PHASCOLARCTOBACTERIUM FAECIUM FOR USE IN THE PREVENTION AND TREATMENT OF OBESITY AND ITS COMORBIDITIES

Abstract

The present invention relates to the strain Phascolarctobacterium faecium DSM 32890 and to its use for the regulation of appetite and the treatment and/or prevention of overweight or the associated metabolic and immunological changes, specifically, hyperglycaemia, glucose intolerance, insulin resistance, dyslipidemia (hypertriglyceridemia, hypercholesterolemia), metabolic syndrome, diabetes and intestinal and/or peripheral tissue inflammation.

Claims

1. A strain of Phascolarctobacterium faecium with deposit number DSM 32890.

2. A strain derived from the strain according to claim 1.

3. The strain according to claim 1, wherein said strain is a genetically modified mutant.

4. The strain according to claim 1, wherein said strain is in the form of viable cells or in the form of non-viable cells.

5. (canceled)

6. A composition comprising the strain according to claim 1.

7. The composition according to claim 6, further comprising at least one bioactive component.

8. The composition according to claim 6, further comprising at least one microorganism other than the strain P. faecium with deposit number DSM 32890.

9. The composition according to claim 8, wherein the microorganism is an intestinal bacterium or a lactic bacterium.

10. The composition according to claim 6, wherein said composition is a pharmaceutical composition.

11. The composition according to claim 10, wherein the composition additionally comprises at least one pharmaceutically acceptable carrier and/or excipient.

12. The composition according to claim 10, wherein said composition is presented in a form adapted for the oral, sublingual, nasal, intrathecal, bronchial, lymphatic, rectal, transdermal, inhaled or parenteral administration thereof.

13. The composition according to claim 6, wherein said composition is a nutritional composition.

14. The composition according to claim 13, wherein said nutritional composition is a food, a supplement, a nutraceutical, a probiotic or a symbiotic.

15. The composition according to claim 14, wherein said food is selected from the list consisting of a dairy product, a plant product, a meat product, a snack, chocolate, beverage or baby food.

16. The composition according to claim 6, wherein said composition has a strain concentration of between 10.sup.3 and 10.sup.14 colony-forming units (cfu) per gram or millilitre of final composition.

17-20. (canceled)

21. A method for the prevention and/or treatment of overweight and/or obesity, or diseases associated with it, comprising administering to a subject a strain according to claim 1.

22. The method according to claim 21, wherein diseases associated with overweight and/or obesity are selected from the list consisting of cardiovascular diseases, metabolic syndrome, diabetes, hyperglycaemia, insulin resistance, cancer, hypertension, dyslipidemia, hypolipidaemia, galactosaemia, phenylketonuria, sitosterolaemia, hyperthyroidism and hypothyroidism.

23. A non-therapeutic method for the regulation of appetite comprising administering to a subject a strain according to claim 1.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0078] FIG. 1. Effect of administering the strain P. faecium (1×10.sup.7-8 cfu/day) to C57BL/6 obese mice (n=10/group) for 14 weeks on body weight gain. (a) Weekly body weight. (b) Body weight gain after 14 weeks of treatment. The data are expressed in grams with means and standard error. Statistically significant differences were established by applying one-way ANOVA followed by the Tukey test (p<0.05). CD, control diet; HFHSD, high-fat high-sucrose diet; HFHSD+P. faecium, high-fat high-sucrose diet+P. faecium.

[0079] FIG. 2. Effect of administering the strain P. faecium (1×10.sup.7-8 cfu/day) to C57BL/6 obese mice (n=10/group) for 14 weeks on basal blood glucose and glucose tolerance. (a) Fasting blood glucose levels (mg/dl) at week 8 and 10. (b) Glucose tolerance test, glycaemia was measured at 15, 30, 60 and 120 minutes after having administered an oral glucose overload (2 g/Kg). The Area Under the Curve (AUC) for the glucose tolerance test results is shown. The data are represented with means and standard error. Statistically significant differences were established by applying one-way ANOVA followed by the Tukey test (p<0.05). CD, control diet; HFHSD, high-fat high-sucrose diet; HFHSD+P. faecium, high-fat high-sucrose diet+P. faecium.

[0080] FIG. 3. Effect of administering the strain P. faecium (1×10.sup.7-8 cfu/day) to C57BL/6 obese mice (n=10/group) for 14 weeks on intake. (a) Relative intake (kcal/day) per animal in weeks 3, 6, 9 and 12. (b) Daily intake (kcal) per animal at week 12 of treatment. (c) Size of white adipose tissue (grams). The data are represented with means and standard error. Statistically significant differences were established by applying one-way ANOVA followed by the Tukey test (p<0.05). CD, control diet; HFHSD, high-fat high-sucrose diet.

[0081] FIG. 4. Effect of administering the strain P. faecium (1×10.sup.7-8 cfu/day) to C57BL/6 obese nice (n=10/group) for 14 weeks on inflammation. (a) Percentage of ILC1 cells in the epithelium, (b) percentage of macrophages with M2 phenotype (Anti-inflammatory), (c) M1/M2 macrophage ratio (Pro-inflammatory/Anti-inflammatory), (d) Mean fluorescence intensity (MFI) of the Gata3 transcription factor and (e) percentage of regulatory T lymphocytes (Treg). The data are represented with means and standard error. Statistically significant differences were established by applying one-way ANOVA followed by the Tukey test (p<0.05). CD, control diet; HFHSD, high-fat high-sucrose diet.

[0082] FIG. 5. Effect of administering the strain P. faecium (1×10.sup.7-8 cfu/day) to C57BL/6 obese mice (n=10/group) for 14 weeks on intraepithelial lymphocytes. (a) Percentage of induced intraepithelial lymphocytes (induced IELs) and (b) natural and induced intraepithelial lymphocyte ratio (nat/ind IEL). The data are represented with means and standard error. Statistically significant differences were established by applying one-way ANOVA followed by the Tukey test (p<0.05). CD, control diet; HFHSD, high-fat high-sucrose diet.

EXAMPLES

Example 1. Isolation and Identification of the Bacterial Strain P. faecium G104 (P. faecium DSM 32890)

[0083] Different intestinal bacteria were isolated from faeces from healthy volunteers. 1.25 grams of faeces were used and diluted in 10 mM phosphate buffer with 0.05% cysteine (1:10 dilution) containing a NaCl concentration of 130 mM (PBS) and homogenised in a Lab-Blender Stomacher 400 (Seward Medical, London, 35 UK). Said dilution was inoculated in 37.5 mL of Intestinal Bacteria Medium (BM), the composition of which is based on the media recommended in previous publications (Gibson, G. R., et al., Appl. Environ. Microbiol., 54 (11): 2750-5, 1988; Lesmes, U et al., J. Agric. Food Chem., 56: 5415-5421, 2008), with some modifications designed by the inventors: [0084] Main ingredients: distilled water (1,600 mL), peptone water (4 g), NaHCO.sub.3 (4 g), CaCl.sub.2 (0.02 g), pectin (4 g), xylan (4 g), wheat bran extract (4 g), arabinogalactans (2 g), gum arabic (2 g), starch (10 g), casein (6 g), inulin (2 g) and NaCl (0.2 g). Autoclaved at 121° C. for 60 minutes and left to cool overnight. [0085] Mucin solution: Mucin (8 g) and distilled water (200 mL). Autoclaved 20 minutes. [0086] Salts and vitamins: Distilled water (100 mL), K.sub.2HPO.sub.4 (0.08 g), KH.sub.2PO.sub.4 (0.08 g), MgSO.sub.4 (0.02 g), hemin (0.01 g) and menadione (0.002 g) [0087] Cysteine solution: L-cysteine-HCl (1 g), distilled water (100 mL)

[0088] The mixture of salts and vitamins and the cysteine solution were combined and 6M KOH was added until the final solution turned translucent brown and was sterilised by filtration. The final BM was obtained by mixing the main ingredients, the mucin solution, salts and vitamins and the cysteine solution, making up a volume of 2 L under sterile conditions.

[0089] The 50 mL of faeces diluted in BM medium were fermented for 24 hours in an anaerobic chamber (Whitley DG250 Workstation, Don Whitley Scientific) under stirring and keeping the pH between 6.9-7.0. The IBM medium fermented for 24 hours was filtered (pore size 0.22 μm) and used as a supplement to Fastidious Anaerobe Agar (FAA) medium agar plates with 0.5% defibrinated blood, in which serial dilutions of the fermented faeces were inoculated (0.1 mL of inoculum of each serial dilution in each plate). This supplement of the fermented IBM medium contains substrates produced by gut microbiota, being a medium enriched with nutrients present in the intestinal ecosystem which allows for a better recovery of autochthonous bacteria under laboratory conditions. The inoculated plates were incubated 72 hours at 37° C. in an anaerobic chamber.

[0090] Among the colonies that grew after 72 hours on the plate, Phascolarctobacterium faecium DSM 32890 was isolated. It was identified by sequencing the 16S rRNA gene (1.26 Kb) using the primers 27f (5′-AGAGTTTGATCCTGGCTCAG-3′ (SEQ ID NO: 1)) and 1401r (5′-CGGTGTGTACAAGACCC-3′ (SEQ ID NO: 2)). The reactions after DNA amplification were purified with the Illustra GFX PCR DNA and Gel Band Purification Kits (GE Healthcare) and sequenced by Sanger technology in an ABI 3730XL sequencer (Stabvida-Caparica-Portugal). By comparing the sequences obtained with the NCBI database and the BLASTn algorithm, the identification of the isolated strain (G104) with the species Phascolarctobacterium faecium strain ACM 3679 (partial sequence, 16S ribosomal RNA) was obtained with 99% percent identity. The new strain Phascolarctobacterium faecium G104 was deposited in the German Collection of Cell Cultures, corresponding to number DSM 32890.

[0091] The 16S sequence used for identifying with the BLASTn algorithm and using the oligos 27F and 1401r for sequencing was the following:

TABLE-US-00001 >16S G104 partial sequence (SEQ ID NO: 3) TCCGACTTCACGCAGGCGGGTTGCAGCCTGCGATCCGAACTGAGATCGGG TTTCTGGGGTTTGCTCTGCCTCGCGGCTTCGCTTCCCTCTGTTTCCGACC ATTGTAGTACGTGTGTAGCCCAAGACATAAGGGGCATGATGACTTGACGT CATCCCCGCCTTCCTCCAGGTTGTCCCTGGCAGTCTCCCATGAGTCCCCA ACTTTACTTGCTGGTAACATAGGATAGGGGTTGCGCTCGTTGCGGGACTT AACCCAACATCTCACGACACGAGCTGACGACAGCCATGCACCACCTGTTT TCTTGTCCCCGAAGGGAAATCTCTATCTCTAGAGCGTTCAATCAATGTCA AGCCTTGGTAAGGTTCTTCGCGTTGCGTCGAATTAAACCACATACTCCAC CGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGCCGT ACTCCCCAGGCGGGGTACTTATTGCGTTAACTCCGGCACAGAAGGGGTCG ATACCTCCTACACCTAGTACCCATCGTTTACGGCCAGGACTACCGGGGTA TCTAATCCCGTTCGCTACCCTGGCTTTCGCATCTCAGCGTCAGACACAGT CCAGAAAGGCGCCTTCGCCACTGGTGTTCCTCCCAATATCTACGCATTTC ACCGCTACACTGGGAATTCCCCTTTCCTCTCCTGCACTCAAGCCTAACAG TTTCCAGCGCCATACGGGGTTGAGCCCCGCATTTTCACGCTCGACTTATT AAGCCGCCTACATGCTCTTTACGCCCAATAATTCCGGACAACGCTCGCCA CCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTCCTCGT TTACTACCGTCATTGCAATGCATTGTTCACACACTGCACGTTCGTCATAA ACAACAGAGCTTTACAGACCGAAATCCTTCATCACTCACGCGGCGTTGCT CCGTCAGACTTTCGTCCATTGCGGAAGATTCCCCACTGCTGCCTCCCGTA GGAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGTTCATCCTCTCAGAC CGGCTACTGATCATCGCCTTGGTAGTCCGTTACACTACCAACTAGCTAAT CAGACGCAGGCCCATCCTTTAGCGATAGCTTACTTGTAGAGGCCATCTTT CTTCATCCTGCCATGCGGCACGATGATCACATCCGGTATTAGCACTCCTT TCGGAATGTTGTCCCCGTCTAAAGGGCAGGTTGCCTACGCGTTACTCACC CGTTCGCCACTAAGAATTCTACCGAAATAA

[0092] The specific growth of this strain was optimised for future assays using the medium recommended by the DSMZ culture collection (Medium No. 104b (PY+8 g/l succinate)).

[0093] For 1 L of medium, 5.0 g trypticase peptone, 5.0 g meat extract, 10.0 g yeast extract, 2.00 g K.sub.2HPO.sub.4, 1.00 mL Tween 80, 40.0 mL salt solution (see below), 1 mg resazurin solution, 0.5 g L-Cysteine-HCl×H.sub.2O, 8.0 g sodium succinate, 950.0 mL distilled water, 10.00 mL hemin solution (see below) and 0.20 mL vitamin K1 solution (see below) were mixed. [0094] Salt solution: 0.25 g CaCl.sub.2×2 H.sub.2O, 0.50 g MgSO.sub.4×7 H.sub.2O, 1.00 g K.sub.2HPO.sub.4, 1.00 g KH.sub.2PO.sub.4, 10.00 g NaHCO.sub.3, 2.00 g NaCl and 1000.00 mL distilled water. [0095] Hemin solution: Dissolve 50 mg of hemin in 1 mL of 1 N NaOH; bring it to 100 mL with distilled water. Keep refrigerated. [0096] Vitamin K1 solution: Dissolve 0.1 mL of vitamin K1 in 20 mL of 95% ethanol and sterilise by filtration. Store refrigerated and protected from light.

[0097] The ingredients were dissolved (except for cysteine, hemin and vitamin K1) and autoclaved for 20 minutes at 121° C. It was left to cool and cysteine, vitamin K1 and hemin were added later. The pH was adjusted to 7.2 and the medium was introduced into an anaerobic chamber to guarantee the anoxic state thereof prior to inoculation of P. faecium G104.

Example 2. Selection of P. faecium Based on its Capacity to In Vitro Modulate Inflammation

[0098] In vitro assays were conducted to comparatively evaluate the immunomodulatory properties of bacteria of human intestinal origin and select the strain capable of inducing the greatest anti-inflammatory response in classical monocytes and therefore with potential therapeutic interest in the treatment of obesity-associated inflammation. To this end, cell suspensions of different bacterial strains were used as a stimulus in cultures of peripheral blood mononuclear cells (PBMCs) and the number of classical monocytes and the levels of the anti-inflammatory cytokine IL-4 with respect to the pro-inflammatory cytokine IFNγ were measured by flow cytometry.

Cultivation and Stimulation of PBMCs.

[0099] From whole blood of healthy volunteers, Peripheral Blood Mononuclear Cells (PBMCs) were isolated using a Ficoll gradient (Ficoll Paque-Plus 17-1440-02, Bioscience). After treating them with a solution to lyse erythrocytes (Lysis Buffer for Red Blood Cells, RBC, Miltenyi Biotec., Spain), they were resuspended in RPM 1640 medium (Gibco, Barcelona, Spain) supplemented with 10% foetal bovine serum (Gibco, Barcelona, Spain), streptomycin (100 μg/mL, Sigma), penicillin (100 U/mL, Sigma) and L-glutamine (Sigma). To perform the experiments, the PBMCs were incubated at a concentration of 106 per mL in 24-well flat-bottom polystyrene plates (Corning, Madrid, Spain) at 37° C., at 5% CO.sub.2. Suspensions of live bacteria were used as a stimulus at a concentration of 10.sup.7 cfu/mL. Purified lipopolysaccharide (LPS) from Salmonella enterica serotype Typhimurium (Sigma Chemical Co, Madrid, Spain) was used as a positive control at a concentration of 1 μg/mL and untreated PBMC samples were used as negative control. The stimulation time was 24 hours at 37° C., at 5% CO.sub.2. After this time elapsed, the cells were collected and centrifuged, separating the cell pellet from the supernatant. Each type of stimulus was assayed in triplicate in 3 independent experiments. The culture supernatants were fractionated and stored in aliquots at −80° C.

Characterisation of the Immunomodulatory Properties of Bacterial Strains on PBMCs by Flow Cytometry.

[0100] The stimulated PBMCs were analysed by flow cytometry in order to determine the levels of classical pro-inflammatory monocytes, using the markers CD14 and CD16. Furthermore, pro-inflammatory cytokine IFNγ levels and anti-inflammatory cytokine IL-4 levels in monocytes were evaluated. To this end, the cells were permeabilised and fixed (Fixation/Permeabilization Solution Kit, BD-Bioscience) and resuspended with the FACS solution (PBS1×+BSA 0.2%). Marker levels were measured using BD LSRFortessa.

[0101] The comparative evaluation of the different bacterial strains made it possible to conclude that the strain of the invention Phascolarctobacterium faecium DSM 32890 was the one that induced the most significant immunomodulatory effects, showing a higher production of the anti-inflammatory cytokine IL-4 with respect to the pro-inflammatory IFNγ (higher IL-4/IFNγ) and a reduction of classical monocytes (CD14.sup.++ CD16.sup.−) with respect to untreated and LPS-treated cells, which is an inducer of obesity-associated inflammation and its complications (Table 1).

TABLE-US-00002 TABLE 1 In vitro characterisation of the immunomodulatory properties of human intestinal bacteria on PBMCs. Bacterial strains Classical Monocytes IL-4/IFNy Untreated 0.71 (0.02)b 1.74 (0.24)d LPS 1.00 (0.05)a 0.80 (0.05)e Alistipes indistinctus 0.69 (0.03)b 3.73 (0.26)b Phascolarctobacterium 0.48 (0.04)c 4.79 (0.08)a faecium Bacteroides dorei 0.70 (0.06)b 3.55 (0.20)b Eubacterium 0.75 (0.04)b 1.61 (0.10)d cylindroides Eubacterium limosum 0.93 (0.03)a 1.39 (0.07)d

[0102] The results are expressed as the mean and standard error thereof of the relative levels of classical monocytes and the IL-4/IFNγ ratio measured by flow cytometry. Significant differences (P<0.05) between groups were established by one-way ANOVA followed by the Tukey post-hoc test. Different letters indicate significant differences between experimental groups (p<0.05).

Example 3. Effects of P. faecium in an Animal Model of Obesity

Development of the Animal Model of Obesity and Sampling.

[0103] Adult male C57BL/6 mice (6-8 weeks, Charles River, Les Oncins, France), kept under controlled temperature (23° C.), relative humidity (40-50%) and 12-hour light/dark cycle conditions, were fed a high-fat (45% Kcal) high-sugar (sucrose) (17% Kcal) high-calorie diet (HFHSD; D12451, Research diet, Brogaarden, Denmark) or a control diet (CD, 10% Kcal from fat, without sucrose; D12450K, Research diet, Brogaarden, Denmark) for 14 weeks. Daily, the mice fed the HFHSD diet received an oral dose of the bacterial strain object of the invention [(1×10.sup.7-1×10.sup.−8) colony-forming units (CFU)] dissolved in 10% skim milk. The carrier (10% skim milk) was administered in the same way to both the control group with the obese phenotype (HFHSD) and the control group with the lean phenotype (CD) (n=10 mice per group). After 14 weeks, the mice were slaughtered by cervical dislocation in order to obtain samples, including blood, intestine, liver, brain, inguinal and epididymal white adipose tissue, brown adipose tissue, faecal content and faeces.

Characterisation of the Metabolic Phenotype

[0104] Body weight was monitored weekly. Fasting basal blood glucose (week 8 and 10) was determined from blood from the saphenous vein using glucose test strips (Contour XT Bayer, Barcelona, Spain) as well as oral glucose tolerance using an oral glucose test (OGTT, week 10) in which glycaemia was measured at 15, 30, 60 and 120 minutes after having administered an oral glucose overload (2 g/Kg) to mice subjected to 4 hours of fasting.

[0105] The bacterium object of intervention reduced weight gain in the diet-induced obesity model (FIGS. 1a and 1b), as well as adiposity in obese mice after 14 weeks of treatment (FIG. 3c). Furthermore, this bacterium improved both basal blood glucose (FIG. 2a) and oral glucose tolerance (FIG. 2b).

Effects on Intake

[0106] The intake recorded weekly in the different cages (5 animals per cage) allowed us to estimate each animal's food intake in kilocalories per day (FIG. 3a). The differences observed were confirmed by evaluating individual intake for 24 hours in week 12 of the experiment. The observed result was a reduction in the amount of kilocalories consumed by the group of animals to which the bacterium had been administered, as compared to the obese group (FIG. 3b). The regulation of appetite appears to be one of the mechanisms by which the bacterium under study reduces weight and body fat.

Effects on Inflammation

[0107] The effects of P. faecium on intestinal inflammation were analysed by flow cytometry. For this, the intestine was subjected to digestion stages together with a mechanical treatment, which enabled the epithelium to be separated from the lamina propria. The clean, cut tissue was incubated under stirring for 30 min at 37° C. with the first pre-digestion solution (5 mM EDTA, 1 mM DTT, 100 μg/mL streptomycin and 100 U/mL penicillin in HBSS [Hank's Balanced Salt Solution]). This process was repeated twice, filtering the tissue with 100 μm filters, thus obtaining the cells of the intestinal epithelium. To obtain the cells of the lamina propria, the remaining tissue was treated with the digestion solution (0.5 mg/mL Collagenase D, 50 U/mL DNase I, 3 mg/mL Dispase II, 100 μg/mL streptomycin and 100 U/mL penicillin in HBSS) under stirring for 30 min at 37° C. The process was repeated twice and filtered using 70 μm filters.

[0108] The cell suspensions obtained were treated with antibodies from different extracellular and intracellular markers. Specifically, in the epithelium, type 1 innate lymphoid cells were determined (using the lineage markers, T-bet and IFNγ). In the lamina propria, the population of M1 macrophages (F4/80, CD80 and iNOS), M2 macrophages (F4/80, CD80 and iNOS) and Treg (CD3, CD4, CD8, CD25 and Foxp3) was determined. The cells were diluted in FACS solution (1×PBS with 0.2% BSA) and analysed on a BD LSRFortessa flow cytometer (Becton Dickinson, NJ, USA).

[0109] In particular, the levels of innate lymphoid cells (LC) in the epithelium were analysed. These cells are responsible for protecting epithelial barriers against pathogens and maintaining tissue homeostasis. Nevertheless, group 1 ILCs (ILC1) can promote inflammation in tissue through the production of IFNγ and they are increased in obese mice. The evaluated bacterium was able to reduce the proportion of ILC1, thus reducing the inflammation triggered by these cells (FIG. 4a).

[0110] The bacterial strain also reduced macrophage polarisation towards an M1 (pro-inflammatory) phenotype that can be induced by an increase in IFNγ production. On the contrary, it increased levels of M2, inducing an anti-inflammatory response (FIG. 4b) by normalising the M1/M2 ratio (FIG. 4c).

[0111] Moreover, P. faecium attenuated the effects of obesity by inducing a Th2/Treg-type response (FIGS. 4d and 4e). This response was evaluated by measuring the levels of Gata3 as a transcription factor mediating a Th2 response and the levels of regulatory T cells with the markers CD25 and FoxP3. The increase in Gata3 could also indicate an increase in the proportion of ILC2 cells and development of ILC3 cells. ILC2 cells are characterised by producing Th2-type cytokines, with an anti-inflammatory effect in the context of obesity. The Gata3 transcription factor has also been described in a subset of intestinal mucosa-associated ILC3 cells that could contribute to the protection thereof.

[0112] Lastly, P. faecium was able to reverse the increase in the proportion of intraepithelial lymphocytes (IEL) induced by the high-calorie diet and normalise the natural/induced IEL ratio (FIG. 5), which would also contribute to re-establishing immune homeostasis and the intestinal barrier, preventing systemic inflammation.