Impact of Colonoscopy Bowel Preparation on Intestinal Microbiota

The gut microbiota is important in maintaining human health, but numerous factors have the potential to alter its composition. Our aim was to examine the impact of a standard bowel preparation on the intestinal microbiota using two different techniques. Fifteen subjects undergoing colonoscopy consumed a bowel preparation comprised of 10 mg bisacodyl and 2 L polyethylene glycol. The microbiota of stool samples, collected one month before, one week before (pre-colonoscopy), and one week, one month, and three to six months after colonoscopy (post-colonoscopy) was evaluated. Two samples were taken three to six months apart from five healthy subjects who did not undergo colonoscopy. Universal primers targeting the V2-V3 region of the 16S rRNA gene were used to PCR amplify all samples for denaturing gradient gel electrophoresis (PCR-DGGE). Pre- and post-colonoscopy samples were compared using Dice's similarity coefficients. Three samples from ten subjects who underwent colonoscopy, and both samples from the five subjects who didn't, were used for high-throughput sequencing of the V1-V3 region of the 16S rRNA gene. Samples were curated and analysed in Mothur. Results of the DGGE analyses show that the fecal microbiota of a small number of subjects had short-term changes. High-throughput sequencing results indicated that the variation between the samples of subjects who underwent colonoscopy was no greater than the variation observed between samples from subjects who did not. We conclude that bowel preparation does not have a lasting effect on the composition of the intestinal microbiota for the majority of subjects.This work was funded by an Australian Post-Graduate Award scholarship. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript

Comparative genomics of Crohn's disease-associated adherent-invasive Escherichia coli

OBJECTIVE Adherent-invasive Escherichia coli (AIEC) are a leading candidate bacterial trigger for Crohn's disease (CD). The AIEC pathovar is defined by in vitro cell-line assays examining specific bacteria/cell interactions. No molecular marker exists for their identification. Our aim was to identify a molecular property common to the AIEC phenotype. DESIGN 41 B2 phylogroup E. coli strains were isolated from 36 Australian subjects: 19 patients with IBD and 17 without. Adherence/invasion assays were conducted using the I-407 epithelial cell line and survival/replication assays using the THP-1 macrophage cell line. Cytokine secretion tumour necrosis factor ((TNF)-α, interleukin (IL) 6, IL-8 and IL-10) was measured using ELISA. The genomes were assembled and annotated, and cluster analysis performed using CD-HIT. The resulting matrices were analysed to identify genes unique/more frequent in AIEC strains compared with non-AIEC strains. Base composition differences and clustered regularly interspaced palindromic repeat (CRISPR) analyses were conducted. RESULTS Of all B2 phylogroup strains assessed, 79% could survive and replicate in macrophages. Among them, 11/41 strains (5 CD, 2 UCs, 5 non-IBD) also adhere to and invade epithelial cells, a phenotype assigning them to the AIEC pathovar. The AIEC strains were phylogenetically heterogeneous. We did not identify a gene (or nucleic acid base composition differences) common to all, or the majority of, AIEC. Cytokine secretion and CRISPRs were not associated with the AIEC phenotype. CONCLUSIONS Comparative genomic analysis of AIEC and non-AIEC strains did not identify a molecular property exclusive to the AIEC phenotype. We recommend a broader approach to the identification of the bacteria-host interactions that are important in the pathogenesis of Crohn's disease.This research was supported by an Australian Academy of Science France-Australia Science Innovation Collaboration early career fellowship; a Gastroenterological Society of Australia (GESA) Clinical Research grant; and funding from the Ministère de la Recherche et de la Technologie, Inserm (UMR1071), INRA (USC-2018); and Nouveau Chercheur EPST from Conseil Régional Auvergne

Gene expression differences between Crohn's disease aphthous ulcers and healthy Peyer's patches highlight novel therapeutic targets

Background and Aim: The earliest macroscopic lesion in Crohn’s disease(CD) is the aphthous ulcer, which overlies Peyer’s patches and lymphoidfollicles. Our aim was to characterize differences in gene expression andthe virome of aphthous ulcers and Peyer’s patches.Methods: Biopsies (n = 24) were obtained from the terminal ileum of 12patients (six with CD and six healthy controls). Aphthous ulcers and adja-cent unaffected mucosa were obtained from patients with CD, and Peyer’spatches and adjacent mucosa from the controls. All patients, except one,were medication free. RNA was extracted using Qiagen kits. NextSeq500 libraries were constructed using NextSeq 500/550 High output kits(Illumina) in a 150 bp paired-end format. Whole genome transcripts wereassessed for quality using FASTQC, trimmed using Trimmomatic, andaligned to the human reference genome using subread mapper. Fragmentcounts were obtained using featureCount, and expression values normal-ized using the trimmed mean of M-values normalization method (TMM).Differential gene expression analyses were performed using generalizedlinear models in edgeR. Cell-specific gene expression was determinedusing ImSig. Reads that did not map to the human genome were used tomine for virus sequences using the VirusSeeker pipeline.Results: We obtained 36 million tags per sample, 87% were retained fordownstream processing, and 93% of these mapped to the human genome.A total of 920 genes were significantly differentially expressed betweenaphthous ulcers and Peyer’s patches (P = <0.001); all were upregulatedin aphthous ulcers. Differential gene expression analysis revealed 34 path-ways that were upregulated in aphthous ulcers relative to Peyer’s patches.Pathways that were over-expressed included those involved in respondingto bacteria, leukocyte chemotaxis, inflammatory response, and creation ofC2 and C4 activators. Receptors for the constant region of IgG were repre-sented in 13 pathways. The cytokine OSM and its receptor (OSMR) werealso over-expressed in aphthous ulcers. ImSig, which is capable of usingtranscriptome data to indicate cell types and their activation state, revealedthat core marker genes for plasma cells were overrepresented in aphthousulcers relative to Peyer’s patches and unaffected or normal mucosa. Therewas no virus common to all aphthous ulcers. We detected human herpesvirus 4 in one aphthous ulcer, human herpes virus 1 in mucosa from apatient with CD, and a novel Totivirus in mucosa of one control patient.Conclusions: A number of gene clusters and immune pathways are over-expressed in aphthous ulcers compared with Peyer’s patches. These bio-logically relevant gene lists highlight a number of new therapeutic targetsand potential biomarkers for early stage disease. Viruses are an unlikelyinitiator of C

Prevalence of Yersinia Species in the Ileum of Crohn's Disease Patients and Controls

Yersinia are common contaminants of food products, but their prevalence in the human gut is poorly documented. Yersinia have been implicated in Crohn's Disease (CD, an inflammatory bowel disease) however their role in CD is controversial. We performed highly sensitive PCR assays of specific sequences for the gyrB gene of Y. aldovae, Y. bercovieri, Y. enterocolitica, Y. intermedia, Y. mollaretii and the inv gene of Y. pseudotuberculosis. We analyzed a total of 470 ileal samples taken from 338 participants (262 CD patients and 76 controls) belonging to three independent cohorts. All patients and controls were phenotyped and genotyped for the main CD susceptibility variants: NOD2, ATG16L1, and IRGM. Yersinia were found in 7.7% of ileal samples (respectively 7.9 and 7.6% in controls and CD patients) corresponding to 10% of participants (respectively 11.8 and 9.5% in controls and CD patients). Y. enterocolitica, Y. pseudotuberculosis and Y. intermedia were the most frequently identified species. The bacteria were more frequent in resected specimens, lymph nodes and Peyer's patches. Yersinia were no more likely to be detected in CD tissues than tissues from inflammatory and non-inflammatory controls. CD patients treated with immunosuppressants were less likely to be Yersinia carriers. In conclusion, this work shows that Yersinia species are frequently found at low levels in the human ileum in health and disease. The role of Yersinia species in this ecosystem should now be explored

Fine-Scale Structure Analysis Shows Epidemic Patterns of Clonal Complex 95, a Cosmopolitan Escherichia coli Lineage Responsible for Extraintestinal Infection

The Escherichia coli lineage known as clonal complex 95 (CC95) is a cosmopolitan human-associated lineage responsible for a significant fraction of extraintestinal infections of humans. Whole-genome sequence data of 200 CC95 strains from various origins enabled determination of the CC95 pangenome. The pangenome analysis revealed that strains of the complex could be assigned to one of five subgroups that vary in their serotype, extraintestinal virulence, virulence gene content, and antibiotic resistance gene profile. A total of 511 CC95 strains isolated from humans living in France, Australia, and the United States were screened for their subgroup membership using a PCR-based method. The CC95 subgroups are nonrandomly distributed with respect to their geographic origin. The relative frequency of the subgroups was shown to change through time, although the nature of the changes varies with continent. Strains of the subgroups are also nonrandomly distributed with respect to source of isolation (blood, urine, or feces) and host sex. Collectively, the evidence indicates that although strains belonging to CC95 may be cosmopolitan, human movement patterns have been insufficient to homogenize the distribution of the CC95 subgroups. Rather, the manner in which CC95 strains evolve appears to vary both spatially and temporally. Although CC95 strains appeared globally as pandemic, fine-scale structure analysis shows epidemic patterns of the CC95 subgroups. Furthermore, the observation that the relative frequency of CC95 subgroups at a single locality has changed over time indicates that the relative fitness of the subgroups has changedThis material is based in part on work supported by Office of Research and Development, Medical Research Service, Department of Veterans Affairs (grant 1 I01 CX000920-01 to J.R.J.). This work was partially supported by a grant from the Fondation Pour la Recherche Médicale (Équipe FRM 2016, DEQ20161136698) to E.D

Analysis of the human monocyte-derived macrophage transcriptome and response to lipopolysaccharide provides new insights into genetic aetiology of inflammatory bowel disease

The FANTOM5 consortium utilised cap analysis of gene expression (CAGE) to provide an unprecedented insight into transcriptional regulation in human cells and tissues. In the current study, we have used CAGE-based transcriptional profiling on an extended dense time course of the response of human monocyte-derived macrophages grown in macrophage colony-stimulating factor (CSF1) to bacterial lipopolysaccharide (LPS). We propose that this system provides a model for the differentiation and adaptation of monocytes entering the intestinal lamina propria. The response to LPS is shown to be a cascade of successive waves of transient gene expression extending over at least 48 hours, with hundreds of positive and negative regulatory loops. Promoter analysis using motif activity response analysis (MARA) identified some of the transcription factors likely to be responsible for the temporal profile of transcriptional activation. Each LPS-inducible locus was associated with multiple inducible enhancers, and in each case, transient eRNA transcription at multiple sites detected by CAGE preceded the appearance of promoter-associated transcripts. LPS-inducible long non-coding RNAs were commonly associated with clusters of inducible enhancers. We used these data to re-examine the hundreds of loci associated with susceptibility to inflammatory bowel disease (IBD) in genome-wide association studies. Loci associated with IBD were strongly and specifically (relative to rheumatoid arthritis and unrelated traits) enriched for promoters that were regulated in monocyte differentiation or activation. Amongst previously-identified IBD susceptibility loci, the vast majority contained at least one promoter that was regulated in CSF1-dependent monocyte-macrophage transitions and/or in response to LPS. On this basis, we concluded that IBD loci are strongly-enriched for monocyte-specific genes, and identified at least 134 additional candidate genes associated with IBD susceptibility from reanalysis of published GWA studies. We propose that dysregulation of monocyte adaptation to the environment of the gastrointestinal mucosa is the key process leading to inflammatory bowel disease

Advances in the management of inflammatory bowel disease

Our understanding of inflammatory bowel diseases (IBD) is constantly evolving, and many new treatment options have emerged recently. This review critically examines the evidence for these new developments and aims to provide an overview for medical professionals involved in the care of patients with IBD. Proposed changes in the use of aminosalicylates, immunosuppressants and biological agents are described, and the evidence for several promising novel agents is reviewed