence for correlation to host diet or phylogeny. This suggests that host-gut microbiome phylosymbiosis depends on factors convergently absent in birds and bats, potentially associated with physiological adaptations to flight. Our findings expose major variations in the behavior of these important symbioses in endothermic vertebrates and may signal fundamental evolutionary shifts in the cost/benefit framework of the gut microbiome.IMPORTANCE In this comprehensive survey of microbiomes of >900 species, including 315 mammals and 491 birds, we find a striking convergence of the microbiomes of birds and animals that fly. In nonflying mammals, diet and short-term evolutionary relatedness drive the microbiome, and many microbial species are specific to a particular kind of mammal, but flying mammals and birds break this pattern with many microbes shared across different species, with little correlation either with diet or with relatedness of the hosts. This finding suggests that adaptation to flight breaks long-held relationships between hosts and their microbes.

Urbanization represents a profound shift in human behaviour, and has considerable cultural and health-associated consequences1,2. Here, we investigate chemical and microbial characteristics of houses and their human occupants across an urbanization gradient in the Amazon rainforest, from a remote Peruvian Amerindian village to the Brazilian city of Manaus. Urbanization was found to be associated with reduced microbial outdoor exposure, increased contact with housing materials, antimicrobials and cleaning products, and increased exposure to chemical diversity. The degree of urbanization correlated with changes in the composition of house bacterial and microeukaryotic communities, increased house and skin fungal diversity, and an increase in the relative abundance of human skin-associated fungi and bacteria in houses. Overall, our results indicate that urbanization has large-scale effects on chemical and microbial exposures and on the human microbiota.

Integrating multiomics datasets is critical for microbiome research; however, inferring interactions across omics datasets has multiple statistical challenges. We solve this problem by using neural networks (https://github.com/biocore/mmvec) to estimate the conditional probability that each molecule is present given the presence of a specific microorganism. We show with known environmental (desert soil biocrust wetting) and clinical (cystic fibrosis lung) examples, our ability to recover microbe-metabolite relationships, and demonstrate how the method can discover relationships between microbially produced metabolites and inflammatory bowel disease.

Over the past decade several studies have reported that the gut microbiomes of mammals with similar dietary niches exhibit similar compositional and functional traits. However, these studies rely heavily on samples from captive individuals and often confound host phylogeny, gut morphology, and diet. To more explicitly test the influence of host dietary niche on the mammalian gut microbiome we use 16S rRNA gene amplicon sequencing and shotgun metagenomics to compare the gut microbiota of 18 species of wild non-human primates classified as either folivores or closely related non-folivores, evenly distributed throughout the primate order and representing a range of gut morphological specializations. While folivory results in some convergent microbial traits, collectively we show that the influence of host phylogeny on both gut microbial composition and function is much stronger than that of host dietary niche. This pattern does not result from differences in host geographic location or actual dietary intake at the time of sampling, but instead appears to result from differences in host physiology. These findings indicate that mammalian gut microbiome plasticity in response to dietary shifts over both the lifespan of an individual host and the evolutionary history of a given host species is constrained by host physiological evolution. Therefore, the gut microbiome cannot be considered separately from host physiology when describing host nutritional strategies and the emergence of host dietary niches.

The gut microbiome likely plays a role in the etiology of multiple health conditions, especially those affecting the gastrointestinal tract. Little consensus exists as to the best, standard methods to collect fecal samples for future microbiome analysis. We evaluated three distinct populations (N = 132 participants) using 16S rRNA gene amplicon sequencing data to investigate the reproducibility, stability, and accuracy of microbial profiles in fecal samples collected and stored via fecal occult blood test (FOBT) or Flinders Technology Associates (FTA) cards, fecal immunochemical tests (FIT) tubes, 70% and 95% ethanol, RNAlater, or with no solution. For each collection method, based on relative abundance of select phyla and genera, two alpha diversity metrics, and four beta diversity metrics, we calculated intraclass correlation coefficients (ICCs) to estimate reproducibility and stability, and Spearman correlation coefficients (SCCs) to estimate accuracy of the fecal microbial profile. Comparing duplicate samples, reproducibility ICCs for all collection methods were excellent (ICCs ≥75%). After 4-7 days at ambient temperature, ICCs for microbial profile stability were excellent (≥75%) for most collection methods, except those collected via no-solution and 70% ethanol. SCCs comparing each collection method to immediately-frozen no-solution samples ranged from fair to excellent for most methods; however, accuracy of genus-level relative abundances differed by collection method. Our findings, taken together with previous studies and feasibility considerations, indicated that FOBT/FTA cards, FIT tubes, 95% ethanol, and RNAlater are excellent choices for fecal sample collection methods in future microbiome studies. Furthermore, establishing standard collection methods across studies is highly desirable.

Although much work has linked the human microbiome to specific phenotypes and lifestyle variables, data from different projects have been challenging to integrate and the extent of microbial and molecular diversity in human stool remains unknown. Using standardized protocols from the Earth Microbiome Project and sample contributions from over 10,000 citizen-scientists, together with an open research network, we compare human microbiome specimens primarily from the United States, United Kingdom, and Australia to one another and to environmental samples. Our results show an unexpected range of beta-diversity in human stool microbiomes compared to environmental samples; demonstrate the utility of procedures for removing the effects of overgrowth during room-temperature shipping for revealing phenotype correlations; uncover new molecules and kinds of molecular communities in the human stool metabolome; and examine emergent associations among the microbiome, metabolome, and the diversity of plants that are consumed (rather than relying on reductive categorical variables such as veganism, which have little or no explanatory power). We also demonstrate the utility of the living data resource and cross-cohort comparison to confirm existing associations between the microbiome and psychiatric illness and to reveal the extent of microbiome change within one individual during surgery, providing a paradigm for open microbiome research and education. IMPORTANCE We show that a citizen science, self-selected cohort shipping samples through the mail at room temperature recaptures many known microbiome results from clinically collected cohorts and reveals new ones. Of particular interest is integrating n = 1 study data with the population data, showing that the extent of microbiome change after events such as surgery can exceed differences between distinct environmental biomes, and the effect of diverse plants in the diet, which we confirm with untargeted metabolomics on hundreds of samples.

The trillions of microbes living in the gut-the gut microbiota-play an important role in human biology and disease. While much has been done to explore its diversity, a full understanding of our microbiomes demands an evolutionary perspective. In this review, we compare microbiomes from human populations, placing them in the context of microbes from humanity's near and distant animal relatives. We discuss potential mechanisms to generate host-specific microbiome configurations and the consequences of disrupting those configurations. Finally, we propose that this broader phylogenetic perspective is useful for understanding the mechanisms underlying human-microbiome interactions.

Our growing awareness of the microbial world's importance and diversity contrasts starkly with our limited understanding of its fundamental structure. Despite recent advances in DNA sequencing, a lack of standardized protocols and common analytical frameworks impedes comparisons among studies, hindering the development of global inferences about microbial life on Earth. Here we present a meta-analysis of microbial community samples collected by hundreds of researchers for the Earth Microbiome Project. Coordinated protocols and new analytical methods, particularly the use of exact sequences instead of clustered operational taxonomic units, enable bacterial and archaeal ribosomal RNA gene sequences to be followed across multiple studies and allow us to explore patterns of diversity at an unprecedented scale. The result is both a reference database giving global context to DNA sequence data and a framework for incorporating data from future studies, fostering increasingly complete characterization of Earth's microbial diversity.

The mammal gut microbiome, which includes host microbes and their respective genes, is now recognized as an essential second genome that provides critical functions to the host. In humans, studies have revealed that lifestyle strongly influences the composition and diversity of the gastrointestinal microbiome. We hypothesized that these trends in humans may be paralleled in mammals subjected to anthropogenic forces such as domestication and captivity, in which diets and natural life histories are often greatly modified. We investigated fecal microbiomes of Przewalski's horse (PH; Equus ferus przewalskii), the only horses alive today not successfully domesticated by humans, and herded, domestic horse (E. f. caballus) living in adjacent natural grasslands. We discovered PH fecal microbiomes hosted a distinct and more diverse community of bacteria compared to domestic horses, which is likely partly explained by different plant diets as revealed by trnL maker data. Within the PH population, four individuals were born in captivity in European zoos and hosted a strikingly low diversity of fecal microbiota compared to individuals born in natural reserves in France and Mongolia. These results suggest that anthropogenic forces can dramatically reshape equid gastrointestinal microbiomes, which has broader implications for the conservation management of endangered mammals.

Recent studies increasingly note the effect of captivity or the built environment on the microbiome of humans and other animals. As symbiotic microbes are essential to many aspects of biology (e.g., digestive and immune functions), it is important to understand how lifestyle differences can impact the microbiome, and, consequently, the health of hosts. Animals living in captivity experience a range of changes that may influence the gut bacteria, such as diet changes, treatments, and reduced contact with other individuals, species and variable environmental substrates that act as sources of bacterial diversity. Thus far, initial results from previous studies point to a pattern of decreased bacterial diversity in captive animals. However, these studies are relatively limited in the scope of species that have been examined. Here we present a dataset that includes paired wild and captive samples from mammalian taxa across six Orders to investigate generalizable patterns of the effects captivity on mammalian gut bacteria. In comparing the wild to the captive condition, our results indicate that alpha diversity of the gut bacteria remains consistent in some mammalian hosts (bovids, giraffes, anteaters, and aardvarks), declines in the captive condition in some hosts (canids, primates, and equids), and increases in the captive condition in one host taxon (rhinoceros). Differences in gut bacterial beta diversity between the captive and wild state were observed for most of the taxa surveyed, except the even-toed ungulates (bovids and giraffes). Additionally, beta diversity variation was also strongly influenced by host taxonomic group, diet type, and gut fermentation physiology. Bacterial taxa that demonstrated larger shifts in relative abundance between the captive and wild states included members of the Firmicutes and Bacteroidetes. Overall, the patterns that we observe will inform a range of disciplines from veterinary practice to captive breeding efforts for biological conservation. Furthermore, bacterial taxa that persist in the captive state provide unique insight into symbiotic relationships with the host.

Advances in sequencing technologies have enabled novel insights into microbial niche differentiation, from analyzing environmental samples to understanding human diseases and informing dietary studies. However, identifying the microbial taxa that differentiate these samples can be challenging. These issues stem from the compositional nature of 16S rRNA gene data (or, more generally, taxon or functional gene data); the changes in the relative abundance of one taxon influence the apparent abundances of the others. Here we acknowledge that inferring properties of individual bacteria is a difficult problem and instead introduce the concept of balances to infer meaningful properties of subcommunities, rather than properties of individual species. We show that balances can yield insights about niche differentiation across multiple microbial environments, including soil environments and lung sputum. These techniques have the potential to reshape how we carry out future ecological analyses aimed at revealing differences in relative taxonomic abundances across different samples. IMPORTANCE By explicitly accounting for the compositional nature of 16S rRNA gene data through the concept of balances, balance trees yield novel biological insights into niche differentiation. The software to perform this analysis is available under an open-source license and can be obtained at https://github.com/biocore/gneiss. Author Video: An author video summary of this article is available.

Many factors affect the microbiomes of humans, mice, and other mammals, but substantial challenges remain in determining which of these factors are of practical importance. Considering the relative effect sizes of both biological and technical covariates can help improve study design and the quality of biological conclusions. Care must be taken to avoid technical bias that can lead to incorrect biological conclusions. The presentation of quantitative effect sizes in addition to P values will improve our ability to perform meta-analysis and to evaluate potentially relevant biological effects. A better consideration of effect size and statistical power will lead to more robust biological conclusions in microbiome studies.

Examining the way in which animals, including those in captivity, interact with their environment is extremely important for studying ecological processes and developing sophisticated animal husbandry. Here we use the Komodo dragon (Varanus komodoensis) to quantify the degree of sharing of salivary, skin, and fecal microbiota with their environment in captivity. Both species richness and microbial community composition of most surfaces in the Komodo dragon's environment are similar to the Komodo dragon's salivary and skin microbiota but less similar to the stool-associated microbiota. We additionally compared host-environment microbiome sharing between captive Komodo dragons and their enclosures, humans and pets and their homes, and wild amphibians and their environments. We observed similar host-environment microbiome sharing patterns among humans and their pets and Komodo dragons, with high levels of human/pet- and Komodo dragon-associated microbes on home and enclosure surfaces. In contrast, only small amounts of amphibian-associated microbes were detected in the animals' environments. We suggest that the degree of sharing between the Komodo dragon microbiota and its enclosure surfaces has important implications for animal health. These animals evolved in the context of constant exposure to a complex environmental microbiota, which likely shaped their physiological development; in captivity, these animals will not receive significant exposure to microbes not already in their enclosure, with unknown consequences for their health. IMPORTANCE Animals, including humans, have evolved in the context of exposure to a variety of microbial organisms present in the environment. Only recently have humans, and some animals, begun to spend a significant amount of time in enclosed artificial environments, rather than in the more natural spaces in which most of evolution took place. The consequences of this radical change in lifestyle likely extend to the microbes residing in and on our bodies and may have important implications for health and disease. A full characterization of host-microbe sharing in both closed and open environments will provide crucial information that may enable the improvement of health in humans and in captive animals, both of which experience a greater incidence of disease (including chronic illness) than counterparts living under more ecologically natural conditions.

Immediate freezing at -20°C or below has been considered the gold standard for microbiome preservation, yet this approach is not feasible for many field studies, ranging from anthropology to wildlife conservation. Here we tested five methods for preserving human and dog fecal specimens for periods of up to 8 weeks, including such types of variation as freeze-thaw cycles and the high temperature fluctuations often encountered under field conditions. We found that three of the methods-95% ethanol, FTA cards, and the OMNIgene Gut kit-can preserve samples sufficiently well at ambient temperatures such that differences at 8 weeks are comparable to differences among technical replicates. However, even the worst methods, including those with no fixative, were able to reveal microbiome differences between species at 8 weeks and between individuals after a week, allowing meta-analyses of samples collected using various methods when the effect of interest is expected to be larger than interindividual variation (although use of a single method within a study is strongly recommended to reduce batch effects). Encouragingly for FTA cards, the differences caused by this method are systematic and can be detrended. As in other studies, we strongly caution against the use of 70% ethanol. The results, spanning 15 individuals and over 1,200 samples, provide our most comprehensive view to date of storage effects on stool and provide a paradigm for the future studies of other sample types that will be required to provide a global view of microbial diversity and its interaction among humans, animals, and the environment. IMPORTANCE Our study, spanning 15 individuals and over 1,200 samples, provides our most comprehensive view to date of storage and stabilization effects on stool. We tested five methods for preserving human and dog fecal specimens for periods of up to 8 weeks, including the types of variation often encountered under field conditions, such as freeze-thaw cycles and high temperature fluctuations. We show that several cost-effective methods provide excellent microbiome stability out to 8 weeks, opening up a range of field studies with humans and wildlife that would otherwise be cost-prohibitive.

As pollinators, bees are cornerstones for terrestrial ecosystem stability and key components in agricultural productivity. All animals, including bees, are associated with a diverse community of microbes, commonly referred to as the microbiome. The bee microbiome is likely to be a crucial factor affecting host health. However, with the exception of a few pathogens, the impacts of most members of the bee microbiome on host health are poorly understood. Further, the evolutionary and ecological forces that shape and change the microbiome are unclear. Here, we discuss recent progress in our understanding of the bee microbiome, and we present challenges associated with its investigation. We conclude that global coordination of research efforts is needed to fully understand the complex and highly dynamic nature of the interplay between the bee microbiome, its host, and the environment. High-throughput sequencing technologies are ideal for exploring complex biological systems, including host-microbe interactions. To maximize their value and to improve assessment of the factors affecting bee health, sequence data should be archived, curated, and analyzed in ways that promote the synthesis of different studies. To this end, the BeeBiome consortium aims to develop an online database which would provide reference sequences, archive metadata, and host analytical resources. The goal would be to support applied and fundamental research on bees and their associated microbes and to provide a collaborative framework for sharing primary data from different research programs, thus furthering our understanding of the bee microbiome and its impact on pollinator health.

Exposure of newborns to the maternal vaginal microbiota is interrupted with cesarean birthing. Babies delivered by cesarean section (C-section) acquire a microbiota that differs from that of vaginally delivered infants, and C-section delivery has been associated with increased risk for immune and metabolic disorders. Here we conducted a pilot study in which infants delivered by C-section were exposed to maternal vaginal fluids at birth. Similarly to vaginally delivered babies, the gut, oral and skin bacterial communities of these newborns during the first 30 d of life was enriched in vaginal bacteria--which were underrepresented in unexposed C-section-delivered infants--and the microbiome similarity to those of vaginally delivered infants was greater in oral and skin samples than in anal samples. Although the long-term health consequences of restoring the microbiota of C-section-delivered infants remain unclear, our results demonstrate that vaginal microbes can be partially restored at birth in C-section-delivered babies.

Multi-omics methods have greatly advanced our understanding of the biological organism and its microbial associates. However, they are not routinely used in clinical or industrial applications, due to the length of time required to generate and analyze omics data. Here, we applied a novel integrated omics pipeline for the analysis of human and environmental samples in under 48 h. Human subjects that ferment their own foods provided swab samples from skin, feces, oral cavity, fermented foods, and household surfaces to assess the impact of home food fermentation on their microbial and chemical ecology. These samples were analyzed with 16S rRNA gene sequencing, inferred gene function profiles, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomics through the Qiita, PICRUSt, and GNPS pipelines, respectively. The human sample microbiomes clustered with the corresponding sample types in the American Gut Project (http://www.americangut.org), and the fermented food samples produced a separate cluster. The microbial communities of the household surfaces were primarily sourced from the fermented foods, and their consumption was associated with increased gut microbial diversity. Untargeted metabolomics revealed that human skin and fermented food samples had separate chemical ecologies and that stool was more similar to fermented foods than to other sample types. Metabolites from the fermented foods, including plant products such as procyanidin and pheophytin, were present in the skin and stool samples of the individuals consuming the foods. Some food metabolites were modified during digestion, and others were detected in stool intact. This study represents a first-of-its-kind analysis of multi-omics data that achieved time intervals matching those of classic microbiological culturing. IMPORTANCE Polymicrobial infections are difficult to diagnose due to the challenge in comprehensively cultivating the microbes present. Omics methods, such as 16S rRNA sequencing, metagenomics, and metabolomics, can provide a more complete picture of a microbial community and its metabolite production, without the biases and selectivity of microbial culture. However, these advanced methods have not been applied to clinical or industrial microbiology or other areas where complex microbial dysbioses require immediate intervention. The reason for this is the length of time required to generate and analyze omics data. Here, we describe the development and application of a pipeline for multi-omics data analysis in time frames matching those of the culture-based approaches often used for these applications. This study applied multi-omics methods effectively in clinically relevant time frames and sets a precedent toward their implementation in clinical medicine and industrial microbiology.

Westernization has propelled changes in urbanization and architecture, altering our exposure to the outdoor environment from that experienced during most of human evolution. These changes might affect the developmental exposure of infants to bacteria, immune development, and human microbiome diversity. Contemporary urban humans spend most of their time indoors, and little is known about the microbes associated with different designs of the built environment and their interaction with the human immune system. This study addresses the associations between architectural design and the microbial biogeography of households across a gradient of urbanization in South America. Urbanization was associated with households' increased isolation from outdoor environments, with additional indoor space isolation by walls. Microbes from house walls and floors segregate by location, and urban indoor walls contain human bacterial markers of space use. Urbanized spaces uniquely increase the content of human-associated microbes-which could increase transmission of potential pathogens-and decrease exposure to the environmental microbes with which humans have coevolved.