WO2016049827A1

WO2016049827A1 - Use of bacteroides in prevention and treatment for coronary artery disease

Info

Publication number: WO2016049827A1
Application number: PCT/CN2014/087850
Authority: WO
Inventors: Qiang FENG; Zhuye JIE; Chuan Liu; Liang Xiao; Huihua XIA; Jun Wang
Original assignee: BGI Shenzhen Co Ltd
Current assignee: BGI Shenzhen Co Ltd
Priority date: 2014-09-30
Filing date: 2014-09-30
Publication date: 2016-04-07
Anticipated expiration: 2017-03-30
Also published as: CN107208037A

Abstract

The use of Bacteroides bacterial strains in the preparation of a formulation for the treatment and/or prevention of coronary artery disease, or for decreasing the level of blood glucose or lipids, or for ameliorating coronary atherosclerotic lesions, in a subject. A formulation (such as a pharmaceutical composition, a nutraceutical composition or a food composition) comprising Bacteroides bacteria. A method for the treatment and/or prevention of coronary artery disease, or for decreasing the level of blood glucose or lipids, or for ameliorating coronary atherosclerotic lesions, in a subject, comprising the step of administrating an effective amount of Bacteroides bacterial strains to a subject in need thereof.

Description

USE OF BACTEROIDES IN THE PREVENTION AND TREATMENT FOR CORONARY ARTERY DISEASE

CROSS-REFERENCE TO RELATED APPLICATION

None

FIELD

The invention relates to microbiology. Specifically, the invention relates to use of Bacteroides bacterial strains in the preparation of a formulation for the treatment and/or prevention of coronary artery disease, or for decreasing the level of blood glucose or lipids, or for ameliorating coronary atherosclerotic lesions, in a subject. The invention also relates to a formulation (such as a pharmaceutical composition, a nutraceutical composition or a food composition) comprising Bacteroides bacteria. The invention also relates to a method for the treatment and/or prevention of coronary artery disease, or for decreasing the level of blood glucose or lipids, or for ameliorating coronary atherosclerotic lesions, in a subject, comprising the step of administrating an effective amount of Bacteroides bacterial strains to a subject in need thereof.

BACKGROUND

Coronary artery disease (CAD) refers to any abnormal condition of the coronary arteries that interferes with the delivery of an adequate supply of blood to the cardiac (i.e. , heart) muscle or any portion thereof. Typically, CAD is caused by the accumulation of plaque on the arterial walls (i.e. , atherosclerosis) , particularly in the large and medium-sized arteries serving the heart. These conditions have similar causes, mechanisms, and treatments. CAD represents the leading cause of death and morbidity worldwide.

Current knowledge indicates that the genetic and environmental factors and their interactions collaboratively induce complex phenotypes and many diseases. Coronary artery disease (CAD) , as one of the most influential complex diseases, has been increasingly investigated by GWAS (Genome-wide association study) in recent years and it was revealed that 10.6％of the inherent cause was represented by 46 common variations (Ehret, G.B. et al. , Genetic variants in novel pathways influence blood pressure and cardiovascular disease risk. Nature 478, 103-109, incorporated herein by reference) . However, our knowledge on the effect of environmental factors like gut microbes as well as the contribution of genes and microbes to the disease still need further.

Our “forgotten organ” ， gut microbiota, plays a crucial role on our health in many aspects, such as intaking energy from food, producing important metabolites, promoting the development and maturity of immune system, and protecting the host from pathogen infection and so on. Recent studies suggested that the flora dysbiosis, chronic inflammatory and metabolic abnormity exist in the intestine of some metabolic diseases like diabetes and obesity. The characteristics for coronary artery disease are inflammation, oxidation and lipid metabolism, which might potentially correlate with the gut microbes and their metabolites. A recent research indicates that gut microbes could metabolize the red meat ingredients (such as L-carnitine, phosphatidyl-choline, cholesterol) into TMA, which would be further oxidized into TMAO in the liver to arise the oxidization reaction in blood vessel to lead inflammatory and lipid deposition, ultimately resulting in atherosclerosis and coronary heart disease. Meanwhile, as compared with healthy subjects, gut microbiota in the patients with symptomatic atherosclerosis exhibits obvious abnormality (Koeth, R. A. et al. Intestinal microbiota metabolism of L-carnitine, a nutrient in red meat, promotes atherosclerosis. Nature medicine 19, 576-585, incorporated herein by reference) . These studies suggested that the dysbiosis of gut microbes may significantly influenced the pathogenesis of coronary artery disease by inducing the human metabolic abnormality. However, the roles of gut flora dysbiosis in pathogenesis of coronary artery disease induced by atherosclerosis and its impact on metabolic system are still puzzling.

SUMMARY

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

The present invention is at least partially based on the following findings by the inventors.

Assessment and characterization of gut microbiota has become a major research area in human disease, including coronary artery disease (CAD) . To carry out analysis on gut microbial content in CAD 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 samples from 165 individuals.

The inventors identified 2 Bacteroides probiotic bacteria. Then the inventors validated the effect of the Bacteroides probiotic bacteria on CAD in an animal experiment, respectively. Results from the animal experiment demonstrated the ability of Bacteroides uniformis and Bacteroides vulgatus to prevent and treat CAD effectively.

Therefore, in one aspect, the present invention provides use of Bacteroides uniformis and/or Bacteroides vulgatus in the preparation of a formulation for the treatment and/or prevention of coronary artery disease, or for decreasing the level of blood glucose or lipids, or for ameliorating coronary atherosclerotic lesions, in a subject.

In a second aspect, the present invention provides a formulation for the treatment and/or prevention of coronary artery disease, or for decreasing the level of blood glucose or lipids, or for ameliorating coronary atherosclerotic lesions, in a subject, comprising an effective amount of Bacteroides uniformis and/or Bacteroides vulgatus.

In a third aspect, the present invention provides a method for the treatment and/or prevention of coronary artery disease, or for decreasing the level of blood glucose or lipids, or for ameliorating coronary atherosclerotic lesions, in a subject, comprising the step of administrating an effective amount of Bacteroides uniformis and/or Bacteroides vulgatus to a subject in need thereof.

In a fourth aspect, the present invention provides Bacteroides uniformis and/or Bacteroides vulgatus, for use in the method for the treatment and/or prevention of coronary artery disease, or for decreasing the level of blood glucose or lipids, or for ameliorating coronary atherosclerotic lesions, in a subject.

BRIEF DISCRIPTION OF DRAWINGS

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

Fig. 1: The 65 most discriminant MLG species in the Random Forest model using 126 MLG markers. The bar length indicated the importance of variable (MLG species) .

Fig. 2: The effects of administration of Bacteroides strain (Bacteroides uniformis ATCC 8492, Bacteroides uniformis CECT 7771, Bacteroides uniformis 0061, Bacteroides vulgatus ATCC 8482, Bacteroides vulgatus PC510 or Bacteroides vulgatus mpk) on OGTT curve at cholesterol-rich HF diet condition. Fig. 2A: Results obtained before administration of Bacteroides strain (on the 16th week) ； Fig. 2B: Results obtained after administration of Bacteroides strain (on the 24th week) . On the 16th week, no significant differences were observed at the level of serum glucose in all HF-fed groups (Fig. 2A) . However, after 8 weeks of gavage treatment (on the 24th week) , as compared with the AS group, a significant decrease in AUC (area under curve) of blood glucose (mmol/l) in Bacteroides uniformis groups and in Bacteroides vulgatus groups was observed (Fig. 2B) .

Fig. 3: Quantification of the coronary atherosclerotic lesion area of mice (n ＝ 8/group) after administration of Bacteroides strain (Bacteroides uniformis ATCC 8492, Bacteroides uniformis CECT 7771, Bacteroides uniformis 0061, Bacteroides vulgatus ATCC 8482, Bacteroides vulgatus PC510 or Bacteroides vulgatus mpk) . Values are shown as the mean ± SEM. Difference between values from groups marked with the same letter is not statistically significant, according to analysis of variance followed by Tukey post hoc test (p> 0.05) . As shown in Fig. 3, the atherosclerotic lesion analysis demonstrated that there was no significant difference between the mice administered with different Bacteroides strains for 8 weeks. By contrast, as compared with AS group, the mean coronary atherosclerotic lesion area of the 6 groups administered with Bacteroides strains was significant reduced.

Fig. 4: The histologic features (Oil red-O stained) of atherosclerotic lesions in the aortic root. A:NC group, B-D: Bacteroides uniformis groups (Bacteroides uniformis ATCC 8492, Bacteroides uniformis CECT 7771, and Bacteroides uniformis 0061, respectively) ； E-G Bacteroides vulgatus groups (Bacteroides vulgatus ATCC 8482, Bacteroides vulgatus PC510, and Bacteroides vulgatus mpk, respectively) ； H: AS group. The results showed that as compared with healthy control group (Fig. 4A, NC group) , mice in AS group developed lipid-rich atherosclerotic lesions in the aortic root after fed with cholesterol-rich HF diet and placebo on the 24th week (Fig. 4H, AS group) ； however, as compared with AS group, the mice administered with Bacteroides strains for 8 weeks displayed substantially decrease of foam cells and lipid deposition in the subintimal area (Fig. 4B～4G) .

Fig. 5: Characterization of atherosclerotic lesion composition by oil red O staining for neutral lipids. Values are shown as the mean ± SEM. Difference between values from groups marked with the same letter is not statistically significant, according to analysis of variance followed by Tukey post hoc test (p> 0.05) . The results showed a significant decrease in lipid accumulation in Bacteroides-treated mice, as compared with AS group, suggesting that the Bacteroides treatment improved the efflux of cholesterol from the lesional macrophages and inhibited the progress of atherosclerosis.

DETAILED DESCRIPTION

Terms used herein have meanings as commonly understood by a person of ordinary skill in the fields to which the present invention is relevant. However, in order to better understand the invention, the definitions and explanations of the relevant terms are provided as follows.

According to the invention, the term “Coronary artery disease (CAD) ” is also known as atherosclerotic heart disease, atherosclerotic cardiovascular disease, coronary heart disease or ischemic heart disease (IHD) , and is the most common type of heart disease and cause of heart attacks. The disease is caused by plaque building up along the inner walls of the arteries of the heart, which narrows the arteries and reduces blood flow to the heart.

According to the invention, the terms “prevent” , “preventing” and “prevention” refer to preventing, suppressing, or delaying the occurrence of a disease (such as CAD) .

According to the invention, the terms “treat” , “treating” and “treatment” refer to treating or curing a disease (such as CAD) , delaying the onset of a symptom of a disease (such as CAD) , and/or retarding the progress of a disease (such as CAD) .

According to the invention, the term “an effective amount” refers to an amount that can effectively achieve the intended purpose. For example, a prophylactically effective amount may be an amount effective or sufficient for preventing, suppressing, or delaying the occurrence of a disease (such as CAD) ； a therapeutically effective amount may be an amount effective or sufficient for treating or curing a disease (such as CAD) , delaying the onset of a symptom of a disease (such as CAD) , and/or retarding the progress of a disease (such as CAD) . Such an effective amount can be readily determined by a person skilled in the art or a physician, and may be in relevant to the intended purpose (preventing or treating) , the general health, age, sex, body weight of a subject, the severity of the disease to be treated, the accompanied disease, the routine of administration and so on. The determination of such an effective amount is well within the ability of a person skilled in the art.

Terms such as “a” ， “an” and “the” are not only intended to refer to a singular entity, but also 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 not intended to delimit the invention, except as outlined in the claims.

In one aspect, the present invention provides use of Bacteroides uniformis and/or Bacteroides vulgatus in the preparation of a formulation for the treatment and/or prevention of coronary artery disease, or for decreasing the level of blood glucose or lipids, or for ameliorating coronary atherosclerotic lesions, in a subject.

In a preferable embodiment, the formulation may comprise any strain from Bacteroides uniformis or any combination thereof. For example, the formulation may comprise one or more strains of Bacteroides uniformis, such as at least 2, 3, 4, 5, or more strains. In a preferable embodiment, Bacteroides uniformis is selected from the group consisting of Bacteroides uniformis ATCC 8492, Bacteroides uniformis CECT 7771, Bacteroides uniformis 0061, and any combination thereof.

In a preferable embodiment, the formulation may comprise any strain from Bacteroides vulgatus or any combination thereof. For example, the formulation may comprise one or more strains of Bacteroides vulgatus, such as at least 2, 3, 4, 5, or more strains. In a preferable embodiment, Bacteroides vulgatus is selected from the group consisting of Bacteroides vulgatus ATCC 8482, Bacteroides vulgatus PC510, Bacteroides vulgatus mpk, and any combination thereof.

In a preferable embodiment, Bacteroides uniformis and Bacteroides vulgatus as described above may be used in combination. Therefore, the formulation may comprise one or more strains from Bacteroides uniformis as well as one or more strains from Bacteroides vulgatus.

In a preferable embodiment, the formulation is a pharmaceutical composition. Such a pharmaceutical composition may comprise a therapeutically or prophylactically effective amount of Bacteroides uniformis and/or Bacteroides vulgatus. In a preferable embodiment, the pharmaceutical composition may be in any form known in the art of medicine. For example, the pharmaceutical composition may be in the form of a tablet, a pill, a suspension, an emulsion, a solution, a gel, a capsule, a powder, or a granule.

In a preferable embodiment, the formulation is a nutraceutical composition or a food composition. Such a composition may be in the form of solid, semi-solid, or liquid. In a preferable embodiment, the formulation is a dairy product, such as milk, milk powder, or yogurt.

In a preferable embodiment, the formulation further comprises an additional agent for treatment and/or prevention of coronary artery disease, or decreasing the level of blood glucose or lipids, or ameliorating coronary atherosclerotic lesions. Such an additional agent may be selected from the group consisting of cholesterol lowering medications, beta-blockers, nitroglycerin, calcium antagonists, statins, nitroglycerin, ACE inhibitors, calcium channel blockers, aspirin, and any combination thereof.

In addition, in case that the formulation comprises Bacteroides uniformis, the additional agent may comprise any strain (s) of Bacteroides vulgatus, such as Bacteroides vulgatus ATCC 8482, Bacteroides vulgatus PC510, Bacteroides vulgatus mpk, or any combination thereof. Similarly, in case that the formulation comprises Bacteroides vulgatus, the additional agent may comprise any strain (s) of Bacteroides uniformis, such as Bacteroides uniformis ATCC 8492, Bacteroides uniformis CECT 7771, Bacteroides uniformis 0061, or any combination thereof.

In a preferable embodiment, the formulation comprises Bacteroides uniformis and/or Bacteroides vulgatus at a unit dosage, for example, at a concentration of at least 1x10⁶ cfu/g, such as at least 1x10⁷ cfu/g, at least 1x10⁸ cfu/g, at least 1x10⁹ cfu/g, at least 1x10¹⁰ cfu/g, at least 1x10¹¹ cfu/g, or at least 1x10¹² cfu/g. In case that the formulation is in the form of liquid (for example, a solution, a suspension, an emulsion) , it may comprise Bacteroides uniformis and/or Bacteroides vulgatus at a concentration of at least 1x10⁶ cfu/ml, such as at least 1x10⁷ cfu/ml, at least 1x10⁸ cfu/ml, at least 1x10⁹ cfu/ml, at least 1x10¹⁰ cfu/ml, at least 1x10¹¹ cfu/ml, or at least 1x10¹² cfu/ml.

In a preferable embodiment, the formulation may be administrated with additional therapy. Such additional therapy may be any known for coronary artery disease, such as Coronary interventions as angioplasty, and Coronary artery bypass grafting.

In a preferable embodiment, the subject is a mammalian, such as a human.

In a preferable embodiment, any strain from Bacteroides uniformis or any combination thereof may be administrated to the subject. For example, one or more strains of Bacteroides uniformis, such as at least 2, 3, 4, 5, or more strains may be administrated to the subject. In a preferable embodiment, Bacteroides uniformis is selected from the group consisting of Bacteroides uniformis ATCC 8492, Bacteroides uniformis CECT 7771, Bacteroides uniformis 0061, and any combination thereof.

In a preferable embodiment, any strain from Bacteroides vulgatus or any combination thereof may be administrated to the subject. For example, one or more strains of Bacteroides vulgatus, such as at least 2, 3, 4, 5, or more strains may be administrated to the subject. In a preferable embodiment, Bacteroides vulgatus is selected from the group consisting of Bacteroides vulgatus ATCC 8482, Bacteroides vulgatus PC510, Bacteroides vulgatus mpk, and any combination thereof.

In a preferable embodiment, Bacteroides uniformis and Bacteroides vulgatus as described above may be used in combination. Therefore, one or more strains from Bacteroides uniformis as well as one or more strains from Bacteroides vulgatus may be administrated to the subject.

In a preferable embodiment, Bacteroides uniformis and/or Bacteroides vulgatus may be formulated and administrated as a pharmaceutical composition. Such a pharmaceutical composition may comprise a therapeutically or prophylactically effective amount of Bacteroides uniformis and/or Bacteroides vulgatus. In a preferable embodiment, the pharmaceutical composition may be in any form known in the art of medicine. For example, the pharmaceutical composition may be in the form of a tablet, a pill, a suspension, an emulsion, a solution, a gel, a capsule, a powder, or a granule.

In a preferable embodiment, Bacteroides uniformis and/or Bacteroides vulgatus may be formulated and administrated as a nutraceutical composition or a food composition. Such a composition may be in the form of solid, semi-solid, or liquid. In a preferable embodiment, such a composition is a dairy product, such as milk, milk powder, or yogurt.

In a preferable embodiment, the method further comprises administrating additional agent for treatment and/or prevention of coronary artery disease, or decreasing the level of blood glucose or lipids, or ameliorating coronary atherosclerotic lesions. Such an additional agent may be administrated before, accompanied with, or after the administration of Bacteroides uniformis and/or Bacteroides vulgatus.

In a preferable embodiment, such an additional agent may be selected from the group consisting of cholesterol lowering medications, beta-blockers, nitroglycerin, calcium antagonists, statins, nitroglycerin, ACE inhibitors, calcium channel blockers, aspirin, and any combination thereof.

In addition, in case that Bacteroides uniformis is administrated to the subject, the additional agent may comprise any strain (s) of Bacteroides vulgatus, such as Bacteroides vulgatus ATCC 8482, Bacteroides vulgatus PC510, Bacteroides vulgatus mpk, or any combination thereof. Similarly, in case that Bacteroides vulgatus is administrated to the subject, the additional agent may comprise any strain (s) of Bacteroides uniformis, such as Bacteroides uniformis ATCC 8492, Bacteroides uniformis CECT 7771, Bacteroides uniformis 0061, or any combination thereof.

In a preferable embodiment, Bacteroides uniformis and/or Bacteroides vulgatus may be administrated at an amount of at least 1x10⁵ cfu/kg, such as at least 1x10⁶ cfu/kg, at least 1x10⁷ cfu/kg, at least 1x10⁸ cfu/kg, at least 1x10⁹ cfu/kg, at least 1x10¹⁰ cfu/kg, at least 1x10¹¹ cfu/kg, or at least 1x10¹² cfu/kg of the body weight of the subject. In a preferable embodiment, Bacteroides uniformis and/or Bacteroides vulgatus may be administrated three times a day, twice a day, once a day, once two days, or once a week.

In a preferable embodiment, the method further comprises administration of additional therapy. Such additional therapy may be any known for coronary artery disease, such as Coronary interventions as angioplasty, and Coronary artery bypass grafting.

In a preferable embodiment, the subject is a mammalian, such as a human.

In a preferable embodiment, any strain from Bacteroides uniformis or any combination thereof may be used for the method. For example, one or more strains of Bacteroides uniformis, such as at least 2, 3, 4, 5, or more strains may be used for the method. In a preferable embodiment, Bacteroides uniformis is selected from the group consisting of Bacteroides uniformis ATCC 8492, Bacteroides uniformis CECT 7771, Bacteroides uniformis 0061, and any combination thereof.

In a preferable embodiment, any strain from Bacteroides vulgatus or any combination thereof may be used for the method. For example, one or more strains of Bacteroides vulgatus, such as at least 2, 3, 4, 5, or more strains may be used for the method. In a preferable embodiment, Bacteroides vulgatus is selected from the group consisting of Bacteroides vulgatus ATCC 8482, Bacteroides vulgatus PC510, Bacteroides vulgatus mpk, and any combination thereof.

In a preferable embodiment, Bacteroides uniformis and Bacteroides vulgatus as described above may be used in combination. Therefore, one or more strains from Bacteroides uniformis as well as one or more strains from Bacteroides vulgatus may be used for the method.

In a preferable embodiment, Bacteroides uniformis and/or Bacteroides vulgatus may be administrated in combination with additional agent for treatment and/or prevention of coronary artery disease, or decreasing the level of blood glucose or lipids, or ameliorating coronary atherosclerotic lesions. Such an additional agent may be administrated before, accompanied with, or after the administration of Bacteroides uniformis and/or Bacteroides vulgatus.

In a preferable embodiment, Bacteroides uniformis and/or Bacteroides vulgatus may be administrated in combination with additional therapy. Such additional therapy may be any known for coronary artery disease, such as Coronary interventions as angioplasty, and Coronary artery bypass grafting.

In a preferable embodiment, the subject is a mammalian, such as a human.

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. The agents as used were all commercially available. 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.

EMBODIMENT

Example 1: Identifying biomarkers relevant to risk of coronary artery disease

1.1: Sample collection

Fecal samples from 165 south Chinese subjects, including 88 patients with atherosclerotic cardiovascular disease (ACVD) and 77 control subjects (training set, Table 1) , were collected by Guangdong Provincial People's Hospital in 2011. ACVD patients were diagnosed and categorized according to pathological features. Subjects were asked to collect fresh fecal samples at hospital. Collected samples were put in sterile tubes and stored at-80 □C immediately until further analysis.

The complete ethical approval has been obtained, and all the patients gave written informed consent. The study was approved by the Institutional Review Board of Guangdong General Hospital.

Table 1: Baseline characteristics of ACVD patients and controls. The fourth column reports results from Wilcoxon rank-sum tests.

Parameter	Patients (n ＝ 88)	Controls (n ＝ 77)	P-value
Parameter	Patients (n ＝ 88)	Controls (n ＝ 77)	P-value	Age	61	59	0.294
Gender (M: F)	68: 19	41: 34	1.97E-05	Age	61	59	0.294
Gender (M: F)	68: 19	41: 34	1.97E-05	Body Mass Index (BMI)	24.54	23.58	0.193

NOTE: For the information of gender, one of the 88 patients’was unknown and two of the 77 controls’were unknown.

1.2: DNA extraction

Fecal samples were thawed on ice and DNA extraction was performed using the Qiagen QIAamp DNA Stool Mini Kit (Qiagen) according to the 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-iTTM dsDNA BR Assay Kit) and gel electrophoresis.

1.3: DNA library construction and sequencing

DNA library construction was performed following the manufacturer`s instruction (Illumina) . The inventors used the routine workflow to perform cluster generation, template hybridization, isothermal amplification, linearization, blocking and denaturation, and hybridization of the sequencing primers. The inventors constructed one paired-end (PE) library with insert size of 350 bp for each sample, followed by a high-throughput sequencing to obtain around 30 million PE reads with length of 2x100bp. High-quality reads were obtained by filtering low-quality reads with ambiguous'N'bases, adapter contamination and human DNA contamination from the Illumina raw reads, and trimming low-quality terminal bases of reads simultaneously.

The inventors totally output about 4.77 Gb of fecal micbiota sequencing data (high quality clean data) per sample (Table 2) for 165 samples (88 patients and 77 controls) on Illumina HiSeq 2000 platform.

Table 2: Summary of metagenomic data. The fourth column reports results from Wilcoxon rank-sum tests.

Parameter	Controls	Patients	P-value
Parameter	Controls	Patients	P-value	Average raw bases (G)	4.85	4.92	0.831
After removing low quality bases	4.76 (98.14％)	4.79 (97.36％)		Average raw bases (G)	4.85	4.92	0.831
After removing low quality bases	4.76 (98.14％)	4.79 (97.36％)		After removing human reads	4.73 (97.53％)	4.78 (97.15％)	0.874

1.4: Processing and analysis of metagenomic data

1.4.1: Gene catalogue construction

Gene catalogue construction. Employing the same parameters that were used to construct the type 2 diabetes gene catalogue (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 performed de novo assembly and gene prediction for the high quality reads of 165 samples using SOAPdenovo v1.06 (Li, R. et al. De novo assembly of human genomes with massively parallel short read sequencing. Genome Research 20, 265-272, doi: 10.1101/gr. 097261.109 (2009) , incorporated herein by reference) and GeneMark v2.7 (Zhu, W., Lomsadze, A. &Borodovsky, M. Ab initio gene identification in metagenomic sequences. Nucleic acids research 38, e132, doi: 10.1093/nar/gkq275 (2010) , incorporated herein by reference) , respectively. All predicted genes were aligned pairwise using BLAT, and genes in which over 90％of their length can be aligned to another one with more than 95％identity (no gaps allowed) , were removed as redundancies, resulting in a non-redundant gene catalogue comprising 4,537,046 genes (4.5 M gene catalogue) .

Taxonomic assignment of genes. Taxonomic assignment of the predicted genes was performed using an in-house pipeline which had described in the published T2D paper (Qin et al. 2012, supra) .

1.4.2: Construction of data profile

Gene profile. The 4,537,046 genes and their measures of relative abundance in 165 samples were used to establish the gene profile for the association study (The inventors use the same method as described in the published T2D paper (Qin et al. 2012, supra) to compute the relative gene abundance. ) .

IMG species and mOTU species profiles. Total fecal clean reads were aligned to the 4,653 reference genomes from IMG v400 (Markowitz, V. M. et al. IMG: the integrated microbial genomes database and comparative analysis system. Nucleic acids research 40, D115-D122 (2012) , incorporated herein by reference) and to the 79,268 sequences of mOTU reference (Sunagawa, S. et al. Metagenomic species profiling using universal phylogenetic marker genes. Nature methods 10, 1196-1199 (2013) , incorporated herein by reference) with default parameters, respectively. 1290 IMG species (species that were shared among at least 10 subjects) and 560 mOTU species were identified.

1.4.3: Analysis of factors influencing gut microbiota gene profile. The inventors used the permutational multivariate analysis of variance (PERMANOVA) to assess the effect of 25 different characteristics, including CAD status, HDLC, CHOL, Gender, FBG, hypertension, APOB, Age, CREA, LDLC, HbA1c, APOA, TP, diabetes, ALB, TRIG, BMI, WHR, Lpa, HBDH, CKMB, AST, CK, ProBNP_E_, and ALT, on gene profiles of 4.5M reference gene catalogue. 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 test. PERMANOA identified two significant factors associated with gut microbe (based on gene profiles) (q <0.05, Table 3) . The analysis indicated that CAD and HDLC status were the most significantly associated markers, supporting that the disease status was the major determinant influencing the composition of gut microbiota. Gender, age and some CAD clinical indices like CHOL, FGB, hypertension and APOB, were also significant factors.

Table 3: PERMANOVA based on euclidean distance analysis of gene profile. The analysis was conducted to test whether clinical parameters and ACVD status have significant impact on the gut microbiota with q-value <0.05.

1.4.4: Identification of ACVD associated markers

Identification of ACVD associated genes. To identify the association between the metagenomic profile and ACVD, a two-tailed Wilcoxon rank-sum test was conducted in 2.1M high occurrence gene (genes that were present in less than 10 samples across all 165 samples were removed) profiles. 438, 750 gene markers (20.48％of 2.1M genes) were obtained, which were enriched in either patients or controls with p-value < 0.01, FDR ＝ 2.23％.

Estimating the false discovery rate (FDR) . Instead of a sequential p-value rejection method, the inventors applied the “q-value” method proposed in a previous study (Storey, J. D. A direct approach to false discovery rates. Journal of the Royal Statistical Society 64, 479-498 (2002) , incorporated herein by reference) to estimate the FDR.

Receiver Operator Characteristic (ROC) analysis. The inventors applied the ROC analysis to assess the performance of the ACVD classification based on metagenomic markers. The inventors then used the “pROC” package in R to draw the ROC curve.

1.4.5: Construction of MLG species and identification of ACVD associated MLG species markers

126 MLG species based on the 438, 750 ACVD associated maker gene profile. The inventors used the 438, 750 gene markers to build the metagenomic linkage group (MLG) by the same method as described in the published T2D paper (Qin et al. 2012, supra) . All the 438, 750 genes were annotated by aligning these genes to the 4, 653 reference genomes in IMG v400. An MLG was assigned to a genome if more than 50％constitutive genes were annotated to that genome, otherwise it was termed as unclassified. Total 136 MLG genomes with > 550 genes were selected. The MLG genomes which belong to a same species were grouped to construct MLG species. Finally the inventors obtained 127 MLGs species. The inventors performed Wilcoxon rank-sum test to the 127 MLGs species with Benjamini-Hochberg adjustment, and 126 MLGs were selected as ACVD-associated MLGs with q < 0.05. 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 (Qin et al. 2012, supra) .

In total, the inventors built 136 metagenomic linkage groups (MLG with >550 genes) based on the distribution and the occurrence rate (Qin et al. 2012, supra) of 438,750 genes. 94.8％of the significant genes (P-value <0.01) were included into MLGs. 136 MLGs (each with >550 genes, >50％coverage and q<0.05) were annotated to NCBI database, and MLGs from the same species were grouped, resulting in 126 MLG species.

Identification of MLG species marker. To identify MLG species makers, the inventors used “randomForest 4.5-36” package in R vision 2.10 for the 126 ACVD associated MLG species. Firstly, the inventors sorted all the 126 MLG species based on the importance given by the “randomForest” method (Liaw, Andy &Wiener, Matthew. Classification and Regression by randomForest, R News (2002) , Vol. 2/3 p. 18, incorporated herein by reference) . MLG marker sets were constructed by creating incremental subsets of the top ranked MLG species, starting from 5 MLG species and ending at all 126 MLG species. For each MLG maker set, the inventors calculated the false predication ratio in our cohort of 165 persons. Finally, the MLG maker set (comprising 65 MLG species) with the lowest false prediction ratio were selected as MLG species makers (Fig. 1) , with false negative (FN) rate of 6.8％and false positive (FP) rate of 3.89％. Furthermore, the inventors drew the ROC curve using the OOB (out of bag) prediction probability of illness from randomForest model based on the selected MLG species markers, and the area under the ROC curve was calculated as 98.17％using R package “pROC” .

Among the 65 MLG species, the control enriched MLG species, Bacteroides uniformis (q ＝ 4.21E-11) and Bacteroides vulgatus (q ＝ 1.80E-09) , were known as SCFAs producing bacteria.

1.4.6: Identification of ACVD associated IMG species and mOTU species. IMG species and mOTU species markers were identified based on the IMG species and mOTU species profiles, The inventors identified the ACVD associated IMG species and mOTU species with q < 0.05 (Wilcoxon rank-sum test with Benjamini-Hochberg adjustment) . Subsequently, IMG species markers and mOTU species markers were selected using the randomForest approach as in MLG species markers selection.

65 IMG species with ROC of 98.52％and 15 mOTU species with ROC of 96.16％also clearly distinguished CAD patients from healthy subjects (q<0.05； see Tables 5 and 6) , according to Wilcoxon rank-sum test and random forest selection. Similarly to the 65 MLG species markers, in the 65 IMG species markers and 15 mOTU species markers, the inventors found that Bacteroides uniformis was significantly enriched in controls.

Example 2: Validation in animal experiments related to CAD

In order to validate the ability of the probiotic bacteria, Bacteroides uniformis and Bacteroides vulgatus, in preventing and treating CAD related diseases, the inventors performed animal experiments.

2.1: Method

To study the inhibition effect of Bacteroides uniformis and Bacteroides vulgatus on the aortic plaque area of murine atherosclerosis, mice were fed with high fat diets for 16 weeks to build the model of atherosclerosis, and then Bacteroides uniformis and Bacteroides vulgatus were given from the 16th week, and the effect on blood glucose and OGTT (oral glucose tolerance test) , serum lipids and atherosclerotic plaque area were observed.

2.1.1: Animals and treatments

64 male specific pathogen-free (SPF) C57BL/6J mice (10-week-old) , with a weight between 22.5～25.9g, were purchased from Laboratory Animal Center of Southern Medical University, China. Mice that used for diet-induced atherosclerosis study have ad libitum access to drinking water and chow diet for 2 weeks acclimatization. Then they were randomly divided into 2 parts: one part (8 mice) was fed with a normal chow diet (NC group, containing 10％kcal from fat, 3.85 total kcal g^-1 from Research Diets, Inc. , New Brunswick, NJ, USA) as healthy controls. The other part (56 mice) was fed with a high fat diet (containing 60％kcal from fat, 5.24 total kcal g^-1, from Research Diets, Inc. ) with 2％cholesterol for induction of atherosclerosis, and 16 weeks later, these 56 mice were randomly divided into 7 groups (8 mice per group) . One group was given the same cholesterol-rich HF diet as atherosclerosis control group (AS group) , receiving 200μl columbia blood medium (DSMZ Medium 693) as placebo. The other six groups (B1～B6 groups) also received the cholesterol-rich HF diet and were administrated with 200μl bacterial suspension containing one of the six candidate probiotic strains, namely Bacteroides uniformis ATCC 8492, Bacteroides uniformis CECT 7771, Bacteroides uniformis 0061, Bacteroides vulgatus ATCC 8482, Bacteroides vulgatus PC510 and Bacteroides vulgatus mpk in columbia blood medium by gavage at a dose of 10⁹-10¹⁰ cfu/ml (freshly prepared weekly) . The mice were fed for further 8 weeks.

Bacteroides uniformis ATCC 8492 and Bacteroides vulgatus ATCC 8482 were purchased from American Type Culture Collection (ATCC) .

Bacteroides uniformis CECT 7771 was purchased from Spanish Type Culture Collection.

Bacteroides uniformis 0061 was obtained from Virginia Polytechnic Institute and State University， Anaerobic Laboratory Culture Collection (Shoemaker, N. B. , &Salyers, A. A. (1988) . Tetracycline-dependent appearance of plasmid like forms in Bacteroides uniformis 0061 mediated by conjugal Bacteroides tetracycline resistance elements. Journal of bacteriology, 170 (4) , 1651-1657) .

Bacteroides vulgatus PC510 was obtained from CSIRO Preventative Health Flagship Research Program and Division of Livestock Industries (Páraic O Cuív, et al. Draft genome sequence of Bacteroides vulgatus PC510, a strain isolated from human feces. Journal of bacteriology, 2011, 193 (15) : 4025-6) .

Bacteroides vulgatus mpk was obtained from Institute of Medical Microbiology and Hygiene, University of Tubingen (Waidmann M, et al. Bacteroides vulgatus protects against Escherichia coli-induced colitis in gnotobiotic interleukin-2-deficient mice. Gastroenterology. 2003 Jul； 125 (1) : 162-77) .

The anaerobic culture technique was used to purify and incubate the strains. The composition of columbia blood medium for incubating was described in DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, German Collection of Microorganisms and Cell Cultures) Medium 693. For experimental purposes, strains were anaerobically propagated at 37 ℃ for 48 hours.

2.1.2: Oral glucose tolerance test (OGTT)

After 8 hours of food deprivation, glucose was administered orally to the mice at a dose of 2.0 g/kg body weight. Blood samples were taken from the tail before and 15, 30, 60, and 120 min after glucose administration, and blood glucose levels were measured with a blood glucose meter (Roche Diagnostics, Mannheim, Germany) . The blood glucose level before glucose administration represented fasting glucose level.

2.1.3: Collection of blood and aorta

At the end of the 24th week, after 12 hours of food deprivation, all blood was collected from the orbital plexus, and serum was isolated by centrifugation at 5000 rpm at 4 ℃ for 15 min and stored at -80℃ for subsequent biochemical testing. All animals were sacrificed by cervical dislocation. Plasma triglycerides (TG) and plasma total cholesterol (TC) were measured using kits coupling enzymatic reaction and spectrophotometric detection of reaction end Plasma products (Nanjing Jiancheng Bio-engineering Institute, China) . The aorta were collected and processed for cross-sectional analyses of plaque area in the aortic root.

2.1.4: Assessment of atherosclerotic plaques

After the collection of blood, heparin was injected into inferior caval vein, and the aorta were removed from the aortic root under the microscope, fixed in paraformaldehyde, then embedded in 10％phosphate buffered formalin, sectioned at 6μm. Light microscopy was performed to evaluate the atherosclerotic changes. Histological sections on the glass slides were scanned to create virtual slides for quantitative evaluation using iVision Software (BioVision Technologies, Exton, PA, USA) . For detection of neutral lipid, histological sections were stained with Oil Red-O stain. After capturing images of the sections, we determined the red area by computer-assisted color-gated measurement on the total section area (SigmaScanPro 5； SPSS Inc. , Chicago, IL, USA) .

2.2: Results

2.2.1: The effect on blood glucose and OGTT

On the 16th week, no significant differences were observed at the level of serum glucose in all HF-fed groups (Fig. 2A) . However, after 8 weeks of gavage treatment (on the 24th week) , as compared with the AS group, a significant decrease in AUC (area under curve) of blood glucose (mmol/l) in Bacteroides uniformis groups and in Bacteroides vulgatus groups was observed in oral glucose tolerance test (Fig. 2B) , as presented in Table 7.

Table 7: AUC value of OGTT curve of each group. Each value is shown as mean ± SEM. Difference between values from groups marked with the same letter is not statistically significant, according to analysis of variance followed by Tukey post hoc test. Among the groups, the same letter marked represents that values were not significantly different, as determined by the Tukey's Test (p> 0.05) .

2.2.2: The effect on serum lipids

The results of administration of Bacteroides uniformis ATCC 8492, CECT 7771, and 0061, and Bacteroides vulgatus ATCC 8482, PC510, and mpk on serum lipids were presented in Table 8. The cholesterol-rich HF diet with placebo gavage significantly increased the plasma triglycerides and plasma total cholesterol, as compared with the NC group. However, the levels of serum lipid were significantly decreased in Bacteroides uniformis and Bacteroides vulgatus gavage groups, as compared with the AS group.

Table 8: The effects of administration of Bacteroides strains on plasma triglycerides (TG) and plasma total cholesterol (TC) . Each value is shown as mean ± SEM. Difference between values from groups marked with the same letter is not statistically significant, according to analysis of variance followed by Tukey post hoc test. Among the groups, the same letter marked represents that values were not significantly different, as determined by the Tukey's Test (p> 0.05) .

2.2.3: Effect of treatment with Bacteroides strains on coronary atherosclerotic lesions in mice

Fig. 3 showed the results of quantification of the coronary atherosclerotic lesion area of mice (n ＝ 8/group) after administration of Bacteroides strain (Bacteroides uniformis ATCC 8492, Bacteroides uniformis CECT 7771, Bacteroides uniformis 0061, Bacteroides vulgatus ATCC 8482, Bacteroides vulgatus PC510 or Bacteroides vulgatus mpk) . Values are shown as the mean ± SEM. Difference between values from groups marked with the same letter is not statistically significant, according to analysis of variance followed by Tukey post hoc test (p> 0.05) .

As shown in Fig. 3, the atherosclerotic lesion analysis demonstrated that there was no significant difference between the mice administered with different Bacteroides strains for 8 weeks. By contrast, as compared with AS group, the mean coronary atherosclerotic lesion area of the 6 groups administered with Bacteroides strains was significant reduced (Fig. 3, Table 9) .

As shown in histological features of longitudinal sections of the aortic root, as compared with healthy control group (Fig. 4A, NC group) , mice in AS group developed lipid-rich atherosclerotic lesions in the aortic root after fed with cholesterol-rich HF diet and placebo on the 24th week (Fig. 4H, AS group) . In contrast, as compared with AS group, the mice administered with Bacteroides strains for 8 weeks displayed substantially decrease of foam cells and lipid deposition in the subintimal area (Fig. 4B～4G) .

The difference in lipid accumulation suggested the relieve effect of B. uniformis ATCC 8492, B. uniformis CECT 7771, B. uniformis 0061, B. vulgatus ATCC 8482, B. vulgatus PC510, and B. vulgatus mpk respectively on atherosclerosis development in cholesterol-rich HF diet induced mouse atherosclerosis models. Quantification of lipid accumulation in atherosclerotic lesions by oil red O staining demonstrated a significant decrease in lipid accumulation in Bacteroides-treated mice, as compared with AS group (Fig. 5, Table 9) . These results indicated that the Bacteroides treatment improved the efflux of cholesterol from the lesional macrophages and inhibited the progress of atherosclerosis.

Table 9: Lesion Area (％) and Oil Red O (％Lesion Area) in all groups

Taken together, the results from the present study demonstrated the ability of Bacteroides uniformis ATCC 8492, Bacteroides uniformis CECT 7771, Bacteroides uniformis 0061, Bacteroides vulgatus ATCC 8482, Bacteroides vulgatus PC510 and Bacteroides vulgatus mpk for ameliorating coronary artery disease and the related diseases, for decreasing the level of blood glucose or lipids, or for ameliorating coronary atherosclerotic lesions.

Although explanatory embodiments have been shown and described in detailed, it would be appreciated by those skilled in the art that the above embodiments are illustrative, and are not intended to limit the present disclosure in any way, and that changes, alternatives, and modifications can be made to the embodiments without departing from the spirit, principles and scope of the present disclosure.

Claims

Use of Bacteroides vulgatus in the preparation of a formulation for the treatment and/or prevention of coronary artery disease, or for decreasing the level of blood glucose or lipids, or for ameliorating coronary atherosclerotic lesions, in a subject.
Use according to claim 1, wherein Bacteroides vulgatus is selected from the group consisting of Bacteroides vulgatus ATCC 8482, Bacteroides vulgatus PC510, Bacteroides vulgatus mpk, and any combination thereof.
Use according to claim 1 or 2, wherein the formulation is a pharmaceutical composition, preferably comprising a therapeutically or prophylactically effective amount of Bacteroides vulgatus, and optionally, is in the form of a tablet, a pill, a suspension, a emulsion, a solution, a gel, a capsule, a powder, or a granule.
Use according to claim 1 or 2, wherein the formulation is a nutraceutical composition or a food composition, and optionally, is in the form of solid, semi-solid, or liquid, and preferably is a dairy product, such as milk, milk powder, or yogurt.
Use according to anyone of claims 1-4, wherein the formulation further comprises an additional agent for treatment and/or prevention of coronary artery disease, or decreasing the level of blood glucose or lipids, or ameliorating coronary atherosclerotic lesions.
Use according to claim 5, wherein the additional agent is selected from the group consisting of Bacteroides uniformis, cholesterol lowering medications, beta-blockers, nitroglycerin, calcium antagonists, statins, nitroglycerin, ACE inhibitors, calcium channel blockers, aspirin, and any combination thereof.
Use according to claim 6, wherein Bacteroides uniformis is selected from the group consisting of Bacteroides uniformis ATCC 8492, Bacteroides uniformis CECT 7771, Bacteroides uniformis 0061, and any combination thereof.
Use according to anyone of claims 1-7, wherein the formulation comprises Bacteroides vulgatus at a concentration of at least 1x10⁶ cfu/g, such as at least 1x10⁷ cfu/g, at least 1x10⁸ cfu/g, at least 1x10⁹ cfu/g, at least 1x10¹⁰ cfu/g, at least 1x10¹¹ cfu/g, or at least 1x10¹² cfu/g, or wherein the formulation is in the form of liquid and comprises Bacteroides vulgatus at a concentration of at least 1x10⁶ cfu/ml, such as at least 1x10⁷ cfu/ml, at least 1x10⁸ cfu/ml, at least 1x10⁹ cfu/ml, at least 1x10¹⁰ cfu/ml, at least 1x10¹¹ cfu/ml, or at least 1x10¹² cfu/ml.
Use according to anyone of claims 1-8, wherein the formulation is administrated with additional therapy, such as Coronary interventions as angioplasty, and Coronary artery bypass grafting.
Use according to anyone of claims 1-9, wherein the subject is a mammalian, such as a human.
A formulation for the treatment and/or prevention of coronary artery disease, or for decreasing the level of blood glucose or lipids, or for ameliorating coronary atherosclerotic lesions, in a subject, comprising an effective amount of Bacteroides vulgatus.
The formulation according to claim 11, wherein Bacteroides vulgatus is selected from the group consisting of Bacteroides vulgatus ATCC 8482, Bacteroides vulgatus PC510, Bacteroides vulgatus mpk, and any combination thereof.
The formulation according to claim 11 or 12, wherein the formulation is a pharmaceutical composition, preferably comprising a therapeutically or prophylactically effective amount of Bacteroides vulgatus, and optionally, is in the form of a tablet, a pill, a suspension, a emulsion, a solution, a gel, a capsule, a powder, or a granule.
The formulation according to claim 11 or 12, wherein the formulation is a nutraceutical composition or a food composition, and optionally, is in the form of solid, semi-solid, or liquid, and preferably is a dairy product, such as milk, milk powder, or yogurt.
The formulation according to anyone of claims 11-14, wherein the formulation further comprises an additional agent for treatment and/or prevention of coronary artery disease, or decreasing the level of blood glucose or lipids, or ameliorating coronary atherosclerotic lesions.
The formulation according to claim 15, wherein the additional agent is selected from the group consisting of Bacteroides uniformis, cholesterol lowering medications, beta-blockers, nitroglycerin, calcium antagonists, statins, nitroglycerin, ACE inhibitors, calcium channel blockers, aspirin, and any combination thereof.
The formulation according to claim 16, wherein Bacteroides uniformis is selected from the group consisting of Bacteroides uniformis ATCC 8492, Bacteroides uniformis CECT 7771, Bacteroides uniformis 0061, and any combination thereof.
The formulation according to anyone of claims 11-17, wherein the formulation comprises Bacteroides vulgatus at a concentration of at least 1x10⁶ cfu/g, such as at least 1x10⁷ cfu/g, at least 1x10⁸ cfu/g, at least 1x10⁹ cfu/g, at least 1x10¹⁰ cfu/g, at least 1x10¹¹ cfu/g, or at least 1x10¹² cfu/g, or wherein the formulation is in the form of liquid and comprises Bacteroides vulgatus at a concentration of at least 1x10⁶ cfu/ml, such as at least 1x10⁷ cfu/ml, at least 1x10⁸ cfu/ml, at least 1x10⁹ cfu/ml, at least 1x10¹⁰ cfu/ml, at least 1x10¹¹ cfu/ml, or at least 1x10¹² cfu/ml.
The formulation according to anyone of claims 11-18, wherein the formulation is administrated with additional therapy, such as Coronary interventions as angioplasty, and Coronary artery bypass grafting.
The formulation according to anyone of claims 11-19, wherein the subject is a mammalian, such as a human.
A method for the treatment and/or prevention of coronary artery disease, or for decreasing the level of blood glucose or lipids, or for ameliorating coronary atherosclerotic lesions, in a subject, comprising the step of administrating an effective amount of Bacteroides vulgatus to a subject in need thereof.
The method according to claim 21, wherein Bacteroides vulgatus is selected from the group consisting of Bacteroides vulgatus ATCC 8482, Bacteroides vulgatus PC510, Bacteroides vulgatus mpk, and any combination thereof.
The method according to claim 21 or 22, wherein the Bacteroides vulgatus is formulated and administrated as a pharmaceutical composition, which is optionally in the form of a tablet, a pill, a suspension, a emulsion, a solution, a gel, a capsule, a powder, or a granule.
The method according to claim 21 or 22, wherein the Bacteroides vulgatus is formulated and administrated as a nutraceutical composition or a food composition, which is optionally in the form of solid, semi-solid, or liquid, and preferably is a dairy product, such as milk, milk powder, or yogurt.
The method according to anyone of claims 21-24, wherein the method further comprises administrating additional agent for treatment and/or prevention of coronary artery disease, or decreasing the level of blood glucose or lipids, or ameliorating coronary atherosclerotic lesions, wherein preferably, said additional agent is administrated before, accompanied with, or after the administration of Bacteroides vulgatus.
The method according to claim 25, wherein the additional agent is selected from the group consisting of Bacteroides uniformis, cholesterol lowering medications, beta-blockers, nitroglycerin, calcium antagonists, statins, nitroglycerin, ACE inhibitors, calcium channel blockers, aspirin, and any combination thereof.
The method according to claim 26, wherein Bacteroides uniformis is selected from the group consisting of Bacteroides uniformis ATCC 8492, Bacteroides uniformis CECT 7771, Bacteroides uniformis 0061, and any combination thereof.
The method according to anyone of claims 21-27, wherein the Bacteroides vulgatus is administrated at an amount of at least 1x10⁵ cfu/kg, such as at least 1x10⁶ cfu/kg, at least 1x10⁷ cfu/kg, at least 1x10⁸ cfu/kg, at least 1x10⁹ cfu/kg, at least 1x10¹⁰ cfu/kg, at least 1x10¹¹ cfu/kg, or at least 1x10¹² cfu/kg of the body weight of the subject, and is preferably administrated three times a day, twice a day, once a day, once two days, or once a week.
The method according to anyone of claims 21-28, wherein the method further comprises administration of additional therapy, such as Coronary interventions as angioplasty, and Coronary artery bypass grafting.
The method according to anyone of claims 21-29, wherein the subject is a mammalian, such as a human.
Bacteroides vulgatus, for use in the method for the treatment and/or prevention of coronary artery disease, or for decreasing the level of blood glucose or lipids, or for ameliorating coronary atherosclerotic lesions, in a subject.
Bacteroides vulgatus for use according to claim 31, wherein Bacteroides vulgatus is selected from the group consisting of Bacteroides vulgatus ATCC 8482, Bacteroides vulgatus PC510, Bacteroides vulgatus mpk, and any combination thereof.
Bacteroides vulgatus for use according to claim 31 or 32, wherein the Bacteroides vulgatus is formulated and administrated as a pharmaceutical composition, which is optionally in the form of a tablet, a pill, a suspension, a emulsion, a solution, a gel, a capsule, a powder, or a granule.
Bacteroides vulgatus for use according to claim 31 or 32, wherein the Bacteroides vulgatus is formulated and administrated as a nutraceutical composition or a food composition, which is optionally in the form of solid, semi-solid, or liquid, and preferably is a dairy product, such as milk, milk powder, or yogurt.
Bacteroides vulgatus for use according to anyone of claims 31-34, wherein the Bacteroides vulgatus is administrated in combination with additional agent for treatment and/or prevention of coronary artery disease, or decreasing the level of blood glucose or lipids, or ameliorating coronary atherosclerotic lesions, wherein preferably, said additional agent is administrated before, accompanied with, or after the administration of Bacteroides vulgatus.
Bacteroides vulgatus for use according to claim 35, wherein the additional agent is selected from the group consisting of Bacteroides uniformis, cholesterol lowering medications, beta-blockers, nitroglycerin, calcium antagonists, statins, nitroglycerin, ACE inhibitors, calcium channel blockers, aspirin, and any combination thereof.
Bacteroides vulgatus for use according to claim 36, wherein Bacteroides uniformis is selected from the group consisting of Bacteroides uniformis ATCC 8492, Bacteroides uniformis CECT 7771, Bacteroides uniformis 0061, and any combination thereof.
Bacteroides vulgatus for use according to anyone of claims 31-37, wherein the Bacteroides vulgatus is administrated at an amount of at least 1x10⁵ cfu/kg, such as at least 1x10⁶ cfu/kg, at least 1x10⁷ cfu/kg, at least 1x10⁸ cfu/kg, at least 1x10⁹ cfu/kg, at least 1x10¹⁰ cfu/kg, at least 1x10¹¹ cfu/kg, or at least 1x10¹² cfu/kg of the body weight of the subject, and is preferably administrated three times a day, twice a day, once a day, once two days, or once a week.
Bacteroides vulgatus for use according to anyone of claims 31-38, wherein the Bacteroides vulgatus is administrated in combination with additional therapy, such as Coronary interventions as angioplasty, and Coronary artery bypass grafting.
Bacteroides vulgatus for use according to anyone of claims 31-39, wherein the subject is a mammalian, such as a human.