Claus T. Christophersen

Diets that differ in their FODMAP content alter the colonic luminal microenvironment

Objective A low FODMAP (Fermentable Oligosaccharides, Disaccharides, Monosaccharides And Polyols) diet reduces symptoms of IBS, but reduction of potential prebiotic and fermentative effects might adversely affect the colonic microenvironment. The effects of a low FODMAP diet with a typical Australian diet on biomarkers of colonic health were compared in a single-blinded, randomised, cross-over trial. Design Twenty-seven IBS and six healthy subjects were randomly allocated one of two 21-day provided diets, differing only in FODMAP content (mean (95% CI) low 3.05 (1.86 to 4.25) g/day vs Australian 23.7 (16.9 to 30.6) g/day), and then crossed over to the other diet with ≥21-day washout period. Faeces passed over a 5-day run-in on their habitual diet and from day 17 to day 21 of the interventional diets were pooled, and pH, short-chain fatty acid concentrations and bacterial abundance and diversity were assessed. Results Faecal indices were similar in IBS and healthy subjects during habitual diets. The low FODMAP diet was associated with higher faecal pH (7.37 (7.23 to 7.51) vs 7.16 (7.02 to 7.30); p=0.001), similar short-chain fatty acid concentrations, greater microbial diversity and reduced total bacterial abundance (9.63 (9.53 to 9.73) vs 9.83 (9.72 to 9.93) log10 copies/g; p<0.001) compared with the Australian diet. To indicate direction of change, in comparison with the habitual diet the low FODMAP diet reduced total bacterial abundance and the typical Australian diet increased relative abundance for butyrate-producing Clostridium cluster XIVa (median ratio 6.62; p<0.001) and mucus-associated Akkermansia muciniphila (19.3; p<0.001), and reduced Ruminococcus torques. Conclusions Diets differing in FODMAP content have marked effects on gut microbiota composition. The implications of long-term reduction of intake of FODMAPs require elucidation. Trial registration number ACTRN12612001185853.

Molecular Diversity of Rumen Methanogens from Sheep in Western Australia

ABSTRACT The molecular diversity of rumen methanogens in sheep in Australia was investigated by using individual 16S rRNA gene libraries prepared from the rumen contents obtained from six merino sheep grazing pasture (326 clones), six sheep fed an oaten hay-based diet (275 clones), and five sheep fed a lucerne hay-based diet (132 clones). A total of 733 clones were examined, and the analysis revealed 65 phylotypes whose sequences (1,260 bp) were similar to those of cultivated methanogens belonging to the order Methanobacteriales. Pasture-grazed sheep had more methanogen diversity than sheep fed either the oaten hay or lucerne hay diet. Methanobrevibacter strains SM9, M6, and NT7 accounted for over 90% of the total number of clones identified. M6 was more prevalent in grazing sheep, and SM9, despite being found in 16 of the 17 sheep, was more prevalent in sheep fed the lucerne-based diet. Five new species were identified. Two of these species exhibited very little sequence similarity to any cultivated methanogens and were found eight times in two of the six sheep that were grazing pasture. These unique sequences appear to represent a novel group of rumen archaea that are atypical for the rumen environment.

Increased abundance of Sutterella spp. and Ruminococcus torques in feces of children with autism spectrum disorder

BackgroundA recent report indicated that numbers of Sutterella spp. are elevated in gastrointestinal biopsies taken from children with autism spectrum disorder (ASD). We have recently reported changes in the numbers of some bacteria within the stool of ASD children, and now examine whether numbers of Sutterella spp. and some other mucosa-associated bacteria linked with gastrointestinal disease (Ruminococcus gnavus and Ruminococcus torques) are also altered in the stool of these children.FindingsWe show that numbers of Sutterella spp. are elevated in feces of ASD children relative to controls, and that numbers of R. torques are higher in the children with ASD with a reported functional gastrointestinal disorder than those without such a disorder.ConclusionsWe show further evidence of changes in the gut microbiota of children with ASD and confirm that the abundance of Sutterella spp. is altered in stool.

Low relative abundances of the mucolytic bacterium Akkermansia muciniphila and Bifidobacterium spp. in feces of children with autism.

ABSTRACT Gastrointestinal disturbance is frequently reported for individuals with autism. We used quantitative real-time PCR analysis to quantify fecal bacteria that could influence gastrointestinal health in children with and without autism. Lower relative abundances of Bifidobacteria species and the mucolytic bacterium Akkermansia muciniphila were found in children with autism, the latter suggesting mucus barrier changes.

A human, double-blind, placebo-controlled, crossover trial of prebiotic, probiotic, and synbiotic supplementation: effects on luminal, inflammatory, epigenetic, and epithelial biomarkers of colorectal cancer

BACKGROUND Diet is an important factor in colorectal carcinogenesis; thus, dietary supplements may have a role in colorectal cancer prevention. OBJECTIVE The objective was to establish the relative luminal, epithelial, and epigenetic consequences of prebiotic, probiotic, and synbiotic dietary supplementation in humans. DESIGN This was a randomized, double-blind, placebo-controlled, 4-wk crossover trial of resistant starch and Bifidobacterium lactis, either alone or as a combined synbiotic preparation, in 20 human volunteers. Rectal biopsy, feces, and serum samples were collected. The rectal mucosal endpoints were DNA methylation at 16 CpG island loci and LINE-1, epithelial proliferation (Ki67 immunohistochemistry), and crypt cellularity. The fecal endpoints were short-chain fatty acid concentrations, pH, ammonia, and microbiological profiles (by denaturing gradient gel electrophoresis and sequencing). Serum endpoints were a panel of cytokines and high-sensitivity C-reactive protein. RESULTS Seventeen subjects completed the entire study. The synbiotic intervention fostered a significantly different fecal stream bacterial community than did either the prebiotic (P = 0.032) or the probiotic (P = 0.001) intervention alone, in part because of a greater proportion of patients harboring fecal Lachnospiraceae spp. These changes developed in the absence of any significant differences in fecal chemistry. There were no differences in epithelial kinetics. CONCLUSIONS This synbiotic supplementation with B. lactis and resistant starch, in the doses used, induced unique changes in fecal microflora but did not significantly alter any other fecal, serum, or epithelial variables. This trial was registered in the Australian New Zealand Clinical Trials Registry at www.anzctr.org.au as ACTRN012606000115538.

Lactobacillus fermentum (PCC®) supplementation and gastrointestinal and respiratory-tract illness symptoms: a randomised control trial in athletes

BackgroundProbiotics purportedly reduce symptoms of gastrointestinal and upper respiratory-tract illness by modulating commensal microflora. Preventing and reducing symptoms of respiratory and gastrointestinal illness are the primary reason that dietary supplementation with probiotics are becoming increasingly popular with healthy active individuals. There is a paucity of data regarding the effectiveness of probiotics in this cohort. The aim of this study was to evaluate the effectiveness of a probiotic on faecal microbiology, self-reported illness symptoms and immunity in healthy well trained individuals.MethodsCompetitive cyclists (64 males and 35 females; age 35 ± 9 and 36 ± 9 y, VO2max 56 ± 6 and 52 ± 6 ml.kg-1.min-1, mean ± SD) were randomised to either probiotic (minimum 1 × 109Lactobacillus fermentum (PCC®) per day) or placebo treatment for 11 weeks in a double-blind, randomised, controlled trial. The outcome measures were faecal L. fermentum counts, self-reported symptoms of illness and serum cytokines.ResultsLactobacillus numbers increased 7.7-fold (90% confidence limits 2.1- to 28-fold) more in males on the probiotic, while there was an unclear 2.2-fold (0.2- to 18-fold) increase in females taking the probiotic. The number and duration of mild gastrointestinal symptoms were ~2-fold greater in the probiotic group. However, there was a substantial 0.7 (0.2 to 1.2) of a scale step reduction in the severity of gastrointestinal illness at the mean training load in males, which became more pronounced as training load increased. The load (duration×severity) of lower respiratory illness symptoms was less by a factor of 0.31 (99%CI; 0.07 to 0.96) in males taking the probiotic compared with placebo but increased by a factor of 2.2 (0.41 to 27) in females. Differences in use of cold and flu medication mirrored these symptoms. The observed effects on URTI had too much uncertainty for a decisive outcome. There were clear reductions in the magnitude of acute exercise-induced changes in some cytokines.ConclusionL. fermentum may be a useful nutritional adjunct for healthy exercising males. However, uncertainty in the effects of supplementation on URTI and on symptoms in females needs to be resolved.Trial registrationThe trial was registered in the Australia and New Zealand Clinical Trials Registry (ACTRN12611000006943).

Resistant starch, large bowel fermentation and a broader perspective of prebiotics and probiotics

The metabolic end products of the large bowel microbiota contribute significantly to human health. After weaning to solid foods, some of the most important of these are the short chain fatty acids (SCFA) produced by the fermentation of undigested dietary components and endogenous secretions. The main SCFA are acetate, propionate and butyrate which have numerous documented effects promoting large bowel function. Of the major acids, butyrate seems especially important. It is a major metabolic fuel for colonocytes and promotes a normal phenotype in these cells, potentially lowering the risk of diseases such as colo-rectal cancer. Imbalances in the microbiota are thought to predispose to large bowel dysfunction and probiotics are being developed to correct this. However, most commercial products contain bacteria (lactobacilli and bifidobacteria) which are dominant species in milk-fed infants but have limited roles in adults. Prebiosis is defined usually by the specific stimulation of these bacteria. However, the end products of most probiotics do not include butyrate or propionate which raises questions about their effectiveness in promoting bowel health in adults. Resistant starch (RS) is a dietary fibre component and its fermentation generally favours butyrate production. Dietary RS intakes and faecal butyrate levels are high in populations at low risk of diet-related large bowel diseases. Conversely, RS intakes and faecal butyrate levels are very low in high risk groups. This raises the possibility that greater RS consumption could be of health benefit. RS is not regarded widely as a prebiotic but (according to the accepted definition) most forms show the requisite features in stimulating specific bacteria, giving raised total SCFA and butyrate levels and a consequent benefit to the host. Current efforts to improve public health through increasing RS consumption could be facilitated by greater recognition of its prebiotic role.

Butyrylated starch intake can prevent red meat induced O6-methyl-2-deoxyguanosine adducts in human rectal tissue : a randomised clinical trial

Epidemiological studies have identified increased colorectal cancer (CRC) risk with high red meat (HRM) intakes, whereas dietary fibre intake appears to be protective. In the present study, we examined whether a HRM diet increased rectal O6-methyl-2-deoxyguanosine (O6MeG) adduct levels in healthy human subjects, and whether butyrylated high-amylose maize starch (HAMSB) was protective. A group of twenty-three individuals consumed 300 g/d of cooked red meat without (HRM diet) or with 40 g/d of HAMSB (HRM+HAMSB diet) over 4-week periods separated by a 4-week washout in a randomised cross-over design. Stool and rectal biopsy samples were collected for biochemical, microbial and immunohistochemical analyses at baseline and at the end of each 4-week intervention period. The HRM diet increased rectal O6MeG adducts relative to its baseline by 21 % (P< 0·01), whereas the addition of HAMSB to the HRM diet prevented this increase. Epithelial proliferation increased with both the HRM (P< 0·001) and HRM+HAMSB (P< 0·05) diets when compared with their respective baseline levels, but was lower following the HRM+HAMSB diet compared with the HRM diet (P< 0·05). Relative to its baseline, the HRM+HAMSB diet increased the excretion of SCFA by over 20 % (P< 0·05) and increased the absolute abundances of the Clostridium coccoides group (P< 0·05), the Clostridium leptum group (P< 0·05), Lactobacillus spp. (P< 0·01), Parabacteroides distasonis (P< 0·001) and Ruminococcus bromii (P< 0·05), but lowered Ruminococcus torques (P< 0·05) and the proportions of Ruminococcus gnavus, Ruminococcus torques and Escherichia coli (P< 0·01). HRM consumption could increase the risk of CRC through increased formation of colorectal epithelial O6MeG adducts. HAMSB consumption prevented red meat-induced adduct formation, which may be associated with increased stool SCFA levels and/or changes in the microbiota composition.

Butyrate esterified to starch is released in the human gastrointestinal tract

BACKGROUND Short-chain fatty acids (SCFAs) maintain human colonic function and may help prevent colonic disease. A study with ileostomists showed that starches acylated with specific SCFAs largely survive passage through the small intestine, but the percentage released in the colon has not been established. OBJECTIVE The objective was to determine the percentage of ingested esterified butyrate released in the human gastrointestinal tract. DESIGN The study was a randomized, crossover, controlled trial consisting of baseline and four 2-wk periods during which 16 volunteers consumed diets low in resistant starch plus 20 and 40 g cooked high-amylose maize starch (HAMS: HAMS20 or HAMS40) or butyrylated HAMS (HAMSB20 or HAMSB40) daily. HAMSB20 contained 31.8 mmol esterified butyrate. Complete 48-h fecal collections were made on days 2-3 and 12-13 of each period. RESULTS Free fecal butyrate concentrations were higher after HAMSB40 than after HAMSB20 (P < 0.005) and HAMS (P < 0.0001) and higher than baseline data (P < 0.0001). Fecal esterified butyrate concentrations were highest in the HAMSB40 (days 12-13; P < 0.0001) group, and concentrations in the HAMSB40 (days 2-3) and HAMSB20 groups were higher than those in the HAMS groups and those at baseline (P < 0.0001). Ingestion of HAMSB20 and HAMSB40 resulted in the release of 26.8 ± 1.0 and 50.2 ± 2.4 mmol butyrate/d (days 12-13) (84.2 ± 3.0% and 79.0 ± 3.1% of total ingested esterified butyrate), respectively, in the gastrointestinal tract. By calculation, ∼57.2% of ingested esterified butyrate was released in the colon. Microbial analysis showed that this release was probably facilitated mainly by Parabacteroides distasonis, which increased in abundance with HAMSB40 (days 12-13) (P < 0.001). CONCLUSIONS This study shows that cooked butyrylated starch delivers esterified butyrate to the human colon effectively and has the potential to improve human bowel health. This trial is registered in the Australian Clinical Trials Registry as ACTRN012606000398505.

Manipulation of the gut microbiota using resistant starch is associated with protection against colitis-associated colorectal cancer in rats

This study evaluated whether dietary resistant starch (RS) and green tea extract (GTE), which have anti-inflammatory and anticancer properties, protect against colitis-associated colorectal cancer (CAC) using a rat model, also investigated potential mechanisms of action of these agents including their effects on the gut microbiota. Rats were fed a control diet or diets containing 10% RS, 0.5% GTE or a combination of the two (RS + GTE). CAC was initiated with 2 weekly azoxymethane (AOM) injections (10mg/kg) followed by 2% dextran sodium sulphate in drinking water for 7 days after 2 weeks on diets. Rats were killed 20 weeks after the first AOM. Colon tissues and tumours were examined for histopathology by H&E, gene/protein expression by PCR and immunohistochemistry and digesta for analyses of fermentation products and microbiota populations. RS and RS + GTE (but not GTE) diets significantly (P< 0.05) decreased tumour multiplicity and adenocarcinoma formation, relative to the control diet. Effects of RS + GTE were not different from RS alone. RS diet caused significant shifts in microbial composition/diversity, with increases in Parabacteroides, Barnesiella, Ruminococcus, Marvinbryantia and Bifidobacterium as primary contributors to the shift. RS-containing diets increased short chain fatty acids (SCFA) and expression of the SCFA receptor GPR43 mRNA, and reduced inflammation (COX-2, NF-kB, TNF-α and IL-1β mRNA) and cell proliferation P< 0.05. GTE had no effect. This is the first study that demonstrates chemopreventive effects of RS (but not GTE) in a rodent CAC model, suggesting RS might have benefit to patients with ulcerative colitis who are at an increased risk of developing CRC.