CA2988579A1

CA2988579A1 - Compositions and methods for increasing the susceptibility of bacteria to antibiotics

Info

Publication number: CA2988579A1
Application number: CA2988579A
Authority: CA
Inventors: Ping-Chuan Christina Tsai
Original assignee: Epibiome Inc
Current assignee: Epibiome Inc
Priority date: 2015-04-21
Filing date: 2016-04-21
Publication date: 2016-10-27
Also published as: AU2016250606A1; EP3285587A1; WO2016172380A1

Abstract

The present invention provides compositions and methods for increasing the susceptibility of bacteria to an antibacterial agent in in vivo or in vitro. The compositions comprise a de- agglomeration agent that can reduce agglomeration of bacteria cells. The compositions and methods may be applied in various fields, including human and veterinary medicine, and food safety applications.

Description

COMPOSITIONS AND METHODS FOR INCREASING THE SUSCEPTIBILITY
OF BACTERIA TO ANTIBIOTICS
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority benefit of U.S. Provisional Patent Applications No.
62/150, 294 filed on April 21, 2015, and No. 62/249, 115 filed on October 30, 2015, the contents of each of which are incorporated herein by reference in their entirety.
FIELD OF THE INVENTION

[0002] The present invention relates to the field of microbiology, antibiotics, human and veterinary medicine, and food safety.
BACKGROUND OF THE INVENTION

[0003] The planet is populated by an estimated 5x103 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.
Sometimes, however, 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.

[0004] Western medicine has historically relied on broad-spectrum disinfectants, 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.
The growing incidence of bacterial resistance to small-molecule antibiotics has led to increased investigation of alternative antibiotic strategies to address bacterial infections, which include bacteriolytic enzymes, bacteriophages, and bacteriocins. Bacterial transplant strategies (e.g., fecal transplants, probiotics) are also being explored.

[0005] Even when in vitro testing indicates that a bacterial strain is susceptible to a particular antibiotic, such susceptibility is not always realized in vivo due to non-genetic resistance mechanisms, usually facilitated by a biological matrix. Agglomeration is one such mechanism whereby bacteria clump together to minimize their surface area-to-volume ratio; this minimizes the total cell surface area that is exposed to exogenously administered antibiotics and often confers a significant survival advantage to those cells located within the interior of the clump.

[0006] Agglomeration is the result of the crosslinking of bacteria cells. In biofilms, for example, organisms adhere to one another due to secretion of extracellular polymers by one or more members of the community. However, crosslinking molecules need not be supplied by the bacteria themselves. In 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. Although an important part of humoral immunity, antibody-mediated agglutination of pathogens can make them less susceptible to antibiotics;
thus, the immune response can hinder, rather than complement, antibiotic therapy.

[0007] Gill et al. (Gill JJ, Sabour PM, Leslie KE, and Griffiths MW. J. Appl.
Microbiol. 2006, 101(2):377-86) have shown that whey proteins in unpasteurized bovine milk inhibit the interaction of Staphylococcus aureus and bacteriophage K. Tanji et al. (Tanji Y, Tanaka A, Tani K, Kurimoto M, and Miyanaga K. Biochem. Eng. J. 2015, 97: 17-24) subsequently demonstrated that the bovine immunoglobulin G (IgG), a component of whey, is responsible for promoting S.
aureus agglutination, lowering the phage¨S. aureus binding constant, and increasing the minimum inhibitory concentration (MIC) of phage. These effects, which were quantified in ex vivo milk media, illuminate the failure mechanism of phage therapy in the treatment of bovine mastitis (Gill JJ, Pacan JC, Carson ME, Leslie KE, Griffiths MW, and Sabour PM. Antimicrob.
Agents Chemother. 2006, 50(9):2912-8).

[0008] The disclosures of all publications, patents, patent applications and published patent applications referred to herein are hereby incorporated herein by reference in their entirety.
BRIEF SUMMARY OF THE INVENTION

[0009] The present invention provides compositions and methods for increasing the susceptibility of bacteria to antibiotic therapy. The subject compositions and methods find use in a variety of different applications, including human and veterinary medicine.

[0010] In one aspect of the present application, there is provided 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.

[0011] In some embodiments according to any one of the compositions provided above, the antibacterial agent comprises a small-molecule antibiotic. In some embodiments, 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, vancomycin, cefaclor, cephapirin, cefadroxil, cephalexin, doxycycline, dicloxacillin, lymecycline, tobramycin, erythromycin, azithromycin, clarithromycin, clindamycin, co-trimoxazole, metronidazole, tinidazole, ciprofloxacin, levofloxacin, norfloxacin, and combinations thereof. In some embodiments, the small-molecule antibiotic is ampicillin. In some embodiments, the small-molecule antibiotic is pirlimycin. In some embodiments, the small-molecule antibiotic is cephapirin.

[0012] In some embodiments according to any one of the compositions provided above, the antibacterial agent comprises a quorum sensing signal molecule.

[0013] In some embodiments according to any one of the compositions provided above, the antibacterial agent comprises a bacteriolytic enzyme.

[0014] In some embodiments according to any one of the compositions provided above, the antibacterial agent comprises a phage-derived protein. In some embodiments, the phage-derived protein is selected from the group consisting of lysozyme, endolysin, lysin, holin, tail fiber protein, and talocin.

[0015] In some embodiments according to any one of the compositions provided above, the antibacterial agent comprises a bacteriophage. In some embodiments, the bacteriophage is bacteriophage K. In some embodiments, the antibacterial agent comprises a cocktail of bacteriophages.

[0016] In some embodiments according to any one of the compositions provided above, the antibacterial agent comprises a bacteriocin. In some embodiments, the bacteriocin is a pyocin. In some embodiments, the bacteriocin is a nisin.

[0017] In some embodiments according to any one of the compositions provided above, the de-agglomeration agent is papain. In some embodiments, the antibacterial agent is bacteriophage K.
In some embodiments, the antibacterial agent is a cocktail of bacteriophages.
In some embodiments, the antibacterial agent is pirlimycin.

[0018] In some embodiments according to any one of the compositions provided above, the de-agglomeration agent is bromelain. In some embodiments, the antibacterial agent is ampicillin. In some embodiments, the antibacterial agent is bacteriophage K. In some embodiments, the antibacterial agent is a cocktail of bacteriophages. In some embodiments, the antibacterial agent is pirlimycin. In some embodiments, the antibacterial agent is cephapirin.

[0019] In another aspect of the present application, there is provided 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. In some embodiments, the probiotic composition comprises lactobacillus or bffidobacterium. In some embodiments, the composition further comprises a bacteriophage. In some embodiments, the composition further comprises a cocktail of bacteriophages.

[0020] In some embodiments according to any one of the compositions described above, the de-agglomeration agent cleaves immunoglobulin (such as IgG, IgA, IgM, IgE, or IgD, or subclasses thereof), protein A, protein G, fibrinogen, or ClfA.

[0021] In some embodiments according to any one of the compositions provided above, the de-agglomeration agent comprises a protease. In some embodiments, the protease is selected from the group consisting of papain, bromelain, IdeS, pepsin, ficain, actinidin, cathepsin-B like protease, plasmin and combinations thereof. In some embodiments, the protease is papain. In some embodiments, the protease is bromelain. In some embodiments, the protease is plasmin.

[0022] In some embodiments according to any one of the compositions provided above, the composition further comprises an adjuvant composition. In some embodiments, the adjuvant composition comprises a chelating agent. In some embodiments, the adjuvant composition comprises a reducing agent. In some embodiments, the adjuvant composition comprises a chelating agent (e.g., EDTA) and a reducing agent (e.g., cysteine). In some embodiments, the adjuvant composition comprises EDTA and cysteine.

[0023] Further provided in one aspect of the present application is 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. In some embodiments, the de-agglomeration agent cleaves immunoglobulin (such as IgG, IgA, IgM, IgE, or IgD, or subclasses thereof), protein A, protein G, fibrinogen, or ClfA.

[0024] In some embodiments according to any one of the above methods for increasing the susceptibility of bacteria cells, the de-agglomeration agent comprises a protease. In some embodiments, the protease is selected from the group consisting of papain, bromelain, IdeS, pepsin, ficain, actinidin, cathepsin-B like protease, plasmin and combinations thereof. In some embodiments, the protease is papain. In some embodiments, the protease is bromelain. In some embodiments, the protease is plasmin.

[0025] In some embodiments according to any one of the above methods for increasing the susceptibility of bacteria cells, the method further comprises adding to the target composition an effective amount of an adjuvant composition. In some embodiments, the adjuvant composition comprises a chelating agent. In some embodiments, the adjuvant composition comprises a reducing agent. In some embodiments, the adjuvant composition comprises a chelating agent (e.g., EDTA) and a reducing agent (e.g., cysteine). In some embodiments, the adjuvant composition comprises EDTA and cysteine.

[0026] In some embodiments according to any one of the above methods for increasing the susceptibility of bacteria cells, the antibacterial agent comprises a small-molecule antibiotic. In some embodiments, 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, oxacillin, vancomycin, cefaclor, cephapirin, cefadroxil, cephalexin, doxycycline, dicloxacillin, lymecycline, tobramycin, erythromycin, azithromycin, clarithromycin, clindamycin, co-trimoxazole, metronidazole, tinidazole, ciprofloxacin, levofloxacin, norfloxacin, and combinations thereof. In some embodiments, 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.

[0028] In some embodiments according to any one of the above methods for increasing the susceptibility of bacteria cells, the antibacterial agent comprises a bacteriolytic enzyme.

[0029] In some embodiments according to any one of the above methods for increasing the susceptibility of bacteria cells, the antibacterial agent comprises a phage-derived protein. In some embodiments, the phage-derived protein is selected from the group consisting of lysozyme, endolysin, lysin, holin, tail fiber protein, and talocin.

[0030] In some embodiments according to any one of the above methods for increasing the susceptibility of bacteria cells, the antibacterial agent comprises a bacteriophage. In some embodiments, the bacteriophage is bacteriophage K. In some embodiments, the antibacterial agent comprises a cocktail of bacteriophages.

[0031] In some embodiments according to any one of the above methods for increasing the susceptibility of bacteria cells, the antibacterial agent comprises a bacteriocin. In some embodiments, the bacteriocin is a pyocin. In some embodiments, the bacteriocin is a nisin.

[0032] In some embodiments according to any one of the above methods for increasing the susceptibility of bacteria cells, the bacteria cells are of a pathogenic bacterium species selected from the group consisting of Staphylococcus aureus, Pseudomonas aeruginosa, Clostridium difficile and Streptococcus. In some embodiments, the bacteria cells are Staphylococcus aureus, Group C or Group G Streptococcus (such as Streptococcus uberis, or Streptococcus dysgalactiae). In some embodiments, the bacteria cells are Bacillus spp. or Clostridium perfringens.

[0033] In some embodiments according to any one of the above methods for increasing the susceptibility of bacteria cells, the bacteria cells are symbiotic bacteria with a pest.

[0034] In some embodiments according to any one of the above methods for increasing the susceptibility of bacteria cells, the target composition is an in vivo composition. In some embodiments, the target composition is present in the mammary gland of an individual. In some embodiments, the target composition is present in the heart valve, lung, blood stream, digestive tract, bone, nose, throat, or skin of the individual. In some embodiments, the individual is a human individual. In some embodiments, the individual is a dairy cow.

[0035] In some embodiments according to any one of the above methods for increasing the susceptibility of bacteria cells, the target composition is an in vitro composition. In some embodiments, the target composition is a serum sample, an extracellular fluid sample, or a food product. In some embodiments, the food product is a dairy product, such as raw milk, for example, mastitic milk.

[0036] 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. In some embodiments, 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. In some embodiments the de-agglomeration agent cleaves immunoglobulin (such as IgG, IgA, IgM, IgE, or IgD, or subclasses thereof), protein A, protein G, fibrinogen, or ClfA.

[0037] In some embodiments according to any one of the above methods of treating a bacterial infection, the de-agglomeration agent comprises a protease. In some embodiments, the protease is selected from the group consisting of papain, bromelain, IdeS, pepsin, ficain, actinidin, cathepsin-B like protease, plasmin and combinations thereof. In some embodiments, the protease is papain. In some embodiments, the protease is bromelain. In some embodiments, the protease is plasmin.

[0038] In some embodiments according to any one of the above methods of treating a bacterial infection, the method further comprises administering to the individual an effective amount of an adjuvant composition. In some embodiments, the adjuvant composition comprises a chelating agent. In some embodiments, the adjuvant composition comprises a reducing agent. In some embodiments, the adjuvant composition comprises a chelating agent (e.g., EDTA) and a reducing agent (e.g., cysteine). In some embodiments, the adjuvant composition comprises EDTA and cysteine.

[0039] In some embodiments according to any one of the above methods of treating a bacterial infection, the antibacterial agent comprises a small-molecule antibiotic. In some embodiments, the small-molecule antibiotic is selected from the group consisting of pirlimycin, ceftiofur, desfurolyceftiofur, amikacin, ampicillin, dihydrostreptomycin, flunixin, gentamicin, neomycin, tilmico sin, oxytetracycline, penicillin, sulfadiazine, sulfadimethoxine, sulfamethazine, sulfamethoxazole, sulfathiazole, tetracycline, tylosin, phenoxymethylpenicillin, flucloxacillin, amoxicillin, amoxicillin-clavulanate, clarithromycin, trimethoprim-sulfamethoxazole, nafcillin, oxacillin, vancomycin, cefaclor, cephapirin, cefadroxil, cephalexin, doxycycline, dicloxacillin, lymecycline, tobramycin, erythromycin, azithromycin, clarithromycin, clindamycin, co-trimoxazole, metronidazole, tinidazole, ciprofloxacin, levofloxacin, norfloxacin, and combinations thereof. In some embodiments, the small-molecule antibiotic is ampicillin. In some embodiments, the small-molecule antibiotic is pirlimycin. In some embodiments, the small-molecule antibiotic is cephapirin.

[0040] In some embodiments according to any one of the above methods of treating a bacterial infection, the antibacterial agent comprises a quorum sensing signal molecule.

[0041] In some embodiments according to any one of the above methods of treating a bacterial infection, the antibacterial agent comprises a bacteriolytic enzyme.

[0042] In some embodiments according to any one of the above methods of treating a bacterial infection, the antibacterial agent comprises a phage-derived protein. In some embodiments, the phage-derived protein is selected from the group consisting of lysozyme, endolysin, lysin, holin, tail fiber protein, and talocin.

[0043] In some embodiments according to any one of the above methods of treating a bacterial infection, the antibacterial agent comprises a bacteriophage. In some embodiments, the bacteriophage is bacteriophage K. In some embodiments, the antibacterial agent comprises a cocktail of bacteriophages.

[0044] In some embodiments according to any one of the above methods of treating a bacterial infection, the antibacterial agent comprises a bacteriocin. In some embodiments, the bacteriocin is a pyocin. In some embodiments, the bacteriocin is a nisin.

[0045] In some embodiments according to any one of the above methods of treating a bacterial infection, the bacterial infection is in mammary gland, heart valve, lung, blood stream, digestive tract, bone, nose, throat, or skin of the individual. In some embodiments, the individual has mastitis. In some embodiments, the de-agglomeration agent is administered by intramammary infusion.

[0046] In some embodiments according to any one of the above methods of treating a bacterial infection, the de-agglomeration agent and the antibacterial agent are administered sequentially.
In some embodiments, the de-agglomeration agent and the antibacterial agent are administered simultaneously, such as in a single composition. In some embodiments, the de-agglomeration agent and the adjuvant composition are administered sequentially. In some embodiments, the de-agglomeration agent and the adjuvant composition are administered simultaneously, such as in a single composition.

[0047] Further provided in one aspect of the present application is 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. In some embodiments, 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. In some embodiments the de-agglomeration agent cleaves immunoglobulin (such as IgG, IgA, IgM, IgE, or IgD, or subclasses thereof), protein A, protein G, fibrinogen, or ClfA.

[0048] In some embodiments according to any one of the above methods of reducing contamination, the de-agglomeration agent comprises a protease. In some embodiments, the protease is selected from the group consisting of papain, bromelain, IdeS, pepsin, ficain, actinidin, cathepsin-B like protease, plasmin and combinations thereof. In some embodiments, the protease is papain. In some embodiments, the protease is bromelain. In some embodiments, the protease is plasmin.

[0049] In some embodiments according to any one of the above methods of reducing contamination, the method further comprises adding to the target composition an effective amount of an adjuvant composition. In some embodiments, the adjuvant composition comprises a chelating agent. In some embodiments, the adjuvant composition comprises a reducing agent.
In some embodiments, the adjuvant composition comprises a chelating agent (e.g., EDTA) and a reducing agent (e.g., cysteine). In some embodiments, the adjuvant composition comprises EDTA and cysteine.

[0050] In some embodiments according to any one of the above methods of reducing contamination, the antibacterial agent comprises a small-molecule antibiotic.
In some embodiments, 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, oxacillin, vancomycin, cefaclor, cephapirin, cefadroxil, cephalexin, doxycycline, dicloxacillin, lymecycline, tobramycin, erythromycin, azithromycin, clarithromycin, clindamycin, co-trimoxazole, metronidazole, tinidazole, ciprofloxacin, levofloxacin, norfloxacin, and combinations thereof. In some embodiments, the small-molecule antibiotic is ampicillin. In some embodiments, the small-molecule antibiotic is pirlimycin. In some embodiments, the small-molecule antibiotic is cephapirin.

[0051] In some embodiments according to any one of the above methods of reducing contamination, the antibacterial agent comprises a quorum sensing signal molecule.

[0052] In some embodiments according to any one of the above methods of reducing contamination, the antibacterial agent comprises a bacteriolytic enzyme.

[0053] In some embodiments according to any one of the above methods of reducing contamination, the antibacterial agent comprises a phage-derived protein. In some embodiments, the phage-derived protein is selected from the group consisting of lysozyme, endolysin, lysin, holin, tail fiber protein, and talocin.

[0054] In some embodiments according to any one of the above methods of reducing contamination, the antibacterial agent comprises a bacteriophage. In some embodiments, the bacteriophage is bacteriophage K. In some embodiments, the antibacterial agent comprises a cocktail of bacteriophages.

[0055] In some embodiments according to any one of the above methods of reducing contamination, the antibacterial agent comprises a bacteriocin. In some embodiments, the bacteriocin is a pyocin. In some embodiments, the bacteriocin is a nisin.

[0056] In some embodiments according to any one of the above methods of reducing contamination, the target composition is a serum sample, an extracellular fluid sample, or a food product. In some embodiments, the food product is a dairy product, such as raw milk, for example, mastitic milk.

[0057] In some embodiments according to any one of the above methods of reducing contamination, 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.

[0058] Further provided in one aspect of the present application is a method for improving gut health of an individual (such as a human 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. In some embodiments, the de-agglomeration agent cleaves immunoglobulin (such as IgG, IgA, IgM, IgE, or IgD, or subclasses thereof), protein A, protein G, fibrinogen, or ClfA.

[0059] In some embodiments according to any one of the above methods for improving gut health, the de-agglomeration agent comprises a protease. In some embodiments, the protease is selected from the group consisting of papain, bromelain, IdeS, pepsin, ficain, actinidin, cathepsin-B like protease, plasmin and combinations thereof. In some embodiments, the protease is papain. In some embodiments, the protease is bromelain. In some embodiments, the protease is plasmin.

[0060] In some embodiments according to any one of the above methods for improving gut health, the method further comprises administering to the individual an effective amount of an adjuvant composition. In some embodiments, the adjuvant composition comprises a chelating agent. In some embodiments, the adjuvant composition comprises a reducing agent. In some embodiments, the adjuvant composition comprises a chelating agent (e.g., EDTA) and a reducing agent (e.g., cysteine). In some embodiments, the adjuvant composition comprises EDTA and cysteine.

[0061] In some embodiments according to any one of the above methods for improving gut health, the probiotic composition comprises lactobacillus or bifidobacterium.

[0062] In some embodiments according to any one of the above methods for improving gut health, the probiotic composition comprises a bacteriophage. In some embodiments, the probiotic composition comprises a cocktail of bacteriophages.

[0063] In some embodiments according to any one of the above methods for improving gut health, the de-agglomeration agent is administered orally.

[0064] In some embodiments according to any one of the above methods for improving gut health, 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.

[0065] Also provided are pharmaceutical compositions, kits and articles of manufacture comprising any of the compositions described herein.

[0066] These and other aspects and advantages of the present invention will become apparent from the subsequent detailed description and the appended claims. It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS

[0067] FIG. 1 depicts a bacteriophage K inhibition assay of Staphylococcus aureus from raw milk with or without papain treatment. In untreated raw milk, bacteriophage K
is not effective at inhibiting the growth of S. aureus, likely due to IgG-mediated agglutination (column 2).
However, in papain-treated raw milk, bacteriophage K markedly inhibits S.
aureus growth after 4 h (column 4).

[0068] FIG. 2 depicts an ampicillin inhibition assay of Staphylococcus aureus cultures from raw milk with or without papain treatment. In untreated raw milk, treatment of S.
aureus with ampicillin (100 .t.g/m1 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).

[0069] FIG. 3 depicts a bacterial inhibition assay by a bacteriophage cocktail enhanced by treatment with papain or bromelain combined with adjuvants (EDTA and cysteine).

[0070] FIG. 4 depicts a bacterial inhibition assay by ampicillin enhanced by treatment with bromelain combined with adjuvants (EDTA and cysteine).

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.
DETAILED DESCRIPTION OF THE INVENTION

[0077] 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. 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.

[0078] 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. In an experiment using raw milk containing a culture of S. aureus isolated from cows with mastitis, it was shown that pretreatment of raw milk with papain substantially enhanced the antibacterial activity of phage K (86% inhibition v. 6% inhibition without pretreatment).

Similarly, in another experiment, when the milk was pretreated with papain, the antibiotic ampicillin inhibited S. aureus growth by >99.9%, a 3 log reduction, at 4 hours, as opposed to 88% growth inhibition in milk without papain treatment. Additionally, bromelain pre-treatment of raw milk supplemented with IgG that simulated the level of IgG found in mastitic milk significantly enhanced S. aureus killing activity of pirlimycin and cephapirin. These findings demonstrate that an agent capable of reducing agglomeration (such as agglutination) of bacteria cells can substantially enhance the efficacy of an antibacterial agent, including bacteriophages and small-molecule antibiotics. It was also discovered that 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. This allows development of effective methods for increasing susceptibility of bacteria in a target composition to antibacterial agents, for inhibiting bacterial contamination in vitro, or treating bacterial infection in vivo, as well as effective compositions suitable for use in such methods.

[0079] Thus, 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"). In some embodiments, the method further comprises adding to the target composition an effective amount of an adjuvant composition (such as chelating agent and/or reducing agent).

[0080] In another aspect, there is provided 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. In some embodiments, the method further comprises administering to the individual an effective amount of an adjuvant composition (such as chelating agent and/or reducing agent).

[0081] In another aspect, there is provided 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. In some embodiments, the method further comprises adding to the target composition an effective amount of an adjuvant composition (such as chelating agent and/or reducing agent).

[0082] Also provided are compositions (including for example anti-bacterial compositions, probiotic compositions, and/or food products) comprising an antibacterial agent and a de-agglomeration agent. In some embodiments, the composition further comprises an adjuvant composition (such as chelating agent and/or reducing agent).
Definitions

[0083] Terms are used herein as generally used in the art, unless otherwise defined as follows.

[0084] As used herein, "bacteria" refers to prokaryotic microorganisms belonging to the domain Bacteria.

[0085] As used herein, "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.

[0086] As used herein, "de-agglomeration agent" refers to an agent that reduces agglomeration of bacteria cells.

[0087] As used herein, "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.

[0088] As used herein, "antibacterial" refers to anything that is destructive to or inhibits the growth of bacteria.

[0089] As used herein, "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.

[0090] As used herein, "bactericidal" 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).

[0091] As used herein, "minimum inhibitory concentration" (MIC) means the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation. Methods for determining MICs for small-molecule antibiotics are disclosed by Andrews (Andrews JM. Journal of Antimicrob. Chemoth. 2001, 48(suppl 1): 5-16), while 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. 2011, 1(2): 75-79). Finally, 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).

[0092] As used herein, 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. In some embodiments, 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. In some embodiments, an effective amount of the de-agglomeration agent is an amount sufficient to reduce agglomeration (such as agglutination) of bacteria cells. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0093] As used herein, "treatment" or "treating" refers to an approach for obtaining beneficial or desired results including clinical results. For purposes of this invention, 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.

[0094] As used herein, "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). In some embodiments, the individual is a human. In some embodiments, the individual is an animal, such as a farm animal, for example a dairy cow.

[0095] As used herein, "mastitic milk" refers to milk from an individual (such as dairy cow) having bacterial infection to the mammary gland.

[0096] As used herein, "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.

[0097] As used herein, "small-molecule" means an organic compound with a molecular weight less than about 900 Da.

[0098] As used herein, "substance" means a particular kind of matter with uniform properties.

[0099] It is understood that aspects and embodiments of the invention described herein include "consisting" and/or "consisting essentially of' aspects and embodiments.

[0100] Reference to "about" a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to "about X"
includes description of "X".

[0101] As used herein, reference to "not" a value or parameter generally means and describes "other than" a value or parameter. For example, the method is not used to treat cancer of type X
means the method is used to treat cancer of types other than X.

[0102] The term "about X-Y" used herein has the same meaning as "about X to about Y."

[0103] As used herein and in the appended claims, the singular forms "a,"
"or," and "the"

include plural referents unless the context clearly dictates otherwise.
Methods of use

[0104] 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. In some embodiments, 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. In some embodiments, the adjuvant composition and the de-agglomeration agent are added to the target composition sequentially. In some embodiments, 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). 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. 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. aureus, Group C or Group G Streptococcus (such as Streptococcus uberis, or Streptococcus dysgalactiae), Pseudomonas aeruginosa, Clostridium difficile, Bacillus spp., or Clostridium perfringens.

[0105] In some embodiments, there is provided 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). In some embodiments, the target composition does not yet contain the bacteria cells. In some embodiments, the target composition already contains the bacteria cells. In some embodiments, the target composition further comprises the antibacterial agent. In some embodiments, the de-agglomeration agent is added to the target composition before the target composition is exposed to the bacteria cells. In some embodiments, 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. 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. In some embodiments, 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. 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. aureus, Group C or Group G
Streptococcus (such as Streptococcus uberis, or Streptococcus dysgalactiae), Pseudomonas aeruginosa, Clostridium difficile, Bacillus spp., or Clostridium perfringens.

[0106] In some embodiments, there is provided 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). In some embodiments, the target composition does not yet contain bacteria cells. In some embodiments, the target composition already contains bacteria cells. In some embodiments, the target composition further comprises the antibacterial agent. In some embodiments, the protease is added to the target composition before the target composition is exposed to the bacteria cells. In some embodiments, 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). In some embodiments, 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. In some embodiments, 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. 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. aureus, Group C or Group G Streptococcus (such as Streptococcus uberis, or Streptococcus dysgalactiae), Pseudomonas aeruginosa, Clostridium difficile, Bacillus spp., or Clostridium perfringens.

[0107] In some embodiments, there is provided a method of reducing bacterial contamination (such as contamination by S. aureus) in a target composition (such as a food composition, for example, milk), comprising adding to the target composition an effective amount of an antibacterial agent and an effective amount of a de-agglomeration agent. In some embodiments, there is provided 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). In some embodiments, 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). In some embodiments, 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. 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.
aureus, Group C or Group G Streptococcus (such as Streptococcus uberis, or Streptococcus dysgalactiae), Pseudomonas aeruginosa, Clostridium difficile, Bacillus spp., or Clostridium perfringens.

[0108] The target composition to which the de-agglomeration agent is added can be an in vitro composition. For example, in some embodiments, the target composition is a food composition, a serum composition, a plant composition, or an ex vivo tissue (such as meat).
In some embodiments, the target composition is a dairy product. In some embodiments, the target composition is milk (such as raw, i.e., unpasteurized, milk). In some embodiments, the target composition is milk from an individual having bacterial infection in the mammary gland. In some embodiments, the target composition is mastitic milk. In some embodiments, 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. In some embodiments, 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.

[0109] For example, in some embodiments, a de-agglomeration agent, such as bromelain, and an antibacterial agent, such as a single bacteriophage, or a cocktail of bacteriophages against several Bacillus spp. spoilage bacteria, are added to pasteurized milk to prolong the shelf life. In another example, a de-agglomeration agent, such as ficain, and an antibacterial agent, such as a bacteriocin against Clostridium perfringens, are added to a cut of meat (such as beef) to prolong its shelf life.

[0110] The methods may find use in food safety applications. In some embodiments, the de-agglomeration agent and the antibacterial agent are applied to processed meat.
In some embodiments, the de-agglomeration agent and the antibacterial agent are sprayed onto an area immediately before food preparation. In some embodiments, 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.

[0111] Thus, for example, in some embodiments, there is provided a method of increasing shelf-life of a food composition (such as a dairy product, for example raw milk), 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. In some embodiments, 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. In some embodiments, 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. Also provided herein are food compositions (such as dairy products, for example raw milk) comprising an antibacterial agent and an effective amount of a de-agglomeration agent that reduces agglomeration (such as agglutination) of bacteria cells.

[0112] In some embodiments, there is provided a method of improving safety of a food composition (such as a dairy product, for example raw milk, or mastitic milk), 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the de-agglomeration agents cleaves immunoglobulin in the food composition. In some embodiments, the de-agglomeration agent is a protease, such as papain, or bromelain. In some embodiments, the antibacterial agent is a bacteriophage, such as bacteriophage K, or a cocktail of bacteriophages.
In some embodiments, the antibacterial agent is a small molecule antibiotic, such as ampicillin, pirlimycin (e.g., PIRSUE ), or cephapirin (e.g., TODAY ). In some embodiments, the adjuvant composition comprises EDTA and cysteine.

[0113] The methods described herein are applicable to a range of food compositions and food products. In some embodiments, the food composition is a liquid composition.
In some embodiments, the food composition is a solid composition, such as meat. In some embodiments, the food composition is in powder form, such as milk powder. In some embodiments, the food composition is a dairy product. In some embodiments, the food composition is milk, such as raw, unpasteurized, or mastitic milk. In some embodiments, the de-agglomeration agent and the antibacterial agent are added to the food composition during the production of the food composition. In some embodiments, the de-agglomeration agent and the antibacterial agent are added to the food composition during the processing of the food composition.
In some embodiments, 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.

[0114] In some embodiments, there is provided 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. In some embodiments, 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. In some embodiments, 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). In some embodiments, the de-agglomeration agents cleaves immunoglobulin (such as IgG) in the mastitic milk composition. In some embodiments, the de-agglomeration agent is a protease, such as papain, or bromelain. In some embodiments, the antibacterial agent is a bacteriophage, such as bacteriophage K, or a cocktail of bacteriophages.
In some embodiments, the antibacterial agent is a small molecule antibiotic, such as ampicillin, pirlimycin (e.g., PIRSUE ), or cephapirin (e.g., TODAY ). In some embodiments, the adjuvant composition comprises EDTA and cysteine.

[0115] In some embodiments, there is provided 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. In some embodiments, 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). In some embodiments, the antibacterial agent 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. In some embodiments, the food composition comprises processed meat. In some embodiments, the food composition comprises a dairy product. In some embodiments, the food composition comprises vegetable or plant parts.

[0116] In some embodiments, the target composition is an in vivo composition.
For example, 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. In some embodiments, 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.

[0117] 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". In some embodiments, 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. In some embodiments, the antibacterial agent and the de-agglomeration agent are added to the target composition sequentially. In some embodiments, 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. In some embodiments, 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.

[0118] Any of the in vitro methods (such as methods of increasing susceptibility of bacteria cells in a target composition to an antibacterial agent, or methods of reducing contamination of an target composition by bacteria cells) described herein may comprise addition 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. In some embodiments, the adjuvant composition comprises a chelating agent, such as EDTA. In some embodiments, the adjuvant composition comprises a reducing agent, such as cysteine. In some embodiments, the adjuvant composition comprises a chelating agent (e.g., EDTA) or a reducing agent (e.g., cysteine). In some embodiments, the adjuvant composition comprises a chelating agent (e.g., EDTA) and a reducing agent (e.g., cysteine). In some embodiments, the adjuvant composition comprises EDTA and cysteine. In some embodiments, 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).

[0119] In some embodiments, 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. In some embodiments, the adjuvant composition and the de-agglomeration agent are admixed prior to the addition. In some embodiments, the adjuvant composition and the de-agglomeration agent are added to the target composition sequentially. In some embodiments, 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. In some embodiments, 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.

[0120] In some embodiments, the concentration of the added de-agglomeration agent in the target composition is between about 0.01 .t.g/m1 and about 10,000 i.t.g/ml, including for example between about 0.1 i.t.g/m1 and about 1,000 i.t.g/ml, such as between about 1 i.t.g/m1 and about 100 jig/ml. In some embodiments, 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. In some embodiments, 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.
In some embodiments, the concentration of the added bacteriophage in the target composition is between about 104 and about 1012 plaque-forming units (pfu) per ml, including for example between about 106 and about 1010 pfu, such as between about 107 and about 109 pfu/ml. In some embodiments, the concentration of the adjuvant composition added to the target composition is between about 1 [I,M to about 100 mM, including for example, about 1 [I,M to about 100 [I,M, about 100 [I,M to about 500 [I,M, about 500 [I,M to about 1 mM, about 1 mM to about 10 mM, or about 10 mM to about 100 mM.

Methods of treatment

[0121] The present application further provides methods of treating bacterial infection, or diseases associated with bacterial infection, such as mastitis.

[0122] In some embodiments, there is provided a method of treating a bacterial infection (such as infection by S. aureus, Group C or Group G Streptococcus, Pseudomonas aeruginosa, or Clostridium difficile) 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 that causes the bacterial infection.
In some embodiments, there is provided 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). In some embodiments, 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). In some embodiments, the adjuvant composition and the de-agglomeration agent (e.g., the protease) are administered to the individual sequentially. In some embodiments, the adjuvant composition and the de-agglomeration agent (e.g., the protease) are administered to the individual 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). 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. 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. aureus, Group C or Group G Streptococcus (such as Streptococcus uberis, or Streptococcus dysgalactiae), Pseudomonas aeruginosa, or Clostridium difficile. In some embodiments, 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.

[0123] The in vivo methods described herein can be useful for treating various diseases, particularly diseases involving bacterial infections. For example, in some embodiments, there is provided 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. In some embodiments, there is provided 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).
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). In some embodiments, 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. In some embodiments, 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.

[0124] 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. For example, 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. In some embodiments, the de-agglomeration agent and the antibacterial agent are administered by intramammary infusion into the teat canal of a dairy cow infected with mastitis. In some embodiments, the de-agglomeration agent and the antibacterial agent are applied topically to a S.
aureus-infected burn on a human patient. For example, 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. In some embodiments, the de-agglomeration agent and the antibacterial agent are delivered by nebulizer to a cystic fibrosis patient infected with Pseudomonas aeruginosa.
For example, Group C or Group G Streptococcus infection can affect farm animals and spread to humans through raw dairy product or contact with farm animals. In some embodiments, Group C or Group G Streptococcus infection can occur in the mammary gland of farm animals. In some embodiments, 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. For example, 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. In some embodiments, de-agglomeration agent and the antibacterial agent are delivered orally to treat a Clostridium difficile infection. For example, Mycobacterium tuberculosis infection can occur in the respiratory tract. The methods and compositions described herein are useful for treating tuberculosis.

[0125] Thus, for example, in some embodiments, there is provided a method of treating mastitis (such as mastitis caused by Staphylococcus aureus, Streptococcus uberis, or Streptococcus dysgalactiae) 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. In some embodiments, the de-agglomeration agent and the antibacterial agent are administered simultaneously. In some embodiments, the de-agglomeration agent and the antibacterial agent are administered as a single composition. In some embodiments, 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). For example, 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.

[0126] In some embodiments, there is provided a method of treating infected skin burn (such as a S. aureus infected skin burn) in an individual, 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. In some embodiments, the de-agglomeration agent and the antibacterial agent are administered as a single composition. In some embodiments, 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 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).

[0127] In some embodiments, there is provided a method of treating infected skin wound (such as a S. aureus infected skin wound) in an individual, comprising administering to the individual an effective amount of an antibacterial agent and an effective amount of a de-agglomeration agent. In some embodiments, the de-agglomeration agent and the antibacterial agent are administered as a single composition. In some embodiments, 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 antibacterial agent is administered orally. In some embodiments, the de-agglomeration agent and optionally the adjuvant composition is administered topically to the site of the skin wound.

[0128] In some embodiments, there is provided 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). In some embodiments, the de-agglomeration agent and the antibacterial agent are administered as a single composition. In some embodiments, 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 inhalation. In some embodiments, the de-agglomeration agent and/or the antibacterial agents and/or the adjuvant composition are administered by using a nebulizer.

[0129] In some embodiments, there is provided a method of treating Clostridium difficile infection (such as colitis or diarrhea) 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. In some embodiments, the de-agglomeration agent and the antibacterial agent are administered as a single composition.
In some embodiments, 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 orally to the individual.

[0130] In some embodiments, 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). In some embodiments, the antibacterial agent and the de-agglomeration agent are administered to the individual sequentially. In some embodiments, 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. In some embodiments, 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.

[0131] 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.

[0132] 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. In one embodiment, the non-bactericidal substance that potentiates the effect of the antibiotic is delivered about 1 h before the antibiotic. In another embodiment, the antibiotic is delivered about 20 min before the non-bactericidal substance that potentiates the effect of the antibiotic. Furthermore 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. In one embodiment, 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.

[0133] 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. In some embodiments, the de-agglomeration agent and the antibacterial agent are delivered orally to promote gut health. In some embodiment, the de-agglomeration agent and the antibacterial agent are sprayed onto a plant to control a bacterial parasite.

[0134] In some embodiments, there is provided 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. 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).

[0135] In some embodiments, there is provided 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. In some embodiments, 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. In some embodiments, the antibacterial agent, the de-agglomeration agent, and optionally the adjuvant composition are applied as a single composition. In some embodiments, the antibacterial agent, the de-agglomeration agent, and optionally the adjuvant composition are applied in separate compositions.

[0136] In some embodiments, 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.
Thus, for example, in some embodiments, there is provided a method of killing a pest (such as an insect), 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.

[0137] In some embodiments, there is provided 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. In some embodiments, the probiotic composition comprises lactobacillus or bifidobacterium. 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 the probiotic composition are administered simultaneously, such as in a single composition. In some embodiments, the de-agglomeration agent and the probiotic composition are administered sequentially.

[0138] Also provided herein are compositions comprising a de-agglomeration agent and a probiotic composition. In some embodiments, the composition further comprises an antibacterial agent (such as a single bacteriophage, or a cocktail of bacteriophages). In some embodiments, the composition further comprises an adjuvant composition (such as chelating agent, e.g., EDTA, and/or reducing agent, e.g., cysteine).

[0139] 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. In some embodiments, the probiotic composition comprises bacterial transplants, such as fecal transplants. In some embodiments, 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). For example, 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. In some embodiments, 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. In some embodiments, the probiotic composition comprises one or more bacteria that produce agents that inhibit the growth of gram negative bacteria. In some embodiments, 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. For example, 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.

[0140] The de-agglomeration agent and the probiotic composition can be administered to the individual simultaneously or sequentially. In some embodiments, 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). In some embodiments, the probiotic composition, and optionally the adjuvant composition are administered sequentially. In some embodiments, 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. In some embodiments, 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.

[0141] Any of the in vivo methods (such as methods of treating a bacterial infection, or methods of improving gut health) described herein 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. In some embodiments, the adjuvant composition comprises a chelating agent, such as EDTA. In some embodiments, the adjuvant composition comprises a reducing agent, such as cysteine. In some embodiments, the adjuvant composition comprises a chelating agent (e.g., EDTA) or a reducing agent (e.g., cysteine). In some embodiments, the adjuvant composition comprises a chelating agent (e.g., EDTA) and a reducing agent (e.g., cysteine). In some embodiments, the adjuvant composition comprises EDTA and cysteine. In some embodiments, 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).

[0142] In some embodiments, 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). In some embodiments, the adjuvant composition and the de-agglomeration agent are admixed prior to the administration. In some embodiments, the adjuvant composition and the de-agglomeration agent are administered to the individual sequentially. In some embodiments, 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.

[0143] The dose of the administered antibacterial agent (such as small-molecule antibiotic) can be calculated from the experimentally determined MIC using pharmacology equations known to persons of skill in the art:
cmax = mw x 2(dose interval/drug half-life), and ), ana dose = (Cnda,, x Vd)/F
wherein Cnda,, is the highest concentration of the antibiotic in the plasma, MIC is the minimum inhibitory concentration, Vd is the volume distribution (i.e. comparison of amount of drug in body versus the plasma concentration), and F is the systemic availability (i.e. fraction of the drug that reaches the blood unchanged).

[0144] 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. Where the pharmacokinetics of the agents or compositions are suitable, 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.
Compositions

[0145] 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. In some embodiments, 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.
In some embodiments, the non-bactericidal agent is a de-agglomeration agent that reduces agglomeration (such as agglutination) of bacteria cells.

[0146] 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. In some embodiments, the antibacterial agent can inhibit the growth of the bacteria cells without killing the bacteria cells (i.e., bacteriostatic agent). In some embodiments, the antibacterial agent kills the bacteria cells (i.e., bactericidal agent). In some embodiments, 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. In some embodiment, the de-agglomeration agent does not exhibit bactericidal activity against the bacteria cells. In some embodiments, the composition further comprises an adjuvant composition, such as chelating agent, e.g., EDTA, and/or reducing agent, for example, cysteine.

[0147] One aspect of the present invention provides a composition comprising a de-agglomeration agent and a probiotic composition, wherein the de-agglomeration agent reduces agglomeration (such as agglutination) of bacteria cells. In some embodiments, the composition further comprises a bacteriophage, such as bacteriophage K. In some embodiments, the composition further comprises a combination (i.e., cocktail) of bacteriophages (such as different bacteriophage strains). In some embodiments, the composition further comprises an adjuvant composition, such as chelating agent, e.g., EDTA, and/or reducing agent, for example, cysteine.

[0148] 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. In some embodiments, the probiotic composition comprises lactobacillus. In some embodiments, the probiotic composition comprises bifidobacterium. In some embodiments, the probiotic composition further comprises an antibacterial agent (such as a bactericidal agent, or a bacteriostatic agent).

[0149] 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.

[0150] In some embodiments, 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). In some embodiments, the de-agglomeration agent is bromelain, and the antibacterial agent is a small-molecule antibiotic (such as ampicillin, pirlimycin, or cephapirin).

[0151] In some embodiments, there is provided 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 (7)), EDTA, and cysteine. In some embodiments, there is provided a composition comprising papain, and ampicillin. In some embodiments, there is provided 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. Any of the 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).

[0152] 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. For example, in some embodiments, 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). In some embodiments, the concentration of the protease in the composition is any of between about 0.01 i.t.g/m1 and about 10,000 i.t.g/ml, between about 0.1 i.t.g/m1 and about 1,000 i.t.g/ml, or between about 1 i.t.g/m1 and about 100 jig/ml. In some embodiments, the antibacterial agent is a bacteriophage. In some embodiments, the concentration of the bacteriophage in the composition is any of between about 104 and about 1012 plaque-forming units (pfu) per ml, between about 106 and about 1010 pfu, or between about 107 and about 109 pfu/ml. In some embodiments, 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. In some embodiments, 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. In some embodiments, the concentration of the adjuvant composition (such as chelating agent, e.g., EDTA, or reducing agent, e.g., cysteine) is between about 1 [I,M to about 100 mM, including for example, about 1 [I,M
to about 100 [I,M, about 100 [I,M to about 500 [I,M, about 500 [I,M to about 1 mM, about 1 mM to about 10 mM, or about 10 mM to about 100 mM.

[0153] Further provided are pharmaceutical 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. Moreover, the use of molecular or particulate coatings such as lecithin, the proper selection of particle size in dispersions, or the use of materials with surfactant properties may be utilized. The pharmaceutical compositions described herein may include other agents, excipients, or stabilizers to improve properties of the composition. Examples of suitable 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. Examples of 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.

[0154] In some embodiments, 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 7Ø In some embodiments, 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.

[0155] In some embodiments, the composition (such as pharmaceutical composition) is suitable for administration to a human individual. In some embodiments, 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. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.

[0156] Also provided are food product compositions comprising any of the compositions provided herein and a food product, such as a dairy product, a processed meat, or a vegetable produce. In some embodiments, the food product is a dairy product, such as milk (including raw milk, unpasteurized milk, or mastitic milk). In some embodiments, 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. In some embodiments, 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.

[0157] In some embodiments, 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.

[0158] In some embodiments, there are provided 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.

[0159] Also provided are 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.
De-agglomeration agents

[0160] The methods and the compositions described herein make use of de-agglomeration agents.

[0161] In some embodiments, the de-agglomeration agent described herein (for both the methods and compositions) reduces agglomeration (such as agglutination) of bacteria cells. In some embodiments, 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. 2015, 97:
17-24. Exemplary methods for measuring bacteria agglomeration (such as agglutination) 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).

[0162] In some embodiments, the de-agglomeration agent reduces agglomeration (such as agglutination) of bacteria cells by reducing crosslinking of the bacteria cells. In some embodiments, the de-agglomeration agent modifies, such as cleaves, the crosslinking molecules that connect bacteria cells. In some embodiments, 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 (C1fA). In some embodiments, 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. In some embodiments, the de-agglomeration agent cleaves immunoglobulin (such as IgG, IgA, IgM, IgE, or IgD, or subclasses thereof), protein A, protein G, fibrinogen, or ClfA.

[0163] 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).
In some embodiments, the bacteria cells express Protein A and/or Protein G.
For example, Group C and Group G Streptococcus bacteria, such as Strep. dysgalactiae, express Protein G. In some embodiments, 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. For example, Protein A or Protein G
expressed by the bacteria cells (such as Staph. aureus or Streptococcus species) 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. For example, 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, and guinea pig IgG. See, for example, Richman D.D. et al.
(1982) "The binding of 1. Staphylococci protein A by the sera of different animal species," J.
Immunol. 128: 2300-2305; and Frank MB (1997) "Antibody Binding to Protein A
and Protein G
beads," 5. In: Frank, MB, ed. Molecular Biology Protocols. Oklahoma City.
Thus, 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. In some embodiments, the bacteria cells (such as S. aureus) express a clumping factor (such as Clumping factor A, or ClfA) that binds to fibrinogen and form agglomeration in animal plasma or tissues.
For example, fibrinogen is found in milk, and ClfA expressed by S. aureus may bind to fibrinogen and lead to agglomeration of S. aureus in the milk. It is understood that the 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. For example, 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.

[0164] In some embodiments, 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. In some embodiments, 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. In some embodiments, the de-agglomeration agent comprises an endopeptidase. In some embodiments, the de-agglomeration agent is a serine-type protease. In some embodiments, the de-agglomeration agent is a cysteine protease. In some embodiments, the de-agglomeration agent is a cysteine protease of the peptidase Ll family. In some embodiments, the de-agglomeration agent is a protease derived from a fruit, such as papaya, pineapple, fig, kiwi, or the like. In some embodiments, 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. In some embodiments, papain is a papaya proteinase I, a cysteine proteinase enzyme present in papaya with Enzyme Commission (EC) number 3.4.22.2. In some embodiments, 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. In some embodiments, IdeS is an immunoglobulin degrading enzyme of Streptococcus pyogenes, a streptococcal cysteine protease with specificity for immunoglobulin G. In some embodiments, pepsin is an endopeptidase in the gastric juice of vertebrates with EC
3.4.23.1. In some embodiments, ficain is a cysteine endopeptidase derived from figs latex with EC 3.4.22.3. In some embodiments, actinidin is a cysteine protease with EC
3.4.22.14. In some embodiments, cathepsin-B like protease is cathepsin-B or a protease of the cathepsin-B family.
In some embodiments, plasmin is a serine protease with EC 3.4.21.7. In some embodiments, a suitable protease is chosen as the de-agglomeration agent by cleaving an agglomeration (such as agglutination) or crosslinking protein. For example, papain or bromelain can be used to cleave immunoglobulin (such as IgG, IgA, IgM, IgE, or IgD, or subclasses thereof). In some embodiments, plasmin is used to cleave fibrinogen that interacts with ClfA
(e.g., expressed by S.
aureus).

[0165] In some embodiments, 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.

[0166] Any of the de-agglomeration agents can be combined with an adjuvant composition.
Exemplary 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. In some embodiments, the adjuvant composition comprises a chelating agent, such as EDTA. In some embodiments, the adjuvant composition comprises a reducing agent, such as cysteine. In some embodiments, the adjuvant composition comprises a chelating agent (e.g., EDTA) or a reducing agent (e.g., cysteine). In some embodiments, the adjuvant composition comprises a chelating agent (e.g., EDTA) and a reducing agent (e.g., cysteine). In some embodiments, the adjuvant composition comprises EDTA and cysteine. In some embodiments, 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).
Antibacterial agents

[0167] 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. In some embodiments, the antibacterial agent is an antibiotic. In some embodiments, the antibacterial agent is a small-molecule antibiotic. In some embodiments, the antibacterial agent is a bacteriolytic enzyme. In some embodiments, the antibacterial agent is a bacteriophage. In some embodiments, the antibacterial agent is a bacteriocin. In some embodiments, the antibacterial agent comprises a quorum sensing signal molecule. In some embodiments, the antibacterial agent comprises a phage-derived protein.

[0168] 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. In some embodiments, 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. In some embodiments, 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. 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 single bacteriophage or a bacteriophage cocktail. In some embodiments, the antibacterial agent comprises a small-molecule antibiotic and a bacteriophage.

[0169] The quorum sensing signal molecules, bacteriolytic enzymes, phage-derived proteins, bacteriophages, and/or bacteriocins may be isolated from a natural source, or produced recombinantly. In some embodiments, the quorum sensing signal molecule, bacteriolytic enzyme, or the bacteriocin is present in a bacterium. For example, in some embodiments, the antibacterial agent is Lactobacillus that produces a bacteriocin that kills protein A or protein G
expressing bacteria (such as Staphylococcus aureus). In some embodiments, the phage-derived protein is present in a phage, a phage vector, or a bacterial host of the phage. In some embodiments, 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.

[0170] In some embodiments, 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, doxycycline, dicloxacillin, lymecycline, tobramycin, erythromycin, azithromycin, clarithromycin, clindamycin, co-trimoxazole, metronidazole, tinidazole, ciprofloxacin, levofloxacin, norfloxacin, and combinations thereof. In some embodiments, 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 .

[0171] In some embodiment, the antibacterial agent comprises a quorum sensing signal molecule.

[0172] In some embodiments, the antibacterial agent comprises a bacteriolytic enzyme.

[0173] In some embodiments, the antibacterial agent comprises a phage-derived protein.
Exemplary phage-derived proteins have been described. For example, see Drulis-Kawa et al.
Carr. Med. Chem. (2015) 22(14): 1757-1773. Any of known phage-derived proteins having antibacterial activities may be used in the present invention. In some embodiments, 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.

[0174] In some embodiments, the antibacterial agent comprises a bacteriophage.
Exemplary bacteriophages include, but are not limited to, bacteriophage K (such as ATCC
strain 19685-B1), bacteriophage 17 (such as ATCC strain 23361-B1), and Stab8. In some embodiments, the antibacterial agent comprises a combination (i.e., cocktail) of bacteriophages. In some embodiments, the combination of bacteriophages comprises bacteriophage K (such as ATCC
strain 19685-B1), bacteriophage 17 (such as ATCC strain 23361-B1), and Stab8.
[0001] In some embodiments, the antibacterial agent comprises a bacteriocin.
In some embodiments, the bacteriocin is a high-molecular weight bacteriocin, such as R-type bacteriocins (e.g., pyocin). In some embodiments, 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. In some embodiments, the bacteriocin is a pyocin. In some embodiments, 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. et al. Current Pharmaceutical Design, 2005, 11:
1067-1075, incorporated herein by reference. In some embodiments, 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. W02007134303, incorporated herein by reference.
EXAMPLES

[0175] The examples, which are intended to be purely exemplary of the invention and should therefore not be considered to limit the invention in any way, also describe and detail aspects and embodiments of the invention discussed above. The examples are not intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (for example, amounts, temperature, etc.), but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
Example 1: Papain enhances bacteria inhibition activity of bacteriophage K

[0176] 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 .t.g/m1 of papain for 30 min at 37 C. Four different mixtures were setup and incubated for 4 h at 37 C: (1) 50 1..1.1 of bacterial culture in mid-log phase growth and 7 ml of untreated raw milk; (2) 50 pi of bacterial culture in mid-log phase growth, 150 pi of bacteriophage K (108 pfu/ml), and 6.85 ml of untreated raw milk; (3) 50 pi of bacterial culture in mid-log phase growth and 7 ml of papain-treated raw milk; and (4) 50 pi of bacterial culture in mid-log phase growth, 150 [11 of bacteriophage K
(108 pfu/ml), and 6.85 ml of papain-treated raw milk. After incubation, 1:10 serial dilutions of each culture were performed. For each dilution, 100 0_, was spread onto a tryptic soy agar plate, which was incubated overnight. The number of colony-forming units was determined for each treatment condition and plotted.

[0177] 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. After papain treatment of the raw milk, bacteriophage K inhibits S. aureus growth by 84% (column 4).
Example 2: Papain enhances bacteria inhibition activity of ampicillin

[0178] 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 .t.g/m1 of papain for 30 min at 37 C.
Three different mixtures were setup and incubated for 4 h at 37 C: (1) 50 pi of bacterial culture in mid-log phase growth and 7 ml of untreated raw milk; (2) 50 pi of bacterial culture in mid-log phase growth, ampicillin (100 .t.g/m1 final concentration), and 7 ml of untreated raw milk; and (3) 50 pi of bacterial culture in mid-log phase growth, ampicillin (100 .t.g/m1 final concentration), and 7 ml of papain-treated raw milk. After incubation, 1:10 serial dilutions of each culture were performed. For each dilution, 100 i.it was spread onto a tryptic soy agar plate, which was incubated overnight. The number of colony-forming units was determined for each treatment condition and plotted.

[0179] FIG. 2 shows that ampicillin treatment results in 88% S. aureus growth inhibition after 4 h in untreated raw milk (column 2) relative to the original inoculum (column 1). Remarkably, when the milk is pretreated with papain, ampicillin inhibits S. aureus growth by >99.9%, a 3 log reduction, at 4 h (column 3, not visible due on this scale). Thus, papain not only enhances the ability of bacteriophage K to infect S. aureus in raw milk, it dramatically enhances the efficacy of ampicillin, a small-molecule antibiotic.
Example 3: Papain or bromelain enhances bacteria inhibition activity of a bacteriophage cocktail

[0180] Papain (Sigma P3125-25mg, at a final concentration of 89 i.t.g/m1) or bromelain (ACROS
Organics AC402830250, at a final concentration of 250 i.t.g/m1) were added to 7mL of raw milk in the presence of 0.5mM 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. Six different conditions were incubated for 12 hours at 37 C: (1) 50 ill of bacterial culture in 7 ml of untreated raw milk; (2) 50 ill of bacterial culture, S. aureus phage cocktail (Phage K(ATCC
19685-B1), 17(ATCC 23361-B1), Stab8 (EpiBiome owned)) (MOI: 30), and 7 ml of untreated raw milk; (3) 50 ill of bacterial culture and 7 ml of papain, EDTA and cysteine-treated raw milk;
(4) 50 ill of bacterial culture, S. aureus phage cocktail (same composition as in (2)) (MOI: 30), and 7 ml of papain, EDTA and cysteine-treated raw milk; (5) 50 ill of bacterial culture and 7 ml of bromelain, EDTA and cysteine-treated raw milk; and (6) 50 ill 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 0_, 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.

[0181] As shown in FIG. 3, addition of the S. aureus phage cocktail (MOI: 30) was unable to eradicate S. aureus and sterilize the culture (column 2 as compared with column 1), leaving 3.0x106 cfu/ml bacterial survivors. The higher observed CFU level in the bromelain treatment (column 5 as compared with column 1) was likely due to dispersion of S. aureus aggregates, leading to an apparent increase in CFU rather than an actual change in the bacterial growth rate.
With the addition of papain (column 4) or bromelain (column 6) and 0.5mM of EDTA and 1mM
of cysteine, the phage cocktail demonstrated enhanced bacterial inhibition, with a much lower amount of bacteria survivors (i.e. about 5.0x103 cfu/ml).

[0182] These results demonstrate the ability to overcome IgG-mediated S.
aureus agglutination in raw milk by treatment with papain or bromelain, which enables a viable therapeutic strategy to address S. aureus mastitis using bacteriophages. The therapeutic strategy is applicable both in the cow udder and in the human mammary gland.
Example 4: Bromelain enhances bacteria inhibition activity of ampicillin

[0183] Bromelain (at a final concentration of 250 i.t.g/m1) 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. Four different conditions were setup and incubated for 2 hours at 37 C: (1) 50 ill of bacterial culture and 7 ml of untreated raw milk; (2) 50 ill of bacterial culture, and 7 ml of untreated raw milk; (3) 50 ill of bacterial culture, and 7 ml of bromelain, EDTA and cysteine-treated raw milk;
and (4) 50 ill of bacterial culture, and 7 ml of bromelain, EDTA and cysteine-treated raw milk.
After incubating (1)-(4) at 37 C for 2 hours, ampicillin (at a final concentration of 14 jig/ml) was added to (2) and (4). (1) and (3) remained untreated. After incubation for an additional 4 hours at 37 C, 1:10 serial dilutions of each culture were performed and 5 0_, 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.
4.

[0184] As shown in FIG. 4, treatment by ampicillin alone resulted in 1.7x105 cfu/ml bacterial survivors after overnight incubation (column 2). When the milk was pre-treated with bromelain, 0.5 mM EDTA and 1 mM cysteine, ampicillin treatment led to enhanced inhibition yielding 1.7x104 cfu/ml bacterial survivors (column 4). Thus, bromelain dramatically enhances the efficacy of ampicillin, a small-molecule antibiotic, in inhibiting S. aureus in raw milk.
Example 5. Microscopic analysis demonstrates de-agglomeration of S. aureus by papain or bromelain treatment.

[0185] The same strain of Staphylococcus aureus as in Examples 3 and 4, which was isolated from cows with symptoms of bovine mastitis, was transformed with a plasmid encoding a green fluorescence protein (GFP) to allow expression of the GFP in the bacteria cells. The transformed bacteria were grown in tryptic soy broth (TSB) to mid-log-phase (¨OD 0.3).
Purified bovine IgG
was added to the TSB at a final concentration of 5 mg/mL. Bromelain (at a final concentration of 250 jig/m1) was added to lmL TSB with bovine IgG in the presence of 0.5mM EDTA
and 1mM
of cysteine prior to the addition of bacteria culture. Three different mixtures were setup and incubated for 24 hours at 37 C: (1) 40 pi of bacterial culture and 960 pi of non-treated TSB with mg/ml bovine IgG; (2) 40 pi of bacterial culture and 960 pi of papain, EDTA
and cysteine-treated TSB with 5 mg/ml bovine IgG; and (3) 40 pi of bacterial culture and 960 [fl of bromelain, EDTA and cysteine-treated TSB with 5 mg/ml bovine IgG. After incubation, the bacterial cultures were fixed by adding 5% of glutarate aldehyde (50%
solution) for 5 minutes. 5 pi of each of bacterial cultures (1), (2) and (3) were added to microscope slides and observed under a fluorescence microscope (Cytell Cell imaging system, GE) at an excitation wavelength of 395 nm to detect the GFP signal.

[0186] As shown in FIG. 5A, Staphylococcus aureus formed aggregates in the presence of 5 mg/ml bovine IgG. Papain (FIG. 5B) or bromelain (FIG. 5C) in combination with 0.5mM
EDTA and 2.5mM cysteine were able to de-agglomerate S. aureus in the presence of 5 mg/ml of bovine IgG. Both treatment conditions showed no or minimal aggregation of S.
aureus. These results suggest that the cysteine proteases papain or bromelain can fragment bovine IgG, which otherwise binds Protein A on the Staphylococcus aureus cell surface to result in agglomeration.

[0187] It was also found that bovine mastitis Strep. uberis formed agglomeration in the presence of 5mg/m1 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.

[0188] These results combined with results from Examples 3 and 4 suggest that the combination of cysteine proteases (such as bromelain) with EDTA and cysteine can be used to break the bacterial agglomeration. Thus, 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.

[0189] Staphylococcus aureus isolated from cows with symptoms of bovine mastitis (ATCC, ATCC No: 31886) was grown in tryptic soy broth (TSB) to mid-log-phase (¨OD
0.3) to obtain a bacteria culture. 100 pi of this bacteria culture and 12 mg/mL of bovine IgG
(Equetech Bio Cat No. SLB66-0010) 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). Four different conditions were tested, each with 7 mL milk plus bacterial culture: (1) untreated; (2) PIRSUE (pirlimycin, antibiotic used for treatment of mastitis, purchased from Zoeitis, final concentration of 0.36 ng/mL); (3) purified bromelain (at a final concentration of 8 iig/m1), 0.5 mM EDTA and 1mM cysteine; and (4) purified bromelain(at a final concentration of 8 jig/ml), 0.5 mM EDTA and 1mM cysteine, followed by addition of PIRSUE (at a final concentration of 0.36 ng/mL). Samples were incubated at 37 C for 12 hours, then 1:10 serial dilutions of each culture were performed, and 5 0_, of each diluted culture was spotted onto CHROMAGARTm S. aureus plates, and incubated overnight at 37 C. Colonies were enumerated, and colony-forming units was determined for each treatment condition and plotted.

[0190] As shown in FIG. 6, treatment of the milk sample (raw milk supplemented with 12 mg/mL of IgG plus S. aureus bacteria culture) treated with PIRSUE alone resulted in 1.9x106 cfu/ml bacterial survivors after 12 hours of incubation (column 2). Pre-treatment of the milk sample with bromelain, 0.5 mM EDTA and 1 mM cysteine, led to enhanced killing of S. aureus grown in the milk sample by PIRSUE , yielding 1.9x104 cfu/ml bacterial survivors (column 4) after 12 hours of incubation. Thus, bromelain enhances the efficacy of pirlimycin, a small-molecule antibiotic, in inhibiting S. aureus growth in raw milk for two orders of magnitude.
Example 7: Bromelain enhances bactericidal activity of cephapirin in raw milk.

[0191] 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 pi of this bacteria culture were added to 7 mL of raw milk and incubated at 37 C for 1 hour. Four different conditions were set up: (1) untreated; (2) TODAY (cephapirin sodium, antibiotic used for treatment of mastitis, Boehringer Ingelheim, final concentration of 0.14 i.t.g/mL) ; (3) purified bromelain (at a final concentration of 8 iig/m1), 0.5 mM EDTA and 1mM cysteine; and (4) purified bromelain (at a final concentration of 8 iig/m1), 0.5 mM EDTA
and 1mM cysteine, followed by addition of TODAY (at a final concentration of 0.14 i.t.g/mL).
Samples were incubated at 37 C for 5 hours, then 1:10 serial dilutions of each culture were performed and 5 0_, of each diluted culture was spotted onto CHROMAGARTm S.
aureus plates, and incubated overnight at 37 C. Colonies were enumerated, and colony-forming units were determined for each treatment condition and plotted.

[0192] As shown in FIG. 7, treatment of the milk sample (raw milk plus S.
aureus bacteria culture) by TODAY alone resulted in 4.8x104 cfu/ml bacterial survivors after 5 hours of incubation (column 2). Pre-treatment of the milk sample with bromelain, 0.5 mM
EDTA and 1 mM cysteine, led to enhanced killing of S. aureus grown in the milk sample by TODAY , yielding 5.3x103 cfu/ml bacterial survivors after 5 hours of incubation (column 4). Thus, bromelain enhances the efficacy of cephapirin, a small-molecule antibiotic, in inhibiting S.
aureus growth in raw milk for one order of magnitude.

Example 8: Bromelain enhances bacteria inhibition activity of cephapirin in simulated mastitic milk.

[0193] Staphylococcus aureus isolated from cows with symptoms of bovine mastitis (ATCC, ATCC No: 31885, 31886, 31887) were grown in tryptic soy broth (TSB) to mid-log-phase (¨OD
0.3) to obtain a bacteria culture. 100 ill of this bacteria culture and 12 mg/mL of bovine IgG
(Equetech Bio Cat No. SLB66-0010) 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). Four different conditions were set up: (1) untreated; (2) TODAY (cephapirin sodium, antibiotic used for treatment of mastitis, Boehringer Ingelheim, final concentration of 0.14 i.t.g/mL) ; (3) purified bromelain (at a final concentration of 8 i.t.g/mL), 0.5 mM EDTA and 1mM cysteine; and (4) purified bromelain(at a final concentration of 8 i.t.g/mL), 0.5 mM EDTA and 1mM cysteine, followed by addition of TODAY (at a final concentration of 0.14 i.t.g/mL). After incubating (1)-(4) at 37 C for 5 hours, 1:10 serial dilutions of each culture were performed, and 5 i.1.1_, of each diluted culture was spotted onto CHROMAGARTm S. aureus plates, and incubated overnight at 37 C.
Colonies were enumerated, and colony-forming units were determined for each treatment condition and plotted.

[0194] As shown in FIG. 8A, treatment of a milk sample (raw milk supplemented with 12 mg/mL IgG) containing S. aureus (ATCC No. 31885) by TODAY alone resulted in 1.9x104 cfu/ml bacterial survivors after 5 hours of incubation (column 2). Pre-treatment of the milk sample with bromelain, 0.5 mM EDTA and 1 mM cysteine, led to enhanced killing of S. aureus (ATCC No. 31885) grown in the milk sample by TODAY , yielding 6.7x102 cfu/ml bacterial survivors (column 4). Thus, bromelain dramatically enhances the efficacy of cephapirin, a small-molecule antibiotic, in inhibiting S. aureus (ATCC No. 31885) growth in raw milk for more than one order of magnitude.

[0195] In FIG. 8B, treatment of a milk sample (raw milk supplemented with 12 mg/mL IgG) containing S. aureus (ATCC No. 31886) by TODAY alone resulted in 2.5x104 cfu/ml bacterial survivors after 5 hours of incubation (column 2). Pre-treatment of the milk sample with bromelain, 0.5 mM EDTA and 1 mM cysteine, led to enhanced killing of S. aureus (ATCC No.
31886) grown in the milk sample by TODAY , yielding 4.1x103 cfu/ml bacterial survivors (column 4). Thus, bromelain dramatically enhances the efficacy of cephapirin, a small-molecule antibiotic, in inhibiting S. aureus (ATCC No. 31886) growth in raw milk for more than one order of magnitude.

[0196] In FIG. 8C, treatment of a milk sample (raw milk supplemented with 12 mg/mL IgG) containing S. aureus (ATCC No. 31887) by TODAY alone resulted in 5.5x104 cfu/ml bacterial survivors after 5 hours of incubation (column 2). Pre-treatment of the milk sample with bromelain, 0.5 mM EDTA and 1 mM cysteine, led to enhanced killing of S. aureus (ATCC No.
31887) grown in the milk sample by TODAY , yielding 3.3x103 cfu/ml bacterial survivors (column 4). Thus, bromelain dramatically enhances the efficacy of cephapirin, a small-molecule antibiotic, in inhibiting S. aureus (ATCC No. 31887) growth in raw milk for more than one order of magnitude.

[0197] Therefore, pre-treatment with bromelain, EDTA and cysteine enhances bacterial killing activities of cephapirin against different strains of S. aureus in simulated mastitic milk.

Claims

What is claimed is:

1. A composition comprising a de-agglomeration agent and an antibacterial agent, wherein the de-agglomeration agent reduces agglomeration of bacteria cells.

2. The composition of claim 1, wherein the antibacterial agent comprises a small-molecule antibiotic.

3. The composition of claim 2, wherein 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, oxacillin, vancomycin, cefaclor, cephapirin, cefadroxil, cephalexin, doxycycline, dicloxacillin, lymecycline, tobramycin, erythromycin, azithromycin, clarithromycin, clindamycin, co-trimoxazole, metronidazole, tinidazole, ciprofloxacin, levofloxacin, norfloxacin, and combinations thereof.

4. The composition of any one of claims 1-3, wherein the antibacterial agent comprises a quorum sensing signal molecule.

5. The composition of any one of claims 1-4, wherein the antibacterial agent comprises a bacteriolytic enzyme.

6. The composition of any one of claims 1-5, wherein the antibacterial agent comprises a phage-derived protein selected from the group consisting of lysozyme, endolysin, lysin, holin, tail fiber protein, and tailocin.

7. The composition of any one of claims 1-6, wherein the antibacterial agent comprises a bacteriophage or a cocktail of bacteriophages.

8. The composition of any one of claims 1-7, wherein the antibacterial agent comprises a bacteriocin.

9. A composition comprising a de-agglomeration agent and a probiotic composition, wherein the de-agglomeration agent reduces agglomeration of bacteria cells.

10. The composition of claim 9, wherein the probiotic composition comprises lactobacillus or bifidobacterium.

11. The composition of claim 9 or claim 10, wherein the composition further comprises a bacteriophage or a cocktail of bacteriophages.

12. The composition of any one of claims 1-11, wherein the bacteria cells are selected from the group consisting of Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, Pseudomonas aeruginosa, Clostridium difficile, Bacillus spp., and Clostridium perfringens.

13. The composition of any one of claims 1-12, wherein the de-agglomeration agent cleaves immunoglobulin, protein A, protein G, fibrinogen, or ClfA.

14. The composition of any one of claims 1-13, wherein the de-agglomeration agent comprises a protease.

15. The composition of claim 14, wherein the protease is selected from the group consisting of papain, bromelain, IdeS, pepsin, ficain, actinidin, cathepsin-B like protease, plasmin and combinations thereof.

16. The composition of any one of claims 1-15, wherein the composition further comprises an adjuvant composition.

17. The composition of claim 16, wherein the adjuvant composition comprises a chelating agent.

18. The composition of claim 16 or claim 17, wherein the adjuvant composition comprises a reducing agent.

19. 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 of the bacteria cells.

20. 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 of the bacteria cells.

21. The method of claim 20, wherein the target composition is a serum sample, an extracellular fluid sample, or a food product.

22. The method of claim 21, wherein the food product is a dairy product.

23. The method of any one of claims 19-22, wherein the method further comprises adding to the target composition an effective amount of an adjuvant composition.

24. A method of treating a bacterial infection in an individual, 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 of bacteria cells that cause the bacterial infection.

25. The method of claim 24, wherein the bacterial infection is in a tissue selected from the group consisting of mammary gland, heart valve, lung, blood stream, digestive tract, bone, nose, throat, or skin of the individual.

26. The method of claim 25, wherein the individual has mastitis.

27. The method of claim 26, wherein the de-agglomeration agent is administered by intramammary infusion.

28. The method of any one of claims 24-27, wherein the de-agglomeration agent and the antibacterial agent are administered sequentially.

29. The method of any one of claims 24-27, wherein the de-agglomeration agent and the antibacterial agent are administered simultaneously.

30. The method of any one of claims 19-29, wherein the antibacterial agent comprises a small-molecule antibiotic.

31. The method of claim 30, wherein 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, oxacillin, vancomycin, cefaclor, cephapirin, cefadroxil, cephalexin, doxycycline, dicloxacillin, lymecycline, tobramycin, erythromycin, azithromycin, clarithromycin, clindamycin, co-trimoxazole, metronidazole, tinidazole, ciprofloxacin, levofloxacin, norfloxacin, and combinations thereof.

32. The method of any one of claims 19-31, wherein the antibacterial agent comprises a quorum sensing signal molecule.

33. The method of any one of claims 19-32, wherein the antibacterial agent comprises a bacteriolytic enzyme.

34. The method of any one of claims 19-33, wherein the antibacterial agent comprises a phage-derived protein selected from the group consisting of lysozyme, endolysin, lysin, holin, tail fiber protein, and tailocin.

35. The method of any one of claims 19-34, wherein the antibacterial agent comprises a bacteriophage or a cocktail of bacteriophages.

36. The method of any one of claims 19-35, wherein the antibacterial agent comprises a bacteriocin.

37. 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 of bacteria cells in the gut of the individual.

38. The method of claim 37, wherein the probiotic composition comprises lactobacillus or bifidobacterium.

39. The method of claim 37 or claim 38, wherein the probiotic composition comprises a bacteriophage or a cocktail of bacteriophages.

40. The method of any one of claims 37-39, wherein the de-agglomeration agent and the probiotic composition are administered sequentially.

41. The method of any one of claims 37-40, wherein the de-agglomeration agent and the probiotic composition are administered simultaneously.

42. The method of any one of claims 24-41, further comprising administering to the individual an effective amount of an adjuvant composition.

43. The method of claim 23 or claim 42, wherein the adjuvant composition comprises a chelating agent.

44. The method of any one of claims 23, 42, or 43, wherein the adjuvant composition comprises a reducing agent.

45. The method of any one of claims 19-44, wherein the de-agglomeration agent cleaves immunoglobulin, protein A, protein G, fibrinogen, or ClfA.

46. The method of any one of claims 19-45, wherein the de-agglomeration agent comprises a protease.

47. The method of claim 46, wherein the protease is selected from the group consisting of papain, bromelain, IdeS, pepsin, ficain, actinidin, cathepsin-B like protease, plasmin and combinations thereof.

48. The method of any one of claims 19-47, wherein the bacteria cells are selected from the group consisting of Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, Pseudomonas aeruginosa, Clostridium difficile, Bacillus spp., and Clostridium perfringens.

49. The method of any one of claims 19-48, wherein the individual is a human individual.

50. The method of any one of claims 19-49, wherein the individual is a dairy cow.