Evaluation of an Adapted Semi-Automated DNA Extraction for Human Salivary Shotgun Metagenomics.

Recent attention has highlighted the importance of oral microbiota in human health and disease, e.g., in Parkinson's disease, notably using shotgun metagenomics. One key aspect for efficient shotgun metagenomic analysis relies on optimal microbial sampling and DNA extraction, generally implementing commercial solutions developed to improve sample collection and preservation, and provide high DNA quality and quantity for downstream analysis. As metagenomic studies are today performed on a large number of samples, the next evolution to increase study throughput is with DNA extraction automation. In this study, we proposed a semi-automated DNA extraction protocol for human salivary samples collected with a commercial kit, and compared the outcomes with the DNA extraction recommended by the manufacturer. While similar DNA yields were observed between the protocols, our semi-automated DNA protocol generated significantly higher DNA fragment sizes. Moreover, we showed that the oral microbiome composition was equivalent between DNA extraction methods, even at the species level. This study demonstrates that our semi-automated protocol is suitable for shotgun metagenomic analysis, while allowing for improved sample treatment logistics with reduced technical variability and without compromising the structure of the oral microbiome.

Evidence of a causal and modifiable relationship between kidney function and circulating trimethylamine N-oxide.

The host-microbiota co-metabolite trimethylamine N-oxide (TMAO) is linked to increased cardiovascular risk but how its circulating levels are regulated remains unclear. We applied "explainable" machine learning, univariate, multivariate and mediation analyses of fasting plasma TMAO concentration and a multitude of phenotypes in 1,741 adult Europeans of the MetaCardis study. Here we show that next to age, kidney function is the primary variable predicting circulating TMAO, with microbiota composition and diet playing minor, albeit significant, roles. Mediation analysis suggests a causal relationship between TMAO and kidney function that we corroborate in preclinical models where TMAO exposure increases kidney scarring. Consistent with our findings, patients receiving glucose-lowering drugs with reno-protective properties have significantly lower circulating TMAO when compared to propensity-score matched control individuals. Our analyses uncover a bidirectional relationship between kidney function and TMAO that can potentially be modified by reno-protective anti-diabetic drugs and suggest a clinically actionable intervention for decreasing TMAO-associated excess cardiovascular risk.

The gut microbiota contributes to the pathogenesis of anorexia nervosa in humans and mice.

Anorexia nervosa (AN) is an eating disorder with a high mortality. About 95% of cases are women and it has a population prevalence of about 1%, but evidence-based treatment is lacking. The pathogenesis of AN probably involves genetics and various environmental factors, and an altered gut microbiota has been observed in individuals with AN using amplicon sequencing and relatively small cohorts. Here we investigated whether a disrupted gut microbiota contributes to AN pathogenesis. Shotgun metagenomics and metabolomics were performed on faecal and serum samples, respectively, from a cohort of 77 females with AN and 70 healthy females. Multiple bacterial taxa (for example, Clostridium species) were altered in AN and correlated with estimates of eating behaviour and mental health. The gut virome was also altered in AN including a reduction in viral-bacterial interactions. Bacterial functional modules associated with the degradation of neurotransmitters were enriched in AN and various structural variants in bacteria were linked to metabolic features of AN. Serum metabolomics revealed an increase in metabolites associated with reduced food intake (for example, indole-3-propionic acid). Causal inference analyses implied that serum bacterial metabolites are potentially mediating the impact of an altered gut microbiota on AN behaviour. Further, we performed faecal microbiota transplantation from AN cases to germ-free mice under energy-restricted feeding to mirror AN eating behaviour. We found that the reduced weight gain and induced hypothalamic and adipose tissue gene expression were related to aberrant energy metabolism and eating behaviour. Our 'omics' and mechanistic studies imply that a disruptive gut microbiome may contribute to AN pathogenesis.

In Vitro Modelling of Oral Microbial Invasion in the Human Colon.

Recent advances in the human microbiome characterization have revealed significant oral microbial detection in stools of dysbiotic patients. However, little is known about the potential interactions of these invasive oral microorganisms with commensal intestinal microbiota and the host. In this proof-of-concept study, we proposed a new model of oral-to-gut invasion by the combined use of an in vitro model simulating both the physicochemical and microbial (lumen- and mucus-associated microbes) parameters of the human colon (M-ARCOL), a salivary enrichment protocol, and whole-metagenome shotgun sequencing. Oral invasion of the intestinal microbiota was simulated by injection of enriched saliva in the in vitro colon model inoculated with a fecal sample from the same healthy adult donor. The mucosal compartment of M-ARCOL was able to retain the highest species richness levels over time, while species richness levels decreased in the luminal compartment. This study also showed that oral microorganisms preferably colonized the mucosal microenvironment, suggesting potential oral-to-intestinal mucosal competitions. This new model of oral-to-gut invasion can provide useful mechanistic insights into the role of oral microbiome in various disease processes. IMPORTANCE Here, we propose a new model of oral-to-gut invasion by the combined use of an in vitro model simulating both the physicochemical and microbial (lumen- and mucus-associated microbes) parameters of the human colon (M-ARCOL), a salivary enrichment protocol, and whole-metagenome shotgun sequencing. Our study revealed the importance of integrating the mucus compartment, which retained higher microbial richness during fermentation, showed the preference of oral microbial invaders for the mucosal resources, and indicated potential oral-to-intestinal mucosal competitions. It also underlined promising opportunities to further understand mechanisms of oral invasion into the human gut microbiome, define microbe-microbe and mucus-microbe interactions in a compartmentalized fashion, and help to better characterize the potential of oral microbial invasion and their persistence in the gut.

Metabolomic and microbiome profiling reveals personalized risk factors for coronary artery disease.

Complex diseases, such as coronary artery disease (CAD), are often multifactorial, caused by multiple underlying pathological mechanisms. Here, to study the multifactorial nature of CAD, we performed comprehensive clinical and multi-omic profiling, including serum metabolomics and gut microbiome data, for 199 patients with acute coronary syndrome (ACS) recruited from two major Israeli hospitals, and validated these results in a geographically distinct cohort. ACS patients had distinct serum metabolome and gut microbial signatures as compared with control individuals, and were depleted in a previously unknown bacterial species of the Clostridiaceae family. This bacterial species was associated with levels of multiple circulating metabolites in control individuals, several of which have previously been linked to an increased risk of CAD. Metabolic deviations in ACS patients were found to be person specific with respect to their potential genetic or environmental origin, and to correlate with clinical parameters and cardiovascular outcomes. Moreover, metabolic aberrations in ACS patients linked to microbiome and diet were also observed to a lesser extent in control individuals with metabolic impairment, suggesting the involvement of these aberrations in earlier dysmetabolic phases preceding clinically overt CAD. Finally, a metabolomics-based model of body mass index (BMI) trained on the non-ACS cohort predicted higher-than-actual BMI when applied to ACS patients, and the excess BMI predictions independently correlated with both diabetes mellitus (DM) and CAD severity, as defined by the number of vessels involved. These results highlight the utility of the serum metabolome in understanding the basis of risk-factor heterogeneity in CAD.

Metagenomic and metabolomic remodeling in nonagenarians and centenarians and its association with genetic and socioeconomic factors.

A better understanding of the biological and environmental variables that contribute to exceptional longevity has the potential to inform the treatment of geriatric diseases and help achieve healthy aging. Here, we compared the gut microbiome and blood metabolome of extremely long-lived individuals (94-105 years old) to that of their children (50-79 years old) in 116 Han Chinese families. We found extensive metagenomic and metabolomic remodeling in advanced age and observed a generational divergence in the correlations with socioeconomic factors. An analysis of quantitative trait loci revealed that genetic associations with metagenomic and metabolomic features were largely generation-specific, but we also found 131 plasma metabolic quantitative trait loci associations that were cross-generational with the genetic variants concentrated in six loci. These included associations between FADS1/2 and arachidonate, PTPA and succinylcarnitine and FLVCR1 and choline. Our characterization of the extensive metagenomic and metabolomic remodeling that occurs in people reaching extreme ages may offer new targets for aging-related interventions.

Combinatorial, additive and dose-dependent drug-microbiome associations.

During the transition from a healthy state to cardiometabolic disease, patients become heavily medicated, which leads to an increasingly aberrant gut microbiome and serum metabolome, and complicates biomarker discovery1-5. Here, through integrated multi-omics analyses of 2,173 European residents from the MetaCardis cohort, we show that the explanatory power of drugs for the variability in both host and gut microbiome features exceeds that of disease. We quantify inferred effects of single medications, their combinations as well as additive effects, and show that the latter shift the metabolome and microbiome towards a healthier state, exemplified in synergistic reduction in serum atherogenic lipoproteins by statins combined with aspirin, or enrichment of intestinal Roseburia by diuretic agents combined with beta-blockers. Several antibiotics exhibit a quantitative relationship between the number of courses prescribed and progression towards a microbiome state that is associated with the severity of cardiometabolic disease. We also report a relationship between cardiometabolic drug dosage, improvement in clinical markers and microbiome composition, supporting direct drug effects. Taken together, our computational framework and resulting resources enable the disentanglement of the effects of drugs and disease on host and microbiome features in multimedicated individuals. Furthermore, the robust signatures identified using our framework provide new hypotheses for drug-host-microbiome interactions in cardiometabolic disease.

Conjugated C-6 hydroxylated bile acids in serum relate to human metabolic health and gut Clostridia species.

Knowledge about in vivo effects of human circulating C-6 hydroxylated bile acids (BAs), also called muricholic acids, is sparse. It is unsettled if the gut microbiome might contribute to their biosynthesis. Here, we measured a range of serum BAs and related them to markers of human metabolic health and the gut microbiome. We examined 283 non-obese and obese Danish adults from the MetaHit study. Fasting concentrations of serum BAs were quantified using ultra-performance liquid chromatography-tandem mass-spectrometry. The gut microbiome was characterized with shotgun metagenomic sequencing and genome-scale metabolic modeling. We find that tauro- and glycohyocholic acid correlated inversely with body mass index (P = 4.1e-03, P = 1.9e-05, respectively), waist circumference (P = 0.017, P = 1.1e-04, respectively), body fat percentage (P = 2.5e-03, P = 2.3e-06, respectively), insulin resistance (P = 0.051, P = 4.6e-4, respectively), fasting concentrations of triglycerides (P = 0.06, P = 9.2e-4, respectively) and leptin (P = 0.067, P = 9.2e-4). Tauro- and glycohyocholic acids, and tauro-a-muricholic acid were directly linked with a distinct gut microbial community primarily composed of Clostridia species (P = 0.037, P = 0.013, P = 0.027, respectively). We conclude that serum conjugated C-6-hydroxylated BAs associate with measures of human metabolic health and gut communities of Clostridia species. The findings merit preclinical interventions and human feasibility studies to explore the therapeutic potential of these BAs in obesity and type 2 diabetes.

Imidazole propionate is increased in diabetes and associated with dietary patterns and altered microbial ecology.

Microbiota-host-diet interactions contribute to the development of metabolic diseases. Imidazole propionate is a novel microbially produced metabolite from histidine, which impairs glucose metabolism. Here, we show that subjects with prediabetes and diabetes in the MetaCardis cohort from three European countries have elevated serum imidazole propionate levels. Furthermore, imidazole propionate levels were increased in subjects with low bacterial gene richness and Bacteroides 2 enterotype, which have previously been associated with obesity. The Bacteroides 2 enterotype was also associated with increased abundance of the genes involved in imidazole propionate biosynthesis from dietary histidine. Since patients and controls did not differ in their histidine dietary intake, the elevated levels of imidazole propionate in type 2 diabetes likely reflects altered microbial metabolism of histidine, rather than histidine intake per se. Thus the microbiota may contribute to type 2 diabetes by generating imidazole propionate that can modulate host inflammation and metabolism.

Impact of laparoscopic Roux-en-Y gastric bypass and sleeve gastrectomy on gut microbiota: a metagenomic comparative analysis.

BACKGROUND: Bariatric surgery is an effective therapeutic procedure for morbidly obese patients. The 2 most common interventions are sleeve gastrectomy (SG) and laparoscopic Roux-en-Y gastric bypass (LRYGB). OBJECTIVES: The aim of this study was to compare microbiome long-term microbiome after SG and LRYGB surgery in obese patients. SETTING: University Hospital, France; University Hospital, United States; and University Hospital, Switzerland. METHODS: Eighty-nine and 108 patients who underwent SG and LRYGB, respectively, were recruited. Stools were collected before and 6 months after surgery. Microbial DNA was analyzed with shotgun metagenomic sequencing (SOLiD 5500 xl Wildfire). MSPminer, a novel innovative tool to characterize new in silico biological entities, was used to identify 715 Metagenomic Species Pan-genome. One hundred forty-eight functional modules were analyzed using GOmixer and KEGG database. RESULTS: Both interventions resulted in a similar increase of Shannon's diversity index and gene richness of gut microbiota, in parallel with weight loss, but the changes of microbial composition were different. LRYGB led to higher relative abundance of aero-tolerant bacteria, such as Escherichia coli and buccal species, such as Streptococcus and Veillonella spp. In contrast, anaerobes, such as Clostridium, were more abundant after SG, suggesting better conservation of anaerobic conditions in the gut. Enrichment of Akkermansia muciniphila was also observed after both surgeries. Function-level changes included higher potential for bacterial use of supplements, such as vitamin B12, B1, and iron upon LRYGB. CONCLUSION: Microbiota changes after bariatric surgery depend on the nature of the intervention. LRYGB induces greater taxonomic and functional changes in gut microbiota than SG. Possible long-term health consequences of these alterations remain to be established.

Commensal Hafnia alvei strain reduces food intake and fat mass in obese mice-a new potential probiotic for appetite and body weight management.

BACKGROUND/OBJECTIVES: Based on the recent identification of E.coli heat shock protein ClpB as a mimetic of the anorexigenic α-melanocyte stimulating hormone (α-MSH), the objective of this study was to preclinically validate Hafnia alvei, a ClpB-producing commensal bacterium as a potential probiotic for appetite and body weight management in overweight and obesity. METHODS: The involvement of enterobacterial ClpB in the putative anti-obesity effects was studied using ClpB-deficient E.coli. A food-grade H. alvei HA4597 strain synthetizing the ClpB protein with an α-MSH-like motif was selected as a candidate probiotic to be tested in ob/ob and high-fat diet (HFD)-fed obese and overweight mice. The relevance of the enterobacterial ClpB gene to human obesity was studied by in silico analysis of fecal metagenomes of 569 healthy individuals from the "MetaHIT" database. RESULTS: Chronic per os administration of native but not ClpB-deficient E.coli strain reduced body weight gain (p < 0.05) and daily meal frequency (p < 0.001) in ob/ob mice. Oral gavage of H.alvei for 18 and 46 days in ob/ob and HFD-fed obese mice, respectively, was well tolerated, reduced body weight gain and fat mass in both obesity models (p < 0.05) and decreased food intake in hyperphagic ob/ob mice (p < 0.001). Elevated fat tissue levels of phosphorylated hormone-sensitive lipase were detected in H.alvei -treated ob/ob mice (p < 0.01). Enterobacterial ClpB gene richness was lower in obese vs. non-obese humans (p < 0.0001) and correlated negatively with BMI in genera of Enterobacter, Klebsiella and Hafnia. CONCLUSIONS: H.alvei HA4597 strain reduces food intake, body weight and fat mass gain in hyperphagic and obese mice. These data combined with low enterobacterial ClpB gene abundance in the microbiota of obese humans provide the rationale for using H.alvei as a probiotic for appetite and body weight management in overweight and obesity.

Effects of Low FODMAP Diet on Symptoms, Fecal Microbiome, and Markers of Inflammation in Patients With Quiescent Inflammatory Bowel Disease in a Randomized Trial.

BACKGROUND & AIMS: There is limited evidence that a diet low in fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) reduces gut symptoms in quiescent inflammatory bowel disease (IBD). We performed a randomized, controlled trial to investigate the effects of a low FODMAP diet on persistent gut symptoms, the intestinal microbiome, and circulating markers of inflammation in patients with quiescent IBD. METHODS: We performed a single-blind trial of 52 patients with quiescent Crohn's disease or ulcerative colitis and persistent gut symptoms at 2 large gastroenterology clinics in the United Kingdom. Patients were randomly assigned to groups that followed a diet low in FODMAPs (n = 27) or a control diet (n = 25), with dietary advice, for 4 weeks. Gut symptoms and health-related quality of life were measured using validated questionnaires. Stool and blood samples were collected at baseline and end of trial. We assessed fecal microbiome composition and function using shotgun metagenomic sequencing and phenotypes of T cells in blood using flow cytometry. RESULTS: A higher proportion of patients reported adequate relief of gut symptoms following the low FODMAP diet (14/27, 52%) than the control diet (4/25, 16%, P=.007). Patients had a greater reduction in irritable bowel syndrome severity scores following the low FODMAP diet (mean reduction of 67; standard error, 78) than the control diet (mean reduction of 34; standard error, 50), although this difference was not statistically significant (P = .075). Following the low FODMAP diet, patients had higher health-related quality of life scores (81.9 ± 1.2) than patients on the control diet (78.3 ± 1.2, P = .042). A targeted analysis revealed that in stool samples collected at the end of the study period, patients on the low FODMAP diet had significantly lower abundance of Bifidobacterium adolescentis, Bifidobacterium longum, and Faecalibacterium prausnitzii than patients on control diet. However, microbiome diversity and markers of inflammation did not differ significantly between groups. CONCLUSIONS: In a trial of the low FODMAP diet vs a control diet in patients with quiescent IBD, we found no significant difference after 4 weeks in change in irritable bowel syndrome severity scores, but significant improvements in specific symptom scores and numbers reporting adequate symptom relief. The low FODMAP diet reduced fecal abundance of microbes believed to regulate the immune response, compared with the control diet, but had no significant effect on markers of inflammation. We conclude that a 4-week diet low in FODMAPs is safe and effective for managing persistent gut symptoms in patients with quiescent IBD. www.isrctn.com no.: ISRCTN17061468.

Prediction of the intestinal resistome by a three-dimensional structure-based method.

The intestinal microbiota is considered to be a major reservoir of antibiotic resistance determinants (ARDs) that could potentially be transferred to bacterial pathogens via mobile genetic elements. Yet, this assumption is poorly supported by empirical evidence due to the distant homologies between known ARDs (mostly from culturable bacteria) and ARDs from the intestinal microbiota. Consequently, an accurate census of intestinal ARDs (that is, the intestinal resistome) has not yet been fully determined. For this purpose, we developed and validated an annotation method (called pairwise comparative modelling) on the basis of a three-dimensional structure (homology comparative modelling), leading to the prediction of 6,095 ARDs in a catalogue of 3.9 million proteins from the human intestinal microbiota. We found that the majority of predicted ARDs (pdARDs) were distantly related to known ARDs (mean amino acid identity 29.8%) and found little evidence supporting their transfer between species. According to the composition of their resistome, we were able to cluster subjects from the MetaHIT cohort (n = 663) into six resistotypes that were connected to the previously described enterotypes. Finally, we found that the relative abundance of pdARDs was positively associated with gene richness, but not when subjects were exposed to antibiotics. Altogether, our results indicate that the majority of intestinal microbiota ARDs can be considered intrinsic to the dominant commensal microbiota and that these genes are rarely shared with bacterial pathogens.

MSPminer: abundance-based reconstitution of microbial pan-genomes from shotgun metagenomic data.

MOTIVATION: Analysis toolkits for shotgun metagenomic data achieve strain-level characterization of complex microbial communities by capturing intra-species gene content variation. Yet, these tools are hampered by the extent of reference genomes that are far from covering all microbial variability, as many species are still not sequenced or have only few strains available. Binning co-abundant genes obtained from de novo assembly is a powerful reference-free technique to discover and reconstitute gene repertoire of microbial species. While current methods accurately identify species core parts, they miss many accessory genes or split them into small gene groups that remain unassociated to core clusters. RESULTS: We introduce MSPminer, a computationally efficient software tool that reconstitutes Metagenomic Species Pan-genomes (MSPs) by binning co-abundant genes across metagenomic samples. MSPminer relies on a new robust measure of proportionality coupled with an empirical classifier to group and distinguish not only species core genes but accessory genes also. Applied to a large scale metagenomic dataset, MSPminer successfully delineates in a few hours the gene repertoires of 1661 microbial species with similar specificity and higher sensitivity than existing tools. The taxonomic annotation of MSPs reveals microorganisms hitherto unknown and brings coherence in the nomenclature of the species of the human gut microbiota. The provided MSPs can be readily used for taxonomic profiling and biomarkers discovery in human gut metagenomic samples. In addition, MSPminer can be applied on gene count tables from other ecosystems to perform similar analyses. AVAILABILITY AND IMPLEMENTATION: The binary is freely available for non-commercial users at www.enterome.com/downloads. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A computational framework to integrate high-throughput '-omics' datasets for the identification of potential mechanistic links.

We recently presented a three-pronged association study that integrated human intestinal microbiome data derived from shotgun-based sequencing with untargeted serum metabolome data and measures of host physiology. Metabolome and microbiome data are high dimensional, posing a major challenge for data integration. Here, we present a step-by-step computational protocol that details and discusses the dimensionality-reduction techniques used and methods for subsequent integration and interpretation of such heterogeneous types of data. Dimensionality reduction was achieved through a combination of data normalization approaches, binning of co-abundant genes and metabolites, and integration of prior biological knowledge. The use of prior knowledge to overcome functional redundancy across microbiome species is one central advance of our method over available alternative approaches. Applying this framework, other investigators can integrate various '-omics' readouts with variables of host physiology or any other phenotype of interest (e.g., connecting host and microbiome readouts to disease severity or treatment outcome in a clinical cohort) in a three-pronged association analysis to identify potential mechanistic links to be tested in experimental settings. Although we originally developed the framework for a human metabolome-microbiome study, it is generalizable to other organisms and environmental metagenomes, as well as to studies including other -omics domains such as transcriptomics and proteomics. The provided R code runs in ~1 h on a standard PC.

Microbial ecology perturbation in human IgA deficiency.

Paradoxically, loss of immunoglobulin A (IgA), one of the most abundant antibodies, does not irrevocably lead to severe infections in humans but rather is associated with relatively mild respiratory infections, atopy, and autoimmunity. IgA might therefore also play covert roles, not uniquely associated with control of pathogens. We show that human IgA deficiency is not associated with massive quantitative perturbations of gut microbial ecology. Metagenomic analysis highlights an expected pathobiont expansion but a less expected depletion in some typically beneficial symbionts. Gut colonization by species usually present in the oropharynx is also reminiscent of spatial microbiota disorganization. IgM only partially rescues IgA deficiency because not all typical IgA targets are efficiently bound by IgM in the intestinal lumen. Together, IgA appears to play a nonredundant role at the forefront of the immune/microbial interface, away from the intestinal barrier, ranging from pathobiont control and regulation of systemic inflammation to preservation of commensal diversity and community networks.

Publisher Correction: Enterotypes in the landscape of gut microbial community composition.

In the version of this Perspective originally published, the first and last name of co-author Manimozhiyan Arumugam were switched. This has now been corrected in all versions of the Perspective.