OBJECTIVES:Exploring associations between the gut microbiota and colonic inflammation and assessing sequential changes during exclusive enteral nutrition (EEN) may offer clues into the microbial origins of Crohn's disease (CD).METHODS:Fecal samples (n=117) were collected from 23 CD and 21 healthy children. From CD children fecal samples were collected before, during EEN, and when patients returned to their habitual diets. Microbiota composition and functional capacity were characterized using sequencing of the 16S rRNA gene and shotgun metagenomics.RESULTS:Microbial diversity was lower in CD than controls before EEN (P=0.006); differences were observed in 36 genera, 141 operational taxonomic units (OTUs), and 44 oligotypes. During EEN, the microbial diversity of CD children further decreased, and the community structure became even more dissimilar than that of controls. Every 10 days on EEN, 0.6 genus diversity equivalents were lost; 34 genera decreased and one increased during EEN. Fecal calprotectin correlated with 35 OTUs, 14 of which accounted for 78% of its variation. OTUs that correlated positively or negatively with calprotectin decreased during EEN. The microbiota of CD patients had a broader functional capacity than healthy controls, but diversity decreased with EEN. Genes involved in membrane transport, sulfur reduction, and nutrient biosynthesis differed between patients and controls. The abundance of genes involved in biotin (P=0.005) and thiamine biosynthesis decreased (P=0.017), whereas those involved in spermidine/putrescine biosynthesis (P=0.031), or the shikimate pathway (P=0.058), increased during EEN.CONCLUSIONS:Disease improvement following treatment with EEN is associated with extensive modulation of the gut microbiome.

EEN impacts on gut microbiota composition and changes fecal metabolic activity. It is difficult to infer a causative association between such changes and disease improvement, but the results do challenge the current perception of a protective role for F. prausnitzii in CD.

BackgroundThe effect that traditional and modern DNA extraction methods have on applications to study the role of gut microbiota in health and disease is a topic of current interest. Genomic DNA was extracted from three faecal samples and one probiotic capsule using three popular methods; chaotropic (CHAO) method, phenol/chloroform (PHEC) extraction, proprietary kit (QIAG). The performance of each of these methods on DNA yield and quality, microbiota composition using quantitative PCR, deep sequencing of the 16S rRNA gene, and sequencing analysis pipeline was evaluated.ResultsThe CHAO yielded the highest and the QIAG kit the lowest amount of double-stranded DNA, but the purity of isolated nucleic acids was better for the latter method. The CHAO method yielded a higher concentration of bacterial taxa per mass (g) of faeces. Sequencing coverage was higher in CHAO method but a higher proportion of the initial sequencing reads were retained for assignments to operational taxonomic unit (OTU) in the QIAG kit compared to the other methods. The QIAG kit appeared to have longer trimmed reads and shorter regions of worse quality than the other two methods. A distinct separation of α-diversity indices between different DNA extraction methods was not observed. When compositional dissimilarities between samples were explored, a strong separation was observed according to sample type. The effect of the extraction method was either marginal (Bray–Curtis distance) or none (unweighted Unifrac distance). Taxon membership and abundance in each sample was independent of the DNA extraction method used.ConclusionsWe have benchmarked several DNA extraction methods commonly used in gut microbiota research and their differences depended on the downstream applications intended for use. Caution should be paid when the intention is to pool and analyse samples or data from studies which have used different DNA extraction methods.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-016-2171-7) contains supplementary material, which is available to authorized users.

Background/AimsStudying the gut microbiota in unaffected relatives of people with Crohn’s disease (CD) may advance our understanding of the role of bacteria in disease aetiology.MethodsFaecal microbiota composition (16S rRNA gene sequencing), genetic functional capacity (shotgun metagenomics) and faecal short chain fatty acids (SCFA) were compared in unaffected adult relatives of CD children (CDR, n = 17) and adult healthy controls, unrelated to CD patients (HUC, n = 14). The microbiota characteristics of 19 CD children were used as a benchmark of CD ‘dysbiosis’.ResultsThe CDR microbiota was less diverse (p = 0.044) than that of the HUC group. Local contribution of β-diversity analysis showed no difference in community structure between the CDR and HUC groups. Twenty one of 1,243 (1.8%) operational taxonomic units discriminated CDR from HUC. The metagenomic functional capacity (p = 0.207) and SCFA concentration or pattern were similar between CDR and HUC (p>0.05 for all SCFA). None of the KEGG metabolic pathways were different between these two groups. Both of these groups (HUC and CDR) had a higher microbiota α-diversity (CDR, p = 0.026 and HUC, p<0.001) with a community structure (β-diversity) distinct from that of children with CD.ConclusionsWhile some alterations were observed, a distinct microbial ‘dysbiosis’, characteristic of CD patients, was not observed in their unaffected, genetically linked kindred.

No abstract

This study aimed to provide evidence on whether children at risk of gastrointestinal inflammation have increased measurements of faecal calprotectin (FC). Faecal calprotectin was measured in 232 children; 55 children (n=11 treatment naïve) with juvenile idiopathic arthritis (JIA), 63 with coeliac disease (CD); 17 with new diagnosis before and after treatment on gluten free diet and 114 controls. None of the treatment-naive children with JIA had raised FC. Four JIA patients on treatment had a raised FC but in all cases a repeat test was normal. In newly diagnosed CD patients, the median (IQR) FC was higher 36.4 (26-61) than in controls 25.0 (23-41) mg/kg (p=0.045) but this significantly decreased 25 (25-25) mg/kg (p=0.012) after six months on gluten free diet. Random measurements of FC are not raised in children with JIA or CD. A significant elevation of FC in these groups is not explained by their diagnosis and therefore needs further investigation.

No abstract

The gut microbiota is implicated in the pathogenesis of Crohn's Disease (CD) (1) . Exclusive enteral nutrition (EEN) is a successful treatment but its mode of action remains unknown (2) . We assessed changes in the fecal microbiota milieu during EEN. Five faecal samples were collected from CD children; 4 during EEN (start, 15, 30, end EEN∼60 days) and a fifth on free habitual diet. Two samples were collected from healthy controls. Faecal pH, bacterial metabolites ( Figure) and quantitative changes (q-PCR) of total and 7 major bacterial groups implicated in CD were measured. 68 samples were from 15 CD children and 40 from 21 controls. Faecal pH and total sulphide increased and butyric acid decreased during EEN (Figure). F. prausnitzii spp. concentration significantly decreased after 30 d on EEN (Figure). In patients who responded to EEN, the magnitude of the observed changes was greater and the concentration of Bacteroides/Prevotella group also decreased. All these changes reverted to pre-treatment levels when the children returned to their free habitual diet.EEN impacts on gut microbiota composition and changes faecal metabolic activity. It is difficult to infer a causative association between such changes and disease improvement but the results do challenge the current perception of a protective role for F. prausnitzii in CD.