Colonic Mucosa-associated Microbiota Research Articles

After Surgically Induced Remission, Ileal and Colonic Mucosa-Associated Microbiota Predicts Crohn’s Disease Recurrence

Investigating the tissue-associated microbiota after surgically induced remission may help to understand the mechanisms initiating intestinal inflammation in Crohn's disease. Patients with Crohn's disease undergoing ileocolic resection were prospectively recruited in 6 academic centers. Biopsy samples from the neoterminal ileum, colon, and rectosigmoid were obtained from colonoscopies performed after surgery. Microbial DNA was extracted for 16S rRNA gene sequencing. Microbial diversity and taxonomic differential relative abundance were analyzed. A random forest model was applied to analyze the performance of clinical and microbial features to predict recurrence. A Rutgeerts score ≥i2 was deemed as endoscopic recurrence. A total of 349 postoperative colonoscopies and 944 biopsy samples from 262 patients with Crohn's disease were analyzed. Ileal inflammation accounted for most of the explained variance of the ileal and colonic mucosa-associated microbiota. Samples obtained from 97 patients who were in surgically induced remission at first postoperative colonoscopy who went on to develop endoscopic recurrence at second colonoscopy showed lower diversity and microbial deviations when compared with patients who remained in endoscopic remission. Depletion of genus Anaerostipes and increase of several genera from class Gammaproteobacteria at the 3 biopsy sites increase the risk of further recurrence. Gut microbiome was able to predict future recurrence better than clinical features. Ileal and colonic mucosa-associated microbiome deviations precede development of new-onset ileal inflammation after surgically induced remission and show good predictive performance for future recurrence. These findings suggest that targeted microbial modulation is a plausible modality to prevent postoperative Crohn's disease recurrence.

The Association between Caffeine Intake and the Colonic Mucosa-Associated Gut Microbiota in Humans-A Preliminary Investigation.

We examined the association between caffeine and coffee intake and the community composition and structure of colonic microbiota. A total of 34 polyp-free adults donated 97 colonic biopsies. Microbial DNA was sequenced for the 16S rRNA gene V4 region. The amplicon sequence variant was assigned using DADA2 and SILVA. Food consumption was ascertained using a food frequency questionnaire. We compared the relative abundance of taxonomies by low (<82.9 mg) vs. high (≥82.9 mg) caffeine intake and by never or <2 cups vs. 2 cups vs. ≥3 cups coffee intake. False discovery rate-adjusted p values (q values) <0.05 indicated statistical significance. Multivariable negative binomial regression models were used to estimate the incidence rate ratio and its 95% confidence interval of having a non-zero count of certain bacteria by intake level. Higher caffeine and coffee intake was related to higher alpha diversity (Shannon index p < 0.001), higher relative abundance of Faecalibacterium and Alistipes, and lower relative abundance of Erysipelatoclostridium (q values < 0.05). After adjustment of vitamin B2 in multivariate analysis, the significant inverse association between Erysipelatoclostridium count and caffeine intake remained statistically significant. Our preliminary study could not evaluate other prebiotics in coffee.

DOP49 Metagenomic Characterisation of The Colonic Mucosa-Associated Microbiota Reveals Novel Microbes and Communities in Health and Crohn’s disease. The ENIGMA study.

Abstract Background Deep functional understanding of the mucosa-associated microbiota (MAM) in Crohn’s disease and health by DNA sequencing approaches is restricted by the dominance of human DNA in samples. We established an ex vivo combination of tissue preservation, microbe culture, and metagenomic sequencing to overcome this knowledge gap1 and applied this approach to characterise the colonic MAM from two populations with different Crohn’s disease epidemiology (Hong Kong and Australia) and in comparison to healthy subjects. Methods Biopsies (right colon) were cryopreserved from 100 participants in the Eastern IBD Gut Microbiota (ENIGMA) study, with 59 Crohn’s disease (29 from Australia) and 41 healthy subjects (20 from Australia). The MAM were recovered from these samples1 and taxonomic and functional assessments of their metagenomes was performed using the HUMANn3 pipeline. Metagenome-assembled genomes (MAG) were produced using the MetaWRAP pipeline, and their taxonomy determined via the Genome Taxonomy Database-Tool kit (GTDB-Tk). Results Our approach supported the recovery of ~1300 high-quality, phylogenetically diverse MAG across 8 bacterial phyla, including 35 “new” species, representing novel pathogenic and protective bacteria. The colonic MAM from healthy subjects possessed greater taxonomic diversity and a larger number of functional genes than the Crohn’s disease subjects from both countries, but fewer of these genes could be assigned to reference taxa. As such, the colonic MAM from healthy subjects possesses novel forms of microbial “dark matter”. There were 27 and 25 species unique to the Australian and Hong Kong Crohn’s disease subjects, respectively, and another 14 species shared by both. Many of the functional gene classifiers differentiating Crohn’s disease from healthy subjects were assigned to low prevalence taxa including Proteus, Acidaminococcus, and Fusobacterium. The dissimilarity scores of the colonic MAM were larger among the Crohn’s disease subjects from both countries, suggesting more heterogeneity in Crohn’s disease than in health. Conclusion Here we use two populations with very different rates of Crohn’s disease incidence to show the colonic MAM of healthy subjects in both countries are more diverse, less heterogenous, and contain a greater amount of functional and taxonomic “dark matter” than Crohn’s disease subjects. Novel microbes have been identified and cultured, bringing genomes to life as microbial consortia and novel axenic isolates, to fully define their role in Crohn’s disease pathogenesis or abrogation. 1Teh JJ, Berendsen EM, et al., ISME J 2021;15:3326-3338. The ENIGMA study is supported by a grant from the Leona M. and Harry B. Helmsley Charitable Trust

DOP48 Key Anti-Inflammatory Faecalibacterium prausnitzii is Heterogeneous and Diverse in the East and West and in Health and Crohn’s disease. The ENIGMA study.

Abstract Background There is insufficient knowledge about the key characteristics of the pathogenic and protective bacteria in inflammatory bowel disease. The ENIGMA study aimed to identify the phylogenetic and functional characteristics of Faecalibacterium prausnitzii, considered a protective “anti-inflammatory” bacterium, in both health and Crohn’s disease in populations in the West (high Crohn’s incidence) and Hong Kong (rapidly increasing Crohn’s incidence). Methods Total DNA was extracted from tissue (right colon and terminal ileum) and stool collected from case-control cohorts in Hong Kong (n=51; 30 Crohn’s, 21 controls) and Australia (n=49; 29 Crohn’s, 20 controls). In parallel, the colonic mucosa-associated microbiota (MAM) for each subject was cultured from anaerobically preserved tissue. Shotgun metagenomic sequencing was used to characterise the stool and colonic MAM, augmented with 16S rRNA gene profiling and F. prausnitzii qPCR. The HUMANn3 and/or QIIME2 packages were used for the taxonomic and/or functional characterisation of the datasets. Metagenome-assembled genomes (MAG) were also produced from colonic MAM datasets using the MetaWRAP pipeline, and taxonomy deduced using the Genome Taxonomy Database-Tool kit (GTDB-tk). Results F. prausnitzii was detected in all case-control cohorts from Hong Kong and Australia. However, the prevalence was greater for the colonic MAM cultures of the Australian subjects, which provided 24/32 high-quality Faecalibacterium-affiliated MAG. No less than 6 different species-level lineages were predicted by GTDB-tk, and a similar phylogeny predicted using the gene encoding immunomodulatory peptides (mam). Functional features for the MAG from Crohn’s disease and control subjects were compared, and genes involved with histidine, riboflavin, purine and pyrimidine metabolism, pentose phosphate pathway, and oxidoreductases were enriched in the control subject MAG. In contrast, MAG derived from Crohn’s disease subjects possessed more functions putatively assigned to antibiotic resistance mechanisms and other protective functions. Differences were also observed in the carbohydrate-active enzyme profiles of the MAG recovered from Crohn’s disease and control subjects. Conclusion The populations of F. prausnitzii recovered from the colonic mucosa associated microbiota are both diverse and vary between Crohn’s disease and control subjects in both countries. The differences include metabolic and physiological characteristics. Therapeutic modification of F. prausnitzii needs to consider phylogenetic and functional characteristics. The ENIGMA study is supported by a grant from the Leona M. and Harry B. Helmsley Charitable Trust

P925 Deep functional assessment of the colonic mucosa-associated microbiota in Crohn’s disease and health identifies pro- and anti- inflammatory secretomes and associated bacterial consortia. The ENIGMA study

Abstract Background Gut microbial dysbiosis and dysregulated host immune responses are key factors implicated in Crohn’s disease pathogenesis. However, the nexus between the mucosa-associated microbiota (MAM) and epithelial cell immune response(s) remains poorly understood. Here, we used in vitro cultures of LS174T human epithelial cells to examine the inflammatory tenor of the secretomes from colonic MAM recovered from healthy and Crohn’s disease subjects in Hong Kong (where Crohn’s disease incidence is low but increasing) and Australia (globally high incidence). Methods Biopsies from healthy (21 HKG, 20 AUS) and Crohn’s disease (30 HKG, 29 AUS) subjects were collected from the right colon and the MAM propagated from tissue using a habitat simulating medium. After 24h of growth, cultures were centrifuged, and the supernatant (secretome) fraction filter sterilised. For the immune assays, the LS174T cell line was transfected with recombinant lentivirus to provide an NF-κB:luc (luciferase) reporter gene fusion (Cellomics Technology, MD, USA) and aliquots used to inoculate 96-well microtiter plates and cultured for 24 h under standard conditions. Cultures were then challenged for 4 hours with 0.1 vol of a MAM secretome. Luc-bioluminescence was measured as a marker of NF-κB transcriptional activity from the individual wells and normalised to the reporter gene response from cells challenged with sterile, uninoculated bacterial medium. Results Across the 100 MAM secretomes tested, the median score for NF-ĸB activation was 104.4% relative to the medium control, suggesting most of the secretomes possessed a net neutral effect, with the 1st and 4th quartiles deemed stimulatory (&amp;gt;126% baseline activity) and suppressive (&amp;lt;92% baseline activity), respectively. There was no significant relationship found between a secretome’s inflammatory score and its corresponding α-diversity metric, nor was there any distinct clustering of the secretomes according to country of origin and/or case-control groups. However, the relative abundances of Roseburia spp. and Clostridium innocuum were significantly enriched in those consortia producing immunostimulatory secretomes. In contrast, the relative abundances of Erysipelatoclostridium ramosum, Ruminococcus gnavus, Parabacteroides distasonis, Blautia sp. CAG257 and Bacteroides xylanisolvens were enriched, and that of Coprococcus comes depleted, in those microbial consortia producing immunosuppressive secretomes. Conclusion Specific bacterial taxa drive the functional signature of the colonic MAM secretome in an immunostimulatory or suppressive direction. The ENIGMA study is supported by a grant from the Leona M. and Harry B. Helmsley Charitable Trust

Analysis of colonic mucosa-associated microbiota using endoscopically collected lavage

The bacterial composition of the gut lumen and mucosa is distinct and the mucosa-associated bacteria are thought to play a more critical role in interactions with the host immune system. However, limited studies of the gut mucosal microbiota in humans have been available due to methodological challenges. Here, we evaluated the potential use of colonic lavage samples for mucosal microbiota analysis in humans. Among the different types of colonic mucosal samples collected from healthy volunteers, the lavage samples contained a higher amount of bacterial DNA and were less contaminated with host DNA compared to mucosal brushing (brush) and biopsy. Although 16S gene amplicon sequencing showed that the bacterial composition of the lavage was intermediate between that of feces and biopsy, mucosal bacteria abundant in the biopsy were also enriched in lavage samples. Furthermore, differences in mucosal microbes between non-smokers and smokers were detectable in lavage samples. Our data emphasize that colonic lavage is suitable for analysis of the mucosal microbiota. Given its minimal invasiveness and high bacterial DNA content, the colonic lavage will promote research on the human mucosal microbiota, especially in gastrointestinal disorders.

P708 Ileal involvement defined by Montreal classification in Crohn’s disease is associated with a distinct microbial composition in ileal and colonic mucosa

Abstract Background The Montreal classification of inflammatory bowel disease (IBD) recognizes distinct subgroups based on location of disease, age at onset and behaviour for Crohn’s disease (CD) and extent in ulcerative colitis (UC). CD with ileal involvement is in part genetically determined and tends to progress to surgery and to be more refractory to medical therapy compared to colonic disease location. We investigated the association of different aspects of the Montreal classification with the ileal and colonic mucosa-associated microbiota. Methods Healthy control (HC) and IBD patients underwent colonoscopy with biopsy samples taken from terminal ileum, ascending and sigmoid colon. Mucosal 16s rRNA gene profiling was analysed and an amplicon sequence variant table was generated and normalized. Patients with prior intestinal resection, IBD-unclassified and recent antibiotic exposure were excluded. Endoscopic activity was defined as a segmental Mayo endoscopic subscore &amp;gt; 0 in UC and a simple endoscopic score &amp;gt; 2 in CD. Blood samples were drawn for genotyping and a weighted genetic risk score (GRS) was built based on 169 IBD risk variants. Pooled ileal and colonic samples were analysed for taxonomic differential abundance (DA) among the variables of the Montreal classification using a linear mixed-effects model (MaAsLin2) with age, sex, biopsy site, endoscopic activity and GRS as covariates. P-values were corrected for false discovery rate (FDR) and &amp;lt; 5% was considered significant. Results A total of 546 biopsy samples from 201 IBD patients and 48 HC were analysed. The characteristics of the cohort and Montreal classification are depicted in the Table. At colonoscopy 168 samples (31.4%) showed endoscopic activity. The multivariate analysis revealed that the genera Agathobacter and Faecalibacterium, as well as the family Ruminococacceae and the order Oscillospirales were significantly reduced in CD with ileal involvement (L1+L3) when compared to either isolated colonic CD (L2), UC (any extent) and HC independent of age, sex, endoscopic activity, biopsy site, and GRS (Figure). Samples from UC and isolated colonic CD patients did not show differences in their microbial profile irrespective of biopsy site. No differences in DA were observed in subgroups based on age at onset and disease behaviour in CD or extent in UC. GRS was not associated with microbial composition. Conclusion These data suggest that CD with ileal involvement (Montreal L1+L3) is a major determinant of the tissue-associated microbial composition in both ileal and colonic mucosa irrespective of variables such as endoscopic activity and GRS. The reduction of several beneficial microbes in CD with ileal involvement might partially explain the more progressive phenotype in such patients.

Galacto-oligosaccharides improve barrier function and relieve colonic inflammation via modulating mucosa-associated microbiota composition in lipopolysaccharides-challenged piglets

BackgroundGalacto-oligosaccharides (GOS) have been shown to modulate the intestinal microbiota of suckling piglets to exert beneficial effects on intestinal function. However, the modulation of intestinal microbiota and intestinal function by GOS in intestinal inflammation injury models has rarely been reported. In this study, we investigated the effects of GOS on the colonic mucosal microbiota composition, barrier function and inflammatory response of lipopolysaccharides (LPS)-challenged suckling piglets.MethodsA total of 18 newborn suckling piglets were divided into three groups, the CON group, the LPS-CON group and the LPS-GOS group. Piglets in the LPS-GOS group were orally fed with 1 g/kg body weight of GOS solution every day. On the d 14, piglets in the LPS-CON and LPS-GOS group were challenged intraperitoneally with LPS solution. All piglets were slaughtered 2 h after intraperitoneal injection and sampled.ResultsWe found that the colonic mucosa of LPS-challenged piglets was significantly injured and shedding, while the colonic mucosa of the LPS-GOS group piglets maintained its structure. Moreover, GOS significantly reduced the concentration of malondialdehyde (MDA) and the activity of reactive oxygen species (ROS) in the LPS-challenged suckling piglets, and significantly increased the activity of total antioxidant capacity (T-AOC). GOS significantly increased the relative abundance of norank_f__Muribaculaceae and Romboutsia, and significantly decreased the relative abundance of Alloprevotella, Campylobacter and Helicobacter in the colonic mucosa of LPS-challenged suckling piglets. In addition, GOS increased the concentrations of acetate, butyrate and total short chain fatty acids (SCFAs) in the colonic digesta of LPS-challenged suckling piglets. GOS significantly reduced the concentrations of interleukin 1β (IL-1β), interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α) and cluster of differentiation 14 (CD14), and the relative mRNA expression of Toll-like receptor 4 (TLR4) and myeloid differentiation primary response 88 (MyD88) in the LPS-challenged suckling piglets. In addition, GOS significantly reduced the relative mRNA expression of mucin2 (MUC2), and significantly increased the protein expression of Claudin-1 and zonula occluden-1 (ZO-1) in LPS-challenged suckling piglets.ConclusionsThese results suggested that GOS can modulate the colonic mucosa-associated microbiota composition and improve the intestinal function of LPS-challenged suckling piglets.

Spatial Characteristics of Colonic Mucosa-Associated Gut Microbiota in Humans.

Limited data exist on the spatial distribution of the colonic bacteria in humans. We collected the colonic biopsies from five segments of 27 polyp-free adults and collected feces from 13 of them. We sequenced the V4 region of the bacterial 16S rRNA gene using the MiSeq platform. The sequencing data were assigned to the amplicon sequence variant (ASV) using SILVA. Biodiversity and the relative abundance of the ASV were compared across the colonic segments and between the rectal and fecal samples. Bacterial functional capacity was assessed using Tax4fun. Each individual had a unique bacterial community composition (Weighted Bray-Curtis P value = 0.001). There were no significant differences in richness, evenness, community composition, and the taxonomic structure across the colon segments in all the samples. Firmicutes (47%), Bacteroidetes (39%), and Proteobacteria (6%) were the major phyla in all segments, followed by Verrucomicrobia, Fusobacteria, Desulfobacterota, and Actinobacteria. There were 15 genera with relative abundance > 1%, including Bacteroides, Faecalibacterium, Escherichia/Shigella, Sutterella, Akkermansia, Parabacteroides, Prevotella, Lachnoclostridium, Alistipes, Fusobacterium, Erysipelatoclostridium, and four Lachnospiraceae family members. Intra-individually, the community compositional dissimilarity was the greatest between the cecum and the rectum. There were significant differences in biodiversity and the taxonomic structure between the rectal and fecal bacteria. The bacterial community composition and structure were homogeneous across the large intestine in adults. The inter-individual variability of the bacteria was greater than inter-segment variability. The rectal and fecal bacteria differed in the community composition and structure.

Habitual Sleep Duration and the Colonic Mucosa-Associated Gut Microbiota in Humans-A Pilot Study.

We examined the association between the colonic adherent microbiota and nocturnal sleep duration in humans. In a cross-sectional study, 63 polyp-free adults underwent a colonoscopy and donated 206 mucosal biopsies. The gut microbiota was profiled using the 16S rRNA gene sequencing targeting the V4 region. The sequence reads were processed using UPARSE and DADA2, respectively. Lifestyle factors, including sleep habits, were obtained using an interviewer-administered questionnaire. We categorized the participants into short sleepers (<6 h per night; n = 16) and normal sleepers (6–8 h per night; n = 47) based on self-reported data. Differences in bacterial biodiversity and the taxonomic relative abundance were compared between short vs. normal sleepers, followed by multivariable analysis. A false discovery rate-adjusted p value (q value) < 0.05 indicated statistical significance. The bacterial community composition differed in short and normal sleepers. The relative abundance of Sutterella was significantly lower (0.38% vs. 1.25%) and that of Pseudomonas was significantly higher (0.14% vs. 0.08%) in short sleepers than in normal sleepers (q values < 0.01). The difference was confirmed in the multivariable analysis. Nocturnal sleep duration was associated with the bacterial community composition and structure in the colonic gut microbiota in adults.

Longitudinal assessment revealed the shifts in rumen and colon mucosal-attached microbiota of dairy calves during weaning transition

The objectives of this study were to investigate the shifts in rumen and colon mucosa-associated microbiota in dairy calves fed a high milk replacer feeding rate before and after weaning and to determine whether such shifts are associated with tissue physiological measures. Longitudinal biopsy was performed to collect rumen and colon mucosal tissues of 4 ruminally cannulated Holstein dairy bull calves (weaned at 6 wk of age) at the end of wk 5 (before weaning), 7 (weaning adaptation) and 12 (after weaning), and were used to assess mucosa-associated microbiota and their changes using amplicon sequencing. Both rumen and colon mucosa-associated bacterial communities shifted during the weaning process, as evidenced by their clear separation among 3 different weaning periods and increased α diversity (Shannon and Chao1 indices) during weaning transition. Among the 3 dominant bacterial phyla identified (relative abundance >1.0%), the relative abundance of Proteobacteria and Bacteroidetes decreased in the rumen mucosa, whereas the relative abundance of Firmicutes increased in both rumen and colon mucosa during weaning transition. In the rumen mucosa, Campylobacter (0.6-22.1%) gradually became prevalent during weaning transition, whereas Succinivibrio (6.2-10.3%) and Prevotella 1 (4.7-10.5%) were dominant regardless of weaning transition. In the colon mucosa, Bacteroides (12.8-25.4%) was dominant during weaning transition, although its relative abundance decreased after weaning. In the meantime, relative abundance of uncultured Lachnospiraceae increased from 2.2% to 25.7% during this period. In addition, genera Pyramidobacter (in the rumen mucosa) and Lachnoclostridium (in the colon mucosa) were positively correlated with rumen papilla surface area and colon mucosal thickness, respectively. Moreover, genera Ruminococcaceae UCG-005 and Sharpea in the rumen mucosa were positively correlated with the molar proportion of propionate and butyrate, respectively. Overall, our findings revealed that rumen and colon mucosa-associated bacterial communities altered in response to the weaning transition, and some bacterial taxa in these communities may have positive effects on rumen and colon mucosa development during this period.

Ileocolonic Histopathological and Microbial Alterations in the Irritable Bowel Syndrome: A Nested Community Case-Control Study.

INTRODUCTION:Histopathological alterations in the ileum and colon in irritable bowel syndrome (IBS) are controversial, and normal values are poorly established. We hypothesized that changes in mucosal immune cells characterize IBS and key changes in immune composition are associated with the mucosa-associated microbiota (MaM).METHODS:A nested case-control study (48 IBS and 106 controls included) from 745 colonoscopy participants in a random population sample. Intraepithelial lymphocytes (IELs)/100 enterocytes and eosinophils/5 nonoverlapping high-power fields counted; mast cells identified by immunocytochemistry (CD117)/5 high-power fields. Paneth cells quantified per 5 crypts. 16S rRNA gene amplicon sequencing performed on available sigmoid MaM, n = 55 and fecal microbiota, n = 20. Microbiota profiles compared between samples with high and low IEL counts.RESULTS:IBS had increased IELs in the terminal ileum (relative risk ratio = 1.70, 95% confidence interval 1.08–2.76, P = 0.022 adjusted for age, sex, and smoking). Cecal IELs were increased in IBS—diarrhea (relative risk ratio = 2.03, 95% confidence interval 1.13–3.63, P = 0.017). No difference was observed in alpha diversity of MaM or fecal microbiota based on IEL count. There was no difference in beta diversity of the MaM according to IEL count in the terminal ileal (TI) (P = 0.079). High TI IEL counts associated with a significant expansion of the genus Blautia (P = 0.024) and unclassified Clostridiales (P = 0.036) in colon MaM.DISCUSSION:A modest but significant increase in IELs was observed in IBS vs. controls in a population-based setting. Subtle TI and cecal inflammation may play a pathogenic role in IBS but needs confirmation. Modest but discernible differences in the colonic MaM were seen according to TI IEL count but not IBS status.

Mucosa-Associated Microbiota in Patients with Irritable Bowel Syndrome: A Comparison of Subtypes

Background: Most studies on gut microbiome of irritable bowel syndrome (IBS) have focused on fecal microbiota, instead of mucosa-associated microbiota (MAM). Aims: The aim of this study wasto investigate the MAM in IBS patients including the difference in subtypes of IBS, namely, diarrhea-predominant IBS (IBS-D) and constipation-predominant IBS (IBS-C). Methods: Endoscopic brush samples were taken from terminal ileum and sigmoid colon of patients with IBS (17 IBS-D patients and 7 IBS-C patients) and 10 healthy controls. The MAM of samples was profiled by 16S rRNA gene amplicon sequencing. Potential changes in the MAM at the functional level were evaluated using PICRUSt software and the KEGG database. Results: There were no differences in MAM composition between terminal ileum and sigmoid colon according to β-diversity based on the UniFrac distance. In view of α-diversity, Shannon (evenness) but not Chao1 (richness) or observed operational taxonomic units tended to be lower in sigmoid colon MAM of IBS-C and IBS-D than the control group. The abundance of 4 genera in the sigmoid colon and 7 genera in the terminal ileum was significantly different among the 3 groups. Linear discriminant analysis effect size (LEfSe) showed that the genera of Ruminococcus, Akkermansia, Butyrivibrio, Methylobacterium, and Microbacterium and the family Erysipelotrichaceae were significantly higher in the IBS-C group, and the abundance of the genera Streptococcus, Acidaminococcus, Butyricicoccus, and Parvimonas was significantly higher in the IBS-D group. In addition, the proportion of genes responsible for the secretion system and LPS biosynthesis was significantly higher and that for methane metabolism, lysine biosynthesis, and enzyme families was significantly lower in the IBS-D group than in the IBS-C group. Conclusion: Dysbiosis pattern and the function of the microbiome seem to be different among subtypes of IBS, and MAM may play a crucial role in IBS symptom generation.

PSIX-32 Assessment of Mucosa-associated Microbiota in the Colon and Rumen of Dairy Calves Fed High Plane of Milk and during Weaning Transition

Abstract The nature of weaning is considered as the most stressful period experienced by dairy calves and has been known to negatively affect calf growth and intake. The objectives of this study were to investigate the shift of rumen and colon mucosa-associated microbiota in dairy calves fed with a high plane of milk pre- and post-weaning, and whether such shifts are associated with rumen fermentation and growth performance. Six Holstein dairy bull calves were ruminally cannulated at the second week of life followed by weaning at the end of week 6. Ruminal and colon tissue samples were biopsied at the end of week 5, 7 and 12 to determine the bacterial community changes, and rumen content was collected at the same time to measure rumen fermentation parameters. QIIME2 was used to analyze microbial profiles and R studio software was used to determine the microbial changes and its relationship with phenotypic measures. Age-dependent profiles were observed for the colon mucosa associated microbiota but not for rumen mucosa associated microbiota, indicating that varied responses to weaning transition exists between colon and rumen mucosa associated microbiota. As calves aged, higher abundance of taxa such as Delftia (associated with hexadecanoic acid production) and lower abundance of Veillonella (associated with short-chain organic acids utilization) were detected in the colon mucosa. The rumen also displayed taxonomic changes with decreases in Pseudobutyrivibrio in older calves. In addition, we identified significantly positive correlations between Prevotella associated with rumen mucosa and average daily gain, Isovalerate and Isobutyrate, suggesting that this genus may promote rumen fermentation during weaning transition. Our findings suggest that weaning transition affects rumen and colon mucosa-associated bacteria, and some of the rumen mucosa-associated microbiota promote rumen fermentation during this period, which may have long-term effects on animal health and productivity.

Oral Health and the Altered Colonic Mucosa-Associated Gut Microbiota.

Systemic diseases have been associated with oral health and gut microbiota. We examined the association between oral health and the community composition and structure of the adherent colonic gut microbiota. We obtained 197 snap-frozen colonic biopsies from 62 colonoscopy-confirmed polyp-free individuals. Microbial DNA was sequenced for the 16S rRNA V4 region using the Illumina MiSeq, and the sequences were assigned to the operational taxonomic unit based on SILVA. We used a questionnaire to ascertain tooth loss, gum disease, and lifestyle factors. We compared biodiversity and relative abundance of bacterial taxa based on the amount of tooth loss and the presence of gum disease. The multivariable negative binomial regression model for panel data was used to estimate the association between the bacterial count and oral health. False discovery rate-adjusted P value (q value) < .05 indicated statistical significance. More tooth loss and gum disease were associated with lower bacterial alpha diversity. The relative abundance of Faecalibacterium was lower (q values < .05) with more tooth loss. The association was significant after adjusting for age, ethnicity, obesity, smoking, alcohol use, hypertension, diabetes, and the colon segment. The relative abundance of Bacteroides was higher in those with gum disease. Oral health was associated with alteration in the community composition and structure of the adherent gut bacteria in the colon. The reduced anti-inflammatory Faecalibacterium in participants with more tooth loss may indicate systemic inflammation. Future studies are warranted to confirm our findings and investigate the systemic role of Faecalibacterium.

Blocking integrin α4β7-mediated CD4 T cell recruitment to the intestine and liver protects mice from western diet-induced non-alcoholic steatohepatitis

The heterodimeric integrin receptor α4β7 regulates CD4 T cell recruitment to inflamed tissues, but its role in the pathogenesis of non-alcoholic steatohepatitis (NASH) is unknown. Herein, we examined the role of α4β7-mediated recruitment of CD4 T cells to the intestine and liver in NASH. Male littermate F11r+/+ (control) and junctional adhesion molecule A knockout F11r-/- mice were fed a normal diet or a western diet (WD) for 8 weeks. Liver and intestinal tissues were analyzed by histology, quantitative reverse transcription PCR (qRT-PCR), 16s rRNA sequencing and flow cytometry. Colonic mucosa-associated microbiota were analyzed using 16s rRNA sequencing. Liver biopsies from patients with NASH were analyzed by confocal imaging and qRT-PCR. WD-fed knockout mice developed NASH and had increased hepatic and intestinal α4β7+ CD4 T cells relative to control mice who developed mild hepatic steatosis. The increase in α4β7+ CD4 T cells was associated with markedly higher expression of the α4β7 ligand mucosal addressin cell adhesion molecule 1 (MAdCAM-1) in the colonic mucosa and livers of WD-fed knockout mice. Elevated MAdCAM-1 expression correlated with increased mucosa-associated Proteobacteria in the WD-fed knockout mice. Antibiotics reduced MAdCAM-1 expression indicating that the diet-altered microbiota promoted colonic and hepatic MAdCAM-1 expression. α4β7 blockade in WD-fed knockout mice significantly decreased α4β7+ CD4 T cell recruitment to the intestine and liver, attenuated hepatic inflammation and fibrosis, and improved metabolic indices. MAdCAM-1 blockade also reduced hepatic inflammation and fibrosis in WD-fed knockout mice. Hepatic MAdCAM-1 expression was elevated in patients with NASH and correlated with higher expression of α4 and β7 integrins. These findings establish α4β7/MAdCAM-1 as a critical axis regulating NASH development through colonic and hepatic CD4 T cell recruitment. Non-alcoholic steatohepatitis (NASH) is an advanced and progressive form of non-alcoholic fatty liver disease (NAFLD), and despite its growing incidence no therapies currently exist to halt NAFLD progression. Herein, we show that blocking integrin receptor α4β7-mediated recruitment of CD4 T cells to the intestine and liver not only attenuates hepatic inflammation and fibrosis, but also improves metabolic derangements associated with NASH. These findings provide evidence for the potential therapeutic application of α4β7 antibody in the treatment of human NASH.

The High-Fat Diet Based on Extra-Virgin Olive Oil Causes Dysbiosis Linked to Colorectal Cancer Prevention.

The present study aims to examine the effects of three different high-fat diet (HFD) on mice gut microbiota in order to analyse whether they create the microenvironmental conditions that either promote or prevent colorectal cancer (CRC). We evaluated colonic mucosa-associated microbiota in CD1 mice fed with HFD, based on 60% kcal from fat-containing coconut, sunflower or extra-virgin olive oil as the only source of fat. The main findings were as follows: (a) All HFD produced a decrease in the richness and diversity of the intestinal microbiota that was independent of mouse weight, (b) HFD switched Lactobacillus to Lactococcus. In general, the results showed that both sunflower- and coconut-HFD generated a pro-inflammatory intestinal microenvironment. In brief, coconut-HFD decreased Akkermansia and increased Staphylococcus, Prevotella and Bacteroides spp. abundance. Sunflower-HFD reduced Akkermansia and Bifidobacterium, while enhancing Sphingomonas and Neisseria spp. abundance. In contrast, EVOO-HFD produced an anti-inflammatory microenvironment characterised by a decreased Enterococcus, Staphylococcus, Neisseria and Pseudomonas spp. abundance. At the same time, it increased the Firmicutes/Bacteroidetes ratio and maintained the Akkermansia population. To conclude, EVOO-HFD produced changes in the gut microbiota that are associated with the prevention of CRC, while coconut and sunflower-HFD caused changes associated with an increased risk of CRC.

Impact of Fermented Corn-Soybean Meal on Gene Expression of Immunity in the Blood, Level of Secretory Immunoglobulin A, and Mucosa-Associated Bacterial Community in the Intestine of Grower-Finisher Pigs.

This study was conducted to determine the effect of a fermented corn–soybean meal [fermented feed (FF)] on the gene expression of immunity in the blood, the level of secretory immunoglobulin A (sIgA), and mucosa-associated bacterial community in the duodenum and colon of grower-finisher pigs. In this study, crossbred barrows (Duroc × Landrace × Large White) were randomly assigned to either an unfermented corn–soybean diet (Ctrl) (n = 6) or an FF diet (n = 6), and then the following were examined: the expression of immunity using real-time reverse transcription polymerase-chain reaction in the blood, sIgA using enzyme-linked immunosorbent assay (ELISA), and changes in the bacterial community using Illumina Hiseq sequencing in the mucosa of the duodenum and colon. Compared with control pigs fed with a standard diet, the results showed that FF caused upregulation of the mRNA expression of Toll-like receptor 3 (TLR3), TLR4, TLR6, and TLR8 in the blood (P < 0.05). Moreover, sequencing of 16S rRNA genes in duodenal mucosa samples indicated that the FF diet had a lower proportion of Tenericutes (P < 0.05) in the duodenal mucosa-associated microbiota, and FF significantly increased the percentage of Rikenellaceae and Christensenellaceae but decreased the abundance of Lachnospiraceae (P < 0.05) in the colonic mucosa-associated microbiota. The ELISA results showed that FF significantly increased the concentration of sIgA in the colonic mucosa (P < 0.05). More importantly, our correlation analysis indicated that the gene expression of immunity in the blood and the concentration of sIgA was associated with colonic mucosa-associated microbiota. Our data provide new knowledge into the adaptation response of the intestine to fermented feeding in monogastric animals.

Probiotics and dietary intervention modulate the colonic mucosa-associated microbiota in high-fat diet populations.

Alterations in the gut microbiota due to a high-fat diet and diet-induced illness have been found in both mouse models and humans. Observational studies suggest that probiotic administration and diet shifts may treat diet-related diseases. However, the effect of these interventions on the colonic mucosa has not yet been elucidated. This study investigated the efficacy of probiotic supplementation and dietary intervention as prophylactic tools under high-fat diet conditions. A total of 36 volunteers that normally consumed a high-fat diet were enrolled and treated with either a control diet, a low-fat dietary intervention, Bifidobacterium triple viable capsule therapy, or a combination of a low-fat diet and Bifidobacterium triple viable capsule therapy. Pyrosequencing of the V3 and V4 regions of the 16S rRNA genes was conducted to determine the extent to which probiotics and dietary intervention altered the mucosal microbiota. This study demonstrated that interventional treatment with probiotics and a low-fat diet increased the diversity of the mucosal microbes, dietary intervention alone produced the most significant effect, whereas the combined intervention exhibited no synergetic improvement. Pyrosequencing demonstrated that probiotics and dietary intervention significantly elevated the abundance of some bacterial taxa assigned to the phylum Firmicutes and the beneficial genera Prevotella, Gemmiger, Coprococcus, and Faecalibacterium and reduced some harmful bacterial taxa assigned to the phylum Proteobacteria and genus Streptophyta. The results of this study suggested that the addition of probiotics and dietary intervention could improve the composition of the colonic mucosal microbiota in high-fat diet populations.

A260 THE LACK OF CHROMOGRANIN-A MODIFIES THE GUT MICROBIOTA COMPOSITION AND REGULATES EXPERIMENTAL COLONIC INFLAMMATION

Abstract Background Ulcerative colitis (UC)is characterized by distinct changes in the gut microbiome and elevated chromogranin-A (CHGA) level, which seem to be a relevant pathogenetic mechanism.CHGA, a prohormone produced by enterochromaffin (EC) cells and cleaved into several bioactive peptides, regulates experimental colonic inflammation. In the rodent, intra-rectal infusion of catestatin, a Chga-derived peptide, alters the distal colonic microbial composition. However, the interplay between CHGA, as a pro-hormone, and the gut microbiome remains elusive. Aims in homoeostatic and pathophysiologic conditions, we investigated the functional consequences of the lack of Chgaon the distal colonic microbiota. Methods Acute colitis (5 % dextran sulfate sodium [DSS], 5 days) was induced in Chga-C57BL/6-deficient (Chga-/-) and wild-type (Chga+/+)mice. Feces and mucosa-associated microbiota (MAM) samples were collected and the V4 region of 16s rRNA was subjected to Miseq Illumina sequencing. Alpha diversity was calculated using Shannon’s diversity index. OTU abundances were summarized using the Bray-Curtis index and non-metric multidimensional scaling (NMDS) analysis to visualize microbiome similarities and a permutational analysis of variance (PERMANOVA) to test the significance of groups were performed respectively. Results In non-colitic homoeostatic condition, the absence of Chga (Chga-/) significantly increased the bacterial richness and modified the bacterial community composition at the genera level between the groups, represented by increased abundance of Lactobacillus species and reduced abundance of Helicobacter&amp; Oscillospira species compared to Chga+/+mice in fecal and colonic MAM. Moreover, the absence of Chga (Chga-/-) resulted in a significant change in the alpha-diversity of fecal and colonic MAM compared to Chga+/+mice. DSS induced-colitis resulted in a significant microbial dysbiosis in Chga+/+mice, however, deletion of Chgaprotected against DSS-induced colitis and reduced the microbial dysbiosis, reduced the family of Rikenellaceaeand maintained the abundance of Bacteroides species, compared to wild-type (Chga+/+). Conclusions The lack of CHGA regulates the biodiversity and the composition of the colonic gut microbiota suggesting a cross-talk between the EC cell and the microbiome. Therefore, targeting CHGA could provide a novel therapeutic strategy by regulating the gut microbiome in physiological and pathophysiological conditions. Funding Agencies CIHR