Bacterial composition and its application in prevention and / or treatment of chronic kidney disease

Abstract

The present disclosure discloses a method for improving, treating and / or preventing mild to moderate chronic kidney disease (CKD) in a subject, comprising administrating one or two bacteria selected from Bacteroides eggerthii and Phocaeicola plebeius (or Bacteroides plebeius).

Description

[0001] BACTERIAL COMPOSITION AND ITS APPLICATION IN PREVENTION AND / OR TREATMENT OF CHRONIC KIDNEY DISEASE

[0002] Priority Information

[0003]

[0001] This application claims benefit of and priority to U.S. Provisional Patent Application No. 63 / 607,957, filed on 8 December 2023, the contents of which is incorporated by reference in its entirety.

[0004] Sequence Listing

[0005]

[0002] The instant application contains a Sequence Listing which has been filed electronically in XML format and is hereby incorporated by reference in its entirety. Said XLM copy, created on 6 December 2024, is named PC0658 SEQ List.xml and is 9 kilobytes in size.

[0006] Field of the Invention

[0007]

[0003] The present disclosure relates to bacterial composition and its applications in treating Chronic Kidney Disease.

[0008] Background of the Invention

[0009]

[0004] Intestinal dysbiosis means there is an imbalance of microorganisms in the intestines. Intestinal dysbiosis is a state in which there is an imbalance of microorganisms on or within our bodies. Intestinal microorganisms, collectively known as gut flora, consist predominantly of various strains of bacteria, and to a lesser extent include fungi and protozoa. The gut flora are essential for digestion and immune functioning and a state of dysbiosis will result in digestive and other systemic symptoms.

[0010]

[0005] Intestinal dysbiosis is present in Chronic Kidney Disease (CKD) patients. Advanced renal failure alters the biochemical milieu of the gastrointestinal tract leading to leak gut. The leak gut leads to bacterial translocation causing micro -inflammation, abnormal immunity and production of noxious metabolites aggravating the uremic toxicity. Therefore, these is a need to improve intestinal dysbiosis in a CKD patient.

[0011] Summary of the Invention

[0012]

[0006] The present disclosure aims to explore the relationship between the specific bacteria and their pathophysiological mechanisms in CKD patients, focusing on the host metabolism and immunity.

[0007] In one embodiment, the present disclosure provides a method for improving, treating and / or preventing mild to moderate chronic kidney disease (CKD) in a subject, comprising administrating one or two bacteria selected from Bacteroides eggerthii and Phocaelcolci plebeius (fonnerly known as Bacteroides plebeius) . Alternatively, the present disclosure provides a composition for use in for improving, treating and / or preventing mild to moderate CKD, wherein the composition comprises one or two bacteria selected from Bacteroides eggerthii and Phocaeicola plebeius (or Bacteroides plebeius). Also provided is use of a composition for use in for improving, treating and / or preventing mild to moderate CKD, wherein the composition comprises one or two bacteria selected from Bacteroides eggerthii and Phocaeicola plebeius (or Bacteroides plebeius).

[0013]

[0008] In another aspect, the present disclosure provides a bacterial combination comprising Bacteroides eggerthii and Phocaeicola plebeiu.

[0014]

[0009] In one embodiment, the bacteria described herein is Bacteroides eggerthii, Phocaeicola plebeius or a combination of Bacteroides eggerthii and Phocaeicola plebeius.

[0015]

[0010] In a further embodimetn, the the bacterial combination comprises Bacteroides eggerthii and Phocaeicola plebeius (Bacteroides plebeius) .

[0016]

[0011] In some embodiments, Bacteroides eggerthii is a strain having a sequence of 16S ribosomal

[0017] RNA (16S rRNA) with higher than 95%, 96%, 97%, 98%, 99% or 99.5% identity to 16S rRNA (SEQ ID NO: 1) of type strain Bacteroides eggerthii strain DSM20697.

[0018]

[0012] In one embodiment, the Bacteroides eggerthiistrain has a sequence of 16S rRNA as set forth in SEQ ID NO: 2.

[0019]

[0013] In some embodiments, Phocaeicola plebeius is a strain having a sequence of 16S ribosomal

[0020] RNA (16S rRNA) with higher than 95%, 96%, 97%, 98%, 99% or 99.5% identity to 16S rRNA (SEQ ID NO:3) of type strain Phocaeicola plebeius strain M12.

[0021]

[0014] In one embodiment, the Phocaeicola plebeius has a sequence of 16S rRNA as set forth in

[0022] SEQ ID NO: 4.

[0015] In some embodiments, the method or composition described herein can improve renal function and / or intestinal epithelial function and / or preserving renal function.

[0023]

[0016] In some embodiments, the method described herein can suppress systemic inflammation.

[0024]

[0017] In some embodiments, the method or composition described herein can improve microalbuminuria.

[0025]

[0018] In one embodiment, the subject described herein is a predialysis CKD patient.

[0026]

[0019] In one embodiment, the method or composition described herein can improve intestinal epithelial function, inflammation, oxidative stress, apoptosis or fibrosis in kidney.

[0027]

[0020] In one embodiment, the composition described herein is in the form suitable for oral administration.

[0028]

[0021] In one embodiment, the composition described herein is a nutritional composition or a pharmaceutical composition.

[0029]

[0022] In some embodiments, the composition described herein further comprises one or more probiotics. Examples of the probiotics include, but are not limited to, lactic acid bacteria, Bifidobacterium, Bifidobacterium longum, Lactobacillus acidophilus, Roseburia faecis, Leuconostoc rnesenteroides, Lactobacillus plantarum, Pediococcus pentosaceus, Lactobacillus brevis, Leuconostoc citreum, Leuconostoc argentmum. Lactobacillus paraplantarum. Lactobacillus coiynifonnis, Weissella spp., Leuconostoc spp . L. pentosus, L. plantarum , Leuconostoc rnesenteroides , L. brevis, L. lactis, L. fermentum, Lactobacillus acidophilus. Bifidobacterium bifidum, Streptococcus thermophilus, Lactobacillus delbruecki subsp. buigaricus. Lactobacillus helveticus. Lactobacillus kefiranofaciens. Lactococcus lactis, Leuconostoc species, and Lactococcus buigaricus.

[0030] Brief Description of the Drawings

[0031]

[0023] Figure 1 shows the morphological characterization of Bacteroides eggerthii strains. Rodshaped Gram negative bacterial cells of Bacteroides eggerthii type strain (B. eggerthii DSM20697) (A) and clinically-isolated strain (B. eggerthii-001 ) (B) are resolved through light microscopy. Scale bars. 5 pm.

[0024] Figure 2 displays the Sequence alignment of 16S ribosomal RNA gene from clinically- isolated strain (Query, B. eggerthii-001) and type strain (Sbjct, B eggerthii strain DSM20697) of Bacteroides eggerthii. The clinically-isolated strain are 99% identical to the type strain.

[0032]

[0025] Figure 3 demonstrates the effects of B. eggerthii-001 in improving renal (evidenced by uACR) and epithelial (evidenced by foot process width) functions in the mouse model of mild CKD induced by HFD feeding. (A) Experimental design (B) Changes in body weight over the experimental period in mice on each diet. CON, chow-fed control; HFD, mice fed with a high-fat diet; CON+B. egg, chow-fed mice given with B. eggerthii-001 for 8 wks; HFD+B. egg, HFD-fed mice given with B. eggerthii-001 for 8 wks. (C) Changes in renal function after gut colonization of B. eggerthii. Urine albumin to creatinine ratio (uACR) was measured after (at wk25) treatment of B. eggerthii-001 by oral gavage and compared by using Student’s t test. *.p <0.05; ***, > <0.001. (D) Quantification of podocyte foot process width. Foot process width was calculated from 5 glomeruli per animal and 5 capillary loops per glomerulus, and compared by using Student's t test. ***,p <0.001. (E) Electron microscopic analysis of kidney tissues from mice on each diet. White arrows, podocyte foot processes.

[0033]

[0026] Figure 4: Suppressive effects of Bacteroides eggerthii treatment on chronic systematic inflammation in the mouse model of mild CKD induced by HFD feeding. Changes in pro-inflammatory cytokine profiles after gut colonization of B. eggerthii. Levels of cytokines were measured after (at wk25) treatment of B. eggerthii-001 by oral gavage and compared by using Student’s t test. *, > <0.05; **, / ? <0.01; *** , <0.001. CON, chow-fed control; HFD, mice fed with a high-fat diet; CON+B. egg, chow-fed mice given with B. eggerthii-001 for 8 wks; HFD+B. egg, HFD-fed mice given with B. eggerthii-001 for 8 wks.

[0034]

[0027] Figure 5: Protective effect of B. eggerthii on E. co / i-mediated induction of epithelial permeability. Fully confluent monolayers of Caco-2 cells were treated with indicated bacterial species alone or in combination for 6 hrs. Permeability was assessed by measuring the parace llular flux of FD-4. *, p <0.05; **, / > <0.01 by using Student’s t test.

[0028] Figure 6: Morphological characterization of Phocaeicola plebeius strains. Rod-shaped

[0035] Gram negative bacterial cells of P. plebeius type strain (Ml 2) (A) and clinically-isolated strain (P. plebeius-

[0036] 001) (B) are resolved through light microscopy. Scale bars, 5 pm.

[0037]

[0029] Figure 7: Sequence alignment of 16S ribosomal RNA gene from clinically-isolated strain (Query, P. plebeius-001) and type strain (Sbjct, P. plebeius strain M12) of Phocaeicola plebeius.

[0038]

[0030] Figure 8: Gut colonization of Phocaeicola plebeius improves renal (evidenced by uACR) and epithelial (evidenced by foot process width) functions in the mouse model of mild CKD induced by HFD feeding. (A) Experimental design (B) Changes in renal function after gut colonization of P. plebeius. Urine albumin to creatinine ratio (uACR) of mice on each diet was measured after (at wk25) treatment of P. plebeius-001 by oral gavage and compared by using Student’s t test. **, p <0.01; ***,p <0.001. CON, chow-fed control; HFD, mice fed with a high-fat diet; CON+P. pleb. chow -fed mice given with P. plebeius- 001 for 8 wks; HFD+P. pleb . HFD-fed mice given with P. plebeius-001 for 8 wks. (C) Quantification of podocyte foot process width. Foot process width was calculated from 5 glomeruli per animal and 5 capillary loops per glomerulus, and compared by using Student's t test. *,p <0.05. (D) Electron microscopic analysis of kidney tissues from mice on each diet. White arrows, podocyte foot processes.

[0039]

[0031] Figure 9: Improvement of metabolic index with P. plebeius treatment in the mouse model of mild CKD induced by HFD feeding. Significant decreases in serum glucose and cholesterol levels were present after oral gavage of P. plebeius-001 for 8 weeks compared to feeding with broth in CKD mouse by using Student’s t test. *, p <0.05.

[0040]

[0032] Figure 10: Protective effect of P. plebeius on E. co / f-mediated induction of epithelial permeability. Fully confluent monolayers of Caco-2 cells were treated with indicated bacterial species alone or in combination for 6 hrs. Permeability was assessed by measuring the paracellular flux of FD-4. *,p <0.05; **, > <0.01 by using Student’s t test.

[0041]

[0033] Figure 11: Effects of conventional probiotic species on renal function compared to . plebeius-001 in the mouse model of mild CKD induced by HFD feeding. (A) Experimental design (B) Changes in body weight over the experimental period in mice on each diet (C) Urine albumin to creatinine ratio (uACR) was measured in mice on each group and compared by using Student’s t test. *,p <0.05; *** > <0.001; . CON, chow-fed control; HFD. mice fed with a high-fat diet; HFD+BL. HFD-fed mice given with Bifidobacterium longum (strain E194b) for 8 wks; HFD+LA, HFD-fed mice given with Lactobacillus acidophilus (strain L917) for 8 wks; HFD+PP, HFD-fed mice given with P. plebeius-OOJ for 8 wks; HFD+RF, HFD-fed mice given with Roseburia faecis (strain M72 / / )for 8 wks.

[0042]

[0034] Figure 12: Gut colonization of different dosing of Phocaeicola plebeius improves renal function in the mouse model of moderate CKD induced by adenine feeding (A) Experimental design (B) Changes of renal function indices with different dosing of P. plebeius. (C) Changes of renal function indices with 4* 10A9 cfu of P. plebeius. Feeding with different dosing of P. plebeius all resulted in improvement of microalbuminuria, serum creatinine and plasma urea nitrogen by using Student's t test; * 7?<O.O5; **, p <0.01; ***. p <0.001; ****, p <0.0001. uACR, urine albumin to creatinine ratio; CREA, serum creatinine; BUN, blood urea nitrogen; Cys-C. cystatin C..

[0043] Detailed Description of the Invention

[0044]

[0035] When items are connected by the conjunction "and," it should not be interpreted as necessitating the presence of each individual item in the group; instead, it should be understood as "and / or," unless explicitly stated otherwise. Likewise, when items are linked by "or," it should not be construed as requiring that the items be mutually exclusive; rather, it should also be interpreted as "and / or," unless explicitly stated otherwise. Additionally, while items, elements, or components of the invention may be referred to in the singular form, the plural form is considered to be included within the scope unless there is a clear indication limiting it to the singular.

[0045]

[0036] As used herein, the terms "a," "an." and "the" should be interpreted to encompass both singular and plural forms, unless specified otherwise. Therefore, "a," "an," and "the" (along with their grammatical variations, as applicable) refer to one or more entities.

[0046]

[0037] The tenn "pharmacally acceptable" as used in this context refers to compounds, materials, compositions, and / or dosage forms that, according to sound medical judgment, are appropriate for contact with the tissues of a subject (whether human or non-human animal) without causing excessive toxicity, irritation, allergic reactions, or other complications, while maintaining a reasonable benefit / risk ratio. Additionally, each carrier, excipient, and similar component must be deemed "acceptable" in terms of compatibility with the other ingredients in the formulation. Suitable carriers and excipients can be identified in standard pharmaceutical references.

[0047]

[0038] The term "edible carrier" denotes substances, materials, compositions, and / or dosage forms that are appropriate for contact with a subject's tissues. Additionally, each carrier must be deemed "acceptable," meaning it should be compatible with the other components of the formulation.

[0048]

[0039] The term "effective amount" as used herein is the amount of colony forming units (cfu) for each strain in the composition that is high enough to significantly modify the condition to be treated in a positive way but low enough to avoid serious side effects (at a reasonable benefit / risk ratio), within the scope of sound medical judgment.

[0049]

[0040] The term "treatment" is understood as meaning to lessen or decrease at least one sign, symptom, indication, or effect of a specific disease or condition. As used herein, "prevention" is understood as to limit, reduce the rate or degree of onset, or inhibit the development of at least one sign or symptom of a disease or condition.

[0050]

[0041] As used herein, the term "subject" is any animal that can benefit from the administration of a compound or composition as disclosed herein. In some embodiments, the subject is a mammal, for example, a human, a primate, a dog, a cat, a horse, a cow, a pig, a rodent, such as for example a rat or mouse. Typically, the mammal is a human.

[0051]

[0042] The term "effective amount" as referenced here refers to the quantity of colony -forming units (cfu) for each strain in the composition that is sufficient to positively impact the condition being treated, while remaining low enough to minimize the risk of serious side effects, all within the bounds of prudent medical judgment.

[0052]

[0043] To elucidate the pathophysiology of core CKD -microbiota exerted in the kidney, the present disclosure investigates the disease mechanisms of tire core-CKD microbiota using multi-omics platforms (metagenomics, metabolomics, immune phenome) in CKD cohorts. Next, complete genome architecture has been defining to state the genomic content pertinent to specific microbe. In addition, rapid testing tools targeting in specific genetic domain of candidate microbial species has been designing (by using qPCR) to facilitate clinical practice and validated in large clinical sample. Finally, treatment of core CKD-microbiota in the model were conducted to prove the therapeutic role of the microbiota on changes of renal function.

[0053]

[0044] Chronic Kidney Disease (CKD) is a long-term condition characterized by a gradual loss of kidney function over time. When the kidneys are damaged or not functioning properly, waste products can accumulate in the body, leading to various health complications. CKD is a serious health condition that requires ongoing management and monitoring. CKD is typically classified into five stages based on the glomerular filtration rate (GFR), which measures how well the kidneys are filtering blood:

[0054] Stage 1: Kidney damage with normal or increased GFR (>90 mL / min). There may be mild symptoms or none at all.

[0055] Stage 2: Mild decrease in GFR (60-89 mL / min). Kidney damage is still present, but symptoms may not be noticeable.

[0056] Stage 3: Moderate decrease in GFR (30-59 mL / min). Symptoms may begin to appear, such as fatigue, swelling, and changes in urination.

[0057] Stage 4: Severe decrease in GFR (15-29 mL / min). More pronounced symptoms and complications may occur, and preparation for dialysis or kidney transplant may be necessary.

[0058] Stage 5: End-stage renal disease (ESRD) (GFR <15 mL / min). The kidneys are no longer able to function adequately, requiring dialysis or a kidney transplant for survival.

[0059]

[0045] Mild CKD refers to the early stages of kidney dysfunction, typically classified as Stage 1 or Stage 2 according to the Kidney Disease: Improving Global Outcomes (KDIGO) guidelines. Moderate chronic kidney disease (CKD) is typically classified as stage 2 or stage 3 of the disease, based on the estimated glomerular filtration rate (eGFR) and other clinical factors. CKD is a progressive condition characterized by a gradual loss of kidney function overtime.

[0046] The present disclosure surprisingly found that CKD microbiota can improve, prevent and / or treat a mild and a moderate CKD.

[0060]

[0047] The core CKD-microbiota described herein include the following.

[0048] In some embodiments, the strains of Bacteroides eggerthii used in the present disclosure have a sequence of 16S ribosomal RNA (16S rRNA) with higherthan 95%, 96%. 97%. 98%. 99% or 99.5% identity to 16S rRNA (SEQ ID NO: 1) of type strain Bacteroides eggerthii strain DSM20697.

[0061]

[0049] In one embodiment, the preferred strain of Bacteroides eggerthii has a sequence of 16S rRNA as set forth in SEQ ID NO: 2.

[0050]

[0051] In some embodiments, the strains of Phocaeicola plebeius have a sequence of 16S ribosomal RNA (16S rRNA) with higher than 95%, 96%, 97%, 98%, 99% or 99.5% identity to 16S rRNA

[0062] (SEQ ID NO:3) of type strain Phocaeicola plebeius strain M12.

[0063]

[0064]

[0052] In one embodiment, the preferred strain of Phocaeicola plebeius has a sequence of 16S rRNA as set forth in SEQ ID NO: 4.

[0065]

[0053] It is considered that (1) the core CKD-microbiota may play role on the specific gut-derived metabolites and host cell immune phcnotypmg. and, consequently affect kidney homeostasis: (2) manipulation of the core CKD-microbiota could affect intestinal epithelial function, inflammation, oxidative stress, apoptosis or fibrosis and ultimately interfere the renal function. Therefore, the specific aims are disclosed in the present disclosure: (1) to validate the CKD-associated gut microbiota by using deep shotgun sequencing in a second cohort and integrate the MGWAS, metabolomics and immune alteration with disease phenotype: (2) to explore pathophysiological mechanisms of CKD-associated gut microbiota using experimental murine models; and (3) to characterize the complete genomic architecture of the core-CKD microbiota for designing of species-specific rapid diagnostic tool enable for clinical translation and prospective validation.

[0066]

[0054] The bacteria described herein can be formulated as a composition in any form suitable for administration, in particular oral administration. This includes for instance solids, semi-solids, liquids, granules and powders.

[0067]

[0055] The composition can be for example a capsule, tablet, drink, powder or dairy product. Optionally, other strains of probiotic (such as Lactic acid bacteria) may be present. Preferably the present composition is a nutraceutical or a phannaccutical product, a nutritional supplement or medical food.

[0068]

[0056] Examples of the compositions of the present disclosure are nutritional compositions, including food products and in particular dairy products. Nutritional compositions of the invention also include food supplements, and functional food. A "food supplement" designates a product made from compounds usually used in foodstuffs, but which is in the form of tablets, powder, capsules, potion or any other form usually not associated with aliments, and which has beneficial effects for one's health. A "functional food" is an aliment which also has beneficial effects for one's health. In particular, food supplements and functional food can have a physiological effect— protective or curative -against a disease.

[0057] If the composition according to the present disclosure is a dietary supplement, it can be administered as such, can be mixed with a suitable drinkable liquid, such as water, yoghurt, milk or fruit juice, or can be mixed with solid or liquid food, hr this context the dietary supplement can be in the form of tablets, pills, capsules, lozenges, granules, powders, suspensions, sachets, pastilles, sweets, bars, syrups and corresponding administration forms, usually in the form of a unit dose. Preferably, the dietary supplement comprising the composition of the present disclosure is administered in tire form of tablets, lozenges, capsules or pow ders, manufactured in conventional processes of preparing dietary supplements.

[0058] The compositions described herein can be pharmaceutically acceptable compositions, which may include one or more pharmaceutically acceptable carriers, excipients, binders, diluents or the like. The instant compositions can be formulated for various routes of administration, for example, by oral administration. They also may be provided in combination with delivery vehicles such as in some encapsulating technology. For oral administration, powders, suspensions, granules, tablets, pills, capsules, gelcaps, and caplets are acceptable as solid dosage forms. These can be prepared, for example, by mixing one or more compounds disclosed herein with at least one additive such as a starch or other additive. Suitable additives are sucrose, lactose, cellulose sugar, mannitol, maltitol, dextran, starch, agar, alginates, chitins, chitosans, pectins, tragacanth gum, gum arabic, gelatins, collagens, casein, albumin, synthetic or semi-synthetic polymers or glycerides. Optionally, oral dosage forms can contain other ingredients to aid in administration, such as an inactive diluent, or lubricants such as magnesium stearate, or preservatives such as paraben or sorbic acid, or anti-oxidants such as ascorbic acid, tocopherol or cysteine, a disintegrating agent, binders, thickeners, buffers, sweeteners, flavoring agents or perfuming agents. Tablets and pills may be further treated with suitable coating materials known in the art.

[0069]

[0059] The compositions of the present disclosure can be presented in various forms suitable for administration, particularly for oral use. These forms include solids, semi -solids, liquids, granules, and powders

[0070]

[0060] The compositions described may be pharmaceutically acceptable and can include one or more acceptable carriers, excipients, binders, diluents, or similar substances. These compositions can be formulated for different routes of administration, particularly oral administration, and may be combined with delivery vehicles, such as encapsulating technologies.

[0071]

[0061] For oral administration, acceptable solid dosage forms include powders, suspensions, granules, tablets, pills, capsules, gelcaps, and caplets. These can be prepared by mixing one or more of the disclosed compounds with at least one additive, such as starch or other suitable substances. Acceptable additives include sucrose, lactose, cellulose sugar, mannitol, maltitol, dextran, starch, agar, alginates, chitin, chitosan, pectin, tragacanth gum, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi -synthetic polymers, or glycerides. Optionally, oral dosage forms may contain additional ingredients to facilitate administration, such as inactive diluents, lubricants (e.g., magnesium stearate), preservatives (e.g., paraben or sorbic acid), antioxidants (e.g., ascorbic acid, tocopherol, or cysteine), disintegrating agents, binders, thickeners, buffers, sweeteners, flavoring agents, or perfuming agents. Tablets and pills may also be coated with suitable materials known in tire field.

[0072]

[0062] Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, syrups, elixirs, suspensions, and solutions, which can contain an inactive diluent like water. Pharmaceutical formulations may be prepared as liquid suspensions or solutions using sterile liquids, including oils, water, alcohol, or combinations thereof. Suitable surfactants, suspending agents, and emulsifying agents may be added for oral or parenteral administration.

[0073]

[0063] The present invention is described in greater detail by the examples presented below, which are preceded by a brief description of the figures. It goes without saying however, that these examples are given by way of illustration of the subject of the invention and do not constitute in any manner a limitation thereto.

[0074] EXAMPLE

[0075]

[0064] Example 1 Clinical isolation of bacterial strains and bacterial treatment

[0076]

[0065] Strains of B. eggerthii and P. plebeius were isolated from feces of two subjects who had neither diagnosed from renal disorders nor had antibiotics for three months prior to the study or family history of common diseases as previously described with some modifications (Kitahara M. Sakamoto M, Ike M. Sakata S. Benno Y: Bacteroides plebeius sp. nov. and Bacteroides coprocola sp. nov. , isolated from human faeces. Int J Syst Evol Microbiol 2005. 55(Pt 5):2143-2147,‘ Holdeman LV. Moore WEC: New Genus, Coprococcus, Twelve New Species, and Emended Descriptions of Four Previously Described Species of Bacteria from Human Feces. Int J Syst Bacterial 1974, 24(2): 260-277). Briefly, fresh feces (1 g) were suspended immediately in 5 mL of sterile PBS, and the suspension diluted to 10'8in Bacteroides minimal medium was then plated anaerobically on Bacteroides Bile Esculin (BBE) Agar or Laked Brucella Blood Agar with Kanamycin and Vancomycin (LKV) at 37 °C until colonies are visualized. Single colonies were collected, and their identity was assessed initially by matrix-assisted laser-desorption / ionization time- of-flight mass spectrometry (MALDI-TOF MS) with a log(score) of >2.0 as the set threshold for a match at the species level and then further confirmed by 16S rRNA gene sequencing with a homology level >98.65 % as considered adequate for species identification (see Figures 1, 2, 6 and 7). For the type strains, P. plebeius (DSM17135), obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) (DSMZ) was propagated in Columbia agar base supplemented with 5% defibrinated sheep blood at 37°C. B. eggerthii (,U( C ' 27754™ , obtained from the American Type Culture Collection, USA) was grown at 37 °C in brain heart infusion (BHI) agar supplemented with 10% horse blood, tryptone -yeast extract -glucose (TYG) medium, or Bacteroides minimal medium (MM) (Bacic MK, Smith CJ: Laboratory maintenance and cultivation of bacteroides species. Curr Protoc Microbiol 2008, Chapter 13:Unit I3C 11) and cultured under anaerobic conditions at 37°C, using a Whitley DG250 anaerobic chamber (Don Whitley, UK) with mixed anaerobic gas (5% carbon dioxide, 5% hydrogen, 90% nitrogen). Anaerobicity was confirmed using an anaerobic indicator (Oxoid. UK).

[0077]

[0066] Bacteria were collected by centrifugation and resuspended in sterile saline. Mice fed with HFD for 16 weeks were treated daily ith sterile saline (n=6) or 4* I ()'' colony -forming units of P. plebeius, B. eggerthii, and the combination of both (n=6) for 8 weeks by oral gavage. Fecal and urine samples from each treatment group for assessing the gut microbiota composition and renal function, respectively, were collected before and after the bacterial treatment in a metabolic chamber under a laminar flow' hood. Kidney and colon tissues as well as whole blood from mice after the treatment were collected for the downstream analysis.

[0078]

[0067] Example 2 Establishment of a modified obesity-related model of CKD

[0079]

[0068] Male 4-week-old C57BL / 6J mice (National Laboratory Animal Center, NARLabs, Taiwan) were kept in groups of four to five per cage and provided with free access to food and sterile drinking water in a temperature -regulated room (21°C±2°C) under a 12 hours dark-light cycle. After a one-week accommodation, mice were given for 16 weeks with standard chow diet (n=6) (chow, 13.5% of energy from fat; LabDiet 5001; LabDiet, USA) orHFD (n=24) (60% of energy from fat; TestDiet 58Y1; TestDiet, USA). Mean energy intake were estimated by converting the amount of food consumed into calories using the data offered by the manufacturer (LabDiet 5001 and TestDiet 58Y1).

[0080]

[0069] Exam pie 3 Electron microscopic analysis

[0081]

[0070] Mouse kidney specimens were fixed in 3% glutaraldehyde in 0. 1 M cacodylate buffer (pH 7.4) for 1 hr at 4°C, then postfixed in 1% osmium tetroxide (pH 7.4), dehydrated in a graded series of ethanol, and embedded in EPON-812 (Sigma-Aldrich, St. Louis, MO, USA). Thin sections (80±5 nm) were cut, stained with uranyl acetate and lead citrate, and examined on a Hitachi H-7500 EM transmission electron microscope (Hitachi, Ltd., Tokyo, Japan). Foot process width was determined as described previously (Dee gens JK, DijkmanHB, Borm GF, Steenbergen EJ, van den Berg JG, Weening JJ. Wetzels JF: Podocyte foot process effacement as a diagnostic tool in focal segmental glomerulosclerosis. Kidney Int 2008, 74(12): 1568-1576). In brief, the number of foot processes per capillary' loop was counted, divided by the length of the glomerular basement membrane, and multiplied by n / 4 in order to correct for presumed random variation in the angle of the section relative to the long axis of the podocyte. Measurements were conducted on 5 glomeruli per animal and the foot process width is expressed as the mean of measurements of 5 capillary loops per glomerulus.

[0082]

[0071] Example s Measurement of microalbuminuria

[0083]

[0072] Mice were placed in metabolic chambers and 24-h urine collection was perfonned after 8 -week treatment of P. plebeius or B. eggerthii. Urine albumin levels were determined using the Murine Microalbuminuria ELISA kit (Exocell, Inc.) and urine creatinine levels were determined using the Microcreatinuria ELISA kit (Exocell, Inc.).

[0084]

[0073] Example 6 Measurement of cytokines / chemokines / growth factors

[0085]

[0074] A panel of 48 hematopoiesis- and immune-related cytokines, chemokines, and growth factors were measured to compare the non-cellular components in the circulation of CKD animals, with the Mouse Immune Monitoring 48-Plex ProcartaPlex Panel (eBioscience) according to the manufacturer's protocol. Each serum sample was tested in duplicate. Plates were read with the Luminex 200 system (Luminex) and analyzed by using ProcartaPlex software (eBioscience). The improvement of renal function results from suppression of systemic inflammation after oral gavage of Bacteroides eggerthii-001 in mouse CKD model (Figure 4).

[0086]

[0075] Example 7 Cel! monolayer permeability assay

[0087]

[0076] Caco-2 cells were seeded on polyester transwell inserts (12mm diameter, 0.4 pm pore size, 1.12 cm2growth area) (Coming Costar Corporation. NY, USA) at 5xl05cells / well and grown for 21 days to reach full confluence. Before the treatment with B. eggerthii, P. plebe ins. or E.coli at 50 MOIs either alone or simultaneously for 6 hrs, cells were washed and placed in antibiotic -free medium. After bacterial treatment, cells were rinsed with PBS and incubated in Hank's balanced salt solution containing 1 mg / ml FITC-dextran 4 kDa (FD4) (Sigma- Aldrich, USA) solution for 2h. Permeability of paracellular flux as an indicator of permeability was assessed by taking 100 pl of medium from the basolateral chamber. Fluorescence was measured using a Multiskan™ FC Microplate Photometer (Thermo Fisher) with excitation and emission at 492 and 520 nm, respectively. A standard curve was generated by a serial dilution of FD4 to determine FD4 concentrations. Fluxes were calculated by using the apparent permeability coefficient (Popp) equation as previously described. Oral gavage of Bacteroides eggerthii-001 for 8 weeks improves renal (evidenced by uACR) and epithelial (evidenced by foot process width) functions in the mouse model of mild CKD induced by HFD feeding (Figure 3, 5). Oral gavage of Phocaeicola plebeius- 001 for 8 weeks improves renal (evidenced by uACR) and epithelial (evidenced by foot process width) functions in the mouse model of mild CKD induced by HFD feeding (Figure 8, 10).

[0077] Example 8 Measurement of renal function and biochemistry parameters

[0088]

[0078] Serum levels of glucose (GLU), total cholesterol (TChol), triglyceride (TG), creatinine (Cre), blood urea nitrogen (BUN), albumin (Alb) and cystatin C (Cys-C) were determined using a Hitachi 7180 biochemistry automatic analyzer (Hitachi, Tokyo, Japan). The improvement of renal function of Phocaeicola plebeius-001 results from improvement of metabolic index, including serum glucose and cholesterol (Figure 9).

[0089]

[0079] Example 9 Bacteria treatment with conventional probiotics

[0090]

[0080] Treatment effects of P. plebeius were compared with conventional probiotic using oral gavage of Bifidobacterium longum (strain El 94b), Lactobacillus acidophilus (strain L917) and Roseburia faecis (strain M72 / 1) for 8 weeks in a modified obesity-related model of CKD. Oral gavage of Phocaeicola plebeius-001 for 8 weeks results in better microalbuminuria improvement compared to conventional probiotics (Figure 11).

[0091]

[0081] Example 10 Establishment of moderate CKD model by adenine feeding

[0092]

[0082] The C57BL / 6J male mice (National Laboratory Animal Center, NARLabs, Taiwan) were housed four to five individuals per cage with free access to food and sterile drinking water (reverse osmosis grade) in a temperature-controlled room (21°C±2°C) under a 12 hours dark -light cycle. All animal experiments procedures received care according to institutional guidelines, and all of the experiments were approved by the Taipei Medical University Institutional Animal Care and Use Committee. After an accommodation period of 1 week with normal chow, seven -week-old C57BL / 6J male mice were fed for 4 weeks with standard chow diet (n=6) (chow, 13.5% of energy from fat: LabDiet 5010; LabDiet, USA) or adenine diet (n=24) (mixture of 1 kg of normal chow powder and 2g of adenine powder. Sigma Aldrich) to induce moderate CKD. Stool, blood and urine samples from each treatment group were collected before and after the treatment and at the time of sacrifice. Tissue samples were harvested at kill. Figure 12 shows gut colonization of different dosing of Phocaeicola plebeius improves renal function in the mouse model of moderate CKD induced by adenine feeding (A) Experimental design (B) Changes of renal function indices with different dosing of P. plebeius. (C) changes of renal function indices with 4* 10A9 cfu of P. plebeius.

Claims

ClaimsWhat is claimed is:

1. A composition for use in for improving, treating and / or preventing mild to moderate CKD, wherein the composition comprises one or two bacteria selected from Bacteroides eggerthii and Phocaeicola plebeius (or Bacteroides plebeius).

2. A bacterial combination comprising Bacteroides eggerthii and Phocaeicola plebeius.

3. The composition of Claim 1 or 2, wherein the composition comprises Bacteroides eggerthii, Phocaeicola plebeius or Bacteroides eggerthii in combination with Phocaeicola plebeius.

4. The composition of any one of proceeding claims, wherein the composition comprising a Bacteroides eggerthii strain having a sequence of 16S ribosomal RNA ( 16S rRNA) with higher than 95%, 96%, 97%, 98%, 99% or 99.5% identity to 16S rRNA (SEQ ID NO: 1) of type strain Bacteroides eggerthii strain DSM20697.

5. The composition of any one of proceeding claims, wherein the composition comprising a Bacteroides eggerthii strain having a sequence of 16S ribosomal RNA (16S rRNA) as set forth in SEQ ID NO:2.

6. The composition of any one of proceeding claims, wherein the composition comprising a Phocaeicola plebeius strain having a sequence of 16S ribosomal RNA (16S rRNA) w ith higher than 95%, 96%, 97%, 98%, 99% or 99.5% identity to 16S rRNA (SEQ ID NO:3) of type strain Phocaeicola plebeius strain M12.

7. The composition of any one of proceeding claims, wherein the composition comprising a Phocaeicola plebeius strain having a sequence of 16S ribosomal RNA (16S rRNA) as set forth in SEQ ID NO:4.

8. The composition of any one of proceeding claims, which can improve renal function and / or intestinal epithelial function and / or preserving renal function.

9. The composition of any one of proceeding claims, which can suppress systemic inflammation.

10. The composition of any one of proceeding claims, which can improve microalbuminuria.

11. The composition of any one of proceeding claims, wherein the subject is a predialysis CKD patient.

12. The composition of any one of proceeding claims, which can improve intestinal epithelial function, inflammation, oxidative stress, apoptosis or fibrosis in kidney.

13. The composition of any one of proceeding claims, which is in the form suitable for oral administration.

14. Tire composition of any one of proceeding claims, which is a nutritional composition or a pharmaceutical composition.

15. Tire composition of any one of proceeding claims, which further comprises one or more probiotics.

16. The composition of any one of proceeding claims, which further comprises one or more Bi fidobacterium longum. Lactobacillus acidophilus and Roseburia faecis.

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