EP3285587A1 - Zusammensetzungen und verfahren zur erhöhung der empfindlichkeit von bakterien gegenüber antibiotika - Google Patents

Zusammensetzungen und verfahren zur erhöhung der empfindlichkeit von bakterien gegenüber antibiotika

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Publication number
EP3285587A1
EP3285587A1 EP16783881.2A EP16783881A EP3285587A1 EP 3285587 A1 EP3285587 A1 EP 3285587A1 EP 16783881 A EP16783881 A EP 16783881A EP 3285587 A1 EP3285587 A1 EP 3285587A1
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EP
European Patent Office
Prior art keywords
composition
agent
agglomeration
antibacterial agent
individual
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP16783881.2A
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English (en)
French (fr)
Inventor
Ping-Chuan Christina TSAI
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Epibiome Inc
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Epibiome Inc
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Publication of EP3285587A1 publication Critical patent/EP3285587A1/de
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/429—Thiazoles condensed with heterocyclic ring systems
    • A61K31/43—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4436—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a heterocyclic ring having sulfur as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/542—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/545—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine
    • A61K31/546—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine containing further heterocyclic rings, e.g. cephalothin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4873—Cysteine endopeptidases (3.4.22), e.g. stem bromelain, papain, ficin, cathepsin H
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/22—Cysteine endopeptidases (3.4.22)
    • C12Y304/22002—Papain (3.4.22.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/22—Cysteine endopeptidases (3.4.22)
    • C12Y304/22004—Bromelain (3.4.22.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10111—Myoviridae
    • C12N2795/10132—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10111—Myoviridae
    • C12N2795/10133—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory

Definitions

  • the present invention relates to the field of microbiology, antibiotics, human and veterinary medicine, and food safety.
  • the planet is populated by an estimated 5x10 bacteria, which represent more biomass than all plants and animals combined. These bacteria perform many beneficial functions, including degradation of decaying organic material, nitrogen fixation, antibiotic production, vitamin synthesis, and fermentation. Through these activities, bacteria participate in many mutualistic and commensal symbiotic relationships with members of other kingdoms.
  • bacteria also engage in parasitic relationships with other organisms, and when this occurs, the bacteria are known as pathogens. These pathogens threaten human and animal health by causing infection, reducing agricultural output, and compromising food safety.
  • preservatives and small-molecule antibiotics as chemical methods to combat bacterial pathogens.
  • Disinfectants and preservatives are useful for preventing infections, but once infection has occurred, antibiotics are typically employed.
  • the effectiveness of small-molecule antibiotics is increasingly diminished by their overuse, especially by their prophylactic and unnecessary use, creating a positive selection pressure for antibiotic-resistant bacteria.
  • These resistant bacteria can transfer resistance genes to other bacteria through horizontal gene transfer.
  • Bacterial transplant strategies e.g. , fecal transplants, probiotics are also being explored.
  • Agglomeration is the result of the crosslinking of bacteria cells.
  • biofilms for example, organisms adhere to one another due to secretion of extracellular polymers by one or more members of the community.
  • crosslinking molecules need not be supplied by the bacteria themselves.
  • agglutination for example, antibodies from an infected host crosslink the pathogens that display the corresponding antigens so that they are more readily eliminated as a clump by phagocytosis.
  • antibody-mediated agglutination of pathogens can make them less susceptible to antibiotics; thus, the immune response can hinder, rather than complement, antibiotic therapy.
  • compositions and methods for increasing the susceptibility of bacteria to antibiotic therapy find use in a variety of different applications, including human and veterinary medicine.
  • a composition comprising a de- agglomeration agent and an antibacterial agent, wherein the de-agglomeration agent reduces agglomeration (such as agglutination) of bacteria cells, such as Staphylococcus aureus, Group C or Group G Streptococcus (such as Streptococcus uberis, or Streptococcus dysgalactiae), Pseudomonas aeruginosa, Clostridium difficile, Bacillus spp., and Clostridium perfringens.
  • agglomeration such as agglutination
  • bacteria cells such as Staphylococcus aureus, Group C or Group G Streptococcus (such as Streptococcus uberis, or Streptococcus dysgalactiae)
  • the antibacterial agent comprises a small-molecule antibiotic.
  • the small- molecule antibiotics is selected from the group consisting of pirlimycin, ceftiofur, desfurolyceftiofur, amikacin, ampicillin, dihydrostreptomycin, flunixin, gentamicin, neomycin, tilmicosin, oxytetracycline, penicillin, sulfadiazine, sulfadimethoxine, sulfamethazine, sulfamethoxazole, sulfathiazole, tetracycline, tylosin, phenoxymethylpenicillin, flucloxacillin, amoxicillin, amoxicillin-clavulanate, clarithromycin, trimethoprim-sulfamethoxazole, nafcillin, oxacillin
  • the antibacterial agent comprises a quorum sensing signal molecule.
  • the antibacterial agent comprises a bacteriolytic enzyme.
  • the antibacterial agent comprises a phage-derived protein.
  • the phage-derived protein is selected from the group consisting of lysozyme, endolysin, lysin, holin, tail fiber protein, and talocin.
  • the antibacterial agent comprises a bacteriophage.
  • the bacteriophage is bacteriophage K.
  • the antibacterial agent comprises a cocktail of bacteriophages.
  • the antibacterial agent comprises a bacteriocin.
  • the bacteriocin is a pyocin.
  • the bacteriocin is a nisin.
  • the de- agglomeration agent is papain.
  • the antibacterial agent is bacteriophage K.
  • the antibacterial agent is a cocktail of bacteriophages.
  • the antibacterial agent is pirlimycin.
  • the de- agglomeration agent is bromelain.
  • the antibacterial agent is ampicillin.
  • the antibacterial agent is bacteriophage K.
  • the antibacterial agent is a cocktail of bacteriophages.
  • the antibacterial agent is pirlimycin.
  • the antibacterial agent is cephapirin.
  • a composition comprising a de-agglomeration agent and a probiotic composition, wherein the de-agglomeration agent reduces agglomeration (such as agglutination) of bacteria cells, such as Staphylococcus aureus, Group C or Group G Streptococcus (such as Streptococcus uberis, or Streptococcus dysgalactiae), Pseudomonas aeruginosa, and Clostridium difficile.
  • the probiotic composition comprises lactobacillus or bifidobacterium.
  • the composition further comprises a bacteriophage.
  • the composition further comprises a cocktail of bacteriophages.
  • the de- agglomeration agent cleaves immunoglobulin (such as IgG, IgA, IgM, IgE, or IgD, or subclasses thereof), protein A, protein G, fibrinogen, or ClfA.
  • immunoglobulin such as IgG, IgA, IgM, IgE, or IgD, or subclasses thereof
  • the de- agglomeration agent comprises a protease.
  • the protease is selected from the group consisting of papain, bromelain, IdeS, pepsin, ficain, actinidin, cathepsin-B like protease, plasmin and combinations thereof.
  • the protease is papain.
  • the protease is bromelain.
  • the protease is plasmin.
  • the composition further comprises an adjuvant composition.
  • the adjuvant composition comprises a chelating agent.
  • the adjuvant composition comprises a reducing agent.
  • the adjuvant composition comprises a chelating agent (e.g., EDTA) and a reducing agent (e.g., cysteine).
  • the adjuvant composition comprises EDTA and cysteine.
  • a method for increasing the susceptibility of bacteria cells in a target composition to an antibacterial agent comprising adding to the target composition an effective amount of a de- agglomeration agent, wherein the de-agglomeration agent reduces agglomeration (such as agglutination) of the bacteria cells.
  • the de- agglomeration agent cleaves immunoglobulin (such as IgG, IgA, IgM, IgE, or IgD, or subclasses thereof), protein A, protein G, fibrinogen, or ClfA.
  • the de-agglomeration agent comprises a protease.
  • the protease is selected from the group consisting of papain, bromelain, IdeS, pepsin, ficain, actinidin, cathepsin-B like protease, plasmin and combinations thereof.
  • the protease is papain.
  • the protease is bromelain.
  • the protease is plasmin.
  • the method further comprises adding to the target composition an effective amount of an adjuvant composition.
  • the adjuvant composition comprises a chelating agent.
  • the adjuvant composition comprises a reducing agent.
  • the adjuvant composition comprises a chelating agent (e.g. , EDTA) and a reducing agent (e.g. , cysteine).
  • the adjuvant composition comprises EDTA and cysteine.
  • the antibacterial agent comprises a small-molecule antibiotic.
  • the small-molecule antibiotic is selected from the group consisting of pirlimycin, ceftiofur, desfurolyceftiofur, amikacin, ampicillin, dihydrostreptomycin, flunixin, gentamicin, neomycin, tilmicosin, oxytetracycline, penicillin, sulfadiazine, sulfadimethoxine, sulfamethazine, sulfamethoxazole, sulfathiazole, tetracycline, tylosin, phenoxymethylpenicillin, flucloxacillin, amoxicillin, amoxicillin-clavulanate, clarithromycin, trimethoprim- sulfamethoxazole, nafcillin,
  • the small-molecule antibiotic is ampicillin. In some embodiments, the small-molecule antibiotic is pirlimycin. In some embodiments, the small-molecule antibiotic is cephapirin. [0027] In some embodiments according to any one of the above methods for increasing the susceptibility of bacteria cells, the antibacterial agent comprises a quorum sensing signal molecule.
  • the antibacterial agent comprises a bacteriolytic enzyme.
  • the antibacterial agent comprises a phage-derived protein.
  • the phage-derived protein is selected from the group consisting of lysozyme, endolysin, lysin, holin, tail fiber protein, and talocin.
  • the antibacterial agent comprises a bacteriophage.
  • the bacteriophage is bacteriophage K.
  • the antibacterial agent comprises a cocktail of bacteriophages.
  • the antibacterial agent comprises a bacteriocin.
  • the bacteriocin is a pyocin.
  • the bacteriocin is a nisin.
  • the bacteria cells are of a pathogenic bacterium species selected from the group consisting of Staphylococcus aureus, Pseudomonas aeruginosa, Clostridium difficile and Streptococcus.
  • the bacteria cells are Staphylococcus aureus, Group C or Group G Streptococcus (such as Streptococcus uberis, or Streptococcus dysgalactiae).
  • the bacteria cells are Bacillus spp. or Clostridium perfringens.
  • the bacteria cells are symbiotic bacteria with a pest.
  • the target composition is an in vivo composition.
  • the target composition is present in the mammary gland of an individual.
  • the target composition is present in the heart valve, lung, blood stream, digestive tract, bone, nose, throat, or skin of the individual.
  • the individual is a human individual.
  • the individual is a dairy cow.
  • the target composition is an in vitro composition.
  • the target composition is a serum sample, an extracellular fluid sample, or a food product.
  • the food product is a dairy product, such as raw milk, for example, mastitic milk.
  • One aspect of the present application provides a method of treating a bacterial infection in an individual (such as a human individual, or a dairy cow), comprising administering to the individual an effective amount of a de-agglomeration agent and an effective amount of an antibacterial agent, wherein the de-agglomeration agent reduces agglomeration (such as agglutination) of bacteria cells that cause the bacterial infection.
  • the bacteria cells are selected from the group consisting of Staphylococcus aureus, Group C or Group G Streptococcus (such as Streptococcus uberis, or Streptococcus dysgalactiae), Pseudomonas aeruginosa, and Clostridium difficile.
  • the de- agglomeration agent cleaves immunoglobulin (such as IgG, IgA, IgM, IgE, or IgD, or subclasses thereof), protein A, protein G, fibrinogen, or ClfA.
  • immunoglobulin such as IgG, IgA, IgM, IgE, or IgD, or subclasses thereof
  • the de- agglomeration agent comprises a protease.
  • the protease is selected from the group consisting of papain, bromelain, IdeS, pepsin, ficain, actinidin, cathepsin-B like protease, plasmin and combinations thereof.
  • the protease is papain.
  • the protease is bromelain.
  • the protease is plasmin.
  • the method further comprises administering to the individual an effective amount of an adjuvant composition.
  • the adjuvant composition comprises a chelating agent.
  • the adjuvant composition comprises a reducing agent.
  • the adjuvant composition comprises a chelating agent (e.g., EDTA) and a reducing agent (e.g., cysteine).
  • the adjuvant composition comprises EDTA and cysteine.
  • the antibacterial agent comprises a small-molecule antibiotic.
  • the small-molecule antibiotic is selected from the group consisting of pirlimycin, ceftiofur, desfurolyceftiofur, amikacin, ampicillin, dihydrostreptomycin, flunixin, gentamicin, neomycin, tilmicosin, oxytetracycline, penicillin, sulfadiazine, sulfadimethoxine, sulfamethazine, sulfamethoxazole, sulfathiazole, tetracycline, tylosin, phenoxymethylpenicillin, flucloxacillin, amoxicillin, amoxicillin-clavulanate, clarithromycin, trimethoprim-sulfamethoxazole, nafcillin, ox
  • the antibacterial agent comprises a quorum sensing signal molecule.
  • the antibacterial agent comprises a bacteriolytic enzyme.
  • the antibacterial agent comprises a phage-derived protein.
  • the phage-derived protein is selected from the group consisting of lysozyme, endolysin, lysin, holin, tail fiber protein, and talocin.
  • the antibacterial agent comprises a bacteriophage.
  • the bacteriophage is bacteriophage K.
  • the antibacterial agent comprises a cocktail of bacteriophages.
  • the antibacterial agent comprises a bacteriocin.
  • the bacteriocin is a pyocin.
  • the bacteriocin is a nisin.
  • the bacterial infection is in mammary gland, heart valve, lung, blood stream, digestive tract, bone, nose, throat, or skin of the individual.
  • the individual has mastitis.
  • the de-agglomeration agent is administered by intramammary infusion.
  • the de-agglomeration agent and the antibacterial agent are administered sequentially.
  • the de- agglomeration agent and the antibacterial agent are administered simultaneously, such as in a single composition.
  • the de-agglomeration agent and the adjuvant composition are administered sequentially.
  • the de- agglomeration agent and the adjuvant composition are administered simultaneously, such as in a single composition.
  • a method of reducing contamination of a target composition by bacteria cells comprising adding to the target composition an effective amount of a de- agglomeration agent and an effective amount of an antibacterial agent, wherein the de-agglomeration agent reduces agglomeration (such as agglutination) of the bacteria cells.
  • the bacteria cells are selected from the group consisting of Staphylococcus aureus, Group C or Group G Streptococcus (such as Streptococcus uberis, or Streptococcus dysgalactiae), Pseudomonas aeruginosa, Clostridium difficile, Bacillus spp.
  • the de-agglomeration agent cleaves immunoglobulin (such as IgG, IgA, IgM, IgE, or IgD, or subclasses thereof), protein A, protein G, fibrinogen, or ClfA.
  • immunoglobulin such as IgG, IgA, IgM, IgE, or IgD, or subclasses thereof
  • the de- agglomeration agent comprises a protease.
  • the protease is selected from the group consisting of papain, bromelain, IdeS, pepsin, ficain, actinidin, cathepsin-B like protease, plasmin and combinations thereof.
  • the protease is papain.
  • the protease is bromelain.
  • the protease is plasmin.
  • the method further comprises adding to the target composition an effective amount of an adjuvant composition.
  • the adjuvant composition comprises a chelating agent.
  • the adjuvant composition comprises a reducing agent.
  • the adjuvant composition comprises a chelating agent (e.g., EDTA) and a reducing agent (e.g., cysteine).
  • the adjuvant composition comprises EDTA and cysteine.
  • the antibacterial agent comprises a small-molecule antibiotic.
  • the small-molecule antibiotic is selected from the group consisting of pirlimycin, ceftiofur, desfurolyceftiofur, amikacin, ampicillin, dihydrostreptomycin, flunixin, gentamicin, neomycin, tilmicosin, oxytetracycline, penicillin, sulfadiazine, sulfadimethoxine, sulfamethazine, sulfamethoxazole, sulfathiazole, tetracycline, tylosin, phenoxymethylpenicillin, flucloxacillin, amoxicillin, amoxicillin-clavulanate, clarithromycin, trimethoprim- sulfamethoxazole, nafcillin, oxacill
  • the antibacterial agent comprises a quorum sensing signal molecule.
  • the antibacterial agent comprises a bacteriolytic enzyme.
  • the antibacterial agent comprises a phage-derived protein.
  • the phage-derived protein is selected from the group consisting of lysozyme, endolysin, lysin, holin, tail fiber protein, and talocin.
  • the antibacterial agent comprises a bacteriophage.
  • the bacteriophage is bacteriophage K.
  • the antibacterial agent comprises a cocktail of bacteriophages.
  • the antibacterial agent comprises a bacteriocin.
  • the bacteriocin is a pyocin.
  • the bacteriocin is a nisin.
  • the target composition is a serum sample, an extracellular fluid sample, or a food product.
  • the food product is a dairy product, such as raw milk, for example, mastitic milk.
  • the de- agglomeration agent and the antibacterial agent are added to the target composition sequentially. In some embodiments, the de- agglomeration agent and the antibacterial agent are added to the target composition simultaneously. In some embodiments, the de- agglomeration agent and the antibacterial agent are added to the target composition as a single composition. In some embodiments, the adjuvant composition and the de- agglomeration agent are added to the target composition sequentially. In some embodiments, the adjuvant composition and the de-agglomeration agent are added to the target composition simultaneously, such as in a single composition.
  • a method for improving gut health of an individual comprising administering to the individual an effective amount of a de- agglomeration agent and an effective amount of a probiotic composition, wherein the de-agglomeration agent reduces agglomeration (such as agglutination) of bacteria cells in the gut of the individual.
  • the de-agglomeration agent cleaves immunoglobulin (such as IgG, IgA, IgM, IgE, or IgD, or subclasses thereof), protein A, protein G, fibrinogen, or ClfA.
  • the de- agglomeration agent comprises a protease.
  • the protease is selected from the group consisting of papain, bromelain, IdeS, pepsin, ficain, actinidin, cathepsin-B like protease, plasmin and combinations thereof.
  • the protease is papain.
  • the protease is bromelain.
  • the protease is plasmin.
  • the method further comprises administering to the individual an effective amount of an adjuvant composition.
  • the adjuvant composition comprises a chelating agent.
  • the adjuvant composition comprises a reducing agent.
  • the adjuvant composition comprises a chelating agent (e.g., EDTA) and a reducing agent (e.g., cysteine).
  • the adjuvant composition comprises EDTA and cysteine.
  • the probiotic composition comprises lactobacillus or bifidobacterium.
  • the probiotic composition comprises a bacteriophage. In some embodiments, the probiotic composition comprises a cocktail of bacteriophages.
  • the de- agglomeration agent is administered orally.
  • the de- agglomeration agent and the probiotic composition are administered sequentially. In some embodiments, the de-agglomeration agent and the probiotic composition are administered simultaneously. In some embodiments, the de- agglomeration agent and the probiotic composition are administered as a single composition. In some embodiments, the adjuvant composition and the de-agglomeration agent are administered to the individual sequentially. In some embodiments, the adjuvant composition and the de-agglomeration agent are administered to the individual simultaneously, such as in a single composition.
  • compositions, kits and articles of manufacture comprising any of the compositions described herein.
  • FIG. 1 depicts a bacteriophage K inhibition assay of Staphylococcus aureus from raw milk with or without papain treatment.
  • bacteriophage K is not effective at inhibiting the growth of S. aureus, likely due to IgG-mediated agglutination (column 2).
  • bacteriophage K markedly inhibits S. aureus growth after 4 h (column 4).
  • FIG. 2 depicts an ampicillin inhibition assay of Staphylococcus aureus cultures from raw milk with or without papain treatment.
  • S. aureus In untreated raw milk, treatment of S. aureus with ampicillin (100 ⁇ g/ml final concentration) inhibits the growth of S. aureus by 88% after 4 h (column 2). Although this is considerable inhibition, the effect of ampicillin is even more pronounced in papain-treated raw milk, inhibiting growth by >99.9% (column 3, not visible on this scale).
  • FIG. 3 depicts a bacterial inhibition assay by a bacteriophage cocktail enhanced by treatment with papain or bromelain combined with adjuvants (EDTA and cysteine).
  • FIG. 4 depicts a bacterial inhibition assay by ampicillin enhanced by treatment with bromelain combined with adjuvants (EDTA and cysteine).
  • FIGs. 5A-5C depict de-agglomeration of S. aureus by treatment with papain or bromelain combined with adjuvants (EDTA and cysteine).
  • FIG. 5A shows S. aureus aggregates in the presence of 5 mg/mL bovine IgG.
  • FIG. 5B shows de- agglomerated S. aureus in the presence of 5 mg/mL bovine IgG after treatment with papain combined with 0.5 mM EDTA and 2.5 mM cysteine.
  • FIG. 5C shows de- agglomerated S. aureus in the presence of 5 mg/mL bovine IgG after treatment with bromelain combined with 0.5 mM EDTA and 2.5 mM cysteine.
  • FIG. 6 depicts an S. aureus killing assay by PIRSUE ® enhanced by pre-treatment with bromelain combined with adjuvants (EDTA and cysteine) in a simulated mastitic milk sample.
  • FIG. 7 depicts an S. aureus killing assay by TODAY ® enhanced by pre-treatment with bromelain combined with adjuvants (EDTA and cysteine) in a raw milk sample.
  • FIG. 8A depicts an S. aureus (ATCC No. 31885) killing assay by TODAY ® enhanced by pre-treatment with bromelain combined with adjuvants (EDTA and cysteine) in a simulated mastitic milk sample.
  • FIG. 8B depicts an S. aureus (ATCC No. 31886) killing assay by TODAY ® enhanced by pre-treatment with bromelain combined with adjuvants (EDTA and cysteine) in a simulated mastitic milk sample.
  • FIG. 8C depicts an S. aureus (ATCC No. 31887) killing assay by TODAY ® enhanced by pre-treatment with bromelain combined with adjuvants (EDTA and cysteine) in a simulated mastitic milk sample.
  • the subject invention relates to compositions and methods for increasing the susceptibility of bacteria in a target composition to an antibacterial agent, such as a small- molecule antibiotic drug, a quorum sensing signal molecule, a bacteriolytic enzyme, a phage- derived protein, a bacteriophage, or a bacteriocin, in an in vivo or in vitro biological context.
  • an antibacterial agent such as a small- molecule antibiotic drug, a quorum sensing signal molecule, a bacteriolytic enzyme, a phage- derived protein, a bacteriophage, or a bacteriocin
  • the compositions comprise a de- agglomeration agent that can reduce agglomeration (such as agglutination) of bacteria cells.
  • the compositions and methods may be applied in various fields, including human and veterinary medicine, and food safety applications.
  • the present application is based on the surprising finding that an agent that reduces agglomeration (such as agglutination) of bacteria cells, for example the protease papain or bromelain, substantially enhances the activity of various antibacterial agents, such as bacteriophages and small-molecule antibiotics, against bacteria, in particular, Staphylococcus aureus.
  • an agent that reduces agglomeration (such as agglutination) of bacteria cells for example the protease papain or bromelain
  • substantially enhances the activity of various antibacterial agents such as bacteriophages and small-molecule antibiotics
  • the efficacy of the antibacterial agent can further be enhanced by including an adjuvant composition comprising chelating agents and/or reducing agents (such as EDTA and cysteine) in the pretreatment with the de- agglomeration agent.
  • an adjuvant composition comprising chelating agents and/or reducing agents (such as EDTA and cysteine) in the pretreatment with the de- agglomeration agent.
  • the present application in one aspect provides a method of increasing the susceptibility of bacteria cells in a target composition to an antibacterial agent, comprising adding to the target composition an effective amount of an agent that reduces agglomeration (such as agglutination) of the bacteria cells (hereinafter also referred to as a "de- agglomeration agent").
  • the method further comprises adding to the target composition an effective amount of an adjuvant composition (such as chelating agent and/or reducing agent).
  • a method of treating a bacterial infection in an individual comprising administering to the individual an effective amount of an antibacterial agent and an effective amount of a de- agglomeration agent.
  • the method further comprises administering to the individual an effective amount of an adjuvant composition (such as chelating agent and/or reducing agent).
  • a method of reducing contamination of a composition by bacteria cells comprising adding to the target composition an effective amount of an antibacterial agent and an effective amount of a de- agglomeration agent.
  • the method further comprises adding to the target composition an effective amount of an adjuvant composition (such as chelating agent and/or reducing agent).
  • compositions including for example anti-bacterial compositions, probiotic compositions, and/or food products comprising an antibacterial agent and a de- agglomeration agent.
  • the composition further comprises an adjuvant composition (such as chelating agent and/or reducing agent).
  • bacteria refers to prokaryotic microorganisms belonging to the domain Bacteria.
  • agglomeration means the aggregation of multiple bacteria into a larger rounded mass. Agglomeration is typically caused by crosslinking of bacteria cells. Such crosslinking may occur through extracellular polymers, such as in a biofilm, or by antibody- mediated agglutination. "Agglutination” refers to the clumping of bacteria in the presence of an antibody, which represents an exemplary mechanism of agglomeration.
  • An antibody is an immunoglobulin (Ig), a protein that binds to a pathogen through one or more antigens, characteristic molecules displayed on the pathogen's surface. Exemplary immunoglobulins include, but are not limited to, IgG, IgA, IgM, IgE, IgD, and subclasses thereof.
  • de- agglomeration agent refers to an agent that reduces agglomeration of bacteria cells.
  • pathogenic bacteria refers to bacteria that can cause an infection.
  • An example of a pathogenic bacterium is Mycobacterium tuberculosis.
  • Pathogenic bacteria do not always cause disease; for example, Staphylococcus and Streptococcus are conditional pathogens, existing as part of the normal human flora and causing disease only under certain conditions.
  • antibacterial refers to anything that is destructive to or inhibits the growth of bacteria.
  • antibiotic refers to an antimicrobial agent that is used in the treatment and prevention of bacterial infections. Antibiotics include but are not limited to small-molecule antibiotics, quorum sensing signal molecules, bacteriolytic enzymes, phage-derived proteins, bacteriophages, bacteriocins, and combinations thereof.
  • bacteriaicidal refers to an agent that results in at least a 3 log reduction in bacteria (i.e. greater than 99.9%) relative to the inoculum (Pankey GA and Sabath LD. Clin. Infect. Dis. 2004, 38(6): 864-870).
  • MIC minimum inhibitory concentration
  • Andrews Andrews JM. Journal of Antimicrob. Chemoth. 2001, 48(suppl 1): 5-16
  • MICs for bacteriophages are disclosed by Gill et al. (2006) and Tanji et al. (2015). Determination of MICs for bacteriocins are described by Hassan et al. (Hassan M, Javadzadeh Y, Lotfipour F, and Badomchi R. Adv. Pharm. Bull.
  • Cisani et al. report methods for determining MIC of bacteriolytic enzymes (Cisani G, Varaldo PE, Grazi G, and Soro O. Antimicrob. Agents Chemother. 1982, 21(4): 531-535).
  • an "effective amount” is an amount of a substance, an agent or a composition sufficient to cause an inhibitory effect on the growth of bacteria cells.
  • an effective amount of the antibacterial agent comprises an amount sufficient to kill a bacterium and/or to decrease the growth rate of the bacterium.
  • an effective amount of the de-agglomeration agent is an amount sufficient to reduce agglomeration (such as agglutination) of bacteria cells.
  • an effective amount of the de- agglomeration agent is an amount sufficient to lower the minimum inhibitory concentration of a bacterium by an antibacterial agent.
  • an effective amount of the de- agglomeration agent is an amount sufficient to reduce agglomeration (such as agglutination) of bacteria cells and to lower the minimum inhibitory concentration of a bacterium by an antibacterial agent.
  • an effective amount of the adjuvant composition is an amount sufficient to reduce agglomeration (such as agglutination) of bacteria cells in combination with a de- agglomeration agent.
  • an effective amount of the adjuvant composition is an amount sufficient to lower the minimum inhibitory concentration of a bacterium by an antibacterial agent in combination with a de- agglomeration agent.
  • an effective amount of the adjuvant composition is an amount sufficient to reduce agglomeration (such as agglutination) of bacteria cells and to lower the minimum inhibitory concentration of a bacterium by an antibacterial agent in combination with a de- agglomeration agent.
  • an effective amount of an agent used in a method of treating is an amount sufficient to delay development of a bacterial infection or a disease associated with the bacterial infection.
  • An effective amount can be administered in one or more administrations.
  • treatment refers to an approach for obtaining beneficial or desired results including clinical results.
  • beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g. , preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease, delaying or slowing the progression of the disease, ameliorating the disease state, and decreasing the dose of one or more other medications required to treat the disease.
  • the methods of the invention contemplate any one or more of these aspects of treatment.
  • individual refers to an animal, such as a mammal, and includes, but is not limited to, domesticated animals (e.g. , cows, sheep, cats, dogs, and horses), primates (e.g. , humans and non-human primates such as monkeys), rabbits, and rodents (e.g. , mice and rats).
  • domesticated animals e.g. , cows, sheep, cats, dogs, and horses
  • primates e.g. , humans and non-human primates such as monkeys
  • rabbits e.g. , mice and rats
  • rodents e.g. , mice and rats
  • the individual is a human.
  • the individual is an animal, such as a farm animal, for example a dairy cow.
  • mastitic milk refers to milk from an individual (such as dairy cow) having bacterial infection to the mammary gland.
  • enzyme means a macromolecular biological catalyst.
  • the enzyme may be isolated from a natural source or it may be synthesized recombinantly. In the latter case, the enzyme may be engineered such that one or more of its properties are changed relative to the wild-type enzyme. Such properties include but are not limited to stability, substrate specificity, binding affinity, immunogenicity, immunotoxicity, and kinetics. In some embodiments, the kinetics of the enzyme is enhanced by directed evolution, and the mutant enzyme is produced recombinantly in yeast.
  • small-molecule means an organic compound with a molecular weight less than about 900 Da.
  • “substance” means a particular kind of matter with uniform properties.
  • reference to "not" a value or parameter generally means and describes "other than” a value or parameter.
  • the method is not used to treat cancer of type X means the method is used to treat cancer of types other than X.
  • the present application in some embodiments provides a method of increasing susceptibility of bacteria cells in a target composition to an antibacterial agent, comprising adding to the target composition an effective amount of a de-agglomeration agent that reduces agglomeration (such as agglutination) of the bacteria cells.
  • the method further comprises adding an adjuvant composition (such as a chelating agent, e.g., EDTA, and/or a reducing agent, e.g., cysteine) to the target composition.
  • an adjuvant composition such as a chelating agent, e.g., EDTA, and/or a reducing agent, e.g., cysteine
  • the adjuvant composition and the de- agglomeration agent are added to the target composition sequentially.
  • the de- agglomeration agent is added prior to (for example at least about any of 1, 2, 3, 4, 5, 6, 7, or 8 hours prior to) the exposure of the target composition to the antibacterial agent. In some embodiments, the de-agglomeration agent is added after (for example at least about any of 1, 2, 3, 4, 5, 6, 7, or 8 hours after) the target composition is exposed to the antibacterial agent. In some embodiments, the adjuvant composition and the de- agglomeration agent are added to the target composition simultaneously, such as in a single composition. In some embodiments, the antibacterial agent comprises a bacteriophage (including, for example, a single bacteriophage, or a cocktail of bacteriophages).
  • the antibacterial agent comprises a small-molecule antibiotic (such as ampicillin, pirlimycin, or cephapirin). In some embodiments, the antibacterial agent comprises a bacteriophage and a small-molecule antibiotic. In some embodiments, the antibacterial agent comprises a quorum sensing signal molecule. In some embodiments, the antibacterial agent comprises a phage-derived protein (such as lysozyme, endolysin, lysin, holin, tail fiber protein, or tailocin). In some embodiments, the antibacterial agent comprises a bacteriocin (such as pyocin, or nisin). In some embodiments, the bacteria cells are selected from the group consisting of S.
  • Streptococcus such as Streptococcus uberis, or Streptococcus dysgalactiae
  • Pseudomonas aeruginosa Pseudomonas aeruginosa
  • Clostridium difficile Bacillus spp.
  • Clostridium perfringens Pseudomonas aeruginosa
  • a method of rendering a target composition more susceptible to the treatment of an antibacterial agent comprising adding to the target composition an effective amount of a de- agglomeration agent that reduces agglomeration (such as agglutination) of bacteria cells (such as bacteria cells targeted by the antibacterial agent).
  • a de- agglomeration agent that reduces agglomeration (such as agglutination) of bacteria cells (such as bacteria cells targeted by the antibacterial agent).
  • the target composition does not yet contain the bacteria cells.
  • the target composition already contains the bacteria cells.
  • the target composition further comprises the antibacterial agent.
  • the de- agglomeration agent is added to the target composition before the target composition is exposed to the bacteria cells.
  • the method further comprises adding an adjuvant composition (such as a chelating agent, e.g., EDTA, and/or a reducing agent, e.g., cysteine) to the target composition.
  • an adjuvant composition such as a chelating agent, e.g., EDTA, and/or a reducing agent, e.g., cysteine
  • the adjuvant composition and the de- agglomeration agent are added to the target composition sequentially.
  • the adjuvant composition and the de- agglomeration agent are added to the target composition simultaneously, such as in a single composition.
  • the antibacterial agent comprises a bacteriophage (including, for example, a single bacteriophage or a cocktail of bacteriophages).
  • the antibacterial agent comprises a small-molecule antibiotic (such as ampicillin, pirlimycin, or cephapirin). In some embodiments, the antibacterial agent comprises a bacteriophage and a small-molecule antibiotic. In some embodiments, the antibacterial agent comprises a quorum sensing signal molecule. In some embodiments, the antibacterial agent comprises a phage-derived protein (such as lysozyme, endolysin, lysin, holin, tail fiber protein, or tailocin). In some embodiments, the antibacterial agent comprises a bacteriocin (such as pyocin, or nisin). In some embodiments, the antibacterial agent inhibits (such as specifically inhibits) the growth of S.
  • a small-molecule antibiotic such as ampicillin, pirlimycin, or cephapirin.
  • the antibacterial agent comprises a bacteriophage and a small-molecule antibiotic. In some embodiments, the antibacterial agent comprises a quorum sensing signal molecule. In some
  • Streptococcus such as Streptococcus uberis, or Streptococcus dysgalactiae
  • Pseudomonas aeruginosa Pseudomonas aeruginosa
  • Clostridium difficile Bacillus spp.
  • Clostridium perfringens Pseudomonas aeruginosa
  • a method of rendering a target composition more susceptible to the treatment of an antibacterial agent comprising adding to the target composition an effective amount of a protease (such as papain, bromelain, or plasmin).
  • a protease such as papain, bromelain, or plasmin.
  • the target composition does not yet contain bacteria cells.
  • the target composition already contains bacteria cells.
  • the target composition further comprises the antibacterial agent.
  • the protease is added to the target composition before the target composition is exposed to the bacteria cells.
  • the method further comprises adding to the target composition an effective amount of an adjuvant composition (for example, a chelating agent, such as EDTA, and/or a reducing agent, such as cysteine).
  • an adjuvant composition for example, a chelating agent, such as EDTA, and/or a reducing agent, such as cysteine.
  • the adjuvant composition and the protease are added to the target composition sequentially. In some embodiments, the adjuvant composition and the protease are added to the target composition simultaneously, such as in a single composition.
  • the antibacterial agent comprises a bacteriophage (including, for example, a single bacteriophage or a cocktail of bacteriophages). In some embodiments, the antibacterial agent comprises a small-molecule antibiotic (such as ampicillin, pirlimycin, or cephapirin). In some embodiments, the antibacterial agent comprises a bacteriophage and a small-molecule antibiotic. In some embodiments, the antibacterial agent comprises a quorum sensing signal molecule.
  • the antibacterial agent comprises a phage-derived protein (such as lysozyme, endolysin, lysin, holin, tail fiber protein, or tailocin).
  • the antibacterial agent comprises a bacteriocin (such as pyocin, or nisin).
  • the antibacterial agent inhibits (such as specifically inhibits) the growth of S. aureus, Group C or Group G Streptococcus (such as Streptococcus uberis, or Streptococcus dysgalactiae), Pseudomonas aeruginosa, Clostridium difficile, Bacillus spp., or Clostridium perfringens.
  • a method of reducing bacterial contamination such as contamination by S. aureus
  • a target composition such as a food composition, for example, milk
  • a method of reducing bacterial contamination in a target composition comprising adding to the target composition an effective amount of an antibacterial agent and an effective amount of a protease (such as papain, bromelain, or plasmin).
  • a protease such as papain, bromelain, or plasmin
  • the method further comprises addition of an effective amount of an adjuvant composition (for example, a chelating agent, such as EDTA, and/or a reducing agent, such as cysteine).
  • an adjuvant composition for example, a chelating agent, such as EDTA, and/or a reducing agent, such as cysteine.
  • the antibacterial agent comprises a bacteriophage (including, for example, a single bacteriophage, or a cocktail of bacteriophages).
  • the antibacterial agent comprises a small-molecule antibiotic (such as ampicillin, pirlimycin, or cephapirin).
  • the antibacterial agent comprises a bacteriophage and a small-molecule antibiotic.
  • the antibacterial agent comprises a quorum sensing signal molecule.
  • the antibacterial agent comprises a phage-derived protein (such as lysozyme, endolysin, lysin, holin, tail fiber protein, or tailocin).
  • the antibacterial agent comprises a bacteriocin (such as pyocin, or nisin).
  • the antibacterial agent inhibits (such as specifically inhibits) the growth of S. aureus, Group C or Group G Streptococcus (such as Streptococcus uberis, or Streptococcus dysgalactiae), Pseudomonas aeruginosa, Clostridium difficile, Bacillus spp., or Clostridium perfringens.
  • the target composition to which the de-agglomeration agent is added can be an in vitro composition.
  • the target composition is a food composition, a serum composition, a plant composition, or an ex vivo tissue (such as meat).
  • the target composition is a dairy product.
  • the target composition is milk (such as raw, i.e., unpasteurized, milk).
  • the target composition is milk from an individual having bacterial infection in the mammary gland.
  • the target composition is mastitic milk.
  • the target composition is a food composition that has been stored for at least about any of 12, 24, 36, 72, or more hours after the making (or harvesting) of the food composition.
  • the de-agglomeration agent and/or the adjuvant composition are added by mixing the agent(s) with the target composition. In some embodiments, the de-agglomeration agent and/or the adjuvant composition are added by spraying the agent(s) onto the target composition.
  • a de-agglomeration agent such as bromelain
  • an antibacterial agent such as a single bacteriophage, or a cocktail of bacteriophages against several Bacillus spp. spoilage bacteria
  • a de-agglomeration agent such as ficain
  • an antibacterial agent such as a bacteriocin against Clostridium perfringens
  • the methods may find use in food safety applications.
  • the de- agglomeration agent and the antibacterial agent are applied to processed meat.
  • the de-agglomeration agent and the antibacterial agent are sprayed onto an area immediately before food preparation.
  • the de-agglomeration agent and the antibacterial agent are added to milk (such as raw, unpasteurized, or mastitic milk) before it is released for sale.
  • a method of increasing shelf- life of a food composition comprising adding to the food composition an effective amount of an antibacterial agent and an effective amount of a de-agglomeration agent that reduces agglomeration (such as agglutination) of bacteria cells.
  • the method further comprises adding an effective amount of an adjuvant composition (such as chelating agent, e.g., EDTA, and/or reducing agent, e.g., cysteine) to the food composition.
  • the antibacterial agent inhibits (such as specifically inhibits) the growth of S.
  • compositions such as dairy products, for example raw milk
  • a de-agglomeration agent that reduces agglomeration (such as agglutination) of bacteria cells.
  • a method of improving safety of a food composition comprising adding to the food composition an effective amount of an antibacterial agent and an effective amount of a de- agglomeration agent that reduces agglomeration (such as agglutination) of bacteria cells.
  • the method further comprises adding an effective amount of an adjuvant composition (such as chelating agent, e.g., EDTA, and/or reducing agent, e.g., cysteine) to the food composition.
  • the antibacterial agent inhibits (such as specifically inhibits) the growth of S.
  • the de- agglomeration agents cleaves immunoglobulin in the food composition.
  • the de- agglomeration agent is a protease, such as papain, or bromelain.
  • the antibacterial agent is a bacteriophage, such as bacteriophage K, or a cocktail of bacteriophages.
  • the antibacterial agent is a small molecule antibiotic, such as ampicillin, pirlimycin ⁇ e.g., PIRSUE ® ), or cephapirin ⁇ e.g., TODAY ® ).
  • the adjuvant composition comprises EDTA and cysteine.
  • the methods described herein are applicable to a range of food compositions and food products.
  • the food composition is a liquid composition.
  • the food composition is a solid composition, such as meat.
  • the food composition is in powder form, such as milk powder.
  • the food composition is a dairy product.
  • the food composition is milk, such as raw, unpasteurized, or mastitic milk.
  • the de- agglomeration agent and the antibacterial agent are added to the food composition during the production of the food composition.
  • the de-agglomeration agent and the antibacterial agent are added to the food composition during the processing of the food composition.
  • the de- agglomeration agent and the antibacterial agent are added to the food composition prior to release of the food composition for sale. In some embodiments, the de- agglomeration agent and the antibacterial agent are added to the food composition prior to storage of the food composition. In some embodiments, the de- agglomeration agent and the antibacterial agent are added to the food composition during the storage of the food composition.
  • a method of reducing bacterial contamination (such as S. aureus, or Group C or Group G Streptococcus) of a mastitic milk composition comprising adding to the mastitic milk composition an effective amount of an antibacterial agent and an effective amount of a de- agglomeration agent that reduces agglomeration (such as agglutination) of bacteria cells that cause the bacterial contamination.
  • the method further comprises adding an effective amount of an adjuvant composition (such as chelating agent, e.g., EDTA, and/or reducing agent, e.g., cysteine) to the mastitic milk composition.
  • an adjuvant composition such as chelating agent, e.g., EDTA, and/or reducing agent, e.g., cysteine
  • the antibacterial agent inhibits (such as specifically inhibits) the growth of S. aureus, Group C or Group G Streptococcus (such as Streptococcus uberis, or Streptococcus dysgalactiae).
  • the de-agglomeration agents cleaves immunoglobulin (such as IgG) in the mastitic milk composition.
  • the de- agglomeration agent is a protease, such as papain, or bromelain.
  • the antibacterial agent is a bacteriophage, such as bacteriophage K, or a cocktail of bacteriophages.
  • the antibacterial agent is a small molecule antibiotic, such as ampicillin, pirlimycin (e.g., PIRSUE ® ), or cephapirin (e.g., TODAY ® ).
  • the adjuvant composition comprises EDTA and cysteine.
  • a method of reducing bacterial contamination of a food composition comprising spraying on the surface of the food composition an effective amount of an antibacterial agent and an effective amount of a de-agglomeration agent that reduces agglomeration (such as agglutination) of bacteria cells that cause the bacterial contamination.
  • the method further comprises spraying on the surface of the food composition an effective amount of an adjuvant composition (such as chelating agent, e.g., EDTA, and/or reducing agent, e.g., cysteine).
  • the antibacterial agent inhibits the growth of S.
  • the food composition comprises processed meat.
  • the food composition comprises a dairy product.
  • the food composition comprises vegetable or plant parts.
  • the target composition is an in vivo composition.
  • the target composition can be milk in the mammary gland of an individual, or a tissue in an individual (for example the mammary tissue, heart valves, lungs, blood stream, digestive tract, bone, nose, throat, or skin), extracellular fluid, etc.
  • the target composition is on the surface of a plant. In some embodiments, the target composition is present in or on the surface of an individual.
  • the methods described herein may use any of the antibacterial agents described in the section "Antibacterial agents", and any of the de- agglomeration agents described in the section "De- agglomeration agents".
  • the antibacterial agent and the de- agglomeration agent are added simultaneously to the target composition, either in a single composition (such as the antibacterial compositions described herein), or in separate compositions.
  • the antibacterial agent and the de- agglomeration agent are added to the target composition sequentially.
  • the antibacterial agent is added to the target composition prior to (e.g. , at least about any of 6, 12, 24, 36, or more hours prior to) the addition of the de-agglomeration agent.
  • the antibacterial agent is added to the target composition after (e.g. , at least about any of 6, 12, 24, 36, or more hours after) the addition of the de- agglomeration agent.
  • any of the in vitro methods may comprise addition of an adjuvant composition in combination with the de- agglomeration agent.
  • adjuvants contemplated herein include, but are not limited to, chelating agents, such as EDTA and salts thereof, and reducing agents, such as cysteine, 2-mercaptoethanol, mercaptoethylamine, TCEP, dithiothreitol (DTT), glutathione, and salts thereof.
  • the adjuvant composition comprises a chelating agent, such as EDTA.
  • the adjuvant composition comprises a reducing agent, such as cysteine.
  • the adjuvant composition comprises a chelating agent (e.g. , EDTA) or a reducing agent (e.g. , cysteine).
  • the adjuvant composition comprises a chelating agent (e.g. , EDTA) and a reducing agent (e.g. , cysteine).
  • the adjuvant composition comprises EDTA and cysteine.
  • the de- agglomeration agent comprises a protease (such as a cysteine protease, for example papain, or bromelain), and the adjuvant composition comprises a chelating agent (e.g. , EDTA) and a reducing agent (e.g. , cysteine).
  • a protease such as a cysteine protease, for example papain, or bromelain
  • the adjuvant composition comprises a chelating agent (e.g. , EDTA) and a reducing agent (e.g. , cysteine).
  • the adjuvant composition (such as chelating agent and/or reducing agent) and the de- agglomeration agent are added simultaneously to the target composition, either in a single composition (such as the antibacterial compositions described herein), or in separate compositions.
  • the adjuvant composition and the de- agglomeration agent are admixed prior to the addition.
  • the adjuvant composition and the de- agglomeration agent are added to the target composition sequentially.
  • the adjuvant composition is added to the target composition prior to (e.g. , at least about any of 1, 2, 3, 4, 5, 6, 12, 24, 36, or more hours prior to) the addition of the de-agglomeration agent.
  • the adjuvant composition is added to the target composition immediately prior to (e.g. , no more than about any of 1, 5, 10, 15, or 30 minutes before) the addition of the de-agglomeration agent. In some embodiments, the adjuvant composition is added to the target composition after (e.g. , at least about any of 1, 2, 3, 4, 5, 6, 12, 24, 36, or more hours after) the addition of the de-agglomeration agent.
  • the concentration of the added de-agglomeration agent in the target composition is between about 0.01 ⁇ g/ml and about 10,000 ⁇ g/ml, including for example between about 0.1 ⁇ g/ml and about 1,000 ⁇ g/ml, such as between about 1 ⁇ g/ml and about 100 ⁇ g/ml.
  • the concentration of the bacteriophage (including, for example, a single bacteriophage, or a cocktail of bacteriophages) as the antibacterial agent in the target composition is at least about any of 0.0001, 0.001, 0.01, 0.1, 1, 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 1000 or more Multiplicity of Infection (MOI) with respect to the bacteria cells.
  • MOI Multiplicity of Infection
  • the effective amount of the bacteriophage (including, for example, a single bacteriophage, or a cocktail of bacteriophages) is about any of 0.0001-0.001, 0.001- 0.01, 0.01-0.1, 0.1-1, 1-2, 2-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50- 100, 1-10, 10- 50, 50-100, 100- 1000, 0.0001- 1, 1-1000, or 0.0001-1000 MOI with respect to the bacteria cells.
  • the concentration of the added bacteriophage in the target composition is
  • the concentration of the adjuvant composition added to the target composition is between about 1 ⁇ to about 100 mM, including for example, about 1 ⁇ to about 100 ⁇ , about 100 ⁇ to about 500 ⁇ , about 500 ⁇ to about 1 mM, about 1 mM to about 10 mM, or about 10 mM to about 100 mM.
  • the present application further provides methods of treating bacterial infection, or diseases associated with bacterial infection, such as mastitis.
  • a method of treating a bacterial infection such as infection by S. aureus, Group C or Group G Streptococcus, Pseudomonas aeruginosa, or Clostridium difficile
  • a bacterial infection such as infection by S. aureus, Group C or Group G Streptococcus, Pseudomonas aeruginosa, or Clostridium difficile
  • administering comprising administering to the individual an effective amount of an antibacterial agent and an effective amount of a de-agglomeration agent that reduces agglomeration (such as agglutination) of bacteria cells that causes the bacterial infection.
  • a method of treating a bacterial infection in an individual comprising administering to the individual an effective amount of an antibacterial agent and an effective amount of a protease (such as papain, bromelain, or plasmin).
  • a protease such as papain, bromelain, or plasmin
  • the method further comprises administering to the individual an effective amount of an adjuvant composition (for example, a chelating agent, such as EDTA, and/or a reducing agent, such as cysteine).
  • an adjuvant composition for example, a chelating agent, such as EDTA, and/or a reducing agent, such as cysteine.
  • the adjuvant composition and the de- agglomeration agent e.g., the protease
  • the adjuvant composition and the de-agglomeration agent e.g., the protease
  • the antibacterial agent comprises a bacteriophage (including, for example, a single bacteriophage, or a cocktail of bacteriophages).
  • the antibacterial agent comprises a small-molecule antibiotic (such as ampicillin, pirlimycin, or cephapirin). In some embodiments, the antibacterial agent comprises a bacteriophage and a small-molecule antibiotic. In some embodiments, the antibacterial agent comprises a quorum sensing signal molecule. In some embodiments, the antibacterial agent comprises a phage-derived protein (such as lysozyme, endolysin, lysin, holin, tail fiber protein, or tailocin). In some embodiments, the antibacterial agent comprises a bacteriocin (such as pyocin, or nisin). In some embodiments, the bacterial infection is caused by S.
  • a small-molecule antibiotic such as ampicillin, pirlimycin, or cephapirin.
  • the antibacterial agent comprises a bacteriophage and a small-molecule antibiotic. In some embodiments, the antibacterial agent comprises a quorum sensing signal molecule. In some embodiments, the antibacterial agent comprises
  • the bacterial infection is in an animal tissue, such as the mammary gland, heart valves, lungs, blood stream, digestive tract, bone, nose, throat, or skin.
  • the in vivo methods described herein can be useful for treating various diseases, particularly diseases involving bacterial infections.
  • a method of treating a disease involving bacterial infection in an individual comprising administering to the individual an effective amount of an antibacterial agent and an effective amount of a de- agglomeration agent that reduces agglomeration (such as agglutination) of bacteria cells.
  • a method of treating a disease involving bacterial infection in an individual comprising administering to the individual an effective amount of an antibacterial agent, an effective amount of a de- agglomeration agent that reduces agglomeration (such as agglutination) of bacteria cells, and an effective amount of an adjuvant composition (such as chelating agent, e.g., EDTA, and/or reducing agent, e.g., cysteine).
  • an adjuvant composition such as chelating agent, e.g., EDTA, and/or reducing agent, e.g., cysteine.
  • Suitable diseases that can be treated using methods of the present application include, but are not limited to, mastitis, infected burns, Clostridium difficile infection, or cystic fibrosis (for example, cystic fibrosis patient infected with Pseudomonas aeruginosa).
  • the bacterial infection is in a tissue of the individual, selected from the group consisting of mammary gland, heart valve, lung, blood stream, digestive tract, bone, nose, throat, or skin.
  • the bacterial infection is caused by Staphylococcus aureus, Group C or Group G Streptococcus (such as Streptococcus uberis, or Streptococcus dysgalactiae), Pseudomonas aeruginosa, or Clostridium difficile.
  • Staphylococcus aureus Group C or Group G Streptococcus (such as Streptococcus uberis, or Streptococcus dysgalactiae), Pseudomonas aeruginosa, or Clostridium difficile.
  • any method that involves the use of any antibacterial agent described in the section "Antibacterial agents”, and any de-agglomeration agent described in the section “De- agglomeration agents”, and optionally an adjuvant composition, to inhibit (including to kill) bacteria is within the scope of the subject invention.
  • the de- agglomeration agent and the antibacterial agent may be administered to humans or other animals to treat a bacterial infection to any tissue or organ, including, but not limited to, mammary gland, heart valve, blood stream, respiratory tract (such as nose, mouth, nasal cavity, throat, larynx, trachea, bronchi, lung, etc.), gastrointestinal tract (such as small intestine, large intestine, stomach, colon, etc.), bone, joints, skin, and mucous surfaces of the body.
  • respiratory tract such as nose, mouth, nasal cavity, throat, larynx, trachea, bronchi, lung, etc.
  • gastrointestinal tract such as small intestine, large intestine, stomach, colon, etc.
  • bone joints
  • skin and mucous surfaces of the body.
  • S. aureus infection may occur on the skin, nose, bloodstream, heart valves, joints, bones, lungs, or reproductive tract.
  • the methods and compositions described herein are useful for treating diseases caused by S.
  • aureus infection including, but not limited to, folliculitis, impetigo, abscesses, cellulitis, toxic epidermal necrolysis, mastitis, pneumonia, bloodstream infections, endocarditis, and osteomyelitis.
  • the de- agglomeration agent and the antibacterial agent are administered by intramammary infusion into the teat canal of a dairy cow infected with mastitis.
  • the de-agglomeration agent and the antibacterial agent are applied topically to a S. aureus-infected burn on a human patient.
  • Pseudomonas aeruginosa infection may occur in respiratory tract, bloodstream, heart, central nervous system, ear, eye, bones, joints, digestive tract, urinary tract, and skin.
  • the methods and compositions described herein are useful for treating diseases caused by Pseudomonas aeruginosa, including, but not limited to, endocarditis, pneumonia, bacteremia, meningitis, brain abscess, otitis, bacterial keratitis, endophthalmitis, osteomyelitis, diarrhea, enteritis, enterocolitis, and ecthyma gangrenosum.
  • the de-agglomeration agent and the antibacterial agent are delivered by nebulizer to a cystic fibrosis patient infected with Pseudomonas aeruginosa.
  • Group C or Group G Streptococcus infection can affect farm animals and spread to humans through raw dairy product or contact with farm animals.
  • Group C or Group G Streptococcus infection can occur in the mammary gland of farm animals.
  • Group C or Group G Streptococcus infection can occur in the throat, skin, or blood stream.
  • the methods and compositions described herein are useful for treating diseases caused by Group C or Group G Streptococcus, including, but not limited to mastitis, skin infection, pharyngitis, and bacteremia.
  • Clostridium Difficile infection can occur in the digestive tract, such as small and large intestines of an individual.
  • the methods and compositions described herein are useful for treating diseases caused by Clostridium Difficile, including, but not limited to diarrhea, and colitis.
  • de-agglomeration agent and the antibacterial agent are delivered orally to treat a Clostridium difficile infection.
  • Mycobacterium tuberculosis infection can occur in the respiratory tract.
  • the methods and compositions described herein are useful for treating tuberculosis.
  • mastitis such as mastitis caused by Staphylococcus aureus, Streptococcus uberis, or Streptococcus dysgalactiae
  • a method of treating mastitis comprising administering to the individual an effective amount of an antibacterial agent and an effective amount of a de-agglomeration agent that reduces agglomeration (such as agglutination) of bacteria cells.
  • the de- agglomeration agent and the antibacterial agent are administered simultaneously.
  • the de-agglomeration agent and the antibacterial agent are administered as a single composition.
  • the de-agglomeration agent and the antibacterial agent are administered as separate compositions (for example, by different administration routes, or the same administration route). In some embodiments, the de- agglomeration agent and the antibacterial agent are administered sequentially. In some embodiments, the method further comprises administering to the individual an effective amount of an adjuvant composition (such as chelating agent, e.g., EDTA, and/or reducing agent, e.g., cysteine). In some embodiments, the de-agglomeration agent and/or the antibacterial agents and/or the adjuvant composition are administered by intramammary infusion. In some embodiments, the individual is a human. In some embodiments, the individual is a cow (such as a dairy cow).
  • an adjuvant composition such as chelating agent, e.g., EDTA, and/or reducing agent, e.g., cysteine.
  • the de-agglomeration agent and/or the antibacterial agents and/or the adjuvant composition are administered
  • a dairy cow, with or without clinical symptoms, whose milk exhibits a high somatic cell count may be treated by intramammary infusion into the affected udder quarter with an agent (with or without an adjuvant composition) that reduces agglomeration (such as agglutination) of bacteria, such as papain, or bromelain, followed by an antibacterial agent, such as pirlimycin, or cephapirin.
  • an agent with or without an adjuvant composition
  • agglomeration such as agglutination
  • an antibacterial agent such as pirlimycin, or cephapirin.
  • a method of treating infected skin burn comprising applying to the site of skin burn an effective amount of an antibacterial agent and an effective amount of a de-agglomeration agent that reduces agglomeration (such as agglutination) of bacteria cells.
  • the de-agglomeration agent and the antibacterial agent are administered as a single composition.
  • the de-agglomeration agent and the antibacterial agent are administered as separate compositions (for example, by different administration routes, or the same administration route).
  • the de- agglomeration agent and the antibacterial agent are administered sequentially.
  • the method further comprises applying to the site of skin burn an effective amount of an adjuvant composition (such as chelating agent, e.g., EDTA, and/or reducing agent, e.g., cysteine).
  • an adjuvant composition such as chelating agent, e.g., EDTA, and/or reducing agent, e.g., cysteine.
  • a method of treating infected skin wound comprising administering to the individual an effective amount of an antibacterial agent and an effective amount of a de- agglomeration agent.
  • the de- agglomeration agent and the antibacterial agent are administered as a single composition.
  • the de- agglomeration agent and the antibacterial agent are administered as separate compositions (for example, by different administration routes, or the same administration route).
  • the de- agglomeration agent and the antibacterial agent are administered sequentially.
  • the method further comprises administering to the individual an effective amount of an adjuvant composition (such as chelating agent, e.g., EDTA, and/or reducing agent, e.g., cysteine).
  • an adjuvant composition such as chelating agent, e.g., EDTA, and/or reducing agent, e.g., cysteine.
  • the antibacterial agent is administered orally.
  • the de-agglomeration agent and optionally the adjuvant composition is administered topically to the site of the skin wound.
  • a method of treating cystic fibrosis in an individual comprising administering to the individual an effective amount of an antibacterial agent and an effective amount of a de-agglomeration agent that reduces agglomeration (such as agglutination) of bacteria cells (such as Pseudomonas aeruginosa).
  • the de-agglomeration agent and the antibacterial agent are administered as a single composition.
  • the de-agglomeration agent and the antibacterial agent are administered as separate compositions (for example, by different administration routes, or the same administration route).
  • the de- agglomeration agent and the antibacterial agent are administered sequentially.
  • the method further comprises administering to the individual an effective amount of an adjuvant composition (such as chelating agent, e.g., EDTA, and/or reducing agent, e.g., cysteine).
  • an adjuvant composition such as chelating agent, e.g., EDTA, and/or reducing agent, e.g., cysteine.
  • the de-agglomeration agent and/or the antibacterial agents and/or the adjuvant composition are administered by inhalation.
  • the de- agglomeration agent and/or the antibacterial agents and/or the adjuvant composition are administered by using a nebulizer.
  • a method of treating Clostridium difficile infection comprising administering to the individual an effective amount of an antibacterial agent and an effective amount of a de- agglomeration agent that reduces agglomeration (such as agglutination) of bacteria cells.
  • the de- agglomeration agent and the antibacterial agent are administered as a single composition.
  • the de- agglomeration agent and the antibacterial agent are administered as separate compositions (for example, by different administration routes, or the same administration route).
  • the de- agglomeration agent and the antibacterial agent are administered sequentially.
  • the method further comprises administering to the individual an effective amount of an adjuvant composition (such as chelating agent, e.g., EDTA, and/or reducing agent, e.g., cysteine).
  • an adjuvant composition such as chelating agent, e.g., EDTA, and/or reducing agent, e.g., cysteine.
  • the de-agglomeration agent and/or the antibacterial agents and/or the adjuvant composition are administered orally to the individual.
  • the antibacterial agent and the de- agglomeration agent are administered simultaneously to the individual, either in a single composition (such as the antibacterial compositions described herein), or in separate compositions (including via the same or different routes).
  • the antibacterial agent and the de- agglomeration agent are administered to the individual sequentially.
  • the antibacterial agent is administered to the individual prior to (e.g. , at least about any of 6, 12, 24, 36, or more hours prior to) the administration of the de-agglomeration agent.
  • the antibacterial agent is administered to the individual after (e.g. , at least about any of 6, 12, 24, 36, or more hours after) the administration of the de- agglomeration agent.
  • the antibacterial agent may be used alone or in combination with another agent, such as a non-bactericidal agent that affects the efficacy of the antibacterial agent.
  • the effective amount of the antibacterial agent when used alone or in combination, may: (i) reduce the number of bacteria cells; (ii) inhibit, retard, slow to some extent and preferably stop bacteria proliferation; (iii) inhibit bacteria growth; (iv) prevent or delay occurrence and/or recurrence of bacterial infection; and/or (v) relieve to some extent one or more of the symptoms associated with the bacterial infection.
  • the non-bactericidal substance (such as the de-agglomeration agent) that potentiates the effect of the antibiotic and the antibiotic itself— need not be administered simultaneously in the subject invention.
  • the non-bactericidal substance that potentiates the effect of the antibiotic is delivered about 1 h before the antibiotic.
  • the antibiotic is delivered about 20 min before the non-bactericidal substance that potentiates the effect of the antibiotic.
  • both components of the composition the non-bactericidal substance that potentiates the effect of the antibiotic and the antibiotic itself— need not be administered by the same route or to the same site to treat an infection.
  • a small-molecule antibiotic is administered orally while the non-bactericidal substance that potentiates the effect of the antibiotic is administered topically to a wound on the patient's arm.
  • Additional applications of the methods and compositions of the present application include, but are not limited to, promoting gut health, controlling bacterial parasite on a plant or animal, and pest control.
  • the de- agglomeration agent and the antibacterial agent are delivered orally to promote gut health.
  • the de-agglomeration agent and the antibacterial agent are sprayed onto a plant to control a bacterial parasite.
  • a method of improving gut health in an individual comprising administering (such as orally administering) to the individual an effective amount of an antibacterial agent and an effective amount of a de-agglomeration agent that reduces agglomeration (such as agglutination) of bacteria cells.
  • the method further comprises administering to the individual an effective amount of an adjuvant composition (such as chelating agent, e.g., EDTA, and/or reducing agent, e.g., cysteine).
  • an adjuvant composition such as chelating agent, e.g., EDTA, and/or reducing agent, e.g., cysteine.
  • a method of controlling bacterial parasite on a plant comprising applying (such as by spraying) onto the plant an effective amount of an antibacterial agent and an effective amount of a de- agglomeration agent that reduces agglomeration (such as agglutination) of bacteria cells.
  • the method further comprises applying onto the plant an effective amount of an adjuvant composition (such as chelating agent, e.g., EDTA, and/or reducing agent, e.g., cysteine) to the composition.
  • an adjuvant composition such as chelating agent, e.g., EDTA, and/or reducing agent, e.g., cysteine
  • the antibacterial agent, the de- agglomeration agent, and optionally the adjuvant composition are applied as a single composition.
  • the antibacterial agent, the de- agglomeration agent, and optionally the adjuvant composition are applied in separate compositions.
  • the target composition is included in bait designed to kill obligate bacterial symbionts in the guts of insect pests like termites, thereby killing the insect pests.
  • a method of killing a pest comprising administering to the pest (such as insect, e.g. by way of bait) a composition comprising an effective amount of an antibacterial agent (such as an agent that inhibits the growth of bacterial symbionts in the gut of the insect) and a de- agglomeration agent that reduces agglomeration (such as agglutination) of symbiotic bacteria cells.
  • a method of improving gut health in an individual comprising administering (e.g., orally administering) to the individual an effective amount of a de- agglomeration agent that reduces agglomeration (such as agglutination) of bacteria cells and a probiotic composition.
  • the probiotic composition comprises lactobacillus or bifidobacterium.
  • the method further comprises administering to the individual an effective amount of an adjuvant composition (such as chelating agent, e.g., EDTA, and/or reducing agent, e.g., cysteine).
  • the de-agglomeration agent and the probiotic composition are administered simultaneously, such as in a single composition.
  • the de- agglomeration agent and the probiotic composition are administered sequentially.
  • compositions comprising a de- agglomeration agent and a probiotic composition.
  • the composition further comprises an antibacterial agent (such as a single bacteriophage, or a cocktail of bacteriophages).
  • the composition further comprises an adjuvant composition (such as chelating agent, e.g., EDTA, and/or reducing agent, e.g., cysteine).
  • the probiotic composition contemplated herein comprises one or more bacteria (e.g., genus, species, or strains) that are beneficial to the gut health of the individual that is administered in combination with the de-agglomeration agent.
  • the probiotic composition comprises bacterial transplants, such as fecal transplants.
  • the probiotic composition comprises one or more bacteria that produce antibacterial agents that inhibit the growth (for example, kill) of bacteria that may agglomerate (such as agglutinate) in the gut of the individual, such as bacteria that express Protein A (e.g., Staphylococcus aureus), or bacteria that express Protein G (e.g., group C or group G Streptococcus bacteria).
  • Lactobacillus produces bacteriocin, and in combination with the de-agglomeration agent (such as papain or bromelain) of the composition, the bacteriocin can kill bacteria that express Protein A or Protein G, such as Staphylococcus aureus, or Streptococcus group A or group G bacteria present in the gut of the individual, thereby improving the gut health of the individual.
  • the probiotic composition comprises one or more bacteria that produce agents that inhibit the growth of certain classes of bacteria that may be harmful for the gut health of the individual.
  • the probiotic composition comprises one or more bacteria that produce agents that inhibit the growth of gram negative bacteria.
  • the probiotic composition comprises one or more bacteria that produce agents that alter the pH of the bacterial growing environment in the gut of the individual.
  • Bifidobacterium produces lactic acid, which can reduce pH of the bacterial growth environment in the gut of the individual, thereby inhibiting growth of gram negative bacteria and improving the gut health of the individual.
  • the de- agglomeration agent and the probiotic composition can be administered to the individual simultaneously or sequentially.
  • the de-agglomeration agent, the probiotic composition, and optionally the adjuvant composition are administered simultaneously to the individual, either in a single composition (such as the antibacterial compositions described herein), or in separate compositions (including via the same or different routes).
  • the probiotic composition, and optionally the adjuvant composition are administered sequentially.
  • the probiotic agent is administered to the individual prior to (e.g., at least about any of 6, 12, 24, 36, or more hours prior to) the administration of the de-agglomeration agent.
  • the probiotic is administered to the individual after (e.g., at least about any of 6, 12, 24, 36, or more hours after) the administration of the de- agglomeration agent.
  • Any of the in vivo methods may comprise administration of an adjuvant composition in combination with the de- agglomeration agent.
  • exemplary adjuvants contemplated herein include, but are not limited to, chelating agents, such as EDTA and salts thereof, and reducing agents, such as cysteine, 2-mercaptoethanol, mercaptoethylamine, TCEP, dithiothreitol (DTT), glutathione, and salts thereof.
  • the adjuvant composition comprises a chelating agent, such as EDTA.
  • the adjuvant composition comprises a reducing agent, such as cysteine.
  • the adjuvant composition comprises a chelating agent ⁇ e.g., EDTA) or a reducing agent ⁇ e.g., cysteine).
  • the adjuvant composition comprises a chelating agent ⁇ e.g., EDTA) and a reducing agent ⁇ e.g., cysteine).
  • the adjuvant composition comprises EDTA and cysteine.
  • the de- agglomeration agent comprises a protease (such as a cysteine protease, for example papain, or bromelain), and the adjuvant composition comprises a chelating agent ⁇ e.g., EDTA) and a reducing agent ⁇ e.g., cysteine).
  • a protease such as a cysteine protease, for example papain, or bromelain
  • the adjuvant composition comprises a chelating agent ⁇ e.g., EDTA) and a reducing agent ⁇ e.g., cysteine).
  • the adjuvant composition (such as chelating agent and/or reducing agent) and the de- agglomeration agent are administered to the individual simultaneously, either in a single composition (such as the antibacterial compositions described herein), or in separate compositions (including via the same or different routes).
  • the adjuvant composition and the de- agglomeration agent are admixed prior to the administration.
  • the adjuvant composition and the de- agglomeration agent are administered to the individual sequentially.
  • the adjuvant composition is administered to the individual prior to ⁇ e.g., at least about any of 1, 2, 3, 4, 5, 6, 12, 24, 36, or more hours prior to) the administration of the de-agglomeration agent. In some embodiments, the adjuvant composition is administered to the individual immediately prior to ⁇ e.g., no more than about any of 1, 5, 10, 15, or 30 minutes before) the administration of the de-agglomeration agent. In some embodiments, the adjuvant composition is administered to the individual after ⁇ e.g., at least about any of 1, 2, 3, 4, 5, 6, 12, 24, 36, or more hours after) the administration of the de- agglomeration agent.
  • the dose of the administered antibacterial agent can be calculated from the experimentally determined MIC using pharmacology equations known to persons of skill in the art:
  • Each of the agents (such as the de- agglomeration agent, and the antibacterial agent) and compositions (such as the probiotic composition, the adjuvant composition, compositions comprising the de- agglomeration agent and the antibacterial agent, and compositions comprising the de- agglomeration agent and the probiotic composition) of the invention may be administered in any suitable form that will provide sufficient levels of the agents or compositions for the intended purpose.
  • Intravenous administration is a useful route of administration, although other parenteral routes can also be employed, where parenteral as used herein includes subcutaneous injections, intravenous injection, intraarterial injection, intramuscular injection, intrasternal injection, intraperitoneal injection, or infusion techniques.
  • the agents or compositions can also be administered orally or enterally, which is a preferred route when compatible with the absorption of the agents or compositions.
  • the agents or compositions can also be administered sublingually, by buccal administration, subcutaneously, by spinal administration, by epidural administration, by administration to cerebral ventricles, by inhalation (e.g. as mists or sprays), rectally, or topically in unit dosage formulations containing conventional nontoxic pharmaceutically acceptable carriers, excipients, adjuvants, and vehicles as desired.
  • the agents or compositions may be administered directly to a specific or affected organ or tissue.
  • the agents or compositions can be mixed with pharmaceutically acceptable carriers, excipients, adjuvants, and vehicles appropriate for the desired route of administration.
  • One aspect of the present invention provides a composition comprising an agent that does not exhibit bactericidal activity against a bacterium (also referred to as a "non-bactericidal agent”), and an antibacterial agent, wherein the antibacterial agent in combination with the non- bactericidal agent results in a lower minimum inhibitory concentration (MIC) against the bacterium than the antibacterial agent alone.
  • a non-bactericidal agent also referred to as a "non-bactericidal agent
  • MIC minimum inhibitory concentration
  • the antibacterial agent in combination with the non-bactericidal agent results in a MIC against the bacterium that is no more than about any of 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, 0.2%, 0.1%, 0.05%, 0.01%, 0.005%, 0.001% or less of the MIC of the antibacterial agent alone.
  • the non-bactericidal agent is a de- agglomeration agent that reduces agglomeration (such as agglutination) of bacteria cells.
  • One aspect of the present invention provides a composition comprising a de- agglomeration agent and an antibacterial agent, wherein the de-agglomeration agent reduces agglomeration (such as agglutination) of bacteria cells.
  • the antibacterial agent can inhibit the growth of the bacteria cells without killing the bacteria cells (i.e., bacteriostatic agent).
  • the antibacterial agent kills the bacteria cells (i.e., bactericidal agent).
  • the bacteria cells are selected from the group consisting of Staphylococcus aureus, Group C or Group G Streptococcus (such as Streptococcus uberis, or Streptococcus dysgalactiae), Pseudomonas aeruginosa, Clostridium difficile, Bacillus spp., and Clostridium perfringens.
  • the de-agglomeration agent does not exhibit bactericidal activity against the bacteria cells.
  • the composition further comprises an adjuvant composition, such as chelating agent, e.g., EDTA, and/or reducing agent, for example, cysteine.
  • compositions comprising a de- agglomeration agent and a probiotic composition, wherein the de-agglomeration agent reduces agglomeration (such as agglutination) of bacteria cells.
  • the composition further comprises a bacteriophage, such as bacteriophage K.
  • the composition further comprises a combination (i.e. , cocktail) of bacteriophages (such as different bacteriophage strains).
  • the composition further comprises an adjuvant composition, such as chelating agent, e.g., EDTA, and/or reducing agent, for example, cysteine.
  • the probiotic composition contemplated in the present invention includes, but is not limited to, bacterial transplants (such as fecal transplants), and a culture of a probiotic bacterial species or a mixture of probiotic bacterial species, which may be isolated from natural sources, or produced in cell culture.
  • exemplary probiotic bacterial species include, but are not limited to lactobacillus and bifidobacterium.
  • the probiotic composition comprises lactobacillus .
  • the probiotic composition comprises bifidobacterium.
  • the probiotic composition further comprises an antibacterial agent (such as a bactericidal agent, or a bacteriostatic agent).
  • any combination of one or more of the de- agglomeration agents and optionally in combination with one or more of the adjuvants or adjuvant compositions as described herein which, when combined with any combination of one or more antibacterial agents described herein, may increase the susceptibility of bacteria cells to the antibacterial agent or the combination of antibacterial agents, relative to the antibacterial agent or the combination of antibacterial agents alone, is within the scope of the subject invention.
  • the non-bactericidal agent (such as the de-agglomeration agent and optionally in combination with the adjuvant composition) that potentiates the effect of the antibacterial agent is papain and the antibacterial agent is a small-molecule antibiotic (such as ampicillin or pirlimycin).
  • the de-agglomeration agent is bromelain, and the antibacterial agent is a small-molecule antibiotic (such as ampicillin, pirlimycin, or cephapirin).
  • a composition comprising bromelain, and pirlimycin (such as PIRSUE ® ). In some embodiments, there is provided a composition comprising bromelain, pirlimycin (such as PIRSUE ® ), EDTA, and cysteine. In some embodiments, there is provided a composition comprising bromelain, and cephapirin (such as TODAY ® ). In some embodiments, there is provided a composition comprising bromelain, cephapirin (such as TODAY ® ), EDTA, and cysteine. In some embodiments, there is provided a composition comprising papain, and ampicillin.
  • a composition comprising papain, ampicillin, EDTA and cysteine. In some embodiments, there is provided a composition comprising papain, and a single bacteriophage or a cocktail of bacteriophages. In some embodiments, there is provided a composition comprising papain, EDTA, cysteine, and a single bacteriophage or a cocktail of bacteriophages. In some embodiments, there is provided a composition comprising bromelain, and a single bacteriophage or a cocktail of bacteriophages. In some embodiments, there is provided a composition comprising bromelain, EDTA, cysteine, and a single bacteriophage or a cocktail of bacteriophages.
  • compositions described herein may be useful for treating mastitis (such as in a dairy cow), or decontaminating a dairy product (such as raw milk, unpasteurized milk, or mastitic milk, for example, milk contaminated with S. aureus).
  • mastitis such as in a dairy cow
  • decontaminating a dairy product such as raw milk, unpasteurized milk, or mastitic milk, for example, milk contaminated with S. aureus
  • the individual concentrations or percentages of the de-agglomeration agent, the adjuvant composition, the antibacterial agent, and/or the probiotic composition in any of the compositions described herein, as well as the relative ratio (e.g. by weight or by volume) of the de- agglomeration agent, the adjuvant composition, the antibacterial agent, and the probiotic composition in any of the compositions described herein may be determined by a person skilled in the art according to the actual application of the composition.
  • the non-bactericidal agent (such as the de- agglomeration agent and optionally in combination with the adjuvant composition) that potentiates the effect of the antibacterial agent is a protease (such as papain, bromelain, or plasmin).
  • the concentration of the protease in the composition is any of between about 0.01 ⁇ g/ml and about 10,000 ⁇ g/ml, between about 0.1 ⁇ g/ml and about 1,000 ⁇ g/ml, or between about 1 ⁇ g/ml and about 100 ⁇ g/ml.
  • the antibacterial agent is a bacteriophage.
  • the concentration of the bacteriophage in the composition is any of between about 10 4 and about 10 12 plaque-forming units (pfu) per ml, between about 10 6 and about 10 10 pfu, or between about
  • the concentration of the bacteriophage (including, for example, a cocktail of bacteriophages) in the composition is at least about any of 0.0001, 0.001, 0.01, 0.1, 1, 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 1000 or more Multiplicity of Infection (MOI) with respect to the bacteria cells.
  • MOI Multiplicity of Infection
  • the concentration of the bacteriophage (including, for example, a cocktail of bacteriophages) in the composition is about any of 0.0001-0.001, 0.001-0.01, 0.01-0.1, 0.1- 1, 1-2, 2-5, 5- 10, 10-15, 15- 20, 20-25, 25-30, 30-40, 40-50, 50-100, 1- 10, 10-50, 50-100, 100- 1000, 0.0001- 1, 1-1000, or 0.0001- 1000 MOI with respect to the bacteria cells.
  • the concentration of the adjuvant composition such as chelating agent, e.g. , EDTA, or reducing agent, e.g.
  • cysteine is between about 1 ⁇ to about 100 mM, including for example, about 1 ⁇ to about 100 ⁇ , about 100 ⁇ to about 500 ⁇ , about 500 ⁇ to about 1 mM, about 1 mM to about 10 mM, or about 10 mM to about 100 mM.
  • compositions comprising any of the compositions described herein, and a pharmaceutically acceptable carrier.
  • suitable pharmaceutical carriers include, but are not limited to, sterile water; saline, dextrose; dextrose in water or saline; condensation products of castor oil and ethylene oxide combining about 30 to about 35 moles of ethylene oxide per mole of castor oil; liquid acid; lower alkanols; oils such as corn oil; peanut oil, sesame oil and the like, with emulsifiers such as mono- or di-glyceride of a fatty acid, or a phosphatide, e.g.
  • lecithin and the like; glycols; polyalkylene glycols; aqueous media in the presence of a suspending agent, for example, sodium carboxymethylcellulose; sodium alginate; poly(vinylpyrolidone) ; and the like, alone, or with suitable dispensing agents such as lecithin; polyoxyethylene stearate; and the like.
  • the carrier may also contain adjuvants such as preserving stabilizing, wetting, emulsifying agents and the like together with the penetration enhancer.
  • the final form may be sterile and may also be able to pass readily through an injection device such as a hollow needle. The proper viscosity may be achieved and maintained by the proper choice of solvents or excipients.
  • compositions described herein may include other agents, excipients, or stabilizers to improve properties of the composition.
  • excipients and diluents include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline solution, syrup, methylcellulose, methyl- and propylhydroxybenzoates, talc, magnesium stearate and mineral oil.
  • the formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents.
  • emulsifying agents include tocopherol esters such as tocopheryl polyethylene glycol succinate and the like, PLURONIC®, emulsifiers based on polyoxy ethylene compounds, Span 80 and related compounds and other emulsifiers known in the art and approved for use in animals or human dosage forms.
  • the compositions (such as pharmaceutical compositions) can be formulated so as to provide rapid, sustained or delayed release of the active ingredient after administration to an individual by employing procedures well known in the art.
  • the composition (such as pharmaceutical composition) is formulated to have a pH in the range of about 4.5 to about 9.0, including for example pH ranges of any one of about 5.0 to about 8.0, about 6.5 to about 7.5, or about 6.5 to about 7.0.
  • the pH of the composition (such as pharmaceutical composition) is formulated to no less than about 6, including for example no less than about any one of 6.5, 7, or 8 (e.g. , about 8).
  • the composition (such as pharmaceutical composition) can also be made to be isotonic with blood by the addition of a suitable tonicity modifier, such as glycerol.
  • the composition (such as pharmaceutical composition) is suitable for administration to a human individual.
  • the composition (such as pharmaceutical composition) is suitable for administration to a human individual by parenteral administration.
  • Formulations suitable for parenteral administration include aqueous and non- aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation compatible with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizing agents, and preservatives.
  • the formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient methods of treatment, methods of administration, and dosage regimens described herein (i.e., water) for injection, immediately prior to use.
  • sterile liquid excipient methods of treatment, methods of administration, and dosage regimens described herein i.e., water
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • compositions comprising any of the compositions provided herein and a food product, such as a dairy product, a processed meat, or a vegetable produce.
  • the food product is a dairy product, such as milk (including raw milk, unpasteurized milk, or mastitic milk).
  • the composition comprising the de-agglomeration agent (for example, optionally in combination with the adjuvant composition) and the antibacterial agent are added to the food product.
  • the composition comprising the de- agglomeration agent (for example, optionally in combination with the adjuvant composition) and the antibacterial agent are sprayed on the surface of the food product.
  • the composition (including the pharmaceutical composition and the food product composition) is contained in a single-use vial, such as a single-use sealed vial. In some embodiments, the composition (including the pharmaceutical composition and the food product composition) is contained in a multi-use vial. In some embodiments, the composition (including the pharmaceutical composition and the food product composition) is contained in bulk in a container. In some embodiments, the composition (including the pharmaceutical composition and the food product composition) is contained in a single container. In some embodiments, the de-agglomeration agent, the antibacterial agent or the probiotic composition, and optionally the adjuvant composition are contained in separate containers.
  • articles of manufacture comprising the compositions (including the pharmaceutical compositions and the food product compositions), formulations, and unit dosages described herein in suitable packaging for use in the methods of treatment, methods of administration, and dosage regimens described herein.
  • suitable packaging for compositions (including the pharmaceutical compositions and the food product compositions) described herein are known in the art, and include, for example, vials (such as sealed vials), vessels (such as sealed vessels), ampules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These articles of manufacture may further be sterilized and/or sealed.
  • kits comprising the compositions (including the pharmaceutical compositions and the food product compositions), formulations, unit dosages, and articles of manufacture described herein for use in the methods of treatment, methods of administration, and dosage regimens described herein.
  • compositions described herein make use of de- agglomeration agents.
  • the de- agglomeration agent described herein reduces agglomeration (such as agglutination) of bacteria cells.
  • the de- agglomeration agent reduces agglomeration (such as agglutination) of bacteria cells by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more.
  • Agglomeration (such as agglutination) of bacteria cells may be measured by any of the methods known in the art, such as the methods described in Gill et al. J. Appl. Microbiol. 2006, 101(2): 377-86; and Tanji et al. Biochem. Eng. J.
  • Exemplary methods for measuring bacteria agglomeration include, but are not limited to, dynamic light scattering, flow cytometry, microscopy (such as electron microscopy and fluorescence microscopy), and quantification of colony forming units (CFU) of bacteria culture by plating the culture and comparison to the CFU of non- agglomerated bacteria culture of the same strain and optical density under the same culturing and plating conditions (for example, as described in Example 1).
  • CFU colony forming units
  • the de-agglomeration agent reduces agglomeration (such as agglutination) of bacteria cells by reducing crosslinking of the bacteria cells.
  • the de- agglomeration agent modifies, such as cleaves, the crosslinking molecules that connect bacteria cells.
  • the crosslinking molecules are derived from whey proteins. Whey proteins include, but are not limited to, beta-lactoglobulin, alpha- lactalbumin, serum albumin, and immunoglobulins (such as IgA, IgD, IgE, IgG, or IgM).
  • Exemplary crosslinking molecules include, but are not limited to, immunoglobulin (such as IgG, IgA, IgM, IgE, or IgD, or subclasses thereof), protein A, protein G, fibrinogen, and Clumping factor A (ClfA).
  • the de-agglomeration agent cleaves an antibody and/or the molecule bound by an antibody on the surface of the bacteria cells that crosslinks the bacteria cells.
  • the de- agglomeration agent cleaves immunoglobulin (such as IgG, IgA, IgM, IgE, or IgD, or subclasses thereof), protein A, protein G, fibrinogen, or ClfA.
  • Bacteria contemplated in the present invention includes, but are not limited to, pathogenic bacteria (including conditional pathogenic bacteria), symbiotic bacteria with a pest (such as an insect), and non-pathogenic bacteria.
  • Exemplary bacteria may include, but are not limited to, Staphylococcus aureus, Pseudomonas aeruginosa, Clostridium difficile, Mycobacterium tuberculosis, and bacteria species of the Streptococcus genus (such as Group C and Group G Streptococcus, for example, Streptococcus uberis and Streptococcus dysgalactiae).
  • the bacteria cells express Protein A and/or Protein G.
  • Group C and Group G Streptococcus bacteria such as Strep, dysgalactiae, express Protein G.
  • the bacteria cells form agglomeration (such as agglutination) in animal tissues, including, for example, mammary gland, heart valves, lungs, blood stream, digestive tract, bone, nose, throat, or skin.
  • Protein A or Protein G expressed by the bacteria cells may interact with IgG in the animal tissues, which leads to agglutination of the bacteria cells. Protein A and protein G have affinity to different immunoglobulin subclasses in various animal species.
  • protein A has strong affinity to human IgGl, human IgG2, human IgG4, mouse IgG2a, mouse IgG2b, rat IgG, rabbit IgG, Guinea pig IgG, and pig IgG; protein A has moderate affinity to human IgA, human IgD, human IgE, human IgM, mouse IgGl, mouse IgG3, hamster IgG, bovine IgG, and horse IgG; protein G has strong affinity to human IgGl, human IgG2, human IgG3, human IgG4, mouse IgGl, mouse IgG2a, mouse IgG2b, mouse IgG3, rat IgG, rat IgG2a, rabbit IgG, hamster IgG, bovine IgG, horse IgG, sheep IgG, goat IgG, and pig IgG; protein G has moderate affinity to rat IgGl, rat IgG2b, rat IgG2c,
  • bacteria cells expressing Protein A or Protein G may interact with any of the immunoglobulin subclasses described herein in the serum, milk, other bodily fluids, or tissues of the animal species expressing the immunoglobulin subclass, and lead to agglutination of the bacteria cells.
  • the bacteria cells such as S.
  • aureus express a clumping factor (such as Clumping factor A, or Of A) that binds to fibrinogen and form agglomeration in animal plasma or tissues.
  • a clumping factor such as Clumping factor A, or Of A
  • fibrinogen is found in milk, and Of A expressed by S. aureus may bind to fibrinogen and lead to agglomeration of S. aureus in the milk.
  • compositions and methods describe herein can be useful for treating bacterial infection or reducing bacteria that agglomerate via any of the agglomeration mechanisms (such as agglutination) described herein or otherwise known in the art.
  • the de- agglomeration agent may cleave a bacterial protein, and/or a host protein that contribute to agglomeration (such as agglutination) of the bacteria cells.
  • exemplary targets of the de- agglomeration agents include, but are not limited to, immunoglobulin (such as IgG, IgA, IgM, IgE, IgD, and subclasses thereof), Protein A, Protein G, fibrinogen, and ClfA.
  • the de-agglomeration agent comprises a protease.
  • the de- agglomeration agent may consist of a single protease, or a combination (i.e. a cocktail) of proteases.
  • the de-agglomeration agent comprises non-protease components in addition to the protease or combination of proteases.
  • Proteases contemplated in the present invention may include, but are not limited to, serine proteases, threonine proteases, cysteine proteases, aspartate proteases, glutamic acid proteases, and metalloproteases.
  • the protease may be a non-specific protease that can hydrolyze a wide range of protein substrates, or a specific protease that only cleaves substrates with a certain sequence.
  • the de-agglomeration agent comprises an endopeptidase.
  • the de- agglomeration agent is a serine-type protease.
  • the de-agglomeration agent is a cysteine protease.
  • the de- agglomeration agent is a cysteine protease of the peptidase LI family.
  • the de-agglomeration agent is a protease derived from a fruit, such as papaya, pineapple, fig, kiwi, or the like.
  • the de-agglomeration agent is a protease selected from the group consisting of papain, bromelain, IdeS, pepsin, ficain, actinidin, cathepsin-B like protease, plasmin and combinations thereof.
  • papain is a papaya proteinase I, a cysteine proteinase enzyme present in papaya with Enzyme Commission (EC) number 3.4.22.2.
  • bromelain is a protease enzyme derived from the plants of the family Bromeliaceae with EC 3.4.22.32 or EC 3.4.22.33.
  • IdeS is an immunoglobulin degrading enzyme of Streptococcus pyogenes, a streptococcal cysteine protease with specificity for immunoglobulin G.
  • pepsin is an endopeptidase in the gastric juice of vertebrates with EC 3.4.23.1.
  • ficain is a cysteine endopeptidase derived from figs latex with EC 3.4.22.3.
  • actinidin is a cysteine protease with EC 3.4.22.14.
  • cathepsin-B like protease is cathepsin-B or a protease of the cathepsin-B family.
  • plasmin is a serine protease with EC 3.4.21.7.
  • a suitable protease is chosen as the de-agglomeration agent by cleaving an agglomeration (such as agglutination) or crosslinking protein.
  • papain or bromelain can be used to cleave immunoglobulin (such as IgG, IgA, IgM, IgE, or IgD, or subclasses thereof).
  • immunoglobulin such as IgG, IgA, IgM, IgE, or IgD, or subclasses thereof.
  • plasmin is used to cleave fibrinogen that interacts with ClfA (e.g., expressed by S. aureus).
  • the protease is isolated from a natural source, such as a fruit. In some embodiments, the protease is synthesized recombinantly. In some embodiments, the protease (such as papain, bromelain, IdeS, pepsin, ficain, actinidin, cathepsin-B like protease, and/or plasmin) is engineered such that one or more of its properties are changed relative to the wild-type protease. Exemplary properties include but are not limited to stability, substrate specificity, binding affinity, immunogenicity, immunotoxicity, and kinetics.
  • any of the de-agglomeration agents can be combined with an adjuvant composition.
  • adjuvants contemplated herein include, but are not limited to, chelating agents, such as ethylenediaminetetraacetic acid (EDTA) and salts thereof, and reducing agents, such as cysteine, 2-mercaptoethanol, mercaptoethylamine, TCEP, dithiothreitol, glutathione and salts thereof.
  • the adjuvant composition comprises a chelating agent, such as EDTA.
  • the adjuvant composition comprises a reducing agent, such as cysteine.
  • the adjuvant composition comprises a chelating agent (e.g.
  • the adjuvant composition comprises a chelating agent (e.g. , EDTA) and a reducing agent (e.g. , cysteine).
  • the adjuvant composition comprises EDTA and cysteine.
  • the de-agglomeration agent comprises a protease (such as a cysteine protease, for example papain, or bromelain), and the adjuvant composition comprises a chelating agent (e.g. , EDTA) and a reducing agent (e.g. , cysteine).
  • the methods and compositions described herein in some embodiments use or comprise antibacterial agents.
  • the antibacterial agents contemplated herein include any substance that is destructive to or inhibits the growth of bacteria.
  • the antibacterial agent is an antibiotic.
  • the antibacterial agent is a small-molecule antibiotic.
  • the antibacterial agent is a bacteriolytic enzyme.
  • the antibacterial agent is a bacteriophage.
  • the antibacterial agent is a bacteriocin.
  • the antibacterial agent comprises a quorum sensing signal molecule.
  • the antibacterial agent comprises a phage-derived protein.
  • the antibacterial agent may be a single substance, or a mixture of substances of the same type (such as small-molecule antibiotic, quorum sensing signal molecule, bacteriolytic enzyme, phage-derived protein, bacteriophage, bacteriocin, and the like) or different types.
  • the antibacterial agent comprises at least any of 1, 2, 3, 4, 5, or 6 agents selected from the group consisting of a small-molecule antibiotic, a quorum sensing signal molecule, a bacteriolytic enzyme, a phage-derived protein, a bacteriophage, and a bacteriocin.
  • the antibacterial agent comprises at least any of 1, 2, 3, 4, 5, or 6 types of agents selected from the group consisting of a small-molecule antibiotic, a quorum sensing signal molecule, a bacteriolytic enzyme, a phage-derived protein, a bacteriophage, and a bacteriocin.
  • the antibacterial agent comprises a small-molecule antibiotic (such as ampicillin, pirlimycin, or cephapirin).
  • the antibacterial agent comprises a single bacteriophage or a bacteriophage cocktail.
  • the antibacterial agent comprises a small-molecule antibiotic and a bacteriophage.
  • the quorum sensing signal molecules, bacteriolytic enzymes, phage-derived proteins, bacteriophages, and/or bacteriocins may be isolated from a natural source, or produced recombinantly.
  • the quorum sensing signal molecule, bacteriolytic enzyme, or the bacteriocin is present in a bacterium.
  • the antibacterial agent is Lactobacillus that produces a bacteriocin that kills protein A or protein G expressing bacteria (such as Staphylococcus aureus).
  • the phage-derived protein is present in a phage, a phage vector, or a bacterial host of the phage.
  • any of the quorum sensing signal molecules, bacteriolytic enzymes, phage- derived proteins, bacteriophages, and/or bacteriocins is engineered such that one or more of its properties are changed relative to the wild-type agent.
  • Exemplary properties include but are not limited to stability, substrate specificity, binding affinity, immunogenicity, immunotoxicity, and kinetics.
  • the antibacterial agent comprises a small-molecule antibiotic.
  • Exemplary small-molecule antibacterial agents contemplated herein include, but are not limited to pirlimycin, ceftiofur, desfurolyceftiofur, amikacin, ampicillin, dihydro streptomycin, flunixin, gentamicin, neomycin, tilmicosin, oxytetracycline, penicillin, sulfadiazine, sulfadimethoxine, sulfamethazine, sulfamethoxazole, sulfathiazole, tetracycline, tylosin, phenoxymethylpenicillin, flucloxacillin, amoxicillin, amoxicillin-clavulanate, clarithromycin, trimethoprim- sulfamethoxazole, nafcillin, oxacillin, vancomycin, cefaclor, cephapirin, cefadroxil, cephalexin,
  • the small-molecule antibiotic is ampicillin. In some embodiments, the small-molecule antibiotic is pirlimycin, such as PIRSUE ® . In some embodiments, the small-molecule antibiotic is cephapirin, such as TODAY ® .
  • the antibacterial agent comprises a quorum sensing signal molecule.
  • the antibacterial agent comprises a bacteriolytic enzyme.
  • the antibacterial agent comprises a phage-derived protein.
  • phage-derived proteins have been described. For example, see Drulis-Kawa et al. Curr. Med. Chem. (2015) 22(14): 1757-1773. Any of known phage-derived proteins having antibacterial activities may be used in the present invention.
  • the phage- derived protein is selected from the group consisting of endolysin, lysin, holin, tail fiber protein, and tailocin. Exemplary lysins have been described, for example, see, Young R (1992). "Bacteriophage lysis: mechanism and regulation". Microbiological Reviews 56 (3): 430-81.
  • the antibacterial agent comprises a bacteriophage.
  • Exemplary bacteriophages include, but are not limited to, bacteriophage K (such as ATCC strain 19685- B l), bacteriophage 17 (such as ATCC strain 23361-B l), and Stab8.
  • the antibacterial agent comprises a combination ⁇ i.e., cocktail) of bacteriophages.
  • the combination of bacteriophages comprises bacteriophage K (such as ATCC strain 19685-B 1), bacteriophage 17 (such as ATCC strain 23361-B l), and Stab8.
  • the antibacterial agent comprises a bacteriocin.
  • the bacteriocin is a high-molecular weight bacteriocin, such as R-type
  • the bacteriocin is a low molecular weight polypeptide bacteriocin, such as nisin.
  • Exemplary bacteriocins include, but are not limited to, Class I-IV LAB antibiotics (such as lantibiotics), colicins, microcins, and pyocins.
  • the bacteriocin is a pyocin.
  • the pyocin is an R-pyocin, F- pyocin, or S-pyocin. Other naturally occurring and engineered bacteriocins have been described; for example, see Gillor O.
  • the bacteriocin is engineered based on a naturally-occurring bacteriocin.
  • Methods of engineering bacteriocins are known in the art, for example, see International Patent Application Publication No. WO2007134303, incorporated herein by reference.
  • Example 1 Papain enhances bacteria inhibition activity of bacteriophage K
  • Staphylococcus aureus isolated from bovine mastitis was grown in tryptic soy broth (TSB) overnight. The overnight culture was diluted and grown to a mid-log-phase optical density ( ⁇ OD 0.4). Raw milk was incubated with 1 ⁇ g/ml of papain for 30 min at 37 °C.
  • FIG. 1 shows that bacteriophage K treatment results in only 6% S. aureus growth inhibition in untreated raw milk (column 2) relative to the original inoculum (column 1).
  • Pretreatment of the raw milk with papain generates more S. aureus cfu after plating (column 3), but this is likely due to dispersion of S. aureus aggregates leading to an apparent increase in cfu rather than an actual change in growth rate.
  • bacteriophage K inhibits S. aureus growth by 84% (column 4).
  • Staphylococcus aureus isolated from bovine mastitis was grown in tryptic soy broth (TSB) overnight. The overnight culture was diluted and grown to a mid-log-phase optical density ( ⁇ OD 0.4). Raw milk was incubated with 10 ⁇ g/ml of papain for 30 min at 37 °C.
  • FIG. 2 shows that ampiciUin treatment results in 88% S. aureus growth inhibition after 4 h in untreated raw milk (column 2) relative to the original inoculum (column 1).
  • ampiciUin inhibits S. aureus growth by >99.9%, a 3 log reduction, at 4 h (column 3, not visible due on this scale).
  • papain not only enhances the ability of bacteriophage K to infect S. aureus in raw milk, it dramatically enhances the efficacy of ampiciUin, a small-molecule antibiotic.
  • Example 3 Papain or bromelain enhances bacteria inhibition activity of a bacteriophage cocktail
  • Papain Sigma P3125-25mg, at a final concentration of 89 ⁇ g/ml
  • bromelain AC402830250, at a final concentration of 250 ⁇ g/ml
  • Staphylococcus aureus isolated from cows with symptoms of bovine mastitis was grown in tryptic soy broth (TSB) to mid-log-phase ( ⁇ OD 0.3) to obtain the bacteria culture.
  • aureus phage cocktail (same composition as in (2)) (MOI: 30), and 7 ml of papain, EDTA and cysteine-treated raw milk; (5) 50 ⁇ of bacterial culture and 7 ml of bromelain, EDTA and cysteine-treated raw milk; and (6) 50 ⁇ of bacterial culture, S. aureus phage cocktail (same composition as in (2)) (MOI: 30), and 7 ml of bromelain, EDTA and cysteine-treated raw milk. After incubation, 1: 10 serial dilutions of each culture were performed and 5 ⁇ ⁇ of each diluted culture was spotted onto CHROMagarTM S. aureus plates, and incubated overnight at 37°C. The number of colony-forming units was determined for each treatment condition and plotted in FIG. 3.
  • the phage cocktail demonstrated enhanced bacterial inhibition, with a much lower amount of bacteria survivors (i.e. about 5.0x10 cfu/ml).
  • Bromelain (at a final concentration of 250 ⁇ g/ml) was added to 7 mL of raw milk in presence of 0.5 mM EDTA and 1 mM of cysteine prior to the addition of bacteria culture.
  • Staphylococcus aureus isolated from cows with symptoms of bovine mastitis was grown in tryptic soy broth (TSB) to mid-log-phase ( ⁇ OD 0.3) to obtain the bacteria culture.
  • Example 5 Microscopic analysis demonstrates de-agglomeration of 5. aureus by papain or bromelain treatment.
  • bovine mastitis Strep, uberis formed agglomeration in the presence of 5mg/ml IgG. Additionally, Staphylococcus aureus and Streptococcus species infections in animals tissues, including heart valves, lungs, blood stream, digestive tract, bone, nose, throat, or skin, etc. that can also form agglomeration due to the interaction between Protein A/G expressed by these two kinds of bacteria and immunoglobulins (such as IgG) in tissues.
  • cysteine proteases such as bromelain
  • cysteine can be used to break the bacterial agglomeration.
  • the combination can be used with bacteriophage (including cocktail of bacteriophages) or small-molecule antibiotics (such as ampicillin) to treat bacterial infections caused by Staphylococcus aureus, and Streptococcus species of Group C and Group G, including Strep, uberis and S. dysgalactiae in animal tissues.
  • Example 6 Bromelain enhances bacteria inhibition activity of pirlimycin in simulated mastitic milk.
  • Staphylococcus aureus isolated from cows with symptoms of bovine mastitis was grown in tryptic soy broth (TSB) to mid-log-phase ( ⁇ OD 0.3) to obtain a bacteria culture.
  • TTB tryptic soy broth
  • ⁇ OD 0.3 mid-log-phase
  • 100 ⁇ of this bacteria culture and 12 mg/mL of bovine IgG were added to 7 mL of raw milk and incubated at 37°C for 1 hour.
  • the addition of bovine IgG could simulate the levels of IgG found in mastitic milk (Tanji et al. Biochem. Eng. J. 2015, 97: 17-24).
  • Example 7 Bromelain enhances bactericidal activity of cephapirin in raw milk.
  • Staphylococcus aureus isolated from cows with symptoms of bovine mastitis was grown (ATCC, ATCC No: 31886) in tryptic soy broth (TSB) to mid-log-phase ( ⁇ OD 0.3) to obtain a bacteria culture. 100 ⁇ of this bacteria culture were added to 7 mL of raw milk and incubated at 37°C for 1 hour.
  • Staphylococcus aureus isolated from cows with symptoms of bovine mastitis were grown in tryptic soy broth (TSB) to mid-log-phase ( ⁇ OD 0.3) to obtain a bacteria culture.
  • TTB tryptic soy broth
  • ⁇ OD 0.3 mid-log-phase
  • 100 ⁇ of this bacteria culture and 12 mg/mL of bovine IgG were added to 7 mL of raw milk and incubated at 37°C for 1 hour.
  • the addition of bovine IgG could simulate the levels of IgG found in mastitic milk (Tanji et al. Biochem. Eng. J. 2015, 97: 17-24).
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