Showing papers by "Rob Knight published in 2013"

Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences

Abstract: Profiling phylogenetic marker genes, such as the 16S rRNA gene, is a key tool for studies of microbial communities but does not provide direct evidence of a community's functional capabilities. Here we describe PICRUSt (phylogenetic investigation of communities by reconstruction of unobserved states), a computational approach to predict the functional composition of a metagenome using marker gene data and a database of reference genomes. PICRUSt uses an extended ancestral-state reconstruction algorithm to predict which gene families are present and then combines gene families to estimate the composite metagenome. Using 16S information, PICRUSt recaptures key findings from the Human Microbiome Project and accurately predicts the abundance of gene families in host-associated and environmental communities, with quantifiable uncertainty. Our results demonstrate that phylogeny and function are sufficiently linked that this 'predictive metagenomic' approach should provide useful insights into the thousands of uncultivated microbial communities for which only marker gene surveys are currently available.

Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing.

Abstract: High-throughput sequencing has revolutionized microbial ecology, but read quality remains a considerable barrier to accurate taxonomy assignment and α-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 and facilitating interpretation of Illumina sequencing results.

Gut Microbiota from Twins Discordant for Obesity Modulate Metabolism in Mice

Abstract: How much does the microbiota influence the host's phenotype? Ridaura et al. ([1241214][1] ; see the Perspective by [ Walker and Parkhill ][2]) 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. [1]: http://www.sciencemag.org/content/341/6150/1241214.full [2]: /lookup/doi/10.1126/science.1243787

The Long-Term Stability of the Human Gut Microbiota

Abstract: 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.

EMPeror: a tool for visualizing high-throughput microbial community data

Abstract: As microbial ecologists take advantage of high-throughput sequencing technologies to describe microbial communities across ever-increasing numbers of samples, new analysis tools are required to relate the distribution of microbes among larger numbers of communities, and to use increasingly rich and standards-compliant metadata to understand the biological factors driving these relationships. In particular, the Earth Microbiome Project drives these needs by profiling the genomic content of tens of thousands of samples across multiple environment types. Features of EMPeror include: ability to visualize gradients and categorical data, visualize different principal coordinates axes, present the data in the form of parallel coordinates, show taxa as well as environmental samples, dynamically adjust the size and transparency of the spheres representing the communities on a per-category basis, dynamically scale the axes according to the fraction of variance each explains, show, hide or recolor points according to arbitrary metadata including that compliant with the MIxS family of standards developed by the Genomic Standards Consortium, display jackknifed-resampled data to assess statistical confidence in clustering, perform coordinate comparisons (useful for procrustes analysis plots), and greatly reduce loading times and overall memory footprint compared with existing approaches. Additionally, ease of sharing, given EMPeror’s small output file size, enables agile collaboration by allowing users to embed these visualizations via emails or web pages without the need for extra plugins. Here we present EMPeror, an open source and web browser enabled tool with a versatile command line interface that allows researchers to perform rapid exploratory investigations of 3D visualizations of microbial community data, such as the widely used principal coordinates plots. EMPeror includes a rich set of controllers to modify features as a function of the metadata. By being specifically tailored to the requirements of microbial ecologists, EMPeror thus increases the speed with which insight can be gained from large microbiome datasets.

Gut microbiomes of Malawian twin pairs discordant for kwashiorkor

Abstract: Kwashiorkor, an enigmatic form of severe acute malnutrition, is the consequence of inadequate nutrient intake plus additional environmental insults. To investigate the role of the gut microbiome, we studied 317 Malawian twin pairs during the first 3 years of life. During this time, half of the twin pairs remained well nourished, whereas 43% became discordant, and 7% manifested concordance for acute malnutrition. Both children in twin pairs discordant for kwashiorkor were treated with a peanut-based, ready-to-use therapeutic food (RUTF). Time-series metagenomic studies revealed that RUTF produced a transient maturation of metabolic functions in kwashiorkor gut microbiomes that regressed when administration of RUTF was stopped. Previously frozen fecal communities from several discordant pairs were each transplanted into gnotobiotic mice. The combination of Malawian diet and kwashiorkor microbiome produced marked weight loss in recipient mice, accompanied by perturbations in amino acid, carbohydrate, and intermediary metabolism that were only transiently ameliorated with RUTF. These findings implicate the gut microbiome as a causal factor in kwashiorkor.

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

Abstract: The human body is home to many different microorganisms, with a range of bacteria, fungi and archaea living on the skin, in the intestine and at various other sites in the body. While many of these microorganisms are beneficial to their human hosts, we know very little about most of them. Early research focused primarily on comparing the microorganisms found in healthy individuals with those found in individuals suffering from a particular illness. More recently researchers have become interested in more general issues, such as understanding how these collections of microorganisms, which are also known as the human microbiota or the human microbiome, become established, and exploring the causes of similarities and differences between the microbiota of individuals. We now know that the communities of microorganisms found in the intestines of genetically related people tend to be more similar than those of people who are not related. Moreover, the communities of microorganisms found in the intestines of non-related adults living in the same household are more similar than those of unrelated adults living in different households. We also know that the range of microorganisms found in the intestine changes dramatically between birth and the age of 3 years. However, these studies have focused on the intestine, and little is known about the effect of relatedness, cohabitation and age on the microbiota at other body sites. Song et al. compared the microorganisms found on the skin, on the tongue and in the intestines of 159 people—and 36 dogs—in 60 families. They found that co-habitation resulted in the communities of microorganisms being more similar to each other, with those on the skin being the most similar. This was true for all comparisons, including human pairs, dog pairs and human–dog pairs. This suggests that humans probably acquire many of the microorganisms on their skin through direct contact with their surroundings, and that humans tend to share more microbes with individuals, including their pets, with which they are in frequent contact. Song et al. also discovered that, unlike what happens in the intestine, the microbial communities on the skin and tongue of infants and children were relatively similar to those of adults. Overall, these findings suggest that the communities of microorganisms found in the intestine changes with age in a way that differs significantly from those found on the skin and tongue.

Advancing our understanding of the human microbiome using QIIME

Abstract: High-throughput DNA sequencing technologies, coupled with advanced bioinformatics tools, have enabled rapid advances in microbial ecology and our understanding of the human microbiome. QIIME (Quantitative Insights Into Microbial Ecology) is an open-source bioinformatics software package designed for microbial community analysis based on DNA sequence data, which provides a single analysis framework for analysis of raw sequence data through publication-quality statistical analyses and interactive visualizations. In this chapter, we demonstrate the use of the QIIME pipeline to analyze microbial communities obtained from several sites on the bodies of transgenic and wild-type mice, as assessed using 16S rRNA gene sequences generated on the Illumina MiSeq platform. We present our recommended pipeline for performing microbial community analysis and provide guidelines for making critical choices in the process. We present examples of some of the types of analyses that are enabled by QIIME and discuss how other tools, such as phyloseq and R, can be applied to expand upon these analyses.

A guide to enterotypes across the human body: meta-analysis of microbial community structures in human microbiome datasets.

Abstract: Recent analyses of human-associated bacterial diversity have categorized individuals into 'enterotypes' or clusters based on the abundances of key bacterial genera in the gut microbiota. There is a lack of consensus, however, on the analytical basis for enterotypes and on the interpretation of these results. We tested how the following factors influenced the detection of enterotypes: clustering methodology, distance metrics, OTU-picking approaches, sequencing depth, data type (whole genome shotgun (WGS) vs.16S rRNA gene sequence data), and 16S rRNA region. We included 16S rRNA gene sequences from the Human Microbiome Project (HMP) and from 16 additional studies and WGS sequences from the HMP and MetaHIT. In most body sites, we observed smooth abundance gradients of key genera without discrete clustering of samples. Some body habitats displayed bimodal (e.g., gut) or multimodal (e.g., vagina) distributions of sample abundances, but not all clustering methods and workflows accurately highlight such clusters. Because identifying enterotypes in datasets depends not only on the structure of the data but is also sensitive to the methods applied to identifying clustering strength, we recommend that multiple approaches be used and compared when testing for enterotypes.

Reconstructing the microbial diversity and function of pre-agricultural tallgrass prairie soils in the United States.

Abstract: Native tallgrass prairie once dominated much of the midwestern United States, but this biome and the soil microbial diversity that once sustained this highly productive system have been almost completely eradicated by decades of agricultural practices. We reconstructed the soil microbial diversity that once existed in this biome by analyzing relict prairie soils and found that the biogeographical patterns were largely driven by changes in the relative abundance of Verrucomicrobia, a poorly studied bacterial phylum that appears to dominate many prairie soils. Shotgun metagenomic data suggested that these spatial patterns were associated with strong shifts in carbon dynamics. We show that metagenomic approaches can be used to reconstruct below-ground biogeochemical and diversity gradients in endangered ecosystems; such information could be used to improve restoration efforts, given that even small changes in below-ground microbial diversity can have important impacts on ecosystem processes.

Global biogeography of highly diverse protistan communities in soil

Abstract: Protists are ubiquitous members of soil microbial communities, but the structure of these communities, and the factors that influence their diversity, are poorly understood. We used barcoded pyrosequencing to survey comprehensively the diversity of soil protists from 40 sites across a broad geographic range that represent a variety of biome types, from tropical forests to deserts. In addition to taxa known to be dominant in soil, including Cercozoa and Ciliophora, we found high relative abundances of groups such as Apicomplexa and Dinophyceae that have not previously been recognized as being important components of soil microbial communities. Soil protistan communities were highly diverse, approaching the extreme diversity of their bacterial counterparts across the same sites. Like bacterial taxa, protistan taxa were not globally distributed, and the composition of these communities diverged considerably across large geographic distances. However, soil protistan and bacterial communities exhibit very different global-scale biogeographical patterns, with protistan communities strongly structured by climatic conditions that regulate annual soil moisture availability.

Meta-analyses of studies of the human microbiota

Abstract: Our body habitat-associated microbial communities are of intense research interest because of their influence on human health. Because many studies of the microbiota are based on the same bacterial 16S ribosomal RNA (rRNA) gene target, they can, in principle, be compared to determine the relative importance of different disease/physiologic/developmental states. However, differences in experimental protocols used may produce variation that outweighs biological differences. By comparing 16S rRNA gene sequences generated from diverse studies of the human microbiota using the QIIME database, we found that variation in composition of the microbiota across different body sites was consistently larger than technical variability across studies. However, samples from different studies of the Western adult fecal microbiota generally clustered by study, and the 16S rRNA target region, DNA extraction technique, and sequencing platform produced systematic biases in observed diversity that could obscure biologically meaningful compositional differences. In contrast, systematic compositional differences in the fecal microbiota that occurred with age and between Western and more agrarian cultures were great enough to outweigh technical variation. Furthermore, individuals with ileal Crohn's disease and in their third trimester of pregnancy often resembled infants from different studies more than controls from the same study, indicating parallel compositional attributes of these distinct developmental/physiological/disease states. Together, these results show that cross-study comparisons of human microbiota are valuable when the studied parameter has a large effect size, but studies of more subtle effects on the human microbiota require carefully selected control populations and standardized protocols.

A meta-analysis of changes in bacterial and archaeal communities with time

Abstract: Ecologists have long studied the temporal dynamics of plant and animal communities with much less attention paid to the temporal dynamics exhibited by microbial communities. As a result, we do not know if overarching temporal trends exist for microbial communities or if changes in microbial communities are generally predictable with time. Using microbial time series assessed via high-throughput sequencing, we conducted a meta-analysis of temporal dynamics in microbial communities, including 76 sites representing air, aquatic, soil, brewery wastewater treatment, human- and plant-associated microbial biomes. We found that temporal variability in both within- and between-community diversity was consistent among microbial communities from similar environments. Community structure changed systematically with time in less than half of the cases, and the highest rates of change were observed within ranges of 1 day to 1 month for all communities examined. Microbial communities exhibited species-time relationships (STRs), which describe the accumulation of new taxa to a community, similar to those observed previously for plant and animal communities, suggesting that STRs are remarkably consistent across a broad range of taxa. These results highlight that a continued integration of microbial ecology into the broader field of ecology will provide new insight into the temporal patterns of microbial and 'macro'-bial communities alike.

Simultaneous amplicon sequencing to explore co-occurrence patterns of bacterial, archaeal and eukaryotic microorganisms in rumen microbial communities.

Abstract: Ruminants rely on a complex rumen microbial community to convert dietary plant material to energy-yielding products. Here we developed a method to simultaneously analyze the community's bacterial and archaeal 16S rRNA genes, ciliate 18S rRNA genes and anaerobic fungal internal transcribed spacer 1 genes using 12 DNA samples derived from 11 different rumen samples from three host species (Ovis aries, Bos taurus, Cervus elephas) and multiplex 454 Titanium pyrosequencing. We show that the mixing ratio of the group-specific DNA templates before emulsion PCR is crucial to compensate for differences in amplicon length. This method, in contrast to using a non-specific universal primer pair, avoids sequencing non-targeted DNA, such as plant- or endophyte-derived rRNA genes, and allows increased or decreased levels of community structure resolution for each microbial group as needed. Communities analyzed with different primers always grouped by sample origin rather than by the primers used. However, primer choice had a greater impact on apparent archaeal community structure than on bacterial community structure, and biases for certain methanogen groups were detected. Co-occurrence analysis of microbial taxa from all three domains of life suggested strong within- and between-domain correlations between different groups of microorganisms within the rumen. The approach used to simultaneously characterize bacterial, archaeal and eukaryotic components of a microbiota should be applicable to other communities occupying diverse habitats.

A microbial clock provides an accurate estimate of the postmortem interval in a mouse model system

Abstract: Our bodies—especially our skin, our saliva, the lining of our mouth and our gastrointestinal tract—are home to a diverse collection of bacteria and other microorganisms called the microbiome. While the roles played by many of these microorganisms have yet to be identified, it is known that they contribute to the health and wellbeing of their host by metabolizing indigestible compounds, producing essential vitamins, and preventing the growth of harmful bacteria. They are important for nutrient and carbon cycling in the environment. The advent of advanced sequencing techniques has made it feasible to study the composition of this microbial community, and to monitor how it changes over time or how it responds to events such as antibiotic treatment. Sequencing studies have been used to highlight the significant differences between microbial communities found in different parts of the body, and to follow the evolution of the gut microbiome from birth. Most of these studies have focused on live animals, so little is known about what happens to the microbiome after its host dies. In particular, it is not known if the changes that occur after death are similar for all individuals. Moreover, the decomposing animal supplies nutrients and carbon to the surrounding ecosystem, but its influence on the microbial community of its immediate environment is not well understood. Now Metcalf et al. have used high-throughput sequencing to study the bacteria and other microorganisms (such as nematodes and fungi) in dead and decomposing mice, and also in the soil beneath them, over the course of 48 days. The changes were significant and also consistent across the corpses, with the microbial communities in the corpses influencing those in the soil, and vice versa. Metcalf et al. also showed that these measurements could be used to estimate the postmortem interval (the time since death) to within approximately 3 days, which suggests that the work could have applications in forensic science.

Genetically dictated change in host mucus carbohydrate landscape exerts a diet-dependent effect on the gut microbiota

Abstract: We investigate how host mucus glycan composition interacts with dietary carbohydrate content to influence the composition and expressed functions of a human gut community. The humanized gnotobiotic mice mimic humans with a nonsecretor phenotype due to knockout of their α1–2 fucosyltransferase (Fut2) gene. The fecal microbiota of Fut2− mice that lack fucosylated host glycans show decreased alpha diversity relative to Fut2+ mice and exhibit significant differences in community composition. A glucose-rich plant polysaccharide-deficient (PD) diet exerted a strong effect on the microbiota membership but eliminated the effect of Fut2 genotype. Additionally fecal metabolites predicted host genotype in mice on a polysaccharide-rich standard diet but not on a PD diet. A more detailed mechanistic analysis of these interactions involved colonization of gnotobiotic Fut2+ and Fut2− mice with Bacteroides thetaiotaomicron, a prominent member of the human gut microbiota known to adaptively forage host mucosal glycans when dietary polysaccharides are absent. Within Fut2− mice, the B. thetaiotaomicron fucose catabolic pathway was markedly down-regulated, whereas BT4241–4247, an operon responsive to terminal β-galactose, the precursor that accumulates in the Fut2− mice, was significantly up-regulated. These changes in B. thetaiotaomicron gene expression were only evident in mice fed a PD diet, wherein B. thetaiotaomicron relies on host mucus consumption. Furthermore, up-regulation of the BT4241–4247 operon was also seen in humanized Fut2− mice. Together, these data demonstrate that differences in host genotype that affect the carbohydrate landscape of the distal gut interact with diet to alter the composition and function of resident microbes in a diet-dependent manner.

Evidence for a persistent microbial seed bank throughout the global ocean

Abstract: Do bacterial taxa demonstrate clear endemism, like macroorganisms, or can one site’s bacterial community recapture the total phylogenetic diversity of the world’s oceans? Here we compare a deep bacterial community characterization from one site in the English Channel (L4-DeepSeq) with 356 datasets from the International Census of Marine Microbes (ICoMM) taken from around the globe (ranging from marine pelagic and sediment samples to sponge-associated environments). At the L4-DeepSeq site, increasing sequencing depth uncovers greater phylogenetic overlap with the global ICoMM data. This site contained 31.7–66.2% of operational taxonomic units identified in a given ICoMM biome. Extrapolation of this overlap suggests that 1.93 × 1011 sequences from the L4 site would capture all ICoMM bacterial phylogenetic diversity. Current technology trends suggest this limit may be attainable within 3 y. These results strongly suggest the marine biosphere maintains a previously undetected, persistent microbial seed bank.

Baseline survey of the anatomical microbial ecology of an important food plant: Solanum lycopersicum (tomato)

Abstract: Research to understand and control microbiological risks associated with the consumption of fresh fruits and vegetables has examined many environments in the farm to fork continuum. An important data gap however, that remains poorly studied is the baseline description of microflora that may be associated with plant anatomy either endemically or in response to environmental pressures. Specific anatomical niches of plants may contribute to persistence of human pathogens in agricultural environments in ways we have yet to describe. Tomatoes have been implicated in outbreaks of Salmonella at least 17 times during the years spanning 1990 to 2010. Our research seeks to provide a baseline description of the tomato microbiome and possibly identify whether or not there is something distinctive about tomatoes or their growing ecology that contributes to persistence of Salmonella in this important food crop. DNA was recovered from washes of epiphytic surfaces of tomato anatomical organs; leaves, stems, roots, flowers and fruits of Solanum lycopersicum (BHN602), grown at a site in close proximity to commercial farms previously implicated in tomato-Salmonella outbreaks. DNA was amplified for targeted 16S and 18S rRNA genes and sheared for shotgun metagenomic sequencing. Amplicons and metagenomes were used to describe “native” bacterial microflora for diverse anatomical parts of Virginia-grown tomatoes. Distinct groupings of microbial communities were associated with different tomato plant organs and a gradient of compositional similarity could be correlated to the distance of a given plant part from the soil. Unique bacterial phylotypes (at 95% identity) were associated with fruits and flowers of tomato plants. These include Microvirga, Pseudomonas, Sphingomonas, Brachybacterium, Rhizobiales, Paracocccus, Chryseomonas and Microbacterium. The most frequently observed bacterial taxa across aerial plant regions were Pseudomonas and Xanthomonas. Dominant fungal taxa that could be identified to genus with 18S amplicons included Hypocrea, Aureobasidium and Cryptococcus. No definitive presence of Salmonella could be confirmed in any of the plant samples, although 16S sequences suggested that closely related genera were present on leaves, fruits and roots.

Phylogenetic stratigraphy in the Guerrero Negro hypersaline microbial mat.

Abstract: The microbial mats of Guerrero Negro (GN), Baja California Sur, Mexico historically were considered a simple environment, dominated by cyanobacteria and sulfate-reducing bacteria. Culture-independent rRNA community profiling instead revealed these microbial mats as among the most phylogenetically diverse environments known. A preliminary molecular survey of the GN mat based on only ∼1500 small subunit rRNA gene sequences discovered several new phylum-level groups in the bacterial phylogenetic domain and many previously undetected lower-level taxa. We determined an additional ∼119 000 nearly full-length sequences and 28 000 >200 nucleotide 454 reads from a 10-layer depth profile of the GN mat. With this unprecedented coverage of long sequences from one environment, we confirm the mat is phylogenetically stratified, presumably corresponding to light and geochemical gradients throughout the depth of the mat. Previous shotgun metagenomic data from the same depth profile show the same stratified pattern and suggest that metagenome properties may be predictable from rRNA gene sequences. We verify previously identified novel lineages and identify new phylogenetic diversity at lower taxonomic levels, for example, thousands of operational taxonomic units at the family-genus levels differ considerably from known sequences. The new sequences populate parts of the bacterial phylogenetic tree that previously were poorly described, but indicate that any comprehensive survey of GN diversity has only begun. Finally, we show that taxonomic conclusions are generally congruent between Sanger and 454 sequencing technologies, with the taxonomic resolution achieved dependent on the abundance of reference sequences in the relevant region of the rRNA tree of life.

Diversity, distribution and sources of bacteria in residential kitchens

Abstract: Summary Bacteria readily colonize kitchen surfaces, and the exchange of microbes between humans and the kitchen environment can impact human health. However, we have a limited understanding of the overall diversity of these communities, how they differ across surfaces and sources of bacteria to kitchen surfaces. Here we used high-throughput sequencing of the 16S rRNA gene to explore biogeo- graphical patterns of bacteria across > 80 surfaces within the kitchens of each of four households. In total, 34 bacterial and two archaeal phyla were identified, with most sequences belonging to the Actinobacteria, Bacteroidetes, Firmicutes and Proteo- bacteria. Genera known to contain common food- borne pathogens were low in abundance but broadly distributed throughout the kitchens, with different taxa exhibiting distinct distribution patterns. The most diverse communities were associated with infre- quently cleaned surfaces such as fans above stoves, refrigerator/freezer door seals and floors. In contrast, the least diverse communities were observed in and around sinks, which were dominated by biofilm- forming Gram-negative lineages. Community compo- sition was influenced by conditions on individual surfaces, usage patterns and dispersal from source environments. Human skin was the primary source of bacteria across all kitchen surfaces, with contribu- tions from food and faucet water dominating in a few specific locations. This study demonstrates that diverse bacterial communities are widely distributed in residential kitchens and that the composition of these communities is often predictable. These results also illustrate the ease with which human- and food- associated bacteria can be transferred in residential settings to kitchen surfaces.

Widespread colonization of the lung by Tropheryma whipplei in HIV infection.

Abstract: Rationale:LunginfectionscausedbyopportunisticorvirulentpathogensareaprincipalcauseofmorbidityandmortalityinHIVinfection. It is unknown whether HIV infection leads to changes in basal lung microflora, which may contribute to chronic pulmonary complications that increasingly are being recognized in individuals infected with HIV. Objectives: To determine whether the immunodeficiency associated with HIV infection resulted in alteration of the lung microbiota. Methods: We used 16S ribosomal RNA targeted pyrosequencing and shotgun metagenomic sequencing to analyze bacterial gene sequences in bronchoalveolar lavage (BAL) and mouths of 82 HIV-positive and 77 HIV-negative subjects. Measurements and Main Results: Sequences representing Tropheryma whipplei, the etiologic agent of Whipple’s disease, were significantly more frequent in BAL of HIV-positive compared with HIV-negative individuals. T. whipplei dominated the community (.50% of sequence reads) in 11 HIV-positive subjects, but only 1 HIV-negative individual (13.4 versus 1.3%; P ¼ 0.0018). In 30 HIV-positive individuals sampled longitudinally, antiretroviral therapy resulted in a significantly reduced relative abundance of T. whipplei in the lung. Shotgun metagenomic sequencing was performed on eight BAL samples dominated by T. whipplei 16S ribosomal RNA. Whole genome assembly of pooled reads showed that uncultured lung

Seasonal Restructuring of the Ground Squirrel Gut Microbiota Over the Annual Hibernation Cycle

Abstract: Many hibernating mammals suspend food intake during winter, relying solely on stored lipids to fuel metabolism. Winter fasting in these species eliminates a major source of degradable substrates to support growth of gut microbes, which may affect microbial community structure and host-microbial interactions. We explored the effect of the annual hibernation cycle on gut microbiotas using deep sequencing of 16S rRNA genes from ground squirrel cecal contents. Squirrel microbiotas were dominated by members of the phyla Bacteroidetes, Firmicutes, and Verrucomicrobia. UniFrac analysis showed that microbiotas clustered strongly by season, and maternal influences, diet history, host age, and host body temperature had minimal effects. Phylogenetic diversity and numbers of operational taxonomic units were lowest in late winter and highest in the spring after a 2-wk period of refeeding. Hibernation increased relative abundance of Bacteroidetes and Verrucomicrobia, phyla that contain species capable of surviving on host-derived substrates such as mucins, and reduced relative abundance of Firmicutes, many of which prefer dietary polysaccharides. Hibernation reduced cecal short-chain fatty acid and ammonia concentrations, and increased and decreased concentrations of acetate and butyrate, respectively. These results indicate that the ground squirrel microbiota is restructured each year in a manner that reflects differences in microbial preferences for dietary vs. host-derived substrates, and thus the competitive abilities of different taxa to survive in the altered environment in the hibernator gut.

The microbiome explored: recent insights and future challenges

Abstract: One of the most exciting scientific advances in recent years has been the realization that commensal microorganisms are not simple 'passengers' in our bodies, but instead have key roles in our physiology, including our immune responses and metabolism, as well as in disease. These insights have been obtained, in part, through the work of large-scale, consortium-driven metagenomic projects. Here, five experts in the field of microbiome research discuss the most surprising and exciting new findings, and outline the future steps that will be necessary to elucidate the numerous roles of the microbiota in human health and disease and to develop viable therapeutic strategies.

A Modular Organization of the Human Intestinal Mucosal Microbiota and Its Association with Inflammatory Bowel Disease

Abstract: Abnormalities of the intestinal microbiota are implicated in the pathogenesis of Crohn's disease (CD) and ulcerative colitis (UC), two spectra of inflammatory bowel disease (IBD). However, the high complexity and low inter-individual overlap of intestinal microbial composition are formidable barriers to identifying microbial taxa representing this dysbiosis. These difficulties might be overcome by an ecologic analytic strategy to identify modules of interacting bacteria (rather than individual bacteria) as quantitative reproducible features of microbial composition in normal and IBD mucosa. We sequenced 16S ribosomal RNA genes from 179 endoscopic lavage samples from different intestinal regions in 64 subjects (32 controls, 16 CD and 16 UC patients in clinical remission). CD and UC patients showed a reduction in phylogenetic diversity and shifts in microbial composition, comparable to previous studies using conventional mucosal biopsies. Analysis of weighted co-occurrence network revealed 5 microbial modules. These modules were unprecedented, as they were detectable in all individuals, and their composition and abundance was recapitulated in an independent, biopsy-based mucosal dataset 2 modules were associated with healthy, CD, or UC disease states. Imputed metagenome analysis indicated that these modules displayed distinct metabolic functionality, specifically the enrichment of oxidative response and glycan metabolism pathways relevant to host-pathogen interaction in the disease-associated modules. The highly preserved microbial modules accurately classified IBD status of individual patients during disease quiescence, suggesting that microbial dysbiosis in IBD may be an underlying disorder independent of disease activity. Microbial modules thus provide an integrative view of microbial ecology relevant to IBD.

Biphasic assembly of the murine intestinal microbiota during early development

Abstract: The birth canal provides mammals with a primary maternal inoculum, which develops into distinctive body site-specific microbial communities post-natally. We characterized the distal gut microbiota from birth to weaning in mice. One-day-old mice had colonic microbiota that resembled maternal vaginal communities, but at days 3 and 9 of age there was a substantial loss of intestinal bacterial diversity and dominance of Lactobacillus. By weaning (21 days), diverse intestinal bacteria had established, including strict anaerobes. Our results are consistent with vertical transmission of maternal microbiota and demonstrate a nonlinear ecological succession involving an early drop in bacterial diversity and shift in dominance from Streptococcus to Lactobacillus, followed by an increase in diversity of anaerobes, after the introduction of solid food. Mammalian newborns are born highly susceptible to colonization, and lactation may control microbiome assembly during early development.

Bacterial Diversity in Two Neonatal Intensive Care Units (NICUs)

Abstract: Infants in Neonatal Intensive Care Units (NICUs) are particularly susceptible to opportunistic infection. Infected infants have high mortality rates, and survivors often suffer life-long neurological disorders. The causes of many NICU infections go undiagnosed, and there is debate as to the importance of inanimate hospital environments (IHEs) in the spread of infections. We used culture-independent next-generation sequencing to survey bacterial diversity in two San Diego NICUs and to track the sources of microbes in these environments. Thirty IHE samples were collected from two Level-Three NICU facilities. We extracted DNA from these samples and amplified the bacterial small subunit (16S) ribosomal RNA gene sequence using ‘universal’ barcoded primers. The purified PCR products were pooled into a single reaction for pyrosequencing, and the data were analyzed using QIIME. On average, we detected 93+/−39 (mean +/− standard deviation) bacterial genera per sample in NICU IHEs. Many of the bacterial genera included known opportunistic pathogens, and many were skin-associated (e.g., Propionibacterium). In one NICU, we also detected fecal coliform bacteria (Enterobacteriales) in a high proportion of the surface samples. Comparison of these NICU-derived sequences to previously published high-throughput 16S rRNA amplicon studies of other indoor environments (offices, restrooms and healthcare facilities), as well as human- and soil-associated environments, found the majority of the NICU samples to be similar to typical building surface and air samples, with the notable exception of the IHEs which were dominated by Enterobacteriaceae. Our findings provide evidence that NICU IHEs harbor a high diversity of human-associated bacteria and demonstrate the potential utility of molecular methods for identifying and tracking bacterial diversity in NICUs.

Distinct cutaneous bacterial assemblages in a sampling of South American Amerindians and US residents

Abstract: The human skin harbors complex bacterial communities. Prior studies showing high inter-individual variation focused on subjects from developed countries. We therefore compared cutaneous bacterial communities of Amerindians in the Venezuelan Amazon with subjects in the United States. Forearm skin specimens were studied from healthy Amerindians in Platanillal village in Amazonas State, and from healthy persons in New York and Colorado. All skin sampling used similar swab/buffer techniques. Multiplexed V2-targeted 16S rRNA gene pyrosequencing yielded high quality sequences from 112 samples. The results show 20 phyla, with three (Proteobacteria, Firmicutes, Actinobacteria) predominating. US residents and Venezuelan Amerindians had significantly different forearm skin bacterial community compositions, with United States dominated by Propionibacterium. Among the Amerindians, there was a deep split based on bacterial community membership, with 30 and 42 samples, respectively, falling into each of the two groups, not associated with age, gender, or body mass index. One Amerindian group had diversity similar to the United States, but was dominated by Staphylococcus rather than Propionibacterium. The other Amerindian group was significantly more diverse and even than the US or the other Amerindian group, and featured a broad range of Proteobacteria. The results provide evidence that ethnicity, lifestyle and/or geography are associated with the structure of human cutaneous bacterial communities.