Clostridium leptum group bacteria abundance and diversity in the fecal microbiota of patients with inflammatory bowel disease: a case–control study in India

BACKGROUND: Alterations in the fecal bacterial flora occur in inflammatory bowel disease (IBD). We examined the abundance and diversity of Clostridium leptum group, an important group of carbohydrate-fermenting bacteria, in the feces of patients with IBD and compared them with healthy controls. METHODS: Seventeen healthy controls (HC), 20 patients with Crohn’s disease (CD) and 22 patients with ulcerative colitis (UC) participated in the study. DNA extracted from fecal samples was amplified by PCR targeting 16S rRNA gene sequences specific to C. leptum group. The PCR product was subjected to temporal temperature gradient electrophoresis (TTGE) and the number and position of individual bands were noted and diversity was estimated. The identity of bands at different positions was confirmed by cloning and sequencing. Real time quantitative PCR with Mesa Green, targeted at specific 16S rRNA gene sequences, was used to quantitate C. leptum group and its most prominent constituent, Faecalibacterium prausnitzii. RESULTS: Twenty five different operational taxonomic units (OTUs, equivalent to species) were identified constituting the C. leptum group in these participants. Their sequences were deposited in GenBank [accession numbers GQ465348 to GQ465370]. OTU number was significantly reduced in CD (7.7±3.7, mean±SD) and UC (9.0±3.0) compared to HC (11.9±2.2) (P=0.0005). The Simpson D index of alpha diversity was not significantly different between the three groups. Total numbers of C. leptum group bacteria and F. prausnitzii were reduced in both CD and UC compared to HC (P=0.0036 and P<0.0001 respectively). Disease activity did not influence numbers of C. leptum or F. prausnitzii in patients with CD or UC. CONCLUSION: C. leptum numbers and diversity were significantly reduced in both CD and UC suggesting that alterations noted were not specific to one disease. This could contribute to reduced short chain fatty acid production in IBD

Common NOD2 mutations are absent in patients with Crohn's disease in India

Background: Crohn's disease is being increasingly diagnosed in the Indian subcontinent. Three apparently common mutations in the NOD2 gene are found in up to 30% of sporadic patients with Crohn's disease in western countries. We examined whether such mutations are also found in Indian patients with Crohn's disease. Methods: Venous blood was collected from 82 patients (age range: 7-65 years, 53 men) with Crohn's disease and 149 control subjects; DNA was extracted and subjected to polymerase chain reaction using specific primers. The amplified fragments of size 185, 163 and 151 bp for R702W, G908R and 1007fs, respectively, were digested with MspI, HhaI and ApaI, and the restriction pattern noted after electrophoresis. Results: Twenty-eight patients had ileocolonic disease, 26 ileal disease, 20 colonic disease and 8 had disease limited to proximal small bowel or stomach. None of the 82 patients showed any of the three NOD2 mutations. The control subjects (93 men) had a variety of chronic gastrointestinal disorders (ulcerative colitis 52, irritable bowel syndrome 30, intestinal tuberculosis 20, colon cancer 7, miscellaneous 37). None of the control subjects showed a mutation in any of the three NOD2 mutation analyses. Conclusion: The three NOD2 gene mutations described above are uncommon in Indian patients with Crohn's disease. This study complements information provided by recent studies on NOD2 mutations in Indians

Differential regulation of cholera toxin-inhibited Na-H exchange isoforms by butyrate in rat ileum

Electroneutral Na absorption occurs in the intestine via sodium-hydrogen exchanger (NHE) isoforms NHE2 and NHE3. Bicarbonate and butyrate both stimulate electroneutral Na absorption through NHE. Bicarbonate- but not butyrate-dependent Na absorption is inhibited by cholera toxin (CT). Long-term exposure to butyrate also influences expression of apical membrane proteins in epithelial cells. These studies investigated the effects of short- and long-term in vivo exposure to butyrate on apical membrane NHE and mRNA, protein expression, and activity in rat ileal epithelium that had been exposed to CT. Ileal loops were exposed to CT in vivo for 5 h and apical membrane vesicles were isolated. 22Na uptake was measured by using the inhibitor HOE694 to identify NHE2 and NHE3 activity, and Western blot analyses were performed. CT reduced total NHE activity by 70% in apical membrane vesicles with inhibition of both NHE2 and NHE3. Reduced NHE3 activity and protein expression remained low following removal of CT but increased to control values following incubation of the ileal loop with butyrate for 2 h. In parallel there was a 40% decrease in CT-induced increase in cAMP content. In contrast, NHE2 activity partially increased following removal of CT and was further increased to control levels by butyrate. NHE2 protein expression did not parallel its activity. Neither NHE2 nor NHE3 mRNA content were affected by CT or butyrate. These results indicate that CT has varying effects on the two apical NHE isoforms, inhibiting NHE2 activity without altering its protein expression and reducing both NHE3 activity and protein expression. Butyrate restores both CT-inhibited NHE2 and NHE3 activities to normal levels but via different mechanisms

Effect of yoghurt containing Bifidobacterium lactis Bb12® on faecal excretion of secretory immunoglobulin A and human beta-defensin 2 in healthy adult volunteers

<p>Abstract</p> <p>Background</p> <p>Probiotics are used to provide health benefits. The present study tested the effect of a probiotic yoghurt on faecal output of beta-defensin and immunoglobulin A in a group of young healthy women eating a defined diet.</p> <p>Findings</p> <p>26 women aged 18-21 (median 19) years residing in a hostel were given 200 ml normal yoghurt every day for a week, followed by probiotic yoghurt containing <it>Bifidobacterium lactis </it>Bb12^®(10⁹in 200 ml) for three weeks, followed again by normal yoghurt for four weeks. Stool samples were collected at 0, 4 and 8 weeks and assayed for immunoglobulin A and human beta-defensin-2 by ELISA. All participants tolerated both normal and probiotic yoghurt well. Human beta-defensin-2 levels in faeces were not altered during the course of the study. On the other hand, compared to the basal sample, faecal IgA increased during probiotic feeding (P = 0.0184) and returned to normal after cessation of probiotic yoghurt intake.</p> <p>Conclusions</p> <p><it>Bifidobacterium lactis </it>Bb12^®increased secretory IgA output in faeces. This property may explain the ability of probiotics to prevent gastrointestinal and lower respiratory tract infections.</p

Protective association of tumor necrosis factor superfamily 15 (TNFSF15) polymorphic haplotype with Ulcerative Colitis and Crohn's disease in an Indian population.

BACKGROUND: Tumor necrosis factor superfamily (TNFSF) proteins are involved in the genesis of inflammatory bowel disease (IBD). We examined the association of seven single nucleotide polymorphisms (SNP) in the TNFSF15 gene with Crohn's disease (CD) and ulcerative colitis (UC) in the Indian population. METHODS: Seven SNPs in the TNFSF15 gene (rs10114470, rs3810936, rs6478108, rs4263839, rs6478109, rs7848647 and rs7869487) were genotyped in 309 CD patients, 330 UC patients and 437 healthy controls using the Sequenom iPLEX MassArray platform. Disease associations were evaluated for allelotypes and for genotypes. RESULTS: The minor T alleles and the TT genotypes of rs10114470 and rs3810936 were significantly protectively associated with both CD and UC. The CC genotype of rs6478108, AA genotype of rs4263839, the AA genotype of rs6478109, the TT genotype of rs7848647 and the CC genotype of rs7869487 were all protectively associated with CD but not with UC. Two haplotype blocks could be discerned, one where SNPs rs10114470 and rs3810936 were in tight LD (D' = 0.8) and the other where rs6478108, rs4263839, rs6478109, rs7848647 and rs7869487 were in tight LD (D' 0.92-1.00). The second block of haplotypes were not associated with CD or with UC. The first block of haplotypes was very significantly associated with both CD and UC. CONCLUSIONS: Strong associations exist between TNFSF15 gene polymorphisms and IBD (both CD and UC) in the Indian population

Alterations of mucosal microbiota in the colon of patients with inflammatory bowel disease revealed by real time polymerase chain reaction amplification of 16S ribosomal ribonucleic acid

Background & objectives: Alterations in microbial communities closely associated with the intestinal mucosa are likely to be important in the pathogenesis of inflammatory bowel disease (IBD). We examined the abundance of specific microbial populations in colonic mucosa of patients with ulcerative colitis (UC), Crohn′s disease (CD) and controls using reverse transcription quantitative polymerase chain reaction (RT-qPCR) amplification of 16S ribosomal ribonucleic acid (16S rRNA). Methods: RNA was extracted from colonic mucosal biopsies of patients with UC (32), CD (28) and patients undergoing screening colonoscopy (controls), and subjected to RT-qPCR using primers targeted at 16S rRNA sequences specific to selected microbial populations. Results: Bacteroides-Prevotella-Porphyromonas group and Enterobacteriaceae were the most abundant mucosal microbiota. Bacteroides and Lactobacillus abundance was greater in UC patients compared with controls or CD. Escherichia coli abundance was increased in UC compared with controls. Clostridium coccoides group and C. leptum group abundances were reduced in CD compared with controls. Microbial population did not differ between diseased and adjacent normal mucosa, or between untreated patients and those already on medical treatment. The Firmicutes to Bacteroidetes ratio was significantly decreased in both UC and CD compared with controls, indicative of a dysbiosis in both conditions. Interpretation & conclusions: Dysbiosis appears to be a primary feature in both CD and UC. Microbiome-directed interventions are likely to be appropriate in therapy of IBD

Association of ATG16L1 gene haplotype with inflammatory bowel disease in Indians.

Inflammatory bowel disease (IBD) is characterized by multigenic inheritance. Defects in autophagy related genes are considered to show genetic heterogeneity between populations. We evaluated the association of several single nucleotide polymorphisms (SNPs) in the autophagy related 16 like 1 (ATG16L1) gene with IBD in Indians. The ATG16L1 gene was genotyped for ten different SNPs using DNA extracted from peripheral blood of 234 patients with Crohn's disease (CD), 249 patients with ulcerative colitis (UC) and 393 healthy controls The SNPs rs2241880, rs4663396, rs3792106, rs10210302, rs3792109, rs2241877, rs6737398, rs11682898, rs4663402 and rs4663421 were genotyped using the Sequenom MassArray platform. PLINK was used for the association analysis and pairwise linkage disequilibrium (LD) values. Haplotype analysis was done using Haploview. All SNPs were in Hardy Weinberg equilibrium in cases and controls. The G allele at rs6737398 exhibited a protective association with both CD and UC. The T allele at rs4663402 and C allele at rs4663421 were positively associated with CD and UC. The T allele at rs2241877 exhibited protective association with UC only. The AA genotype at rs4663402 and the GG genotype at rs4663421 were protectively associated with both CD and UC. Haplotype analysis revealed that all the SNPs in tight LD (D' = 0.76-1.0) and organized in a single haplotype block. Haplotype D was positively associated with IBD (P = 5.8 x 10-6 for CD and 0.002 for UC). SNPs in ATG16L1 were associated with IBD in Indian patients. The relevance to management of individual patients requires further study

Association of <i>IRGM</i> Gene Mutations with Inflammatory Bowel Disease in the Indian Population

<div><p>Background</p><p>Mutations in the <i>IRGM</i> gene have been associated with Crohn's disease in several populations but have not been explored in Indian patients with this disease. This study examined the association of <i>IRGM</i> mutations with ulcerative colitis and Crohn's disease in Indian patients with inflammatory bowel disease.</p><p>Methods</p><p>The <i>IRGM</i> gene was amplified in four segments and Sanger-sequenced in 101 participants (42 Crohn's disease, 39 ulcerative colitis, and 20 healthy controls). Ten single nucleotide polymorphisms (SNP) were genotyped in 1200 participants (352 Crohn's disease, 400 ulcerative colitis, and 448 healthy controls) using Sequenom MassARRAY iPLEX. Disease associations were evaluated for each of the ten SNPs.</p><p>Results</p><p>Thirty one mutations were identified in the <i>IRGM</i> gene, of which two had not hitherto been reported (150226250- ss947429272 & 150227858- ss947429273). Ten SNPs (6 from the above and 4 from the literature) were evaluated. Significant associations with Crohn's disease were noted with the T allele of rs1000113 (OR 1.46, 95% CI 1.12–1.90), T allele of rs9637876 (OR 1.25, 95% CI 1.005–1.561) and C allele of rs 13361189 (OR 1.33, 95% CI 1.07–1.669). Two SNPs – rs11747270 and rs180802994 – did not exhibit Hardy-Weinberg equilibrium but were associated with both Crohn's disease and ulcerative colitis in this population. The remaining SNPs did not show significant associations with either Crohn's disease or ulcerative colitis.</p><p>Conclusions</p><p>Association of <i>IRGM</i> gene SNPs with Crohn's disease is reported for the first time in Indian patients. We also report, for the first time, an association of rs 9637876 in the <i>IRGM</i> gene with Crohn's disease.</p></div

Primers and products in PCR amplification of <i>IRGM</i> gene.

<p>52°C was the annealing temperature for all four overlapping PCRs. Primers 2 & 3 were chosen from a previously published article (17) and primers 1 & 4 were newly designed using GeneFisher2 (20).</p><p>Primers and products in PCR amplification of <i>IRGM</i> gene.</p