Johan Dicksved

A Pyrosequencing Study in Twins Shows That Gastrointestinal Microbial Profiles Vary With Inflammatory Bowel Disease Phenotypes

BACKGROUND & AIMS The composition of the gastrointestinal microbiota is thought to have an important role in the etiology of inflammatory bowel diseases (IBDs) such as Crohns disease (CD) and ulcerative colitis (UC). Interindividual variation and an inability to detect less abundant bacteria have made it difficult to correlate specific bacteria with disease. METHODS We used 454 pyrotag sequencing to determine the compositions of microbial communities in feces samples collected from a cohort of 40 twin pairs who were concordant or discordant for CD or UC, and in mucosal samples from a subset of the cohort. The cohort primarily comprised patients who were in remission, but also some with active disease. RESULTS The profiles of the microbial community differed with disease phenotypes; relative amounts of bacterial populations correlated with IBD phenotypes. The microbial compositions of individuals with CD differed from those of healthy individuals, but were similar between healthy individuals and individuals with UC. Profiles from individuals with CD that predominantly involved the ileum differed from those with CD that predominantly involved the colon; several bacterial populations increased or decreased with disease type. Changes specific to patients with ileal CD included the disappearance of core bacteria, such as Faecalibacterium and Roseburia, and increased amounts of Enterobacteriaceae and Ruminococcus gnavus. CONCLUSIONS Bacterial populations differ in abundance among individuals with different phenotypes of CD. Specific species of bacteria are associated with ileal CD; further studies should investigate their role in pathogenesis.

Twin studies reveal specific imbalances in the mucosa-associated microbiota of patients with ileal Crohn's disease

Background: Large interindividual variation in the composition of the intestinal microbiota between unrelated individuals has made it challenging to identify specific aspects of dysbiosis that lead to Crohns disease (CD). Methods: To reduce variations in exposure during establishment of the gut flora and the influence of genotype, we studied the mucosa‐associated microbiota of monozygotic twin pairs that were discordant (n = 6) or concordant (n = 4) for CD. DNA was extracted from biopsies collected from 5 locations between the ileum and rectum. Bacterial 16S ribosomal RNA genes were amplified and community composition assessed by terminal‐restriction fragment length polymorphism, cloning and sequencing, and quantitative real‐time polymerase chain reaction (PCR). Results: The microbial compositions at all biopsy locations for each individual were similar, regardless of disease state, but there were differences between individuals. In particular, individuals with predominantly ileal CD had a dramatically lower abundance (P < 0.001) of Faecalibacterium prausnitzii and increased abundance (P < 0.03) of Escherichia coli compared to healthy co‐twins and those with CD localized in the colon. This dysbiosis was significantly correlated to the disease phenotype rather than genotype. Conclusions: The reduced abundance of F. prausnitzii and increased abundance of E. coli are indicative of an ileal CD phenotype, distinct from colonic CD, and the relative abundances of these specific bacterial populations are promising biomarker candidates for differential diagnosis of CD and eventually customized treatment.

Molecular analysis of the gut microbiota of identical twins with Crohn's disease

Increasing evidence suggests that a combination of host genetics and the composition of the gut microbiota are important for development of Crohns disease (CD). Our aim was to study identical twins with CD to determine microbial factors independent of host genetics. Fecal samples were studied from 10 monozygotic twin pairs with CD (discordant n=6 and concordant n=4) and 8 healthy twin pairs. DNA was extracted, 16S rRNA genes were PCR amplified and T-RFLP fingerprints generated using general bacterial and Bacteroides group-specific primers. The microbial communities were also profiled based on their percentage G+C contents. Bacteroides 16S rRNA genes were cloned and sequenced from a subset of the samples. The bacterial diversity in each sample and similarity indices between samples were estimated based on the T-RFLP data using a combination of statistical approaches. Healthy individuals had a significantly higher bacterial diversity compared to individuals with CD. The fecal microbial communities were more similar between healthy twins than between twins with CD, especially when these were discordant for the disease. The microbial community profiles of individuals with ileal CD were significantly different from healthy individuals and those with colonic CD. Also, CD individuals had a lower relative abundance of B. uniformis and higher relative abundances of B. ovatus and B. vulgatus. Our results suggest that genetics and/or environmental exposure during childhood, in part, determine the gut microbial composition. However, CD is associated with dramatic changes in the gut microbiota and this was particularly evident for individuals with ileal CD.

Metabolomics Reveals Metabolic Biomarkers of Crohn's Disease

The causes and etiology of Crohns disease (CD) are currently unknown although both host genetics and environmental factors play a role. Here we used non-targeted metabolic profiling to determine the contribution of metabolites produced by the gut microbiota towards disease status of the host. Ion Cyclotron Resonance Fourier Transform Mass Spectrometry (ICR-FT/MS) was used to discern the masses of thousands of metabolites in fecal samples collected from 17 identical twin pairs, including healthy individuals and those with CD. Pathways with differentiating metabolites included those involved in the metabolism and or synthesis of amino acids, fatty acids, bile acids and arachidonic acid. Several metabolites were positively or negatively correlated to the disease phenotype and to specific microbes previously characterized in the same samples. Our data reveal novel differentiating metabolites for CD that may provide diagnostic biomarkers and/or monitoring tools as well as insight into potential targets for disease therapy and prevention.

Molecular characterization of the stomach microbiota in patients with gastric cancer and in controls

Persistent infection of the gastric mucosa by Helicobacter pylori can initiate an inflammatory cascade that progresses into atrophic gastritis, a condition associated with reduced capacity for secretion of gastric acid and an increased risk of developing gastric cancer. The role of H. pylori as an initiator of inflammation is evident but the mechanism for development into gastric cancer has not yet been proven. A reduced capacity for gastric acid secretion allows survival and proliferation of other microbes that normally are killed by the acidic environment. It has been postulated that some of these species may be involved in the development of gastric cancer; however, their identities are poorly defined. In this study, the gastric microbiota from ten patients with gastric cancer was characterized and compared with that from five dyspeptic controls using the molecular profiling approach terminal restriction fragment length polymorphism (T-RFLP), in combination with 16S rRNA gene cloning and sequencing. T-RFLP analysis revealed a complex bacterial community in the cancer patients that was not significantly different from that in the controls. Sequencing of 140 clones revealed 102 phylotypes, with representatives from five bacterial phyla (Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria and Fusobacteria). The data revealed a relatively low abundance of H. pylori and showed that the gastric cancer microbiota was instead dominated by different species of the genera Streptococcus, Lactobacillus, Veillonella and Prevotella. The respective role of these species in development of gastric cancer remains to be determined.

Molecular Fingerprinting of the Fecal Microbiota of Children Raised According to Different Lifestyles

ABSTRACT In this population-based study, 90 children from three European countries were examined to determine the impact of lifestyle on the fecal microbiota. The study was designed to assess the impact of two extreme lifestyles that we hypothesized could impact the microbial composition in the gut: i.e., an anthroposophic lifestyle (restricted use of antibiotics, greater consumption of fermented vegetables, etc.) versus living on a farm (greater consumption of farm milk, contact with animals, etc.). In previous studies, these lifestyles correlated with lower prevalence of allergies. Terminal restriction fragment length polymorphism (T-RFLP) was used to assess the bacterial composition in fecal samples since recent studies have shown that the majority of this community cannot be cultivated. The T-RFLP data were used to calculate richness and evenness of the fecal microbiota. Children that were attending Steiner schools (anthroposophic children) had a significantly higher diversity of microbes in their feces than farm children, who in turn also had lower diversity than the control groups. Specific primers were also used to focus on the Lactobacillus-like community (lactic acid bacteria [LAB]). Large differences were found in the LAB subpopulations in the sampled groups. In some children, the LAB subpopulation was dominated by a species that has not yet been cultivated.

Metabolic Profiles and Genetic Diversity of Denitrifying Communities in Activated Sludge after Addition of Methanol or Ethanol

ABSTRACT External carbon sources can enhance denitrification rates and thus improve nitrogen removal in wastewater treatment plants. The effects of adding methanol and ethanol on the genetic and metabolic diversity of denitrifying communities in activated sludge were compared using a pilot-scale plant with two parallel lines. A full-scale plant receiving the same municipal wastewater, but without external carbon source addition, was the reference. Metabolic profiles obtained from potential denitrification rates with 10 electron donors showed that the denitrifying communities altered their preferences for certain compounds after supplementation with methanol or ethanol and that methanol had the greater impact. Clone libraries of nirK and nirS genes, encoding the two different nitrite reductases in denitrifiers, revealed that methanol also increased the diversity of denitrifiers of the nirS type, which indicates that denitrifiers favored by methanol were on the rise in the community. This suggests that there might be a niche differentiation between nirS and nirK genotypes during activated sludge processes. The composition of nirS genotypes also varied greatly among all samples, whereas the nirK communities were more stable. The latter was confirmed by denaturing gradient gel electrophoresis of nirK communities on all sampling occasions. Our results support earlier hypotheses that the compositions of denitrifier communities change during predenitrification processes when external carbon sources are added, although no severe effect could be observed from an operational point of view.

454 Pyrosequencing Analysis on Faecal Samples from a Randomized DBPC Trial of Colicky Infants Treated with Lactobacillus reuteri DSM 17938

Objective To analyze the global microbial composition, using large-scale DNA sequencing of 16 S rRNA genes, in faecal samples from colicky infants given L. reuteri DSM 17938 or placebo. Methods Twenty-nine colicky infants (age 10–60 days) were enrolled and randomly assigned to receive either Lactobacillus reuteri (108 cfu) or a placebo once daily for 21 days. Responders were defined as subjects with a decrease of 50% in daily crying time at day 21 compared with the starting point. The microbiota of faecal samples from day 1 and 21 were analyzed using 454 pyrosequencing. The primers: Bakt_341F and Bakt_805R, complemented with 454 adapters and sample specific barcodes were used for PCR amplification of the 16 S rRNA genes. The structure of the data was explored by using permutational multivariate analysis of variance and effects of different variables were visualized with ordination analysis. Results The infants’ faecal microbiota were composed of Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes as the four main phyla. The composition of the microbiota in infants with colic had very high inter-individual variability with Firmicutes/Bacteroidetes ratios varying from 4000 to 0.025. On an individual basis, the microbiota was, however, relatively stable over time. Treatment with L. reuteri DSM 17938 did not change the global composition of the microbiota, but when comparing responders with non-responders the group responders had an increased relative abundance of the phyla Bacteroidetes and genus Bacteroides at day 21 compared with day 0. Furthermore, the phyla composition of the infants at day 21 could be divided into three enterotype groups, dominated by Firmicutes, Bacteroidetes, and Actinobacteria, respectively. Conclusion L. reuteri DSM 17938 did not affect the global composition of the microbiota. However, the increase of Bacteroidetes in the responder infants indicated that a decrease in colicky symptoms was linked to changes of the microbiota. Trial Registration ClinicalTrials.gov NCT00893711

Lactobacillus reuteri Maintains a Functional Mucosal Barrier during DSS Treatment Despite Mucus Layer Dysfunction

Treatment with the probiotic bacterium Lactobacillus reuteri has been shown to prevent dextran sodium sulfate (DSS)-induced colitis in rats. This is partly due to reduced P-selectin-dependent leukocyte- and platelet-endothelial cell interactions, however, the mechanism behind this protective effect is still unknown. In the present study a combination of culture dependent and molecular based T-RFLP profiling was used to investigate the influence of L. reuteri on the colonic mucosal barrier of DSS treated rats. It was first demonstrated that the two colonic mucus layers of control animals had different bacterial community composition and that fewer bacteria resided in the firmly adherent layer. During DSS induced colitis, the number of bacteria in the inner firmly adherent mucus layer increased and bacterial composition of the two layers no longer differed. In addition, induction of colitis dramatically altered the microbial composition in both firmly and loosely adherent mucus layers. Despite protecting against colitis, treatment with L. reuteri did not improve the integrity of the mucus layer or prevent distortion of the mucus microbiota caused by DSS. However, L. reuteri decreased the bacterial translocation from the intestine to mesenteric lymph nodes during DSS treatment, which might be an important part of the mechanisms by which L. reuteri ameliorates DSS induced colitis.

Inclusion of Chicory (Cichorium intybus L.) in Pigs' Diets Affects the Intestinal Microenvironment and the Gut Microbiota

ABSTRACT The content and composition of prebiotic plant fiber in the diet is important in promoting gut-related health. This study investigated the effects of the dietary inclusion of chicory forage and roots on the intestinal microenvironment of pigs. Thirty-seven-week-old pigs were fed 1 of 5 diets for 18 days, including a cereal-based control diet and 4 diets with the inclusion of 80 and 160 g kg−1 of body weight chicory forage (CF80 and CF160), 80 g kg−1 chicory root (CR80), and a mix of 80 g kg−1 forage and 80 g kg−1 chicory root (CFR). The animals maintained good performance and health irrespective of diet. Bacterial community structure and diversity in ileal and colonic samples was assessed using terminal restriction fragment length polymorphism (T-RFLP), combined with cloning and sequencing. Samples clustered perfectly according to gut segment with a higher bacterial diversity in colon than ileum. Distal ileum was dominated by lactic acid bacteria (LAB), and the relative amount of this group was increased by the CF160 and CFR diets. The colonic bacterial community was dominated by butyrate-producing bacteria and Prevotella. The increased relative abundance of butyrate-producing bacteria in the colon was positively correlated with the molar proportion of acetic acid and furthermore linked to the chicory forage diets (CF80 and CF160). Diets including chicory roots (CR80 and CFR) were correlated with a higher colonic abundance of Megasphaera elsdenii. The fermentation products and pH in digesta responded to diet type and were correlated with shifts in the microbiota, showing that chicory influences the intestinal microenvironment of pigs.