WO2012039615A2 - Commensal rat ileum bacterium (crib) - Google Patents

Abstract

The invention relates to an isolated commensal rat ileum bacterium (CRIB) that comprises one or more of the following sequences: a. SEQ ID NO: 1 or the complement sequence thereof; b. SEQ ID NO: 2 or the complement sequence thereof; c. SEQ ID NO: 3 or the complement sequence thereof; d. SEQ ID NO: 21 or the complement sequence thereof; e. a sequence having a sequence identity of more than 97,2 % with SEQ ID NO:1 or its complement; f. a sequence having a sequence identity of more than 97,2 % with SEQID NO:2 or its complement; g. a sequence having a sequence identity of more than 97,2 % with SEQ ID NO: 3 or its complement; h. a sequence having a sequence identity of more than 97,2 % with SEQ ID NO:21 or its complement; i. a sequence selected form SEQ ID NO: 22 to SEQ ID NO: 130; j. a sequence that has an identity of more than 70% with a stretch of 1000 consecutive nucleotides from any of the sequences from SEQ ID NO: 22 to SEQ ID NO: 130; k. a sequence that has an identity of more than 70% with any of the sequences from SEQ ID NO: 22 to SEQ ID NO: 130, provided that said sequence has a length of more than 1000 nucleotides 1. a sequence that has an identity of more than 70% with the total of sequences formed by SEQ ID NO: 22 to SEQ ID NO: 130. The invention further relates to use of said bacterium in a probiotic composition and/or for prevention or treatment of pancreatitis, inflammatory bowel disease (IBD) like Crohn's disease, pouchitis, necrotizing enterocolitis, proctitis, ulcerative colitis, or irritable bowel syndrome (IBS). It can also be used as treatment of Celiac disease, food allergy, dysbacteriosis, cholera and diabetes type 1.

Description

Title: Commensal rat ileum bacterium (CRIB)

The invention relates to the fields of medicine, bacteriology and health science.

BACKGROUND OF THE INVENTION

Probiotics, are defined by the FAO and the WHO as living microorganisms which, when administered in adequate amounts, confer a beneficial health effect on the host. Addition of these probiotic bacteria to the diet is an approach for increasing the health of the individual human or animal by promoting the presence of beneficial bacteria in the intestine. Basically probiotics act on three different levels in the intestine; they influence the microbial environment (microbe- microbe interaction), they influence the gut barrier function (microbe- gut epithelium interaction) and they are able to modulate the immune system ( microbe-immune interaction).

The roles, which probiotics have on microbe- microbe interactions include among others competition for nutrients and colonization areas with harmful micro- organisms including pathogenic bacteria but also Candida. Probiotics can act for example by production of antimicrobial substances, such as bacteriocins and/or the generation of restrictive physiological conditions, including acidification, caused by lactic acid and other fermentations (Teitelbaum et al. 2002. Nutritional impact of pre- and probiotics as protective gastrointestinal organisms. Annual Reviews Nutrition 22:107-138).

On improving the intestinal barrier function probiotics increase mucus production and strengthen tight junctions and preserve them in structure and function. Improving barrier function is desirable since it prevents leakage of larger molecules from the gut into the system.

Probiotics also have shown to be able to modulate the immune system.

Activating the immune response depends on recognition of microbe-associated molecular patterns (MAMPs) through pattern recognition receptors like Toll-like receptors. Numerous studies have shown that probiotics can increase antiinflammatory and suppress pro -inflammatory cytokines. Probiotics have been used as prevention and/or treatment of several disorders like diarrhoea, constipation, IBD, IBS, allergy, etc.

Use of probiotics as dietary intervention is partly based on the concept that specific strains selected from the healthy gut microbiota may have powerful anti- pathogenic and anti-inflammatory properties, and therefore may provide resistance to intestinal diseases (Isolauri et al. 2002. Probiotics: a role in the treatment of intestinal infection and inflammation? Gut 50:54-59). In monogastric animals, including humans, commensal microbiota contribute to intestinal protection against pathogens by competition for nutrients and pathogen binding sites, and/or regulation of immune response.

Many probiotic compositions are known, since early history. The oldest probiotics are components of products we now recognize as 'normal' food, such as yoghurt, tempeh and some juices and soy beverages. An overview of medically used probiotics has been given by Blandino, G. et al., 2008, Probiotics: overview of microbiological and immunological characteristics, Expert Rev. Anti Infect. Ther. 6:497-508.

However, although many beneficial probiotic compositions are already known, there is a continuing interest both from the (bio)medical as well as from the industrial perspective in the identification of additional gut microbiota with a health promoting effect.

SUMMARY OF THE INVENTION

The invention is directed to a bacterium that comprises one or more of the following sequences:

a. SEQ ID NO: 1 or the complement sequence thereof;

b. SEQ ID NO: 2 or the complement sequence thereof;

c. SEQ ID NO: 3 or the complement sequence thereof;

d. SEQ ID NO: 21 or the complement sequence thereof;

e. a sequence having a sequence identity of more than 97,2 % with SEQ

ID NO:l or its complement;

f. a sequence having a sequence identity of more than 97,2 % with SEQ ID NO:2 or its complement; a sequence having a sequence identity of more than 97,2 % with SEQ ID NO: 3 or its complement;

h. a sequence having a sequence identity of more than 97,2 % with SEQ ID NO:21 or its complement;

1. a sequence selected form SEQ ID NO: 22 to SEQ ID NO: 130;

J- a sequence that has an identity of more than 70% with a stretch of 1000 consecutive nucleotides from any of the sequences from SEQ ID NO: 22 to SEQ ID NO: 130;

k, a sequence that has an identity of more than 70% with any of the

sequences from SEQ ID NO: 22 to SEQ ID NO: 130, provided that said sequence has a length of more than 1000 nucleotides

1. a sequence that has an identity of more than 70% with the total of sequences formed by SEQ ID NO: 22 to SEQ ID NO: 130.

A preferred bacterium according to the invention is the commensal rat ileum bacterium (CRIB) deposited under the Budapest Treaty with the DSMZ, on date 29 June 2007 under the accession number 19498. Further preferred is a bacterium that has a DNA-

DNA hybridisation of more than 70% with a bacterium according to the invention. The bacterium according to the invention is preferably used probiotic and/or for maintenance of the normal physiological barrier function and/or maintenance of normal regulation of the immune system and/or for the treatment, treatment- sparing and/or prevention of diseases, disorders, or syndromes related to disturbed barrier function and/or which are immune-mediated

Specifically in the last case, the bacterium is used in the treatment, treatment- sparing and/or prevention of an inflammatory disease, preferably an inflammatory intestinal disease, more preferably, inflammatory bowel disease (IBD) such as Crohn's disease, pouchitis and ulcerative colitis, irritable bowel syndrome (IBS), pancreatitis, celiac disease, food allergy, dysbacteriosis, cholera, diabetes type 1, necrotizing enterocolitis or proctitis.

Also part of the invention is a probiotic composition comprising a probiotic culture having a probiotic count of between 10^s and 10¹¹ cfu/ml or cfu/g comprising a bacterium according to the invention and a carrier medium. In another embodiment, the invention comprises a food suitable for a mammal, preferably a human, comprising a bacterium according to the invention or a probiotic composition according to the invention.

Also comprised in the invention is a method for detecting a bacterium according to the invention comprising detecting the presence of any of the sequences chosen from SEQ ID NO:l, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 21 and their complement sequences and a sequence having a sequence identity of more than 97,2 % with these sequences. In another embodiment, the invention comprises a method for detecting a bacterium according to the invention comprising detecting the presence of any of the sequences chosen from the group of SEQ ID NO:22 to SEQ ID NO: 130, the sequences that have an identity of more than 70% with a stretch of 1000 consecutive nucleotides from any of the sequences from SEQ ID NO: 22 to SEQ ID NO: 130, and sequences that have an identity of more than 70% with the total of the sequences from SEQ ID NO: 22 to SEQ ID NO: 130. In yet another embodiment, the invention comprises a method for detecting a bacterium according to the invention comprising measuring the DNA-DNA hybridisation with a bacterium deposited under the Budapest Treaty with the DSMZ, on date 29 June 2007 under the accession number 19498and testing whether this DNA-DNA hybridisation is more than 70%.

Further, the invention comprises a kit of parts comprising a combination of a first and a second primer selected from the group of:

a. a first primer crib-61f having SEQ ID NO: 4 and a second primer crib- 155r having SEQ ID NO:6 or crib-220r having SEQ ID NO:7 or crib- 235r having a sequence SEQ ID NO:8 or crib-193r having SEQ ID NO:9; or

b. a first primer crib-132f having SEQ ID NO:5 and a second primer crib-

220r having SEQ ID NO:7.

The invention also comprises an isolated and/or recombinant nucleic acid comprising SEQ ID NO:l or the complement sequence thereof, or SEQ ID NO:2 or the complement sequence thereof, or SEQ ID NO:3 or the complement sequence thereof, or SEQ ID NO: 21 or the complement thereof, or a sequence having a sequence identity of more than 97,2 % with SEQ ID NO:l, 2, 3 or 21 or the complement thereof or a sequence according to any of the sequences of SEQ ID NO: 22 to SEQ ID NO: 130. or any of the sequences that have an identity of more than 70% with a stretch of 1000 consecutive nucleotides from any of the sequences from SEQ ID NO: 22 to SEQ ID NO: 130.

Also part of the invention is a method for evaluating the immune status of an animal, comprising determining the presence of a bacterium according to the invention and/or a nucleic acid as defined above in a sample of said animal

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Correlations between the T-RFLP profiles obtained from the ileal samples mapped onto the first three principle components of PCA. Hatched dots indicate healthy rats. Diseased rats are indicated by black dots (placebo treated animals) or gray dots (probiotic treated animals), respectively.

Figure 2. Effect of pancreatitis and treatment with either placebo or the probiotic mixture on the relative ileal abundance of T-RF 457, corresponding to the yet uncharacterized bacterial phylotype referred to as CRIB. Indicated are healthy control (transparent bars) and diseased animals, treated either with placebo (light gray bars) or the probiotic mixture (dark gray bars), respectively. Box- whisker plots represent median with interquartile range (boxes) and minimum and maximum non- outlier values (whiskers). * p<0.05 compared to median.

Figure 3. Correlation between the percentage relative abundance of CRIB

determined by T-RFLP analysis (relative peak intensity of T-RF 457 bp) and qPCR analysis (relative abundance of 16S rRNA gene copies amplified with CRIB-specific primers).

Figure 4. Associations between different measures of disease outcome and the relative ileal abundance of CRIB in diseased rats after seven days of acute

pancreatitis. Animals were treated with either placebo (gray dots) or probiotics (black - lined squares). Indicated are (A) total bacterial counts in the duodenum, (B) bacterial translocation to the MLNs and (C) total pathology of the pancreas. Relative abundance of CRIB was determined by qPCR analysis. Bacterial counts are expressed in loglO colony forming units per gram sample (loglO CFU/g). Pearson's correlation coefficients are provided, with corresponding p-values. The dotted lines indicate the division between the samples with a low (<7.5%) or high (>7.5%) relative ileal abundance of CRIB

587 (>7.5%).).

Figure 5. Association between bacterial translocation to remote organs during acute pancreatitis and the relative ileal abundance of CRIB in diseased rats. Bacterial translocation is indicated by the total bacterial counts in the (a) mesenteric lymph nodes (MLNs) (b) spleen (c) liver and (d) pancreas. Relative abundance of CRIB was determined by qPCR analysis. Bacterial counts are expressed in logio colony forming units per gram sample (logio CFU/g).

Figure 6. Associations between different measures of disease outcome and the relative ileal abundance of CRIB in diseased rats after seven days of acute

pancreatitis. Animals were treated with either placebo (gray dots) or probiotics (black- lined squares). Indicated are bacterial translocation to the (a) spleen, (b) liver and (c) pancreas, and histopathological scores of the pancreas of (d) acinar cell pathology and (e) inflammatory infiltrate. Relative abundance of CRIB was determined by qPCR analysis. Bacterial counts are expressed in logio colony forming units per gram sample (logio CFU/g). Spearman's correlation coefficients are provided, with corresponding p- values. The dotted lines indicate the division between samples containing a low (<7.5%) or high (>7.5%) relative ileal abundance of CRIB (>7.5%).

Figure 7. Effect of low (<11%; transparent bars) or high (>11%; gray bars) relative ileal abundance of CRIB in diseased rats on plasma cytokine levels of (a) CXCL1, (b) IL-16, (c) IL-18, (d) IL-10, (e) TNF-a and (f) IL-6. Box- whisker plots represent median with interquartile range (boxes) and minimum and maximum non-outlier values (whiskers).† p < 0.10, * p < 0.05 compared to median.

Figure 8. Neighbor joining tree based on the 16S rRNA gene sequence of CRIB and other related Clostridia. E. coli was included as an outgroup. Alignment and phylogenetic analysis were performed with the ARB software (Ludwig et al., 2004). Tree was calculated for E. coli positions 55— 926. The reference bar indicates 10% sequence divergence. GenBank accession numbers of 16S rRNA gene reference sequences are given in parentheses.

Figure 9. Experimental design Example 1. Eight days prior to induction of acute pancreatitis, a permanent gastric cannula was inserted. Probiotics or placebo were administered intragastrically through the permanent gastric cannula once daily, starting five days prior to induction of acute pancreatitis, and twice daily from day zero until six days after induction of acute pancreatitis. Seven days after induction of acute pancreatitis, surviving rats were anesthetized to allow aseptic removal of organ and blood samples. After sample collection, rats were euthanized by blood loss,

Figure 10. Alignment of the 16S rRNA gene sequence of CRIB with selected reference sequences of closely related Clostridium spp. and environmental clones. Nucleotides identical to the CRIB sequence are indicated by dots; gaps are shown as dashes. The positions of the primers CRIB-61F and CRIB-235R, developed to specifically detect CRIB, are indicated in yellow.

Figure 11. T-RFLP patterns of the duodenal microbiota of 8 representative diseased rats, treated with either placebo (upper 4 traces) or the probiotic mixture (lower 4 traces). Duodenal samples were obtained 7 days after induction of pancreatitis. Major TR-F peaks with corresponding bacterial species are: 58 - Lactobacillus spp., 61 - Myxococcus spp., 91 - Prevotella spp., 106 - unknown, 145 - L. thermophilus, 188 - L. johnsonii, 372 - Escherichia coli, 559 - Streptococcus spp. and 581 - Pediococcus pentosaceus. TR-F peaks matching with the 6 applied probiotic strains, as determined experimentally (data not shown), are indicated with dotted lines and can be divided into four groups: A - Lactococcus lactis, B - Bifidobacterium bifidum and B. lactis, C - Lactobacillus acidophilus, D - L. casei and L. salivarius.

Figure 12. T-RFLP patterns of ileal microbiota of healthy control (1-4) and diseased animals, treated with either placebo (5- 13) or the probiotic mixture (14-25). Ileal samples from the diseased animals were obtained 7 days after induction of pancreatitis. The arrowhead indicates the position of T-RF 457, corresponding to a yet uncharacterized bacterial phylotype (referred to as commensal rat ileum bacterium; CRIB). T-RF peaks matching with the 6 applied probiotic strains are indicated with thin boxes and can be divided into four groups: A - Lactococcus lactis, B - Bifidobacterium bifidum and B. lactis, C -Lactobacillus acidophilus and D - L. casei and L. salivarius.

Figure 13. Transepithelial electrical resistance of Caco-2 cells after treatment with ethanol, CRIB or a combination of both.

Figure 14. Graphical representation of the data in Figure 13.

Figure 15 Sequences of CRIB

Figure 15a SEQ ID NO: l, 16S rRNA gene sequence, 437 nucleotides

Figure 15b SEQ ID NO:2, 16S rRNA gene sequence, 407 nucleotides

Figure 15c SEQ ID NO:3 16S rRNA sequence, 1466 nucleotides

Figure 16 Probes and primers targeting the 16S ribosomal RNA and the encoding gene

Primers: Figure 16, 1)— 6)

Probes: Figure 16, a) probe a and b) probe b

For specific detection of CRIB, probe a can be used in combination with probe b, or probe b can be used alone.

Probe a is a previously published probe, specific for some members of the Clostridium lituseburense group (Clostridium cluster XI). Fig. 17 shows an example of the PCR results of example 3.

Fig. 18 shows the percentage of CRIB 16S rRNA gene over total bacterial counts after 1 and 2 days of incubation. Fig. 19 shows the DGGE results of the 16S rRNA analysis of original rat ileum bacteria samples.

Fig. 20 shows the percentage of CRIB 16S rRNA gene over total bacterial counts after 1 and 2 days of incubation. Fig. 21 shows the percentage of CRIB 16S rRNA gene over total bacterial counts after dilutions series, NP is non pasteurized, P is pasteurized. Figure 16 reveals how pasteurization was not helping with CRIB isolation, because the ratio is lower with pasteurization than without. We chose the highest dilution where CRIB was still detectable, NP 10-8, to do a next dilution series (Fig. 17).

Fig. 22 shows the percentage of CRIB 16S rRNA gene over total bacterial counts after second dilution series.

Fig. 23 shows the dilution series of colony 1.

Fig. 24 shows the dilution series of colony 5 Fig. 25 shows the DGGE of the dilution series of colonies 1 and 5

Fig. 26 shows the growth curve of CRIB

Fig. 27 shows the relative expression levels of FOXP3, Tbet, GATA3 and RORyT after treatment with CRIB.

Fig. 28 UV photograph of the agarose gel with PCR product, using different template variants and rat CRIB specific primer. The template variants were loaded in the lanes (1 till 26) as follow: Lane l=Marker; Lane 2= (pre treatment) variant 1; Lane 3= (pre treatment) variant 5; Lane 4= (pre treatment) variant 1; Lane 5= (pre treatment) variant 5; Lane 6 = (pre treatment) variant 1; Lane 7= (pre treatment) variant 6; Lane 8= (pre treatment) variant 1; Lane 9= (pre treatment) variant 6; Lane 10= (pre treatment) variant 2; Lane ll=isolated rat CRIB DNA; Lane 12= (pre treatment) variant 2; Lane 13 =isolated rat CRIB DNA; Lane 14= (pre treatment) variant 3; Lane 15= isolated, amplified and purified 16S rRNA DNA PCR product from rat

CRIB; Lane 16= (pre treatment) variant 3;Lane 17= isolated, amplified and purified 16S rRNA DNA PCR product from rat CRIB; Lane 18= (pre treatment) variant 4; Lane 19=isolated rat CRIB DNA; Lane 20= (pre treatment) variant 4; Lane 21= isolated, amplified and purified 16S rRNA DNA PCR product from rat CRIB; Lane 22, 23, 24= bacterial culture (without pre treatment); Lane 25= negative control; Lane 26=Marker (explanation of template variants in Examples)

Fig. 29 UV photograph of the agarose gel with PCR product, using different template variants, rat CRIB specific primers and extended PCR program. The template variants were loaded in the lanes (1 till 26) as follow: Lane l=Marker; Lane 2= (pre treatment) variant 1; Lane 3= (pre treatment) variant 5;Lane 4= (pre treatment) variant 1; Lane 5= (pre treatment) variant 5; Lane 6 = (pre treatment) variant 1; Lane 7= (pre treatment) variant 6; Lane 8= (pre treatment) variant 1; Lane 9= (pre treatment) variant 6; Lane 10= (pre treatment) variant 2; Lane 11= bacterial culture (without pre treatment); Lane 12= (pre treatment) variant 2; Lane 13= bacterial culture (without pre treatment); Lane 14= (pre treatment) variant 3; Lane 15= isolated, amplified and purified 16S rRNA DNA PCR product from rat CRIB; Lane 16= (pre treatment) variant 3;Lane 17 = bacterial culture (without pre treatment); Lane 18= (pre treatment) variant 4;Lane 19= bacterial culture (without pre treatment); Lane 20= (pre treatment) variant 4; Lane 21= not loaded; Lane 22= negative control; Lane 23= not loaded; Lane 24= negative control; Lane 25= not loaded; Lane 26=Marker (explanation of template variants in Examples). Fig. 30 UV photograph of the agarose gel with PCR product, using different template variants and 16S rRNA gene general primers. The template variants were loaded in the lanes (1 till 21) as follow: Lane l=Marker; Lane 2= (pre treatment) variant 7;Lane 3= bacterial culture (without pre treatment); Lane 4= (pre treatment) variant 7; Lane 5 = bacterial culture (without pre treatment); Lane 6= (pre treatment) variant 8; Lane 7= bacterial culture (without pre treatment); Lane 8= (pre treatment) variant 9; Lane 9= (pre treatment) variant 13; Lane 10= (pre treatment) variant 10; Lane 11= (pre treatment) variant 14; Lane 12= (pre treatment) variant 11; Lane 13=isolated rat CRIB DNA; Lane 14= (pre treatment) variant 12; Lane 15= negative control; Lane 16=isolated rat CRIB DNA; Lane 17 = not loaded; Lane 18= negative control; Lane 19= not loaded; Lane 20= (pre treatment) variant 15; Lane 21=Marker (explanation of template variants in Examples).

Fig. 31 Human isolate related to CRIB, in chains (liquid culture). DETAILED DESCRIPTION OF THE INVENTION Definitions

The term "% sequence identity" is defined herein as the percentage of nucleotides in a nucleic acid sequence that is identical with the nucleotides in a nucleic acid sequence of interest, after aligning the sequences and optionally introducing gaps, if necessary, to achieve the maximum percent sequence identity. Methods and computer programs for alignments are well known in the art, such as by using the ARB software package as described in example 2. As used herein, the terms "nucleic acid sequence" and "nucleotides" also encompass non-natural molecules based on and/or derived from nucleic acid sequences, such as for instance artificially modified nucleic acid sequences, peptide nucleic acids, as well as nucleic acid sequences comprising at least one modified nucleotide and/or non-natural nucleotide such as for instance inosine.

The term "probiotic composition" as used herein refers to a composition comprising one or more probiotic organisms and one or more acceptable excipients suitable for application to a mammal. It will be appreciated that acceptable excipients will be well known to the person skilled in the art of probiotic composition

preparation. Examples of such acceptable excipients include: sugars such as sucrose, isomerized sugar, glucose, fructose, palatinose, trehalose, lactose and xylose; sugar alcohols such as sorbitol, xylitol, erythritol, lactitol, palatinol, reduced glutinous starch syrup and reduced glutinous maltose syrup; polysaccharides as maltodextrins, starches like maize starch, rice starch, potato starch and wheat starch, emulsifiers such as sucrose esters of fatty acid, glycerin esters of fatty acid and lecithin;

thickeners (stabilizers) such as carrageenan, xanthan gum, guar gum, pectin and locust bean gum; acidifiers such as citric acid, lactic acid and malic acid; fruit juices such as lemon juice, orange juice and berry juice; vitamins such as vitamin A, vitamin B, vitamin C, vitamin D and vitamin E; and minerals such as calcium, iron, manganese and zinc.

The term "modulation of differentiation of immune cells by a CRIB" as used herein refers to an increase or decrease in the number of a specific differentiated immune cell type as a result of the presence of a CRIB compared to the number of the same differentiated immune cell type in the absence of CRIB. The term "dysbiosis" is defined as a state in which the microbiota produces harmful effects via (a) qualitative and quantitative changes in the intestinal microbiota itself, (b) changes in their metabolic activities; and (c) changes in their local distribution.

The term "microbiota" as used herein denominates the community of commensal microorganisms that colonise the lumen and surfaces of the gastrointestinal tract, together with the food-ingested, or transient microorganisms.

The term "permeability of the gut" as used herein refers to a characteristic of the gut mucosa to transport certain compounds or molecules from the lumen of the gut to systemic circulation. In case permeability is increased, more molecules can pass the barrier and will come into the systemic circulation, from which it can be spread all over the body. In a healthy situation the gut barrier is tight and only specific molecules are able to pass. Probiotics have the function to enhance the barrier function and thus decrease the permeability of the intestinal mucosa.

The term "primer" as used herein refers to a stretch of nucleic acids which is complementary to a given target nucleic acid sequence and that is needed to initiate replication by a polymerase.

The term "probe" as used herein refers to a labelled segment of a nucleic acid, preferably DNA or RNA, or synthetic nucleic acids, used to find a specific sequence of nucleotides in a DNA or RNA sample, including also other types of biological samples in which DNA or RNA are present, such as for example samples of gut tissue or intestinal content. Probes may be synthesized in the laboratory, with a sequence complementary to the target DNA or RNA sequences.

The term "DNA-DNA hybridization" indicates a molecular technique that measures the degree of genetic similarity between a pool of DNA sequences. When genomic DNA sequences of two bacterial isolates are compared the similarity values can be used to determine the genetic distances between these two micro-organisms. Such a method basically is performed by labelling the DNA of one organism and mixing it with the unlabelled DNA to which it should be compared. The mixture is incubated to allow DNA strands to dissociate and re-anneal, forming hybrid double- stranded DNA. Hybridized sequences with a high degree of similarity will bind more firmly, and require more energy to separate them: i.e. they separate when heated at a higher temperature than dissimilar sequences. To assess the melting profile of the hybridized DNA, the double -stranded DNA is bound to a column and the mixture is heated in small steps. At each step, the column is washed; sequences that melt become single-stranded and wash off the column. The temperatures at which labelled DNA comes off the column reflects the amount of similarity between sequences (and the self-hybridization sample serves as a control). These results are combined to determine the degree of genetic similarity between organisms. A value of 70% DNA- DNA hybridization was proposed by Wayne, L.G. et al.. (International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics Int J Syst Bacteriol 37, 463-464.1987) as a recommended standard for delineating species.)

Embodiments

The inventors have identified and isolated a new anaerobic and spore forming bacterium according to the invention which is herein named CRIB. CRIB stands for commensal rat ileal bacterium, as the bacterium was first found by the present inventors in the ileum of a rat. Commensal refers to the beneficial interrelationship that appears to exist between the bacterium and a host of this bacterium. As demonstrated in the experimental part, CRIB is also present in other animals, including human.

The invention therefore provides a CRIB which was deposited under the

Budapest Treaty with the DSMZ, on 29 June 2007 under the accession number 19498.

Sequencing of the 16S rRNA gene of said CRIB showed that CRIB is a bacterium closely related to the Clostridiaceae (Fig. 11). It is generally known that within a bacterium species, mutations in the genetic information occur which do not affect the probiotic characteristics. Therefore, all bacteria having at least a sequence SEQ ID NO: l or the complement sequence thereof, or SEQ ID NO:2 or the complement sequence thereof, preferably the sequence coding for 16S RNA as shown in SEQ ID NO: 3, or a sequence having a sequence identity of more than 97,2 % with SEQ ID NO: l or its complement or a sequence having a sequence identity of more than 97,2 % with SEQ ID NO:2 or its complement, preferably a sequence having a sequence identity of 97.2% or more with SEQ ID NO: 3 are considered to be a CRIB and would fall within the scope of the invention. More preferably, said sequence identity is more than 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%. Most preferably, said sequence identity is 100%.

Also a bacterium which has a very high homology with the 16S rRNA of the human CRIB bacterium, as specified in SEQ ID NO: 21. Preferably, a bacterium having a 16S rRNA gene sequence that has a sequence identity of 97.2% or more with SEQ ID NO: 21. More preferably, said sequence identity is more than 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%. Most preferably, said sequence identity is 100%.

Next to the 16S rRNA sequence many other genomic sequences are available, as presented in SEQ ID NO: 22 to SEQ ID NO: 130. These are genomic sequences from the rat isolated CRIB, and it is submitted that the invention comprises bacteria having these sequences and/or sequences that are highly similar to those. In respect to high similarity with regards to SEQ ID NOs:22-130, high similarity is defined as having a sequence identity of more than 70%, preferably of more than 75%, of more than 80%, of more than 85%, of more than 90%, of more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99% over a stretch of 1000 or more nucleotides, or— if the sequence in the SEQ ID is longer than 1000 nucleotides— over the complete sequence as disclosed in the SEQ ID.

In particular bacteria which have a sequence which is more than 70%, preferably of more than 75%, of more than 80%, of more than 85%, of more than 90%, of more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99% identical to the total of sequences of SEQ ID NO: 22 to SEQ ID NO: 130 are preferred.

Another way to characterize the similarity between DNA (gene) sequences is

DNA-DNA hybridisation. As is shown in example 6 of the present specification, the strains that were the closest with respect to similarity of 16S rRNA gene sequence, gave a very low DNA-DNA hybridisation. In an alternative embodiment, the invention thus comprises a bacterium that has a relative DNA-DNA similarity as provided by DNA-DNA hybridization of 70% or more, preferably of 75%, of 80%, of 85%, of 90%, of 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more with the CRIB bacterium as deposited under the Budapest Treaty with the DSMZ, on 29 June 2007 under the accession number 19498. Preferably said bacterium is used as a probiotic, optionally in combination with other probiotic organisms.

The combinations referred to above may conveniently be presented for use in the form of a probiotic composition and thus probiotic compositions comprising a combination as defined above together with one or more excipients comprise a further aspect of the invention. The individual components of such combinations may be administered either sequentially or simultaneously in separate or combined probiotic compositions.

In a preferred embodiment, CRIB is combined with bifidobacteria, (like Bifidobacterium bifidum, B. infantis, B. lactis, B. longum) and / or bacteria belonging to the genus Lactobacillus (such as Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus gallinarum, Lactobacillus amylovorus, Lactobacillus brevis, Lactobacillus rhamnosus, Lactobacillus kefir, Lactobacillus paracasei, Lactobacillus crispatus, Lactobacillus delbrueckii subsp. delbrueckii, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus, Lactobacillus zeae, Lactobacillus plantarum and Lactobacillus salivarius) and / or bacteria belonging to the genus Streptococcus (such as Streptococcus thermophilus and Streptococcus salivarius) and / or bacteria belonging to genus Lactococcus (such as Lactococcus lactis subsp. cremoris and Lactococcus lactis subsp. Lactis) and / or bacteria belonging to the genus Bacillus (such as Bacillus subtilis) and /or yeast belonging to the genus Saccharomyces, Torulaspora and Candida (such as Saccharomyces cerevisiae, Torulaspora delbrueckii and Candida kefyr).

It is preferred that CRIB is administered in a suitable probiotic composition comprising a probiotic culture having preferably a probiotic count of between 10^s and 10¹¹ cfu/ml or cfu/g and a carrier medium. Compositions of the invention may be prepared by admixture, suitably at ambient temperature and atmospheric pressure and preferably under anaerobic conditions. Usually said compositions are adapted for oral administration, such as the administration forms discussed below.

In general, it can be said that administration of CRIB decreases dysbiosis. Furthermore, it is also contemplated that also parts of the CRIB bacterium (specific proteins formed in or excreted by CRIB, specific binding molecules on the surface of the CRIB bacteria, specific carbohydrates and so on) can be used for exerting the effects as specified in the current invention.

Studies with rats suffering from acute pancreatitis and receiving a

multispecies probiotic mixture have demonstrated that although probiotic treatment did not reverse the change in microbiota, CRIB was significantly increased in relative abundance. The number of CRIB was positively correlated with an improved histology of the pancreas, with a decreased number of bacteria in the duodenum, mesenteric lymph nodes, spleen-, liver- and pancreatic necrosis suggesting an improved barrier function. Secondly CRIB was correlated with reduced plasma pro-inflammatory cytokines, indicating an effect of CRIB on immune modulation too.

The inventors have shown that said CRIB modulates the differentiation of naive T-cells which play a role in the immune system. It was found that CRIB has a stimulating effect on a subset of regulatory T cells, the so-called Thl7 cells, which play a role in infectious diseases. Thl7 cells are required for control and elimination of invasive infections, such as pneumococcal infections. Furthermore, said CRIB has a stimulating effect on Thl cells, which are involved in the defence against intracellular pathogens. In addition, said CRIB enhances the integrity of the intestinal mucosa and compensates for the deterioration of the barrier function of the gut by ethanol.

Therefore, said CRIB according the invention can advantageously be used in a treatment comprising both cure or inhibition of a disease and prevention of a disease.

Accordingly, CRIB or a probiotic composition comprising CRIB may be administered to diseased subjects, but also to healthy subjects. Thus, CRIB or a probiotic composition comprising CRIB can advantageously be used in the treatment, inhibition or prevention of a disease, disorder or syndrome, preferably in the treatment, treatment- sparing or prevention of disease, disorder or syndrome related to decreased barrier function and/or immune-mediated condition, more preferably pancreatitis, inflammatory bowel disease (IBD) such as Crohn's disease, pouchitis, necrotizing enterocolitis, proctitis, ulcerative colitis or irritable bowel syndrome (IBS). It can also be used as treatment, treatment- sparing or for the prevention of celiac disease, food allergy, dysbacteriosis, cholera and diabetes type 1. Target groups for prophylactic treatment with CRIB are healthy subjects, but especially subjects that are prone to infection and subjects that run a risk of gut barrier function detoriation. Subjects are mammals, but especially humans, live stock, such as cattle, horses and sheep, and pet animals.

The invention further provides a method of treatment and/or prophylaxis of the above disorders, in a human or animal subject, which comprises administering to the subject a therapeutically effective amount of CRIB, which may be used in combination with other probiotic organisms or with therapeutic agents, for example, other medicaments known to be useful in the treatment and/or prophylaxis of gastrointestinal diseases (e.g. diarrhoea), cholesterol excesses, allergies and infection.

Thus, as a further aspect of the invention, there is provided a combination comprising said CRIB together with one or more further agents.

The compositions may be in the form of tablets, capsules, oral liquid preparations, conventional food products, powders, granules, lozenges, reconstitutable powders or suspensions or oral liquid preparations.

Tablets and capsules for oral administration may be in unit dose form, and may contain one or more conventional excipients, such as binding agents, fillers, tabletting lubricants, disintegrants, and acceptable wetting agents. The tablets may be coated according to methods well known in pharmaceutical practice.

Oral liquid preparations may be in the form of, for example, aqueous or oily suspension, solutions, emulsions, syrups or elixirs, or may be in the form of a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils),

preservatives, and if desired, conventional flavourings or colorants. Preferably, said liquid preparation is suitable for anaerobic bacteria.

In one preferred embodiment, the composition of the invention is formulated as a conventional food product, more preferably, a dairy based product (e.g. fermented milk, vegetable milk, soybean milk, butter, cheese or yoghurt) or fruit juice. The composition is preferably formulated as a food or drink for adult and infant humans and animals. In an alternatively preferred embodiment, the composition is formulated as a lyophilised or spray- dried powder.

CRIB is anaerobic, that is why the isolation and culturing of CRIB has to be done under anaerobic circumstances. However, some presence of oxygen in the medium is tolerated. The relative absence of oxygen and maintaining a low redox potential of the culture media by addition of a reducing agent are, next to the component composition of the medium, two important parts of culturing and isolation of CRIB. The removal of oxygen is usually achieved by boiling the medium, and subsequent cooling under a stream of anoxic gas.

The invention also provides a method for detecting CRIB, based on the detection of the presence of the 16S RNA of CRIB, preferably of SEQ ID NO:l or the complement sequence thereof, or SEQ ID NO:2 or the complement sequence thereof, preferably the sequence coding for 16S RNA as shown in SEQ ID NO: 3, or a sequence having a sequence identity of more than 97,2 % with SEQ ID NO:l or its complement or a sequence having a sequence identity of more than 97,2 % with SEQ ID NO:2 or its complement, preferably a sequence having a sequence identity of 97.2% or more with SEQ ID NO: 3. Of course, other parts of the genome may be used as well. More preferably, said sequence identity is more than 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%.

Detection techniques based on the detection of specific sequences are well known in the art. It is preferred that sensitive PCR techniques are used, using specific primers which enable specific amplification of CRIB nucleotides. Therefore, the invention provides a kit comprising said primers, usually consisting of a first and a second primer, alternatively indicated as forward and reverse primer. These primers may be of DNA, RNA or synthetic oligonucleotides. The CRIB specific 16S RNA is preferably detected using a first primer crib-61f having SEQ ID NO: 4 and a second primer crib-235r having a sequence SEQ ID NO:8 .

Alternatively, detection of CRIB can be performed with a first primer crib-61f having SEQ ID NO: 4 and a second primer crib- 155r having SEQ ID NO:6 or crib-220r having SEQ ID NO:7 or crib- 193r having SEQ ID NO:9; or with a first primer crib- 132f having SEQ ID NO:5 and a second primer crib-220r having SEQ ID NO:7. Detection with these primers, however, is less specific and may result in false positive detection of other bacteria, like Clostridium species.

In some embodiments, said kit may further comprise a buffer and/or an enzyme.

The invention further provides an isolated and/or recombinant nucleic acid comprising SEQ ID NO: l or the complement sequence thereof, or SEQ ID NO:2 or the complement sequence thereof, preferably the sequence coding for 16S RNA as shown in SEQ ID NO: 3, or a sequence having a sequence identity of more than 97,2 % with SEQ ID NO:l or its complement or a sequence having a sequence identity of more than 97,2 % with SEQ ID NO:2 or its complement, preferably a sequence having a sequence identity of 97.2% or more with SEQ ID NO: 3. Preferably, said sequence identity is higher than 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9. Said isolated and/or recombinant nucleic acid can suitably be used as a probe to detect a CRIB derived nucleic acid by allowing a labelled probe to hybridize under stringent conditions with a nucleic acid from said CRIB and detect the presence of a CRIB derived nucleic acid hybridized to said labelled probe. Alternatively, a shorter sequence of SEQ ID NO:l or SEQ ID NO: 3 can be used as a probe. Preferably such a probe has SEQ ID NO: 11.

The inventors have furthermore found that CRIB is capable of directly and/or indirectly influencing at least three components of an immune status of an individual. The first component is the percentage of time that an animal is free of a disease of the gastrointestinal tract, which is increased if CRIB is present. According to the invention CRIB promotes a balanced gastrointestinal microbiota, where a balanced microbiota denotes a composition of microbiota which acts symbiotically with the host especially in the production of vitamins. A balanced gastrointestinal microbiota at least in part protects the host from unwanted colonization or overgrowth by

(opportunistic) pathogens. The second component is the passage through the mucosal barrier. The mucosal barrier is a layer of epithelial cells and mucus which separates the external environment of the digestive tract from the internal environment. A mucosal barrier needs to exist between the internal and external environment of the gastrointestinal tract, as otherwise pathogens could enter the internal environment of the body, which could lead to infections. According to the invention, presence of CRIB at least in part counteracts undesired passage of the mucosal barrier by undesired components, such as pathogens. The last aspect is the capability of CRIB to directly and/or indirectly influence gastrointestinal infections by an effect on the mucosal and/or systemic immune system.

The present invention therefore provides a method for evaluating the immune status of an individual by determining whether a sample of said individual comprises a CRIB bacterium and/or a nucleic acid sequence thereof. Therefore, preferably, it is determined whether a sample of said individual comprises SEQ ID NO:l or the complement sequence thereof, or SEQ ID NO:2 or the complement sequence thereof, preferably the sequence coding for 16S RNA as shown in SEQ ID NO: 3, or a sequence having a sequence identity of more than 97,2 % with SEQ ID NO:l or its complement or a sequence having a sequence identity of more than 97,2 % with SEQ ID NO:2 or its complement, preferably a sequence having a sequence identity of 97.2% or more with SEQ ID NO: 3. Preferably said sequence identity is higher than 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%. In this way, the invention provides a solution to the difficulty of determining an immune status, particularly in the determination of gastrointestinal-related risks. The presence of CRIB in an individual, preferably in the gastrointestinal tract of an individual, indicates that said individual is of a lower risk of obtaining or suffering from a gastrointestinal disorder, and/or that the individual has better chances of recovery from the disorder, as compared to individuals that do not comprise CRIB.

A sample as used in the invention comprises any sample containing biological material. A sample is preferably a sample of an animal. An animal as used in the invention comprises any animal with a digestive tract. Said animal preferably comprises a vertebrate, more preferably a mammal, such as for instance a human or a domestic animal kept for company or for production purposes. Non-limiting examples of animals kept for production purposes are cows, pigs and poultry. In one preferred embodiment an animal of the invention comprises a human individual. The terms animal and individual as used herein, are interchangeable.

An immune status as used in the invention, is defined as a condition of the animal with respect to the degree in which the animal is resistant to infectious threats, particularly gastrointestinal disorders and/or complications thereof.

Since the amount of nucleic acid of a CRIB bacterium in a sample of an animal is related to the amount of CRIB bacteria present in said animal, the amount of CRIB-derived nucleic acid in a sample is indicative for an immune status of an animal. For instance, an elevated amount of nucleic acid of a CRIB bacterium in a sample is positively correlated to an improved immune status of an individual.

Therefore, the invention in a further embodiment provides a method according to the invention for determining the immune status of an animal, further comprising quantifying the amount of detected nucleic acid. An amount of nucleic acid is determined in absolute or relative quantities. A quantity is for instance compared with at least one reference value of a comparable healthy animal, with at least one reference value obtained from at least one sample with a known amount of CRIB, and/or CRIB derived nucleic acid, and/or with at least one reference value derived from at least one animal with a specific disease. Reference values are for instance plotted to give a reference curve. Determining an amount of nucleic acid of the invention is for example a good method for monitoring an individual patient. An immune status of a patient can be followed over time by repeatedly determining the amount of CRIB- derived nucleic acids in a sample of said patient. If the amount of CRIB bacteria decreases, it indicates that the immune status of an individual declines, and thus that the risk increases that the individual develops a disorder and/or that the disorder of the individual aggravates. And in the opposite case, when an amount of a nucleic acid of a CRIB bacterium in consecutive samples of an individual increases, it indicates that the immune status of an individual improves. Therefore the risk of developing a disorder and/or developing complications decreases. In this way, a decline or recovery of an immune status is assessed. In one

embodiment, the risk for an individual of getting infected by a gut pathogen is evaluated with a method according to the invention.

According to the present invention, CRIB is capable of at least in part counteracting the danger of bacterial translocation. Bacterial translocation often leads to sepsis, a serious health threatening condition. Another danger that is at least in part counteracted by the CRIB microorganism is infection by external pathogens.

Specifically individuals that are somehow immuno-compromised are susceptible for gastrointestinal disorders, for infection by bacterial translocation and/or infection by external pathogens. Hence, it is particularly preferred to determine an immune status of an immuno-compromised individual with a method according to the invention, so that suitable measures and/or precautions can be taken. Therefore, in one

embodiment the invention provides a method according to the invention for determining the immune status of an animal, wherein said animal is known to have a compromised immune system. A compromised immune system as used in the invention is any condition of an animal that makes the animal more susceptible to a gastrointestinal disorder, complications thereof and/or infection by a pathogen. A compromised immune system is for instance present when an animal is undergoing a disease from any kind. Furthermore, an animal that is in circumstances that are known to be sub-optimal, is highly suspected to have a compromised immune system. An example is a human being or a pet such as a cat that has received intensive antibiotic therapy, an immunosuppressive therapy and/or anti-inflammatory drugs. Further examples are non-human animals living under sub-optimal conditions, such as pigs in the bio-industry or poultry living in a high density community. A further example of animals having a compromised immune system are calves that are separated from the mother cow at an early stage. The housing conditions of these calves are often a further risk factor. Non-limiting examples of diseases that cause a compromised immune system are allergy, lung disease, heart disease, shock, including haemorrhagic shock, severe trauma, burn, auto-immune disorders and gastro-intestinal disorders.

An immune status of an individual is preferably determined when an individual is known to have health complaints and thus to have a compromised immune system. A method of the invention is preferably applied when an individual is known to have a compromised immune system. For example, when a veterinarian has been called in for an ill individual animal or a flock of animals, or when a human patient visits the physician with health complaints. A preferred circumstance wherein a method according to the invention is applied is when a patient is admitted to hospital, specifically when a patient has a severe condition. Treatment can be beneficially adjusted to the outcome of the determination of the immune status of the individual. For instance, if the amount of CRIB-derived nucleic acid in a sample of said individual appears to be low or zero, additional precautions and/or safety measures are preferred.

The organ system that is most directly affected by the presence of CRIB is the gastro-intestinal tract. Specifically in case of suspected or assessed gastro-intestinal disorders it is therefore beneficial to determine whether CRIB is present in a sample of an animal having the suspected or assessed disorder, thus determining the immune status of said animal. The risk of an individual for developing a more serious condition and/or getting disease complications is assessed. Therefore the invention provides a method according to the invention, for determining the immune status of an animal, wherein said animal is suffering from, or at risk of suffering from, a gastro-intestinal disorder. Gastro-intestinal disorders for instance include, but are not limited to, the inflammatory bowel diseases (IBD like Crohn's disease and ulcerative colitis), pancreatitis, bacterial, viral, chemical or drug-related gastro- enteritis, antibiotic-associated colitis, malabsorption syndromes (celiac disease, tropical sprue, Whipple's disease, intestinal lymphangiectasia), necrotizing enterocolitis, proctitis, tumors of the gastrointestinal tract, irritable bowel syndrome (IBS), constipation, diarrhoea, abdominal bloating or pain, incontinence and biliary disorders, gastritis, pouchitis and hepatitis. The invention can also be used as treatment, treatment— sparing or prevention of food allergy, dysbacteriosis, cholera and diabetes type 1. In the examples it is for instance demonstrated that the amount wherein CRIB is present in an animal is indicative for the risk of occurrence of complications of pancreatitis. The predictive value of the amount of CRIB in a sample of an individual is of great advantage in the treatment of an individual as a treatment can now be better adjusted to the individual needs.

As the CRIB bacterium mainly resides in the intestines, a sample is preferably derived from the intestines. In one embodiment of the invention, the invention therefore provides a method for determining the immune status of an animal according to the invention, wherein said sample comprises contents of an intestine. A sample in one embodiment comprises contents of an intestine of the animal as such a sample directly demonstrates the presence of the CRIB bacterium in the intestines. Alternatively, a sample for instance comprises ileostoma effluent. Such samples are preferably derived from human patients with a stoma in the ileum. A sample further for example comprises contents of an intestine obtained during a biopsy or surgery. In order to determine the immune status of an animal, CRIB is in a preferred embodiment of the invention detected by means of a primer and/or a probe of the invention. Therefore, the invention provides a method for determining the immune status of an animal according to the invention, wherein said detecting comprises providing a primer and/or a probe according to the invention to nucleic acid of said sample, and determining whether said primer and/or a probe specifically hybridizes to nucleic acid of said sample. In one embodiment said primer and/or probe is administered to said sample without prior purification procedures. Preferably however, nucleic acid of a sample is firstly at least in part isolated. For instance, nucleic acid is at least in part isolated using a chaotropic agent. Further, the invention comprises the use of administration of CRIB to an individual when it was found according to the above described methods that said individual had a lowered immune status, to alleviate said immune status.

The invention further provides the use of a CRIB according to the invention for the modulation of immune cells. Preferably, said immune cells comprise T cells, more preferably CD4+ T cells. Even more preferably, said immune cells comprise Thl or Thl7 positive T cells. Thus, CRIB can be used in prevention or for the treatment or treatment- sparing of various immune-mediated disorders, such as allergy, autoimmune disorders and immune deficiency disorders, especially when these disorders have an intestinal component.

The invention further provides the use of a CRIB according to the invention for maintenance or restoration of the normal permeability of the gut barrier. Thus, CRIB can be used in the prevention or for the treatment of various disorders related to permeability of the gut, such as inflammatory bowel diseases, metabolic syndrome, hepatic encephalopathy, mental disorders and auto-immune disorders.

The invention is further illustrated by the following examples. The examples are not to be interpreted as limiting the scope of the invention in any way.

EXAMPLES

Example 1

In order to get a better insight in the role of gut microbiota in the process of bacterial translocation during experimental pancreatitis, a detailed analysis of the bacterial communities present in the various parts of the intestine was made.

Molecular analysis of intestinal samples of rats treated with probiotics has led to a new hypothesis on how probiotics can improve the clinical course of acute

pancreatitis. The clinical success of prophylactic treatment with a multispecies probiotic mixture in rats strongly correlates with the stimulation of a yet uncultured bacterial phylotype in the ileum of these animals. The most closely related bacterial species of this novel bacterial phylotype is Clostridium lituseburense. For the detection of this novel bacterial phylotype a specific and quantitative PCR (qPCR) was developed using 16S ribosomal RNA (rRNA) targeted primers. This qPCR can be used in future studies to detect this novel bacterium in clinically relevant samples.

MATERIALS AND METHODS

Animals

Male specific pathogen-free Sprague-Dawley rats, 250-350 grams (Harlan, Horst, The Netherlands) were kept under constant housing conditions with a 12-hour light/dark cycle and free access to water and food (RMH 1110; Hope Farms, Woerden, The Netherlands) throughout the experiment. All animals were allowed to adjust to these conditions for one week prior to surgery. Rats were randomized between two experimental groups: 17 rats were included in the group prophylactically treated with probiotics, and 21 rats were assigned to the placebo group. Both groups were subjected to the surgical procedures described below. In addition 6 rats, which did not receive any surgical intervention or treatment, were used as healthy controls. All animal procedures were performed in accordance with institutional guidelines and with approval from the institutional animal care committee of the University Medical Center, Utrecht, The Netherlands.

Probiotics and Placebo

The multispecies probiotic mixture consisted of equal amounts of 6 different viable, freeze-dried probiotic strains, Bifidobacterium bifidum (W23), Bifidobacterium animalis subsp. lactis (W52) Lactobacillus acidophilus (W70), Lactobacillus casei (W56), Lactobacillus salivarius (W24), Lactococcus lactis (W58), blended in a carrier material consisting of maize starch, maltodextrins and a mineral mix (Ecologic^® 641, Winclove Bio Industries, Amsterdam, The Netherlands). The placebo product consisted of carrier material only. Directly before administration, both the freeze- dried placebo product and the probiotic mixture were reconstituted in sterile water for 15 minutes at 37 °C. A single probiotic dose of 1.0 ml contained a total of about 5 x 10⁹ colony forming units (CFU) of bacteria. Probiotics or placebo were administered intragastrically through a permanent gastric cannula once daily, starting five days prior to induction of acute pancreatitis, and twice daily for six days after induction of acute pancreatitis (Figure 9). Surgical procedures

Surgical procedures were performed as described previously (van Minnen et al., 2007). Briefly, all procedures were performed under general anesthesia using a combination of 2% isoflurane gas and 0.3 ml 10% buprenorphine intramuscular (Temgesic; Reckitt Benckiser Healthcare Ltd., Hull, UK). At the start of the experiment, a permanent gastric cannula was fitted by tunneling a silicone cannula subcutaneously from the abdominal wall to the back of the animal. The gastric end of the cannula was inserted into the stomach through a puncture within a purse-string suture on the greater curvature. Animals were allowed to recover for 3 days prior to the start of daily probiotics or placebo administration. Five days after starting daily administration of probiotics or placebo, acute pancreatitis was induced as described previously, with minor adaptations (Schmidt et al., 1992). The common bile duct was clamped and 0.5 ml sterilized glycodeoxycholic acid in 10 mM glycylglycine-NaOH-buffered solution (pH 8.0, 37 °C; chemicals obtained from Sigma- Aldrich Chemie B.V., Zwijndrecht, The Netherlands) was infused, after which hepato- duodenal bile flow was restored. Next, the right jugular vein was cannulated for continuous intravenous infusion of cerulein (5 g/kg/hr for six hours; chemicals obtained from Sigma- Aldrich Chemie B.V.).

Tissue and fluid samples

Seven days after induction of pancreatitis, surviving rats were anesthetized to allow aseptic removal of tissue samples and sampling of peritoneal fluid and blood. After sample collection, rats were euthanized by blood loss. Mesenteric lymph nodes, liver, spleen, pancreas and duodenum were removed for microbiological analysis. After carefully removing all pancreatic and mesenteric tissue from the proximal duodenum and distal part of the terminal ileum, sections of approximately 2 cm were excised at both locations. Both segments were then transferred to a sterile vial, snap-frozen in liquid nitrogen and stored at -80 °C. A portion of pancreatic tissue was fixed in 4% formalin and analyzed histopathologically, using standard hematoxylin and eosin (H&E) staining. Histopathological severity of acute pancreatitis was assessed based on a scoring system modified from Schmidt et al. (1992) as previously described (van Minnen et al., 2007). Several aspects of (peri)pancreatic histopathology were assessed (peritonitis, edema, ductal pathology, inflammatory infiltrate, acinar cells, acinar dilatation and hemorrhagic changes). These aspects were scored on a scale, varying from maximal 2 to maximal 5 points per aspect.

All tissue samples were weighed and processed immediately for quantitative and qualitative culturing of aerobic and anaerobic microorganisms on appropriate media as described previously (van Minnen et al., 2007). Tissue samples were homogenized in cysteine broth and cultured in 10-fold dilution series. The samples were cultured aerobically on blood agar, MacConkey-agar (Gram-negative bacteria) and Columbia Colistin Nalidixic Acid (CNA) agar (staphylococci and streptococci), micro-aerobically on Man-Rogosa-Sharpe-agar (lactobacilli) and anaerobically on Schaedler agar. Bacterial counts are presented in CFU/g. Threshold detection level of bacterial growth was >10² CFU/g

Multiplex cytokine assays

Plasma cytokine levels of interleukin-la (IL-la), IL-6, IL-12p70, IL-18, interferon- γ (IFN-γ), CXCLl (growth related oncogene; GRO/KC) and CCL2 (Monocyte chemoattractant protein- 1; MCP-1), all from Linco Research, Inc. (St. Louis, MO, USA), and IL-lB, IL-2, IL-10, tumor necrosis factor-a (TNF-a), all from Bio-Rad

Laboratories (Hercules CA, USA), were analyzed using rat cytokine multiplex assays according to the manufacturer's instructions.

DNA extraction

Duodenum and ileum samples were taken from the freezer (-20 °C) and a 1 cm section from the centre of the sample was excised on a sterile field. A longitudinal incision over the full 1 cm section was made and the sample was left to thaw on a piece of sterile aluminum foil placed directly on a cooled metal plate at 4 °C. After thawing, the intestinal content including the mucosa was scraped off with the back of a sterile surgical blade and collected in a 2 ml screw cap tube containing a buffer solution of 6M guanidine thyocyanate, 0.6% Tween-20 (v/v), 10 mM EDTA, 50 mM Tris-HCl (pH 6.5) and 2 g of zirconia beads (<0.1 mM; Biospec, Bartlesville, OK, USA). For DNA extraction from pure bacterial cultures and the complete probiotic mixture, approximately 10⁸ CFUs were used. Cells were physically disrupted by shaking for 4 min in a MiniBeadbeater-96™ (Biospec), followed by heating for 5 min at 90 °C.

Samples were centrifuged for 1 min at 10,000 x g. From each tube 200 μΐ of the supernatant was subsequently used for DNA isolation. DNA isolation was performed as described previously (Carter & Milton, 1993), except for omitting the final washing step with acetone.

T-RFLP analysis

Terminal-restriction fragment length polymorphism (T-RFLP) analysis was performed essentially as described previously (Liu et al., 1997). For PCR amplification of 16S rRNA gene fragments the universal oligonucleotide primers 8F and 926R (Table 1), respectively with fluorescent dyes 6-FAM and NED at the 5' end, were used. PCR reaction mixtures (15 μΐ) contained PCR buffer (Applied Biosystems, Foster City, CA, USA), 62.5 μΜ of each deoxynucleoside triphosphate (dNTP; Applied Biosystems), 1.5 mM MgC , 0.5 μΜ of each primer, 0.5 U of Taq DNA polymerase (Promega,

Madison, WI, USA) and 1 μΐ of DNA sample. DNA amplification was performed with a 9700 thermal cycler (Perkin- Elmer, Norwalk, CT, USA) using the following program: 94 °C for 5 min, followed by 35 cycles of 30 s at 94 °C, 45 s at 56 °C and 2 min at 72 °C, and a final extension for 5 min at 72 °C. PCR products were digested with restriction endonucleases Mspl and HinplI (New England Biolabs, Ipswich, MA, USA) with both enzymes in the same reaction. Resulting fragments were size separated by capillary electrophoresis (CE) using an ABI 3100 genetic analyzer (Applied Biosystems) equipped with 36 cm capillaries using POP4 gel matrix. A custom size standard with ROX labeled fragments, MapMarker 30— 1,000

(Bioventures, Murfreesboro, TN, USA) was added to each sample prior to CE for accurate sizing of terminal-restriction fragments (T-RFs). After electrophoresis, the lengths of fluorescently labeled T-RFs were determined using GeneScan software (Applied Biosystems).

Observed T-RFs were assigned to microbial taxa using the MCPC database (Dr. Van Haeringen Laboratorium B.V., Wageningen, The Netherlands), which was built using in silico T-RF predictions from data that were extracted from prokaryote sections of the EMBL sequence database (release 82) using the Patscan program (Dsouza et al., 1997). For each of the extracted sequences the T-RF size using the aforementioned combination of primers and restriction enzymes was calculated for both primers, and calculated values were validated for a number of pure cultures, including E. coli, Clostridium perfringens and Staphylococcus aureus (data not shown). The relative peak intensity of each T-RF (referred to as relative abundance) was defined as the height of a specific peak as percentage of the total sum of all peaks heights for a given sample. Relative abundance values were calculated for all the T- RFs between 50 and 600 basepairs (bp) long and with peak heights of more than 50 fluorescence units, which was well above the background noise level (10-20 FU) of non-template control reactions. This procedure was followed for both primers, the data obtained with primer 8F was used in the figures.

Identification of the 16S rRNA gene sequence of CRIB

PCR reaction mixtures (50 μΐ) were prepared using Taq DNA polymerase kit from Invitrogen (Gaithersburg, MD, USA) and contained 0.5 μΐ of Th DNA polymerase (1.25 U), 20 mM Tris-HCl (pH 8.5), 50 mM KC1, 3.0 mM MgCl₂, 5 pmol of the primers 8F and 926R (Table 1), 200 μΜ of each dNTP and 1 μΐ DNA of the T-RFLP PCR reactions (diluted to ~1 ng/μΐ). DNA amplification was performed with a Tl thermocycler (Whatman Biometra, Gottingen, Germany) using the following program: 94 °C for 5 min, followed by 35 cycles of 30 s at 94 °C, 20 s at 56 °C and 40 s at 68 °C, and a final extension for 7 min at 68 °C. Aliquots (5 μΐ) were analyzed by agarose electrophoresis to check for product size and quantity.

The PCR products were purified with the QIAquick PCR purification kit (Westburg, Leusden, The Netherlands) according to the manufacturer's instructions, and cloned into the pGEM-T easy vector system (Promega) using competent E. coli JM109 as a host. The colonies of ampicillin-resistant transformants were transferred with a sterile toothpick to 15 μΐ of Tris-EDTA buffer and boiled for 15 min at 95 °C. PCR was performed using the vector- specific primers T7 and SP6 (Table 1) to check the sizes of the inserts using the cell lysate as a template. PCR products of ~0.95 kb were purified as described above, and digested at 37 °C for 1.5 hr with 20 U of Cfol and 20 U of Mspl (Boehringer, Mannheim, Germany) in 20 μΐ reaction mixtures containing the respective buffer. One clone containing an insert yielding a fragment of approximately 450 bp after restriction analysis, were grown in Luria Broth liquid medium (5 ml) with ampicillin (100 μg/ml) and used for further 16S rRNA gene sequence analysis. Plasmid DNA was isolated using the Wizard Plus purification system (Promega) and sequenced by using the Sequenase (T7) sequencing kit

(Amersham Life Sciences, Slough, UK) according to the manufacturer's specifications using primers T7, SP6, 519R and 533F (Table 1) labeled with IRD-800. Sequences were automatically analyzed on a LiCor DNA Sequencer 4000L (LiCor, Lincoln, NB, USA). The sequences were compared to sequences deposited in publicly accessible databases using the NCBI BLAST search tool (McGinnis & Madden, 2004). The partial CRIB 16S rRNA gene sequence determined in this study has been submitted to the GenBank database under accession number HQ224563.

Phylogenetic analysis and primer design

The 16S rRNA gene sequence from a clone selected as described above was aligned with the 16S rRNA sequences from closely related bacterial species (Figure 10) using the ARB software package and release 90 of the ARB-SILVA reference database (Ludwig et al., 2004). Different primer sets were developed using the ProbeDesign and ProbeMatch functions in ARB, and tested for specificity by conventional PCR. The primer set CRIB-61F/CRIB-235R (Table 1) was further tested by qPCR. For phylogenetic analyses as presented here, the sequence was realigned using the SINA WebAligner (h ttp ://www. arb - sil v . de) and compared to published sequences using ARB- Sil va release 102.

Quantitative PCR analysis

Quantitative PCR reactions were performed using the iQ5 Real-Time PCR Detection System (Bio-Rad). PCR reaction mixtures (25 μΐ) contained 12.5 μΐ iQ SYBR Green Supermix (Biorad), 0.2 μΜ of each primer and 5 μΐ of template DNA. Quantification of 16S rRNA gene copies in each sample was performed in triplicate, and average values were calculated. Standard curves were generated from a dilution series of 16S rRNA gene fragments amplified from the target sequence (10⁸-10° copies μL^■1). The universal primer set Bactl369/Prokl492 (Table 1) (Suzuki et al., 2000) was used for

quantification of total bacteria using the following program: 3 min at 95 °C, followed by 40 cycles of 15 s at 95 °C, 30 s at 56 °C and 30 s at 72 °C. The CRIB-specific primers CRIB-61F/CRIB-235R were used to determine the relative abundance of CRIB 16S rRNA gene copies in the samples. Amplification conditions included 3 min at 95 °C, followed by 40 cycles of 20 s at 95 °C and 1 min at 68 °C. Gradient PCR was initially used to determine the optimal annealing temperature with amplification efficiency, range of linearity and lowest detectable concentration as criteria. The specificity of the CRIB-specific qPCR primer set was tested using the 16S rRNA gene amplified from genomic DNA of closely related bacterial species: Clostridium lituseburense (DSM 797^T), Clostridium irregulare (DSM 2635^T), Clostridium hiranonis (DSM 13275^T) and Clostridium bartlettii (DSM 16795^T). An approximately 1500 bp 16S rRNA gene fragment from each strain was amplified by conventional PCR using primer pair 8F/Prokl492 (Table 1).

Relative abundance of CRIB was calculated by dividing the CRIB 16S rRNA gene copies amplified using the primer set CRIB-61F/CRIB-235R by the total 16S rRNA gene copies amplified using the primer set Bactl369/Prokl492 per μΐ of isolated DNA (Table 1).

Table 1 Primers and their targets used in this study

Primer Sequence 5'-3' Target Reference

AGA GTT TGA TCC TGG CTC

8F Universal 16S rRNA gene (Lane, 1991)

(AG)

CCG TCA ATT CCT TTR AGT (Muyzer et al.,

926R Universal 16S rRNA gene

TT 1995)

TAA TAC GAC TCA CTA TAG

T7 pGEM-T vector specific Promega

G

GAT TTA GGT GAC ACT ATA

SP6 pGEM-T vector specific Promega

G

519R GWA TTA CCG CGG CKG CTG Universal 16S rRNA gene (Lane, 1991)

GTG CCA GCA GCC GCG GTA (Weisburg et al.,

533F Universal 16S rRNA gene

A 1991)

(Suzuki et al.,

Bactl369F CGG TGA ATA CGT TCY CGG Universal 16S rRNA gene

2000)

GGW TAC CTT GTT ACG ACT (Suzuki et al., Prokl492R Universal 16S rRNA gene

T 2000)

GTC GAG CGA TTT ACT TCG CRIB-specific 16S rRNA This study (SEQ ID

CRIB-61F

GTA gene NO: 4)

CRIB- GGG TCC ATC CTG TAC CGC CRIB-specific 16S rRNA This study (SEQ ID 235R AAA gene NO: 8) Statistical analysis

T-RFLP profiles were compared by visual inspection and Principal Component Analysis (PCA) using custom built VHL Analysis software (Dr. Van Haeringen Laboratorium B.V.). The VHL Analysis software was built in collaboration with Dalicon B.V. (Wageningen, The Netherlands) using IDL (RSI, Boulder, CO, USA). The number of T-RFs is presented as mean ± SEM. The non-parametric Kruskal-Wallis test, followed by post-hoc Mann-Whitney £7-tests with Bonferroni correction were used to test for statistical differences in relative abundance values of CRIB between the differenet independent treatment groups. The intraclass correlation coeffecicient (ICC) was calculated to compare the T-RFLP to qPCR analysis. The correlation between two variables was described with Spearman's rank correlation coefficients and corresponding p-values. Differences in bacterial counts, histopathological scores and plasma cytokine levels between different groups of animals were analyzed using the Mann-Whitney Latest. Differences with p-values <0.05 were considered statistically significant.

RESULTS

Acute pancreatitis induces changes in the duodenal and ileal microbiota To study the changes in duodenal and ileal microbiota as result of acute pancreatitis, duodenal and ileal samples were studied by T-RFLP analysis of PCR-amplified 16S rRNA gene fragments.

Previously we have demonstrated that there was a small increase in the total bacterial counts in the duodenum and a significant increase in culturable

opportunistic pathogens seven days after induction of acute pancreatitis (van Minnen et al., 2007). In this study, T-RFLP analysis of duodenal microbial profiles

demonstrated that the number of T-RFs almost doubled in the diseased animals (seven days after induction of the acute pancreatitis) compared to healthy control animals (25 ± 3.5 vs. 41 ± 8.4; healthy control vs. placebo, respectively). T-RFLP analysis of the bacterial communities in the terminal ileum, however, showed that induction of acute pancreatitis resulted only in a modest increase in the number of T- RFs in the ileum of the diseased animals compared to healthy control animals (60.3 ± 7.2 vs 69.0 ± 7.5; healthy control vs. placebo, respectively). The composition of the ileal microbiota, as evaluated by Principal Component Analysis (PCA), was altered in the diseased animals compared to the healthy controls. PCA revealed that each healthy rat displayed a unique microbiata fingerprint, visible as a characteristic scattering of the samples in the 3-dimensional space of the first three principal components (Figure 1). In contrast, the samples of the diseased animals show a strong clustering demonstrating that the diseased animals apparently acquired a rather uniform 'acute pancreatitis associated microbiota' independent of which treatment was administered. Probiotics stimulate a not described bacterium in the terminal ileum

T-RFLP analysis demonstrated that T-RFs of identical length as predicted for the probiotic bacteria could also be detected in placebo-treated animals indicating that the endogenous microbiota shares similar T-RFs with the probiotic bacteria.

Probiotic-induced changes in the duodenal microbiota could not be detected based on T-RFLP analysis (Figure 11). However, in the ileal samples a significant increase of an unknown bacterial phylotype (T-RF 457 bp, primer 8F, Mspl and HinPI digested) was observed in the probiotic treated animals compared to the placebo- treated animals (Figure 2 and Figure 12). This T-RF could also be detected in a number of healthy control animals. It could be excluded that this bacterial phylotype was administered inadvertently, because it was absent in both the multispecies probiotic mixture and the carrier material (placebo).

In order to further characterize this phylotype, a 16S rRNA gene clone library was generated from ileal DNA samples containing the 457 bp fragment. Clones were screened by RF analysis using the same enzymes as those applied for T-RFLP analysis, and a representative clone containing an insert leading to an RF of 450-500 bp was used for further sequence analysis. The cloned 16S rRNA gene fragment, yielding a predicted T-RF of 457 bp, showed less than 97% sequence similarity to the 16S rRNA gene sequences of bacterial isolates (Table 2), suggesting that it might represent a new bacterial species, and which in the following is referred to as

'commensal rat ileum bacterium' (CRIB). Higher sequence similarities were observed with environmental sequences retrieved from mammalian intestinal samples, including rat, mouse and human (Figure 8). Table 2 16 S rRNA gene sequence identities of the CRIB-clone with published sequences of bacterial isolates

* the percentage of identity was determined by comparison of the partial 16S rENA gene sequence of CEIB with sequences present in the database using the BLAST tool from NCBI.

Based on the 16S rRNA gene sequence of the CRIB-clone a primer set was designed for the specific quantitative detection of CRIB in environmental samples by qPCR. In order to demonstrate that the CRIB-specific 16S rRNA gene primers indeed specifically detect the same 16S rRNA gene sequences as the 457 bp T-RF, qPCR was performed on the ileal DNA samples and compared to the T-RFLP results, showing overall good agreement between the two techniques for calculation of the relative abundance of CRIB in the ileal samples on basis of the highly significant correlation between the two datasets (Figure 3).

Relative ileal abundance of CRIB is associated with decreased disease severity

Different measures of disease outcome were analyzed in order to assess disease severity, including duodenal bacterial overgrowth, bacterial translocation to remote organs and pancreas pathology. The relative abundance of CRIB in the ileum of the diseased rats was inversely correlated with the degree of bacterial overgrowth in the duodenum (Figure 4a). Total bacterial counts were lower in the duodenum of animals with high relative ileal abundance of CRIB. In addition, bacterial infection of the MLNs (Figure 4b), as well as the liver, spleen and pancreas (Figure 6) was also inversely correlated with the relative ileal abundance of CRIB. Furthermore, histopathological evaluation of pancreatic tissue obtained seven days after induction of acute pancreatitis, demonstrated that pancreas pathology was significantly and inversely correlated with the relative ileal abundance of CRIB (Figure 4c). Acinar cell pathology and inflammatory cell infiltrate were the aspects which contributed most to this correlation (Figure 6). It should be noted that the relative ileal numbers of the administered probiotic strains, or endogenous strains with identical T-RFs, did not correlate with the clinical and histological severity of the pancreatitis. Above data suggest that the clinical effects of intervention with probiotics is mediated by CRIB. In order to substantiate this association, the diseased animals were divided in two groups with either a low (<7.5%) or a high (>7.5%) relative ileal abundance of CRIB, based on their average relative ileal abundance of CRIB as determined by qPCR analysis. A significantly lower total number of duodenal bacteria was detected in the animals with >7.5% CRIB compared to the animals with <7.5% CRIB (p<0.05). In addition, a relative abundance of CRIB above 7.5% was also significantly correlated (p<0.05) with less bacterial infection of the MLNs and lower total histopathological scores of the pancreas.

Relative ileal abundance of CRIB is associated with altered cytokine levels during acute pancreatitis

To determine the possible association between high relative abundance of CRIB in the ileum and cytokine plasma levels, a cytokine multiplex assay was performed on the plasma samples obtained seven days after induction of acute pancreatitis. In a previous study, we identified high plasma levels of IL-16, IL-6, IL-10, CXCL1 and TNF-a as strong predictors of mortality and bacteremia during the course of acute pancreatitis in rats, and it was found that treatment with a multispecies probiotic mixture causes a mild reduction of these cytokines (manuscript in preparation). In line with this observation, the animals with high relative ileal abundance of CRIB

(>11%) showed significantly lower plasma levels of IL- 16 and CXCL1, and a tendency to reduced levels of IL- 10 and TNF-a (Figure 7). In addition, elevated plasma levels of IL-18, a pro -inflammatory cytokine, which is significantly increased in acute pancreatitis patients with local or systemic complications (Rau et al., 2001), were only detected in animals having a low relative ileal abundance of CRIB (<11%).

DISCUSSION

A yet uncharacterized bacterial phylotype (CRIB) was identified in this study and it was associated with reduced severity of pancreatitis and associated sepsis. A higher than average relative ileal abundance of CRIB (i.e. >7.5%) was significantly correlated with decreased duodenal bacterial overgrowth, reduced bacterial translocation to remote organs, reduced infection of pancreatic necrosis and improved pancreas histology. In addition, high relative abundance of this bacterial phylotype was associated with less severe immune responses during acute pancreatitis as demonstrated by lower plasma levels of pro-inflammatory cytokines. Moreover, we demonstrated that there is an association between the presence of CRIB in the ileum of rats and the administration of a multispecies probiotic mixture. Together, these results show that effects of this multispecies probiotic mixture (Ecologic^® 641) are mediated by stimulation of a novel gut commensal bacterium (CRIB), which protects the host from severe sepsis. The most closely related bacterial species of this not previously described bacterial phylotype is Clostridium lituseburense. For the detection of this bacterial phylotype a specific and quantitative PCR (qPCR) assay was developed using 16S rRNA gene-targeted primers. This qPCR can be used in future studies to detect this phylotype in clinically relevant samples.

Dysbiosis of the small intestinal microbiota is often seen in critically ill patients and can be a cause for the development of sepsis. For example, small intestinal bacterial overgrowth can be detected in patients with hepatic cirrhosis and is associated with systemic endotoxemia (Bauer et al., 2002; Yang et al., 1998). In the case of acute pancreatitis, the occurrence of bacterial overgrowth is a pathological event during the course of acute pancreatitis due to impaired intestinal motility and is quantitatively and qualitatively correlated with bacterial translocation and infection of pancreatic necrosis (van Felius et al., 2003; van Minnen et al., 2007). In addition, it has recently been demonstrated in a rat model that during acute pancreatitis bacterial translocation occurs from the small intestine rather than from the colon (Fritz, S. et al.,Am. J. Surg. 200:111- 117, 2010)). The duodenal bacterial counts found in this study confirm our previous findings that acute pancreatitis leads to duodenal bacterial overgrowthDespite the occurrence of bacterial overgrowth in the duodenum, there was no change in composition of the duodenal microbiota. In contrast, the composition of the ileal microbiota was considerably changed upon induction of acute pancreatitis. PCA revealed that each healthy rat displayed a unique ileal microbiota fingerprint, as previously observed for intestinal microbiota of healthy humans (Booijink et al., 2010). In contrast, increased similarity of the T- RFLP profiles of diseased animals indicated that these animals acquired an 'acute pancreatitis associated microbiota' independent of which treatment deployed.

Apparently, pancreatitis induces such a strong modification of the ileal microbiota that the unique individual microbial inhabitants are replaced by other, potentially pathogenic bacteria.

Here we show that one bacterial phylotype (referred to as CRIB) was significantly more abundant in the ileum of animals prophylacticly treated with a probiotic mixture compared to the placebo treated animals. However, both T-RFLP and qPCR analysis showed large variation in the relative abundance of CRIB in the terminal ileum, which suggests that this bacterium is not an equally dominant member of the normal microbiota in every animal. This might explain why the administration of the multispecies probiotic mixture could not prevent pancreatitis - associated infectious complications in every animal. In fact, in animals that reacted poorly to probiotic treatment (e.g. the animals with high pancreatic bacterial counts), CRIB was only present in relatively low numbers (<7.5%). The chosen threshold value was set at 7.5%, based on the average relative abundance of CRIB in the total group of diseased animals. These results illustrate an important role of CRIB in the effectiveness of probiotic treatment.

The terminal ileum is the main site for interaction of commensal microbiota with the host immune system. This is reflected by the fact that the Peyer's patches, organized lymphoid tissue important for immune surveillance and initiating of immune responses in the gut, are mainly located in the ileum (Iwasaki, 2007). In our study we demonstrated that CRIB can be a dominant member of the ileal microbiota in rats, reaching up to 20% of the total microbiota in some animals. The mechanisms by which CRIB confers protective effects includes modulation of the mucosal immune system. This is demonstrated by the observation that the relative abundance of CRIB was correlated with altered plasma cytokine levels during acute pancreatitis. Recently important roles in counterbalancing dysbiosis and regulation of immune responses have been suggested for several specific members of the gut microbiota. Faecalibacterium prausnitzii has been described to have antiinflammatory properties in both cellular and animal models (Sokol et al., 2008), and underrepresentation of this bacterial species in the intestinal microbiota was suggested to be involved in IBD pathogenesis in both animals and humans (Sokol et al., 2009). In addition, segmented filamentous bacteria (SFB) were shown to have a crucial role in maturation of T cell responses in the gut (Gaboriau-Routhiau et al., 2009; Ivanov et al., 2009). Faecalibacterium prausnitzii and SFBs are members of the same taxonomic class as CRIB, the Clostridia, however, they belong to different subgroups (Figure 8). In addition, a recent study has demonstrated a significant induction of colonic regulatory T cells as result of the colonization by a specific mix of indigenous Clostridium species in a mouse experiment (Atarashi, K. et al., Science 331:337-341, 2011). In that study, early inoculation of these Clostridium species resulted in the amelioration of the effects of experimental colitis and a lower IgE production Altogether, these studies demonstrate that specific members of the Clostridia can have health-promoting effects.

REFERENCES for example 1

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Beger HG, Rau BM (2007). Severe acute pancreatitis: Clinical course and management. World J. Gastroenterol. 13: 5043-51.

Booijink CC, El-Aidy S, Rajilic-Stojanovic M, Heilig HG, Troost FJ, Smidt H et al (2010). High temporal and inter- individual variation detected in the human ileal microbiota. Environ. Microbiol.: (in press). Carter MJ, Milton ID (1993). An inexpensive and simple method for DNA purifications on silica particles. Nucleic Acids Res. 21: 1044.

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Gaboriau-Routhiau V, Rakotobe S, Lecuyer E, Mulder I, Lan A, Bridonneau C et al (2009). The key role of segmented filamentous bacteria in the coordinated maturation of gut helper T cell responses. Immunity 31: 677-89.

Ivanov, II, Atarashi K, Manel N, Brodie EL, Shima T, Karaoz U et al (2009). Induction of intestinal Thl7 cells by segmented filamentous bacteria. Cell 139: 485- 98.

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Liu WT, Marsh TL, Cheng H, Forney LJ (1997). Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl. Environ. Microbiol. 63: 4516-22.

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McGinnis S, Madden TL (2004). BLAST: at the core of a powerful and diverse set of sequence analysis tools. Nucleic Acids Res. 32: W20-5.

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Rau B, Baumgart K, Paszkowski AS, Mayer JM, Beger HG (2001). Clinical relevance of caspase-1 activated cytokines in acute pancreatitis: high correlation of serum interleukin-18 with pancreatic necrosis and systemic complications. Crit. Care Med. 29: 1556-62.

Schmid SW, Uhl W, Friess H, Malfertheiner P, Buchler MW (1999). The role of infection in acute pancreatitis. Gut 45: 311-6.

Schmidt J, Rattner DW, Lewandrowski K, Compton CC, Mandavilli U, Knoefel WT et al (1992). A better model of acute pancreatitis for evaluating therapy. Ann. Surg. 215: 44-56. Sokol H, Pigneur B, Watterlot L, Lakhdari O, Bermudez-Humaran LG, Gratadoux JJ et al (2008). Faecalibacterium prausnitzii is an anti- inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients. Proc. Natl. Acad. Set. U. S. A. 105: 16731-6.

Sokol H, Seksik P, Furet JP, Firmesse O, Nion-Larmurier I, Beaugerie L et al (2009). Low counts of Faecalibacterium prausnitzii in colitis microbiota. Inflamm. Bowel Dis. 15: 1183-9.

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van Minnen LP, Timmerman HM, Lutgendorff F, Verheem A, Harmsen W, Konstantinov SR et al (2007). Modification of intestinal flora with multispecies probiotics reduces bacterial translocation and improves clinical course in a rat model of acute pancreatitis. Surgery 141: 470-80.

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Example 2

Isolation and characterization of CRIB

Isolation was done with help of dilution series with a specific medium. To check the purity of the cultures we isolated DNA of the bacteria in the ileal samples and mixed cultures and used techniques as DGGE and real time PCR to analyze the DNA. A pure culture of CRIB was achieved after several dilution series and a dilution series with solid medium. This was checked by DGGE, real time PCR, microscope and sequencing. After the isolation we started with the characterization, by trying to find a specific medium for CRIB by using diverse combinations of substrates. Also we made a growth curve by measuring the optical density of the culture at 660 nm during time. We can conclude that CRIB was isolated by comparing the sequence of the pure culture with the known sequence. Method:

Bacteria are grown in 50ml liquid medium in 120ml bottles. The gas phase of the bottles is replaced by a gas mixture of N2/CO2 (80/20 % v/v) with a flushing system. The bottles are sterilized at 120°C for 20 minutes before use. To isolate CRIB we used a dilution series approach (Fig. 11). The first bottle (dilutionO) is inoculated with 1 ml of either a rat sample or a previous culture. Of this first bottle 0.5ml are taken and inoculated into the second bottle (dilution 10 ²). From the second bottle 0,5ml are taken and inoculated into the third bottle (dilution 10 ⁴) and this was repeated until dilution 10^~10. After the cultures are fully grown, DNA isolation and qPCRs are done in order to detect CRIB growth. We chose the highest dilution where CRIB is still present as inoculum for a new dilution series. When an almost pure culture was achieved in liquid medium, we made a dilution series with solid medium. In solid medium bacteria grew as colonies. We picked one colony and inoculated it in liquid medium again to obtain a pure culture.

Cell morphology of strain CRIB was examined by using a light microscope (DM2000, Leica) at lOOOx, with cells grown for 24 h or 48 h at 37 °C in CRIB and PYG medium. To obtain ultrathin sections for transmission electron microscopy, harvested cells were fixed with glutaraldehyde and then with osmium tetroxide, stained with uranyl acetate (Ryter & Kellenberger, 1958), dehydrated in an ethanol series and embedded in epoxy resin. Flagella and endospores were examined according to the methods of Leifson and Schaeffer- Fulton, respectively (Smibert & Krieg, 1994). After Gram- staining cells were observed by light microscope. Colony morphology was examined by the eye.

Oxygen tolerance was examined by growth of CRIB in thioglucolate Cell motility was examined by detection of turbidity throughout tubes containing semisolid medium (Leifson, I960). General strain characteristics such as colony colour, size and shape were determined after 24 h and 48 h of growth on solid PYG medium. Growth was tested at various temperatures (20-50 °C, in increments of 5 °C) and pH's (5.0—9.0, in increments of 0.5 pH units). Tolerance of NaCl was tested at different NaCl concentrations [0-3 % (w/v), in increments of 0.5 %, and 1-7 % (w/v), in increments of 1%] . Tolerance of bile (Oxgall, Difco) was tested at different bile salt concentrations [0-25 % (v/v), in increments of 5 %]. The growth of strain CRIB in the presence of Tween-80 was examined using different Tween-80 concentrations [0-0.04 % (v/v), in increments of 0.01 %]. The influence of shaking during incubation of liquid cultures on the growth of strain CRIB was determined by comparing the growth while shaking at 100 rpm with growth while not shaking.

Substrate utilization was determined by adding one of the following compounds to PY medium to a final concentration of 0.5 % (w/v): D-adonitol, D-arabinose, L-arabinose, D-arabitol, L-arabitol, D-cellobiose, cellulose, erythritol, D-fructose, D-fucose, L- fucose, D-galactose, D-glucose, glycerol, glycogen, inositol, inulin, D-lactose, D- maltose, D-mannitol, D-mannose, D-melibiose, methyl-a-D-glycopyranoside, methyl-a- D-mannopyranoside, mucin, D-raffinose, L-rhamnose, D-ribose, D-saccharose, soluble starch, D-sorbitol, D-trehalose, D-turanose, xylitol, D-xylose, L-xylose. Growth was measured spectropho to metrically at ODeoonm. Acid formation was observed by measuring the pH of the media up till 30 days of incubation. The substrate utilization properties of strain CRIB were compared to those of 4 closely related Clostridium species using the API 50CH and API 20A systems (bioMerieux) according to the manufacturer's instructions except that PY medium was used for inoculation.

MIC-values ^g/ml) of several antimicrobial agents (clindamycin, penicillin G and metronidazole) against strain CRIB were determined using Etest gradient strips (bioMerieux).

Chemotaxomic characterization of strain CRIB was carried out by DSMZ

(Braunschweig, Germany). For analysis of the cellular fatty acid composition, peptidoglycan structure, cell wall sugars, polar lipid and quinone composition, cells of strain CRIB were cultured in CRIB medium for 15 h at 37 °C.

For determination of the cellular fatty acid composition of strain CRIB fatty acid methyl esters were obtained from cells of strain CRIB by saponification, methylation and extraction using minor modifications of the previously described methods

(Kuykendall et aL , 1988; Miller, 1982). The fatty acid methyl esters mixtures were separated by GC and analysed using the Sherlock Microbial Identification System (MIS) as previously described (Adachi et. a! , 2007). Peaks were automatically integrated and fatty acid names and percentages were calculated by the MIS

Standard Software (Microbial ID).

The peptidoglycan of strain CRIB was isolated and purified according to the method of Schleifer (1985). Peptidoglycan preparations were obtained after disruption of cells by shaking with glass beads and subsequent trypsin digestion. The peptidoglycans were hydrolysed in 4N HC1 (16 h at 100 °C). The amino acids and peptides in the cell-wall hydrolysates were analysed by two-dimensional TLC on cellulose plates by using a previously described solvent system (Rhuland et al., 1955). After derivatization, according to previously described methods (MacKenzie, 1987) the molar ratios of amino acids were determined by GC. The iV-heptafluorobutyryl amino acid isobutyl esters obtained by derivatization were subjected to GC-MS. For cell wall sugar analysis the peptidoglycan of strain CRIB^T was hydrolysed in IN H2SO4 (2 h at 100 °C). H2SO4 was removed by 20 % N,N- dioctylmethylamine in chloroform according to Whiton et al. (1985) and the sugars in the hydrolysate were analysed by TLC on cellulose plates according to Staneck & Roberts (1974).

Respiratory lipoquinones and polar lipids were extracted from freeze dried cell material using the two stage method described by Tindall et al. (1990). For analysis of the respiratory lipoquinones, the lipoquinones were separated by silica gel TLC using hexane : ieri-butylmethylether (9 : 1, v/v) as solvent. UV absorbing bands

corresponding to the different quinone clases (e.g. menaquinones or ubiquinones) were removed from the plate and further analysed by HPLC using methanol : heptane (9 : 1, v/v) as the mobile phase. Respiratory lipoquinones were detected at 269 nm. For analysis of the polar lipids, the polar lipids were separated by two-dimensional TLC on silica gel. The first direction was developed in chloroform : methanol : water (65 : 25 : 4, v/v/v), and the second in chloroform : methanol : acetic acid : water (80 : 12 : 15 : 4, v/v/v/v). Total lipid material and specific functional groups were detected using dodecamolybdophosphoric acid (total lipids), Zinzadze reagent (phosphate), ninhydrin (free amino groups), periodate-Schiff (a-glycols), Dragendorff (quaternary nitrogen), and a-naphthol-sulphuric acid (glycolipids). Based on phylogenetic, genotypic, chemotaxonomic and phenotypic differences between strain CRIB and previously described species, we suggest that strain CRIB should be assigned as type strain of a novel species in the genus Clostridium, for which we propose the name Clostridium romboutsii sp. Nov.

Results of phenotypical characterization

The type strain CRIB, deposited under the Budapest Treaty with the DSMZ, on date 29 June 2007 under the accession number 19498 was isolated from the gastro- intestinal tract of rats at the University of Wageningen, the Netherlands.

Bacteria are obligate anaerobe. Cells are non-motile, spore-forming rods. Typical cells are 1.0-2.0 μπι x 1.0-5.3 μπι in size and occur in single, in pairs or in chains. Cells stain Gram-positive, but rapidly become Gram- stain-negative as cultures reach maximum stationary phase. Free spores are seen occasionally after prolonged incubation. Surface colonies on PYG agar plates incubated anaerobically for 24 hours are 0.5- 1 mm in diameter, circular, flat to low convex, opaque with translucent margins, white, shiny and smooth, and an entire to indulate margin. Good growth occurs on CRIB and PYG broth or agar. Temperature range for growth is 30-45 °C with an optimum temperature of 37 °C. The pH range for growth is 6.5-8.0 with and optimum pH of 7.0-7.5. Growth occurs at NaCl concentrations of 0-1 % (w/v). Growth is inhibited by 5-20 % bile. Growth is stimulated by addition of 0.01 % Tween 80 (v/v) to PYG medium and shaking of the cultures at 100 rpm. Cultures in PYG broth are turbid with a smooth sediment and a pH of 5.5-6.0 after incubation for a week.

Abundant gas is produced in PYG deep agar cultures. Strain CRIB was able to grow mainly on carbohydrates. Weak growth was observed on yeast extract and bacterial peptone as the sole carbon source. The strain produces acid from L-fucose, galactose, glucose, raffinose and sucrose when grown in PY medium. Acid is not produced from D-adonitol, D-arabinose, L-arabinose, D-arabitol, L-arabitol, cellobiose, erytritol, D- fucose, glycerol, glycogen, inositol, lactose, maltose, mannitol, mannose, melibiose, methyl-a-D-mannopyranoside, methyl-a-D-glucopyranoside, mucin, rhamnose, ribose, sorbitol, soluble starch, sorbose, trehalose, turanose, D-xylose and L-xylose. Starch is not hydrolysed. SCFA produced are acetate, butyrate and lactate. ¾ is produced. The type strain is sensitive to clindamycin (MIC 0.125 μg/ml), penicillin G (MIC >0.016 μg/ml), and metronidazole >0.016 μg/ml).

The major cellular fatty acids of strain CRIB were found to be saturated and unsaturated Cie and Cis fatty acids, with Ci6:o and Ci8:i cis 9 being the predominant fatty acids. The peptidoglycan of strain CRIB contains alanine and glutamic acid (molar ratio 1.4 : 1.0) and a di-iV-heptafluorobutyryl-lanthionine-diisobutylester. The peptidoglycan type of strain CRIB was concluded to be Alo lantionine- direct, which has, to our knowledge, not been previously described for Clostridium species. The cell wall sugars are glucose and galactose. The major respiratory lipoquinone present is the menaquinone MK-6 (100%). The polar lipids of strain CRIB comprise a

diphosphatidylglycerol, a phosphatidylglycerol, five phospolipids, five glycolipids and a lipid. The G+C content of strain CRIB is 28.1 %. Strain CRIB showed low relative DNA- DNA similarity to the type strains C lituseburense DSM 797^T (15.5±0.8 %); C.

irregulare DSM 2635^T (18.1±1.3 %); C hiranonis DSM 13275^T (31.4±2.5 %) and C. bartlettii DSM 16795^T (20.4±3.5 %).

DNA isolation

DNA isolation was done with the FastDNA SPIN Kit for soil MP

Biomedicals). Bacteria were first lysed by shaking tubes with the sample at very high speed. These tubes contained silica particles and a DNA stabilizing solution which is a mixture of detergents and salts. The detergents have two functions: a) they contribute to inactivate nucleases and b) they provide lubrication during the lysing step to control the degree of shearing of the DNA. After lysation the DNA was purified by several washing and centrifugation steps.

Subsequently the DNA was dissolved in water to be application ready.

DGGE (denaturing gradient gel electrophoresis)

Materials: - 0% denaturing solution (acrylamide, T.A.E. buffer, glycerol)

- 100% denaturing solution (acrylamide, T.A.E. buffer, formamide, glycerol, urea)

- Ammonium persulfate (APS)

- Tetramethylethylenediamine (TEMED)

- T.A.E. buffer (TRIS base, glacial acetic acid, EDTA)

- lx Cairn's fixation solution

- Silverstaining solution (AgNOe)

- Developing solution (NaBEU, NaOH, formaldehyde)

- Cairn's preservation solution (ethanol, glycerol)

One of the primers used with for the 16S rRNA PCR had a GC clamp. This clamp consisted of approximately 40base pairs with G's and C's, so it has such a high GC% that it did not denature in the gel. Because of this clamp a 'fork' originated while migrating through the gel, with the 2 single stranded strains of DNA attached to the clamp. As soon as this fork originated, migration of the DNA through the gel stopped. Because the samples contain different bacteria species, a specific pattern of bands occurred. The place of a band was specific for a certain bacterium. The gradient gel was made with two solutions, a 0% denaturing solution and a 100% denaturing solution. Usually the gel was made with a gradient from 30 to 60% denaturing chemicals. First the 30% and 60% denaturing solutions were made. These solutions were used to make the gradient with help of a gradient pump. A 0% denaturing stacking gel was poured above the gradient gel and a comb was placed. The stacking gel was necessary for loading the samples in the gel, the samples will reach the denaturing gel at the same time, with better results later.

The gel needed to polymerize for at least an hour, chemicals used for the

polymerization were APS and TEMED. After the polymerization the comb could be removed from the gel and the gel was ready for use. The samples and markers were loaded and subsequently the electrophoresis took place overnight (16 hours) at 60°C in lx TAE buffer. After the electrophoresis the gel was silver stained. First the DNA was fixated in the gel with the fixation solution. After that the gel was stained with the silverstaining solution. Subsequently the gel was developed with the developer solution, and preserved with preservation solution covered with cellophane. Then the gel needed to be dried overnight at 60°C.

Real time PCR

The results of the real time PCR are shown in a graph (Fig. 17). The graph shows the amount of fluorescence visible in each cycle of the real time PCR. Every line is a different sample or standard. To achieve reliable results every sample was measured in triplicate and the standards in duplicate. To calculate the ratio of CRIB versus total bacteria in the cultures, we used two different programs and primer sets. One specific for CRIB and one universal real time PCR for all bacteria present in the culture. The two different programs applied are shown in table 3 (for total bacteria) and 4 (for CRIB quantification). Table 3: Real time PCR program for total bacteria

Cycle - repetitions Time and temperature

1 - (lx) 3 min - 95°C

2 - (40x) 15 sec - 95°C

45 sec - 56°C

Real time data

3 - (lx) 1 min - 95°C

4 - (lx) 1 min - 65°C

5 - (60x) 10 sec - 65°C Table 4: Real time PCR program for CRIB

Cycle - repetitions Time and temperature

1 - (lx) 3 min - 95°C

2 - (40x) 20 sec - 95°C

1 min - 68°C

Real time data

3 - (lx) 1 min - 95°C

4 - (lx) 1 min - 68°C

5 - (55x) 10 sec - 68°C Isolation

The purpose of our study was to isolate CRIB from ileal rat samples. We used 8 samples, from 4 different rats. First we measured the percentage of 16S rRNA gene copies of CRIB in comparison with the total amount of bacteria by qPCR (Fig. 18). This way we could select the samples with the highest percentage for further use. The percentage of CRIB of samples 3.1 and 4.2 resulted in more than 100%. This was probably due to bias in the quantification process. Figure 19 shows the DGGE gel of the starting rat samples. There were many different bands, each most probably coding for a different bacterium. There were also differences between the samples. The positive control was Clostridium difficile, one of the closest cultured relatives of CRIB. We chose samples 2.2 and 3.1 to make a dilution series with CRIB medium I, which contained glucose, maltose, cellobiose, casitone, yeast extract and starch. We tried growing the samples for 1, 2 or 3 days, but the amount of CRIB decreased over time. Because CRIB did not grow well on CRIB medium I, we designed a new medium,

CRIB medium II, which contained the same ingredients as medium I plus bile salts, cystein, rumen fluid, beef extract, hemin, vitamin Kl and peptone (see Table 6) . We used sample 4.2 to make the new dilution series. After 1 day of incubation CRIB grew, but after 2 days the number of CRIB went down again. In figure 20 we show some examples of the decrease of CRIB numbers over time (the ratio of CRIB versus total bacteria after 1 and after 2 days of incubation). The samples are dilutions 10^~2 and 10^{" 10} non pasteurized (NP) and 10^~2 and 10^~6 pasteurized (P). The amount of CRIB drastically decreased after 2 days of incubation at 37°C.

Because of the loss of CRIB from sample 4.2, we made two new dilution series with sample 3.2 (Fig. 21), one series we pasteurized (P) and one was non-pasteurized (NP).

Figure 22 shows that the cultures were getting more pure. After this dilution series we made a dilution series with solid medium, so we could pick colonies to hopefully obtain pure culture. We made 2 dilution series from 0 to 10^~10, one with sample 10^~8 and one with 10^~8-10^~4.We randomly picked 15 colonies in total, of which 9 of the 10^~4 dilution of sample 10^~8-10^~4. Of these 9 colonies, 8 colonies grew on liquid medium. After inspection of the culture through the microscope, we chose colony 1 and 5 to make a dilution series on liquid medium (Figs. 23 and 24). The ratios of the dilution series of colony 1 and 5 showed that the culture was almost pure. We also checked the dilution series with DGGE (Fig. 25). The dilution series on the left from 0 to 10-6 was from colony 1, and the dilution series on the right from colony 5. As positive control we used Clostridium difficile. The gel showed that the profiles of all the cultures were the same, so they contained the same

bacteria/bacterium. To check whether the cultures were pure, we sent samples 1 10 ¹, 1 10^"4, lxlO^"5, 5xl0^"4 and 5xl0^~6 for direct sequencing (no previous cloning into commercial vectors). We analyzed the results of the sequences obtained to see whether there was only DNA from one bacterium or if we could detect background signals from other species. Subsequently we compared the sequences to the known 900bp clones previously obtained for CRIB (unpublished). The data showed that samples lxlO ⁴, lx 10 ⁴, 1 xlO ⁵ and 5 xlO ⁴ were pure cultures of CRIB. BLAST analyses of the 1500bp sequence of CRIB on 16S rRNA gene databases are shown in table 5

Table 5: Closest cultured relatives of CRIB

Closest cultured relative Sequence identity (%)

Clostridium lituseburense 96

Clostridium bartlettii 95

Clostridium metallolevans 95

Characterization

After the isolation we started to characterize CRIB. First we investigated different media to see which substrates were necessary for the growth of this bacterium Cell culturing media without starch, bile salts or cystein did not support growth of CRIB. We also did a test with oxygen to see how strictly anaerobic CRIB is. For this we centrifuged a culturing medium (in presence of oxygen) and inoculated the pellet into fresh medium. After a day we checked if the bacteria could still grow, even after oxygen exposure. We made a growth curve of CRIB by measuring the optical density at 660nm (Fig. 26). The first measurement was after 18 hours and since then we measured every hour. After 22 hours CRIB started dying and the optical density decreased. Table 6. CRIB medium

Vitamin Kl: 0.15ml in 30ml ethanol 95%. Stored at 4°C in dark.

Hemin: 50mg in 50ml water + 1ml NaOH IN. Stored at 4°C.

Before inoculation, the medium had to be supplemented with 5% bicarbonate solution (containing the reducing agent Na2S, or alternatively cystein HC1) and 5% salts solution (containing the vitamins).

Bicarbonate solution:

COMPONENT/SOLUTION QUANTITY

Solution 4 (NaHCOs) 100 mL Solution 5 (Na₂S.9 H₂0) 2 mL

Salts and vitamins solution:

Stock solutions:

Stock solutions to make the basal medium (grams/milligrams are given for dilution in 1L):

Solution 1 27.2 g KH2PO4

Solution 2 35.6 g Na₂HP04.2H₂0

Solution 3 24 g NH4CI; 24 g NaCl; 8 g MgCl₂.6H₂0: 8.8 g CaCl₂.2H₂0

Solution 4 80 g NaHCOs

Solution 5 240.2 g Na₂S.9 H₂0 (store under N₂ and in the dark)

Trace elements (solutions 6 & 7): Acid Stock Solution (Solution 6)

0.5 mM MnCb 61.25 mg

7.5 mM FeCl₂ 943.5 mg

0.5 mM CoCl₂ 64.5 mg

0.1 mM NiCl₂ 12.86 mg

0.5 mM ZnCl₂ 67.7 mg

Alkaline Stock solution (Solution 7)

10 mM NaOH 400 mg

0.1 mM Na₂SeO₃ 17.3 mg

0.1 mM Na₂W0₄ 29.4 mg

0.1 mM Na₂Mo0₄ 20.5 mg

Solution 8: 0.5 g Resazurin

Vitamins: Biotin 20 mg

Nicotinamid 200 mg p-Aminobenzoic acid 100 mg

Thiamin (Vit Bl) 200 mg

Panthotenic acid 100 mg

Pyridoxamine 500 mg

Cyanocobalamine (Vit B12) 100 mg

Riboflavin 100 mg

(Folate 50 mg)

(Lipoate 50 mg) When the isolated, pure CRIB culture was obtained, we set off to sequence the complete 16S RNA gene, by cloning the gene, using primers 27F and 1492R, into a PGEM-T-Easy vector, and sequencing the gene with vector primers T7, SP6 and 650. The sequence that was found was (SEQ ID NO: 3):

GTTTGATCCTGGCTCAGGATGAACGCTGGCGGCGTGCCTAACACATGCAAGTCGAGCGAT TTACTTCGGTAAAGAGCGGCGGACGGGTGAGTAACGCGTGGGTAACCTGCCCTGTACACA CGGATAACGTACCGAAAGGTATGCTAATACGAGATAAAATACTTTTGTCGCATGGTAGAA GTATCAAAGCTTTTGCGGTACAGGATGGACCCGCGTCTGATTAGCTAGTTGGTAAGGTAA CGGCTTACCAAGGCGACGATCAGTAGCCGACCTGAGAGGGTGATCGGCCACATTGGAACT GAGACACGGTCCAAACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGAAA GCCTGATGCAGCAACGCCGCGTGAGCGATGAAGGCCTTCGGGTCGTAAAGCTCTGTCCTC AAGGAAGATAATGACGGTACTTGAGGAGGAAGCCCCGGCTAAC TACGTGCCAGCAGCCGC GGTAATACGTAGGGGGCTAGCGTTATTCCGAAATTACTGGGCGAAAAGGGTGCGTAGGGT GGTTTCTAAAGTCAGAGGTGAAAGGCTACGGCTCAACCGTAGTAAGCCTTTGAAACTGGG GAACTTGAGTGCAGGAGAGGAGAGTGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATAT TAGGAGGAACACCAGTTGCGAAGGCGGCTCTCTGGACTGTAACTGACACTGAGGCACGAA AGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTACT AGCTGTCGGAGGTTACCCCCTTCGGTGGCGCAGCTAACGCATTAAGTACTCCGCCTGGGA AGTACGCTCGCAAGAGTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGTAGCGGAGC ATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTAAGCTTGACATCCTTTTGACCG ATGCCTAATCGCATCTTTCCCTTCGGGGACAGAAGTGACAGGTGGTGCATGGTTGTCGTC AGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGCCTTTAGTT GCCAGCATTAAGTTGGGCACTCTAGAGGGACTGCCAGGGATAACCTGGAGGAAGGTGGGG ATGACGTCAAATCATCATGCCCCTTATGCTTAGGGCTACACACGTGCTACAATGGGTGGT ACAGAGGGCAGCCAAGTCGTGAGGCGGAGCTAATCCCTTAAAGCCATTCTCAGTTCGGAT TGTAGGCTGAAACTCGCCTACATGAAGCTGGAGTTAC TAGTAATCGCAGATCAGAATGCT GCGGTGAATGCGTTCCCGGGTCTTGTACACACCGCCCGTCACACCACGGAAGTTGGGGGC GCCCGAAGCCACTTAGCTAACCCTTTTGGGAAGCGAGTGTCGAAGGTGAAATCAATAACT GGGGTGAAGTCGTAACAAGGTAGCCG

For genome sequencing, cells of strain CRIB were cultured in CRIB medium for 15 h at 37 °C. Cells were harvested and genomic DNA was isolated by the CTAB method proposed by the Joint Genome Institute (JGI) with minor modifications. High- molecular-weight genomic DNA was provided to GATC Biotech (Germany) for sequencing. First the genomic DNA was sequenced on an Illumina Genome

Analyzer IIx using the 36-cycle SBS Kit v5 in paired end mode, and the 8kb mate pair library was sequenced on a Roche GS FLX Titanium with the Lib-L kit. The contigs that have been sequenced can be found in the sequence listing which is filed together with the application under SEQ ID NO: 22 - SEQ ID NO: 130.

Example 3

Aim: The aim of this study was to investigate the effect of CRIB on the tight junctions integrity of Caco-2 cells responding to ethanol challenge. It is increasingly recognized that ethanol induces damage of the intestinal epithelial barrier function and increases intestinal permeability. Increased intestinal permeability can lead to increased translocation of bacterial products (endotoxin) from the intestine to the liver and general circulation where endotoxin may trigger inflammatory changes in the liver contributing to alcohol-induced liver injury. Nutritional products that are able to improve gut health might prevent alcohol-induced increased intestinal permeability and subsequent liver injury.

Methods: Caco-2 cells were plated as monolayers at a density of 1.5 x 10⁵ on a 0.4-μπι pore cell culture insert with a surface area of 0.33 cm². Monolayers were treated with either ethanol (20 mM, 40 mM), CRIB (50

of l.OxlO⁹ and 1.0x10^s) or CRIB + ethanol for 2 hrs. Monolayers incubated with ethylene glycol tetra acetic acid (EGTA), known to disrupt tight junctions or with only medium were served as +ve and— ve control, respectively. Transepithelial electrical resistance (TER), an indicator of tight junctions integrity was monitored using epithelial voltohmmeter.

Results: This study demonstrated that the transepithelial electrical resistance (TER) decreased with time after treatment with ethanol. With CRIB treatment, the changes of TER were improved as compared with ethanol groups (see Fig. 13 and 14).

Conclusion: CRIB exerted a protective effect against the damage of Caco-2 monolayer integrity by ethanol.

Example 4

Method for stimulating T cell differentiation by CRIB

Isolated Peripheral blood mononuclear cells (PBMC) were cultured together with CRIB. Subsequently, CD4 positive T cells were isolated and lysed. By using quantitative PCR, mRNA levels of FOXP3, Tbet, GATA3 and RORyT were

determined. The amount of these mRNAs was compared to a household gene product, 62M. The relative amounts of mRNA were compared to a positive control (tetanus stimulated cells) and the relative amount of mRNA of FOXP3, Tbet, GATA3 and RORyT after treatment with CRIB is compared with the amounts of the same gene products in medium without CRIB.

The results are shown in Figure 27. The expression levels of Tbet and RORyT are respectively 1.5 and 2.7 times increased compared to medium. Example 5

I. Design of specific primers for CRIB

For the design of specific primers for the detection of CRIB, sequences from a CRIB clone (900bp) were aligned with its closest relatives.

Different primer sets were first analyzed by traditional PCR. The primers tested were:

1) crib-61f (S-*-crib-0061-a-S-21): Tm 62°C

Sequence 5'-3': GTCGAGCGATTTACTTCGGTA

2) 132f (S-*-Clit-0132-a-S-24): Tm 72°C

Sequence 5'-3': CTG C C CTGTAC AC ACGGATAAC AT

3) 155r (S-*-Clit-0155-a-A-24): Tm 72°C

Sequence 5'-3': ATGTTATCCGTGTGTACAGGGCAG

4) 220r (S-*-crib-0220-a-A-25): Tm 70°C

Sequence 5'-3': CGCAAAAGCTTTGATACTTCTACCA

PCR products of CRIB were detected only with the primer combination 61f— 155r. This combination also amplified DNAs from the following Clostridia clinical isolates:

• UC-2: C difficile

• UC-7: C. sp.

• UC- 11: C. limosum

• UC-20: C difficile

· UC-21: C. sporogenes

• UC-23: C difficile

• UC-24: C difficile

New reverse primers were designed to use in combination with crib-61f:

5) 193r (S-*-crib-0193-a-A-25): Tm 64°C

Sequence 5'-3': TATTCTACCATGCGATAAAAGTATT

6) 235r (S-*-crib-0235-a-A-21): Tm 66°C

Sequence 5'-3': GGGTCCATCCTGTACCGCAAA

7) 234r (S-*- crib-0234-a-A-21): Tm 64°C

Sequence 5'-3': GGTCCATCCTGTACCGCAAAA

DNAs of the closest relatives according to 16S rRNA sequencing analysis as well as some of the previously amplified clinical isolates were included in the PCR reaction. The samples used were:

· CRIB clone (positive control)

• UC-2: Clostridium difficile

• UC-20: Clostridium difficile

• Clostridium lituseburense (type strain 797)

• Clostridium bartlettii (type strain 16795)

· UC- 11: Clostridium limosum

• Negative control To identify the best condition for the primers used, gradient PCRs were performed from 50°C - 70°C on all DNAs:

Total 24.5 2009

0.5μ1 DNA

The following primer combinations gave the best positive results:

61f / 235r

The PCR products of the samples are shown in Fig. 22 and 23.

Product was obtained from CRIB and C. lituseburense.

C. lituseburense type strain 797 could be detected up to 53.3°C and CRIB was detected up to 64°C.

Product was obtained from CRIB and C. limosum (UC-11).

C. limosum could be detected up to 58.6°C and CRIB was detected up to 70.4°C.

II. Testing of primers for CRIB by qPCR

II.l. The primer combination crib-61f / SEQ ID NO:193r was tested by iQ5 real time PCR, in gradient from 50°C to 70°C.

Samples:

As templates we used DNA from CRIB and C. lituseburense type strain 797 since they were the only that gave a PCR product. NT: no template

BioEad mix 562.5 Protocol:

F (ΙΟμΜ) 22.5 Cycle 1: (IX)

E (ΙΟμΜ) 2§.5 Step 1: 95.0 °C for 03:00. Water 292.5 Cycle 2: (40X)

Total 900 Step 1: 95.0 °C for 00:20.

Step 2: 50.0 °C- 70.0 °C for 00:30.

5μ1 DNA Step 3: 72.0 °C for 00:30.

Data collection and real-time analysis enabled.

Cycle 3: (IX)

Step 1: 95.0 °C for 01:00.

Cycle 4: (IX)

Step 1: 50.0 °C for 01:00.

Cycle 5: (9 IX)

Step 1: 50.0 °C-95.0 °C for 00:10.

Increase set point temperature after cycle 2 by 0.5 °C

Melt curve data collection and analysis enabled.

797 15 23.42 23.56 0.206 51.5

797 15 23.71 23.56 0.206 51.5

797 16 23.72 23.84 0.171 50

797 16 23.96 23.84 0.171 50

II.2. The primer combination 61f/235r was tested by iQ5 real time PCR, in gradient from 50°C to 70°C.

Samples:

As templates we used DNA from CRIB and C. limosum UC-11 since they were the only that gave a PCR product. NT: no template

Protocol:

BioEad mix 562.5 Cycle 1: (IX)

F (10μΜ) Step 1: 95.0 °C for 03:00. E (10μΜ) 22.5 Cycle 2: (40X)

Water 292.5 Step 1: 95.0 °C for 00:20.

Total 900 Step 2: 50.0 °C-70.0 °C for 00:30.

Step 3: 72.0 °C for 00:30.

5μ1 DNA

20 Data collection and real-time analysis enabled.

Cycle 3: (IX)

Step 1: 95.0 °C for 01:00.

Cycle 4: (IX)

Step 1: 50.0 °C for 01:00.

Cycle 5: (9 IX)

Step 1: 50.0 °C-95.0 °C for 00:10.

Increase set point temperature after cycle 2 by 0.5 °C

Melt curve data collection and analysis enabled.

Results:

We selected primers 61f / 235r at 68°C for future analysis since C. limosum that amplifies with this primer set is not so phylogenetically close to CRIB as C.

lituseburense.

Example 5 - Isolation and characterisation of human CRIB

Material en methods

Liquid media preparation

CRIB medium consisted of a basal bicarbonate -buffered medium (Stams et al., 1993) and the following supplements (L): 30 g bacteriological peptone, 5 g yeast extract, 5 g beef extract, 4 g glucose, lg cellobiose, 1 g maltose, 1 soluble starch, 0.5 L- cysteine hydrochloride, 0.4 g bile salts, 0.25 mg hemin, 0.0001 % (v/v) vitamin Ki. The pH was adjusted to 7. MIN CRIB medium was modified CRIB medium, supplied with 1/10 quantity of peptone, yeast extract and beef extract. PY medium consisted of basal peptone-yeast extract medium (Holdeman et aL, 1977). The media were autoclave sterilized. The vitamins were filter sterilized.

Inoculation of ileal effluent was done under strict anaerobic conditions. Tenfold serial dilutions of the ileal effluent were used for the isolation with MPN approach (Sutton, 2010). The incubations were done in a climate room at 37°C.

Solid medium preparation, inoculation and single colony isolation for PCR screening of the bacterial colonies

DSMZ medium is a modified version of medium 104 of DSMZ. Tenfold dilution series were prepared in liquid CRIB medium starting with bacterial culture containing diverse species with 0.33% relative abundance of human CRIB. The dilution series and the inoculations on solid medium were done under strict anaerobic conditions in an anaerobic plastic tent. Of the tenfold dilution series (range 10°- 10^~8), ΙΟΟμΙοί the culture was plated on solid medium using sterile glass beads. The petri dishes were incubated in anaerobic jars with oxygen scavengers (Anaerocult A (Merck, 1.13829.0001) and oxygen indicators (Anaerotest (Merck, 1.15112.0001), at 37°C. After 15h incubation in anaerobic jars, 288 colonies were separately inoculated in a 96-wells Polypropylene MASTERBLOCK® (Greiner Bio One) in 0.5 ml CRIB medium under sterile aerobic conditions (Biological Safety Cabinet, Thermo Scientific MS C -Advantage) using 1 μΐ and 10 μΐ flexible plastic inoculation loops (Copan). The 96-wells plates were covered with BREATHseal™(Greiner Bio One) and incubated in anaerobic plastic bags with oxygen scavengers and oxygen indicators at 37°C for 24h. The next day a second series of 96-wells Polypropylene MASTERBLOCK® (containing 0.5ml medium per well) was re-inoculated using 100 μΐ of the cultures from the first day. The plates were incubated for 22h in the same way. Glycerol stock solution (0.5ml per well) was added to the first series masterblocks and stored at -20°C until further use. On the third day the second series was transferred into separate eppendorf tubes, centrifuged for 10 min at lOOOOrpm and decanted. The cells were dissolved in the residual medium (approx. 50μ1), stored at 8°C and used for PCR screening of the separate colonies. Ileostomy effluent One sample of ileostomy effluent taken from a healthy individual with an ileostomy. The ileostomy effluent was processed on the same day as the day of collection. One part of the ileostomy effluent sample was stored as a glycerol stock at - 80°C for future use. Another part was stored at -20°C for DNA isolation. Obtaining of single colonies and pure culture of the human CRIB

DSMZ medium was made as mentioned above with the following modification: 0.9ml rumen fluid was added to the medium before boiling; resazurin was skipped; the pH was adjusted to 7.6.

Tenfold dilution series of bacterial culture, containing presumably 64% human CRIB were performed using CRIB liquid medium. Using micropipette, 0.1ml of the culture and the 10 fold dilution series (range 10 °- 10 ^~7) were inoculated on petri dishes with solid medium using Ιμΐ and 10 μΐ flexible plastic inoculation loops (Copan). The dilution series and the inoculations on solid medium were done under strict anaerobic conditions in an anaerobic plastic tent. The petri dishes were incubated in anaerobic jars with oxygen scavengers and oxygen indicators, in climate room at 37°C. Six single colonies were re-inoculated, each on a separate petri dish and re-inoculated 3 times (3 following days). From the last inoculation one colony was used for DNA isolation and second colony from the same dish for continuation on liquid medium. This 12h culture was used to make a glycerol stock.

Glycerol stocks Glycerol stocks were prepared from bacterial cultures immediately after they were removed from the incubator by adding 4 ml culture to the bottles containing glycderol stock solution (a 50% v/v solution of glycerol and 20 mM phosphate solution with 500 mg/1 resazurine). The procedure was done under sterile conditions, using syringe and needle. The culture stocks were kept at -80°C until further use. When using the culture glycerol stocks, they were first defrosted and heated to room temperature. Probiotic mixture

The probiotic mixture contained 10¹⁰ CFU/g of six probiotic species:

Bifidobacterium bifidum, B. lactis, Lactobacillus acidophilus, L. casei, L. salivarius, Lactococcus lactis (Winclove Bio industries B.V.).

Microscopy

Investigation of the morphology of the vegetative bacterial cells was done by using phase contrast microscopy (Leica DM 2000). The images were handled with the software Leica Application Suite, Version 2.5.0 Rl; Leica Microsystems (Switzerland) Limited; Leica Microsystems CMS GmbH.

Gram staining The gram staining of the cultured bacteria was done with the Bacto® 3- STEP

GRAM STAIN, Difco Laboratories kit, following the producer's instruction protocol.

DNA isolation DNA was isolated from 1 ml of bacterial culture. For DNA isolation the

FastDNA® spin kit for soil (MP Biomedicals, Solon, United States) was used, with a modified version of the producer provided protocol, where the first step of the protocol was replaced by 15 min centrifugation (xlOOOO) of the culture for obtaining of cell pellet, using Eppendorf Centrifuge 5417R. The DNA concentration was

spectrophotometrically quantified (The Thermo Scientific NanoDrop 2000: Micro- Volume UV-Vis Spectrophotometer for Nucleic Acid and Protein Quantitation, NanoDrop® Technologies, Wilmington, USA). Real-time PCR detection and quantification

Real time PCRs were performed in a total reaction volume of 25 μΐ containing 12.5 μΐ 2X iQ SYBR Green Supermix (Biorad), 0.2 μΜ of each of the primers, and 5μ1 of tenfold diluted template DNA. Reactions were done in duplicate or triplicate. For quantification of the total bacteria the universal primer set was used: Bact-1369f (Biolegio) /Prok-1492r (Biolegio) (Suzuki et al, 2000). For quantification of CRIB, a specific primer set was used: CRIB-61f (Euregentec) /CRIB-235r (Biolegio). The DNA used for the standard was extracted from rat CRIB culture. Standard curves were generated from dilution series of PCR amplified and purified DNA product of 16S rRNA gene sequences. The primers used for PCR amplification of the 16S rRNA gene were: Bact-27f/Uni-1492r (Lane, 1991, (Table 7). The sample used for the standard curve contained 101.9 ng/μΐ DNA. The DNA concentration was used to calculate the number of the 16rRNA gene copies in the dilution series used to generate the standard curves. The dilution series were obtained, containing 10°- 10⁸ copies/μΐ of the 16S rRNA gene.

Real time PCR reactions were performed with the iCycler iQ5 (BioRad). Data analysis was conducted with iQ5 Optical System Software Version 2.0. The quantification of the 16S rRNA gene copies in a sample was performed using the following program: 3 min at 95 °C, followed by 40 cycles of 15 s at 95 °C, 30 s at 56 °C and 30 s at 72 °C. The quantification of the human CRIB in a sample was done using the following program: 3 min at 95 °C, followed by 40 cycles of 20 s at 95 °C and 1 min at 68°C.

The relative abundance of the human CRIB was evaluated by means of simple calculation of the percentage of the DNA copies per sample obtained with the CRIB specific primers, from the total DNA copies obtained with the universal 16S rRNA primers.

PCR amplification For screening of individual colonies for the presence of human CRIB, PCR reactions were performed using a GoTaq® DNA Polymerase (M3175) kit from

Promega Benelux b.v. (Leiden, The Netherlands). The kit contains 5X Green GoTaq™ Reaction Buffer (M791A), GoTaq® Polymerase (M830B), Deoxynucleotide

Triphosphates (dNTPs). The used primers were CRIB-61f and CRIB-235r. For the PCR reactions cultures started from individual colonies obtained after culturing on solid medium and re- inoculated on liquid medium were used as template,. The reactions consisted of 0.2 μΜ of each primer, 1.25U Taq polymerase, 0.2mM (dNTPs), 1.5mM MgC . The PCR reactions were performed in a total reaction volume of 25 μΐ. As template 2 μΐ bacterial culture was used. The DNA amplifications were done using G- STORM GS1 Thermocycler and Tl Thermocycler Biometra. The following PCR program was used: 3 min at 95 °C, followed by 35 cycles of 20 s at 95 °C, 30 s at 64 °C and 30 s at 72 °C, and finally 72 °C for 5 min.

Table 7. Primers used in this experiment.

Primer Sequence (5'-3') Target Reference

CRIB-61F GTC GAG CGA TTT ACT TCG Rat CRIB- this

GTA specific 16S application rRNA gene

CRIB-235R GGG TCC ATC CTG TAC CGC Rat CRIB- this

AAA specific 16S application rRNA gene

Bactl369F CGG TGA ATA CGT TCY CGG Universal 16S Suzuki et al., rRNA gene 2000

Prokl492R GGW TAC CTT GTT ACG ACT Universal 16S Suzuki et al.,

T rRNA gene 2000

T7prom- TGA ATT GTA ATA C GA CTC Universal 16S Lane, 1991 Bact-27-f ACT ATA GGG GTT TGA TCC rRNA gene

TGG CTC AG Uni- 1492-r CGG CTA CCT TGT TAC GAC Universal 16S Lane,1991

rRNA gene

Positive control for the PCR reaction

Because after the first PCR run no product was obtained, different (pre- treatment) variants of positive control were tested (listed below), as well different annealing temperatures for the PCR reaction.

List of the positive control variants tested for optimization of the PCR

Variant 1.

1. Centrifuge at lOOOOrpm for 5 minutes. Decant. Re-suspend the cell pellet in 1 ml of SPB and repellet by spinning for 5 minutes. Repeat. After the last wash re- suspend the cell pellet in 500ul of water. Freeze and thaw (37°C) sample three times with vigorous vortexing between repetitions to break the bacterial cell wall.

2. Place the sample in a 95°C heating block for 5 minutes.

Variant 2.

1. Centrifuge at lOOOOrpm for 5 minutes. Decant. Resuspend the cell pellet in 500ul of water.

2. Place the sample in a 95°C heating block for 15 minutes. Variant 3.

Centrifuge at lOOOOrpm for 5 minutes. Decant. Resuspend the cell pellet in 1 ml of SPB and repellet by spinning for 5 minutes. Repeat. After the last wash resuspend the cell pellet in 500ul of water. Freeze and thaw sample three times with vigorous shaking or vortexing between repetitions to break the bacterial cell wall.

Variant 4.

Centrifuge at lOOOOrpm for 5 minutes. Decant. Resuspend the cell pellet in 500ul of water. Freeze and thaw sample three times with vigorous shaking or vortexing between repetitions to break the bacterial cell wall. Variant 5.

1. Centrifuge at lOOOOrpm for 5 minutes. Decant. Resuspend the cell pellet in 500ul of water.

2. Place the sample in a 95°C heating block for 5 minutes.

Variant 6.

1. Centrifuge at lOOOOrpm for 5 minutes. Decant. Resuspend the cell pellet in 1 ml of SPB.

2. Place the sample in a 95°C heating block for 5 minutes

Variant 7.

1. Centrifuge at lOOOOrpm for 5 minutes. Decant. Resuspend the cell pellet in 0.978 ml SPB and 0.122ml MT Buffer and repellet by spinning for 5 minutes. Repeat. After the last wash resuspend the cell pellet in 500ul of water. Freeze and thaw (37°C) sample three times with vigorous vortexing between repetitions to break the bacterial cell wall.

2. Place the sample in a 95°C heating block for 5 minutes.

Variant 8.

1. Centrifuge at lOOOOrpm for 5 minutes. Decant. Resuspend the cell pellet in 0.978 ml SPB and 0.122ml MT Buffer and repellet by spinning for 5 minutes. Repeat. After the last wash resuspend the cell pellet in 500ul of water.

2. Place the sample in a 95°C heating block for 5 minutes. This Variant 9.

1. Centrifuge at lOOOOrpm for 5 minutes. Decant. Resuspend the cell pellet in 0.978 ml SPB and repellet by spinning for 5 minutes. Repeat. After the last wash resuspend the cell pellet in 500ul of water. Freeze and thaw (37°C) sample three times with vigorous vortexing between repetitions to break the bacterial cell wall.

2. Place the sample in a 95°C heating block for 5 minutes.

Variant 10. 1. Centrifuge at lOOOOrpm for 5 minutes. Decant. Resuspend the cell pellet in 0.978 ml SPB and repellet by spinning for 5 minutes. Repeat. After the last wash resuspend the cell pellet in 500ul of water. Freeze and thaw (37°C) sample once with vigorous vortexing to break the bacterial cell wall.

2. Place the sample in a 95°C heating block for 5 minutes.

Variant 11.

1. Centrifuge at lOOOOrpm for 5 minutes. Decant. Resuspend the cell pellet in 500ul of water.

2. Place the sample in a 95°C heating block for 5 minutes.

Variant 12.

Centrifuge at lOOOOrpm for 5 minutes. Decant. Resuspend the cell pellet in 500ul of water. Freeze and thaw (37°C) sample three times with vigorous vortexing between repetitions to break the bacterial cell wall.

Variant 13.

1. Centrifuge at lOOOOrpm for 5 minutes. Decant. Resuspend the cell pellet in 500ul of water.

2. Place the sample in a 95°C heating block for 30 minutes. Variant 14.

CRIB culture was boiled in the microwave oven for 30 sec. Variant 15.

CRIB culture was boiled in the microwave oven for 30 sec. Centrifuge at lOOOOrpm for 5 minutes. Decant. Resuspend the cell pellet in 500ul of water.

Finally rat CRIB culture and the PCR program mentioned above were used for the colony screening.

Gel electrophoresis In order to check the quality of the PCR products, gel electrophoresis was performed. The PCR products were visualized with SIBR® Safe DNA gel stain (Invitrogen) on agarose gel with agarose concentration of 0.9% or 2% for separation of expected products with different size (lower agarose concentration for the larger DNA molecules), and run at 100V for respectively 30 min and 20 min. The DNA product was observed using the Molecular Imager® Gel Doc™ XR+ System and Quantity One Analysis Software Version 4.6.6 was used for working with the images. The size of the DNA product was compared with DNA markers: GeneRuler™ lOObp Plus DNA ladder (SM0321, Fermentas) for the short DNA products and GeneRuler™ 1 kb Plus DNA ladder (SM1331, Fermentas) for the long DNA products.

16S rRNA gene sequencing

PCR on the 16S rRNA gene was performed using the universal primers Bact- 27f/Uni-1492r (Lane, 1991), (Table 7). The following PCR program was used: 2 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 40 s at 52 °C and 1.30 min at 72 °C, and finally 72 °C for 5 min. PCR products were purified using the High Pure PCR Cleanup MicroKit Version 4.0 (04.983.912.001, Roche) following the producer's instruction protocol. The purified PCR product was sent for sequencing (BaseClear, Leiden, The Netherlands) using the same primers used for the amplification of the 16S rRNA gene and one additional primer (650f) to comprise the complete 1500b p sequence. The consensus sequence alignment was done automatically using CLC Main Workbench software package (Genostar Bioinformatics Solutions, France). The consensus sequence of the 16S rRNA gene was compared with the sequences from the GenBank using the program BLAST (Basic Local Alignment Search Tool) of The National Centre for Biotechnology Information (2), NCBI, 2011).

Results Evaluation of the suitability of three liquid media for culturing of the human CRIB Three media, each with different specific substrates were tested for culturing of rat CRIB. It was assumed that bacterial strains of a specific species isolated from different hosts would have similar nutritional requirements: the growth of CRIB isolated from rat and also from humans would be supported by the same medium. Raffinose, supplied as the sole carbon source (PY medium) was evaluated for supporting the growth of rat CRIB. Abundant versus sparse supply of nitrogen sources (CRIB medium versus min CRIB medium) was used to evaluate the utilization of the supplied nitrogen sources. At that moment the characterization of this novel bacterial species (rat CRIB) was still ongoing and no full data were available yet. The media were inoculated with rat CRIB glycerol stock and incubated for culturing. The optical density of the 15 h culture was measured (Table 8) .

Table 8. The most important differences between the three compared media

Microscopically (lOOOx magnification) observed vegetative bacterial cells obtained by culturing in CRIB and Min- CRIB medium, were rod shaped, single, in chains, or in pairs. The vegetative cells cultured in PY medium were present at very low concentrations and were morphologically abnormal (lost structure and turgor). Those bottles were incubated for another 12 h. After observation at lOOOx magnification there no vegetative cells were observed and the culture OD was not changed. It means that this medium is not supporting the growth of the rat CRIB and, moreover, the vegetative cells are not able to survive this environment. From these results the conclusion was made that the most appropriate medium for culturing of rat CRIB was CRIB medium.

The next step was evaluating the suitability of the three media for culturing of human ileal microbiota. There were serial 10 fold dilutions of the inoculum (ileostomy effluent) made with 9 samples each of the media (10^~5 -10 ^~14). The 10^~5 sample was made using 1 ml of ileostomy effluent. The following measurements were done: OD of 15 h culture, DNA yield concentration (after DNA isolation). The relative abundance of the human CRIB was calculated using the data from the qPCR quantification analysis (Table 9).

Table 9. The suitability (summarized) of the three media for culturing of human microbiota

Based on these results the following conclusions were made: the CRIB medium was the most suitable to culture the ileal microbiota and trials aiming the isolation of the human CRIB using Most Probable Number enrichment dilutions approach. The next experiments were performed with the use of CRIB medium; the other two media were omitted from the trials.

Continuation of the samples with the highest relative abundance of the human CRIB After a 7.78% relative abundance of human CRIB was obtained in one of the enrichment dilutions (table 9) another trial was planned with enrichment dilutions continuing with this sample. Three serial dilutions of 9 samples each (KH-IO"¹⁰) were made.

The relative abundance of the human CRIB in all 27 samples after 15 h culturing time was calculated. The variation of the relative abundance of the human CRIB was between 0.00% and 0.33%, which was not a satisfactory result.

In the next trial another approach was evaluated. Evaluating the possibilities to isolate the target organism using time- dependent growth pattern, enrichment dilutions and medium additives

This trial was theoretically based on "the most probable number" approach, in combination with species specific growth pattern (growth curve). Termination of the culturing at different culturing times in different inoculum dilutions was supposed to support, either the most abundant (high dilutions) or the fast growing organisms (low dilutions). Because there was no information available about the target species (for example growth curve), the relative abundance of the human CRIB was calculated at different culturing times and dilution series.

It was hypothesized that the probiotic mixture would directly support the growth of the human CRIB, and make a shift in the relative abundance of the species community in benefit of the human CRIB.

Rumen fluid is used as additive of unknown factors for supporting the growth of anaerobic bacterial species (Zoetendal et al., 2003).

Evaluating the suitability of the CRIB medium for culturing of probiotic species

The first step in the trial was evaluating the suitability of the CRIB medium for culturing the bacterial species included in the probiotic mixture. Dilutions were made with concentration- 10⁷; 10⁵; 10³; 10² CFU per sample. After 15 h culturing time a high OD of all cultures was observed by eye. Also observation at lOOOx magnification confirmed high density of vegetative bacterial cells belonging to different species (cocci and rod shaped cells). The final concentration of the probiotic mixture added to the human microbiota samples was 10² CFU. Filter sterilized rumen fluid (5ml per liter medium) was added before boiling of the medium. The trial set up is summarized in table 10.

Table 10. Trial set up including inoculum dilution series, different culturing times and medium additives

In this trial the OD of the cultures was measured and the relative abundance of the human CRIB was calculated. The OD of all samples was close to or at saturation level of the culture (above 2.4). The relative abundance of none of the 154 samples was above 0.3%. Another approach for isolation of the target organism was planned, using fresh

(used direct after delivery in the laboratory) ileostomy material. This trial was made to avoid the impact of the freezing step to the viability of the target species. Serial dilution (8 variants) of the inoculum (10 ⁵- 10 ¹³) was performed. CRIB liquid medium with cow rumen fluid as additive was used for the culturing. Three different culturing times were included in the trial: 13h, 14h, and 15h. The calculation of the relative abundance of human CRIB in 24 samples resulted in unsatisfactory concentrations (below or equal to 1%). Screening of bacterial cultures obtained from single bacterial colonies from human ileostomy effluent, using PCR as a tool for identification

Obtaining single colonies by plating of inoculum on rich solid medium is a standard procedure used for isolation of bacterial species. After separation on solid medium, the colonies were separately inoculated in liquid medium.

Using the specific rat CRIB primers, PCR amplification was performed. As template for the PCR reaction, CRIB medium culture from the isolated single colonies was used. Eighty five PCR reactions (85 pure cultures obtained from single colonies) in the first trial resulted in no PCR products. In this trial also the positive control (rat CRIB culture) resulted in no PCR product.

Choosing the positive control

The next step to be done was obtaining a reliable positive control. To evaluate whether a different quantity of culture (the medium is known as obstructive PCR reaction factor) would result in product with expected size (174bp, using the rat CRIB specific primers 61F-235R), PCR reaction series with different culture quantities (1.0 μΐ -3.0 μΐ, with 0.25μ1νοΚιπιβ difference in between the reactions) of the positive control were run. The positive control was 15h rat CRIB culture. The PCR reactions resulted in no product.

To evaluate whether the PCR reaction would result in product, after removing the medium (thought to be the obstructive factor), a new trial was planned with different culture quantity (0.25μ1 -2.5 μΐ, with a volume difference of 0.25 μΐ) and variant with removed medium. This variant was obtained as follow:

Centrifuge at 12000 for 2 min, decant, mix the cell pellet with 60 μΐ Sodium Phosphate Buffer (SPB) from The FastDNA® spin kit for soil, centrifuge at 12000 for 2 min, repeat twice. After the second wash step re-suspend the cell pellet in 60 μΐ DES from the kit.

The obtained cell suspension was used as template for the PCR reactions in the same quantities as the variants using culture. To evaluate if the problem is related to the use of the rat CRIB primers, two sets of primers were tested in this trial: the rat CRIB specific primers (61F-235R) and the 16S rRNA gene primers (27F and 1492R). The PCR program used for amplification of a 1365 bp long DNA molecule was as follows: 2 min at 94 °C, followed by 35 cycles of 30 s at 94 °C, 40 s at 52 °C and 1.30 min at 72 °C, followed by 5.0 min at 72°C.

None of the variants resulted in obtaining of PCR product.

The next step was evaluating the role of template accessibility (intact bacterial wall) for the PCR reaction. Different variants were included: removal of the medium, mechanical and physical treatment of the cells. In total six variants mentioned in Material and Methods of this example as Variant 1 to Variant 6.

None of the variants of this trial resulted in PCR product.

In the next trial more potential positive control variants were included: isolated DNA as well as the cell wall disruption variants from the previous trial. This was done to check the sensitivity of the PCR reaction with isolated DNA. This would answer the question at which step of the process the occurring problem should be searched. Two isolated DNA variants were included: isolated rat CRIB DNA and isolated, amplified and purified 16S rRNA DNA PCR product from rat CRIB. A band from the amplification product with a size of 174 bp was expected as result of the PCR reaction. The trial gave a reliable result with a band of the expected size only with the variant using amplified 16S rRNA DNA molecule as template (Figure 28). Band traces were also visible in the samples containing isolated rat CRIB DNA, but not in all repetitions, which was evaluated as not reliable result. There was no PCR product from the test variants 1 to 6.

The next tested variant included an extended PCR program with an extra temperature step. The latter was providing satisfactory template accessibility (bacterial cell wall disruption). The program was as follow:

3 cycles of 3 min at 94 ° C, 3 min at 55 °C, followed by 3 min at 95 °C, followed by 35 cycles of 20 s at 95 °C, 30 s at 68 °C and 30 s at 72 °C, followed by 50 min at 72°C.

Here also only the variant using amplified 16S rRNA DNA molecule (Figure 29) as template gave a reliable result Next, PCR reactions using the 16S rRNA gene primers were performed, to compare with the outcome of the previous trial. The result would give direction to troubleshooting the amplification problem: pre-treatment of the bacterial cell wall, or the sensitivity of the PCR reaction using rat CRIB primers.

Nine different template treatment variants were included in this trial, mentioned in Material and Methods (Positive control for the PCR reaction) as Variant 7 till Variant 15.

This trial resulted in obtaining PCR products of expected size (1465 bp) with most template variants. Only the variants using culture as template and pre- treatment variants 11, 13, 14 did not result in PCR product (Figure 30).

A shift in the search direction for PCR reaction troubleshooting was made. In the previous study of this research group, targeting the use of real-time PCR for rat CRIB quantification analysis, the development of the primers and the specificity tests were done using amplified 16S rRNA DNA molecule and different amplification temperatures. In order to test whether a change in annealing temperature would result in a better PCR reaction output, three different annealing temperatures were tested, 61°C, 64°C and 65°C. At 61°C all variants of the positive control (same as in the PCR reactions using 16S rRNA gene primers and template variants as described above) resulted in a PCR product with the expected size. However, all PCR reactions using single colony liquid cultures from human ileostomy effluent as template (in total 20) resulted in a PCR product. This was not a satisfactory result (PCR specificity was not optimal). Next, 64°C amplification temperature and the same template variants as above were tested. In this trial all positive control variants resulted in one band with the expected size. There were no products from PCR reactions using single colony liquid cultures from human ileostomy effluent as template. Finally also 65°C annealing temperature was tested. There was no product when rat CRIB culture was used as template. In summarized form the results from these experiments are given in Table 11.

Table 11. Summary of the output from PCR reactions using PCR program with different annealing temperatures Annealing Obtained PCR product from template:

T (°C) isolated rat rat CRIB bacterial rat single colony culture,

CRIB DNA cells (pre-treatment CRIB obtained from

variants) culture human ileostomy effluent

61 yes yes yes yes

64 yes yes yes no

65 yes no no no

An annealing temperature of 64°Cwas chosen as the most suitable for further screening of the single colony cultures.

Using the approach of PCR colony screening, in total 125 colonies were screened, which did, however, not result in a positive PCR reaction. From these results it can be concluded that none of the singe colonies can be assumed to be human CRIB.

Meanwhile, another approach for isolation of the human CRIB, using pH as selective factor was tested. Evaluating the possibilities to isolate human CRIB using medium pH as selective factor pH is an important factor for the survival and functioning of all bacterial species and is one of the most important factors for determining the presence/abundance of the species in certain environment (together with nutritional requirements such as availability of accessible feeding elements, vitamins; presence of intoxicants etc). In this trial the pH of the medium was evaluated as potential selective factor. It was hypothesized that changing the pH of the medium would give opportunities to only some species present in the sample to survive/multiply. As a consequence of the latter, the isolation of the target species would become easier.

Two variants of medium were used in this trial, containing in total four samples: CRIB medium and CRIB medium with cow rumen fluid (CRF) as additive. The culturing time was 8h (Table 12). The used inoculum consisted of 1 ml glycerol stock from ileostomy effluent culture, containing 0.19% human CRIB. Table 12. Influence of the pH of the relative abundance of the human CRIB in the liquid cultures

All four samples were continued on media with different pH-values and two variants of culturing times: 8h and 48h.One milliliter glycerol stocks from the previous trial were used to inoculate the media. For some samples there is no collected data related to the relative abundance of CRIB. Only a selection of samples was used to calculate the relative abundance of CRIB (Table 13). The samples with lowest OD from the shortest culturing time(*) were excluded from this calculation, as well as the samples with high OD in the shortest culturing time (**).

The trial resulted in obtaining high relative abundance of the human CRIB in the short culturing time, high pH and CRIB medium with CRF.

Obtaining pure cultures

In the previous trial the relative abundance of the target species achieved high percentage, facilitating its isolation in pure culture. The glycerol stock from the culture with the highest relative abundance of human CRIB from the previous experiment (64.34%) was used to obtain single colonies. Two types of colonies appeared after 15h culturing time on solid medium: big and small. At lOOOx magnification two kinds of morphologically different vegetative bacterial cells were observed: duplo cocci and rod shaped. The rod shaped bacterial cells were corresponding to the bigger colony type. To obtain pure culture of the human CRIB, thirty single colonies were transferred individually to liquid medium. The procedure was done as described in material and methods. Table 13. Influence of the pH, type medium and the culturing time on the relative abundance of the human CRIB in the liquid cultures

Selection of the pure cultures with the human CRIB

After incubation for 15h, DNA was extracted from the cultures and qPCR was performed to calculate the relative abundance of the human CRIB in the bottles. Two bottles with 100% relative abundance of CRIB were chosen for continuation and sequencing.

Some characteristics of the human CRIB

As expected, CRIB appeared to be a strict anaerobic, Gram positive bacterium. The isolated bacterial cells appeared morphologically as rods, single, in duplo or in chains, 2.32-4,59 μπι long (Figure 31). 16S rRNA gene sequencing

Six single colonies were sent for sequencing. A sequence consensus of the 16S rRNA gene was constructed (see below, SEQ ID NO: 21), based on 1404bp from the 16S rRNA gene. There is one mismatch in the sequence of the forward primer and no mismatch in the sequence of the reverse primer.

1G∞ TM:AGGAT¾GA^^ fi]SG.¾SAA ϊ AFT CACAA j

ICGT A CTCTG^^

CAGCCG£^^

aTiCAAGTCAGGAGTG AA

GAG A A J ^

'¾_:¾GG^'CJQ^' ¾AC

AGTACTGCGCCTGGGA GT^

CGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCTAAGCTTGACATCCTTT^

rc C TCTTTCmimG GACAG

G ii;Gi¾ ];T A A i ("CGJ: A A

Ajn _: G AA -j¾G

•• C '¾T ; C T

C^rrCGG IM CTGJ^A

CGGATAGCTAACCTTTTGGAAGCGTCCGTCGAAGGTGAAATCAATAACTGGGGTGAAGTCGTAACAAGGT AGC The 16S rRNA gene sequence was compared to the 16S rRNA gene sequence of other isolated species. The BLAST search resulted in hits with the highest sequence similarity of 97% to four species belonging to genus Clostridium (Table 14). Table 14. List of the isolated species with 16S rRNA gene sequence with the highest similarity hits of 97% to the human CRIB 16S rRNA gene sequence, available in GenBank.

The calculation of the sequence similarity between the human CRIB (1411 bp from 16S rRNA gene sequence) and the rat CRIB (1466 bp from the 16S rRNA gene sequence, SEQ ID NO:3) resulted in 96%, with 97% bases identity, 1% gaps and with a coverage of 100%.

Example 6 - DNA-DNA hybridization.

Methodology:

Preparation of wet cell mass:

For DNA-DNA hybridizations, cells of strain CRIB as deposited under the Budapest Treaty with the DSMZ, on 29 June 2007 under the accession number 19498, were cultured in CRIB medium for 15 h at 37 °C. The type strains of four

phylogenetically related species of the genus Clostridium cluster XI (C. lituseburense DSM 797^T, C irregulare DSM 2635^T, C hiranonis DSM 13275^T and C bartlettii DSM 16795¹) were obtained from DSMZ (Braunschweig, Germany) and cultured in the media suggested by DSMZ.

DNA-DNA hybridizations done by DSMZ:

DNA-DNA hybridizations of strain CRIB with related species was carried out by DSMZ (Braunschweig, Germany). Cells were disrupted by using a French pressure cell (Thermo Spectronic) and the DNA in the crude lysates was purified by chromatography on hydroxyapatite as described by Cashion, P. et al. (Anal. Biochem. 81:461-466, 1977). DNA-DNA hybridization was carried out as described by de Ley, J. et al. (Eur. J. Biochem. 12:133-142, 1970) under consideration op the modifications described by Huss, V. et al. (Syst. Appl. Microbiol. 4:184- 192, 1983) using a model Cary 100 Bio UV/VIS-spectrophotometer equipped with a Peltier-thermostatted 6x6 multicell changer and a temperature controller with in-situ temperature probe (Varian).

Results DDH of CRIB with closely related species:

Strain CRIB showed low relative DNA-DNA similarity (means±SD, n=2) to the type strains C. lituseburense DSM 797^T (15.5±0.8 %); C. irregulare DSM 2635^T (18.1±1.3 %); C. hiranonis DSM 13275^T (31.4±2.5 %) and C. bartlettii DSM 16795^T (20.4±3.5 %). These similarity values were below the cut-off point of 70 % for species delineation that was recommended by Wayne et al. (1987).

Claims

Claims

1. A bacterium that comprises one or more of the following sequences:

m. SEQ ID NO: 1 or the complement sequence thereof;

n. SEQ ID NO: 2 or the complement sequence thereof;

o. SEQ ID NO: 3 or the complement sequence thereof;

p. SEQ ID NO: 21 or the complement sequence thereof;

q. a sequence having a sequence identity of more than 97,2 % with SEQ

ID NO:l or its complement;

r. a sequence having a sequence identity of more than 97,2 % with SEQ ID NO:2 or its complement;

s. a sequence having a sequence identity of more than 97,2 % with SEQ

ID NO: 3 or its complement;

t. a sequence having a sequence identity of more than 97,2 % with SEQ

ID NO:21 or its complement;

u. a sequence selected form SEQ ID NO: 22 to SEQ ID NO: 130;

v. a sequence that has an identity of more than 70% with a stretch of 1000 consecutive nucleotides from any of the sequences from SEQ ID NO: 22 to SEQ ID NO: 130;

w. a sequence that has an identity of more than 70% with any of the

sequences from SEQ ID NO: 22 to SEQ ID NO: 130, provided that said sequence has a length of more than 1000 nucleotides

x. a sequence that has an identity of more than 70% with the total of sequences formed by SEQ ID NO: 22 to SEQ ID NO: 130.

2. A bacterium according to claim 1, characterized in that it is the commensal rat ileum bacterium (CRIB) deposited under the Budapest Treaty with the DSMZ, on date 29 June 2007 under the accession number 19498.

3. A bacterium that has a DNA-DNA hybridisation of more than 70% with a bacterium according to claim 1.

4. The bacterium according to any of claims 1 - 3 for use as probiotic.

5. The bacterium according to any of claims 1 - 3 for use in maintenance of the normal physiological barrier function and/or maintenance of normal regulation of the immune system

6. The bacterium according to any of claims 1 - 3 for use in the treatment, treatment-sparing and/or prevention of diseases, disorders, or syndromes related to disturbed barrier function and/or which are immune- mediated

7. The bacterium according to claim 6 for use in the treatment, treatment-sparing and/or prevention of an inflammatory disease, preferably an inflammatory intestinal disease, more preferably, inflammatory bowel disease (IBD) such as Crohn's disease, pouchitis and ulcerative colitis, irritable bowel syndrome (IBS), pancreatitis, celiac disease, food allergy, dysbacteriosis, cholera, diabetes type 1, necrotizing

enterocolitis or proctitis.

8. A probiotic composition comprising a probiotic culture having a probiotic count of between 10^s and 10¹¹ cfu/ml or cfu/g comprising a bacterium according to anyone of claims 1-3 and a carrier medium.

9. Food suitable for a mammal, preferably a human, comprising a bacterium according to anyone of claims 1-3 or a composition according to claim 8.

10. A method for detecting a bacterium according to anyone of claims 1-3

comprising detecting the presence of any of the sequences chosen from SEQ ID NO:l, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 21 and their complement sequences and a sequence having a sequence identity of more than 97,2 % with these sequences.

11. A method for detecting a bacterium according to anyone of claims 1-3

comprising detecting the presence of any of the sequences chosen from the group of SEQ ID NO:22 to SEQ ID NO: 130, the sequences that have an identity of more than 70% with a stretch of 1000 consecutive nucleotides from any of the sequences from SEQ ID NO: 22 to SEQ ID NO: 130, and sequences that have an identity of more than 70% with the total of the sequences from SEQ ID NO: 22 to SEQ ID NO: 130.

12. A method for detecting a bacterium according to anyone of claims 1-3

comprising measuring the DNA-DNA hybridisation with a baceterium according to claim 2 and testing whether this DNA-DNA hybridisation is more than 70%.

13. A kit of parts comprising a combination of a first and a second primer selected from the group of:

a. a first primer crib-61f having SEQ ID NO: 4 and a second primer crib- 155r having SEQ ID NO:6 or crib-220r having SEQ ID NO:7 or crib- 235r having a sequence SEQ ID NO:8 or crib-193r having SEQ ID NO:9; or

b. a first primer crib-132f having SEQ ID NO:5 and a second primer crib- 220r having SEQ ID NO:7.

14. An isolated and/or recombinant nucleic acid comprising SEQ ID NO:l or the complement sequence thereof, or SEQ ID NO:2 or the complement sequence thereof, or SEQ ID NO:3 or the complement sequence thereof, or SEQ ID NO: 21 or the complement thereof, or a sequence having a sequence identity of more than 97,2 % with SEQ ID NO:l, 2, 3 or 21 or their complement or a sequence according to any of the sequences of SEQ ID NO: 22 to SEQ ID NO: 130. or any of the sequences that have an identity of more than 70% with a stretch of 1000 consecutive nucleotides from any of the sequences from SEQ ID NO: 22 to SEQ ID NO: 130

15. A method for evaluating the immune status of an animal, comprising

determining the presence of a bacterium according to anyone of claims 1-3 and/or a nucleic acid according to claim 14 in a sample of said animal.