Colonisation of the colonic mucus gel layer with butyrogenic and hydrogenotropic bacteria in health and ulcerative colitis.

Butyrate is the primary energy source for colonocytes and is essential for mucosal integrity and repair. Butyrate deficiency as a result of colonic dysbiosis is a putative factor in ulcerative colitis (UC). Commensal microbes are butyrogenic, while others may inhibit butyrate, through hydrogenotropic activity. The aim of this study was to quantify butyrogenic and hydrogenotropic species and determine their relationship with inflammation within the colonic mucus gel layer (MGL). Mucosal brushings were obtained from 20 healthy controls (HC), 20 patients with active colitis (AC) and 14 with quiescent colitis (QUC). Abundance of each species was determined by RT-PCR. Inflammatory scores were available for each patient. Statistical analyses were performed using Mann-Whitney-U and Kruskall-Wallis tests. Butyrogenic R. hominis was more abundant in health than UC (p < 0.005), prior to normalisation against total bacteria. Hydrogenotropic B. wadsworthia was reduced in AC compared to HC and QUC (p < 0.005). An inverse correlation existed between inflammation and R. hominis (ρ - 0.460, p < 0.005) and B. wadsworthia (ρ - 0.646, p < 0.005). Other hydrogenotropic species did not widely colonise the MGL. These data support a role for butyrogenic bacteria in UC. Butyrate deficiency in UC may be related to reduced microbial production, rather than inhibition by microbial by-products.

A case-control study examining the association of smad7 and TLR single nucleotide polymorphisms on the risk of colorectal cancer in ulcerative colitis.

AIM: Ulcerative colitis (UC) is characterized by chronic mucosal inflammation and an increased risk of colorectal cancer. smad7, TLR2 and TLR4 modulate intestinal inflammation and their polymorphisms affect the risk of development of sporadic colorectal cancer. The aim of the current study was to examine the association between single nucleotide polymorphisms (SNPs) in smad7, TLR2 and TLR4 and the development of colorectal cancer in patients with UC. METHOD: DNA was extracted from formalin-fixed, paraffin-embedded tissue from 90 patients with UC who had undergone panproctocolectomy between 1985 and 2013 (30 with UC-associated colorectal cancer and 60 control UC patients). Control cases were matched 2:1 for age at diagnosis of colitis, duration of disease and gender. Genotyping was performed for the smad7 rs4464148, rs11874392, rs12953717 and rs4939827 SNPs, the TLR2 rs5743704 and rs5743708 SNPs and the TLR4 rs4986790 and rs4986791 SNPs. RESULTS: Sixty three of the 90 patients (70%) were men and the mean age at diagnosis of UC was 38.6 ± 1.6 years. The mean time to the diagnosis of UC-associated colorectal cancer was 13.5 ± 1.9 years. The 5-year recurrence-free and cancer-specific survival rates were 76% and 88%, respectively. All eight SNPs were in Hardy-Weinberg equilibrium. None of the eight SNPs assessed in smad7, TLR2 or TLR4 were associated with the development of UC-associated colorectal cancer at an allelic or genotypic level. CONCLUSIONS: These data do not support an association between polymorphisms in smad7, TLR2 or TLR4 and the development of UC-associated colorectal cancer.

The abundance of Akkermansia muciniphila and its relationship with sulphated colonic mucins in health and ulcerative colitis.

Akkermansia muciniphila utilises colonic mucin as its substrate. Abundance is reduced in ulcerative colitis (UC), as is the relative proportion of sulphated mucin in the mucus gel layer (MGL). It is unknown if these phenomena are related, however reduced sulphated mucins could contribute to reduced abundance, owing to a lack of substrate. The aim of this study was to quantify A. muciniphila within the MGL and to relate these findings with markers of inflammation and the relative proportion of sulphomucin present. Colonic biopsies and mucus brushings were obtained from 20 patients with active UC (AC), 14 with quiescent UC (QUC) and 20 healthy controls (HC). A. muciniphila abundance was determined by RT-PCR. High iron diamine alcian-blue staining was performed for histological analysis. Patients with AC had reduced abundance of A. muciniphila compared to HC and QUC. A positive association was found between A. muciniphila abundance and higher percentage of sulphated mucin (ρ 0.546, p = 0.000). Lower abundances of A. muciniphila correlated with higher inflammatory scores (ρ = 0.294 (p = 0.001)). This study confirms an inverse relationship between A. muciniphila and inflammation and a positive association between A. muciniphila abundance and percentage of sulfated mucin in the MGL.

Isolation and gene expression profiling of intestinal epithelial cells: crypt isolation by calcium chelation from in vivo samples.

AIM: The epithelial layer within the colon represents a physical barrier between the luminal contents and its underlying mucosa. It plays a pivotal role in mucosal homeostasis, and both tolerance and anti-pathogenic immune responses. Identifying signals of inflammation initiation and responses to stimuli from within the epithelial layer is critical to understanding the molecular pathways underlying disease pathology. This study validated a method to isolate and analyze epithelial populations, enabling investigations of epithelial function and response in a variety of disease setting. MATERIALS AND METHODS: Epithelial cells were isolated from whole mucosal biopsies harvested from healthy controls and patients with active ulcerative colitis by calcium chelation. The purity of isolated cells was assessed by flow cytometry. The expression profiles of a panel of epithelial functional genes were investigated by reverse transcription-polymerase chain reaction (PCR) in isolated epithelial cells and corresponding mucosal biopsies. The expression profiles of isolated cells and corresponding mucosal biopsies were evaluated and compared between healthy and inflamed colonic tissue. RESULTS: Flow cytometry identified 97% of cells isolated as intestinal epithelial cells (IECs). Comparisons of gene expression profiles between the mucosal biopsies and isolated IECs demonstrated clear differences in the gene expression signatures. Sixty percent of the examined genes showed contrasting trends of expression between sample types. CONCLUSION: The calcium chelation isolation method provided a reliable method for the isolation of a pure population of cells with preservation of epithelial cell-specific gene expression. This demonstrates the importance of sample choice when investigating functions directly affecting the colonic epithelial layer.

Colonic biogeography in health and ulcerative colitis.

The relevance of biogeography to the distal gut microbiota has been investigated in both health and inflammatory bowel disease (IBD), however multiple factors, including sample type and methodology, microbiota characterization and interpersonal variability make the construction of a core model of colonic biogeography challenging. In addition, how phylogenetic classification relates to immunogenicity and whether consistent alterations in the microbiota are associated with ulcerative colitis (UC) remain open questions. This addendum seeks to review the human colonic microbiota in health and UC as currently understood, in the broader context of the human microbiome.

A Preliminary Study Examining the Binding Capacity of Akkermansia muciniphila and Desulfovibrio spp., to Colonic Mucin in Health and Ulcerative Colitis.

BACKGROUND: Akkermansia muciniphila and Desulfovibrio spp. are commensal microbes colonising the mucus gel layer of the colon. Both species have the capacity to utilise colonic mucin as a substrate. A. muciniphila degrades colonic mucin, while Desulfovibrio spp. metabolise the sulfate moiety of sulfated mucins. Altered abundances of these microorganisms have been reported in ulcerative colitis (UC). However their capacity to bind to human colonic mucin, and whether this binding capacity is affected by changes in mucin associated with UC, remain to be defined. METHODS: Mucin was isolated from resected colon from control patients undergoing resection for colonic cancer (n = 7) and patients undergoing resection for UC (n = 5). Isolated mucin was purified and printed onto mucin microarrays. Binding of reference strains and three clinical isolates of A. muciniphila and Desulfovibrio spp. to purified mucin was investigated. RESULTS: Both A. muciniphila and Desulfovibro spp. bound to mucin. The reference strain and all clinical isolates of A. muciniphila showed increased binding capacity for UC mucin (p < .005). The Desulfovibrio reference strain showed increased affinity for UC mucin. The mucin binding profiles of clinical isolates of Desulfovibrio spp. were specific to each isolate. Two isolates showed no difference in binding. One UC isolate bound with increased affinity to UC mucin (p < .005). CONCLUSION: These preliminary data suggest that differences exist in the mucin binding capacity of isolates of A. muciniphila and Desulfovibrio spp. This study highlights the mucin microarray platform as a means of studying the ability of bacteria to interact with colonic mucin in health and disease.

Changing patterns of rotavirus strains circulating in Ireland: re-emergence of G2P[4] and identification of novel genotypes in Ireland.

Worldwide, Group A Rotavirus (RVA) is recognized as the most common aetiological agent of acute diarrheal disease in children. One hundred and ninety seven positive faecal samples were obtained from patients between 2006 and 2008. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to amplify the VP7 and VP4 gene segments of these samples, and G and P typing was carried out subsequently. The most common strain type was G1P[8], and the emergent global G9-type was identified in both years. RVA strain type G2P[4], previously reported in Ireland in 1999, was also detected. Genotypes G2 and G3 in combination with P[4] were detected in 2006-2007 only. There was also an emergence of strain types including G3P[4], G9P[4], G2P[4 + 8] and G2G4P[8] in this study. Molecular analysis of the VP7 genes revealed G1 strains circulating within lineage Ic as previously reported in Ireland. In addition, new sublineage within lineage I of G1 strains was also identified. Analysis of G4 strain NRVL-Hum-49 revealed similarity with other human G4 viruses in lineage Ib. G9 strain NRVL-Hum-74 clustered with a unique G9 strain, CIT-254, in lineage IIIc. This data supports the observations made that the profile of RVA strains in Ireland appears to be dynamic. This study demonstrates that the circulation of human rotavirus is changing continuously in Ireland, and continued surveillance of the circulating strains is needed to detect the appearance of new strains, or new variants which may lead to vaccine breakthrough.

Detection and characterisation of bovine rotavirus in Ireland from 2006-2008.

BACKGROUND: Worldwide, Group A bovine rotavirus (RVA boRV) is one of the main causes of neonatal calf diarrhoea. Currently, limited epidemiological and sequence data exists on the RVA disease in bovines in Southern Ireland only. The aim of the study was to generate epidemiological and sequence data of RVA boRV distributed over a wide geographical area in Ireland. FINDINGS: 272 stool samples were obtained from symptomatic calves and analysed to identify the prevalent G and P genotypes. Viral type combinations including G6P[5], G6P[11] and G10P[11] genotype were the most frequently identified. The G6P[5] combination was predominant throughtout the study, accounting for 70% (n = 191). Sequence analysis of the VP7 gene revealed that Irish G6 strains fell within Lineage IV, similiar to previous reports in Ireland. CONCLUSION: The detection of unusual G and P combinations may have an impact on rotavirus control programmes and current vaccines may need to incorporate new strains, as the current vaccine available may not offer protection against all of these circulating types.

Influences of the colonic microbiome on the mucous gel layer in ulcerative colitis.

The colonic mucus gel layer (MGL) is a critical component of the innate immune system acting as a physical barrier to microbes, luminal insults, and toxins. Mucins are the major component of the MGL. Selected microbes have the potential to interact with, bind to, and metabolize mucins. The tolerance of the host to the presence of these microbes is critical to maintaining MGL homeostasis. In disease states such as ulcerative colitis (UC), both the mucosa associated microbes and the constituent MGL mucins have been shown to be altered. Evidence is accumulating that implicates the potential for mucin degrading bacteria to negatively impact the MGL and its stasis. These effects appear more pronounced in UC.   This review is focused on the host-microbiome interactions within the setting of the MGL. Special focus is given to the mucolytic potential of microbes and their interactions in the setting of the colitic colon.

Depth-dependent differences in community structure of the human colonic microbiota in health.

OBJECTIVE: The aims of this study were to develop techniques for spatial microbial assessment in humans and to establish colonic luminal and mucosal spatial ecology, encompassing longitudinal and cross-sectional axes. DESIGN: A microbiological protected specimen brush was used in conjunction with a biopsy forceps to sample the colon in nine healthy volunteers undergoing colonoscopy. Terminal Restriction Fragment Length Polymorphism analysis was used to determine the major variables in the spatial organization of the colonic microbiota. RESULTS: Protected Specimen Brush sampling retrieved region-specific, uncontaminated samples that were enriched for bacterial DNA and depleted in human DNA when compared to biopsy samples. Terminal Restriction Fragment Length Polymorphism analysis revealed a segmentation of bacterial communities between the luminal brush and biopsy-associated ecological niches with little variability across the longitudinal axis of the colon and reduced diversity in brush samples. CONCLUSION: These results support the concept of a microbiota with little longitudinal variability but with some degree of segregation between luminal and mucosal communities.

The influence of CTGF single-nucleotide polymorphisms on outcomes in Crohn's disease.

OBJECTIVE: To examine the association between single-nucleotide polymorphisms (SNPs) in CTGF (connective tissue growth factor) and patient outcomes after terminal ileal resection for Crohn's disease. BACKGROUND: The primary indication for intestinal resection in Crohn's disease is fibrostenotic terminal ileal disease. CTGF is a cytokine overexpressed in the intestine of patients with Crohn's disease that influences outcomes in other disease processes. METHODS: DNA was extracted from formalin-fixed, paraffin-embedded tissue from 147 patients with Crohn's disease who had undergone terminal ileal resection between 1981 and 2009. Genotyping was performed for 4 CTGF SNPs (rs9402373, rs12526196, rs6918698, and rs9399005), which modulate nuclear factor binding and CTGF production, and a smad3 SNP (rs17293632) involved in the CTGF pathway. Patients were phenotyped using the Montreal Disease Classification. RESULTS: Sixty-seven of 147 patients (45.6%) were male; the mean age at diagnosis was 30.3 ± 12.6 years and the mean follow-up duration was 8.3 ± 7.1 years. Genotype-phenotype analysis demonstrated that the rs6918698GG genotype was associated with an older age of disease onset [>40 years; 30.6% vs 13.2%; odds ratio (OR): 2.891; 95% confidence interval (CI): 1.170-7.147). The rs9402373CC genotype was positively associated with type B1 disease (50.7% vs 26.3%; OR: 2.876; 95% CI: 1.226-6.743) and negatively associated with B2 disease (37.0% vs 65.0%; OR: 0.317; 95% CI: 0.144-0.699). None of the 5 SNPs assessed influenced clinical or surgical recurrence of Crohn's disease after intestinal resection. On multivariate analysis, male sex odds ratio (OR): 0.235; 95% CI: 0.073-0.755; P = 0.015] and never having smoked tobacco (OR: 0.249; 95% CI: 0.070-0.894; P = 0.033) reduced the risk, whereas having a prior appendectomy increased the risk (OR: 5.048; 95% CI: 1.632-15.617; P = 0.005) of surgical recurrence. CONCLUSIONS: These data implicate the rs6918698GG genotype with an age of disease onset of greater than 40 years in Crohn's disease whereas the rs9402373CC genotype is associated with a nonstricturing, nonpenetrating disease phenotype. CTGF SNPs do not influence the rate of recurrence after terminal ileal resection for Crohn's disease.

Genetic relatedness of Pseudomonas aeruginosa isolates among a paediatric cystic fibrosis patient cohort in Ireland.

Pseudomonas aeruginosa is one of the primary pathogens in the cystic fibrosis (CF) lung and a significant cause of morbidity and mortality. Reports of the spread of epidemic or transmissible strains of P. aeruginosa within and across CF centres in Europe have raised concern regarding the possibility of clonal spread among and within CF centres in Ireland. P. aeruginosa isolates (313 isolates from 142 sputum samples and 53 throat swabs) from 68 CF patients were examined using PFGE to explore the diversity of P. aeruginosa isolates among CF patients in a Dublin paediatric hospital. Only 57 different P. aeruginosa genotypes were identified among the 313 isolates. Forty-three of the genotypes were observed only in individual patients (distinct genotypes) while 13 cluster strains (present in two to four patients) were observed. Typing of P. aeruginosa isolates identified one indistinguishable clonal isolate of P. aeruginosa present in 13 CF patients (13/68; 19.1 %) which displayed higher levels of antibiotic resistance than those displayed by P. aeruginosa isolates of distinct genotype.

Evaluation of the efficacy of real-time polymerase chain reaction for the routine early detection of Pseudomonas aeruginosa in cystic fibrosis sputum and throat swab specimens.

A longitudinal study of 2099 sputa and throat swabs received from 183 pediatric cystic fibrosis patients over a 29-month period was used to evaluate the efficacy of real-time polymerase chain reaction (PCR) for the early detection of Pseudomonas aeruginosa as compared to microbiologic culture. Real-time PCR resulted in an increased number of specimens identified as P. aeruginosa positive. The sensitivity of culture was 82% (373/453) and of PCR was 93% (420/453) when considering both positive culture and PCR results as true positives. Of the 80 specimens identified as PCR positive/culture negative for P. aeruginosa, the subsequent patient sample in 32.5% (26/80) of specimens concerned was identified as P. aeruginosa culture positive, suggesting that PCR has the potential to detect P. aeruginosa earlier than the microbiologic culture. Real-time PCR analysis found no evidence of the Liverpool and Manchester epidemic P. aeruginosa strains in the cohort examined. The findings of this study highlight the importance of specimen collection protocols to ensure that adequate samples are received at the laboratory for testing, thereby minimizing the potential for reporting of false-negative P. aeruginosa culture results.