Induction of cytokine formation by human intestinal bacteria in gut epithelial cell lines.

AIMS: To investigate the effects of human gut micro-organisms on cytokine production by human intestinal cell lines. METHODS AND RESULTS: Quantitative real-time PCR assays were developed to measure the production of pro-inflammatory (IL-1α, IL-6, IL-18 and TNFα) and anti-inflammatory (TGF-ß1, TGF-ß2, TGF-ß3, IL-4 and IL-10) cytokines in HT-29 and Caco-2 cell lines. They were co-cultured with a range of mucosal bacteria isolated from ulcerative colitis patients, together with lactobacilli and bifidobacteria obtained from healthy people. HT-29 cells were also co-cultured with Campylobacter jejuni, enterotoxigenic Escherichia coli (ETEC), enteropathogenic E. coli and Salmonella typhimurium. The majority of commensal bacteria tested suppressed the expression of anti-inflammatory cytokine mRNA, increased IL-18, reduced IL-1α, and with the exception of nonpathogenic E. coli, reduced TNF-α. All overtly pathogenic species increased both pro-inflammatory and anti-inflammatory cytokine mRNA. CONCLUSION: Commensal and pathogenic species induced fundamentally different cytokine responses in human intestinal epithelial cell lines. SIGNIFICANCE AND IMPACT OF THE STUDY: Interactions between commensal bacteria tested in this study and the innate immune system were shown to be anti-inflammatory in nature, in contrast to the pathogenic organisms investigated. These data contribute towards our understanding of how potential probiotic species can be used to suppress the pro-inflammatory response in inflammatory bowel disease.

Acquisition, evolution and maintenance of the normal gut microbiota.

The gut is sterile at birth, but is rapidly colonised by faecal and vaginal bacteria of maternal origin. Over the succeeding weeks, months and years, a complex microbiota develops that plays a major role in host physiology. While the digestive tract is colonised to varying degrees by micro-organisms throughout its length, due to acid pH and the short retention time of gastric contents, bacterial numbers in the stomach are usually low. The rapid passage of digestive materials through the upper gut does not provide time for significant bacterial growth to occur, but cell numbers increase considerably in the distal ileum. The rate of movement of intestinal contents slows in the colon, which facilitates the development of complex bacterial communities. The large intestine is an intricate ecosystem that contains a complex microbiota composed of several hundred different types of bacteria. The growth and metabolism of microbial communities in the large intestine are determined by many factors, such as diet, environment and host physiological processes, as well as the anatomic structure of the digestive tract, disease, immunity, host genetics, drugs and ageing. Modifications in diet and host immune system activity, as well as physiological changes in the digestive tract affect microbiota composition in older people. The elderly have fewer bifidobacteria and higher numbers of enterobacteria and clostridia than young adults. Increased antibiotic use in older people and simply going into hospital have been shown to change bacterial community structure in the colonic microbiota, although the metabolic significance of this is unclear.

Bacterial metabolism and health-related effects of galacto-oligosaccharides and other prebiotics.

Most studies involving prebiotic oligosaccharides have been carried out using inulin and its fructo-oligosaccharide (FOS) derivatives, together with various forms of galacto-oligosaccharides (GOS). Although many intestinal bacteria are able to grow on these carbohydrates, most investigations have demonstrated that the growth of bifidobacteria, and to a lesser degree lactobacilli, is particularly favoured. Because of their safety, stability, organoleptic properties, resistance to digestion in the upper bowel and fermentability in the colon, as well as their abilities to promote the growth of beneficial bacteria in the gut, these prebiotics are being increasingly incorporated into the Western diet. Inulin-derived oligosaccharides and GOS are mildly laxative, but can result in flatulence and osmotic diarrhoea if taken in large amounts. However, their effects on large bowel habit are relatively minor. Although the literature dealing with the health significance of prebiotics is not as extensive as that concerning probiotics, considerable evidence has accrued showing that consumption of GOS and FOS can have significant health benefits, particularly in relation to their putative anti-cancer properties, influence on mineral absorption, lipid metabolism, and anti-inflammatory and other immune effects such as atopic disease. In many instances, prebiotics seem to be more effective when used as part of a synbiotic combination.

Review article: prebiotics in the gastrointestinal tract.

BACKGROUND: Prebiotics are short-chain carbohydrates that alter the composition, or metabolism, of the gut microbiota in a beneficial manner. It is therefore expected that prebiotics will improve health in a way similar to probiotics, whilst at the same time being cheaper, and carrying less risk and being easier to incorporate into the diet than probiotics. AIM: To review published evidence for prebiotic effects on gut function and human health. METHODS: We searched the Science Citation Index with the terms prebiotic, microbiota, gut bacteria, large intestine, mucosa, bowel habit, constipation, diarrhoea, inflammatory bowel disease, Crohn's disease, ulcerative colitis, pouchitis, calcium and cancer, focussing principally on studies in humans and reports in the English language. Search of the Cochrane Library did not identify any clinical study or meta-analysis on this topic. RESULTS: Three prebiotics, oligofructose, galacto-oligosaccharides and lactulose, clearly alter the balance of the large bowel microbiota by increasing bifidobacteria and Lactobacillus numbers. These carbohydrates are fermented and give rise to short-chain fatty acid and intestinal gas; however, effects on bowel habit are relatively small. Randomized-controlled trials of their effect in a clinical context are few, although animal studies show anti-inflammatory effects in inflammatory bowel disease, while calcium absorption is increased. CONCLUSIONS: It is still early days for prebiotics, but they offer the potential to modify the gut microbial balance in such a way as to bring direct health benefits cheaply and safely.

Production of immune response mediators by HT-29 intestinal cell-lines in the presence of Bifidobacterium-treated infant microbiota.

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.

Prebiotic digestion and fermentation.

Prebiotics, as currently conceived of, are all carbohydrates of relatively short chain length. To be effective they must reach the cecum. Present evidence concerning the 2 most studied prebiotics, fructooligosaccharides and inulin, is consistent with their resisting digestion by gastric acid and pancreatic enzymes in vivo. However, the wide variety of new candidate prebiotics becoming available for human use requires that a manageable set of in vitro tests be agreed on so that their nondigestibility and fermentability can be established without recourse to human studies in every case. In the large intestine, prebiotics, in addition to their selective effects on bifidobacteria and lactobacilli, influence many aspects of bowel function through fermentation. Short-chain fatty acids are a major product of prebiotic breakdown, but as yet, no characteristic pattern of fermentation acids has been identified. Through stimulation of bacterial growth and fermentation, prebiotics affect bowel habit and are mildly laxative. Perhaps more importantly, some are a potent source of hydrogen in the gut. Mild flatulence is frequently observed by subjects being fed prebiotics; in a significant number of subjects it is severe enough to be unacceptable and to discourage consumption. Prebiotics are like other carbohydrates that reach the cecum, such as nonstarch polysaccharides, sugar alcohols, and resistant starch, in being substrates for fermentation. They are, however, distinctive in their selective effect on the microflora and their propensity to produce flatulence.

Bacterial milieu and mucosal bacteria in ulcerative colitis.

The aetiology of ulcerative colitis (UC) is unknown, but there is evidence that bacteria are needed for initiation and maintenance of the disease. A number of organisms have been associated with UC, but evidence for a specific transmissible agent is weak. Despite this, there is a good case for mucosal bacterial involvement, either through pathogens colonizing the epithelial surface, by non-pathogenic commensal species occupying adhesion sites on the mucosa and preventing invasion by harmful bacteria, or by inappropriate host immune responses to members of the normal microflora. Since mucosal bacteria exist in close juxtaposition to host tissues, it might be expected that they interact to a greater extent with the immune and neuroendocrine systems than their luminal counterparts. For this reason, comparative bacteriological analyses were done on rectal biopsies from patients with active colitis, and individuals who had no inflammatory bowel disease. Complex bacterial communities colonized the rectal mucosa in all subjects and great interindividual variabilities in mucosal bacterial populations were observed in both groups. These organisms often occurred in microcolonies, which may have implications for UC, since it would result in higher localized concentrations of bacterial antigens, or toxins, than would be the case if the organisms were diffusely spread across the mucosa.

Comparison of carbohydrate substrate preferences in eight species of bifidobacteria.

Eight species of bifidobacteria were tested for their abilities to grow on a range of monosaccharides (glucose, arabinose, xylose, galactose and mannose). In contrast to the other sugars, glucose and galactose were utilized by all species and, in general, specific growth rates were highest on these sugars. Different substrate preferences were observed between species when the bacteria were grown in the presence of all five monosaccharides. For example, glucose and xylose were coutilized by Bifidobacterium longum, whereas glucose repressed uptake of all other sugars in B. bifidum and B. catenulatum. Galactose was the preferred substrate with B. pseudolongum. In B. angulatum, glucose and galactose were utilized simultaneously. B. breve did not grow on arabinose when this sugar provided the sole source of energy. However, glucose and arabinose were preferentially taken up during growth on sugar mixtures.

Formation of glycoprotein degrading enzymes by Bacteroides fragilis.

Bacteroides fragilis NCDO 2217 produced a wide range of cell-associated hydrolytic enzymes (neuraminidase, alpha-fucosidase, alpha-N-acetylgalactosaminidase, beta-galactosidase, beta-N-acetylglucosaminidase) that could potentially degrade the carbohydrate moieties of mucin, a complex glycoprotein. The type of substrate used for growth markedly influenced their formation in batch cultures. Synthesis of neuraminidase, alpha-fucosidase, alpha-N-acetylgalactosaminidase and to a lesser extent, beta-N-acetylglucosaminidase, was inversely related to growth rate in continuous cultures (D = 0.03 h-1-0.23 h-1) in which porcine gastric mucin provided the sole source of carbon and nitrogen.

Formation of a dipeptidyl arylamidase by Bacteroides splanchnicus NCTC 10825 with specificities towards glycylprolyl-x and valylalanine-x substrates.

Bacteroides splanchnicus in common with several members of the B. fragilis group constitutively produced a number of protein and peptide hydrolysing enzymes. Amongst the most active was an arylamidase, which specifically hydrolysed the dipeptidyl chromogenic substrates glycylprolyl p-nitroanilide (GPRPNA), glycylprolyl beta-naphthylamide (GP beta NA) and valylalanine p-nitroanilide (VAPNA), and had some proteolytic activity towards azocasein. No activity was detected against proline beta-naphthylamide, glycine, valanine or alanine p-nitroanilides. Physiological studies showed that the enzyme was largely cell-associated during exponential growth in batch culture, but was progressively released by the bacteria before the cells entered stationary phase. Glycylprolyl arylamidase (GPA) was completely cell-bound during growth in continuous culture, where synthesis increased concomitantly with dilution rate (specific growth rate) in both carbon- and nitrogen-limited chemostats. Gel-filtration chromatography of B. splanchnicus cell extracts yielded a single peak of GPA activity, with an apparent molecular mass of c. 160 kDa, while one peak of enzyme activity was eluted by 0.3 M NaCl during cation-exchange chromatography. Activity staining of SDS polyacrylamide gels showed a single GPA band at 80 kDa, suggesting that the enzyme was a dimer. Two fractions of GPA activity were recorded during preparative isoelectric focusing with apparent isoelectric points of pH 3.51 (fraction 3) and 3.95 (fraction 6), indicating the possible existence of GPA isoenzymes. GPRPNA, VAPNA and azocasein were hydrolysed by the major fraction (fraction 3), while only the p-nitroanilide substrates were hydrolysed by fraction 6. Studies with the partially purified enzyme obtained from gel filtration columns showed a relatively broad pH optimum at 7.5-8.2. Inhibition experiments demonstrated that while aspartic (pepstatin A), thiol (iodoacetate) and metalloprotease (EDTA, cysteine) inhibitors had little effect on hydrolysis of glycylproline p-nitroanilide, GPA was strongly inhibited (c. 80%) by 5 mM phenylmethylsulphonyl fluoride (PMSF), indicating it to be a serine enzyme.

Ecological and physiological studies on large intestinal bacteria in relation to production of hydrolytic and reductive enzymes involved in formation of genotoxic metabolites.

Several hydrolytic and reductive bacterial enzymes (beta-glucuronidase, GN; beta-glucosidase, GS; arylsulphatase, AS; azoreductase, AR; nitroreductase, NR) involved in production of mutagenic or genotoxic metabolites were measured in human colonic contents. Cell-associated AS and extracellular GS were approximately twice as high in the distal colon compared with the proximal bowel, while AR changed little throughout the gut. Measurements of these enzymes in faeces from seven healthy donors confirmed that the majority were cell-associated, and demonstrated high levels of inter-individual variability. NR decreased four-fold between the proximal and distal colon while extracellular GN was reduced by 50%. Most probable number (MPN) analysis on faeces obtained from six healthy donors showed that counts of intestinal bacteria producing GS and AR were c. 10(10) and 10(11)/g, respectively, in all samples tested. Numbers of GN- and AS-forming organisms were between two and three orders of magnitude lower. Inter-individual carriage rates of bacterial populations synthesising NR were highly variable. Screening of 20 pure cultures of intestinal bacteria, belonging to six different genera, showed that Bacteroides ovatus, in particular, synthesised large amounts of GS, whereas B. fragilis, B. vulgatus and Bifidobacterium pseudolongum formed the highest cell-associated levels of GN. In general, bifidobacteria and Lactobacillus acidophilus did not produce significant amounts of AR. All five clostridia studied (Clostridium bifermentans, C. septicum, C. perfringens, C. sporogenes and C. butyricum) produced NR and AR, as did the bacteroides (B. fragilis, B. ovatus and B. vulgatus). Escherichia coli and C. perfringens formed large amounts of NR. Levels of AS production were invariably low and few of the organisms screened synthesised this enzyme. In-vitro studies investigating the effect of intestinal transit time on enzyme production, in a three-stage (V1-V3) continuous culture model of the colon operated at system retention times (R) of either 31.1 or 68.4 h, showed that specific activities of GS were up to four-fold higher (V3) at R = 31.1 h. Bacteriological analysis demonstrated that representative populations of colonic micro-organisms were maintained in the fermentation system, and indicated that changes in GS activity were not related to numbers of the predominant anaerobic or facultative anaerobic species within the model, but were explainable on the basis of substrate-induced modulation of bacterial metabolism.

Modulation of genotoxic enzyme activities by non-digestible oligosaccharide metabolism in in-vitro human gut bacterial ecosystems.

Supplementation of the human diet with prebiotic substances such as inulin and non-digestible oligosaccharides (NDO), e.g., galacto-oligosaccharides (GOS), has been associated with various health benefits. However, little information is available regarding the spatial location of their metabolism in human gut bacterial ecosystems. Therefore, the present study investigated the metabolism of inulin and GOS with respect to bacterial growth, bifidobacterial stimulatory properties and anti-mutagenicity potential, in a three-stage continuous culture model of the colon which reproduces the physicochemical characteristics of the proximal (V1) and distal (V2, V3) colons. Fermentation of both carbohydrates was rapid, and occurred primarily in V1, as evidenced by acid formation. Inulin metabolism was associated with 10-fold stimulation of lactobacillus populations, together with smaller increases in bifidobacterial cell counts in V1. However, peptostreptococci, enterococci and Clostridium perfringens also increased in this fermentation vessel. In contrast, GOS was only weakly bifidogenic in V1, although these bacteria did proliferate in V2. GOS also increased lactobacilli by an order of magnitude in V1. However, overall changes in microbial populations resulting from inulin or GOS addition were minimal in V2 and V3. Potential beneficial effects of inulin metabolism included minor reductions in beta-glucosidase and beta-glucuronidase, whereas GOS strongly suppressed these enzymes, together with arylsulphatase (AS). Growth of putatively health promoting micro-organisms was not only associated with reductions in enzymes linked to genotoxicity. For example, both carbohydrates stimulated synthesis of nitroreductase and azoreductase, throughout the fermentation system, while inulin increased AS. Colonic transit time is an important factor in bacterial metabolism in the large bowel, and these data suggest that, in some circumstances, NDO fermentation will occurprincipally in the proximal colon.

Changes in predominant bacterial populations in human faeces with age and with Clostridium difficile infection.

The bacterial composition of human faeces can vary greatly with factors such as age and disease, although relatively few studies have monitored these events, particularly at species level. In this investigation, bacteria were isolated from faecal samples from healthy young adults and elderly subjects, and elderly patients with Clostridium difficile-associated diarrhoea (CDAD). The organisms were identified to species level on the basis of their cellular fatty acid profiles with the MIDI system. In some groups of bacteria, species diversity was found to change with age despite the overall numbers of organisms being similar at genus level. Bacteroides thetaiotaomicron, B. ovatus and Prevotella tannerae were common gram-negative anaerobes isolated from young adults. Bacteroides species diversity increased in the faeces of healthy elderly people. Bifidobacterial species diversity decreased with age, with Bifidobacterium adolescentis and Bif. angulatum being the most common isolates. CDAD patients were characterised by greater diversity of facultative species, lactobacilli and clostridia, but greatly reduced numbers of bacteroides, prevotella and bifidobacteria. Such bacterial population changes in the normal microbiota could result in metabolic conditions favourable for the establishment of pathogenic micro-organisms, such as clostridia, and would have considerable effects on the biochemical capacity of the large intestine as a whole. Alterations in the community structure of bifidobacteria and lactobacilli have relevance for dietary and therapeutic interventions such as the use of pre- or probiotics that aim to modify the composition or metabolic activities of the intestinal microflora in a beneficial way, particularly in elderly people or individuals at risk of CDAD.

Effect of short-chain carbohydrates on human intestinal bifidobacteria and Escherichia coli in vitro.

Plate counts and small subunit (SSU) rRNA abundance were used to study the effects of fructo-oligosaccharides (FOS), fructose, or galacto-oligosaccharides (GOS) on bifidobacterial populations in human faecal microbiotas. The bacteria were grown in pH-controlled anaerobic fermentation vessels. Untreated cultures and fructose-amended fermenters were used as controls. Bifidobacterium longum, B. adolescentis and B. angulatum comprised the dominant bifidobacterial populations throughout the experiment. No major differences were found in the four treatments, in terms of viable counts of the organisms or of total populations of bifidobacteria at any time point. However, large differences were observed with respect to the abundance of bifidobacterial SSU rRNA between the treatments. Greatest bifidobacterial SSU rRNA abundance was seen in FOS cultures, with the lowest in the untreated control fermentation. GOS and fructose also increased bifidobacterial SSU rRNA. Cultures supplemented with FOS and GOS were also associated with lower colony counts and SSU rRNA abundance for Escherichia coli, compared with fructose-supplemented and control fermenters. At the 24-h time point, the untreated control contained 19.8 microg of enterobacterial SSU rRNA/ml of culture fluid, compared with 11.4 microg/ml for the fructose fermentation, and 2.6 and 0.5 microg/ml for the FOS and GOS culture vessels, respectively.

Variation in human intestinal microbiota with age.

The large intestinal microbiota plays an important role in normal bowel function and the maintenance of host health, through the formation of short chain fatty acids, modulation of immune system reactivity and development of colonisation resistance. However, the effects of ageing on bacterial community structure in the colon are not well documented. Aim of this study is to assess bacterial species diversity in the human faecal microbiota with respect to age and Clostridium difficile infection. Bacterial populations were quantified from stool samples obtained from children (16 months to seven years), young adults (21-34 years), healthy elderly people (67-88 years) and patients diagnosed with Clostridium difficile-associated diarrhoea (68-73 years). Microbial diversity was assessed to species level for samples from the latter three subject groups. Marked interindividual variations occurred in microbial composition at genus and species levels. The faecal microbiota of children was found to be bacteriologically less complex whilst advancing age was associated with decreased bifidobacteria and increased bacteroides species diversity. Changes in microbial composition with age or disease will alter the metabolic capacity of the gut microbiota and has important implications for therapies aimed at modulating the large intestinal microbiota.

Role of intestinal bacteria in nutrient metabolism.

The human large intestine contains a microbiota, the components of which are generically complex and metabolically diverse. Its primary function is to salvage energy from carbohydrate not digested in the upper gut. This is achieved through fermentation and absorption of the major products, short chain fatty acids (SCFA), which represent 40-50% of the available energy of the carbohydrate. The principal SCFA, acetate, propionate and butyrate, are metabolized by the colonic epithelium (butyrate), liver (propionate) and muscle (acetate). Intestinal bacteria also have a role in the synthesis of vitamins B and K and the metabolism of bile acids, other sterols and xenobiotics. The colonic microflora are also responsive to diet. In the presence of fermentable carbohydrate substrates such as non-starch polysaccharides, resistant starch and oligosaccharides, bacteria grow and actively synthesize protein. The amount of protein synthesis and turnover within the large intestine is difficult to determine, but around 15 g biomass is excreted in faeces each day containing 1 g bacterial-N. Whether bacterially synthesized amino acids are ever absorbed from the colon remains unclear. Finally, individual colonic micro-organisms such as sulphate-reducing bacteria, bifidobacteria and clostridia, respond selectively to specific dietary components in a way that may be important to health.

Human colonic microbiota: ecology, physiology and metabolic potential of intestinal bacteria.

In both health and disease, the colonic microbiota plays an important role in several areas of human physiology. This complex assemblage of microorganisms endows great metabolic potential on the large intestine, primarily through its degradative abilities. Many hundreds of different types of bacteria, varying widely in physiology and biochemistry, exist in a multitude of different microhabitats in the lumen of the large gut, the mucin layer and on mucosal surfaces. Both microbiota and host obtain clear benefits from association. For example, growth substrates from diet and body tissues, together with a relatively stable environment for bacteria to proliferate are provided by the host, which in turn has evolved to use butyrate, a bacterial fermentation product, as its principal source of energy for epithelial cells in the distal bowel. The main sources of carbon and energy for intestinal bacteria are complex carbohydrates (starches, non-starch polysaccharides). Carbohydrate metabolism is of great importance in the large intestine, since generically, and in terms of absolute numbers, the vast majority of culturable microorganisms are saccharolytic. The amounts and types of fermentation products formed by colonic bacteria depend on the relative amounts of each substrate available, their chemical structures and compositions, as well as the fermentation strategies (biochemical characteristics and catabolite regulatory mechanisms) of bacteria participating in depolymerization and fermentation of the substrates. Protein breakdown and dissimilatory amino acid metabolism result in the formation of a number of putatively toxic metabolites, including phenols, indoles and amines. Production of these substances is inhibited or repressed in many intestinal microorganisms by a fermentable source of carbohydrate. Owing to the anatomy and physiology of the colon, putrefactive processes become quantitatively more important in the distal bowel, where carbohydrate is more limiting.

Investigations of bifidobacterial ecology and oligosaccharide metabolism in a three-stage compound continuous culture system.

BACKGROUND: Several different types of in vitro fermentation systems are currently employed to investigate pro- and prebiotic activities in the human large intestinal microbiota, ranging from simple batch cultures, with or without stirring and pH control, to more complex models involving pH controlled single and multiple-component continuous culture systems. METHODS: In this investigation, we used a three-stage continuous culture model to study the activities of colonic bacteria. This fermentation system reproduces several of the nutritional and environmental characteristics of the proximal large intestine (vessel 1) and the distal colon (vessels 2 and 3), and was validated using bacteriological, metabolic and chemical measurements made with intestinal material obtained from different regions of the large bowel. In this paper, we report studies on prospective probiotic effects of Bifidobacterium longum NCFB 2259 in relation to other bacterial populations, production of tyrosine and phenylalanine metabolites, and bacterial synthesis of enzymes involved in the formation of putatively genotoxic metabolites, including beta-glucosidase (GS), arylsulphatase (AS), beta-glucuronidase (GN), nitroreductase (NR) and azoreductase (AR). RESULTS: Bacterial activities at two different retention times were studied (31.1 and 68.4 h), which correspond to large intestinal transit times. At R = 31.4 h, significant probiotic effects were observed with respect to reductions in GS and GN, upon adding B. longum. However, despite the fact that this organism does not ferment aromatic amino acids or produce significant amounts of genotoxic enzymes, dysbiotic manifestations occurred in that both NR synthesis and dissimilatory tyrosine metabolism were stimulated. In contrast, at R = 68.4 h, GS formation increased between five and 20-fold, while GN and NR activities increased by a factor of two after adding the bifidobacterium. These data are reviewed in relation to potential health hazards that may be encountered with long-term probiotic administration. In the prebiotic experiments, the three-stage fermentation system was operated at R = 65 h. Oligofructose was added to V1 to give an initial concentration of 30 grams per litre, when the system was in steady state, to study its effects on a number of experimental parameters including bifidogenicity, bacterial growth, fermentation product formation and mutagenicity. After addition of the oligosaccharide, a multiplicity of effects were observed in V1, where synthesis of NR and AR, bifidobacterial populations and overall fermentation processes were stimulated, although these influences progressively diminished in V2 and V3. CONCLUSIONS: These studies indicate that bacterial metabolism and putative beneficial consequences associated with the breakdown of readily fermentable prebiotics in the large intestine may in some circumstances be spatially and temporally limited to the proximal bowel.

Synbiotic consumption changes the metabolism and composition of the gut microbiota in older people and modifies inflammatory processes: a randomised, double-blind, placebo-controlled crossover study.

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.

Oesophageal bacterial biofilm changes in gastro-oesophageal reflux disease, Barrett's and oesophageal carcinoma: association or causality?

SUMMARY: 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. AIM: To define the oesophageal microbiota of patients with GERD, BO and OAC compared with controls and to investigate mucosal responses related to the microbiota. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.