The colonisation and establishment of the intestinal microbiota starts immediately at birth and is essential for the development of the intestine and the immune system. This microbial community gradually increases in number and diversity until the age of two or three years when it becomes a stable ecosystem resembling that of adults. This period constitutes a unique window of opportunity to modulate it through probiotic action, with a potential impact in later health. In the present work we have investigated how putative bifidobacterial probiotics modify the metabolic profiles and immune-modulatory properties of faecal microbiotas. An in vitro pH-controlled single-stage continuous-culture system (CCS) inoculated with infant faeces was employed to characterise the effects of two Bifidobacterium species on the intestinal microbiotas in three children, together with the effects of these modified microbiotas on cytokine production by HT-29 cells. Intestinal bacterial communities, production of short-chain fatty acids and lactate were determined by quantitative PCR and gas chromatography, respectively. Cytokines production by HT-29 cells was measured by ELISA. The combination of CCS with infant faeces and human intestinal cells provided a suitable model to evaluate the specific modulation of the intestinal microbiota and immune system by probiotics. In the CCS, infant faecal microbiotas were influenced by the addition of bifidobacteria, resulting in changes in their ability to induce the production of immune mediators by HT-29 cells. The different metabolic and immunological responses induced by the bifidobacterial species tested indicate the need to assess potential probiotics in model systems including complex intestinal microbiotas. Potential probiotic bifidobacteria can modulate the infant microbiota and its ability to induce the production of mediators of the immune response by intestinal cells.

Antibiotic therapies are important in combating disease-causing microorganisms and maintaining host health. It is widely accepted that exposure of the gut microbiota to antibiotics can lead to decreased susceptibility and the development of multi-drug-resistant disease-causing organisms, which can be a major clinical problem. It is also important to consider that antibiotics not only target pathogenic bacteria in the gut, but also can have damaging effects on the ecology of commensal species. This can reduce intrinsic colonization resistance and contribute to problems with antibiotic resistance, including lateral transfer of resistance genes. Our knowledge of the impact of antibiotic treatment on the ecology of the normal microbiota has been increased by recent advances in molecular methods and use of in vitro model systems to investigate the impact of antibiotics on the biodiversity of gut populations and the spread of antibiotic resistance. These highlight the need for more detailed structural and functional information on the long-term antibiotic-associated alterations in the gut microbiome, and spread of antibiotic resistance genes. This will be crucial for the development of strategies, such as targeted therapeutics, probiotics, prebiotics and synbiotics, to prevent perturbations in the gut microbiota, the restoration of beneficial species and improvements in host health.

Human beings live in close association with vast numbers of microorganisms that are present on the skin, mouth, and gastrointestinal tract. Although the small bowel and stomach can become heavily colonized by pathogenic bacteria and yeasts under some circumstances, the colon is the principal region of bacterial colonization in the healthy gastrointestinal tract, owing to the antimicrobial effects of gastric acid and bile salts in the upper gut and the rapid passage of digestive materials that prevents microbial overgrowth. While the effects are often subtle, intestinal microorganisms exert their influence on the host in many ways. Culturing studies have shown that the microbiota comprises several hundred bacterial species, subspecies, and biotypes, and that some organisms occur in higher numbers than others, although about 40 species constitute approximately 99% of all isolates. Pathogenic bacteria invading the body are affected by, and deal with, mucus barriers in different ways. Ulcerative colitis (UC) is one of the two major forms of idiopathic inflammatory bowel disease, and is an acute and chronic disabling condition that is essentially incurable and treated primarily with anti-inflammatory drugs and steroids. Many reactions are known in which enzymes produced by intestinal microorganisms form carcinogens from dietary precursors. Fecal bile acids are also related to the risk of colon cancer, because they are converted to steroids by intestinal microorganisms, which have procarcinogenic properties.

The majority of bacteria live not planktonically, but as residents of sessile biofilm communities. Such populations have been defined as 'matrix-enclosed microbial accretions, which adhere to both biological and nonbiological surfaces'. Bacterial formation of biofilm is implicated in many chronic disease states. Growth in this mode promotes survival by increasing community recalcitrance to clearance by host immune effectors and therapeutic antimicrobials. The human gastrointestinal (GI) tract encompasses a plethora of nutritional and physicochemical environments, many of which are ideal for biofilm formation and survival. However, little is known of the nature, function, and clinical relevance of these communities. This review summarizes current knowledge of the composition and association with health and disease of biofilm communities in the GI tract.

Barrett's oesophagus (BO) and gastro-oesophageal reflux disease (GERD) are precursors of oesophageal adenocarcinoma (OAC). There is an oesophageal biofilm, which changes in disease, but its role in aetiopathogenesis remains unclear.To define the oesophageal microbiota of patients with GERD, BO and OAC compared with controls and to investigate mucosal responses related to the microbiota.Cultural analysis identified the dominant bacterial species from a subset of each disease group. Based on this, molecular techniques were used to define the cohort. Host responses were analysed in tissues and co-culture experiments.A total of 111 species belonging to 26 genera were isolated. There was a significant decrease in bacterial counts in the GERD and BO groups for all genera except Campylobacter, which colonised GERD and Barrett's patients in increasing numbers. Campylobacter concisus was the dominant species. This relationship was not seen in the cancer group. Significant increases in IL-18 were seen in GERD and BO colonised by Campylobacter.This study defines differences in the oesophageal biofilm in disease states, revealing the emergence of C. concisus as the dominant new colonist in the refluxed oesophagus. We also associate the presence of these bacteria with increased expression of cytokines related to carcinogenesis.

The composition and metabolic activities of the human colonic microbiota are modulated by a number of external factors, including diet and antibiotic therapy. Changes in the structure and metabolism of the gut microbiota may have long-term consequences for host health. The large intestine harbors a complex microbial ecosystem comprising several hundreds of different bacterial species, which complicates investigations on intestinal physiology and ecology. To facilitate such studies, a highly simplified microbiota consisting of 14 anaerobic and facultatively anaerobic organisms (Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium pseudolongum, Bifidobacterium adolescentis, Clostridium butyricum, C. perfringens, C. bifermentans, C. innocuum, Escherichia coli, Enterococcus faecalis, Enterococcus faecium, Lactobacillus acidophilus) was used in this investigation. Ampicillin [9.2 μg (ml culture)(-1)] was added to two chemostats operated at different dilution rates (D; 0.10 h(-1) and 0.21 h(-1)), and metronidazole [76.9 μg (ml culture)(-1)] was added to a third vessel (D = 0.21 h(-1)). Perturbations in bacterial physiology and metabolism were sampled over a 48-h period. Lactobacillus acidophilus and C. bifermentans populations did not establish in the fermentors under the imposed growth conditions. Ampicillin resulted in substantial reductions in bacteroides and C. perfringens populations at both dilution rates. Metronidazole strongly affected bacteroides communities but had no effect on bifidobacterial communities. The bacteriostatic effect of ampicillin on bifidobacterial species was growth rate dependent. Several metabolic activities were affected by antibiotic addition, including fermentation product formation and enzyme synthesis. The growth of antibiotic-resistant bifidobacteria in the large bowel may enable them to occupy ecological niches left vacant after antibiotic administration, preventing colonization by pathogenic species.

It was with great excitement that we planned the combined meetings of Northeast Aquaculture Conference and Exposition (NACE) and the International Conference on Shellfish Restoration (ICSR) with the 33rd Milford Aquaculture Seminar (MAS). Bringing these different but complementary audiences together resulted in a meeting that combined many different aspects of fisheries restoration and aquaculture under one umbrella.Four hundred attendees including government representatives, research scientists, industry, and academia at both the university and vocational high school levels attended this event. The meeting commenced on Wednesday December 12th 2012, with over eighty people attending seven field trips to area aquaculture farms and research facilities. The formal program began on Thursday, December 13th with a plenary session including invited speakers Eric Schwabb, Acting Assistant Secretary for Conservation and Management at NOAA; Sebastian Belle, Executive Director of the Maine Aquaculture Association and Boze Hancock from the Nature Conservancy, who discussed the role of aquaculture in fisheries restoration. John Bullard, the Northeast Regional Administrator of NOAA Fisheries Service, addressed the group during the luncheon the following day. The technical papers and workshops presented were divided into 35 sessions over three days of the meeting and included topics such as the history of aquaculture, aquaculture hatchery innovations, siting and planning issues, risk management, aquaculture business management, ocean acidification, climate change, as well as aquaculture disease issues and potential remedies. Having persons present who have experienced problems and successes, along with those in manufacturing and government responsible for addressing concerns and sharing best practices, was invaluable. Discussions among this cross-section of persons who represent different aspects of aquaculture were as important as the well-presented and interesting formal papers and poster sessions. These discussions were facilitated by the many opportunities presented during the large trade show, reception, breaks, lunches, banquet and lobster bake held throughout the meeting.The importance of and need for aquaculture were made evident by all who were in attendance. We are grateful to the twenty four meeting sponsors and to all those who participated and made this such an important and memorable meeting.While oyster restoration efforts have been under way in the Chesapeake Bay for more than two decades, until recently, varying reporting methods and success criteria made it difficult to determine how much progress had been made. A goal to "Restore native oyster habitat and populations in 20 out of 35 to 40 candidate tributaries by 2025" was set in the 2010 strategy to implement the Chesapeake Bay Protection and Restoration Executive Order (signed by President Obama in 2009). This goal made it a priority for state and federal fishery managers, academics, and scientists working on oyster restoration to collaboratively define a "restored tributary" and a "restored reef" to enable them to track progress. A team of these people, led by NOAA staff, agreed on "oyster metrics" in 2011 (http//preview.tinyurl.com/8kmbdpm) that specify key metrics and target ranges for them, including tributary size, how to determine how much restorable bottom a tributary contains, how much of that restorable bottom needs to be restored, and the minimum oyster density and biomass in that restored bottom to count a tributary as restored. These new metrics enable experts to clearly see how oyster restoration efforts are working and use adaptive management to improve these efforts. These metrics serve as a tool to plan and evaluate oyster restoration consistently across the Chesapeake Bay, and the consensus-based framework used to develop them may have broader application to other restoration activities.In the past, oyster restoration in Maryland was performed primarily

This chapter contains sections titled: Probiotics Prebiotics Synbiotics

BACKGROUND:Ageing can result in major changes in the composition and metabolic activities of bacterial populations in the large gut and an impaired immune system. AIM:To investigate the effects of synbiotic consumption on the colonic microbiota, immune function and health status in older people. METHODS:A randomised, double-blind placebo-controlled, 4-week crossover study was carried out, involving 43 older volunteers, using a synbiotic comprising the probiotic Bifidobacterium longum and an inulin-based prebiotic Synergy 1 (SudZucker, Mannheim, Germany). Faecal and blood samples were collected, and clinical status scored at the start, and at 2- and 4-week intervals, with a 4-week washout between each feeding period. Faecal bacteria were determined by fluorescent in situ hybridisation. Short-chain fatty acid concentrations, cytokine production, bowel habit and a range of clinical parameters were measured. RESULTS:The synbiotic increased bifidobacterial numbers by 1.4 log units (P < 0.0001) and also increased members of the phyla Actinobacteria and Firmicutes (P = 0.0004, P < 0.0001). Proteobacteria were reduced by 1.0 log units (P < 0.0001). Synbiotic feeding was associated with increased butyrate production (P = 0.0399). The pro-inflammatory response was modified by the synbiotic, with significantly reduced pro-inflammatory cytokine TNF-α in peripheral blood after 2 and 4 weeks of synbiotic consumption (P = 0.02, P = 0.0406). The synbiotic had no effect on bowel habit or any clinical parameters. CONCLUSION:Short-term synbiotic use can be effective in improving the composition and metabolic activities of colonic bacterial communities and immune parameters in older people. This study was registered at clinicaltrials.gov as NCT01226212.

ABSTRACT Bacteria belonging to the normal colonic microbiota are associated with the etiology of ulcerative colitis (UC). Although several mucosal species have been implicated in the disease process, the organisms and mechanisms involved are unknown. The aim of this investigation was to characterize mucosal biofilm communities over time and to determine the relationship of these bacteria to patient age and disease severity and duration. Multiple rectal biopsy specimens were taken from 33 patients with active UC over a period of 1 year. Real-time PCR was used to quantify mucosal bacteria in UC patients compared to 18 noninflammatory bowel disease controls, and the relationship between indicators of disease severity and bacterial colonization was evaluated by linear regression analysis. Significant differences were detected in bacterial populations on the UC mucosa and in the control group, which varied over the study period. High clinical activity indices (CAI) and sigmoidoscopy scores (SS) were associated with enterobacteria, desulfovibrios, type E Clostridium perfringens, and Enterococcus faecalis, whereas the reverse was true for Clostridium butyricum, Ruminococcus albus, and Eubacterium rectale. Lactobacillus and bifidobacterium numbers were linked with low CAI. Only E. rectale and Clostridium clostridioforme had a high age dependence. These findings demonstrated that longitudinal variations in mucosal bacterial populations occur in UC and that bacterial community structure is related to disease severity.

Patients with dysphagia require long-term nutritional support. This can be delivered by the enteral route via a percutaneous endoscopic gastrostomy (PEG) tube. Enteral nutrition (EN) bypasses the body's innate defences that prevent the microbial colonization of the proximal gut, which predisposes to microbial overgrowth. A continuous culture model simulating the upper gastrointestinal tract microbiota of EN patients was used to investigate the effects of a synbiotic (Lactobacillus acidophilus DUN-311, Bifidobacterium bifidum BB-02, Bifidobacterium lactis BL-01, Synergy 1) on microbial community structure and metabolism. A PEG tube was inserted into the fermenters to study biofilm formation. The synbiotic delivered in sterile semi-skimmed milk (SSSM) was introduced either 48 h prior to or after PEG tube insertion. The synbiotic reduced biofilm formation on PEG tube surfaces, with suppression of Escherichia coli and Klebsiella pneumoniae when it was added subsequent to PEG insertion. When synbiotic feeding was commenced prior to PEG insertion, colonization by Staphylococcus aureus, Candida albicans and Candida famata was also inhibited. Lactate production increased in response the synbiotic or control (SSSM). These results indicate that the use of a synbiotic has the potential to reduce pathogen colonization on PEG tube surfaces in vivo, thereby reducing the incidence of biofilm-related infectious complications.

The colonic microbiota plays an important role in human digestive physiology and makes a significant contribution to homeostasis in the large bowel. The microbiome probably comprises thousands of different bacterial species. The principal metabolic activities of colonic microorganisms are associated with carbohydrate and protein digestion. Nutrients of dietary and host origin support the growth of intestinal organisms. Short-chain fatty acids (SCFAs), predominantly acetate, propionate, and butyrate, are the principal metabolites generated during the catabolism of carbohydrates and proteins. In contrast, protein digestion yields a greater diversity of end products, including SCFAs, amines, phenols, indoles, thiols, CO2, H2, and H2S, many of which have toxic properties. The majority of SCFAs are absorbed from the gut and metabolized in various body tissues, making a relatively small but significant contribution to the body's daily energy requirements. Carbohydrate fermentation is, for the most part, a beneficial process in the large gut, because the growth of saccharolytic bacteria stimulates their requirements for toxic products associated with putrefaction, for incorporation into cellular proteins, thereby protecting the host. However, as digestive materials move along the gut, carbohydrates become depleted, which may be linked to the increased prevalence of colonic disease in the distal bowel.

ABSTRACT Adherence of bacteria to epithelial cells is an important step in colonization and immune modulation in the large bowel. The aims of this study were to use a three-stage continuous-culture system (CCS) to investigate how environmental factors affect bacterial attachment to Caco-2 cells and modulation of cytokine expression by gut microorganisms, including a probiotic Bifidobacterium longum strain, DD2004. The CCS simulated environmental conditions in the proximal large intestine (vessel 1 [V1]) and distal colon (V2 and V3) at two different system retention times ( R ) within the range of normal colonic transits (20 and 60 h). The model was inoculated with human fecal material, and fluorescence in situ hybridization (FISH) was used to characterize microbial populations and to assess bacterial attachment to Caco-2 cells. Real-time quantitative PCR (qPCR) was employed to measure cytokine gene expression following challenge with bacteria from different components of the CCS in the presence and absence of B. longum . At an R of 60 h, bacterial adherence increased from V1 to V3, but this trend was reversed at an R of 20 h. Atopobia were the predominant adherent organisms detected at both system retention times in each culture vessel. Modulation of transforming growth factor β1 (TGF-β1), interleukin 6 (IL-6), and IL-18 gene expression by CCS bacteria was marked at an R of 60 h, while at an R of 20 h, IL-4, IL-10, TGF-β2, IL-1α, and tumor necrosis factor alpha (TNF-α) were significantly affected. The addition of B. longum affected cytokine expression significantly at both retention times. This study demonstrates that environmental determinants regulate the adherence properties of intestinal bacteria and their abilities to regulate cytokine synthesis.

Enteral feeding via a percutaneous endoscopic gastrostomy tube is required for nutritional support in patients with dysphagia. Enteral tube feeding bypasses the innate defence mechanisms in the upper gastrointestinal tract. This study examined the surface-associated microbial populations and immune response in the gastric and duodenal mucosae of eight enteral nutrition (EN) patients and ten controls. Real-time PCR and fluorescence in situ hybridization were employed to assess microbiota composition and mucosal pro-inflammatory cytokine expression. The results showed that EN patients had significantly higher levels of bacterial DNA in mucosal biopsies from the stomach and duodenum ( P <0.05) than the controls, and that enterobacteria were the predominant colonizing species on mucosal surfaces in these individuals. Expression of the pro-inflammatory cytokines interleukin (IL)-1 α , IL-6 and tumour necrosis factor- α was significantly higher in gastric and small intestinal mucosae from patients fed normal diets in comparison with those receiving EN ( P <0.05). These results indicate that EN can lead to significant bacterial overgrowth on upper gastrointestinal tract mucosae and a significantly diminished pro-inflammatory cytokine response.

Complex and highly variable site-dependent bacterial ecosystems exist throughout the length of the human gastrointestinal tract. Until relatively recently, the majority of our information on intestinal microbiotas has come from studies on feces, or from aspirates taken from the upper gut. However, there is evidence showing that mucosal bacteria growing in biofilms on surfaces lining the gut differ from luminal populations, and that due to their proximity to the epithelial surface, these organisms may be important in modulating the host's immune system and contributing to some chronic inflammatory diseases. Over the past decade, increasing interest in mucosal bacteria, coupled with advances in molecular approaches for assessing microbial diversity, has begun to provide some insight into the complexity of these mucosa-associated communities. In gastrointestinal conditions such as inflammatory bowel diseases (ulcerative colitis, Crohn's disease), it has been shown that a dysbiosis exists in microbial community structure, and that there is a reduction in putatively protective mucosal organisms such as bifidobacteria. Therefore, manipulation of mucosal communities may be beneficial in restoring normal functionality in the gut, thereby improving the immune status and general health of the host. Biofilm structure and function has been studied intensively in the oral cavity, and as a consequence, mucosal communities in the mouth will not be covered in this chapter. This review addresses our current knowledge of mucosal populations in the gastrointestinal tract, changes that can occur in community structure in disease, and therapeutic modulation of biofilm composition by antibiotics, prebiotics, and probiotics.

Introduction An altered colonic microbiota contributes to inflammatory bowel disease (IBD) pathogenesis. Adult studies suggest modification of the microbiota by synbiotics (probiotics+prebiotics) can improve ulcerative colitis (UC)1 and Crohn9s disease (CD)2. Aim To assess the feasibility of using a synbiotic in children with IBD. Methods Patients with IBD aged 6 to 16 years were approached. Children were excluded if they used any prebiotic/probiotic agent in the preceding 14 days, were using antibiotics or had a severe IBD exacerbation. Patients were withdrawn if they started antibiotics or another prebiotic/probiotic or they wished to discontinue. Participation involved taking the probiotic ( Bifidobacterium longum in a capsule) and prebiotic (3.5 g Synergy 1 inulin oligofructose) twice daily for 12 weeks. A pre-study questionnaire and physician9s global assessment (PGA) were completed. Questionnaires were provided for return at 1, 4, 8 and 12 weeks. Trial completion involved finishing the synbiotic course and returning all questionnaires. Results 23 children were recruited. 11 (48%) were male. Median age was 13.4 years (7.8–16.6 years). 11 had CD, 6 UC and 4 IBD-unspecified. Initial PGA was ‘inactive’ in 19, ‘mild’ in 3, ‘moderate’ in 1 and ‘severe’ in 1. Only 3 of the first 17 patients completed the study. Feedback indicated the prebiotic was poorly tolerated, therefore the last six participants undertook a tapered probiotic course: none in week 1, once daily in week 2, then twice daily from week 3. Only two of six completed the modified study. Completion overall was 5/23 (22%). Reasons for withdrawal were: six diarrhoea/IBD flare, six stopped returning questionnaires, four required antibiotics, one advised by GP because of rash, one unwell on prebiotic but completed probiotic and one parent stopped after noticing ‘no difference. Conclusion Although synbiotics have shown promise in adult IBD this feasibility study in fairly well paediatric IBD patients has shown poor tolerability. We suspect the drop-out rate was due to the prebiotic agent, but further studies are necessary to assess the tolerance of different prebiotics/probiotics in paediatric IBD before larger efficacy trials.

10 12 14 16 18 20 22 24 Running title: Intestinal bacterial attachment and cytokine production 26