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Background: Standard pediatric growth curves cannot be used to impute missing height or weight measurements in individual children. The Michaelis-Menten equation, used for characterizing substrate-enzyme saturation curves, has been shown to model growth in many organisms including nonhuman vertebrates. We investigated whether this equation could be used to interpolate missing growth data in children in the first three years of life and compared this interpolation to several common interpolation methods and pediatric growth models., Methods: We developed a modified Michaelis-Menten equation and compared expected to actual growth, first in a local birth cohort (N = 97) then in a large, outpatient, pediatric sample (N = 14,695)., Results: The modified Michaelis-Menten equation showed excellent fit for both infant weight (median RMSE: boys: 0.22 kg [IQR:0.19; 90% < 0.43]; girls: 0.20 kg [IQR:0.17; 90% < 0.39]) and height (median RMSE: boys: 0.93 cm [IQR:0.53; 90% < 1.0]; girls: 0.91 cm [IQR:0.50;90% < 1.0]). Growth data were modeled accurately with as few as four values from routine well-baby visits in year 1 and seven values in years 1-3; birth weight or length was essential for best fit. Interpolation with this equation had comparable (for weight) or lower (for height) mean RMSE compared to the best performing alternative models., Conclusions: A modified Michaelis-Menten equation accurately describes growth in healthy babies aged 0-36 months, allowing interpolation of missing weight and height values in individual longitudinal measurement series. The growth pattern in healthy babies in resource-rich environments mirrors an enzymatic saturation curve., (© 2024. The Author(s).)

We sought to better understand how intestinal microbiota confer protection against Clostridioides difficile ( C. difficile ) infection (CDI). We utilized gnotobiotic altered Schaedler flora (ASF) mice, which lack the abnormalities of germfree (GF) mice as well as the complexity and heterogeneity of antibiotic-treated mice. Like GF mice, ASF mice were highly prone to rapid lethal CDI, without antibiotics, while very low infectious doses resulted in chronic CDI. Administering such chronic CDI mice an undefined preparation of Clostridia lowered C. difficile levels by several logs. Importantly, such resolution of CDI was associated with colonization of Lachnospiraceae. Fractionation of the Clostridia population to enrich for Lachnospiraceae led to the appreciation that its CDI-impeding property strongly associated with a specific Lachnospiraceae strain, namely uncultured bacteria and archaea (UBA) 3401. UBA3401 was recalcitrant to being propagated as a pure culture but could be maintained in ASF mice, wherein it comprised up to about 50% of the intestinal microbiota, which was sufficient to generate a high-quality genomic sequence of this bacterium. Sequence analysis and ex vivo study of UBA3401 indicated that it had the ability to secrete substance(s) that directly impeded C. difficile growth. Moreover, in vivo administration of UBA3401/ASF feces provided strong protection to C. difficile challenge. Thus, UBA3401 may contribute to and/or provide a means to study microbiota-mediated CDI resistance.

The human gut virome and its early life development are poorly understood. Prior studies have captured single-point assessments with the evolution of the infant virome remaining largely unexplored. We performed viral metagenomic sequencing on stool samples collected longitudinally from a cohort of 53 infants from age 2 weeks to 3 years (80.7 billion reads), and from their mothers (9.8 billion reads) to examine and compare viromes. The asymptomatic infant virome consisted of bacteriophages, nonhuman dietary/environmental viruses, and human-host viruses, predominantly picornaviruses. In contrast, human-host viruses were largely absent from the maternal virome. Previously undescribed, sequence-divergent vertebrate viruses were detected in the maternal but not infant virome. As infants aged, the phage component evolved to resemble the maternal virome, but by age 3, the human-host component remained dissimilar from the maternal virome. Thus, early life virome development is determined predominantly by dietary, infectious, and environmental factors rather than direct maternal acquisition., Competing Interests: Declaration of interests S.F., D.S., and C.Y.C. are co-inventors on US patent 11,380,421, “Pathogen Detection using Next Generation Sequencing,” under which algorithms for taxonomic classification, filtering, and pathogen detection are used by SURPI+ software for virus identification from metagenomic data., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Fiber-rich foods promote health, but mechanisms by which they do so remain poorly defined. Screening fiber types, in mice, revealed psyllium had unique ability to ameliorate 2 chronic inflammatory states, namely, metabolic syndrome and colitis. We sought to determine the mechanism of action of the latter., Methods: Mice were fed grain-based chow, which is naturally rich in fiber or compositionally defined diets enriched with semi-purified fibers. Mice were studied basally and in models of chemical-induced and T-cell transfer colitis., Results: Relative to all diets tested, mice consuming psyllium-enriched compositionally defined diets were markedly protected against both dextran sulfate sodium- and T-cell transfer-induced colitis, as revealed by clinical-type, histopathologic, morphologic, and immunologic parameters. Such protection associated with stark basal changes in the gut microbiome but was independent of fermentation and, moreover, maintained in mice harboring a minimal microbiota (ie, Altered Schaedler Flora). Transcriptomic analysis revealed psyllium induced expression of genes mediating bile acids (BA) secretion, suggesting that psyllium's known ability to bind BA might contribute to its ability to prevent colitis. As expected, psyllium resulted in elevated level of fecal BA, reflecting their removal from enterohepatic circulation but, in stark contrast to the BA sequestrant cholestyramine, increased serum BA levels. Moreover, the use of BA mimetics that activate the farnesoid X receptor (FXR), as well as the use of FXR-knockout mice, suggested that activation of FXR plays a central role in psyllium's protection against colitis., Conclusions: Psyllium protects against colitis via altering BA metabolism resulting in activation of FXR, which suppresses pro-inflammatory signaling., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Travelers' diarrhea (TD) is the most prevalent illness encountered by deployed military personnel and has a major impact on military operations, from reduced job performance to lost duty days. Frequently, the etiology of TD is unknown and, with underreporting of cases, it is difficult to accurately assess its impact. An increasing number of ailments include an altered or aberrant gut microbiome. To better understand the relationships between long-term deployments and TD, we studied military personnel during two nine-month deployment cycles in 2015-2016 to Honduras. To collect data on the prevalence of diarrhea and impact on duty, a total of 1173 personnel completed questionnaires at the end of their deployment. 56.7% reported reduced performance and 21.1% reported lost duty days. We conducted a passive surveillance study of all cases of diarrhea reporting to the medical unit with 152 total cases and a similar pattern of etiology. Enteroaggregative E. coli (EAEC, 52/152), enterotoxigenic E. coli (ETEC, 50/152), and enteropathogenic E. coli (EPEC, 35/152) were the most prevalent pathogens detected. An active longitudinal surveillance of 67 subjects also identified diarrheagenic E. coli as the primary etiology (7/16 EPEC, 7/16 EAEC, and 6/16 ETEC). Eleven subjects were recruited into a nested longitudinal substudy to examine gut microbiome changes associated with deployment. A 16S rRNA amplicon survey of fecal samples showed differentially abundant baseline taxa for subjects who contracted TD versus those who did not, as well as detection of taxa positively associated with self-reported gastrointestinal distress. Disrupted microbiota was also qualitatively observable for weeks preceding and following the incidents of TD. These findings illustrate the complex etiology of diarrhea amongst military personnel in deployed settings and its impacts on job performance. Potential factors of resistance or susceptibility can provide a foundation for future clinical trials to evaluate prevention and treatment strategies., Competing Interests: One of the coauthors, Jamie Fraser, is affiliated with the non-profit Henry M. Jackson Foundation. The Henry M. Jackson Foundation only provided Fraser with a salary, and did not have any role in our study design, execution, or manuscript preparation.

Soil microbes that colonize plant roots and are responsive to differences in plant genotype remain to be ascertained for agronomically important crops. From a very large-scale longitudinal field study of 27 maize inbred lines planted in three fields, with partial replication 5 y later, we identify root-associated microbiota exhibiting reproducible associations with plant genotype. Analysis of 4,866 samples identified 143 operational taxonomic units (OTUs) whose variation in relative abundances across the samples was significantly regulated by plant genotype, and included five of seven core OTUs present in all samples. Plant genetic effects were significant amid the large effects of plant age on the rhizosphere microbiome, regardless of the specific community of each field, and despite microbiome responses to climate events. Seasonal patterns showed that the plant root microbiome is locally seeded, changes with plant growth, and responds to weather events. However, against this background of variation, specific taxa responded to differences in host genotype. If shown to have beneficial functions, microbes may be considered candidate traits for selective breeding., Competing Interests: Conflict of interest statement: J.L.D. is a cofounder, shareholder, and member of the Scientific Advisory Board of AgBiome, a corporation with the goal to use plant-associated microbes to improve plant productivity. R.E.L. is a shareholder and member of the Scientific Advisory Board of AgBiome. All other authors declare no competing financial interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Bisphenol A (BPA) accumulates in the maturing gut and liver in utero and is known to alter gut bacterial profiles in offspring. Gut bacterial dysbiosis may contribute to chronic colonic and systemic inflammation. We hypothesized that perinatal BPA exposure-induced intestinal (and liver) inflammation in offspring is due to alterations in the microbiome and colonic metabolome. The 16S rRNA amplicon sequencing analysis revealed differences in beta diversity with a significant reduction in the relative abundances of short-chain fatty acid (SCFA) producers such as Oscillospira and Ruminococcaceae due to BPA exposure. Furthermore, BPA exposure reduced fecal SCFA levels and increased systemic lipopolysaccharide (LPS) levels. BPA exposure-increased intestinal permeability was ameliorated by the addition of SCFA in vitro . Metabolic fingerprints revealed alterations in global metabolism and amino acid metabolism. Thus, our findings indicate that perinatal BPA exposure may cause gut bacterial dysbiosis and altered metabolite profiles, particularly SCFA profiles, leading to chronic colon and liver inflammation. IMPORTANCE Emerging evidence suggests that environmental toxicants may influence inflammation-promoted chronic disease susceptibility during early life. BPA, an environmental endocrine disruptor, can transfer across the placenta and accumulate in fetal gut and liver. However, underlying mechanisms for BPA-induced colonic and liver inflammation are not fully elucidated. In this report, we show how perinatal BPA exposure in rabbits alters gut microbiota and their metabolite profiles, which leads to colonic and liver inflammation as well as to increased gut permeability as measured by elevated serum lipopolysaccharide (LPS) levels in the offspring. Also, perinatal BPA exposure leads to reduced levels of gut bacterial diversity and bacterial metabolites (short-chain fatty acids [SCFA]) and elevated gut permeability-three common early biomarkers of inflammation-promoted chronic diseases. In addition, we showed that SCFA ameliorated BPA-induced intestinal permeability in vitro . Thus, our study results suggest that correcting environmental toxicant-induced bacterial dysbiosis early in life may reduce the risk of chronic diseases later in life.

Inflammatory bowel disease (IBD) is characterized by flares of inflammation with a periodic need for increased medication and sometimes even surgery. The aetiology of IBD is partly attributed to a deregulated immune response to gut microbiome dysbiosis. Cross-sectional studies have revealed microbial signatures for different IBD subtypes, including ulcerative colitis, colonic Crohn's disease and ileal Crohn's disease. Although IBD is dynamic, microbiome studies have primarily focused on single time points or a few individuals. Here, we dissect the long-term dynamic behaviour of the gut microbiome in IBD and differentiate this from normal variation. Microbiomes of IBD subjects fluctuate more than those of healthy individuals, based on deviation from a newly defined healthy plane (HP). Ileal Crohn's disease subjects deviated most from the HP, especially subjects with surgical resection. Intriguingly, the microbiomes of some IBD subjects periodically visited the HP then deviated away from it. Inflammation was not directly correlated with distance to the healthy plane, but there was some correlation between observed dramatic fluctuations in the gut microbiome and intensified medication due to a flare of the disease. These results will help guide therapies that will redirect the gut microbiome towards a healthy state and maintain remission in IBD.

Bacterial species composition in the gut has emerged as an important factor in obesity and its related metabolic diseases such as type 2 diabetes. Out of thousands of bacterial species-level phylotypes inhabiting the human gut, the majority belong to two dominant phyla, the Bacteroidetes and Firmicutes. Members of the Bacteroidetes in particular have been associated with human metabolic diseases. However, their associations with disease are not always consistent between studies. Delving deeper into the diversity within the Bacteroidetes reveals a vast diversity in genomes and capacities, which partly explain how not all members respond equally to similar environmental conditions in their hosts. Here, we discuss the Bacteroidetes phylum, associations of its members with metabolic phenotypes, and efforts to characterize functionally their interactions with their hosts. Harnessing the Bacteroidetes to promote metabolic health will require a nuanced understanding of how specific strains interact with their microbial neighbors and their hosts under various conditions.

While high-throughput sequencing methods are revolutionizing fungal ecology, recovering accurate estimates of species richness and abundance has proven elusive. We sought to design internal transcribed spacer (ITS) primers and an Illumina protocol that would maximize coverage of the kingdom Fungi while minimizing nontarget eukaryotes. We inspected alignments of the 5.8S and large subunit (LSU) ribosomal genes and evaluated potential primers using PrimerProspector. We tested the resulting primers using tiered-abundance mock communities and five previously characterized soil samples. We recovered operational taxonomic units (OTUs) belonging to all 8 members in both mock communities, despite DNA abundances spanning 3 orders of magnitude. The expected and observed read counts were strongly correlated (r = 0.94 to 0.97). However, several taxa were consistently over- or underrepresented, likely due to variation in rRNA gene copy numbers. The Illumina data resulted in clustering of soil samples identical to that obtained with Sanger sequence clone library data using different primers. Furthermore, the two methods produced distance matrices with a Mantel correlation of 0.92. Nonfungal sequences comprised less than 0.5% of the soil data set, with most attributable to vascular plants. Our results suggest that high-throughput methods can produce fairly accurate estimates of fungal abundances in complex communities. Further improvements might be achieved through corrections for rRNA copy number and utilization of standardized mock communities., Importance: Fungi play numerous important roles in the environment. Improvements in sequencing methods are providing revolutionary insights into fungal biodiversity, yet accurate estimates of the number of fungal species (i.e., richness) and their relative abundances in an environmental sample (e.g., soil, roots, water, etc.) remain difficult to obtain. We present improved methods for high-throughput Illumina sequencing of the species-diagnostic fungal ribosomal marker gene that improve the accuracy of richness and abundance estimates. The improvements include new PCR primers and library preparation, validation using a known mock community, and bioinformatic parameter tuning., (Copyright © 2016 Taylor et al.)

Skin and soft tissue infections (SSTIs) are common in the general population, with increased prevalence among military trainees. Previous research has revealed numerous nasal microbial signatures that correlate with SSTI development and Staphylococcus aureus colonization. Thus, we hypothesized that the ecology of the inguinal, oropharynx, and perianal regions may also be altered in response to SSTI and/or S. aureus colonization. We collected body site samples from 46 military trainees with purulent abscess (SSTI group) as well as from 66 asymptomatic controls (non-SSTI group). We also collected abscess cavity samples to assess the microbial composition of these infections. Samples were analyzed by culture, and the microbial communities were characterized by high-throughput sequencing. We found that the nasal, inguinal, and perianal regions were similar in microbial composition and significantly differed from the oropharynx. We also observed differences in Anaerococcus and Streptococcus abundance between the SSTI and non-SSTI groups for the nasal and oropharyngeal regions, respectively. Furthermore, we detected community membership differences between the SSTI and non-SSTI groups for the nasal and inguinal sites. Compared to that of the other regions, the microbial compositions of the nares of S. aureus carriers and noncarriers were dramatically different; we noted an inverse correlation between the presence of Corynebacterium and the presence of Staphylococcus in the nares. This correlation was also observed for the inguinal region. Culture analysis revealed elevated methicillin-resistant S. aureus (MRSA) colonization levels for the SSTI group in the nasal and inguinal body sites. Together, these data suggest significant microbial variability in patients with SSTI as well as between S. aureus carriers and noncarriers. IMPORTANCE While it is evident that nasal colonization with S. aureus increases the likelihood of SSTI, there is a significant lack of information regarding the contribution of extranasal colonization to the overall risk of a subsequent SSTI. Furthermore, the impact of S. aureus colonization on bacterial community composition outside the nasal microbiota is unclear. Thus, this report represents the first investigation that utilized both culture and high-throughput sequencing techniques to analyze microbial dysbiosis at multiple body sites of healthy and diseased/colonized individuals. The results described here may be useful in the design of future methodologies to treat and prevent SSTIs.

Background: Gastrointestinal disturbances are among symptoms commonly reported by individuals diagnosed with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). However, whether ME/CFS is associated with an altered microbiome has remained uncertain. Here, we profiled gut microbial diversity by sequencing 16S ribosomal ribonucleic acid (rRNA) genes from stool as well as inflammatory markers from serum for cases (n = 48) and controls (n = 39). We also examined a set of inflammatory markers in blood: C-reactive protein (CRP), intestinal fatty acid-binding protein (I-FABP), lipopolysaccharide (LPS), LPS-binding protein (LBP), and soluble CD14 (sCD14)., Results: We observed elevated levels of some blood markers for microbial translocation in ME/CFS patients; levels of LPS, LBP, and sCD14 were elevated in ME/CFS subjects. Levels of LBP correlated with LPS and sCD14 and LPS levels correlated with sCD14. Through deep sequencing of bacterial rRNA markers, we identified differences between the gut microbiomes of healthy individuals and patients with ME/CFS. We observed that bacterial diversity was decreased in the ME/CFS specimens compared to controls, in particular, a reduction in the relative abundance and diversity of members belonging to the Firmicutes phylum. In the patient cohort, we find less diversity as well as increases in specific species often reported to be pro-inflammatory species and reduction in species frequently described as anti-inflammatory. Using a machine learning approach trained on the data obtained from 16S rRNA and inflammatory markers, individuals were classified correctly as ME/CFS with a cross-validation accuracy of 82.93 %., Conclusions: Our results indicate dysbiosis of the gut microbiota in this disease and further suggest an increased incidence of microbial translocation, which may play a role in inflammatory symptoms in ME/CFS.

Background: The operational taxonomic unit (OTU) is widely used in microbial ecology. Reproducibility in microbial ecology research depends on the reliability of OTU-based 16S ribosomal subunit RNA (rRNA) analyses., Results: Here, we report that many hierarchical and greedy clustering methods produce unstable OTUs, with membership that depends on the number of sequences clustered. If OTUs are regenerated with additional sequences or samples, sequences originally assigned to a given OTU can be split into different OTUs. Alternatively, sequences assigned to different OTUs can be merged into a single OTU. This OTU instability affects alpha-diversity analyses such as rarefaction curves, beta-diversity analyses such as distance-based ordination (for example, Principal Coordinate Analysis (PCoA)), and the identification of differentially represented OTUs. Our results show that the proportion of unstable OTUs varies for different clustering methods. We found that the closed-reference method is the only one that produces completely stable OTUs, with the caveat that sequences that do not match a pre-existing reference sequence collection are discarded., Conclusions: As a compromise to the factors listed above, we propose using an open-reference method to enhance OTU stability. This type of method clusters sequences against a database and includes unmatched sequences by clustering them via a relatively stable de novo clustering method. OTU stability is an important consideration when analyzing microbial diversity and is a feature that should be taken into account during the development of novel OTU clustering methods.

Recent studies have linked human gut microbes to obesity and inflammatory bowel disease, but consistent signals have been difficult to identify. Here we test for indicator taxa and general features of the microbiota that are generally consistent across studies of obesity and of IBD, focusing on studies involving high-throughput sequencing of the 16S rRNA gene (which we could process using a common computational pipeline). We find that IBD has a consistent signature across studies and allows high classification accuracy of IBD from non-IBD subjects, but that although subjects can be classified as lean or obese within each individual study with statistically significant accuracy, consistent with the ability of the microbiota to experimentally transfer this phenotype, signatures of obesity are not consistent between studies even when the data are analyzed with consistent methods. The results suggest that correlations between microbes and clinical conditions with different effect sizes (e.g. the large effect size of IBD versus the small effect size of obesity) may require different cohort selection and analysis strategies., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

We present a performance-optimized algorithm, subsampled open-reference OTU picking, for assigning marker gene (e.g., 16S rRNA) sequences generated on next-generation sequencing platforms to operational taxonomic units (OTUs) for microbial community analysis. This algorithm provides benefits over de novo OTU picking (clustering can be performed largely in parallel, reducing runtime) and closed-reference OTU picking (all reads are clustered, not only those that match a reference database sequence with high similarity). Because more of our algorithm can be run in parallel relative to "classic" open-reference OTU picking, it makes open-reference OTU picking tractable on massive amplicon sequence data sets (though on smaller data sets, "classic" open-reference OTU clustering is often faster). We illustrate that here by applying it to the first 15,000 samples sequenced for the Earth Microbiome Project (1.3 billion V4 16S rRNA amplicons). To the best of our knowledge, this is the largest OTU picking run ever performed, and we estimate that our new algorithm runs in less than 1/5 the time than would be required of "classic" open reference OTU picking. We show that subsampled open-reference OTU picking yields results that are highly correlated with those generated by "classic" open-reference OTU picking through comparisons on three well-studied datasets. An implementation of this algorithm is provided in the popular QIIME software package, which uses uclust for read clustering. All analyses were performed using QIIME's uclust wrappers, though we provide details (aided by the open-source code in our GitHub repository) that will allow implementation of subsampled open-reference OTU picking independently of QIIME (e.g., in a compiled programming language, where runtimes should be further reduced). Our analyses should generalize to other implementations of these OTU picking algorithms. Finally, we present a comparison of parameter settings in QIIME's OTU picking workflows and make recommendations on settings for these free parameters to optimize runtime without reducing the quality of the results. These optimized parameters can vastly decrease the runtime of uclust-based OTU picking in QIIME.

Human microbiome research is an actively developing area of inquiry, with ramifications for our lifestyles, our interactions with microbes, and how we treat disease. Advances depend on carefully executed, controlled, and reproducible studies. Here, we provide a Primer for researchers from diverse disciplines interested in conducting microbiome research. We discuss factors to be considered in the design, execution, and data analysis of microbiome studies. These recommendations should help researchers to enter and contribute to this rapidly developing field., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Eukaryotic microbes (protists) residing in the vertebrate gut influence host health and disease, but their diversity and distribution in healthy hosts is poorly understood. Protists found in the gut are typically considered parasites, but many are commensal and some are beneficial. Further, the hygiene hypothesis predicts that association with our co-evolved microbial symbionts may be important to overall health. It is therefore imperative that we understand the normal diversity of our eukaryotic gut microbiota to test for such effects and avoid eliminating commensal organisms. We assembled a dataset of healthy individuals from two populations, one with traditional, agrarian lifestyles and a second with modern, westernized lifestyles, and characterized the human eukaryotic microbiota via high-throughput sequencing. To place the human gut microbiota within a broader context our dataset also includes gut samples from diverse mammals and samples from other aquatic and terrestrial environments. We curated the SILVA ribosomal database to reflect current knowledge of eukaryotic taxonomy and employ it as a phylogenetic framework to compare eukaryotic diversity across environment. We show that adults from the non-western population harbor a diverse community of protists, and diversity in the human gut is comparable to that in other mammals. However, the eukaryotic microbiota of the western population appears depauperate. The distribution of symbionts found in mammals reflects both host phylogeny and diet. Eukaryotic microbiota in the gut are less diverse and more patchily distributed than bacteria. More broadly, we show that eukaryotic communities in the gut are less diverse than in aquatic and terrestrial habitats, and few taxa are shared across habitat types, and diversity patterns of eukaryotes are correlated with those observed for bacteria. These results outline the distribution and diversity of microbial eukaryotic communities in the mammalian gut and across environments.

Quantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genus Bacteroides are among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters.

High-throughput sequencing technology, coupled with the use of conserved marker genes, has allowed for the understanding of communities of microbes (both culturable and unculturable) as well as their phylogenetic placement. The recent explosion of sequencing data prompted the development of software that could process the vast amount of data generated and phylogenetically differentiate groups of samples. Host-associated microbial studies have revealed that microbes are highly varied between individuals and fluctuate within an individual. Large-scale studies are being undertaken that include collection of extensive environmental data to help uncover the forces that shape microbial communities.

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.

Microbial ecology is flourishing, and in the process, is making contributions to how the ecology and biology of large organisms is understood. Ongoing advances in sequencing technology and computational methods have enabled the collection and analysis of vast amounts of molecular data from diverse biological communities. While early studies focused on cataloguing microbial biodiversity in environments ranging from simple marine ecosystems to complex soil ecologies, more recent research is concerned with community functions and their dynamics over time. Models and concepts from traditional ecology have been used to generate new insight into microbial communities, and novel system-level models developed to explain and predict microbial interactions. The process of moving from molecular inventories to functional understanding is complex and challenging, and never more so than when many thousands of dynamic interactions are the phenomena of interest. We outline the process of how epistemic transitions are made from producing catalogues of molecules to achieving functional and predictive insight, and show how those insights not only revolutionize what is known about biological systems but also about how to do biology itself. Examples will be drawn primarily from analyses of different human microbiota, which are the microbial consortia found in and on areas of the human body, and their associated microbiomes (the genes of those communities). Molecular knowledge of these microbiomes is transforming microbiological knowledge, as well as broader aspects of human biology, health and disease.

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.

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.

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.

Variation in the composition of the human oral microbiome in health and disease has been observed. We have characterized inter- and intra-individual variation of microbial communities of 107 individuals in one of the largest cohorts to date (264 saliva samples), using culture-independent 16S rRNA pyrosequencing. We examined the salivary microbiome in up to three time-points during 10 yr spanning adolescence, and determined the influence of human genotype, gender, age, and weight class. Participants, including 27 monozygotic and 18 dizygotic twin pairs, were sampled mainly at ages 12-13, 17-18, and 22-24, with a few sampled as early as 8 yr of age. In contrast to gut or skin microbiomes, there is a core genus-level salivary microbiome. Individuals are more similar to themselves and their co-twins in the 12-17 and in the 17-22 cohorts than to the whole sample population, but not over the 10 yr from 12 to 22; and monozygotic twin pairs are statistically not more similar than dizygotic twin pairs. The data are most consistent with shared environment serving as the main determinant of microbial populations. Twins resemble each other more closely than the whole population at all time-points, but become less similar to each other when they age and no longer cohabit. Several organisms have age-specific abundance profiles, including members of the genera Veillonella, Actinomyces, and Streptococcus. There is no clear effect of weight class and gender. The results of this work will provide a basis to further study oral microbes and human health.

QIIME (canonically pronounced "chime") is a software application that performs microbial community analysis. It is an acronym for Quantitative Insights Into Microbial Ecology, and has been used to analyze and interpret nucleic acid sequence data from fungal, viral, bacterial, and archaeal communities. The following protocols describe how to install QIIME on a single computer and use it to analyze microbial 16S sequence data from nine distinct microbial communities., (© 2012 by John Wiley & Sons, Inc.)

Colitis results from breakdown of homeostasis between intestinal microbiota and the mucosal immune system, with both environmental and genetic influencing factors. Flagellin receptor TLR5-deficient mice (T5KO) display elevated intestinal proinflammatory gene expression and colitis with incomplete penetrance, providing a genetically sensitized system to study the contribution of microbiota to driving colitis. Both colitic and noncolitic T5KO exhibited transiently unstable microbiotas, with lasting differences in colitic T5KO, while their noncolitic siblings stabilized their microbiotas to resemble wild-type mice. Transient high levels of proteobacteria, especially enterobacteria species including E. coli, observed in close proximity to the gut epithelium were a striking feature of colitic microbiota. A Crohn's disease-associated E. coli strain induced chronic colitis in T5KO, which persisted well after the exogenously introduced bacterial species had been eliminated. Thus, an innate immune deficiency can result in unstable gut microbiota associated with low-grade inflammation, and harboring proteobacteria can drive and/or instigate chronic colitis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

DNA sequencing continues to decrease in cost with the Illumina HiSeq2000 generating up to 600 Gb of paired-end 100 base reads in a ten-day run. Here we present a protocol for community amplicon sequencing on the HiSeq2000 and MiSeq Illumina platforms, and apply that protocol to sequence 24 microbial communities from host-associated and free-living environments. A critical question as more sequencing platforms become available is whether biological conclusions derived on one platform are consistent with what would be derived on a different platform. We show that the protocol developed for these instruments successfully recaptures known biological results, and additionally that biological conclusions are consistent across sequencing platforms (the HiSeq2000 versus the MiSeq) and across the sequenced regions of amplicons.

Background: The extent to which numerous strains of genetically engineered mice, including mice lacking Toll-like receptor 5 (T5KO), display colitis is environment dependent. Gut microbiota underlie much of the variation in phenotype. Accordingly, embryonic rederivation of T5KO mice ameliorated their spontaneous colitis despite only partially correcting elevated proinflammatory gene expression. It was postulated that endogenous anti-inflammatory pathways mediated the absence of overt inflammation in these mice when their gut microbiota were reset. Consequently, it was hypothesised that neutralisation of the anti-inflammatory cytokine interleukin 10 (IL-10) might induce uniform colitis in T5KO mice, and thus provide a practical means to study mechanisms underlying their inflammation., Methods: Two distinct strains of non-colitic T5KO mice, as well as mice lacking MyD88, Toll-like receptor 4 (TLR4), IL-1 receptor (IL-1R) and various double knockouts (DKOs) were treated weekly for 4  weeks with 1 mg/mouse of IL-10 receptor neutralising antibody (IL-10R mAb) and colitis assayed 1 week later. The composition of the caecal microbiota was determined by 454 pyrosequencing of 16S rRNA genes., Results: Anti-IL-10R mAb treatment led to severe uniform intestinal inflammation in both strains of T5KO mice. Such neutralisation of IL-10 signalling did not cause colitis in wild-type littermates nor mice lacking TLR4, MyD88 or IL-1R. The susceptibility of T5KO mice to this colitis model was not rescued by absence of TLR4 in that T4/T5 DKO mice displayed severe colitis in response to anti-IL-10R mAb treatment. IL-1β signalling was crucial for this colitis model in that IL-1R/T5 DKOs were completely protected from colitis in response to IL-10R mAb treatment. Lastly, it was observed that blockade of IL-10R function was associated with changes in the composition of gut microbiota, which were observed in mice that were susceptible and resistant to IL-10R mAb-induced colitis., Conclusion: Regardless of whether they harbour a colitogenic microbiota, loss of TLR5 predisposes mice to colitis triggered by immune dysregulation via an IL-1β-dependent pathway.

Obesity has been linked to the human gut microbiota; however, the contribution of gut bacterial species to the obese phenotype remains controversial because of conflicting results from studies in different populations. To explore the possible dysbiosis of gut microbiota in obesity and its metabolic complications, we studied men and women over a range of body mass indices from the Old Order Amish sect, a culturally homogeneous Caucasian population of Central European ancestry. We characterized the gut microbiota in 310 subjects by deep pyrosequencing of bar-coded PCR amplicons from the V1-V3 region of the 16S rRNA gene. Three communities of interacting bacteria were identified in the gut microbiota, analogous to previously identified gut enterotypes. Neither BMI nor any metabolic syndrome trait was associated with a particular gut community. Network analysis identified twenty-two bacterial species and four OTUs that were either positively or inversely correlated with metabolic syndrome traits, suggesting that certain members of the gut microbiota may play a role in these metabolic derangements.

Taxonomic classification of the thousands-millions of 16S rRNA gene sequences generated in microbiome studies is often achieved using a naïve Bayesian classifier (for example, the Ribosomal Database Project II (RDP) classifier), due to favorable trade-offs among automation, speed and accuracy. The resulting classification depends on the reference sequences and taxonomic hierarchy used to train the model; although the influence of primer sets and classification algorithms have been explored in detail, the influence of training set has not been characterized. We compared classification results obtained using three different publicly available databases as training sets, applied to five different bacterial 16S rRNA gene pyrosequencing data sets generated (from human body, mouse gut, python gut, soil and anaerobic digester samples). We observed numerous advantages to using the largest, most diverse training set available, that we constructed from the Greengenes (GG) bacterial/archaeal 16S rRNA gene sequence database and the latest GG taxonomy. Phylogenetic clusters of previously unclassified experimental sequences were identified with notable improvements (for example, 50% reduction in reads unclassified at the phylum level in mouse gut, soil and anaerobic digester samples), especially for phylotypes belonging to specific phyla (Tenericutes, Chloroflexi, Synergistetes and Candidate phyla TM6, TM7). Trimming the reference sequences to the primer region resulted in systematic improvements in classification depth, and greatest gains at higher confidence thresholds. Phylotypes unclassified at the genus level represented a greater proportion of the total community variation than classified operational taxonomic units in mouse gut and anaerobic digester samples, underscoring the need for greater diversity in existing reference databases.

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., Competing Interests: statement The authors declare no competing financial interests.

QIIME (canonically pronounced "chime") is a software application that performs microbial community analysis. It is an acronym for Quantitative Insights Into Microbial Ecology, and has been used to analyze and interpret nucleic acid sequence data from fungal, viral, bacterial, and archaeal communities. The following protocols describe how to install QIIME on a single computer and use it to analyze microbial 16S sequence data from nine distinct microbial communities., (© 2011 by John Wiley & Sons, Inc.)

Diet strongly affects human health, partly by modulating gut microbiome composition. We used diet inventories and 16S rDNA sequencing to characterize fecal samples from 98 individuals. Fecal communities clustered into enterotypes distinguished primarily by levels of Bacteroides and Prevotella. Enterotypes were strongly associated with long-term diets, particularly protein and animal fat (Bacteroides) versus carbohydrates (Prevotella). A controlled-feeding study of 10 subjects showed that microbiome composition changed detectably within 24 hours of initiating a high-fat/low-fiber or low-fat/high-fiber diet, but that enterotype identity remained stable during the 10-day study. Thus, alternative enterotype states are associated with long-term diet.

High throughput sequencing technology has opened a window into the vast communities of bacteria that live on and in humans, demonstrating tremendous variability, and that they play a large role in health and disease. The eukaryotic component of the human gut microbiome remains relatively unexplored with these methods, but turning these tools toward microbial eukaryotes in the gut will likely yield myriad insights into disease as well as the ecological and evolutionary principles that govern the gut microbiota. Microbial eukaryotes are common inhabitants of the human gut worldwide and parasitic taxa are a major source of morbidity and mortality, especially in developing countries, though there are also taxa that cause no harm or are beneficial. While the role microbial eukaryotes play in healthy individuals is much less clear, there are likely many complex interactions between the bacterial, archaeal, and eukaryotic microbiota that influence human health. Integrating eukaryotic microbes into a broad view of microbiome function requires an integrated ecological approach rather than one focused on specific, disease-causing taxa. Moving forward, we expect broad surveys of the eukaryotic microbiota and associated bacteria from geographically and socioeconomically diverse populations to paint a more complete picture of the human gut microbiome in health and disease.

Verrucomicrobia are ubiquitous in soil, but members of this bacterial phylum are thought to be present at low frequency in soil, with few studies focusing specifically on verrucomicrobial abundance, diversity, and distribution. Here we used barcoded pyrosequencing to analyze verrucomicrobial communities in surface soils collected across a range of biomes in Antarctica, Europe, and the Americas (112 samples), as well as soils collected from pits dug in a montane coniferous forest (69 samples). Data collected from surface horizons indicate that Verrucomicrobia average 23% of bacterial sequences, making them far more abundant than had been estimated. We show that this underestimation is likely due to primer bias, as many of the commonly used PCR primers appear to exclude verrucomicrobial 16S rRNA genes during amplification. Verrucomicrobia were detected in 180 out of 181 soils examined, with members of the class Spartobacteria dominating verrucomicrobial communities in nearly all biomes and soil depths. The relative abundance of Verrucomicrobia was highest in grasslands and in subsurface soil horizons, where they were often the dominant bacterial phylum. Although their ecology remains poorly understood, Verrucomicrobia appear to be dominant in many soil bacterial communities across the globe, making additional research on their ecology clearly necessary.

Background: There appears to be a rise in the rate of emergency peripartum hysterectomy (EPH) in the developed world., Aims:   To determine the incidence, indications, risk factors, complications and management of EPH in our tertiary level teaching hospital, the Royal Hospital for Women (RHW) in Sydney, over the last decade., Methods: A retrospective analysis was conducted of all cases of EPH performed at the RHW between the years 1999-2008 inclusive. EPH was defined as one performed after 20 weeks gestation for uncontrollable uterine bleeding not responsive to conservative measures occurring at any time after delivery but within the first 6 weeks post-partum. Cases were ascertained via our hospital obstetric database., Results: There were 33 EPH among 38,998 births, a rate of 0.85 per 1000 births. Indications for EPH were morbid adherence of the placenta (54.8%), placenta praevia (19.4%), uterine atony (12.9%) and uterine rupture or cervical laceration (9.7%). A significant association between previous caesarean section (CS) and abnormal placentation was confirmed (P=0.011), especially for morbid adherence of the placenta (P=0.004). There was one maternal death. Maternal morbidity was significant, with disseminated intravascular coagulation and urinary tract injury among the most common complications. All women required blood transfusions, and over a quarter were admitted to the intensive care unit., Conclusions: In our series, abnormal placentation causing severe haemorrhage was the commonest indication for EPH. Previous CS is a risk factor for abnormal placentation and particularly for morbid adherence of the placenta. The morbidity associated with EPH is considerable., (© 2011 The Authors. Australian and New Zealand Journal of Obstetrics and Gynaecology © 2011 The Royal Australian and New Zealand College of Obstetricians and Gynaecologists.)

Archaea, primarily Crenarchaeota, are common in soil; however, the structure of soil archaeal communities and the factors regulating their diversity and abundance remain poorly understood. Here, we used barcoded pyrosequencing to comprehensively survey archaeal and bacterial communities in 146 soils, representing a multitude of soil and ecosystem types from across the globe. Relative archaeal abundance, the percentage of all 16S rRNA gene sequences recovered that were archaeal, averaged 2% across all soils and ranged from 0% to >10% in individual soils. Soil C:N ratio was the only factor consistently correlated with archaeal relative abundances, being higher in soils with lower C:N ratios. Soil archaea communities were dominated by just two phylotypes from a constrained clade within the Crenarchaeota, which together accounted for >70% of all archaeal sequences obtained in the survey. As one of these phylotypes was closely related to a previously identified putative ammonia oxidizer, we sampled from two long-term nitrogen (N) addition experiments to determine if this taxon responds to experimental manipulations of N availability. Contrary to expectations, the abundance of this dominant taxon, as well as archaea overall, tended to decline with increasing N. This trend was coupled with a concurrent increase in known N-oxidizing bacteria, suggesting competitive interactions between these groups.