METHODS OF USE & COMPOSITIONS OF ANTIBIOTIC ALTERNATIVES IN LIVESTOCK

Abstract

This invention relates to the development of products that are alternatives to antibiotics with medically important human therapeutic uses for livestock meat and protein production efficiency. These products will be used to replace antibiotics used to rapidly increase lean muscle mass with specific feed regimens. The present invention is for the identification of non-human antibiotic products to be used with different feed regimens for livestock growth and meat promotion that include prebiotic supplements and keystone species probiotics that produce the desired growth promotion phenotypic effects. The present invention includes the identification of enterotypes that can be used as targets for recapitulation by compounds that are screened to produce the desired phenotype.

Claims

1-5. (canceled)

6. A method for determining keystone bacterial species in the gut of a livestock animal comprising the steps of: determining one or more natural bacterial enterotypes of a livestock; administering one or more antibiotics to the livestock; identifying one or more antibiotic administration-induced bacterial enterotypes; and comparing the antibiotic administration-induced bacterial enterotypes to the natural bacterial enterotypes to identify one or more keystone bacterial species.

7. The method of claim 6 wherein each of the bacterial enterotypes represent the gastrointestinal microbiomes in one or more of the stomach, rumen, small intestines, or colon; or wherein the one or more antibiotics are selected from the group consisting of aminoglycosides, cephalosporins, cyclic peptides, diterpines, fluoroquinolones, hydrazines, ionophores, lincosamides, macrolides, organoarsenics, nitroimidazoles, penicillins, streptogramins, and sulfonamides; or wherein the antibiotics are co-administered with a feed regimen.

8. The method of claim 6, further comprising the step of defining a probiotic containing at least one of the keystone bacterial species for administration to the livestock.

9. The method of claim 6, wherein the livestock is selected from the group consisting of cattle, poultry, swine, fish, sheep, goat, venison, and bison; or wherein each enterotype is determined by one or more of metagenomic, proteomic, transcriptomic, and metabolomic analyses; or wherein the one or more keystone bacterial species are associated with a phenotypic effect on the livestock and wherein the phenotypic effect is deposition of lean muscle rather than fat.

10. The method of claim 8, further comprising administering the probiotic to the livestock; or optionally wherein the probiotic promotes growth by adding lean muscle mass for meat production in the livestock; or wherein the probiotic containing one or more keystone bacterial species produces metabolic end products that result in the phenotypic effect of deposition of lean muscle rather than fat when administered to the livestock.

11. The method of claim 6, wherein the at least one keystone bacterial species is used as a biological factory in a bioreactor to produce prebiotic metabolic products.

12. The method of claim 11, further comprising the step of administering the prebiotic metabolic products to the livestock, wherein the livestock demonstrate the phenotypic effect of deposition of lean muscle rather than fat.

13. A screening method for recapitulating a target bacterial enterotype in the gut of a livestock animal comprising the steps of: determining a natural bacterial enterotype of a livestock; administering one or more antibiotics to the livestock to yield a desired phenotype; identifying an antibiotic administration-induced target bacterial enterotype associated with the desired phenotype; and screening for one or more non-antibiotic compounds that recapitulate the target bacterial enterotype.

14. The method of claim 13, wherein the one or more antibiotics are selected from the group consisting of aminoglycosides, cephalosporins, cyclic peptides, diterpines, fluoroquinolones, hydrazines, ionophores, lincosamides, macrolides, organoarsenics, nitroimidazoles, penicillins, streptogramins, and sulfonamides.

15. The method of claim 13 wherein the bacterial enterotype represents the gastrointestinal microbiome in one or more of the stomach, rumen, small intestines, or colon; or optionally wherein the desired phenotype is deposition of lean muscle rather than fat in the livestock.

16. The method of claim 13, further comprising the step of administering the one or more non-antibiotic compounds as a prebiotic to the livestock.

17. The method of claim 13, wherein the administration of the prebiotic results in deposition of lean muscle rather than fat in the livestock; or optionally wherein the livestock is selected from the group consisting of cattle, poultry, swine, fish, sheep, goat, venison, and bison.

18. A method for optimizing the production efficiency in a livestock animal by administering a metabolic composition, the method comprising the steps of: determining one or more natural bacterial enterotypes of a livestock; administering one or more antibiotics to the livestock; identifying one or more antibiotic administration-induced bacterial enterotypes; comparing the antibiotic administration-induced bacterial enterotypes to the natural bacterial enterotypes to identify one or more keystone bacterial species; and administering a metabolic composition to the livestock.

19. The method of claim 18, wherein the metabolic composition comprises one or more of an identified keystone bacterial species, a probiotic containing one or more identified keystone bacterial species, metabolic products from the one or more identified keystone bacterial species, or combinations thereof; or optionally wherein the optimized production efficiency results in the deposition of lean muscle rather than fat in the livestock.

20. A metabolic composition for increasing production efficiency in a livestock animal comprising one or more of a keystone bacterial species, a probiotic containing one or more keystone bacterial species, metabolic products from the one or more identified keystone bacterial species, or combinations thereof.

21. The composition of claim 21, further comprising components selected from the group consisting of vitamins, proteins, fatty acids, sugars, and compounds screened to modulate microbiota in various gastrointestinal microbiomes.

22. A screening-based method according to claim 13 of producing lean muscle in a livestock animal comprising the steps of: determining a natural bacterial enterotype of a livestock; administering one or more antibiotics to the livestock to yield a desired phenotype; identifying an antibiotic administration-induced target bacterial enterotype associated with the desired phenotype; screening for one or more non-antibiotic compounds that recapitulate the target bacterial enterotype; and administering the one or more non-antibiotic compounds to obtain the desired phenotype.

23. The method of claim 23 wherein the non-antibiotic compounds are administered in a tablet, in feed pellets, in a formulation administered simultaneously or nearly simultaneously in feed, water, or milled feed combination, in a probiotic with colony formation units deposited on feed, or in a formulation administered at separate times of day from feed.

24. A prebiotic composition for optimizing the deposition of lean muscle in a livestock animal comprising one or more non-antibiotic compounds that recapitulate a gut microbiome enterotype associated with the deposition of lean muscle rather than fat in the livestock.

25. The composition of claim 33, further comprising components selected from the group consisting of vitamins, proteins, fatty acids, and sugars.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0046] All publications, patents and patent applications, including any drawings and appendices therein are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent or patent application, drawing, or appendix was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0047] Where appropriate, any one or more of the other active agents may be in the form of a pharmaceutically acceptable salt.

[0048] Suitable pharmaceutically acceptable salts include, but are not limited to, salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulfuric, phosphoric, nitric, carbonic, boric, sulfuric, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, malic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benzenesulphonic, salicylic, sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.

[0049] It is intended that the aspects and embodiments of this invention encompasses all solid forms, including amorphous forms, as well as crystalline forms, and polymorphs thereof.

[0050] Throughout this specification the term ‘in combination’ means that one or more other actives are both administered to the fish or livestock animal over the same period of treatment. They may be administered together, i.e. at the same time. In this case they may be administered in a single formulation, (e.g. as a single tablet or capsule or feed pellets) or in separate formulations administered simultaneously or nearly simultaneously in feed or water or milled feed combination, or probiotic with colony formation units (CFUs) deposited on feed. Alternatively, they may be administered at separate times of day.

[0051] The combinations of the invention provide benefits which are at least additive compared to the use of either agent alone. In many embodiments, the combinations are something more than additive, e.g. synergistic, compared to the use of either agent alone.

[0052] The definition of the term ‘treatment’ in this specification encompasses both growth promotion and deposition of lean muscle mass for production efficiency as well as this purpose plus disease treatment, prophylaxis and prevention (i.e. reducing or eliminating the risk of contracting the disease). As well as meaning curing animals of the disease, ‘treatment’ also includes preventing the onset of symptoms, controlling (e.g. by slowing or eliminating) progression of disease, preventing the spread of the disease to other parts of the body and/or to other persons, reducing the spread of the disease and other facets of veterinary practice which will be readily understood by the person skilled in the art to fall within the meaning of the term ‘treatment’.

[0053] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

FORMULATIONS

[0054] For the above-mentioned therapeutic uses, the dosage administered will, of course, vary with the compound employed, the mode of administration, the treatment desired and the production purpose or disorder indicated.

[0055] Compositions may be administered systemically, e.g. by oral administration in the form of mixed or milled feeds, tablets, capsules, syrups, powders or granules; or by parenteral administration in the form of a sterile solution, suspension or emulsion for injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion); or by rectal administration in the form of suppositories.

[0056] For oral administration, one or more active agents may be admixed with an adjuvant or a carrier, for example, lactose, saccharose, sorbitol, mannitol; a starch, for example, potato starch, corn starch or amylopectin; a cellulose derivative; a binder, for example, gelatine or polyvinylpyrrolidone; and/or a lubricant, for example, magnesium stearate, calcium stearate, polyethylene glycol, a wax, paraffin, and the like, and then compressed into tablets. If coated tablets are required, the cores, prepared as described above, may be coated with a concentrated sugar solution which may contain, for example, gum arabic, gelatine, talcum and titanium dioxide. Alternatively, the tablet may be coated with a suitable polymer dissolved in a readily volatile organic solvent.

[0057] For the preparation of soft gelatine capsules, one or more active agents may be admixed with, for example, a vegetable oil or polyethylene glycol. Hard gelatine capsules may contain granules of the compound using either the above-mentioned excipients for tablets. Also liquid or semisolid formulations of the compound of the invention may be filled into hard gelatine capsules. Liquid preparations for oral application may be in the form of sprayed depositions on solid feeds, syrups or suspensions, for example, solutions containing the compound of the invention, the balance being sugar and a mixture of ethanol, water, glycerol and propylene glycol. Optionally such liquid preparations may contain colouring agents, flavouring agents, sweetening agents (such as saccharine), preservative agents and/or carboxymethylcellulose as a thickening agent or other excipients known to those skilled in art.

Methods of Bioinformatics

[0058] The bioinformatics of the present invention employs for metagenomic microbiota analysis a base of both 18S ribosomal RNA fingerprinting as well as whole genome shotgun sequencing (WGS) analysis of metagenomic data. The WGS in the present invention includes techniques that use iterative scanning of small motifs, including 12 amino-acid (36 bp) motifs, that are then compared for a comprehensive taxonomy against all 280,000 named organisms in public databases and are benchmarked against other pipelines (e.g., MetaPhlan, Phylosift, GOTTCHA and Kraken).

[0059] The present invention includes metagenomic, proteomic, transcriptomic, and metabolomic analyses of the various GI microbiomes (e.g., stomach, rumen, small intestines, colon) for each livestock production species, including, but not limited to cattle, poultry (including raised game birds), swine, fish (including farmed fish), venison, bison, sheep, and goats, both before and after administration of an antibiotic, including, but not limited to, those in the class of aminoglycosides, cephalosporins, cyclic peptides, diterpines, fluoroquinolones, hydrazines, ionophores, lincosamides, macrolides, organoarsenics, nitroimidazoles, penicillins, streptogramins, and sulfonamides in combination with various feeds used for those production species.

[0060] The present invention also includes metagenomic, proteomic, transcriptomic, and metabolomic analyses of the various GI microbiomes (e.g., stomach, rumen, small intestines, colon) for each livestock production species, including, but not limited to cattle, poultry (including raised game birds), swine, fish (including farmed fish), venison, bison, sheep, and goats, both before and after administration of combinations of antibiotics, including, but not limited to, those in the class of aminoglycosides, cephalosporins, cyclic peptides, diterpines, fluoroquinolones, hydrazines, ionophores, lincosamides, macrolides, organoarsenics, nitroimidazoles, penicillins, streptogramins, and sulfonamides in combination with various feeds used for those production species.

[0061] The present invention also includes metagenomic, proteomic, transcriptomic, and metabolomic analyses of the various GI microbiomes (e.g., stomach, rumen, small intestines, colon) for each livestock production species, including, but not limited to cattle, poultry (including raised game birds), swine, fish (including farmed fish), venison, bison, sheep, and goats, both before and after administration of combinations of antibiotics and antiprotozoal agents, including, but not limited to, those in the class of coccidiostats, aminoglycosides, cephalosporins, cyclic peptides, diterpines, fluoroquinolones, hydrazines, ionophores, lincosamides, macrolides, organoarsenics, nitroimidazoles, penicillins, streptogramins, and sulfonamides in combination with various feeds used for those production species.

[0062] The present invention includes the different enterotypes (including their use as informational recipes) associated with feed regimens determined by metagenomic analysis of the various GI microbiomes (e.g., stomach, rumen, small intestines, colon) for each livestock production species, including, but not limited to cattle, poultry (including raised game birds), swine, fish (including farmed fish), venison, bison, sheep, and goats, both before and after administration of an antibiotic, including, but not limited to, those in the class of aminoglycosides, cephalosporins, cyclic peptides, diterpines, fluoroquinolones, hydrazines, ionophores, lincosamides, macrolides, organoarsenics, nitroimidazoles, penicillins, streptogramins, and sulfonamides used for those production species.

[0063] The present invention also the different enterotypes (including their use as informational recipes) associated with feed regimens determined by metagenomic analysis of the various GI microbiomes (e.g., stomach, rumen, small intestines, colon) for each livestock production species, including, but not limited to cattle, poultry (including raised game birds), swine, fish (including farmed fish), venison, bison, sheep, and goats, both before and after administration of combinations of antibiotics, including, but not limited to, those in the class of aminoglycosides, cephalosporins, cyclic peptides, diterpines, fluoroquinolones, hydrazines, ionophores, lincosamides, macrolides, organoarsenics, nitroimidazoles, penicillins used for those production species.

[0064] The present invention also includes the different enterotypes (including their use as informational recipes) associated with feed regimens determined by metagenomic analysis of the various GI microbiomes (e.g., stomach, rumen, small intestines, colon) for each livestock production species, including, but not limited to cattle, poultry (including raised game birds), swine, fish (including farmed fish), venison, bison, sheep, and goats, both before and after administration of combinations of antibiotics and antiprotozoal agents, including, but not limited to, those in the class of coccidiostats, aminoglycosides, cephalosporins, cyclic peptides, diterpines, fluoroquinolones, hydrazines, ionophores, lincosamides, macrolides, organoarsenics, nitroimidazoles, penicillins, streptogramins, and sulfonamides used for those production species.

[0065] The present invention includes the keystone species in enterotypes, and products derived from their administration or administration of their metabolic products, determined by comparison of the metagenomic, proteomic, transcriptomic, and metabolomic analyses of the various GI microbiomes (e.g., stomach, rumen, small intestines, colon), analysed in sequence of the passage of food in the digestive tract (first stomach or rumen, then small intestines, then colon) of the different livestock production species, including, but not limited to cattle, poultry (including raised game birds), swine, fish (including farmed fish), venison, bison, sheep, and goats, both before and after administration of an antibiotic, including, but not limited to, those in the class of aminoglycosides, cephalosporins, cyclic peptides, diterpines, fluoroquinolones, hydrazines, ionophores, lincosamides, macrolides, organoarsenics, nitroimidazoles, penicillins, streptogramins, and sulfonamides in combination with various feeds used for those production species, to determine a metabolic code for digestion through the solving of simultaneous equations with variables being bacterial strains with genes encoding metabolic products for food breakdown.

[0066] The present invention encompasses combinations of enterotype probiotic products or artificially optimized metabolic solutions (Bolsols) that are non-naturally occurring and involving inventive steps as contemplated in the intellectual property scheme described in Exhibit 1: Novel IP Regime: Patenting Microbial Ecologies and Exhibit 2: Metabols and Bolsols to Process Food or Feed.

[0067] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings, such as attached FIG. 1 Syntheses), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0068] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.