Mucosa-associated Microbiota Research Articles

P1217 The gut microbiota as a determinant in the development of post-surgical recurrence in Crohn`s disease

Abstract Background Crohn's disease (CD), is a chronic condition with a growing incidence in recent decades. Despite the development of new biologic therapies it is estimated that up to 70% of CD patients will require surgical intervention during their lifetime. However, surgery is not considered curative even in cases where a resection of all macroscopic involvement is performed, and the management of post-surgical recurrence remains a therapeutic challenge. Bacterial dysbiosis has been postulated as a precipitating factor in CD and may play a significant role in the development of post-surgical recurrence (PSR) of this condition. We prospectively studied the characteristics of bacterial diversity in patients with CD following ileocecal resection to identify the predictive value of intestinal microbiota in the onset of PSR. Methods We collected fecal and healthy and affected intestinal mucosal samples from 12 patients with CD who underwent surgery during the course of their disease. PSR was defined using a Rutgeerts index ≥i2b. The microbiota was assessed using 16S rRNA sequencing, and a correlation analysis was conducted between the intestinal microbiota and clinical variables of the patients. The study was conducted in accordance with the Helsinki Declaration, and all patients were recruited from among those undergoing scheduled colonoscopy for clinical indications at the Gastroenterology Unit of the University Clinical Hospital San Cecilio in Granada, Spain. Results In patients with CD and severe recurrence, mucosa-associated microbiota was characterized by a significant reduction in alpha and beta diversity compared to subjects in clinical remission. An enrichment of bacteria belonging to the phyla Bacillota and Pseudomonadota was observed, along with a reduction in Bacteroidota in subjects with PSR. We observed a negative correlation between the presence of E. coli and Shigella in patients in clinical remission and those actively undergoing treatment with biological therapy. Conclusion The recolonization of microbiota after ileocecal resection differs between patients with and without recurrence, with Fusobacteria and butyrate-producing bacteria being the most prominent contributors to driving PSR. The reduction in the abundance of short-chain fatty acid-producing bacteria and stimulators of anti-inflammatory cytokines like IL-10 could create a proinflammatory environment conducive to the development of PSR. These findings may provide valuable insights into the disease prognosis, allowing for the identification of patients at higher risk of recurrence and laying the groundwork for more targeted therapeutic approaches after surgery

Nutritional and functional roles of β-mannanase on intestinal health and growth of newly weaned pigs fed two different types of feeds.

This study aimed to investigate the nutritional and functional roles of β-mannanase on the intestinal health and growth of newly weaned pigs fed a typical or low-cost formulated feeds (LCF). Twenty-four newly weaned pigs at 6.2 kg ± 0.4 body weight (BW) were allotted to three dietary treatments based on a randomized complete block design with sex and initial BW as blocks. Three dietary treatments are as follows: Control, typical nursery feeds including animal protein supplements and enzyme-treated soybean meal; LCF with increased amounts of soybean meal, decreased amounts of animal protein supplements, and no enzyme-treated soybean meal; LCF+, low-cost formulated feed with β-mannanase at 100 g/t, providing 800 thermostable β-mannanase unit (TMU) per kg of feed. Pigs were fed based on a three-phase feeding program for a total of 37 d. On day 37 of feeding, all pigs were euthanized and the gastrointestinal tract was removed for sample collection to analyze intestinal health parameters, mucosa-associated microbiota, and gene expression of tight junction proteins. Pigs fed LCF increased (P < 0.05) the relative abundance of Proteobacteria and Helicobacter in the jejunal mucosa, tended to decrease (P = 0.097; P = 0.098) the concentration of malondialdehyde (MDA) and the expression of zona occluden 1 (ZO-1) gene in the jejunum, tended to decrease average daily gain (ADG; P = 0.084) and final BW (P = 0.090), and decreased (P < 0.05) average daily feed intake. Pigs fed LCF + tended to decrease (P = 0.088) digesta viscosity, decreased (P < 0.05) the relative abundance of Helicobacter, and increased (P < 0.05) Lactobacillus in the jejunal mucosa compared to LCF. Additionally, LCF + tended to increase final BW (P = 0.059) and ADG (P = 0.054), increased (P < 0.05) gain to feed ratio (G:F), and reduced (P < 0.05) fecal score compared to LCF. LCF with decreased amounts of animal protein supplements and increased amounts of soybean meal had negative effects on the composition of the mucosa-associated microbiota, intestinal integrity, and growth performance of nursery pigs. Beta-mannanase supplementation to LCF decreased digesta viscosity, increased the relative abundance of potentially health-benefitting microbiota such as Lactobacillus, and improved growth and fecal score, thus reflecting its efficacy in low-cost formulated feeds with increased amounts of soybean meal.

Effects of dietary xylanase supplementation on growth performance, intestinal health, and immune response of nursery pigs fed diets with reduced metabolizable energy.

This study aimed to investigate the effects of xylanase on growth performance and intestinal health of nursery pigs fed diets with reduced metabolizable energy (ME). One hundred ninety-two pigs at 8.7 kg ± 0.7 body weight (BW) after 7 d of weaning were allotted in a randomized complete block design with initial BW and sex as blocks. Eight dietary treatments consisted of 5 ME levels (3,400, 3,375, 3,350, 3,325, and 3,300 kcal ME/kg) below the NRC (2012) requirement and 4 levels of xylanase (0, 1,200, 2,400, and 3,600 XU/kg) to a diet with 3,300 kcal ME/kg. All pigs received their respective treatments for 35 d in 2 phases, pre-starter (14 d) and starter (21 d). On day 35, eight pigs in 3,400 kcal/kg (CON), 3,300 kcal/kg (LE), and 3,300 kcal/kg + 3,600 XU xylanase/kg (LEX) were euthanized to collect jejunal tissues and digesta for the evaluation of mucosa-associated microbiota, intestinal immune response, oxidative stress status, intestinal morphology, crypt cell proliferation, and digesta viscosity as well as ileal digesta to measure apparent ileal digestibility. Data were analyzed using the MIXED procedure on SAS 9.4. The LE increased (P < 0.05) jejunal digesta viscosity, tended to have decreased (P = 0.053) relative abundance of Prevotella, and tended to increase (P = 0.055) Lactobacillus. The LE also increased (P < 0.05) the concentration of protein carbonyl whereas malondialdehyde, villus height (VH), villus height to crypt depth ratio (VH:CD), apparent ileal digestibility (AID) of nutrients, and finally average daily feed intake were decreased (P < 0.05). The LE did not affect average daily gain (ADG). The LEX decreased (P < 0.05) digesta viscosity, increased (P < 0.05) the relative abundance of Prevotella, decreased (P < 0.05) Helicobacter, decreased (P < 0.05) the concentration of protein carbonyl, tended to increase (P = 0.065) VH, and decreased (P < 0.05) VH:CD and crypt cell proliferation. Moreover, LEX increased (P < 0.05) the AID of dry matter and gross energy and tended to increase (P = 0.099; P = 0.076) AID of crude protein, and ether extract. The LEX did not affect ADG but did tend to decrease (P = 0.070) fecal score during the starter phase. Overall, reducing ME negatively affected intestinal health parameters and nutrient digestibility without affecting growth. Supplementation of xylanase mitigated some of the negative effects observed by ME reduction on intestinal health and digestibility of nutrients without affecting growth.

Modeling the effects of prebiotic interventions on luminal and mucosa-associated gut microbiota without and with Clostridium difficile challenge in vitro.

Prebiotics can modulate the gut microbial community composition and function for improved (gut) health and increase resilience against infections. In vitro models of the gut facilitate the study of intervention effects on the gut microbial community relevant to health. The mucosa-associated gut microbiota, which thrives in close contact with the host plays a pivotal role in colonization resistance and health. Therefore, we here introduce the Mi-screen, an experimental approach implementing a 96-well plate equipped with a mucus agar layer for the additional culturing of mucosa-associated microbiota in vitro. In this study, we screened the effects of 2'-Fucosyllactose (2'-FL), fructooligosaccharides (FOS), and inulin within a complex microbiota without and with infection with the C. difficile strains ATCC 43599 (Ribotype 001) or ATCC BAA-1870 (Ribotype 027). We analyzed the microbial community composition and short-chain fatty acid levels after 48 h of incubation. The inclusion of an additional substrate and surface in the form of the mucus agar layer allowed us to culture a microbial richness ranging between 100-160 in Chao index, with Shannon indices of 5-6 across culture conditions, indicative of a microbial diversity of physiological relevance. The mucus agar layer stimulated the growth of characteristic mucosa-associated bacteria such as Roseburia inulinovorans. The prebiotic interventions affected luminal and mucosal microbial communities cultured in vitro and stimulated short-chain fatty acid production. FOS, inulin and 2'-FL promoted the growth of Bifidobacterium adolescentis within the mucosa-associated microbiota cultured in vitro. When spiking the untreated conditions with pathogenic C. difficile, the strains thrived within the luminal and the mucosal sample types, whereas prebiotic treatments exhibited inhibitory effects on C. difficile growth and prevented colonization. In conclusion, the Mi-screen facilitates the screening of luminal and mucosa-associated gut microbial community dynamics in vitro and therefore fills an important gap in the field of in vitro modeling.

Effects of food-grade iron(III) oxide nanoparticles on cecal digesta- and mucosa-associated microbiota and short-chain fatty acids in rats.

Although iron(III) oxide nanoparticles (IONPs) are widely used in diverse applications ranging from food to biomedicine, the effects of IONPs on different locations of gut microbiota and short-chain fatty acids (SCFAs) are unclear. So, a subacute repeated oral toxicity study on Sprague Dawley (SD) rats was performed, administering low (50 mg/kg·bw), medium (100 mg/kg·bw), and high (200 mg/kg·bw) doses of IONPs. In this study, we found that a high dose of IONPs increased animal weight, and 16S rRNA sequencing revealed that IONPs caused intestinal flora disorders in both the cecal digesta- and mucosa-associated microbiota. However, only high-dose IONP exposure changed the abundance and composition of the mucosa-associated microbiota. IONPs increased the relative abundances of Firmicutes, Ruminococcaceae_UCG-014, Ruminiclostridium_9, Romboutsia, and Bilophila and decreased the relative abundance of Bifidobacterium, and many of these microorganisms are associated with weight gain, obesity, inflammation, diabetes, and mucosal damage. Functional analysis showed that changes in the gut microbiota induced by a high dose of IONPs were mainly related to metabolism, infection, immune, and endocrine disease functions. IONPs significantly elevated the levels of valeric, isobutyric, and isovaleric acid, promoting the absorption of iron. This is the first description of intestinal microbiota dysbiosis in SD rats caused by IONPs, and the effects and mechanisms of action of IONPs on intestinal and host health need to be further studied and confirmed.

Supplementation with high-GABA-producing Lactobacillus plantarum L5 ameliorates essential tremor triggered by decreased gut bacteria-derived GABA

BackgroundThe γ-aminobutyric acid (GABA) hypothesis posits a role of GABA deficiency in the central nervous system in the pathogenesis and progression of essential tremor (ET). However, the specific causative factor for GABA deficiency is not clear. The gut microbiota in mammals has recently been considered as a significant source of GABA. Furthermore, the GABA-based signals originating from the intestine can be transmitted to the brain through the “enteric nervous system–vagus nerve–brain” axis. However, the plausible contribution of gut microbiota to ET seems inspiring but remains obscure.MethodsFecal samples from patients with ET and healthy controls were examined by metagenomic sequencing to compare the composition of gut microbiota and the expression of genes involved in GABA biosynthesis. The impact of gut microbiota on ET was explored through transplantation of fecal microbiota from patients with ET into the murine ET model. Lactic acid bacteria producing high amounts of GABA were identified through whole-genome sequencing and ultra-performance liquid chromatography-tandem mass spectrometry. Subsequently, mice were treated with the high-GABA-producing strain Lactobacillus plantarum L5. Tremor severity, behavioral tests, pro-inflammatory cytokines, GABA concentration, and gut microbiota composition were examined in these mice.ResultsThe gut microbiota of patients with ET demonstrated an impaired GABA-producing capacity and a reduced fecal GABA concentration. Transplantation of the gut microbiota from patients with ET induced an extension of tremor duration and impaired mobility in the murine model of ET. L5 exhibited an augmented GABA-producing capacity, with the De Man-Rogosa-Sharpe culture broth containing 262 mg/l of GABA. In addition, administration of L5 significantly decreased the tremor severity and enhanced the movement capability and grasping ability of ET mice. In vivo mechanistic experiments indicated that L5 reshaped the gut microbial composition, supplemented the mucosa-associated microbiota with GABA-producing capacity, increased the GABA concentrations in the cerebellum, and diminished inflammation in the central nervous system.ConclusionsThese findings highlight that deficiency of GABA-producing gut microbes plays an essential role in the pathogenesis of ET and that L5 is a promising candidate for treating ET.

Alterations of ileal mucosa-associated microbiota in hypercholesterolemia patients

Many metabolic diseases have been demonstrated to be associated with changes in the microbiome. However, no studies have yet been conducted to examine the characteristics of the mucosal microbiota of patients with hypercholesterolemia. We aimed to examine mucosa-associated microbiota in subjects with hypercholesterolemia. We conducted a case-control study, in which ileal mucosal samples were collected from 13 hypercholesterolemia patients and 13 controls for 16S rRNA gene sequencing. There were differences in the composition of ileal mucosal microbiota based on beta diversity between the hypercholesterolemia and control groups (P < 0.05). Compared with the control group, the phylum Bacteroidetes and the genera Bacteroides, Butyricicoccus, Parasutterella, Candidatus_Soleaferrea, and norank_f__norank_o__Izemoplasmatales were less abundant in the hypercholesterolemia group (P < 0.05), while the genus Anaerovibrio was enriched in the hypercholesterolemia group (P < 0.05). The relative abundance of Bacteroides was negatively correlated with total cholesterol and low-density lipoprotein cholesterol (P < 0.01). The relative abundance of Coprococcus was negatively correlated with triglycerides and body mass index (all P < 0.05). PICRUSt functional prediction analysis showed that pathways related to Glycerophospholipid metabolism, ABC transporters, Phosphotransferase system, and Biofilm formation - Escherichia coli, and infectious diseases of pathogenic Escherichia coli were enriched in the hypercholesterolemia group. This work suggests a potential role of ileal mucosal microbiota in the development of hypercholesterolemia.

Mucosa-Associated Microbiota Dysbiosis in the Terminal Ileum Correlates With Bowel Symptoms in Diarrhea-Predominant Irritable Bowel Syndrome.

The mucosa-associated microbiota (MAM) is not as frequently studied in diarrhea-predominant irritable bowel syndrome (IBS-D) compared with the fecal microbiota. In this study, we examined the MAM in the terminal ileum and its correlation with bowel symptoms in IBS-D. Mucosal biopsies of the terminal ileum from 25 patients with IBS-D and 25 healthy controls were collected for 16S ribosomal RNA gene sequencing. Correlation analysis was performed. Compared with healthy controls, the MAM in the terminal ileum showed a decreased alpha diversity in the IBS-D cohort (Chao1 and Shannon indexes, P < 0.05). And the overall MAM profile clustered separately into 2 groups (ADONIS [PERMANOVA, permutational multivariate analysis of variance], P < 0.05). At the phylum level, the relative abundance of Proteobacteria was significantly higher in the ileal MAM of patients with IBS-D while that of Firmicutes was significantly lower. At the genus level, the relative abundance of Pseudomonas was significantly higher in the IBS-D cohort, with lower Bacteroides and Ruminococcus . Moreover, 40.0% of patients with IBS-D had multiple small nodules (nodular lymphoid hyperplasia) on the mucosal surface of the terminal ileum, which indicated a low-grade inflammation. In patients with IBS-D with nodular lymphoid hyperplasia, the changes of Pseudomonas and Bacteroides were more overt. Correlation analysis revealed that the relative abundance of Pseudomonas positively correlated with abdominal pain and the severity of IBS. Patients with IBS-D showed a dysbiosis of MAM in the terminal ileum, which may be associated with bowel symptoms. Moreover, 40.0% of them displayed mucosal low-grade inflammation, with a more severe mucosal microbial disturbance.

273 Effects of Xylanase Supplementation in Diets with Reduced Metabolizable Energy and Supplemental Fat on Intestinal Health, Mucosa Associated Microbiota and Growth Performance of Nursery Pigs

Abstract This study aimed to investigate the effects of reducing metabolizable energy (ME) below the requirement by reducing supplemental fat on intestinal health and growth performance of nursery pigs. One hundred and ninety-two newly weaned pigs at 7.3 ± 0.6 kg BW were allotted in a RCBD with sex and initial BW as blocks. Pigs were assigned to one of 8 dietary treatments with varying ME levels (3,400, 3,375, 3,350, 3,325, and 3,300 kcal/kg ME) by reducing supplemental fat and increasing xylanase (X0: 0, X40: 1,200, X80: 2,400 and X120: 3,600 XU/kg of feed) in ME deficient diets by 100 kcals/kg. Pigs were fed diets for 35 d in 2 phases (14 d and 21 d, respectively) and growth performance was measured at the end of each phase. Phase 2 diet had titanium dioxide (0.4%) as indigestible external marker to measure ileal digestibility of nutrients. At d 35, a pig representing the median BW within each pen from 3 dietary treatments (ME at 3,400 and 3,300 kcal/kg ME with or without xylanase) were euthanized for sampling jejunal mucosa, jejunal tissue, and ileal digesta to analyze intestinal health parameters, mucosa associated microbiota, and apparent ileal digestibility (AID) of nutrients. Data were analyzed using Mixed procedure of SAS 9.4. Overall, reducing ME level had no effect on growth performance of nursery pigs. However, reducing ME by 100 kcal/kg ME below the requirement increased (P &amp;lt; 0.05) digesta viscosity (1.7 to 2.2 cP) and protein carbonyl (0.18 to 0.40 nmol/mg), tended to change the relative abundance of Prevotellaceae (P = 0.062, 49 to 23%) and Lactobacillaceae (P = 0.086, 7 to 19%), reduced (P &amp;lt; 0.05) villus height (467 to 433 µm) and villus height to crypt depth ratio (1.80 to 1.64) in the jejunum, and reduced (P &amp;lt; 0.05) AID of DM (59.5 to 47.3%), gross energy (59.1 to 43.0%), crude protein (63.4 to 44.6%), and ether extract (67.7 to 55.2%). Xylanase supplementation decreased (P &amp;lt; 0.05) digesta viscosity (2.2 to 1.82 cP) and protein carbonyl (0.40 to 0.17 nmol/mg), increased the relative abundance of Prevotellaceae (20.22 to 61.56%), increased villus height (433 to 462 µm) and villus height to crypt depth ratio (1.64 to 1.99) in the jejunum, and increased (P &amp;lt; 0.05) AID of DM (47.3 to 58.7%), and gross energy (42.9 to 56.0%). In conclusion, reducing ME (100 kcal/kg below the requirement) by reducing supplemental fat (by 2% unit) impaired intestinal health, reducing ability of the pigs to digest feeds whereas xylanase supplementation appeared to mediate some of the negative effects seen by energy reduction by improving intestinal morphology, reducing oxidative damage products, and diversity of the mucosa-associated microbiota.

145 Dose-Response and Functional Roles of Zinc Glycinate on Growth and Intestinal Health of Nursery Pigs Challenged with F18+Escherichia Coli

Abstract This study was to evaluate dose-response and supplemental effects of zinc glycinate (ZnGly) on growth and intestinal health of nursery pigs challenged with F18+Escherichia coli. Nursery pigs (n = 40; BW = 6.5 ± 0.5 kg) were allotted to 5 treatments in a randomized complete block design with BW and sex as blocks: 1) NC: no-challenge/no-supplement; (2) PC: F18+E. coli challenge/no-supplement; (3 to 5) F18+E. coli challenge/supplementations of ZnGly at 400, 800, and 1,200 mg/kg. On d 7, challenged groups were orally inoculated with F18+E. coli at 6 × 109 CFU/mL, whereas NC received saline solution. Pigs were fed for 28 d based on 2 phases (Phase 1: 14 d and phase 2: 14 d), and experimental diets were formulated to meet or exceed nutrient requirements suggested by NRC (2012). Pigs were euthanized on d 28 to collect tissues to evaluate TNF-α, IL-8, malondialdehyde, protein carbonyl, gene expression of Zn transporters, and mucosa-associated microbiota in the jejunum. Data were analyzed using contrasts in the MIXED procedure of SAS. The E. coli challenge tended to reduce (P = 0.065) ADG (308 to 244 g/d) and increase (P &amp;lt; 0.05) fecal score (3.2 to 4.1) of the pigs during the first week of post-challenge. Supplementation of ZnGly from 0 to 1,200 mg/kg linearly (P = 0.092) increased G:F (0.69 to 0.81) during phase 1 and decreased (P &amp;lt;0.05) fecal score (4.1 to 3.5) of the pigs during the first week of post-challenge. Supplementation of ZnGly from 0 to 1,200 mg/kg had quadratic effects on TNF-α (P = 0.065; minimum 1.51 pg/mg at 405 mg/kg of ZnGly), IL-8 (P = 0.093; minimum 0.53 ng/mg at 494 mg/kg), and protein carbonyl (P = 0.054; minimum 2.30 pg/mg at 675 mg/kg) and linearly increased mRNA expressions of (P = 0.057) ZIP4 (0.69 to 1.03) and (P = 0.075) ZnT5 (0.92 to 1.22) in the jejunum of the pigs. Supplementation of ZnGly from 0 to 1,200 mg/kg tended to linearly decrease (P = 0.065) Chao1 index (150.6 to 144.6) and linearly increased (P &amp;lt; 0.05) relative abundance of Actinobacteria (13.10 to 16.26%) and had quadratic effects (P &amp;lt; 0.05) on the relative abundance of Cyanobacteria (minimum 0.67 % at 625 mg/kg) and Proteobacteria (minimum 45.6% at 735 mg/kg) at the phylum level with Enterobacteriaceae (minimum 4.79% at 660 mg/kg) at family level in jejunal mucosa-associated microbiota of the pigs. In conclusion, supplementation of ZnGly at a range of 400 to 675 mg/kg could reduce the negative impacts of F18+E. coli on growth performance and diarrhea of nursery pigs. These positive changes are related to reducing jejunal inflammation and oxidative stress by enhancing jejunal Zn absorption with positive changes in jejunal mucosa-associated microbiota in the pigs.

146 Efficacy of Zinc Glycinate Reducing Zinc Oxide on Growth and Intestinal Health of Nursery Pigs Challenged with F18+Escherichia Coli

Abstract The objective of this study was to investigate the effect of ZnGly supplementation reducing ZnO in feeds on growth performance and intestinal health of nursery pigs challenged with F18+Escherichia coli (E. coli). A total of 56 nursery pigs (initial BW = 6.5 ± 0.5 kg) were allotted to 7 treatments based on a randomized complete block design using BW and sex as blocks. Treatment groups were: 1) NC: no-challenged with E. coli; 2) PC: challenged with F18+E. coli; 3) challenged with F18+ E. coli and fed ZnO at 2,500 mg/kg in the diet; (4 to 7) challenged with F18+E. coli and fed ZnGly at 400 mg/kg and ZnO at 0, 700, 1,400, and 2,357 mg/kg in the diets. Pigs were fed for 28 days based on 2 phases (Phase 1: 14 d and phase 2: 14 d) and 48 pigs were orally inoculated with F18+ E. coli (6 × 109 CFU/mL) on d 7. Pigs were euthanized on d 28 to collect tissues to evaluate TNF-α, IL-8, malondialdehyde, protein carbonyl, gene expression of Zn transporters, and mucosa-associated microbiota in the jejunum. Data were analyzed by Mixed procedure of SAS. The E. coli challenge tended to decrease (P = 0.076) ADG (200 to 155 g/d) and (P = 0.055) G:F (0.82 to 0.69) during phase 1, and increased (P &amp;lt; 0.05) fecal score (3.2 to 4.1) of nursery pigs during d 7 to 14. There was no difference on the growth performance during the overall period, although pigs fed with the pharmaceutical level of ZnO had greater (P &amp;lt; 0.05) ADG (343 vs. 256 g/d) than pigs fed with ZnGly at 400 mg/kg during the first week of post-challenge. Supplementation of ZnGly with reducing the levels of ZnO from 2,357 to 0 mg/kg tended to have a quadratic response on the fecal score (maximum 4.2 at 667 mg/kg of ZnO) during post-challenge period. Supplementation of ZnGly with reducing levels of ZnO from 2,357 to 0 mg/kg linearly decreased (P &amp;lt; 0.05) IL-8 (0.94 to 0.58 ng/mg) and malondialdehyde (0.49 to 0.34 μM/mg) in jejunum of the pigs. Supplementation of ZnGly reducing the levels of ZnO from 2,357 to 0 mg/kg linearly increased (P &amp;lt; 0.05) Chao1 (142.2 to 148.7) and decreased (P &amp;lt; 0.05) Shannon (4.08 to 3.99) with quadratic effects (P &amp;lt; 0.05) on relative abundance of Pseudomonadaceae (minimum 6.15% at 1,000 mg/kg) and Streptomycetaceae (minimum 5.91% at 800 mg/kg) at family level in jejunum of the pigs. In conclusion, ZnGly supplementation at 400 mg/kg could effectively reduce the use of ZnO in nursery feeds by having similar effects to the pharmaceutical use of ZnO without negatively affecting growth performance and intestinal health of pigs challenged with F18+E. coli.

281 Effects of Corynebacterium Glutamicum Cell Mass Supplementation on Modulation of Mucosa-Associated Microbiota to Intestinal Health of Nursery Pigs

Abstract The objectives were to evaluate the effects of Corynebacterium glutamicum cell mass (CGCM) supplementation, replacing blood plasma in diets, on the gene expressions of biomarkers associated with peptidoglycan and their correlation with intestinal health in nursery pigs. Weaned pigs (n = 32) were allotted to 4 dietary treatments based on the randomized complete block design with initial BW as blocks. The diets with supplementation of CGCM (0, 0.7, 1.4, and 2.1%) replacing blood plasma up to 1.5% were fed for 21 d. Pigs were euthanized on d 21 to collect proximal and distal jejunal mucosa to measure intestinal health markers. Gene expression of receptors associated with peptidoglycan (PGN) and intestinal health were evaluated by PCR. Correlations between gene expressions of receptors associated with PGN and intestinal health markers in jejunal mucosa were assessed by Pearson’s analysis. Data were analyzed using the Mixed procedure of SAS 9.4. Increasing CGCM supplementation affected (quadratic) the relative gene expression of peptidoglycan recognition protein (PGLYRP) 1 (P = 0.080, max: 1.85% at 1.1% CGCM), PGLYRP4 (P &amp;lt; 0.05, max: 3.24% at 1.2% CGCM), and interferon-γ (P = 0.088, max: 3.25% at 1.1% CGCM). Increasing CGCM supplementation did not affect the relative gene expression of PGLYRP2A, PGLYRP2B, PGLYRP3, Toll-like receptor (TLR) 2, TLR4, Nucleotide-binding oligomerization domain-containing protein (NOD) 1, NOD2, cluster of differentiation (CD) 14, and nuclear factor-kappa B. In proximal jejunal mucosa, PGLYRP1 was positively correlated with immunoglobulin A (IgA) and interleukin 8 (r = 0.50 and 0.45, respectively; P &amp;lt; 0.05). PGLYRP4 was positively correlated with protein carbonyl and IgA (r = 0.65 and 0.60, respectively; P &amp;lt; 0.05). Interferon-γ was positively correlated with Campylobacter coli (r = 0.61; P &amp;lt; 0.05) and IgA (r = 0.52; P &amp;lt; 0.05). PGLYRP2A was negatively correlated (r = -0.44; P &amp;lt; 0.05) with Pseudomonadaceae. TLR2 was positively correlated with Corynebacteriaceae, Pasteurellaceae, and Propionibacteriaceae (r = 0.55, 0.51, and 0.52, respectively; P &amp;lt; 0.05). NOD1 was positively correlated (r = 0.50; P &amp;lt; 0.05) with Propionibacteriaceae. NOD2 were positively correlated with Pasteurellaceae and Propionibacteriaceae (r = 0.73 and 0.53, respectively; P &amp;lt; 0.05). CD14 was negatively correlated (r = -0.47; P &amp;lt; 0.05) with IgA. Campylobacteraceae was positively correlated with tumor necrosis factor-alpha (r = 0.50; P &amp;lt; 0.05). Pseudomonadaceae was negatively correlated with IgG (r = -0.39; P &amp;lt; 0.05). In conclusion, supplementing 1.1% to 1.2% CGCM, replacing 0.8 to 0.9% blood plasma, increased the relative gene expressions of PGLYRP1, PGLYRP4, and interferon-γ, which were related to the modulation of the mucosa-associated microbiota, immune response, and oxidative stress markers caused by CGCM supplementation. Supplementing 2.1% CGCM showed to have a similar expression of receptors associated with PGN and intestinal health by promoting the growth of less inflammatory microbiota.

Characteristics of Gastric Mucosa-Associated Microbiota in Patients with Early Gastric Cancer After Successful Helicobacter pylori Eradication.

Helicobacter pylori (H. pylori) is widely recognized as a definite carcinogen in gastric cancer (GC). Although H. pylori eradication reduces the risk of GC, GC recurrence has been detected even after successful H. pylori eradication. Recently, the analysis of gut microbiota was reported. This study aimed to evaluate the correlation between gastric mucosa-associated microbiota (G-MAM) and early gastric cancer (EGC) after successful H. pylori eradication. In this pilot study, G-MAM were collected during the esophagogastroduodenoscopy of 17 patients, receiving H. pylori eradication therapy at least 5years ago. The patients were divided into those with EGC (the EGC group, 8 patients) and those without EGC (the NGC group, 9 patients). Microbial samples in the greater curvature of the pyloric site were obtained using an endoscopic cytology brush, and the G-MAM profiles of each sample were analyzed using 16S rRNA V3-V4 gene sequencing. Between the two groups, there was no significant difference in the median age, sex, median period after successful eradication of H. pylori, the α diversity, and the average abundance at the phylum level. At the genus level, the average abundance of Unclassified Oxalobacteraceae, Capnocytophaga, and Haemophilus was significantly lower in the EGC group than in the NGC group (0.89 vs. 0.14%, P < 0.01, 0.28 vs. 0.00%, P < 0.01 and 5.84 vs. 2.16%, P = 0.034, respectively). We demonstrated alternations in the profiles of G-MAM between the two groups. Our results suggest that G-MAM may influence carcinogenesis after successful H. pylori eradication.

Efficacy of soy protein concentrate replacing animal protein supplements in mucosa-associated microbiota, intestinal health, and growth performance of nursery pigs.

This study investigated the effects of using soy protein concentrate (SPC) to replace animal protein supplements on mucosa-associated microbiota, intestinal health, and growth performance of nursery pigs. Fifty-six newly weaned pigs (BW= 6.4±0.6kg) were allotted to 5 treatments in a randomized complete block design. Pigs were fed for 35d in 3 phases (P; 1, 2, 3) for 10, 12, 13d, respectively. Dietary treatments were: (1) basal diet with fish meal (P1: 4%, P2: 2%, and P3: 1%), poultry meal (P1: 10%, P2: 8%, and P3: 4%), and blood plasma (P1: 4%, P2: 2%, and P3: 1%), where SPC replacing none (NC); (2) basal diet with SPC replacing fish meal (RFM); (3) basal diet with SPC replacing poultry meal (RPM); (4) basal diet with SPC replacing blood plasma (RBP); and (5) basal diet with SPC replacing all animal protein supplements (PC). Growth performance was recorded for each phase. Pigs were euthanized on d 35 to collect jejunal mucosa and tissue to evaluate intestinal health and microbiota, and ileal digesta to measure apparent ileal digestibility (AID) of nutrients. Data were analyzed using the MIXED procedure of SAS. Overall, RFM, RPM, and RBP did not affect growth performance, whereas PC decreased (P<0.05) ADG and ADFI. The RPM increased (P<0.05) Prevotella stercorea and decreased (P<0.05) Helicobacter rappini. The PC decreased (P<0.05) H.rappini, whilst increasing (P<0.05) Prevotella copri, Propionibacterium acnes, and Pelomonas aquatica. The RFM tended to increase (P=0.096) immunoglobulin A in the jejunum. The PC tended to decrease (P=0.078) jejunal crypt cell proliferation. There were no differences in the villus height, AID of nutrients, intestinal inflammation, and intestinal oxidative stress among treatments. In conclusion, SPC can replace fish meal, poultry meal, or blood plasma individually without affecting growth performance and intestinal health, and AID of nutrients of nursery pigs. Particularly SPC replacing poultry meal benefitted intestinal health by reducing H.rappini and increasing P.stercorea. However, SPC replacing all three animal protein supplements reduced growth of nursery pigs mainly by reducing feed intake.

Relationship Between Rectal Swab and Tissue Samples in Mucosa-associated Microbiota in Patients With Inflammatory Bowel Disease.

Gut mucosa-associated microbiota is more closely correlated with disease phenotypes than fecal microbiota; however sampling via tissue biopsy is more invasive and uncomfortable. Rectal swab may be a suitable substitute for tissue biopsy, but its effectiveness is controversial. This study aimed to evaluate differences in the microbiota at these sites in patients with inflammatory bowel disease (IBD). Inflammatory bowel disease patients and a control group were enrolled when surveillance colonoscopy was scheduled. Samples of colon biopsy tissues, rectal swabs during colonoscopy, and feces before bowel preparation were collected to analyze microbial composition. To explore the short-term effects of bowel preparation on swab microbiota, prepreparation swab samples were also collected from 27 IBD patients. A total of 33 Crohn's disease, 54 ulcerative colitis, and 21 non-IBD patients were enrolled. In beta diversity analysis, fecal microbiota clearly differed from swab and tissue microbiota in the 3 disease groups. The swab microbiota was closer to, but still different from, the tissue microbiota. Consistently, we identified that swab samples differed more in abundant genera from feces than from tissue. Beta diversity analysis did not reveal a difference in swab microbiota before and after bowel preparation, but the genus composition of most individuals varied markedly. Swab microbiota more closely resembled tissue microbiota relative to fecal microbiota, but there were still differences. Bowel preparation did not alter the overall swab microbiota in the short term but markedly changed the microbial composition in most patients.

Diverticulosis is not associated with altered gut microbiota nor is it predictive of future diverticulitis: a population-based colonoscopy study

Background The etiopathogenesis of diverticular disease is unknown. Objective To compare the fecal and mucosa-associated microbiota between participants with and without diverticulosis and participants who later developed diverticulitis versus those that did not from a population-based study. Methods The PopCol study, conducted in Stockholm, Sweden, invited a random sample of 3556 adults to participate, of which 745 underwent colonoscopy. Overall, 130 participants (17.5%) had diverticulosis. 16S rRNA gene sequencing was conducted on available sigmoid biopsy samples from 529 and fecal samples from 251 individuals. We identified individuals who subsequently developed acute diverticulitis up to 13 years after sample collection. In a case-control design matching for gender, age (+/−5 years), smoking and antibiotic exposure, we compared taxonomic composition, richness and diversity of the microbiota between participants with or without diverticulosis, and between participants who later developed acute diverticulitis versus those who did not. Results No differences in microbiota richness or diversity were observed between participants with or without diverticulosis, nor for those who developed diverticulitis compared with those who did not. No bacterial taxa were significantly different between participants with diverticulosis compared with those without diverticulosis. Individuals who later developed acute diverticulitis (2.8%) had a higher abundance of genus Comamonas than those who did not (p = .027). Conclusions In a population-based cohort study the only significant difference was that those who later develop diverticulitis had more abundance of genus Comamonas. The significance of Comamonas is unclear, suggesting a limited role for the gut microbiota in the etiopathogenesis of diverticular disease.

Reduction of butyric acid-producing bacteria in the ileal mucosa-associated microbiota is associated with the history of abdominal surgery in patients with Crohn’s disease

ABSTRACTFecal microbiota is a significant factor determining the cause, course, and prognosis of Crohn’s disease (CD). However, the factors affecting mucosa-associated microbiota (MAM) remain unclear. This retrospective study examined the differences in ileal MAM between CD patients and healthy controls and investigated the factors affecting MAM in CD patients to clarify potential therapeutic targets. Ileal MAM was obtained using brush forceps during endoscopic examination from 23 healthy controls and 32 CD patients (most were in remission). The samples’ microbiota was profiled using the Illumina MiSeq platform. Compared to controls, CD patients had significantly reduced α-diversity in the ileum and a difference in β-diversity. The abundance of butyric acid-producing bacteria in the ileal MAM was significantly lower in CD patients with a history of abdominal surgery than in those without. Because butyric acid is a major energy source in the intestinal epithelium, its metabolism via β-oxidation increases oxygen consumption in epithelial cells, reducing oxygen concentration in the intestinal lumen and increasing the abundance of obligate anaerobic bacteria. The suppression of obligate anaerobes in CD patients caused an overgrowth of facultative anaerobes. Summarily, reducing the abundance of butyric acid-producing bacteria in the ileal MAM may play an important role in CD pathophysiology.

Comparative effects of soy protein concentrate, enzyme-treated soybean meal, and fermented soybean meal replacing animal protein supplements in feeds on growth performance and intestinal health of nursery pigs

BackgroundSoy protein supplements, with high crude protein and less antinutritional factors, are produced from soybean meal by different processes. This study evaluated the comparative effects of various soy protein supplements replacing animal protein supplements in feeds on the intestinal immune status, intestinal oxidative stress, mucosa-associated microbiota, and growth performance of nursery pigs.MethodsSixty nursery pigs (6.6 ± 0.5 kg BW) were allotted to five treatments in a randomized complete block design with initial BW and sex as blocks. Pigs were fed for 39 d in 3 phases (P1, P2, and P3). Treatments were: Control (CON), basal diet with fish meal 4%, 2%, and 1%, poultry meal 10%, 8%, and 4%, and blood plasma 4%, 2%, and 1% for P1, P2, and P3, respectively; basal diet with soy protein concentrate (SPC), enzyme-treated soybean meal (ESB), fermented soybean meal with Lactobacillus (FSBL), and fermented soybean meal with Bacillus (FSBB), replacing 1/3, 2/3, and 3/3 of animal protein supplements for P1, P2, and P3, respectively. Data were analyzed using the MIXED procedure in SAS 9.4.ResultsThe SPC did not affect the BW, ADG, and G:F, whereas it tended to reduce (P = 0.094) the ADFI and tended to increase (P = 0.091) crypt cell proliferation. The ESM did not affect BW, ADG, ADFI, and G:F, whereas tended to decrease (P = 0.098) protein carbonyl in jejunal mucosa. The FSBL decreased (P < 0.05) BW and ADG, increased (P < 0.05) TNF-α, and Klebsiella and tended to increase MDA (P = 0.065) and IgG (P = 0.089) in jejunal mucosa. The FSBB tended to increase (P = 0.073) TNF-α, increased (P < 0.05) Clostridium and decreased (P < 0.05) Achromobacter and alpha diversity of microbiota in jejunal mucosa.ConclusionsSoy protein concentrate, enzyme-treated soybean meal, and fermented soybean meal with Bacillus could reduce the use of animal protein supplements up to 33% until 7 kg body weight, up to 67% from 7 to 11 kg body weight, and entirely from 11 kg body weight without affecting the intestinal health and the growth performance of nursery pigs. Fermented soybean meal with Lactobacillus, however, increased the immune reaction and oxidative stress in the intestine consequently reducing the growth performance.

Metabarcoding of colonic cleansing fluid reveals unique bacterial members of mucosal microbiota associated with Inflammatory Bowel Disease

Background Inflammatory Bowel Disease (IBD) is a group of chronic idiopathic inflammatory diseases of the gastrointestinal (GI) tract associated with the dysbiosis of gut microbiota. Metabarcoding-based profiling of the gut microbiota of IBD patients is generally based on the stool samples collected from individual patients which rarely represent the mucosa-associated microbiota. The ideal sampling strategy for routine monitoring of the mucosal component of IBD has yet to be determined. Methods We hereby compare the microbiota composition of the colonic cleansing fluid (CCF) collected during colonoscopy with stool samples from IBD patients. The relationship between IBD and gut microbiota was revealed through the application of the 16S rRNA amplicon sequencing-based metabarcoding approach. CCF and stool samples were collected from IBD patients with Crohn’s disease and ulcerative colitis. Results The present study shows significant differences in the microbial composition of CCF samples, presumably indicating changes in the mucosal microbiota of IBD patients as compared to the control group. Short-chain fatty acid-producing bacteria under the family Lachnospiraceae, the actinobacterial genus Bifidobacterium, the proteobacterial Sutterella and Raoultella are found to contribute to the microbial dysbiosis of the mucosal flora in IBD patients. Conclusions CCF microbiota has the capacity to distinguish IBD patients from healthy controls and, thus, may constitute an alternative analysis strategy for the early diagnosis and disease progression in IBD biomarker research.

Effects of dosing non-toxigenic Clostridia on the bacterial populations and immunological responses in the intestinal tract of lactating dairy cows.

Understanding the effects of dosing non-toxigenic Clostridia to cows is rare and has received little attention so far. In the present study, a total of eight lactating dairy cows were divided in two groups: control (n = 4) or Clostridia challenged (oral supplementation of five diverse strains of Paraclostridium bifermentans, n = 4). Bacterial communities were analyzed by qPCR and next-generation sequencing (NGS) in the buccal mucosa as well as digesta and mucosal samples of the gastrointestinal (GI) tract from rumen to rectum (10 compartments), as well as fecal samples. Transcriptomic analysis of barrier and immune-related gene expression was performed on rumen, jejunum, and liver samples. We observed increased microbial populations with the Clostridial challenge in the buccal tissues and the proximal GI tract (forestomach), correlating with Clostridial loads in the feed. Otherwise, there were no significant differences in microbial populations (p > 0.05) throughout the distal part of the GI tract. The NGS approach, however, revealed that the Clostridial challenge changed the relative abundance of gut and fecal microbiota. In particular, in the challenge group, no Bifidobacterium was observed in the mucosa-associated microbiota and abundance of Pseudomonadota increased in the feces. These results indicated potential adverse effects of Clostridia to cow health. In general, immune responses to the Clostridial challenge were weak. However, transcriptional analysis revealed the down-regulation of junction adhesion molecule encoding gene (-1.44 of log2 fold-change), which might impact intestinal permeability.