Jose C. Clemente

UCHIME improves sensitivity and speed of chimera detection

Motivation: Chimeric DNA sequences often form during polymerase chain reaction amplification, especially when sequencing single regions (e.g. 16S rRNA or fungal Internal Transcribed Spacer) to assess diversity or compare populations. Undetected chimeras may be misinterpreted as novel species, causing inflated estimates of diversity and spurious inferences of differences between populations. Detection and removal of chimeras is therefore of critical importance in such experiments. Results: We describe UCHIME, a new program that detects chimeric sequences with two or more segments. UCHIME either uses a database of chimera-free sequences or detects chimeras de novo by exploiting abundance data. UCHIME has better sensitivity than ChimeraSlayer (previously the most sensitive database method), especially with short, noisy sequences. In testing on artificial bacterial communities with known composition, UCHIME de novo sensitivity is shown to be comparable to Perseus. UCHIME is >100× faster than Perseus and >1000× faster than ChimeraSlayer. Contact: robert@drive5.com Availability: Source, binaries and data: http://drive5.com/uchime. Supplementary information: Supplementary data are available at Bioinformatics online.

Human gut microbiome viewed across age and geography

Gut microbial communities represent one source of human genetic and metabolic diversity. To examine how gut microbiomes differ among human populations, here we characterize bacterial species in fecal samples from 531 individuals, plus the gene content of 110 of them. The cohort encompassed healthy children and adults from the Amazonas of Venezuela, rural Malawi and US metropolitan areas and included mono- and dizygotic twins. Shared features of the functional maturation of the gut microbiome were identified during the first three years of life in all three populations, including age-associated changes in the genes involved in vitamin biosynthesis and metabolism. Pronounced differences in bacterial assemblages and functional gene repertoires were noted between US residents and those in the other two countries. These distinctive features are evident in early infancy as well as adulthood. Our findings underscore the need to consider the microbiome when evaluating human development, nutritional needs, physiological variations and the impact of westernization.

Gut microbiota from twins discordant for obesity modulate metabolism in mice.

Introduction Establishing whether specific structural and functional configurations of a human gut microbiota are causally related to a given physiologic or disease phenotype is challenging. Twins discordant for obesity provide an opportunity to examine interrelations between obesity and its associated metabolic disorders, diet, and the gut microbiota. Transplanting the intact uncultured or cultured human fecal microbiota from each member of a discordant twin pair into separate groups of recipient germfree mice permits the donors’ communities to be replicated, differences between their properties to be identified, the impact of these differences on body composition and metabolic phenotypes to be discerned, and the effects of diet-by-microbiota interactions to be analyzed. In addition, cohousing coprophagic mice harboring transplanted microbiota from discordant pairs provides an opportunity to determine which bacterial taxa invade the gut communities of cage mates, how invasion correlates with host phenotypes, and how invasion and microbial niche are affected by human diets. Cohousing Ln and Ob mice prevents increased adiposity in Ob cage mates (Ob). (A) Adiposity change after 10 days of cohousing. *P < 0.05 versus Ob controls (Student’s t test). (B) Bacteroidales from Ln microbiota invade Ob microbiota. Columns show individual mice. Methods Separate groups of germfree mice were colonized with uncultured fecal microbiota from each member of four twin pairs discordant for obesity or with culture collections from an obese (Ob) or lean (Ln) co-twin. Animals were fed a mouse chow low in fat and rich in plant polysaccharides, or one of two diets reflecting the upper or lower tertiles of consumption of saturated fats and fruits and vegetables based on the U.S. National Health and Nutrition Examination Survey (NHANES). Ln or Ob mice were cohoused 5 days after colonization. Body composition changes were defined by quantitative magnetic resonance. Microbiota or microbiome structure, gene expression, and metabolism were assayed by 16S ribosomal RNA profiling, whole-community shotgun sequencing, RNA-sequencing, and mass spectrometry. Host gene expression and metabolism were also characterized. Results and Discussion The intact uncultured and culturable bacterial component of Ob co-twins’ fecal microbiota conveyed significantly greater increases in body mass and adiposity than those of Ln communities. Differences in body composition were correlated with differences in fermentation of short-chain fatty acids (increased in Ln), metabolism of branched-chain amino acids (increased in Ob), and microbial transformation of bile acid species (increased in Ln and correlated with down-regulation of host farnesoid X receptor signaling). Cohousing Ln and Ob mice prevented development of increased adiposity and body mass in Ob cage mates and transformed their microbiota’s metabolic profile to a leanlike state. Transformation correlated with invasion of members of Bacteroidales from Ln into Ob microbiota. Invasion and phenotypic rescue were diet-dependent and occurred with the diet representing the lower tertile of U.S. consumption of saturated fats, and upper tertile of fruits and vegetables, but not with the diet representing the upper tertile of saturated fats, and lower tertile of fruit and vegetable consumption. These results reveal that transmissible and modifiable interactions between diet and microbiota influence host biology. Transforming Fat to Thin How much does the microbiota influence the hosts phenotype? Ridaura et al. (1241214 ; see the Perspective by Walker and Parkhill) obtained uncultured fecal microbiota from twin pairs discordant for body mass and transplanted them into adult germ-free mice. It was discovered that adiposity is transmissible from human to mouse and that it was associated with changes in serum levels of branched-chain amino acids. Moreover, obese-phenotype mice were invaded by members of the Bacteroidales from the lean mice, but, happily, the lean animals resisted invasion by the obese microbiota. Mice carrying gut bacteria from lean humans protect their cage mates from the effects of gut bacteria from fat humans. [Also see Perspective by Walker and Parkhill] The role of specific gut microbes in shaping body composition remains unclear. We transplanted fecal microbiota from adult female twin pairs discordant for obesity into germ-free mice fed low-fat mouse chow, as well as diets representing different levels of saturated fat and fruit and vegetable consumption typical of the U.S. diet. Increased total body and fat mass, as well as obesity-associated metabolic phenotypes, were transmissible with uncultured fecal communities and with their corresponding fecal bacterial culture collections. Cohousing mice harboring an obese twin’s microbiota (Ob) with mice containing the lean co-twin’s microbiota (Ln) prevented the development of increased body mass and obesity-associated metabolic phenotypes in Ob cage mates. Rescue correlated with invasion of specific members of Bacteroidetes from the Ln microbiota into Ob microbiota and was diet-dependent. These findings reveal transmissible, rapid, and modifiable effects of diet-by-microbiota interactions.

The Impact of the Gut Microbiota on Human Health: An Integrative View

The human gut harbors diverse microbes that play a fundamental role in the well-being of their host. The constituents of the microbiota--bacteria, viruses, and eukaryotes--have been shown to interact with one another and with the host immune system in ways that influence the development of disease. We review these interactions and suggest that a holistic approach to studying the microbiota that goes beyond characterization of community composition and encompasses dynamic interactions between all components of the microbiota and host tissue over time will be crucial for building predictive models for diagnosis and treatment of diseases linked to imbalances in our microbiota.

Diet Drives Convergence in Gut Microbiome Functions Across Mammalian Phylogeny and Within Humans

The normal range of physiological and metabolic phenotypes has been shaped by coevolution with microbial symbionts. Coevolution of mammals and their gut microbiota has profoundly affected their radiation into myriad habitats. We used shotgun sequencing of microbial community DNA and targeted sequencing of bacterial 16S ribosomal RNA genes to gain an understanding of how microbial communities adapt to extremes of diet. We sampled fecal DNA from 33 mammalian species and 18 humans who kept detailed diet records, and we found that the adaptation of the microbiota to diet is similar across different mammalian lineages. Functional repertoires of microbiome genes, such as those encoding carbohydrate-active enzymes and proteases, can be predicted from bacterial species assemblages. These results illustrate the value of characterizing vertebrate gut microbiomes to understand host evolutionary histories at a supraorganismal level.

The Long-Term Stability of the Human Gut Microbiota

Background Understanding the dynamics and stability of the human gut microbiota is important if its characterization is to play a role in the diagnosis, treatment, and prevention of disease. To characterize stability in related and unrelated individuals and its responsiveness to physiologic change (weight loss), we developed a method for bacterial 16S rRNA amplicon sequencing at high depth with high precision. We also sequenced the genomes of anaerobic bacteria represented in culture collections prepared from fecal samples collected from individuals over time. Methods Low-error amplicon sequencing (LEA-Seq) is a quantitative method based on redundant sequencing of bacterial 16S rRNA genes. A dilute, barcoded, oligonucleotide primer solution is used to create ~150,000 linear PCR extensions of the template DNA. The labeled, bottlenecked linear PCR pool is amplified with exponential PCR, using primers that specifically amplify only the linear PCR molecules. The exponential PCR pool is sequenced at sufficient depth to obtain ~20× coverage. Multiple reads enable the generation of an error-corrected consensus sequence for each barcoded template molecule. LEA-Seq can be used for a variety of other applications. Relationship among time, physiology, and microbiota stability. (A) Stability of fecal microbiota follows a power-law function (n = 37 females sampled over time; >1 week to <5 years). Dashed lines show 95% confidence bounds over 10- and 50-year extrapolations (inset). (B) Microbiota stability is inversely related to the stability of each individual’s body mass index. Results and Discussion LEA-Seq of fecal samples from 37 healthy U.S. adults sampled 2 to 13 times up to 296 weeks apart revealed that they harbored 195 ± 48 bacterial strains, representing 101 ± 27 species. On average, their individual microbiota was remarkably stable, with 60% of strains remaining over the course of 5 years. Stability followed a power law, which, when extrapolated, suggests that most strains in an individual’s intestine are residents for decades (figure, panel A). Members of Bacteroidetes and Actinobacteria are significantly more stable components than the population average. LEA-Seq of four individuals sampled during an 8- to 32-week period during a calorie-restricted dietary study showed that weight stability is a significantly better predictor of microbiota stability than the time interval between samples (figure, panel B). After generating clonally arrayed collections of anaerobic bacteria from frozen fecal samples collected from six weight-stable individuals sampled 7 to 69 weeks apart, we produced draft genome sequences for 534 isolates representing 188 strains and 75 species. A targeted approach focused on Methanobrevibacter smithii isolates from two sets of twin pairs and their mothers and Bacteroides thetaiotaomicron strains from nine donors including sister-sister and mother-daughter pairs. Strains, defined as isolates sharing >96% of their genome content, were maintained over time within an individual and between family members but not between unrelated individuals. Thus, early gut colonizers, such as those acquired from our parents and siblings, have the potential to exert their physiologic, metabolic, and immunologic effects for most, and perhaps all, of our lives. Inheritance Guts We know little about the stability of the constituent microbiota in the human gut or the extent to which the gut microbiota are a potential target for long-term health interventions. Faith et al. (p. 10.1126/science.1237439) analyzed the fecal microbiota of 37 individuals and found that, on average, 60% of bacterial strains remained stable for up to 5 years and many were estimated to remain stable for decades. Low-error sequencing data suggest that initial microbial colonizers of infant guts could persist over the life span of an individual. A low-error 16S ribosomal RNA amplicon sequencing method, in combination with whole-genome sequencing of >500 cultured isolates, was used to characterize bacterial strain composition in the fecal microbiota of 37 U.S. adults sampled for up to 5 years. Microbiota stability followed 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 of individuals who consumed a monotonous liquid diet for up to 32 weeks indicated that changes in strain composition were better predicted by changes in weight than by differences in 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.

Experimental and analytical tools for studying the human microbiome

The human microbiome substantially affects many aspects of human physiology, including metabolism, drug interactions and numerous diseases. This realization, coupled with ever-improving nucleotide sequencing technology, has precipitated the collection of diverse data sets that profile the microbiome. In the past 2 years, studies have begun to include sufficient numbers of subjects to provide the power to associate these microbiome features with clinical states using advanced algorithms, increasing the use of microbiome studies both individually and collectively. Here we discuss tools and strategies for microbiome studies, from primer selection to bioinformatics analysis.

Cohabiting family members share microbiota with one another and with their dogs.

Human-associated microbial communities vary across individuals: possible contributing factors include (genetic) relatedness, diet, and age. However, our surroundings, including individuals with whom we interact, also likely shape our microbial communities. To quantify this microbial exchange, we surveyed fecal, oral, and skin microbiota from 60 families (spousal units with children, dogs, both, or neither). Household members, particularly couples, shared more of their microbiota than individuals from different households, with stronger effects of co-habitation on skin than oral or fecal microbiota. Dog ownership significantly increased the shared skin microbiota in cohabiting adults, and dog-owning adults shared more ‘skin’ microbiota with their own dogs than with other dogs. Although the degree to which these shared microbes have a true niche on the human body, vs transient detection after direct contact, is unknown, these results suggest that direct and frequent contact with our cohabitants may significantly shape the composition of our microbial communities. DOI: http://dx.doi.org/10.7554/eLife.00458.001

Partial restoration of the microbiota of cesarean-born infants via vaginal microbial transfer

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.

The microbiome of uncontacted Amerindians

Fecal, oral, and skin biomes of isolated Amerindians show higher human bacterial diversity including antibiotic resistance genes. Most studies of the human microbiome have focused on westernized people with life-style practices that decrease microbial survival and transmission, or on traditional societies that are currently in transition to westernization. We characterize the fecal, oral, and skin bacterial microbiome and resistome of members of an isolated Yanomami Amerindian village with no documented previous contact with Western people. These Yanomami harbor a microbiome with the highest diversity of bacteria and genetic functions ever reported in a human group. Despite their isolation, presumably for >11,000 years since their ancestors arrived in South America, and no known exposure to antibiotics, they harbor bacteria that carry functional antibiotic resistance (AR) genes, including those that confer resistance to synthetic antibiotics and are syntenic with mobilization elements. These results suggest that westernization significantly affects human microbiome diversity and that functional AR genes appear to be a feature of the human microbiome even in the absence of exposure to commercial antibiotics. AR genes are likely poised for mobilization and enrichment upon exposure to pharmacological levels of antibiotics. Our findings emphasize the need for extensive characterization of the function of the microbiome and resistome in remote nonwesternized populations before globalization of modern practices affects potentially beneficial bacteria harbored in the human body.