CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/265,172, filed December 9, 2021, which is incorporated by reference herein in its entirety.

BACKGROUND

[0002] Bacteriophages can be used to target a number of bacteria. However, if a produced bacteriophage is not stored properly it can be rendered inactive. There is therefore a need to develop methods for producing bacteriophage that can be stored for reasonable periods of time.

SUMMARY OF THE INVENTION

[0003] Various aspects of the present disclosure relate to a bacteriophage composition. The bacteriophage composition includes a bacteriophage component in a range of from 1.0 x 10 ⁴ to 1.0 x 10 ¹² PFU/g of the bacteriophage composition. The bacteriophage component retains 50% to 95% biological activity relative to a theoretical biological activity. The bacteriophage composition further includes a solid carrier component in a range of from 0.5% (w/v) to 20% (w/v) of the bacteriophage composition. A moisture content of the bacteriophage composition is less than 10 wt%.

[0004] Various aspects of the present disclosure relate to a bacteriophage composition. The bacteriophage component in a range of from 1.0 x 10 ⁶ to 5.0 x 10 ⁸ PFU/g of the bacteriophage composition. The bacteriophage component retains 80% to 95% biological activity relative to a theoretical biological activity. A carrier component is in a range of from 1% (w/v) to 4% (w/v) of the bacteriophage composition. The carrier component includes lactose, a mixture of lactose and leucine, maltodextrin, or a mixture thereof. A moisture content of the bacteriophage composition is less than 10 wt%.

[0005] Various aspects of the present disclosure relate to a method of making a bacteriophage composition. The bacteriophage composition includes a bacteriophage component in a range of from 1.0 x 10 ⁴ to 1.0 x 10 ¹² PFU/g of the bacteriophage composition. The bacteriophage component retains 50% to 95% biological activity relative to a theoretical biological activity. The bacteriophage composition further includes a solid carrier component in a range of from 0.5% (w/v) to 20% (w/v) of the bacteriophage composition. A moisture content of the bacteriophage composition is less than 10 wt%. The method includes removing water from a mixture. The mixture includes a lysate that includes the bacteriophage component and the carrier component.

[0006] Various aspects of the present disclosure relate to a freeze-drying method of making a bacteriophage composition. The bacteriophage composition includes a bacteriophage component in a range of from 1.0 x 10 ⁴ to 1.0 x 10 ¹² PFU/g of the bacteriophage composition. The bacteriophage component retains 50% to 95% biological activity relative to a theoretical biological activity. The bacteriophage composition further includes a solid carrier component in a range of from 0.5% (w/v) to 20% (w/v) of the bacteriophage composition. A moisture content of the bacteriophage composition is less than 10 wt%. The method includes freezing a mixture including a lysate that includes the bacteriophage component. A concentration of the bacteriophage component in the mixture is in a range of from 1 X 10 ⁷ to 1 X 10 ⁹ PFU/mL. The mixture also includes a carrier component. The carrier component is in a range of from 1% (w/v) to 4% (w/v) of the bacteriophage composition. The mixture further includes removing water from the frozen mixture.

[0007] Various aspects of the present disclosure further relate to a kit. The kit includes a bacteriophage composition. The bacteriophage composition includes a bacteriophage component in a range of from 1.0 x 10 ⁴ to 1.0 x 10 ¹² PFU/g of the bacteriophage composition. The bacteriophage component retains 50% to 95% biological activity relative to a theoretical biological activity. The bacteriophage composition further includes a solid carrier component in a range of from 0.5% (w/v) to 20% (w/v) of the bacteriophage composition. A moisture content of the bacteriophage composition is less than 10 wt%. The bacteriophage composition is disposed within a container.

[0008] Various aspects of the present disclosure relate to a method of using a bacteriophage composition. The bacteriophage composition includes a bacteriophage component in a range of from 1.0 x 10 ⁴ to 1.0 x 10 ¹² PFU/g of the bacteriophage composition. The bacteriophage component retains 50% to 95% biological activity relative to a theoretical biological activity. The bacteriophage composition further includes a solid carrier component in a range of from 0.5% (w/v) to 20% (w/v) of the bacteriophage composition. A moisture content of the bacteriophage composition is less than 10 wt%. The method includes administering the bacteriophage composition to a subject (e.g., a human or animal subject).

[0009] Various aspects of the present disclosure relate to a composition including a bacteriophage composition. The bacteriophage composition includes a bacteriophage component in a range of from 1.0 x 10 ⁴ to 1.0 x 10 ¹² PFU/g of the bacteriophage composition. The bacteriophage component retains 50% to 95% biological activity relative to a theoretical biological activity. The bacteriophage composition further includes a solid carrier component in a range of from 0.5% (w/v) to 20% (w/v) of the bacteriophage composition. A moisture content of the bacteriophage composition is less than 10 wt%. The composition including the bacteriophage composition can be a comprising a pharmaceutical composition, a nutraceutical composition, a food composition, a beverage composition, a water source, a top dressing, a food pellet, an extruded solid, topical cream ointment or solution, or a mixture thereof.

[0010] There are many benefits to using the instantly described bacteriophage composition. For example, once the bacteriophage composition is obtained it can be stored for long periods of time. The amount of time it can be stored relates to the dry nature of the bacteriophage composition, which substantially prevents the bacteriophage component from being contaminated and rendered inactive.

BRIEF DESCRIPTION OF THE FIGURES

[0011] The drawings illustrate generally, by way of example, but not by way of limitation, various examples of the present invention.

[0012] FIG. 1 is a series of plots showing the statistical significance of four variables on the percent biological activity retention.

[0013] FIG. 2 is a graph showing bacteriophage titer in a bacteriophage composition.

[0014] FIG. 3 shows the percent biological activity retention in a bacteriophage composition.

[0015] FIG. 4 shows a bacteriophage titer of various spray dried bacteriophage compositions compared to freeze-dried bacteriophage compositions.

[0016] FIG. 5 shows percent biological activity retention or various spray dried bacteriophage compositions compared to freeze-dried bacteriophage compositions.

DETAILED DESCRIPTION OF THE INVENTION

[0017] Reference will now be made in detail to certain examples of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter. [0018] Throughout this document, values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of “about 0.1% to about 5%” or “about 0.1% to 5%” should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,” unless indicated otherwise. Likewise, the statement “about X, Y, or about Z” has the same meaning as “about X, about Y, or about Z,” unless indicated otherwise.

[0019] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. The statement “at least one of A and B” or “at least one of A or B” has the same meaning as “A, B, or A and B.” In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section.

[0020] In the methods described herein, the acts can be carried out in any order without departing from the principles of the invention, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0021] The term “about” as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range, and includes the exact stated value or range. The term “substantially” as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%. The term “substantially free of’ as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that about 0 wt% to about 5 wt% of the composition is the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than or equal to about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt%.

[0022] According to various examples of the present disclosure, a bacteriophage composition can be formed. The bacteriophage composition can be a component of a pharmaceutical composition, a nutraceutical composition, a food composition, a beverage composition, a water source, a top dressing, a food pellet, an extruded solid, topical cream ointment or solution, or a mixture thereof. The bacteriophage composition includes a bacteriophage component that includes a bacteriophage. A bacteriophage, also referred to as a phage, is a virus that infects and replicates within bacteria and archaea. Bacteriophages include proteins that encapsulate a DNA or RNA genome, and may have structures that are either simple or elaborate. Their genomes may encode as few as four genes and as many as hundreds of genes. Phages replicate within the bacterium following the injection of their genome into its cytoplasm. [0023] Bacteriophages are obligate intracellular parasites that multiply inside bacteria by co-opting some or all of the host biosynthetic machinery. Phages contain nucleic acid and protein, and may be enveloped by a lipid membrane. Depending upon the phage, the nucleic acid genome can be either DNA or RNA but not both, and can exist in either circular or linear forms. The size of the phage genome varies depending upon the phage. The simplest phages have genomes that are only a few thousand nucleotides in size, while the more complex phages may contain more than 100,000 nucleotides in their genome, and in rare instances more than 1,000,000. The number and amount of individual types of protein in phage particles will vary depending upon the phage. The proteins function in infection and to protect the nucleic acid genome from environmental nucleases.

[0024] Phage genomes come in a variety of sizes and shapes (e.g., linear or circular). Most phages range in size from 24-200 nm in diameter. The capsid is composed of many copies of one or more phage proteins, and acts as a protective envelope around the phage genome. Many phages have tails attached to the phage capsid. The tail is a hollow tube through which the phage nucleic acid passes during infection. The size of the tail can vary and some phages do not even have a tail structure. In the more complex phages, the tail is surrounded by a contractile sheath which contracts during infection of the bacterial host cell. At the end of the tail, phages have a base plate and one or more tail fibers attached to it. The base plate and tail fibers are involved in the binding of the phage to the host cell.

[0025] Lytic or virulent phages are phages which can only multiply in bacteria and lyse the bacterial host cell at the end of the life cycle of the phage. The lifecycle of a lytic phage begins with an eclipse period. During the eclipse phase, no infectious phage particles can be found either inside or outside the host cell. The phage nucleic acid takes over the host biosynthetic machinery and phage specific mRNAs and proteins are produced. Early phage mRNAs code for early proteins that are needed for phage DNA synthesis and for shutting off host DNA, RNA and protein biosynthesis. In some cases, the early proteins actually degrade the host chromosome. After phage DNA is made late mRNAs and late proteins are made. The late proteins are the structural proteins that include the phage as well as the proteins needed for lysis of the bacterial cell. In the next phase, the phage nucleic acid and structural proteins are assembled and infectious phage particles accumulate within the cell. The bacteria begin to lyse due to the accumulation of the phage lysis protein, leading to the release of intracellular phage particles. The number of particles released per infected cell can be as high as 1000 or more. Lytic phage may be enumerated by a plaque assay. The assay is performed at a low enough concentration of phage such that each plaque arises from a single infectious phage. The infectious particle that gives rise to a plaque is called a PFU (plaque forming unit).

[0026] Lysogenic phages are those that can either multiply via the lytic cycle or enter a quiescent state in the host cell. In the quiescent state, the phage genome exists as a prophage (i.e., it has the potential to produce phage). In most cases, the phage DNA actually integrates into the host chromosome and is replicated along with the host chromosome and passed on to the daughter cells. The host cell harboring a prophage is not adversely affected by the presence of the prophage and the lysogenic state may persist indefinitely. The lysogenic state can be terminated upon exposure to adverse conditions. Conditions which favor the termination of the lysogenic state include: desiccation, exposure to UV or ionizing radiation, exposure to mutagenic chemicals, etc. Adverse conditions lead to the production of proteases (rec A protein), the expression of the phage genes, reversal of the integration process, and lytic multiplication. [0027] In some examples, a phage genome includes at least 5 kilobases (kb), at least 10 kb, at least 15 kb, at least 20 kb, at least 25 kb, at least 30 kb, at least 35 kb, at least 40 kb, at least 45 kb, at least 50 kb, at least 55 kb, at least 60 kb, at least 65 kb, at least 70 kb, at least 75 kb, at least 80 kb, at least 85 kb, at least 90 kb, at least 95 kb, at least 100 kb, at least 105 kb, at least 110 kb, at least 115 kb, at least 120 kb, at least 125 kb, at least 130 kb, at least 135 kb, at least 140 kb, at least 145 kb, at least 150 kb, at least 175 kb, at least 200 kb, at least 225 kb, at least 250 kb, at least 275 kb, at least 300 kb, at least 325 kb, at least 350 kb, at least 375 kb, at least 400 kb, at least 425 kb, at least 450 kb, at least 475 kb, or at least 500 kb of nucleic acids. [0028] Over the past decade, great efforts have been put into delivering therapeutic dosage forms of phage for treatment of respiratory infections. Successful phage therapy requires phages to remain viable during the production and delivery in aerosolized form or aqueous form so that clinically significant dose can be administered. Powder or dry bacteriophage compositions have the potential to provide easy storage, transport and administration with long shelf-life over liquid formulations.

[0029] The bacteriophage composition of the instant disclosure is a dry composition. For example, a moisture content of the bacteriophage composition can be in a range of from about 0 wt% to 10 wt%, from about 0.05 to about 6 wt%, less than, equal to, or greater than about 0 wt%, 1, 2, 3, 4, 5, 6, 7, 8, 9, or about 10 wt%. The bacteriophage composition can be a powder that can take on many types of morphologies. For example, the morphology can include spherical particles with smooth surfaces, corrugated particles with rough surfaces, or irregular agglomerates. Smooth spherical particles can help to solubilize the bacteriophage composition upon reconstitution or can make the bacteriophage composition suitable to be used as an aerosol. As non-limiting examples, a particle size in the bacteriophage composition (measured as a Dso value) can range from about 0.01 pm to 6 pm, about 0.01 pm to 2 pm, less than, equal to, or greater than about 0.01 pm, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or about 6.0 pm.

[0030] The bacteriophage composition can include a bacteriophage component in a range of from 1.0 x 10 ⁴ to 1.0 x 10 ¹² plaque-forming units/g (“PFU/g”) of the bacteriophage composition, about 1.0 x 10 ⁶ to 1.0 x 10 ¹⁰ PFU/g, about 1.0 x 10 ⁶ to 5.0 x 10 ⁸ PFU/g, less than, equal to, or greater than about 1.0 x 10 ⁴ PFU/g, 1.0 x 10 ⁵ , 1.0 x 10 ⁶ , 1.0 x 10 ⁷ , 1.0 x 10 ⁸ , 1.0 x 10 ⁹ , or about 1.0 x 10 ¹⁰ PFU/g.

[0031] The bacteriophage can be a non-recombinant bacteriophage or a recombinant bacteriophage that is obtained as a lysate, by lysing a microorganism. A method for generating a recombinant bacteriophage genome can inlcude: (a) contacting a non-recombinant bacteriophage genome with a sgRNA-CRISPR enzyme conjugate in vitro under conditions where the sgRNA- CRISPR enzyme conjugate cleaves a protospacer sequence within the non-recombinant bacteriophage genome to produce a cleaved non-recombinant bacteriophage genome; and (b) recombining in vitro the cleaved non-recombinant bacteriophage genome with a heterologous nucleic acid in the presence of a recombination system under conditions to produce a recombinant bacteriophage genome. The cleaved non-recombinant bacteriophage genome includes a first cleaved bacteriophage genomic fragment and a second cleaved bacteriophage genomic fragment. In some embodiments, the heterologous nucleic acid comprises a 5' flanking region that is homologous to the 3' end of the first cleaved bacteriophage genomic fragment, and a 3' flanking region that is homologous to the 5' end of the second cleaved bacteriophage genomic fragment.

[0032] Additionally or alternatively, in some embodiments, the method further comprises propagating the recombinant bacteriophage genome in a bacterial host. The bacterial host may be a non-natural bacterial host cell or a natural bacterial host cell. Additionally or alternatively, in some embodiments, the CRISPR enzyme is a Cas protein selected from the group consisting of Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Casio, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, and Csf4. In certain embodiments, the CRISPR enzyme is Cas9. [0033] The bacteriophage of the composition is biologically active. As explained further herein, the drying techniques used to produce the bacteriophage composition yield a high degree of bacteriophage components that retain biological activity. As used herein “biological activity” means an antibacterial activity. As an example, the bacteriophage component can retain 50% to 95% biological activity relative to a theoretical biological activity (alternatively referred to as a percent biological activity retention), about 75% to about 95%, about 80 % to about 95%, less than, equal to, or greater than about 50% biological activity, 55, 60, 65, 70, 75, 80, 85, 90, or about 95% biological activity. As understood herein the theoretical biological activity refers to the activity of the bacteriophage component that would be observed in the bacteriophage composition if there was no loss of bacteriophage component during the drying technique. The total amount of loss was measured by comparing the titer of the bacteriophage before drying with the titer of the bacteriophage after drying. Thus, the measurement of retained biological activity relates to the amount of bacteriophage component remaining in the final composition. [0034] The bacteriophage component can include a single type of bacteriophage or a plurality of types of bacteriophages. As an example, the bacteriophage component can include at least one bacteriophage to target any desirable bacteria. Non-limiting examples of suitable bacteria targets include Aeromonas hydrophila, Clostridium perfringens, Escherichia coli, Edwardsiella tarda, Staphylococcus aureus, Vibrio anguillarum, Vibrio harveyi, Vibrio salmonicida, Vibrio parahemolyticus, Cytophaga marin, Pseudomonas plecoglossicida, Pseudomonas fluorescens, Campylobacter jejuni, Campylobacter coli, Salmonella enteriditis, Salmonella Typhimurium, Salmonella gallinarum, Pectobacterium atrosepticum, Pantoea stewartia, Ralstonia solanacearum, Xylella fastidiosa, Erwinia amylovora, Piscirickettsia salmonis, Yersinia ruckeri, Nocardia cummidelens, Nocardia fluminea, Moritella viscosa, Streptococcus uberis, Streptococcus iniae, Streptococcus parauberis, Klebsiella pneumoniae, Listeria monocytogenes, Streptomyces luridiscabiei, Streptomyces cf. albidoflavus , or a mixture thereof. In some particular examples the bacteriophage can target a Salmonella enteriditis. In some examples, bacteria of particular interest to target with bacteriophages include multiple drug resistance (MDR) bacteria

[0035] Bacteriophage therapy has been gaining renewed interests for its ability to eradicate bacteria. Bacteriophage therapy exploits the lytic life cycle of phages, which causes bacteriolysis followed by subsequent release of progenies. The released phage progenies then target nearby bacteria and the cycle is repeated. Potential advantages of phage therapy over conventional antibiotic treatment are due to the facts that phages are (i) naturally occurring antibacterials with low inherent toxicity, (ii) self-amplifying agents, and (iii) highly specific limiting unnecessary damage to non-targeted bacteria. The activity of phages against bacteria has been shown in in vitro studies as well as their efficacy in animals and humans.

[0036] The bacteriophage composition, according to the present disclosure, includes a solid carrier component. The solid carrier component can range from about 0.5% (w/v) to 20% (w/v) of the bacteriophage composition, about 0.5% (w/v) to 10% (w/v), about 1% (w/v) to 4% (w/v), less than, equal to, or greater than about 0.5% (w/v), 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,

6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17,

17.5, 18, 18.5, 19, 19.5, or about 20% (w/v).

[0037] The solid carrier component can include many suitable materials such as a protein, a saccharide, an amino acid, a mineral, or a mixture thereof. Specific examples of materials for the solid carrier component can include maltodextrin, dextrin 35, dextrose, trehalose, sucrose, lactose, leucine, glycine, tyloxapol, ammonium bicarbonate, casein, trehalose, gelatin, whey protein, skim milk, mannitol, sorbitol, a mixture of lactose and leucine, lactoferrin, glutamine, peptone, polyethylene glycol, hypromellose, poly lactic-co-gly colic acid, poly vinyl alcohol, dextran 35, polyvinylpyrrolidone or a mixture thereof.

[0038] The solid carrier component can act to protect and stabilize bacteriophage components from temperature and shear stresses during drying. In some examples carriers that act as protein protectors are shown to function well. In either freeze-drying or spray drying, the added carriers are responsible to protect the phage from stresses generated during water removal, yet the carriers serve different functions during each process. In freeze-drying, carriers are used to cryoprotect the phage, whereas in spray drying, carriers protect phage from thermal and mechanical stresses. Also, in the case that spray drying is used for preparing inhalable phage powders, carriers must confer desirable aerodynamic properties needed for the powders. Some solid carrier components such as lactose or leucine are already FDA-approved.

[0039] The bacteriophage composition can include additional components. For example, the bacteriophage composition can include a dry buffer component, a dry probiotic component, a dry prebiotic component, or a mixture thereof. Any one of the probiotic component, the prebiotic component, or the mixture thereof can range from about 0.05 wt% to 30 wt% of the bacteriophage composition, about 1 wt% to 15 wt%, less than, equal to, or greater than about 0.05 wt%, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12,

12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22,

22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, or about 30 wt%.

[0040] In some examples, the bacteriophage component can be at least partially encapsulated. For example, the bacteriophage component can be at least partially encapsulated in a polymer matrix, a membrane, or in an emulsification. In examples where the bacteriophage composition is encapsulated in a polymer matrix, the polymer matrix can include alginate, chitosan, pectin, polyethylene oxide, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene oxide, poly(lactic-co-gly colic acid, cellulose diacetate, hyaluronic acid methacrylate, or a mixture thereof.

[0041] The methods of making the bacteriophage composition are beneficial in that a high degree of biological activity of the bacteriophage component is retained. Methods of making the bacteriophage composition include removing water from a mixture. The mixture can include the bacteriophage component or a lysate the includes the bacteriophage component as well as the carrier component. Other components such as the dry buffer component, dry probiotic, dry prebiotic component, or mixture thereof can also be added to the mixture. In some examples, the lysate that includes the bacteriophage component and the carrier component can be mixed before water is removed.

[0042] In general, removing water from the mixture can be accomplished using freeze- drying or spray drying. With respect to spray drying, the degree to which the biological activity of the bacteriophage component is preserved is generally a result of controlling the inlet temperature of a nozzle used for spray drying, a flow rate of air through the nozzle, and a feed rate of the mixture to the nozzle.

[0043] With respect to the inlet temperature, the inlet temperature of a nozzle used for spray drying is in a range of from about 30 °C to 80 °C, about 40 °C to 60 °C, less than, equal to, or greater than about 30 °C, 35, 40, 45, 50, 55, 60, 65, 70, 75, or about 80 °C. With respect to the flow or air through the nozzle, the flow of air through the nozzle can be in a range of from about 300 L/h to 900 L/h, about 400 L/h to 750 L/h, less than, equal to, or greater than about 300 L/h, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, or about 750 L/h. With respect to the feed rate of the mixture to the nozzle, the feed rate of the mixture can be in a range of from about 0.1 g/min to 8 g/min, about 0.5 g/min to 4 g/min, less than, equal to, or greater than about 0.1 g/min, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or about 8 g/min.

[0044] Freeze-drying includes freezing the mixture described herein above. For the purposes of this disclosure, the mixture is considered to be frozen, when the mixture is in a frozen amorphous/crystalline phase. Once sufficiently frozen, the frozen mixture is dried.

Drying occurs by exposing the frozen mixture to a condenser to remove moisture therefrom. The frozen mixture can be further exposed to a vacuum for further drying.

[0045] Irrespective of the drying technique, a factor that influences the final biological activity of the bacteriophage composition is the concentration of the bacteriophage component in the mixture before drying. For example, it has been demonstrated that a higher biological activity is found if the mixture, before drying, has a bacteriophage component in a range of from about 1 X 10 ⁵ to 1 X 10 ¹⁰ PFU/g, about 1 X 10 ⁷ to 1 X 10 ⁹ PFU/g, less than, equal to, or greater than about 1 X 10 ⁵ PFU/g, 1 X 10 ⁶ , 1 X 10 ⁷ , 1 X 10 ⁸ , 1 X 10 ⁹ , or about 1 X 10 ¹⁰ PFU/g. It has been found the concentrations outside of these ranges does not yield bacteriophage compositions having correspondingly high biological activities.

[0046] Once the dry bacteriophage composition is obtained it can be stored for long periods of time. The dry nature of the bacteriophage composition substantially prevents the bacteriophage component from being contaminated and rendered inactive. For example, the absence of water in the bacteriophage composition helps to prevent microbe growth, helps to prevent the accumulation of soluble impurities in the composition, helps to prevent sticking and caking of the bacteriophage composition, helps to ensure uniform application of the bacteriophage composition, or helps to minimize the storage space required for the bacteriophage composition.

[0047] The dry bacteriophage composition can be stored in any suitable container such as an Eppendorf tube, vial, or the like and can be readily reconstituted in water or an aqueous buffer solution ahead of use. An example of an aqueous buffer can include a Tris-base, MgSOE. NaCl, and water. Another example of an aqueous buffer can include Na2HPO4, KH2PO4, NaCl, MgSO4, CaCh, and water. The aqueous buffer has a pH in a range of from about 5.5 to about 8.5, about 7 to about 8, less than, equal to, or greater than about 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, or about 8.5. The bacteriophage composition can be stored at room temperature or refrigerated. In some examples, the bacteriophage composition is not reconstituted in a liquid but instead is aerosolized to allow for inhalation.

[0048] As described hereinabove, the bacteriophage composition can be used in many different contexts. As an illustrative example, the long term stability of the bacteriophage composition makes it particularly useful to use as a component of a pharmaceutical composition for phage therapy. As an example of use in phage therapy a bacteriophage composition can include one phage isolate or a phage cocktail. In some examples, the composition includes at least two different isolated strains of phage. In another aspect, the composition includes a phage cocktail and a pharmaceutically acceptable carrier. In some examples, the pharmaceutical composition is formulated for systemic or local application. In some examples, the pharmaceutical composition includes a sterile buffer. In some examples, the composition further includes an additional agent, e.g., an agent selected from the group consisting of an antibiotic agent, an anti-inflammatory agent, an antiviral agent, a local anesthetic agent, and a corticosteroid. In some examples, the composition is for use in treating a bacterial infection. The daily dosage may be administered in one or more doses. In some examples, the treatment includes administering a tablet or other orally compatible delivery vehicle having the bacteriophage composition.

[0049] In one aspect, cocktail compositions of different phage strains are administered to a human with a disease. The “cocktail” may include at least two different isolated strains of phage, for example, two, three, four, five, six, seven, eight, nine, ten, or more different isolated bacteriophage strains. The cocktail may be used alone or in further combination with other therapies, e.g., antibiotic agents and/or growth factors. In some examples, the phage cocktail includes at least 2 phage strains, at least 3 phage strains, at least 4 phage strains, at least 5 phage strains, at least 6 phage strains, at least 7 phage strains, at least 8 phage stains, at least 9 phage strains, at least 10 phage strains, or more. In some examples, the phage cocktail includes 2-20 phage strains, 2-15 phage strains, 2-10 phage strains, 3-8 phage strains, or 4-6 phage strains. In more examples, the combination does not impair or reduce (or does not substantially or significantly impair or reduce) infecting ability and/or lytic activity of the individual bacteriophage in the presence of distinct bacteriophage strains

[0050] The phage or phage cocktails are incorporated into a composition for the use in treatment of a disease. A cocktail of different phage strains, or a single phage isolate, may be combined with a pharmaceutically acceptable carrier, such as an excipient or stabilizer, e.g., to form a tablet. Examples of pharmaceutically acceptable carriers, excipients, and stabilizers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin and gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium.

[0051] The bacteriophage or cocktail compositions may also be combined with one or more non-phage therapeutic and/or prophylactic agents, useful for the treatment and/or prevention of bacterial infections, as known in the art (e.g., one or more antibiotic agents). Other therapeutic and/or prophylactic agents that may be used in combination with the phage or phage cocktails include, but are not limited to, antibiotic agents, anti-inflammatory agents, antiviral agents, prebiotics, probiotics, and corticosteroids. In some examples, the phage or phage cocktail is administered in the absence of a non-phage based antibiotic agent.

[0052] Standard antibiotics that may be used with pharmaceutical compositions including a phage cocktail include, but are not limited to, amikacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin, streptomycin, tobramycin, apramycin, rifamycin, naphthomycin, mupirocin, geldanamycin, ansamitocin, carbacephems, imipenem, meropenem, ertapenem, faropenem, doripenem, panipenem/betamipron, biapenem, PZ-601, cephalosporins, cefacetrile, cefadroxil, cefalexin, cefaloglycin, cefalonium, cefaloridine, cefalotin, cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cefradine, cefroxadine, ceftezole, cefaclor, cefonicid, cefprozil, cefuroxime, cefuzonam, cefmetazole, cefotetan, cefoxitin, cefcapene, cefdaloxime, cefdinir, cefditoren, cefetamet, cefixime, cefmenoxime, cefteram, ceftibuten, ceftiofur, ceftiolene, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime latamoxef, cefclidine, cefepime, cefluprenam, cefoselis, cefozopran, cefpirome, cefquinome, flomoxef. ceftobiprole, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, aztreonam, pencillin and penicillin derivatives, actinomycin, bacitracin, colistin, polymyxin B, cinoxacin, flumequine, nalidixic acid, oxolinic acid, piromidic acid, pipemidic acid, rosoxacin, ciprofloxacin, enoxacin, fleroxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin, gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin, pazufloxacin, sparfloxacin, temafloxacin, tosufloxacin, clinafloxacin, garenoxacin, gemifloxacin, stifloxacin, trovalfloxacin, prulifloxacin, acetazolamide, benzolamide, bumetanide, celecoxib, chlorthalidone, clopamide, dichlorphenamide, dorzolamide, ethoxyzolamide, furosemide, hydrochlorothiazide, indapamide, mafendide, mefruside, metolazone, probenecid, sulfacetamide, sulfadimethoxine, sulfadoxine, sulfanilamides, sulfamethoxazole, sulfasalazine, sultiame, sumatriptan, xipamide, tetracycline, chi ortetracy cline, oxy tetracycline, doxycycline, lymecycline, meclocycline, methacycline, minocycline, rolitetracy cline, methicillin, nafcillin, oxacilin, cioxacillin, vancomycin, teicoplanin, clindamycin, co-trimoxazole, flucioxacillin, dicloxacillin, ampicillin, amoxicillin and any combination thereof in amounts that are effective to additively or synergistically enhance the therapeutic effect of a composition having phage for a given infection.

[0053] In one example, the compositions generally may include a sterile buffer, such as a sterile PBS, water, or saline buffer. One particular buffer includes Tris-HCl, NaCl, and/or MgSO47H2O, e.g., about 0.05 M Tris-HCl (pH 7.4-7.5), about 0.1 M NaCl, and/or about 10 mM MgSO47H2O. In other examples, the formulation further includes a buffer and 10 mM MgCh. In other examples, the phage containing formulation further includes a buffer having about 5 mM to about 15 mM CaCh, e.g., about 10 mM CaCh.

[0054] In some examples, compositions are provided in a hermetically sealed container. In one example, the dry bacteriophage composition can be reconstituted so as to be formulated as an aqueous solution or gel. The composition may include water; esters, isopropyl myristate and isopropyl palmitate; ethers such as dicapryl ether and dimethyl isosorbide; alcohols such as ethanol and isopropanol; fatty alcohols such as cetyl alcohol, cetearyl alcohol, stearyl alcohol and biphenyl alcohol; isoparaffins such as isooctane, isododecane and is hexadecane; silicone oils such as cyclomethicone, dimethicone, dimethicone cross-polymer, polysiloxanes and their derivatives, e.g., organomodified derivatives; polyols such as propylene glycol, glycerin, butylene glycol, pentylene glycol and hexylene glycol; or any combinations or mixtures of the foregoing. Aqueous vehicles may include one or more solvents miscible with water, including lower alcohols, such as ethanol, isopropanol, and the like.

[0055] The phage or cocktails thereof, can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, e.g., orally or parenterally, by intravenous, intramuscular, or subcutaneous routes. In one example, the phage or cocktails thereof may be administered as a tablet.

[0056] In one example, the phage or cocktails thereof may be administered by infusion or injection. Solutions of the phage or cocktails thereof can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0057] The pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it may include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. [0058] The phage or cocktails thereof optionally in combination with another active compound may be administered parenterally, for example, intravenously, orally, intraperitoneally, intramuscularly, or subcutaneously. Such administration may be as a single bolus injection, multiple injections, or as a short- or long-duration infusion. Implantable devices (e.g., implantable infusion pumps) may also be employed for the periodic parenteral delivery over time of equivalent or varying dosages of the particular formulation. For such parenteral administration, the compounds (a conjugate or other active agent) may be formulated as a sterile solution in water or another suitable solvent or mixture of solvents. The solution may contain other substances such as salts, sugars (particularly glucose or mannitol), to make the solution isotonic with blood, buffering agents such as acetic, citric, and/or phosphoric acids and their sodium salts, and preservatives.

[0059] Thus, the phage or cocktails thereof or in combination with another active agent, may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the phage or cocktails thereof optionally in combination with an active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of conjugate and optionally other active compound in such useful compositions is such that an effective dosage level will be obtained. [0060] The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, com starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as com starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of Wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the phage or cocktails thereof optionally in combination with another active compound may be incorporated into sustained-release preparations and devices. [0061] The phage or cocktails thereof optionally in combination with another active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the phage or cocktails thereof optionally in combination with another active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0062] The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms during storage can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it may be useful to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0063] Various examples according to the instant disclosure include administering the bacteriophage composition to a subject as a component of a nutritional supplement, feed, beverage, or a mixture thereof. For example, the bacteriophage composition can be administered to a subject such as poultry, fish (e.g., an aquaculture), swine, domestic pet, or a human) as a component of disclosure include administering the bacteriophage composition to a subject as a component of a nutritional supplement, feed, beverage, or a mixture thereof. EXAMPLES

[0064] Various examples of the present invention can be better understood by reference to the following Examples which are offered by way of illustration. The present invention is not limited to the Examples given herein.

1. SPRAY DRIED BACTERIOPHAGE COMPOSITION

[0065] Spray dried bacteriophage compositions were prepared. The process of preparing the spray dried bacteriophage composition includes feeding a bacteriophage lysate and carrier into a spray drier. The spray drier was a Buchi, Mini Spray Drier Model B-290, available from BUCHI Corporation, New Castle DE. The bacteriophage lysate included a plurality of bacteriophages that were active against salmonella. The concentration of the bacteriophage in the lysate was 10 ⁷ PFU/g. The carrier was a mix of lactose and leucine. The carrier mix included 27.2 g/L lactose and 12.8 g/L leucine.

[0066] Four variables were controlled during the spray drying. Those variables included the spray drier inlet temperature, the spray drier nozzle airflow, the feed rate of the bacteriophage lysate to the spray drier, and the carrier concentration. The spray drier inlet temperature was varied in a range of from 40 °C to 60 °C. The spray drier nozzle airflow was varied in a range of from 400 to 750 L/h. The feed rate of the bacteriophage lysate to the spray drier was varied in a range of from 0.5 g/min to 4 g/min. The carrier concentration was varied in a range of from 1 wt% to 4 wt% of the mixture of the bacteriophage composition and carrier.

[0067] Varying the spray drier inlet temperature, the spray drier nozzle airflow, the feed rate of the bacteriophage lysate to the spray drier, and the carrier concentration was studied with respect to their impact on the degree of percent biological activity retention in the produced bacteriophage composition, measured bacteriophage PFU/g of the bacteriophage composition, and yield % of the bacteriophage composition. As described herein above, the percent biological activity retention is a measure of the degree to which the bacteriophage in the lysate is retained in the bacteriophage composition.

[0068] It was found that only the percent biological activity retention was affected by maintaining a concentration of the carrier in a range of from 1 wt% to 4 wt%. None of the other variables had a statistically significant effect on any of the other outcomes. FIG. 1 is a series of plots showing the statistical significance of the four variables on the percent biological activity retention. 2. FREEZE-DRIED BACTERIOPHAGE COMPOSITION

[0069] Freeze-dried bacteriophage compositions were prepared. The process of preparing the freeze-dried bacteriophage composition includes using a freezer for freezing a bacteriophage lysate and carrier. The bacteriophage lysate included a plurality of bacteriophages that were active against salmonella. The frozen mixture of the bacteriophage lysate and carrier are then subjected to a condenser to remove moisture from the freeze-dried bacteriophage composition. The condenser was run for three days in an environment having a temperature at - 50 °C with a vacuum maintaining a pressure of 0.030 mBar. The condenser was a Labconco Model: FreeZone 2.5 Liter Benchtop Freeze Dryer and the freezer was a FreeZone 6 Liter Benchtop Freeze Dryer, both available from Labconoco, Kansas City, MO.

[0070] A variety of different carriers were used and the resulting bacteriophage titer in the bacteriophage composition and percent biological activity retention in the bacteriophage composition was studied. Carriers included maltodextrin, sucrose, lactose, a mixture of lactose and leucine, casein, mannitol, dextrin, trehalose, gelatin, whey, and mannitol. The concentration of the carriers was at 4% w/v. FIG. 2 is a graph showing the bacteriophage titer in the bacteriophage composition. FIG. 3 shows the percent biological activity retention in the bacteriophage composition.

[0071] The concentration of the bacteriophage in the lysate was varied along with different carriers to study the impact of those factors on titer in the bacteriophage composition and percent biological activity retention in the bacteriophage composition. The carriers were in the bacteriophage composition at a concentration of 4% (w/v) and the carriers were maltodextrin or a mixture of lactose and leucine. As shown in FIGs. 4 and 5, a bacteriophage concentration of 109 PFU/g in the lysate produced the best titer in the bacteriophage composition and percent biological activity retention in the bacteriophage composition.

3. COMPARISON OF FREEZE-DRIED AND SPRAY DRIED BACTERIOPHAGE COMPOSITIONS

[0072] Four spray dried bacteriophage compositions (SpDr #s 4, 5, 8, and 9) were prepared according to the protocol of Example 1. The bacteriophage concentration in the lysate was 10 ⁷ PUF/g. The carrier was a mixture of lactose and leucine. In SpDr #4 the carrier concentration was 1 %(w/v), in SpDr #5 the carrier concentration was 2 %(w/v), in SpDr #8 the carrier concentration was 3 %(w/v), and in SpDr #9 the carrier concentration was 4 %(w/v). [0073] Four freeze-dried bacteriophage compositions (FrDr #s 4, 5, 8, and 9) were prepared according to the protocol of Example 2. The bacteriophage concentration in the lysate was 10 ⁷ PUF/g. The carrier was a mixture of lactose and leucine. In FrDr #4 the carrier concentration was 1 %(w/v), in FrDr #5 the carrier concentration was 2 %(w/v), in FrDr #8 the carrier concentration was 3 %(w/v), and in FrDr #9 the carrier concentration was 4 %(w/v). [0074] FIG. 6 is a graph showing the bacteriophage titer in spray dried bacteriophage compositions and freeze-dried bacteriophage compositions. FIG. 7 is a graph showing the percent biological activity retention in spray dried bacteriophage compositions and freeze-dried bacteriophage compositions. As shown in FIGs. 6 and 7, freeze-dried bacteriophage compositions tend to perform better than spray dried bacteriophage compositions.

[0075] The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the examples of the present invention. Thus, it should be understood that although the present invention has been specifically disclosed by specific examples and optional features, modification and variation of the concepts herein disclosed may be resorted to by those of ordinary skill in the art, and that such modifications and variations are considered to be within the scope of examples of the present invention.

Exemplary Aspects.

[0076] The following exemplary Aspects are provided, the numbering of which is not to be construed as designating levels of importance:

[0077] Aspect 1 provides a bacteriophage composition comprising: a bacteriophage component in a range of from 1.0 x 10 ⁴ to 1.0 x 10 ¹² PFU/g of the bacteriophage composition, the bacteriophage component retaining 50% to 95% biological activity relative to a theoretical biological activity of the bacteriophage component; and a solid carrier component in a range of from 0.5% (w/v) to 20% (w/v) of the bacteriophage composition; wherein a moisture content of the bacteriophage composition is less than 10 wt%. [0078] Aspect 2 provides the bacteriophage composition of Aspect 1, wherein the bacteriophage component comprises a plurality of types of bacteriophages [0079] Aspect 3 provides the bacteriophage composition of any one of Aspects 1 or 2, wherein the bacteriophage component comprises at least one bacteriophage that targets a bacteria chosen from the group comprising Aeromonas hydrophila, Clostridium perfringens, Escherichia coli, Edwardsiella tarda, Staphylococcus aureus, Vibrio anguillarum, Vibrio harveyi, Vibrio salmonicida, Vibrio parahemolyticus , Cytophaga marin, Pseudomonas plecoglossicida, Pseudomonas fluorescens, Campylobacter jejuni, Campylobacter coli, Salmonella enteriditis, Salmonella Typhimurium, Salmonella Gallinarum, P ectobacterium atrosepticum, Pantoea stewartia, Ralstonia solanacearum, Xylella fastidiosa, Erwinia amylovora, Piscirickettsia salmonis, Yersinia ruckeri, Nocardia cummidelens, Nocardia fluminea, Moritella viscosa, Streptococcus uberis, Streptococcus iniae, Streptococcus parauberis, Klebsiella pneumoniae, Listeria monocytogenes, Streptomyces luridiscabiei, Streptomyces cf. albidoflavus , or a mixture thereof.

[0080] Aspect 4 provides the bacteriophage composition of any one of Aspects 1-3, wherein the bacteriophage component comprises at least one bacteriophage that targets a Salmonella enteriditis.

[0081] Aspect 5 provides the bacteriophage composition of any one of Aspects 1-4, wherein the bacteriophage component is in a range of from 1.0 x 10 ⁶ to 1.0 x 10 ¹⁰ PFU/g of the bacteriophage composition.

[0082] Aspect 6 provides the bacteriophage composition of any one of Aspects 1-5, wherein the bacteriophage component is in a range of from 1.0 x 10 ⁶ to 5.0 x 10 ⁸ PFU/g of the bacteriophage composition.

[0083] Aspect 7 provides the bacteriophage composition of any one of Aspects 1-6, wherein the bacteriophage composition retains 75% to 95% biological activity relative to the theoretical biological activity.

[0084] Aspect 8 provides the bacteriophage composition of any one of Aspects 1-7, wherein the bacteriophage composition retains 80% to 95% biological activity relative to a theoretical biological activity.

[0085] Aspect 9 provides the bacteriophage composition of any one of Aspects 1-8, wherein the carrier is in a range of from 0.5% (w/v) to 10% (w/v) of the bacteriophage composition.

[0086] Aspect 10 provides the bacteriophage composition of any one of Aspects 1-9, wherein the carrier is in a range of from 1% (w/v) to 4% (w/v) of the bacteriophage composition. [0087] Aspect 11 provides the bacteriophage composition of any one of Aspects 1-10, wherein the carrier comprises a protein, a saccharide, an amino acid, or a mixture thereof.

[0088] Aspect 12 provides the bacteriophage composition of any one of Aspects 1-11, wherein the carrier comprises maltodextrin, dextrin 35, dextrose, trehalose, sucrose, lactose, leucine, casein, trehalose, gelatin, whey protein, skim milk, mannitol, sorbitol, a mixture of lactose and leucine, lactoferrin, glutamine, peptone, or a mixture thereof.

[0089] Aspect 13 provides the bacteriophage composition of any one of Aspects 1-12, wherein the moisture content of the bacteriophage composition is less than 6 wt%.

[0090] Aspect 14 provides the bacteriophage composition of any one of Aspects 1-13, wherein the moisture content of the bacteriophage composition is in a range of from 0 wt% to 10 wt%.

[0091] Aspect 15 provides the bacteriophage composition of any one of Aspects 1-14, wherein the moisture content of the bacteriophage composition is in a range of from 0.05 wt% to 6 wt%.

[0092] Aspect 16 provides the bacteriophage composition of any one of Aspects 1-15, further comprising a buffer component, a probiotic component, a prebiotic component, or a mixture thereof.

[0093] Aspect 17 provides the bacteriophage composition of Aspect 16, wherein the buffer component, the probiotic component, the prebiotic component, or the mixture thereof independently range from 0.05 wt% to 30 wt% of the bacteriophage composition.

[0094] Aspect 18 provides the bacteriophage composition of any one of Aspects 16 or 17, wherein the dry buffer component, the dry probiotic component, the dry prebiotic component, or the mixture thereof independently range from 1 wt% to 15 wt% of the bacteriophage composition.

[0095] Aspect 19 provides the bacteriophage composition of any one of Aspects 1-18, wherein the bacteriophage component is at least partially encapsulated.

[0096] Aspect 20 provides the bacteriophage composition of Aspect 19, wherein the bacteriophage component is at least partially encapsulated in a polymer matrix.

[0097] Aspect 21 provides the bacteriophage composition of Aspect 20, wherein the polymer matrix comprises alginate, chitosan, pectin, polyethylene oxide, polyvinyl alcohol, polyvinylpyrrolidone, polyethylene oxide, poly(lactic-co-glycolic acid, cellulose diacetate, hyaluronic acid methacrylate, or a mixture thereof.

[0098] Aspect 22 provides a bacteriophage composition comprising: a bacteriophage component in a range of from 1.0 x 10 ⁶ to 5.0 x 10 ⁸ PFU/g of the bacteriophage composition, the bacteriophage component retaining 80% to 95% biological activity relative to a theoretical biological activity; and a carrier component in a range of from 1% (w/v) to 4% (w/v) of the bacteriophage composition, the carrier component comprising lactose, a mixture of lactose and leucine, maltodextrin, or a mixture thereof; wherein a moisture content of the bacteriophage composition is less than 10 wt%.

[0099] Aspect 23 provides a method of making the bacteriophage composition of any one of Aspects 1-22, the method comprising: removing water from a mixture comprising a lysate comprising the bacteriophage component; and the carrier component.

Aspect 24 provides the method of Aspect 23, further comprising mixing the lysate comprising the bacteriophage component and the carrier component before removing water from the mixture.

[0100] Aspect 25 provides the method of any one of Aspects 23 or 24, wherein removing water from the mixture comprises freeze-drying or spray drying.

[0101] Aspect 26 provides the method of Aspect 25, wherein removing water from the mixture comprises spray drying and: an inlet temperature of a nozzle used for spray drying is in a range of from 30 °C to 80 °C; a flow of air through the nozzle is in a range of from 300 L/h to 900 L/h; and a feed rate of the mixture to the nozzle is in a range of from 0.1 g/min to 8 g/min.

[0102] Aspect 27 provides the method of Aspect 26, wherein the inlet temperature of a nozzle used for spray drying is in a range of from 40 °C to 60 °C; the flow of air through the nozzle is in a range of from 400 L/h to 750 L/h; and the feed rate of the mixture to the nozzle is in a range of from 0.5 g/min to 4 g/min. [0103] Aspect 28 provides the method of Aspect 25, wherein removing water from the mixture comprises freeze-drying comprising: freezing the mixture to form a frozen mixture; and drying the frozen mixture. [0104] Aspect 29 provides the method of Aspect 28, wherein freezing the mixture occurs until the mixture is in a frozen amorphous/crystalline phase.

[0105] Aspect 30 provides the method of any one of Aspects 28 or 29, wherein drying the frozen mixture comprises exposing the frozen mixture to a condenser to remove moisture therefrom.

[0106] Aspect 31 provides the method of Aspect 30, further comprising exposing the frozen mixture to a vacuum.

[0107] Aspect 32 provides the method of any one of Aspects 23-31, wherein a concentration of the bacteriophage component in the mixture is in a range of from 1 X 10 ⁵ to 1 X 10 ¹⁰ PFU/g.

[0108] Aspect 33 provides the method of any one of Aspects 23-32, wherein a concentration of the bacteriophage component in the mixture is in a range of from 1 X 10 ⁷ to 1 X 10 ⁹ PFU/g.

[0109] Aspect 34 provides a freeze-drying method of making the bacteriophage composition of any one of Aspects 1-33, the method comprising: freezing a mixture comprising a lysate comprising the bacteriophage component, wherein a concentration of the bacteriophage component in the mixture is in a range of from 1 X 10 ⁷ to 1 X 10 ⁹ PFU/mL, and the carrier component, wherein the carrier component is in a range of from 1% (w/v) to 4% (w/v) of the bacteriophage composition; and removing water from the frozen mixture.

[0110] Aspect 35 provides a kit comprising the bacteriophage composition of any one of Aspects 1-34, the kit comprising: a container; and the bacteriophage composition disposed within the container.

[0111] Aspect 36 provides a method of using the bacteriophage composition of any one of Aspects 1-35, the method comprising administering the bacteriophage composition to a subject.

[0112] Aspect 37 provides the method of Aspect 36, further comprising reconstituting the bacteriophage composition in a buffer before administering the bacteriophage composition. [0113] Aspect 38 provides the method of Aspect 36, further comprising mixing the bacteriophage composition into a solid matrix composition before administering to a subject. [0114] Aspect 38 provides a composition comprising the bacteriophage composition of any one of Aspects 1-37, the composition comprising a pharmaceutical composition, a nutraceutical composition, a food composition, a beverage composition, a water source, a top dressing, a food pellet, an extruded solid, topical cream ointment or solution, or a mixture thereof.