Longitudinal Multi-omics Reveals Subset-Specific Mechanisms Underlying Irritable Bowel Syndrome.

The gut microbiome has been implicated in multiple human chronic gastrointestinal (GI) disorders. Determining its mechanistic role in disease has been difficult due to apparent disconnects between animal and human studies and lack of an integrated multi-omics view of disease-specific physiological changes. We integrated longitudinal multi-omics data from the gut microbiome, metabolome, host epigenome, and transcriptome in the context of irritable bowel syndrome (IBS) host physiology. We identified IBS subtype-specific and symptom-related variation in microbial composition and function. A subset of identified changes in microbial metabolites correspond to host physiological mechanisms that are relevant to IBS. By integrating multiple data layers, we identified purine metabolism as a novel host-microbial metabolic pathway in IBS with translational potential. Our study highlights the importance of longitudinal sampling and integrating complementary multi-omics data to identify functional mechanisms that can serve as therapeutic targets in a comprehensive treatment strategy for chronic GI diseases. VIDEO ABSTRACT.

US Immigration Westernizes the Human Gut Microbiome.

Many US immigrant populations develop metabolic diseases post immigration, but the causes are not well understood. Although the microbiome plays a role in metabolic disease, there have been no studies measuring the effects of US immigration on the gut microbiome. We collected stool, dietary recalls, and anthropometrics from 514 Hmong and Karen individuals living in Thailand and the United States, including first- and second-generation immigrants and 19 Karen individuals sampled before and after immigration, as well as from 36 US-born European American individuals. Using 16S and deep shotgun metagenomic DNA sequencing, we found that migration from a non-Western country to the United States is associated with immediate loss of gut microbiome diversity and function in which US-associated strains and functions displace native strains and functions. These effects increase with duration of US residence and are compounded by obesity and across generations.

Advancing the microbiome research community.

The human microbiome has become a recognized factor in promoting and maintaining health. We outline opportunities in interdisciplinary research, analytical rigor, standardization, and policy development for this relatively new and rapidly developing field. Advances in these aspects of the research community may in turn advance our understanding of human microbiome biology.

LMdist: Local Manifold distance accurately measures beta diversity in ecological gradients.

MOTIVATION: Differentiating ecosystems poses a complex, high-dimensional problem constrained by capturing relevant variation across species profiles. Researchers use pairwise distances and subsequent dimensionality reduction to highlight variation in a few dimensions. Despite popularity in analysis of ecological data, these low-dimensional visualizations can contain geometric abnormalities such as "arch" and "horseshoe" effects, potentially obscuring the impact of environmental gradients. These abnormalities appear in ordination but are in fact a product of oversaturated large pairwise distances. RESULTS: We present Local Manifold distance (LMdist), an unsupervised algorithm which adjusts pairwise beta diversity measures to better represent true ecological distances between samples. Beta diversity measures can have a bounded dynamic range in depicting long environmental gradients with high species turnover. Using a graph structure, LMdist projects pairwise distances onto a manifold and traverses the manifold surface to adjust pairwise distances at the upper end of the beta diversity measure's dynamic range. This allows for values beyond the range of the original measure. Not all datasets will have oversaturated pairwise distances, nor will capture variation that resembles a manifold, so LMdist adjusts only those pairwise values which may be undervalued in the presence of a sampled gradient. The adjusted distances serve as input for ordination and statistical testing. We demonstrate on real and simulated data that LMdist effectively recovers distances along known gradients and along complex manifolds such as the Swiss roll dataset. LMdist enables more powerful statistical tests for gradient effects and reveals variation orthogonal to the gradient. AVAILABILITY AND IMPLEMENTATION: Available on GitHub at https://github.com/knights-lab/LMdist.

Effects of Diet-Modulated Autologous Fecal Microbiota Transplantation on Weight Regain.

BACKGROUND & AIMS: We evaluated the efficacy and safety of diet-modulated autologous fecal microbiota transplantation (aFMT) for treatment of weight regain after the weight-loss phase. METHODS: In the DIRECT PLUS (Dietary Intervention Randomized Controlled Trial Polyphenols-Unprocessed) weight-loss trial (May 2017 through July 2018), abdominally obese or dyslipidemic participants in Israel were randomly assigned to healthy dietary guidelines, Mediterranean diet, and green-Mediterranean diet weight-loss groups. All groups received free gym membership and physical activity guidelines. Both isocaloric Mediterranean groups consumed 28 g/d walnuts (+440 mg/d polyphenols provided). The green-Mediterranean dieters also consumed green tea (3-4 cups/d) and a Wolffia globosa (Mankai strain, 100 g/d) green shake (+800 mg/d polyphenols provided). After 6 months (weight-loss phase), 90 eligible participants (mean age, 52 years; mean weight loss, 8.3 kg) provided a fecal sample that was processed into aFMT by frozen, opaque, and odorless capsules. The participants were then randomly assigned to groups that received 100 capsules containing their own fecal microbiota or placebo until month 14. The primary outcome was regain of the lost weight over the expected weight-regain phase (months 6-14). Secondary outcomes were gastrointestinal symptoms, waist circumference, glycemic status, and changes in the gut microbiome, as measured by metagenomic sequencing and 16s ribosomal RNA. We validated the results in a parallel in vivo study of mice specifically fed with Mankai compared with control chow diet. RESULTS: Of the 90 participants in the aFMT trial, 96% ingested at least 80 of 100 oral aFMT or placebo frozen capsules during the transplantation period. No aFMT-related adverse events or symptoms were observed. For the primary outcome, although no significant differences in weight regain were observed among the participants in the different lifestyle interventions during months 6-14 (aFMT, 30.4% vs placebo, 40.6%; P = .28), aFMT significantly attenuated weight regain in the green-Mediterranean group (aFMT, 17.1%, vs placebo, 50%; P = .02), but not in the dietary guidelines (P = .57) or Mediterranean diet (P = .64) groups (P for the interaction = .03). Accordingly, aFMT attenuated waist circumference gain (aFMT, 1.89 cm vs placebo, 5.05 cm; P = .01) and insulin rebound (aFMT, -1.46 ± 3.6 µIU/mL vs placebo, 1.64 ± 4.7 µIU/mL; P = .04) in the green-Mediterranean group but not in the dietary guidelines or Mediterranean diet (P for the interaction = .04 and .03, respectively). The green-Mediterranean diet was the only intervention to induce a significant change in microbiome composition during the weight-loss phase, and to prompt preservation of weight-loss-associated specific bacteria and microbial metabolic pathways (mainly microbial sugar transport) after the aFMT. In mice, Mankai-modulated aFMT in the weight-loss phase compared with control diet aFMT, significantly prevented weight regain and resulted in better glucose tolerance during a high-fat diet-induced regain phase (all, P < .05). CONCLUSIONS: Autologous FMT, collected during the weight-loss phase and administrated in the regain phase, might preserve weight loss and glycemic control, and is associated with specific microbiome signatures. A high-polyphenols, green plant-based or Mankai diet better optimizes the microbiome for an aFMT procedure. ClinicalTrials.gov number, NCT03020186.

Multi-Omics Analyses Show Disease, Diet, and Transcriptome Interactions With the Virome.

BACKGROUND & AIMS: The gut virome includes eukaryotic viruses and bacteriophages that can shape the gut bacterial community and elicit host responses. The virome can be implicated in diseases, such as irritable bowel syndrome (IBS), where gut bacteria play an important role in pathogenesis. We provide a comprehensive and longitudinal characterization of the virome, including DNA and RNA viruses and paired multi-omics data in a cohort of healthy subjects and patients with IBS. METHODS: We selected 2 consecutive stool samples per subject from a longitudinal study cohort and performed metagenomic sequencing on DNA and RNA viruses after enriching for viral-like particles. Viral sequence abundance was evaluated over time, as well as in the context of diet, bacterial composition and function, metabolite levels, colonic gene expression, host genetics, and IBS subsets. RESULTS: We found that the gut virome was temporally stable and correlated with the colonic transcriptome. We identified IBS-subset-specific changes in phage populations; Microviridae, Myoviridae, and Podoviridae species were elevated in diarrhea-predominant IBS, and other Microviridae and Myoviridae species were elevated in constipation-predominant IBS compared to healthy controls. We identified correlations between subsets of the virome and bacterial composition (unclassifiable "dark matter" and phages) and diet (eukaryotic viruses). CONCLUSIONS: We found that the gut virome is stable over time but varies among subsets of patients with IBS. It can be affected by diet and potentially influences host function via interactions with gut bacteria and/or altering host gene expression.

Placentas delivered by pre-pregnant obese women have reduced abundance and diversity in the microbiome.

Maternal pre-pregnancy obesity may have an impact on both maternal and fetal health. We examined the microbiome recovered from placentas in a multi-ethnic maternal pre-pregnant obesity cohort, through an optimized microbiome protocol to enrich low bacterial biomass samples. We found that the microbiomes recovered from the placentas of obese pre-pregnant mothers are less abundant and less diverse when compared to those from mothers of normal pre-pregnancy weight. Microbiome richness also decreases from the maternal side to the fetal side, demonstrating heterogeneity by geolocation within the placenta. In summary, our study shows that the microbiomes recovered from the placentas are associated with pre-pregnancy obesity. IMPORTANCE: Maternal pre-pregnancy obesity may have an impact on both maternal and fetal health. The placenta is an important organ at the interface of the mother and fetus, and supplies nutrients to the fetus. We report that the microbiomes enriched from the placentas of obese pre-pregnant mothers are less abundant and less diverse when compared to those from mothers of normal pre-pregnancy weight. More over, the microbiomes also vary by geolocation within the placenta.

Genetic effects on the commensal microbiota in inflammatory bowel disease patients.

Several bacteria in the gut microbiota have been shown to be associated with inflammatory bowel disease (IBD), and dozens of IBD genetic variants have been identified in genome-wide association studies. However, the role of the microbiota in the etiology of IBD in terms of host genetic susceptibility remains unclear. Here, we studied the association between four major genetic variants associated with an increased risk of IBD and bacterial taxa in up to 633 IBD cases. We performed systematic screening for associations, identifying and replicating associations between NOD2 variants and two taxa: the Roseburia genus and the Faecalibacterium prausnitzii species. By exploring the overall association patterns between genes and bacteria, we found that IBD risk alleles were significantly enriched for associations concordant with bacteria-IBD associations. To understand the significance of this pattern in terms of the study design and known effects from the literature, we used counterfactual principles to assess the fitness of a few parsimonious gene-bacteria-IBD causal models. Our analyses showed evidence that the disease risk of these genetic variants were likely to be partially mediated by the microbiome. We confirmed these results in extensive simulation studies and sensitivity analyses using the association between NOD2 and F. prausnitzii as a case study.

Interaction of bacterial metagenome and virome in patients with cirrhosis and hepatic encephalopathy.

OBJECTIVE: Altered bacterial composition is associated with disease progression in cirrhosis but the role of virome, especially phages, is unclear. DESIGN: Cross-sectional and pre/post rifaximin cohorts were enrolled. Cross-sectional: controls and cirrhotic outpatients (compensated, on lactulose (Cirr-L), on rifaximin (Cirr-LR)) were included and followed for 90-day hospitalisations. Pre/post: compensated cirrhotics underwent stool collection pre/post 8 weeks of rifaximin. Stool metagenomics for bacteria and phages and their correlation networks were analysed in controls versus cirrhosis, within cirrhotics, hospitalised/not and pre/post rifaximin. RESULTS: Cross-sectional: 40 controls and 163 cirrhotics (63 compensated, 43 Cirr-L, 57 Cirr-LR) were enrolled. Cirr-L/LR groups were similar on model for end-stage liver disease (MELD) score but Cirr-L developed greater hospitalisations versus Cirr-LR (56% vs 30%, p=0.008). Bacterial alpha/beta diversity worsened from controls through Cirr-LR. While phage alpha diversity was similar, beta diversity was different between groups. Autochthonous bacteria linked negatively, pathobionts linked positively with MELD but only modest phage-MELD correlations were seen. Phage-bacterial correlation network complexity was highest in controls, lowest in Cirr-L and increased in Cirr-LR. Microviridae and Faecalibacterium phages were linked with autochthonous bacteria in Cirr-LR, but not Cirr-L hospitalised patients had greater pathobionts, lower commensal bacteria and phages focused on Streptococcus, Lactococcus and Myoviridae. Pre/post: No changes in alpha/beta diversity of phages or bacteria were seen postrifaximin. Phage-bacterial linkages centred around urease-producing Streptococcus species collapsed postrifaximin. CONCLUSION: Unlike bacteria, faecal phages are sparsely linked with cirrhosis characteristics and 90-day outcomes. Phage and bacterial linkages centred on urease-producing, ammonia-generating Streptococcus species were affected by disease progression and rifaximin therapy and were altered in patients who experienced 90-day hospitalisations.

Functional Genomics of Host-Microbiome Interactions in Humans.

The human microbiome has been linked to various host phenotypes and has been implicated in many complex human diseases. Recent genome-wide association studies (GWASs) have used microbiome variation as a complex trait and have uncovered human genetic variants that are associated with the microbiome. Here we summarize results from these studies and illustrate potential regulatory mechanisms by which host genetic variation can interact with microbiome composition. We argue that, similar to human GWASs, it is important to use functional genomics techniques to gain a mechanistic understanding of causal host-microbiome interactions and their role in human disease. We highlight experimental, functional, and computational genomics methodologies for the study of the genomic basis of host-microbiome interactions and describe how these approaches can be utilized to explain how human genetic variation can modulate the effects of the microbiome on the host.

SHOGUN: a modular, accurate and scalable framework for microbiome quantification.

SUMMARY: The software pipeline SHOGUN profiles known taxonomic and gene abundances of short-read shotgun metagenomics sequencing data. The pipeline is scalable, modular and flexible. Data analysis and transformation steps can be run individually or together in an automated workflow. Users can easily create new reference databases and can select one of three DNA alignment tools, ranging from ultra-fast low-RAM k-mer-based database search to fully exhaustive gapped DNA alignment, to best fit their analysis needs and computational resources. The pipeline includes an implementation of a published method for taxonomy assignment disambiguation with empirical Bayesian redistribution. The software is installable via the conda resource management framework, has plugins for the QIIME2 and QIITA packages and produces both taxonomy and gene abundance profile tables with a single command, thus promoting convenient and reproducible metagenomics research. AVAILABILITY AND IMPLEMENTATION: https://github.com/knights-lab/SHOGUN.

Colorectal cancer mutational profiles correlate with defined microbial communities in the tumor microenvironment.

Variation in the gut microbiome has been linked to colorectal cancer (CRC), as well as to host genetic variation. However, we do not know whether, in addition to baseline host genetics, somatic mutational profiles in CRC tumors interact with the surrounding tumor microbiome, and if so, whether these changes can be used to understand microbe-host interactions with potential functional biological relevance. Here, we characterized the association between CRC microbial communities and tumor mutations using microbiome profiling and whole-exome sequencing in 44 pairs of tumors and matched normal tissues. We found statistically significant associations between loss-of-function mutations in tumor genes and shifts in the abundances of specific sets of bacterial taxa, suggestive of potential functional interaction. This correlation allows us to statistically predict interactions between loss-of-function tumor mutations in cancer-related genes and pathways, including MAPK and Wnt signaling, solely based on the composition of the microbiome. In conclusion, our study shows that CRC microbiomes are correlated with tumor mutational profiles, pointing towards possible mechanisms of molecular interaction.

US Immigration Is Associated With Rapid and Persistent Acquisition of Antibiotic Resistance Genes in the Gut.

Little is known about the effect of human migration on gut microbiome antibiotic resistance gene (ARG) carriage. Using deep shotgun stool metagenomics analysis, we found a rapid increase in gut microbiome ARG richness and abundance in women from 2 independent ethnic groups relocating from Thailand to the United States.

Characterization of Campylobacter jejuni, Campylobacter upsaliensis, and a novel Campylobacter sp. in a captive non-human primate zoological collection.

BACKGROUND: The aim of this study was to longitudinally investigate the prevalence and characterization of Campylobacter spp. from non-human primates primate (NHP) with a history of endemic diarrhea housed at Como Park Zoo. METHODS: Fecal samples from 33 symptom-free NHP belonging to eight different species were collected weekly for 9 weeks. Species-level characterization and phylogenetic analysis of isolates included biochemical testing and 16S rRNA sequencing. RESULTS: Campylobacter spp. were isolated from the feces of 42% (14/33) of the primates. Three Campylobacter spp. (C upsaliensis, C jejuni, and novel Campylobacter sp.) were identified from three NHP species. A possible positive host Campylobacter species-specificity was observed. However, no statistical association was observed between the isolation of Campylobacter spp. and age and sex of the animal. CONCLUSIONS: The study revealed the value of conducting repeated fecal sampling to establish the overall prevalence of Campylobacter in zoo-maintained NHP; it also importantly identifies a novel Campylobacter sp. isolated from white-faced saki monkeys.

Bacterial community structure and function distinguish gut sites in captive red-shanked doucs (Pygathrix nemaeus).

The mammalian order primates contains wide species diversity. Members of the subfamily Colobinae are unique amongst extant primates in that their gastrointestinal systems more closely resemble those of ruminants than other members of the primate order. In the growing literature surrounding nonhuman primate microbiomes, analysis of microbial communities has been limited to the hindgut, since few studies have captured data on other gut sites, including the foregut of colobine primates. In this study, we used the red-shanked douc (Pygathrix nemaeus) as a model for colobine primates to study the relationship between gastrointestinal bacterial community structure and gut site within and between subjects. We analyzed fecal and pregastric stomach content samples, representative of the hindgut and foregut respectively, using 16S recombinant DNA (rDNA) sequencing and identified microbiota using closed-reference operational taxonomic unit (OTU) picking against the GreenGenes database. Our results show divergent bacterial communities clearly distinguish the foregut and hindgut microbiomes. We found higher bacterial biodiversity and a higher Firmicutes:Bacteroides ratio in the hindgut as opposed to the foregut. These gut sites showed strong associations with bacterial function. Specifically, energy metabolism was upregulated in the hindgut, whereas detoxification was increased in the foregut. Our results suggest a red-shanked douc's foregut microbiome is no more concordant with its own hindgut than it is with any other red-shanked douc's hindgut microbiome, thus reinforcing the notion that the bacterial communities of the foregut and hindgut are distinctly unique. OPEN PRACTICES: This article has been awarded Open Materials and Open Data badges. All materials and data are publicly accessible via the IRIS Repository at https://www.iris-database.org/iris/app/home/detail?id=york:934328. Learn more about the Open Practices badges from the Center for Open Science: https://osf.io/tvyxz/wiki.

Captivity humanizes the primate microbiome.

The primate gastrointestinal tract is home to trillions of bacteria, whose composition is associated with numerous metabolic, autoimmune, and infectious human diseases. Although there is increasing evidence that modern and Westernized societies are associated with dramatic loss of natural human gut microbiome diversity, the causes and consequences of such loss are challenging to study. Here we use nonhuman primates (NHPs) as a model system for studying the effects of emigration and lifestyle disruption on the human gut microbiome. Using 16S rRNA gene sequencing in two model NHP species, we show that although different primate species have distinctive signature microbiota in the wild, in captivity they lose their native microbes and become colonized with Prevotella and Bacteroides, the dominant genera in the modern human gut microbiome. We confirm that captive individuals from eight other NHP species in a different zoo show the same pattern of convergence, and that semicaptive primates housed in a sanctuary represent an intermediate microbiome state between wild and captive. Using deep shotgun sequencing, chemical dietary analysis, and chloroplast relative abundance, we show that decreasing dietary fiber and plant content are associated with the captive primate microbiome. Finally, in a meta-analysis including published human data, we show that captivity has a parallel effect on the NHP gut microbiome to that of Westernization in humans. These results demonstrate that captivity and lifestyle disruption cause primates to lose native microbiota and converge along an axis toward the modern human microbiome.

Pretransplant Gut Colonization with Intrinsically Vancomycin-Resistant Enterococci (E. gallinarum and E. casseliflavus) and Outcomes of Allogeneic Hematopoietic Cell Transplantation.

Pretransplant gut colonization with intrinsically vancomycin-resistant enterococci (iVRE) (Enterococcus gallinarum and Enterococcus casseliflavus) is uncommon and with unknown clinical impact. In a matched-pairs analysis of patients with versus without iVRE colonization (n = 18 in each group), we demonstrated significantly higher 2-year overall survival (86% [95% confidence interval, 52% to 96%] versus 35% [95% confidence interval, 8% to 65]; P <.01) and lower nonrelapse mortality (P <.01) among colonized patients. Putative metabolomes differentiated iVRE from E. faecalis/faecium and may contribute to a healthier gut microbiome in iVRE-colonized patients.

Urinary microbiome associated with chronic allograft dysfunction in kidney transplant recipients.

BACKGROUND: We performed a study to identify differences in the urinary microbiome associated with chronic allograft dysfunction (CAD) and compared the urinary microbiome of male and female transplant recipients with CAD. METHODS: This case-control study enrolled 67 patients within the Deterioration of Kidney Allograft Function (DeKAF) Genomics cohort at two transplant centers. CAD was defined as a greater than 25% rise in serum creatinine relative to a 3 month post-transplant baseline. Urine samples from patients with and without CAD were analyzed using 16S V4 bacterial ribosomal DNA sequences. RESULTS: Corynebacterium was more prevalent in female and male patients with CAD compared to non-CAD female patients (P = 0.0005). A total 21 distinct Operational Taxonomic Unit (OTUs) were identified as significantly different when comparing CAD and non-CAD patients using Kruskal-Wallis (P < 0.01). A subset analysis of female patients with CAD compared to non-CAD females identified similar differentially abundant OTUs, including the genera Corynebacterium and Staphylococcus (Kruskal-Wallis; P = 0.01; P = 0.004, respectively). Male CAD vs female CAD analysis showed greater abundance of phylum Proteobacteria in males. CONCLUSION: There were differences in the urinary microbiome when comparing female and male CAD patients with their female non-CAD counterparts and these differences persisted in the subset analysis limited to female patients only.

Human gut microbiome viewed across age and geography.

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