Mucosa-associated Microbiota Research Articles

Co-frequency or contrary? The effects of Qiwei Baizhu Powder and its bioactive compounds on mucosa-associated microbiota of mice with antibiotic-associated diarrhea.

Qiwei Baizhu Powder (QWBZP) has been proven effective in treating antibiotic-associated diarrhea (AAD), and the mechanism is associated with regulating the gut microbiota. However, the role of the bioactive compounds of QWBZP in regulating the gut microbiota is still unclear. In this study, 24 mice were divided into a normal control group (N), a model group (R), a QWBZP decoction group (TW), and a QWBZP-TG group (TG). AAD mouse models were established by mixed antibiotic administration. After modeling, mice in the TW group and TG group were treated with QWBZP decoction and QWBZP-TG, respectively. Mice in the N group and R group were gavaged with sterile water. 16S rRNA gene sequencing was used to investigate the changes of mucosa-associated microbiota (MAM) in the small intestine of mice. Moreover, the levels of diamine oxidase (DAO), D-Lactate, secretory immunoglobulin A (sIgA), interleukin 6 (IL-6), IL-10, and tumor necrosis factor-α (TNF-α) were detected using enzyme-linked immunosorbent assay (ELISA) kits. The results showed that QWBZP-TG significantly altered the diversity, structure, and abundance of MAM in the AAD mice. QWBZP-TG exerted a stronger suppression effect on Escherichia and Clostridium compared with QWBZP decoction. Meanwhile, QWBZP-TG downregulated the abundance of Lactobacillus, which elicited an opposite effect to QWBZP decoction. Prevotella was the signature bacteria that responded to the QWBZP-TG intervention. Furthermore, both QWBZP decoction and QWBZP-TG decreased the levels of DAO, D-Lactate, sIgA, IL-6, and TNF-α in the AAD mice. The role of glycosides is to help QWBZP ameliorate diarrhea symptoms by inhibiting the proliferation of diarrhea-associated bacteria, reducing inflammation and regulating immunity.

Effects of dietary Lactobacillus postbiotics and bacitracin on the modulation of mucosa-associated microbiota and pattern recognition receptors affecting immunocompetence of jejunal mucosa in pigs challenged with enterotoxigenic F18+ Escherichia coli

BackgroundEnterotoxigenic Escherichia coli (E. coli) is a threat to humans and animals that causes intestinal disorders. Antimicrobial resistance has urged alternatives, including Lactobacillus postbiotics, to mitigate the effects of enterotoxigenic E. coli.MethodsForty-eight newly weaned pigs were allotted to NC: no challenge/no supplement; PC: F18+ E. coli challenge/no supplement; ATB: F18+ E. coli challenge/bacitracin; and LPB: F18+ E. coli challenge/postbiotics and fed diets for 28 d. On d 7, pigs were orally inoculated with F18+ E. coli. At d 28, the mucosa-associated microbiota, immune and oxidative stress status, intestinal morphology, the gene expression of pattern recognition receptors (PRR), and intestinal barrier function were measured. Data were analyzed using the MIXED procedure in SAS 9.4.ResultsPC increased (P < 0.05) Helicobacter mastomyrinus whereas reduced (P < 0.05) Prevotella copri and P. stercorea compared to NC. The LPB increased (P < 0.05) P. stercorea and Dialister succinatiphilus compared with PC. The ATB increased (P < 0.05) Propionibacterium acnes, Corynebacterium glutamicum, and Sphingomonas pseudosanguinis compared to PC. The PC tended to reduce (P = 0.054) PGLYRP4 and increased (P < 0.05) TLR4, CD14, MDA, and crypt cell proliferation compared with NC. The ATB reduced (P < 0.05) NOD1 compared with PC. The LPB increased (P < 0.05) PGLYRP4, and interferon-γ and reduced (P < 0.05) NOD1 compared with PC. The ATB and LPB reduced (P < 0.05) TNF-α and MDA compared with PC.ConclusionsThe F18+ E. coli challenge compromised intestinal health. Bacitracin increased beneficial bacteria showing a trend towards increasing the intestinal barrier function, possibly by reducing the expression of PRR genes. Lactobacillus postbiotics enhanced the immunocompetence of nursery pigs by increasing the expression of interferon-γ and PGLYRP4, and by reducing TLR4, NOD1, and CD14.

Characteristics of Mucosa-Associated Microbiota in Ulcerative Colitis Patients with 5-Aminosalicylic Acid Intolerance.

5-Aminosalicylic acid (5-ASA) is a first-line therapy for ulcerative colitis (UC). This study examined the mucosa-associated microbiota (MAM) in UC patients, distinguishing between those who were 5-ASA tolerant and intolerant. Brushing samples were collected from the sigmoid and ileal end of patients with UC during endoscopic procedures. The samples were profiled by using the Illumina MiSeq platform. The V3-V4 regions of the 16S rRNA gene (460 bp) were amplified by using tailed PCR. A total of 15 patients with 5-ASA intolerance, 38 patients with 5-ASA tolerance, and 19 healthy controls were recruited in this study. The α-diversity indices were remarkably different among the three groups in the ileum mucosa but not in the sigmoid colon. In the ileum mucosa, Alistipes, Ruminococcaceae, and Odoribacter were less abundant in the 5-ASA-intolerant group than in the control and 5-ASA-tolerant groups. On the contrary, Merdibacter, Brevundimonas, and Porphyromonas were more abundant in the 5-ASA-intolerant group than in other groups. The present study showed that the changes in MAM were characterized by a decrease in mucoprotective bacteria rather than an increase in harmful bacteria.

Reduced Abundance of Phocaeicola in Mucosa-associated Microbiota Is Associated with Distal Colorectal Cancer Metastases Possibly through an Altered Local Immune Environment.

The aim of this study was to identify the microbiota whose decrease in tumor area was associated with the metastatic process of distal colorectal cancer (CRC). Twenty-eight consecutive patients with distal CRC undergoing surgical resection in our hospital were enrolled. Microbiota in 28 specimens from surgically resected colorectal cancers were analyzed using 16S ribosomal ribonucleic acid gene amplicon sequencing and the relative abundance (RA) of microbiota was evaluated. The densities of tumor-infiltrating lymphocytes (TIL) and tumor associated macrophages (TAM) in the colorectal cancers were immunohistochemically evaluated. Phocaeicola was the most abundant microbiota in normal mucosa. The RA of Phocaeicola in tumor tissues tended to be lower than that in normal mucosa although the difference was not significant (p=0.0732). The RA of Phocaeicola at tumor sites did not correlate either with depth of tumor invasion (pT-stage) or tumor size, however they were significantly reduced in patients with nodal metastases (p<0.05) and those with distant metastases (p<0.001). The RA of Phocaeicola at tumor sites showed positive correlation with the densities of CD3(+) or CD8(+) TIL. Since P. vulgatus was the most dominant species (47%) of the Phocaeicola, the RA of P. vulgatus and CRC metastasis and its association with TIL and TAM were also investigated. P. vulgatus showed a similar trend to genus Phocaeicola but was not statistically significant. A relative reduction of Phocaeicola attenuates the local anti-tumor immune response in distal CRC, which may facilitate metastatic spread.

Integration of metagenomics and metaproteomics in the intestinal lavage fluids benefits construction of discriminative model and discovery of biomarkers for HBV liver diseases.

Intestinal lavage fluid (IVF) containing the mucosa-associated microbiota instead of fecal samples was used to study the gut microbiota using different omics approaches. Focusing on the 63 IVF samples collected from healthy and hepatitis B virus-liver disease (HBV-LD), a question is prompted whether omics features could be extracted to distinguish these samples. The IVF-related microbiota derived from the omics data was classified into two enterotype sets, whereas the genomics-based enterotypes were poorly overlapped with the proteomics-based one in either distribution of microbiota or of IVFs. There is lack of molecular features in these enterotypes to specifically recognize healthy or HBV-LD. Running machine learning against the omics data sought the appropriate models to discriminate the healthy and HBV-LD IVFs based on selected genes or proteins. Although a single omics dataset is basically workable in such discrimination, integration of the two datasets enhances discrimination efficiency. The protein features with higher frequencies in the models are further compared between healthy and HBV-LD based on their abundance, bringing about three potential protein biomarkers. This study highlights that integration of metaomics data is beneficial for a molecular discriminator of healthy and HBV-LD, and reveals the IVF samples are valuable for microbiome in a small cohort.

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.

Do fish gut microbiotas vary across spatial scales? A case study of Diplodus vulgaris in the Mediterranean Sea

BackgroundBiogeography has been linked to differences in gut microbiota in several animals. However, the existence of such a relationship in fish is not clear yet. So far, it seems to depend on the fish species studied. However, most studies of fish gut microbiotas are based on single populations. In this study, we investigated the gut microbiota of fish from three wild populations of the two-banded sea bream Diplodus vulgaris (Geoffroy Saint-Hilaire, 1817) to determine whether its diversity, structure and potential functionality reflect the geographic origin of the fish, at large and small geographical scale. Additionally, we explored the host- and environmental-related factors explaining this relationship.ResultsWe showed that the taxonomy and potential functionality of the mucosa-associated gut microbiota of Diplodus vulgaris differ to varying degrees depending on the spatial scale considered. At large scale, we observed that both the taxonomical structure and the potential functionality of the fish microbiota differed significantly between populations. In contrast, the taxonomical diversity of the microbial community displayed a significant relationship with factors other than the geographic origin of the fish (i.e. sampling date). On the other hand, at small scale, the different composition and diversity of the microbiota differ according to the characteristics of the habitat occupied by the fish. Specifically, we identified the presence of Posidonia oceanica in the benthic habitat as predictor of both the microbiota composition and diversity. Lastly, we reported the enrichment of functions related to the metabolism of xenobiotics (i.e. drugs and 4-aminobenzoate) in a population and we indicated it as a potential target of future monitoring.ConclusionsWith this study, we confirmed the importance of investigating the gut microbiota of wild fish species using multiple populations, taking into account the different habitats occupied by the individuals. Furthermore, we underscored the use of the biodegradation potential of the gut microbiota as an alternative means of monitoring emerging contaminants in Mediterranean fish.

Celiac Disease: The Importance of Studying the Duodenal Mucosa-Associated Microbiota.

There is increasing evidence indicating that changes in both the composition and functionality of the intestinal microbiome are closely associated with the development of several chronic inflammatory diseases, with celiac disease (CeD) being particularly noteworthy. Thanks to the advent of culture-independent methodologies, the ability to identify and quantify the diverse microbial communities residing within the human body has been significantly improved. However, in the context of CeD, a notable challenge lies in characterizing the specific microbiota present on the mucosal surfaces of the intestine, rather than relying solely on fecal samples, which may not fully represent the relevant microbial populations. Currently, our comprehension of the composition and functional importance of mucosa-associated microbiota (MAM) in CeD remains an ongoing field of research because the limited number of available studies have reported few and sometimes contradictory results. MAM plays a crucial role in the development and progression of CeD, potentially acting as both a trigger and modulator of the immune response within the intestinal mucosa, given its proximity to the epithelial cells and direct interaction. According to this background, this review aims to consolidate the existing literature specifically focused on MAM in CeD. By elucidating the complex interplay between the host immune system and the gut microbiota, we aim to pave the way for new interventions based on novel therapeutic targets and diagnostic biomarkers for MAM in CeD.

Exercise Affects Mucosa-Associated Microbiota and Colonic Tumor Formation Induced by Azoxymethane in High-Fat-Diet-Induced Obese Mice.

The only reliable factor that reduces the risk of colorectal carcinogenesis is physical activity. However, the underlying mechanisms remain unclear. In this study, we examined the effects of physical activity against gut microbiota, including mucosa-associated microbiota (MAM) on azoxymethane-induced colorectal tumors in obese mice. We divided the subjects into four groups: normal diet (ND), high-fat diet (HFD), ND + exercise (Ex), and HFD + Ex groups. The Ex group performed treadmill exercise for 20 weeks. Thereafter, fecal and colonic mucus samples were extracted for microbiota analysis. DNA was collected from feces and colonic mucosa, and V3-V4 amplicon sequencing analysis of the 16SrRNA gene was performed using MiSeq. The HFD group had significantly more colonic polyps than the ND group (ND 6.5 ± 1.3, HFD 11.4 ± 1.5, p < 0.001), and the addition of Ex suppressed the number of colonic polyps in ND and HFD groups (ND 6.5 ± 1.3, ND + Ex 2.8 ± 2.5, p < 0.05). The HFD group showed significantly lower concentrations of succinic, acetic, butyric, and propionic acids (mg/g) in feces, compared with the ND group (succinic acid HFD 0.59, ND 0.17; acetic acid HFD 0.63, ND 2.41; propionic acid HFD 0.10, ND 0.47; and N-butyric acid HFD 0.31, ND 0.93). In the case of ND, succinic acid and butyric acid tended to decrease with Ex (succinic acid ND 0.17, ND + Ex 0.12; N-butyric acid ND 0.93, ND + Ex 0.74 0.74). Succinic acid, acetic acid, butyric acid, and propionic acid levels in feces were significantly lower in the HFD group than in the ND group; in both feces and mucus samples, Butyricicoccus and Lactobacillus levels were significantly lower in the HFD group. Akkermansia was significantly increased in ND + Ex and HFD + Ex groups. Diet and exercise affected the number of colorectal tumors. Furthermore, diet and exercise alter intestinal MAM, which may be involved in colorectal tumor development.

Investigation of the nutritional and functional roles of a combinational use of xylanase and β-glucanase on intestinal health and growth of nursery pigs

BackgroundXylanase and β-glucanase combination (XG) hydrolyzes soluble non-starch polysaccharides that are anti-nutritional compounds. This study aimed to evaluate the effects of increasing levels of XG on intestinal health and growth performance of nursery pigs.MethodsForty pigs (6.5 ± 0.4 kg) were assigned to 5 dietary treatments and fed for 35 d in 3 phases (11, 9, and 15 d, respectively). Basal diets mainly included corn, soybean meal, and corn distiller’s dried grains with solubles, contained phytase (750 FTU/kg), and were supplemented with 5 levels of XG at (1) 0, (2) 280 TXU/kg xylanase and 125 TGU/kg β-glucanase, (3) 560 and 250, (4) 840 and 375, or (5) 1,120 and 500, respectively. Growth performance was measured. On d 35, all pigs were euthanized and jejunal mucosa, jejunal digesta, jejunal tissues, and ileal digesta were collected to determine the effects of increasing XG levels and XG intake on intestinal health.ResultsIncreasing XG intake tended to quadratically decrease (P = 0.059) viscosity of jejunal digesta (min: 1.74 mPa·s at 751/335 (TXU/TGU)/kg). Increasing levels of XG quadratically decreased (P < 0.05) Prevotellaceae (min: 0.6% at 630/281 (TXU/TGU)/kg) in the jejunal mucosa. Increasing XG intake quadratically increased (P < 0.05) Lactobacillaceae (max: 40.3% at 608/271 (TXU/TGU)/kg) in the jejunal mucosa. Increasing XG intake quadratically decreased (P < 0.05) Helicobacteraceae (min: 1.6% at 560/250 (TXU/TGU)/kg) in the jejunal mucosa. Increasing levels of XG tended to linearly decrease (P = 0.073) jejunal IgG and tended to quadratically increase (P = 0.085) jejunal villus height to crypt depth ratio (max: 2.62 at 560/250 (TXU/TGU)/kg). Increasing XG intake tended to linearly increase the apparent ileal digestibility of dry matter (P = 0.087) and ether extract (P = 0.065). Increasing XG intake linearly increased (P < 0.05) average daily gain.ConclusionsA combinational use of xylanase and β-glucanase would hydrolyze the non-starch polysaccharides fractions, positively modulating the jejunal mucosa-associated microbiota. Increased intake of these enzyme combination possibly reduced digesta viscosity and humoral immune response in the jejunum resulting in improved intestinal structure, and ileal digestibility of nutrients, and finally improving growth of nursery pigs. The beneficial effects were maximized at a combination of 550 to 800 TXU/kg xylanase and 250 to 360 TGU/kg β-glucanase.

183 Evaluation of phytogenic formulations on intestinal health and the performance of nursery pigs challenged with F18+Escherichia coli.

Abstract The objective of this study was to evaluate phytogenic formulations on intestinal health and growth performance of nursery pigs challenged with F18+Escherichia coli. Weaned pigs [n = 40; initial body weight (BW) = 6.4 ± 0.1 kg] at 21 d of age were individually housed and assigned to 4 dietary treatments in a randomized complete block design, with initial BW and sex as blocking factors. Basal diets were formulated following NRC (2012) and fed to pigs for 28 d in 3 phases (P1:11 d, P2:10 d and P3:7 d). Treatments were NC: basal diet, non-challenge; PC: basal diet, challenged with F18+E. coli; AV1: basal diet supplemented with 1% phytobiotic containing herbal extracts and essential oils in powder form, challenged with F18+E. coli; AV2: basal diet + 1% phytobiotic containing herbal extracts and essential oils in liquid form, challenged with F18+E. coli. Growth performance and fecal score (FS) were measured weekly. On d 7, the challenged groups were orally inoculated with F18+E. coli (2.0 × 1010), whereas unchallenged pigs received a sterile saline solution. On d 28, all pigs were euthanized to collect jejunal samples to evaluate inflammatory status, oxidative stress, and diversity and relative abundance of mucosa-associated microbiota. Data were analyzed using the MIXED procedure on SAS 9.4. During the post-challenge period, PC decrease (P &amp;lt; 0.05) average daily gain (ADG) and average daily feed intake (ADFI) when compared with NC. The PC increased (P &amp;lt; 0.05) FS during d 7 to 11 when compared with NC, whereas AV1 and AV2 decreased (P &amp;lt; 0.05) FS during d 7 to 11 when compared with PC. The AV1 decreased (P &amp;lt; 0.05) Chao1 and Shannon indexes when compared with PC. The PC increased (P &amp;lt; 0.05) the relative abundance of Prevotellaceae, Lachnospiraceae, and Ruminococcaceae compared with NC. The AV1 reduced (P &amp;lt; 0.05) the relative abundance of Veillonellaceae, Prevotellaceae, Lachnospiraceae and tended to reduce (P = 0.062) the relative abundance of Ruminococcaceae when compared with PC. The AV2 tended to reduce the relative abundance of Streptococcaceae (P = 0.073) and Ruminococcaceae (P = 0.074) when compared with PC. The PC increased (P &amp;lt; 0.05) occludin and tended to increase (P = 0.096) TLR4 when compared with NC. The AV1 tended to increase (P = 0.053) protein carbonyl when compared with PC. In conclusion, F18+E. coli challenge increased FS, disrupted the mucosa-associated microbiota, increased TLR4 expression, consequently reduced growth performance of pigs. The AV1 showed promise in reducing oxidative stress during F18+E. coli challenge, seen through decreased protein carbonyl levels possibly due to alterations on mucosa-associated microbiota, although without affecting growth performance. The AV2 had a moderate impact on reducing FS and altering mucosa-associated microbiota, without effect on growth performance.

63 Impacts of dietary myristic acids on mucosa-associated microbiota in relation to intestinal health and growth parameters of nursery pigs

Abstract This study investigated the evaluate the role of dietary myristic acid on mucosa-associated microbiota for their relevance to intestinal health and growth performance of nursery pigs. A total of 36 newly weaned pigs [6.6 ± 0.4 kg body weight (BW)] were assigned to 3 treatments (n = 12/treatment) using a randomized complete block design with initial BW and sex as blocks. Pigs were fed for 35 d in 3 phases (10, 10, and 15 d, respectively). Treatments were 1) NC: basal diet; 2) PC: NC + 0.03% bacitracin in all phases; and 3) MA: NC + myristic acid complex at 0.20% in phases 1 and 2 and 0.12% in phase 3. Pigs were euthanized to collect jejunal mucosa and jejunal tissues for microbiome sequencing and measuring intestinal health parameters. Data were analyzed using preplanned contrasts (NC vs. PC and NC vs. MA) in the Proc MIXED procedure and correlation coefficients between microbiota and the intestinal health parameters were determined by the CORR procedure of SAS. The PC increased (P &amp;lt; 0.05) Lactobacillaceae (22.2 to 55.1%) compared with NC. The MA increased (P &amp;lt; 0.05) Veillonellaceae (1.7 to 5.2%), Lachnospiraceae (0.3 to 3.6%), Coriobacteriaceae (0.6 to 1.5%), and Ruminococcaceae (0.5 to 2.1%) and tended to increase (P = 0.072) Bifidobacteriaceae (6.9 to 18.2%) compared with NC. The PC increased (P &amp;lt; 0.05) Simpson (0.72 to 0.89) index and the MA increased (P &amp;lt; 0.05) Chao1 (124.3 to 221.0), Shannon (3.4 to 5.0), and Simpson (0.7 to 0.9) indexes compared with NC. The relative abundance (RA) of Helicobacteraceae was positively correlated with IL-8 (r = 0.63; P &amp;lt; 0.05) and protein carbonyl (r = 0.65; P &amp;lt; 0.05). The RA of Bifidobacteriaceae was negatively correlated with IgG (r = -0.51; P &amp;lt; 0.05), IL-8 (r = -0.58; P &amp;lt; 0.05), TNF-α (r = -0.46; P &amp;lt; 0.05), and protein carbonyl (r = -0.64; P &amp;lt; 0.05) whereas it was positively correlated with average daily gain (ADG; r = 0.51; P &amp;lt; 0.05) and average daily feed intake (ADFI; r = 0.49; P &amp;lt; 0.05). The RA of Lactobacillaceae negatively correlated with IL-8 (r = -0.59; P &amp;lt; 0.05) and protein carbonyl (r = -0.54; P &amp;lt; 0.05) whereas it was positively correlated with ADFI (r = 0.43; P &amp;lt; 0.05) and MDA (r = 0.44; P &amp;lt; 0.05). The RA of Veilonellaceae and Megasphaera sp were negatively correlated (r = -0.49; r = -0.61; P &amp;lt; 0.05) with IgG, respectively. In conclusion, MA mainly increased RA of Bifidobacterium and Megasphaera, whereas PC increased RA of Lactobacillus and Mitsuokella in the jejunal mucosa. The positive modulations of RA of mucosa-associated microbiota by PC and MA were strongly correlated with improved intestinal health and growth of nursery pigs.

45 Awardee Talk: Investigation of the nutritional and functional roles of a combinational use of xylanase and β-glucanase on intestinal health and growth of nursery pigs

Abstract The objective of this study was to evaluate the intestinal health and growth performance of nursery pigs affected by dietary supplementation of increasing levels of xylanase and b-glucanase combinations. Pigs [n = 40; initial body weight (BW) = 6.5 ± 0.4 kg] were assigned to 5 dietary treatments and fed for 35 d in 3 phases (11, 9, and 15 d, respectively). Basal diets mainly included corn, soybean meal, and distiller’s dried grains with solubles with phytase (750 FTU/kg) supplemented with 5 levels of xylanase and b-glucanase combinations (NSPase) at 1) 0, 2) 280 TXU/kg xylanase and 125 TGU/kg b-glucanase, 3) 560 and 250, 4) 840 and 375, or 5) 1,120 and 500, respectively. Growth performance and fecal score were measured for each phase. On d 35, all pigs were euthanized and collected jejunal mucosa, jejunal digesta, jejunal tissues, and ileal digesta to determine the effects of increasing NSPase levels (%) and NSPase intake on jejunal mucosa-associated microbiota, jejunal digesta viscosity, immune responses, intestinal morphology, feed digestibility, and growth performance. Increasing NSPase level quadratically decreased (P &amp;lt; 0.05) Bacteroidetes (min: 0.7% at 636 TXU/kg and 284 TGU/kg), Prevotellaceae (min: 0.6% at 630 TXU/kg and 281 TGU/kg) in the jejunal mucosa. Increasing NSPase intake quadratically increased (P &amp;lt; 0.05) Firmicutes (max: 72.7% at 604 TXU/kg and 270 TGU/kg), Lactobacillaceae (max: 40.3% at 608 TXU/kg and 271 TGU/kg), and Lactobacillus (max: 40.3% at 608 TXU/kg and 271 TGU/kg) in the jejunal mucosa. Increasing NSPase intake also quadratically decreased (P &amp;lt; 0.05) Proteobacteria (min: 3.8% at 562 TXU/kg and 251 TGU/kg), Helicobacteraceae (min: 1.6% at 560 TXU/kg and 250 TGU/kg), and Helicobacter (min: 1.6% at 560 TXU/kg and 250 TGU/kg) in the jejunal mucosa. Increasing NSPase intake tended to quadratically decrease (P = 0.059) viscosity of jejunal digesta reaching the minimum (1.74 mPa·s) at 751 TXU/kg and 335 TGU/kg. Increasing NSPase level tended to linearly decrease (P = 0.073) jejunal IgG and tended to quadratically increase (P = 0.085) jejunal villus height to crypt depth ratio reaching the maximum (2.62) at 560 TXU/kg and 250 TGU/kg. Increasing NSPase intake tended to linearly increase the apparent ileal digestibility of dry matter (P = 0.087) and ether extract (P = 0.065). Increasing NSPase intake linearly increased (P &amp;lt; 0.05) average daily gain (ADG). In conclusion, increasing the combinational use of xylanase and β-glucanase positively modulated jejunal mucosa-associated microbiota, reduced jejunal digesta viscosity, reduced intestinal humoral immune response, enhanced villus morphology, increased ileal digestibility of nutrients, and increased growth of nursery pigs. The beneficial effects were maximized when xylanase and β-glucanase were supplemented at a range of 550 to 800 TXU/kg and 246 to 357 TGU/kg, respectively.

Altered mucosal bacteria and metabolomics in patients with Peutz–Jeghers syndrome

BackgroundPeutz–Jeghers syndrome (PJS) is a rare genetic disorder characterized by the development of pigmented spots, gastrointestinal polyps and increased susceptibility to cancers. Currently, most studies have investigated intestinal microbiota through fecal microbiota, and there are few reports about mucosa-associated microbiota. It remains valuable to search for the key intestinal microbiota or abnormal metabolic pathways linked to PJS.AimThis study aimed to assess the structure and composition of mucosa-associated microbiota in patients with PJS and to explore the potential influence of intestinal microbiota disorders and metabolite changes on PJS.MethodsThe bacterial composition was analyzed in 13 PJS patients and 12 controls using 16S rRNA gene sequencing (Illumina MiSeq) for bacteria. Differential analyses of the intestinal microbiota were performed from the phylum to species level. Liquid chromatography-tandem mass spectrometry (LC‒MS) was used to detect the differentially abundant metabolites of PJS patients and controls to identify different metabolites and metabolic biomarkers of small intestinal mucosa samples.ResultsHigh-throughput sequencing confirmed the special characteristics and biodiversity of the mucosa microflora in patients with PJS. They had lower bacterial biodiversity than controls. The abundance of intestinal mucosal microflora was significantly lower than that of fecal microflora. In addition, lipid metabolism, amino acid metabolism, carbohydrate metabolism, nucleotide metabolism and other pathways were significantly different from those of controls, which were associated with the development of the enteric nervous system, intestinal inflammation and development of tumors.ConclusionThis is the first report on the mucosa-associated microbiota and metabolite profile of subjects with PJS, which may be meaningful to provide a structural basis for further research on intestinal microecology in PJS.

Isolation of mucosa-associated microbiota dysbiosis in the ascending colon in hepatitis C virus post-sustained virologic response cirrhotic patients.

Achieving sustained virologic response (SVR) in patients infected with hepatitis C virus (HCV) reduces all-cause mortality. However, the mechanisms and risk factors for liver fibrosis and portal hypertension post-SVR remain incompletely understood. In the gut-liver axis, mucosa-associated microbiota (MAM) substantially influence immune and metabolic functions, displaying spatial heterogeneity at the anatomical intestinal site. We analyzed MAM composition and function to isolate the locoregional MAM involved in chronic liver disease progression in HCV post-SVR patients. We collected MAM samples from three intestinal sites (terminal ileum, ascending colon, and sigmoid colon) via brushing during colonoscopy in 23 HCV post-SVR patients and 25 individuals without liver disease (controls). The 16S rRNA of bacterial DNA in specimens collected with a brush and in feces was sequenced. The molecular expression of intestinal tissues and hepatic tissues were evaluated by quantitative real-time PCR. In the post-SVR group, the microbial β-diversity of MAM, especially in the ascending colon, differed from the control group and was associated with liver fibrosis progression. In PICRUSt analysis, MAM in the ascending colon in the liver cirrhosis (LC) group showed compromised functions associated with the intestinal barrier and bile acid production, and FGF19 expression was markedly decreased in the terminal ileum biopsy tissue in the LC group. At the genus level, six short-chain fatty acid (SCFA)-producing bacterial genera, Blautia, Alistipes, Roseburia, Agathobaculum, Dorea, and Pseudoflavonifractor were reduced in the ascending colon of post-SVR LC patients. In patients of HCV post-SVR, we identified the association between the degree of liver fibrosis and dysbiosis of mucosa-associated SCFA-producing bacterial genera that may be related to intestinal barrier and bile acid production in the ascending colon.

Heat stress and feeding effects on the mucosa-associated and digesta microbiome and their relationship to plasma and digesta fluid metabolites in the jejunum of dairy cows

The intestinal microbiota plays a pivotal role in digestive processes and maintains gut health and intestinal homeostasis. These functions may be compromised by increased environmental heat which in turn reduces feed intake and gut integrity, while activating the intestinal immune system. It remains unknown whether high ambient temperatures, causing heat stress (HS) to dairy cows, disturb the eubiosis of the microbial community and if so, to which extent the reduction in feed intake and the impairment of circulating and intestinal metabolites account for the alterations of the jejunal microbiota. To address these questions, jejunal digesta, mucosa, and plasma samples from cows exposed to heat stress (HS: 28°C, temperature-humidity-index (THI) = 76, n = 10), control conditions (CON: 16°C, THI = 60, n = 10), or pair-feeding (PF: 16°C, THI = 60, n = 10) for 7 d were collected. Digesta fluids were examined for pH, acetate, nonesterified fatty acids (NEFA), glucose, and lactate, while plasma samples were analyzed for glucose, lactate, β-hydroxybutyrate (BHB), triglycerides, NEFA, creatinine and urea. The microbiota of digesta and mucosa samples were analyzed by 16S rRNA sequencing. The α diversity was higher in mucosa than digesta, but not affected by high ambient temperatures. However, the mucosa-associated microbiota appears more responsive to ambient heat than the digesta microbiome. The adaptive responses under HS conditions comprised an increased mucosal abundance of Bifidobacteriaceae, Succinivibrionaceae UCG-001, Clostridia and Lactobacillus. In the digesta, HS has exerted effects on microbial abundance of Colidextribacter and Lachnospiraceae UCG-008. Several correlations between plasma or intestinal metabolites and microbiota were elucidated, including Methanobacteriaceae correlating positively with plasma BHB and digesta glucose concentrations. Moreover, the reduction in feed intake during HS had non-negligible effects on microbial diversity and the abundance of certain taxa, underpinning the importance of nutrient supply on maintaining intestinal homeostasis.

Effect of a Lactobacilli-Based Direct-Fed Microbial Product on Gut Microbiota and Gastrointestinal Morphological Changes.

The calf's gastrointestinal tract (GIT) microbiome undergoes rapid shifts during early post-natal life, which can directly affect calf performance. The objectives of this study were to characterise and compare differences in the establishment and succession of GIT microbiota, GIT morphological changes, and the growth of dairy calves from birth until weaned. Forty-four newborn Holstein-Friesian calves were randomly selected and assigned to Treatment (TRT) and Control (CON) groups. The TRT group calves received a once-daily dose of a direct-fed microbial (DFM) liquid product containing Lacticaseibacillus paracasei, Lentilactobacillus buchneri, and Lacticaseibacillus casei, all formerly known as Lactobacillus. Fresh faecal samples were manually taken from the rectum of all calves, and gross necropsy was performed on the forestomachs and gastrointestinal tracts. Bacterial DNA was extracted from frozen faecal samples for 16S rRNA gene amplicon sequencing. Calves in the TRT group had greater live weights (p = 0.02) at weaning compared with calves in the CON group (mean = 69.18 kg, SD = 13.37 kg). The average daily live weight gain (ADG) and total feed intake were similar between the two groups. Calves in the TRT group had greater duodenum, abomasum, and reticulum weights (p = 0.05). Rumen and intestinal development (p < 0.05) and faecal microbial diversity (p < 0.05) were more pronounced in the TRT group. The relative abundances of eight genera differed (p < 0.001) between the groups. Supplementing calves with the LAB-based DFM increased live weight at weaning and had a more pronounced effect on the development of rumen and the gastrointestinal tract and on microbiota diversity and evenness. Future work is needed to better understand the potential association of LAB-DFM products on gut mucosa-associated microbiota.

Chronic intestinal pseudo-obstruction: associations with gut microbiota and genes expression of intestinal serotonergic pathway

BackgroundPediatric chronic intestinal pseudo-obstruction (PIPO) is a rare disease characterized by symptoms and radiological signs suggestive of intestinal obstruction, in the absence of lumen-occluding lesions. It results from an extremely severe impairment of propulsive motility. The intestinal endocrine system (IES) jointly with the enteric nervous system (ENS) regulates secreto-motor functions via different hormones and bioactive messengers/neurotransmitters. The neurotransmitter 5-hydroxytryptamine (5-HT) (or serotonin) is linked to intestinal peristalsis and secretory reflexes. Gut microbiota and its interplay with ENS affect 5-HT synthesis, release, and the subsequent serotonin receptor activation. To date, the interplay between 5-HT and gut microbiota in PIPO remains largely unclear. This study aimed to assess correlations between mucosa associated microbiota (MAM), intestinal serotonin-related genes expression in PIPO. To this purpose, biopsies of the colon, ileum and duodenum have been collected from 7 PIPO patients, and 7 age-/sex-matched healthy controls. After DNA extraction, the MAM was assessed by next generation sequencing (NGS) of the V3-V4 region of the bacterial RNA 16 S, on an Illumina Miseq platform. The expression of genes implicated in serotoninergic pathway (TPH1, SLC6A4, 5-HTR3 and 5-HTR4) was established by qPCR, and correlations with MAM and clinical parameters of PIPO have been evaluated.ResultsOur results revealed that PIPO patients exhibit a MAM with a different composition and with dysbiosis, i.e. with a lower biodiversity and fewer less connected species with a greater number of non-synergistic relationships, compared to controls. qPCR results revealed modifications in the expression of serotonin-related intestinal genes in PIPO patients, when compared to controls. Correlation analysis do not reveal any kind of connection.ConclusionsFor the first time, we report in PIPO patients a specific MAM associated to underlying pathology and an altered intestinal serotonin pathway. A possible dysfunction of the serotonin pathway, possibly related to or triggered by an altered microbiota, may contribute to dysmotility in PIPO patients. The results of our pilot study provide the basis for new biomarkers and innovative therapies targeting the microbiota or serotonin pathways in PIPO patients.

OP04 Endoscopic healing in paediatric IBD perpetuates a persistent signature characterized by pathogenic Th17 cells and tissue-associated molecular and microbial drivers of disease

Abstract Background Endoscopic healing (EH) is the major long-term treatment target for inflammatory bowel diseases (IBD) in adults and paediatric patients. However, EH may not represent disease clearance. Hence, risk of relapse remains high and treatment discontinuation after reaching EH often results in disease exacerbation. We aimed to identify persistent cellular, molecular and microbial drivers of IBD under EH and longitudinally collected endoscopic biopsies from paediatric patients for analysis of single T-cell and bulk transcriptomics as well as mucosa-associated microbiota. Methods We followed disease trajectories of paediatric IBD patients treated according to international guidelines and collected mucosal biopsies from the terminal ileum (TI) and sigmoid colon (SC) at baseline (T1) and after approximately 3-12 months (T2) to assess EH. Non-IBD controls showed no macroscopic or histologic signs of inflammation. Host RNA and bacterial DNA were simultaneously extracted from single biopsies for bulk RNA-seq, and 16S rRNA seq. A second biopsy was used for isolation of lamina propria mononuclear cells (LPMCs), followed by CD45+, CD3+ cell sorting and single T-cell analysis (10X Genomics). Data were quality controlled and integrated. Results 217 biopsies (135 simultaneous extraction of host RNA and bacterial DNA; 82 single T-cell analysis) from 32 paediatric IBD patients (mean age 13 ± 3.5 years, 21 Crohn’s disease [CD], 11 ulcerative colitis [UC]; T1 = 32, T2 = 32, T &amp;gt; 2 = 6) and 5 non-IBD controls were analysed. Time between T1 and T2 averages 40 ± 22 weeks. At baseline, 15 patients (14 CD, 1 UC) were newly diagnosed and 31/32 patients had active mucosal inflammation. Twenty-two patients (71%) achieved EH at T2 (SES-CED ≤2 and absence of ulcerations), mostly on anti-TNF therapy. One patient (EH at T1) experienced relapsing disease within 10 weeks after discontinuing medication. In the bulk RNA-seq, we identified 299 differential expressed genes (DEGs; corrected p-value &amp;lt;0.05, FC &amp;gt;2.0), such as DUOX2, SAA2-SAA4, FCGR3B and NOS2, that are associated with active IBD and remained upregulated in EH in contrast to non-IBD controls. In addition single cell analysis revealed an IBD-specific pathogenic Th17 cluster that continued to exist in EH and showed high correlation with top DEGs after data integration. 16S rRNA gene seq highlighted highly individual mucosa-associated bacterial profiles and a reduced α-diversity in active and EH compared to non-IBD controls. Conclusion A persisting IBD signature in EH reflects ongoing cellular, molecular and microbial activity in comparison to non-IBD controls despite EH and mucosal regeneration. These markers may provide targets for future or sequential therapies.

P424 Analysis of the intestinal microbiome using an Endoscopic Brush in Ulcerative colitis

Abstract Background The precise pathogenesis of the Ulcerative colitis is still yet unknown, but one of its cause is known to be microbial dysbiosis. The mucosa-associated microbiota are more deeply involved in the pathogenesis of UC. However, the optimal sampling of mucosa-associated microbiome has yet to be investigated. In this study, we investigated the mucosa-associated microbiome in patients with ulcerative colitis, using endoscopic brush samples. We hypothesized that endoscopic brushing is precise and noninvasive method to get sample of mucosa-associated microbiome. Methods Patients with UC who visited the gastroenterology department of Korea University Anam hospital were screened for this study. Clinical data such as medical records, colonoscopy and fecal samples were reviewed. Using a stool and saliva sample collector kit respectively, the subjects provided stool and saliva samples. Brushing samples were collected during the sigmoidoscopy procedure with 3-4 brush strokes on the colon mucosa using the cytology brush. The samples were analyzed for microbiome in the Korea University Medical Center. Results From July 2022 to January 2023, we prospectively enrolled 19 patients with UC. Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria were the most common species in microbiota of brush, stool and saliva.(Fig.1-1) The microbiome between stool and brush was no significant difference in alpha and beta diversities.(Fig.1-2) However, Oral microbiome was different from stool and brush in beta diversities.(Fig.1-3) Patients were categorized into to analyze the oral microbiome. A trend was observed where increased disease severity was associated with an increase in Firmicutes.(Fig.1-4) Conclusion The microbiome of stool and brush was no significantly different. However, the novel sampling of mucosa-associated microbiome, endoscopic brush, is not inferior compared to currently used sampling of stool. Also the analysis of the oral microbiome suggested that Firmicutes could be considered a useful biomarker for assessing disease severity. Therefore, it is necessary to conduct followup research by increasing the number of subjects.