The importance of 16S rRNA gene amplicon profiles for understanding the influence of microbes in a variety of environments coupled with the steep reduction in sequencing costs led to a surge of ...microbial sequencing projects. The expanding crowd of scientists and clinicians wanting to make use of sequencing datasets can choose among a range of multipurpose software platforms, the use of which can be intimidating for non-expert users. Among available pipeline options for high-throughput 16S rRNA gene analysis, the R programming language and software environment for statistical computing stands out for its power and increased flexibility, and the possibility to adhere to most recent best practices and to adjust to individual project needs. Here we present the Rhea pipeline, a set of R scripts that encode a series of well-documented choices for the downstream analysis of Operational Taxonomic Units (OTUs) tables, including normalization steps,
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-diversity analysis, taxonomic composition, statistical comparisons, and calculation of correlations. Rhea is primarily a straightforward starting point for beginners, but can also be a framework for advanced users who can modify and expand the tool. As the community standards evolve, Rhea will adapt to always represent the current state-of-the-art in microbial profiles analysis in the clear and comprehensive way allowed by the R language. Rhea scripts and documentation are freely available at https://lagkouvardos.github.io/Rhea.
The SRA (Sequence Read Archive) serves as primary depository for massive amounts of Next Generation Sequencing data, and currently host over 100,000 16S rRNA gene amplicon-based microbial profiles ...from various host habitats and environments. This number is increasing rapidly and there is a dire need for approaches to utilize this pool of knowledge. Here we created IMNGS (Integrated Microbial Next Generation Sequencing), an innovative platform that uniformly and systematically screens for and processes all prokaryotic 16S rRNA gene amplicon datasets available in SRA and uses them to build sample-specific sequence databases and OTU-based profiles. Via a web interface, this integrative sequence resource can easily be queried by users. We show examples of how the approach allows testing the ecological importance of specific microorganisms in different hosts or ecosystems, and performing targeted diversity studies for selected taxonomic groups. The platform also offers a complete workflow for de novo analysis of users' own raw 16S rRNA gene amplicon datasets for the sake of comparison with existing data. IMNGS can be accessed at www.imngs.org.
Bacteria within family S24-7 (phylum Bacteroidetes) are dominant in the mouse gut microbiota and detected in the intestine of other animals. Because they had not been cultured until recently and the ...family classification is still ambiguous, interaction with their host was difficult to study and confusion still exists regarding sequence data annotation.
We investigated family S24-7 by combining data from large-scale 16S rRNA gene analysis and from functional and taxonomic studies of metagenomic and cultured species.
A total of 685 species was inferred by full-length 16S rRNA gene sequence clustering. While many species could not be assigned ecological habitats (93,045 samples analyzed), the mouse was the most commonly identified host (average of 20% relative abundance and nine co-occurring species). Shotgun metagenomics allowed reconstruction of 59 molecular species, of which 34 were representative of the 16S rRNA gene-derived species clusters. In addition, cultivation efforts allowed isolating five strains representing three species, including two novel taxa. Genome analysis revealed that S24-7 spp. are functionally distinct from neighboring families and versatile with respect to complex carbohydrate degradation.
We provide novel data on the diversity, ecology, and description of bacterial family S24-7, for which the name Muribaculaceae is proposed.
The gut microbiome lies at the intersection between the environment and the host, with the ability to modify host responses to disease-relevant exposures and stimuli. This is evident in how enteric ...microbes interact with the immune system, e.g., supporting immune maturation in early life, affecting drug efficacy via modulation of immune responses, or influencing development of immune cell populations and their mediators. Many factors modulate gut ecosystem dynamics during daily life and we are just beginning to realise the therapeutic and prophylactic potential of microbiome-based interventions. These approaches vary in application, goal, and mechanisms of action. Some modify the entire community, such as nutritional approaches or faecal microbiota transplantation, while others, such as phage therapy, probiotics, and prebiotics, target specific taxa or strains. In this review, we assessed the experimental evidence for microbiome-based interventions, with a particular focus on their clinical relevance, ecological effects, and modulation of the immune system.
Lifestyle, obesity, and the gut microbiome are important risk factors for metabolic disorders. We demonstrate in 1,976 subjects of a German population cohort (KORA) that specific microbiota members ...show 24-h oscillations in their relative abundance and identified 13 taxa with disrupted rhythmicity in type 2 diabetes (T2D). Cross-validated prediction models based on this signature similarly classified T2D. In an independent cohort (FoCus), disruption of microbial oscillation and the model for T2D classification was confirmed in 1,363 subjects. This arrhythmic risk signature was able to predict T2D in 699 KORA subjects 5 years after initial sampling, being most effective in combination with BMI. Shotgun metagenomic analysis functionally linked 26 metabolic pathways to the diurnal oscillation of gut bacteria. Thus, a cohort-specific risk pattern of arrhythmic taxa enables classification and prediction of T2D, suggesting a functional link between circadian rhythms and the microbiome in metabolic diseases.
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•Human gut microbiome exhibits diurnal rhythmicity across populations and individuals•Obese and T2D individuals show disrupted circadian rhythms in the gut microbiome•Arrhytmic bacterial signatures contribute to risk classification and prediction of T2D•These risk signatures show regional differences in applicability across three cohorts
Reitmeier et al. show that specific gut microbes exhibit rhythmic oscillations in relative abundance and identified taxa with disrupted rhythmicity in individuals with type 2 diabetes (T2D). This arrhythmic signature contributed to the classification and prediction of T2D, suggesting functional links between circadian rhythmicity and the microbiome in metabolic diseases.
Interactions between the gut microbial ecosystem and host lipid homeostasis are highly relevant to host physiology and metabolic diseases. We present a comprehensive multi-omics view of the effect of ...intestinal microbial colonization on hepatic lipid metabolism, integrating transcriptomic, proteomic, phosphoproteomic, and lipidomic analyses of liver and plasma samples from germfree and specific pathogen-free mice. Microbes induce monounsaturated fatty acid generation by stearoyl-CoA desaturase 1 and polyunsaturated fatty acid elongation by fatty acid elongase 5, leading to significant alterations in glycerophospholipid acyl-chain profiles. A composite classification score calculated from the observed alterations in fatty acid profiles in germfree mice clearly differentiates antibiotic-treated mice from untreated controls with high sensitivity. Mechanistic investigations reveal that acetate originating from gut microbial degradation of dietary fiber serves as precursor for hepatic synthesis of C16 and C18 fatty acids and their related glycerophospholipid species that are also released into the circulation.
Recent findings suggest an association between obesity, loss of gut barrier function and changes in microbiota profiles. Our primary objective was to examine the effect of caloric restriction and ...subsequent weight reduction on gut permeability in obese women. The impact on inflammatory markers and fecal microbiota was also investigated. The 4-week very-low calorie diet (VLCD, 800 kcal/day) induced a mean weight loss of 6.9 ± 1.9 kg accompanied by a reduction in HOMA-IR (Homeostasis model assessment-insulin resistance), fasting plasma glucose and insulin, plasma leptin, and leptin gene expression in subcutaneous adipose tissue. Plasma high-molecular weight adiponectin (HMW adiponectin) was significantly increased after VLCD. Plasma levels of high-sensitivity C-reactive protein (hsCRP) and lipopolysaccharide-binding protein (LBP) were significantly decreased after 28 days of VLCD. Using three different methods, gut paracellular permeability was decreased after VLCD. These changes in clinical parameters were not associated with major consistent changes in dominant bacterial communities in feces. In summary, a 4-week caloric restriction resulted in significant weight loss, improved gut barrier integrity and reduced systemic inflammation in obese women.
Summary
Cultivation via classical agar plate (CAP) approaches is widely used to study microbial communities, but they are time‐consuming. An alternative approach is the application of single‐cell ...dispensing (SCD), which allows high‐throughput, label‐free sorting of microscopic particles. We aimed to develop a new anaerobic SCD workflow to cultivate human gut bacteria and compared it with CAP using faecal communities on three rich culture media. We found that the SCD approach significantly decreased the experimental time to obtain pure cultures from 17 ± 4 to 5 ± 0 days, while the isolate diversity and relative abundance coverage were comparable for both approaches. We further tested the total captured fraction by sequencing the sorted bacteria directly after growth as bulk biomass from 2400 dispensed single cells without downstream identification of individual strains. In this approach, the cultured fraction increased from 35.2% to 52.2% for SCD, highlighting the potential for deeper cultivation projects from single samples. SCD‐based cultivation also captured species not detected by sequencing (16 ± 5 per sample, including seven novel taxa). From this work, 82 human gut bacterial species across five phyla (Actinobacteriota, Bacteroidota, Desulfobacterota, Firmicutes and Proteobacteria) and 24 families were obtained, including the first cultured member of 11 novel genera and 10 novel species that were fully characterized taxonomically.
The intestinal microbiota is known to regulate host energy homeostasis and can be influenced by high-calorie diets. However, changes affecting the ecosystem at the functional level are still not well ...characterized. We measured shifts in cecal bacterial communities in mice fed a carbohydrate or high-fat (HF) diet for 12 weeks at the level of the following: (i) diversity and taxa distribution by high-throughput 16S ribosomal RNA gene sequencing; (ii) bulk and single-cell chemical composition by Fourier-transform infrared- (FT-IR) and Raman micro-spectroscopy and (iii) metaproteome and metabolome via high-resolution mass spectrometry. High-fat diet caused shifts in the diversity of dominant gut bacteria and altered the proportion of Ruminococcaceae (decrease) and Rikenellaceae (increase). FT-IR spectroscopy revealed that the impact of the diet on cecal chemical fingerprints is greater than the impact of microbiota composition. Diet-driven changes in biochemical fingerprints of members of the Bacteroidales and Lachnospiraceae were also observed at the level of single cells, indicating that there were distinct differences in cellular composition of dominant phylotypes under different diets. Metaproteome and metabolome analyses based on the occurrence of 1760 bacterial proteins and 86 annotated metabolites revealed distinct HF diet-specific profiles. Alteration of hormonal and anti-microbial networks, bile acid and bilirubin metabolism and shifts towards amino acid and simple sugars metabolism were observed. We conclude that a HF diet markedly affects the gut bacterial ecosystem at the functional level.
Growing interest in microbial contributions to human health and disease has increasingly led investigators to examine the microbiome in both healthy skin and cutaneous disorders, including acne, ...psoriasis, and atopic dermatitis. The need for common language, effective study design, and validated methods is critical for high-quality standardized research. Features, unique to skin, pose particular challenges when conducting microbiome research. This review discusses microbiome research standards and highlights important factors to consider, including clinical study design, skin sampling, sample processing, DNA sequencing, control inclusion, and data analysis.
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