A COMPOSITION COMPRISING A COHORT OF BACTERIA
20200345051 ยท 2020-11-05
Inventors
Cpc classification
A23L33/40
HUMAN NECESSITIES
A61P1/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A23V2002/00
HUMAN NECESSITIES
International classification
A23L33/135
HUMAN NECESSITIES
A23L33/00
HUMAN NECESSITIES
Abstract
A composition comprises a cohort of at least five isolated unique strains of a single species of bacteria, wherein the species of bacteria is represented in the mammalian microbiome, and wherein the cohort of isolated strains exhibit different and diverse catabolic capability thereby increasing the chances that one or more of the strains in the composition will find a nutritional niche in the infant GI tract.
Claims
1. A composition comprising a cohort of at least five isolated strains of Bifidobacterium longum selected from the group consisting of: Bifidobacterium longum ssp suis APC1461 as deposited with the National Collection of Industrial and Marine Bacteria under the Accession No. NCIMB 42832 on 29 Sep. 2017; Bifidobacterium longum ssp longum APC1473 as deposited with the National Collection of Industrial and Marine Bacteria under the Accession No. NCIMB 42824 on 29 Sep. 2017; Bifidobacterium longum ssp longum APC1476 as deposited with the National Collection of Industrial and Marine Bacteria under the Accession No. NCIMB 42825 on 29 Sep. 2017; Bifidobacterium longum ssp longum APC1478 as deposited with the National Collection of Industrial and Marine Bacteria under the Accession No. NCIMB 42826 on 29 Sep. 2017; Bifidobacterium longum ssp longum APC1480 as deposited with the National Collection of Industrial and Marine Bacteria under the Accession No. NCIMB 42827 on 29 Sep. 2017; Bifidobacterium longum ssp longum APC1503 as deposited with the National Collection of Industrial and Marine Bacteria under the Accession No. NCIMB 42828 on 29 Sep. 2017; Bifidobacterium longum ssp longum APC289 as deposited with the National Collection of Industrial and Marine Bacteria under the Accession No. NCIMB 42829 on 29 Sep. 2017; Bifidobacterium longum ssp longum APC293 as deposited with the National Collection of Industrial and Marine Bacteria under the Accession No. NCIMB 42830 on 29 Sep. 2017; Bifidobacterium longum ssp longum APC295 as deposited with the National Collection of Industrial and Marine Bacteria under the Accession No. NCIMB 42831 on 29 Sep. 2017; Bifidobacterium longum ssp longum APC1462 as deposited with the National Collection of Industrial and Marine Bacteria under the Accession No. NCIMB 42833 on 29 Sep. 2017; Bifidobacterium longum ssp longum APC1465 as deposited with the National Collection of Industrial and Marine Bacteria under the Accession No. NCIMB 42834 on 29 Sep. 2017; Bifidobacterium longum ssp longum APC1464 as deposited with the National Collection of Industrial and Marine Bacteria under the Accession No. NCIMB 42839 on 5 Oct. 2017; Bifidobacterium longum ssp longum APC1468 as deposited with the National Collection of Industrial and Marine Bacteria) under the Accession No. NCIMB 42840 on 5 Oct. 2017; and Bifidobacterium longum ssp longum APC1504 as deposited with the National Collection of Industrial and Marine Bacteria under the Accession No. NCIMB 42841 on 5 Oct. 2017.
2. The composition according to claim 1, in which the cohort of isolated strains includes at least one of: Bifidobacterium longum ssp longum APC1478; and Bifidobacterium longum ssp longum APC295.
3. The composition according to claim 2, in which the cohort of strains includes: Bifidobacterium longum ssp longum APC1478; and Bifidobacterium longum ssp longum APC295.
4. The composition according to claim 1, in which the cohort of strains includes Bifidobacterium longum ssp longum APC1477.
5. A composition according to claim 1, in which the cohort of strains includes: Bifidobacterium longum ssp longum APC1478; Bifidobacterium longum ssp longum APC295; and Bifidobacterium longum ssp longum APC1477.
6. The composition according to claim 1, in which the cohort of isolated strains of Bifidobacterium longum together contain at least 60% of the total gene repertoire of the Bifidobacterium longum pangenome.
7. A composition according to claim 1, in which the cohort of isolated strains comprises Bifidobacterium longum ssp longum APC1477 and at least 5 isolated strains of Bifidobacterium longum selected from the group of claim 1 including Bifidobacterium longum ssp longum APC1478 and Bifidobacterium longum ssp longum APC295.
8. The composition according to claim 1, in which the cohort of isolated strains comprises at least 6 isolated strains of Bifidobacterium longum selected from the group of claim 1.
9. The composition according to claim 1, in which the cohort of isolated strains comprises at least 8 isolated strains of Bifidobacterium longum selected from the group of claim 1.
10. The composition according to claim 1, in which the composition is an infant milk formula.
11-15. (canceled)
16. The composition according to claim 1 selected from a food or dietary supplement composition.
17. The composition according to claim 1, comprising at least 10.sup.6 cfu per gram.
Description
BRIEF DESCRIPTION OF THE FIGURES
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DETAILED DESCRIPTION OF THE INVENTION
[0073] All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full.
[0074] Definitions and General Preferences
[0075] Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art:
[0076] Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term a or an used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms a (or an), one or more, and at least one are used interchangeably herein.
[0077] As used herein, the term comprise, or variations thereof such as comprises or comprising, are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term comprising is inclusive or open-ended and does not exclude additional, unrecited integers or method/process steps.
[0078] Treatment
[0079] As used herein, the term disease is used to define any abnormal condition that impairs physiological function and is associated with specific symptoms. The term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition or syndrome in which physiological function is impaired irrespective of the nature of the aetiology (or indeed whether the aetiological basis for the disease is established). It therefore encompasses conditions arising from infection, trauma, injury, surgery, radiological ablation, poisoning or nutritional deficiencies. The strains of the invention may be employed to treat or prevent disease or conditions, for example diseases or conditions characterised by a dysregulated microbiome, and other diseases or conditions including metabolic disease (for example Type I or Type II diabetes), inflammatory disease, cardiovascular disease, proliferative diseases, autoimmune disease, or degenerative conditions.
[0080] As used herein, the term dysregulated microbiome as applied to a mammal, for example a human, for example an infant human, should be understood to mean a microbiome that is depleted in one or more species of bacteria normally present in the microbiome. An example is the microbiome of an infant (i.e. up to six months old) that was born prematurely, born by Caesarian section, or has been treated with antibiotics, and which has a microbiome which is depleted in Bifidobacterium longum strains of bacteria. Other examples include elderly patients, patients undergoing antibiotic or chemo-therapy, autism patients, all of whom are recognised as having a microbiome depleted in Bifidobacteria and would be suitable for treatment with a composition of the invention. Patients with poor metabolic health, for example patients with metabolic disease, are recognised as having a microbiome depleted in Akkermansia, and would be suitable for treatment with a composition of the invention comprising a cohort of isolated strains of Akkermansia (for example Akkermansia muciniphila) comprising a composite genetic profile that is representative of a human microbiome-specific pangenome of an Akkermansia. species.
[0081] As used herein, the term treatment or treating refers to an intervention (e.g. the administration of an agent to a subject) which cures, ameliorates or lessens the symptoms of a disease or removes (or lessens the impact of) its cause(s) (for example, the reduction in accumulation of pathological levels of lysosomal enzymes). In this case, the term is used synonymously with the term therapy.
[0082] Additionally, the terms treatment or treating refers to an intervention (e.g. the administration of an agent to a subject) which prevents or delays the onset or progression of a disease or reduces (or eradicates) its incidence within a treated population. In this case, the term treatment is used synonymously with the term prophylaxis.
[0083] As used herein, an effective amount or a therapeutically effective amount of an agent defines an amount that can be administered to a subject without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio, but one that is sufficient to provide the desired effect, e.g. the treatment or prophylaxis manifested by a permanent or temporary improvement in the subject's condition. The amount will vary from subject to subject, depending on the age and general condition of the individual, mode of administration and other factors. Thus, while it is not possible to specify an exact effective amount, those skilled in the art will be able to determine an appropriate effective amount in any individual case using routine experimentation and background general knowledge. A therapeutic result in this context includes eradication or lessening of symptoms, reduced pain or discomfort, prolonged survival, improved mobility and other markers of clinical improvement. A therapeutic result need not be a complete cure.
[0084] In the context of treatment and effective amounts as defined above, the term subject (which is to be read to include individual, animal, patient or mammal where context permits) defines any subject, particularly a mammalian subject, for whom treatment is indicated. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; and rodents such as mice, rats, hamsters and guinea pigs. In preferred embodiments, the subject is a human.
[0085] Strains and Species
[0086] Bifidobacterium longum ssp suis APC1461 strain refers to the strain of bacteria deposited with the National Collection of Industrial and Marine Bacteria (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB219YA, UK) under the Accession No. NCIMB 42832 on 29 Sep. 2017. Characteristics of the strain are provided in the Tables below and the accompanying figures.
[0087] Bifidobacterium longum ssp longum APC1473 strain refers to the strain of bacteria deposited with the National Collection of Industrial and Marine Bacteria (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB219YA, UK) under the Accession No. NCIMB 42824 on 29 Sep. 2017. Characteristics of the strain are provided in the Tables below and the accompanying figures.
[0088] Bifidobacterium longum ssp longum APC1476 strain refers to the strain of bacteria deposited with the National Collection of Industrial and Marine Bacteria (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB219YA, UK) under the Accession No. NCIMB 42825 on 29 Sep. 2017. Characteristics of the strain are provided in the Tables below and the accompanying figures.
[0089] Bifidobacterium longum ssp longum APC1478 strain refers to the strain of bacteria deposited with the National Collection of Industrial and Marine Bacteria (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB219YA, UK) under the Accession No. NCIMB 42826 on 29 Sep. 2017. Characteristics of the strain are provided in the Tables below and the accompanying figures.
[0090] Bifidobacterium longum ssp longum APC1480 strain refers to the strain of bacteria deposited with the National Collection of Industrial and Marine Bacteria (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB219YA, UK) under the Accession No. NCIMB 42827 on 29 Sep. 2017. Characteristics of the strain are provided in the Tables below and the accompanying figures.
[0091] Bifidobacterium longum ssp longum APC1503 strain refers to the strain of bacteria deposited with the National Collection of Industrial and Marine Bacteria (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB219YA, UK) under the Accession No. NCIMB 42828 on 29 Sep. 2017. Characteristics of the strain are provided in the Tables below and the accompanying figures.
[0092] Bifidobacterium longum ssp longum APC289 strain or DPC 6317 refers to the strain of bacteria deposited with the National Collection of Industrial and Marine Bacteria (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB219YA, UK) under the Accession No. NCIMB 42829 on 29 Sep. 2017. Characteristics of the strain are provided in the Tables below and the accompanying figures.
[0093] Bifidobacterium longum ssp longum APC293 strain or DPC 6321 refers to the strain of bacteria deposited with the National Collection of Industrial and Marine Bacteria (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB219YA, UK) under the Accession No. NCIMB 42830 on 29 Sep. 2017. Characteristics of the strain are provided in the Tables below and the accompanying figures.
[0094] Bifidobacterium longum ssp longum APC295 strain or DPC 6362 refers to the strain of bacteria deposited with the National Collection of Industrial and Marine Bacteria (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB219YA, UK) under the Accession No. NCIMB 42831 on 29 Sep. 2017. Characteristics of the strain are provided in the Tables below and the accompanying figures.
[0095] Bifidobacterium longum ssp longum APC1462 strain refers to the strain of bacteria deposited with the National Collection of Industrial and Marine Bacteria (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB219YA, UK) under the Accession No. NCIMB 42833 on 29 Sep. 2017. Characteristics of the strain are provided in the Tables below and the accompanying figures.
[0096] Bifidobacterium longum ssp longum APC1465 strain refers to the strain of bacteria deposited with the National Collection of Industrial and Marine Bacteria (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB219YA, UK) under the Accession No. NCIMB 42834 on 29 Sep. 2017. Characteristics of the strain are provided in the Tables below and the accompanying figures.
[0097] Bifidobacterium longum ssp longum APC1464 strain refers to the strain of bacteria deposited with the National Collection of Industrial and Marine Bacteria (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB219YA, UK) under the Accession No. NCIMB 42839 on 5 Oct. 2017. Characteristics of the strain are provided in the Tables below and the accompanying figures.
[0098] Bifidobacterium longum ssp longum APC1468 strain refers to the strain of bacteria deposited with the National Collection of Industrial and Marine Bacteria (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB219YA, UK) under the Accession No. NCIMB 42840 on 5 Oct. 2017. Characteristics of the strain are provided in the Tables below and the accompanying figures.
[0099] Bifidobacterium longum ssp longum APC1504 strain refers to the strain of bacteria deposited with the National Collection of Industrial and Marine Bacteria (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB219YA, UK) under the Accession No. NCIMB 42841 on 5 Oct. 2017. Characteristics of the strain are provided in the Tables below and the accompanying figures.
[0100] Bifidobacterium longum APC 1472 refers to the strain of bacteria deposited with the National Collection of Industrial and Marine Bacteria (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB219YA, UK) under the Accession No. NCIMB 42795 on 1 August 2017. Characteristics of the strain are provided in the Tables below and the accompanying figures.
[0101] Bifidobacterium longum APC 1466 refers to a strain of Bifidobacterium longum spp. longum obtained from the neonatal gut having the characteristics provided in Table 1 and the Figures, especially
[0102] Bifidobacterium longum APC 1477 refers to a strain of Bifidobacterium longum spp. longum obtained from the neonatal gut having the characteristics provided in Table 1 and the Figures, especially
[0103] Bifidobacterium longum DPC 6316 refers to a strain of Bifidobacterium longum spp. longum obtained from the neonatal gut having the characteristics provided in Table 1 and the Figures, especially
[0104] Bifidobacterium longum DPC 6320 refers to a strain of Bifidobacterium longum spp. longum obtained from the neonatal gut having the characteristics provided in Table 1 and the Figures, especially
[0105] Annoted sequences for strains APC 1466, 1477, DPC 6316 and 6320 are available from Genebank at the following address: https://www.ncbi.nlm.nih.gov/nuccore?term=(((bifidobacterium%20longum)/%20AND%20arbboleya%5BAuthor%5BAuthor%5D))%20AND%20APC%201466, the complete contents are incorporated herein by reference.
[0106] Deposits of the deposited strains were made by Teagasc Food Research Centre, a Centre forming part of Agriculture and Food and Development Authority (Teagacs), on 29 Sep. 2017 and 5 Oct. 2017. All of the above strains are characterised to species level by 16s rRNA-internally transcribed spacer (ITS) gene sequence analysis and to strain level by pulsed field gel electrophoresis (PFGE). The strains were all isolated from faecal samples from healthy, human infants. All strains were culturable. The strains were confirmed as unique due to distinct PFGE profiles and varying phenotypic traits, The strain may be in a viable or non-viable form, or mixtures of viable and non-viable bacteria. The strain may be provided in any format, for example as a liquid culture (or supernatant derived from a liquid culture), or in a dried or freeze-dried format. The invention may also employ growth media in which the strain of the invention was grown, or cell lysates generated using the strain of the invention. The invention also includes mutants and variants of the deposited strain that are substantially identical, genetically and phenotypically, to the deposited strain and retain the activity of the deposited strain. Thus, the invention includes derivatives of the strain that have been genetically engineered to modify the genome of the strain, typically without fundamentally altering the functionality of the organism, for example engineered for heterologous expression of a nucleic acid, or engineered to overexpress an endogenous gene, or engineered to silence a gene, to produce a recombinant or transformed strain of the invention. Genetic modifications may be carried out using recombinant DNA techniques and reagents (see below). The term also includes variants of the strain having natural or spontaneous genetic alterations. The term is also intended to encompass variant strains obtained by serial passage of the isolated strain of the invention. The variant generally has a 16S rRNA amplicon (fragment) sequence that is identical or substantially identical with the deposited strain, for example at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical with the deposited strain. Sequence homology can be determined using an online homology algorithm BLAST, publicly available at http://www.ncbi.nlm.nih.gov/BLAST/.
[0107] In this specification, the term isolated as applied to a strain of bacteria means that the strain is isolated from other strains of bacteria. The individual isolated strains are then combined together to form the cohort of isolated bacteria comprising unique isolated strains.
[0108] In this specification, the term unique as applied to the strains making up the cohort of bacteria means that each strain in the cohort has a distinct PFGE profile. Methods for determining the PFGE profile of a strain of bacteria are described below.
[0109] In this specification, the term cohort of isolated strains refers to at least five strains of the same species, and ideally of the same sub-species. In one embodiment, the term refers to at least 6, 7, 8, 9, 10 strains of the same species.
[0110] In this specification, the term pangenome as applied to a specific species of bacteria means the total gene repertoire determined across a representative number of unique strains of the specific species of bacteria that are represented in a specific microbiome, for example a human microbiome or a bovine microbiome. The pangenome is therefore species specific and microbiome specific; the Bifidobacterium longum pangenome described below is a specific to the human gut microbiome, and more specifically the infant human gut microbiome. The number of unique strains required to compile a species-specific and microbiome-specific pangenome depends on the species concerned, and will be known to a person skilled in the art, and in the case of some species of bacteria 10-15 unique strains may be sufficient and in the case of other species (Bifidobacterium longum pangenome for infant human gut microbiome) more than 20 unique strains are considered to be required to provide an adequate and representative pangenome. Methods of computation of the pangenome of a bacterial species are described below, and are exemplified herein with respect to the pangenome of Bifidobacterium longum (and also described in Arboleya et al BMC Genomics 2018: 19; 33). Pangenomes for other species of bacteria are described in the literature, for example Lactobacilli (O'Toole et al. Nature Communications (2015) 6:8322; DOI: 10.1038), Akkermansia (Guo et al. BMC Genomics (2017) 18:800), Rhodococcus equi (Anastasi et al (2016) Genome Biol. Evol. 8(10):3140-3148, and Bifidobacterium longum (O'Callaghan et al. BMC Genomics (2015) 16:832).
[0111] In this specification, the term representative of as applied to a microbiome-specific pangenome of a species of bacteria means a cohort of strains of the species of bacteria that together contain at least 60% of the total gene repertoire of the pangenome of that species as determined using the methods described herein. In one embodiment, the term means a cohort of strains of the species that together contain at least 70%, 80% or 90% of the total gene repertoire of the pangenome.
[0112] In this specification, the term capable of survival in a simulated intestinal environment as applied to a strain of bacteria should be understood to mean that the strain is resistant to low pH, resistant to bile, and/or capable of adhering to intestinal epithelial cells as defined in the Guidelines for the Evaluation of Probiotics in Food (FAO/WHO Working Group Report Apr. 30 and May 1 2002).
[0113] In this specification, the term variation in sugar utilisation profiles as applied to the cohort of isolated strains means that the cohort of strains is together capable of utilising a number of different sugars including human milk oligosaccharides and preferably plant derived carbohydrates. In one embodiment, the cohort of strains is together capable of utilising 3, 4 or all of xylo-oligosaccharides, arabinan, arabinoxylan, galactan, and fucosyllactose.
[0114] In this specification, the term intestinal origin as applied to a strain of bacteria means that the bacteria is derived from the intestine of a mammal.
[0115] Compositions and Foods
[0116] The invention also relates to a composition comprising a cohort of strains according to the invention, or a composition comprising one or more strains of the invention. The composition may be a pharmaceutical composition, or a food composition, or a dietary supplement composition. The term food refers to a man-made food product including beverages, food additives and food supplements. Examples of foods include dairy products such as milk, yoghurt, cheese, cheese food, dairy powders, probiotic formulations, infant formula powders, follow-on milk formula, food for special medicinal purposes, meat products, soups, vegetable products, fruit juices, fruit products, breads, confectionary, cakes, sports supplements, nutritional supplements and the like.
[0117] In one embodiment, the composition includes a probiotic material. In one embodiment, the composition comprises a prebiotic material.
[0118] Probiotic refers to a microbial cell preparation, typically a bacterial cell preparation, that exerts a beneficial effect on the health or well-being of a host. They are described in Salminen et al (Trends Food Sci. Technol. 1999:10 107-110).
[0119] Prebiotic refers to a material or composition that can promote the growth of probiotic microbes or bacteria, especially bacterial growth in the mammalian gastrointestinal tract. Examples include oligosaccharides, dietary fibres, or mixtures thereof. Exemplary prebiotics are described in WO2011/039176, pages 12 to 15.
[0120] The invention also relates to pharmaceutical compositions which comprise pharmaceutical carriers.
[0121] As used herein, the term pharmaceutical composition refers to a therapeutically effective amount of the strain of the invention, and a pharmaceutically acceptable carrier. In a specific embodiment, the term pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. In the case of the present invention, the term therapeutically effective amount should be taken to mean an amount of therapeutic which results in a clinically significant increase in proliferation of target cells, for example gut epithelial cells or skin epithelial cells.
[0122] As used herein, the term adjuvant means an agent that enhances the recipient's immune response to an immunogenic peptide or protein. Details of suitable adjuvant compositions are well known to those skilled in the art.
[0123] As used herein, the term pharmaceutically acceptable carrier refers to a diluent, adjuvant, excipient, or vehicle with which the Therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like.
[0124] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E. W. Martin. Such compositions will contain a therapeutically effective amount of the therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
[0125] In a preferred embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to, ease pain at the, site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
[0126] Pharmaceutical compositions formulated or configured for oral administration and gastric transit are known in the art and include those described below: [0127] Sathish et al (Int. J. Pharm. Sci. 2013; 258-269); [0128] Kushal et al (Int. Res. J. Pharm. 2013, 4(3)); [0129] Philip et al (Oman Med J. 2010, 25(2)); [0130] Polymers for controlled drug deliveryPeter Tarcha (CRC Press, 21 Nov. 1990); [0131] Pharmaceutical coating technologyMichael Aulton et al (Taylor & Francis 27 Oct. 1995); [0132] http://www.slideshare.net/Balimusale/oral-controlled-drug-delivery-system; [0133] European Patent No: 2418968 (Teagasc); and [0134] European Patent No: 2097072 (RCSI). [0135] Brayden et al. (European Journal of Pharmaceutical Sciences 79 (2015), 102-111. [0136] Tambuwala et al. (Journal of Controlled Release 217 (2015) 221-227. [0137] Zhang et al. Evaluation of alginate-whey protein microcapsules for intestinal delivery of lipophilic compounds in pigs (J. Sci. Food Agric. (2015). [0138] Lamprecht et al. (Journal of Controlled Release 104 (2005) 337-346. [0139] Hua et al. (Nanomedicine: Nanotechnology, Biology and Medicine 11 (2015) 1117-1132. [0140] Drug Delivery: Fundamentals and Applications (Chapter 7, Oral Drug Delivery, Hillary and Brayden)
[0141] As used herein, the term food refers to a man-made food product including beverages, food additives and food supplements. Examples of foods include dairy products such as milk, yoghurt, cheese, cheese food, dairy powders, probiotic formulations, infant formula powders, follow-on milk formula, food for special medicinal purposes, meat products, soups, vegetable products, fruit juices, fruit products, breads, confectionary, cakes, sports supplements, nutritional supplements and the like.
[0142] Dosage
[0143] It is preferable that the strain or composition is administered at least once per week over a treatment period of at least 4 weeks, and preferably at least 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18 or 20 week period. Preferably, the strain or composition is administered several times a week, and ideally once a day. Compositions of the invention generally comprise between 10.sup.3 and 10.sup.12 cfu of the strain of the invention per gram of dry weight of the composition. In one embodiment, the composition comprises10.sup.3 and 10.sup.12 cfu, or 10.sup.4 and 10.sup.12 cfu, or 10.sup.6 and 10.sup.10 cfu of the strain of the invention per gram of dry weight of the composition. A daily dose generally comprises between 10.sup.3 and 10.sup.12 cfu of the strain. In one embodiment, the daily dose comprises10.sup.3 and 10.sup.12 cfu, or 10.sup.4 and 10.sup.12 cfu, or 10.sup.6 and 10.sup.10 cfu of the strain.
[0144] Recombinant DNA Techniques and Reagents
[0145] In one aspect, the invention relates to isolated Bifidobacterium longum strains provided above. The invention also relates to mutants and variants of the strain that are substantially identical to the deposited strain and exhibit the same weight modification functionality. This includes strains that are genetically engineered to alter the genome of the deposited strain (i.e. strained engineered for heterologous expression of nucleic acid), and variants obtained through natural genetic alterations such as spontaneous mutations, adaption and serial passage the term engineered as applied to a cell means genetically engineered using recombinant DNA technology, and generally involves the step of synthesis of a suitable expression vector (see below) and then transfecting (i.e. stably or transiently) the expression vector into a host cell (generally stable transfection). The term heterologous expression refers to expression of a nucleic acid in a host cell that does not naturally have the nucleic acid. Insertion of the nucleic acid into the heterologous host is performed by recombinant DNA technology. The term heterologous in-situ expression as applied to a bacterium of the invention means that the bacterium is capable of expressing the nucleic acid in-situ in the mammalian gut, especially in-situ expression when adhered to the epithelial layer of the gut. As used herein, the term recombinant bacterium or transformed bacterium refers to a bacterium comprising an exogenous nucleic acid stably integrated into the cellular genome. In another embodiment, the present invention provides a cell comprising a non-integrated (i.e., episomal) exogenous nucleic acid, such as a plasmid, cosmid, phagemid, or linear expression element, which comprises a sequence coding suitable for expression of an exogenous nucleic acid. In other embodiments, the present invention provides a cell line produced by stably transfecting a host cell with a plasmid comprising an expression vector of the invention.
[0146] As used herein, the term expression vector may be any suitable vector, including chromosomal, non-chromosomal, and synthetic nucleic acid vectors (a nucleic acid sequence comprising a suitable set of expression control elements) suitable for heterologous expression of a nucleic acid. Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the Tad pilus encoding nucleic acid molecule is comprised in a naked DNA or RNA vector, including, for example, a linear expression element (as described in, for instance, Sykes and Johnston, Nat Biotech 12, 355-59 (1997)), a compacted nucleic acid vector (as described in for instance U.S. Pat. No. 6,077,835 and/or WO 00/70087), or a plasmid vector such as pBR322, pUC 19/18, or pUC 118/119. Such nucleic acid vectors and the usage thereof are well known in the art (see, for instance, U.S. Pat. Nos. 5,589,466 and 5,973,972). In one embodiment, the DNA comprises an expression control sequence.
[0147] In one embodiment, the vector is suitable for heterologous expression of a nucleic acid in a bacterial cell. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, 1989, J Biol Chem 264, 5503-5509), pET vectors (Novagen, Madison, Wis.) and the like. In one embodiment, the expression vector may also or alternatively be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be employed. Suitable vectors include, for example, vectors comprising constitutive or inducible promoters such as yeast alpha factor, alcohol oxidase and PGH (reviewed in: F. Ausubel et al., ed., 1987, Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York; and Grant et al., 1987, Methods in Enzymol 153, 516-544). In other embodiments, the expression vector is suitable for expression in baculovirus-infected insect cells. (Kost, T; and Condreay, J P, 1999, Current Opinion in Biotechnology 10 (5): 428-33.)
[0148] Expression control sequences are engineered to control and drive the transcription of genes of interest, and subsequent expression of proteins in various cell systems. Plasmids combine an expressible gene of interest with expression control sequences (i.e. expression cassettes) that comprise desirable elements such as, for example, promoters, enhancers, selectable markers, operators, etc. In an expression vector of the invention, Tad pilus-encoding nucleic acid molecules may comprise or be associated with any suitable promoter, enhancer, selectable marker, operator, repressor protein, polyA termination sequences and other expression-facilitating elements.
[0149] Promoter as used herein indicates a DNA sequence sufficient to direct transcription of a DNA sequence to which it is operably linked, i.e., linked in such a way as to permit transcription of the exogenous nucleotide sequence when the appropriate signals are present. The expression of a nucleotide sequence may be placed under control of any promoter or enhancer element known in the art. Examples of such elements include strong expression promoters (e.g., human CMV IE promoter/enhancer or CMV major IE (CMV-MIE) promoter, as well as RSV, SV40 late promoter, SL3-3, MMTV, ubiquitin (Ubi), ubiquitin C (UbC), and HIV LTR promoters). In some embodiments, the vector comprises a promoter selected from the group consisting of SV40, CMV, CMV-IE, CMV-MIE, RSV, SL3-3, MMTV, Ubi, UbC and HIV LTR.
[0150] Nucleic acid molecules of the invention may also be operably linked to an effective poly (A) termination sequence, an origin of replication for plasmid product in E. coli, an antibiotic resistance gene as selectable marker, and/or a convenient cloning site (e.g., a polylinker). Nucleic acids may also comprise a regulatable inducible promoter (inducible, repressable, developmentally regulated) as opposed to a constitutive promoter such as CMV IE (the skilled artisan will recognize that such terms are actually descriptors of a degree of gene expression under certain conditions).
[0151] Selectable markers are elements well-known in the art. Under the selective conditions, only cells that express the appropriate selectable marker can survive. Commonly, selectable marker genes express proteins, usually enzymes, that confer resistance to various antibiotics in cell culture. In other selective conditions, cells that express a fluorescent protein marker are made visible, and are thus selectable. Embodiments include beta-lactamase (bla) (beta-lactam antibiotic resistance or ampicillin resistance gene or ampR), bls (blasticidin resistance acetyl transferase gene), bsd (blasticidin-S deaminase resistance gene), bsr (blasticidin-S resistance gene), Sh ble (Zeocin resistance gene), hygromycin phosphotransferase (hpt) (hygromycin resistance gene), tetM (tetracycline resistance gene or tetR), neomycin phosphotransferase II (npt) (neomycin resistance gene or neoR), kanR (kanamycin resistance gene), and pac (puromycin resistance gene).
[0152] In certain embodiments, the vector comprises one or more selectable marker genes selected from the group consisting of bla, bls, BSD, bsr, Sh ble, hpt, tetR, tetM, npt, kanR and pac. In other embodiments, the vector comprises one or more selectable marker genes encoding green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), cyano fluorescent protein (CFP), enhanced cyano fluorescent protein (eCFP), or yellow fluorescent protein (YFP).
[0153] For the purposes of this invention, gene expression in eukaryotic cells may be tightly regulated using a strong promoter that is controlled by an operator that is in turn regulated by a regulatory protein, which may be a recombinant regulatory fusion protein (RFP). The RFP consists essentially of a transcription blocking domain, and a ligand-binding domain that regulates its activity. Examples of such expression systems are described in US20090162901A1, which is herein incorporated by reference in its entirety.
[0154] As used herein operator indicates a DNA sequence that is introduced in or near a gene in such a way that the gene may be regulated by the binding of the RFP to the operator and, as a result, prevents or allow transcription of the gene of interest, i.e. a nucleotide encoding a polypeptide of the invention. A number of operators in prokaryotic cells and bacteriophage have been well characterized (Neidhardt, ed., Escherichia coli and Salmonella; Cellular and Molecular Biology 2d. Vol 2 ASM Press, Washington D.C. 1996). These include, but are not limited to, the operator region of the LexA gene of E. coli, which binds the LexA peptide, and the lactose and tryptophan operators, which bind the repressor proteins encoded by the Lad and trpR genes of E. coli. These also include the bacteriophage operators from the lambda PR and the phage P22 ant/mnt genes, which bind the repressor proteins encoded by lambda cl and P22 arc. In some embodiments, when the transcription blocking domain of the RFP is a restriction enzyme, such as NotI, the operator is the recognition sequence for that enzyme. One skilled in the art will recognize that the operator must be located adjacent to, or 3 to the promoter such that it is capable of controlling transcription by the promoter. For example, U.S. Pat. No. 5,972,650, which is incorporated by reference herein, specifies that tetO sequences be within a specific distance from the TATA box. In specific embodiments, the operator is preferably placed immediately downstream of the promoter. In other embodiments, the operator is placed within 10 base pairs of the promoter.
[0155] In an exemplary cell expression system, cells are engineered to express the tetracycline repressor protein (TetR) and a protein of interest is placed under transcriptional control of a promoter whose activity is regulated by TetR. Two tandem TetR operators (tetO) are placed immediately downstream of a CMV-MIE promoter/enhancer in the vector. Transcription of the gene encoding the protein of interest directed by the CMV-MIE promoter in such vector may be blocked by TetR in the absence of tetracycline or some other suitable inducer (e.g. doxycycline). In the presence of an inducer, TetR protein is incapable of binding tetO, hence transcription then translation (expression) of the protein of interest occurs. (See, e.g., U.S. Pat. No. 7,435,553, which is herein incorporated by reference in its entirety.)
[0156] The vectors of the invention may also employ Cre-lox recombination tools to facilitate the integration of a gene of interest into a host genome. A Cre-lox strategy requires at least two components: 1) Cre recombinase, an enzyme that catalyzes recombination between two loxP sites; and 2) loxP sites (e.g. a specific 34-base pair by sequence consisting of an 8-bp core sequence, where recombination takes place, and two flanking 13-bp inverted repeats) or mutant lox sites. (See, e.g. Araki et al., 1995, PNAS 92:160-4; Nagy, A. et al., 2000, Genesis 26:99-109; Araki et al., 2002, Nuc Acids Res 30(19):e103; and US20100291626A1, all of which are herein incorporated by reference). In another recombination strategy, yeast-derived FLP recombinase may be utilized with the consensus sequence FRT (see also, e.g. Dymecki, S. M., 1996, PNAS 93(12): 6191-6196).
Exemplification
[0157] The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention.
[0158] Methods
[0159] Bacterial Strains and Growth Conditions
[0160] The twenty Bifidobacterium longum strains used (Table 3) had previously been isolated from infant faeces, except in two cases where the strains originate from adult faeces, and deposited in the APC Culture Collection (APC Microbiome Institute, Ireland). Strains coded as DPC were isolated by Barrett and coworkers (Barrett E, Ross R P, Fitzgerald G F, & Stanton C (2007) Rapid screening method for analyzing the conjugated linoleic acid production capabilities of bacterial cultures. Applied and environmental microbiology 73(7):2333-2337.). Bifidobacteria were routinely grown on de Man-Rogose-Sharpe (MRS) medium (Difco Laboratories, Detroit, Mich.) supplemented with 0.05% w/v cysteine-HCl (Sigma, St. Louis, Mo.) (MRSC) and incubated at 37 C. under anaerobic conditions (10% H2, 10% CO2, and 80% N2) in a chamber Mac 500 (Don Whitley Scientific, West Yorkshire, UK). For solid medium, 2% (w/v) agar (Oxoid, Basingstoke, UK) was added. Prior to each DNA extraction bacteria were sub-cultured twice and overnight cultures were used.
[0161] Genomic DNA Extraction, Sequencing and Data Assembly
[0162] DNA was isolated by using DNeasy Blood & Tissue Kit (Qiagen, Sussex, UK) following the manufacturer's instructions. Briefly, the overnight culture were centrifuged, washed with PBS (Sigma, St. Louis, Mo.) and incubated for 1 hour at 37 C. in an enzymatic lysis buffer with lysozyme (50 mg/ml) (Sigma, St. Louis, Mo.). Then, manufacturer's protocol was followed with the extra addition of RNAse (Sigma, St. Louis, Mo.). Sequencing and assembly was performed by Eurofins Genetic Services Ltd. (Ebersberg, Germany). The genomic DNA was sequenced on an Illumina MiSeq platform using chemistry v3 with paired-end sequencing. The draft genomes were de novo assembled following a pipeline that incorporates Velvet software (v1.2.10) (50) and a multi-kmer approach (51).
[0163] Contig Analysis and General Feature Prediction
[0164] A preliminary comparative genomic analysis (methods explained in the next section) was conducted to align the twenty B. longum APC/DPC genomes with all complete genomes of B. longum available in the NCBI public database at the moment of the study (Table 5), in order to select the most appropriate genome of reference for ordering the contigs prior to annotation. The alignment of the twenty B. longum draft genome sequences was performed against a number of closely related, fully sequenced, currently and publicly available B. longum genomes (Table 5). The B. longum JDM301, B. longum BBMN68, B. longum NCIMB 8809, B. longum DJ010A and B. longum GT15 genomes were thus selected as reference genomes so as to determine presumed contig order and orientation of each draft genome. MAUVE (v2.3.1) was used to reorder contigs based on the reference genome along with Artemis software (v.14) for visualization and manual editing of the beginning of each genome at the dnaA gene. Prediction of putative open reading frames (ORFs) was carried out using Prodigal predictor v2.50 software (http://prodigal.ornl.gov). Identified ORFs were then automatically annotated on the basis of BLASTP analysis (Altschul S F, Gish W, Miller W, Myers E W, & Lipman D J (1990) Basic local alignment search tool. Journal of molecular biology 215(3):403-410.). Functional assignment was performed against the non-redundant protein data base provided by the National Centre for Biotechnology (https://www.ncbi.nlm.nih.gov). Artemis was also used for inspecting and editing, where necessary, the ORF finding outputs and the associated BLASTP results. Moreover, the annotations were further refined and verified using the protein family (Pfam) (http://pfam.xfam.org) database. Glycosyl hydrolases were predicted and annotated based on similarity to the Carbohydrate-Active enZYmes (CAZy) (http://www.cazy.org/) database, Enzyme Commission number (EC) database (http://enzyme.expasy.org) using annot8r pipeline (http://www.nematodes.org/bioinformatics/annot8r) and Pfam alignments
[0165] Comparative Genomics
[0166] An all-versus-all BLASTP alignment (Altschul et al.) (50% identity; e-value 1e-4 cut-off) was performed on the extracted protein sequences from each strain. The BLAST output were used as an input for the clustering into protein families sharing the same function using the Markov Cluster Algorithm (MCL) with an inflation index of 2.5, as previously described (Bottacini F, et al. (2014) Comparative genomics of the Bifidobacterium breve taxon. BMC genomics 15:170.). The obtained gene families were classified as belonging to either the core or to the variable genome based on their presence in either all strains or in a subset of the investigated strains, respectively. In the orthologue extraction an additional filter for paralogues was applied by selecting only those families that were shown to contain a single protein member for each genome.
[0167] Pangenome Analyses
[0168] In order to predict the possible dynamic changes of genome size at the genus, the sizes of pangenome, core genome and unique gene were computed. For all the genomes used in this study, a pangenome calculation was done using PGAP (Zhao Y, et al. (2012) PGAP: pan-genomes analysis pipeline. Bioinformatics (Oxford, England) 28(3):416-418.), as previously described (Bottacini et al.). The ORF content of each genome was organized in functional gene clusters using the gene family (GF) method implemented in the PGAP pipeline. A pangenome profile and a core genome profile were built using all possible BLAST combinations for each genome being sequentially added.
[0169] Phylogenetic Analyses
[0170] The supertree computation was performed from the alignment of a set of orthologous genes obtained from a BLAST-based comparative approach as indicated above, with our APC/DPC strains plus all the B. longum genomes available in the NCBI public database at the time of writing. Each set of orthologous proteins was aligned using MUSCLE (Edgar R C (2004) MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC bioinformatics 5:113.) and phylogenetic trees were constructed using the maximum-likelihood in PhyML (Guindon S, et al. (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Systematic biology 59(3):307-321.). The resulting consensus tree was computed using the Consense module from Phylip package v3.69 using the majority rule method (http://evolution.genetics.washington.edu/phylip.html) and phylogenetic data were submitted to TreeBASE database (http://treebase.org/treebase-web/home.html).
[0171] Bifidobacterial growth on different carbohydrate sources and GTM associations For evaluation of growth on the different carbohydrates sources, strains were cultured on modified MRS (mMRS) medium manually prepared with the following composition: tryptone (10.0 g/L), yeast extract (5 g/L), beef extract (10.0 g/L), K2HPO4 (3.0 g/L), KH2PO4 (3.0 g/L), tri-ammonium citrate (2.0 g/L), pyruvic acid (0.2 g/L), cysteine-HCl (0.3 g/L), Tween-80 (1 mL), MgSO4.7H2O (0.575 g/L), MnSO4.4H2O (0.12 g/L), FeSO4.7H2O (0.034 g/L). Prior to autoclaving, mMRS medium was adjusted to pH 6.8.
[0172] Thirty-one carbohydrates were tested in this study (Table 7). Solutions of most of them were prepared by dissolution at 5% (w/v) in distilled water and sterilized by filtration (0.45 pm). They were then added to mMRS medium at final concentration of 0.5% (v/v). Since certain carbohydrates do not easily dissolve in water (pullulan, starch, amylopectin, inulin, arabinoxylan (wheat and rye), pectin galactan, mucin, galactan potato, pectin apple, arabinan, arabinogalactan, xylan and amylopectin), they were added directly to mMRS medium at a final concentration of 0.5% (w/v) and autoclaved at 121 C. for 15 min. For growth assays Bifidobacterium strains were cultured at 37 C. under anaerobic conditions in 10 ml of MRSC. Afterwards they were sub-cultured twice, first at 2% (v/v) into 10 mL of fresh medium during 8 hours, and then at 1% (v/v) into 10 mL of MRSC fresh medium overnight. The next day, strains were inoculated at 1% (v/v) into 10 mL of mMRS medium, previously supplemented with the soluble carbohydrates at a final concentration of 0.5% (v/v) or directly to mMRS with non-soluble carbohydrate autoclaved. Prior to inoculation both media were supplemented with cysteine-HCl at final concentration of 0.05%.
[0173] Growth of the bacterial strains was evaluated by optical density (OD600 nm) using UV-1280 spectrophotometer (Schimadzu Corporation, Kyoto, Japan). Measurements were taken manually over a 24-hour period at different time points: 0, 6, 9, 12 and 24 hours. mMRS supplemented with 0.05% w/v cysteine-HCl and without the addition of a carbohydrate source served as a negative control. Based on the OD600 nm values at 12 hours, bacterial growth was categorized as good growth (+) with an OD600 nm higher than 0.5 and moderate growth (+) with an OD600 nm between 0.4 and 0.5. A cut-off value of 0.4 OD600 nm was used to discriminate strains which were not able to grow in the given carbohydrates.
[0174] Following completion of the fermentation assessment of B. longum strains, an in-silico genotype/phenotype gene-trait matching (GTM) exercise was performed, correlating the presence/absence of genes obtained from comparative analysis with an observed phenotype (Table 4
[0175] The analysis was performed on a subset of gene families obtained after exclusion of the ones present in all the strains (core-genome). A further filter was included which excluded from the analysis that fraction of genes not involved in carbon sources utilization (e.g. transposases, R/M systems, CRISPR systems and prophages). After obtaining the final number of families a further reduction of the dataset in clusters of unique combination of occurrence was performed which allowed to obtain the genotype binary matrix (values 0 for absence and 1 for presence of a gene family). This obtained matrix contained 372 clusters on the rows and 20 strains on the columns. A second binary matrix was also similarly generated containing the fermentation profile and constituting the phenotype. In this case a value of 0 was assigned for OD600 <0.3 and value 1 for OD600 >0.4 (taken at 12 hours of growth curve). The phenotype binary matrix contained 16 carbohydrates on the rows and 20 strains on the columns, organized in the same order as in the genotype.
[0176] Following alignment of these two matrices the matching percentage between each row of the two matrices and the result was represented as a heatmap. The positive matches obtained from the analysis (>95% of match between genotype and phenotype) as well as the relative genomic surrounded regions were further inspected and compared with additional information retrieved from alternative available databases such as EC (Enzyme Classification) database (Bairoch A (2000) The ENZYME database in 2000. Nucleic Acids Research 28(1):304-305.) and PFAM (http://pfam.sanger.ac.uk) alignments, in order to further support the obtained results
[0177] Acid Tolerance
[0178] Bifidobacterium strains were tested for their ability to survive under acidic conditions according to the method by Liong and Shah (2005) with some minor adjustments as follows: Fresh cultures were grown for 48 hours in 10 ml mMRS, harvested by centrifugation (2263 g10 min) and then washed once in an equal amount of Dulbecco's Phosphate buffered Saline (PBS) (Sigma Aldrich, Wicklow, Ireland). Bacterial cells were then immediately resuspended in mMRS, adjusted to pH 2.5 with 5 M HCL, and then incubated under anaerobic conditions at 37 C. Aliquots were withdrawn at time point 0 and 30, 60, 90 and 120 minutes. A 10 fold dilution of each aliquot was performed in maximum recovery diluent (MRD) (Oxoid, Basingstoke, Hampshire, England) followed by pour plating onto RCM. The plates were incubated in 37 C. for 72 h under anaerobic conditions.
[0179] Bile Tolerance
[0180] To determine the ability of 29 B. longum strains to survive in the presence of 0.3% (w/v) bovine bile (Sigma Aldrich), bacterial cells from cultures were grown for 48 h in 10 ml mMRS, harvested by centrifugation (2263 g10 min) and washed once in PBS and were resuspended in mMRS media containing 0.3% (w/v) bovine bile and adjusted to pH 7.0 with 4 M NaOH. Bacteria were then incubated under anaerobic conditions. Serial 10-fold dilutions were made in MRD followed by pour plating on RCM agar. Bacterial growth was monitored by viable cell counts after an incubation period of 72 hours in 37 C under anaerobic conditions.
[0181] Adhesion to HT-29 Cells
[0182] Preparation of HT-29 Cells
[0183] HT-29 cells were obtained from ATCC, Virginia, USA. The cell line was cultured in McCoy's 5A medium (Sigma Aldrich) supplemented with 10% (v/v) fetal bovine serum (FBS) (Sigma Aldrich) and 1% of antibiotic mixture (penicillin-streptomycin; Sigma Aldrich), seeded (2105 cells/ml-1) into 12-well plates (Sigma Aldrich) and incubated for 71 days in 37 C., 5% CO2 until a confluent monolayer was reached (approx 2106 cells/ml-1). One day prior to assay, the media was changed to antibiotic-free McCoy's 5A medium supplemented with 2% FBS.
[0184] Adhesion Assay
[0185] Based on the ability of B. longum strains to tolerate various concentrations of acid and bile, the best surviving strains were tested for their ability to adhere to HT-29 cells. Prior to the adhesion assay, the confluent monolayers of HT-29 cells grown on 12-well plates were washed twice by swirling the plates with PBS, pre-warmed to 37 C. Fresh bacterial cultures, grown for 48 hours in mMRS, were harvested by centrifugation (2263g for 10 minutes), washed 3 times in PBS and then resuspended in 10 ml antibiotic-free and serum-free McCoy's 5A medium. Aliquots of bacterial suspension (approx. 8.5108-1109) were added to the HT-29 cells and incubated for 4 h at 37 C. in 5% CO2. After 4 h, cells were rinsed 3 times in order to remove all non-adherent bacteria and were then incubated together with 0.5 ml/well of trypsin-EDTA (Sigma Aldrich) in 37 C. for 15 minutes in order to release the cells. Thereafter, 1.5 ml/well of PBS was added to inactivate the trypsin. The level of adhesion was estimated by 10-fold dilution series in MRD and pour plating with RCM after incubation in 37 C. for 72 h. Each strain was tested in triplicate and assay was then repeated on an independent occasion.
[0186] Statistical Analysis
[0187] Data from acid tolerance assay and adhesion assay are presented as meanstandard error mean (SEM).
[0188] C-Section Model
[0189] C-section delivery has recently been associated with decreased colonization rates of Bifidobacterium, Bacteroides and Lactobacillus, with a decrease in diversity and richness of the microbiota. It has also been associated with an increased risk of developing obesity, type 1 diabetes, as well as immune disorders such as asthma or allergies. The Cesarian section (C-section) mouse model is a method of neonate/infant extraction from the mothers uterus, as opposed to a natural birth, or per vaginum. At full term (Gestation day 21-22 in a mouse), the mother is euthanized by cervical dislocation. To reduce bacterial contamination of the abdominal cavity, the abdominal skin is prepped by application of isopropyl alcohol and the abdominal skin is retracted. The abdomen is incised using a clean scalpel or sharp scissors. The uterus is removed and placed on sterile gauze sponges. To prevent hypothermia of the fetus in the uterus, the gauze sponges are placed on clean tissue and a heating pad is placed beneath to provide thermal support. The individual fetus is removed by cutting the uterus gently with a sharp scissors. The pups (Gestation day 21-22) are then expelled by gentle pressure, and the umbilical cord is cut approximately 1-3 mm distal to the umbilical attachment. Cotton swabs are used to tear the amniotic membrane and massage each pup gently until spontaneous breathing is noted. If a pup does not breathe spontaneously within 30-60 s after initiating physical stimulation, a cotton swab is applied gently to the oral mucous membrane of the affected animal for 1-3 s, and physical stimulation is continued until spontaneous respiration was observed. Additional pregnant females are allowed to deliver spontaneously and the litters are used as full-term natural delivery control and as foster mothers. The pups may be dried by smearing them with the bedding material in the cage of foster mother. This model allows the comparing and contrasting of differing microbiota expression profiles between the two forms of birth (in humans) and examine negative health effects as a result of an unnatural birthing process. It allows interventional therapies to be developed to mitigate the adverse health effects from the lack of an initial per vaginum microbiota inoculation. C-section delivered babies seem to be dominated by bacteria typically present on the skin and in the environment, predominantly from non-maternal sources.
TABLE-US-00003 TABLE 3 Bifidobacterium longum genomes sequenced and object of analysis in this study. GC No. of Source of Isolation No. of Genome content unique Genomes Subject Age Delivery Feeding Contigs ORFs Size (bp) (%) genes Reference B. longum APC 1461 A 27 w CS BM 38 2020 2424450 60.0 60 This study B. longum APC 1462 B 1 w ND BM 28 2016 2423258 60.2 4 This study B. longum APC 1464 B 1 w ND BM 32 1939 2351990 60.0 3 This study B. longum APC 1465 C 4 w ND BM 58 2076 2457685 59.6 6 This study B. longum APC 1466 C 27 w ND BM 52 2057 2425450 59.8 0 This study B. longum APC 1468 C 27 w ND BM 46 2033 2400632 60.1 12 This study B. longum APC 1472 E 1 w CS BM 51 1965 2369515 60.1 3 This study B. longum APC 1473 F 27 w CS BM 40 1901 2322545 59.8 6 This study B. longum APC 1476 G 27 w ND BM 49 2180 2538014 60.0 1 This study B. longum APC 1477 H 1 w ND BM 25 1775 2234263 59.8 0 This study B. longum APC 1478 H 4 w ND BM 22 1777 2228826 59.8 1 This study B. longum APC 1480 J 4 w ND BM 28 2103 2483224 59.9 6 This study B. longum APC 1482 D 1 w ND FM 73 1955 2342906 60.1 17 This study B. longum APC 1503 D 1 w ND FM 40 2211 2568177 59.7 23 This study B. longum APC 1504 I 1 w ND BM 52 1923 2315762 60.2 14 This study B. longum DPC 6316 K 25 y NA NA 33 2014 2399437 60.4 23 (49) B. longum DPC 6317 L 3 d NA NA 21 2009 2454098 60.2 8 (49) B. longum DPC 6320 M 64 y NA NA 26 1865 2335839 59.9 9 (49) B. longum DPC 6321 N 3 d NA NA 29 1967 2387834 59.9 24 (49) B. longum DPC 6323 O 4 d NA NA 53 2016 2407907 60.2 7 (49) w: weeks; y: years; d: days; CS: C-Section; ND: natural birth; BM: breast-milk; FM:formula milk; NA: not applicable
TABLE-US-00004 TABLE 4 Gene-trait matching with functions resulting from hierarchical clustering analysis. Gene Carbohydrate cluster Functions XYLO- A Hypothetical protein OLIGOSACCHARIDES Putative outer membrane protein (XOS) Galactoside O-acetyltransferase Alpha-L-arabinofuranosidase Beta-1,4-xylosidase ABC transporter permease ABC transporter permease Lactose ABC transporter substrate-binding protein LacI family transcriptional regulator NADH-dependent butanol dehydrogenase 1 ARABINAN B Endo-1,4-beta-xylanase Endo-1,4-beta-xylanase Beta-xylosidase Endo-1,4-beta-xylanase ARABINOXYLAN C ABC transporter, permease protein, probably fructooligosaccharide porter ABC transporter, permease protein, probably fructooligosaccharide porter ABC transporter, extracellular SBP, probably fructooligosaccharide porter Exo-alpha-L-arabinofuranosidase II Lipase LacI family transcriptional regulator Beta-xylosidase Beta-xylosidase Beta-xylosidase o endo-arabinase Alpha-arabinofuranosidase I GALACTAN D ATP-binding and permease modules of ABC transporter system Putative transport protein Solute-binding protein of ABC transporter system for sugars galactan metabolism ABC transporter permease ABC transporter permease Beta-galactosidase galactan metabolism Transcriptional regulator LacI family galactan metabolism Glycosyl hydrolases family 53 Endogalactanase galactan metabolism 25-diketo-D-gluconic acid reductase FUCOSYLLACTOSE E LacI family transcriptional regulator (FL) putative ABC transporter permease ABC transporter permease ABC transporter substrate binding component Mandelate racemase/muconate lactonizing protein Short chain dehydrogenase Hypothetical protein Dihydrodipicolinate synthase Predicted fucose isomerase Alpha-1,3/4-fucosidase Hypothetical protein (Glycosyl hydrolases family 95)
TABLE-US-00005 TABLE 5 Bifidobacterium longum genomes publicly available used for different analysis along the study. Used in Used in Used in Used in Used in Used in Status Reference Comparative the ORF prediction Pangenome Phylogenetics of the Genomes Code Selection Analysis prediction of GHs computation analysis genome* Reference B. longum JDM301 BLJ x x x x x x complete NCBI database B. longum BBMN68 BBMN68 x x x x x x complete NCBI database B. longum NCIMB 8809 B8809 x x x x x x complete NCBI database B. longum DJO10A BLD x x x x x x complete NCBI database B. longum GT15 BLGT x x x x x x complete NCBI database B. longum 105A BL105A x x x x x x complete NCBI database B. longum F8 BIL x x x x x x complete NCBI database B. longum NCC 2705 BL2705 x x x x x x complete NCBI database B. longum 157F BLIF x x x x x x complete NCBI database B. longum ATCC 15697 BLIJ x x x x x x complete NCBI database B. longum KACC 91563 BLNIAS x x x x x x complete NCBI database B. longum JCM1217 BLLJ x x x x x x complete NCBI database B. longum CCUG 30698 BBL306 x x x x x x complete NCBI database B. longum BXY01 BXY01 x x complete NCBI database B. longum BT1 BT1 x complete NCBI database B. longum BG7 BG7 x x complete NCBI database B. longum E18 AUYD01 x x complete NCBI database B. longum BT1 x draft NCBI database B. longum EK3 x draft NCBI database B. longum x draft NCBI database BIB1401242951 B. longum x draft NCBI database BIB1401272845b B. longum x draft NCBI database BIC1401212621a B. longum x draft NCBI database BIC1401272845a B. longum x draft NCBI database BIC1401212621b B. longum x draft NCBI database BIC1401111250 B. longum x draft NCBI database BIC1307292462 B. longum x draft NCBI database BIC1206122787 B. longum CMCC P001 x draft NCBI database B. longum AGR 2137 x draft NCBI database B. longum DSM 20211 x draft NCBI database B. longum LMG 21814 x draft NCBI database B. longumVMKB44 x draft NCBI database B. longum BLO12 x draft NCBI database B. longum 72B x draft NCBI database B. longum 9 x draft NCBI database B. longum 7 x draft NCBI database B. longum 17-1B x draft NCBI database B. longum 1-6B x draft NCBI database B. longum 44B x draft NCBI database B. longum 379 x draft NCBI database B. longum 1-5B x draft NCBI database B. longum LMG 13197 x draft NCBI database B. longum D2957 x draft NCBI database B. longum CECT 7210 x draft NCBI database B. longum CECT 7347 x draft NCBI database B. longum MC-42 x draft NCBI database B. longum CMW7750 x draft NCBI database B. longum CCUG 52486 x draft NCBI database B. longum EK13 x draft NCBI database B. longum ATCC 55813 x draft NCBI database B. longum 7-1B x draft NCBI database B. longum 35B x draft NCBI database B. longum 2-2B x draft NCBI database B. longum EK5 x draft NCBI database *At the moment of the analysis.
TABLE-US-00006 TABLE 6 EC annotation numbers of the in silico glycosyl hydrolases predicted. GH EC Enzyme family family numbers (Ara-f)(3)-Hypbeta-L-arabinobiosidase GH121 3.2.1.187 (Ara-f)(3)-Hypbeta-L-arabinobiosidase GH43 3.2.1.187 1,4-alpha-glucanbranchingenzyme GH13 2.4.1.18 Alpha-amylase GH13 3.2.1.1 Alpha-D-xylosidexylohydrolase GH31 3.2.1.177 Alpha-galactosidase GH36 3.2.1.22 Alpha-galactosidase GH27 3.2.1.22 Alpha-glucosidase GH13 3.2.1.20 Alpha-L-fucosidase GH95 3.2.1.51 Alpha-mannosidase GH38 3.2.1.24 Amylosucrase GH13 2.4.1.4 Arabinanendo-1,5-alpha-L- GH43 3.2.1.99 arabinosidase Arabinogalactanendo-beta-1,4- GH53 3.2.1.89 galactanase Beta-glucosidase GH1 3.2.1.38 Beta-fructofuranosidase GH32 3.2.1.26 Beta-galactosidase GH42 3.2.1.23 Beta-galactosidase GH2 3.2.1.23 Beta-glucosidase GH3 3.2.1.21 Beta-glucosidase GH1 3.2.1.21 Beta-N-acetylhexosaminidase GH3 3.2.1.52 Beta-N-acetylhexosaminidase GH20 3.2.1.52 Cyclomaltodextrinase GH13 3.2.1.54 Dextransucrase GH25 2.4.1.5 Endo-1,4-beta-xylanase GH5 3.2.1.8 Endo-alpha-N-acetylgalactosaminidase GH101 3.2.1.97 Galactosylceramidase GH59 3.2.1.46 Gellanlyase GH43 4.2.2.25 Glucan1,3-beta-glucosidase GH5 3.2.1.58 Glucanendo-1,6-beta-glucosidase GH30 3.2.1.75 Isoamylase GH13 3.2.1.68 L-arabinofuranosidase GH51 3.2.1.55 L-arabinofuranosidase GH43 3.2.1.55 L-arabinofuranosidase GH127 3.2.1.185 Oligo-1,6-glucosidase GH13 3.2.1.10 Pullulanase GH13 3.2.1.41 Xylan1,4-beta-xylosidase GH43 3.2.1.37
TABLE-US-00007 TABLE 7 Carbohydrates used for in vitro assays. Carbohydrates Company Source 2-O-Fucosyllactose Glycom 3-O-Fucosyllactose Glycom Amylopectin Fluka Maize Arabinan Megazyme Sugar Beet Arabinogalactan Megazyme Larch Wood L-Arabinose Sigma Arabinoxylan (rye) Megazyme Rye Flour Arabinoxylan (wheat) Megazyme Wheat Flour FOS Fucose Galactan Megazyme Potato D-Galactose Carbosynth D-Glucose monohydrate Sigma GOS Inulin Sigma -Lactose monohydrate Sigma Lacto-N-neotretaose Glycom D-Mannose Sigma Mucin N-acetyl glucosamine N-acetyl mannosamine N-acetyl galactosamine Pectic Galactan Megazyme Potato Pectin Sigma Apple Pullulan Sialic acid Starch Sigma Sucrose Sigma Xylo-oligosaccharide P95 Longlife Xylan Megazyme Beechwood D-Xylose Fluka
Equivalents
[0190] The foregoing description details presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto.