Terminal restriction fragment length polymorphism analysis of the diversity of fecal microbiota in patients with ulcerative colitis

Background: Terminal restriction fragment length polymorphism (T‐RFLP) analysis is a powerful tool to assess the diversity of complexed microbiota. This permits rapid comparison of microbiota from many samples. In this study, we performed T‐RFLP analysis of the fecal microbiota from patients with ulcerative colitis (UC). Methods: Forty‐four patients with UC (23 women and 21 men, median age 25 years) and 46 healthy individuals (25 women and 21 men, median age 34 years) were enrolled in this study. DNA was extracted from their stool samples, and the 16S rRNA genes were amplified by PCR. The PCR products were then digested with HhaI and/or MspI restriction enzymes, and the length of the T‐RF was determined. Results: The fecal microbial communities were classified in 8 clusters. Almost all the healthy individuals (39 of 46) were included in cluster I, and most of the UC patients could be divided into the other 7 clusters, indicating that fecal bacterial communities are different between healthy individuals and active UC patients. Some T‐RFs, derived from the unclassified bacteria, Ruminococcus, Eubacterium, Fusobacterium, gammaproteobacteria, unclassified Bacteroides, and unclassified Lactobacillus, were detected in the UC patients, but not in the healthy individuals. The T‐RFLP patterns were also different between the active patients and inactive (remission) patients. The T‐RF derived from the unclassified bacteria, Ruminococcus and Eubacterium, and the T‐RFs derived from the unclassified bacteria, Eubacterium, and Fusobacterium were predominantly detected in the active patients not the inactive patients. In contrast, the T‐RFs derived from Lactobacillus and unclassified Lactobacillus were more predominant in the inactive (remission) patients. In 4 patients with proctitis, the pattern of fecal microbial diversity was very similar. Conclusions: T‐RFLP analyses showed that the diversity of fecal microbiota in patients with UC was different from that in healthy individuals. Unclassified bacteria, as well as known bacteria, can contribute to alterations in the bacterial diversity of UC patients.

[1]  C. Cerniglia,et al.  PCR detection of Ruminococcus spp. in human and animal faecal samples. , 1997, Molecular and cellular probes.

[2]  C. Manichanh,et al.  Reduced diversity of faecal microbiota in Crohn’s disease revealed by a metagenomic approach , 2005, Gut.

[3]  S. Cucchiara,et al.  Gut-associated bacterial microbiota in paediatric patients with inflammatory bowel disease , 2006, Gut.

[4]  P. Saxman,et al.  Terminal Restriction Fragment Length Polymorphism Analysis Program, a Web-Based Research Tool for Microbial Community Analysis , 2000, Applied and Environmental Microbiology.

[5]  M. Wilkinson,et al.  Quantitative fluorescence in situ hybridization of Bifidobacterium spp. with genus-specific 16S rRNA-targeted probes and its application in fecal samples , 1995, Applied and environmental microbiology.

[6]  H. Hayashi,et al.  Molecular Analysis of Fecal Microbiota in Elderly Individuals Using 16S rDNA Library and T‐RFLP , 2003, Microbiology and immunology.

[7]  J. Doré,et al.  Biodiversity of the Mucosa‐Associated Microbiota Is Stable Along the Distal Digestive Tract in Healthy Individuals and Patients With Ibd , 2005, Inflammatory bowel diseases.

[8]  F. Shanahan,et al.  Probiotic impact on microbial flora, inflammation and tumour development in IL‐10 knockout mice , 2001, Alimentary pharmacology & therapeutics.

[9]  Joël Doré,et al.  Gut flora and inflammatory bowel disease , 2004 .

[10]  S. Miehlke,et al.  Prevalence of Bacteroides and Prevotella spp. in ulcerative colitis. , 2006, Journal of medical microbiology.

[11]  M. Sakamoto,et al.  Application of terminal RFLP analysis to characterize oral bacterial flora in saliva of healthy subjects and patients with periodontitis. , 2003, Journal of medical microbiology.

[12]  Manfred Dietel,et al.  Mucosal flora in inflammatory bowel disease. , 2002, Gastroenterology.

[13]  Philippe Marteau,et al.  Specificities of the fecal microbiota in inflammatory bowel disease , 2006, Inflammatory bowel diseases.

[14]  J. Doré,et al.  Review article: gut flora and inflammatory bowel disease. , 2004, Alimentary pharmacology & therapeutics.

[15]  C. Neut,et al.  Self inflicted rectal ulcer: hearing is believing , 2003, Gut.

[16]  J. Doré,et al.  Direct Analysis of Genes Encoding 16S rRNA from Complex Communities Reveals Many Novel Molecular Species within the Human Gut , 1999, Applied and Environmental Microbiology.

[17]  D. Rampton,et al.  Molecular Characterization of Rectal Mucosa‐Associated Bacterial Flora in Inflammatory Bowel Disease , 2005, Inflammatory bowel diseases.

[18]  C. Kitts,et al.  Terminal restriction fragment patterns: a tool for comparing microbial communities and assessing community dynamics. , 2001, Current issues in intestinal microbiology.

[19]  R. Sartor,et al.  Resident Enteric Bacteria Are Necessary for Development of Spontaneous Colitis and Immune System Activation in Interleukin-10-Deficient Mice , 1998, Infection and Immunity.

[20]  A. Andoh,et al.  Characterization of antibody responses against rectal mucosa‐associated bacterial flora in patients with ulcerative colitis , 2000, Journal of gastroenterology and hepatology.

[21]  E. Zoetendal,et al.  Temperature Gradient Gel Electrophoresis Analysis of 16S rRNA from Human Fecal Samples Reveals Stable and Host-Specific Communities of Active Bacteria , 1998, Applied and Environmental Microbiology.

[22]  D. Karl,et al.  A computer-simulated restriction fragment length polymorphism analysis of bacterial small-subunit rRNA genes: efficacy of selected tetrameric restriction enzymes for studies of microbial diversity in nature , 1996, Applied and environmental microbiology.

[23]  Ami,et al.  Coated mesalazine (5-aminosalicylic acid) versus sulphasalazine in the treatment of active ulcerative colitis: a randomised trial. , 1989, BMJ.

[24]  R. Hammer,et al.  The germfree state prevents development of gut and joint inflammatory disease in HLA-B27 transgenic rats , 1994, The Journal of experimental medicine.

[25]  H. Hayashi,et al.  Terminal Restriction Fragment Length Polymorphism Analysis for Human Fecal Microbiota and Its Application for Analysis of Complex Bifidobacterial Communities , 2003, Microbiology and immunology.

[26]  H. Hayashi,et al.  Diversity of the Clostridium coccoides group in human fecal microbiota as determined by 16S rRNA gene library. , 2006, FEMS microbiology letters.

[27]  Y. Mahida,et al.  Host-bacterial interactions in inflammatory bowel disease. , 2004, Clinical science.

[28]  R. Bibiloni,et al.  The bacteriology of biopsies differs between newly diagnosed, untreated, Crohn's disease and ulcerative colitis patients. , 2006, Journal of medical microbiology.

[29]  K. Nagashima,et al.  Application of New Primer-Enzyme Combinations to Terminal Restriction Fragment Length Polymorphism Profiling of Bacterial Populations in Human Feces , 2003, Applied and Environmental Microbiology.

[30]  H. Kiyono,et al.  Alteration of Vβ Usage and Cytokine Production of CD4+ TCR ββ Homodimer T Cells by Elimination of Bacteroides vulgatus Prevents Colitis in TCR α-Chain-Deficient Mice1 , 2000, The Journal of Immunology.

[31]  Gerwin C. Raangs,et al.  Variations of Bacterial Populations in Human Feces Measured by Fluorescent In Situ Hybridization with Group-Specific 16S rRNA-Targeted Oligonucleotide Probes , 1998, Applied and Environmental Microbiology.

[32]  J. Hampe,et al.  Reduction in diversity of the colonic mucosa associated bacterial microflora in patients with active inflammatory bowel disease , 2004, Gut.

[33]  H. Kiyono,et al.  Alteration of V beta usage and cytokine production of CD4+ TCR beta beta homodimer T cells by elimination of Bacteroides vulgatus prevents colitis in TCR alpha-chain-deficient mice. , 2000, Journal of immunology.

[34]  D. Jewell,et al.  Role of the faecal stream in the maintenance of Crohn's colitis. , 1985, Gut.

[35]  E. Zoetendal,et al.  Mucosa-Associated Bacteria in the Human Gastrointestinal Tract Are Uniformly Distributed along the Colon and Differ from the Community Recovered from Feces , 2002, Applied and Environmental Microbiology.

[36]  R Balfour Sartor,et al.  Therapeutic manipulation of the enteric microflora in inflammatory bowel diseases: antibiotics, probiotics, and prebiotics. , 2004, Gastroenterology.

[37]  Mitsuo Sakamoto,et al.  Molecular analysis of jejunal, ileal, caecal and recto-sigmoidal human colonic microbiota using 16S rRNA gene libraries and terminal restriction fragment length polymorphism. , 2005, Journal of medical microbiology.

[38]  H. Hayashi,et al.  Phylogenetic Analysis of the Human Gut Microbiota Using 16S rDNA Clone Libraries and Strictly Anaerobic Culture‐Based Methods , 2002, Microbiology and immunology.

[39]  Mitsuo Sakamoto,et al.  Novel phylogenetic assignment database for terminal-restriction fragment length polymorphism analysis of human colonic microbiota. , 2005, Journal of microbiological methods.