The Skin Microbiome: Current Techniques, Challenges, and Future Directions.

Skin acts as a barrier that promotes the colonization of bacteria, fungi, archaea, and viruses whose membership and function may differ depending on the various specialized niches or micro-environments of the skin. The group of microorganisms inhabiting the skin, also known as the skin microbiome, offers protection against pathogens while actively interacting with the host's immune system. Some members of the skin microbiome can also act as opportunistic pathogens. The skin microbiome is influenced by factors such as skin site, birth mode, genetics, environment, skin products, and skin conditions. The association(s) of the skin microbiome with health and disease has (have) been identified and characterized via culture-dependent and culture-independent methods. Culture-independent methods (such as high-throughput sequencing), in particular, have expanded our understanding of the skin microbiome's role in maintaining health or promoting disease. However, the intrinsic challenges associated with the low microbial biomass and high host content of skin microbiome samples have hindered advancements in the field. In addition, the limitations of current collection and extraction methods and biases derived from sample preparation and analysis have significantly influenced the results and conclusions of many skin microbiome studies. Therefore, the present review discusses the technical challenges associated with the collection and processing of skin microbiome samples, the advantages and disadvantages of current sequencing approaches, and potential future areas of focus for the field.

Viral Metagenomics as a Tool to Track Sources of Fecal Contamination: A One Health Approach.

The One Health framework recognizes that human, animal, and environmental health are linked and highly interdependent. Fecal contamination of water, soil, foodstuff, and air may impact many aspects of One Health, and culture, PCR-based, and sequencing methods are utilized in the detection of fecal contamination to determine source, load, and risk to inform targeted mitigation strategies. Viruses, particularly, have been considered as fecal contamination indicators given the narrow host range many exhibit and their association with other biological contaminants. Culture- and molecular-based methods are considered the gold-standards for virus detection and for determining specific sources of fecal contamination via viral indicators. However, viral metagenomics is also being considered as a tool for tracking sources of fecal contamination. In the present review, studies tracking potential sources of fecal contamination in freshwaters, marine waters, foodstuff, soil, and air using viral metagenomics are discussed to highlight the potential of viral metagenomics for optimizing fecal source tracking. Limitations of the use of viral metagenomics to track fecal contamination sources, including sample processing, nucleic acid recovery, sequencing depth, and bioinformatics are also discussed. Finally, the present review discusses the potential of viral metagenomics as part of the toolbox of methods in a One Health approach.

Unraveling the viral dark matter through viral metagenomics.

Viruses are part of the microbiome and have essential roles in immunology, evolution, biogeochemical cycles, health, and disease progression. Viruses influence a wide variety of systems and processes, and the continued discovery of novel viruses is anticipated to reveal new mechanisms influencing the biology of diverse environments. While the identity and roles of viruses continue to be discovered and understood through viral metagenomics, most of the sequences in virome datasets cannot be attributed to known viruses or may be only distantly related to species already described in public sequence databases, at best. Such viruses are known as the viral dark matter. Ongoing discoveries from the viral dark matter have provided insights into novel viruses from a variety of environments, as well as their potential in immunological processes, virus evolution, health, disease, therapeutics, and surveillance. Increased understanding of the viral dark matter will continue with a combination of cultivation, microscopy, sequencing, and bioinformatic efforts, which are discussed in the present review.

Fecal Microbiota Transplantation Commonly Failed in Children With Co-Morbidities.

OBJECTIVES: Fecal microbiota transplantation (FMT) is arguably the most effective treatment for recurrent Clostridioides difficile infection (rCDI). Clinical reports on pediatric FMT have not systematically evaluated microbiome restoration in patients with co-morbidities. Here, we determined whether FMT recipient age and underlying co-morbidity influenced clinical outcomes and microbiome restoration when treated from shared fecal donor sources. METHODS: Eighteen rCDI patients participating in a single-center, open-label prospective cohort study received fecal preparation from a self-designated (single case) or two universal donors. Twelve age-matched healthy children and four pediatric ulcerative colitis (UC) cases from an independent serial FMT trial, but with a shared fecal donor were examined as controls for microbiome restoration using 16S rRNA gene sequencing of longitudinal fecal specimens. RESULTS: FMT was significantly more effective in rCDI recipients without underlying chronic co-morbidities where fecal microbiome composition in post-transplant responders was restored to levels of healthy children. Microbiome reconstitution was not associated with symptomatic resolution in some rCDI patients who had co-morbidities. Significant elevation in Bacteroidaceae, Bifidobacteriaceae, Lachnospiraceae, Ruminococcaceae, and Erysipelotrichaceae was consistently observed in pediatric rCDI responders, while Enterobacteriaceae decreased, correlating with augmented complex carbohydrate degradation capacity. CONCLUSION: Recipient background disease was a significant risk factor influencing FMT outcomes. Special attention should be taken when considering FMT for pediatric rCDI patients with underlying co-morbidities.

Multi 'omic data integration: A review of concepts, considerations, and approaches.

The application of 'omic techniques including, but not limited to genomics/metagenomics, transcriptomics/meta-transcriptomics, proteomics/meta-proteomics, and metabolomics to generate multiple datasets from a single sample have facilitated hypothesis generation leading to the identification of biological, molecular and ecological functions and mechanisms, as well as associations and correlations. Despite their power and promise, a variety of challenges must be considered in the successful design and execution of a multi-omics study. In this review, various 'omic technologies applicable to single- and meta-organisms (i.e., host + microbiome) are described, and considerations for sample collection, storage and processing prior to data generation and analysis, as well as approaches to data storage, dissemination and analysis are discussed. Finally, case studies are included as examples of multi-omic applications providing novel insights and a more holistic understanding of biological processes.

Probiotic VSL#3 Treatment Reduces Colonic Permeability and Abdominal Pain Symptoms in Patients With Irritable Bowel Syndrome.

Background: Little is known regarding the clinical impact of treatment and treatment duration of probiotic VSL#3 on gut and microbiome function in irritable bowel syndrome (IBS). As part of a safety trial, we assessed the effect of VSL#3 treatment duration on abdominal pain, stooling, gut permeability, microbiome composition and function. Methods: Adults with IBS were randomized into an open label trial to receive the probiotic VSL#3 for 4 or 8 weeks. Adverse events, abdominal pain, and stooling patterns were recorded daily. Gut permeability, fecal bile acid levels, and microbiome composition were profiled at baseline and after treatment. Results: Fifteen subjects completed the trial (4-week: n = 8; 8-week: n = 7). Number of pain episodes decreased in both groups (P = 0.049 and P = 0.034; 4- vs. 8-week, respectively). Probiotic organisms contained in VSL#3 were detected in feces by whole shotgun metagenomic sequencing analysis and relative abundances of Streptococcus thermophilus, Bifidobacterium animalis, Lactobacillus plantarum, and Lactobacillus casei subsp. paraccasei correlated significantly with improved abdominal pain symptoms and colonic permeability at study completion. Although abdominal pain correlated significantly with the detection of probiotic species at study completion, a composite view of gut microbiome structure showed no changes in community diversity or composition after VSL#3 treatment. Conclusions: Probiotic organisms identified in stool correlated significantly with improvement in colonic permeability and clinical symptoms, prompting future studies to investigate the mechanistic role of VSL#3 and colonic permeability in IBS pathophysiology in a larger randomized controlled trial. Clinical Trial Registration: www.clinicaltrials.gov, Identifier: NCT00971711.

Metagenomic Information Recovery from Human Stool Samples Is Influenced by Sequencing Depth and Profiling Method.

Sequencing of the 16S rRNA gene (16S) has long been a go-to method for microbiome characterization due to its accessibility and lower cost compared to shotgun metagenomic sequencing (SMS). However, 16S sequencing rarely provides species-level resolution and cannot provide direct assessment of other taxa (e.g., viruses and fungi) or functional gene content. Shallow shotgun metagenomic sequencing (SSMS) has emerged as an approach to bridge the gap between 16S sequencing and deep metagenomic sequencing. SSMS is cost-competitive with 16S sequencing, while also providing species-level resolution and functional gene content insights. In the present study, we evaluated the effects of sequencing depth on marker gene-mapping- and alignment-based annotation of bacteria in healthy human stool samples. The number of identified taxa decreased with lower sequencing depths, particularly with the marker gene-mapping-based approach. Other annotations, including viruses and pathways, also showed a depth-dependent effect on feature recovery. These results refine the understanding of the suitability and shortcomings of SSMS, as well as annotation tools for metagenomic analyses in human stool samples. Results may also translate to other sample types and may open the opportunity to explore the effect of sequencing depth and annotation method.

Potential Applications of Human Viral Metagenomics and Reference Materials: Considerations for Current and Future Viruses.

Viruses are ubiquitous particles comprising genetic material that can infect bacteria, archaea, and fungi, as well as human and other animal cells. Given that determining virus composition and function in association with states of human health and disease is of increasing interest, we anticipate that the field of viral metagenomics will continue to expand and be applied in a variety of areas ranging from surveillance to discovery and will rely heavily upon the continued development of reference materials and databases. Information regarding viral composition and function readily translates into biological and clinical applications, including the rapid sequence identification of pathogenic viruses in various sample types. However, viral metagenomic approaches often lack appropriate standards and reference materials to enable cross-study comparisons and assess potential biases which can be introduced at the various stages of collection, storage, processing, and sequence analysis. In addition, implementation of appropriate viral reference materials can aid in the benchmarking of current and development of novel assays for virus identification, discovery, and surveillance. As the field of viral metagenomics expands and standardizes, results will continue to translate into diverse applications.

Relationships of Microbiome Markers With Extraintestinal, Psychological Distress and Gastrointestinal Symptoms, and Quality of Life in Women With Irritable Bowel Syndrome.

INTRODUCTION: Altered microbial diversity has been associated with gastrointestinal (GI) symptoms in persons with irritable bowel syndrome (IBS). Less is known about the relationship of microbiome with extraintestinal pain and psychological distress symptoms and quality of life (QOL) in persons with IBS. We aimed to evaluate the relationship of fecal microbiota to GI symptoms, stool consistency, psychological distress, extraintestinal pain, and QOL in participants meeting Rome III criteria for IBS. METHODS: Seventy-six women completed a 28-day diary that included GI, stool consistency, psychological distress, and extraintestinal pain ratings. Participants completed the IBS-Specific Quality of Life questionnaire. Stool samples were collected and analyzed by 16S rRNA gene sequencing. Principal component analysis was performed and the first 2 components (PC1, PC2) were used to test relationships among bacterial families and clinical measures. RESULTS: Participants were categorized as IBS constipation (n=22), IBS diarrhea (n=39), IBS mixed (n=13), and IBS unsubtyped (n=2). There was a significant group effect for the Firmicutes to Bacteroidetes ratio and PC1. Lower microbial diversity and richness were associated with increased urgency and extraintestinal pain, worse QOL, and looser stools. Lower extraintestinal pain was associated with increased Rikenellaceae, Christensenellaceae, Dehalobabacteriaceae, Oscillospiraceae, Mogibacteriaceae, Ruminococcaceae, Sutterellaceae, Desulfovibrionaceae, and Erysipelotrichaceae abundances. QOL was positively associated with many of these same bacterial families. Higher Firmicutes to Bacteroidetes ratio was positively associated with loose stools. There were no statistically significant relationships between daily psychological distress or abdominal pain and bacterial families. CONCLUSIONS: Stool microbial diversity and composition are linked to daily extraintestinal symptoms, stool consistency, and QOL in women with IBS.

Human Virome and Disease: High-Throughput Sequencing for Virus Discovery, Identification of Phage-Bacteria Dysbiosis and Development of Therapeutic Approaches with Emphasis on the Human Gut.

The virome is comprised of endogenous retroviruses, eukaryotic viruses, and bacteriophages and is increasingly being recognized as an essential part of the human microbiome. The human virome is associated with Type-1 diabetes (T1D), Type-2 diabetes (T2D), Inflammatory Bowel Disease (IBD), Human Immunodeficiency Virus (HIV) infection, and cancer. Increasing evidence also supports trans-kingdom interactions of viruses with bacteria, small eukaryotes and host in disease progression. The present review focuses on virus ecology and biology and how this translates mostly to human gut virome research. Current challenges in the field and how the development of bioinformatic tools and controls are aiding to overcome some of these challenges are also discussed. Finally, the present review also focuses on how human gut virome research could result in translational and clinical studies that may facilitate the development of therapeutic approaches.

Microbiota stratification identifies disease-specific alterations in neuro-Behçet's disease and multiple sclerosis.

OBJECTIVES: Altered gut microbiota community dynamics are implicated in diverse human diseases including inflammatory disorders such as neuro-Behçet's disease (NBD) and multiple sclerosis (MS). Traditionally, microbiota communities are analysed uniformly across control and disease groups, but recent reports of subsample clustering indicate a potential need for analytical stratification. The objectives of this study are to analyse and compare faecal microbiota community signatures of ethno-geographical, age and gender matched adult healthy controls (HC), MS and NBD individuals. METHODS: Faecal microbiota community compositions in adult HC (n=14), NBD patients (n=13) and MS (n=13) were analysed by 16S rRNA gene sequencing and standard bioinformatics pipelines. Bipartite networks were then used to identify and re-analyse dominant compositional clusters in respective groups. RESULTS: We identified Prevotella and Bacteroides dominated subsample clusters in HC, MS, and NBD cohorts. Our study confirmed previous reports that Prevotella is a major dysbiotic target in these diseases. We demonstrate that subsample stratification is required to identify significant disease-associated microbiota community shifts with increased Clostridiales evident in Prevotella-stratified NBD and Bacteroides-stratified MS patients. CONCLUSIONS: Patient cohort stratification may be needed to facilitate identification of common microbiota community shifts for causation testing in disease.

Fungal Community Structural and Microbial Functional Pattern Changes After Soil Amendments by Oilseed Meals of Jatropha curcas and Camelina sativa: A Microcosm Study.

The meals after oil extraction from many oilseed crops have nutrition and biofumigation potential for land application. Oilseed meal (SM) from the dedicated bioenergy crop Jatropha curcas were implicated to contain compounds that have antibacterial properties on some soil pathogens. However, little is known about its effect on non-targeted soil microbial community, especially on fungi. SM from Camelina sativa contains moderate level of glucosinolates (GLS) and was under studied. To investigate soil fungal community responses to jatropha and camelina SMs, we conducted a lab based microcosm study, amending soil with 1% SMs of jatropha, camelina, flax, and biomass of wheat straw. Fungal community abundance and structure were analyzed based on the ITS region using qPCR and tag-pyrosequencing. Microbial functional changes were examined by community level physiological profile (CLPP) using Biolog assay. Both SMs from jatropha and camelina showed biofumigant properties and inhibited fungal proliferation. Jatropha SM significantly altered soil fungal community structures with lower fungal biodiversity and higher Chaetomium composition. Camelina SM amended soil promoted Fusarium proliferation. CLPP indicated sequential hierarchy for C metabolism in the oilseed-amended microcosms was generally complex C > phosphate-associated C > carboxylic acids > carbohydrates > amines > amino acids. No significant difference in CLPP was detected due to the type of SM treatment. Our data indicate that both SMs of jatropha and camelina have biofumigant properties and can differentially impact soil microbial communities, and the changes were relatively persistent over time. Microbial functional patterns on the other side were not impacted by SM type. Our study revealed biofumigant and nutritional influence of SMs from dedicated biofuel plants on soil microbial community. This information will help properly using jatropha and camelina SMs for pathogen control while minimizing their negative impacts on non-target microorganisms. However, further studies in the field are demanded to investigate their influences in real practice.

Leveraging Human Microbiome Features to Diagnose and Stratify Children with Irritable Bowel Syndrome.

Accurate diagnosis and stratification of children with irritable bowel syndrome (IBS) remain challenging. Given the central role of recurrent abdominal pain in IBS, we evaluated the relationships of pediatric IBS and abdominal pain with intestinal microbes and fecal metabolites using a comprehensive clinical characterization and multiomics strategy. Using rigorous clinical phenotyping, we identified preadolescent children (aged 7 to 12 years) with Rome III IBS (n = 23) and healthy controls (n = 22) and characterized their fecal microbial communities using whole-genome shotgun metagenomics and global unbiased fecal metabolomic profiling. Correlation-based approaches and machine learning algorithms identified associations between microbes, metabolites, and abdominal pain. IBS cases differed from controls with respect to key bacterial taxa (eg, Flavonifractor plautii and Lachnospiraceae bacterium 7_1_58FAA), metagenomic functions (eg, carbohydrate metabolism and amino acid metabolism), and higher-order metabolites (eg, secondary bile acids, sterols, and steroid-like compounds). Significant associations between abdominal pain frequency and severity and intestinal microbial features were identified. A random forest classifier built on metagenomic and metabolic markers successfully distinguished IBS cases from controls (area under the curve, 0.93). Leveraging multiple lines of evidence, intestinal microbes, genes/pathways, and metabolites were associated with IBS, and these features were capable of distinguishing children with IBS from healthy children. These multi-omics features, and their links to childhood IBS coupled with nutritional interventions, may lead to new microbiome-guided diagnostic and therapeutic strategies.

Considering the Supraorganism: Harnessing the Gut Microbiome for Cancer Prevention, Detection, and Treatment.

Although causal relationships between specific microbes and cancer are well recognized, a growing body of evidence suggests that the broader community of gut microbes may influence cancer risk, provide diagnostic insight, shape clinical course, and impact treatment success in the gastrointestinal tract and beyond. In this issue, Murphy et al. reviewed evidence for the role of the microbiome in clinical oncology. Given that the gut microbiome may be modified by treatments ranging from diet and live biotherapeutics to microbial transplantation, microbiome-directed therapies hold tremendous promise for personalized therapies and improved treatment outcomes.

Biochemical but not compositional recovery of skin mucosal microbiome communities after disruption.

BACKGROUND: The microbiomes of animals are complex communities that strongly affect the health of the hosts. Microbiomes on mucosal surfaces have the highest densities and most extensive biochemical exchanges with the hosts. Although antibiotics are potent tools to manage infections, they can disrupt the normal microbiota, causing numerous side effects. MATERIALS AND METHODS: Taking a community ecology approach, mucosal microbiome community responses to five disruptive conditions (two broad-spectrum antibiotics, a biocide, elevated temperature, and rinsing) were analyzed. Skin of the fish Gambusia affinis was the mucosal model. Microbiome recovery was measured by culturable counts, community biochemical profiles, genetic fingerprinting, and community 16S gene sequencing (rinsing condition only). RESULTS: Following all disruptions, the total counts rose and then returned to the pre-treatment (PT) level. This overgrowth was confirmed via direct staining and community metabolic activity measurements. After rinsing, diversity decreased and one taxon dominated (family Aeromonadaceae) temporarily, the findings similar to numerous other studies with antibiotics. While the community did not return to the PT taxonomic composition, the biochemical profile did. CONCLUSION: This suggests that the biochemical pathways in a community are important during recovery, and a return to the original composition is not required to restore original function.

Airway Microbiome and Development of Bronchopulmonary Dysplasia in Preterm Infants: A Systematic Review.

OBJECTIVES: To summarize evidence regarding microbial dysbiosis of the airway associated with bronchopulmonary dysplasia (BPD) and to explore heterogeneity among studies. STUDY DESIGN: We included studies that evaluated the airway microbiome in preterm infants who developed BPD using culture-independent molecular techniques and reported alpha- and beta-diversity metrics and microbial profiles. RESULTS: The 6 included studies had substantial clinical and methodological heterogeneity. Most studies reported the presence of an airway microbiome early after birth and an evolution in the first weeks of life with increasing bacterial loads. The early airway microbiome was dominated by Staphylococcus and Ureaplasma spp. Two studies reported differences in alpha- and beta- diversity indices in preterm infants with BPD compared with those who did not develop BPD. Increased microbial community turnover, changes in the relative abundance of Proteobacteria and Firmicutes, and decreased Lactobacilli were reported with BPD progression. Most included infants were born by cesarean delivery, and a majority were exposed to postnatal antibiotics. No data regarding feeding human milk or correlations with the development of gut microbiota (gut-lung axis) were available. CONCLUSIONS: Microbial dysbiosis may be associated with BPD progression and severity, and further study of microbiome optimization in preterm infants at risk for BPD is warranted.

Re-purposing 16S rRNA gene sequence data from within case paired tumor biopsy and tumor-adjacent biopsy or fecal samples to identify microbial markers for colorectal cancer.

Microbes colonizing colorectal cancer (CRC) tumors have the potential to affect disease, and vice-versa. The manner in which they differ from microbes in physically adjacent tissue or stool within the case in terms of both, taxonomy and biological activity remains unclear. In this study, we systematically analyzed previously published 16S rRNA sequence data from CRC patients with matched tumor:tumor-adjacent biopsies (n = 294 pairs, n = 588 biospecimens) and matched tumor biopsy:fecal pairs (n = 42 pairs, n = 84 biospecimens). Procrustes analyses, random effects regression, random forest (RF) modeling, and inferred functional pathway analyses were conducted to assess community similarity and microbial diversity across heterogeneous patient groups and studies. Our results corroborate previously reported association of increased Fusobacterium with tumor biopsies. Parvimonas and Streptococcus abundances were also elevated while Faecalibacterium and Ruminococcaceae abundances decreased in tumors relative to tumor-adjacent biopsies and stool samples from the same case. With the exception of these limited taxa, the majority of findings from individual studies were not confirmed by other 16S rRNA gene-based datasets. RF models comparing tumor and tumor-adjacent specimens yielded an area under curve (AUC) of 64.3%, and models of tumor biopsies versus fecal specimens exhibited an AUC of 82.5%. Although some taxa were shared between fecal and tumor samples, their relative abundances varied substantially. Inferred functional analysis identified potential differences in branched amino acid and lipid metabolism. Microbial markers that reliably occur in tumor tissue can have implications for microbiome based and microbiome targeting therapeutics for CRC.

Leveraging sequence-based faecal microbial community survey data to identify a composite biomarker for colorectal cancer.

OBJECTIVE: Colorectal cancer (CRC) is the second leading cause of cancer-associated mortality in the USA. The faecal microbiome may provide non-invasive biomarkers of CRC and indicate transition in the adenoma-carcinoma sequence. Re-analysing raw sequence and metadata from several studies uniformly, we sought to identify a composite and generalisable microbial marker for CRC. DESIGN: Raw 16S rRNA gene sequence data sets from nine studies were processed with two pipelines, (1) QIIME closed reference (QIIME-CR) or (2) a strain-specific method herein termed SS-UP (Strain Select, UPARSE bioinformatics pipeline). A total of 509 samples (79 colorectal adenoma, 195 CRC and 235 controls) were analysed. Differential abundance, meta-analysis random effects regression and machine learning analyses were carried out to determine the consistency and diagnostic capabilities of potential microbial biomarkers. RESULTS: Definitive taxa, including Parvimonas micra ATCC 33270, Streptococcus anginosus and yet-to-be-cultured members of Proteobacteria, were frequently and significantly increased in stools from patients with CRC compared with controls across studies and had high discriminatory capacity in diagnostic classification. Microbiome-based CRC versus control classification produced an area under receiver operator characteristic (AUROC) curve of 76.6% in QIIME-CR and 80.3% in SS-UP. Combining clinical and microbiome markers gave a diagnostic AUROC of 83.3% for QIIME-CR and 91.3% for SS-UP. CONCLUSIONS: Despite technological differences across studies and methods, key microbial markers emerged as important in classifying CRC cases and such could be used in a universal diagnostic for the disease. The choice of bioinformatics pipeline influenced accuracy of classification. Strain-resolved microbial markers might prove crucial in providing a microbial diagnostic for CRC.

Characterization of the Stool Microbiome in Hispanic Preschool Children by Weight Status and Time.

BACKGROUND: Variations in gut microbiota composition and diversity have been associated with childhood adiposity, although most studies describing this have been cross-sectional in nature. Our objective was to evaluate associations between body weight and the gut microbiota over time in obese preschool-age children. METHODS: Obese, preschool-age, Hispanic children provided stool samples at baseline and following a 6-month behavioral intervention. Normal-weight (NW) children also provided stool samples. Stool microbial community composition was characterized using 16S rRNA gene sequencing. Estimates of within-sample diversity were calculated on operational taxonomic unit (OTU) count data, and the Firmicutes:Bacteroidetes (F:B) ratio was determined on per-sample basis. Estimates of between-sample diversity were generated using the weighted Unifrac metric, differential abundances were evaluated using Wilcoxon rank-sum tests, and associations of microbiome features with clinical data were quantified using Spearman rank correlations. RESULTS: For the 30 obese children sampled preintervention and postintervention, a decrease in body mass index (BMI) z-score from 2.55 to 2.34 (p = 0.004, paired t-test) was observed. Bacteroides massiliensis was significantly enriched in obese children, while B. plebius was significantly enriched in NW controls. We identified significant correlations between multiple Bacteroides-like OTUs and BMI z-score, but neither F:B ratios nor OTU-level abundances were altered in conjunction with weight change in the obese children. Rather, highly individualized OTU-level responses were observed. CONCLUSIONS: Although differences exist between the gut microbiota of obese and NW children, we detected highly individualized responses of the gut microbiota of obese children over time and following weight loss.