Freda Farquharson

Impact of diet and individual variation on intestinal microbiota composition and fermentation products in obese men

There is growing interest in understanding how diet affects the intestinal microbiota, including its possible associations with systemic diseases such as metabolic syndrome. Here we report a comprehensive and deep microbiota analysis of 14 obese males consuming fully controlled diets supplemented with resistant starch (RS) or non-starch polysaccharides (NSPs) and a weight-loss (WL) diet. We analyzed the composition, diversity and dynamics of the fecal microbiota on each dietary regime by phylogenetic microarray and quantitative PCR (qPCR) analysis. In addition, we analyzed fecal short chain fatty acids (SCFAs) as a proxy of colonic fermentation, and indices of insulin sensitivity from blood samples. The diet explained around 10% of the total variance in microbiota composition, which was substantially less than the inter-individual variance. Yet, each of the study diets induced clear and distinct changes in the microbiota. Multiple Ruminococcaceae phylotypes increased on the RS diet, whereas mostly Lachnospiraceae phylotypes increased on the NSP diet. Bifidobacteria decreased significantly on the WL diet. The RS diet decreased the diversity of the microbiota significantly. The total 16S ribosomal RNA gene signal estimated by qPCR correlated positively with the three major SCFAs, while the amount of propionate specifically correlated with the Bacteroidetes. The dietary responsiveness of the individual’s microbiota varied substantially and associated inversely with its diversity, suggesting that individuals can be stratified into responders and non-responders based on the features of their intestinal microbiota.

Altered intestinal microbiota and blood T cell phenotype are shared by patients with Crohn's disease and their unaffected siblings

Objective Crohns disease (CD) is associated with intestinal dysbiosis, altered blood T cell populations, elevated faecal calprotectin (FC) and increased intestinal permeability (IP). CD-associated features present in siblings (increased risk of CD) but not in healthy controls, provide insight into early CD pathogenesis. We aimed to (1) Delineate the genetic, immune and microbiological profile of patients with CD, their siblings and controls and (2) Determine which factors discriminate between groups. Design Faecal microbiology was analysed by quantitative PCR targeting 16S ribosomal RNA, FC by ELISA, blood T cell phenotype by flow cytometry and IP by differential lactulose-rhamnose absorption in 22 patients with inactive CD, 21 of their healthy siblings and 25 controls. Subjects genotype relative risk was determined by Illumina Immuno BeadChip. Results Strikingly, siblings shared aspects of intestinal dysbiosis with patients with CD (lower concentrations of Faecalibacterium prausnitzii (p=0.048), Clostridia cluster IV (p=0.003) and Roseburia spp. (p=0.09) compared with controls). As in CD, siblings demonstrated a predominance of memory T cells (p=0.002) and elevated naïve CD4 T cell β7 integrin expression (p=0.01) compared with controls. FC was elevated (>50 μg/g) in 8/21 (38%) siblings compared with 2/25 (8%) controls (p=0.028); whereas IP did not differ between siblings and controls. Discriminant function analysis determined that combinations of these factors significantly discriminated between groups (χ2=80.4, df=20, p<0.001). Siblings were separated from controls by immunological and microbiological variables. Conclusions Healthy siblings of patients with CD manifest immune and microbiological abnormalities associated with CD distinct from their genotype-related risk and provide an excellent model in which to investigate early CD pathogenesis.

Extending colonic mucosal microbiome analysis - Assessment of colonic lavage as a proxy for endoscopic colonic biopsies

BackgroundSequencing-based analysis has become a well-established approach to deciphering the composition of the gut microbiota. However, due to the complexity of accessing sufficient material from colonoscopic biopsy samples, most studies have focused on faecal microbiota analysis, even though it is recognised that differences exist between the microbial composition of colonic biopsies and faecal samples. We determined the suitability of colonic lavage samples to see if it had comparable microbial diversity composition to colonic biopsies as they are without the limitations associated with sample size. We collected paired colonic biopsies and lavage samples from subjects who were attending for colorectal cancer screening colonoscopy.ResultsNext-generation sequencing and qPCR validation were performed with multiple bioinformatics analyses to determine the composition and predict function of the microbiota. Colonic lavage samples contained significantly higher numbers of operational taxonomic units (OTUs) compared to corresponding biopsy samples, however, diversity and evenness between lavage and biopsy samples were similar. The differences seen were driven by the presence of 12 OTUs which were in higher relative abundance in biopsies and were either not present or in low relative abundance in lavage samples, whilst a further 3 OTUs were present in higher amounts in the lavage samples compared to biopsy samples. However, predicted functional community profiling based on 16S ribosomal ribonucleic acid (rRNA) data indicated minimal differences between sample types.ConclusionsWe propose that colonic lavage samples provide a relatively accurate representation of biopsy microbiota composition and should be considered where biopsy size is an issue.

Specific substrate-driven changes in human faecal microbiota composition contrast with functional redundancy in short-chain fatty acid production

The diet provides carbohydrates that are non-digestible in the upper gut and are major carbon and energy sources for the microbial community in the lower intestine, supporting a complex metabolic network. Fermentation produces the short-chain fatty acids (SCFAs) acetate, propionate and butyrate, which have health-promoting effects for the human host. Here we investigated microbial community changes and SCFA production during in vitro batch incubations of 15 different non-digestible carbohydrates, at two initial pH values with faecal microbiota from three different human donors. To investigate temporal stability and reproducibility, a further experiment was performed 1 year later with four of the carbohydrates. The lower pH (5.5) led to higher butyrate and the higher pH (6.5) to more propionate production. The strongest propionigenic effect was found with rhamnose, followed by galactomannans, whereas fructans and several α- and β-glucans led to higher butyrate production. 16S ribosomal RNA gene-based quantitative PCR analysis of 22 different microbial groups together with 454 sequencing revealed significant stimulation of specific bacteria in response to particular carbohydrates. Some changes were ascribed to metabolite cross-feeding, for example, utilisation by Eubacterium hallii of 1,2-propanediol produced from fermentation of rhamnose by Blautia spp. Despite marked inter-individual differences in microbiota composition, SCFA production was surprisingly reproducible for different carbohydrates, indicating a level of functional redundancy. Interestingly, butyrate formation was influenced not only by the overall % butyrate-producing bacteria in the community but also by the initial pH, consistent with a pH-dependent shift in the stoichiometry of butyrate production.

Chlorogenic acid versus amaranth's caffeoylisocitric acid – Gut microbial degradation of caffeic acid derivatives

The almost forgotten crop amaranth has gained renewed interest in recent years due to its immense nutritive potential. Health beneficial effects of certain plants are often attributed to secondary plant metabolites such as phenolic compounds. As these compounds undergo significant metabolism after consumption and are in most cases not absorbed very well, it is important to gain knowledge about absorption, biotransformation, and further metabolism in the human body. Whilst being hardly found in other edible plants, caffeoylisocitric acid represents the most abundant low molecular weight phenolic compound in many leafy amaranth species. Given that this may be a potentially bioactive compound, gastrointestinal microbial degradation of this substance was investigated in the present study by performing in vitro fermentation tests using three different fecal samples as inocula. The (phenolic) metabolites were analyzed using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Furthermore, quantitative polymerase chain reaction (qPCR) analyses were carried out to study the influence on the microbiome and its composition. The in vitro fermentations led to different metabolite profiles depending on the specific donor. For example, the metabolite 3-(4-hydroxyphenyl)propionic acid was observed in one fermentation as the main metabolite, whereas 3-(3-hydroxyphenyl)propionic acid was identified in the other fermentations as important. A significant change in selected microorganisms of the gut microbiota however was not detected. In conclusion, caffeoylisocitric acid from amaranth, which is a source of several esterified phenolic acids in addition to chlorogenic acid, can be metabolized by the human gut microbiota, but the metabolites produced vary between individuals.

Porcine Small and Large Intestinal Microbiota Rapidly Hydrolyze the Masked Mycotoxin Deoxynivalenol-3-Glucoside and Release Deoxynivalenol in Spiked Batch Cultures In Vitro

ABSTRACT Mycotoxin contamination of cereal grains causes well-recognized toxicities in animals and humans, but the fate of plant-bound masked mycotoxins in the gut is less well understood. Masked mycotoxins have been found to be stable under conditions prevailing in the small intestine but are rapidly hydrolyzed by fecal microbiota. This study aims to assess the hydrolysis of the masked mycotoxin deoxynivalenol-3-glucoside (DON3Glc) by the microbiota of different regions of the porcine intestinal tract. Intestinal digesta samples were collected from the jejunum, ileum, cecum, colon, and feces of 5 pigs and immediately frozen under anaerobic conditions. Sample slurries were prepared in M2 culture medium, spiked with DON3Glc or free deoxynivalenol (DON; 2 nmol/ml), and incubated anaerobically for up to 72 h. Mycotoxin concentrations were determined using liquid chromatography-tandem mass spectrometry, and the microbiota composition was determined using a quantitative PCR methodology. The jejunal microbiota hydrolyzed DON3Glc very slowly, while samples from the ileum, cecum, colon, and feces rapidly and efficiently hydrolyzed DON3Glc. No further metabolism of DON was observed in any sample. The microbial load and microbiota composition in the ileum were significantly different from those in the distal intestinal regions, whereas those in the cecum, colon and feces did not differ. IMPORTANCE Results from this study clearly demonstrate that the masked mycotoxin DON3Glc is hydrolyzed efficiently in the distal small intestine and large intestine of pigs. Once DON is released, toxicity and absorption in the distal intestinal tract likely occur in vivo. This study further supports the need to include masked metabolites in mycotoxin risk assessments and regulatory actions for feed and food.

Mutual Interaction of Phenolic Compounds and Microbiota: Metabolism of Complex Phenolic Apigenin-C- and Kaempferol-O-Derivatives by Human Fecal Samples

Human colonic bacteria have an important impact on the biotransformation of flavonoid glycosides and their conversion can result in the formation of bioactive compounds. However, information about the microbial conversion of complex glycosylated flavonoids and the impact on the gut microbiota are still limited. In this study, in vitro fermentations with selected flavonoid O- and C-glycosides and three different fecal samples were performed. As a result, all flavonoid glycosides were metabolized via their aglycones yielding smaller substances. Main metabolites were 3-(4-hydroxyphenyl)propionic acid, 3-phenylpropionic acid, and phenylacetic acid. Differences in the metabolite formation due to different time courses between the donors were determined. Therefore, from all fermentations, the ones with a specific donor were always slower resulting in a lower number of metabolites compared to the others. For example, tiliroside was totally degraded from 0 h (105 ± 13.2 μM) within the first 24 h, while in the fermentations with fecal samples from other donors, tiliroside (107 ± 52.7 μM at 0 h) was not detected after 7 h anymore. In general, fermentation rates of C-glycosides were slower compared to the fermentation rates of O-glycosides. The O-glycoside tiliroside was degraded within 4 h while the gut microbiota converted the C-glycoside vitexin within 13 h. However, significant changes (p < 0.05) in the microbiota composition and short chain fatty acid levels as products of carbohydrate fermentation were not detected between incubations with different phenolic compounds. Therefore, microbiota diversity was not affected and a significant prebiotic effect of phenolic compounds cannot be assigned to flavonoid glycosides in food-relevant concentrations.

Dietary fibers inhibit obesity in mice, but host responses in the cecum and liver appear unrelated to fiber-specific changes in cecal bacterial taxonomic composition

Dietary fibers (DF) can prevent obesity in rodents fed a high-fat diet (HFD). Their mode of action is not fully elucidated, but the gut microbiota have been implicated. This study aimed to identify the effects of seven dietary fibers (barley beta-glucan, apple pectin, inulin, inulin acetate ester, inulin propionate ester, inulin butyrate ester or a combination of inulin propionate ester and inulin butyrate ester) effective in preventing diet-induced obesity and links to differences in cecal bacteria and host gene expression. Mice (n = 12) were fed either a low-fat diet (LFD), HFD or a HFD supplemented with the DFs, barley beta-glucan, apple pectin, inulin, inulin acetate ester, inulin propionate ester, inulin butyrate ester or a combination of inulin propionate ester and inulin butyrate ester for 8 weeks. Cecal bacteria were determined by Illumina MiSeq sequencing of 16S rRNA gene amplicons. Host responses, body composition, metabolic markers and gene transcription (cecum and liver) were assessed post intervention. HFD mice showed increased adiposity, while all of the DFs prevented weight gain. DF specific differences in cecal bacteria were observed. Results indicate that diverse DFs prevent weight gain on a HFD, despite giving rise to different cecal bacteria profiles. Conversely, common host responses to dietary fiber observed are predicted to be important in improving barrier function and genome stability in the gut, maintaining energy homeostasis and reducing HFD induced inflammatory responses in the liver.

OC-017 A Discriminant Analysis Demonstrates that Siblings of Patients with Crohn’S Disease have a Distinct Microbiological and Immune Phenotype Compared with Healthy Controls: Insights into Disease Pathogenesis

Introduction Crohn’s disease (CD) is associated with genetic risk, intestinal dysbiosis, altered blood T-cell phenotype, increased faecal calprotectin (FC) and intestinal permeability (IP). Factors shared by CD patients and unaffected siblings may be implicated in CD pathogenesis. Aims Delineate the genetic, immune and microbial phenotype of patients, siblings and healthy controls (HC); identify factors associated with CD that discriminate siblings from HC. Methods Faecal microbiota, FC, blood T-cell phenotype, IP and genotype risk over 72 CD risk loci, were measured by qPCR, ELISA, flow cytometry, sugar permeability and Illumina Bead Array respectively, in 22 patients with inactive CD, 21 of their healthy siblings and 25 HC. Results In addition to genotype risk, siblings shared aspects of the phenotype of CD patients, distinct from HC, as previously reported.1 Direct discriminant function analysis revealed that the variables maximally separating siblings from HC (Function 2) were: increased β7 integrin expression by circulating naïve CD4+ T-cells and an increased proportion of memory CD4+ T-cells as well as reduced faecal Roseburia spp. (Image 1). In contrast, the variables differentiating CD patients from HC (Function 1) were: elevated FC and altered faecal microbiota (reduced Faecalibacterium prausnitzii, Cluster IV Ruminococcus spp. Bacteroides-Prevotella and Clostridial cluster IV). Abstract OC-017 Figure 1 Conclusion Healthy siblings of CD patients manifest immune and microbiological abnormalities associated with CD, distinct from their genetic risk. Unaffected siblings of CD patients are an excellent model in which to investigate early CD pathogenesis. Disclosure of Interest None Declared Reference Hedin et al. Gut 2012; 61: Suppl. 2 A22-A23.

Tu1729 Evaluation of Fecal Aspirate As a Proxy for Colonoscopy Biopsy Sampling to Assess Microbial Diversity

Introduction The gut microbiota plays a key role in Inflammatory Bowel Disease (IBD) pathogenesis and is also involved in colorectal neoplastic progression. In particular, the “dysbiosis” theory has evolved from work which has shown IBD sufferers to have different microbiologic profiles to normal controls. There is strong evidence to demonstrate that individuals harbour a unique microbiota with faecal and mucosal ecosystems being distinct. This would indicate that faecal samples are not appropriate surrogate markers for mucosal samples and other alternative sampling options should be considered. The aim of the study was to assess the microbial diversity of paired biopsies and faecal aspirates to determine whether faecal aspirate can be used as a suitable surrogate for colonic biopsies. Methods Bacterial DNA was extracted from 21 paired mucosal biopsies and faecal aspirates, and subjected to denaturing gradient gel electrophoresis (DGGE) and q-PCR analysis to quantify the relative abundance of the following major bacterial groups: Bacteroidetes, Ruminococcaceae , Lachnospiraceae and Enterobacteriaceae . The relative abundance of bacterial groups was expressed as a percentage of total bacteria. Results Increased variation was found across the faecal aspirates compared to biopsies with regards to relative abundance of the four bacterial groups analysed by q-PCR (Table 1). This indicates that microbial diversity of faecal aspirates is more variable than mucosal biopsies. When the mean abundance of each bacterial group across the biopsies and across the faecal aspirates was compared using a Paired t-test, there was a significant difference in levels of Bacteroides (62.3% vs 44.4%; p = Conclusion Substantial microbial diversity differences exist between faecal aspirate and biopsy samples. Faecal aspirates demonstrate considerably more variation than biopsies, and there would appear to be no correlation between paired samples. These results suggest that faecal aspirate is not a suitable microbiological surrogate for mucosal biopsies. Disclosure of Interest None Declared.