WO2011043654A1 - Methods for diagnosing irritable bowel syndrome - Google Patents

Methods for diagnosing irritable bowel syndrome Download PDF

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Publication number
WO2011043654A1
WO2011043654A1 PCT/NL2010/050645 NL2010050645W WO2011043654A1 WO 2011043654 A1 WO2011043654 A1 WO 2011043654A1 NL 2010050645 W NL2010050645 W NL 2010050645W WO 2011043654 A1 WO2011043654 A1 WO 2011043654A1
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rel
ibs
uncultured
uncultured bacterium
bacteria
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PCT/NL2010/050645
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French (fr)
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Lambertus Tuk
Willem Meindert De Vos
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Aak Patent B.V.
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Priority to US13/500,194 priority Critical patent/US20120238468A1/en
Priority to CA2776420A priority patent/CA2776420A1/en
Priority to EP10763069A priority patent/EP2486143A1/en
Publication of WO2011043654A1 publication Critical patent/WO2011043654A1/en
Priority to US14/796,652 priority patent/US20150307924A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/10—Enterobacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS

Definitions

  • the present invention is in the field of microbiology and gastrointestinal health, and relates to the use of the gastrointestinal microbiota as a biomarker for intestinal aberrations, notably Irritable Bowel Syndrome.
  • the gastro-intestinal tract is colonized since birth by complex communities of microbes, including bacteria, archaea and fungi, that develop in time and space. These microbial communities were collectively termed gut microflora in previous times but are now known as gut microbiota that is of a highly complex nature. (Rajilic-Stojanovic et al. 2007. Environ Microbiol 9: 2125-2136)
  • the gut microbiota is involved in a variety of metabolic functions, such as the processing of food components that are not digested by the host, the synthesis of vitamins and the production of short chain fatty acids.
  • IBD Inflammatory Bowel Disease
  • IBS Irritable Bowel Syndrome
  • IBS is a multifactorial and complex disorder clinically characterized by recurrent episodes of abdominal discomfort or pain, altered bowel habit and urge.
  • IBD and IBS also other diseases are known to be associated with aberrations in microbiota and these include obesity, the various types of diabetes such as type I diabetes and type II diabetes, Autistic Spectrum Disorder (ASD) related diseases, celiac disease and some forms of cancer (Zoetendal et al, 2008, supra).
  • ASD Autistic Spectrum Disorder
  • IBS is the most prevalent functional bowel disorder, that affects up to 20 percent of the general population in the world. Furthermore, IBS is associated with a high rate of absenteeism from work, a significant impairment in quality of life and substantial health care costs.
  • the diagnosis of IBS is based on aberrant bowel functions using the so called Rome criteria and three subtypes of IBS are discriminated, including the constipation (IBS-C), diarrhea (IBS-D) and alternating constipation/diarrhea (IBS-A) subtypes (Thompson et al, 1989. Gastroenterology 130: 1552-1556; Longstreth et al, 2006. Gastroenterology 130: 1480-1491).
  • IBD is based on non-invasive diagnostic procedures as the presence of inflammatory biomarkers in the blood, imaging diagnostics and endoscopic observations (including histology of mucosal specimens), IBS is much harder to diagnose.
  • IBS can only be diagnosed by exclusion of IBD and other bowel disorders (such as celiac disease, colorectal cancer and lactose malabsorption) and is dependent on an anamnesis as laid down in the Rome criteria. This makes the diagnosis of IBS a rather undefined 'exclusion diagnosis' and relatively expensive.
  • biomarkers that are indicative of IBS, as is confirmed by the US National Institute of Health that states that no test for IBS is known (http://digestive.niddk.nih.gov/ddiseases/pubs/ibs/).
  • reliable non- invasive biomarkers are needed to develop a diagnostic test for IBS.
  • These biomarkers can be used to diagnose IBS but also will be instrumental in defining IBS or sub- classifying IBS as well as monitoring the pharmacological responses to a therapeutic intervention.
  • the identification of such biomarkers may lead to the discovery and development of new and innovative therapeutic interventions for IBS.
  • IBS The pathophysiologic pathway of IBS is unknown, and diagnostic procedures, among other by blood analysis, endoscopy, histology and radiologic procedures, do not reveal any common structural abnormalities in the digestive tract. While for a long time IBS has been considered a psychosomatic abberation, in recent years support has been provided for the involvement of biological and hereditary factors concerning the hypersensitivity of the brain-gut axis. Recent studies provide several lines of evidence that support a relation between intestinal microbiota and IBS. In various cases IBS is triggered in previously healthy individuals by acute GI tract infection (gastro-enteritis) by external microbiota resulting in the so called post-infective IBS: up to 25% of patients with acute GI tract infection develop IBS.
  • GI tract infection gastro-enteritis
  • US 2008/182291 describes a method of diagnosing constipation in a subject by analysing a breath, flatus, blood or saliva sample from a subject for the presence of methane.
  • a stool sample may be analysed for the presence of at least one methanogenic organism, selected from Ruminococcus sp., Methanobrevibacter sp., Bacteroides sp., Clostridium sp., and Methanobacter sp.
  • methanogenic organism selected from Ruminococcus sp., Methanobrevibacter sp., Bacteroides sp., Clostridium sp., and Methanobacter sp.
  • Ruminococcus sp., Bacteroides sp., and Clostridium sp. are methane-producing organisms.
  • Methanobrevibacter sp. and Methanobacter sp. are methane-producing organisms, but they do not belong to the Kingdom Bacteria but rather to the Kingdom Archeae.
  • CoUinsella aerofaciens belongs to the Actinobacteria, Gram-positive bacteria with a high G+C content.
  • Clostridium cocleatum-related bacteria constitute a small group in the Clostridium cluster XVIII while Coprococcus eutactus-related bacteria form a minor group in the Clostridium coccoides/Eubacterium rectale (Clostridium cluster XlVa) cluster, including also Eubacterium ruminantium and several not yet cultured phylotypes (see Table 3).
  • FISH fluorescent in situ hybridization
  • biomarkers that are indicative of IBS preferably non-invasive biomarkers, that can be used to develop a diagnostic test for IBS.
  • biomarkers indicative of IBS may be instrumental in defining IBS and/or subtyping IBS, as well in monitoring pharmaceutical responses to a therapeutic intervention.
  • biomarkers may allow discovery and development of new and innovative therapeutic interventions for IBS.
  • Figure 1 shows Redundancy Analysis of all HITChip datasets collected from Study 1 and Study 2, including in total 95 IBS subjects and 90 healthy controls.
  • Figure 2 shows a decision tree for classifying IBS subjects (U) and Healthy controls (H) using hybridization to 4 probes with the indicated Probe ID. Numbers indicate number of subjects in the order H/U reflecting Healthy/IBS. Summary of the Invention
  • the present invention provides for a method for diagnosing and/or subtyping Irritable Bowel Syndrome (IBS) in a test sample, said method comprising the steps of: a) determining the levels of two or more bacteria which are present in statistically significantly different levels between IBS subjects and healthy subjects, said bacteria being selected from IBS-decreased bacteria and IBS-increased bacteria, said IBS- decreased bacteria being selected from bacteria belonging to the supertaxon Bacteroidetes, selected from the taxa Prevotella melaninogenica et rel, Prevotella oralis et rel, Uncultured Bacteroidetes, Tanner ella et rel, Parabacteroides distasonis et rel, AUistipes et rel, Bacteroides plebeius et rel, Bacteroides splachnicus et rel, or to the supertaxon Clostridium cluster IV, selected from the taxa Subdoligranulum variabile et rel, Faecalibacterium praus
  • step cl) is performed, whereas step c2) is not performed.
  • step c2) is performed, whereas step cl) is not performed.
  • both steps cl) and c2) are performed.
  • said method is for diagnosing IBS, wherein in step a) at least the levels of two or more bacteria which are present in statistically significantly different levels between IBS subjects and healthy subjects, said bacteria being selected from IBS-decreased bacteria and IBS-increased bacteria, said IBS-decreased bacteria being selected from bacteria belonging to the supertaxon Bacteroidetes, selected from the taxa Prevotella melaninogenica et rel, Prevotella oralis et rel, Uncultured Bacteroidetes, Tannerella et rel.,; or to the supertaxon Clostridium cluster XVII, said bacteria belonging to the taxon Catenibacterium mitsuokai et rel; or to the supertaxon Proteobacteria, said bacteria belonging to the taxon Xanthomonadaceae; or to the supertaxon Uncultured Clostridiales, said bacteria belonging to the taxon Uncultured Clostridiales I; and said IBS-increased bacteria being selected from
  • said method is for diagnosing IBS, wherein in step a) the levels of at least one IBS-increased bacteria selected from bacteria belonging to the taxa Dorea formicigenerans et rel., Ruminococcus obeum et rel., and Lachnospira pectinoschiza et rel, and the level of at least one IBS-decreased bacteria selected from bacteria belonging to the taxa Prevotella melaninogenica et rel, Prevotella oralis et rel, and Catenibacterium mitsuokai et rel, are determined.
  • said method is for subtyping IBS-A, wherein in step a) the levels of two or more bacteria which are present in statistically significantly different levels between IBS subjects and healthy subjects, said bacteria being selected from IBS-decreased bacteria and IBS-increased bacteria, said IBS-decreased bacteria being selected from bacteria belonging to the supertaxon Bacteroidetes, selected from the taxa Uncultured Bacteroidetes, Tannerella et rel., Parabacteroides distasonis et rel., Allistipes et rel., Bacteroides plebeius et rel., Bacteroides splachnicus et rel., or to the supertaxon Clostridium cluster IV, selected from the taxa Subdoligranulum variabile et rel, Faecalibacterium prausnitzii et rel, Oscillospira guillermondii et rel, Sporobacter termitidis et rel, Ruminococcus
  • said method is for subtyping IBS-C, wherein in step a) at least the levels of two or more bacteria belonging to the taxa Prevotella oralis et rel, Bacteroides plebeius et rel, Clostridium stercorarium et rel, Dorea formicigenerans et rel, Clostridium nexile et rel, Catenibacterium mitsuokai et rel, or Xanthomonadaceae in a test sample are determined.
  • said method is for subtyping IBS-D, wherein in step a) at least the levels of two or more bacteria belonging to the taxa Dorea formicigenerans et rel, Ruminococcus obeum et rel, Clostridium nexile et rel, Ruminococcus lactaris et rel, Lachnospira pectinoschiza et rel, Catenibacterium mitsuokai et rel, or the uncultured Clostridiales I in a test sample are determined.
  • step a) of the method of the invention the levels of at least one IBS-increased bacteria and at least one IBS-decreased bacteria in said test sample are determined.
  • the levels of at least one IBS-increased bacteria selected from bacteria belonging to the taxa Dorea formicigenerans et rel., Ruminococcus obeum et rel., and Lachnospira pectinoschiza et rel and the level of at least one IBS-decreased bacteria selected from bacteria belonging to the taxa Prevotella melaninogenica et rel, Prevotella oralis et rel, and Catenibacterium mitsuokai et rel, in said test sample are determined.
  • step a) at least the levels of bacteria belonging to the taxa Dorea formicigenerans et rel., Ruminococcus obeum et rel., and Lachnospira pectinoschiza et rel., and the level of bacteria belonging to the taxa Prevotella melaninogenica et rel, Prevotella oralis et rel, and Catenibacterium mitsuokai et rel, in said test sample are determined.
  • the level of said one or more bacteria may be measured by determining the level of nucleic acid sequences, amino acid sequences and/or metabolites specific for said one or more bacteria, preferably the level of nucleic acid sequences specific for said one or more bacteria, e.g. 16S rRNA gene sequences or unique genomic sequences of said one or more bacteria.
  • the level of said 16S rRNA gene sequences of said one or more bacteria is measured by determining one or more variable regions of said 16S rRNA gene sequences, e.g., one or more of the variable regions VI and/or V6 of said 16S rRNA gene sequences.
  • the levels of nucleic acid sequences specific for said two or more bacteria are determined using PCR or LCR.
  • the present invention is also directed to a method for diagnosing and/or subtyping Irritable Bowel Syndrome (IBS) in a test sample, said method comprising the steps of: i) providing a test sample; ii) determining the level of at least three nucleic acids capable of hybridising to at least three nucleic acid sequences selected from the nucleic acid sequences of SEQ ID Nos: 1-100, or derivatives or fragments thereof deviating by at most 2 nucleotides, and complements, reverse, and reverse complements thereof, under stringent hybridization conditions, in said test sample; ii) comparing the level of said at least three nucleic acids from said test sample to the level of said at least three nucleic acids from a control sample; and iiia) relating the level of said at least three nucleic acids from said test sample to a diagnosis of whether the test sample is from a subject suffering from Irritable Bowel Syndrome; and/or iiib) relating the level of said at least three nu
  • the present invention pertains to a method for diagnosing and/or subtyping Irritable Bowel Syndrome (IBS) in a test sample, said method comprising the steps of: i) providing a test sample; ii) determining the level of at least three nucleic acids capable of hybridising to 16S rRNA nucleic acid sequences hybridizing to the complementary strand of any of the nucleic acid sequences SEQ ID NO.: 1-100 or fragments of said 16S rRNA nucleic acid sequences hybridizing to the complementary strand of any of the nucleic acid sequences SEQ ID NO.: 1-100, and complements, reverse, and reverse complements thereof, under stringent hybridization conditions, in said test sample; ii) comparing the level of said at least three nucleic acids from said test sample to the level of said at least three nucleic acids from a control sample; and iiia) relating the level of said at least three nucleic acids from said test sample to a diagnosis of whether the test sample is
  • an increased level of nucleic acids from said test sample said nucleic acids being capable of hybridising to nucleic acid sequences selected from the nucleic acid sequences of SEQ ID Nos: l-27, 70-71, 73-77, 99-100, or derivatives or fragments thereof deviating by at most 2 nucleotides, and complements, reverse, and reverse complements thereof, under stringent hybridization conditions, compared to the level of said nucleic acids from said control sample relates to the diagnosis that the subject is suffering from IBS.
  • a decreased level of nucleic acids from said test sample said nucleic acids being capable of hybridising to nucleic acid sequences selected from the nucleic acid sequences of SEQ ID Nos:28-69, 72,78-98, or derivatives or fragments thereof deviating by at most 2 nucleotides, and complements, reverse, and reverse complements thereof, under stringent hybridization conditions, compared to the level of said nucleic acids from said control sample relates to the diagnosis that the subject is suffering from IBS.
  • the level of at least 6 nucleic acid sequences from said test sample is determined.
  • Significance Analysis of Microarrays may be used in comparing the levels of said three or more nucleic acid sequence from said test sample with the levels of said three or more nucleic acid sequence from a control sample.
  • Prediction Analysis of Microarray may be used in comparing the levels of said three or more nucleic acid sequence from said test sample with the levels of said three or more nucleic acid sequence from a control sample.
  • Redundancy Analysis is used in comparing the levels of said three or more nucleic acid sequence from said test sample with the levels of said three or more nucleic acid sequence from a control sample.
  • the level is determined using a method selected from: hybridization of the nucleic acids in a sample to the nucleic acid sequences having SEQ ID NO.: 1-100, and complements, reverse, and reverse complements thereof, under stringent hybridization conditions; a Polymerase Chain reaction (PCR) or a Ligase Chain Reaction (LCR).
  • PCR Polymerase Chain reaction
  • LCR Ligase Chain Reaction
  • the present invention relates to an array for diagnosing IBS and/or subtyping IBS-A, IBS-C, or IBS-D, said array comprising at least two nucleic acid sequences specifically hybridize to one or more of SEQ ID NOs: 1-100, or derivatives or fragments thereof deviating by at most 2 nucleotides, and complements, reverse, and reverse complements thereof.
  • Said array may comprise at least two nucleic acid sequences selected from the nucleic acid sequences having SEQ ID Nos: 1-100.
  • the at least two nucleic acid sequences may be bound to a solid phase matrix.
  • the array may be a DNA or RNA array, and may be a micro-array.
  • the present invention is concerned with use of an array of the present invention for diagnosing IBS and/or subtyping IBS-A, IBS-C, or IBS-D.
  • IBS subjects In the present invention, in a first study a detailed comparison was made between the microbiota of 62 subjects suffering from IBS (defined according to Rome II or III criteria) and 46 healthy subjects. In a second study, a detailed comparison was made between a further 33 IBS subjects and 43 healthy subjects. It has been demonstrated that based on HITChip profiling of DNA extracted from intestinal samples, a distinction can be made between healthy subjects and subjects suffering from IBS (hereinafter also referred to as "IBS subjects"). Subsequently, a detailed comparison was made between the HITChip data from healthy subjects and subjects suffering from IBS using Redundancy Analysis (RDA). This revealed significant differences between healthy subjects and subjects suffering from IBS.
  • RDA Redundancy Analysis
  • the present invention relates to a method for diagnosing and/or subtyping Irritable Bowel Syndrome (IBS) in a test sample, said method comprising the steps of: a) determining the levels of two or more bacteria which are present in statistically significantly different levels between IBS subjects and healthy subjects, said bacteria being selected from IBS-decreased bacteria and IBS-increased bacteria, said IBS- decreased bacteria being selected from bacteria belonging to the supertaxon Bacteroidetes, selected from the taxa Prevotella melaninogenica et rel, Prevotella oralis et rel, Uncultured Bacteroidetes, Tanner ella et rel, Parabacteroides distasonis et rel, AUistipes et rel, Bacteroides plebeius et rel, Bacteroides splachnicus et rel., or to the supertaxon Clostridium cluster IV, selected from the taxa Subdoligranulum variabile et rel, Faecalibacter
  • IBS-increased bacteria refers to bacteria that are statistically significantly present more abundantly in IBS subjects compared to healthy subjects.
  • IBS-decreased bacteria refers to bacteria that are statistically significantly present more abundantly in healthy subjects compared to IBS subjects.
  • IBS-increased bacteria encompass, without limitation, bacteria belonging to the supertaxon Clostridium cluster XlVa, selected from the taxa Dorea formicigenerans et rel., Ruminococcus obeum et rel., Clostridium nexile et rel., Clostridium symbiosum et rel, Outgrouping Clostridium cluster XlVa, Ruminococcus lactaris et rel, Lachnospira pectinoschiza et rel, Ruminococcus gnavus et rel.
  • IBS- decreased bacteria encompass, without limitation, bacteria belonging to the supertaxon Bacteroidetes, selected from the taxa Prevotella melaninogenica et rel, Prevotella oralis et rel, Uncultured Bacteroidetes, Tannerella et rel, Parabacteroides distasonis et rel, Allistipes et rel, Bacteroides plebeius et rel, Bacteroides splachnicus et rel, Bacteroides uniformis et rel, Clostridium stercorarium et rel.., or to the supertaxon Clostridium cluster IV, selected from the taxa Subdoligranulum variabile et rel, Faecalibacterium prausnitzii et rel, Oscillospira guillermondii et rel, Sporobacter termitidis et rel, Ruminococcus callidus et rel, Eubacterium siraeum e
  • the level of one or more bacteria belonging to the taxa Ruminococcus gnavus et rel. , Bacteroides uniformis et rel, and Clostridium stercorarium et rel. are further determined.
  • step a) the level of one or more bacteria belonging to the taxa Ruminococcus gnavus et rel., Dorea formicigenerans et rel., Ruminococcus obeum et rel., Clostridium nexile et rel., Clostridium symbiosum et rel., Outgrouping Clostridium cluster XlVa, Prevotella oralis et rel., Prevotella melaninogenica et rel., Uncultured Bacteroidetes, Parabacteroides distasonis et rel, Allistipes et rel.
  • test sample refers to an intestinal sample.
  • Intestinal samples refer to all samples that originate from the intestinal tract, including, without limitation, feces samples, rectal swap samples, but also samples obtained from other sites in the intestinal tract, such as mucosal biopsies, as was shown previously (Zoetendal et al 2002 . Appl. Environ. Microbiol. 68:3401-7 and Kerkhoffs et al, 2009, supra).
  • a test sample may be obtained from an IBS subject, from a healthy individual, from a subject with unknown diagnosis of IBS, or from a person with complaints related to the gastro-intenstinal tract.
  • a test sample may be obtained from a subject known to suffer from IBS, or may be from a a subject with unknown diagnosis of IBS.
  • the test sample may have been processed; for example, DNA and/or RNA may have been isolated from feces samples, rectal swap samples, or samples obtained from other sites in the intestinal tract.
  • mRNA is isolated from feces samples, rectal swap samples, or samples obtained from other sites in the intestinal tract to provide a test sample comprising mRNA.
  • the level of said one or more bacteria may be determined using any method known in the art. Such method includes, without limitation, hybridization, and amplification reactions such as polymerase chain reaction (PCR) and ligase chain reaction (LCR).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • nucleic acid arrays are ordered sequences of DNA or RNA that can be used to selectively isolate and later on quantify specific nucleic acid sequences in complex mixtures - by changing the hybridization and washing conditions the specificity of the detected nucleic acid duplexes can be modulated.
  • oligonucleotide sequences used to detect a target sequence will be referred to hereinbelow as a "probe".
  • Suitable hybridisation conditions i.e. buffers used, salt strength, temperature, duration
  • these conditions may vary, depending on factors such the size of the probes, the G+C-content of the probes and whether the probes are bound to an array as described below.
  • hybridisation conditions are preferably chosen such that each probe will only form a hybrid (duplex) with a target sequence with which the probe is essentially complementary, if such a target sequence is present, and otherwise will not form any hybrid.
  • the term "essentially complementary” as used herein does not mean that the complementarity of a probe to a target sequence such as the 16S rRNA gene should be perfect, and mismatches up to 2 nucleotides can be envisaged.
  • the probe should at least in part be complementary to a specific target sequence.
  • the probe may be any nucleic acid (i.e. DNA or RNA) but is preferably DNA.
  • the probe will generally have a size of about 10 to 100 base pairs, preferably about 10 to 40 base pairs.
  • the probes may all be of the same size, or may be of different sizes.
  • the probes can be obtained in any suitable manner. For example, knowing the 16S RNA gene sequences of the bacteria identified herein, probes may be synthesized that are complementary to any part of the sequence of such 16S RNA gene sequence, i.e. using an automated DNA-synthesizer or in any other manner known per se. Also, solid phase nucleic acid synthesis techniques may be used, which may result directly in an array with the desired probes. Furthermore, the probes may be obtained using techniques of genetic engineering, for instance by primer extension using the target sequence as a template, and/or by using one or more restriction enzymes, optionally using amplification.
  • the probes may comprise one or more "alternative nucleosides".
  • alternative nucleosides include the bases Inosine (I) and Uracil (U), as well as dUTP and dITP, and these are included within the term "labeled nucleotide analog". It is to be understood that the presence of such alternative nucleosides does not prevent the probe and its target sequence to be essentially complementary to one another as defined above.
  • Quantitative nucleic acid-based amplification reactions may also be used to detect and quantify specific nucleic acid sequences in complex mixtures as in the present invention. These include the well known Polymerase Chain Reaction (PCR) and Ligase Chain Reaction (LCR) and modifications thereof (see McPherson & Moller, 2006. PCR, second edition. Taylor & Francis Group; Wiedman et al, 1994. PCR Meth Appl; 3:S51-S64). LCR is a method of DNA amplification similar to PCR but differs from PCR because it amplifies the probe molecule rather than producing amplicons through polymerization of nucleotides. Two probes are used per each DNA strand and are ligated together to form a single polynucleotide.
  • PCR Polymerase Chain Reaction
  • LCR Ligase Chain Reaction
  • LCR uses both a DNA polymerase enzyme and a DNA ligase enzyme to drive the reaction.
  • the resulting polynucleotide can be amplified by PCR and analysed separately or, notably when in multiplex samples, hybridized to arrays.
  • nucleic acids include, without limitation, the 16S RNA gene as well as the 16S rRNA itself, directly or after conversion into DNA via the reverse transcriptase reaction.
  • nucleic acid sequences can be used provided they are sufficiently different and diagnostic between IBS subjects and healthy individuals. These may include DNA sequences, both coding and non-coding, in the genomes of specific microbes that differ in prevalence between healthy and IBS subjects. Comparative genome or transciptome analysis may be a useful tool to identify such DNA sequences.
  • nucleic acid sequences are identified in intestinal microbiota that can be used to discriminate IBS subjects from healthy individuals, allowing IBS subjects to be diagnosed.
  • Numerous nucleic acid isolation methods are available that differ in their approach that includes mechanical or enzymatic lysis and specific purification methods. While all these methods are applicable to intestinal samples, the repeated bead beating method as described by Yu & Morrison (2004. BioTechniques 36:808-812) is among the most efficient ones while enzymatic methods such as those described recently by Ahroos & Tynkynnen (2009. J. Appl. Microbiol. 106:506-514) can be used in combination with automated methods. All methods introduce specific biases but for comparative purposes all methods can be used if used consistently.
  • the obtained nucleic acids may be used as template for PCR or LCR and/or hybridization reactions described above, e.g. using nucleic acid arrays.
  • sequences of the probes provided in Tables 2 and 4 can also be used to identify in the 16S rRNA databases all complete or partial 16S rRNA gene sequences that give a match, either completely or even partially. In this way a catalogue of 16S rRNA gene sequences can be obtained that can be used as targets for the development of specific PCR primers or LCR probes to detect these.
  • step b) of the method of the present invention the level of said one or more bacteria in said test sample is compared to a level of said one or more bacteria in a control sample.
  • the control sample may advantageously be derived from a healthy subject, and is preferably treated in the same way as is the test sample.
  • the control sample is sampled in the same way as is the test sample, if applicable, nucleic acid is isolated in the same way as is the test sample, and, if applicable, hybridization or quantitative amplification is performed under the same conditions to allow a fair comparison of the test sample and control sample.
  • the level values may be stored, e.g., in a computer, and used for the comparative purposes herein set forth.
  • the level of said one or more bacteria in a test sample is compared to the same bacteria in a control sample, for example, the level of Ruminococcus obeum et rel. in a test sample is compared to the level of Ruminococcus obeum et rel. in a control sample, the level of Bacteroides splachnicus et rel. in a test sample is compared to the level of Bacteroides splachnicus et rel. in a control sample, and the like.
  • an increased level of IBS- increased bacteria and/or a decreased level of IBS-decreased bacteria is related to a diagnosis that the test sample is from a subject suffering from Irritable Bowel Syndrome.
  • an increased level of IBS- increased bacteria and/or a decreased level of IBS-decreased bacteria is related to a diagnosis of whether the test sample is from a subject suffering from IBS-A, IBS-C, or IBS-D.
  • the level of one or more bacteria in a test sample is increased when it is significantly higher than the level of said one or more bacteria in a control sample. It is also considered increased when the level of one or more bacteria in the test sample is at least 5%, such as 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% higher than the corresponding one or more bacteria in the control sample.
  • the level of one or more bacteria in a test sample is decreased when it is significantly lower than the level of said one or more bacteria in a control sample. It is also considered decreased when the level of one or more bacteria in the test sample is at least 5%, such as 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% lower than the corresponding one or more bacteria in the control sample.
  • step cl) is performed, whereas step c2) is not performed.
  • step c2) is performed, whereas step cl) is not performed.
  • both steps cl ) and c2) are performed.
  • steps a), b) and cl may be performed to diagnose IBS.
  • steps a), b) and cl may be performed to diagnose IBS.
  • steps a), b) and cl may be performed to diagnose IBS.
  • steps a), b) and cl) may be performed to diagnose IBS.
  • steps a), b), and c2) may be sufficient to perform steps a), b), and c2) in order to subtype the IBS.
  • said method is for diagnosing IBS, wherein in step a) at least the levels of two or more bacteria which are present in statistically significantly different levels between IBS subjects and healthy subjects, said bacteria being selected from IBS-decreased bacteria and IBS-increased bacteria, said IBS-decreased bacteria being selected from bacteria belonging to the supertaxon Bacteroidetes, selected from the taxa Prevotella melaninogenica et rel, Prevotella oralis et rel, Uncultured Bacteroidetes, Tannerella et rel.,; or to the supertaxon Clostridium cluster XVII, said bacteria belonging to the taxon Catenibacterium mitsuokai et rel; or to the supertaxon Proteobacteria, said bacteria belonging to the taxon Xanthomonadaceae; or to the supertaxon Uncultured Clostridiales, said bacteria belonging to the taxon Uncultured Clostridiales I; and said IBS-increased bacteria being selected from
  • said method is for diagnosing IBS, wherein in step a) the levels of at least one IBS-increased bacteria selected from bacteria belonging to the taxa Dorea formicigenerans et rel., Ruminococcus obeum et rel., and Lachnospira pectinoschiza et rel, and the level of at least one IBS-decreased bacteria selected from bacteria belonging to the taxa Prevotella melaninogenica et rel, Prevotella oralis et rel., and Catenibacterium mitsuokai et rel., are determined.
  • said method is for subtyping IBS-A, wherein in step a) the levels of two or more bacteria which are present in statistically significantly different levels between IBS subjects and healthy subjects, said bacteria being selected from IBS-decreased bacteria and IBS-increased bacteria, said IBS-decreased bacteria being selected from bacteria belonging to the supertaxon Bacteroidetes, selected from the taxa Uncultured Bacteroidetes, Tanner ella et rel, Parabacteroides distasonis et rel, Allistipes et rel, Bacteroides plebeius et rel, Bacteroides splachnicus et rel., or to the supertaxon Clostridium cluster IV, selected from the taxa Subdoligranulum variabile et rel, Faecalibacterium prausnitzii et rel, Oscillospira guillermondii et rel, Sporobacter termitidis et rel, Ruminococcus callidus
  • said method is for subtyping IBS-A, wherein in step a) the levels of two or more bacteria which are present in statistically significantly different levels between IBS subjects and healthy subjects, said bacteria being selected from IBS-decreased bacteria and IBS-increased bacteria, said IBS-decreased bacteria being selected from bacteria belonging to the supertaxon Bacteroidetes, selected from the taxa Parabacteroides distasonis et rel, Allistipes et rel, Bacteroides splachnicus et rel .
  • Clostridium cluster IV selected from the taxa Subdoligranulum variabile et rel, Faecalibacterium prausnitzii et rel, Oscillospira guillermondii et rel, Sporobacter termitidis et rel, Ruminococcus callidus et rel, Eubacterium siraeum et rel., Anaerotruncus colihominis et rel., Clostridium cellulosi et rel., Clostridium leptum et rel., Ruminococcus bromii et rel., or to the supertaxon Clostridium cluster IX, said bacteria belonging to the taxon Phascolarctobacterium faecium et rel; or to the supertaxon Clostridium cluster XVI, said bacteria belonging to the taxon Eubacterium biforme et rel.; or to the supertaxon Uncultured Clostridium
  • the bacteria belonging to these taxa are unique for IBS-A subtyping.
  • said method is for subtyping IBS-C, wherein in step a) at least the levels of two or more bacteria belonging to the taxa Prevotella oralis et rel., Bacteroides plebeius et rel, Dorea formicigenerans et rel, Clostridium nexile et rel, Catenibacterium mitsuokai et rel, or Xanthomonadaceae in a test sample are determined.
  • said method is for subtyping IBS-D, wherein in step a) at least the levels of two or more bacteria belonging to the taxa Dorea formicigenerans et rel, Ruminococcus obeum et rel, Clostridium nexile et rel, Ruminococcus lactaris et rel, Lachnospira pectinoschiza et rel, Catenibacterium mitsuokai et rel, or the uncultured Clostridiales I in a test sample are determined.
  • the levels of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or more bacteria which are present in statistically significantly different levels between IBS subjects and healthy subjects, said bacteria being selected from IBS-decreased bacteria and IBS-increased bacteria, said IBS-decreased bacteria as defined hereinabove are determined to allow an even more reliable diagnosis of IBS and/or subtyping of IBS-A, IBS-C and/or IBS-D. Furthermore, any other statistical operation to the levels of said microbial groups available to persons skilled in the art also may allow for a more reliable diagnosis of IBS.
  • the level of said one or more bacteria may be measured by determining the levels of nucleic acid sequences, amino acid sequence and/or metabolites specific for said one or more bacteria, preferably the level of nucleic acid sequences specific for said one or more bacteria.
  • 16S rRNA also known as small subunit (SSU) RNA
  • SSU small subunit
  • the nucleotide sequence of the 16S rRNA genes is frequently used in diagnostics as it shows differences between microbial species.
  • 16S rRNA gene sequences are instrumental in defining the taxonomic position of microbes.
  • these 16S rRNA sequences may also identify microbes that have not yet been cultured but are only known because of the presence of a 16S rRNA gene sequence.
  • V1-V8 A growing database of over a million microbial 16S rRNA sequences can be found in publicly available databases such as http://www.arb-silva.de (Pruesse et al, 2007. Nucleic Acid Res. 35 :7188) and http://rdp.cmu.mse.edu (Cole et al, 2008. Nucleic Acids Res. 35 (Database issue): D169-D172). It has been well-established that the 16S rRNA sequence contains a limited number of variable regions of several dozens of nucleotides, termed V1-V8, that are targets for developing nucleic acid probes, PCR primers or LCR probes.
  • HIT Human Intestinal Tract
  • Hybridization to the HIT probes can be used to deduce what microbe is present and allows its taxonomic identification at different level, the most important ones including genus-like groups (sequence similarity > 90% - so called level 2 groups) and phylotype-like groups (sequence similarity > 98% - so called level 3 groups) (Rajilic-Stojanovic et al 2009, supra).
  • Table 3 defines the identified groupings even when the systematic names of the involved bacterial species is changing due to advanced taxonomic insight.
  • Percentages (%) sequence identity refers to the percentage identical nucleotides between two sequences and can be determined using for example pairwise local alignment tools such as the program “water” of EmbossWIN (version 2.10.0) using default parameters, (gap opening penalty 10.0 and gap extension penalty 0.5, using Blosum62 for proteins and DNAFULL matrices for nucleic acids) or "Bestfit" of GCG Wisconsin Package, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, using default parameters. Alternatively, BLAST analysis using default settings may also be used, such as nucleotide Blast of NCIMB, with a gap creation penalty 11 and gap extension penalty 1.
  • the level of said one or more bacteria is preferably measured by determining the level of specific nucleic acid sequences in said test sample, which nucleic acid sequences are preferably 16S rRNA gene sequences of said one or more bacteria, more preferably one or more variable regions of said 16S rRNA gene sequences, e.g., one or more of the variable regions VI and/or V6 of said 16S rRNA gene sequences.
  • biomarkers can serve alone or in combination as biomarkers for IBS subjects.
  • a biomarker, or biological marker is in general a substance used as an indicator of a biologic state.
  • Biomarkers can include a variety of stable macromolecular molecules, including nucleic acids, proteins or lipids but also metabolites or a combination thereof.
  • nucleic acids including DNA and RNA, that are present in the intestinal microbiota as they are stable but can be isolated easily.
  • proteins encoded by the said DNA can be considered useful biomarkers, notably when they are stable.
  • the identification of the microbial groups that are specifically affected also allows new classification of IBS and its subsequent therapy.
  • This therapy may consist of the consumption of correcting microbes, conforming to the definition of probiotics (see http://www.isapp.net/).
  • consumption of prebiotics can be envisaged that affect the microbial composition (http://www.isapp.net/).
  • pharmaceutical preparations can be envisaged that affect the microbiota in such a way that the identified defects are corrected.
  • 'defects' are defined as 'deviating from healthy subjects with regard to gastro-intestinal microbiota'.
  • IBS-C, IBS-D and IBS-A types are mainly based on form and frequency of stool samples and hence subjective, undefined and biased (Thompson et al., 1989. Gastroenterol Int 2:92-95; Longstreth et al., 2006, supra; Thompson, 2006. Gastroenterology 130: 1552-1556).
  • the traditional classification of IBS subjects based on the Rome criteria does not provide a solid basis for therapy and this hampers treatment of the IBS subjects.
  • the invention Based on the microbiota analysis and detection of the identified oligonucleotides specific for IBS (probes having SEQ ID Nos: l-27, 70-71, 73-77, 99-100) and Healthy subjects (probes having SEQ ID Nos:28- 69, 72, 78-98) (see Tables 2 and 4) of the invention new, rational and unbiased differentiation of the IBS subjects can be realized. It is envisaged that this results in classifications that are useful in combination with specific treatments and thus improving the efficacy of therapies. As such, the invention will allow for differentiating IBS subjects based upon the microbiota in their GI tract. Hence, the classification of IBS following microbiota analysis is a preferred embodiment of the invention. Inspection of the major differences in microbial composition in the IBS-C, IBS-D and IBS-A allows the definition of IBS subtypes based on specific microbial composition.
  • an alternative way of diagnosing and/or subtyping IBS is to use the selective hybridization probes of SEQ ID NO.: 1-100 identified herein, or complements, reverse, or reverse-complements thereof.
  • the hybridization probes of SEQ ID NO.: 1-100 may be used as such for hybridization with nucleic acids isolated from a test sample to provide a diagnosis of IBS and/or to subtype IBS.
  • probes with up to 2 nucleotide mismatches in comparison to SEQ ID NO.: 1-100, or complements, reverse, or reverse-complements thereof, may be used.
  • the probes may be used to identify 16S rRNA nucleic acid sequences useful for diagnosing IBS and/or subtyping IBS.
  • the nucleic acid sequences of SEQ ID NO.: 1-100, or derivatives or fragments thereof deviating by at most 2 nucleotides, or complements, reverse, or reverse-complements thereof may be used to perform a search in well-known public nucleic acid sequence databases in order to identify those 16S rRNA sequences that are useful in diagnosing IBS and/or subtyping IBS.
  • the SILVA and RDP databases were searched for 16S rRNA gene sequences using the nucleic acid sequences of SEQ ID NO.: 1-100 allowing up to 2 mismatches from these nucleic acid sequences. This resulted in multiple hits for each of the nucleic acid sequences.
  • the 16S rRNA sequences thus identified, as well as sequences derived therefrom may also be used to diagnose IBS and/or subtype IBS.
  • nucleic acid sequences suitable for hybridization reactions herein also referred to as "probes" useful to diagnose IBS and/or subtype IBS may be identified starting from the 16S rRNA sequences identified using nucleic acid sequences of SEQ ID NO.
  • the 16S rRNA sequences identified using nucleic acid sequences of SEQ ID NO.: 1-100, or derivatives or fragments thereof deviating by at most 2 nucleotides, or complements, reverse, or reverse-complements thereof may be used to develop amplification primers for use in amplification reactions, e.g., for use in PCR or LCR reactions. Such amplification reactions may also be used to diagnose IBS and/or subtype IBS.
  • Sequences which are the complement, reverse or reverse- complement of the nucleic acid sequences of SEQ ID Nos: 1-100, derivatives or fragments thereof deviating by at most 2 nucleotides 16S rRNA sequences identified using nucleic acid sequences of SEQ ID NO.: 1-100, or derivatives or fragments thereof deviating by at most 2 nucleotides, may also be used in the methods of the invention.
  • the present invention is also directed to a method for diagnosing and/or subtyping Irritable Bowel Syndrome (IBS) in a test sample, said method comprising the steps of: i) providing a test sample; ii) determining the level of at least three nucleic acids capable of hybridising to at least three nucleic acid sequences selected from the nucleic acid sequences of SEQ ID Nos: l-100, or derivatives or fragments thereof deviating by at most 2 nucleotides, and complements, reverse, and reverse complements thereof, under stringent hybridization conditions, in said test sample; ii) comparing the level of said at least three nucleic acids from said test sample to the level of said at least three nucleic acids from a control sample; and iiia) relating the level of said at least three nucleic acids from said test sample to a diagnosis of whether the test sample is from a subject suffering from Irritable Bowel Syndrome; and/or iiib) relating the level of said at least
  • step i) in step i) the level of at least three nucleic acids capable of hybridising to 16S rRNA nucleic acid sequences hybridizing to the complementary strand of any of the nucleic acid sequences SEQ ID NO.: 1-100 or fragments of said 16S rRNA nucleic acid sequences hybridizing to the complementary strand of any of the nucleic acid sequences SEQ ID NO.: 1-100, and complements, reverse, and reverse complements thereof, under stringent hybridization conditions, in said test sample, is determined.
  • level as used in combination with nucleic acids or nucleic acid sequences may refer to expression level as determined using mRNA, or the amount of genomic DNA present in a sample.
  • Stringent hybridisation conditions can be used to identify nucleotide sequences, which are substantially identical to a given nucleotide sequence. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequences at a defined ionic strength and pH. The T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridises to a perfectly matched probe. Typically stringent conditions will be chosen in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least 60°C. Lowering the salt concentration and/or increasing the temperature increases stringency.
  • Stringent conditions for RNA-DNA hybridisations are for example those which include at least one wash in 0.2X SSC at 63°C for 20min, or equivalent conditions.
  • Stringent conditions for DNA-DNA hybridisation are for example those which include at least one wash (usually 2) in 0.2X SSC at a temperature of at least 50°C, usually about 55°C, for 20 min, or equivalent conditions. See also Sambrook et al. (1989) and Sambrook and Russell (2001).
  • step iiia) is performed, whereas step iiib) is not performed.
  • step iiib) is performed, whereas step iiia) is not performed.
  • both steps iiia) and iiib) are performed.
  • steps i), ii) and iiia) may be performed to diagnose IBS.
  • steps i), ii) and iiia) may be performed to diagnose IBS.
  • steps i), ii), and iiib) may be sufficient to perform steps i), ii), and iiib) in order to subtype the IBS.
  • an increased level of nucleic acids from said test sample said nucleic acids being capable of hybridising to nucleic acid sequences selected from the nucleic acid sequences of SEQ ID Nos: l-27, 70-71, 73-77, 99-100, or derivatives or fragments thereof deviating by at most 2 nucleotides, and complements, reverse, and reverse complements thereof, under stringent hybridization conditions, compared to the level of said nucleic acids from said control sample relates to the diagnosis that the subject is suffering from IBS.
  • a decreased level of nucleic acids from said test sample said nucleic acids being capable of hybridising to nucleic acid sequences selected from the nucleic acid sequences of SEQ ID Nos:28-69, 72, 78-98, or derivatives or fragments thereof deviating by at most 2 nucleotides, and complements, reverse, and reverse complements thereof, under stringent hybridization conditions, compared to the level of said nucleic acids from said control sample relates to the diagnosis that the subject is suffering from IBS.
  • nucleic acid or nucleotide sequences of SEQ ID NO. : 1-100, or derivatives or fragments thereof deviating from SEQ ID NO. : 1-100 by at most 2 nucleotides, or the complement, reverse, or reverse-complement thereof, may be used to discriminate between healthy subjects and subjects suffering from IBS, as well as between subject suffering from the various subtypes of IBS: IBS-A, IBS-C and IBS-D.
  • nucleic acid sequences selected from the group consisting of SEQ ID NO.: 1-100 may suffice for diagnosing IBS and/or subtyping IBS-A, IBS-C and/or IBS- D, it is preferred that at least 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, 30, 35, 40, or more nucleic acid sequences selected from the group consisting of SEQ ID Nos.: l-100 are employed in the method of the present invention.
  • all nucleic acid sequences of SEQ ID NO.: 1-100, or derivatives or fragments thereof deviating by at most 2 nucleotides, or the complement, reverse, or reverse-complement thereof, are employed for diagnosing and/or subtyping IBS in a test sample.
  • the levels of the nucleic acid sequences in a test sample may be subjected to statistical and/or bioinformatical analysis to obtain analyzed data; and the analyzed data of said test sample may be compared to analyzed data from a control sample, to provide a diagnosis of whether the test sample is from a subject suffering from Irritable Bowel Syndrome.
  • hybridization patterns on a micro-array comprising the nucleic acid sequences having SEQ ID NO: 1-100.
  • the hybridization data generated using SEQ ID Nos.: l-100 may be processed using statistical and/or bioinformatical analysis such as Principal Component Analysis (PCA) and/or Redundancy Analysis (RDA).
  • PCA Principal Component Analysis
  • RDA Redundancy Analysis
  • the analyzed data may then be compared to analyzed data from a control sample which has been subject to the same statistical and/or bioinformatical analysis, which may relate to a diagnosis of whether the test sample is from a subject suffering from IBS.
  • Significance Analysis of Microarrays is used in comparing the levels of said three or more nucleic acid sequence from said test sample with the levels of said three or more nucleic acid sequence from a control sample.
  • SAM Significance Analysis of Microarrays
  • Prediction Analysis of Microarray is used in comparing the levels of said three or more nucleic acid sequence from said test sample with the levels of said three or more nucleic acid sequence from a control sample.
  • PAM Prediction Analysis of Microarray
  • Redundancy Analysis is used in comparing the levels of said three or more nucleic acid sequence from said test sample with the levels of said three or more nucleic acid sequence from a control sample.
  • RDA Redundancy Analysis
  • the level may be determined using a method selected from: hybridization of the nucleic acids in a sample to the nucleic acid sequences having SEQ ID NO.: 1-100, and complements, reverse, and reverse complements thereof, under stringent hybridization conditions; a Polymerase Chain reaction (PCR) or a Ligase Chain Reaction (LCR).
  • PCR Polymerase Chain reaction
  • LCR Ligase Chain Reaction
  • the invention pertains to a method for diagnosing and/or subtyping Irritable Bowel Syndrome (IBS) in a test sample, said method comprising the steps of: i) determining the level of amplification of at least three nucleic acid sequences from a test sample using one or more of the nucleic acid sequences of SEQ ID NO.: 1-100, or derivatives or fragments thereof deviating by at most 2 nucleotides, or nucleic acids capable of hybridising to 16S rRNA nucleic acid sequences hybridizing to the complementary strand of any of the nucleic acid sequences SEQ ID NO.: 1-100 or fragments of said 16S rRNA nucleic acid sequences hybridizing to the complementary strand of any of the nucleic acid sequences SEQ ID NO.: 1-100, and complements, reverse, and reverse complements thereof; ii) comparing the level of amplification of said at least three nucleic acid sequences from said test sample to the level of amplification of said at least three
  • the levels of one or more bacteria belonging to the taxa Collinsella may be used for diagnosing and subtyping IBS in the method of the present invention.
  • they may be used for subtyping IBS-A in the methods of the present invention.
  • a decreased level of two or more bacteria belonging to the taxa Collinsella in the test sample relates to a diagnosis that the test sample is from a subject suffering from IBS-A.
  • the present invention provides for an array for diagnosing IBS and/or subtyping IBS-A, IBS-C, or IBS-D, said array comprising at least two nucleic acid sequences having the nucleic acid sequence of SEQ ID NOs: 1-100, or derivatives or fragments thereof deviating by at most 2 nucleotides, or complements, reverse, and reverse complements thereof. It was found that the nucleotide sequences mentioned were highly suitable for diagnosing IBS from 3,699 unique nucleotide sequences that were tested.
  • said array comprises at least two nucleic acid sequences selected from the nucleic acid sequences having SEQ ID Nos: 1-100.
  • the at least two nucleic acid sequences may be bound to a solid phase matrix.
  • the array may be a DNA or RNA array, and may be a micro-array.
  • the present invention is concerned with the use of an array of the invention for diagnosing IBS and/or subtyping IBS-A, IBS-C, or IBS-D.
  • the verb "to comprise” and its conjugations is used in its non- limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.
  • the verb "to consist” may be replaced by "to consist essentially of meaning that a composition of the invention may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristics of the invention.
  • indefinite article “a” or “an” does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements.
  • the indefinite article “a” or “an” thus usually means “at least one”.
  • a level in a test sample is increased or decreased when it is at least 5%, such as 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% higher or lower, respectively, than the corresponding level in a control sample.
  • Level 1 corresponds to the phylum, or in case of Firmicutes to the Clostridium cluster; Level 2 includes groups of sequences with 90% or more sequence similarity; Level 3 represents unique phylotypes that were defined as species for cultivated microorganisms, or representatives of each monophyletic group with > 98% sequence identity for clones corresponding to uncultured microorganisms (herein identified as "relatives" or "et rel.”).
  • Propionibacterium Propionibacterium acnes AB041617
  • Asteroleplasma Asteroleplasma et rel. Uncultured bacterium UC7-1 1 AJ608228
  • Lactobacillus helveticus X61 141 uncultured Lactobacillus sp. LabF368 AF335876 uncultured Lactobacillus sp. LabF93 AF33591 1
  • Streptococcus intermedius et Streptococcus intermedius AF 104671 rel.
  • Veillonella parvula AF439640 Veillonella atypica AF439641 uncultured bacterium ABLCfS AF499900
  • Clostridium difficile AF072473 Clostridium bifermentans AF320283 Clostridium glycolicum AY007244 Clostridium sticklandii L04167
  • Clostridium felsineum Clostridium felsineum X77851
  • Ruminococcus sp. CJ60 AB080891 uncultured human gut bacterium JWlH4a AB080862 uncultured bacterium (human infant) L37A AF253389 uncultured bacterium Adhufecl 17rbh AY471716 uncultured bacterium Muc2-3 AY451997
  • Ruminococcus obeum L76601 uncultured Ruminococcus sp. N067 AB064763 uncultured bacterium KZ22 AY916216 uncultured bacterium NL49 AY916245 uncultured bacterium NQ96 AY916255 uncultured bacterium VI 27 AY916274
  • Example 1 Comparison of the fecal microbiota of IBS and healthy subjects (Study 1) Fecal samples were obtained from a first study (Study 1) of a total of 62 IBS subjects including 19 with IBS-C, 25 with IBS-D and 18 with IBS-A, and a total of 46 healthy individuals that were age and gender matched. Microbial DNA was isolated from these fecal samples following the method of Ahlroos & Tynkynnen (2009, supra) and used for profiling using the HITChip phylogenetic microarray using 3699 distinct HIT probes as described (Rajilic-Stojanovic et al, 2009, supra) .
  • a total of 34 HIT probes (oligonucleotides having SEQ ID Nos: 1-27, 70-71 , 73-77, 99-100) showed a significantly higher hybridization signal in the IBS subjects than the healthy individuals, while a total of 66 (oligonucleotideshaving SEQ ID Nos:28-69, 72, 78-98) showed less hybridization in the IBS subjects than the healthy subjects, respectively.
  • the sequences of these oligonucleotides are disclosed in Tables 2 and 4 and allow the development of specific probes as described above.
  • these probes can be used to screen the 16S rDNA databases for complete 16S rRNA sequences that subsequently can be used as target for the development of specific probes as described above.
  • the data sets obtained from Study 1 and Study 2 were combined. Subsequently, a training data set, consisting of 2/3 of the data, and a test data set, consisting of 1/3 of the data, were randomly selected.
  • the rationale behind this division of the data sets is that the test data are not used at all in the modeling or selection process but only in the final testing. This should protect from over-fitting of the models into the data (i.e. from an inferior generalization).
  • the training data was used to filter out the most discriminating HIT probes using a t-test. These are listed in Table 3.
  • LDA stepwise linear discriminant analysis
  • a multivariant analysis system see Venables, W. N. and Ripley, B. D. (2002) Modern Applied Statistics with S. Fourth edition. Springer Publishers.
  • the subsequent classification was done in two nested cross-validation loops, where the inner one was used to select the discriminating features in a stepwise- LDA, and the outer loop to validate the performance of the classifiers for unseen data.
  • the final test simulation was done by applying the stepwise-LDA to all of the training data, and then classifying the 1/3 of the binded test data, and comparing it to the 10 randomized classifications.
  • Table 4 Identification, sequence and analysis of the HIT probes coded SEQ ID 68- 100 that were obtained in the stepwise linear discriminant analysis of various parts of the datasets of Study 1 and Study 2.
  • the oligonucleotides are indicated with their nucleotide sequence (3 'to 5 ').
  • the oligonucleotides with SEQ ID Nos:70-71 , 73-77, 99-100 showed a significantly higher hybridization signal in the IBS than the healthy subjects, whereas the oligonucleotides with SEQ ID Nos:68-69, 72, 78-98 showed the opposite.
  • HIT probes can be used in a diagnostic test to differentiate IBS subjects from healthy controls using all 185 subjects derived from Study 1 and Study 2, a number of these were analysed in a hierarchical analysis.
  • the power of combining four discriminating HIT probes could be easily illustrated in a hierarchial decision tree (Fig. 2). It could be shown that hybridization to HIT probe with ID Seq 80 and its cut off at a certain hybridization value allowed to assign correctly 34 of healthy controls as healthy and 3 IBS subjects falsely. Similarly, a second HIT probe with ID Seq 77 could be used for further differentiating the remaining 148 subjects and could assign 18 healthy controls correctly and 5 IBS ones falsely.
  • a third HIT probe with ID Seq 72 could be used to differentiate the remaining 125 subjects and could assign 63 IBS subjects correctly and 17 healthy controls incorrectly.
  • ID Seq 90 could be applied to differentiate the remaining 45 subjects and this resulted in the correct assignment of 13 Healthy controls and 18 IBS subjects, while 6 IBS subjects and 8 healthy controls were falsely assigned.
  • the probes that added significant value to the first classification were the probes 72, 77 and 90 that are specific for the bacterial taxa including Eubacterium sireaeum et rel., Lachnospira pectinoschiza et rel. and Subdoligranulum variabile et rel. , respectively. These bacterial taxa already had been identified in a separate analysis when addressing Study 1 (see Table 1). This result testifies for the power of diagnosing IBS by determining the level of various and different groups of IBS- increased or IBS- decreased bacteria and using these in a decision tree as described here.

Abstract

The present invention discloses a method for diagnosing Irritable Bowel Syndrome (IBS) in a test sample by determining the level of several bacterial taxa in the test sample, comparing this level with the levels of those bacterial taxa in a control sample, and relating the level to a diagnosis of IBS. Additionally, the present invention provides a method for treatment of IBS based on said diagnosis. Also, the invention provides a method for subtyping IBS in a test sample.

Description

METHODS FOR DIAGNOSING IRRITABLE BOWEL SYNDROME
Field of the invention
The present invention is in the field of microbiology and gastrointestinal health, and relates to the use of the gastrointestinal microbiota as a biomarker for intestinal aberrations, notably Irritable Bowel Syndrome.
Background
The gastro-intestinal tract is colonized since birth by complex communities of microbes, including bacteria, archaea and fungi, that develop in time and space. These microbial communities were collectively termed gut microflora in previous times but are now known as gut microbiota that is of a highly complex nature. (Rajilic-Stojanovic et al. 2007. Environ Microbiol 9: 2125-2136) The gut microbiota is involved in a variety of metabolic functions, such as the processing of food components that are not digested by the host, the synthesis of vitamins and the production of short chain fatty acids. However, in recent years it has been established that gut microbes interact with the host cells resulting in modulation of host processes including gut motility, gut barrier and immune function (Zoetendal et al, 2008. Gut 57: 1605-1615). Hence, aberrations in the gut microbiota can be associated with a variety of functional intestinal disorders, including Inflammatory Bowel Disease (hereinafter also referred to as "IBD") and Irritable Bowel Syndrome (hereinafter also referred to as "IBS"). IBD includes mainly Crohn's Disease and Ulcerative Colitis that are manifested by recurrent severe bouts of inflammation of various parts of the intestinal tract. IBS is a multifactorial and complex disorder clinically characterized by recurrent episodes of abdominal discomfort or pain, altered bowel habit and urge. Apart from IBD and IBS also other diseases are known to be associated with aberrations in microbiota and these include obesity, the various types of diabetes such as type I diabetes and type II diabetes, Autistic Spectrum Disorder (ASD) related diseases, celiac disease and some forms of cancer (Zoetendal et al, 2008, supra).
From all the diseases that affect the gastro-intestinal tract, IBS is the most prevalent functional bowel disorder, that affects up to 20 percent of the general population in the world. Furthermore, IBS is associated with a high rate of absenteeism from work, a significant impairment in quality of life and substantial health care costs. The diagnosis of IBS is based on aberrant bowel functions using the so called Rome criteria and three subtypes of IBS are discriminated, including the constipation (IBS-C), diarrhea (IBS-D) and alternating constipation/diarrhea (IBS-A) subtypes (Thompson et al, 1989. Gastroenterology 130: 1552-1556; Longstreth et al, 2006. Gastroenterology 130: 1480-1491). While the diagnosis of IBD is based on non-invasive diagnostic procedures as the presence of inflammatory biomarkers in the blood, imaging diagnostics and endoscopic observations (including histology of mucosal specimens), IBS is much harder to diagnose. Nowadays, IBS can only be diagnosed by exclusion of IBD and other bowel disorders (such as celiac disease, colorectal cancer and lactose malabsorption) and is dependent on an anamnesis as laid down in the Rome criteria. This makes the diagnosis of IBS a rather undefined 'exclusion diagnosis' and relatively expensive. Hence there is a great need to develop biomarkers that are indicative of IBS, as is confirmed by the US National Institute of Health that states that no test for IBS is known (http://digestive.niddk.nih.gov/ddiseases/pubs/ibs/). Specifically, reliable non- invasive biomarkers are needed to develop a diagnostic test for IBS. These biomarkers can be used to diagnose IBS but also will be instrumental in defining IBS or sub- classifying IBS as well as monitoring the pharmacological responses to a therapeutic intervention. Moreover, the identification of such biomarkers may lead to the discovery and development of new and innovative therapeutic interventions for IBS.
The pathophysiologic pathway of IBS is unknown, and diagnostic procedures, among other by blood analysis, endoscopy, histology and radiologic procedures, do not reveal any common structural abnormalities in the digestive tract. While for a long time IBS has been considered a psychosomatic abberation, in recent years support has been provided for the involvement of biological and hereditary factors concerning the hypersensitivity of the brain-gut axis. Recent studies provide several lines of evidence that support a relation between intestinal microbiota and IBS. In various cases IBS is triggered in previously healthy individuals by acute GI tract infection (gastro-enteritis) by external microbiota resulting in the so called post-infective IBS: up to 25% of patients with acute GI tract infection develop IBS. During these infections the intestinal function and microbiota composition is affected. In several cases successful treatment of IBS has been shown by the consumption of pre- and probiotics that are all known to affect the intestinal microbiota composition and function (Spiller, 2009. Aliment Pharmacol Ther 28: 385-396). Finally, there are observations that IBS subjects in comparison with healthy individuals show deviations in intestinal microbiota composition or metabolites. However, no clear picture emerges from these studies as to what are the specific microbes or microbial groups that differ between IBS and healthy subjects. This is partly caused by the fact that in many cases use is made of culturing techniques to identify microbes, where it is well known that many of the intestinal microbes can not been cultured, and cultivation therefore is known to give significant biases.
US 2008/182291 describes a method of diagnosing constipation in a subject by analysing a breath, flatus, blood or saliva sample from a subject for the presence of methane. Alternatively, a stool sample may be analysed for the presence of at least one methanogenic organism, selected from Ruminococcus sp., Methanobrevibacter sp., Bacteroides sp., Clostridium sp., and Methanobacter sp. However, none of
Ruminococcus sp., Bacteroides sp., and Clostridium sp. are methane-producing organisms. Methanobrevibacter sp. and Methanobacter sp. are methane-producing organisms, but they do not belong to the Kingdom Bacteria but rather to the Kingdom Archeae.
Recently, molecular methods have been used in attempts to determine differences between IBS and healthy subjects. Approaches based on quantitative polymerase chain reaction (qPCR) of small parts (usually less than 100 nucleotides) of the 16S rRNA gene gave some indication of differences between a variable set of microbial groups without leading to consistent outcomes. Initial studies were done with limited microbiological and statistical power and showed that in comparison with fecal samples from healthy individuals, IBS subjects contain more Clostridium coccoides and Bifidobacterium catenulatum (Malinen et al, 2005. Am J Gastroenterol. 100:373- 82). However, in another study, 6 IBS-C subjects showed a reduced number of bacteria belonging to the Clostridium coccoides/Eubacterium rectale cluster in comparison with healthy controls (Maukonen et al, 2006. J Med Microbiol 55: 625-633). The C. coccoides/E. rectale group is the largest and most dominant bacterial group in the intestinal tract representing up to half of the total microbiota. Hence it can not as such be used in diagnostics as is also indicated by the authors of this study who note that the target C. coccoides-E. rectale group (phylogenetic clusters XlVa and XlVb) is too large to detect subtle variations between the microbiota of control and IBS subjects. Therefore, this group needs to be divided into smaller subgroups in further studies (Maukonen et al., 2006, supra). In a recent study, DNA extracted from pooled fecal samples derived from 23 healthy and 24 subjects with different IBS types was fractionated according to its guanine and cytosine (G+C) content followed by sequence analysis of 16S rDNA clone libraries (Kassinen et al., 2007. Gastroenterology 2007; 133: 24-33). While some differences were observed in 3 of the over 15 fractions, this approach is not quantitative and known to be affected by cloning bias. Moreover, the used approach includes a density gradient centrifugation step to fractionate the DNA samples according to their G+C content that is not applicable for routine diagnostics. However, in the same study also specific qPCRs were performed that showed statistically significant but only slightly larger and highly variable numbers of CoUinsella aerofaciens, Clostridium cocleatum-related and Coprococcus eutactus- related bacteria as compared to samples from healthy controls (Kassinen et al., 2007, supra). This study also indicated that differences for other members of Firmicutes remained statistically non-significant. CoUinsella aerofaciens belongs to the Actinobacteria, Gram-positive bacteria with a high G+C content. The other two groups are part of the Firmicutes, Gram-positive bacteria with a low G+C content and Clostridium cocleatum-related bacteria constitute a small group in the Clostridium cluster XVIII while Coprococcus eutactus-related bacteria form a minor group in the Clostridium coccoides/Eubacterium rectale (Clostridium cluster XlVa) cluster, including also Eubacterium ruminantium and several not yet cultured phylotypes (see Table 3).
In conclusion, the qPCR approaches provided no clear signature of IBS dysbiosis and it has been stated recently that the results reported so far are conflicting and likely explained by variations in experimental design (Codling et al, Dig Dis Sci 2010 Feb; 55(2):392-397). Moreover, these conflicting results can also be caused by the heterogeneity of IBS with respect to etiology, pathophysiology and symptomatology. Indeed, in many cases only a limited number of intestinal samples from IBS and healthy subjects is analyzed and in some cases these are derived from the same study (Malinen et al, 2005, supra; Matto et al., 2005. FEMS Immunol Med Microbiol 43: 213-222; Maukonen et al, 2006, supra; Kassinen et al., 2007, supra). Moreover, in some cases only a specific subtype of IBS is addressed or samples are pooled prior to analysis which precludes analysis of variations. In a recent study specific groups of bacteria were enumerated using fluorescent in situ hybridization (FISH) with specific 16S rR A gene probes or qPCR analysis of part of the 16S rR A gene (Kerckhoffs et al, 2009. World J Gastroenterol 2009 June 21 ; 15(23): 2887- 2892). A lower number of Bifidobacteria and no other differences in the major intestinal groups was found in 41 IBS subjects as compared to healthy controls - this included the C.coccoides/E. rectale {Clostridium cluster XIV a) cluster that showed no differences. However, careful analysis of the reported data shows that the lower number of Bifidobacteria was restricted to only the 14 IBS-D subjects and specifically included the Bifidobacterium catenulatum group. These results were corroborated with brush samples from duodenal mucosa, indicating that fecal samples constitute useful material for assessing the state of the microbiota in the gastro -intestinal tract.
The highest number of IBS subjects analysed in a single comparative study reported so far is a recent comparison that included 47 IBS and 33 healthy subjects (Codling et al, 2009, supra). By using a rather qualitative method revealing sequence variations in 16S rRNA genes, ie separating 16S rRNA gene amplicons by Denaturing Gradient Gel Electrophoresis (DGGE), global differences were observed between fecal samples from IBS subjects and healthy controls (Codling et al, 2009, supra). This study supported the possibility to differentiate between IBS and healthy subjects but failed to reveal any specific microbial group or species that could be associated with this difference.
A limited number of studies addressed the dynamics over time of the fecal microbiota in IBS subjects in comparison with that of healthy individuals. A study based on DGGE analysis suggested reduced temporal stability in IBS subjects but used visual inspection and did not correct for the use of antibiotics (Matto et al., 2005, supra). A follow up study with the appropriate corrections for the use of antibiotics showed that for periods of 3 months in 16 IBS subjects compared to 16 matched healthy subjects, the temporal stability of the Clostridium histolyticum group (also known as Clostridium cluster I and II) was higher in the IBS-c type than in the healthy subjects (Maukonen et al. 2006, supra). The methods of DGGE analysis due to their low resolution however lead to inconsistent results and outcomes that are notoriously difficult to reproduce. In addition, only a profile is generated without any link to taxonomic information. Moreover, as these methods can be best applied on small amplicons (around a few hundred bp) they have been only applied in addressing the sequence variation in the VI -V3 region of the 16S rRNA genes. Finally, the methods based on DGGE are laborious, time-consuming and have significant gel to gel variations and require relatively long processing times - hence they can not be used as a routine diagnostic tool. A summary of the drawbacks of the so far used methods is provided in a recent review that also indicates the need for IBS diagnostics and clinical algorithms that would identify subjects with differing causes of IBS as a way to improve the results of therapies, varying from pharmaceutical treatments to dietary, probiotics and prebiotics interventions (Parkes et al, 2008. Am J Gastroenterol 2008;103: 1557-1567).
Recently, a human-intestine specific phylogenetic microarray has been developed and validated that provides a way to provide high throughput data of the intestinal microbiota in an accurate way over a large dynamic range (Zoetendal et al, 2008, supra; Rajilic-Stojanovic et al, 2009. Environ Microbiol 11 : 1736-1743). In a preliminary study using a first version of the HITChip, 20 IBS and 20 healthy subjects were compared - apart from an increased level of Bacillus spp and reduced level of Bacteroides spp in IBS subjects that could not be specified, no other significant differences were observed between IBS and healthy subjects (M. Rajilic-Stojanovic, Diversity of the human gastro -intestinal microbiota, PhD thesis Wageningen University 2007, pp 116-134). This can be attributed to a limited number of subjects and use of a first version of the HITChip with redundant probes. In this study only significant differences between healthy subjects and subjects with subtypes of IBS, i.e. IBS-A, IBS-C, IBS-D, were observed for some bacterial groups. This limits any clinical application as a general diagnostic tool for IBS.
Hence, there is a need in the art to identify biomarkers that are indicative of IBS, preferably non-invasive biomarkers, that can be used to develop a diagnostic test for IBS. Moreover, such biomarkers indicative of IBS may be instrumental in defining IBS and/or subtyping IBS, as well in monitoring pharmaceutical responses to a therapeutic intervention. Moreover, such biomarkers may allow discovery and development of new and innovative therapeutic interventions for IBS.
Figures
The invention will be illustrated using the appended Figure, in which:
Figure 1 shows Redundancy Analysis of all HITChip datasets collected from Study 1 and Study 2, including in total 95 IBS subjects and 90 healthy controls. Figure 2 shows a decision tree for classifying IBS subjects (U) and Healthy controls (H) using hybridization to 4 probes with the indicated Probe ID. Numbers indicate number of subjects in the order H/U reflecting Healthy/IBS. Summary of the Invention
The present invention provides for a method for diagnosing and/or subtyping Irritable Bowel Syndrome (IBS) in a test sample, said method comprising the steps of: a) determining the levels of two or more bacteria which are present in statistically significantly different levels between IBS subjects and healthy subjects, said bacteria being selected from IBS-decreased bacteria and IBS-increased bacteria, said IBS- decreased bacteria being selected from bacteria belonging to the supertaxon Bacteroidetes, selected from the taxa Prevotella melaninogenica et rel, Prevotella oralis et rel, Uncultured Bacteroidetes, Tanner ella et rel, Parabacteroides distasonis et rel, AUistipes et rel, Bacteroides plebeius et rel, Bacteroides splachnicus et rel, or to the supertaxon Clostridium cluster IV, selected from the taxa Subdoligranulum variabile et rel, Faecalibacterium prausnitzii et rel, Oscillospira guiUermondii et rel, Sporobacter termitidis et rel, Ruminococcus callidus et rel, Eubacterium siraeum et rel, Anaerotruncus colihominis et rel, Clostridium cellulosi et rel, Clostridium leptum et rel, Ruminococcus bromii et rel, or to the supertaxon Clostridium cluster IX, said bacteria belonging to the taxon Phascolarctobacterium faecium et rel. ; or to the supertaxon Clostridium cluster XVI, said bacteria belonging to the taxon Eubacterium biforme et rel; or to the supertaxon Clostridium cluster XVII, said bacteria belonging to the taxon Catenibacterium mitsuokai et rel; or to the supertaxon Proteobacteria, said bacteria belonging to the taxon Xanthomonadaceae; or to the supertaxon Uncultured Clostridiales, selected from the taxa Uncultured Clostridiales I and Uncultured Clostridiales II; or to the supertaxon Uncultured Mollicutes, said bacteria belonging to the taxon Uncultured Mollicutes, and said IBS-increased bacteria being selected from bacteria belonging to the supertaxon Clostridium cluster XlVa, selected from the taxa Dorea formicigenerans et rel, Ruminococcus obeum et rel, Clostridium nexile et rel, Clostridium symbiosum et rel., Outgrouping Clostridium cluster XlVa, Ruminococcus lactaris et rel, Lachnospira pectinoschiza et rel; in a test sample; b) Comparing said level of said two or more IBS-decreased and/or IBS-increased bacteria in said test sample to a level of said two or more IBS-decreased and/or IBS-increased bacteria in a control sample; and cl) relating a decreased level of said IBS-decreased bacteria and/or an increased level of said IBS-increased bacteria in the test sample compared to the control sample to a diagnosis that the test sample is from a subject suffering from Irritable Bowel Syndrome; and/or c2) relating an increased level of said IBS-increased bacteria or a decreased level of said IBS-decreased bacteria in the test sample compared to the control sample to a diagnosis of whether the test sample is from a subject suffering from IBS-A, IBS-C, or IBS-D.
In an embodiment, step cl) is performed, whereas step c2) is not performed. In another embodiment, step c2) is performed, whereas step cl) is not performed. In yet another embodiment, both steps cl) and c2) are performed.
In an embodiment, said method is for diagnosing IBS, wherein in step a) at least the levels of two or more bacteria which are present in statistically significantly different levels between IBS subjects and healthy subjects, said bacteria being selected from IBS-decreased bacteria and IBS-increased bacteria, said IBS-decreased bacteria being selected from bacteria belonging to the supertaxon Bacteroidetes, selected from the taxa Prevotella melaninogenica et rel, Prevotella oralis et rel, Uncultured Bacteroidetes, Tannerella et rel.,; or to the supertaxon Clostridium cluster XVII, said bacteria belonging to the taxon Catenibacterium mitsuokai et rel; or to the supertaxon Proteobacteria, said bacteria belonging to the taxon Xanthomonadaceae; or to the supertaxon Uncultured Clostridiales, said bacteria belonging to the taxon Uncultured Clostridiales I; and said IBS-increased bacteria being selected from bacteria belonging to the supertaxon Clostridium cluster XlVa, selected from the taxa Dorea formicigenerans et rel, Ruminococcus obeum et rel, Clostridium nexile et rel, Clostridium symbiosum et rel, Outgrouping Clostridium cluster XlVa, Ruminococcus lactaris et rel, Lachnospira pectinoschiza et rel; in a test sample are determined.
In an embodiment, said method is for diagnosing IBS, wherein in step a) the levels of at least one IBS-increased bacteria selected from bacteria belonging to the taxa Dorea formicigenerans et rel., Ruminococcus obeum et rel., and Lachnospira pectinoschiza et rel, and the level of at least one IBS-decreased bacteria selected from bacteria belonging to the taxa Prevotella melaninogenica et rel, Prevotella oralis et rel, and Catenibacterium mitsuokai et rel, are determined.
In an embodiment, said method is for subtyping IBS-A, wherein in step a) the levels of two or more bacteria which are present in statistically significantly different levels between IBS subjects and healthy subjects, said bacteria being selected from IBS-decreased bacteria and IBS-increased bacteria, said IBS-decreased bacteria being selected from bacteria belonging to the supertaxon Bacteroidetes, selected from the taxa Uncultured Bacteroidetes, Tannerella et rel., Parabacteroides distasonis et rel., Allistipes et rel., Bacteroides plebeius et rel., Bacteroides splachnicus et rel., or to the supertaxon Clostridium cluster IV, selected from the taxa Subdoligranulum variabile et rel, Faecalibacterium prausnitzii et rel, Oscillospira guillermondii et rel, Sporobacter termitidis et rel, Ruminococcus callidus et rel, Eubacterium siraeum et rel, Anaerotruncus colihominis et rel, Clostridium cellulosi et rel, Clostridium leptum et rel, Ruminococcus bromii et rel., or to the supertaxon Clostridium cluster IX, said bacteria belonging to the taxon Phascolarctobacterium faecium et rel; or to the supertaxon Clostridium cluster XVI, said bacteria belonging to the taxon Eubacterium biforme et rel; or to the supertaxon Uncultured Clostridiales, selected from the taxa Uncultured Clostridiales I and Uncultured Clostridiales II; or to the supertaxon Uncultured MoUicutes, said bacteria belonging to the taxon Uncultured MoUicutes, and said IBS-increased bacteria being selected from bacteria belonging to the supertaxon Clostridium cluster XlVa, selected from the taxa Dorea formicigenerans et rel, Ruminococcus obeum et rel, Outgrouping Clostridium cluster XlVa, in a test sample are determined.
In a further embodiment, said method is for subtyping IBS-C, wherein in step a) at least the levels of two or more bacteria belonging to the taxa Prevotella oralis et rel, Bacteroides plebeius et rel, Clostridium stercorarium et rel, Dorea formicigenerans et rel, Clostridium nexile et rel, Catenibacterium mitsuokai et rel, or Xanthomonadaceae in a test sample are determined.
In another embodiment, said method is for subtyping IBS-D, wherein in step a) at least the levels of two or more bacteria belonging to the taxa Dorea formicigenerans et rel, Ruminococcus obeum et rel, Clostridium nexile et rel, Ruminococcus lactaris et rel, Lachnospira pectinoschiza et rel, Catenibacterium mitsuokai et rel, or the uncultured Clostridiales I in a test sample are determined.
In a preferred embodiment, in step a) of the method of the invention the levels of at least one IBS-increased bacteria and at least one IBS-decreased bacteria in said test sample are determined. In another preferred embodiment, in step a) of the method of the invention the levels of at least one IBS-increased bacteria selected from bacteria belonging to the taxa Dorea formicigenerans et rel., Ruminococcus obeum et rel., and Lachnospira pectinoschiza et rel, and the level of at least one IBS-decreased bacteria selected from bacteria belonging to the taxa Prevotella melaninogenica et rel, Prevotella oralis et rel, and Catenibacterium mitsuokai et rel, in said test sample are determined.
In yet another preferred embodiment, in step a) at least the levels of bacteria belonging to the taxa Dorea formicigenerans et rel., Ruminococcus obeum et rel., and Lachnospira pectinoschiza et rel., and the level of bacteria belonging to the taxa Prevotella melaninogenica et rel, Prevotella oralis et rel, and Catenibacterium mitsuokai et rel, in said test sample are determined.
The level of said one or more bacteria may be measured by determining the level of nucleic acid sequences, amino acid sequences and/or metabolites specific for said one or more bacteria, preferably the level of nucleic acid sequences specific for said one or more bacteria, e.g. 16S rRNA gene sequences or unique genomic sequences of said one or more bacteria.
In an embodiment, the level of said 16S rRNA gene sequences of said one or more bacteria is measured by determining one or more variable regions of said 16S rRNA gene sequences, e.g., one or more of the variable regions VI and/or V6 of said 16S rRNA gene sequences.
In a suitable embodiment, the levels of nucleic acid sequences specific for said two or more bacteria are determined using PCR or LCR.
The present invention is also directed to a method for diagnosing and/or subtyping Irritable Bowel Syndrome (IBS) in a test sample, said method comprising the steps of: i) providing a test sample; ii) determining the level of at least three nucleic acids capable of hybridising to at least three nucleic acid sequences selected from the nucleic acid sequences of SEQ ID Nos: 1-100, or derivatives or fragments thereof deviating by at most 2 nucleotides, and complements, reverse, and reverse complements thereof, under stringent hybridization conditions, in said test sample; ii) comparing the level of said at least three nucleic acids from said test sample to the level of said at least three nucleic acids from a control sample; and iiia) relating the level of said at least three nucleic acids from said test sample to a diagnosis of whether the test sample is from a subject suffering from Irritable Bowel Syndrome; and/or iiib) relating the level of said at least three nucleic acids from said test sample to a diagnosis of whether the test sample is from a subject suffering from IBS-A, IBS-C, or IBS-D.
In a further aspect, the present invention pertains to a method for diagnosing and/or subtyping Irritable Bowel Syndrome (IBS) in a test sample, said method comprising the steps of: i) providing a test sample; ii) determining the level of at least three nucleic acids capable of hybridising to 16S rRNA nucleic acid sequences hybridizing to the complementary strand of any of the nucleic acid sequences SEQ ID NO.: 1-100 or fragments of said 16S rRNA nucleic acid sequences hybridizing to the complementary strand of any of the nucleic acid sequences SEQ ID NO.: 1-100, and complements, reverse, and reverse complements thereof, under stringent hybridization conditions, in said test sample; ii) comparing the level of said at least three nucleic acids from said test sample to the level of said at least three nucleic acids from a control sample; and iiia) relating the level of said at least three nucleic acids from said test sample to a diagnosis of whether the test sample is from a subject suffering from Irritable Bowel Syndrome; and/or iiib) relating the level of said at least three nucleic acids from said test sample to a diagnosis of whether the test sample is from a subject suffering from IBS-A, IBS-C, or IBS-D.
In an embodiment, in step iiia) an increased level of nucleic acids from said test sample, said nucleic acids being capable of hybridising to nucleic acid sequences selected from the nucleic acid sequences of SEQ ID Nos: l-27, 70-71, 73-77, 99-100, or derivatives or fragments thereof deviating by at most 2 nucleotides, and complements, reverse, and reverse complements thereof, under stringent hybridization conditions, compared to the level of said nucleic acids from said control sample relates to the diagnosis that the subject is suffering from IBS.
In another embodiment, in step iiia) a decreased level of nucleic acids from said test sample, said nucleic acids being capable of hybridising to nucleic acid sequences selected from the nucleic acid sequences of SEQ ID Nos:28-69, 72,78-98, or derivatives or fragments thereof deviating by at most 2 nucleotides, and complements, reverse, and reverse complements thereof, under stringent hybridization conditions, compared to the level of said nucleic acids from said control sample relates to the diagnosis that the subject is suffering from IBS.
In an embodiment, the level of at least 6 nucleic acid sequences from said test sample is determined. Significance Analysis of Microarrays (SAM) may be used in comparing the levels of said three or more nucleic acid sequence from said test sample with the levels of said three or more nucleic acid sequence from a control sample. Alternatively, Prediction Analysis of Microarray (PAM) may be used in comparing the levels of said three or more nucleic acid sequence from said test sample with the levels of said three or more nucleic acid sequence from a control sample. In another embodiment, Redundancy Analysis is used in comparing the levels of said three or more nucleic acid sequence from said test sample with the levels of said three or more nucleic acid sequence from a control sample.
In an embodiment, the level is determined using a method selected from: hybridization of the nucleic acids in a sample to the nucleic acid sequences having SEQ ID NO.: 1-100, and complements, reverse, and reverse complements thereof, under stringent hybridization conditions; a Polymerase Chain reaction (PCR) or a Ligase Chain Reaction (LCR).
In another aspect, the present invention relates to an array for diagnosing IBS and/or subtyping IBS-A, IBS-C, or IBS-D, said array comprising at least two nucleic acid sequences specifically hybridize to one or more of SEQ ID NOs: 1-100, or derivatives or fragments thereof deviating by at most 2 nucleotides, and complements, reverse, and reverse complements thereof. Said array may comprise at least two nucleic acid sequences selected from the nucleic acid sequences having SEQ ID Nos: 1-100. The at least two nucleic acid sequences may be bound to a solid phase matrix. The array may be a DNA or RNA array, and may be a micro-array.
In a further aspect, the present invention is concerned with use of an array of the present invention for diagnosing IBS and/or subtyping IBS-A, IBS-C, or IBS-D. Detailed Description of the Invention
In the present invention, in a first study a detailed comparison was made between the microbiota of 62 subjects suffering from IBS (defined according to Rome II or III criteria) and 46 healthy subjects. In a second study, a detailed comparison was made between a further 33 IBS subjects and 43 healthy subjects. It has been demonstrated that based on HITChip profiling of DNA extracted from intestinal samples, a distinction can be made between healthy subjects and subjects suffering from IBS (hereinafter also referred to as "IBS subjects"). Subsequently, a detailed comparison was made between the HITChip data from healthy subjects and subjects suffering from IBS using Redundancy Analysis (RDA). This revealed significant differences between healthy subjects and subjects suffering from IBS. These results with a large group of over 150 human subjects, for the first time provided evidence for the use of microbiota to differentiate between healthy subjects and subjects suffering from IBS. Hence, advanced comparisons were made between the HITChip data of healthy subjects and subjects suffering from IBS resulting in the identification of a series of microbial taxa (phylotype-like and genus-like groups) that can be used to differentiate IBS and healthy subjects. Moreover, detailed analysis of the HIT probes showed that a set of 100 HIT probes of each 16-30 nucleotides were found to be significantly different and hybridized to a higher (27) or lower (40) extent in the IBS subjects than in the healthy subjects.
Thus, the present invention relates to a method for diagnosing and/or subtyping Irritable Bowel Syndrome (IBS) in a test sample, said method comprising the steps of: a) determining the levels of two or more bacteria which are present in statistically significantly different levels between IBS subjects and healthy subjects, said bacteria being selected from IBS-decreased bacteria and IBS-increased bacteria, said IBS- decreased bacteria being selected from bacteria belonging to the supertaxon Bacteroidetes, selected from the taxa Prevotella melaninogenica et rel, Prevotella oralis et rel, Uncultured Bacteroidetes, Tanner ella et rel, Parabacteroides distasonis et rel, AUistipes et rel, Bacteroides plebeius et rel, Bacteroides splachnicus et rel., or to the supertaxon Clostridium cluster IV, selected from the taxa Subdoligranulum variabile et rel, Faecalibacterium prausnitzii et rel, Oscillospira guiUermondii et rel, Sporobacter termitidis et rel, Ruminococcus callidus et rel, Eubacterium siraeum et rel, Anaerotruncus colihominis et rel, Clostridium cellulosi et rel, Clostridium leptum et rel, Ruminococcus bromii et rel., or to the supertaxon Clostridium cluster IX, said bacteria belonging to the taxon Phascolarctobacterium faecium et rel. ; or to the supertaxon Clostridium cluster XVI, said bacteria belonging to the taxon Eubacterium biforme et rel; or to the supertaxon Clostridium cluster XVII, said bacteria belonging to the taxon Catenibacterium mitsuokai et rel; or to the supertaxon Proteobacteria, said bacteria belonging to the taxon Xanthomonadaceae; or to the supertaxon Uncultured Clostridiales, selected from the taxa Uncultured Clostridiales I and Uncultured Clostridiales II; or to the supertaxon Uncultured Mollicutes, said bacteria belonging to the taxon Uncultured Mollicutes, and said IBS-increased bacteria being selected from bacteria belonging to the supertaxon Clostridium cluster XlVa, selected from the taxa Dorea formicigenerans et rel., Ruminococcus obeum et rel., Clostridium nexile et rel., Clostridium symbiosum et rel., Outgrouping Clostridium cluster XlVa, Ruminococcus lactaris et rel., Lachnospira pectinoschiza et rel.; in a test sample; b) Comparing said level of said two or more IBS-decreased and/or IBS-increased bacteria in said test sample to a level of said two or more IBS-decreased and/or IBS-increased bacteria in a control sample; and cl) relating a decreased level of said IBS-decreased bacteria and/or an increased level of said IBS-increased bacteria in the test sample compared to the control sample to a diagnosis that the test sample is from a subject suffering from Irritable Bowel Syndrome; and/or c2) relating an increased level of said IBS-increased bacteria or a decreased level of said IBS-decreased bacteria in the test sample compared to the control sample to a diagnosis of whether the test sample is from a subject suffering from IBS-A, IBS-C, or IBS-D.
As used herein, the term "IBS-increased bacteria" refers to bacteria that are statistically significantly present more abundantly in IBS subjects compared to healthy subjects. The term "IBS-decreased bacteria" as used herein refers to bacteria that are statistically significantly present more abundantly in healthy subjects compared to IBS subjects. IBS-increased bacteria as used herein encompass, without limitation, bacteria belonging to the supertaxon Clostridium cluster XlVa, selected from the taxa Dorea formicigenerans et rel., Ruminococcus obeum et rel., Clostridium nexile et rel., Clostridium symbiosum et rel, Outgrouping Clostridium cluster XlVa, Ruminococcus lactaris et rel, Lachnospira pectinoschiza et rel, Ruminococcus gnavus et rel. IBS- decreased bacteria as used herein encompass, without limitation, bacteria belonging to the supertaxon Bacteroidetes, selected from the taxa Prevotella melaninogenica et rel, Prevotella oralis et rel, Uncultured Bacteroidetes, Tannerella et rel, Parabacteroides distasonis et rel, Allistipes et rel, Bacteroides plebeius et rel, Bacteroides splachnicus et rel, Bacteroides uniformis et rel, Clostridium stercorarium et rel.., or to the supertaxon Clostridium cluster IV, selected from the taxa Subdoligranulum variabile et rel, Faecalibacterium prausnitzii et rel, Oscillospira guillermondii et rel, Sporobacter termitidis et rel, Ruminococcus callidus et rel, Eubacterium siraeum et rel., Anaerotruncus colihominis et rel, Clostridium cellulosi et rel, Clostridium leptum et rel, Ruminococcus bromii et rel, or to the supertaxon Clostridium cluster IX, said bacteria belonging to the taxon Phascolarctobacterium faecium et rel; or to the supertaxon Clostridium cluster XVI, said bacteria belonging to the taxon Eubacterium biforme et rel.; or to the supertaxon Clostridium cluster XVII, said bacteria belonging to the taxon Catenibacterium mitsuokai et rel.; or to the supertaxon Proteobacteria, said bacteria belonging to the taxon Xanthomonadaceae; or to the supertaxon Uncultured Clostridiales, selected from the taxa Uncultured Clostridiales I and Uncultured Clostridiales II; or to the supertaxon Uncultured Mollicutes, said bacteria belonging to the taxon Uncultured Mollicutes
It has been shown in the present study that the levels of these bacteria in an intestinal sample from IBS subjects differ significantly from levels of these bacteria in an intestinal sample from healthy individuals (Table 1 below shows the ratio of the level of the bacteria in healthy subjects over IBS subjects; the grey background indicates bacteria for which the levels are statistically significantly different between IBS subjects and healthy subjects (p<0.05)).
In an embodiment, the level of one or more bacteria belonging to the taxa Ruminococcus gnavus et rel. , Bacteroides uniformis et rel, and Clostridium stercorarium et rel. are further determined.
In step a), the level of one or more bacteria belonging to the taxa Ruminococcus gnavus et rel., Dorea formicigenerans et rel., Ruminococcus obeum et rel., Clostridium nexile et rel., Clostridium symbiosum et rel., Outgrouping Clostridium cluster XlVa, Prevotella oralis et rel., Prevotella melaninogenica et rel., Uncultured Bacteroidetes, Parabacteroides distasonis et rel, Allistipes et rel. Subdoligranulum variabile et rel, Faecalibacterium prauznitzii et rel, Sporobacter termitidis et rel, Ruminococcus callidus et rel, Eubacterium biforme et rel, Eubacterium sireaum et rel, Oscillospira guillermondii et rel, the uncultured Clostridiales I and II, Tannerella et rel, Bacteroides plebeius et rel, Bacteroides splachnicus et rel, Bacteroides uniformis et rel, Clostridium stercorarium et rel, Anaerotruncus colihominis et rel, Clostridium cellulosi et rel, Clostridium leptum et rel, Ruminococcus bromii et rel, Phascolarctobacterium faecium et rel, Ruminococcus lactaris et rel, Lachnospira pectinoschiza et rel, Catenibacterium mitsuokai et rel, Xanthomonadaceae, or Uncultured Mollicutes in a test sample is determined.
The term "test sample" as used herein refers to an intestinal sample. Intestinal samples refer to all samples that originate from the intestinal tract, including, without limitation, feces samples, rectal swap samples, but also samples obtained from other sites in the intestinal tract, such as mucosal biopsies, as was shown previously (Zoetendal et al 2002 . Appl. Environ. Microbiol. 68:3401-7 and Kerkhoffs et al, 2009, supra). A test sample may be obtained from an IBS subject, from a healthy individual, from a subject with unknown diagnosis of IBS, or from a person with complaints related to the gastro-intenstinal tract. In case of subtyping of IBS, a test sample may be obtained from a subject known to suffer from IBS, or may be from a a subject with unknown diagnosis of IBS. The test sample may have been processed; for example, DNA and/or RNA may have been isolated from feces samples, rectal swap samples, or samples obtained from other sites in the intestinal tract. Preferably, mRNA is isolated from feces samples, rectal swap samples, or samples obtained from other sites in the intestinal tract to provide a test sample comprising mRNA.
The level of said one or more bacteria may be determined using any method known in the art. Such method includes, without limitation, hybridization, and amplification reactions such as polymerase chain reaction (PCR) and ligase chain reaction (LCR).
For clinical diagnostics the use of nucleic acid arrays is highly advantageous as it couples accuracy and speed to quantitative analysis. Nucleic acid arrays are ordered sequences of DNA or RNA that can be used to selectively isolate and later on quantify specific nucleic acid sequences in complex mixtures - by changing the hybridization and washing conditions the specificity of the detected nucleic acid duplexes can be modulated.
The oligonucleotide sequences used to detect a target sequence, whether on nucleic acid arrays or in solution, will be referred to hereinbelow as a "probe".
Suitable hybridisation conditions (i.e. buffers used, salt strength, temperature, duration) can be selected by the skilled person, on the basis of experience or optionally after some preliminary experiments. These conditions may vary, depending on factors such the size of the probes, the G+C-content of the probes and whether the probes are bound to an array as described below.
Suitable hybridisation conditions are for instance described in Sambrook et al, Molecular Cloning: A Laboratory manual, (1989) 2nd. Ed. Cold Spring Harbour, N.Y.; Berger and Kimmel, "Guide to Molecular Cloning Techniques", Methods in Enzymology", (1987), Volume 152, Academic Press Inc., San Diego, CA; Young and Davis (1983) Proc. Natl. Acad. Sci. flJSA) 80: 1194; Laboratory Techniques in Biochemistry and Molecular Biology, Vol.24, Hybridization with Nucleic Acid Probes, P. Thijssen, ed., Elsevier, N.Y. (1993).
The hybridisation conditions are preferably chosen such that each probe will only form a hybrid (duplex) with a target sequence with which the probe is essentially complementary, if such a target sequence is present, and otherwise will not form any hybrid. The term "essentially complementary" as used herein does not mean that the complementarity of a probe to a target sequence such as the 16S rRNA gene should be perfect, and mismatches up to 2 nucleotides can be envisaged.
Each probe should at least in part be complementary to a specific target sequence. The probe may be any nucleic acid (i.e. DNA or RNA) but is preferably DNA. The probe will generally have a size of about 10 to 100 base pairs, preferably about 10 to 40 base pairs. The probes may all be of the same size, or may be of different sizes. The probes can be obtained in any suitable manner. For example, knowing the 16S RNA gene sequences of the bacteria identified herein, probes may be synthesized that are complementary to any part of the sequence of such 16S RNA gene sequence, i.e. using an automated DNA-synthesizer or in any other manner known per se. Also, solid phase nucleic acid synthesis techniques may be used, which may result directly in an array with the desired probes. Furthermore, the probes may be obtained using techniques of genetic engineering, for instance by primer extension using the target sequence as a template, and/or by using one or more restriction enzymes, optionally using amplification.
Also, the probes may comprise one or more "alternative nucleosides". Examples thereof include the bases Inosine (I) and Uracil (U), as well as dUTP and dITP, and these are included within the term "labeled nucleotide analog". It is to be understood that the presence of such alternative nucleosides does not prevent the probe and its target sequence to be essentially complementary to one another as defined above.
Quantitative nucleic acid-based amplification reactions may also be used to detect and quantify specific nucleic acid sequences in complex mixtures as in the present invention. These include the well known Polymerase Chain Reaction (PCR) and Ligase Chain Reaction (LCR) and modifications thereof (see McPherson & Moller, 2006. PCR, second edition. Taylor & Francis Group; Wiedman et al, 1994. PCR Meth Appl; 3:S51-S64). LCR is a method of DNA amplification similar to PCR but differs from PCR because it amplifies the probe molecule rather than producing amplicons through polymerization of nucleotides. Two probes are used per each DNA strand and are ligated together to form a single polynucleotide. LCR uses both a DNA polymerase enzyme and a DNA ligase enzyme to drive the reaction. In a specific application of LCR, the resulting polynucleotide can be amplified by PCR and analysed separately or, notably when in multiplex samples, hybridized to arrays.
The target for DNA arrays and quantitative nucleic acid-based amplification reactions such as PCR or LCR are nucleic acids, so DNA or RNA. Such nucleic acids include, without limitation, the 16S RNA gene as well as the 16S rRNA itself, directly or after conversion into DNA via the reverse transcriptase reaction. However, also other nucleic acid sequences can be used provided they are sufficiently different and diagnostic between IBS subjects and healthy individuals. These may include DNA sequences, both coding and non-coding, in the genomes of specific microbes that differ in prevalence between healthy and IBS subjects. Comparative genome or transciptome analysis may be a useful tool to identify such DNA sequences.
In the invention described here specific nucleic acid sequences are identified in intestinal microbiota that can be used to discriminate IBS subjects from healthy individuals, allowing IBS subjects to be diagnosed. Numerous nucleic acid isolation methods are available that differ in their approach that includes mechanical or enzymatic lysis and specific purification methods. While all these methods are applicable to intestinal samples, the repeated bead beating method as described by Yu & Morrison (2004. BioTechniques 36:808-812) is among the most efficient ones while enzymatic methods such as those described recently by Ahroos & Tynkynnen (2009. J. Appl. Microbiol. 106:506-514) can be used in combination with automated methods. All methods introduce specific biases but for comparative purposes all methods can be used if used consistently. The obtained nucleic acids may be used as template for PCR or LCR and/or hybridization reactions described above, e.g. using nucleic acid arrays.
The addition "et rel." behind the genus-like group name (level 2 group name) stands for et relatives, indicating all relatives of this phylogenetic group, i.e., those indicated in Table 3, in the column headed "level 3". This information, including the indicated 16S rRNA gene sequences, can be used to develop specific PCR primers or LCR probes to detect the one or more members of these groups. In some literature the addition "et rel." is replaced by "-like" to indicate the fact that the group includes more than one related species. However, this is a rather ambiguous designation and hence all terms with "et rel." are cleary defined in Table 3, which has been published by Rajilic- Stojaniovic et al. 2009 vide supra. Moreover, the sequences of the probes provided in Tables 2 and 4 can also be used to identify in the 16S rRNA databases all complete or partial 16S rRNA gene sequences that give a match, either completely or even partially. In this way a catalogue of 16S rRNA gene sequences can be obtained that can be used as targets for the development of specific PCR primers or LCR probes to detect these.
In step b) of the method of the present invention, the level of said one or more bacteria in said test sample is compared to a level of said one or more bacteria in a control sample. The control sample may advantageously be derived from a healthy subject, and is preferably treated in the same way as is the test sample. Thus, preferably the control sample is sampled in the same way as is the test sample, if applicable, nucleic acid is isolated in the same way as is the test sample, and, if applicable, hybridization or quantitative amplification is performed under the same conditions to allow a fair comparison of the test sample and control sample. It is not necessary to determine the level of said one or more bacteria in a control sample each time a test sample is measured; once the level of said one or more bacteria is reliably determined in a control sample, the level values may be stored, e.g., in a computer, and used for the comparative purposes herein set forth.
The level of said one or more bacteria in a test sample is compared to the same bacteria in a control sample, for example, the level of Ruminococcus obeum et rel. in a test sample is compared to the level of Ruminococcus obeum et rel. in a control sample, the level of Bacteroides splachnicus et rel. in a test sample is compared to the level of Bacteroides splachnicus et rel. in a control sample, and the like.
In step cl) of the method of the present invention, an increased level of IBS- increased bacteria and/or a decreased level of IBS-decreased bacteria is related to a diagnosis that the test sample is from a subject suffering from Irritable Bowel Syndrome.
In step c2) of the method of the present invention, an increased level of IBS- increased bacteria and/or a decreased level of IBS-decreased bacteria is related to a diagnosis of whether the test sample is from a subject suffering from IBS-A, IBS-C, or IBS-D.
As used herein, the level of one or more bacteria in a test sample is increased when it is significantly higher than the level of said one or more bacteria in a control sample. It is also considered increased when the level of one or more bacteria in the test sample is at least 5%, such as 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% higher than the corresponding one or more bacteria in the control sample.
As used herein, the level of one or more bacteria in a test sample is decreased when it is significantly lower than the level of said one or more bacteria in a control sample. It is also considered decreased when the level of one or more bacteria in the test sample is at least 5%, such as 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% lower than the corresponding one or more bacteria in the control sample.
In an embodiment, step cl) is performed, whereas step c2) is not performed. In another embodiment, step c2) is performed, whereas step cl) is not performed. In yet another embodiment, both steps cl ) and c2) are performed. For test samples of unknown origin, i.e. of which it is not known whether it is from an IBS subject or from a healthy individual, steps a), b) and cl) may be performed to diagnose IBS. In such case, it may be advantageous to perform both steps cl) and c2) to simultaneously diagnose and subtype IBS. For test samples obtained from an IBS subject, it may be sufficient to perform steps a), b), and c2) in order to subtype the IBS.
In an embodiment, said method is for diagnosing IBS, wherein in step a) at least the levels of two or more bacteria which are present in statistically significantly different levels between IBS subjects and healthy subjects, said bacteria being selected from IBS-decreased bacteria and IBS-increased bacteria, said IBS-decreased bacteria being selected from bacteria belonging to the supertaxon Bacteroidetes, selected from the taxa Prevotella melaninogenica et rel, Prevotella oralis et rel, Uncultured Bacteroidetes, Tannerella et rel.,; or to the supertaxon Clostridium cluster XVII, said bacteria belonging to the taxon Catenibacterium mitsuokai et rel; or to the supertaxon Proteobacteria, said bacteria belonging to the taxon Xanthomonadaceae; or to the supertaxon Uncultured Clostridiales, said bacteria belonging to the taxon Uncultured Clostridiales I; and said IBS-increased bacteria being selected from bacteria belonging to the supertaxon Clostridium cluster XlVa, selected from the taxa Dorea formicigenerans et rel, Ruminococcus obeum et rel, Clostridium nexile et rel, Clostridium symbiosum et rel, Outgrouping Clostridium cluster XlVa, Ruminococcus lactaris et rel, Lachnospira pectinoschiza et rel; in a test sample are determined.
In an embodiment, said method is for diagnosing IBS, wherein in step a) the levels of at least one IBS-increased bacteria selected from bacteria belonging to the taxa Dorea formicigenerans et rel., Ruminococcus obeum et rel., and Lachnospira pectinoschiza et rel, and the level of at least one IBS-decreased bacteria selected from bacteria belonging to the taxa Prevotella melaninogenica et rel, Prevotella oralis et rel., and Catenibacterium mitsuokai et rel., are determined.
In an embodiment, said method is for subtyping IBS-A, wherein in step a) the levels of two or more bacteria which are present in statistically significantly different levels between IBS subjects and healthy subjects, said bacteria being selected from IBS-decreased bacteria and IBS-increased bacteria, said IBS-decreased bacteria being selected from bacteria belonging to the supertaxon Bacteroidetes, selected from the taxa Uncultured Bacteroidetes, Tanner ella et rel, Parabacteroides distasonis et rel, Allistipes et rel, Bacteroides plebeius et rel, Bacteroides splachnicus et rel., or to the supertaxon Clostridium cluster IV, selected from the taxa Subdoligranulum variabile et rel, Faecalibacterium prausnitzii et rel, Oscillospira guillermondii et rel, Sporobacter termitidis et rel, Ruminococcus callidus et rel, Eubacterium siraeum et rel, Anaerotruncus colihominis et rel, Clostridium cellulosi et rel, Clostridium leptum et rel, Ruminococcus bromii et rel., or to the supertaxon Clostridium cluster IX, said bacteria belonging to the taxon Phascolarctobacterium faecium et rel; or to the supertaxon Clostridium cluster XVI, said bacteria belonging to the taxon Eubacterium biforme et rel.; or to the supertaxon Uncultured Clostridiales, selected from the taxa Uncultured Clostridiales I and Uncultured Clostridiales II; or to the supertaxon Uncultured Mollicutes, said bacteria belonging to the taxon Uncultured Mollicutes, and said IBS-increased bacteria being selected from bacteria belonging to the supertaxon Clostridium cluster XlVa, selected from the taxa Dorea formicigenerans et rel, Ruminococcus obeum et rel, Outgrouping Clostridium cluster XlVa, in a test sample are determined.
In another embodiment, said method is for subtyping IBS-A, wherein in step a) the levels of two or more bacteria which are present in statistically significantly different levels between IBS subjects and healthy subjects, said bacteria being selected from IBS-decreased bacteria and IBS-increased bacteria, said IBS-decreased bacteria being selected from bacteria belonging to the supertaxon Bacteroidetes, selected from the taxa Parabacteroides distasonis et rel, Allistipes et rel, Bacteroides splachnicus et rel . , or to the supertaxon Clostridium cluster IV, selected from the taxa Subdoligranulum variabile et rel, Faecalibacterium prausnitzii et rel, Oscillospira guillermondii et rel, Sporobacter termitidis et rel, Ruminococcus callidus et rel, Eubacterium siraeum et rel., Anaerotruncus colihominis et rel., Clostridium cellulosi et rel., Clostridium leptum et rel., Ruminococcus bromii et rel., or to the supertaxon Clostridium cluster IX, said bacteria belonging to the taxon Phascolarctobacterium faecium et rel; or to the supertaxon Clostridium cluster XVI, said bacteria belonging to the taxon Eubacterium biforme et rel.; or to the supertaxon Uncultured Clostridiales, selected from the taxa Uncultured Clostridiales I and Uncultured Clostridiales Ilin a test sample are determined.
The bacteria belonging to these taxa are unique for IBS-A subtyping.
In a further embodiment, said method is for subtyping IBS-C, wherein in step a) at least the levels of two or more bacteria belonging to the taxa Prevotella oralis et rel., Bacteroides plebeius et rel, Dorea formicigenerans et rel, Clostridium nexile et rel, Catenibacterium mitsuokai et rel, or Xanthomonadaceae in a test sample are determined.
In another embodiment, said method is for subtyping IBS-D, wherein in step a) at least the levels of two or more bacteria belonging to the taxa Dorea formicigenerans et rel, Ruminococcus obeum et rel, Clostridium nexile et rel, Ruminococcus lactaris et rel, Lachnospira pectinoschiza et rel, Catenibacterium mitsuokai et rel, or the uncultured Clostridiales I in a test sample are determined.
It is preferred that the levels of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or more bacteria which are present in statistically significantly different levels between IBS subjects and healthy subjects, said bacteria being selected from IBS-decreased bacteria and IBS-increased bacteria, said IBS-decreased bacteria as defined hereinabove are determined to allow an even more reliable diagnosis of IBS and/or subtyping of IBS-A, IBS-C and/or IBS-D. Furthermore, any other statistical operation to the levels of said microbial groups available to persons skilled in the art also may allow for a more reliable diagnosis of IBS.
The level of said one or more bacteria may be measured by determining the levels of nucleic acid sequences, amino acid sequence and/or metabolites specific for said one or more bacteria, preferably the level of nucleic acid sequences specific for said one or more bacteria.
One of the most researched microbial nucleic acids is that of the 16S rRNA. This 16S rRNA, also known as small subunit (SSU) RNA, is encoded by an approximately 1500 bp gene that is present in a variable number of copies, usually 1-10 per microbial genome. The nucleotide sequence of the 16S rRNA genes is frequently used in diagnostics as it shows differences between microbial species. In fact 16S rRNA gene sequences are instrumental in defining the taxonomic position of microbes. Moreover, these 16S rRNA sequences may also identify microbes that have not yet been cultured but are only known because of the presence of a 16S rRNA gene sequence. In case this gene sequence differs significantly (usually less than 98 % similarity) from the 16S rRNA gene sequence of a known species, this is indicated as a new phylotype (a microbe that has not been cultured yet). However, a growing number of microbes are brought into culture and otherwise described by sequence analysis of their complete or partial genomes. Up to now over several thousands of microbial genomes have been sequenced and are publicly available (see http://genomesonline.org or http://www.ncbi.nlm.nih.gov). Many more are to follow either after their isolation or from metagenome projects that aim to sequence the entire microbial DNA present in an ecosystem, such as Human Microbiome Project aiming to determine the metagenome of the human microbiota (see http://nihroadmap.nih.gov/hmp/).
A growing database of over a million microbial 16S rRNA sequences can be found in publicly available databases such as http://www.arb-silva.de (Pruesse et al, 2007. Nucleic Acid Res. 35 :7188) and http://rdp.cmu.mse.edu (Cole et al, 2008. Nucleic Acids Res. 35 (Database issue): D169-D172). It has been well-established that the 16S rRNA sequence contains a limited number of variable regions of several dozens of nucleotides, termed V1-V8, that are targets for developing nucleic acid probes, PCR primers or LCR probes. By analyzing the variable regions in the microbes that are found in the human intestinal tract, it was observed that the most diagnostic information for developing nucleic acid probes were the VI and V6 regions (Rajilic- Stojanovic et al, 2009, supra). Hence, based on the sequences of these variable regions a total of over 3,699 unique oligonucleotide probes of around 16-30 nucleotides have been developed that are present on the so called Human Intestinal Tract (HIT) Chip, a phylogenetic microarray (Rajilic-Stojanovic et al 2009, supra). These oligonucleotides are called HIT probes. Hybridization to the HIT probes can be used to deduce what microbe is present and allows its taxonomic identification at different level, the most important ones including genus-like groups (sequence similarity > 90% - so called level 2 groups) and phylotype-like groups (sequence similarity > 98% - so called level 3 groups) (Rajilic-Stojanovic et al 2009, supra). Table 3 defines the identified groupings even when the systematic names of the involved bacterial species is changing due to advanced taxonomic insight.
"Percentages (%) sequence identity" refers to the percentage identical nucleotides between two sequences and can be determined using for example pairwise local alignment tools such as the program "water" of EmbossWIN (version 2.10.0) using default parameters, (gap opening penalty 10.0 and gap extension penalty 0.5, using Blosum62 for proteins and DNAFULL matrices for nucleic acids) or "Bestfit" of GCG Wisconsin Package, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, using default parameters. Alternatively, BLAST analysis using default settings may also be used, such as nucleotide Blast of NCIMB, with a gap creation penalty 11 and gap extension penalty 1.
Thus, the level of said one or more bacteria is preferably measured by determining the level of specific nucleic acid sequences in said test sample, which nucleic acid sequences are preferably 16S rRNA gene sequences of said one or more bacteria, more preferably one or more variable regions of said 16S rRNA gene sequences, e.g., one or more of the variable regions VI and/or V6 of said 16S rRNA gene sequences.
The disclosed microbial groups as well as the differentiating oligonucleotide probes can serve alone or in combination as biomarkers for IBS subjects. A biomarker, or biological marker, is in general a substance used as an indicator of a biologic state. Biomarkers can include a variety of stable macromolecular molecules, including nucleic acids, proteins or lipids but also metabolites or a combination thereof. Of particular interest are nucleic acids, including DNA and RNA, that are present in the intestinal microbiota as they are stable but can be isolated easily. However, also proteins encoded by the said DNA can be considered useful biomarkers, notably when they are stable.
Starting from the microbial groups, bacteria and probes described herein, persons skilled in the art can deduce LCR, PCR or hybridization probes to specifically discriminate IBS subjects from healthy subjects using intestinal microbiota as target. In some cases even discriminatory microbial groups are identified that are specifically affected in one or more specific types of IBS. Affected in this context means either more or less prevalent in IBS subjects, allowing for biomarker development for specific IBS-subtypes such as IBS-C, IBS-A and IBS-D.
The identification of the microbial groups that are specifically affected also allows new classification of IBS and its subsequent therapy. This therapy may consist of the consumption of correcting microbes, conforming to the definition of probiotics (see http://www.isapp.net/). In addition, consumption of prebiotics can be envisaged that affect the microbial composition (http://www.isapp.net/). Finally, pharmaceutical preparations can be envisaged that affect the microbiota in such a way that the identified defects are corrected. Here 'defects' are defined as 'deviating from healthy subjects with regard to gastro-intestinal microbiota'.
It is evident that the present diagnosis of IBS should be improved and analysis of the gut microbiota is an important diagnostic tool. However, the classification of IBS into the IBS-C, IBS-D and IBS-A types according to the Rome criteria is mainly based on form and frequency of stool samples and hence subjective, undefined and biased (Thompson et al., 1989. Gastroenterol Int 2:92-95; Longstreth et al., 2006, supra; Thompson, 2006. Gastroenterology 130: 1552-1556). The traditional classification of IBS subjects based on the Rome criteria does not provide a solid basis for therapy and this hampers treatment of the IBS subjects.
Based on the microbiota analysis and detection of the identified oligonucleotides specific for IBS (probes having SEQ ID Nos: l-27, 70-71, 73-77, 99-100) and Healthy subjects (probes having SEQ ID Nos:28- 69, 72, 78-98) (see Tables 2 and 4) of the invention new, rational and unbiased differentiation of the IBS subjects can be realized. It is envisaged that this results in classifications that are useful in combination with specific treatments and thus improving the efficacy of therapies. As such, the invention will allow for differentiating IBS subjects based upon the microbiota in their GI tract. Hence, the classification of IBS following microbiota analysis is a preferred embodiment of the invention. Inspection of the major differences in microbial composition in the IBS-C, IBS-D and IBS-A allows the definition of IBS subtypes based on specific microbial composition.
Starting from the present invention, it may be possible to determine the level of the bacterial taxa as described hereinabove. However, an alternative way of diagnosing and/or subtyping IBS is to use the selective hybridization probes of SEQ ID NO.: 1-100 identified herein, or complements, reverse, or reverse-complements thereof. The hybridization probes of SEQ ID NO.: 1-100 may be used as such for hybridization with nucleic acids isolated from a test sample to provide a diagnosis of IBS and/or to subtype IBS. Alternatively, probes with up to 2 nucleotide mismatches in comparison to SEQ ID NO.: 1-100, or complements, reverse, or reverse-complements thereof, may be used. Alternatively, the probes may be used to identify 16S rRNA nucleic acid sequences useful for diagnosing IBS and/or subtyping IBS. To this end, the nucleic acid sequences of SEQ ID NO.: 1-100, or derivatives or fragments thereof deviating by at most 2 nucleotides, or complements, reverse, or reverse-complements thereof, may be used to perform a search in well-known public nucleic acid sequence databases in order to identify those 16S rRNA sequences that are useful in diagnosing IBS and/or subtyping IBS. In the present case, the SILVA and RDP databases were searched for 16S rRNA gene sequences using the nucleic acid sequences of SEQ ID NO.: 1-100 allowing up to 2 mismatches from these nucleic acid sequences. This resulted in multiple hits for each of the nucleic acid sequences. It is to be understood that the 16S rRNA sequences thus identified, as well as sequences derived therefrom, may also be used to diagnose IBS and/or subtype IBS. For example, nucleic acid sequences suitable for hybridization reactions (herein also referred to as "probes") useful to diagnose IBS and/or subtype IBS may be identified starting from the 16S rRNA sequences identified using nucleic acid sequences of SEQ ID NO. : 1-100, or derivatives or fragments thereof deviating by at most 2 nucleotides, or complements, reverse, or reverse- complements thereof. Alternatively, the 16S rRNA sequences identified using nucleic acid sequences of SEQ ID NO.: 1-100, or derivatives or fragments thereof deviating by at most 2 nucleotides, or complements, reverse, or reverse-complements thereof, may be used to develop amplification primers for use in amplification reactions, e.g., for use in PCR or LCR reactions. Such amplification reactions may also be used to diagnose IBS and/or subtype IBS. Sequences which are the complement, reverse or reverse- complement of the nucleic acid sequences of SEQ ID Nos: 1-100, derivatives or fragments thereof deviating by at most 2 nucleotides, 16S rRNA sequences identified using nucleic acid sequences of SEQ ID NO.: 1-100, or derivatives or fragments thereof deviating by at most 2 nucleotides, may also be used in the methods of the invention.
The present invention is also directed to a method for diagnosing and/or subtyping Irritable Bowel Syndrome (IBS) in a test sample, said method comprising the steps of: i) providing a test sample; ii) determining the level of at least three nucleic acids capable of hybridising to at least three nucleic acid sequences selected from the nucleic acid sequences of SEQ ID Nos: l-100, or derivatives or fragments thereof deviating by at most 2 nucleotides, and complements, reverse, and reverse complements thereof, under stringent hybridization conditions, in said test sample; ii) comparing the level of said at least three nucleic acids from said test sample to the level of said at least three nucleic acids from a control sample; and iiia) relating the level of said at least three nucleic acids from said test sample to a diagnosis of whether the test sample is from a subject suffering from Irritable Bowel Syndrome; and/or iiib) relating the level of said at least three nucleic acids from said test sample to a diagnosis of whether the test sample is from a subject suffering from IBS-A, IBS-C, or IBS-D.
In an alternative method of the invention, in step i) the level of at least three nucleic acids capable of hybridising to 16S rRNA nucleic acid sequences hybridizing to the complementary strand of any of the nucleic acid sequences SEQ ID NO.: 1-100 or fragments of said 16S rRNA nucleic acid sequences hybridizing to the complementary strand of any of the nucleic acid sequences SEQ ID NO.: 1-100, and complements, reverse, and reverse complements thereof, under stringent hybridization conditions, in said test sample, is determined.
The term "level" as used in combination with nucleic acids or nucleic acid sequences may refer to expression level as determined using mRNA, or the amount of genomic DNA present in a sample.
"Stringent hybridisation conditions" can be used to identify nucleotide sequences, which are substantially identical to a given nucleotide sequence. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequences at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridises to a perfectly matched probe. Typically stringent conditions will be chosen in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least 60°C. Lowering the salt concentration and/or increasing the temperature increases stringency. Stringent conditions for RNA-DNA hybridisations (Northern blots using a probe of e.g. lOOnt) are for example those which include at least one wash in 0.2X SSC at 63°C for 20min, or equivalent conditions. Stringent conditions for DNA-DNA hybridisation (Southern blots using a probe of e.g. lOOnt) are for example those which include at least one wash (usually 2) in 0.2X SSC at a temperature of at least 50°C, usually about 55°C, for 20 min, or equivalent conditions. See also Sambrook et al. (1989) and Sambrook and Russell (2001).
In an embodiment, step iiia) is performed, whereas step iiib) is not performed. In another embodiment, step iiib) is performed, whereas step iiia) is not performed. In yet another embodiment, both steps iiia) and iiib) are performed. For test samples of unknown origin, i.e. of which it is not known whether it is from an IBS subject or from a healthy individual, steps i), ii) and iiia) may be performed to diagnose IBS. In such case, it may be advantageous to perform both steps iiia) and iiib) to simultaneously diagnose and subtype IBS. For test samples obtained from an IBS subject, it may be sufficient to perform steps i), ii), and iiib) in order to subtype the IBS.
In an embodiment, in step iiia) an increased level of nucleic acids from said test sample, said nucleic acids being capable of hybridising to nucleic acid sequences selected from the nucleic acid sequences of SEQ ID Nos: l-27, 70-71, 73-77, 99-100, or derivatives or fragments thereof deviating by at most 2 nucleotides, and complements, reverse, and reverse complements thereof, under stringent hybridization conditions, compared to the level of said nucleic acids from said control sample relates to the diagnosis that the subject is suffering from IBS.
In a further embodiment, in step iiia) a decreased level of nucleic acids from said test sample, said nucleic acids being capable of hybridising to nucleic acid sequences selected from the nucleic acid sequences of SEQ ID Nos:28-69, 72, 78-98, or derivatives or fragments thereof deviating by at most 2 nucleotides, and complements, reverse, and reverse complements thereof, under stringent hybridization conditions, compared to the level of said nucleic acids from said control sample relates to the diagnosis that the subject is suffering from IBS.
As such, the nucleic acid or nucleotide sequences of SEQ ID NO. : 1-100, or derivatives or fragments thereof deviating from SEQ ID NO. : 1-100 by at most 2 nucleotides, or the complement, reverse, or reverse-complement thereof, may be used to discriminate between healthy subjects and subjects suffering from IBS, as well as between subject suffering from the various subtypes of IBS: IBS-A, IBS-C and IBS-D. Although two nucleic acid sequences selected from the group consisting of SEQ ID NO.: 1-100 may suffice for diagnosing IBS and/or subtyping IBS-A, IBS-C and/or IBS- D, it is preferred that at least 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 25, 30, 35, 40, or more nucleic acid sequences selected from the group consisting of SEQ ID Nos.: l-100 are employed in the method of the present invention. In an embodiment, all nucleic acid sequences of SEQ ID NO.: 1-100, or derivatives or fragments thereof deviating by at most 2 nucleotides, or the complement, reverse, or reverse-complement thereof, are employed for diagnosing and/or subtyping IBS in a test sample.
The levels of the nucleic acid sequences in a test sample may be subjected to statistical and/or bioinformatical analysis to obtain analyzed data; and the analyzed data of said test sample may be compared to analyzed data from a control sample, to provide a diagnosis of whether the test sample is from a subject suffering from Irritable Bowel Syndrome. For example, hybridization patterns on a micro-array comprising the nucleic acid sequences having SEQ ID NO: 1-100. In this method, the hybridization data generated using SEQ ID Nos.: l-100 may be processed using statistical and/or bioinformatical analysis such as Principal Component Analysis (PCA) and/or Redundancy Analysis (RDA). The analyzed data may then be compared to analyzed data from a control sample which has been subject to the same statistical and/or bioinformatical analysis, which may relate to a diagnosis of whether the test sample is from a subject suffering from IBS.
In an embodiment, Significance Analysis of Microarrays (SAM) is used in comparing the levels of said three or more nucleic acid sequence from said test sample with the levels of said three or more nucleic acid sequence from a control sample. The person skilled in the art is capable of performing SAM analysis. SAM analysis is described in detail by Tusher et al. (Proc Natl Acad Sci U S A, 2001, vol 98:5116- 5121), which is herein incorporated by reference.
In another embodiment, Prediction Analysis of Microarray (PAM) is used in comparing the levels of said three or more nucleic acid sequence from said test sample with the levels of said three or more nucleic acid sequence from a control sample. The person skilled in the art is capable of performing PAM analysis. PAM analysis is described in detail by Tibshirani et al. (Proc Natl Acad Sci U S A, 2002, vol 99:6567- 6572), which is herein incorporated by reference.
In yet another embodiment, Redundancy Analysis (RDA) is used in comparing the levels of said three or more nucleic acid sequence from said test sample with the levels of said three or more nucleic acid sequence from a control sample. The person skilled in the art is capable of performing RDA analysis. RDA analysis is described in detail by Leps and Smilauer (2003. Cambridge University Press: Multivariate analysis of ecological 780 data using CANOCO), which is herein incorporated by reference.
The level may be determined using a method selected from: hybridization of the nucleic acids in a sample to the nucleic acid sequences having SEQ ID NO.: 1-100, and complements, reverse, and reverse complements thereof, under stringent hybridization conditions; a Polymerase Chain reaction (PCR) or a Ligase Chain Reaction (LCR).
In yet another aspect, the invention pertains to a method for diagnosing and/or subtyping Irritable Bowel Syndrome (IBS) in a test sample, said method comprising the steps of: i) determining the level of amplification of at least three nucleic acid sequences from a test sample using one or more of the nucleic acid sequences of SEQ ID NO.: 1-100, or derivatives or fragments thereof deviating by at most 2 nucleotides, or nucleic acids capable of hybridising to 16S rRNA nucleic acid sequences hybridizing to the complementary strand of any of the nucleic acid sequences SEQ ID NO.: 1-100 or fragments of said 16S rRNA nucleic acid sequences hybridizing to the complementary strand of any of the nucleic acid sequences SEQ ID NO.: 1-100, and complements, reverse, and reverse complements thereof; ii) comparing the level of amplification of said at least three nucleic acid sequences from said test sample to the level of amplification of said at least three nucleic acid sequences from a control sample; and iiia) relating the level of amplification of said at least three nucleic acid sequences from said test sample compared to the level of amplification of said at least three nucleic acid sequences from a control sample to a diagnosis of whether the test sample is from a subject suffering from Irritable Bowel Syndrome; and/or iiib) relating the level of amplification of said at least three nucleic acid sequences from said test sample compared to the level of amplification of said at least three nucleic acid sequences from a control sample to a diagnosis of whether the test sample is from a subject suffering from IBS-A, IBS-C, or IBS-D.
It is to be noted that also the levels of one or more bacteria belonging to the taxa Collinsella (see Table 1) may be used for diagnosing and subtyping IBS in the method of the present invention. In particular, they may be used for subtyping IBS-A in the methods of the present invention. A decreased level of two or more bacteria belonging to the taxa Collinsella in the test sample relates to a diagnosis that the test sample is from a subject suffering from IBS-A. In another aspect, the present invention provides for an array for diagnosing IBS and/or subtyping IBS-A, IBS-C, or IBS-D, said array comprising at least two nucleic acid sequences having the nucleic acid sequence of SEQ ID NOs: 1-100, or derivatives or fragments thereof deviating by at most 2 nucleotides, or complements, reverse, and reverse complements thereof. It was found that the nucleotide sequences mentioned were highly suitable for diagnosing IBS from 3,699 unique nucleotide sequences that were tested.
Preferably, said array comprises at least two nucleic acid sequences selected from the nucleic acid sequences having SEQ ID Nos: 1-100. The at least two nucleic acid sequences may be bound to a solid phase matrix. The array may be a DNA or RNA array, and may be a micro-array.
In a final aspect, the present invention is concerned with the use of an array of the invention for diagnosing IBS and/or subtyping IBS-A, IBS-C, or IBS-D.
In this document and in its claims, the verb "to comprise" and its conjugations is used in its non- limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, the verb "to consist" may be replaced by "to consist essentially of meaning that a composition of the invention may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristics of the invention.
In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".
The terms "increased level" and "decreased level" as used throughout this document refers to a significantly increased level or significantly decreased level. Generally, a level in a test sample is increased or decreased when it is at least 5%, such as 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% higher or lower, respectively, than the corresponding level in a control sample.
All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.
It will be clear that the above description and figures is included to illustrate some embodiments of the invention, and not to limit the scope of protection. Starting from this disclosure, many more embodiments will be evident to a skilled person which are within the scope of protection and the essence of this invention and which are obvious combinations of prior art techniques and the disclosure of this patent.
Table 1. Significantly different level 2 groups between IBS and healthy subjects. The ratio of the average hybridization signal of healthy controls and IBS subjects (all together and grouped according to IBS-c, IBS-d and IBS-a) is presented together with the significance level (as indicated by a t-test; grey indicates significance at the p < 0.05 level).
ratio ratio
Healthy/IBS ratioHealthy/IBSa Healthy/IBSc ratio Healthy/1 BSd level 2 grouping
Collinsella 1.067422638 1.30818031 Θ 0.861611883 1.102839021
Prevotella oralis et rel. 2.50645837 1 885652604 3.468264544: 2.63988894 Prevotella melaninogenica et rel. 6,103165028 4.547786751 α59225815' 5.839139496 Uncultured Bacteroidetes 1.594242942 5.53188383 2.870739989 0.856600392 Tannerella et rel. 1.458425328 2.255614261 1.491450292 1.135363064
Parabacteroides distasonis et rel. 25ΪΗθ083 ' 1.896660973 1.47461125 0.91548181 Allistipes et rel. 1.231160253 2.320606069 1.483347865 0.832313145 Bacteroides plebeius et rel. 1.177594224 1.458299882 1.221359335: 1.004695263 Bacteroides splachnicus et rel. 1.35233461 1 1.856163251 650408006 1.01 1978637 Bacteroides uniformis et rel. 1.2619212 1.930363518 1.187514956 1.036010656 Clostridium stercorarium et rel. 1.06482326 1.44982711 1,301672763: 0.798919757 Subdoligranulum variable et rel. 1.489785141 1.856106447 1.237418199 1.485102714 Faecalibacterium prausnitzii et rel. 1.452699466 2.206842163 1.59021522 1.098883946 Oscillospira guillermondii et rel. 1.325377497 1.963710788 1.162504598 1.156349757 Sporobacter termitidis et rel. 1.26301141 2.009029079 0.981832432 1.173783084 Ruminococcus callidus et rel. 1.21932763 1.974907643 0.991798789 1.083275743 Eubacterium siraeum et rel. 1.067167586 2,25627375 1.026148792 0.777957372 Anaerotruncus colihominis et rel. 1.178665395 1.58067704 1.054874471 1.063012882 Clostridium cellulosi et rel. 1.075722177 1.488351486 0.878190227 1.025716793 Clostridium leptum et rel. 1.098185868 1.629177102 0.901210594 1.007200192 Ruminococcus bromii et rel. 0.875180165 1.343617533 0.659613943 0.849966096 Phascolarctobacterium faecium et rel. 1.264148569 1.872740868 1.267509097 1.012218313 Ruminococcus gnavus et rel. 0.656049569 0.626052848 0.60605995 0.725559954 Dorea formicigenerans et rel. 0.72762871 0.730100931 0.71 1 10021 0.738073733 Ruminococcus obeum et rel. 0.714295591 0.644128485 0790523423" 0,723969208 Clostridium nexile et rel. 0.758714834 ( 834702967 0.7Q7423528 0.74126632 Clostridium symbiosum et rel. 0.820499635 0.870947601 0790559736 ' θΈ 79608 Ruminococcus lactaris et rel. 0.849613518 1.029989947 0.906476977
Lachnospira pectinoschiza et rel. 0.846714788 0.926194039 0.857579737
Outgrouping Clostridium cluster XlVa 0,8 1246186 0.726331202 0.980219023
Figure imgf000035_0001
Eubacterium biforme et rel. 1 19690258 1.372442978 0.9791 15703 1.277301644 Catenibacterium mitsuokai et rel. 1.778170534 0 956656049 2.975624876 2.791019698 Xanthomonadaceae 1.351269902 1.29347056 1.819903677 244906428'' Uncultured Clostridiales I 1.494223068 2.488266625 0.89 433344 1.829478268 Uncultured Clostridiales II ' 50526469''' 2,35864502 0.922623162 TT356 »086 Uncultured Mollicutes 1.183429709 1.3071924 1.088190195 1.1729432 Table 2. Identification, sequence and analysis of the HIT probes that differ significantly at the p < 0.05 level between IBS subjects and healthy controls. The oligonucleotides with S EQ ID NO: 1-27 that showed a significantly higher hybridization signal in the IBS than the healthy subjects and the oligonucleotides with SEQ ID 28-67 that showed the opposite, are indicated with their nucleotide sequence (3 'to 5')·
Sequence 5' to 3 ' direction (T=U in RNA) SEQ ID NO.
GCCGCTCAGTCACAATCCTC 1
GCCACTAGAAATAGATCAAATCCAC 2
GCCGCTCAGTCACAAAACTCTTCA 3
CCGAAGTTTCAATAAAGTAATTCCCG 4
GCCACTAGAATTAAATTAAATCGACCG 5
CGAAGTCTCAATGAAATATTTCCCG 6
CACTAGAAATAGATCAAATCCACCG 7
GCCACTCAGTCACAGTCTCTC 8
GCCGCTCAGTCACCAAGG 9
GCCGCTCAGTCACAACACTC 10
GCCGCTCAGTCACAAAACC 11
GCCGCTCAGTCACAAACGGA 12
GCCGCTCAGTCACTGTCC 13
GCCACTAGAATTAAATTATATCGACCG 1
GCCACTAGAATTAAATCATATCGACC 15
TGTCTCCGCTGCCCCGAA 16
TAAATCATATCGACCGAAGTTTCAATAAAA 17
AAATTATATCGACCGAAGTTTCAATAAAG 18
GCCACTAGAAATAAATCAAATCCACC 19
AGCAAGCTCCTCCTTCAGCG 20
ATCCTCTTCATCCGAAGAATCTAAG 21
GCCGCTTTCCACTCTTAACTTCAA 22
AGAAATCCGTCAAGGTGCTTCGC 23
GAAGTTTCAATAAAATAATTCCCGTTCG 24
TGTCCTCTTCCTCCGAAGATTCTG 25
CCGAAGTTTCAATAAAATAATTCCCG 26
GATCCGTTTAAGGTGCTTCGTTCG 27
TGTCTCTGCGTCCCGAAGGAAAA 28
TGTCTCTGCGTCCCGAAGGAATA 29
TGTCTCTGCGTCCCGAAGGAAA 30
GCCACTGTCCTCTGCTTCAC 31
ATCGTCGCAGGATGTCAAGACTTG 32
CAAGCTCCTCTCAGCTCCG 33
GGCTGACATGTCTCCACATCATTC 34
CGTCGCAGGATGTCAAGACTTG 35
ACCGTCGCAGGATGTCAAGAC 36
TGTCTCTGCTGTCCCGAAGGAAA 37
GCCACTGTCCTCTGCTTCGAA 38
ATCGTCAAGGGATGTCAAGACTTG 39
TGCGTCGCAGGATGTCAAGAC 40
CATTCAGTTGCAATTCAAGCCCGG 41
GCCACTTTCCTCTACATCCATTG 42
GGATTTCACACATCTCTGTGCTA 43
TTCGTCAAGGGATGTCAAGACTTG 44
GTTCGTCAAGGGATGTCAAGAC 45 GCCACTCGATTTGAAGAGCAAGC 4(5
GCCACTAACCGCTCCAATAGTAAA 47
GATTTGAAGAGCAAGCTCCTCATC 48
GCCACTCGATCAAGGAAGCAAG 49
TTCACAACTGCCTTGCGGCTGA 50
CCTCTTTCCACAGATTCTCGTTCG 51
CGATTTGAAGAGCAAGCTCCTCA 52
GAATCCGTAATCAAGCTTCGTTCG 53
TTCTCCTGCAATTCAAGCCCGG 54
TCGTTAGCAGGATGTCAAACCCTG 55
ATGCACCTGCAATTCAAGCCCG 56
CAAGCTCCTCATCTCTCGTTCG 57
TGTCTCCTTGCTCCGAAGAGAAA 58
TGTCTCCTTGCTCCGAAGAGAAAA 59
TGTCTCCTTGCTCCGAAGAGATTA 60
TGTCTCGATGTCCCGAAGGATTTC 61
AGAGCAAGCTCCTCATCTCTCG 62
GCCACTAGATTGTAGAAAAAGCAAG 63
GCACCTAATGCATCTCTGCTTCG 64
GAAGCAAGCTTCCTCTCTCTCG 65
CAAGCTCCTCTTGATTCCGTTCG 66
AGAGAATTATTAGCAAGCTAGCAATTC 67
Table 3. Classification of phylotypes identified in the present invention based on the 16S rRNA gene sequence similarity with accession number of the 16S rRNA gene sequence. Level 1 corresponds to the phylum, or in case of Firmicutes to the Clostridium cluster; Level 2 includes groups of sequences with 90% or more sequence similarity; Level 3 represents unique phylotypes that were defined as species for cultivated microorganisms, or representatives of each monophyletic group with > 98% sequence identity for clones corresponding to uncultured microorganisms (herein identified as "relatives" or "et rel.").
Accession
Level 1 Level 2 Level 3
number
Actinobacteria Actinomycetaceae Arcanobacterium pyogenes M29552
Actinomyces naeslundii M3391 1
Uncultured bacterium clone Eldhufec234 AY920109
Uncultured bacterium clone Eldhufec081 AY919956 uncultured bacterium Z650 AY979340 uncultured bacterium NH01 AY978941
Atopobium Atopobium parvulum AF292372
Atopobium minutum M59059
Bifidobacterium Bifidobacterium breve AB006658
Bifidobacterium thermophilum ABO 16246
Bifidobacterium angulatum D86182
Bifidobacterium dentium D86183
Bifidobacterium infantis D86184
Bifidobacterium pseudocatenulatum D86187
Bifidobacterium gallicum D86189 Bifidobacterium pseudolongum D86194
Bifidobacterium bifidum M38018
Bifidobacterium adolescentis M58729
Bifidobacterium catenulatum M58732
Bifidobacterium longum M58739
Bifidobacterium sp. CB8 AB064925
Uncultured bacterium clone Eldhufec082 AY919957 uncultured bacterium (human infant) L14E AF253371 uncultured bacterium (human infant) N14A AF253397 uncultured bacterium Adhufec069rbh AY471706 uncultured Bifidobacterium sp. 15D AF275886 uncultured Bifidobacterium sp. 13D AF275884
Bifidobacterium sp. PL1 AF306789
Collinsella Collinsella aerofaciens ABO 11814
Collinsella sp. CB52 AB064936
Uncultured bacterium clone Eldhufec074 AY919949
Collinsella stercoris AB031062
Collinsella intestinalis AB031063
Corynebacterium Corynebacterium xerosis AF024653
Corynebacterium ulcer ans X81911
Corynebacterium ammoniagenes X82056
Corynebacterium pseudodiphtheriticum X84258 uncultured bacterium LI92 AY978122 uncultured bacterium N337 AY980429
Eggerthella lenta et rel. Eggerthella lenta
uncultured Gram-positive bacterium N01H5 uncultured bacterium ME67
Uncultured bacterium clone Eldhufec078
Uncultured bacterium clone Eldhufec076
Uncultured bacterium clone Eldhufec075
Denitrobacterium sp. CCUG 45665
uncultured bacterium Adhufec036abh
Micrococcaceae Micrococcus luteus AJ276811
Rothia dentocariosa M59055
Uncultured bacterium clone Eldhufec080 AY919955 uncultured bacterium HuJJ72 AY684419
Propionibacterium Propionibacterium acnes AB041617
Propionibacterium avidum AJ003055 Propionibacterium granulosum AJ003057 Propionibacterium propionicum X53216 Propionibacterium jensenii X53219 Propionibacterium acidipropionici X53221
Bacteroidetes Alistipes et rel. Alistipes putredinis LI 6497
Bacteroides sp. CJ44 AB080886 uncultured bacterium C706 AY916343 uncultured bacterium D080 AY916354 uncultured bacterium Ml 62 AY916149 uncultured bacterium MG06 AY916286 uncultured bacterium NH37 AY916174 uncultured bacterium NN46 AY916247
Uncultured bacterium clone Eldhufec050 AY919925
Uncultured bacterium clone Eldhufec022 AY919897 uncultured bacterium cadhufec076h7 AF530308 uncultured bacterium adhufec52.25 AF153864
Alistipes finegoldii AJ518874
Bacteroides sp. DSM 12148 AJ518876 uncultured bacterium Adhufec002rbh AY471693
Alistipes oderdonkii AY974072
Alistipes shahii AY974071
Bacteroides fragilis et rel. bacterium adhufec23
bacterium adhufec355
Bacteroides thetaiotaomicron
Bacteroides fragilis
uncultured bacterium MR34 uncultured bacterium Z091
Uncultured bacterium clone Eldhufec021
uncultured bacterium LCRC79
Bacteroides finegoldii
Bacteroides nordii
Bacteroides salyersiae
Bacteroides intestinalis et rel. uncultured bacterium OLD A- A 1 ] AB099761 uncultured bacterium HuCA21 AJ409009 Bacteroides intestinalis AB214329
Bacteroides ovatus et rel. Bacteroides ovatus
Bacteroides caccae
uncultured bacterium NC94 uncultured bacterium NP35
uncultured bacterium HuCA34
imcultured bacterium HuCC30
Uncultured bacterium clone Eldhufec030
Bacteroides plebeius et rel. bacterium adhufec367
Bacteroides sp. COl 1
uncultured bacterium D790
Uncultured bacterium clone Eldliufec045
Uncultured bacterium clone Eldhufec335
Bacteroides coprocola
Bacteroides plebeius
imcultured bacterium Adhufec025abh uncultured bacterium Adhufec086rbh AY471710
Bacteroides splachnicus et rel. bacterium adhufec84 AF132281
Bacteroides splanchnicus LI 6496 uncultured bacterium C268 AY916330 uncultured bacterium M048 AY916145 uncultured bacterium MN96 AY916307 uncultured bacterium NK71 AY916241 uncultured bacterium NK90 AY916243 uncultured bacterium NN42 AY916246 uncultured bacterium NN84 AY916248 uncultured bacterium NP53 AY916254 uncultured bacterium NX93 AY916310
Uncultured bacterium clone Eldhufec044 AY919919
Uncultured bacterium clone Eldhufec048 AY919923
Bacteroides stercoris et rel. bacterium adhufec303 AF132259
Bacteroides eggerthii L16485
Bacteroides stercoris X83953
Uncultured bacterium clone Eldhufec057 AY919932
Uncultured bacterium clone Eldhufec025 AY919900
Bacteroides uniformis et rel. Bacteroides uniformis L16486 uncultured Bacteroides sp. NS2A1 1 AB064816
Bacteroides vulgatus et rel. Bacteroides vulgatus M58762
Bacteroides dorei AB242142
Parabacteroides distasonis et Parabacteroides distasonis M25249 rel. Parabacteroides merdae X83954 uncultured bacterium OLDA-B 10 AB099754 uncultured bacterium M270 AY916152 uncultured bacterium MH76 AY916297
Uncultured bacterium clone Eldhufec042 AY919917 uncultured bacterium LCLC20 AF499837 uncultured bacterium ABLCf 15 AF499899
Parabacteroides goldsteinii AY974070
Prevotella melaninogenica et bacterium adhufec235 AF132249 rel. Prevotella intermedia AF414821
Prevotella albensis AJ01 1683
Prex'otella melaninogenica LI 6469
Prevotella veroralis L16473
Prevotella disiens L16483 uncultured bacterium B 176 AY916316 uncultured bacterium Ml 07 AY916148
Uncultured bacterium clone Eldhufec008 AY919883
Uncultured bacterium clone Eldhufec007 AY919882
Uncultured bacterium clone Eldhufec033 AY919908
Uncultured bacterium clone Eldhufec038 AY919913 Uncultured bacterium clone Eldhufec037 AY919912
Uncultured bacterium clone Eldhufec036 AY91991 1
Uncultured bacterium clone Eldhufec035 AY919910
Uncultured bacterium clone Eldhufec034 AY919909
Uncultured bacterium clone Eldhufec005 AY919880
Uncultured bacterium clone Eldhufec009 AY919884
Uncultured bacterium clone Eldhufec024 AY919899
Uncultured bacterium clone Eldhufec019 AY919894 uncultured bacterium HuJJ84 AY684413
Prevotella sp. BI-42 AJ581354
Prevotella oralis et rel. Prevotella oralis LI 6480
Figure imgf000041_0001
uncultured bacterium HuCC28 AJ315483
Uncultured bacterium clone EldhufecOl 1 AY919886
Uncultured bacterium clone Eldhufec043 AY919918
Uncultured bacterium clone EldhufecOl 5 AY919890
Uncultured bacterium clone EldhufecO 17 AY919892
Uncultured bacterium clone EldhufecO 12 AY919887 uncultured bacterium HuJJ29 AY684415 uncultured bacterium Adhufec036rbh AY471699
Prevotella ruminicola et rel. Prevotella ruminicola AF218618
Prex'otella brevis AJO 1 1682
Uncultured bacterium clone Eldhufec028 AY919903
Prevotella tannerae et rel. uncultured bacterium OLDC-G2 AB099769 uncultured bacterium OLDC-D5 AB099768 uncultured bacterium ME28 AY916231
Uncultured bacterium clone EldhufecO 18 AY919893
Uncultured bacterium clone EldhufecO 14 AY919889
Uncultured bacterium clone Eldhufec003 AY919878 uncultured bacterium cadhufec40cl0 AF530373
Tannerella et rel. bacterium adhufec77.25 AF 153865 uncultured bacterium D487 AY916372 uncultured bacterium D761 AY916386 uncultured bacterium M070 AY916146 uncultured bacterium NG45 AY916172 uncultured bacterium NI77 AY916176 uncultured bacterium N037 AY916249 uncultured bacterium NO50 AY916251
Uncultured bacterium clone EldhufecO 10 AY919885
Uncultured bacterium clone Eldhufec041 AY919916
Uncultured bacterium clone Eldhufec006 AY919881
Uncultured bacterium clone Eldhufec004 AY919879
Uncultured bacterium clone Eldhufec023 AY919898 uncultured bacterium Adhufec048rbh AY471701 Unclutured Bacteroidetes Bacteroides sp. CB40 AB064919
Asteroleplasma Asteroleplasma et rel. Uncultured bacterium UC7-1 1 AJ608228
Bacilli Aerococcus Aerococcus viridans M58797
Bacillus et rel. Bacillus halodurans AB013373
Bacillus subtilis ABO 18484
Bacillus pumilus AB020208
Bacillus flexus AB021 185
Bacillus cereus AF076031
Bacillus sphaericus AF 169495
Brevibacillus brevis AF424048
Bacillus megaterium D16273
Bacillus circulans D78312
Bacillus coagulans D78313
Aneurinibacillus aneurinolyticus D78455
Paenibacillus lautus D78472
Bacillus badius X77790
Paenibacillus durus X77846
Enterococcus Enterococcus faecalis AB012212
Enterococcus faecium AB012213
Enterococcus gallinarum AF039898
Enterococcus casseliflavus AF039899
Enterococcus durans AF061000
Enterococcus avium AF061008
Enterococcus hirae AF06101 1 uncultured bacterium cadhufec093h7 AF530310 uncultured bacterium (human infant) D8E AF253331
Gemella Gemella morbillorum L14327
Granulicatella Uncultured bacterium clone Eldhufecl98 AY920073
Lactobacillus gasseri et rel. Lactobacillus gasseri AF243142
Lactobacillus jensenii AF243159
Lactobacillus crispatus AF257096
Lactobacillus johnsonii AJ002515
Lactobacillus delbrueckii AY050173
Lactobacillus acidophilus M58802
Lactobacillus amylovorus M58805
Lactobacillus helveticus X61 141 uncultured Lactobacillus sp. LabF368 AF335876 uncultured Lactobacillus sp. LabF93 AF33591 1
Lactobacillus ultunensis AY253660
Lactobacillus kalixensis AY253657
Lactobacillus plantarum et rel. Pediococcus acidilactici AB018213
Lactobacillus brevis AB024299
Lactobacillus mucosae AF126738
Lactobacillus rhamnosus AF243146 Lactobacillus paracasei AF243147
Lactobacillus fermentum AF243149
Lactobacillus vaginalis AF243177
Lactobacillus plantarum AJ271852
Lactobacillus casei AJ272201
Lactobacillus pentosus D7921 1
Lactobacillus reuteri L23507
Lactobacillus buchneri M5881 1
Pediococcus pentosaceus M58834
Lactobacillus oris X61 131 uncultured Lactobacillus sp. LabS14 AF335913
Lactobacillus antri AY253659
Lactobacillus gastricus AY253658
Lactobacillus parabuchneri AB205056
Lactobacillus sakei et rel. Lactobacillus sakei M58829
Lactobacillus salivarius et rel. Lactobacillus salivarius AF42031 1
Lactobacillus ruminis M58828
Lactococcus Lactococcus lactis AJ271851
Lactococcus sp. 451 AY762109
Staphylococcus Staphylococcus aureus AF015929
Staphylococcus epidermidis D83362
Staphylococcus saccharolyticus L37602
Streptococcus bovis et rel. Streptococcus equinus AB002514
Streptococcus uberis AB023573
Streptococcus agalactiae AB023574
Streptococcus pyogenes AF076028
Streptococcus bovis AF104109
Streptococcus infantarius AF177729
Streptococcus lutetiensis AF429763
Streptococcus salivarius M58839
Streptococcus thermophilus X59028 uncultured bacterium 0LDA-B7 AB099789
Streptococcus equi subsp. zooepidemicus AB 104843
Streptococcus equisimilis AJ31461 1
Streptococcus intermedius et Streptococcus intermedius AF 104671 rel. Streptococcus constellatus AF 104676
Streptococcus anginosus AF 145240
Streptococcus parasanguinis X53652
Uncultured bacterium clone Eldhufecl95 AY920070
Streptococcus mitis et rel. Streptococcus sanguis AF003928
Streptococcus mitis AF003929
Streptococcus oralis AF003932
Streptococcus viridans AF076036
Streptococcus mutans AJ243965 uncultured Streptococcus sp. NB5C1 AB064839 bacterium ucfecDB2 ARB B5C8D
A
Weissella et rel. Weissella cibaria AJ295989
Leuconostoc mesenteroides M23035
Weissella confusa M23036 uncultured Leuconostoc sp. LabF165 AF335897
Clostridium Clostridium Eubacterium multiforme AB018184 cluster I Clostridium paraputrificum AB032556
Clostridium perfringens AB045282
Clostridium botulinum AF 105402
Sarcina ventriculi AF 1 10272
Clostridium putrefaciens AF 127024
Clostridium subterminale AF241842
Clostridium butyricum AJ002592
Clostridium tertium AJ245413
Clostridium tyrobutyricum L08062
Eubacterium moniliforme L34622
Clostridium cadaveris M59086
Clostridium fallax M59088
Clostridium cochlearium M59093
Clostridium limosum M59096
Clostridium malenominatum M59099
Clostridium paraperfringens M59102
Clostridium sporogenes M59115
Clostridium acetobutylicum S46735
Clostridium septicum U59278
Clostridium barati X68174
Clostridium beijerinckii X68179
Clostridium celatum X77844
Clostridium sartagoformum Y18175
Uncultured bacterium clone Eldhufec341 AY920216
Eubacterium budayi AB018183
Eubacterium nitritogenes AB018185
Clostridium Clostridium stercorarium et uncultured bacterium B839 AY916322 cluster III rel. uncultured bacterium D145 AY916358 uncultured bacterium LEI 7 AY916205
Uncultured bacterium clone Eldhufec339 AY920214
Uncultured bacterium UC7-82 AJ608246
Clostridium thermocellum et uncultured bacterium C288 AY916331 rel. Uncultured bacterium clone Eldhufec338 AY920213
Clostridium Anaerotruncus colihominis et bacterium adhufec 101 AF132235 cluster IV rel. uncultured Gram-positive bacterium N02-2 AB064805 uncultured bacterium D577 AY916375 uncultured bacterium LF02 AY916207 uncultured bacterium LL29 AY916260 uncultured bacterium LL87 AY916261 uncultured bacterium HuCAl AJ408957
Uncultured bacterium clone Eldhufec246 AY920121
Uncultured bacterium clone Eldhufec21 1 AY920086
Uncultured bacterium clone Eldhufec214 AY920089
Uncultured bacterium clone Eldhufec215 AY920090
Uncultured bacterium clone Eldhufec265 AY920140
Uncultured bacterium clone Eldhufec270 AY920145
Anaerotruncus colihominis AJ315980
Clostridium cellulosi rel. uncultured human gut bacterium JW1B12 AB080849 uncultured bacterium OLDB-E4 AB099734 uncultured bacterium C342 AY916333 uncultured bacterium D036 AY916351 uncultured bacterium K507 AY916200 uncultured bacterium LZ45 AY916188 uncultured bacterium M490 AY916159 uncultured bacterium M51 1 AY916162 uncultured bacterium MH24 AY916292 uncultured bacterium Z456 AY916179 uncultured bacterium D626 AY916378
Uncultured bacterium clone Eldhufec236 AY9201 1 1
Uncultured bacterium clone Eldhufec212 AY920087
Uncultured bacterium clone Eldhufec213 AY920088
Uncultured bacterium clone Eldhufec273 AY920148
Uncultured bacterium clone Eldhufec249 AY920124
Uncultured bacterium UC7-44 AJ608241
Uncultured bacterium UC7-69 AJ608244 uncultured bacterium cadhufec022h7 AF530299 uncultured bacterium ABLCf36 AF499903 uncultured bacterium HuAC35 AY684394 uncultured bacterium Adhufecl06abh AY471691
Clostridium leptum et rel. Clostridium leptum M59095
Clostridium sporosphaeroides M591 16 uncultured human gut bacterium JW1C7 AB080848 uncultured bacterium C464 AY916336 uncultured bacterium C735 AY916345 uncultured bacterium K288 AY916193 uncultured bacterium HuCA24 AJ408976
Uncultured bacterium clone Eldhufec221 AY920096
Uncultured bacterium UC7-14 AJ608230 uncultured bacterium adhufecl68 AF 132242
Ruminococcus sp. 16442 AJ318889 Clostridium orbiscindens et Clostridium orbiscindens Y18187 rel. human intestinal firmicute CJ36 AB080896 human intestinal firmicute C J31 AB080897 uncultured human gut bacterium JW1D6 AB080858 uncultured human gut bacterium JW2G1 AB080857 uncultured human gut bacterium JW1G9 AB080856 uncultured human gut bacterium JW2A8 AB080855 uncultured bacterium OLDA-F4 AB099727 uncultured bacterium B632 AY916320 uncultured bacterium D330 AY916365 uncultured bacterium D465 AY916371 uncultured bacterium D588 AY916376 uncultured bacterium G267 AY916285 uncultured bacterium K351 AY916196 uncultured bacterium LV67 AY916184 uncultured bacterium M510 AY916161 uncultured bacterium W074 AY916213 uncultured bacterium HuCB24 AJ408998
Uncultured bacterium clone Eldhufec218 AY920093
Uncultured bacterium clone Eldhufec272 AY920147
Uncultured bacterium clone Eldhufec262 AY920137
Uncultured bacterium clone Eldhufec264 AY920139
Uncultured bacterium clone Eldhufec267 AY920142
Uncultured bacterium clone Eldhufec229 AY920104 uncultured bacterium cadhufec074h7 AF530307
Bacteroides capillosus AY136666 uncultured bacterium Adhufec 102rbh AY471712
Eubacterium siraeum et rel. Eubacterium siraeum L34625 uncultured bacterium B025 AY916313
Uncultured bacterium clone Eldhufec237 AY9201 12
Uncultured bacterium clone Eldhufec239 AY920114
Uncultured bacterium UC7- 117 AJ608247 uncultured bacterium Adhufec058abh AY471683
Faecalibacterium prausnitzii bacterium adhufec 113 AF132236 et rel. but rate-producing bacterium A2-165 AJ270469 butyrate-producing bacterium L2-6 AJ270470
Faecalibacterium prausnitzii AJ413954 uncultured bacterium KM82 AY916180 uncultured bacterium KP66 AY916136 uncultured bacterium HuCA25 AJ408973 uncultured bacterium HuCAl 1 AJ408966
Uncultured bacterium clone Eldhufec238 AY920113
Uncultured bacterium clone Eldhufec226 AY920101
Uncultured bacterium clone Eldhufec227 AY920102 Uncultured bacterium clone Eldliufec288 AY920163 Uncultured bacterium clone Eldhufec228 AY920103 Uncultured bacterium clone Eldhufec259 AY920134 Uncultured bacterium clone Eldhufec261 AY920136 Uncultured bacterium clone Eldhufec276 AY920151 Uncultured bacterium clone Eldhufec282 AY920157 Uncultured bacterium clone Eldhufec256 AY920131 Uncultured bacterium clone Eldhufec255 AY920130 Uncultured bacterium clone Eldhufec252 AY920127 Uncultured bacterium clone Eldhufec281 AY920156 Uncultured bacterium clone Eldhufec251 AY920126 imcultured bacterium adhufec08.25 AF153871 uncultured bacterium A10 AF05241 1 uncultured bacterium AdhufecOlOabh AY471671 uncultured bacterium Adhufec055abh AY471682 uncultured bacterium Adhufec052abh AY471681 imcultured bacterium Adhufec064rbh AY471704 uncultured bacterium Adhufec057rbh AY471702 uncultured bacterium Adhufecl07rbh AY471714
Oscillospira guillermondii et bacterium adhufec269 AF132255 rel. imcultured human gut bacterium JWl C 1 1 AB080854 uncultured bacterium OLDA-D 1 1 AB099726 uncultured bacterium OLDC-D12 AB099725 uncultured bacterium OLDA-H2 AB099721 imcultured bacterium A051 AY916256 uncultured bacterium B81 1 AY916321 uncultured bacterium C574 AY916337 uncultured bacterium D 134 AY916357 imcultured bacterium D288 AY916364 imcultured bacterium D440 AY916370 uncultured bacterium LE02 AY916204 uncultured bacterium MA30 AY916224 imcultured bacterium MM71 AY916303 imcultured bacterium V239 AY916276 uncultured bacterium HuCB7 AJ408991 Uncultured bacterium clone Eldhufec241 AY920116 Uncultured bacterium clone Eldhufec223 AY920098 Uncultured bacterium clone Eldhufec257 AY920132 Uncultured bacterium clone Eldhufec301 AY920176 Uncultured bacterium clone Eldhufec285 AY920160 Uncultured bacterium clone Eldhufec283 AY920158 imcultured bacterium cadhufecl21h7 AF530315 uncultured bacterium Adhufec002abh AY471669 uncultured bacterium Adhufec044abh AY471679 Outgrouping Clostridium uncultured bacterium C747 AY916347 cluster IV uncultured bacterium LD25 AY916202 uncultured bacterium V366 AY916279
Uncultured bacterium clone Eldhufec318 AY920193
Uncultured bacterium clone Eldhufec320 AY920195
Uncultured bacterium clone Eldhufec321 AY920196
Uncultured bacterium clone Eldhufec319 AY920194
Papillibacter cinnamivorans et bacterium adhufec296 AF132258 rel. butyrate-producing bacterium A2-207 AJ270471 uncultured Gram-positive bacterium NB5F9 AB064783 uncultured bacterium Z015 AY916177
Uncultured bacterium clone Eldhufec233 AY920108
Uncultured bacterium clone Eldhufec245 AY920120
Uncultured bacterium clone Eldliufec258 AY920133 uncultured bacterium cadhufec32cl0 AF530372
Ruminococcus bromii et rel. Ruminococcus bromii L76600 uncultured bacterium HuCB2 AJ408987
Uncultured bacterium clone Eldhufec230 AY920105
Uncultured bacterium clone Eldhufec291 AY920166
Uncultured bacterium clone Eldhufec225 AY920100
Uncultured bacterium clone Eldhufec291 AY920166 uncultured bacterium cadhufec02 lh7 AF530298 uncultured bacterium Adhufec014rbh AY471694
Ruminococcus callidus et rel. Ruminococcus flavefaciens AF030446
Ruminococcus albus AF030451
Ruminococcus callidus L76596
Clostridium methylpentosum Y18181 uncultured Gram-positive bacterium NS4G9 AB06481 1 uncultured Ruminococcus sp. NOl 1 AB064808 uncultured bacterium D005 AY916350 uncultured bacterium D739 AY916385 uncultured bacterium D789 AY916389 uncultured bacterium MF20 AY916235 uncultured bacterium MH26 AY916293
Uncultured bacterium clone Eldhufec235 AY9201 10
Uncultured bacterium clone Eldhufec284 AY920159
Uncultured bacterium clone Eldhufec250 AY920125
Sporobacter termitidis rel. bacterium adhufec31 1 AF132261 bacterium adhufecl08 AF132283 uncultured bacterium OLDC-E8 AB099728 uncultured bacterium C352 AY916334 uncultured bacterium C354 AY916335 uncultured bacterium C727 AY916344 uncultured bacterium D762 AY916387 uncultured bacterium L495 AY916281 uncultured bacterium L041 AY916265 uncultured bacterium LQ71 AY916268 uncultured bacterium LY18 AY916187 Uncultured bacterium clone Eldhufec210 AY920085 Uncultured bacterium clone Eldhufec290 AY920165 Uncultured bacterium clone Eldhufec274 AY920149 Uncultured bacterium clone Eldhufec231 AY920106 Uncultured bacterium clone Eldhufec294 AY920169 Uncultured bacterium clone Eldhufec216 AY920091 Uncultured bacterium clone Eldhufec217 AY920092 Uncultured bacterium clone Eldhufec287 AY920162 Uncultured bacterium clone Eldhufec220 AY920095 Uncultured bacterium clone Eldhufec232 AY920107 Uncultured bacterium UC7- 1 AJ608220
Subdoligranulum variable at bacterium adhufecl3
rel. uncultured Gram-positive bacterium N02- uncultured Gram-positive bacterium NB5C6 human intestinal firmicute CJ7
uncultured human gut bacterium JW1D4
uncultured bacterium LC79
uncultured bacterium M479
uncultured bacterium HuCB5
Uncultured bacterium clone Eldliufec243
Uncultured bacterium clone Eldhufec222
Uncultured bacterium clone Eldhufec224
Uncultured bacterium clone Eldhufec260
Uncultured bacterium clone Eldhufec302
Uncultured bacterium clone Eldliufec268
uncultured bacterium cadhufec068h7
uncultured bacterium cadhufec066h7
uncultured bacterium ABLCf22
Subdoligranulum variabile
Clostridium Dialister Dialister pneumosintes
cluster IX uncultured Gram-positive bacterium NS2B1
Uncultured bacterium clone Eldhufec091
Uncultured bacterium clone Eldhufec093
Uncultured bacterium clone Eldhufec089
Uncultured bacterium clone Eldhufec096
uncultured bacterium B856
uncultured bacterium MG10
Megamonas hypermegale et Megamonas hypermegale AJ420107 rel. human intestinal firmicute CB 15 AB064931 uncultured bacterium cadhufec43cl0 AF530374 Megasphaera elsdenii et rel. Megasphaera elsdenii AF283705 uncultured bacterium OLDC-D 10 AB099774 uncultured bacterium HuCB85 AJ409007
Uncultured bacterium clone Eldhufec098 AY919973 uncultured bacterium inhufecA- 11 AY328359
Mitsuokella multiacida et rel. Selenomonas ruminantium ABO 17195
Mitsuokella multiacida X81878 uncultured Gram-positive bacterium NB5E1 AB064853 uncultured bacterium OLDC-C6 AB099772
Peptococcus niger et rel. Peptococcus niger X55797 uncultured bacterium D393 AY916367 uncultured bacterium MH31 AY916294 uncultured bacterium V247 AY916277
Uncultured bacterium clone Eldhufec095 AY919970 uncultured bacterium HuDIlO AY862394
Phascolarctobacterium bacterium adhufec395
faecium et rel. Acidaminococcus fermentans
uncultured Gram-positive bacterium NB4G9 uncultured bacterium OLDB-D6
uncultured bacterium OLDB-B2
uncultured bacterium Dl 15
Uncultured bacterium clone Eldhufec097
Uncultured bacterium clone Eldhufec094
uncultured bacterium cadhufecl37cl0
Uncultured Selenomonadaceae uncultured bacterium HuAC20 AY684401
Veillonella Veillonella dispar AF439639
Veillonella parvula AF439640 Veillonella atypica AF439641 uncultured bacterium ABLCfS AF499900
Clostridium Anaerovorax odorimutans rel. uncultured Gram-positive bacterium N02-6 AB064863 cluster XI uncultured human gut bacterium JW1G2 AB080883 uncultured bacterium LN56 AY916263 uncultured bacterium MO 17 AY916142 uncultured bacterium MH36 AY916295 uncultured bacterium P615 AY916312
Uncultured bacterium clone Eldhufecl85 AY920060
Uncultured bacterium clone Eldhufecl87 AY920062
Uncultured bacterium clone Eldhufecl86 AY920061 uncultured bacterium HuJJ43 AY684403 uncultured bacterium HuRC86 AY684402
Clostridium difficile et rel. Clostridium hiranonis AB023970
Clostridium difficile AF072473 Clostridium bifermentans AF320283 Clostridium glycolicum AY007244 Clostridium sticklandii L04167
Clostridium sordellii M59105
Eubacterium tenue M59118
Clostridium irregularis X73447
Clostridium ghoni X73451 uncultured Gram-positive bacterium NS1E9 AB064876 uncultured Clostridium sp. NB4D7 AB064872 uncultured bacterium OLDB-G12 AB099796 uncultured bacterium M364 AY916153
Uncultured bacterium clone Eldhufecl89 AY920064 uncultured bacterium LCLC73 AF499844 uncultured bacterium LCLC21 AF499843
Clostridium bartlettii AY438672
Clostridium felsineum Clostridium felsineum X77851
Peptostreptococcus Peptostreptococcus anaerobius D14150 anaerobius et rel. uncultured bacterium CI 20 AY916327
Clostridium Peptostreptococcus micros et Peptoniphilus asaccharolyticus D14138 cluster XIII rel. Anaerococcus prevotii D14139
Anaerococcus hydrogenalis D14140
Peptostreptococcus micros D14143
Peptoniphilus indolicus D14147
Finegoldia magna D14149 uncultured bacterium G170 AY981208
Tissierella Tissierella praeacuta X80833
Clostridium Acetitomaculum ruminis rel. bacterium adhufec250 AF132253 cluster XlVa uncultured bacterium D416 AY916368 uncultured bacterium LP40 AY916266 uncultured bacterium M977 AY916221
Uncultured bacterium clone Eldhufecl57 AY920032
Uncultured bacterium clone Eldhufecl20 AY919995
Uncultured bacterium clone Eldhufecl 17 AY919992
Uncultured bacterium clone Eldhufecl 10 AY919985
Uncultured bacterium clone Eldhufecl 03 AY919978 uncultured bacterium HuDI84 AY684365
Anaerostipes caccae et rel. Clostridium indolis AF028351 bacterium adhufec25 AF132254
Anaerostipes caccae AJ270487 uncultured Gram-positive bacterium NB2G8 AB064714 uncultured Gram-positive bacterium N02-5 AB064713 uncultured human gut bacterium JW2C7 AB080875 uncultured bacterium HuCA20 AJ408972
Bryantella formatexigens et bacterium adhufec40 AF132270 rel. Eubacterium cellulosolvens L34613 uncultured Gram-positive bacterium NS2F9 AB064773 Ruminococcus sp. C028 AB064891 uncultured bacterium M629 AY916166 uncultured bacterium M963 AY916220 uncultured bacterium ME57 AY916233 uncultured bacterium MF29 AY916238 uncultured bacterium P315 AY91631 1
Uncultured bacterium clone Eldhufecl 35 AY920010
Uncultured bacterium clone Eldhufecl 52 AY920027
Uncultured bacterium UC7-3 AJ608221
Uncultured bacterium UC7-50 AJ608242 uncultured bacterium cadhufec56cl0 AF530376 uncultured bacterium ABLCf44 AF499907
Bryantella formatexigens AJ318527 uncultured bacterium HuRC75 AY684376 uncultured bacterium Adhufecl24abh AY471692
Butyrivibrio crossotus et rel. bacterium adhufec406 AF 132269
Eubacterium ramulus AJO 1 1522
Butyrivibrio crossotus X89981 uncultured bacterium D680 AY916379 uncultured bacterium D692 AY916380 uncultured bacterium D726 AY916383 uncultured bacterium D738 AY916384 uncultured bacterium MG71 AY916289
Uncultured bacterium clone Eldhufecl 38 AY920013
Uncultured bacterium clone Eldhufecl 55 AY920030
Uncultured bacterium clone Eldhufecl 16 AY919991
Uncultured bacterium clone Eldhufecl 14 AY919989
Uncultured bacterium clone Eldhufecl 12 AY919987
Uncultured bacterium clone Eldhufecl 47 AY920022
Uncultured bacterium clone Eldhufec244 AY9201 19 uncultured bacterium Adhufec023abh AY471673 uncultured bacterium Adliufec 1 12rbh AY471715 uncultured bacterium Muc3-1 AY451999
Clostridium uncultured human gut bacterium JW1G3 AB080863 glycyrrhizinilyticum et rel. uncultured human gut bacterium J 1A12 AB080860 uncultured bacterium NP09 AY916252 uncultured bacterium HuCC43 AJ315487
Uncultured bacterium clone Eldhufecl25 AY920000
Uncultured bacterium clone Eldhufecl23 AY919998 uncultured bacterium cadhufec69cl0 AF530380 uncultured bacterium cadhufecl01h7 AF530314 uncultured bacterium HuRC 12 AY684370
Clostridium glycyrrhizinilyticum AB233029
Clostridium lactifermentans et uncultured bacterium G075 AY916283 rel. uncultured bacterium K305 AY916194 uncultured bacterium NK21 AY916240
Uncultured bacterium clone Eldhufecl41 AY920016
Uncultured bacterium clone Eldhufecl 82 AY920057
Uncultured bacterium clone Eldliufecl83 AY920058 uncultured bacterium HuDI72 AY684405 uncultured bacterium HuDI23 AY684406
Clostridium lactatifermentans AY033434
Clostridium nexile et rel. butyrate-producing bacterium A2-231 AJ270484
Clostridium nexile X73443 uncultured Gram-positive bacterium NB4C3 AB064747 uncultured Gram-positive bacterium N02-4 AB064746 uncultured Gram-positive bacterium N031 AB064743 uncultured Gram-positive bacterium N081 AB064742 uncultured bacterium OLDB-F3 AB099735 uncultured bacterium cadhufec20a04 AF530331 uncultured bacterium LCRC24 AF499855 uncultured bacterium ABLC1 AF499881 uncultured bacterium ABLCf89 AF499909
Clostridium sphenoides et rel. bacterium A21 AF052418 bacterium A54 AF052421 bacterium adhufec382 AF132267
Clostridium sphenoides X73449 uncultured Gram-positive bacterium NB2A8 AB064730 uncultured Gram-positive bacterium N02-2 AB064727 uncultured bacterium HuCA27 AJ408978 uncultured bacterium HuCA19 AJ408971 uncultured bacterium HuCA17 AJ408969 uncultured bacterium LCLC63 AF499839 uncultured bacterium LCLC23 AF499838 uncultured bacterium ABLC30 AF499880 uncultured bacterium ABLCfl 1 AF499906
Clostridium hathewayi AJ31 1620 uncultured bacterium Adhufec088khh AY471662
Clostridium symbiosum et rel. Clostridium clostridiiformes M59089
Clostridium symbiosum M59112
Clostridium sp. CJ23 AB080893 uncultured bacterium B147 AY916315 uncultured bacterium B395 AY916317 uncultured bacterium B840 AY916323 uncultured bacterium K375 AY916197 uncultured bacterium L812 AY916282 uncultured bacterium MB66 AY916225 uncultured bacterium MD61 AY916228 uncultured bacterium MI29 AY916299 uncultured bacterium HuCC34 AJ315486
Uncultured bacterium clone Eldhufecl49 AY920024
Uncultured bacterium clone Eldhufecl 15 AY919990
Uncultured bacterium clone EldhufeclOO AY919975 uncultured bacterium inhufecA-32 AY328366 uncultured bacterium LCTI22 AF499870
Clostridium asparagiforme AJ582080
Clostridium bolteae AJ508452 butyrate-producing bacterium M62/1 AY305309 uncultured bacterium M985 AY983861
Coprococcus catus et rel. butyrate-producing bacterium L2-10 AJ270486 uncultured human gut bacterium JW1B8 AB080861 uncultured bacterium K089 AY916135 uncultured bacterium NW71 AY916309
Uncultured bacterium UC7-62 AJ608243 uncultured bacterium cadhufec098h7 AF530312
Coprococcus catus AB038359
Coprococcus eutactus et rel. Eubacterium ruminantium AB008552 bacterium A57 AF052422 bacterium adhufecl 57 AF132241 butyrate-producing bacterium A2-166 AJ270489
Coprococcus eutactus D14148 uncultured Ruminococcus sp. NB2B8 AB064761
Uncultured bacterium UC7-8 AJ608226
Dorea formicigenerans et rel. Clostridium scindens AB020727
Clostridium hylemonae AB023972 bacterium A71 AF052423
Dorea formicigenerans L34619 uncultured Gram-positive bacterium NS2C1 AB064738 human intestinal firmicute C039 AB064889 uncultured human gut bacterium JWlH4b AB080873 uncultured bacterium KW79 AY916215 uncultured bacterium N874 AY916190 uncultured bacterium HuCB21 AJ408996
Dorea longicatena AJ132842
Eubacterium hallii et rel. Eubacterium hallii L34621 uncultured bacterium HuCB26 AJ409000 uncultured bacterium HuCC15 AJ315482 uncultured bacterium Adhufecl 06khh AY471665 uncultured bacterium Adhufecl 27rbh AY471720 bacterium ucfecDC6
Eubacterium rectale et rel. Butyrivibrio fibrisolvens AB004910
Eubacterium rectale L34627 uncultured bacterium D522 AY916373 uncultured bacterium M372 AY916154 uncultured bacterium HuCB37 AJ409004 uncultured bacterium HuCA8 AJ408964
Uncultured bacterium clone Eldhufecl30 AY920005
Uncultured bacterium clone Eldhufecl21 AY919996
Lachnobacterium sp. wal 14165 AJ518873 uncultured bacterium A22 AF052419
Eubacterium ventriosum et rel. bacterium adliufec335 AF132262
Eubacterium ventriosum L34421 uncultured bacterium D177 AY916360
Lachnobacillus bovis et rel. bacterium Al l AF052412 bacterium adhufec68 AF132278 uncultured bacterium B558 AY916318 uncultured bacterium D695 AY916382 uncultured bacterium ME 1 1 AY916230
Uncultured bacterium clone Eldhufecl39 AY920014
Uncultured bacterium clone Eldhufecl37 AY920012
Uncultured bacterium clone Eldhufecl53 AY920028
Uncultured bacterium clone Eldhufecl 18 AY919993
Lachnospira pectinoschiza et Lachnospira pectinoschiza LI 4675 rel. Eubacterium eligens L34420 uncultured bacterium LZ58 AY916189
Uncultured bacterium clone Eldhufecl 40 AY920015
Uncultured bacterium clone Eldhufecl05 AY919980
Uncultured bacterium UC7- 131 AJ608250 uncultured bacterium ABLCf6 AF499905
Outgrouping Clostridium bacterium adliufec236 AF132250 cluster XlVa bacterium adhufec295 AF132257 bacterium adhufec405 AF132268 bacterium adhufec52 AF132274
Clostridium aminovalericum M23929 uncultured human gut bacterium JW1C1 AB080872 uncultured human gut bacterium JW1D8 AB080871 uncultured bacterium LL95 AY916262 uncultured bacterium MK42 AY916301 uncultured bacterium N322 AY916273 uncultured bacterium NL43 AY916244 uncultured bacterium V213 AY916275 uncultured bacterium HuCB56 AJ409006
Uncultured bacterium clone Eldhufecl 29 AY920004
Uncultured bacterium clone Eldhufecl 84 AY920059
Uncultured bacterium clone Eldhufecl 1 1 AY919986 butyrate-producing bacterium SS3/4 AY305316 uncultured bacterium HuAC36 AY684386 uncultured bacterium Adhufec004abh AY471670 uncultured bacterium Adhufec07 lrbh AY471707 uncultured bacterium Muc3 - 13 AY452004
Roseburia intestinalis et rel. butyrate-producing bacterium A2-183 AJ270482
Uncultured bacterium clone Eldhufecl22 AY919997 butyrate-producing bacterium M72/1 AY305310
Roseburia intestinalis AJ312385
Ruminococcus gnavus et rel. Eubacterium contortum L34615
Ruminococcus gnavus L76597
Ruminococcus torques L76604
Clostridium oroticum M59109
Ruminococcus sp. CJ60 AB080891 uncultured human gut bacterium JWlH4a AB080862 uncultured bacterium (human infant) L37A AF253389 uncultured bacterium Adhufecl 17rbh AY471716 uncultured bacterium Muc2-3 AY451997
Ruminococcus hansenii et rel. Ruminococcus productus D14144
Clostridium coccoides M59090
Ruminococcus hansenii M591 14
Ruminococcus hydrogenotrophicus X95624 uncultured bacterium KS62 AY916137
Ruminococcus lactaris et rel. bacterium adhufec80.25 AF153858
Ruminococcus lactaris L76602 uncultured bacterium Gl 87 AY916284 uncultured bacterium LI 60 AY916218 uncultured bacterium HuRC19 AY684372
Ruminococcus luti et rel. butyrate-producing bacterium T2-132 AJ270483 uncultured Ruminococcus sp. N03 AB064755 uncultured Ruminococcus sp. NB2F4 AB064753 uncultured Ruminococcus sp. N02-22 AB064751 uncultured bacterium El 77 AY916259 uncultured bacterium KS90 AY916138 uncultured bacterium L068 AY916217 uncultured bacterium HuCA5 AJ408961
Uncultured bacterium clone Eldhufecl06 AY919981
Uncultured bacterium UC7-36 AJ608238
Uncultured bacterium UC7-7 AJ608225
Ruminococcus luti AJ133124 uncultured bacterium adhufec30.25 AF153854 uncultured bacterium Adhufec086abh AY471687 uncultured bacterium Adhufec048abh AY471680
Ruminococcus obeum et rel. bacterium adhufec35.25 AF153853
Ruminococcus obeum L76601 uncultured Ruminococcus sp. N067 AB064763 uncultured bacterium KZ22 AY916216 uncultured bacterium NL49 AY916245 uncultured bacterium NQ96 AY916255 uncultured bacterium VI 27 AY916274
Uncultured bacterium UC7-35 AJ608237 uncultured bacterium Muc 1 -21 AY451996 uncultured bacterium Muc 1-1 1 AY451995 uncultured bacterium Muc3-10 AY452003 uncultured bacterium Muc3-5 AY452001 uncultured bacterium Muc6-16 AY452019 uncultured bacterium Muc6-13 AY452017 bacterium ucfecDB7
Unclutured Ruminococci uncultured Ruminococcus sp. NS2E3 AB064750 uncultured human gut bacterium JW1 B 1 1 AB080869 uncultured human gut bacterium JW1H7 AB080868 uncultured bacterium K379 AY916198 uncultured bacterium ME 10 AY916229 uncultured bacterium HuCB25 AJ408999 uncultured bacterium HuCA26 AJ408977 uncultured bacterium HuCA2 AJ408958
Uncultured bacterium clone Eldhufecl32 AY920007
Uncultured bacterium clone Eldhufecl33 AY920008
Uncultured bacterium clone Eldhufecl02 AY919977
Uncultured bacterium UC7-23 AJ608235 uncultured bacterium cadhufecl02cl0 AF530364 uncultured bacterium cadhufec028h7 AF530301 uncultured bacterium A20 AF052417 uncultured bacterium A14 AF052415 uncultured bacterium HuDI20 AY684379 uncultured bacterium (human infant) LI 27 AF253374 uncultured bacterium (human infant) P36G AF253346 uncultured bacterium (human infant) P36H AF253344 uncultured bacterium Adhufecl 23khh AY471668 uncultured bacterium Muc3-9 AY452002 uncultured bacterium Muc4-13 AY452010 bacterium ucfecDB 13
Clostridium Eubacterium limosum et rel. Pseudoramibacter alactolyticus AB036759 cluster XV Eubacterium limosum AF064242
Eubacterium barkeri M23927
Anaerofustis stercorihominis AJ518871
Eubacterium sp. CS1 Van AJ518868
Clostridium Eubacterium biforme et rel. uncultured bacterium D 196 AY916362 cluster XVI Uncultured bacterium clone Eldhufec204 AY920079 Uncultured bacterium clone Eldhufec206 AY920081 butyrate-producing bacterium SM7/1 1 AY305313
Eubacterium biforme M59230 uncultured Gram-positive bacterium NB2C7 AB064867
Eubacterium cvlindroides et Eubacterium cylindroides L34616 rel. Eubacterium dolichum L34682
Eubacterium tortuosum L34683 Clostridium innocuum M23732
Solobacterium moorei et rel. Holdemania flliformis Yl 1466 uncultured bacterium M615 AY916164
Uncultured bacterium clone Eldhufec205 AY920080
Solobacterium moorei AY044916
Clostridium Catenibacterium Lactobacillus vitulinus M23727 cluster XVII Lactobacillus catenaformis M23729 human intestinal firmicute CB 12 AB064934
Uncultured bacterium clone Eldhufec203 AY920078
Catenibacterium mitsuokai AB030226
Clostridium Clostridium ramosum et rel. Clostridium cocleatum AF028350 cluster XVIII Clostridium ramosum M23731
Clostridium spiroforme X73441
Uncultured bacterium clone Eldhufec200 AY920075
Clostridium sp. 14774 AJ315981
Coprobacillus catenaformis et Coprobacillus catenaformis AB030218 rel. uncultured bacterium KU74 AY916140 uncultured bacterium NI20 AY916175 uncultured bacterium LCLC 16 AF499845
Uncultured Uncultured Clostridiales I uncultured human gut bacterium JW2B4 AB080852
Clostridiales uncultured bacterium OLDA-F7 AB099784 uncultured bacterium OLDB-A9 AB099783 uncultured bacterium OLDCA-1 AB099781 uncultured bacterium C 118 AY916326 uncultured bacterium C257 AY916329 uncultured bacterium C627 AY916340 uncultured bacterium D049 AY916352 uncultured bacterium D279 AY916363 uncultured bacterium D693 AY916381 uncultured bacterium LH65 AY916208 uncultured bacterium M220 AY916150 uncultured bacterium M233 AY916151 uncultured bacterium M412 AY916156 uncultured bacterium M621 AY916165 uncultured bacterium MF22 AY916236 uncultured bacterium MF35 AY916239 uncultured bacterium MG86 AY916291 uncultured bacterium NH06 AY916173
Uncultured bacterium clone Eldliufec312 AY920187
Uncultured bacterium clone Eldhufec309 AY920184
Uncultured bacterium clone Eldhufec31 1 AY920186
Uncultured bacterium clone Eldhufec308 AY920183
Uncultured bacterium clone Eldhufec310 AY920185
Uncultured bacterium clone Eldhufec314 AY920189
Uncultured bacterium UC7-9 AJ608227
Uncultured bacterium UC7-127 AJ608249
Uncultured Clostridiales Ila uncultured human gut bacterium JW2H12 AB080880 uncultured bacterium OLDB-C2 AB099778 uncultured bacterium C736 AY916346 uncultured bacterium LQ86 AY916269 uncultured bacterium M501 AY916160
Uncultured bacterium clone Eldhufec333 AY920208
Uncultured bacterium clone Eldhufec322 AY920197
Uncultured bacterium clone Eldhufec332 AY920207
Uncultured Clostridiales lib uncultured human gut bacterium JWl HI 1 AB080881 uncultured human gut bacterium JW1B2 AB080879 uncultured bacterium OLDB-H1 AB099779 uncultured bacterium OLDB-F4 AB099777 uncultured bacterium C583 AY916338 uncultured bacterium C655 AY916341 uncultured bacterium D1 1 AY916361 uncultured bacterium 342 AY916195 uncultured bacterium M403 AY916155 uncultured bacterium MH87 AY916298 uncultured bacterium MM92 AY916304 uncultured bacterium HuCA6 AJ408962
Uncultured bacterium clone Eldhufec328 AY920203
Uncultured bacterium clone Eldhufec323 AY920198
Uncultured bacterium clone Eldhufec334 AY920209
Uncultured bacterium clone Eldhufec330 AY920205
Uncultured bacterium clone Eldhufec331 AY920206
Uncultured bacterium clone Eldhufec336 AY92021 1
Uncultured bacterium clone Eldhufec327 AY920202
Uncultured bacterium clone Eldhufec325 AY920200
Uncultured bacterium clone Eldhufec324 AY920199
Uncultured bacterium clone Eldhufec326 AY920201 uncultured bacterium cadhufec008h7 AF530296 uncultured bacterium cadhufecl8c08 AF530351 uncultured bacterium cadhufecl7tO5 AF530343 uncultured bacterium AdhufecO 15rbh AY471695 uncultured bacterium Adhufecl02abh AY471690 uncultured bacterium Adhufecl23rbh AY471719
Uncultured Uncultured Mollicutes bacterium adhufec202 AF132232
Mollicutes bacterium adhufec279 AF132233 uncultured bacterium C027 AY916325 uncultured bacterium CI 33 AY916328 uncultured bacterium C611 AY916339 uncultured bacterium C754 AY916348 uncultured bacterium D051 AY916353 uncultured bacterium D423 AY916369 uncultured bacterium LW88 AY916186 uncultured bacterium MCI 2 AY916226 uncultured bacterium NB 12 AY916191
Uncultured bacterium clone Eldhufec209 AY920084
Uncultured bacterium clone Eldhufec207 AY920082
Uncultured bacterium clone Eldhufec208 AY920083
Cyanobacteria Uncultured Chroococcales uncultured bacterium MO 19 AY916143
Fusobacteria Cetobacterium Cetobacterium somerae AJ438155
Fusobacterium Fusobacterium necrophorum AF044948
Fusobacterium naviforme AJ006965
Fusobacterium gonidoformans M58679
Fusobacterium mortiferum M58680
Fusobacterium varium M58686
Fusobacterium nucleatum X55404
Fusobacterium necrogenes X55408
Fusobacterium russii X55409
Clostridium rectum X77850 uncultured bacterium HuJJIO AY684429
Leptotrichia Leptotrichia bucallis L37788
Alpha- Methylobacterium uncultured bacterium ABLCfl4 AF499910
Proteobacteria Novosphingobium uncultured bacterium ABLCf85 AF499911
Oceanospirillum uncultured bacterium D623 AY916377 uncultured bacterium D784 AY916388 uncultured bacterium MK72 AY916302 uncultured bacterium V326 AY916278
Beta- Alcaligenes faecalis et rel. Achromobacter denitrificans AF232712
Proteobacteria uncultured bacterium ABLC 15 AF499888
Alcaligenes faecalis DQ 110882
Kerstersia gyiorum AY131213
Aquabacterium uncultured bacterium ABLC71 AF499885
Burkholderia uncultured bacterium LCLC40 AF499842
Neisseria uncultured bacterium HuJJ55 AY684428
Oxalobacter formigenes et rel. Oxalobacter formigenes U49749 uncultured bacterium ABLC55 AF499887 Sutterella wadsworthia et rel. Sutterella wadsworthia
uncultured bacterium D093
uncultured bacterium Ml 05
uncultured bacterium HuCA4
uncultured bacterium HuCC33
Uncultured bacterium clone Eldhufec064
Uncultured bacterium clone Eldhufec063
uncultured bacterium ABLC72
uncultured bacterium HuDI12
Gamma- Aeromonas veronii AF099024 Proteobacteria Aeromonas Aeromonas enteropelogenes S42871
Anaerobiospirillum Anaerobiospirillum thomasii AJ420985
Anaerobiospirillum succiniciproducens U96412
Enterobacter aerogenes et rel. Enterobacter aerogenes AB004750
Citrobacter freundii AF025365 Citrobacter koseri AF025366 Citrobacter braakii AF025368 Citrobacter werkmanii AF025373 Tatumella ptyseos AJ233437 Raoultella terrigena Y17658 Klebsiella oxytoca Y17660 Raoultella planticola Y17663 Enterobacter cancerogenus Z96078 uncultured bacterium OLDA-E9 AB099791 Citrobacter gillenii AF025367 Citrobacter murliniae AF025369 Averyella dalhousiensis DQ481464
Escherichia coli et rel. Escherichia coli
Edwardsiella tarda
Citrobacter sedlakii
Citrobacter farmeri
Salmonella enterica
Shigella flexneri
Shigella dysenteriae
Uncultured bacterium clone Eldhufec069
Cedecea davisae
Escherichia fergusonii
Trabulsiella guamensis
Citrobacter amalonaticus
uncultured bacterium Muc4- 17
Haemophilus Haemophilus haemolyticus M75045
Haemophilus parainfluenzae M75081
Klebsiella pneumoniae et rel. Pantoea agglomerans AB004691
Serratia liquefaciens AB004752 Klebsiella pneumoniae AB004753
Enterobacter cloacae AF157695
Yokenella regensburgei AY269192
Enterobacter asburiae AB004744
Leminorella Leminorella grimontii AJ233421
Moraxellaceae Moraxella catarrhalis A27627
Acinetobacter calcoaceticus AF 159045
Acinetobacter johnsonii AF188300
Acinetobacter haemolyticus Z93437 uncultured bacterium HuJJ26 AY684425 uncultured bacterium HuJJ19 AY684423
Proteus et rel. Providencia stuartii AF008581
Proteus mirabilis AF008582
Proteus vulgaris AJ233425
Morganella morganii AJ301681
Providencia alcalifaciens AJ301684
Providencia rettgeri AM040492
Providencia rustigianii AM040489
Moellerella wisconsensis AM040754
Proteus penneri AJ634474
Pseudomonas Pseudomonas aeruginosa AB037545
Pseudomonas stutzeri AF038653
Pseuodomonas Pseudomonas monteilii AF064458
Pseudomonas fluorescens AJ278813
Pseudomonas putida D84020
Serratia Serratia marcescens M59160
Vibrio Vibrio parahaemolyticus M59161
Grimontia hollisae S83393
Vibrio fluvialis X74703
Vibrio furnissii X74704
Xanthomonadaceae uncultured bacterium ABLCG 1 AF499898 uncultured bacterium ABLC16 AF499891
Yersinia et rel. Yersinia pseudotuberculosis AF282307
Yersinia enterocolitica AF282308
Hafnia alvei M59155
Yersinia frederiksenii X75273
Yersinia rohdei X75276
Yersinia kristensenii X75278
Yersinia bercovieri X75281
Bilophila Bilophila wadsworthia L35148
Proteobacteria Desulfovibrio et rel. Desulfovibrio desulfuricans AF098671
Desulfvibrio piger AF192152 uncultured bacterium D168 AY916359 uncultured bacterium LE30 AY916206
Uncultured bacterium clone Eldhufec073 AY919948
Desulfovibrio fairfieldensis U42221 bacterium ucfecDB 10
bacterium ucfecDB 12
Epsilon- Arcobacter Arcobacter cryaerophilus LI 4624
Proteobacteria Arcobacter butzleri U34386
Campylobacter Campylobacter hominis AF062490
Campylobacter fetus AJ306568
Campylobacter jejuni AL139074
Campylobacter coli L04312
Campylobacter lari L04316
Campylobacter rectus L04317
Campylobacter gracilis L04320
Bacteroides ureolyticus L04321
Campylobacter concisus L04322
Campylobacter upsaliensis L14628
Helicobacter Helicobacter pylori AE000511
Flexispira rappini AF034135
Helicobacter canadensis AF262037
Helicobacter cinaedi AF396082
Helicobacter pullorum L36141
Helicobacter winghamensis AF246984
Lentisphaerae Victivallis Victivallis vadensis AY049713
Spirochaetes Brachyspira Brachyspira aalborgi AF395882
Brachyspira pilosicoli AY155458
Verruco-microbia Akkermansia Uncultured bacterium clone Eldhufec002 AY919877
Akkermansia muciniphila AY271254 uncultured bacterium HuRC51 AY684431
Examples
Example 1. Comparison of the fecal microbiota of IBS and healthy subjects (Study 1) Fecal samples were obtained from a first study (Study 1) of a total of 62 IBS subjects including 19 with IBS-C, 25 with IBS-D and 18 with IBS-A, and a total of 46 healthy individuals that were age and gender matched. Microbial DNA was isolated from these fecal samples following the method of Ahlroos & Tynkynnen (2009, supra) and used for profiling using the HITChip phylogenetic microarray using 3699 distinct HIT probes as described (Rajilic-Stojanovic et al, 2009, supra) . Based on the intenstity of the hybridization signals obtained in the HITChip analysis from the 62 IBS subjects and 46 healthy individuals a total of 36 level 2 microbial groups from the total of over 100 groups was found to be reacting significantly different between IBS and healthy subjects (see Table 1 above). The identified microbial groups can be developed as biomarker as described above. Moreover, the differences in microbiota can be corrected to the healthy level. This can be directly realized by consuming the microbes and/or their proteins or metabolites that are reduced in the IBS subjects, as if they were probiotics. This has already been suggested for Faecalibacterium prauznitzii in the case of IBD and here we extend this approach for said bacteria to the case of IBS (Sokol et al, 2008. Proc Natl Acad Sci U S A 105: 16731-36). In addition, indirect modulation of the presence or absence of specific microbial groups can also be realized by the consumption of pre- and probiotics or its combination. Lastly, for the in the invention identified microbiota that are related to bioactive pathways, these pathways too can be used or targeted for the treatment of IBS. Example 2. Identification of IBS- and Healthy-specific oligonucleotides
In order to further define the specific oligonucleotide probes that were reacting different in the IBS subjects as compared to the healthy controls, the hybridization of all 3,699 HIT probes of the HITChip in Study 1 (Example 1) was analyzed, resulting in a total of 100 HIT probes were found to be differentially hybridizing (Tables 2 and 4). A total of 34 HIT probes (oligonucleotides having SEQ ID Nos: 1-27, 70-71 , 73-77, 99-100) showed a significantly higher hybridization signal in the IBS subjects than the healthy individuals, while a total of 66 (oligonucleotideshaving SEQ ID Nos:28-69, 72, 78-98) showed less hybridization in the IBS subjects than the healthy subjects, respectively. The sequences of these oligonucleotides are disclosed in Tables 2 and 4 and allow the development of specific probes as described above. Moreover, these probes can be used to screen the 16S rDNA databases for complete 16S rRNA sequences that subsequently can be used as target for the development of specific probes as described above. This has been done using the SILVA and RDP databases using the ProbeCheck program (http://131.130.66.200/cgi- bin/probecheck/probecheck.pl). As the discriminating oligonucleotides are used in a hybridization assay, their complementarity to a 16S rRNA gene should not necessarily be perfect and mismatches up to 2 nucleotides can be envisaged. Hence the SILVA and RDP databases were searched for 16S rRNA gene sequences using the discriminating IBS- and Health-specific oligonucleotides allowing up to 2 mismatches. This resulted in multiple hits for each of the oligonucleotides showing the feasibility of this approach. Example 3. Further analysis of the differences in fecal microbiota of IBS and healthy subjects
To further substantiate the differentiation of IBS subjects and healthy controls based on fecal microbiota, a second set of samples was analyzed that included a total of 33 IBS subjects that were not further differentiated and 43 healthy controls that were age and gender matched (Study 2). Fecal samples were obtained from these 77 individuals and microbial DNA was isolated from these following the repeated bead beating method as described (Yu & Morrison, 2004, supra). This DNA was used for profiling using the HITChip phylogenetic micro array using 3699 distinct HIT probes as described (Rajilic- Stojanovic et al, 2009, supra). As the DNA extraction method differed between Study 1 (Example 1) and Study 2 (the results presented here) as an enzymatic and mechanical lysis method was used, respectively, it was of interest to see the differentiation of the datasets obtained from the HITChip analysis in both tests. A Redundancy Analysis (RDA) was performed using all data from both Study 1 and Study 2. The results (Fig. 2) show a remarkable separation between samples from IBS subjects and healthy controls.
This indicates that in spite of being derived from 2 different studies and 2 different DNA extraction methods, the obtained data sets are sufficiently robust to show a clear separation between IBS subjects and healthy controls. Moreover, this analysis demonstrates that it is possible to differentiate IBS subjects from health controls based on biomarkers derived from their intestinal microbiota.
Example 4. Detection and benchmarking diagnostic probes
To further detect and benchmark specific HIT probes that were potential diagnostic markers to differentiate between fecal microbiota of IBS subjects and healthy controls, the data sets obtained from Study 1 and Study 2 were combined. Subsequently, a training data set, consisting of 2/3 of the data, and a test data set, consisting of 1/3 of the data, were randomly selected. The rationale behind this division of the data sets is that the test data are not used at all in the modeling or selection process but only in the final testing. This should protect from over-fitting of the models into the data (i.e. from an inferior generalization). The training data was used to filter out the most discriminating HIT probes using a t-test. These are listed in Table 3. They were used to classify the training set with different classifiers, including stepwise linear discriminant analysis (LDA), a multivariant analysis system (see Venables, W. N. and Ripley, B. D. (2002) Modern Applied Statistics with S. Fourth edition. Springer Publishers). The subsequent classification was done in two nested cross-validation loops, where the inner one was used to select the discriminating features in a stepwise- LDA, and the outer loop to validate the performance of the classifiers for unseen data. The final test simulation was done by applying the stepwise-LDA to all of the training data, and then classifying the 1/3 of the binded test data, and comparing it to the 10 randomized classifications. A clinically meaningful separation could be obtained that When this stepwise LDA was applied to the 1/3 of the blinded test data, a correct classification was realized of 81 % of the samples derived from the IBS subjects. When the obtained result was compared to the randomized classifications (repeated 10 times) using t-test, the difference between the non-randomized classification and the randomized classifications was found to be statistically highly significant (p-value 6.697e-09). This result was obtained with the HIT probes with the SEQ ID No 83 and 88 (Table 4). Hence, this example shows that a clinically meaningful diagnosis could be already realized with the the lowest number of multiple HIT probes, namely two probes.
Table 4. Identification, sequence and analysis of the HIT probes coded SEQ ID 68- 100 that were obtained in the stepwise linear discriminant analysis of various parts of the datasets of Study 1 and Study 2. The oligonucleotides are indicated with their nucleotide sequence (3 'to 5 '). The oligonucleotides with SEQ ID Nos:70-71 , 73-77, 99-100 showed a significantly higher hybridization signal in the IBS than the healthy subjects, whereas the oligonucleotides with SEQ ID Nos:68-69, 72, 78-98 showed the opposite.
SEQ ID
sequence 5' to 3 ' direction (T=U in RNA) NO:
CACCCCTCCTTTTCGGGAG 68
TAAACTACTTCCCGCTGCCGC 69
GCCGCTAATCCACTTCCCGAA 70
TGTCTCATTACGAGCAAGCTCACG 71
GGTCACTCGATGTCAAGACCTG 72
GTCAAAGGAGCAAGCTCCTCG 73
TACGTCACTCGATGTCAAGACCTG 74
TTCGTCACTCGATGTCAAGACCTG 75
AACGTCACTCGATGTCAAGACCTG 76 GCCACTCAGTCATAAAAAACTTCATC 77
GCCACTCAGTCATAAAAAACTTCATTC 78
GCCGCTCAGTCACTTAAGAAATCA 79
CGAAGTCCGTGCTGCCG 80
GCCGCTCAGTCACAAAGACTTCAA 81
AAATCCATCCGAAAACTTCATTTTAATTGC 82
GCCACTCGCCACCAGACC 83
TGTCTCCTCTGTCCGTAGAAAAAA 84
GCCGGTCGCCATCTTTAGTTTG 85
CAAGCTCCCTTTGGTCCGC 86
TGTCACTCTGCTCCCGAAGGA 87
TGTCTCTCTGTTCCCGAAGGAAA 88
TGTCTTCCTGCCCCGAAGC 89
GACATCATGCACCTCTGCACTATG 90
GCCACTCGTCACCGAAGGA 91
AGCAAGCTCCCTTCATCCGC 92
CACCGCCTCATCTCCGAG 93
GCCACTCGCCACCAGGTG 94
TGTCTCTCTGTTCCCGAAGGAAAC 95
TGTCACTCTGTTCCCGAAGAAC 96
GCCACCCAGTCACTTGAGC 97
CCACTCGCCACCAGGG 98
CCGCCAGGATTGCTCCCG 99
TGTCTCGTATTGAGCAAGCTCACA 100
Example 5
To further substantiate that combinations of HIT probes can be used in a diagnostic test to differentiate IBS subjects from healthy controls using all 185 subjects derived from Study 1 and Study 2, a number of these were analysed in a hierarchical analysis. The power of combining four discriminating HIT probes could be easily illustrated in a hierarchial decision tree (Fig. 2). It could be shown that hybridization to HIT probe with ID Seq 80 and its cut off at a certain hybridization value allowed to assign correctly 34 of healthy controls as healthy and 3 IBS subjects falsely. Similarly, a second HIT probe with ID Seq 77 could be used for further differentiating the remaining 148 subjects and could assign 18 healthy controls correctly and 5 IBS ones falsely. Subsequently, a third HIT probe with ID Seq 72 could be used to differentiate the remaining 125 subjects and could assign 63 IBS subjects correctly and 17 healthy controls incorrectly. Finally, ID Seq 90 could be applied to differentiate the remaining 45 subjects and this resulted in the correct assignment of 13 Healthy controls and 18 IBS subjects, while 6 IBS subjects and 8 healthy controls were falsely assigned. Altogether the use of these 4 HIT probes resulted in the correct classification of 85 % of the IBS subjects. For those experienced in the art it will be evident that a strict classification can be obtained by using combinations of several of the HIT probes in conjunction with different cut-off values.
The probes that added significant value to the first classification (Fig. 2) were the probes 72, 77 and 90 that are specific for the bacterial taxa including Eubacterium sireaeum et rel., Lachnospira pectinoschiza et rel. and Subdoligranulum variabile et rel. , respectively. These bacterial taxa already had been identified in a separate analysis when addressing Study 1 (see Table 1). This result testifies for the power of diagnosing IBS by determining the level of various and different groups of IBS- increased or IBS- decreased bacteria and using these in a decision tree as described here.

Claims

1. A method for diagnosing and/or subtyping Irritable Bowel Syndrome (IBS) in a test sample, said method comprising the steps of:
a) determining the levels of two or more bacteria which are present in statistically significantly different levels between IBS subjects and healthy subjects, said bacteria being selected from IBS-decreased bacteria and IBS-increased bacteria, said IBS-decreased bacteria being selected from bacteria belonging to the supertaxon Bacteroidetes, selected from the taxa Prevotella melaninogenica et rel, Prevotella oralis et rel, Uncultured Bacteroidetes, Tannerella et rel., Parabacteroides distasonis et rel, Allistipes et rel, Bacteroides plebeius et rel., Bacteroides splachnicus et rel, or to the supertaxon Clostridium cluster IV, selected from the taxa Subdoligranulum variabile et rel, Faecalibacterium prausnitzii et rel, Oscillospira guillermondii et rel, Sporobacter termitidis et rel, Ruminococcus callidus et rel, Eubacterium siraeum et rel, Anaerotruncus colihominis et rel, Clostridium cellulosi et rel, Clostridium leptum et rel, Ruminococcus bromii et rel, or to the supertaxon Clostridium cluster IX, said bacteria belonging to the taxon Phascolarctobacterium faecium et rel; or to the supertaxon Clostridium cluster XVI, said bacteria belonging to the taxon Eubacterium biforme et rel; or to the supertaxon Clostridium cluster XVII, said bacteria belonging to the taxon Catenibacterium mitsuokai et rel; or to the supertaxon Proteobacteria, said bacteria belonging to the taxon Xanthomonadaceae; or to the supertaxon Uncultured Clostridiales, selected from the taxa Uncultured Clostridiales I and Uncultured Clostridiales II; or to the supertaxon Uncultured Mollicutes, said bacteria belonging to the taxon Uncultured Mollicutes, and said IBS-increased bacteria being selected from bacteria belonging to the supertaxon Clostridium cluster XlVa, selected from the taxa Dorea formicigenerans et rel, Ruminococcus obeum et rel, Clostridium nexile et rel, Clostridium symbiosum et rel, Outgrouping Clostridium cluster XlVa, Ruminococcus lactaris et rel, Lachnospira pectinoschiza et rel; in a test sample; b) Comparing said level of said two or more IBS-decreased and/or IBS-increased bacteria in said test sample to a level of said two or more IBS-decreased and/or IBS-increased bacteria in a control sample; and
cl) relating a decreased level of said IBS-decreased bacteria and/or an increased level of said IBS-increased bacteria in the test sample compared to the control sample to a diagnosis that the test sample is from a subject suffering from Irritable Bowel Syndrome; and/or
c2) relating an increased level of said IBS-increased bacteria or a decreased level of said IBS-decreased bacteria in the test sample compared to the control sample to a diagnosis of whether the test sample is from a subject suffering from IBS-A,
IBS-C, or IBS-D.
2. A method according to claim 1, wherein in step a) the levels of at least one IBS- increased bacteria and at least one IBS-decreased bacteria are determined.
3. A method according to claim 2, wherein in step a) the level of at least one IBS- increased bacteria selected from bacteria belonging to the taxa Dorea formicigenerans et rel., Ruminococcus obeum et rel., and Lachnospira pectinoschiza et rel., and the level of at least one IBS-decreased bacteria selected from bacteria belonging to the taxa Prevotella melaninogenica et rel, Prevotella oralis et rel, and Catenibacterium mitsuokai et rel, are determined.
4. A method according to claim 3, wherein in step a) at least the level of bacteria belonging to the taxa Dorea formicigenerans et rel., Ruminococcus obeum et rel., and Lachnospira pectinoschiza et rel., and the level of bacteria belonging to the taxa Prevotella melaninogenica et rel, Prevotella oralis et rel, and Catenibacterium mitsuokai et rel, are determined.
5. A method according to any of the preceding claims, wherein the level of said one or more bacteria is measured by determining the level of nucleic acid sequences, amino acid sequences and/or metabolites specific for said one or more bacteria in said test sample.
6. A method according to claim 5, wherein the level of nucleic acid sequences specific for said one or more bacteria are determined using PCR or LCR.
7. A method for diagnosing and/or subtyping Irritable Bowel Syndrome (IBS) in a test sample, said method comprising the steps of:
i) providing a test sample;
ii) determining the level of at least three nucleic acids capable of hybridising to at least three nucleic acid sequences selected from the nucleic acid sequences of SEQ ID Nos: 1-100, or derivatives or fragments thereof deviating by at most 2 nucleotides, and complements, reverse, and reverse complements thereof, under stringent hybridization conditions, in said test sample;
ii) comparing the level of said at least three nucleic acids from said test sample to the level of said at least three nucleic acids from a control sample; and
iiia) relating the level of said at least three nucleic acids from said test sample to a diagnosis of whether the test sample is from a subject suffering from Irritable Bowel
Syndrome; and/or
iiib) relating the level of said at least three nucleic acids from said test sample to a diagnosis of whether the test sample is from a subject suffering from IBS-A, IBS-C, or IBS-D.
8. A method according to claim 7, wherein in step iiia) an increased level of nucleic acids from said test sample, said nucleic acids being capable of hybridising to nucleic acid sequences selected from the nucleic acid sequences of SEQ ID Nos: 1-27, 70-71, 73-77, 99-100, or derivatives or fragments thereof deviating by at most 2 nucleotides, and complements, reverse, and reverse complements thereof, under stringent hybridization conditions, compared to the level of said nucleic acids from said control sample relates to the diagnosis that the subject is suffering from IBS.
9. A method according to any one of claim 7 or 8, wherein in step iiia) a decreased level of nucleic acids from said test sample, said nucleic acids being capable of hybridising to nucleic acid sequences selected from the nucleic acid sequences of SEQ ID Nos:28-69, 72,78-98, or derivatives or fragments thereof deviating by at most 2 nucleotides, and complements, reverse, and reverse complements thereof, under stringent hybridization conditions, compared to the level of said nucleic acids from said control sample relates to the diagnosis that the subject is suffering from IBS.
10. A method according to any one of claims 7-9, wherein the level of at least 6 nucleic acid sequences from said test sample is determined.
11. A method according to any one of claims 7-10, wherein Significance Analysis of Microarrays (SAM) is used in comparing the levels of said three or more nucleic acid sequence from said test sample with the levels of said three or more nucleic acid sequence from a control sample.
12. A method according to any one of claims 7-10, wherein Prediction Analysis of Microarray (PAM) is used in comparing the levels of said three or more nucleic acid sequence from said test sample with the levels of said three or more nucleic acid sequence from a control sample.
13. A method according to any one of claims 7-10, wherein Redundancy Analysis is used in comparing the levels of said three or more nucleic acid sequence from said test sample with the levels of said three or more nucleic acid sequence from a control sample.
14. A method for diagnosing and/or subtyping Irritable Bowel Syndrome (IBS) in a test sample, said method comprising the steps of:
i) providing a test sample;
ii) determining the level of at least three nucleic acids capable of hybridising to
16S rRNA nucleic acid sequences hybridizing to the complementary strand of any of the nucleic acid sequences SEQ ID NO.: 1-100 or fragments of said 16S rRNA nucleic acid sequences hybridizing to the complementary strand of any of the nucleic acid sequences SEQ ID NO.: 1-100, and complements, reverse, and reverse complements thereof, under stringent hybridization conditions, in said test sample;
ii) comparing the level of said at least three nucleic acids from said test sample to the level of said at least three nucleic acids from a control sample; and iiia) relating the level of said at least three nucleic acids from said test sample to a diagnosis of whether the test sample is from a subject suffering from Irritable Bowel Syndrome; and/or
iiib) relating the level of said at least three nucleic acids from said test sample to a diagnosis of whether the test sample is from a subject suffering from IBS-A, IBS-C, or IBS-D.
15. A method according to any one of claims 7-14, wherein the level is determined using a method selected from: hybridization of the nucleic acids in a sample to the nucleic acid sequences having SEQ ID NO. : 1-100, and complements, reverse, and reverse complements thereof, under stringent hybridization conditions; a Polymerase Chain reaction (PCR) or a Ligase Chain Reaction (LCR).
16. An array for diagnosing IBS and/or subtyping IBS-A, IBS-C, or IBS-D, said array comprising at least two nucleic acid sequences having the nucleic acid sequence of SEQ ID NOs: 1-100, or derivatives or fragments thereof deviating by at most 2 nucleotides, or complements, reverse, and reverse complements thereof.
17. An array according to claim 16, which comprises at least two nucleic acid sequences selected from the nucleic acid sequences having SEQ ID Nos: 1-100.
18. An array according to any one of claims 16 or 17, wherein the at least two nucleic acid sequences are bound to a solid phase matrix.
19. An array according to any one of claims 16-18, wherein the array is a DNA or RNA array.
20. An array according to any one of claims 16-19,which is a micro-array.
21. Use of an array according to any one of claims 16-20 for diagnosing IBS and/or subtyping IBS-A, IBS-C, or IBS-D.
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