Identification of an Intestinal Microbiota Signature Associated With Severity of Irritable Bowel Syndrome.

BACKGROUND & AIMS: We have limited knowledge about the association between the composition of the intestinal microbiota and clinical features of irritable bowel syndrome (IBS). We collected information on the fecal and mucosa-associated microbiota of patients with IBS and evaluated whether these were associated with symptoms. METHODS: We collected fecal and mucosal samples from adult patients who met the Rome III criteria for IBS at a secondary/tertiary care outpatient clinics in Sweden, as well as from healthy subjects. The exploratory set comprised 149 subjects (110 with IBS and 39 healthy subjects); 232 fecal samples and 59 mucosal biopsy samples were collected and analyzed by 16S ribosomal RNA targeted pyrosequencing. The validation set comprised 46 subjects (29 with IBS and 17 healthy subjects); 46 fecal samples, but no mucosal samples, were collected and analyzed. For each subject, we measured exhaled H2 and CH4, oro-anal transit time, and the severity of psychological and gastrointestinal symptoms. Fecal methanogens were measured by quantitative polymerase chain reaction. Numerical ecology analyses and a machine learning procedure were used to analyze the data. RESULTS: Fecal microbiota showed covariation with mucosal adherent microbiota. By using classic approaches, we found no differences in fecal microbiota abundance or composition between patients with IBS vs healthy patients. A machine learning procedure, a computational statistical technique, allowed us to reduce the 16S ribosomal RNA data complexity into a microbial signature for severe IBS, consisting of 90 bacterial operational taxonomic units. We confirmed the robustness of the intestinal microbial signature for severe IBS in the validation set. The signature was able to discriminate between patients with severe symptoms, patients with mild/moderate symptoms, and healthy subjects. By using this intestinal microbiota signature, we found IBS symptom severity to be associated negatively with microbial richness, exhaled CH4, presence of methanogens, and enterotypes enriched with Clostridiales or Prevotella species. This microbiota signature could not be explained by differences in diet or use of medications. CONCLUSIONS: In analyzing fecal and mucosal microbiota from patients with IBS and healthy individuals, we identified an intestinal microbiota profile that is associated with the severity of IBS symptoms. TRIAL REGISTRATION NUMBER: NCT01252550.

Brain Structure and Response to Emotional Stimuli as Related to Gut Microbial Profiles in Healthy Women.

OBJECTIVE: Brain-gut-microbiota interactions may play an important role in human health and behavior. Although rodent models have demonstrated effects of the gut microbiota on emotional, nociceptive, and social behaviors, there is little translational human evidence to date. In this study, we identify brain and behavioral characteristics of healthy women clustered by gut microbiota profiles. METHODS: Forty women supplied fecal samples for 16S rRNA profiling. Microbial clusters were identified using Partitioning Around Medoids. Functional magnetic resonance imaging was acquired. Microbiota-based group differences were analyzed in response to affective images. Structural and diffusion tensor imaging provided gray matter metrics (volume, cortical thickness, mean curvature, surface area) as well as fiber density between regions. A sparse Partial Least Square-Discrimination Analysis was applied to discriminate microbiota clusters using white and gray matter metrics. RESULTS: Two bacterial genus-based clusters were identified, one with greater Bacteroides abundance (n = 33) and one with greater Prevotella abundance (n = 7). The Prevotella group showed less hippocampal activity viewing negative valences images. White and gray matter imaging discriminated the two clusters, with accuracy of 66.7% and 87.2%, respectively. The Prevotella cluster was associated with differences in emotional, attentional, and sensory processing regions. For gray matter, the Bacteroides cluster showed greater prominence in the cerebellum, frontal regions, and the hippocampus. CONCLUSIONS: These results support the concept of brain-gut-microbiota interactions in healthy humans. Further examination of the interaction between gut microbes, brain, and affect in humans is needed to inform preclinical reports that microbial modulation may affect mood and behavior.

Lactulose Challenge Determines Visceral Sensitivity and Severity of Symptoms in Patients With Irritable Bowel Syndrome.

BACKGROUND & AIMS: Patients with irritable bowel syndrome (IBS) can be assigned to groups with different gastrointestinal (GI) symptoms based on results from a combined nutrient and lactulose challenge. We aimed to identify factors that predict outcomes to this challenge and to determine whether this can be used in noninvasive assessment of visceral sensitivity in patients with IBS. METHODS: We performed a prospective study of 100 patients with IBS diagnosed according to Rome III criteria (all subtypes) and seen at a secondary or tertiary care center. After an overnight fast, subjects were given a liquid breakfast (400 mL; Nutridrink) that contained 25 g lactulose. Before the challenge, we assessed visceral sensitivity (via rectal barostat), oro-anal transit time, and fecal microbiota composition (via 16S ribosomal RNA pyrosequencing); we determined IBS severity using questionnaires. The intensity of 8 GI symptoms, the level of digestive comfort, and the amount of exhaled H2 and CH4 in breath were measured before and during a 4-hour period after the liquid breakfast. RESULTS: Based on the intensity of 8 GI symptoms and level of digestive comfort during the challenge, patients were assigned to groups with high-intensity GI symptoms (HGS; n = 39) or low-intensity GI symptoms (LGS; n = 61); patients with HGS had more severe IBS (P < .0001), higher somatization (P < .01), and lower quality of life (P < .05-.01) than patients with LGS. Patients with HGS also had significantly higher rectal sensitivity to random phasic distensions (P < .05-.001, compared with patients with LGS). There were no significant differences between groups in fecal microbiota composition, exhaled gas in breath, or oro-anal transit time. CONCLUSIONS: We found, in a prospective study, that results from a lactulose challenge test could be used to determine visceral sensitivity and severity of IBS. The intensity of patient symptoms did not correlate with the composition of the fecal microbiota. The lactulose challenge test may help better characterize patients with IBS and evaluate the efficacy of new treatments. ClinicalTrial.gov no: NCT01252550.

Calcium requirements from dairy foods in France can be met at low energy and monetary cost.

Inadequate Ca intakes are a concern for global public health. In France, most dietary Ca is provided by dairy products: milks, fermented milks (mostly yogurts), dairy desserts and cheeses. The present dairy database (n 837) included milks (n 101), fermented milks, yogurts and other fresh dairy products (n 326), desserts (n 162) and a wide variety of cheeses (n 248). Energy and nutrient values were obtained from industry sources and the French national nutrient composition database. Retail prices were from Paris supermarkets. Products in each group were aggregated into twenty-one categories using clustering analyses. The costs in energy (kJ (kcal)), euros (€), and in SFA, added sugar and Na (defined here as nutrients to LIMit) associated with providing 120 mg of Ca (equivalent to 15 % daily value (15 % DV)) were calculated for each product group and category. The milk group supplied Ca at the lowest energy, monetary and LIM cost. Fresh plain and 'light' yogurts and fermented milks were next, followed by sweetened yogurts and flavoured milks. Light dairy desserts provided Ca with relatively few energy but were more expensive. Cheeses were a heterogeneous group. Hard cheeses (Comté) provided the most Ca per serving. Semi-hard cheeses (Camembert) and cream and blue cheeses (Roquefort) provided Ca at a cost comparable with sweetened yogurts and flavoured milks. Double cream, soft and goat cheeses were not optimal Ca sources. New value metrics can help identify affordable dairy foods that provide Ca without excessive energy or nutrients to limit. These conditions were satisfied by a wide variety of dairy products in France.

Fermented dairy products modulate Citrobacter rodentium-induced colonic hyperplasia.

We evaluated the protective effects of fermented dairy products (FDPs) in an infection model, using the mouse pathogen Citrobacter rodentium (CR). Treatment of mice with FDP formulas A, B, and C or a control product did not affect CR colonization, organ specificity, or attaching and effacing lesion formation. Fermented dairy product A (FDP-A), but neither the supernatant from FDP-A nor ß-irradiated (IR) FDP-A, caused a significant reduction in colonic crypt hyperplasia and CR-associated pathology. Profiling the gut microbiota revealed that IR-FDP-A promoted higher levels of phylotypes belonging to Alcaligenaceae and a decrease in Lachnospiraceae (Ruminococcus) during CR infection. Conversely, FDP-A prevented a decrease in Ruminococcus and increased Turicibacteraceae (Turicibacter). Importantly, loss of Ruminococcus and Turicibacter has been associated with susceptibility to dextran sodium sulfate-induced colitis. Our results demonstrate that viable bacteria in FDP-A reduced CR-induced colonic crypt hyperplasia and prevented the loss of key bacterial genera that may contribute to disease pathology.

Fasting breath H2 and gut microbiota metabolic potential are associated with the response to a fermented milk product in irritable bowel syndrome.

OBJECTIVES: Aim of this study was to assess the effect of a fermented milk product containing Bifidobacterium lactis CNCM I-2494 (FMP) on gastrointestinal (GI) symptoms and exhaled H2 and CH4 during a nutrient and lactulose challenge in patients with irritable bowel syndrome (IBS). METHODS: We included 125 patients with IBS (Rome III). Fasted subjects were served a 400ml liquid test meal containing 25g lactulose. The intensity of eight GI symptoms and the amount of exhaled H2 and CH4 were assessed before and during 4h after meal intake. The challenge was repeated after 14 days consumption of FMP or a control product in a double-blind, randomized, parallel design. The metabolic potential of fecal microbiota was profiled using 16S MiSeq analysis of samples obtained before and after the intervention. RESULTS: 106 patients with IBS were randomized. No difference between FMP or control groups was found on GI symptoms or breath H2 and CH4 in the whole cohort. A post-hoc analysis in patients stratified according to their fasting H2 levels showed that in high H2 producers (fasting H2 level≥10ppm, n = 35), FMP consumption reduced fasting H2 levels (p = 0.003) and H2 production during the challenge (p = 0.002) and tended to decrease GI discomfort (p = 0.05) vs. control product. The Prevotella/Bacteroides metabolic potential at baseline was higher in high H2 producers (p<0.05) vs. low H2 producers and FMP consumption reduced this ratio (p<0.05) vs. control product. CONCLUSIONS: The response to a fermented milk product containing Bifidobacterium lactis CNCM I-2494 (FMP) in patients with IBS seems to be associated with the metabolic potential of the gut microbiota. TRIAL REGISTRATION: ClinicalTrial.gov NCT01252550. These results were presented as congress posters at Digestive Disease Week 2016 in San Diego, USA and United European Gastroenterology Week 2016 in Vienna, Austria.

A Data Integration Multi-Omics Approach to Study Calorie Restriction-Induced Changes in Insulin Sensitivity.

Background: The mechanisms responsible for calorie restriction (CR)-induced improvement in insulin sensitivity (IS) have not been fully elucidated. Greater insight can be achieved through deep biological phenotyping of subjects undergoing CR, and integration of big data. Materials and Methods: An integrative approach was applied to investigate associations between change in IS and factors from host, microbiota, and lifestyle after a 6-week CR period in 27 overweight or obese adults (ClinicalTrials.gov: NCT01314690). Partial least squares regression was used to determine associations of change (week 6 - baseline) between IS markers and lifestyle factors (diet and physical activity), subcutaneous adipose tissue (sAT) gene expression, metabolomics of serum, urine and feces, and gut microbiota composition. ScaleNet, a network learning approach based on spectral consensus strategy (SCS, developed by us) was used for reconstruction of biological networks. Results: A spectrum of variables from lifestyle factors (10 nutrients), gut microbiota (10 metagenomics species), and host multi-omics (metabolic features: 84 from serum, 73 from urine, and 131 from feces; and 257 sAT gene probes) most associated with IS were identified. Biological network reconstruction using SCS, highlighted links between changes in IS, serum branched chain amino acids, sAT genes involved in endoplasmic reticulum stress and ubiquitination, and gut metagenomic species (MGS). Linear regression analysis to model how changes of select variables over the CR period contribute to changes in IS, showed greatest contributions from gut MGS and fiber intake. Conclusion: This work has enhanced previous knowledge on links between host glucose homeostasis, lifestyle factors and the gut microbiota, and has identified potential biomarkers that may be used in future studies to predict and improve individual response to weight-loss interventions. Furthermore, this is the first study showing integration of the wide range of data presented herein, identifying 115 variables of interest with respect to IS from the initial input, consisting of 9,986 variables. Clinical Trial Registration: clinicaltrials.gov (NCT01314690).

MixMC: A Multivariate Statistical Framework to Gain Insight into Microbial Communities.

Culture independent techniques, such as shotgun metagenomics and 16S rRNA amplicon sequencing have dramatically changed the way we can examine microbial communities. Recently, changes in microbial community structure and dynamics have been associated with a growing list of human diseases. The identification and comparison of bacteria driving those changes requires the development of sound statistical tools, especially if microbial biomarkers are to be used in a clinical setting. We present mixMC, a novel multivariate data analysis framework for metagenomic biomarker discovery. mixMC accounts for the compositional nature of 16S data and enables detection of subtle differences when high inter-subject variability is present due to microbial sampling performed repeatedly on the same subjects, but in multiple habitats. Through data dimension reduction the multivariate methods provide insightful graphical visualisations to characterise each type of environment in a detailed manner. We applied mixMC to 16S microbiome studies focusing on multiple body sites in healthy individuals, compared our results with existing statistical tools and illustrated added value of using multivariate methodologies to fully characterise and compare microbial communities.

Ecological robustness of the gut microbiota in response to ingestion of transient food-borne microbes.

Resident gut microbes co-exist with transient bacteria to form the gut microbiota. Despite increasing evidence suggesting a role for transient microbes on gut microbiota function, the interplay between resident and transient members of this microbial community is poorly defined. We aimed to determine the extent to which a host's autochthonous gut microbiota influences niche permissivity to transient bacteria using a fermented milk product (FMP) as a vehicle for five food-borne bacterial strains. Using conventional and gnotobiotic rats and gut microbiome analyses (16S rRNA genes pyrosequencing and reverse transcription qPCR), we demonstrated that the clearance kinetics of one FMP bacterium, Lactococcus lactis CNCM I-1631, were dependent on the structure of the resident gut microbiota. Susceptibility of the resident gut microbiota to modulation by FMP intervention correlated with increased persistence of L. lactis. We also observed gut microbiome configurations that were associated with altered stability upon exposure to transient bacteria. Our study supports the concept that allochthonous bacteria have transient and subject-specific effects on the gut microbiome that can be leveraged to re-engineer the gut microbiome and improve dysbiosis-related diseases.

Dietary patterns differently associate with inflammation and gut microbiota in overweight and obese subjects.

BACKGROUND: Associations between dietary patterns, metabolic and inflammatory markers and gut microbiota are yet to be elucidated. OBJECTIVES: We aimed to characterize dietary patterns in overweight and obese subjects and evaluate the different dietary patterns in relation to metabolic and inflammatory variables as well as gut microbiota. DESIGN: Dietary patterns, plasma and adipose tissue markers, and gut microbiota were evaluated in a group of 45 overweight and obese subjects (6 men and 39 women). A group of 14 lean subjects were also evaluated as a reference group. RESULTS: Three clusters of dietary patterns were identified in overweight/obese subjects. Cluster 1 had the least healthy eating behavior (highest consumption of potatoes, confectionary and sugary drinks, and the lowest consumption of fruits that was associated also with low consumption of yogurt, and water). This dietary pattern was associated with the highest LDL cholesterol, plasma soluble CD14 (p = 0.01) a marker of systemic inflammation but the lowest accumulation of CD163+ macrophages with anti-inflammatory profile in adipose tissue (p = 0.05). Cluster 3 had the healthiest eating behavior (lower consumption of confectionary and sugary drinks, and highest consumption of fruits but also yogurts and soups). Subjects in this Cluster had the lowest inflammatory markers (sCD14) and the highest anti-inflammatory adipose tissue CD163+ macrophages. Dietary intakes, insulin sensitivity and some inflammatory markers (plasma IL6) in Cluster 3 were close to those of lean subjects. Cluster 2 was in-between clusters 1 and 3 in terms of healthfulness. The 7 gut microbiota groups measured by qPCR were similar across the clusters. However, the healthiest dietary cluster had the highest microbial gene richness, as evaluated by quantitative metagenomics. CONCLUSION: A healthier dietary pattern was associated with lower inflammatory markers as well as greater gut microbiota richness in overweight and obese subjects. TRIAL REGISTRATION: ClinicalTrials.gov NCT01314690.

Changes of the human gut microbiome induced by a fermented milk product.

The gut microbiota (GM) consists of resident commensals and transient microbes conveyed by the diet but little is known about the role of the latter on GM homeostasis. Here we show, by a conjunction of quantitative metagenomics, in silico genome reconstruction and metabolic modeling, that consumption of a fermented milk product containing dairy starters and Bifidobacterium animalis potentiates colonic short chain fatty acids production and decreases abundance of a pathobiont Bilophila wadsworthia compared to a milk product in subjects with irritable bowel syndrome (IBS, n = 28). The GM changes parallel improvement of IBS state, suggesting a role of the fermented milk bacteria in gut homeostasis. Our data challenge the view that microbes ingested with food have little impact on the human GM functioning and rather provide support for beneficial health effects.