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New treatment targets are needed for colorectal cancer (CRC). We define expression of High Mobility Group Box 1 (HMGB1) protein throughout colorectal neoplastic progression and examine the biological consequences of aberrant expression. HMGB1 is a ubiquitously expressed nuclear protein that shuttles to the cytoplasm under cellular stress. HMGB1 impacts cellular responses, acting as a cytokine when secreted. A total of 846 human tissue samples were retrieved; 6242 immunohistochemically stained sections were reviewed. Subcellular epithelial HMGB1 expression was assessed in a CRC Tissue Microarray ( n = 650), normal colonic epithelium ( n = 75), adenomatous polyps ( n = 52), and CRC polyps (CaP, n = 69). Stromal lymphocyte phenotype was assessed in the CRC microarray and a subgroup of CaP. Normal colonic epithelium has strong nuclear and absent cytoplasmic HMGB1. With progression to CRC, there is an emergence of strong cytoplasmic HMGB1 ( p < 0.001), pronounced at the leading cancer edge within CaP ( p < 0.001), and reduction in nuclear HMGB1 ( p < 0.001). In CRC, absent nuclear HMGB1 is associated with mismatch repair proteins ( p = 0.001). Stronger cytoplasmic HMGB1 is associated with lymph node positivity ( p < 0.001) and male sex ( p = 0.009). Stronger nuclear ( p = 0.011) and cytoplasmic ( p = 0.002) HMGB1 is associated with greater CD4 + T-cell density, stronger nuclear HMGB1 is associated with greater FOXP3 + ( p < 0.001) and ICOS + ( p = 0.018) lymphocyte density, and stronger nuclear HMGB1 is associated with reduced CD8 + T-cell density ( p = 0.022). HMGB1 does not directly impact survival but is associated with an 'immune cold' tumour microenvironment which is associated with poor survival ( p < 0.001). HMGB1 may represent a new treatment target for CRC.

Campylobacter showae a bacterium historically linked to gingivitis and periodontitis, has recently been associated with inflammatory bowel disease and colorectal cancer. Our aim was to generate genome sequences for new clinical C. showae strains and identify functional properties explaining their pathogenic potential. Eight C. showae genomes were assessed, four strains isolated from inflamed gut tissues from paediatric Crohn’s disease patients, three strains from colonic adenomas, and one from a gastroenteritis patient stool. Genome assemblies were analyzed alongside the only 3 deposited C. showae genomes. The pangenome from these 11 strains consisted of 4686 unique protein families, and the core genome size was estimated at 1050 ± 15 genes with each new genome contributing an additional 206 ± 16 genes. Functional assays indicated that colonic strains segregated into 2 groups: adherent/invasive vs. non-adherent/non-invasive strains. The former possessed Type IV secretion machinery and S-layer proteins, while the latter contained Cas genes and other CRISPR associated proteins. Comparison of gene profiles with strains in Human Microbiome Project metagenomes showed that gut-derived isolates share genes specific to tongue dorsum and supragingival plaque counterparts. Our findings indicate that C. showae strains are phenotypically and genetically diverse and suggest that secretion systems may play an important role in virulence potential.

Extracorporeal Shock Wave Therapy (ESWT) is used clinically in various disorders including chronic wounds for its pro-angiogenic, proliferative, and anti-inflammatory effects. However, the underlying cellular and molecular mechanisms driving therapeutic effects are not well characterized. Macrophages play a key role in all aspects of healing and their dysfunction results in failure to resolve chronic wounds. We investigated the role of ESWT on macrophage activity in chronic wound punch biopsies from patients with non-healing venous ulcers prior to, and two weeks post-ESWT, and in macrophage cultures treated with clinical shockwave intensities (150–500 impulses, 5 Hz, 0.1 mJ/mm2). Using wound area measurements and histological/immunohistochemical analysis of wound biopsies, we show ESWT enhanced healing of chronic ulcers associated with improved wound angiogenesis (CD31 staining), significantly decreased CD68-positive macrophages per biopsy area and generally increased macrophage activation. Shockwave treatment of macrophages in culture significantly boosted uptake of apoptotic cells, healing-associated cytokine and growth factor gene expressions and modulated macrophage morphology suggestive of macrophage activation, all of which contribute to wound resolution. Macrophage ERK activity was enhanced, suggesting one mechanotransduction pathway driving events. Collectively, these in vitro and in vivo findings reveal shockwaves as important regulators of macrophage functions linked with wound healing. This immunomodulation represents an underappreciated role of clinically applied shockwaves, which could be exploited for other macrophage-mediated disorders.

Introduction High Mobility Group Box-1 (HMGB1) is a ubiquitous nuclear protein that regulates gene expression. When phosphorylated, it translocates to the cytoplasm and extracellular space to impact immune responses and epithelial cell behaviour. Our previous data revealed that dynamic subcellular localisation of HMGB1 is associated with colorectal neoplastic progression, with cytoplasmic HMGB1 expression a feature of early carcinogenesis. The leading edge of cancer invasion in polyp cancers (CaP) had strong expression in both nuclear and cytoplasmic compartments. Our aim was to define the biological impact of this expression profile. Methods CD4+ helper T cells, CD8+ cytotoxic T cells, FOXP3+ regulatory T cells, CD20+ B cells and CD68+ macrophages were assessed by immunohistochemistry on endoscopically retrieved paraffin embedded CaP lesions sourced from the Grampian Tissue Biorepository (n=25). Ethical approval was granted by the Grampian Biorepository Scientific Access Group. The invasive cancer margin was identified by a gastrointestinal pathologist. The immune cell infiltrate was expressed as number of positive cells per high power field (x20) in the adjacent stroma alongside intensity of nuclear and cytoplasmic HMGB1 expression. The impact of HMGB1 on colonic epithelial cell behaviour was assessed by stimulation of Caco-2 cells (ATCC HTB-37TM) for 48–72 hours with 0, 50 and 100 ng/ml recombinant HMGB1 with (1) analysis of wound healing by scratch assay (images captured at time 0 and 24 hour intervals and analysed with ImageJ), (2) proliferation by incorporation of tritiated thymidine and (3) expression of permeability genes (CLDN2, CLDN4, OCLN, CDH1, TJP1) using TaqMan RT-qPCR, normalised to expression of GAPDH and B2M and analysed by the Livak method. Results A robust inflammatory infiltrate was identified adjacent to the invasive cancer margin with a preponderance of CD4+ T cells and CD68+ macrophages. The subcellular localisation of HMGB1 (nuclear versus cytoplasmic) did not result in phenotypically distinct populations. There was no difference in epithelial restitution in response to HMGB1 over 48 hours, although high concentration at 72 hours was associated with reduced wound healing (p Conclusions Cytoplasmic HMGB1 expression did not impact the phenotype of adjacent immune cell infiltrate at the cancer margin. HMGB1 influences growth dynamics of colonic epithelial cells and reduces the expression of the pore closing tight junction gene, CLDN4. Understanding HMGB1 driven biology in CaP could identify an important mechanism for early carcinogenesis.

Introduction There is a clinical need for new and safer treatment options for inflammatory bowel disease (IBD). Interleukin-27 (IL-27) is an endogenous immunosuppressive cytokine through inhibition of Th2 and Th17 T cell responses and promotion of IL-10 secreting Tr1 T regulatory cells. Oral IL-27 clinically attenuates disease in several T cell and innate cell driven pre-clinical models of IBD. We need to translate this pre-clinical data to human IBD to underpin proof of concept for IL-27 as a new therapeutic for IBD. Our aim was to define IL-27 evoked responses in the human colonic epithelial barrier. Methods A human colon derived epithelial organoid model was established from fresh colon tissue obtained through the Grampian Tissue Biorepository. Ethical approval was granted by the Biorepository Scientific Access Group. Isolated colonic crypts were grown in a 3D matrix with conditioned media and growth factors to maturity. RT-qPCR using TaqMan probes characterised organoid permeability gene expression (CLDN2, CLDN4, OCLN, CDH1, TJP1, ECM1) normalised to GAPDH and B2M following a 48 hour stimulation with recombinant IL-27 (rIL-27) (0, 50, 100 ng/ml). Data was analysed with the Livak method and one-way ANOVA. The impact of rIL-27 on colonic epithelial growth dynamics was established on Caco-2 and HT-29 cell lines, stimulated with 0, 50 and 100 ng/ml rIL-27, defined by (1) wound scratch assays over 72 hours with digital images of wound areas captured at 24 hour intervals and analysed with ImageJ software, and (2) tritiated thymidine incorporation proliferation assays stimulated for 48 hours. Results rIL-27 evoked a differential epithelial permeability gene expression in human colonic organoids, with reduced expression of CLDN2 (p Conclusion Our data demonstrates that IL-27 enhances epithelial barrier wound healing. Gene expression data suggests that cell-cell adhesion is enhanced through increased E-cadherin expression, with a reduction in permeability through decreased expression of claudin-2 (pore forming) and increase in claudin-4 (pore closing). Tight junction function is enhanced through increased expression of occludin and tight junctional protein-1. Further studies will define the IL-27 driven permeability related protein expression profile and impact on functional permeability in organoids and whether IL-27 is a potential new treatment for IBD.

In recent years, an increasing number of Campylobacter species have been associated with human gastrointestinal (GI) diseases including gastroenteritis, inflammatory bowel disease, and colorectal cancer. Campylobacter concisus, an oral commensal historically linked to gingivitis and periodontitis, has been increasingly detected in the lower GI tract. In the present study, we generated robust genome sequence data from C. concisus strains and undertook a comprehensive pangenome assessment to identify C. concisus virulence properties and to explain potential adaptations acquired while residing in specific ecological niche(s) of the GI tract. Genomes of 53 new C. concisus strains were sequenced, assembled, and annotated including 36 strains from gastroenteritis patients, 13 strains from Crohn's disease patients and four strains from colitis patients (three collagenous colitis and one lymphocytic colitis). When compared with previous published sequences, strains clustered into two main groups/genomospecies (GS) with phylogenetic clustering explained neither by disease phenotype nor sample location. Paired oral/faecal isolates, from the same patient, indicated that there are few genetic differences between oral and gut isolates which suggests that gut isolates most likely reflect oral strain relocation. Type IV and VI secretion systems genes, genes known to be important for pathogenicity in the Campylobacter genus, were present in the genomes assemblies, with 82% containing Type VI secretion system genes. Our findings indicate that C. concisus strains are genetically diverse, and the variability in bacterial secretion system content may play an important role in their virulence potential.

Objectives The objectives of this study were to comprehensively assess mRNA expression of 84 drug transporters in human colorectal biopsies and six representative cell lines, and to investigate the alteration of drug transporter gene expression after exposure to three candidate microbicidal antiretroviral (ARV) drugs (tenofovir, darunavir and dapivirine) in the colorectal epithelium. The outcome of the objectives informs development of optimal ARV-based microbicidal formulations for prevention of HIV-1 infection. Methods Drug transporter mRNA expression was quantified from colorectal biopsies and cell lines by quantitative real-time PCR. Relative mRNA expression was quantified in Caco-2 cells and colorectal explants after induction with ARVs. Data were analysed using Pearson's product moment correlation (r), hierarchical clustering and principal component analysis (PCA). Results Expression of 58 of the 84 transporters was documented in colorectal biopsies, with genes for CNT2, P-glycoprotein (P-gp) and MRP3 showing the highest expression. No difference was noted between individual subjects when analysed by age, gender or anatomical site (rectum or recto-sigmoid) (r = 0.95-0.99). High expression of P-gp and CNT2 proteins was confirmed by immunohistochemical staining. Similarity between colorectal tissue and cell-line drug transporter gene expression was variable (r = 0.64-0.84). PCA showed distinct clustering of human colorectal biopsy samples, with the Caco-2 cells defined as the best surrogate system. Induction of Caco-2 cell lines with ARV drugs suggests that darunavir-based microbicides incorporating tenofovir may result in drug-drug interactions likely to affect distribution of individual drugs to sub-epithelial target cells. Conclusions These findings will help optimize complex formulations of rectal microbicides to realize their full potential as an effective approach for pre-exposure prophylaxis against HIV-1 infection.

Inflammatory bowel disease (IBD) is characterised by an inappropriate chronic immune response against resident gut microbes. This may be on account of distinct changes in the gut microbiota termed as dysbiosis. The role of fungi in this altered luminal environment has been scarcely reported. We studied the fungal microbiome in de-novo paediatric IBD patients utilising next generation sequencing and compared with adult disease and normal controls. We report a distinct difference in fungal species with Ascomycota predominating in control subjects compared to Basidiomycota dominance in children with IBD, which could be as a result of altered tolerance in these patients.

The human gut microbiota plays a major role in the development of colorectal cancer (CRC). Many studies have attempted to define links between microbiota residents, their function and disease development. We now have incredible molecular tools to allow us to study the gut microbiome however in order to make best use of these sophisticated approaches we need to ensure that samples are collected and processed using standardized and reproducible protocols. Here we provide an overview of molecular analysis methods and describe protocols for collecting and processing clinical samples for subsequent microbiome analysis.

The human gut microbiota plays a major role in the development of colorectal cancer (CRC). Many studies have attempted to define links between microbiota residents, their function and disease development. We now have incredible molecular tools to allow us to study the gut microbiome however in order to make best use of these sophisticated approaches we need to ensure that samples are collected and processed using standardized and reproducible protocols. Here we provide an overview of molecular analysis methods and describe protocols for collecting and processing clinical samples for subsequent microbiome analysis.

Introduction In recent years, an increasing number of Campylobacter species have been associated with human gastrointestinal diseases. Campylobacter showae, an oral commensal that was historically linked to gingivitis and periodontitis, has recently been associated with newly diagnosed and established inflammatory bowel disease, hepatolithiasis, and colorectal cancer. The aim of this study was to generate robust genome sequence data for a number of clinical C. showae strains and identify functional properties that could explain their pathogenic potential. Method Genomes of eight C. showae strains were sequenced, assembled, annotated and compared including four strains from paediatric Crohn’s disease patients, three strains from colonic adenomas and one from a gastroenteritis patient. A series of functional analyses were also undertaken including adhesion and invasion assays, toxicity assays and inflammatory cytokine production assessment. Results The 8 new genome assemblies were of high contiguity and completeness and compared favourably with the existing 3 C. showae genomes. Functional analysis indicated that colonic strains could be classified into 2 groups - adherent/invasive vs non-adherent strains with differing virulence factors between the groups, which may be explained by specific genes. Overall, the 11 strains contained 4591 unique gene centroids. Adherent/invasive strains had 807 unique centroids, while non-adherent/non-invasive strains had 497 unique centroids (in 2 or more strains). The oral strain – RM3277 also contained 270 centroids not shared with the colonic strains. Adherent strains possessed a number of bacterial secretion system proteins including Type I and IV machinery that were absent in non-adherent strains. These differences may contribute to the distinct phenotypes observed between strains relating to motility, metal ion transport and host cell interaction. All strains contained unique plasmid sequences as well as a number of predicted genomic islands including some prophage clusters. Conclusion Our findings indicate that C. showae strains are phenotypically and genetically diverse, and suggest the genomes of this bacterium contain modifications in secretion systems that may play an important role in their virulence potential. Additionally, we have greatly added to the current C. showae resources with genome and plasmid assemblies of 8 strains. Disclosure of Interest None Declared

Introduction Campylobacter concisus is an oral commensal, which has historically been linked to gingivitis and periodontitis but more recently has been linked with gastrointestinal diseases including inflammatory bowel disease and gastroenteritis. Functional and genomic data for C. concisus is limited with little evidence to define potential differences, in terms of pathogenicity, between oral and intestinal isolates. The aim of the current study was to generate robust genome sequence data for a number of clinical C. concisus strains and compare pathogenic potential between oral, faecal and mucosal biopsy derived isolates. Method Genomes from 53 C. concisus strains were sequenced, assembled and annotated including thirty-four strains from gastroenteritis patients, fourteen strains from Crohn’s disease patients, 4 strains from colitis patients (3 collagenous colitis and 1 lymphocytic colitis), and one from a colonic adenoma patient. Six patients had both oral and faecal isolates available. Library construction was performed using the Illumina Nextera XT DNA library prep kit and sequenced using Illumina MiSeq. Selected strains were also subjected to long-read sequencing (PacBio). Genome assemblies were annotated with Prokka and quality assessment tools including QUAST, BUSCO and REAPR were used. Results Genome assemblies compared very favourably to existing publicly available C. concisus genome assemblies (10 available from previous studies). Isolates clustered into two main groups/genomospecies (GS) based on comparison of the 16S rRNA and 23S rRNA gene sequences. Clinical characterisation or location of the strains (oral vs intestinal) did not stratify according to GS typing. Of the 6 sets of paired faecal-oral strains, derived from Crohn’s patients most paired samples were separated based on GS typing. Interestingly the oral strains did not form a GS and did not strongly cluster together relatively to the faecal and biopsy strains in the study. PCR investigation for the virulence factor exotoxin 9 indicated that positive strains were more likely to belong to genomospecies II. Comparison of exotoxin 9 positivity between patient paired samples indicated that oral strains were PCR positive whilst faecal strains were predominantly negative for the virulence factor exotoxin 9. Conclusion Our findings indicate that C. concisus strains are phenotypically and genetically diverse, and suggest that there may well be differences in virulence potential depending on where strains are located. Disclosure of Interest None Declared

Recurrent aphthous stomatitis (RAS) is the most common disease affecting oral mucosae. Etiology is unknown, but several factors have been implicated, all of which influence the composition of microbiota residing on oral mucosae, which in turn modulates immunity and thereby affects disease progression. Although no individual pathogens have been conclusively shown to be causative agents of RAS, imbalanced composition of the oral microbiota may play a key role. In this study, we sought to determine composition profiles of bacterial microbiota in the oral mucosa associated with RAS. Using high-throughput 16S rRNA gene sequencing, we characterized the most abundant bacterial populations residing on healthy and ulcerated mucosae in patients with RAS (recruited using highly stringent criteria) and no associated medical conditions; we also compared these to the bacterial microbiota of healthy controls (HCs). Phylum-level diversity comparisons revealed decreased Firmicutes and increased Proteobacteria in ulcerated sites, as compared with healthy sites in RAS patients, and no differences between RAS patients with healthy sites and HCs. Genus-level analysis demonstrated higher abundance of total Bacteroidales in RAS patients with healthy sites over HCs. Porphyromonadaceae comprising species associated with periodontal disease and Veillonellaceae predominated in ulcerated sites over HCs, while no quantitative differences of these families were observed between healthy sites in RAS patients and HCs. Streptococcaceae comprising species associated with oral health predominated in HCs over ulcerated sites but not in HCs over healthy sites in RAS patients. This study demonstrates that mucosal microbiome changes in patients with idiopathic RAS—namely, increased Bacteroidales species in mucosae of RAS patients not affected by active ulceration. While these changes suggest a microbial role in initiation of RAS, this study does not provide data on causality. Within this limitation, the study contributes to the understanding of the potential role of mucosal microbiome changes in oral mucosal disease.

Rotaviruses (RV) are the leading cause of gastroenteritis in children less than five years of age worldwide. Rotarix®, a live attenuated monovalent vaccine containing a RV strain of G1P[8] specificity has been included in the childhood immunisation schedule from June 2013 in Scotland. This study aimed to characterise the prevalent RV strains in Scotland before and after the introduction of the RV vaccine.RV positive faecal samples from Scottish virology laboratories covering the years 2012-2015 were genotyped. Viral RNA was extracted from faecal suspensions. VP7 and VP4 gene specific primers were used for multiplex hemi-nested PCRs and sequencing. Mann-Whitney U test and Chi-square test were used for statistical comparison.There was a decrease in RV positive samples from the Scottish virology laboratories from 7409 samples in the pre-vaccination years (2009-2013) to 760 in 2014-2015, with an annual reduction of RV infections by 74.4% (RR-3.95; 95%-CI, 3.53-4.42, p0.001). 362 samples from the pre-vaccination period and 278 samples from the post-vaccination were genotyped. There was a drop in prevalence of G1P[8] strains (72.1%, 95%-CI, 67.42-76.33 to 15%, 95%-CI, 11.38-19.79) after introduction of the vaccine. In the post-vaccination period G2P[4] was the dominant strain in Scotland (21.9%, 95%-CI, 17.48-27.17) with increase in G9P[8] (12.9%, 95%-CI, 9.50-7.41), G12P[8] (12.2%, 95%-CI, 8.89-16.60) and G3P[8] (11.9%, 95%-CI, 8.58-16.20) infections. Phylogenetic analysis of the VP7 and VP4 genes showed no major differences between the pre and post-vaccination G1P[8] strains.This laboratory based surveillance study shows significant reduction in reported RV cases and a shift in proportion from G1P[8] to G2P[4] strains after introduction of RV vaccination in Scotland. The genotyping data from a subset of the total reported RV cases will be used to ascertain cross protection against strains and identify vaccine induced RV strain shifts in the years to come.

Background Sequencing-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. Results Next-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. Conclusions We propose that colonic lavage samples provide a relatively accurate representation of biopsy microbiota composition and should be considered where biopsy size is an issue. Electronic supplementary material The online version of this article (doi:10.1186/s40168-016-0207-9) contains supplementary material, which is available to authorized users.

The gastrointestinal microbiota is considered important in inflammatory bowel disease (IBD) pathogenesis. Discoveries from established disease cohorts report reduced bacterial diversity, changes in bacterial composition, and a protective role for Faecalibacterium prausnitzii in Crohn's disease (CD). The majority of studies to date are however potentially confounded by the effect of treatment and a reliance on established rather than de-novo disease.Microbial changes at diagnosis were examined by biopsying the colonic mucosa of 37 children: 25 with newly presenting, untreated IBD with active colitis (13 CD and 12 ulcerative colitis (UC)), and 12 pediatric controls with a macroscopically and microscopically normal colon. We utilized a dual-methodology approach with pyrosequencing (threshold10,000 reads) and confirmatory real-time PCR (RT-PCR).Threshold pyrosequencing output was obtained on 34 subjects (11 CD, 11 UC, 12 controls). No significant changes were noted at phylum level among the Bacteroidetes, Firmicutes, or Proteobacteria. A significant reduction in bacterial α-diversity was noted in CD vs. controls by three methods (Shannon, Simpson, and phylogenetic diversity) but not in UC vs. controls. An increase in Faecalibacterium was observed in CD compared with controls by pyrosequencing (mean 16.7% vs. 9.1% of reads, P=0.02) and replicated by specific F. prausnitzii RT-PCR (36.0% vs. 19.0% of total bacteria, P=0.02). No disease-specific clustering was evident on principal components analysis.Our results offer a comprehensive examination of the IBD mucosal microbiota at diagnosis, unaffected by therapeutic confounders or changes over time. Our results challenge the current model of a protective role for F. prausnitzii in CD, suggesting a more dynamic role for this organism than previously described.

Recently, two large genome-wide association studies, conducted in Chinese populations, have reported associations between upper gastrointestinal cancer and the rs2274223, rs13042395 and rs4072037 polymorphisms in PLCE1, C20orf54 and MUC1, respectively. We aimed to determine whether similar associations existed for Caucasian populations. We genotyped two population-based, case-control studies of upper gastrointestinal cancer; the first study included 290 gastric cancer (GC) cases and 376 controls and the second study included 306 GC cases, 107 oesophageal adenocarcinoma cancer cases, 52 oesphageal squamous cell cancer cases and 211 controls. Odds ratios (OR) and 95% confidence intervals (CIs) were computed from logistic models and adjusted for confounding variables. The rs4072037 polymorphism in MUC1 was associated with a reduced risk of GC of intestinal histological type (OR 0.4; 95% CI 0.2-0.9) and a reduced risk of oesophageal squamous cell cancer (OR 0.5; 95% CI 0.2-1.0), but not oesphageal adenocarcinoma. Similarly, rs2274223 in PLCE1 was associated with a reduced risk of oesophageal squamous cell cancer (OR 0.5; 95% CI 0.3-1.0), but not oesphageal adenocarcinoma. We observed no association between rs13042395 in Corf54 and the risk of gastric or oesphageal cancer in either of the two studies. Our findings for rs4072037 and the risk of GC are in agreement with one previous report for a Caucasian population. To the best of our knowledge, this is the first study to report an association between rs2274223 and rs4072037 and the risk of oesophageal squamous cell carcinoma in a Caucasian population.

Background\ud \ud Considerable effort has been made to categorise the bacterial composition of the human gut and correlate findings with gastrointestinal disease. The infant gut has long been considered sterile at birth followed by rapid colonisation; however, this view has recently been challenged. We examined first-pass meconium from healthy term infants to confirm or refute sterility.\ud \ud Methods\ud \ud Healthy mothers were approached following vaginal delivery. First-pass meconium stools within 24 hours of delivery were obtained from healthy, breastfed infants with tight inclusion/exclusion criteria including rejecting any known antibiotic exposure - mother within 7 days preceding delivery or infant after birth. Stools were processed in triplicate for fluorescent in-situ hybridisation (FISH) with 16S rRNA-targeted probes including Bifidobacterium; Bacteroides-Prevotella; Lactobacillaceae/Enterococcaceae; Enterobacteriaceae; Streptococcaceae; Staphylococcaceae and Enterococcaceae. Absolute counts of all bacteria and proportional identification of each bacterial group were calculated. Confirmation of bacterial presence by PCR was undertaken on FISH-positive samples.\ud \ud Results\ud \ud The mothers of 31 newborn infants were recruited, 15 met inclusion/exclusion criteria and provided a sample within 24 hours of birth, processed in the lab within 4 hours. All babies were 37–40 weeks gestation. 8/15 were male, mean birth weight was 3.4kg and mean maternal age was 32 years. Meconium samples from 10/15 (66%) infants had evidence of bacteria based on FISH analysis. Of these, PCR was positive in only 1. Positive FISH counts ranged from 2.2 - 41.8 x 104 cells/g with a mean of 15.4 x 104 cells/g. (The limit of detection for automated counting is 106 cells/g). Cell counts were too low to allow formal diversity analysis. Amplification by PCR was not possible despite positive spiked samples demonstrating the feasibility of reaction. One baby was dominated by Enterobacteriaceae. The others contained 2-5 genera, with Bifidobacterium, Enterobacteriaceae, Enterococcaceae and Bacteroides-Prevotella the most prevalent. There was no association between bacterial counts and rupture of membrane duration, time to passage of meconium or time to lab.\ud \ud Conclusion\ud \ud This study provides evidence that low numbers of bacteria are present in first-pass meconium samples from healthy, vaginally-delivered, breastfed term infants. Only two-thirds of meconium samples had detectable bacteria, though at levels too low for automated counting or for reliable confirmation by PCR. This study suggests that gut bacterial colonisation is extremely limited at birth and occurs rapidly thereafter.

Cytokine gene polymorphisms modify expression and their circulating protein levels reflect inflammatory response. Chronic inflammation plays a key role in the pathogenesis of colorectal neoplasia (CRN) associated with inflammatory bowel disease, but it is not clear whether inflammation is a cause or an effect of tumours in sporadic CRN. We therefore investigated the association of cytokine gene polymorphisms and circulating cytokine levels on the risk of CRN in Northeast Scotland, which has a high incidence of CRN. We recruited two groups of patients from a screening colonoscopy cohort, either preprocedure or 3-24 months postprocedure. Participants with CRN were compared with participants with no evidence of CRN (controls). Blood-derived DNA was used to genotype polymorphisms in IL1B, IL1-RN, IL6, IL8, IL10, PTGS2 and TNFA genes. Circulating levels of high-sensitivity C-reactive protein and six cytokines [interleukin-1β (IL-1β), IL-4, IL-6, IL-8, IL-10 and tumour necrosis factor-α (TNF-α)] were measured. To examine the effect of CRN resection on marker levels, we used propensity score matching. A total of 884 patients were eligible for analysis, including 388 CRN cases and 496 controls. Cases were older (mean age 64 vs. 62 years, P0.01) and more likely to be men (67 vs. 55%, P0.001). Controls were more likely to be regular users of NSAID (P0.0001). Compared with homozygous carriage of respective common alleles, proinflammatory CC genotypes of IL1B-31CT [odds ratio (OR) (95% confidence interval (CI) 1.68 (1.03-2.73)] and PTGS2-765 CG [OR (95% CI) 2.97 (1.05-8.46)] were each associated with an increased risk of CRN. Conversely, carriage of the A allele of IL8-251AT was associated with a lower risk of CRN compared with the TT genotype [ORs (95% CI) 0.60 (0.41-0.86) for heterozygous, 0.88 (0.57-1.37) for homozygous, and 0.68 (0.48-0.95) for heterozygous and homozygous combined]. Compared with postprocedure cases, IL8, TNF-α and C-reactive protein levels were significantly higher in preprocedure cases, but IL4 and IL10 protein levels were significantly lower. Proinflammatory cytokine gene polymorphisms in IL1B-31 and PTGS2-765 increase the risk of developing CRN. Levels of proinflammatory IL8, TNF-α and C-reactive protein markers are significantly higher while CRN is in situ. Along with the NSAID findings, these data point to inflammation as an underlying pathogenetic mechanism in CRN.

Host genetic factors play an important role in modifying the risk of human disease, including cancers of the upper gastrointestinal tract, with increasing interest in Toll-like receptor (TLR) signaling and the impact of genetic polymorphisms in these systems. The CD14-159C/T and the TLR9-1237T/C promoter polymorphisms have previously been shown to be associated with various inflammatory conditions including Helicobacter pylori-induced gastritis in Caucasian populations. In this study, we assessed the association of these two functional single nucleotide polymorphisms with gastric cancer in two independent Caucasian population-based case–control studies of upper gastrointestinal tract cancer, initially in 312 noncardia gastric carcinoma cases and 419 controls and then in 184 noncardia gastric carcinomas, 123 cardia carcinomas, 159 esophageal cancers, and 211 frequency-matched controls. Odds ratios were computed from logistic models and adjusted for potential confounding factors. No significant association was found between the CD14-159C/T and the TLR9-1237T/C promoter polymorphisms and increased risk of gastric cancer. Neither single nucleotide polymorphism has been assessed in a Caucasian gastric cancer case–control study before; although the CD14-159C/T polymorphism has been reported to show no apparent association with H. pylori-related gastric malignancy in a Taiwanese Chinese population. In conclusion, although our earlier preliminary studies suggested that the CD14-159C/T and the TLR9-1237T/C promoter polymorphisms increase the risk of precancerous outcomes, they do not seem to increase the risk of gastric cancer itself. This discrepancy merits further examination.

Introduction Children presenting for the first time with inflammatory bowel disease (IBD) offer a unique opportunity to study aetiological agents before the confounders of treatment. Microaerophilic bacteria can exploit the ecological niche of the intestinal epithelium; Helicobacter and Campylobacter are previously implicated in IBD pathogenesis. We set out to study these and other microaerophilic bacteria in de-novo paediatric IBD. Patients and Methods 100 children undergoing colonoscopy were recruited including 44 treatment naïve de-novo IBD patients and 42 with normal colons. Colonic biopsies were subjected to microaerophilic culture with Gram-negative isolates then identified by sequencing. Biopsies were also PCR screened for the specific microaerophilic bacterial groups: Helicobacteraceae, Campylobacteraceae and Sutterella wadsworthensis. Results 129 Gram-negative microaerophilic bacterial isolates were identified from 10 genera. The most frequently cultured was S. wadsworthensis (32 distinct isolates). Unusual Campylobacter were isolated from 8 subjects (including 3 C. concisus, 1 C. curvus, 1 C. lari, 1 C. rectus, 3 C. showae). No Helicobacter were cultured. When comparing IBD vs. normal colon control by PCR the prevalence figures were not significantly different (Helicobacter 11% vs. 12%, p = 1.00; Campylobacter 75% vs. 76%, p = 1.00; S. wadsworthensis 82% vs. 71%, p = 0.312). Conclusions This study offers a comprehensive overview of the microaerophilic microbiota of the paediatric colon including at IBD onset. Campylobacter appear to be surprisingly common, are not more strongly associated with IBD and can be isolated from around 8% of paediatric colonic biopsies. S. wadsworthensis appears to be a common commensal. Helicobacter species are relatively rare in the paediatric colon. Trial Registration This study is publically registered on the United Kingdom Clinical Research Network Portfolio (9633).

Introduction Emerging evidence links the gut microbiota to colonic inflammation and colorectal cancer (CRC). Studies have shown over-representation of oral bacterial species including Campylobacter spp. in CRC tissue and also njc-Campylobacter are over-represented in IBD wherein normal homeostasis has been lost. Understanding the role of njc-Campylobacter as an aetiological agent in CRC and elucidating their pathogenicity characteristics is of immense clinical importance. Aim To perform whole genome sequencing (WGS) of clinically derived njc-Campylobacter to compare/identify virulence traits potentially linked to disease presentation. Methods WGS of 21 unique clinically derived Campylobacter isolates was performed (7 Campylobacter showae, 3 Campylobacter ureolyticus and 11 Campylobacter concisus) from biopsy/faecal samples of subjects with malignancy, acute enteritis and IBD. DNA was extracted using the QIAamp DNA Mini Kit. Libraries were prepared using Illumina Nextera XT DNA Library Prep Kit and Nextera XT Index Kit v2, multiplexed, and paired-end sequenced on the Illumina MiSeq. De novo read assembly used the A5-miseq pipeline with QUAST. Genome annotation was performed using the rapid prokaryotic genome annotation tool Prokka. Results Genome assembly of C. showae strains resulted in 8 to 59 contigs per strain (mean number = 21; average genome size 2,199,446 nucleotides); C. concisus isolates had an average contig number of 72 and average genome size of 1,982,586 nucleotides. C. ureolyticus strains average contig number was 134 and average genome size of 1,608,008 nucleotides. Through Prokka annotation, the average number of coding sequences was 2215 for C. showae isolates (range 2050–2368) with function predicted for ~71.3%. For C. concisus the average number of coding sequences was 1985 (range 1833–2087) with function predicted for ~ 71.9%. C. ureolyticus had a lower number of coding sequences (average 1613) with function predicted for ~72.6%. We focused on the VirB4/D10 operon homologs, which are components of the Type IV secretion system. Contrary to previously published sequence data from oral strains, the majority of colonic C. showae isolated and 2 C. concisus strains possessed almost complete T4SS systems and additional virulence traits, including the exotoxin 9 gene. Conclusion The findings clearly demonstrate genetic differences among colonic Campylobacter isolates as compared to oral strains and sheds light on the pathogenic potential of these emerging colonic pathogens. The findings also further highlight the need for further sequencing studies to be undertaken. Disclosure of Interest None Declared

Sinorhizobium meliloti forms a symbiosis with the legume alfalfa, whereby it differentiates into a nitrogen-fixing bacteroid. The lipid A species of S. meliloti are modified with very long-chain fatty acids (VLCFAs), which play a central role in bacteroid development. A six-gene cluster was hypothesized to be essential for the biosynthesis of VLCFA-modified lipid A. Previously, two cluster gene products, AcpXL and LpxXL, were found to be essential for S. meliloti lipid A VLCFA biosynthesis. In this paper, we show that the remaining four cluster genes are all involved in lipid A VLCFA biosynthesis. Therefore, we have identified novel gene products involved in the biosynthesis of these unusual lipid modifications. By physiological characterization of the cluster mutant strains, we demonstrate the importance of this gene cluster in the legume symbiosis and for growth in the absence of salt. Bacterial LPS species modified with VLCFAs are substantially less immunogenic than Escherichia coli LPS species, which lack VLCFAs. However, we show that the VLCFA modifications do not suppress the immunogenicity of S. meliloti LPS or affect the ability of S. meliloti to induce fluorescent plant defense molecules within the legume. Because VLCFA-modified lipids are produced by other rhizobia and mammalian pathogens, these findings will also be important in understanding the function and biosynthesis of these unusual fatty acids in diverse bacterial species.

Inflammatory bowel disease (IBD) arises in genetically susceptible individuals as a result of an unidentified environmental trigger, possibly a hitherto unknown bacterial pathogen. Twenty-six clinical isolates of Sutterella wadsworthensis were obtained from 134 adults and 61 pediatric patients undergoing colonoscopy, of whom 69 and 29 respectively had IBD. S. wadsworthensis was initially more frequently isolated from IBD subjects, hence this comprehensive study was undertaken to elucidate its role in IBD. Utilizing these samples, a newly designed PCR was developed, to study the prevalence of this bacterium in adult patients with ulcerative colitis (UC). Sutterella wadsworthensis was detected in 83.8% of adult patients with UC as opposed to 86.1% of control subjects (p = 0.64). Selected strains from IBD cases and controls were studied to elicit morphological, proteomic, genotypic and pathogenic differences. This study reports Scanning Electron Microscopy (SEM) appearances and characteristic MALDI-TOF MS protein profiles of S. wadsworthensis for the very first time. SEM showed that the bacterium is pleomorphic, existing in predominantly two morphological forms, long rods and coccobacilli. No differences were noted in the MALDI-TOF mass spectrometry proteomic analysis. There was no distinct clustering of strains identified from cases and controls on sequence analysis. Cytokine response after monocyte challenge with strains from patients with IBD and controls did not yield any significant differences. Our studies indicate that S. wadsworthensis is unlikely to play a role in the pathogenesis of IBD. Strains from cases of IBD could not be distinguished from those identified from controls.

Background Changes in bacterial populations termed “dysbiosis” are thought central to ulcerative colitis (UC) pathogenesis. In particular, the possibility that novel Helicobacter organisms play a role in human UC has been debated but not comprehensively investigated. The aim of this study was to develop a molecular approach to investigate the presence of Helicobacter organisms in adults with and without UC. Methodology/Principal Findings A dual molecular approach to detect Helicobacter was developed. Oligonucleotide probes against the genus Helicobacter were designed and optimised alongside a validation of published H. pylori probes. A comprehensive evaluation of Helicobacter genus and H. pylori PCR primers was also undertaken. The combined approach was then assessed in a range of gastrointestinal samples prior to assessment of a UC cohort. Archival colonic samples were available from 106 individuals for FISH analysis (57 with UC and 49 non-IBD controls). A further 118 individuals were collected prospectively for dual FISH and PCR analysis (86 UC and 32 non-IBD controls). An additional 27 non-IBD controls were available for PCR analysis. All Helicobacter PCR-positive samples were sequenced. The association between Helicobacter and each study group was statistically analysed using the Pearson Chi Squared 2 tailed test. Helicobacter genus PCR positivity was significantly higher in UC than controls (32 of 77 versus 11 of 59, p = 0.004). Sequence analysis indicated enterohepatic Helicobacter species prevalence was significantly higher in the UC group compared to the control group (30 of 77 versus 2 of 59, p, Broad Medical Research Program (Grant number IBD-0178), Aberdeen Royal Infirmary. GI Research Unit, NHS Scotland (Clinical Academic Training Fellowship, from the Chief Scientist Office (CAF/08/01))

Introduction Rotaviruses (RV) are the leading cause of severe gastroenteritis in children less than five years of age worldwide. Rotarix®, a live attenuated monovalent vaccine containing a RV strain of G1P[8] specificity has been included in the routine childhood immunisation schedule from June 2013 in Scotland and is offered to infants under the age of four months in two doses. This study aimed to characterise the prevalent rotavirus strains in Scotland prior to the introduction of the vaccine. Method Rotavirus positive faecal samples from various Scottish regional virology laboratories covering the years 2012–2013, before the introduction of the vaccine, were genotyped. Viral RNA was extracted from faecal suspensions using guanidine isothiocyanate – silica gel extraction method. VP7 and VP4 gene specific multiplex hemi-nested PCRs were used for genotyping. Surveillance systems were established by Health Protection Scotland to monitor the impact of the vaccine. Results A total of 387 samples were genotyped from the regional virology laboratories in Aberdeen, Dundee, Inverness, Glasgow and Edinburgh. The commonest strain in Scotland was G1P[8] accounting for 72.1% of cases. The other strains that were identified were G2P[4] in 7.2%, G4P[8] in 6.9%, G9P[8] in 3.4% and G3P[8] in a further 2.3% of cases. Single cases of G12P[8] and G9P[4] were also found (0.26% respectively). Mixed infections were seen in 5.4% of cases. The provisional uptake for the first dose of Rotarix® vaccine in Scotland for children born between 1 st of July to 30 th Sept 2013 was 92.7%% for the completed course by 12 months. The laboratory confirmed cases of RV reported to HPS via ECOSS (The Electronic Communication of Surveillance in Scotland) showed a marked reduction in confirmed reports of RV compared to a three year average of the years 2011–13. Surrogate markers, e.g. calls to NHS24 and GP consultations with childhood diarrhoea and vomiting, have also shown a downward trend. Conclusion The pre-vaccine surveillance of RV strains confirms that G1P[8] is the predominant strain in Scotland. There has been a promising reduction in laboratory confirmed cases of RV in Scotland following the introduction of the vaccine. The baseline genotyping data will be used to ascertain cross protection against strains and also identify vaccine induced RV strain shifts and an overall evaluation of the programme. Disclosure of interest None Declared.

Introduction Drug transporter expression in the colorectal epithelium is likely to play a role in the mucosal disposition of anti-retrovirals (ARVs) in rectal microbicidal preparations and impact their efficacy in prevention of HIV-1 infection. However, there is limited information on expression levels available. This study aims to assess expression of 84 drug transporter genes in human colorectal tissue and representative cell lines pre and post ARV exposure. Method Drug transporter mRNA expression was quantified from colorectal biopsies (n = 12) and 6 colorectal cell lines using real time PCR. Relative mRNA expression was quantified in Caco-2 cells after induction with tenofovir (TFV; 5 mM), dapivirine (DPV; 10 µM) and darunavir (DRV; 250 µM) for up to 3 days. Data were analysed using Pearson’s correlation (r), hierarchical clustering and principal component analysis. Results Fifty-eight of the 84 transporters were expressed in colorectal tissue. SLC28A2/CNT2 was the most expressed uptake transporter (>25 fold increase) followed by efflux transporters ABCB1/P-gp and ABCC3/MRP3 (4–5 fold increase). No difference was noted between individual patients, either sexes or biopsy sites (rectum or sigmoid) (r = 0.95–0.99). Similarities between tissue and cell lines were low (r values 0.67–0.81). Principal component analysis showed distinct clustering of colorectal tissue and cell lines with Caco-2 cells showing least variance with tissues. Immunohistochemical staining of 7 colorectal biopsy sections confirmed high expression of Pgp and CNT2 drug transporters. TFV stimulation of Caco-2 cells resulted in > 2 fold increase in expression of MRP5, LAT2, OATPE and GLUT1 mRNA. DPV stimulation resulted in > 2 fold increase in expression of GLUT1, PEPT1 and ABCA1. DRV stimulation resulted in > 2 fold increase in expression of SLC3A2, OATPE and MCT3 mRNA. Conclusion This study has enumerated drug transporter mRNA expression in colorectal tissue and cell lines. There was partial correlation between cell lines and tissue limiting cell line use as in vivo surrogate. TFV and DPV did not induce majority of efflux transporters in Caco-2 cells. This would entail increased retention of drug within the mucosa and as a result enhanced delivery to CD4 T cells. Further studies will elucidate whether combination of ARVs will be similarly effective. Disclosure of interest None Declared.

Aims Considerable effort has been made to categorise the bacterial composition of the human gut and correlate findings with gastrointestinal disease. The infant gut has long been considered sterile at birth followed by rapid colonisation by pioneer microbiota. We examined first-pass meconium from healthy term infants to confirm/refute sterility. Methods Healthy mothers were approached following vaginal delivery. First-pass meconium within 24 h of delivery were obtained from healthy, breastfed infants. Antibiotic use was an exclusion criterion- mother within 7 days or infant after birth. Stools were processed in triplicate for fluorescent in-situ hybridisation (FISH) with16S rRNA-targeted probes against all bacteria; Bifidobacterium; Bacteroides-Prevotella; Lactobacillaceae/Enterococcaceae; Enterobacteriaceae; Streptococcaceae; Staphylococcaceae and Enterococcaceae. Absolute counts of all bacteria and proportional identification for each bacterial group were calculated. DNA extraction followed by universal bacterial RT-PCR was performed on FISH-positive samples. Results 15 babies met inclusion/exclusion criteria. All babies were 37–40 weeks gestation. 8/15 were male, mean birth weight was 3.36kg and mean maternal age was 31.9 years. 10/15 (66%) infants had evidence of bacteria on FISH. Of these, RT-PCR was positive in only 1. Positive FISH counts ranged from 2.2 to 41.8*104 cells/g with the mean of positive samples being 15.4*104 cells/g. (Limit of detection for automated counting is 106 cells/g). Cell counts were too low to allow formal diversity analysis. Amplification by RT-PCR was not possible despite positive spiked samples demonstrating the feasibility of reaction. Three babies were dominated by a single family, either Enterobacteriaceae or Enterococcaceae. The others contained 2–5 genera. Bifidobacterium, Enterobacteriaceae and Bacteroides-Prevotella were the most dominant bacteria identified. There was no association between rupture of membrane duration, time to passage of meconium or time to lab with bacterial counts. Conclusion Evidence of bacteria in first-pass meconium samples from healthy, vaginally-delivered, breastfed term infants is scant with only two-thirds having demonstrable bacteria at levels too low for automated counting. Bacterial RT-PCR failed to amplify 9/10 FISH-positive samples. This study suggests that gut bacterial diversity is extremely limited at birth and supports the hypothesis that the neonatal gut is sterile and colonised rapidly thereafter.

Human thrombi vary in their susceptibility to lysis and this is clinically important. Several potential contributory factors were examined in this study by using model thrombi, created under flow; these provide a robust, reproducible and easily-manipulated system. Here we identify the plasminogen activators (PA) active in model thrombi of known age and define the cellular and plasma contribution to activity in different areas. The cell-rich head of model thrombi had strong thrombin and PA activity, with coagulant activity also at the tail. Thrombin activity decreased as model thrombi were aged. PA activity in the thrombus head also decreased on ageing of thrombi but activity emerged around the thrombi, including the tail. Activity in the head of fresh model thrombi was primarily due to uPA, with some contribution from tPA. Experiments with thrombi prepared from platelet-rich plasma and added leucocytes showed that uPA activity at the head of fresh thrombi was derived from PMN. Older thrombi had tPA activity around the tail of the thrombus; this activity occurred in the absence of cells. This study highlights the importance of PMN-derived uPA activity in the lysis of fresh thrombi, with activity originating in the leucocyte-rich head. It also shows that thrombi are dynamic structures in which fibrin can be repeatedly laid down and lysed, observations that are relevant to therapeutic lysis and potential rethrombosis.

Introduction Dysbiosis may contribute to inflammatory bowel disease (IBD) pathogenesis along with a reduced bacterial diversity. Limited bacterial diversity studies have been performed at the onset of disease in adults but rarely in children. High-throughput, parallel sequencing technology (next-generation sequencing) provides the means of assessing microbial diversity in samples from diverse ecosystems such as the colonic mucosa. Methods Paediatric patients undergoing colonoscopy were recruited to two groups: those with a new diagnosis of IBD at their first presentation and controls with a macroscopically normal colon and no evidence of IBD on biopsy. All subjects were free from systemic antibiotics, steroids and immunosuppression for 3 months. 5 extensive UC patients (E3) by Montreal criteria and 5 controls with macroscopically/microscopically normal colons were selected for assessment. The median age was 11.5 years in the UC group and 10.7 years in the controls. All patients were male. Colonic mucosal biopsies were taken from the rectum/sigmoid. DNA extraction was performed by a modified Qiagen QiAMP mini-kit method. The presence of bacteria was confirmed by universal eubacterial primers before next-generation PCR utilising V3 Forward/V6 Reverse primers. Bacterial diversity was assessed by 454 Titanium sequencing. Sequencing data was filtered, chimera and error checked and denoised before rarefaction to 13 000 reads per sample. Statistical comparisons were made by Mann–Whitney U tests using Sigma Plot 11. Results All biopsies were positive for bacterial DNA with universal eubacterial primers. The most commonly identified bacterial phyla (comprising 95.4% of sequence reads) were Bacteroidetes (45.3%), Firmicutes (40.5%) and Proteobacteria (9.7%). Bacteroidetes were significantly more common in the control colon than in UC (7641 median reads versus 4062, p=0.032) whereas Firmicutes were significantly more common in the UC colon than in controls (5471 median reads versus 3892, p=0.016). The difference between Proteobacteria was not significant (p=0.421). Bacterial diversity assessed by the Shannon index was similar in both groups (Medians of 6.1 in UC and 6.5 in controls, p=0.841). Conclusion Colonic mucosal bacteria differ between paediatric patients with extensive UC at diagnosis and controls. UC microbiota was typified by a reduction in Bacteroidetes and an increase in Clostridia . Surprisingly, a reduction in bacterial diversity is not present in extensive UC at diagnosis. This is contrary to findings from previous studies in established disease and warrants further investigation.

Introduction Crohn’s disease (CD) is now understood to be caused by the interaction between genetic and environmental factors with dysregulation of gut microbiota playing a pivotal role. NOD2 , the strongest genetic risk factor for CD, encodes a pattern recognition receptor and plays an important role in epithelial defence. Studies of NOD2 -knockout mice have demonstrated shifts in gut microbiota. Human studies to date have been limited by relatively small numbers of individuals homozygous for NOD2 mutations without accurate matching of controls. Methods Individuals with CD of known NOD2 status were identified from the UK IBD genetics consortium. Patients in clinical remission were selected if they carried 2 of the common NOD2 variants (homozygotes or compound heterozygotes). Each NOD2 mutant patient was matched to a NOD2 wild-type patient. For all CD patients a household control was approached. Healthy volunteers stratified by NOD2 genotype were recruited from the Cambridge Bioresource. Faecal samples were frozen within 24h of collection. DNA was extracted using the FASTDNA Spin Kit for Soil. The V1–3 region of the 16S rRNA gene was amplified and amplicons were sequenced with Illumina MiSeq. Sequence data was processed in Mothur. Calprotectin was measured in all samples by ELISA. Results 97/107 individuals were included in the primary analysis (40 CD patients [58% NOD2 mutant], 32 bioresource volunteers [50% NOD2 mutant], 25 household controls). The minimum reads per sample were 3953, mean 21216. There was a significant reduction in diversity (inverse Simpson index), Ruminococcaceae including Faecalibacteria and increase in Enterobacteriaceae in samples from CD patients vs. controls (all p NOD2 status, either within the CD patients or bioresource controls. Conclusion This study confirms previously identified shifts in gut microbiota in CD patients. However, no significant differences in gut microbiota were seen when analysed by NOD2 status. This may be a reflection of sample size or of studying gut bacteria in stool as opposed to the mucosally-associated compartment. We are presently recruiting additional cases and controls to increase study power for additional analysis. Disclosure of Interest None Declared.

Introduction Children presenting for the first time with inflammatory bowel disease (IBD) offer a unique opportunity to study etiological agents before the confounders of treatment. Microaerophilic bacteria can exploit the ecological niche of the intestinal epithelium; Helicobacter and Campylobacter are previously implicated in IBD pathogenesis. The aim of the study was to assess these and other microaerophilic bacteria in de-novo paediatric IBD. Methods 100 children undergoing colonoscopy were recruited including 44 treatment naive de-novo IBD patients and 42 with normal colons. Colonic biopsies were subjected to microaerophilic culture with Gram-negative isolates then identified by sequencing. Biopsies were also PCR screened for the specific microaerophilic bacterial groups: Helicobacteraceae, Campylobacteraceae and Sutterella wadsworthensis. Results 129 Gram-negative microaerophilic bacterial isolates were identified from 10 genera. The most frequently cultured was S. wadsworthensis (32 distinct isolates). Unusual Campylobacter were isolated from 8 subjects (including 3 C. concisus, 1 C. curvus, 1 C. lari, 1 C. rectus, 3 C. showae). No Helicobacter were cultured. When comparing IBD vs. normal colon control by PCR the prevalence figures were not significantly different (Helicobacter 11% vs. 12%, p = 1.00; Campylobacter 75% vs. 76%, p = 1.00; S. wadsworthensis 82% vs. 71%, p = 0.312). Conclusion This study offers a comprehensive overview of the microaerophilic microbiota of the paediatric colon including at IBD onset. Campylobacter appear to be surprisingly common, are not more strongly associated with IBD and can be isolated from around 8% of paediatric colonic biopsies. S. wadsworthensis appears to be a common commensal. Helicobacter species are relatively rare in the paediatric colon. Disclosure of Interest None Declared

Introduction Great progress has been made in understanding the pathophysiology of Crohn’s disease (CD) and ulcerative colitis (UC), and it is now widely believed that dysregulation of the relationship between the gut microbiota and the host immune system is central to both diseases. Most published studies to date have focused on assessing the microbiota found in faecal samples, but it is increasingly recognised that this may not be an accurate reflection of what is happening at the mucosal level. The Aims of this study were to establish a bioresource of IBD patients and controls, and to use this to assess differences in gut microbiota between cases and controls and between different IBD subphenotypes. Methods Patients undergoing colonoscopy and flexible sigmoidoscopy were recruited to the South East Scotland IBD bioresource. Mucosal biopsies were obtained and snap-frozen using dry ice. DNA was then extracted using a QIAGEN spin column-based method. Blood and serum samples were also biobanked for genetic, epigenetic and metabolomics analysis. Quantitative PCR was used to measure the relative abundance of different bacterial classes compared to the total measured with universal primers. Statistical analysis was done using Mann Whitney U, chi squared and Fisher’s exact tests using R 2.15.2. Results After quality control, results were available from 147 biopsies taken from 84 patients. There were 20 patients with CD, 26 with UC, 2 IBD-U, 20 healthy controls (HC) and 16 with other diagnoses. Median age was 45 years (interquartile range [IQR] 28–50) in the CD group, 51 y (38–63) in the UC group and 39 (30–49) in the HC group. There were no significant differences in age or sex distribution between the CD, UC and HC groups. There was a statistically significant reduction in CD compared with HC in Ruminococcaceae (p = 0.002, figure 1), Lachnospiraceae (p = 0.046) and Bacteroides (0.024) and an increase in Enterobacteriaceae (p = 0.046). Comparing UC with HC, there was a significant increase in Enterobacteriaceae (p = 0.025), but no difference in the other classes. There were no significant differences in any bacterial class between biopsies taken from inflamed and non-inflamed sites in IBD patients. Conclusion This study confirms reductions in Ruminococcaceae described previously in CD, and increased Enterobacteriaceae in both CD and UC. The presence of paired DNA and serum samples offers the exciting potential to explore the relationship between the gut microbiota, epigenetic and metabolomics changes. Disclosure of Interest None Declared.

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.

Introduction Paediatric Inflammatory bowel disease (IBD) incidence is rising worldwide. Recently the role of the gut microbiota has been recognised as pivotal in disease pathogenesis. IBD microbial studies to date have focused on bacterial diversity assessment in established disease cohorts, with limited studies in treatment naive patients. In contrast to bacteria, the exact role of the colonising fungi and their pathogenic potential has not been fully explored. The aim of the study was to examine candidate fungal triggers at disease onset in children with IBD using pyrosequencing, utilising the Bacteria in Inflammatory bowel disease in Scottish Children Undergoing Investigation before Treatment (BISCUIT) study cohort. Methods 128 children undergoing colonoscopy were approached from three Scottish paediatric centres (Aberdeen, Glasgow and Dundee) with 100 ultimately recruited and biopsied; 44 IBD (comprising Crohn’s disease (CD; 29), ulcerative colitis (UC; 13) and IBD-type unspecified (2)), 42 normal colon controls (NCC) and 14 “others”. All IBD patient samples were taken from inflamed tissue. Fungal DNA was amplified on a reduced cohort of 37 recruits (13 CD, 12 UC, 12 NCC) using 18S rDNA primers. Roche 454 Titanium sequencing was conducted by NewGene (Newcastle, UK). Data analysis was performed using QIIME version 1.3.0 workflow. Taxonomy assignment of operational taxonomic units (OTUs) was performed according to ribosomal database project taxonomy. OTU tables were rarefied at 3,000 reads. Results Fungal DNA was amplifiable from 7 patient samples, 6 children with a diagnosis of IBD – 4 with CD (BISCUIT1, 31, 62 and 89), 2 children with UC (BISCUIT33 and 104) and 1 NCC (BISCUIT 27). Fungal diversity was assessed in all paediatric samples alongside three adult samples to act as comparison. The adult samples comprised 1 patient with UC (2UC21Aa) and 2 NCC (GH4 and GH9). Phylum level analysis indicated that all fungal sequences belonged to the Ascomycota and Basidiomycota phyla. Control patients contained predominantly Ascomycota sequences ( > 80% of sequences in all patients) whilst 6/7 IBD patients contained exclusively Basidiomycota species. Genus level analysis was undertaken and there was no similarity between fungal profiles from the paediatric and adult samples. Conclusion By using robust methodology we have characterised the IBD “fungal microbiota” at diagnosis in children. Based on the current study, it would appear that a distinctly altered fungal species profile is present at IBD disease presentation. Further work should now focus on expanding this study and identifying how to beneficially modify the microbiota using established and novel IBD treatments. Disclosure of Interest None Declared

Introduction Determining faecal sample bacterial community structure through sequence analysis of DNA has become a very important facet of inflammatory bowel disease (IBD) research. The possible impact of different commercially available DNA extraction kits and the influence of different lab environments on the data generated has however received relatively little attention. The study compared bacterial communities in faecal samples extracted using commercial DNA kits in established gastrointestinal research laboratories. Methods Faecal samples from 2 healthy volunteers and 2 IBD patients with relapse were investigated. DNA extraction was undertaken using MoBio and Fastprep DNA extraction kits in two established labs. Two protocols were followed for processing samples using the Fastprep kit. Each DNA sample was then split and an aliquot transferred to the other lab. Pyrosequencing PCR of bacterial 16S rRNA genes was performed in both labs on all samples. Quantitative PCR analysis (q-PCR) to validate sequencing data was also performed. Hierarchical clustering was done using the Jaccard and Theta Yue & Clayton similarity coefficients on the pyrosequencing data. Results DNA extracted using methods FastPrep1, FastPrep2 and the MoBio kit yielded median DNA concentrations of 476 (interquartile range 290–518), 453 (IQR 228–689) and 22 (IQR 9–36) ng/µL respectively. Those obtained with MoBio were significantly lower than FastPrep (p Ruminococcaceae and Bacteroidetes were significantly increased in MoBio extracted samples, while Lachnospiraceae and Enterobacteriaceae were significantly reduced (p Enterobacteriaceae , Bacteroides , Ruminococcaceae and Lachnospiraceae respectively. Conclusion This study demonstrates significant differences in DNA yield and bacterial DNA composition seen when comparing DNA extracted from the same faecal sample with different extraction kits. This highlights the importance of ensuring that all samples to be analysed together are prepared with the same DNA extraction method, and the need for caution when comparing studies that have used different methods. Disclosure of Interest None Declared

Introduction The human intestinal tract is a complex microbial ecosystem unique to every individual and contributing sufficient metabolic equivalents to be considered an organ in its own right. Dysbiosis is thought to contribute to the chronic inflammation which is involved in colorectal cancer (CRC) development. We have shown that the premalignant adenoma stage of CRC is underpinned by chronic inflammation and hypothesise that the colonic microbiota is an important driver of neoplastic progression. The aim of the study was to compare bacterial diversity in adenomas and paired adjacent normal colonic tissue. Methods 72 patients undergoing colonoscopy as part of the national bowel cancer screening programme were recruited. All patients underwent polypectomy for large adenomas (> 1cm). Denaturing gradient gel electrophoresis (DGGE on all subjects) and pyrosequencing (18 subjects) were used to compare the bacterial diversity present between the adenoma and adjacent normal paired tissue samples. Results 46% of patients showed differences in microbial diversity by DGGE analysis between adenoma and adjacent normal tissue. 21% had only limited differences whilst 79% showed dramatically altered diversity profiles. Phylum-level comparisons across the study cohort, based on sequencing, revealed no statistically significant differences for the Bacteroidetes, Firmicutes and Proteobacteria phyla. When samples were split based on DGGE analysis (i.e. similar or different DGGE profiles between paired polyp and normal mucosa), eight of the possible 10 phyla showed statistically significant differences. Collectively samples with similar profiles between polyp and normal tissue (DGGE ‘same’ group) had higher levels of Bacteroidetes and lower numbers of Actinobacteria, Firmicutes and Proteobacteria than the DGGE ‘different’ group. However when genus-level comparisons were assessed the DGGE ‘different’ sample pairs demonstrated a much greater level of dysbiosis. The levels of normal commensal genera were reduced and there was evidence of large numbers of potentially pathogenic bacteria including Shigella, Enterobacter and Fusobacteria spp. Conclusion Differences in microbial diversity harboured by a significant number of polyps compared to their immediately adjacent normal mucosa point to an important role for these bacteria in CRC progression. We hypothesise that the disturbed microbial homeostasis in these polyps, in genetically predisposed individuals and taking into account multiple environmental factors, underpins the pathogenesis of colorectal neoplasia. The identification of a “harmful” pro-inflammatory microbiota that is associated with precancerous stages (i.e. adenomas) offers the potential for manipulations of this microbiota with probiotic, prebiotic and indeed antibiotic means. Disclosure of Interest None Declared.

Introduction The critical role of luminal bacteria in the pathogenesis of ulcerative colitis (UC) is well recognised, but an individual causative microorganism has not been singled out so far. Campylobacter concisus and other non-jejuni species of Campylobacter have been implicated as putative aetiological agents of inflammatory bowel disease in children. The prevalence of these bacteria in adult patients with IBD would give a better insight into their role as immune triggers that initiate and perpetuate chronic inflammation. This study aims to investigate the prevalence of C concisus in colonic biopsy samples from adult patients with UC and compare that with control patients. Methods Adult patients who were undergoing diagnostic colonoscopy were recruited for the study, which included 68 patients with histologically proven UC and 79 control patients with no macroscopic or microscopic evidence of IBD. Biopsies were obtained during colonoscopy and immediately snap frozen in liquid nitrogen and transferred to a −80°C freezer. DNA was extracted from the biopsy samples and subjected to PCR utilising Campylobacter genus-specific primers. Positive Campylobacter samples were then subjected to nested PCR using C concisus–specific primers. Fisher9s exact test was used to examine any statistical difference in the presence of Campylobacter and C concisus DNA between the study groups. Results Detection of all Campylobacter DNA utilising genus specific primers was significantly higher in cases of UC, with a prevalence of 72.1% (49/68) compared to 24.1% (19/79) in control patients (p=0.0001). Nested PCR for C concisus DNA was positive in 30.9% (21/68) of biopsy samples from patients with UC, which was significantly higher than the prevalence rate of 7.6% (6/79) from control patients (p=0.0005). Conclusion The higher prevalence of Campylobacter genus and more specifically C concisus in biopsy samples from adult patients with UC suggest a key role of this genus of bacteria in initiating the chronic inflammation that is characteristically seen in UC. To the best of our knowledge this is the first report of this association of Cconcisus with UC in adult patients.