CROSS-REFERENCE TO RELATED APPLICATION

None

FIELD

The invention relates to microbiology, specifically, this invention relates to Eubacterium bacterial strains in the treatment and prevention of colorectal cancer -related diseases in the application, and also involves the composition comprising Eubacterium bacteria and its application.

BACKGROUND

Colorectal cancer (CRC) is the third most common form of cancer and the second leading cause of cancer-related death in the Western world (Schetter AJ, Harris CRC (2011) Alterations of microRNAs contribute to colon carcinogenesis. Semin Oncol 38:734-742, incorporated herein by reference). A lot of people are diagnosed with CRC and many patients die of this disease each year worldwide. Although current strategies, including surgery, radiotherapy, and chemotherapy, have a significant clinical value for CRC, the relapses and metastases of cancers after surgery have hampered the success of those treatment modalities.

Chronic intestinal inflammation occurring in inflammatory bowel disease (IBD) induces persistent damage and enhanced mucosal permeability along the gastrointestinal tract, playing an important role in the development of colorectal cancer (CRC). Together with the hereditary syndromes of familial adenomatous polyposis and hereditary nonpolyposis, IBD is among the top three high-risk conditions for CRC (Xie J, Itzkowitz SH (2008) Cancer in inflammatory bowel disease.World J Gastroenterol 14: 378-389, incorporated herein by reference). Among ulcerative colitis (UC) patients, one of the two main manifestations of IBD, the relative risk of developing CRC correlates with the extent and duration of disease (Eaden JA, Abrams KR, Mayberry JF (2001) The risk of colorectal cancer in ulcerative colitis: a meta-analysis. Gut 48: 526-535, incorporated herein by reference). In patients with IBD, this risk increases by 0.5-1.0% yearly after 8-10 years (Munkholm P (2003) Review article: the incidence and prevalence of colorectal cancer in inflammatory bowel disease. Aliment Pharmacol Ther 18 Suppl 2: 1-5, incorporated herein by reference).

The human gut microflora, which contains about 100 trillion microbial organisms, plays a critical role in maintaining host health, both in the gastrointestinal tract and systemically through the absorption of metabolites (e.g. vitamins and short chain fatty acids) (Moore WE, Holdeman LV (1974) Human fecal flora: the normal flora of 20 Japanese-Hawaiians. Appl Microbiol 27: 961-979, incorporated herein by reference). Recent studies have demonstrated that specific strains of bacteria are implicated in the regulation of the intestinal homeostasis, delivering regulatory signals to the epithelium, the mucosal immune system and to the neuromuscular activity of the gut (Shanahan F (2004) Probiotics in inflammatory bowel disease-therapeutic rationale and role. Adv Drug Deliv Rev 56: 809-818, incorporated herein by reference). Moreover, some commensal and pathogenic organisms of the human enteric microbiome are essential in the pathogenesis of IBD and CRC. Therefore, manipulating the gut bacterial composition and local metabolite production by using probiotic bacteria has been explored as a promising avenue for therapeutic intervention against CRC. Probiotics are live microbial feed supplements which beneficially impact on host health. They rely on introducing particular exogenous strains into the gut microflora. For the study of CRC, the inventors performed analysis in the metagenomics field, presenting data from deep metagenomic profiling of CRC gut microbiome,.

SUMMARY

Embodiments of the present disclosure seek to solve at least one of the problems existing in the prior art to at least some extent.

The present invention is based on the following findings by the inventors:

Assessment and characterization of gut microbiota has become a major research area in human disease, including colorectal cancer (CRC), one of the commonest causes of death among all types of cancers. To carry out analysis on gut microbial content in CRC patients, the inventors carried out a protocol for a Metagenome-Wide Association Study (MGWAS) (Qin, J. et al. A metagenome-wide association study of gut microbiota in type 2 diabetes. Nature 490, 55-60 (2012), incorporated herein by reference) based on deep shotgun sequencing of the gut microbial DNA from 128 Chinese individuals. The inventors identified 2 probiotic bacteria. Then the inventors validated the probiotic bacteria in animal experiment related to colitis and CRC respectively. Results from the animal experiment demonstrated the ability of Eubacterium ventriosum and Eubacterium eligens to prevent and treat both colitis and colorectal cancer effectively.

BRIEF DISCRIPTION OF DRAWINGS

These and other aspects and advantages of the present disclosure will become apparent and more readily appreciated from the following descriptions taken in conjunction with the drawings, in which:

Fig.l shows distribution of P-value association statistics of all microbial genes in this study. The association analysis of CRC p-value distribution identified a disproportionate over-representation of strongly associated markers at lower P-values, with the majority of genes following the expected P-value distribution under the null hypothesis. This suggests that the significant markers likely represent true rather than spurious associations.

Fig.2 Protective effects of intragastric administration of Eubacterium ventriosum ATCC 27560 , Eubacterium ventriosum L2-12 , Eubacterium ventriosum STAFF 1042, Eubacterium eligens ATCC 27750 , Eubacterium eligens STAFF 1020 , Eubacterium eligens TSDC 10.2- 1.1 respectively on DSS-induced colitis in C57B1/ 6J considering (A) weight loss, (B) disease activity index and (C) Colon length. Data are represented as mean ± standard error. Different asterisks (*) indicate significant differences (*P<0.05, **P<0.01, ***p<0.001).

Fig.3 Quantification using ELISA of (A)TNF-aand (B) IL-10 in colons obtained at day 15 after induction of colitis. Data are represented as mean ± standard error. Different asterisks (*) indicate significant differences (*P<0.05, **P<0.01, ***p<0.001)

Fig.4 Effect of Eubacterium ventriosum ATCC 27560, Eubacterium ventriosum L2-12 , Eubacterium ventriosum STAFF 1042, Eubacterium eligens ATCC 27750 , Eubacterium eligens STAFF 1020, Eubacterium eligens TSDC 10.2- 1.1 respectively treatment on (A)_Disease activity indices and (B) colon histopathology. Different asterisks (*) indicate significant differences (*P<0.05, **P<0.01, ***p<0.001)

Fig.5 Eubacterium ventriosum ATCC 27560 , Eubacterium ventriosum L2-12 , Eubacterium ventriosum STAFF 1042, Eubacterium eligens ATCC 27750 , Eubacterium eligens STAFF 1020, Eubacterium eligens TSDC 10.2- 1.1 respectively modulate colonic gene expression in BABL/C mice (relatived with -actin). Data are represented as mean±standard error, Different asterisks (*) indicate significant differences (*P<0.05, **P<0.01, ***p<0.001).

DETAILED DESCRIPTION

Terms used herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as "a", "an" and "the" are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.

In one aspect, the present application provides Eubacterium eligens or Eubacterium ventriosum for use in the prevention or treatment of colitis and/or colorectal cancer. In another aspect, the present application provides use of Eubacterium eligens or Eubacterium ventriosum for manufacture of a medicament for prevention or treatment of colitis and/or colorectal cancer. In another aspect, the present application provides a method for the prevention or treatment of colitis and/or colorectal cancer comprising administering an effective amount of Eubacterium eligens or Eubacterium ventriosum to a patient in need thereof. In yet another aspect, the present application provides a kit comprising Eubacterium eligens or Eubacterium ventriosum and instructions for its use as a medicament. The present application further provides a pharmaceutical composition comprising Eubacterium eligens or Eubacterium ventriosum and a pharmaceutically acceptable vehicle.

Preferably, the Eubacterium eligens is a strain selected from the group consisting of Eubacterium eligens ATCC 27750, Eubacterium eligens STAFF 1020 and Eubacterium eligens TSDC 10.2- 1.1. Preferably, the Eubacterium ventriosum is a strain selected from the group consisting of Eubacterium ventriosum ATCC27560, Eubacterium ventriosum L2-12, and Eubacterium ventriosum STAFF 1042. In one embodiment, the Eubacterium eligens or Eubacterium ventriosum is administered in the form of living Eubacterium eligens or Eubacterium ventriosum or metabolites thereof.

The present invention is further exemplified in the following non-limiting Examples. Unless otherwise stated, parts and percentages are by weight and degrees are Celsius. As apparent to one of ordinary skill in the art, these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only, and the agents were all commercially available. EMBODIMENT

Example 1. Identifying probiotic bacteria from 128 Chinese individuals

1.1 Sample collection and DNA extraction

Stool samples from 128 subjects, including 74 colorectal cancer patients and 54 healthy controls (Table 1) were collected in the Prince of Wales Hospital with informed consent. To be eligible for inclusion in this study, individuals have to fit the following criteria for stool sample collection: 1) no taking of antibiotics or other medications with no particular diets (diabetics, vegetarians, etc) and with normal lifestyle (without extra stress) for a minimum of 3 months; 2) a minimum of 3 months after any medical intervention; 3) no history of colorectal surgery, any kind of cancer, or inflammatory or infectious diseases of the intestine. Subjects were asked to collect stool samples before colonoscopy examination in standardized containers at home and store samples in their home freezer immediately. Frozen samples were then delivered to the Prince of Wales Hospital in insulating polystyrene foam containers and stored at -80°C immediately until use.

Stool samples were thawed on ice and DNA extraction was performed using the

QiagenQIAamp DNA Stool Mini Kit according to manufacturer's instructions. Extracts were treated with DNase-free RNase to eliminate RNA contamination. DNA quantity was determined using NanoDrop spectrophotometer, Qubit Fluorometer (with the Quant-iTTMdsDNA BR Assay

Kit) and gel electrophoresis.

Table 1 | Baseline characteristics of colorectal cancer cases and controls in the cohort I. BMI: body mass index; eGFR: epidermal growth factor receptor; DM: diabetes mellitus type 2.

Parameter Controls (n=54) Cases (n=74)

Age 61.76 66.04

Sex (M:F) 33:21 48:26

BMI 23.47 23.9

eGFR 72.24 74.15

DM (%) 16 (29.6%) 29 (39.2%)

Entero type (1 :2:3) 26:22:6 37:31 :6

Stage of disease (1 :2:3:4) n.a. 16:21 :30:7

Location (proximal: distal) n.a. 13:61

1.2 DNA library construction and sequencing

DNA library construction was performed following the manufacturer's instruction (Illumina HiSeq 2000 platform). The inventors used the same workflow as described previously to perform cluster generation, template hybridization, isothermal amplification, linearization, blocking and denaturation, and hybridization of the sequencing primers (Qin, J. et al. A metagenome-wide association study of gut microbiota in type 2 diabetes. Nature 490, 55-60 (2012), incorporated herein by reference).

The inventors constructed one paired-end (PE) library with insert size of 350bp for each sample, followed by a high- throughput sequencing to obtain around 30 million PE reads of length 2xl00bp. High quality reads were extracted by filtering low quality reads with 'N' base, adapter contamination and human DNA contamination from the raw data, and by trimming low quality terminal bases of reads at the same time. 751 million metagenomic reads (high quality reads) were generated (5.86 million reads per individual on average, Table 2).

1.3 Reads mapping

The inventors mapped the high quality reads to the gene catalogue (Table 2) to a published reference gut gene catalogue established from European and Chinese adults (Qin, J. et al. A metagenome-wide association study of gut microbiota in type 2 diabetes. Nature 490, 55-60 (2012), incorporated herein by reference) (identity >= 90%), based on which the inventors derived the gene profiles using the same method of the published T2D paper in Qin et al. 2012, supra. From the reference gene catalogue as Qin et al. 2012, supra, the inventors derived a subset of 2,110,489 (2.1M) genes that appeared in at least 6 samples in all 128 Hong Kong samples.

Table 2. Summary of metagenomic data and mapping to reference gene catalogue. Fourth column reports results from Wilcoxon rank-sum tests.

Parameter Controls Cases j P-value

Average raw reads 60162577 60496561 j 0.8082 j After removing low quality reads 59423292 (98.77%) 59715967 (98.71%) j 0.831

After removing human reads 59380535 ± 7378751 58112890 ± 10324458 j 0.419

Mapping rate 66.82% 66.27% j 0.252

1.4 Analysis of factors influencing gut microbiota gene profiles

To ensure robust comparison of the gene content of 128 metagenomes, the inventors created a set of 2,110,489 (2.1M) genes that were present in at least 6 subjects, and generated 128 gene abundance profiles using these 2.1 million genes. The inventors used the permutational multivariate analysis of variance (PERMANOVA) test to assess the effect of different characteristics, including age, BMI, eGFR, TCHO, LDL, HDL, TG, gender, DM, CRC status, smoking status and location, on gene profiles of 2.1M genes. The inventors performed the analysis using the method implemented in package "vegan" in R, and the permuted p-value was obtained by 10,000 times permutations. The inventors also corrected for multiple testing using "p. adjust" in R with Benjamini-Hochberg method to get the q-value for each gene.

When the inventors performed permutational multivariate analysis of variance (PERMANOVA) on 13 different covariates, only CRC status was significantly associated with these gene profiles (q = 0.0028, Table 3), showing a stronger association than the second best determinant body mass index (q = 0.15). Thus the data suggest an altered gene composition in CRC patient microbiomes.

Table 3. PERMANOVA analysis using microbial gene profile. The analysis was conducted to test whether clinical parameters and colorectal cancer (CRC) status have significant impact on the gut microbiota with q<0.05. BMI: body mass index; DM: diabetes mellitus type 2; HDL: high density lipoprotein; TG: triglyceride; eGFR: epidermal growth factor receptor; TCHO: total cholesterol; LDL; low density lipoprotein.

1.5 CRC-associated genes identified by MGWAS

1.5.1 Identification of colorectal cancer associated genes. The inventors performed a metagenome wide association study (MGWAS) to identify the genes contributing to the altered gene composition in CRC. To identify the association between the metagenomic profile and colorectal cancer, a two-tailed Wilcoxon rank-sum test was used in the 2.1M (2,110,489) gene profiles. The inventors got 140,455 gene markers ,_which were enriched in either case or control with O.01 (Fig. 1).

1.5.2 Estimating the false discovery rate (FDR). Instead of a sequential P-value rejection method, the inventors applied the "qvalue" method proposed in a previous study(J. D. Storey, R. Tibshirani, Statistical significance for genomewide studies. Proceedings of the National Academy of Sciences of the United States of America 100, 9440 (Aug 5, 2003), incorporated herein by reference) to estimate the FDR. In the MGWAS, the statistical hypothesis tests were performed on a large number of features of the 140,455 genes. The false discovery rate (FDR) was 11.03%.

1.6 Data profile construction

Basing on the sequencing reads of the 128 microbiomes abtained above, the inventors examined the taxonomic differences between control and CRC -associated microbiomes to identify microbial taxa contributing to the dysbiosis. For this, the inventors used taxonomic profiles derived from three different methods, as supporting evidence from multiple methods would strengthen an association. First, the inventors mapped metagenomic reads to 4650 microbial genomes in the IMG database (version 400) and estimated the abundance of microbial species included in that database (denoted IMG species). Second, the inventors estimated the abundance of species-level molecular operational taxonomic units (mOTUs) using universal phylogenetic marker genes. Third, the inventors organized the 140,455 genes identified by MGWAS into metagenomic linkage groups (MLGs) that represent clusters of genes originating from the same genome, annotated the MLGs at species level using IMG database whenever possible, grouped MLGs based on these species annotations, and then estimated the abundance of these species (denoted MLG species).

1.6.1 Species annotation of IMG genomes

For each IMG genome, using the NCBI taxonomy identifier provided by IMG, the inventors identified the corresponding NCBI taxonomic classification at species and genus levels using NCBI taxonomy dump files. The genomes without corresponding NCBI species names were left with its original IMG names, most of which were unclassified.

1.6.2 Gene profiles

The inventors mapped our high-quality reads to the gene catalogue to a published reference gut gene catalogue established from European and Chinese adults (Qin, J. et al. A metagenome-wide association study of gut microbiota in type 2 diabetes. Nature 490, 55-60 (2012), incorporated herein by reference) (identity >= 90%), based on which the inventors derived the gene profiles using the same method of the published T2D paper as Qin et al. 2012, supra.

1.6.3 mOTU profile

Clean reads (high quality reads in Example 1) were aligned to mOTU reference (total 79268 sequences) with default parameters (S. Sunagawa et al, Metagenomic species profiling using universal phylogenetic marker genes. Nature methods 10, 1196 (Dec, 2013) , incorporated herein by reference). 549 species level mOTUs were identified, including 307 annotated species and 242 mOTU linkage groups without representative genomes, which were putatively Firmicutes or Bacteroidetes.

1.6.4 IMG-species and IMG-genus profiles

Bacterial, archaeal and fungal sequences were extracted from IMG v400 reference database (V. M. Markowitz et al, IMG: the Integrated Microbial Genomes database and comparative analysis system. Nucleic acids research 40, D115 (Jan, 2012) , incorporated herein by reference) downloaded from http: //ftp.jgi-psf.org. 522,093 sequences were obtained in total, and SOAP reference index was constructed based on 7 equal size chunks of the original file. Clean reads were aligned to reference using SOAP aligner (R. Li et al, SOAP2: an improved ultrafast tool for short read alignment. Bioinformatics 25, 1966 (Aug 1, 2009), incorporated herein by reference) version 2.22, with parameters "-m 4 -s 32 -r 2 -n 100 -x 600 -v 8 -c 0.9 -p 3". Then, SOAP coverage software was used to calculate read coverage of each genome, normalized with genome length, and further normalized to relative abundance for each individual sample. The profile was generated based on uniquely mapped reads only.

1.7 Identification of colorectal cancer associated species

Based on the identified 140,455 colorectal cancer associated maker genes profile, the inventors constructed the colorectal cancer associated MLGs using the method described in the previous type 2 diabetes study as Qin et al. 2012, supra. All genes were aligned to the reference genomes of IMG database v400 to get genome level annotation. An MLG was assigned to a genome if > 50% constitutive genes were annotated to that genome, otherwise it was termed as unclassified. Total 87 MLGs with gene number over than 100 were selected as colorectal cancer associated MLGs. These MLGs were grouped based on the species annotation of these genomes to construct MLG species.

To estimate the relative abundance of an MLG species, the inventors estimated the average abundance of the genes of the MLG species, after removing the 5% lowest and 5% highest abundant genes. Relative abundance of IMG species was estimated by summing the abundance of IMG genomes belonging to that species.

These analysis identified 30 IMG species, 21 mOTUs and 86 MLG species that were significantly associated with CRC status (Wilcoxon rank-sum test, q<0.05; see Tables 4, 5). Eubacterium ventriosum was consistently enriched in the control microbiomes across all three methods (Wilcoxon rank-sum tests - IMG: q = 0.0414; mOTU: q = 0.012757; MLG: q = 5.446xl0 ^"4 ), and Eubacterium eligens was enriched according to two methods (Wilcoxon rank-sum tests - IMG: q = 0.069; MLG: q = 0.00031). These results indicated that both of these two species may have great ability as probiotic bacteria in preventing and treating CRC related diseases.

On the other hand, Parvimonas micra (q<1.80xl0 ^"5 ), Peptostreptococcus stomatis (q<1.80xl0 ^"5 ), Solobacterium moorei (q<0.004331) and Fusobacterium nucleatum (q<0.004565) were consistently enriched in CRC patient microbiomes across all three methods. P. stomatis has been associated with oral cancer (S. Pushalkar et al, Comparison of oral microbiota in tumor and non-tumor tissues of patients with oral squamous cell carcinoma. BMC microbiology 12, 144 (2012), incorporated herein by reference), and S. moorei has been associated with bacteremia (R. M. Pedersen, H. M. Holt, U. S. Justesen, Solobacterium moorei bacteremia: identification, antimicrobial susceptibility, and clinical characteristics. Journal of clinical microbiology 49, 2766 (Jul, 2011), incorporated herein by reference). Recent work with 16S rRNA sequencing has observed significant enrichment of F. nucleatum in CRC tumor samples (M. Castellarin et al., Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma. Genome research 22, 299 (Feb, 2012), incorporated herein by reference), and this bacteria has been shown to possess adhesive (G. Bachrach, C. lanculovici, R. Naor, E. I. Weiss, Fluorescence based measurements of Fusobacterium nucleatum coaggregation and of fusobacterial attachment to mammalian cells. FEMS microbiology letters 248, 235 (Jul 15, 2005), incorporated herein by reference), invasive (Y. W. Han et al., Interactions between periodontal bacteria and human oral epithelial cells: Fusobacterium nucleatum adheres to and invades epithelial cells. Infection and immunity 68, 3140 (Jun, 2000), incorporated herein by reference) and pro-inflammatory (A. D. Kostic et al., Fusobacterium nucleatum Potentiates Intestinal Tumorigenesis and Modulates the Tumor-Immune Microenvironment. Cell Host Microbe 14, 207 (2013), incorporated herein by reference) properties. These results confirmed this association in a new cohort with different genetic and cultural origins. However, a highly significant enrichment of P. micra - an obligate anaerobic bacterium that can cause oral infections like F. nucleatum (G. Sundqvist, Taxonomy, ecology, and pathogenicity of the root canal flora. Oral surgery, oral medicine, and oral pathology 78, 522 (Oct, 1994), incorporated herein by reference) - in CRC-associated microbiomes is a novel finding. P. micra is involved in the etiology of periodontis (B. H. Kremer et al., Peptostreptococcus micros smooth and rough genotypes in periodontitis and gingivitis. Journal of periodontology 71, 209 (Feb, 2000) , incorporated herein by reference), and it produces a wide range of proteolytic enzymes and uses peptones and amino acids as energy source (D. A. Murdoch, H. N. Shah, Reclassification of Peptostreptococcus magnus (Prevot 1933) Holdeman and Moore 1972 as Finegoldia magna comb. nov. and Peptostreptococcus micros (Prevot 1933) Smith 1957 as Micromonas micros comb. nov. Anaerobe 5, 555 (10//, 1999), incorporated herein by reference). It is known to produce hydrogen sulphide (J. Carlsson, J. T. Larsen, M. B. Edlund, Peptostreptococcus micros has a uniquely high capacity to form hydrogen sulfide from glutathione. Oral microbiology and immunology 8, 42 (Feb, 1993), incorporated herein by reference), which promotes tumor growth and proliferation of colon cancer cells (C. Szabo et al., Tumor-derived hydrogen sulfide, produced by cystathionine -beta-synthase, stimulates bioenergetics, cell proliferation, and angiogenesis in colon cancer. Proceedings of the National Academy of Sciences of the United States of America 110, 12474 (Jul 23, 2013), incorporated herein by reference). Further research is required to verify whether P. micra is involved in the pathogenesis of CRC, or its enrichment is a result of CRC associated changes in the colon and/or rectum. Nevertheless, it may represent opportunities for non- invasive diagnostic biomarkers for CRC.

Example 2. Validation in animal experiment

In order to validate the ability of the probiotic bacteria, Eubacterium ventriosum and Eubacterium eligens, in preventing and treating CRC related diseases, the inventors performed animal experiments.

2.1 Validation in animal experiment related to colitis

2.1.1 Method Ten to twelve weeks old C57BL/6J mice used for DSS-induced colitis study were prepared in SPF condition. After acclimatization, part of mice have ad libitum access to drinking water containing 3.5 - 5% DSS (dextran sodium sulfate) for five days for colitis induction. Bacterial suspensions of Eubacterium ventriosum ATCC 27560 , Eubacterium ventriosum L2-12 , Eubacterium ventriosum STAFF 1042, Eubacterium eligens ATCC 27750 , Eubacterium eligens STAFF 1020 , Eubacterium eligens TSDC 10.2-1.1 (10 ⁹ -10 ¹⁰ cfu/ml , freshly prepared daily) ,or medium was administered respectively to mice daily by intragastric gavage from day 1 to day 14 after induction of colitis. No colitis and colitis control groups received medium intragastric ally (500μl/50g.BW every day , n=10 in each group).

C57BL/6J mice were purchased from Laboratory Animal Center of Southern Medical University, China. Eubacterium ventriosum ATCC 27560 and Eubacterium eligens ATCC 27750 were purchased from American Type Culture Collection (ATCC).

Eubacterium ventriosum L2-12: Estelle Devillard, Freda M. Mcintosh, Sylvia H. Duncan, and R. John Wallace (March 2007). Metabolism of Linoleic Acid by Human Gut Bacteria: Different Routes for Biosynthesis of Conjugated Linoleic Acid. Journal of Bacteriology 189 (6): 2566-2570.

Eubacterium ventriosum STAFF 1042, Eubacterium eligens STAFF 1020: Kageyama, A. et al. Rapid detection of human fecal Eubacterium species and related genera by nested PCR method, Microbiology and Immunology (2001) 45(4): 315-318.

Eubacterium eligens TSDC 10.2- 1.1 was purchased from Washington University in St. Louis (USA) http://www.genomesonline.org/projects?id=47012.

Mice were observed daily and recorded clinical measurements. Body weights and the disease activity index (Table 6) (Alex P, Zachos NC, Nguyen T, Gonzales L, Chen TE, Conklin ,LS, et al. 2009 Distinct cytokine patterns identified from multiplexprofiles of murine DSS and TNBS-induced colitis. Inflamm Bowel Dis; 15(3): 341-52. incorporated herein by reference.), a composite score reflecting clinical signs of the disease (i.e. perianal soiling, rectal bleeding and diarrhea) , were assessed for mice undergoing the DSS challenge , before DSS administration and weekly after intragastric gavage Inflammation was monitored at day 15 after induction of colitis when the mice were sacrificed by cervical dislocation.

Table 6 Clinical scoring standard

i score i Body weight loss Stool consistency Fecal blood j 0 i None Normal No bleeding j i i 0-5% weight loss Wet stools Occult bleeding Soft stools with sticking

5%~10% weight loss Slight bleeding

surface

10%~15% weight loss Moderate bleeding Loose stools

More than 15% weight loss Severe bleeding Waterv diarrhea

The colon was removed, dissected free of fat and mesentery, carefully opened, and cleaned with PBS (phosphate buffered saline). Colon length was measured. Colonic damage and inflammation were assessed blindly according to the Wallace criteria. Colon was also collected into liquid nitrogen and stored at -80 °C for ELISA (Enzyme Linked Immunosorbent Assay) examinations.

2.1.2 Results

2.1.2.1 Severity of DSS-lnduced Colitis in Mice Was Reduced by Eubacterium ventriosum ATCC 27560 , Eubacterium ventriosum L2-12 , Eubacterium ventriosum STAFF 1042, Eubacterium eligens ATCC 27750 , Eubacterium eligens STAFF 1020 , Eubacterium eligens TSDC 10.2- 1.1 respectively.

The inventors explored the anti-inflammatory effects in vivo by testing the ability of E. eligens ATCC 27750, STAFF 1020, TSDClO.2-1.1 and E. ventriosum L2-12, STAFF 1042, ATCC 27560 respectively to recover acute colitis induced by DSS. A severe acute inflammatory was observed in the colitis control group after DSS treated, and no significant protective effect was observed in mice receiving sterile culture medium. In contrast, daily intragastric administration of 6 living Eubacterium strains respectively all resulted in a marked attenuation of colitis with reduced weight loss (Table 7, Fig.2A), and decreasing disease activity index associated with colitis at the 7 ^th and 14 ^th day after intragastric gavage (the profiles of the data at 7 ^th day were similar with those at the 14 ^th day which was not shown here.). And partial colon length were normalized in comparison to the medium treated group (Table 8, Fig.2B _v 2C). There was no difference in body weight and disease activity index at the beginning of intragastric gavage. Data were represented as mean ± standard error.

Table 7 Data of Body weight ( W0: body weight before DSS-treated ; Wl : body weight at the 7th day after intragastric gavage )

Groups * WO Wl W1-W0 (wi-woywo*ioo% medium 28.06±0.63 22.98±0.95 -(5.08±1.01) -(18.12±3.635)

ATCC 27750 27.83±0.52 23.58±1.73 -(4.25±0.88) -(15.29±3.167)

L2-12 28.64±0.65 24.00±1.25 -(4.63±0.62) -(16.184zh2.178 ) STAFF 1042 27.53±0.98 23.48±1.17 -(4.04±1.29) -(14.693±4.701 )

ATCC 27560 29.04±0.79 24.77±1.02 -(4.27±1.24) -(14.69±4.267)

STAFF 1020 27.81±1.03 24.20±1.46 -(3.61±1.11) -(12.967±4.009 )

TSDClO.2-1.1 27.36±.088 23.18±1.10 -(4.18±0.72) -(15.278±2.667 )

No colitis control 26.91±0.44 29.45±0.56 2.54±0.30 8.62±1.124

Colitis control 28.7±0.60 23.23±1.39 -(5.48±1.23) -(19.08±4.27)

* ATCC 27560 represents Eubacterium ventriosum ATCC 27560; L2-12 represents Eubacterium ventriosum L2-12 , STAFF 1042 represents Eubacterium ventriosum STAFF 1042, ATCC 27750 represents Eubacterium eligens ATCC 27750, STAFF 1020 represents Eubacterium eligens STAFF 1020, TSDClO.2-1.1 represents Eubacterium eligens TSDC10.2-1.1.

Table 8 Data of clinical measurements related to colitis

2.1.2.2 Eubacterium ventriosum ATCC 27560 , Eubacterium ventriosum L2-12 , Eubacterium ventriosum STAFF 1042, Eubacterium eligens ATCC 27750 , Eubacterium eligens STAFF 1020, Eubacterium eligens TSDC 10.2- 1.1 modulate expression of colonic inflammatory respectively

Colonic proinflammatory TNF-a (tumor necrosis factor alpha) cytokines and anti-inflammatory IL-10 (interleukin-10) were quantified by ELISA. Compared with the no-colitis control group, TNF-a levels was increased, whereas IL-10 levels were barely modified in the colitis mice. After treated with suspensions of E. eligens ATCC 27750 , the secretion of TNF-a (Fig.3A , Table 8) was significantly lower than in the colitis control and medium groups. Meanwhile, IL-10 secretion was induced in the colon of mice treated with ATCC 27750 (Fig.3B , Table 8). Similar phenomenon '? were observed in C57B1/ 6J mice administration with E. ventriosum ATCC 27560 . E. ventriosum L2-12 , E. ventriosum STAFF 1042 , E. eligens STAFF 1020 and E. eligens TSDC 10.2- 1.1 (Fig.3 , Table 8 ).

2.2 Validation in animal experiment related to colorectal cancer

2.2.1 Method

The BAB L/c mice were challenged with azoxymethane (AOM, 10 mg/kg) at the age of 10 weeks followed by a 2.0% dextran sodium sulfate (DSS) treatment in the drinking water for 7 days beginning on week 2, week 5 and week 8 to induce colitis-associated colorectal cancer (CRC). Then mice were treated with Eubacterium ventriosum ATCC 27560 , L2-12 , STAFF 1042, and Eubacterium eligens ATCC 27750 , STAFF 1020, TSDC 10.2- 1.1 (10 ⁹ -10 ⁱ⁰ cm/mi , freshly prepared daily) , or control medium respectively from week 9 of the study until killing by cervical dislocation on week 11 (0.1ml /50 g.BW every day, n = 15 in each group). BABL/c mice were purchased from Laboratory Animal Center of Southern Medical University, China.

Disease activity index was calculated as previous described, which was assessed for mice undergoing the AOM+DSS challenge , before and weekly after intragastric gavage.

For histopathology examination, after the necropsy, colonic sections were fixed in 10% buffered neutral formalin, later embedded in paraffin, and then sectioned (6 μτη) and stained with H&E ( Hematoxylin and eosin). Tissue slides were examined in an Olympus microscope (lympus America Inc., Dulles, VA). Specimens underwent blinded histological examination and were scored 1-4 on adenomas and adenocarcinomas.

For RNA isolation and real-time polymerase chain reaction of cytokines, tTotal RNA from colon was isolated using the Qiagen RNA isolation kit (Qiagen) according to the manufacturer's instructions, and then was used to generate the cDNA template using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) and real-time RT-PCR was performed as previously described (Bassaganya-Riera J, Reynolds K, Martino-Catt S, Cui Y, Hennighausen L, et al. (2004) Activation of PPAR gamma and delta by conjugated linoleic acid mediates protection from experimental inflammatory bowel disease. Gastroen- terology 127: 777-791 , incorporated herein by reference). mRNA expression of CD36, and PPARy (peroxisome pro liferator- activated receptor γ ) were assessed by real-time quantitative PGR.

2.2.2 Results

2.2.2.1 After daily administrated with E. ventriosum L2-12 or E. ventriosum STAFF 1042 or E. ventriosum ATCC 27560 or E. eligens STAFF 1020 or E. eligens TSDClO.2-1.1 or E. eligens ATCC 27750 for 2 weeks, the disease activity index were significantly lower than CRC control group (Table 9), while no difference was shown in the models of inflammation-driven colorectal cancer before intragastric gavage (data was not shown here). And the effect of six Euhacterium strains treatments respectively on colon histopathology of experimental azoxymethane-induced colorectal cancer were detected, and all these treatments diminished adenoma and adenocarcinoma formation when compared to the control (Fig.4, Table 9 ).

2.2.2.2 E. ventriosum L2-12 or E. ventriosum STAFF 1042 or is, ventriosum ATCC 27560 or E. eligens STAFF 1020 or E. eligens TSDC 10.2-1.1 or E. eligens ATCC 27750 modulate expression of colonic inflammatory and carcinogenesis markers respectively.

Mice challenged with azoxymethane and DSS were euthanized in the tumor-bearing phase of the disease. Those mice treated with E. eligens ATCC 27750 or E. eligens STAFF 1020 or E. eligens TSDC 10.2- 1.1 or E. ventriosum L2-12 or /·. ^' ventriosum STAFF 1042 or E. ventriosum ATCC 27560 showed increased mRNA expression of CD36 and PPARy in the colon compared with medium-treated control group (Fig.5 , Table 9).

Table 9 Data of clinical measurements related to CRC

Above all, results from this study demonstrate the ability of E. ventriosum L2-12 or E. ventriosum STAFF 1042 or E. ventriosum ATCC 27560 or E. eligens STAFF 1020 or E. eligens TSDClO.2-1.1 or E. eligens ATCC 27750 to ameliorate both colitis and colorectal cancer. Table 4. IMG and mOTU species associated with CRC with q-value < 0.05

Table 5. List of 86 MLG species formed after grouping MLGs with more than 100 genes using species annotation when available.

Although explanatory embodiments have been shown and described, it would be appreciated by those skilled in the art that the a

embodiments can not be construed to limit the present disclosure, and changes, alternatives, and modifications can be made in the embodi without departing from spirit, principles and scope of the present disclosure.