New bacterial strain that produces butyrate and several independent factors with Anti-inflammatory properties

Abstract

One bacterial strain, designated HC1M1C21T, was isolated from human faeces and analyzed using phylogenetic, morphologic and biochemical approaches. Phylogenetic analyses based on 16S rRNA sequences and a set of 92 bacterial core genes indicated that this strain belongs to the Lachnospiraceae family and clusters near Pilosibacter fragilis (96.9% 16S rRNA sequence identity). HC1M1C21T has a DNA G+C content of 48.7 mol%. This strain is anaerobic, Gram- stain-positive, non-motile and non-spore-forming. HC1M1C21T cells appear as single rods or chained rods with tapered ends. Optimal growth was observed at 37°C, at pH between 5.5 and 6.5 and at salinity below 10 g / L. HC1M1C21T is a potent butyrate producer. The invention pertains to a new bacterial strain that produces butyrate and several independent factors with anti-inflammatory properties.

Description

[0001] NEW BACTERIAL STRAIN THAT PRODUCES BUTYRATE AND SEVERAL INDEPENDENT FACTORS WITH ANTI-INFLAMMATORY PROPERTIES

[0002] FIELD OF THE INVENTION:

[0003] The present invention is in the field of medicine, in particular microbiology.

[0004] BACKGROUND OF THE INVENTION:

[0005] The human gut microbiota is a complex ecosystem comprising a huge diversity of microorganisms. These microorganisms play key roles in host digestion, metabolism, immune responses and behavior (1-4). Improvements in next-generation sequencing have greatly expanded our understanding of the gut microbiome's diversity over the past two decades. However, a significant portion of the gut microbiota still remains recalcitrant to culture (5). To overcome the limitations of traditional culture methods, culturomics has recently emerged as a powerful technique to isolate, cultivate and identify novel bacterial species playing potential important roles in host physiology (6-8). For instance, Lachnospiraceae is an abundant family of the human gut microbiota, producing a large variety of metabolites (9-12). The main Lachnospiraceae genera identified in the human gut microbiota are Agathobacter , Blaulia. Coprococcus. Dorea. and Roseburia (9). Certain strains within this family, particularly those that produce butyrate, have shown considerable promise as probiotics. Butyrate is a short-chain fatty acid with e.g. anti-inflammatory and anti-carcinogenic properties, making butyrate- producing bacteria potential candidates for treating a range of diseases, including inflammatory bowel disease, colorectal cancer, metabolic disorders and eating disorders. Of note, other bacterial species from the gut microbiota may secrete effectors or express surface-bound factors exhibiting anti-inflammatory properties, independently from butyrate production.

[0006] SUMMARY OF THE INVENTION:

[0007] The invention pertains to a new bacterial strain that produces butyrate and several independent factors with anti-inflammatory properties, as defined by the claims.

[0008] DETAILED DESCRIPTION OF THE INVENTION:

[0009] One bacterial strain, designated HC1M1C21T, was isolated from human faeces and analyzed using phylogenetic, morphologic and biochemical approaches. Phylogenetic analyses based on 16S rRNA sequences and a set of 92 bacterial core genes indicated that this strain belongs to the Lachnospiraceae family and clusters near Pilosibacter fragilis (96.9% 16S rRNA sequence identity) (13). HC1M1C21Thas a DNA G+C content of 48.7 mol%. This strain is anaerobic, Gram-stain-positive, non-motile and non-spore-forming. HC1M1C21Tcells appear as single rods or chained rods with tapered ends. Optimal growth was observed at 37°C, at pH between 5.5 and 6.5 and at salinity below 10 g / L. HC1M1C21Tis a potent producer of butyrate and several independent factors with anti-inflammatory properties. On the basis of these data, HC1M1C21Trepresents a novel species from the Pilosibacter genus, for which the name Pilosibacter rotomagensis sp. nov. is proposed. The type strain of P. rotomagensis is HC1M1C21T.

[0010] The first object of the present invention relates to the isolated bacterial strain deposited in accordance with the Budapest Treaty, on June 6, 2024 at the COLLECTION NATION ALE DE CULTURES DE MICROORGANISMES (CNCM) under the accession number CNCM I- 6077.

[0011] In some embodiments, the bacterial strain described herein is genetically engineered to express a heterologous polynucleotide. This polynucleotide may encode enzymes or other proteins that enhance the bacterium's capacity to produce butyrate or other beneficial metabolites. Genetic modifications can be achieved through various recombinant DNA techniques, including but not limited to CRISPR-Cas9, homologous recombination, or plasmid-based transformation.

[0012] For example, CRISPR-Cas9 technology permits precise editing of the bacterial genome, enabling the insertion, deletion, or modification of specific genetic sequences. This precision ensures optimal expression of the introduced polynucleotide. Homologous recombination allows seamless integration of new genetic material into the bacterium's chromosome, which is particularly useful for stable gene expression over extended periods.

[0013] Alternatively, plasmid-based transformation involves introducing plasmids — small, circular DNA molecules — into bacterial cells. These plasmids can carry multiple genes necessary for butyrate production and other advantageous traits. Designed to replicate independently within the bacterial cell, plasmids ensure consistent expression of the introduced genes.

[0014] Beyond enhancing the production of butyrate and other bacterial factors with anti-inflammatory properties, genetic modifications can improve the bacterium's resilience in various environments. For instance, genes conferring resistance to harsh gastrointestinal conditions, such as low pH or high bile concentrations, can be introduced, ensuring the bacterial strain remains viable and effective throughout its passage through the digestive tract. Moreover, genetic engineering can enable the bacterial strain to produce additional health-promoting compounds. These compounds could include vitamins, antimicrobial peptides, or antiinflammatory molecules. Such multifunctional strains would provide comprehensive benefits, addressing various health concerns concurrently. Another potential application of genetic modification is developing strains capable of degrading harmful substances within the gut. For instance, introducing genes encoding enzymes that break down toxic metabolites or pathogens would enhance the bacterium's role in maintaining gut health and preventing diseases.

[0015] The bacterial strain of the present invention can be included in a variety of formulations designed to deliver therapeutic benefits to the host. These formulations may include, but are not limited to, capsules, tablets, powders, or liquid suspensions. The chosen formulation would depend on the intended use, whether for nutritional supplementation, treatment of gastrointestinal disorders, or enhancement of general health. Additionally, the bacterial strain can be incorporated into functional foods or beverages, providing an easy and palatable way for individuals to consume the probiotic. Examples of such functional foods include yogurts, smoothies, and snack bars. The incorporation of the bacterial strain into these products would require ensuring the stability and viability of the bacteria throughout the product's shelf life.

[0016] In some embodiments, the composition typically comprises carriers or vehicles. As used herein, the term "carriers" or "vehicles" mean materials suitable for administration and include any such material known in the art such as, for example, any liquid, gel, solvent, liquid diluent, solubilizer, or the like, which is non-toxic and which does not interact with any components of the composition in a deleterious manner. Examples of nutritionally acceptable carriers include, for example, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.

[0017] In some embodiments, the bacterial strain may be combined with prebiotics to create synbiotic formulations. Prebiotics are non-digestible food components that beneficially affect the host by stimulating the growth and / or activity of beneficial bacteria in the colon. Non-limiting examples of prebiotics include: oligosaccharides optionally containing fructose, galactose, mannose; dietary fibers, in particular soluble fibers, soy fibers; inulin; and combinations thereof. Preferred prebiotics are fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), isomaltooligosaccharides (IMO), xylo-oligosaccharides (XOS), arabino-xylo oligosaccharides (AXOS), mannan-oligosaccharides (MOS), oligosaccharides of soy, glycosyl sucrose (GS), lactosucrose (LS), lactulose (LA), palatinose-oligosaccharides (PAO), malto-oligosaccharides, gums and / or hydrolysates thereof, pectins and / or hydrolysates thereof, and combinations of the foregoing. The synbiotic formulations would be designed to optimize the growth and activity of the bacterial strain of the present invention within the gut, thereby maximizing its therapeutic effects.

[0018] In some embodiments, the composition comprises any other ingredients or excipients known to be employed in the type of composition in question. Non-limiting examples of such ingredients include proteins, amino acids, carbohydrates, oligosaccharides, lipids, prebiotics or probiotics, nucleotides, nucleosides, other vitamins, minerals, and other micronutrients.

[0019] In some embodiments, the composition comprises one or more vitamins. Vitamins may be folic acid, vitamin B12 and vitamin B6, in particular folic acid and vitamin B12, in particular folic acid. In some embodiments, the composition comprises one or more vitamin which is lipid- soluble, for example one or more of vitamin A, vitamin D, vitamin E and vitamin K.

[0020] In some embodiments, the composition further comprises one or more minerals. Examples of minerals are sodium, potassium, chloride, calcium, phosphate, magnesium, iron, zinc, copper, selenium, manganese, fluoride, iodine, chromium, or molybdenum. The minerals are usually added in salt form. The minerals may be added alone or in combination.

[0021] In some embodiments, the composition contains emulsifiers. Examples of food grade emulsifiers typically include diacetyl tartaric acid esters of mono- and di-glycerides, lecithin and mono- and di-glycerides. Similarly suitable salts and stabilisers may be included.

[0022] In some embodiments, the composition contains protective hydrocolloids (such as gums, proteins, modified starches), binders, film forming agents, encapsulating agents / materials, wall / shell materials, matrix compounds, coatings, emulsifiers, surface active agents, solubilizing agents (oils, fats, waxes, lecithins etc.), adsorbents, carriers, fillers, co-compounds, dispersing agents, wetting agents, processing aids (solvents), flowing agents, taste masking agents, weighting agents, jellifying agents, gel forming agents, antioxidants and antimicrobials. The composition may also contain conventional additives and adjuvants, excipients and diluents, including, but not limited to, water, gelatine of any origin, vegetable gums, ligninsulfonate, talc, sugars, starch, gum arabic, vegetable oils, polyalkylene glycols, flavouring agents, preservatives, stabilizers, emulsifying agents, buffers, lubricants, colorants, wetting agents, fillers, and the like. In all cases, such further components will be selected having regard to their suitability for the intended recipient.

[0023] In some embodiments, the composition of the present invention further comprises one or more probiotics. As used herein the term “probiotic” is meant to designate live microorganisms which, when administered in a sufficient amount, exert a positive effect on health, comfort and wellness beyond traditional nutritional effects. Probiotic microorganisms have been defined as live microorganisms which when administered in adequate amounts confer a health benefit on the host” (FAO / WHO 2001). Non limiting examples of probiotics include: Bifidobacterium, Lactobacillus, Lactococcus, Enterococcus, Streptococcus, Kluyveromyces, Saccharoymces, Candida, in particular selected from the group consisting of Bifidobacterium longum, Bifidobacterium lactis, Bifidobacterium animalis, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium adolescentis, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus salivarius, Lactobacillus lactis, Lactobacillus rhamnosus, Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillus salivarius, Lactococcus lactis, Enterococcus faecium, Saccharomyces cerevisiae, Saccharomyces boulardii or mixtures thereof,

[0024] In some embodiment, the bacterial strain of the present invention can be freeze-dried or lyophilized to extend its shelf life and ease of transportation. This process involves removing water from the bacterial cells while maintaining their viability and functionality. Freeze-dried bacterial strains can be rehydrated and activated upon consumption, ensuring their effectiveness.

[0025] A further object of the present invention relates to use of the bacterial strain of the present invention as a probiotic. According to the present invention, the bacterial strain of the present invention is particularly suitable for the treatment of various diseases wherein the production of butyrate or antiinflammatory factors is desired. Butyrate, a short-chain fatty acid, plays a crucial role in maintaining gut health by serving as an energy source for colonocytes, reducing inflammation, and enhancing the gut barrier function.

[0026] Examples of diseases that may benefit from the bacterial strain of the present invention include inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis. These conditions are characterized by chronic inflammation of the gastrointestinal tract, leading to symptoms such as abdominal pain, diarrhea, weight loss, and fatigue. Traditional treatments for IBD often involve the use of anti-inflammatory drugs, immune system suppressors, and biologies, which can have significant side effects and may not be effective for all patients. Patients with IBD may need surgery to remove damaged parts of their intestine. Ileal pouch- anal anastomosis is a common procedure used to treat severe and refractory colitis, which can lead to a complication called “pouchitis”, characterized by high levels of inflammation in the formed pouch. The innovative approach of utilizing probiotics producing butyrate and several independent factors with anti-inflammatory properties offers a promising alternative or complementary therapy for IBD and associated complications such as pouchitis. By introducing beneficial bacterial strains that produce butyrate and other anti-inflammatory factors within the gut, the inflammation can be reduced naturally, and the gut barrier integrity can be restored.

[0027] In addition to IBD, other conditions such as irritable bowel syndrome (IBS), colorectal cancer, Nectrotizing Enterocolitis (NEC) and antibiotic-associated diarrhea could also see therapeutic benefits from probiotics producing butyrate and several independent factors with antiinflammatory properties. For instance, IBS, a disorder affecting the large intestine, can cause cramping, abdominal pain, bloating, gas, and diarrhea or constipation. NEC is a disease with a high mortality rate affecting neonates. NEC is characterized by intestinal inflammation leading to cell death and necrosis in the intestine and colon, ultimately triggering intestinal perforation, peritonitis and sepsis. NEC-associated inflammation can be therefore mitigated by the butyrate- producing bacteria of the present invention. Colorectal cancer patients often suffer from gut dysbiosis and inflammation, which can be mitigated by bacteria producing butyrate and other anti-inflammatory factors. Furthermore, butyrate has been observed to improve insulin sensitivity and reduce the risk of metabolic disorders, indicating its broader implications beyond gut health. The application of butyrate-producing probiotics could thus extend to treating Type 2 diabetes and obesity-related inflammation. Along the same line, a decrease in butyrate-producing bacteria has been observed in eating disorders such as Anorexia Nervosa. Butyrate-producing probiotics could thus be useful to treat this type of disorders affecting the gut-brain axis.

[0028] Accordingly, a further object of the present invention relates to a method of therapy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a bacterial strain according to the present invention.

[0029] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

[0030] As used herein, the term “therapeutically effective amount” is used to refer to an amount of a compound that results in prevention, delay of onset of symptoms, or amelioration of symptoms of a condition, e.g., inflammation. A therapeutically effective amount may, for example, be sufficient to treat, prevent, reduce the severity, delay the onset, and / or reduce the risk of occurrence of one or more symptoms of an autoimmune a disorder and / or a disease or condition associated with gut inflammation and / or compromised gut barrier function. A therapeutically effective amount, as well as a therapeutically effective frequency of administration, can be determined by methods known in the art and discussed below.

[0031] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0032] FIGURES:

[0033] Figure 1: Phylogenetic trees. (A) Optimal tree, based on 16S rRNA sequences, and inferred using the Neighbor-Joining method. (B) Phylogenetic tree inferred using concatenated alignments of 92 core genes from whole genome sequences. Percentage bootstrap values are given at branching points (1000 replicates). The phylogenetic distances are in the units of the number of base substitutions per site. The species Oscillibacter valericigenes (Oscillospiraceae) was used to root the trees.

[0034] Figure 2: Time, temperature, pH and salinity-dependent growth of HC1M1C211. (A) Growth kinetics of HC1M1C21Tin MRS broth (37°C; pH 5.7). (B) Temperature-dependent growth of HC 1M1C21Tin MRS broth (pH 5.7; 48h incubation). (C) pH-dependent growth of HC 1M1C21Tin MRS broth (37°C; 24h incubation). (D) Salinity-dependent growth of HC 1M1C21Tin MRS broth (37°C; pH 5.7; 72h incubation). Values are expressed as means ± s.d.; n=3-7; one-way ANOVA with Tukey’s correction; Labeled plots without a common letter differ; P < 0.05. Figure 3: Electron micrographs of HC1M1C211. Bacterial cells were grown on MRS broth in anaerobic conditions at 37°C for 1 day. Images were obtained by scanning electron microscopy (A) or transmission electron microscopy (B).

[0035] Figure 4: HC1M1C211reduces IL8 expression in response to TNFa in HT-29 cells. (A) Quantification of IL8 mRNA levels in HT-29 cells pre-treated with culture supernatants (SN) of HC1M1C21Tfor Ih (HC1M1C21Twas grown in MRS medium for either 24h or 48h) or MRS alone, and then incubated or not for 5h with 100 ng / mL TNFa. (B) Quantification of IL8 levels in HT-29 cells pre-treated with whole HC1M1C21Tcells for Ih and then incubated or not for 5h with 100 ng / mL TNFa. Values are expressed as fold-change compared to cells exposed to TNFa only (mean ± s.d.; n=3-4; *, <0.05; ***, P< 0.001; One-way ANOVA with Tukey’s correction). (C) Quantification of ZC3H12A and STDB1 levels in HT-29 cells pretreated with HC1M1C21Tculture supernatants or butyrate (5 mM) and then incubated or not for 5h with 100 ng / mL TNFa. Values are expressed as the log2 fold-change relative to untreated cells (mean ± s.e.; n=4; NS, not significant; ***, P< 0.001; Wald test).

[0036] Figure 5: Oral administration of HC1M1C211does not induce weight loss or increased intestinal inflammation in mice. C57Bl / 6JRj male mice were administered with HC1M1C21Tvia oral gavage, once daily, during 14 days (2.109live bacteria / mouse / day). (A) body weight of mice administered or not with HC1M1C21T. No significant changes in body weight were observed in response to HC1M1C21Tadministration (mean ± s.d.; n=7; 2-way ANOVA with Sidak’s correction). (B) Quantification of calprotectin levels in faeces collected from the colon of mice after 14 days of HC1M1C21Tadministration (mean ± s.d.; n=7; NS, not significant; unpaired two-tailed t test).

[0037] Figure 6: Oral administration of HC1M1C211decreases inflammation in a murine colitis model. C57Bl / 6JRj male mice were exposed or not to 1% DSS in their drinking water for 7 days, followed by 3 days of regular water. Half of the animals were administered with HC1M1C21Tvia oral gavage, once daily (2.109live bacteria / mouse / day). HC1M1C21Tadministration began 5 days before DSS treatment and continued throughout the whole experiment. Plasmatic levels of TNFa at the end of the experiment was quantified by ELISA (mean ± s.d.; n=4-7; Labeled means without a common letter differ; Kruskal-Wallis test with Dunn’s correction). EXAMPLE 1:

[0038] The HC1M1C21Tstrain was isolated from a fresh human stool sample from a healthy woman. The stool sample was collected and stored anaerobically at 4°C for 24 hours. 5 g of stools were transferred into an anaerobic chamber and resuspended in 4.5 mL of a 9 g / L NaCl solution. The obtained suspension was then supplemented with sodium L-ascorbate and L-cysteine hydrochloride monohydrate, at a final concentration of 5 mg / mL and 1 mg / mL, respectively. The obtained solution was then serially diluted and plated onto MRS agar plates (de Man, Rogosa and Sharpe medium, BD Difco™ 288130), supplemented with 0.5 mg / mL L-cysteine hydrochloride monohydrate. After incubation at 37°C for 48h in anaerobic conditions, individual colonies were picked, grown anaerobically at 37°C in MRS broth supplemented with 0.5 mg / mL L-cysteine hydrochloride monohydrate, and re-streaked to confirm purity. Identification of each isolate was performed by PCR amplification and sequencing of the 16S rRNA gene. The isolate HC1M1C21Twas selected since its 16S rRNA gene was distinct from all referenced 16S rRNA gene in the NCBI rRNA / ITS database.

[0039] Genomic DNA of the HC1M1C21Tstrain was extracted from pelleted bacteria grown in MRS broth using Qiagen Genomic-tips, according to the manufacturer’s instructions, and sequenced using long reads sequencing technology from Oxford Nanopore Technologies (ONT) (Microsynth, Balgach, Switzerland).

[0040] Phylogenetic trees based on 16S rRNA sequences or a set of 92 bacterial core genes from HC1M1C21Tand reference Lachnospiraceae and Clostridiaceae species were reconstructed using the Neighbour-Joining method analysis, with 1000 bootstrap replicates (14,15) (Figures The phylogenetic distances were computed using the Maximum Composite

[0041] Likelihood method (16). Phylogenetic analyses were conducted in MEGA 11 (17).

[0042] The closest known relative to HC1M1C21Tis Pilosibacter fragilis (96.9% sequence identity for the 16s rRNA gene), a gram-stain-positive bacterium isolated from human faeces (13). The POCP (Percentage Of Conserved Proteins) between HC1M1C21Tand Pilosibacter fragilis, calculated with the POCP-nf pipeline (18), is 69.0%. This value, which is above the threshold of 50%, indicates that these two bacteria belong to the same genus (19). Average Nucleotide Identity (ANI) was estimated with the OrthoANI using USEARCH (OrthoANIu) tool (20). Ortho ANIu value between HC1 Ml C21Tand Pilosibacter fragilis is 88.2%. This value, which is below the threshold of 95%, indicates that HC1M1C21Tand P. fragilis belong to different species (20,21). We used the Protologger pipeline to analyze the genome sequence of HC1M1C21T(22). The genome size of HC1M1C21Tis 3.46 Mbp (with a completeness of 92.41% and 0% contamination) and the DNA G+C content is 48.7%mol. No CRISPR arrays was identified. The genome contains 3058 potential coding sequences.

[0043] To examine bacterial growth, the HC1M1C21Tstrain was incubated in MRS broth supplemented with 0.5 mg / mL L-cysteine hydrochloride monohydrate, in anaerobic conditions, at different temperatures, pH and salinity. The HC1M1C21Tstrain grows at temperature from 30 to 40°C, with optimum growth at 37°C. The HC1M1C21Tstrain grows at pH 5.5-6.5 (Figures 2A-2C) and does not grow in broth with salinity above 10 g / L (Figure 2D).

[0044] To examine motility, HC1M1C21Twas stab-inoculated on semi-solid MRS medium containing 0.35 g / 100 mL agar, and incubated in anaerobic conditions at 37°C. HC1M1C21Tappears as non-motile, in contrast to a motile E. coli strain (DSM 300831). This result is consistent with the lack of flagella observed by electron microscopy (Figures 3A and 3B). The growth of HC1M1C21Tis inhibited on Bile Esculine sodium Azide (BEA) agar, in the presence of sodium azide (0.15 g / L) and beef bile (10 g / L) (Bio-rad, #63994). No catalase activity was detected using 3% (v / v) hydrogen peroxide solution, and no oxidase activity was detected using Bactidrop oxidase test (Remel, Thermo Fisher Scientific). The ability of HC1M1C21Tto form spores was then evaluated. A culture of HC1M1C21Twas centrifugated and treated with 70 % (v / v) ethanol to kill vegetative cells. Treated bacteria were then inoculated on MRS agar supplemented with 0.1% sodium taurocholate to stimulate the germination of the potential remaining spores (6). No colonies were observed after treatment, indicating that HC1M1C21Tis not forming spores. This result is consistent with the lack of typical spore structures observed by electron microscopy (Figures 3A and 3B). For all these different assays, we used, as positive controls, a bile-resistant and esculin-hydrolyzing Enterococcus faecalis strain, the catalasepositive Staphylococcus aureus RN4220 strain, the oxidase-positive E. coli DSM 30083Tstrain and the spore-forming Clostridium sporogenes ATCC 15579 strain.

[0045] Colonies formed by HC1M1C21Ton MRS plates after 48h of incubation at 37 °C under anaerobic conditions are 1-3 mm in diameter, ivory, circular, entire, convex, mucoid and opaque. HC1M1C21Tcells are 4.4 ± 0.4 pm long and 1.4 ± 0.2 pm wide. Gram staining of HC1M1C21Tshow that this species is a Gram-positive bacterium, which is consistent with phylogenetic analyses. Of note, some HC1M1C21Tcells exhibit only a patchy purple staining. Cell morphology was then evaluated using both scanning (SEM) and transmission (TEM) electron microscopy (Figures 3A and 3B). For this, HC1M1C21Tgrown in MRS broth was centrifugated and washed in IX PBS (Phosphate Buffered Saline). Cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.0) containing 0.4 mg / ml ruthenium red for 15 hours at 4°C. For SEM, the cells were rinsed in 0.1 M cacodylate buffer (pH 7.0) containing 0.4 mg / ml ruthenium red, sedimented during a week on Thermanox® coverslip coated with poly-L-lysine and then dehydrated in progressive bath of ethanol (70-100%) before being critical point dryed (CPD 030 LEICA Microsystem). The cells were sputtered with platinum and observed with a JEOL 7200F scanning electron microscope. For TEM, the cells were rinsed in 0.1 M cacodylate buffer (pH 7.0) containing 0.4 mg / ml ruthenium red and post-fixed for 2 hours with 1% osmium tetroxyde in cacodylate buffer 0.1 M pH 7.0 supplemented with 0.4 mg / ml ruthenium red (at 4°C in the dark). The cells were rinsed again in 0.1 M cacodylate buffer (pH 7.0) containing 0.4 mg / ml ruthenium red, pelleted in 7.5% gelatin at 40°C and then dehydrated in progressive bath of ethanol (70-100%). Cells were embedded in resin Embed 812 followed by a step of polymerization at 60°C during 48h. Ultrathin sections were performed and contrasted with uranyl acetate and lead citrate. Bacteria were observed with a JEOL 1011 transmission electron microscope and image were taken with an ORIUS 200 camera and a digital micrograph software.

[0046] Since many Lachnospiraceace are butyrate producers, we evaluated the ability of HC1M1C21Tto produce butyrate using gas chromatography-mass spectrometry. For this, we collected supernatants from cultures of HC1M1C21Tgrown in MRS or BHI broth. Supernatants were acidified using hydrochloric acid to adjust the pH to 2-3. Organic acids contained in the supernatants were extracted by liquid-liquid extraction with methyl tert-butyl ether and directly analyzed by gas chromatography-mass spectrometry, without derivation, using a high-polarity polyethylene glycol type column (Agilent J&W DB-FFAP GC column) (23). Quantification was performed using selected ion monitoring mode. Deuterated butyric acid (butyric-d7 acid) was used as an internal standard. We observed that HC1M1C21Tproduced high levels of butyrate after 24 and 48h of growth at 37°C in anaerobic conditions, in both MRS and BHI broth. Butyrate production in Lachnospiraceae from carbohydrates can be mediated by two independent pathways: the butyrate kinase pathway (24) and the butyryl-coA: acetate CoA- transferase (BCoAT) pathway (25). We searched the HC IM 1C21Tgenome and identified one putative BCoAT enzyme, sharing 84% homology with the BCoAT protein from Agathobacter rectalis VPI 0990Tstrain. No homolog to butyrate kinase could be identified in the HC1M1C21Tgenome (using the butyrate kinase from Coprococcus comes ATCC 27758Tstrain as a reference).

[0047] Finally, we characterized various biochemical features of HC1M1C21Tby using the API 20A anaerobe test kit and the API Rapid ID 32A anaerobe identification kit (bioMerieux), according to the manufacturer’s instructions (Table 1) Dorea formicigenerans (DSM 3992T) and Enterocloster bolteae (DSM 15670T) grown on YCFAC (Yeast Casitone Fatty Acids Agar with Carbohydrates) plates were used as reference strains in these tests.

[0048] On the basis of these data including cell morphology, growth parameters, enzymatic features and phylogenetic analyses based on 16S rRNA and whole genome sequences, HC1M1C21Tstrain represents a novel species, for which the name Pilosibacter rotomagensis sp. nov. is proposed.

[0049] EXAMPLE 2:

[0050] Material and methods:

[0051] HT-29 (American Type Culture Collection (ATCC)- HTB-38) cells were cultivated in McCoy's 5a Medium Modified (Gibco), supplemented with 10% Fetal Bovine Serum (FBS, Eurobio) and a mixture of penicillin (10000 U / mL) and streptomycin (10 mg / mL), at 37°C in a 5% CO2 atmosphere. The day before treatments, HT-29 cells were seeded in wells at a density of 4.0 x 104cells / well in a 96 well plate.

[0052] HC1M1C21Twas grown in parallel anaerobically at 37°C in MRS broth supplemented with 0.5 mg / mL L-cysteine hydrochloride monohydrate for 24h or 48h. Each bacterial culture was then centrifugated. The supernatants were collected and the pelleted bacteria were resuspended in sterile IX PBS.

[0053] The day of the experiment, HT-29 cells were incubated in HBSS (Hanks’ Balanced Salt Solution; Sigma-Aldrich) supplemented with either whole HC1M1C21Tcells (resuspended in PBS), HC1M1C21Tsupernatant or MRS (5%, v / v) for 1 hour, and then incubated with 100 ng / mL of recombinant human TNFa (PeproTech) for 5 hours. Viability of cells was checked using the CellTiter-Glo® luminescent cell viability assay (Promega), according to the manufacturer’s protocol.

[0054] RNAs were then extracted from HT-29 cells using the RNeasy Plus Mini kit (Qiagen), following manufacturer’s instructions. Gene expression levels were then quantified either by RNA-Seq or by qRT-PCR. To assess specific gene expression by qRT-PCR, for each condition, 0.8-1 pg of total RNAs were reverse transcribed using random hexamers and M-MLV reverse transcriptase (Invitrogen). Specific cDNAs were quantified by qPCR using Itaq Universal SYBR Green Supermix (Bio-Rad). GAPDH was used as an internal reference for normalization. Serial dilution of target cDNAs was included on each plate to generate relative curves and integrate primer efficiency in the calculations of mRNA quantities.

[0055] Results:

[0056] TNFa induces a strong increase in the expression level of IL8 in HT-29, as expected. We observed thatHClMlC21Tsupernatant significantly downregulates IL8 expression in response to TNFa (Figure 4A). This result indicates that the HC1M1C21Tculture supernatant contains molecules with anti-inflammatory properties. In addition, we observed that whole bacteria also significantly inhibit IL8 expression (Figure 4B). This result suggests that some non-secreted molecules from HC1M1C21Talso exhibit anti-inflammatory properties.

[0057] Interestingly, we identified genes for which the HC1M1C21Tsupernatant blunts TNFa-induced up- or down-regulations, whereas butyrate has no effect (Figure 4C). This suggests that the HC1M1C21Tsupernatant blunts the regulation of specific TNFa-targeted genes, using butyrate- independent mechanisms.

[0058] Together, these results suggest that HC1M1C21Tpossess anti-inflammatory properties, which may be mediated by both secreted and non-secreted bacterial effectors.

[0059] EXAMPLE 3:

[0060] Material and methods:

[0061] To evaluate the impact of HC1M1C21Tadministration on mouse body weight and intestinal physiology, 9-weeks-old C57Bl / 6JRj male mice were administered with HC1M1C21Tvia oral gavage, once daily, during 14 days (2.109bacteria / mouse / day). The bacteria administered to the mice were first grown anaerobically at 37°C in MRS broth supplemented with 0.5 mg / mL L-cysteine hydrochloride monohydrate for 24h. They were then centrifuged and resuspended in sterile IX PBS. The body weight of each mouse was determined on a daily basis. On day 15, the mice were sacrificed and their faeces were collected from the colon to quantify the level of calprotectin by ELISA, as previously described (R&D Systems, Minneapolis) (26).

[0062] To evaluate the impact of HC1M1C21Tadministration on chemically-induced colitis in mice, 9-weeks-old C57Bl / 6JRj male mice were given access to drinking water containing 1% DSS (Dextran Sulfate Sodium) for 7 days, followed by 2 days of regular water. Half of the animals were administered with HC1M1C21Tvia oral gavage, once daily (2.109bacteria / mouse / day). HC1M1C21Tadministration began 5 days before DSS treatment and continued throughout the whole experiment. On day 15, mice were sacrificed and their blood collected to quantify the plasmatic levels of TNFa by ELISA (R&D Systems, Minneapolis).

[0063] Results:

[0064] In order to assess the lack of toxicity of HC1M1C21T, we administered this bacterial strain to mice via oral gavage for 14 consecutive days. The administration of HC1M1C21Tdid not impact body weight nor induce intestinal inflammation in mice (Figures 5A and 5B).

[0065] We then tested whether an oral administration of the HC1M1C21Tstrain may alleviate inflammation in a chemically-induced colitis mouse model. For this, mice were given access to drinking water containing 1% DSS for 7 consecutive days. DSS triggers an erosion of the intestinal epithelium, which leads to intestinal and systemic inflammation, which can be monitored by quantifying the plasmatic level of pro-inflammatory cytokines such as TNFa. In parallel to the DSS-treatment, mice received or not the HC1M1C21Tstrain via oral gavage. We observed that mice treated with both DSS and HC IM 1C21Texhibited significantly lower levels of plasmatic TNFa than mice treated with DSS only (Figure 6). This result demonstrates that giving the HClMlC21Tbacterial strain orally significantly reduces inflammation in an in vivo colitis model.

[0066] TABLES:

[0067] Table 1: Biochemical features of HC1M1C21T. Symbols: +, positive; w+, weakly positive; -, negative; f, similar results in both API20A and API Rapid 32A test; *, different results in API20A and API Rapid 32A tests. Each test was performed in triplicates.

[0068] Characteristic HC1M1C21TDorea formicigenerans Enterocloster bolteae

[0069] DSM 3992TDSM 15670t Catalase

[0070] Oxidase

[0071] Indole production -f

[0072] Acid produced from:

[0073] D-Glucose

[0074] D-Mannitol

[0075] D-Lactose

[0076] D-Saccharose (sucrose)

[0077] D-Maltose

[0078] Salicin

[0079] D-Xylose

[0080] L-Arabinose

[0081] Glycerol

[0082] D-Cellobiose

[0083] D-Mannose -f

[0084] D-Melezitose

[0085] D-Raffinose -f

[0086] D-Sorbitol

[0087] L-Rhamnose

[0088] D-Trehalose

[0089] Enzyme activities:

[0090] Urease -f

[0091] Arginine dihydrolase

[0092] Gelatin protease a-galactosidase p-galactosidase +

[0093] 6 phosphate-a-galactosidase a-glucosidase p-glucosidase (4NBDGP) p-glucosidase (esculin) a-arabinosidase p-glucuronidase

[0094] N-acetyl-p-glucosaminidase

[0095] Glutamic acid decarboxylase a-fucosidase

[0096] Nitrate reduction

[0097] Alkaline phosphatase w+

[0098] Arginine arylamidase +

[0099] Proline arylamidase

[0100] Leucyl-glycine arylamidase w+

[0101] Phenylalanine arylamidase

[0102] Leucine arylamidase

[0103] Pyroglutamic acid arylamidase w+

[0104] Tyrosine arylamidase

[0105] Alanine arylamidase

[0106] Glycine arylamidase +

[0107] Histidine arylamidase +

[0108] Glutamyl-Glutamic acid arylamidase

[0109] Serine arylamidase REFERENCES:

[0110] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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[0136] (This sheet is not part of and does not count as a sheet of the international application)

[0137] FOR RECEIVING OFFICE USE ONLY

[0138] FOR INTERNATIONAL BUREAU USE ONLY

Claims

CLAIMS:

1. An isolated bacterial strain deposited in accordance with the Budapest Treaty, on June 6, 2024 at the COLLECTION NATIONALE DE CULTURES DE MICROORGANISMES (CNCM) under the accession number CNCM 1-6077.

2. The bacterial strain according to claim 1 that is genetically engineered to express a heterologous polynucleotide.

3. A composition comprising the isolated bacterial strain according to claim 1 or 2 and one or more carriers.

4. The composition according to claim 3 that further comprises one or more prebiotics.

5. The composition according to claim 3 or 4 that further comprises one or more probiotics.

6. Use of the bacterial strain according to claim 1 or 2 as a probiotic.

7. A method of treating a disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the bacterial strain according to claim 1 or 2.

8. The method according to claim 7 wherein the disease is selected from the group consisting of inflammatory bowel disease, colorectal cancer, metabolic disorders and eating disorders / 9. The method according to claim 8 wherein the inflammatory bowel disease (IBD) is Crohn's disease or ulcerative colitis.

10. The method according to claim 7 wherein disease is selected from the group consisting of pouchitis, irritable bowel syndrome (IBS), Nectrotizing Enterocolitis (NEC) and antibiotic-associated diarrhea.

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