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Inflammatory bowel diseases (IBD) are affected by dietary factors, including nondigestible carbohydrates (fibers), which are fermented by colonic microbes. Fibers are overall beneficial, but not all fibers are alike, and some patients with IBD report intolerance to fiber consumption. Given reproducible evidence of reduced fiber-fermenting microbes in patients with IBD, we hypothesized that fibers remain intact in select patients with reduced fiber-fermenting microbes and can then bind host cell receptors, subsequently promoting gut inflammation.Colonic biopsies cultured ex vivo and cell lines in vitro were incubated with oligofructose (5 g/L), or fermentation supernatants (24-hour anaerobic fermentation) and immune responses (cytokine secretion [enzyme-linked immunosorbent assay/meso scale discovery] and expression [quantitative polymerase chain reaction]) were assessed. Influence of microbiota in mediating host response was examined and taxonomic classification of microbiota was conducted with Kraken2 and metabolic profiling by HUMAnN2, using R software.Unfermented dietary β-fructan fibers induced proinflammatory cytokines in a subset of IBD intestinal biopsies cultured ex vivo, and immune cells (including peripheral blood mononuclear cells). Results were validated in an adult IBD randomized controlled trial examining β-fructan supplementation. The proinflammatory response to intact β-fructan required activation of the NLRP3 and TLR2 pathways. Fermentation of β-fructans by human gut whole microbiota cultures reduced the proinflammatory response, but only when microbes were collected from patients without IBD or patients with inactive IBD. Fiber-induced immune responses correlated with microbe functions, luminal metabolites, and dietary fiber avoidance.Although fibers are typically beneficial in individuals with normal microbial fermentative potential, some dietary fibers have detrimental effects in select patients with active IBD who lack fermentative microbe activities. The study is publicly accessible at the U.S. National Institutes of Health database (clinicaltrials.gov identification number NCT02865707).

Persistent inflammation can trigger altered epigenetic, inflammatory, and bioenergetic states. Inflammatory bowel disease (IBD) is an idiopathic disease characterized by chronic inflammation of the gastrointestinal tract, with evidence of subsequent metabolic syndrome disorder. Studies have demonstrated that as many as 42% of patients with ulcerative colitis (UC) who are found to have high-grade dysplasia, either already had colorectal cancer (CRC) or develop it within a short time. The presence of low-grade dysplasia is also predictive of CRC. Many signaling pathways are shared among IBD and CRC, including cell survival, cell proliferation, angiogenesis, and inflammatory signaling pathways. Current IBD therapeutics target a small subset of molecular drivers of IBD, with many focused on the inflammatory aspect of the pathways. Thus, there is a great need to identify biomarkers of both IBD and CRC, that can be predictive of therapeutic efficacy, disease severity, and predisposition to CRC. In this study, we explored the changes in biomarkers specific for inflammatory, metabolic, and proliferative pathways, to help determine the relevance to both IBD and CRC. Our analysis demonstrated, for the first time in IBD, the loss of the tumor suppressor protein Ras associated family protein 1A (RASSF1A), via epigenetic changes, the hyperactivation of the obligate kinase of the NOD2 pathogen recognition receptor (receptor interacting protein kinase 2 [RIPK2]), the loss of activation of the metabolic kinase, AMP activated protein kinase (AMPKα1), and, lastly, the activation of the transcription factor and kinase Yes associated protein (YAP) kinase, that is involved in proliferation of cells. The expression and activation status of these four elements are mirrored in IBD, CRC, and IBD-CRC patients and, importantly, in matched blood and biopsy samples. The latter would suggest that biomarker analysis can be performed non-invasively, to understand IBD and CRC, without the need for invasive and costly endoscopic analysis. This study, for the first time, illustrates the need to understand IBD or CRC beyond an inflammatory perspective and the value of therapeutics directed to reset altered proliferative and metabolic states within the colon. The use of such therapeutics may truly drive patients into remission.

A relationship between ulcerative colitis (UC) and diet has been shown in epidemiological and experimental studies. In a 6-month, open-label, randomized, placebo-controlled trial, adult UC patients in clinical remission were randomized to either an “Anti-inflammatory Diet (AID)” or “Canada’s Food Guide (CFG)”. Menu plans in the AID were designed to increase the dietary intake of dietary fiber, probiotics, antioxidants, and omega-3 fatty acids and to decrease the intake of red meat, processed meat, and added sugar. Stool was collected for fecal calprotectin (FCP) and microbial analysis. Metabolomic analysis was performed on urine, serum, and stool samples at the baseline and study endpoint. In this study, 53 patients were randomized. Five (19.2%) patients in the AID and 8 (29.6%) patients in the CFG experienced a clinical relapse. The subclinical response to the intervention (defined as FCP < 150 µg/g at the endpoint) was significantly higher in the AID group (69.2 vs. 37.0%, p = 0.02). The patients in the AID group had an increased intake of zinc, phosphorus, selenium, yogurt, and seafood versus the control group. Adherence to the AID was associated with significant changes in the metabolome, with decreased fecal acetone and xanthine levels along with increased fecal taurine and urinary carnosine and p-hydroxybenzoic acid levels. The AID subjects also had increases in fecal Bifidobacteriaceae, Lachnospiraceae, and Ruminococcaceae. In this study, we found thatdietary modifications involving the increased intake of anti-inflammatory foods combined with a decreased intake of pro-inflammatory foods were associated with metabolic and microbial changes in UC patients in clinical remission and were effective in preventing subclinical inflammation.

BackgroundUlcerative colitis (UC) is associated with altered intestinal microbiome (‘dysbiosis’), most significantly, reduced strict butyrate-producing anaerobes and increased facultative anaerobes. Inulin-type prebiotics appear to reduce and prevent colitis in preclinical studies and small clinical trials. However, these results need to be validated in randomized controlled clinical trial (RCT) studies.AimsThe aim of this RCT study was to assess the efficacy of β-fructans (oligofructose and inulin) in preventing relapses in UC patients in clinical remission as well as identify potential mechanisms of activity.MethodsAdult UC patients in clinical remission (total Mayo score ≤ 2) were randomized to supplement their diet with 15g/d of either β-fructans (oligofructose and inulin; Synergy1/Prebiotin) or placebo (maltodextrin) for 6 months. Partial Mayo scores, medications, adverse events and intervention compliance were monitored monthly. Fecal specimens were collected throughout the study to measure fecal calprotectin (FCP), along with stool metabolites.ResultsEighty-nine UC patients in clinical remission were randomized to β-fructans (n=43) or placebo (n=46). Of those, 11 participants never started, 2 were withdrawn, and 76 were included in the study population (β-fructans n=35; placebo n=41). Although there was no difference in symptomatic clinical flare (β-fructans n=11 flare; placebo n=10 flare, P=0.60), patients randomized to oligofructose and inulin showed only a 3-fold median FCP increase versus 17-fold in the placebo group (P=0.038). Interestingly, antibiotics and serotonin reuptake inhibitors significantly increased the relative risk (RR) of flare (RR 3.321, 95% Cl 2.005 to 5.344, P < 0.0001). β-Fructan intake significantly increased anti-inflammatory fecal metabolites (arabinose, L-arabitol, 5-oxo-D-proline).ConclusionsAlthough oligofructose and inulin did not prevent symptomatic relapses in UC patients, their oral administration significantly reduced the severity of biochemical relapse compared to placebo which was further associated with increased anti-inflammatory metabolites.

Background Dietary fibers pass through the bowel undigested and are fermented within the intestine by microbes, typically promoting gut health. However, many IBD patients describe experiencing sensitivity to fibers. β-glucan, found on the surface of fungal cells during fungal infection, has been shown to bind to fiber receptors, such as Dectin-1, on host immune cells, resulting in a pro-inflammatory response. These fungal fibres share properties with dietary fibers. Aims As an altered gut microbial composition has been associated with IBD, we hypothesized that the loss of fiber-fermenting microbes populating the gut in IBD could lead to dietary fibers not being efficiently broken down into their beneficial biproducts (e.g. short chain fatty acids; SCFA), resulting in binding of intact fibers to pro-inflammatory host cell receptors. Methods Immune and epithelial cell lines and colonic biopsies cultured ex vivo were incubated with oligofructose or inulin (5g/L), or pre-fermented fibers (24hr anaerobic fermentation). Immune responses were measured by cytokine secretion (ELISA), and expression (qPCR). Barrier integrity was measured by transepithelial resistance (TEER). Food frequency questionnaire (FFQ) data of patient fiber consumption were correlated with gut microbes (shotgun sequencing) and immune responses to fiber in patient biopsies. Results Unfermented oligofructose induced IL-1β secretion in leukocytes (macrophage, T cell, neutrophil) and in colon biopsies from pediatric Crohn disease (CD; n=38) and ulcerative colitis (UC; n=20) patients cultured ex vivo, but not in non-IBD patients (n=21). IL-1β secretion was greater in patients with more severe disease. Pre-fermentation of oligofructose by whole-microbe intestinal washes from non-IBD patients or remission patients reduced secretion of IL-1β, while whole microbe intestinal washes from severe IBD patients were unable to ferment oligofructose or reduce cytokine secretion. Fiber effects on IL-1β secretion in biopsies positively correlated with effects on barrier integrity in T84 cells. Fiber-associated immune responses in patient biopsies cultured ex vivo (ELISA) correlated with fiber avoidance (FFQ) and gut microbiome (sequencing) in matching patient samples. Conclusions Our findings demonstrate that intolerance and avoidance of prebiotic fibers in select IBD patients is associated with the inability to ferment these fibers, leading to pro-inflammatory immune responses and intestinal barrier disruption. This highlights select disease state scenarios, in which administration of fermentable fibers should be avoided and tailored dietary interventions should be considered in IBD patients. Funding Agencies CIHRWeston Foundation

The intestinal microbiota is involved in ulcerative colitis (UC) pathogenesis. Prebiotics are hypothesized to improve health through alterations to gut microbiota composition and/or activity. Our aim was therefore to determine if inulin-type fructans induce clinical benefits in UC, and identify if benefits are linked to compositional and/or functional shifts of the luminal (fecal) and mucosal (biopsy) bacterial communities. Patients (n = 25) with mild/moderately active UC received 7.5 g (n = 12) or 15 g (n = 13) daily oral oligofructose-enriched inulin (Orafti®Synergy1) for 9 weeks. Total Mayo score, endoscopic activity and fecal calprotectin were assessed. Fecal and mucosal bacterial communities were characterized by 16S rRNA tag sequencing, and short chain fatty acids (SCFA) production were measured in fecal samples. Fructans significantly reduced colitis in the high-dose group, with 77% of patients showing a clinical response versus 33% in the low-dose group (P = 0.04). Fructans increased colonic butyrate production in the 15 g/d dose, and fecal butyrate levels were negatively correlated with Mayo score (r = −0.50; P = 0.036). The high fructan dose led to an increased Bifidobacteriaceae and Lachnospiraceae abundance but these shifts were not correlated with improved disease scores. In summary, this pilot study revealed that 15 g/d dose inulin type fructans in UC produced functional but not compositional shifts of the gut microbiota, suggesting that prebiotic-induced alterations of gut microbiota metabolism are more important than compositional changes for the benefits in UC. The findings warrant future well-powered controlled studies for the use of β-fructans as adjunct therapy in patients with active UC.

AIM: To identify demographic, clinical, metabolomic, and lifestyle related predictors of relapse in adult ulcerative colitis (UC) patients.METHODS: In this prospective pilot study, UC patients in clinical remission were recruited and followed-up at 12 mo to assess a clinical relapse, or not. At baseline information on demographic and clinical parameters was collected. Serum and urine samples were collected for analysis of metabolomic assays using a combined direct infusion/liquid chromatography tandem mass spectrometry and nuclear magnetic resolution spectroscopy. Stool samples were also collected to measure fecal calprotectin (FCP). Dietary assessment was performed using a validated self-administered food frequency questionnaire.RESULTS: Twenty patients were included (mean age: 42.7 ± 14.8 years, females: 55%). Seven patients (35%) experienced a clinical relapse during the follow-up period. While 6 patients (66.7%) with normal body weight developed a clinical relapse, 1 UC patient (9.1%) who was overweight/obese relapsed during the follow-up (P= 0.02). At baseline, poultry intake was significantly higher in patients who were still in remission during follow-up (0.9 ozvs0.2 oz,P= 0.002). Five patients (71.4%) with FCP > 150 μg/g and 2 patients (15.4%) with normal FCP (≤ 150 μg/g) at baseline relapsed during the follow-up (P= 0.02). Interestingly, baseline urinary and serum metabolomic profiling of UC patients with or without clinical relapse within 12 mo showed a significant difference. The most important metabolites that were responsible for this discrimination were trans-aconitate, cystine and acetamide in urine, and 3-hydroxybutyrate, acetoacetate and acetone in serum.CONCLUSION: A combination of baseline dietary intake, fecal calprotectin, and metabolomic factors are associated with risk of UC clinical relapse within 12 mo.

Background The etiology of inflammatory bowel diseases (IBD) remains unknown, although gut microorganisms and diet have been implicated. Dietary fibers pass through the bowel undigested and are fermented within the intestine by microbes, promoting gut health. However, many IBD patients describe experiencing sensitivity to fibres. Interestingly, fiber receptors on immune cells are able to interact with fibers typically found on the surface of fungal cells (which share properties with dietary fibers), for example, resulting in a paradoxical pro-inflammatory response. Aims As an altered microbial composition is a hallmark of IBD, we hypothesized that the loss of fiber fermenting-microbes populating the IBD gut could lead to dietary fibers not being efficiently broken down into their beneficial biproducts, resulting in binding of intact fibers to pro-inflammatory host cell receptors. This can ultimately drive pro-inflammatory responses and a microenvironment that promotes continued dysbiosis and increased pathogenicity of select microbes, as observed in IBD. Methods Fiber receptor expression gut was examined using immunohistochemistry and flow cytometry and demonstrated elevated receptor expression due to increased presence of immune cells in IBD patient biopsies. Cytokine secretion, in response to fiber (5mg/mL) or pre-fermented fibers, cultured with microbes of interest, was measured by ELISAs in cell lines in vitro and biopsy tissues cultured ex vivo. Results Whole-fibers induced pro-inflammatory cytokine production in macrophage, monocytes, and neutrophils. Specific microbes were capable of fermenting fiber, measured by gas chromatography. Pre-fermentation of fibers by these microbes reduced inflammatory cytokine production. The fiber oligofructose increased IL-1β in pediatric CD (n=44) and UC (n=29) biopsies cultured ex vivo but not in non-IBD (n=25). The increase was greater in patients with more severe disease. Pre-fermentation of oligofructose by bacteria reduced this secretion of IL-1β. Whole-microbe intestinal washes from severe IBD patients were unable to ferment oligofructose or reduce fiber-associated inflammation in macrophage cells compared to remission or non-IBD children. Statistical analysis of food frequency questionnaire (FFQ) data on fiber consumption demonstrated that fiber-associated inflammation in patient biopsies cultured ex vivo (ELISA and qPCR) correlated with fiber avoidance (FFQ). Conclusions Comparing in vitro findings to our patient FFQs, intestinal washes (microbe abundance), and detailed patient history will better define the relationship between microbes, dietary fibers, and gut inflammation in IBD. This will allow for tailored dietary intervention through dietary recommendations, prebiotic, and/or probiotic therapies. Funding Agencies CCCWeston Foundation, WCHRI

BACKGROUND: Inflammatory bowel diseases (IBD), including Crohn disease and ulcerative colitis, are chronic and severely debilitating gastrointestinal disorders, with rising prevalence, especially in children. While the etiology of IBD remains poorly understood, gut microorganisms and diet have been implicated in IBD pathogenesis. Interestingly, dietary fibers pass through the small bowel undigested and are fermented within the large intestine by specific gut microbes, promoting gut health by altering the gut microenvironment, modulation of the immune system, and altered energy metabolism. Our previous work indicates an altered balance between commensal and pathobiont microbes in IBD, suggesting one opportunity for the misregulation of fiber fermentation within the IBD gut. Interestingly, specific carbohydrate receptors on the surface of immune cells interact with fibers, such as those expressed by certain species of fungi, driving an inflammatory response. AIMS: Here we hypothesize that the altered microbiota in IBD causes significant changes in fiber fermentation, ultimately driving intestinal inflammation. METHODS: Bacterial growth rates in response to specific fibers were examined by OD600 growth curves. Bacterial fiber fermentation patterns were evaluated by mass spectrometry of SCFAs. The effects of specific fibers on bacterial pathogenicity when co-cultured with human HT29 epithelial cells was measured by gentamicin protection assays and visualized by microscopy. Immunohistochemistry of biopsy tissues collected from pediatric IBD (n=14) and control patients (n=7) was utilised to examine expression levels of fiber receptors expressed on human cells. RESULTS: We have demonstrated that commensal microorganism, isolated live from IBD patients, display a different pattern of fiber fermentation compared to pathobiont species. Specific fibers also increase the invasive potential of these pathobionts. Preliminary immunohistochemistry suggests that there is increased expression of specific carbohydrate receptors in IBD, suggesting an increased sensitivity to unfermented dietary fibers in these patients. CONCLUSIONS: These results suggest that some patients may display a sensitivity to specific dietary fibers due to a lack of appropriate fiber fermenting microbes in their gut. Profiling the patient specific microbiota of IBD patients may allow us to make more specific dietary recommendations to assist in reducing intestinal inflammation in IBD. Comparing in vitro findings to our readily available patient food frequency questionnaires (FFQs) and detailed patient history will allow us to define key disease features of IBD and also identify new and improved treatment options, such as microbe-altering treatments, and tailored nutritional recommendations. FUNDING AGENCIES: CCCCCFC

BACKGROUND: The association between diet and inflammatory bowel disease remains largely unknown. To date, few studies have been conducted to investigate the association between the inflammatory potential of a diet and disease activity in patients with inflammatory bowel disease. AIMS: We conducted this study to evaluate the relationship between dietary inflammatory index (DII) and fecal calprotectin (FCP) levels in a cohort of ulcerative colitis (UC) patients. METHODS: This was a cross-sectional study on adult UC patients who were in clinical remission (partial Mayo250 µg/g which is associated with higher disease activity and increased probability of UC relapse was considered as high. RESULTS: Fifty-nine UC patients were recruited. Their mean age was 40.3 ± 14.2 years and 61% of them were females. Their mean DII score was -0.3 ± 1.9. Patients with high FCP had higher DII scores than patients with low FCP (0.8 ± 1.7 vs. -0.8 ± 1.8, P=0.004). UC patients with a more pro-inflammatory diet (DII score > -0.5 (median value)) were more likely to have high fecal calprotectin in comparison to patients with a less pro-inflammatory diet (44.8 vs. 16.7%, P=0.02, Odds ratio: 4.1, 95% confidence interval: 1.2–13.6). In addition, Spearman analysis suggested a significant correlation between FCP and DII scores (r=0.35, P=0.007). Among different components of DII, FCP was correlated significantly with dietary intake of alcohol (r= -0.29), β-Carotene (r= -0.44), fiber (r= -0.23), polyunsaturated fatty acids (r= -0.22), and vitamin E (r= -0.42). CONCLUSIONS: We found that a pro-inflammatory diet was related to increased FCP in UC patients. These findings suggest that an association between diet and increased disease activity or relapse might be mediated through inflammatory properties of the diet. FUNDING AGENCIES: Alberta Innovates - Bio Solutions

Background Inflammatory bowel diseases (IBD) are a group of complex and multifactorial disorders with unknown etiology. Chronic intestinal inflammation develops against resident intestinal bacteria in genetically susceptible hosts. We hypothesized that host intestinal immunoglobulin (Ig) G can be used to identify bacteria involved in IBD pathogenesis. Results IgG-bound and -unbound microorganisms were collected from 32 pediatric terminal ileum aspirate washes during colonoscopy [non-IBD (n = 10), Crohn disease (n = 15), and ulcerative colitis (n = 7)], and composition was assessed using the Illumina MiSeq platform. In vitro analysis of invasive capacity was evaluated by fluorescence in situ hybridization and gentamicin invasion assay; immune activation was measured by qPCR. Despite considerable inter-individual variations, IgG binding favored specific and unique mucosa-associated species in pediatric IBD patients. Burkholderia cepacia, Flavonifractor plautii, and Rumminococcus sp. demonstrated increased IgG binding, while Pseudomonas ST29 demonstrated reduced IgG binding, in IBD. In vitro validation confirmed that B. cepacia, F. plautii, and Rumminococcus display invasive potential while Pseudomonas protogens did not. Conclusion Using IgG as a marker of pathobionts in larger patient cohorts to identify microbes and elucidate their role in IBD pathogenesis will potentially underpin new strategies to facilitate development of novel, targeted diagnostic, and therapeutic approaches. Interestingly, this method can be used beyond the scope of this manuscript to evaluate altered gut pathobionts in a number of diseases associated with altered microbiota including arthritis, obesity, diabetes mellitus, alcoholic liver disease, cirrhosis, metabolic syndrome, and carcinomas. Electronic supplementary material The online version of this article (10.1186/s40168-018-0604-3) contains supplementary material, which is available to authorized users.

The increase in chronic metabolic and immunologic disorders in the modern society is linked to major changes in the dietary patterns. These chronic conditions are associated with intestinal microbiota dysbiosis where important groups of carbohydrate fermenting, short-chain fatty acids-producing bacteria are reduced. Dietary prebiotics are defined as a selectively fermented ingredients that result in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon host health. Application of prebiotics may then restore the gut microbiota diversity and activity. Unlike the previously accepted prebiotics definition, where a limited number of bacterial species are involved in the prebiotic activity, new data from community-wide microbiome analysis demonstrated a broader affect of the prebiotics on the intestinal microbiota. These new findings require a revision of the current definition. In addition, prebiotics may exert immunomodulatory effects through microbiota-independent mechanisms that will require future investigations involving germ-free animal disease models.

Background and Aims: Ulcerative colitis [UC] is associated with colonic mucosa barrier defects and bacterial dysbiosis, but these features may simply be the result of inflammation. Therefore, we sought to assess whether these features are inherently abrogated in the terminal ileum [TI] of UC patients, where inflammation is absent. Methods: TI biopsies from paediatric inflammatory bowel disease [IBD] subsets [Crohn’s disease [CD; n = 13] and UC [ n = 10]], and non-IBD disease controls [ n = 12] were histologically graded, and alcian blue/periodic acid-Schiff stained biopsies were quantified. The mucosal barrier was assessed for mucin [MUC2], immunoglobulin [Ig]A, IgG, and total bacteria (fluorescence in-situ hybridisation [FISH probe EUB338]) by immunofluorescence. The regulation of mucin secretion was investigated by NLRP6 gene expression and immunofluorescence. The composition of the active mucosa-associated microbiota was explored by sequencing the 16S rRNA amplicon generated from total RNA. Results: Despite the absence of ileitis, UC patients displayed ileal barrier depletion illustrated by reductions in mucin-containing goblet cells and mucin production and altered epithelial NLRP6 expression. In both CD patients with ileitis and UC patients with normal histology, bacteria coated with IgA and IgG penetrated the TI mucin layer. Biopsy 16S rRNA sequencing revealed a reduction in α-diversity by three methods [Shannon, Simpson, and Equitability indices] between UC and non-IBD paediatric patients. Conclusions: These findings suggest an underlying defect in the UC-afflicted intestinal tract even in the absence of inflammation, implicating barrier and microbial changes as primary abnormalities in UC that may play a causative role in disease development.

Background: Prebiotic fibers stimulate the growth and activity ofthe gut microbiota. Interleukin 10‐deficient (IL-10 2/2 ) mice develop a colitis that is influenced by the gut microbial composition. Objective: The purpose of this study was to determine the effect of prebiotic fibers on the intestinal microbiota and immune function in IL-10 2/2 mice. Methods: At 4 wk of age, male IL-10 2/2 mice (n = 8/group) were randomly assigned to 5 diets: unpurified diet with cellulose (4%;control),corn-derivedhydroxypropylatednew resistantstarch(NRS)(2%NRS+ 2%cellulose),solublefiber dextrinfrom tapioca (SFD-t) (4%), soluble fiber dextrin from corn (SFD-c) (4%), or soluble corn fiber (4%) for 12 wk. Growth, small intestinal permeability, histologic injury, intestinal cytokine secretion, and microbiota composition by 16S ribosomal RNA pyrosequencing of stool were measured. ANOVA and principal component analysis were applied to assess the fibers’ effects. Results: There were no significant differences in mouse growth, intestinal weight, length, or gut permeability over the 12 wk feeding period. Mice fed dextrin-based diets secreted 47‐88% less colonic IL-1b, tumor necrosis factor a, and IL-23 (SFD-t diet) and IL-12 heterodimer p70, IL-6, and chemokine ligand 1 (CXCL1) (SFD-c diet) (P < 0.05) than did the control group, whereas NRS-fed mice secreted 55‐77% less IL-6 and CXCL1 (P < 0.05). Both SFD-t‐ and SFD-c‐fed mice had a 70‐75% lower abundance of Lactobacillaceae than control mice. The SFD-t diet group had a lower enterocyte injury score ( P< 0.04) than did control mice, and this was associated with increased abundance of butyrate producers, including Incertae sedis XIV, Lachnospiraceae, and Ruminococcaceae (P < 0.001). Conclusions: These results demonstrate that soluble prebiotic fibers selectively stimulate the growth of a distinctive gut microbiota in IL-10 2/2 mice. SFD-t induced the growth of butyrate-producing microbes and was effective in reducing proinflammatory cytokine secretion and enterocyte injury in this mouse model of colitis. JN utr2015;145:2060‐6.

BACKGROUND: Several studies support a role for gut microorganisms in the development of the common and severely debilitating chronic inflammatory bowel diseases (IBD), Crohn disease (CD) and ulcerative colitis (UC). To date, most studies have examined only samples from stool or inflamed areas, limiting the ability to differentiate between cause and effect. Our recent work examined the uninflamed terminal ileum (TI) in pediatric UC, and showed changes in diversity and composition of bacteria, suggesting that microbes proximal to diseased areas may drive inflammation distally. AIMS: As immunoglobulin (Ig)G is increased in IBD and has been shown to bind more selectively to pathogens than its counterpart, IgA, we hypothesized that IgG is formed in response to invasive microbes, and could be used to identify specific taxa involved in IBD pathogenesis. To test this, we isolated IgG-bound intestinal bacteria, identified by 16S rDNA sequencing, and tested their virulence in vitro. METHODS: TI luminal washes were collected during endoscopy of pediatric IBD patients and non-IBD controls. IgG bound (IgG+) and unbound (IgG-) bacteria were separated and collected using FACS and DNA was extracted for 16S rDNA sequencing (Ilumina MiSeq platform). Virulence of specific IgG-bound bacteria was tested in-vitro via gentamycin protection assay, microscopy and qPCR for markers of immune response. RESULTS: Species diversity and microbial composition were reduced in UC compared to non-IBD. Furthermore, the ratio of IgG+/IgG- bacteria increased in CD and UC patients by 2 and 1.5 fold, respectively. Although total abundance of Burkholderia cepacia, was comparable in IBD and non-IBD, there was a 2.5 and 4.1 fold increase in the ratio of IgG+/IgG- binding to B. cepacia in remission/mild or moderate/severe UC, respectively, compared to non-IBD. B. cepacia was previously identified in the ileum of IBD cohorts however, it is not typically recognized as an intestinal pathogen. B. cepacia displays pro-inflammatory effects and invasive potential in an in-vitro model, supporting its pathobiont potential and further supporting the use of IgG as a marker of pathobionts in IBD. CONCLUSIONS: We have demonstrated the ability of IgG-binding to selectively identify previously unrecognized mucosa-associated pathobionts, validated in-vitro. Elucidating the role of specific bacterial species in UC pathogenesis will underpin new strategies to improve our ability to direct therapies to those patients most likely to respond. FUNDING AGENCIES: CCCCCFA

BACKGROUND: Inflammatory bowel diseases (IBD) are chronic inflammatory conditions of the intestines likely induced by abnormal immune response to resident intestinal bacteria in genetically susceptible hosts. Probiotics, prebiotics and dietary fibres alter the gut microbiota and improve its function, thus potentially counteracting the development of inflammation. Although the role of these compounds in the prevention and management of IBD is relatively understudied, anecdotal evidence suggests widespread, undocumented use of these supplements by patients. Investigating the use of probiotics and prebiotics by IBD patients and association with disease severity may allow for optimization of therapy and improved clinical outcomes. AIMS: To assess if the self-motivated intake of probiotics, prebiotics and dietary fibre supplements is associated with the disease severity in patients with IBD. METHODS: We conducted a cross-sectional study of patients with a diagnosis of IBD in the University of Alberta IBD clinic. Using a 20-item questionnaire we collected data from patients on demographics, disease characteristics and knowledge and use of probiotics, prebiotics and dietary fibre supplements. We used a chart review to ascertain the occurrence of flares, disease duration and objective markers of inflammation such as fecal calprotectin (FCP) as indicators of disease severity. RESULTS: In this study, 100 participants (45% females) with a known diagnosis of IBD (47% Crohn’s disease, 34% ulcerative colitis, 19% IBD-unclassified) completed questionnaires. Large proportions of participants were knowledgeable about probiotics (88%) and dietary fibres (76%), but less about prebiotics (42%). The majority of users had Crohn’s disease (42% CD vs. 35% UC, p=0.33). 66% of surveyed patients had used these products as an alternate therapy. Disease flares in the last two years did not have an effect on usage (73% users vs. 71% non-users, p=0.8). The use of alternative therapies was higher in patients with longer history of IBD (77% in those with a duration >5 years vs. 22% in those with a duration 250 µg/g (an indicator of acute disease) during the preceding six months reported supplement use, compared to 52% with FCP values below this value (P=0.11). CONCLUSIONS: This study shows that a large proportion of IBD patients are interested in alternative therapies for IBD. Increased flare frequency and objective markers of inflammation did not have an effect on usage. However, longer disease history was the main factor influencing the search for a “cure” by the patients. These microbiota-altering strategies have the ability to affect disease outcomes, therefore clinicians and researchers should identify and document their use. FUNDING AGENCIES: CIHR

BACKGROUND: Inflammatory bowel diseases (IBD), including Crohn disease (CD) and ulcerative colitis (UC), are a group of chronic and severely debilitating gastrointestinal disorders, with an exacerbated immune response to the gut microbiota. AIMS: We hypothesized that pathogenic bacteria are more likely to be bound by immunoglobulin (Ig) G antibodies and identification of these strains could help identify mechanisms of immune activation. METHODS: Aspirate washes were obtained from the terminal ileum of pediatric IBD and non-IBD patients during endoscopy. Samples were fixed in paraformaldehyde and stringently washed to separate bacteria. Prior to fluorescence-activated cell sorting (FACS), samples were stained with propidium iodide (PI) and an anti-IgG fluorescent antibody with proper controls to differentiate bacteria bound by IgG (IgG+) from all others (IgG-). Validation in a select set of samples involved image cytometry. DNA was extracted from sorted bacteria using bead beating extraction and phenol/chloroform purification. Analysis of bacterial DNA samples was completed using the Ilumina MiSeq platform for 16S rRNA gene sequencing. RESULTS: Quality of patient bacterial DNA samples was used to identify those suitable to be sequenced resulting in 36 total washes from children without IBD (n=10), and with CD (n=17) or UC (n=9). Firmicutes and Bacteroidetes phyla were most commonly identified; however, no significant differences were found between IBD and non-IBD controls upon examining the composition of the ileal mucosa-associated microbiome. There was a 2 and 1.5 fold increase in the overall ratio of IgG+/IgG- bacteria in CD and UC, respectively. In CD, there was an increase in IgG+/IgG- ratio of both Bacteroidetes and Proteobacteria phyla. In UC this ratio was increased for Actinobacteria. IgG binding favored specific family level strains including Porphyromonadaceae (Bacteroidetes) and Enterobacteriaceae (Proteobacteria) in CD; Barnesiellaceae (Bacteroidetes) and Bifidobacteriaceae (Actinobacteria) in UC; and Clostridiaceae and Veillonellaceae (Firmicutes) in non-IBD, although considerable variation was noted between patients. CONCLUSIONS: This study demonstrated selective increase of mucosa associated bacteria bound by IgG in the ileum of pediatric IBD patients compared to non-IBD. Further use of this method in larger cohort studies can identify individual microbes as therapeutic targets facilitating the development of targeted diagnostic and improved therapeutic approaches for IBD. FUNDING AGENCIES: CCCCCFC, AIHS

BACKGROUND: A relationship between ulcerative colitis and diet has been shown in epidemiological and experimental studies. AIMS: To investigate the effectiveness of an anti-inflammatory diet for maintenance of remission in UC patients. METHODS: In this 6-month randomized control trial, adult UC patients in clinical remission (partial Mayo score

BACKGROUND: Inflammatory bowel diseases (IBD) are chronic inflammatory conditions of the intestines that are believed to be induced by abnormal activation of the immune cells in response to resident intestinal bacteria in genetically susceptible hosts. Probiotics, prebiotics and dietary fibres alter the gut microbiota and improve its function, thus potentially counteracting the development of inflammation. Unlike the current standard medications for IBD, these supplements are relatively safe. As such they are suggested as promising novel adjuvant modalities in the treatment of IBD. Although the role of these compounds in the prevention and treatment of IBD is understudied, there seems to be a widespread, undocumented use of these supplements by patients. Identification of usage of probiotics and prebiotics in IBD patients, will allow for optimization of therapy, and improved clinical outcomes. AIMS: To assess the intake of probiotics, prebiotics and dietary fibre supplements in patients with IBD in Edmonton, Alberta. METHODS: A cross-sectional, observational study using a 20-item survey questionnaire was used in patients with a diagnosis of IBD in the IBD Clinic at the University of Alberta. Data regarding demographics, disease characteristics, use and knowledge of probiotics, prebiotics and dietary fibre supplements was collected and analyzed using Fisher’s exact test. RESULTS: In this pilot study, 23 participants with a known diagnosis of IBD (57% ulcerative colitis, 39% Crohn’s disease, 4% indeterminate colitis) completed survey questionnaires. Statistical analysis demonstrated that a large number of participants were knowledgeable about probiotics and dietary fibres, but less about prebiotics (87% probiotics, 74% fibres, 43% prebiotics, P < 0.38). Sixty five per cent of surveyed patients have used these products in the past. However, regular usage (in the last year), was much lower (48% probiotics, 28% fibres, 4% prebiotics). Of those who used these alternative treatments in the past, 31% of patients experienced an increase in quality of life (QoL). Those without a university degree were more likely to use these products (67%, P < 0.37). The effect of gender on usage was negligible (53% males, 47% females, P < 1.0). Preliminary results did not reach statistical significance. CONCLUSIONS: This pilot study shows that a large proportion of IBD patients have used probiotics, prebiotics and dietary fibre supplements in the past despite the lack of well-proven efficacy. Since these microbiota targeting strategies have a great potential to improve disease outcomes, it is important that clinicians and researchers document their use. FUNDING AGENCIES: CIHR

The choice of the cereal substrate determines sourdough microbiota, however, the substrate-associated ecological factors for this phenomenon have not been elucidated. This study investigated the competitiveness of Lactobacillus sanfranciscensis LTH 2590, a wheat sourdough isolate, and four isolates from sorghum sourdoughs (ting), Lactobacillus casei FUA3166, Lactobacillus harbinensis FUA3199, Lactobacillus parabuchneri FUA3169, and Lactobacillus coryniformis FUA3307, in sorghum sourdoughs, sorghum sourdoughs supplemented with maltose, or wheat sourdoughs. Fermentations were characterised by determination of cell counts, pH, and quantification of metabolites. Maltose was the main carbon source in wheat sourdoughs whereas glucose was the major carbon source in sorghum. L. coryniformis and L. parabuchneri produced 1,3- and 1,2-propanediol from glycerol and lactate, respectively, metabolites that were previously not observed in sourdough. To determine the competitiveness of strains, wheat and sorghum slurries were inoculated with equal cells counts of L. sanfranciscensis, L. parabuchneri, and L. casei fermented at 28°C or 34°C and propagated by back-slopping every 24h. Lactobacilli in sourdough were quantified by plating and species-specific quantitative PCR (qPCR). Generally, sorghum and wheat sourdoughs inoculated with isolates from ting gave no appreciable differences in the metabolites produced during the fermentation process. L. sanfranciscensis grew in wheat but not in sorghum sourdoughs, or sorghum sourdoughs supplemented with 2% maltose, 1% tryptone, 0.1% l-cysteine and 2% sucrose. Furthermore, L.sanfranciscensis decreased progressively during propagation of sorghum sourdoughs but ting isolates were overgrown by L. sanfranciscensis after three propagations in wheat sourdoughs independent of the incubation temperature. The anti-microbial activity of four different types of sorghum extracts was tested against L. sanfranciscensis, L. parabuchneri, and L. casei to correlate the resistance to phenolic compounds to growth in wheat or sorghum sourdoughs. L. sanfranciscensis was inhibited by phenolic extracts from sorghum flours whereas ting isolates were resistant. In conclusion, microbiota of sorghum sourdough differ from wheat and rye because sorghum contains active concentrations of antimicrobial phenolic compounds, and offers glucose as major carbon source.

Modulation of intestinal microbiota by non-digestible carbohydrates may reduce inflammation in inflammatory bowel disease (IBD). The aim of the present study was to assess the effects of inulin and fructo-oligosaccharides (FOS) on intestinal microbiota and colitis in HLA-B27 transgenic rats, a well-validated rodent model for IBD. In this study, 4-week-old rats were fed 8 g/kg body weight inulin or FOS for 12 weeks, or not. Faeces were collected at 4 and 16 weeks of age; and caecal samples were collected at necropsy. The effects of inulin and FOS on chronic intestinal inflammation were assessed using a gross gut score, histology score and levels of mucosal IL-1β. Intestinal microbiota were characterised by quantitative PCR and denaturing gradient gel electrophoresis. Colitis was significantly reduced in all FOS-fed rats compared to the control diet, whereas inulin decreased chronic intestinal inflammation in only half the number of animals. Quantitative analysis of caecal microbiota demonstrated that inulin increased the numbers of total bacteria and theBacteroides-Prevotella-Porphyromonasgroup, FOS increased bifidobacteria, and both fructans decreasedClostridiumcluster XI. In the faecal samples, both inulin and FOS decreased total bacteria,Bacteroides-Prevotella-Porphyromonasgroup, andClostridiumclusters XI and XIVa. FOS increasedBifidobacteriumspp., and mediated a decrease of gene copies of Enterobacteriaceae andClostridium difficiletoxin B in faeces. SCFA concentrations in the faecal and caecal samples were unaffected by the diets. In conclusion, FOS increased the abundance ofBifidobacteriumspp., whereas both fructans reducedClostridiumcluster XI andC. difficiletoxin gene expression, correlating with a reduction of chronic intestinal inflammation.

The aim of the present research is to identify rapidly the lactic acid bacteria (LAB) microflora of four natural French sourdoughs (GO, BF, VB and RF), applying a biphasic (restriction length polymorphism (RFLP) and sequencing) approach for bacterial identification. For this purpose, a database with the RFLP patterns of 30 lactobacilli type strains was created. So-developed ISR-RFLP algorithm was further applied for the differentiation and identification of 134 sourdough isolates. The 16S-23S rDNA intergenic spacer region was amplified by primers tAla and 23S/10, and then digested by HindIII, HinfI and α-TaqI enzymes. Nucleotide sequences of the cloned 16S-23S intergenic spacer region (ISR) were determined by the dideoxynucleotide chain termination method. The T7Prom and M13rev primers flanking the multiple cloning site of pCR2.1 DNA were used to sequence both DNA strands. The RFLP profile obtained upon digestion with HindIII, HinfI and α-TaqI enzymes can be used to discriminate Lactobacillus sanfranciscensis (66%), Lactobacillus panis (17%), Lactobacillus nantensis (11%) and Lactobacillus hammesii(6%) in sourdough GO, Lactobacillus sanfranciscensis (80%), Lactobacillus spicheri (14%) and Lactobacillus pontis(6%) in sourdoughs BF. In sourdoughs VB, which differed in the process temperature, we can differentiate Lactobacillus sanfranciscensis (89%) and Leuconostoc mesenteroidessubsp. mesenteroides (11%). Lactobacillus frumenti(47%), Lactobacillus hammesii (8%), and Lactobacillus paralimentarius (45%) were differentiated in sourdough RF.

A polyphasic taxonomic study of the bacterial flora isolated from traditional French wheat sourdough, using phenotypic characterization and phylogenetic as well as genetic methods, revealed a consistent group of isolates that could not be assigned to any recognized species. These results were confirmed by randomly amplified polymorphic DNA and amplified fragment length polymorphism fingerprinting analyses. Cells were Gram-positive, homofermentative rods. Comparative 16S rRNA gene sequence analysis of the representative strain LP33T indicated that these strains belong to the genus Lactobacillus and that they formed a branch distinct from their closest relatives Lactobacillus farciminis, Lactobacillus alimentarius, Lactobacillus paralimentarius and Lactobacillus mindensis. DNA–DNA reassociation experiments with the three phylogenetically closest Lactobacillus species confirmed that LP33T (=DSM 16982T=CIP 108546T=TMW 1.1265T) represents the type strain of a novel species, for which the name Lactobacillus nantensis sp. nov. is proposed.

Twenty morphologically different strains were chosen from French wheat sourdough isolates. Cells were Gram-positive, non-spore-forming, non-motile rods. The isolates were identified using amplified-fragment length polymorphism, randomly amplified polymorphic DNA and 16S rRNA gene sequence analysis. All isolates were members of the genus Lactobacillus. They were identified as representing Lactobacillus plantarum, Lactobacillus paralimentarius, Lactobacillus sanfranciscensis, Lactobacillus spicheri and Lactobacillus sakei. However, two isolates (LP38T and LP39) could be clearly discriminated from recognized Lactobacillus species on the basis of genotyping methods. 16S rRNA gene sequence similarity and DNA–DNA relatedness data indicate that the two strains belong to a novel Lactobacillus species, for which the name Lactobacillus hammesii is proposed. The type strain is LP38T (=DSM 16381T=CIP 108387T=TMW 1.1236T).

Background & Aims Many first-degree relatives of patients with Crohn's disease (CD) have increased intestinal permeability. Video capsule endoscopy (VCE) is the most sensitive imaging test to identify small bowel mucosal lesions that could indicate subclinical CD. We aimed to estimate the association of increased intestinal permeability with small bowel ulcerations detectable by VCE in healthy first-degree relatives of patients with CD. Methods We conducted a cross-sectional study of 223 healthy, asymptomatic first-degree relatives of patients with CD (parents, siblings, and children; 9–45 years old) enrolled at the University of Alberta between 2009 and 2012. Patients were given the lactulose and mannitol test to measure small bowel permeability; we used high-performance liquid chromatography to measure concentrations of lactulose and mannitol in urine samples (increased permeability defined as a ratio of lactulose/mannitol 0.025 or greater). Patients with increased permeability (n = 39) and randomly selected subjects with normal permeability (n = 59) were then examined by VCE for signs of small bowel inflammation and subclinical CD. The prevalence of small bowel lesions was compared among groups. We performed logistic regression analyses to estimate odds ratios for the association of small bowel ulcerations with intestinal permeability. Results Among 223 first-degree relatives of patients with CD, 30% were found to have increased intestinal permeability; VCE examination found 24% of subjects to have 3 or more small bowel ulcers. Three or more small bowel ulcers were detected in 28% of patients with increased intestinal permeability and 20% of patients with normal intestinal permeability ( P = .37). The adjusted odds ratio for the association of 3 or more small bowel ulcers with increased intestinal permeability was 1.5 (95% confidence interval, 0.6–3.8; P = .46). Conclusions Thirty percent of healthy, asymptomatic first-degree relatives of patients with CD have increased intestinal permeability. However, a strong association of small bowel ulceration seen on VCE with increased intestinal permeability was not observed.

Twenty-six strains of lactic acid bacteria (LAB) isolated from French and Bulgarian sourdoughs were screened for their enzymatic activities, to elucidate their possible roles during the fermentatio...

CPT-11 is a drug used as chemotherapy for colorectal cancer. CPT-11 causes toxic side-effects in patients. CPT-11 toxicity has been attributed to the activity of intestinal microbiota, however, intestinal microbiota may also have protective effects in CP!-11 chemotherapy. This study aimed to elucidate mechanisms through which microbiota and dietary fibres could modify host health. Rats bearing a Ward colon carcinoma were treated with a two-cycle CPT-11/5-fluorouracil therapy recapitulating clinical therapy of colorectal cancer. Animals were fed with a semi-purified diet or a semi-purified diet was supplemented with non-digestible carbohydrates (isomalto-oligosaccharides, resistant starch, fructo-oligosaccharides, or inulin) in 3 independent experiments. Changes in intestinal microbiota, bacteria translocating to mesenteric lymphnodes, cecal GUD activity, and cecal SCFA production, and the intestinal concentration of CPT-11 and its metabolites were analysed. Non-digestible carbohydrates significantly influenced feed intake, body weight and other indicators of animal health. The identification of translocating bacteria and their quantification in cecal microbiota indicated that overgrowth of the intestine by opportunistic pathogens was not a major contributor to CPT-11 toxicity. Remarkably, fecal GUD activity positively correlated to body weight and feed intake but negatively correlated to cecal SN-38 concentrations and IL1-β. The reduction in CPT-11 toxicity by non-digestible carbohydrates did not correlate to stimulation of specific bacterial taxa. However, cecal butyrate concentrations and feed intake were highly correlated. The protective role of intestinal butyrate production was substantiated by a positive correlation of the host expression of MCT1 (monocarboxylate transporter 1) with body weight as well as a positive correlation of the abundance of bacterial butyryl-CoA gene with cecal butyrate concentrations. These correlations support the interpretation that the influence of dietary fibre on CPT-11 toxicity is partially mediated by an increased cecal production of butyrate.

Benjamin JL, Hedin CRH, Koutsoumpas A, et al. Randomised, double-blind, placebo-controlled trial of fructo-oligosaccharides in active Crohn's disease. (Inflamm Bowel Dis 2011)

The microbiota of four industrial French sourdoughs (BF, GO, VB and RF) was characterized by PCR temporal temperature gel electrophoresis (TTGE). The TTGE technique reveals differences in the 16S rDNA V6–V8 regions of these bacteria. DNA was extracted directly from sourdough samples. A specific TTGE fingerprint was determined for 30 bacterial species, including members of the genera Lactobacillus, Leuconostoc and Weissella, all known to be present in sourdough. These sourdoughs contain different species of lactic acid bacteria (LAB) depending on ecological conditions prevailing in the different sourdough fermentations. Only a few LAB species were found to be competitive and became dominant. Lactobacillus sanfranciscensis was observed as the most frequently found species. In sourdough GO, L. sanfranciscensis, Lactobacillus panis and two new species, Lactobacillus nantensis and Lactobacillus hammesii, were detected. Sourdough BF contain L. sanfranciscensis, Lactobacillus spicheri and Lactobacillus pontis. In sourdough VB, which differed in the process temperature, we identified exclusively L. sanfranciscensis and Leuconostoc mesenteroides subsp. mesenteroides. Lactobacillus frumenti, L. hammesii and Lacobacillus paralimentarius became the predominant species in sourdough RF. Compared with conventional bacteriological methods, the use of this new molecular approach to analyze the sourdough ecosystem should therefore allow a more complete and rapid assessment of its specific microbiota.

Aims: The organization of ribosomal RNA (rrn) operons in Lactobacillus sanfranciscensis was studied in order to establish an easy-to-perform method for identification of L. sanfranciscensis strains, based on the length and sequence polymorphism of the 16S-23S rDNA intergenic spacer region (ISR). Methods and Results: PCR amplification of the 16S-23S rDNA ISRs of L. sanfranciscensis gave three products distinguishing this micro-organism from the remaining Lactobacillus species. Sequence analysis revealed that two of the rrn operons were organized as in previously reported lactobacilli: large spacer (L-ISR), containing tRNAIle and tRNAAla genes; small spacer (S-ISR) without tRNA genes. The third described spacer (medium, M-ISR), original for L. sanfranciscensis, harboured a tRNA-like structure. An oligonucleotide sequence targeting the variable region between tDNAIle and tDNAAla of L. sanfranciscensis L-ISR was approved to be suitable in species-specific identification procedure. Analysis by pulse-field gel electrophoresis of the chromosomal digest with the enzyme I-CeuI showed the presence of seven rrn clusters. Lactobacillus sanfranciscensis genome size was estimated at c. 1·3 Mb. Conclusions: Direct amplification of 16S-23S ISRs or PCR with specific primer derived from L-ISR showed to be useful for specific typing of L. sanfranciscensis. This was due to the specific rrn operon organization of L. sanfranciscensis strains. Significance and Impact of the Study: In this paper, we have reported a rapid procedure for L. sanfranciscensis identification based on specific structures found in its rrn operon.

International audience; Aims: To characterize lactobacilli isolated from the intestines of ducks or pigs with respect to the production of extracellular homopolysaccharides (HoPS) and oligosaccharides. Methods and Results: Lactobacillus strains of duck or pig origin were screened for HoPS synthesis and > 25% of the isolates produced fructans or glucans from sucrose. Glucan-forming strains were found within the species Lactobacillus reuteri and Lactobacillus animalis and fructan-forming strains were found within Lactobacillus mucosae, Lactobacillus crispatus and Lactobacillus acidophilus. The glucan-forming strains of L. reuteri but not L. animalis produced glucose-oligosaccharides in additon to the respective polymers, and two fructan-forming strains of L. acidophilus produced kestose. Genes coding for glycosyltransferases were detected by PCR and partially characterized by sequence analysis. Conclusions: A large proportion of lactobacilli from intestinal habitats produce HoPS from sucrose and polysaccharide formation is generally associated with the formation of glucose- and fructose oligosaccharides. Significance and Impact of the Study: The characterization of the metabolic potential of intestinal lactobacilli contributes to the understanding of the molecular basis of autochthony in intestinal habitats. Moreover, this is the first report of glucose-oligosaccharide production during growth of lactobacilli, and one novel fructosyltransferase and one novel glucansucrase were partially characterized on the genetic level.

The production of a novel broad-spectrum antimicrobial peptide enterococcin A 2000, active against Gram-positive and Gram-negative microorganisms including Listeria subsp. and Escherichia coli, by Enterococcus faecium strain A 2000 isolated from the surface of traditional Bulgarian yellow cheese "kash-kaval" is considerably influenced by complex nitrogen sources in the production medium. Medium components, especially peptone and yeast extract, and their concentration contributed to the increase in bacteriocin production during the stationary phase (16-46 h) of cultivation even in the absence of one of the components present in the basal cultivation MRS medium.

Lactic acid bacterium isolated from Bulgarian cheese and identified as Enterococcus faecium produces a small hydrophobic peptide substance (enterococcin A 2000) with broad spectrum of antimicrobial activity. The wide range of enterococcin antibacterial activity of this compound against Gram-positive, as well as against some Gram-negative bacteria, suggests a single mechanism of action. The mode of action of enterococcin A 2000 was studied in intact liver mitochondria and synthetic phospholipid liposomes used as model systems. Enterococcin A 2000 stimulated the ATPase activity in intact mitochondria. The kinetic curve of ATP hydrolysis differed from that obtained in presence of dinitrophenol (DNP) and showed a character similar to the ATP hydrolysis in the presence of classic ionophores. Enterococcin A 2000, when bound to synthetic phospholipid liposomes, permeabilized liposomes liberating the marker carboxyfluorescein (CF).

Endoscopic disease recurrence after ileocolonic resection and re-anastomosis in Crohn's disease (CD) patients occurs in a majority of patients. The aim of this study was to examine environmental exposures, the gut microbiome and urinary metabolites in CD patients following ileocolonic resection to determine if specific gut microbes, their metabolites or toxins or environmental factors could be linked with either relapse or remission. Methods: Biopsies were obtained from the neo-terminal ileum after ileocecal resection from CD patients in endoscopic remission (n=20) or in relapse (n=18). Microbial DNA was extracted from biopsies using the QIAamp DNA stool mini kit. Multitag pyrosequencing of the 16S gene was used to characterize the mucosa-associated microbiota, and the RDP 10 classifier used to assign taxonomic groups. Genes encoding for colibactin, C. difficile toxin B, C. perfringens alpha toxin, shiga-like toxin 1 were quantified by qPCR. Urine samples were collected and metabolites measured using 1H NMR. Peak identification was done using Chenomx NMRSuite v7.5 and multivariate analysis performed using MetaboAnalyst 2.0. An environmental questionnaire was obtained that examined early environmental exposures, family history, diet, smoking behaviours, animal exposure and current medication. Principle component analysis (PCA) and support vector machines were used to determine differences between patients with endoscopic relapse and those that remained in remission. Results: Support vector machines applied to total microbial abundance at the genus level and PCA did not show a clear separation between CD patients in relapse or remission. In addition, there were no differences in any toxin encoding genes. In contrast, patients with endoscopic disease recurrence could be separated from patients without ileal inflammation through partial least squares discriminant analysis of urinary metabolites. In addition, correlation analysis showed a positive correlation between urinary metabolites involved in essential processes such as energy production and amino acid metabolism and Bacteroidaceae and negative correlations between amino acid metabolism and Clostridia in the inflamed group. In contrast, the non-inflamed group showed positive correlations between metabolites involved in energy production and Enterobacteriaceae. The most important environmental features associated with disease relapse included smoking cigarettes and/or pipes. Age, gender, years since last surgery, domicile location, or use of immunosuppressants did not differ between CD patients suffering relapse or remaining in endoscopic remission. Conclusions: Recurrence of Crohn's disease after surgery was associated with a unique urinary metabolomics signature that could be linked with specific gut microbes. The major risk factor associated with recurrence of disease was smoking.