A Pyrosequencing Study in Twins Shows that Gastrointestinal Microbial Profiles Vary with Inflammatory Bowel Disease Phenotypes
Ben P. Willing; Johan Dicksved; Jonas Halfvarson; Anders F. Andersson;, Marianna Lucio; Zongli Zheng; Gunnar Järnerot; Curt Tysk; Janet K. Jansson; Lars Engstrand
Gastroenterology. 2010;139(6):1844-1854. © 2010 AGA Institute
Abstract and Introduction
Background & Aims: The composition of the gastrointestinal microbiota is thought to have an important role in the etiology of inflammatory bowel diseases (IBDs) such as Crohn's disease (CD) and ulcerative colitis (UC). Interindividual variation and an inability to detect less abundant bacteria have made it difficult to correlate specific bacteria with disease.
Methods: We used 454 pyrotag sequencing to determine the compositions of microbial communities in feces samples collected from a cohort of 40 twin pairs who were concordant or discordant for CD or UC, and in mucosal samples from a subset of the cohort. The cohort primarily comprised patients who were in remission, but also some with active disease.
Results: The profiles of the microbial community differed with disease phenotypes; relative amounts of bacterial populations correlated with IBD phenotypes. The microbial compositions of individuals with CD differed from those of healthy individuals, but were similar between healthy individuals and individuals with UC. Profiles from individuals with CD that predominantly involved the ileum differed from those with CD that predominantly involved the colon; several bacterial populations increased or decreased with disease type. Changes specific to patients with ileal CD included the disappearance of core bacteria, such as Faecalibacterium and Roseburia, and increased amounts of Enterobacteriaceae and Ruminococcus gnavus.
Conclusions: Bacterial populations differ in abundance among individuals with different phenotypes of CD. Specific species of bacteria are associated with ileal CD; further studies should investigate their role in pathogenesis.
Introduction
Inflammatory bowel diseases (IBDs) including Crohn's disease (CD) and ulcerative colitis (UC), result from inappropriate activation of the gastrointestinal mucosal immune system by the intestinal microbiota. Although the exact role of the microbiota in etiology is not yet known, genetic susceptibility and environmental triggers have been shown to play an important role.[1] The current hypothesis is that the breakdown in the host–microbial mutualism is the consequence of an imbalance between protective and harmful bacteria (dysbiosis),[2] and there is growing interest in identification of microbes that kindle the inflammation. Some changes in the microbial community are shared in CD and UC including reduced diversity (particularly Firmicutes),[3] presence of noncommensals,[2,4] and increased concentrations of Escherichia coli including pathogenic strains.[5,6] However, some changes are specific, including reduced presence of the Clostridium leptum group, particularly Faecalibacterium prausnitzii in CD.[2,4,6–9] This reduction is particularly evident for individuals with CD localized in the ileum (ICD), compared with those with primarily colonic involvement (CCD).[3,6,10,11] A reduction of the Clostridium coccoides group, but no specific members, was reported for individuals with UC.[4,12]
Establishment of the correct diagnosis is important because treatment strategies for CD and UC differ. Recently, a gene expression assay for inflamed colonic biopsies was proposed.[13] However, many patients with IBD are in remission[14] and maintenance therapies differ depending on the initial diagnosis.[15] Thus, biomarkers are needed for patients both in remission and with active disease for correct diagnosis of UC and CD (both ICD and CCD). There is also a need for better diagnostic tools for CD involving both the ileum and the colon, which is denoted ileocolonic Crohn's disease (ICCD).
Profiling the fecal microbiome using methods based on the 16S ribosomal RNA gene is less biased than cultivationbased approaches. In particular, pyrosequencing using parallel bar-coded sequence tags enables deep probing of multiple samples and provides high taxonomic resolution.[16,17] The power of pyrotag sequencing for exploration of the human microbiome has been shown in different body sites[16,18] and in response to antibiotic treatment.[19]
The aim of this study was to deeply explore the composition of the IBD gut microbiome using pyrotag sequencing and to identify specific bacterial signatures associated with IBD phenotypes. Because of the known interindividual variation in the human microbiome it is difficult to correlate specific bacteria with disease. Therefore, we studied a set of twin pairs to specifically focus on disease influence in genetically matched individuals. The cohort included twins who were concordant for disease and those who were discordant. A subset of the twins, assessed using low-resolution molecular fingerprinting techniques, previously were found to have significant differences in their gut microbiomes according to CD disease status.[6,20] Here, we applied pyrotag sequencing to a large twin cohort (40 twin pairs), including twins with UC, ICD, CCD, and ICCD, with the ultimate goal of defining bacterial signatures as potential diagnostic and monitoring targets.
Materials and Methods
Human Subjects
A set of 40 twin pairs (29 monozygotic, 11 dizygotic) obtained from a previously described Swedish population[21] were studied (Supplementary Table 1). Twin pairs with one twin previously hospitalized for IBD were identified by running the Swedish twin registry against the Swedish Hospital Discharge Register. After written consent from each twin, medical notes were scrutinized to verify the diagnosis of IBD[22] and to phenotype the disease according to the Montreal classification.[23] Twin pairs of the same sex, and if both twins in each pair had approved further contact and not undergone extensive IBD-related surgical resection (ie, colectomy), were invited to undergo colonoscopy. The twins were asked to send fecal samples 7–10 days before the colonoscopy and to fill in a questionnaire regarding environmental exposure, dietary habits, antibiotics, and drug use as previously described[20] (Supplementary Table 1). Antibiotic use beyond a year before sampling was not recorded. In UC, disease activity was classified using the Mayo score[24] in patients undergoing colonoscopy (n = 13) and using the Investigator Global Evaluation in patients who did not undergo colonoscopy (n = 3). In CD, the Harvey Bradshaw score[25] was calculated in patients undergoing colonoscopy (n = 24) and a modified Harvey Bradshaw score (without evaluation of a possible abdominal mass) was used in patients who did not undergo colonoscopy (n = 5). UC twins included 14 discordant pairs and 1 concordant pair. CD twins included 6 concordant pairs and 17 discordant pairs. Within the CD cohort, 15 individuals had ICD, 12 had CCD, and 2 had ICCD. Two healthy monozygotic twin pairs were included as controls and indicators of microbial similarities between healthy twins. Patient groups, as defined by disease, were similar in age (mean ± standard deviation [SD]) as follows: UC, 54.8 ± 12.4 (n = 16); CCD, 47.2 ± 7.6 (n = 12); healthy, 51.9 ± 11.5 (n = 35); ICD, 55.9 ± 16.1 (n = 15); and ICCD 49–67 (n = 2). Twins who were concordant for disease displayed phenotypic similarity. The use of human subjects for this study was approved by the Örebro County Ethical Committee (Dnr167/03).
Biopsy Collection
Biopsy specimens were analyzed from a subset of the twin cohort, which included a total of 9 twin pairs who were discordant or concordant for CD. The collection of biopsy specimens was described previously.[6]
DNA Extraction
DNA was extracted from 250 mg of feces using the MoBio Power Soil DNA Kit (Solana Beach, CA) according to the manufacturer's instructions. DNA from biopsies was isolated from the entire biopsy using the QIAamp DNA Mini Kit (Qiagen, Inc, Hilden, Germany) with the additional bead-beating steps on a FastPrep-24 (MP Biomedicals, Solon, OH) as previously described.[6]
454 FLX Sequencing
To investigate bacterial community composition in extracted DNA, V5 and V6 variable regions of the 16S ribosomal RNA gene were amplified by polymerase chain reaction (PCR) using forward primer (784f 5'AGGATTAGATACCCTGGTA3') and reverse primer (1061r 5'CRRCACGAGCTGACGAC3'). The reverse primer was tagged with 1 of 4 labels at the 5' end along with the adaptor sequence (5'GCCTCCCTCGCGCCATCAG3') to allow 4 samples to be included in a single 454 FLX sequencing lane as previously described.[16] Detailed PCR and purification procedures are described in the Supplementary Materials and Methods section. Pyrotag sequencing was performed on a 454 Life Sciences Genome Sequencer FLX machine (Roche, Broma, Sweden), at the Royal Institute of Technology (KTH, Stockholm, Sweden).
Taxonomic Analysis
Sequences were checked for quality and those that were less than 200 base pairs in length, contained incorrect primer sequences, or contained more than 1 ambiguous base were discarded. Remaining sequences then were subjected to complete linkage clustering in the Ribosomal Database Project II (RDPII) by using a conservative 5% dissimilarity to define operational taxonomic units (OTUs) because of the short sequence length. The most abundant sequence from each OTU was selected as a representative sequence and was taxonomically classified by Basic Local Alignment Search Tool (BLAST) searching against a local BLAST database composed of 269,420 bacterial 16S ribosomal RNA gene sequences longer than 1,200 bp with good Pintail scores from RDP v. 10.7. The OTU inherited the taxonomy (down to genus level) of the best scoring RDP hit fulfilling the criteria of 95% or more identity over an alignment length of 180 base pairs or more.
Ben P. Willing; Johan Dicksved; Jonas Halfvarson; Anders F. Andersson;, Marianna Lucio; Zongli Zheng; Gunnar Järnerot; Curt Tysk; Janet K. Jansson; Lars Engstrand
Gastroenterology. 2010;139(6):1844-1854. © 2010 AGA Institute
Abstract and Introduction
Background & Aims: The composition of the gastrointestinal microbiota is thought to have an important role in the etiology of inflammatory bowel diseases (IBDs) such as Crohn's disease (CD) and ulcerative colitis (UC). Interindividual variation and an inability to detect less abundant bacteria have made it difficult to correlate specific bacteria with disease.
Methods: We used 454 pyrotag sequencing to determine the compositions of microbial communities in feces samples collected from a cohort of 40 twin pairs who were concordant or discordant for CD or UC, and in mucosal samples from a subset of the cohort. The cohort primarily comprised patients who were in remission, but also some with active disease.
Results: The profiles of the microbial community differed with disease phenotypes; relative amounts of bacterial populations correlated with IBD phenotypes. The microbial compositions of individuals with CD differed from those of healthy individuals, but were similar between healthy individuals and individuals with UC. Profiles from individuals with CD that predominantly involved the ileum differed from those with CD that predominantly involved the colon; several bacterial populations increased or decreased with disease type. Changes specific to patients with ileal CD included the disappearance of core bacteria, such as Faecalibacterium and Roseburia, and increased amounts of Enterobacteriaceae and Ruminococcus gnavus.
Conclusions: Bacterial populations differ in abundance among individuals with different phenotypes of CD. Specific species of bacteria are associated with ileal CD; further studies should investigate their role in pathogenesis.
Introduction
Inflammatory bowel diseases (IBDs) including Crohn's disease (CD) and ulcerative colitis (UC), result from inappropriate activation of the gastrointestinal mucosal immune system by the intestinal microbiota. Although the exact role of the microbiota in etiology is not yet known, genetic susceptibility and environmental triggers have been shown to play an important role.[1] The current hypothesis is that the breakdown in the host–microbial mutualism is the consequence of an imbalance between protective and harmful bacteria (dysbiosis),[2] and there is growing interest in identification of microbes that kindle the inflammation. Some changes in the microbial community are shared in CD and UC including reduced diversity (particularly Firmicutes),[3] presence of noncommensals,[2,4] and increased concentrations of Escherichia coli including pathogenic strains.[5,6] However, some changes are specific, including reduced presence of the Clostridium leptum group, particularly Faecalibacterium prausnitzii in CD.[2,4,6–9] This reduction is particularly evident for individuals with CD localized in the ileum (ICD), compared with those with primarily colonic involvement (CCD).[3,6,10,11] A reduction of the Clostridium coccoides group, but no specific members, was reported for individuals with UC.[4,12]
Establishment of the correct diagnosis is important because treatment strategies for CD and UC differ. Recently, a gene expression assay for inflamed colonic biopsies was proposed.[13] However, many patients with IBD are in remission[14] and maintenance therapies differ depending on the initial diagnosis.[15] Thus, biomarkers are needed for patients both in remission and with active disease for correct diagnosis of UC and CD (both ICD and CCD). There is also a need for better diagnostic tools for CD involving both the ileum and the colon, which is denoted ileocolonic Crohn's disease (ICCD).
Profiling the fecal microbiome using methods based on the 16S ribosomal RNA gene is less biased than cultivationbased approaches. In particular, pyrosequencing using parallel bar-coded sequence tags enables deep probing of multiple samples and provides high taxonomic resolution.[16,17] The power of pyrotag sequencing for exploration of the human microbiome has been shown in different body sites[16,18] and in response to antibiotic treatment.[19]
The aim of this study was to deeply explore the composition of the IBD gut microbiome using pyrotag sequencing and to identify specific bacterial signatures associated with IBD phenotypes. Because of the known interindividual variation in the human microbiome it is difficult to correlate specific bacteria with disease. Therefore, we studied a set of twin pairs to specifically focus on disease influence in genetically matched individuals. The cohort included twins who were concordant for disease and those who were discordant. A subset of the twins, assessed using low-resolution molecular fingerprinting techniques, previously were found to have significant differences in their gut microbiomes according to CD disease status.[6,20] Here, we applied pyrotag sequencing to a large twin cohort (40 twin pairs), including twins with UC, ICD, CCD, and ICCD, with the ultimate goal of defining bacterial signatures as potential diagnostic and monitoring targets.
Materials and Methods
Human Subjects
A set of 40 twin pairs (29 monozygotic, 11 dizygotic) obtained from a previously described Swedish population[21] were studied (Supplementary Table 1). Twin pairs with one twin previously hospitalized for IBD were identified by running the Swedish twin registry against the Swedish Hospital Discharge Register. After written consent from each twin, medical notes were scrutinized to verify the diagnosis of IBD[22] and to phenotype the disease according to the Montreal classification.[23] Twin pairs of the same sex, and if both twins in each pair had approved further contact and not undergone extensive IBD-related surgical resection (ie, colectomy), were invited to undergo colonoscopy. The twins were asked to send fecal samples 7–10 days before the colonoscopy and to fill in a questionnaire regarding environmental exposure, dietary habits, antibiotics, and drug use as previously described[20] (Supplementary Table 1). Antibiotic use beyond a year before sampling was not recorded. In UC, disease activity was classified using the Mayo score[24] in patients undergoing colonoscopy (n = 13) and using the Investigator Global Evaluation in patients who did not undergo colonoscopy (n = 3). In CD, the Harvey Bradshaw score[25] was calculated in patients undergoing colonoscopy (n = 24) and a modified Harvey Bradshaw score (without evaluation of a possible abdominal mass) was used in patients who did not undergo colonoscopy (n = 5). UC twins included 14 discordant pairs and 1 concordant pair. CD twins included 6 concordant pairs and 17 discordant pairs. Within the CD cohort, 15 individuals had ICD, 12 had CCD, and 2 had ICCD. Two healthy monozygotic twin pairs were included as controls and indicators of microbial similarities between healthy twins. Patient groups, as defined by disease, were similar in age (mean ± standard deviation [SD]) as follows: UC, 54.8 ± 12.4 (n = 16); CCD, 47.2 ± 7.6 (n = 12); healthy, 51.9 ± 11.5 (n = 35); ICD, 55.9 ± 16.1 (n = 15); and ICCD 49–67 (n = 2). Twins who were concordant for disease displayed phenotypic similarity. The use of human subjects for this study was approved by the Örebro County Ethical Committee (Dnr167/03).
Biopsy Collection
Biopsy specimens were analyzed from a subset of the twin cohort, which included a total of 9 twin pairs who were discordant or concordant for CD. The collection of biopsy specimens was described previously.[6]
DNA Extraction
DNA was extracted from 250 mg of feces using the MoBio Power Soil DNA Kit (Solana Beach, CA) according to the manufacturer's instructions. DNA from biopsies was isolated from the entire biopsy using the QIAamp DNA Mini Kit (Qiagen, Inc, Hilden, Germany) with the additional bead-beating steps on a FastPrep-24 (MP Biomedicals, Solon, OH) as previously described.[6]
454 FLX Sequencing
To investigate bacterial community composition in extracted DNA, V5 and V6 variable regions of the 16S ribosomal RNA gene were amplified by polymerase chain reaction (PCR) using forward primer (784f 5'AGGATTAGATACCCTGGTA3') and reverse primer (1061r 5'CRRCACGAGCTGACGAC3'). The reverse primer was tagged with 1 of 4 labels at the 5' end along with the adaptor sequence (5'GCCTCCCTCGCGCCATCAG3') to allow 4 samples to be included in a single 454 FLX sequencing lane as previously described.[16] Detailed PCR and purification procedures are described in the Supplementary Materials and Methods section. Pyrotag sequencing was performed on a 454 Life Sciences Genome Sequencer FLX machine (Roche, Broma, Sweden), at the Royal Institute of Technology (KTH, Stockholm, Sweden).
Taxonomic Analysis
Sequences were checked for quality and those that were less than 200 base pairs in length, contained incorrect primer sequences, or contained more than 1 ambiguous base were discarded. Remaining sequences then were subjected to complete linkage clustering in the Ribosomal Database Project II (RDPII) by using a conservative 5% dissimilarity to define operational taxonomic units (OTUs) because of the short sequence length. The most abundant sequence from each OTU was selected as a representative sequence and was taxonomically classified by Basic Local Alignment Search Tool (BLAST) searching against a local BLAST database composed of 269,420 bacterial 16S ribosomal RNA gene sequences longer than 1,200 bp with good Pintail scores from RDP v. 10.7. The OTU inherited the taxonomy (down to genus level) of the best scoring RDP hit fulfilling the criteria of 95% or more identity over an alignment length of 180 base pairs or more.
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