Staphylococcal phage compositions and cocktails thereof

Abstract

Disclosed herein are phage compositions for infecting and / or killing Staphylococcus spp.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 379,807, filed October 17, 2022, which is incorporated herein by reference in its entirety.

[0002] INCORPORATION BY REFERENCE OF SEQUENCE LISTING This application is filed with an electronic Sequence Listing, which is provided as a file named 53240-754601_SL.xml, created on September 21, 2023, and is 4,258,034 bytes in size. The electronic format information in the Sequence Listing is incorporated by reference in its entirety. Summary of the Invention

[0003] In certain aspects herein, compositions are described comprising at least two bacteriophages, wherein a first bacteriophage is at least 80% identical to p1378 or p4815 and a second bacteriophage is at least 80% identical to p1494e002 or p2808. In some embodiments, the first bacteriophage is at least 80% identical to p1378 and the second bacteriophage is at least 80% identical to p1494e002. In some embodiments, the first bacteriophage is at least 80% identical to p1378 and the second bacteriophage is at least 80% identical to p2808. In some embodiments, the first bacteriophage is at least 80% identical to p4815 and the second bacteriophage is at least 80% identical to p1494e002. In some embodiments, the first bacteriophage is at least 80% identical to p4815 and the second bacteriophage is at least 80% identical to p2808. In some embodiments, the composition further comprises a third bacteriophage that is at least 80% identical to p4815. In some embodiments, the composition further comprises a fourth bacteriophage that is at least 80% identical to p2808. In some embodiments, the composition further comprises a third bacteriophage that is at least 80% identical to p4815. In some embodiments, the composition further comprises a third bacteriophage that is at least 80% identical to p2808. In certain aspects, described herein are compositions comprising a plurality of bacteriophages, including a first bacteriophage and a second bacteriophage, wherein bacteria treated with the plurality of bacteriophages exhibit reduced regrowth compared to treatment with the first bacteriophage or the second bacteriophage alone, and wherein the first bacteriophage comprises a Rosenblumvirus and the second bacteriophage comprises a Phietavirus or a Kayvirus. In some embodiments, the regrowth is measured by optical density (OD) at a wavelength of 600 nm.In some embodiments, the amount of regrowth of bacteria treated with a plurality of bacteriophages 12 hours after treatment is about 50%, 40%, 30%, 20%, or 10% or less than the amount of regrowth of bacteria treated with a first bacteriophage. In some embodiments, the amount of regrowth of bacteria treated with a plurality of bacteriophages 12 hours after treatment is about 50%, 40%, 30%, 20%, or 10% or less than the amount of regrowth of bacteria treated with a second bacteriophage. In some embodiments, the bacteria is a Staphylococcus bacterium. In some embodiments, the bacteria comprises Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus salivarius, Staphylococcus argenteus, Staphylococcus hemolyticus, or Staphylococcus schweitzeri, or any combination of two or more thereof. In some embodiments, the bacteria comprises Staphylococcus aureus.

[0004] In certain aspects, described herein are bacteriophages engineered to be lytic by removal, replacement, or inactivation of a lysogenic sequence, wherein the lysogenic sequence comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of a sequence selected from SEQ ID NOs: 49-58. In some embodiments, the bacteriophage is an engineered Phytophthora virus. In some embodiments, described herein are compositions comprising a bacteriophage described herein and further comprising a Rosenblum virus and / or a Kai virus. In some embodiments, described herein are methods for treating a disease or condition associated with Staphylococcus aureus, the method comprising administering a bacteriophage described herein or a composition described herein to a subject in need of treatment for the disease or condition. In some embodiments, the Staphylococcus aureus causes and / or contributes to the disease or condition.

[0005] Certain embodiments described herein include bacteriophages that share at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1378 (PTA-127329). Certain embodiments described herein include bacteriophages that share at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p4815 (PTA-127331). Certain embodiments described herein include bacteriophages that share at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1494e002 (PTA-127345). In certain embodiments herein, described are bacteriophages that contain at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p2808 (PTA-127332).

[0006] In certain embodiments herein, a composition comprising at least two bacteriophages, wherein a first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1378, and a second bacteriophage comprises at least one of p1381, p4815, p1378e062, p1378e074, p4815e037, p1 Compositions are described that contain at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, p2808, or p1473.

[0007] In certain embodiments herein, a composition comprising at least two bacteriophages, wherein a first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p4815, and a second bacteriophage comprises at least one of p1381, p1378, p1378e062, p1378e074, p4815e037, p1 Compositions are described in which the second bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p4815, 01494e002, or p2808. In some embodiments, the composition further comprises a third bacteriophage that comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p4815, 01494e002, or p2808. In some embodiments, the composition further comprises a fourth bacteriophage that comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p4815, 01494e002, or p2808.

[0008] In certain embodiments herein, a composition comprising at least two bacteriophages, wherein a first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1494e002, and a second bacteriophage comprises at least one of p1381, p1378, p4815, p1378e062, p1378e074, p4 Compositions are described that contain at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1468e003, p1478e003, p2808, or p1473.

[0009] In certain embodiments herein, a composition comprising at least two bacteriophages, wherein a first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p2808, and a second bacteriophage comprises at least one of p1381, p1378, p4815, p1378e062, p1378e074, p4815e Compositions are described, comprising at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, or p1473. In some embodiments, at least two bacteriophages of the composition infect at least about 90% of a population of at least about 30 Staphylococcus bacteria. In some embodiments, at least about 90% infection is determined by plaque assay or growth inhibition assay. In some embodiments, at least about 90% is at least about 95%. In some embodiments, at least about 90% is at least about 98%. In some embodiments, at least about 90% is at least about 99%. In some embodiments, the population of Staphylococcus bacteria is 8, 5, 22, 15, 1, 30, 398, 105, 45, 672, 2250, 582, 72, 97, 239, 34, 87, 101, 109, 1159, 1165, 1181, 12, 121, 152, 1750, 20, 225, 25, 291, 3628, 59, 7, 779, 88, 10, 1011, 1049, 1156, The population of Staphylococcus bacteria includes Staphylococcus bacteria having an MLST of 1351, 149, 1637, 1649, 1757, 1842, 188, 1970, 2066, 256, 2867, 2945, 3149, 3182, 3510, 39, 395, 4317, 47, 4730, 50, 508, 573, 6, 630, 737, 828, 848, 923, or 93, or a combination of two or more thereof. In some embodiments, the population of Staphylococcus bacteria includes bacteria isolated from a bloodstream infection. In some embodiments, at least about 40% of the population of Staphylococcus bacteria are multidrug resistant.

[0010] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawings]

[0011] The novel features of the invention are set forth with particularity in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings.

[0012] [Figure 1] FIG. 1 shows the predicted lysogenic region of Phytavirus bacteriophage p1473. [Figure 2] Dilution series of wild-type (WT) p1473 and several mutants plated onto a lawn of S. aureus using the double agar overlay method. WT phage and mutants Var002 and Var006 produced small, cloudy plaques, while mutants Var009, Var010, and Var012 produced larger, clear plaques. [Figure 3] Zoomed-in images of larger plaque morphology of wild-type p1473, VarO10, and VarO12, with red arrows indicating individual plaques. [Figure 4] FIG. 1 depicts a bacteriophage killing assay using strain b4063 (USA300 strain FPR3757) in LB challenged with wild-type p1473, VarO12, or VarO42, as measured by bacterial counts as a function of time. [Figure 5] FIG. 1 shows growth curves of three S. aureus clinical isolates (b2991, b3022, and b3202) in LB loaded with p1473WT or p1473 VarO12. [Figure 6] FIG. 1 depicts the sequence of the Var010 deletion (SEQ ID NO: 30). [Figure 7] FIG. 1 shows the sequence of the Var012 deletion (SEQ ID NO: 31), which is also the first deletion of Var042. [Figure 8] FIG. 10 depicts the second deletion of Var042 (SEQ ID NO: 32). [Figure 9] Figure 1 shows PCR screening of Kaivirus phages engineered to contain Type I CRISPR System IB from Listeria monocytogenes (LMIB). [Figure 10] FIG. 1 shows PCR screening of the InqO promoter against phages. [Figure 11] Figure 1 shows the concentration of b2655 over time inoculated with engineered and wild-type p4815 kaivirus phages. Each engineered p4815 kaivirus contains one of three promoter variants used to drive expression of lacticin Q (InqQ): Pcat-lnqQ, PsarA-lnqQ, or PtmpG-lnqQ promoter. [Figure 12] FIG. 1 depicts an alignment of the genome of the parent Kaivirus phage with engineered mutants containing DNase I and Pcat promoters. [Figure 13A] FIG. 10 depicts deletions of p1494e002 phage (SEQ ID NO: 58, also referred to as e002 or var0022). [Figure 13B] FIG. 10 depicts the concentration over time of FPR3757 inoculated with engineered (p1494e002) and wild-type (p1494WT) p1494. [Figure 13C] FIG. 1 depicts the growth curve of Staphylococcus isolate b2655 over time after infection with wild-type phage (p1498 WT) or engineered phage (p1498e001), or without phage. [Figure 14A] FIG. 11 depicts growth curves of b4735 inoculated with individual phages (p1378, p1494e002, p2808, p4815) and a cocktail (CK811). [Figure 14B]FIG. 11 depicts growth curves of b2745 inoculated with individual phages (p1378, p1494e002, p2808, p4815) and phage cocktail (CK811). [Figure 14C] FIG. 1 shows the growth curves of b4681 inoculated with individual phages (p1378, p1494e002, p2808, p4815) and the phage cocktail (CK811). [Figure 15] FIG. 1 is a Venn diagram showing various bacterial strains targeted by combinations of Phytavirus, Rosenblumvirus, and Kaivirus, and bacteriophages. [Figure 16A] FIG. 1 depicts the growth curve of Staphylococcus isolate b4681 after infection with p1378 or without phage. [Figure 16B] FIG. 1 depicts the growth curve of staphylococcal isolate b4681 after infection with p4815 or without phage. [Figure 16C] FIG. 1 depicts the growth curve of staphylococcal isolate b4681 after infection with p2808 or without phage. [Figure 16D] FIG. 10 depicts the growth curve of Staphylococcus isolate b4681 after infection with p1494e002 or without phage. [Figure 16E] Figure 1 depicts the growth curve of Staphylococcus isolate b4681 after infection with a three-genera cocktail (CK811 containing p1378, p1494e002, p2808, and p4815) or without phages. [Figure 17A] FIG. 1 depicts the growth curve of Staphylococcus isolate b4681 after infection with p1378 or without phage. [Figure 17B] FIG. 1 depicts the growth curve of staphylococcal isolate b4681 after infection with p2808 or without phage. [Figure 17C] Figure 1 depicts the growth curve of Staphylococcus isolate b4681 after infection with a two-generic cocktail (CK664 containing p1378, p2808, and p4815) or without phages. [Figure 17D]FIG. 1 depicts the growth curve of staphylococcal isolate b4681 after infection with p4815 or without phage. [Figure 17E] FIG. 10 depicts the growth curve of Staphylococcus isolate b4681 after infection with p1498e001 or without phage. [Figure 17F] Figure 1 depicts the growth curve of Staphylococcus isolate b4681 after infection with a three-genera cocktail (CK766 containing p1378, p1498e001, p2808, and p4815) or without phages. [Figure 18A] FIG. 10 depicts the growth curve of Staphylococcus isolate b4787 after infection with p1378 or without phage. [Figure 18B] FIG. 1 depicts the growth curve of Staphylococcus isolate b4787 after infection with p2808 or without phage. [Figure 18C] FIG. 1 depicts the growth curve of staphylococcal isolate b4787 after infection with p4815 or without phage. [Figure 18D] FIG. 10 depicts the growth curve of Staphylococcus isolate b4787 after infection with p1498e001 or without phage. [Figure 18E] FIG. 1 depicts the growth curve of Staphylococcus isolate b4787 after infection with the two-genera cocktail (CK665) or without phages. [Figure 18F] FIG. 1 depicts the growth curve of Staphylococcus isolate b4787 after infection with the three-genera cocktail (CK766) or without phages. [Figure 19A] FIG. 1 depicts the growth curve of Staphylococcus isolate b4665 after infection with p1378 or without phage. [Figure 19B] FIG. 1 depicts the growth curve of Staphylococcus isolate b4665 after infection with p2808 or without phage. [Figure 19C] FIG. 1 depicts the growth curve of Staphylococcus isolate b4665 after infection with the two-genera cocktail (CK665) or without phages. [Figure 19D] FIG. 1 depicts the growth curve of staphylococcal isolate b4665 after infection with p4815 or without phage. [Figure 19E] FIG. 10 depicts the growth curve of Staphylococcus isolate b4665 after infection with p1498e001 or without phage. [Figure 19F] FIG. 1 depicts the growth curve of Staphylococcus isolate b4665 after infection with the three-genera cocktail (CK766) or without phages. [Figure 20A] FIG. 10 depicts the growth curve of Staphylococcus isolate b4689 after infection with p1378 or without phage. [Figure 20B] FIG. 1 depicts the growth curve of Staphylococcus isolate b4689 after infection with p2808 or without phage. [Figure 20C] FIG. 1 depicts the growth curve of Staphylococcus isolate b4689 after infection with the two-genera cocktail (CK665) or without phages. [Figure 20D] FIG. 1 depicts the growth curve of Staphylococcus isolate b4689 after infection with p4815 or without phage. [Figure 20E] FIG. 10 depicts the growth curve of Staphylococcus isolate b4689 after infection with p1498e001 or without phage. [Figure 20F] FIG. 1 depicts the growth curve of Staphylococcus isolate b4689 after infection with the three-genera cocktail (CK766) or without phages. DETAILED DESCRIPTION OF THE INVENTION

[0013] In certain aspects herein, compositions and methods for killing and / or infecting staphylococci using bacteriophages are disclosed. In certain aspects herein, compositions are described that include a plurality of bacteriophages, including a first bacteriophage and a second bacteriophage, wherein the plurality of bacteriophages infect at least about 90% of a population of at least about 30 staphylococcus bacteria. In certain aspects herein, compositions are described that include a plurality of bacteriophages, including a first bacteriophage that binds to a first receptor on staphylococcus bacteria in a population of staphylococcus bacteria and a second bacteriophage that binds to a second receptor on staphylococcus bacteria in the population of staphylococcus bacteria. In some embodiments, the bacteriophage specifically binds to a first receptor when the binding of the bacteriophage to the second receptor is less than about 10% of the binding of the bacteriophage to the first receptor. In some embodiments, the first bacteriophage specifically binds to a first receptor on a Staphylococcus bacterium in the population of Staphylococcus bacteria, hi some embodiments, the second bacteriophage specifically binds to a second receptor on a Staphylococcus bacterium in the population of Staphylococcus bacteria.

[0014] In certain aspects herein, described are compositions comprising a plurality of bacteriophages, including a first bacteriophage and a second bacteriophage, wherein bacteria treated with the plurality of bacteriophages exhibit reduced amounts of regrowth compared to treatment with either the first bacteriophage or the second bacteriophage alone.

[0015] In some embodiments, the bacteriophage described herein targets or infects Staphylococcus aureus. In some embodiments, the bacteriophage is engineered to eliminate lysogeny. In some embodiments, the bacteriophage comprises a CRISPR / Cas system described herein. In some embodiments, the bacteriophage comprises an antimicrobial agent or a peptide. In some embodiments, the bacteriophage does not comprise a CRISPR / Cas system. In some embodiments, the bacteriophage does not comprise an antimicrobial agent or a peptide.

[0016] In certain embodiments, compositions are disclosed herein that include a plurality of bacteriophages. In some cases, the plurality includes one or more engineered bacteriophages, e.g., engineered to remove lysogeny genes and / or to include a payload such as a CRISPR-Cas component and / or an antimicrobial peptide. In some embodiments, the plurality of bacteriophages targets a broad host range. For example, the composition targets at least about 90% of a population of at least about 30 staphylococcus bacteria. In some embodiments, the plurality of bacteriophages includes a first bacteriophage that binds to a first receptor on the staphylococcus bacteria and a second bacteriophage that binds to a second receptor on the staphylococcus bacteria, and the plurality of bacteriophages is more resilient to staphylococci than the first or second bacteriophage alone.

[0017] Bacteriophage In certain aspects herein, bacteriophages are described that target Staphylococcus species. In some embodiments, the bacteriophages target Staphylococcus aureus (S. aureus). In some embodiments, the bacteriophages specifically target Staphylococcus species over other bacterial species. In some embodiments, the bacteriophages target Staphylococcus species in the absence of a CRISPR-Cas system.

[0018] In some embodiments, the bacteriophage targets Staphylococcus species. In some embodiments, the bacteriophage is a Kaivirus, a Twortvirus, a Rosenblumvirus, a Fietavirus, a Triavirus, a Dubowvirus, a Beceayunavirus, a Peeveelvirus, a Coventryvirus, or a Rockefellervirus. As used herein, a Fietavirus may include a Fietavirus or a Dubowvirus. In some embodiments, the bacteriophage is a Kaivirus. In some embodiments, the bacteriophage is a Twortvirus. In some embodiments, the bacteriophage is a Rosenblumvirus. In some embodiments, the bacteriophage is a Fietavirus. In some embodiments, the bacteriophage is a Triavirus. In some embodiments, the bacteriophage is a Dubowvirus. In some embodiments, the bacteriophage is a Bessiejuna virus. In some embodiments, the bacteriophage is a Peavirus. In some embodiments, the bacteriophage is a Coventry virus. In some embodiments, the bacteriophage is a Rockefeller virus. In some embodiments, the bacteriophage encodes a CRISPR-Cas system. In some embodiments, the bacteriophage encodes a peptide. In some embodiments, the bacteriophage does not comprise or encode a CRISPR-Cas system. In some embodiments, the bacteriophage does not comprise or encode a peptide.

[0019] In some embodiments, the bacteriophage is a Phytovirus. In some embodiments, the Phytovirus is engineered to remove, replace, or inactivate a lysogeny gene or a promoter of a lysogeny gene. As a non-limiting example, the lysogeny gene encodes a suppressor. In some embodiments, removal of the lysogeny gene includes removing between about 1% and 100% of the lysogeny gene, or between about 10 and about 1,200 base pairs of the lysogeny gene are removed. In some embodiments, the lysogeny gene encodes an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 47 or 48. In some embodiments, the bacteriophage comprises or is engineered to encode one or more components of a CRISPR-Cas system and / or an antimicrobial peptide. In some embodiments, the bacteriophage does not comprise or encode one or more components of a CRISPR-Cas system. In some embodiments, the bacteriophage does not comprise or encode an antimicrobial peptide.

[0020] In some embodiments, the fetavirus is p1473. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1473. In some embodiments, p1473 is engineered to remove, replace, or inactivate a lysogeny gene or a promoter of a lysogeny gene. In some embodiments, the fetavirus is p1473 comprising a nucleic acid encoding a CRISPR-Cas system disclosed herein. In some embodiments, the fetavirus is p1473 comprising a nucleic acid encoding a peptide disclosed herein.

[0021] In some embodiments, the Phytavirus is p1498. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1498. In some embodiments, p1498 is engineered to remove, replace, or inactivate a lysogeny gene or a promoter of a lysogeny gene. In some embodiments, p1498 is engineered to remove, replace, or inactivate at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of SEQ ID NO: 52. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1498e001 (PTA-127336). In some embodiments, the Phytovirus is p1498, which comprises the nucleic acid encoding the CRISPR-Cas system disclosed herein.In some embodiments, the Phytovirus is p1498, which comprises the nucleic acid encoding the peptide disclosed herein.

[0022] In some embodiments, the Phytavirus is p3693. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p3693. In some embodiments, p3693 is engineered to remove, replace, or inactivate a lysogeny gene or a promoter of a lysogeny gene. In some embodiments, p3693 is engineered to remove, replace, or inactivate at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of SEQ ID NO: 53. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p3693e001 (PTA-127337). In some embodiments, the Phytavirus is p3693, which comprises the nucleic acid encoding the CRISPR-Cas system disclosed herein.In some embodiments, the Phytavirus is p3693, which comprises the nucleic acid encoding the peptide disclosed herein.

[0023] In some embodiments, the phytovirus is p3224. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p3224. In some embodiments, p3224 is engineered to remove, replace, or inactivate a lysogeny gene or a promoter of a lysogeny gene. In some embodiments, p3224 is engineered to remove, replace, or inactivate at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of SEQ ID NO: 54. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p3224e054. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p3224e002 (PTA-127341). In some embodiments, the fetavirus is p3224 comprising a nucleic acid encoding a CRISPR-Cas system disclosed herein. In some embodiments, the fetavirus is p3224 comprising a nucleic acid encoding a peptide disclosed herein.

[0024] In some embodiments, the phytovirus is p5593. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p5593. In some embodiments, p5593 is engineered to remove, replace, or inactivate a lysogeny gene or a promoter of a lysogeny gene. In some embodiments, p5593 is engineered to remove, replace, or inactivate at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of SEQ ID NO: 55. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p5593e001 (PTA-127342). In some embodiments, the Phytovirus is p5593, which comprises the nucleic acid encoding the CRISPR-Cas system disclosed herein.In some embodiments, the Phytovirus is p5593, which comprises the nucleic acid encoding the peptide disclosed herein.

[0025] In some embodiments, the phytovirus is p1468. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1468. In some embodiments, p1468 is engineered to remove, replace, or inactivate a lysogeny gene or a promoter of a lysogeny gene. In some embodiments, p1468 is engineered to remove, replace, or inactivate at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of SEQ ID NO: 56. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1468e003 (PTA-127343). In some embodiments, the Phytovirus is p1468, which comprises the nucleic acid encoding the CRISPR-Cas system disclosed herein.In some embodiments, the Phytovirus is p1468, which comprises the nucleic acid encoding the peptide disclosed herein.

[0026] In some embodiments, the phytovirus is p1478. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1478. In some embodiments, p1478 is engineered to remove, replace, or inactivate a lysogeny gene or a promoter of a lysogeny gene. In some embodiments, p1478 is engineered to remove, replace, or inactivate at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of SEQ ID NO: 57. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1478e003 (PTA-127344). In some embodiments, the Phytavirus is p1478, which comprises the nucleic acid encoding the CRISPR-Cas system disclosed herein.In some embodiments, the Phytavirus is p1478, which comprises the nucleic acid encoding the peptide disclosed herein.

[0027] In some embodiments, the phytovirus is p1494. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1494. In some embodiments, p1494 is engineered to remove, replace, or inactivate a lysogeny gene or a promoter of a lysogeny gene. In some embodiments, p1494 is engineered to remove, replace, or inactivate at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of SEQ ID NO: 58. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1494e002 (PTA-127345). In some embodiments, the phytovirus is p1494, which comprises the nucleic acid encoding the CRISPR-Cas system disclosed herein.In some embodiments, the phytovirus is p1494, which comprises the nucleic acid encoding the peptide disclosed herein.

[0028] In some embodiments, the Phytavirus is p1498. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1498. In some embodiments, p1498 is engineered to remove, replace, or inactivate a lysogeny gene or a promoter of a lysogeny gene. In some embodiments, p1498 is engineered to remove, replace, or inactivate at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of SEQ ID NO: 52. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1498e001 (PTA-127336). In some embodiments, p1498 is engineered to remove, replace, or inactivate at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of SEQ ID NO: 52. In some embodiments, the fetavirus is p1498 comprising a nucleic acid encoding a CRISPR-Cas system disclosed herein. In some embodiments, the fetavirus is p1498 comprising a nucleic acid encoding a peptide disclosed herein.

[0029] In some embodiments, the bacteriophage is a Rosenblum virus. In some embodiments, the Rosenblum virus comprises a nucleic acid encoding a CRISPR-Cas system. In some embodiments, the Rosenblum virus comprises a nucleic acid encoding a peptide. In some embodiments, the bacteriophage does not comprise or encode one or more components of a CRISPR-Cas system. In some embodiments, the bacteriophage does not comprise or encode an antimicrobial peptide.

[0030] In some embodiments, the Rosenblum virus is p2808 (PTA-127332). In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p2808. In some embodiments, p2808 is engineered to remove, replace, or inactivate a lysogeny gene or a promoter of a lysogeny gene. In some embodiments, the Rosenblum virus is p2808 comprising a nucleic acid encoding a CRISPR-Cas system disclosed herein. In some embodiments, the Rosenblum virus is p2808 comprising a nucleic acid encoding a peptide disclosed herein.

[0031] In some embodiments, the Rosenblum virus is Staphylococcal bacteriophage rv44AHJD. In some embodiments, the Rosenblum virus is Staphylococcal bacteriophage pabna. In some embodiments, the Rosenblum virus is Staphylococcal bacteriophage 66. In some embodiments, the Rosenblum virus is selected from bacteriophages identified under NCBI taxonomy ID 690287. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with the Rosenblum virus. In some embodiments, the Rosenblum virus is engineered to remove, replace, or inactivate a lysogeny gene or a promoter of a lysogeny gene. In some embodiments, the Rosenblum virus comprises a nucleic acid encoding a CRISPR-Cas system disclosed herein. In some embodiments, the Rosenblum virus comprises a nucleic acid encoding a peptide disclosed herein.

[0032] In some embodiments, the bacteriophage is a kaivirus. In some embodiments, the kaivirus comprises a nucleic acid encoding a CRISPR-Cas system. In some embodiments, the kaivirus comprises a nucleic acid encoding a peptide. In some embodiments, the bacteriophage does not comprise or encode one or more components of a CRISPR-Cas system. In some embodiments, the bacteriophage does not comprise or encode an antimicrobial peptide.

[0033] In some embodiments, the Kaivirus is p1378 (PTA-127329). In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1378. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1378e075 (PTA-127338). In some embodiments, p1378 is engineered to remove, replace, or inactivate a lysogeny gene or a promoter of a lysogeny gene. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1378e062 (PTA-127333). In some embodiments, p1378 is engineered to remove, replace, or inactivate at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of SEQ ID NO: 49. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1378e074 (PTA-127334). In some embodiments, p1378 is engineered to remove, replace, or inactivate at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of SEQ ID NO: 50. In some embodiments, p1378 is engineered to remove, replace, or inactivate at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of SEQ ID NO: 51. In some embodiments, p1378 is engineered to remove, replace, or inactivate at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of SEQ ID NO: 50 and at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of SEQ ID NO: 51. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1378e074.In some embodiments, the Kaivirus is p1378 that comprises a nucleic acid encoding a CRISPR-Cas system disclosed herein. In some embodiments, the Kaivirus is p1378 that comprises a nucleic acid encoding a peptide disclosed herein.

[0034] In some embodiments, the Kaivirus is p1381 (PTA-127330). In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1381. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1381e017 (PTA-127339). In some embodiments, p1381 is engineered to remove, replace, or inactivate a lysogeny gene or a promoter of a lysogeny gene. In some embodiments, the Kaivirus is p1381 comprising a nucleic acid encoding a CRISPR-Cas system disclosed herein. In some embodiments, the Kaivirus is p1381 comprising a nucleic acid encoding a peptide disclosed herein.

[0035] In some embodiments, the Kaivirus is p4815 (PTA-127331). In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p4815. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p4815e037 (PTA-127335). In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p4815e053 (PTA-127340). In some embodiments, p4815 is engineered to remove, replace, or inactivate a lysogeny gene or a promoter of a lysogeny gene. In some embodiments, the Kaivirus is p4815 that comprises a nucleic acid encoding a CRISPR-Cas system disclosed herein. In some embodiments, the Kaivirus is p4815 that comprises a nucleic acid encoding a peptide disclosed herein.

[0036] In some embodiments, the bacteriophage of interest is obtained from an environmental source or a commercial research vendor. In some embodiments, the obtained bacteriophage is screened against a library of bacteria and related strains for lytic activity. In some embodiments, the bacteriophage is screened against a library of bacteria and related strains for the ability to generate primary resistance in the screened bacteria.

[0037] In some embodiments, the nucleic acid sequence is inserted into a bacteriophage, e.g., a nucleic acid sequence encoding one or more components of a CRISPR-Cas system, and / or a peptide. In some embodiments, insertion of the nucleic acid sequence into the bacteriophage preserves the lytic activity of the bacteriophage. In some embodiments, the nucleic acid sequence is inserted into the bacteriophage genome. In some embodiments, the nucleic acid sequence is inserted into the bacteriophage genome at a transcription terminator site at the end of an operon of interest. In some embodiments, the nucleic acid sequence is inserted into the bacteriophage genome as a replacement for one or more removed non-essential genes. In some embodiments, the nucleic acid sequence is inserted into the bacteriophage genome as a replacement for one or more removed lysogenic genes. In some embodiments, replacement of non-essential and / or lysogenic genes with the nucleic acid sequence does not affect the lytic activity of the bacteriophage. In some embodiments, replacement of non-essential and / or lysogenic genes with the nucleic acid sequence preserves the lytic activity of the bacteriophage. In some embodiments, the replacement of a non-essential and / or lysogenic gene with a nucleic acid sequence enhances the lytic activity of the bacteriophage, hi some embodiments, the replacement of a non-essential and / or lysogenic gene with a nucleic acid sequence renders a lysogenic bacteriophage lytic.

[0038] Bacteriophage cocktail Also disclosed herein are cocktails comprising two or more bacteriophages. In some embodiments, the two or more bacteriophages are selected from the family consisting of Kaivirus, Tuautovirus, Rosenblumvirus, Phyetavirus, or Toriavirus. In some embodiments, at least one bacteriophage of the cocktail comprises a CRISPR-Cas system. In some embodiments, at least two bacteriophages of the cocktail comprise a CRISPR-Cas system. In some embodiments, at least three bacteriophages of the cocktail comprise a CRISPR-Cas system. In some embodiments, at least four bacteriophages of the cocktail comprise a CRISPR-Cas system. In some embodiments, at least one bacteriophage of the cocktail does not comprise a CRISPR-Cas system. In some embodiments, at least two bacteriophages of the cocktail do not comprise a CRISPR-Cas system. In some embodiments, at least one bacteriophage of the cocktail comprises an antimicrobial peptide described herein. In some embodiments, at least two bacteriophages of the cocktail comprise a nucleic acid encoding an antimicrobial peptide described herein. In some embodiments, at least one bacteriophage comprises a nucleic acid encoding a CRISPR-Cas system and at least one bacteriophage comprises a nucleic acid encoding an antimicrobial peptide. In some embodiments, at least one bacteriophage of the cocktail comprises a nucleic acid encoding a CRISPR-Cas system. In some embodiments, no bacteriophage of the cocktail comprises a nucleic acid encoding a CRISPR-Cas system or an antimicrobial peptide.

[0039] In some embodiments, the cocktail comprises a Fietavirus. Optionally, the Fietavirus is engineered to remove, replace, or inactivate a lysogeny gene or a promoter of the lysogeny gene. In some example cocktails, the cocktail comprises a Fietavirus and a Rosenblum virus. In some example cocktails, the cocktail comprises a Fietavirus and a Kaivirus. In some example cocktails, the cocktail comprises a Fietavirus, a Rosenblum virus, and a Kaivirus. Optionally, the Fietavirus comprises a nucleic acid encoding a CRISPR-Cas system. Optionally, the Fietavirus comprises a nucleic acid encoding an antimicrobial peptide. Optionally, the Fietavirus binds to a different bacterial receptor than another bacteriophage in the cocktail. Optionally, the Fietavirus is able to infect bacteria if the bacteria develop resistance and / or have a mutation that prevents infection by another bacteriophage. In some such cases, the cocktail is more resistant to bacterial development than a single bacteriophage.

[0040] In some embodiments, the cocktail includes a Rosenblum virus. In some example cocktails, the cocktail includes a Rosenblum virus and a Fietavirus. Optionally, the Fietavirus is engineered to remove, replace, or inactivate a lysogeny gene or a promoter of a lysogeny gene. In some example cocktails, the cocktail includes a Rosenblum virus and a Kaivirus. In some example cocktails, the cocktail includes a Fietavirus, a Rosenblum virus, and a Kaivirus. Optionally, the Rosenblum virus includes a nucleic acid encoding a CRISPR-Cas system. Optionally, the Rosenblum virus includes a nucleic acid encoding an antimicrobial peptide. Optionally, the Rosenblum virus binds to a different bacterial receptor than another bacteriophage in the cocktail. Optionally, the Rosenblum virus is able to infect bacteria if the bacteria develop resistance and / or have a mutation that prevents infection by another bacteriophage. In some such cases, the cocktail is more resistant to bacterial resistance than a single bacteriophage.

[0041] In some embodiments, the cocktail includes a Kaivirus. In some example cocktails, the cocktail includes a Kaivirus and a Rosenblum virus. In some example cocktails, the cocktail includes a Kaivirus and a Fietavirus. Optionally, the Fietavirus is engineered to remove, replace, or inactivate a lysogeny gene or a promoter of a lysogeny gene. In some example cocktails, the cocktail includes a Fietavirus, a Rosenblum virus, and a Kaivirus. Optionally, the Kaivirus includes a nucleic acid encoding a CRISPR-Cas system. Optionally, the Kaivirus includes a nucleic acid encoding an antimicrobial peptide. Optionally, the Kaivirus binds to a different bacterial receptor than another bacteriophage in the cocktail. Optionally, the Kaivirus is able to infect a bacterium if the bacterium develops resistance and / or has a mutation that prevents infection by another bacteriophage. In some such cases, the cocktail is more resistant to bacterial development than a single bacteriophage.

[0042] In some embodiments, multiple bacteriophages are used together. In some embodiments, the multiple bacteriophages used together target the same or different bacteria in a sample or subject. In some embodiments, a cocktail comprising multiple bacteriophages is used together. In some embodiments, the cocktail comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 phages. In some embodiments, the cocktail comprises two phages. In some embodiments, the cocktail comprises three phages. In some embodiments, the cocktail comprises four phages. In some embodiments, the cocktail comprises five phages. In some embodiments, the cocktail comprises six phages. In some embodiments, at least one bacteriophage in the cocktail comprises a CRISPR array. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 bacteriophages present in the cocktail comprise a CRISPR array. In some embodiments, at least one bacteriophage in the cocktail comprises a nucleic acid sequence encoding a cascade polypeptide. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 bacteriophages present in the cocktail comprise a nucleic acid sequence encoding a cascade polypeptide. In some embodiments, at least one bacteriophage in the cocktail comprises a nucleic acid sequence encoding a Cas3 polypeptide. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 bacteriophages present in the cocktail comprise a nucleic acid sequence encoding a Cas3 polypeptide, hi some embodiments, at least one bacteriophage in the cocktail comprises a nucleic acid sequence encoding an antimicrobial peptide.

[0043] In some embodiments, the cocktail comprises at least two bacteriophages, and the bacteriophages comprise p1378, p4815, p1494e002, or p2808. In some embodiments, the first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1378. In some embodiments, the second bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p4815. In some embodiments, the third bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1494e002. In some embodiments, the fourth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p2808. In some embodiments, at least 1, 2, 3, or 4 bacteriophages comprise a CRISPR-Cas system. In some embodiments, at least 1, 2, 3, or 4 bacteriophages do not comprise a CRISPR-Cas system.

[0044] In some embodiments, the first bacteriophage comprises at least 70%, 75%, 70%, 85%, 90%, 95%, or 100% sequence identity with p1378. In some embodiments, the cocktail comprises a second bacteriophage, wherein the second bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1381, p4815, p1378e062, p1378e074, p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, p2808, or p1473. In some embodiments, the cocktail comprises a third bacteriophage, wherein the third bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1381, p4815, p1378e062, p1378e074, p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, p2808, or p1473. In some embodiments, the cocktail comprises a fourth bacteriophage, wherein the fourth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1381, p4815, p1378e062, p1378e074, p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, p2808, or p1473.In some embodiments, the cocktail comprises a fifth bacteriophage, wherein the fifth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1381, p4815, p1378e062, p1378e074, p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, p2808, or p1473. In some embodiments, the cocktail comprises a sixth bacteriophage, wherein the sixth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1381, p4815, p1378e062, p1378e074, p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, p2808, or p1473.

[0045] In some embodiments, the first bacteriophage comprises at least 70%, 75%, 70%, 85%, 90%, 95%, or 100% sequence identity with p4815. In some embodiments, the cocktail comprises a second bacteriophage, wherein the second bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1378, p1381, p1378e062, p1378e074, p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, p2808, or p1473. In some embodiments, the cocktail comprises a third bacteriophage, wherein the third bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1378, p1381, p1378e062, p1378e074, p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, p2808, or p1473. In some embodiments, the cocktail comprises a fourth bacteriophage, wherein the fourth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1378, p1381, p1378e062, p1378e074, p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, p2808, or p1473.In some embodiments, the cocktail comprises a fifth bacteriophage, wherein the fifth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1378, p1381, p1378e062, p1378e074, p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, p2808, or p1473. In some embodiments, the cocktail comprises a sixth bacteriophage, wherein the sixth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1378, p1381, p1378e062, p1378e074, p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, p2808, or p1473.

[0046] In some embodiments, the first bacteriophage comprises at least 70%, 75%, 70%, 85%, 90%, 95%, or 100% sequence identity with p1494e002. In some embodiments, the cocktail comprises a second bacteriophage, wherein the second bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1378, p1381, p4815, p1378e062, p1378e074, p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p2808, or p1473. In some embodiments, the cocktail comprises a third bacteriophage, wherein the third bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1378, p1381, p4815, p1378e062, p1378e074, p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p2808, or p1473. In some embodiments, the cocktail comprises a fourth bacteriophage, wherein the fourth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1378, p1381, p4815, p1378e062, p1378e074, p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p2808, or p1473.In some embodiments, the cocktail comprises a fifth bacteriophage, wherein the fifth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1378, p1381, p4815, p1378e062, p1378e074, p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p2808, or p1473. In some embodiments, the cocktail comprises a sixth bacteriophage, wherein the sixth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1378, p1381, p4815, p1378e062, p1378e074, p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p2808, or p1473.

[0047] In some embodiments, the first bacteriophage comprises at least 70%, 75%, 70%, 85%, 90%, 95%, or 100% sequence identity with p2808. In some embodiments, the cocktail comprises a second bacteriophage, wherein the second bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1378, p1381, p4815, p1378e062, p1378e074, p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, or p1473. In some embodiments, the cocktail comprises a third bacteriophage, wherein the third bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1378, p1381, p4815, p1378e062, p1378e074, p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, or p1473. In some embodiments, the cocktail comprises a fourth bacteriophage, wherein the fourth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1378, p1381, p4815, p1378e062, p1378e074, p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, or p1473.In some embodiments, the cocktail comprises a fifth bacteriophage, wherein the fifth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1378, p1381, p4815, p1378e062, p1378e074, p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, or p1473. In some embodiments, the cocktail comprises a sixth bacteriophage, wherein the sixth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1378, p1381, p4815, p1378e062, p1378e074, p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, or p1473.

[0048] In some embodiments, the first bacteriophage comprises at least 70%, 75%, 70%, 85%, 90%, 95%, or 100% sequence identity to p1378, p1381, p4815, p1378e062, p1378e074, p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, p2808, or p1473. In some embodiments, the cocktail comprises a second bacteriophage, wherein the second bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1378, p1381, p4815, p1378e062, p1378e074, p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, p2808, or p1473. In some embodiments, the cocktail comprises a third bacteriophage, wherein the third bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1378, p1381, p4815, p1378e062, p1378e074, p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, p2808, or p1473. In some embodiments, the cocktail comprises a fourth bacteriophage, wherein the fourth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1378, p1381, p4815, p1378e062, p1378e074, p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, p2808, or p1473.In some embodiments, the cocktail comprises a fifth bacteriophage, wherein the fifth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1378, p1381, p4815, p1378e062, p1378e074, p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, p2808, or p1473. In some embodiments, the cocktail comprises a sixth bacteriophage, wherein the sixth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to p1378, p1381, p4815, p1378e062, p1378e074, p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, p2808, or p1473.

[0049] In some embodiments, the bacteriophage cocktail has a host range that is greater than the host range of the individual bacteriophages. The increased host range may allow for targeting multiple strains of Staphylococcus aureus. In some embodiments, the bacteriophage cocktail targets at least 70%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more than 99% of strains of Staphylococcus aureus. In some embodiments, the strains of Staphylococcus aureus include b004604, b004605, b004606, b004607, b004608, b004609, b004610, b004611, b004612, b004613, b004614, b004615, b004616, b004617, b004618, b004619, b004620, b004621, b004622, b004623, b004624, b004625, b004626, b004627, b004628, b004629, b004630, b004631, b004632, b004633, b004634, b004635, b004636, b004637, b004638, b004639, b004640, b004641, b004642, b004643, b004644, b004645, b004646, b004647, b004648, b004649, b004650, b004651, b004652, b004653, b004654, b004655, b004656, b004657, b004658, b004660, b004661, b004662 04626, b004627, b004628, b004629, b004630, b004631, b004632, b004633, b004634, b004635, b004636, b004637, b004638 , b004639, b004640, b004641, b004642, b004643, b004644, b004645, b004646, b004647, b004648, b004649, b004650, b004 651, b004652, b004653, b004654, b004655, b004656, b004657, b004658, b004659, b004660, b004661, b004662, b004663, b 004664, b004665, b004666, b004667, b004668, b004669, b004670, b004671, b004672, b004673, b004674, b004675, b00467 6, b004677, b004678, b004679, b004680, b004681, b004682, b004683, b004684, b004685, b004686, b004687, b004688, b00 4689, b004690, b004691, b004692, b004693, b004694, b004695, b004696, b004697, b004698, b004699, b004700, b004701,b004702、b004703、b004704、b004705、b004706、b004707、b004708、b00470 9、b004710、b004711、b004712、b004713、b004714、b004715、b004716、b0047 17, b004718, b004719, b004720, b004721, b004722, b004723, b004724, b004725, b004726, b004727, b004728, b004729, b004730, b004731, b004732, b0 04733, b004734, b004735, b004736, b004737, b004738, b004739, b004740, b004741, b004742, b004743, b004744, b004745, b004746, b004747, b004748 b004749, b004750, b004751, b004752, b004753, b004754, b004755, b004756, b004757, b004758, b004759, b004760, b004761, b004762, b004763, b004 764、b004765、b004766、b004767、b004768、b004769、b004770、b004771、b004772、b004773、b004774、b004775、b004776、b004777、b004778、b004779、b 004780、b004781、b004782、b004783、b004784、b004785、b004786、b004787、b004788、b004789、b004790、b004791、b004792、b004793、b004794、b00479 5、b004796、b004797、b004798、b004799、b004800、b004801、b004802、b004 803、b004804、b004805、b004806、b004807、b004808、b004809、b004810、b00 4811、b004812、b004813、b004814、b004815、b004816、b004817、b004818、b 004819、b004820、b004821、b004822、b004823、b004824、b004825、b004826、b004827, b004828, b004829, b004830, b004831, b004832, b004833, b004834, b004835, b004836, b00483 7, b004838, b004839, b004840, b004841, b004842, b004843, b004844, b004845, b004846, b004847, b004 848, b004849, b004850, b004851, b004852, b004853, b004854, b004855, b004856, b004857, b004858, b0 04859, b004860, b004861, b004862, b004863, b004864, b004865, b004866, b004867, b004868, b004869, b004870, b004871, b004872, b004873, b004874, b004875, b004876, b004877, b004878, b004879, b00488 0, b004881, b004882, b004883, b004884, b004885, b004886, b004887, b004888, b004889, b004890, b004 891, b004892, b004893, b004894, b004895, b004896, b004897, b004898, b004899, b004900, b004901, b004902, b004903, b004904, b004905, b004906, b004907, b004908, b004909, b004910, and b004911. In some embodiments, the strain of Staphylococcus aureus is 8, 5, 22, 15, 1, 30, 398, 105, 45, 672, 2250, 582, 72, 97, 239, 34, 87, 101, 109, 1159, 1165, 1181, 12, 121, 152, 1750, 20, 225, 25, 291, 3628, 59, 7, 779, 8 8, 10, 1011, 1049, 1156, 1351, 149, 1637, 1649, 1757, 1842, 188, 1970, 2066, 256, 2867, 2945, 3149, 3182, 3510, 39, 395, 4317, 47, 4730, 50, 508, 573, 6, 630, 737, 828, 848, 923,or 93 different MLSTs. In some embodiments, the Staphylococcus bacteria comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 different MLSTs. In some embodiments, the Staphylococcus bacteria are isolated from bloodstream infections. In some embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the Staphylococcus strains are multidrug-resistant. In some embodiments, the bacteriophage cocktail specifically targets Staphylococcus aureus but not other bacterial species. In some embodiments, the bacteriophage cocktail targets Staphylococcus aureus but not other Staphylococcus species. The increased host range may allow for targeting multiple strains of Staphylococcus spp. In some embodiments, the bacteriophage cocktail targets at least 70%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more than 99% of strains of Staphylococcus spp. In some embodiments, the bacteriophage cocktail specifically targets Staphylococcus spp. but not other bacterial species. In some embodiments, the bacteriophage cocktail targets Staphylococcus spp. but not non-Staphylococcus spp. In some embodiments, the bacteriophage cocktail includes two or more of Phyetavirus, Rosenblum virus, and Kaivirus.

[0050] In some embodiments, the bacteriophages in the cocktail are selected to minimize the ability of the target bacteria to develop resistance. In some embodiments, the cocktail includes a first bacteriophage that binds to a first receptor on Staphylococcus bacteria and a second bacteriophage that binds to a second receptor on Staphylococcus bacteria, and the multiple bacteriophages are more resistant to Staphylococcus than the first or second bacteriophage alone. For example, if Staphylococcus bacteria develop resistance to the first bacteriophage, the bacteria remain susceptible to infection by the second bacteriophage, and vice versa. In some embodiments, a cocktail of at least two bacteriophages from different genera targets at least 70%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more than 99% of Staphylococcus aureus strains. In some embodiments, the cocktail comprises at least two bacteriophages, wherein a first bacteriophage binds to a first receptor on Staphylococcus aureus and a second bacteriophage binds to a second receptor on Staphylococcus aureus, hi some embodiments, the bacteriophage cocktail comprises two or more of Phytavirus, Rosenblumvirus, and Kaivirus.

[0051] Staphylococcus aureus In some embodiments, the bacteria comprise one or more Staphylococcus species. In some embodiments, the bacteria comprise one or more Staphylococcus strains. In some embodiments, the target bacteria is Staphylococcus aureus. In some embodiments, the target bacteria is Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Streptococcus salivary, Staphylococcus argenteus, Staphylococcus hemolyticus, or Staphylococcus schweitzeri, or any combination of two or more thereof.

[0052] In some embodiments, the target bacteria cause an infection or disease. In some embodiments, the infection or disease is acute or chronic. In some embodiments, the infection or disease is localized or systemic. In some embodiments, the infection or disease is idiopathic. In some embodiments, the infection or disease is acquired through means including, but not limited to, respiratory inhalation, ingestion, skin and wound infections, bone infections, bloodstream infections, middle ear infections, gastrointestinal infections, peritoneal infections, urinary tract infections, genitourinary tract infections, oral soft tissue infections, intraperitoneal infections, epidermal or mucosal absorption, eye infections (including contact lens contamination), endocarditis, infections in cystic fibrosis and non-cystic fibrosis bronchiectasis (NCFB), infections of indwelling medical devices such as joint prostheses, dental implants, catheters, and cardiac implants, sexual contact, and / or hospital-acquired and ventilator-associated bacterial pneumonia. In some embodiments, the target bacteria cause a urinary tract infection. In some embodiments, the target bacteria cause and / or exacerbate an inflammatory disease. In some embodiments, the target bacteria cause and / or exacerbate an autoimmune disease. In some embodiments, the target bacteria cause and / or exacerbate eczema. In some embodiments, the target bacteria cause and / or exacerbate atopic dermatitis. In some embodiments, the target bacteria cause and / or exacerbate inflammatory bowel disease (IBD). In some embodiments, the target bacteria cause and / or exacerbate psoriasis. In some embodiments, the target bacteria cause and / or exacerbate psoriatic arthritis (PA). In some embodiments, the target bacteria cause and / or exacerbate rheumatoid arthritis (RA). In some embodiments, the target bacteria cause and / or exacerbate systemic lupus erythematosus (SLE). In some embodiments, the target bacteria cause and / or exacerbate multiple sclerosis (MS). In some embodiments, the target bacteria cause and / or exacerbate Graves' disease. In some embodiments, the target bacteria causes and / or exacerbates Hashimoto's thyroiditis. In some embodiments, the target bacteria causes and / or exacerbates myasthenia gravis.In some embodiments, the target bacteria cause and / or exacerbate vasculitis. In some embodiments, the target bacteria cause and / or exacerbate cancer. In some embodiments, the target bacteria cause and / or exacerbate cancer progression. In some embodiments, the target bacteria cause and / or exacerbate cancer metastasis. In some embodiments, the target bacteria cause and / or exacerbate resistance to cancer treatment. In some embodiments, the treatments used to address cancer include, but are not limited to, chemotherapy, immunotherapy, hormone therapy, targeted drug therapy, and / or radiation therapy. In some embodiments, the cancer manifests in organs including, but not limited to, the anus, bladder, blood and blood components, bone, bone marrow, brain, breast, cervix, colon and rectum, esophagus, kidney, larynx, lymphatic system, muscle (i.e., soft tissue), oral cavity and pharynx, ovaries, pancreas, prostate, skin, small intestine, stomach, testes, thyroid, uterus, and / or vulva. In some embodiments, the target bacteria cause and / or exacerbate a central nervous system (CNS) disorder. In some embodiments, the target bacteria cause and / or exacerbate attention deficit / hyperactivity disorder (ADHD). In some embodiments, the target bacteria cause and / or exacerbate autism. In some embodiments, the target bacteria cause and / or exacerbate bipolar disorder. In some embodiments, the target bacteria cause and / or exacerbate major depressive disorder. In some embodiments, the target bacteria cause and / or exacerbate epilepsy. In some embodiments, the target bacteria cause and / or exacerbate a neurodegenerative disorder, including, but not limited to, Alzheimer's disease, Huntington's disease, and / or Parkinson's disease.

[0053] In some embodiments, the target bacteria is b004604, b004605, b004606, b004607, b004608, b004609, b004610, b004611, b004612, b004613, b004614, b004615, b004616, b004617, b004618, b004619, b004620, b004621, b004622, b004623, b004624, b004625, b004626, b004627, b004628, b004629, b004630, b004631, b004632, b004633, b004634, b004635, b004636, b004637, b004638, b004639, b004640, b004641, b004642, b004643, b004644, b004645, b004646, b004647, b004648, b004649, b004650, b004651, b004652, b004653, b004654, b004655, b004656, b004657, b004658, b004659, b004660, b004661 04633, b004634, b004635, b004636, b004637, b004638, b004639, b004640, b004641, b004642, b004643, b004644, b004645, b004646, b004647, b004648 , b004649, b004650, b004651, b004652, b004653, b004654, b004655, b0046 56, b004657, b004658, b004659, b004660, b004661, b004662, b004663, b004 664, b004665, b004666, b004667, b004668, b004669, b004670, b004671, b0 04672, b004673, b004674, b004675, b004676, b004677, b004678, b004679, b 004680, b004681, b004682, b004683, b004684, b004685, b004686, b004687 , b004688, b004689, b004690, b004691, b004692, b004693, b004694, b00469 5, b004696, b004697, b004698, b004699, b004700, b004701, b004702, b004 703, b004704, b004705, b004706, b004707, b004708, b004709, b004710, b00 4711, b004712, b004713, b004714, b004715, b004716, b004717, b004718, b 004719, b004720, b004721, b004722, b004723, b004724, b004725, b004726,b004727, b004728, b004729, b004730, b004731, b004732, b004733, b004734, b004735, b004736, b004737, b004738, b004739, b004740, b004741, b0047 42、b004743、b004744、b004745、b004746、b004747、b004748、b004749、b004750、b004751、b004752、b004753、b004754、b004755、b004756、b004757、b0 04758、b004759、b004760、b004761、b004762、b004763、b004764、b004765、b004766、b004767、b004768、b004769、b004770、b004771、b004772、b004773 b004774, b004775, b004776, b004777, b004778, b004779, b004780, b004781, b004782, b004783, b004784, b004785, b004786, b004787, b004788, b004 789、b004790、b004791、b004792、b004793、b004794、b004795、b004796、b0 04797、b004798、b004799、b004800、b004801、b004802、b004803、b004804、b 004805、b004806、b004807、b004808、b004809、b004810、b004811、b004812 、b004813、b004814、b004815、b004816、b004817、b004818、b004819、b00482 0、b004821、b004822、b004823、b004824、b004825、b004826、b004827、b004 828、b004829、b004830、b004831、b004832、b004833、b004834、b004835、b00 4836、b004837、b004838、b004839、b004840、b004841、b004842、b004843、b 004844、b004845、b004846、b004847、b004848、b004849、b004850、b004851、b004852, b004853, b004854, b004855, b004856, b004857, b004858, b004859 , b004860, b004861, b004862, b004863, b004864, b004865, b004866, b004867 , b004868, b004869, b004870, b004871, b004872, b004873, b004874, b00487 5, b004876, b004877, b004878, b004879, b004880, b004881, b004882, b00488 b004896, b004897, b004898, b004899, b004900, b004901, b004902, b004903, b004904, b004905, b004906, b004907, b004908, b004909, b004910, and b004911. In some embodiments, the Staphylococcus bacteria is 8, 5, 22, 15, 1, 30, 398, 105, 45, 672, 2250, 582, 72, 97, 239, 34, 87, 101, 109, 1159, 1165, 1181, 12, 121, 152, 1750, 20, 225, 25, 291, 3628, 59, 7, 779, 88, 10, 1011, 1049 , 1156, 1351, 149, 1637, 1649, 1757, 1842, 188, 1970, 2066, 256, 2867, 2945, 3149, 3182, 3510, 39, 395, 4317, 47, 4730, 50, 508, 573, 6, 630, 737, 828, 848, 923, or 93. In some embodiments, the Staphylococcus bacteria comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 different MLSTs. In some embodiments, the Staphylococcus bacteria are isolated from a bloodstream infection.

[0054] Lysogeny removal Disclosed herein in certain embodiments are bacteriophages engineered to remove, replace, or inactivate a lysogeny gene or a promoter of a lysogeny gene. As a non-limiting example, the lysogeny gene encodes a suppressor. In some embodiments, the bacteriophage is further engineered to include one or more components of a CRISPR-Cas system and / or an antimicrobial peptide. In some embodiments, the lysogeny gene encodes a suppressor. In some embodiments, removal of the lysogeny gene includes removing between about 1% and 100% of the lysogeny gene, or between about 10 and about 1,200 base pairs of the lysogeny gene.

[0055] In some embodiments, the bacteriophage is an obligately lytic bacteriophage. In some embodiments, the bacteriophage is a temperate bacteriophage in which the lysogenic genes are retained. In some embodiments, the bacteriophage is a temperate bacteriophage in which some of the lysogenic genes have been removed, replaced, or inactivated. In some embodiments, the bacteriophage is a temperate bacteriophage in which some of the lysogenic genes have been removed, replaced, or inactivated, thereby making it lytic. In some embodiments, the bacteriophage is made lytic by removing at least a portion of the lysogenic gene or the promoter of the lysogenic gene. In some embodiments, this portion is at least about 1% to 100% of the nucleotides of the lysogenic gene. In some embodiments, this portion is less than about 1%. In some embodiments, the portion removed is a single base. In some embodiments, this portion is from about 10 base pairs to all of the lysogenic gene. For example, the portion to be removed may be about 10-1200, 10-1100, 10-1000, 10-900, 10-800, 10-700, 10-600, 10-500, 10-400, 10-300, 10-200, 10-100, 50-1200, 50-1100, 50-1000, 50-900, 50-800, 50-70 0, 50-600, 50-500, 50-400, 50-300, 50-200, 50-100, 100-1200, 100-1100, 100-1000, 100-900, 100-800, 100-700, 100-600, 100-500, 100-400, 100-300, or 100-200 base pairs. In some embodiments, the lysogeny gene encodes an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 47 or 48.In some embodiments, the lysogenic region comprises a sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 49-58. In some embodiments, the lysogenic region comprises a sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 49. In some embodiments, the lysogenic region comprises a sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 50. In some embodiments, the lysogenic region comprises a sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:51. In some embodiments, the lysogenic region comprises a sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 52. In some embodiments, the lysogenic region comprises a sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 53. In some embodiments, the lysogenic region comprises a sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:54.In some embodiments, the lysogenic region comprises a sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 55. In some embodiments, the lysogenic region comprises a sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:56. In some embodiments, the lysogenic region comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 57. In some embodiments, the lysogenic region comprises a sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 58. In some embodiments, at least a portion of any one of SEQ ID NOs: 49-58 is removed. For example, at least a portion is at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of any one of SEQ ID NOs: 49-58.

[0056] In some embodiments, a nucleic acid sequence is introduced into a bacteriophage genome at a first position, while one or more non-essential and / or lysogenic genes are separately removed and / or inactivated from the bacteriophage genome at separate locations. In some embodiments, a nucleic acid sequence is introduced into a bacteriophage at a first position, while one or more non-essential and / or lysogenic genes are separately removed and / or inactivated from the bacteriophage genome at multiple separate locations. In some embodiments, the removal and / or inactivation of one or more non-essential and / or lysogenic genes does not affect the lytic activity of the bacteriophage. In some embodiments, the removal and / or inactivation of one or more non-essential and / or lysogenic genes preserves the lytic activity of the bacteriophage. In some embodiments, the removal of one or more non-essential and / or lysogenic genes renders a lysogenic bacteriophage into a lytic bacteriophage.

[0057] In some embodiments, the bacteriophage is a temperate bacteriophage that has been made lytic by any of the aforementioned means. In some embodiments, the temperate bacteriophage is made lytic by removal, replacement, or inactivation of one or more lysogenic genes. In some embodiments, the lytic activity of the bacteriophage results from the removal, replacement, or inactivation of at least one lysogenic gene. In some embodiments, the lysogenic gene plays a role in maintaining the lysogenic cycle in the bacteriophage. In some embodiments, the lysogenic gene plays a role in establishing the lysogenic cycle in the bacteriophage. In some embodiments, the lysogenic gene plays a role in both establishing the lysogenic cycle in the bacteriophage and maintaining the lysogenic cycle in the bacteriophage.

[0058] In some embodiments, a bacteriophage is rendered lytic by removing a sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 49-58. In some embodiments, at least a portion of any one of SEQ ID NOs: 49-58 is removed. For example, the at least a portion is at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of any one of SEQ ID NOs: 49-58.

[0059] In some embodiments, the lysogeny gene is a repressor gene. In some embodiments, the bacteriophage is made lytic by removal of the "varO10" region annotated in Figure 6 as the "varO10" deletion (SEQ ID NO: 30). In some embodiments, the bacteriophage is made lytic by removal of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 nucleotides of the varO10 region. In some embodiments, the bacteriophage is made lytic by removal of the "varO12" region annotated in Figure 7 as the "varO12" deletion (SEQ ID NO: 31). In some embodiments, a bacteriophage is made lytic by removing at least 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides of the var012 region. In some embodiments, a bacteriophage is made lytic by removing the "var042" region, annotated in Figure 8 as the "var042" deletion (SEQ ID NO: 32). In some embodiments, a bacteriophage is made lytic by removing at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 nucleotides of the var042 region. In some embodiments, a bacteriophage is made lytic by removing the "var009" deletion (SEQ ID NO: 30). In some embodiments, the bacteriophage is made lytic by removing at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 nucleotides of the var009 region. In some embodiments, the bacteriophage is made lytic by removing at least 10, 20, 30, or 40 nucleotides of the var0022 deletion (SEQ ID NO: 58).In some embodiments, the bacteriophage is rendered lysed by removing at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 nucleotides encoding a suppressor having an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 47 or 48. In some embodiments, the bacteriophage is made lytic by deletion of the anti-repressor gene integrase, a Pem-K-like phage protein gene, or a combination thereof. In some embodiments, the bacteriophage is made lytic by deletion of a gene depicted in Figure 1. In some embodiments, the bacteriophage is made lytic by deletion of the anti-repressor gene integrase, a Pem-K-like phage protein gene, or a combination thereof, the genes depicted in Figure 1. In some embodiments, the bacteriophage is at least 10-2000, 20-2000, 30-2000, 40-2000, 50-2000, 60-2000, 70-2000, 80-2000, 90-2000, 100-2000, 200-2000, 300-2000, 400-2000, 500-2000, 600-2000, 700-2000, 800-2000, 90 ...0-2000, 2000-2000, 3000-2000, 4000-2000, 500-2000, 600-2000, 7000-2000, 8000-2000, 9000-2000, 10000-2000, 10000-2000, 10000-2000, 10000-2000, 10000-2000, 10000-2000, 10000-2000, 10000-2000, 10000-2000, 10000-2000, 10000-2000, 10000-2 In some embodiments, the bacteriophage is rendered lytic by deletion of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the antirepressor gene. In some embodiments, the lysogeny gene is a cl repressor gene. In some embodiments, the lysogeny gene is an activator gene.In some embodiments, the lysogeny gene is a cII gene. In some embodiments, the lysogeny gene is a Cro gene. In some embodiments, the lysogeny gene is an int (integrase) gene. In some embodiments, two or more lysogeny genes are removed, replaced, or inactivated to cause the bacteriophage lysogenic cycle to cease and / or the lytic cycle to begin. In some embodiments, a temperate bacteriophage is made lytic by the insertion of one or more lytic genes. In some embodiments, the lytic gene encodes a holin, an endolysin, an endopeptidase, pinoline, spanin, or an Rz / Rz1-like protein. In some embodiments, the lytic gene encodes a holin. In some embodiments, the lytic gene encodes an endolysin. In some embodiments, the lytic gene encodes an endopeptidase. In some embodiments, the lytic gene encodes a pinoline. In some embodiments, the lytic gene encodes a spanin. In some embodiments, the lytic gene encodes an Rz / Rz-1-like protein. In some embodiments, temperate bacteriophage are made lytic by inserting one or more genes that contribute to the induction of the lytic cycle. In some embodiments, temperate bacteriophage are made lytic by altering the expression of one or more genes that contribute to the induction of the lytic cycle. In some embodiments, temperate bacteriophage are phenotypically changed from a lysogenic bacteriophage to a lytic bacteriophage. In some embodiments, temperate bacteriophage are made lytic by an environmental change. In some embodiments, the environmental change includes, but is not limited to, a change in temperature, pH, or nutrients; exposure to antibiotics, hydrogen peroxide, foreign DNA, or DNA damaging agents; the presence of organic carbon; and the presence of heavy metals (e.g., in the form of chromium(VI)). In some embodiments, temperate bacteriophage that have been made lytic are prevented from reverting to a lysogenic state. In some embodiments, the temperate bacteriophage that has been made lysogenic is prevented from reverting to a lysogenic state by the self-targeting activity of the first introduced CRISPR array.In some embodiments, the temperate bacteriophage that has been rendered lytic is prevented from reverting to a lysogenic state by introducing an additional CRISPR array. In some embodiments, the bacteriophage does not confer any new properties to the target bacterium beyond cell death caused by the lytic activity of the bacteriophage and / or the activity of the first or second CRISPR array.

[0060] In some embodiments, replacement, removal, inactivation, or any combination thereof, of one or more non-essential and / or lysogenic genes is achieved by chemical, biochemical, and / or any suitable method, hi some embodiments, insertion of one or more lytic genes is achieved by any suitable chemical, biochemical, and / or physical method by homologous recombination.

[0061] Further disclosed in some embodiments herein is a temperate bacteriophage comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, provided that the bacteriophage is rendered lytic by removing the region annotated as "var010" in FIG. 6, "var012" in FIG. 7, "var042" in FIG. 8, "e002" or "var0022" in FIG. 13A (SEQ ID NO: 58), at least a portion of SEQ ID NO: 47 or 48, or a combination thereof. Further disclosed herein in some embodiments is a temperate bacteriophage comprising a first spacer sequence or a first nucleic acid sequence encoding a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, provided that the bacteriophage is rendered lytic by removing a sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 49-58. In some embodiments, at least a portion of any one of SEQ ID NOs: 49-58 is removed. For example, the at least portion is at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of any one of SEQ ID NOs: 49-58. In some embodiments, the bacteriophage infects multiple bacterial strains. In some embodiments, the target nucleotide sequence comprises all or a portion of a promoter sequence of the target gene. In some embodiments, the target nucleotide sequence comprises all or a portion of a nucleotide sequence located on the coding strand of a transcribed region of the target gene. In some embodiments, the target nucleotide sequence comprises at least a portion of an essential gene required for survival of the target bacterium.In some embodiments, the essential gene is Tsf, acpP, gapA, infA, secY, csrA, trmD, ftsA, fusA, glyQ, eno, nusG, dnaA, pheS, rplB, gltX, hisS, rplC, aspS, gyrB, dnaE, rpoA, rpoB, pheT, infB, rpsC, rplF, alaS, leuS, serS, rplD, gyrA, glmS, fus, adk, rpsK, rplR, ctrA, parC, tRNA-Ser, tRNA-Asn, or metK. In some embodiments, the target nucleotide sequence is in a non-essential gene or other genomic locus. In some embodiments, the target nucleotide sequence is in a non-essential gene. In some embodiments, the target nucleotide sequence is in a non-essential locus. In some embodiments, the target nucleotide sequence is a non-coding sequence. In some embodiments, the non-coding sequence is an intergenic sequence. In some embodiments, the spacer sequence is complementary to a target nucleotide sequence of a highly conserved sequence in the target bacterium. In some embodiments, the spacer sequence is complementary to a target nucleotide sequence of a sequence present in the target bacterium. In some embodiments, the spacer sequence is complementary to a target nucleotide sequence comprising all or part of the promoter sequence of an essential gene. In some embodiments, the first nucleic acid sequence comprises a first CRISPR array further comprising at least one repeat sequence. In some embodiments, the at least one repeat sequence is operably linked to the first spacer sequence at either its 5' end or 3' end. In some embodiments, the target bacterium is Staphylococcus aureus.

[0062] Antimicrobials and Peptides In some embodiments, the bacteriophage disclosed herein are further genetically modified to express an antimicrobial peptide, a functional fragment of an antimicrobial peptide, and / or a lytic gene. In some embodiments, the bacteriophage disclosed herein expresses at least one antimicrobial agent or peptide disclosed herein. In some embodiments, the bacteriophage comprises a nucleic acid encoding a peptide that prevents phage degradation or that assists in the disruption or degradation of the biofilm matrix.

[0063] In some embodiments, the bacteriophage described herein comprises a nucleic acid encoding a peptide that prevents phage degradation or enables the phage to evade host defenses. In some embodiments, the bacteriophage disclosed herein comprises a nucleic acid sequence encoding an enzybiotic, where the protein product of the nucleic acid sequence targets phage-resistant bacteria. In some embodiments, the peptide comprises TreA (e.g., a sequence at least 80% identical to SEQ ID NO: 10). In some embodiments, the peptide comprises Ipi (e.g., a sequence at least 80% identical to SEQ ID NO: 11).

[0064] In some embodiments, the bacteriophage comprises a nucleic acid encoding an enzyme that assists in the disruption or degradation of the biofilm matrix. In some embodiments, the bacteriophage disclosed herein comprises a nucleic acid encoding dispersin D aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, α-galactosidase, β-galactosidase, glucoamylase, α-glucosidase, β-glucosidase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease, xylanase, or lyase. In some embodiments, the enzyme is selected from the group consisting of cellulases, e.g., the glycosyl hydroxylase family of cellulases, such as the glycosyl hydroxylase 5 family of enzymes, also known as cellulase A; polyglucosamine (PGA) depolymerase; and colanic acid depolymerase, e.g., 1,4-L-fucodise hydrolase, colanic acid, depolymerizing arginase, DNase I, or a combination thereof. In some embodiments, the bacteriophage disclosed herein secretes the enzyme disclosed herein. In some embodiments, the peptide interferes with quorum sensing and biofilm formation. In some embodiments, the peptide increases the sensitivity of bacterial cells to antibiotics. In some embodiments, the enzyme comprises DNAse I (e.g., a sequence at least 80% identical to SEQ ID NO:9). In some embodiments, the enzyme comprises a RIP (e.g., a sequence at least 80% identical to SEQ ID NO:14). In some embodiments, the enzyme comprises FS3 (e.g., a sequence at least 80% identical to SEQ ID NO:12).

[0065] In some embodiments, the antimicrobial agent or peptide is expressed and / or secreted by a bacteriophage disclosed herein. In some embodiments, the antimicrobial agent or peptide comprises PLNC8α. In some embodiments, the antimicrobial agent or peptide comprises PLNC8β. In some embodiments, the antimicrobial agent or peptide comprises LytM. In some embodiments, the antimicrobial agent or peptide comprises an anti-restriction-modification enzyme. In some embodiments, the antimicrobial agent or peptide comprises lacticin Q (LnqQ, e.g., a sequence at least 80% identical to SEQ ID NO: 13 or 15). In some embodiments, the LnqQ peptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 13 or 15. In some embodiments, the LnqQ peptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 13. In some embodiments, the LnqQ peptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 15.

[0066] In some embodiments, an antimicrobial agent or peptide is expressed and / or secreted by a bacteriophage disclosed herein. In some embodiments, a bacteriophage disclosed herein secretes and expresses an antibiotic such as ampicillin, penicillin, penicillin derivatives, cephalosporins, monobactams, carbapenems, ofloxacin, ciprofloxacin, levofloxacin, gatifloxacin, norfloxacin, lomefloxacin, trovafloxacin, moxifloxacin, sparfloxacin, gemifloxacin, pazafloxacin, or any antibiotic disclosed herein. In some embodiments, a bacteriophage disclosed herein comprises a nucleic acid sequence encoding an antimicrobial peptide, expresses an antimicrobial peptide, or secretes a peptide that aids or enhances killing of a target bacterium. In some embodiments, the bacteriophage disclosed herein comprises a nucleic acid sequence that encodes a peptide, encodes an antimicrobial peptide, expresses an antimicrobial peptide, or secretes a peptide that assists or enhances the activity of the first and / or second Type I CRISPR-Cas system.

[0067] In some embodiments, the antimicrobial agent or peptide is encoded by a sequence comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the antimicrobial agent or peptide is encoded by a sequence comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the antimicrobial agent or peptide is encoded by a sequence comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3. In some embodiments, the antimicrobial agent or peptide is encoded by a sequence comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the antimicrobial agent or peptide is encoded by a sequence comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the antimicrobial agent or peptide is encoded by a sequence comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:7.

[0068] In some embodiments, the antimicrobial agent or peptide comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 9. In some embodiments, the antimicrobial agent or peptide comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 10. In some embodiments, the antimicrobial agent or peptide comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 11. In some embodiments, the antimicrobial agent or peptide comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 12. In some embodiments, the antimicrobial agent or peptide comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 13. In some embodiments, the antimicrobial agent or peptide comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 14. In some embodiments, the antimicrobial agent or peptide comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:15.

[0069] CRISPR / CAS system The CRISPR-Cas system is a natural adaptive immune system found in bacteria and archaea. CRISPR systems are nuclease systems involved in defense against invasive phages and plasmids, providing a form of acquired immunity. Diverse CRISPR-Cas systems exist based on the set of cas genes and their phylogenetic relationships. There are at least six different types (I-VI), with Type I representing over 50% of all systems identified in both bacteria and archaea. In some embodiments, Type I, Type II, Type III, Type IV, Type V, or Type VI CRISPR-Cas systems are used herein.

[0070] Type I systems are divided into seven subtypes, including types IA, IB, IC, ID, IE, IF, and IU. Type I CRISPR-Cas systems contain a multisubunit complex called Cascade (related to the complex involved in antiviral defense), Cas3 (a protein with nuclease, helicase, and exonuclease activities responsible for target DNA degradation), and a CRISPR array encoding crRNA (which stabilizes the Cascade complex and guides Cascade and Cas3 to the DNA target). Cascade forms a complex with crRNA, and the protein-RNA pair recognizes its genomic target through complementary base pairing between the 5' end of the crRNA sequence and a predetermined protospacer. This complex is directed to a homologous locus in pathogen DNA via a region encoded within the crRNA and a protospacer adjacent motif (PAM) in the pathogen genome. Base pairing occurs between the crRNA and the target DNA sequence, resulting in a conformational change. In the IE system, the PAM is recognized by the CasA protein in the Cascade, which then unwinds the flanking DNA to assess the degree of base pairing between the target and the spacer portion of the crRNA. Upon sufficient recognition, the Cascade recruits and activates Cas3, which then nicks the non-target strand and begins degrading the strand in a 3' to 5' direction.

[0071] In the IC system, the proteins Cas5, Cas8c, and Cas7 form a cascade effector complex. Cas5 processes the pre-crRNA (which can take the form of a multi-spacer array or a single spacer between two repeats) to produce individual crRNAs composed of a hairpin structure formed from the remaining repeat sequence and a linear spacer. The effector complex then binds to the processed crRNA and scans the DNA to identify a PAM site. In the IC system, the PAM is recognized by the Cas8c protein, which then acts to unwind the DNA duplex. If the sequence 3' of the PAM matches the crRNA spacer that binds to the effector complex, a conformational change occurs in the complex, and Cas3 is recruited to the site. Cas3 then nicks the non-target strand and begins degrading the DNA.

[0072] In the type IB system, the proteins Cas8b1, Cas7, and Cas5 form a cascade effector complex. Cas5 processes the pre-crRNA (which can be in the form of a multi-spacer array or a single spacer between two repeats) to produce individual crRNAs composed of a hairpin structure formed from the remaining repeat sequence and a linear spacer. The effector complex then binds to the processed crRNA and scans the DNA to identify a PAM site. In the type IB system, the PAM is recognized by the Cas8b1 protein, which then acts to unwind the DNA duplex. If the sequence 3' of the PAM matches the crRNA spacer that binds to the effector complex, a conformational change occurs in the complex, and Cas3 is recruited to the site. Cas3 then nicks the non-target strand and begins DNA degradation. In some embodiments, the type IB system is derived from Listeria monocytogenes (LMIB) (e.g., SEQ ID NO: 22). In some embodiments, the Type IB system is modified from Listeria monocytogenes (LMIB) (e.g., SEQ ID NO: 23). In some embodiments, the Type IB system comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 25-29.

[0073] In some embodiments, the CRISPR-Cas system is endogenous to the target bacterium. In some embodiments, when the CRISPR-Cas system is endogenous to the target bacterium, the target bacterium comprises at least one gene encoding a Cas polypeptide. In some embodiments, when the CRISPR-Cas system is endogenous to the target bacterium, the target bacterium comprises a nucleic acid encoding a Cas3 polypeptide. In some embodiments, when the CRISPR-Cas system is endogenous to the target bacterium, the target bacterium comprises a nucleic acid encoding a Cascade complex. In some embodiments, when the CRISPR-Cas system is endogenous to the target bacterium, the target bacterium comprises a nucleic acid sequence encoding a Cas3 polypeptide and a Cascade complex.

[0074] In some embodiments, the CRISPR-Cas system is exogenous to the target bacterium. In some embodiments, when the CRISPR-Cas system is exogenous to the target bacterium, the bacteriophage comprises at least one gene encoding a Cas polypeptide. In some embodiments, when the CRISPR-Cas system is exogenous to the target bacterium, the bacteriophage comprises a nucleic acid encoding a Cas3 polypeptide. In some embodiments, when the CRISPR-Cas system is exogenous to the target bacterium, the bacteriophage comprises a nucleic acid encoding a Cascade complex. In some embodiments, when the CRISPR-Cas system is exogenous to the target bacterium, the bacteriophage comprises a nucleic acid sequence encoding a Cas3 polypeptide and a Cascade complex. In some embodiments, when the CRISPR-Cas system is exogenous to the target bacterium, the target bacterium does not comprise a nucleic acid encoding a Cas3 polypeptide. In some embodiments, when the CRISPR-Cas system is exogenous to the target bacterium, the target bacterium does not express a Cas3 polypeptide. In some embodiments, when the CRISPR-Cas system is exogenous to the target bacterium, the target bacterium does not contain a nucleic acid encoding a Cascade complex. In some embodiments, when the CRISPR-Cas system is exogenous to the target bacterium, the target bacterium does not express a Cascade complex. In some embodiments, when the CRISPR-Cas system is exogenous to the target bacterium, the target bacterium does not contain a nucleic acid sequence encoding a Cas3 polypeptide and a Cascade complex. In some embodiments, when the CRISPR-Cas system is exogenous to the target bacterium, the target bacterium does not express a Cas3 polypeptide and a Cascade complex.

[0075] In some embodiments, the CRISPR-Cas system is a type I CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is a type IA CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is a type IB CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is a type IB CRISPR-Cas system derived from Listeria monocytogenes. In some embodiments, the CRISPR-Cas system is a type IC CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is a type ID CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is a type IE CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is an IF CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is a type IU CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is a type II CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is a type III CRISPR-Cas system.

[0076] In some embodiments, processing of the CRISPR array disclosed herein includes, but is not limited to, the following processes: 1) transcription of nucleic acid encoding pre-crRNA, 2) recognition of pre-crRNA by the cascade and / or a specific member of the cascade, such as Cas6, and (3) processing of pre-crRNA into mature crRNA by the cascade or a member of the cascade, such as Cas6. In some embodiments, the mode of action of Type I CRISPR systems includes, but is not limited to, the following processes: 4) mature crRNA complex formation with the cascade, 5) target recognition by the complexed mature crRNA / cascade complex, and 6) nuclease activity at the target resulting in DNA degradation.

[0077] In some embodiments, the type I CRISPR-Cas system is a type IA system, a type IB system, a type IC system, a type ID system, a type IE system, a type IF system, or a type IU system. In some embodiments, the type I CRISPR-Cas system is a type IA system. In some embodiments, the type I CRISPR-Cas system is a type IB system. In some embodiments, the type I CRISPR-Cas system is a type IC system. In some embodiments, the type I CRISPR-Cas system is a type ID system. In some embodiments, the type I CRISPR-Cas system is an IE system. In some embodiments, the type I CRISPR-Cas system is an IF system. In some embodiments, the type I CRISPR-Cas system is an IU system. In some embodiments, the type I CRISPR-Cas system comprises a cascade polypeptide. The type I cascade polypeptide processes the CRISPR array to produce processed RNA, which is then used to bind the complex to a target sequence complementary to the spacer in the processed RNA. In some embodiments, the type I cascade complex is a type IA cascade polypeptide, a type IB cascade polypeptide, a type IC cascade polypeptide, a type ID cascade polypeptide, a type IE cascade polypeptide, a type IF cascade polypeptide, or a type IU cascade polypeptide.

[0078] In some embodiments, a type I Cascade complex comprises (a) a nucleotide sequence encoding a Cas6b polypeptide, a nucleotide sequence encoding a Cas8b (Csh1) polypeptide, a nucleotide sequence encoding a Cas7 (Csh2) polypeptide, and a nucleotide sequence encoding a Cas5 polypeptide (type IB); (b) a nucleotide sequence encoding a Cas5d polypeptide, a nucleotide sequence encoding a Cas8c (Csd1) polypeptide, and a nucleotide sequence encoding a Cas7 (Csd2) polypeptide (type IC); (c) a nucleotide sequence encoding a Cse1 (CasA) polypeptide, a nucleotide sequence encoding a Cse2 (CasB) polypeptide, a nucleotide sequence encoding a Cas7 (CasC) polypeptide, a nucleotide sequence encoding a Cas5 (CasD) polypeptide, and a Cas6e (CasE) polypeptide. (d) a nucleotide sequence encoding a Cys1 polypeptide, a nucleotide sequence encoding a Cys2 polypeptide, a Cas7 (Cys3) polypeptide, and a nucleotide sequence encoding a Cas6f polypeptide (type IF); (e) a nucleotide sequence encoding a Cas7 (Csa2) polypeptide, a Cas8a1 (Csx13) polypeptide or a Cas8a2 (Csx9) polypeptide, a nucleotide sequence encoding a Cas5 polypeptide, a nucleotide sequence encoding a Csa5 polypeptide, a nucleotide sequence encoding a Cas6a polypeptide, a Cas3' polypeptide, and a nucleotide sequence encoding a Cas3' polypeptide without nuclease activity (type IA); (f) a nucleotide sequence encoding a Casl Od (Csc3) polypeptide, a nucleotide sequence encoding a Csc2 polypeptide, a Csc1 polypeptide, and a nucleotide sequence encoding a Cas6d polypeptide (type ID); and / or (g) a Cas8u2 polypeptide, a Cas7 polypeptide, and a fusion Cas5-Cas6 polypeptide (type IU).In some embodiments, the type I cascade complex comprises a cascade polypeptide disclosed herein.

[0079] In some embodiments, the type I CRISPR-Cas system comprises a cascade polypeptide. The type I cascade polypeptide processes the CRISPR array to produce processed RNA, which is then used to bind the complex to a target sequence complementary to the spacer in the processed RNA. In some embodiments, the type I cascade complex is a type IA cascade polypeptide, a type IB cascade polypeptide, a type IC cascade polypeptide, a type ID cascade polypeptide, a type IE cascade polypeptide, a type IF cascade polypeptide, or a type IU cascade polypeptide. In some embodiments, the CRISPR-Cas system is a type IB CRISPR-Cas system (LMIB) derived from Listeria monocytogenes.

[0080] In some embodiments, the CRISPR-Cas system is encoded by a sequence comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:22. In some examples, the CRISPR-Cas system is encoded by a sequence that includes at least a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more than 50 nucleotides of SEQ ID NO:22. In some examples, the CRISPR-Cas system is encoded by a sequence that includes at least a portion having at least or about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, or more than 215 nucleotides of SEQ ID NO:22. In some embodiments, the CRISPR-Cas system is encoded by a sequence comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:23. In some examples, the CRISPR-Cas system is encoded by a sequence that includes at least a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more than 50 nucleotides of SEQ ID NO:23.In some examples, the CRISPR-Cas system is encoded by a sequence that includes at least a portion having at least or about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, or more than 215 nucleotides of SEQ ID NO:23.

[0081] In some embodiments, the CRISPR-Cas system comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:25 (e.g., Cas6). In some embodiments, the CRISPR-Cas system comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:29 (e.g., Cas8). In some embodiments, the CRISPR-Cas system comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 26 (e.g., Cas7). In some embodiments, the CRISPR-Cas system comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 27 (e.g., Cas5). In some embodiments, the CRISPR-Cas system comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:28 (e.g., Cas3). In some examples, the CRISPR-Cas system comprises at least or about 95% homology to any one of SEQ ID NOs:25-29. In some examples, the CRISPR-Cas system comprises at least or about 97% homology to any one of SEQ ID NOs:25-29. In some examples, the CRISPR-Cas system comprises at least or about 99% homology to any one of SEQ ID NOs:25-29. In some examples, the CRISPR-Cas system comprises 100% homology to any one of SEQ ID NOs:25-29.In some examples, the CRISPR-Cas system includes at least a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more than 50 amino acids of any one of SEQ ID NOs:25-29. In some examples, the CRISPR-Cas system includes at least a portion having at least or about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, or more than 215 amino acids of any one of SEQ ID NOs:25-29.

[0082] In some embodiments, the CRISPR-Cas system comprises a Cas6 polypeptide encoded by a sequence having at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 33. In some embodiments, the CRISPR-Cas system comprises a Cas8 polypeptide encoded by a sequence having at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 34. In some embodiments, the CRISPR-Cas system comprises a Cas7 polypeptide encoded by a sequence having at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 35. In some embodiments, the CRISPR-Cas system comprises a Cas5 polypeptide encoded by a sequence having at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 36. In some embodiments, the CRISPR-Cas system comprises a Cas3 polypeptide encoded by a sequence having at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:37.

[0083] In some embodiments, the CRISPR-Cas system comprises a Cas6 polypeptide encoded by a sequence having at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 38. In some embodiments, the CRISPR-Cas system comprises a Cas8 polypeptide encoded by a sequence having at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 39. In some embodiments, the CRISPR-Cas system comprises a Cas7 polypeptide encoded by a sequence having at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 40. In some embodiments, the CRISPR-Cas system comprises a Cas5 polypeptide encoded by a sequence having at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 41. In some embodiments, the CRISPR-Cas system comprises a Cas3 polypeptide encoded by a sequence having at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:42.

[0084] CRISPR arrays Some embodiments described herein provide a CRISPR array (crArray) comprising a spacer sequence and at least one repeat sequence. In some embodiments, the CRISPR array encodes a processed mature crRNA. In some embodiments, the mature crRNA is introduced into a phage or target bacterium described herein. In some embodiments, the phage comprises a nucleic acid encoding the processed mature crRNA. In some embodiments, endogenous or exogenous Cas6 processes the CRISPR array into mature crRNA. In some embodiments, exogenous Cas6 is introduced into a phage. In some embodiments, the phage comprises exogenous Cas6. In some embodiments, exogenous Cas6 is introduced into a target bacterium.

[0085] In some embodiments, processing of the CRISPR array disclosed herein includes, but is not limited to, the following processes: 1) transcription of nucleic acid encoding pre-crRNA, 2) recognition of pre-crRNA by the cascade and / or a specific member of the cascade, such as Cas6, and (3) processing of pre-crRNA into mature crRNA by the cascade or a member of the cascade, such as Cas6. In some embodiments, the mode of action of Type I CRISPR systems includes, but is not limited to, the following processes: 4) mature crRNA complex formation with the cascade, 5) target recognition by the complexed mature crRNA / cascade complex, and 6) nuclease activity at the target resulting in DNA degradation.

[0086] In some embodiments, the CRISPR array comprises a spacer sequence. In some embodiments, the CRISPR array further comprises at least one repeat sequence. In some embodiments, the at least one repeat sequence is operably linked to the spacer sequence at either its 5'-end or 3'-end. In some embodiments, the CRISPR array is of any length and comprises any number of spacer nucleotide sequences alternating with the repeat nucleotide sequences necessary to target one or more target sequences to achieve a desired level of killing of target bacteria. In some embodiments, the CRISPR array comprises, consists essentially of, or consists of 1 to about 100 spacer nucleotide sequences, each linked to the repeat nucleotide sequence at the 5'-end and 3'-end, respectively. In some embodiments, the CRISPR arrays disclosed herein comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, 22 In some embodiments, the CRISPR array comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:24. In some embodiments, the CRISPR array comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to amino acids 41-267 of SEQ ID NO:24.In some embodiments, the repeat sequence comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 43. In some embodiments, the spacer sequence comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 44-46. In some embodiments, the spacer sequence comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 44. In some embodiments, the spacer sequence comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 45. In some embodiments, the spacer sequence comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 46.

[0087] Spacer sequence In some embodiments, the spacer sequence is complementary to a target nucleotide sequence in the target bacterium. In some embodiments, the target nucleotide sequence is a coding region. In some embodiments, the coding region is an essential gene. In some embodiments, the coding region is a non-essential gene. In some embodiments, the target nucleotide sequence is a non-coding sequence. In some embodiments, the non-coding sequence is an intergenic sequence. In some embodiments, the spacer sequence is complementary to a target nucleotide sequence of a highly conserved sequence in the target bacterium. In some embodiments, the spacer sequence is complementary to a target nucleotide sequence of a sequence present in the target bacterium. In some embodiments, the spacer sequence is complementary to a target nucleotide sequence comprising all or a portion of a promoter sequence of an essential gene. In some embodiments, the spacer sequence comprises 1, 2, 3, 4, or 5 mismatches compared to the target nucleotide sequence. In some embodiments, the mismatches are contiguous. In some embodiments, the mismatches are non-contiguous. In some embodiments, the spacer sequence has 70% complementarity to the target nucleotide sequence. In some embodiments, the spacer sequence has 80% complementarity to the target nucleotide sequence. In some embodiments, the spacer sequence is 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to the target nucleotide sequence. In some embodiments, the spacer sequence is 100% complementary to the target nucleotide sequence. In some embodiments, the spacer sequence is fully complementary or substantially complementary over a region of the target nucleotide sequence that is at least about 8 nucleotides to about 150 nucleotides in length. In some embodiments, the spacer sequence is fully complementary or substantially complementary over a region of the target nucleotide sequence that is at least about 20 nucleotides to about 100 nucleotides in length. In some embodiments, the 5' region of the spacer sequence is 100% complementary to the target nucleotide sequence, while the 3' region of the spacer is substantially complementary to the target nucleotide sequence, such that the overall complementarity of the spacer sequence to the target nucleotide sequence is less than 100%.For example, in some embodiments, the first 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides in the 3' region (seed region) of a 20-nucleotide spacer sequence are 100% complementary to the target nucleotide sequence, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target nucleotide sequence. In some embodiments, the first 7-12 nucleotides at the 3' end of the spacer sequence are 100% complementary to the target nucleotide sequence, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary to the target nucleotide sequence (e.g., at least about 50% complementary (e.g., 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)). In some embodiments, the first 7-10 nucleotides at the 3' end of the spacer sequence are 75% to 99% complementary to the target nucleotide sequence, while the remaining nucleotides in the 5' region of the spacer sequence are at least about 50% to about 99% complementary to the target nucleotide sequence. In some embodiments, the first 7-10 nucleotides at the 3' end of the spacer sequence are 100% complementary to the target nucleotide sequence, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target nucleotide sequence. In some embodiments, the first 10 nucleotides (within the seed region) of the spacer sequence are 100% complementary to the target nucleotide sequence, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target nucleotide sequence.In some embodiments, the 5' region of the spacer sequence (e.g., the first 8 nucleotides at the 5' end, the first 10 nucleotides at the 5' end, the first 15 nucleotides at the 5' end, the first 20 nucleotides at the 5' end) has about 75% or more complementarity (75% to about 100% complementarity) to the target nucleotide sequence, while the remainder of the spacer sequence has about 50% or more complementarity to the target nucleotide sequence. In some embodiments, the first 8 nucleotides at the 5' end of the spacer sequence have 100% complementarity to the target nucleotide sequence or have one or two mutations such that they are about 88% or about 75% complementary to the target nucleotide sequence, respectively, while the remainder of the spacer nucleotide sequence is at least about 50% or more complementary to the target nucleotide sequence.

[0088] In some embodiments, the spacer nucleotide sequence is about 15 to about 150 nucleotides in length. In some embodiments, the spacer nucleotide sequence is about 15 to about 100 nucleotides in length (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 nucleotides or more in length). In some embodiments, the spacer nucleotide sequence is about 8 to about 150 nucleotides in length, about 8 to about 100 nucleotides in length, about 8 to about 50 nucleotides in length, about 8 to about 40 nucleotides in length, about 8 to about 30 nucleotides in length, about 8 to about 25 nucleotides in length, about 8 to about 20 nucleotides in length, about 10 to about 150 nucleotides in length, about 10 to about 100 nucleotides in length, about 10 to about 80 nucleotides in length, about 10 to about 50 nucleotides in length, about 10 to about 40, about 10 to about 30, about 10 to about 25, about 10 to about 20, about 15 to about 150, about 15 to about 100, about 15 to about 50, about 15 to about 40, about 15 to about 30, about 20 to about 150 nucleotides in length, about 20 to about 100 nucleotides in length. The nucleotide length may be about 20 to about 80 nucleotides, about 20 to about 50 nucleotides, about 20 to about 40, about 20 to about 30, about 20 to about 25, at least about 8, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 32, at least about 35, at least about 40, at least about 44, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150 or more nucleotides in length, and any value or range therein.In some embodiments, the Listeria monocytogenes type IB Cas system has a spacer length of about 30 to 39 nucleotides, about 31 to about 38 nucleotides, about 32 to about 37 nucleotides, about 36 to about 37 nucleotides, or about 37 nucleotides. In some embodiments, the Listeria monocytogenes type IB system has a spacer length of about 37 nucleotides. In some embodiments, the Listeria monocytogenes type IB Cas system has a spacer length of at least about 10, at least about 15, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 29, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, at least about, at least about 35, at least about 36, at least about 37, at least about 38, at least about 39, at least about 20, at least about 41, at least about 42, at least about 43, at least about 44, at least about 45 nucleotides, or more than about 45 nucleotides.

[0089] In some embodiments, the identities of two or more spacer sequences in a CRISPR array are the same. In some embodiments, the identities of two or more spacer sequences in a CRISPR array are different. In some embodiments, the identities of two or more spacer sequences in a CRISPR array are different but are complementary to one or more target nucleotide sequences. In some embodiments, the identities of two or more spacer sequences in a CRISPR array are different but are complementary to one or more target nucleotide sequences that are overlapping sequences. In some embodiments, the identities of two or more spacer sequences in a CRISPR array are different but are complementary to one or more target nucleotide sequences that are not overlapping sequences. In some embodiments, the target nucleotide sequence is about 10 to about 40 contiguous nucleotides in length located immediately adjacent to a PAM sequence (a PAM sequence located immediately 3' of the target region) in the genome of the organism. In some embodiments, the target nucleotide sequence is located next to or adjacent to a PAM (protospacer adjacent motif).

[0090] The PAM sequence is found in the target gene adjacent to the region to which the spacer sequence binds as a result of its complementarity to the region, identifying the point at which base pairing with the spacer nucleotide sequence begins. The exact PAM sequence required varies for different CRISPR-Cas systems and is identified through established bioinformatics and experimental procedures. Non-limiting examples of PAMs include CCA, CCT, CCG, TTC, AAG, AGG, ATG, GAG, and / or CC. In Type I systems, the PAM is located immediately 5' of the sequence that matches the spacer and therefore 3' of the sequence that base pairs with the spacer nucleotide sequence, allowing it to be directly recognized by the Cascade. Upon recognition of the protospacer, the Cascade typically recruits the endonuclease Cas3, which cleaves and degrades the target DNA. In Type II systems, the PAM is required for the Cas9 / sgRNA to form an R-loop to interrogate a specific DNA sequence through Watson-Crick pairing of its guide RNA with the genome. PAM specificity is a function of the DNA binding specificity of the Cas9 protein (e.g., the protospacer adjacent motif recognition domain at the C-terminus of Cas9).

[0091] In some embodiments, the target nucleotide sequence in the bacteria to be killed is any essential target nucleotide sequence of interest. In some embodiments, the target nucleotide sequence is a non-essential sequence. In some embodiments, the target nucleotide sequence comprises, consists essentially of, or consists of all or a portion of a nucleotide sequence encoding a promoter of an essential gene or its complement. In some embodiments, the spacer nucleotide sequence is complementary to a promoter of an essential gene or a portion thereof. In some embodiments, the target nucleotide sequence comprises all or a portion of a nucleotide sequence located on the coding or non-coding strand of an essential gene. In some embodiments, the target nucleotide sequence comprises all or a portion of a nucleotide sequence located on the coding of a transcribed region of an essential gene.

[0092] In some embodiments, an essential gene is any gene important for the survival of an organism. However, essentiality largely depends on the conditions in which the organism lives. For example, a gene required for starch digestion is only essential when starch is the only energy source. In some embodiments, the target nucleotide sequence comprises all or part of the promoter sequence of the target gene. In some embodiments, the target nucleotide sequence comprises all or part of a nucleotide sequence located on the coding strand of the transcribed region of the target gene. In some embodiments, the target nucleotide sequence comprises at least a portion of an essential gene required for the survival of the target bacterium. In some embodiments, an essential gene is Tsf, acpP, gapA, infA, secY, csrA, trmD, ftsA, fusA, glyQ, eno, nusG, dnaA, pheS, rplB, gltX, hisS, rplC, aspS, gyrB, dnaE, rpoA, rpoB, pheT, infB, rpsC, rplF, alaS, leuS, serS, rplD, gyrA, glmS, fus, adk, rpsK, rplR, ctrA, parC, tRNA-Ser, tRNA-Asn, or metK. In some embodiments, a non-essential gene is any gene that is not important for the survival of an organism. However, non-essentiality will largely depend on the context in which the organism lives.

[0093] In some embodiments, non-limiting examples of target nucleotide sequences of interest include target nucleotide sequences encoding transcriptional regulators, translational regulators, polymerase genes, metabolic enzymes, transporters, RNases, proteases, DNA replication enzymes, DNA modification or degradation enzymes, regulatory RNAs, transfer RNAs, or ribosomal RNAs. In some embodiments, the target nucleotide sequences are derived from genes involved in cell division, cell structure, metabolism, motility, pathogenicity, virulence, or antibiotic resistance. In some embodiments, the target nucleotide sequences are derived from hypothetical genes whose functions have not yet been characterized. Thus, for example, these genes can be any genes from any bacteria.

[0094] Suitable spacer sequences for the complete construct phage can be identified by locating a search set of representative genomes, searching the genomes with relevant parameters, and determining the quality of the spacers used in the CRISPR-engineered phage.

[0095] First, a suitable search set of representative genomes is identified and acquired for the organism / species / target of interest. The set of representative genomes can be found in various databases, including but not limited to the NCBI GenBank or PATRIC database. NCBI GenBank is one of the largest databases available and contains a mixture of reference and submitted genomes for nearly every organism sequenced to date. Specifically, for pathogenic prokaryotes, the PATRIC (Pathosystems Resource Integration Center) database provides an additional comprehensive resource of genomes, focusing on clinically relevant strains and genomes relevant to pharmaceuticals. Both of the above databases allow bulk download of genomes via FTP (File Transfer Protocol) servers, allowing for rapid and programmatic acquisition of datasets.

[0096] The genome is then searched for relevant parameters to identify the location of appropriate spacer sequences. The genome can be read from start to finish in both forward and reverse complementary directions, identifying the location of contiguous stretches of DNA containing PAM (protospacer adjacent motif) sites. The spacer sequence is an N-length DNA sequence adjacent to the 3' or 5' of the PAM site (depending on the type of CRISPR system), where N is specific to the Cas system of interest and is known in advance. Characterization of PAM and spacer sequences may be performed during Cas system discovery and initial research. Any observed PAM-adjacent spacers may be saved to a file and / or database for downstream use. The exact PAM sequence required varies for different CRISPR-Cas systems and is identified through established bioinformatics and experimental procedures.

[0097] Next, the quality of the spacers used in the CRISPR-engineered phage is determined. Each observed spacer can be evaluated to determine how many times it occurs in each evaluated genome. Observed spacers may be evaluated to determine how many times they are likely to occur in each given genome. Spacers occurring at more than one location per genome may be advantageous because, if a mutation occurs, the Cas system may not be able to recognize the target site, and each additional "backup" site increases the likelihood that a suitable non-mutated target location exists. Observed spacers may also be evaluated to determine whether they occur in functionally annotated regions of the genome. If such information is available, the functional annotation may be further evaluated to determine whether those genomic regions are "essential" for the organism's survival and function. By focusing on spacers that occur in all or nearly all evaluated genomes of interest (up to 99%), spacer selection can be broadly applicable to many targeted genomes. Given the existence of a large selection pool of conserved spacers, preference is given to spacers that occur in regions of the genome with known functions; higher priority may be given if these genomic regions are "essential" for survival and occur more than once per genome.

[0098] In some embodiments, the spacer comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 44-46. In some embodiments, the spacer sequence comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 44. In some embodiments, the spacer sequence comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 45. In some embodiments, the spacer sequence comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:46.

[0099] Repetitive nucleotide sequences In some embodiments, the repetitive nucleotide sequences of the CRISPR array comprise the nucleotide sequences of any known repetitive nucleotide sequences of a CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is a type I CRISPR-Cas system. In some embodiments, the repetitive nucleotide sequences are synthetic sequences that include the secondary structure (e.g., internal hairpin) of a naturally occurring repeat from a type I CRISPR-Cas system. In some embodiments, the repetitive nucleotide sequences are different from each other based on known repetitive nucleotide sequences of a CRISPR-Cas system. In some embodiments, the repetitive nucleotide sequences are each composed of a different secondary structure (e.g., internal hairpin) of a naturally occurring repeat from a CRISPR-Cas system. In some embodiments, the repetitive nucleotide sequences are a combination of an operable distinct repetitive nucleotide sequence and a CRISPR-Cas system.

[0100] In some embodiments, the spacer sequence is linked at its 5' end to the 3' end of the repeat sequence. In some embodiments, the spacer sequence is linked at its 5' end to about 1 to about 8, about 1 to about 10, or about 1 to about 15 nucleotides of the 3' end of the repeat sequence. In some embodiments, about 1 to about 8, about 1 to about 10, or about 1 to about 15 nucleotides of the repeat sequence are part of the 3' end of the repeat sequence. In some embodiments, the spacer nucleotide sequence is linked at its 3' end to the 5' end of the repeat sequence. In some embodiments, the spacer is linked at its 3' end to about 1 to about 8, about 1 to about 10, or about 1 to about 15 nucleotides of the 5' end of the repeat sequence. In some embodiments, about 1 to about 8, about 1 to about 10, or about 1 to about 15 nucleotides of the repeat sequence are part of the 5' end of the repeat sequence.

[0101] In some embodiments, the spacer nucleotide sequence is linked at its 5' end to a first repeat sequence and at its 3' end to a second repeat sequence to form a repeat-spacer-repeat sequence. In some embodiments, the spacer sequence is linked at its 5' end to the 3' end of the first repeat sequence and at its 3' end to the 5' end of the second repeat sequence, where the spacer sequence and second repeat sequence are repeated to form a repeat-(spacer-repeat)n sequence, where n is any integer between 1 and 100. In some embodiments, the repeat-(spacer-repeat) n sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 , 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more spacer nucleotide sequences.

[0102] In some embodiments, repeat sequence is identical or substantially identical to the repeat sequence from wild-type CRISPR locus.In some embodiments, repeat sequence is the sequence described herein.In some embodiments, repeat sequence comprises part of wild-type repeat sequence (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more consecutive nucleotides of wild-type repeat sequence). In some embodiments, the repeat sequence comprises, consists essentially of, or consists of at least one nucleotide (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more nucleotides, or any range therein). In some embodiments, the repeat sequence comprises, consists essentially of, or consists of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 or less nucleotides. In some embodiments, the repeat sequence comprises about 20-40, 21-40, 22-40, 23-40, 24-40, 25-40, 26-40, 27-40, 28-40, 29-40, 30-30, 31-40, 32-40, 33-40, 34-40, 35-40, 36-40, 37-40, 38-40, 39-40, 20-39, 20-38, 20-37, 20-36, 20-35, 20-34, 20-33, 20-32, 20-31, 20-30, 20-29, 20-28, 20-26, 20-25, 20-24, 20-23, 20-22, or 20-21 nucleotides.In some embodiments, the repeat sequence comprises about 20-35, 21-35, 22-35, 23-35, 24-35, 25-35, 26-35, 27-35, 28-35, 29-35, 30-30, 31-35, 32-35, 33-35, 34-35, 25-40, 25-39, 25-38, 25-37, 25-36, 25-35, 25-34, 25-33, 25-32, 25-31, 25-30, 25-29, 25-28, or 25-26 nucleotides. In some embodiments, the system is a Listeria monocytogenes (L. monocytogenes) Type IB Cas system. In some embodiments, the Listeria monocytogenes Type IB Cas system has a repeat length of about 25-38 nucleotides.

[0103] In some embodiments, the repeats comprise at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:43.

[0104] transcription activator In some embodiments, the nucleic acid sequence further comprises a transcriptional activator. In some embodiments, the encoded transcriptional activator regulates expression of a gene of interest in Staphylococcus spp. In some embodiments, the transcriptional activator activates expression of a gene of interest in Staphylococcus spp., whether exogenous or endogenous. In some embodiments, the transcriptional activator activates an expressed gene of interest in Staphylococcus spp. by disrupting the activity of one or more inhibitory elements in the Staphylococcus spp. In some embodiments, the inhibitory element comprises a transcriptional repressor. In some embodiments, the inhibitory element comprises a global transcriptional repressor. In some embodiments, the inhibitory element is a histone-like nucleoid structuring (H-NS) protein or a homolog or functional fragment thereof. In some embodiments, the inhibitory element is a leucine-responsive regulatory protein (LRP). In some embodiments, the inhibitory element is a CodY protein.

[0105] In some bacteria, the CRISPR-Cas system is poorly expressed and considered silent under most environmental conditions. In these bacteria, regulation of the CRISPR-Cas system is the result of the activity of transcriptional regulators, such as histone-like nucleoid structuring (H-NS) proteins, which are widely involved in regulating host genome transcription. H-NS exerts control over host transcriptional regulation by multimerizing along AT-rich sites, resulting in DNA bending.

[0106] Similarly, in some bacteria, repression of the CRISPR-Cas system is controlled by inhibitory elements, such as the leucine-responsive regulatory protein (LRP). LRP is involved in binding to regions upstream and downstream of the transcription start site. Notably, the activity of LRP in regulating the expression of the CRISPR-Cas system varies among bacteria. Unlike H-NS, which has broad interspecies repression activity, LRP has been shown to differentially regulate the expression of host CRISPR-Cas systems. Thus, in some instances, LRP reflects a host-specific means of regulating CRISPR-Cas system expression in different bacteria.

[0107] In some cases, repression of CRISPR-Cas systems is also controlled by the inhibitory element CodY. CodY is a GTP-sensing transcriptional repressor that acts through DNA binding. The intracellular concentration of GTP acts as an indicator of environmental nutrient status. Under normal culture conditions, GTP is abundant and binds to CodY, repressing transcriptional activity. However, as GTP concentrations decrease, CodY becomes less active in binding to DNA, thereby allowing transcription of previously repressed genes to occur. Thus, CodY acts as a strict global transcriptional repressor.

[0108] In some embodiments, the transcriptional activator is a LeuO polypeptide, any homolog or functional fragment thereof, a leuO coding sequence, or an agent that upregulates LeuO. In some embodiments, the transcriptional activator includes any ortholog or functional equivalent of LeuO. In some bacteria, LeuO counteracts H-NS by acting as a global transcriptional regulator that responds to the environmental nutritional status of the bacteria. Under normal conditions, LeuO expression is insufficient. However, upon amino acid starvation and / or reaching stationary phase in the bacterial life cycle, LeuO is upregulated. Increased LeuO expression antagonizes H-NS in overlapping promoter regions, resulting in gene expression. Overexpression of LeuO upregulates the expression of CRISPR-Cas systems.

[0109] In some embodiments, expression of LeuO results in the destruction of inhibitory elements. In some embodiments, destruction of inhibitory elements by expression of LeuO eliminates transcriptional repression of the CRISPR-Cas system. In some embodiments, expression of LeuO eliminates transcriptional repression of the CRISPR-Cas system by the activity of H-NS. In some embodiments, destruction of inhibitory elements by expression of LeuO causes increased expression of the CRISPR-Cas system. In some embodiments, increased expression of the CRISPR-Cas system due to destruction of inhibitory elements caused by expression of LeuO causes increased CRISPR-Cas processing of nucleic acid sequences comprising a CRISPR array. In some embodiments, increased expression of the CRISPR-Cas system due to destruction of inhibitory elements by expression of LeuO causes increased CRISPR-Cas processing of nucleic acid sequences comprising a CRISPR array, such that the level of lethality of the CRISPR array against bacteria is increased. In some embodiments, the transcriptional activator causes increased activity of a bacteriophage and / or a CRISPR-Cas system.

[0110] Adjustment element In some embodiments, the nucleic acid sequence is operably associated with various promoters, terminators, and other regulatory elements for expression in various organisms or cells. In some embodiments, the nucleic acid sequence further comprises a leader sequence. In some embodiments, the nucleic acid sequence further comprises a promoter sequence. In some embodiments, at least one promoter and / or terminator is operably linked to a CRISPR array. Any promoter useful in the present disclosure may be used, including, for example, promoters functional in the organism of interest, as well as constitutive, inducible, developmentally regulated, tissue-specific / preferred promoters disclosed herein. Regulatory elements used herein may be endogenous or heterologous. In some embodiments, an endogenous regulatory element from a target organism is inserted into a genetic background in which it does not naturally occur (e.g., a location in the genome different from that found in nature), thereby producing a recombinant or non-naturally occurring nucleic acid.

[0111] In some embodiments, expression of the nucleic acid sequence is constitutive, inducible, temporally regulated, developmentally regulated, or chemically regulated. In some embodiments, expression of the nucleic acid sequence is made constitutive, inducible, temporally regulated, developmentally regulated, or chemically regulated by operably linking the nucleic acid sequence to a promoter that is functional in the organism of interest. In some embodiments, repression is made reversible by operably linking the nucleic acid sequence to an inducible promoter that is functional in the organism of interest. The choice of promoter disclosed herein will vary depending on the amount, temporal, and spatial requirements for expression, as well as the host cell to be transformed.

[0112] Exemplary promoters for use with the methods, bacteriophages, and compositions disclosed herein include promoters that are functional in bacteria, such as L-arabinose-inducible (araBAD, P BAD) promoter, any lac promoter, L-rhamnose inducible (rhaPBAD) promoter, T7 RNA polymerase promoter, trc promoter, tac promoter, λ phage promoter (p L p L -9 G-50), anhydrotetracycline-inducible (tetA) promoter, trp, Ipp, phoA, recA, proU, cst-1, cadA, nar, Ipp-lac, cspA, 11-lac operator, T3-lac operator, T4 gene 32, T5-lac operator, nprM-lac operator, Vhb, protein A, corynebacterium-E. coli-like promoter, thr, horn, diphtheria toxin promoter, sig A, sig B, nusG, SoxS, katb, α-amylase (Pamy), Ptms, P43 (composed of two overlapping RNA polymerase σ factor recognition sites, σA and σB), Ptms, P43, rplK-rplA, ferredoxin promoter, and / or xylose promoter. In some embodiments, the promoter is the BBa_J23102 promoter. In some embodiments, the promoter functions in a broad range of bacteria, such as BBa_J23104, BBa_J23109, etc. In some embodiments, the promoter is derived from the target bacterium, such as an endogenous CRISPR promoter, an endogenous Cas operon promoter, or a promoter from sarA, lipA, ptsH, or cap1 of Staphylococcus aureus.

[0113] In some embodiments, the promoter comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 16-19. In some examples, the promoter comprises at least a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more than 50 nucleotides of any one of SEQ ID NOs: 16-19. In some examples, the promoter comprises at least a portion having at least or about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, or more than 215 nucleotides of any one of SEQ ID NOs: 16-19.

[0114] In some embodiments, the nucleic acid comprises a ribosome binding site (RBS) downstream of a promoter, e.g., a promoter described herein. Optionally, the RBS is native to the gene with which it is operably associated. Optionally, the RBS is not native to the gene with which it is operably associated. Optionally, the RBS is native to the promoter with which it is operably associated. Optionally, the RBS is not native to the promoter with which it is operably associated. As a non-limiting example, the RBS of the sarA promoter is an RBS from sodB rather than sarA. For example, an RBS used with a promoter for translation of a protein herein comprises SEQ ID NO: 21. Optionally, the promoter is sarA.

[0115] Expression cassette In some embodiments, the nucleic acid sequence is or is within an expression cassette. In some embodiments, the expression cassette is designed to express a nucleic acid sequence disclosed herein. In some embodiments, the nucleic acid sequence is an expression cassette encoding components of a CRISPR-Cas system and / or a peptide. In some embodiments, the nucleic acid sequence is an expression cassette encoding components of a type I CRISPR-Cas system. In some embodiments, the nucleic acid sequence is an expression cassette encoding an operable CRISPR-Cas system. In some embodiments, the nucleic acid sequence is an expression cassette encoding operable components of a type I CRISPR-Cas system, including Cascade and Cas3. In some embodiments, the nucleic acid sequence is an expression cassette encoding operable components of a type I CRISPR-Cas system, including crRNA, Cascade, and Cas3. In some embodiments, the nucleic acid sequence is an expression cassette encoding a peptide (e.g., an antimicrobial peptide).

[0116] In some embodiments, the expression cassette containing the nucleic acid sequence of interest is chimeric, meaning that at least one of its components is heterologous to at least one of its other components, hi some embodiments, the expression cassette occurs naturally but has been obtained in a recombinant form useful for heterologous expression.

[0117] In some embodiments, the expression cassette comprises a transcriptional and / or translational termination region (i.e., a termination region) that is functional in the selected host cell. In some embodiments, the termination region is responsible for the termination of transcription beyond the heterologous nucleic acid sequence of interest and for accurate mRNA polyadenylation. In some embodiments, the termination region originates from the transcriptional initiation region, from the operably linked nucleic acid sequence of interest, from the host cell, or from another source (i.e., foreign or heterologous to the promoter, to the nucleic acid sequence of interest, to the host, or any combination thereof). In some embodiments, a terminator is operably linked to a nucleic acid sequence disclosed herein.

[0118] In some embodiments, the expression cassette comprises a nucleotide sequence for a selectable marker, hi some embodiments, the nucleotide sequence encodes either a selectable or a screenable marker, depending on whether the marker confers a trait that is selected for by chemical means, such as by the use of a selective agent (e.g., an antibiotic), or whether the marker is simply a trait that is identified by observation or testing, such as by screening (e.g., fluorescence).

[0119] vector In addition to expression cassettes, the nucleic acid sequences disclosed herein (e.g., nucleic acid sequences comprising CRISPR arrays, CRISPR-Cas, peptides, and antimicrobial agents) are used in conjunction with vectors. A vector comprises a nucleic acid molecule containing a nucleotide sequence to be transferred, delivered, or introduced. Non-limiting examples of general classes of vectors include viral vectors, plasmid vectors, phage vectors, phagemid vectors, cosmid vectors, fosmid vectors, bacteriophages, artificial chromosomes, or Agrobacterium binary vectors, which may or may not be self-transmissible or mobilizable, and are in double- or single-stranded, linear, or circular form. Vectors transform prokaryotic or eukaryotic hosts by integration into the cellular genome or exist extrachromosomally (e.g., autonomously replicating plasmids with an origin of replication). Additionally, shuttle vectors are included, meaning DNA vehicles capable of replicating in two different host organisms, either naturally or by design. In some embodiments, shuttle vectors replicate in actinomycetes as well as bacteria and / or eukaryotes. In some embodiments, the nucleic acid in the vector is under the control of, and operably linked to, a promoter or other regulatory elements suitable for transcription in a host cell, hi some embodiments, the vector is a bifunctional expression vector that functions in multiple hosts.

[0120] Array Optimization In some embodiments, the nucleic acid sequence encoding the payload (e.g., nucleic acid insert) is optimized for stable expression in the phage genome. In some embodiments, the insert is stable through at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 generations of passage. In some embodiments, the nucleic acid sequence is optimized by optimizing the insertion site, modifying the secondary structure, modifying the DNA modification site, modifying the restriction enzyme motif, codon optimization, GC% optimization, or a combination thereof. In some embodiments, the insertion site of the nucleic acid sequence is optimized. In some embodiments, the nucleic acid sequence is modified to remove secondary structure.

[0121] In certain embodiments, a bacteriophage comprises a nucleic acid insert modified from an exogenous nucleic acid described herein, wherein the nucleic acid comprises a first plurality of codons encoding a first protein, and the nucleic acid insert comprises a second plurality of codons encoding a second protein, wherein the first protein and the second protein have at least 90% amino acid sequence identity, and at least 50% of the second plurality of codons are frequent codons in the bacteriophage genome. Certain aspects herein describe a method for inserting an exogenous sequence comprising a plurality of codons encoding a first protein into a bacteriophage, the method comprising substituting one or more of the plurality of codons with codons derived from the bacteriophage to generate a nucleic acid insert encoding a second protein, wherein the first protein and the second protein have at least 90% amino acid sequence identity. In certain aspects herein, a nucleic acid insert is described that is modified from an exogenous nucleic acid described herein, wherein the nucleic acid comprises a first plurality of codons encoding a first protein, and the nucleic acid insert comprises a second plurality of codons encoding a second protein, wherein the first protein and the second protein have at least 90% amino acid sequence identity, and at least 50% of the second plurality of codons are frequently occurring codons in a bacteriophage genome. In some embodiments, the first protein and the second protein have at least 95%, 97.5%, 99%, or 99.5% sequence identity. In some embodiments, at least 50% of the second plurality of codons, but less than 60%, 70%, 80%, 90%, or more than 90%, are frequently occurring codons in a bacteriophage genome. In some embodiments, the second plurality of codons matches the codon profile in a bacteriophage genome.

[0122] In some embodiments, the nucleic acid sequence is modified to remove a DNA modification site. In some embodiments, the DNA modification site comprises a DNA methylation site.

[0123] In some embodiments, the nucleic acid sequence is modified to remove restriction enzyme motifs. In some embodiments, the nucleic acid sequence is modified to remove restriction enzyme motifs from restriction enzymes derived from the bacterial species described herein. In some embodiments, the nucleic acid insert does not contain or contains fewer than 10 sites recognized by bacterial enzymes.

[0124] In some embodiments, the nucleic acid sequence is codon-optimized for expression in any species of interest. Codon optimization involves modifying the nucleotide sequence for codon usage bias using a species-specific codon usage table. The codon usage table is generated based on sequence analysis of the most highly expressed genes in the species of interest. If the nucleotide sequence is to be expressed in the nucleus, the codon usage table is generated based on sequence analysis of highly expressed nuclear genes in the species of interest. The modification of the nucleotide sequence is determined by comparing the species-specific codon usage table with the codons present in the native polynucleotide sequence. Codon optimization of a nucleotide sequence results in a nucleotide sequence that has less than 100% identity (e.g., 50%, 60%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc.) to a native nucleotide sequence, but still encodes a polypeptide having the same function as that encoded by the original nucleotide sequence. In some embodiments, the nucleic acid sequences of the present disclosure are codon optimized for expression in an organism / species of interest.

[0125] In some embodiments, nucleic acid sequences are modified to optimize percent GC content. In some embodiments, the percent GC content is modified so that at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% of the nucleotides contain guanine or cytosine. In some embodiments, the percent GC content is modified so that no more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% of the nucleotides contain guanine or cytosine.

[0126] In some embodiments, the exogenous nucleic acid is a bacterial nucleic acid. In some embodiments, the nucleic acid insert and the bacterial nucleic acid have less than 100%, 95%, 90%, 80%, 70%, 60%, or 50% sequence identity. In some embodiments, the first bacterial protein is a CRISPR-Cas protein described herein. In some embodiments, the first bacterial protein is an antimicrobial agent and / or peptide described herein.

[0127] transformation In some embodiments, the nucleic acid sequences and / or expression cassettes disclosed herein are transiently expressed and / or stably integrated into the genome of a host organism. In some embodiments, the nucleic acid sequences and / or expression cassettes disclosed herein are introduced into cells by any method known to those skilled in the art. Exemplary methods of transformation include electroporation of competent cells, passive uptake by competent cells, chemical transformation of competent cells, and transformation by any other electrical, chemical, physical (mechanical), and / or biological mechanism (including any combination thereof) that introduces nucleic acids into cells. In some embodiments, transformation of cells includes nuclear transformation. In some embodiments, transformation of cells includes plasmid transformation and conjugation.

[0128] In some embodiments, when more than one nucleic acid sequence is introduced, the nucleotide sequences are constructed as part of a single nucleic acid construct or as separate nucleic acid constructs, and are located on the same or different nucleic acid constructs. In some embodiments, the nucleotide sequences are introduced into the cell of interest in a single transformation event or in separate transformation events.

[0129] How to use In certain embodiments, disclosed herein are methods of killing a target bacterium, the methods comprising contacting or introducing any of the bacteriophages disclosed herein into the target bacterium, hi some embodiments, the target bacterium is a Staphylococcus species.

[0130] Further, in certain embodiments herein, disclosed is a method of modifying a mixed population of bacterial cells having a first bacterial species that contains a target nucleotide sequence in an essential gene and a second bacterial species that does not contain the target nucleotide sequence in an essential gene, the method comprising introducing into the mixed population of bacterial cells any of the bacteriophages disclosed herein.

[0131] Also disclosed herein in certain embodiments is a method of treating a disease in an individual in need thereof, the method comprising administering to the individual any of the bacteriophages disclosed herein.

[0132] In some embodiments, the target bacteria are killed solely by the lytic activity of the bacteriophage. In some embodiments, the target bacteria is Staphylococcus. In some embodiments, a population of Staphylococcus bacteria is targeted by the bacteriophage. In some embodiments, a population of Staphylococcus bacteria is killed by the bacteriophage. In some embodiments, the bacteriophage cocktail described herein targets multiple Staphylococcus strains. In some embodiments, the bacteriophage cocktail targets at least 70%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more than 99% of Staphylococcus strains. In some embodiments, the bacteriophage cocktail targets at least 70%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more than 99% of Staphylococcus aureus strains. In some embodiments, the bacteriophage cocktail kills at least 70%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more than 99% of strains of Staphylococcus. In some embodiments, the bacteriophage cocktail kills at least 70%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more than 99% of strains of Staphylococcus aureus. In some embodiments, the bacteriophage cocktail specifically targets Staphylococcus species but not other bacterial species. In some embodiments, the bacteriophage cocktail targets Staphylococcus species but not non-Staphylococcus species. In some embodiments, the bacteriophage cocktail comprises two or more of Phyetavirus, Rosenblum virus, and Kaivirus.

[0133] In some embodiments, Staphylococcus bacteria are killed only by the lytic activity of bacteriophage.In some embodiments, Staphylococcus bacteria are killed only by the activity of CRISPR-Cas system.In some embodiments, Staphylococcus bacteria are killed by the processing of CRISPR array by CRISPR-Cas system to produce processed crRNA that can direct CRISPR-Cas-based endonuclease activity and / or cleavage at target nucleotide sequence in target gene of bacteria.In some embodiments, Staphylococcus bacteria are killed only by antimicrobial peptide.

[0134] In some embodiments, Staphylococcus bacteria are killed by the lytic activity of bacteriophage combined with the activity of type I CRISPR-Cas system.In some embodiments, Staphylococcus bacteria are killed by the activity of type I CRISPR-Cas system regardless of the lytic activity of bacteriophage.In some embodiments, the activity of type I CRISPR-Cas system complements or enhances the lytic activity of bacteriophage.In some embodiments, the activity of type I CRISPR-Cas system and the lytic activity of bacteriophage are additive.

[0135] In some embodiments, Staphylococcus bacteria are killed by the activity of the type I CRISPR-Cas system and the lytic activity of the bacteriophage combined with the antimicrobial peptide.In some embodiments, Staphylococcus bacteria are killed by the activity of the type I CRISPR-Cas system, regardless of the lytic activity of the bacteriophage and the antimicrobial peptide.In some embodiments, the activity of the type I CRISPR-Cas system complements or enhances the lytic activity of the bacteriophage and the antimicrobial peptide.In some embodiments, the activity of the type I CRISPR-Cas system, the lytic activity of the bacteriophage, and the activity of the antimicrobial peptide are additive.

[0136] In some embodiments, the lytic activity of bacteriophage and the activity of type I CRISPR-Cas system are synergistic.In some embodiments, synergistic activity is defined as the activity of killing phage at a level greater than the additive combination of the lytic activity of bacteriophage and type I CRISPR-Cas system.In some embodiments, the lytic activity of bacteriophage is regulated by the concentration of bacteriophage.In some embodiments, the activity of type I CRISPR-Cas system is regulated by the concentration of bacteriophage.

[0137] In some embodiments, synergistic killing of bacteria is adjusted to favor killing by the lytic activity of bacteriophage over the activity of CRISPR-Cas system by increasing the concentration of bacteriophage administered to the bacteria. In some embodiments, synergistic killing of bacteria is adjusted to favor killing by the lytic activity of bacteriophage over the activity of CRISPR-Cas system by decreasing the concentration of bacteriophage administered to the bacteria. In some embodiments, lytic replication at low concentrations allows amplification and killing of target bacteria. In some embodiments, phage amplification is not required at high concentrations. In some embodiments, synergistic killing of bacteria is adjusted to favor killing by the lytic activity of bacteriophage over the activity of CRISPR-Cas system by altering the number, length, composition, identity, or any combination thereof of spacers to increase the lethality of the CRISPR array. In some embodiments, the synergistic killing of bacteria is modulated to favor killing by the activity of the CRISPR-Cas system over the lytic activity of the bacteriophage by altering the number, length, composition, identity, or any combination thereof, of the spacers to reduce the lethality of the CRISPR array.

[0138] In some embodiments, the lytic activity of bacteriophage, the activity of type I CRISPR-Cas system, and the activity of antimicrobial peptide are synergistic.In some embodiments, synergistic activity is defined as the activity of killing phage at a level greater than the additive combination of the lytic activity of bacteriophage, type I CRISPR-Cas system, and antimicrobial peptide.In some embodiments, the lytic activity of bacteriophage is regulated by the concentration of bacteriophage and antimicrobial peptide.In some embodiments, the activity of type I CRISPR-Cas system is regulated by the concentration of bacteriophage.

[0139] Route of administration and dosage The dosage and duration of administration of the compositions disclosed herein will depend on various factors, including the age of the subject, the weight of the subject, and the resistance of the phage. In some embodiments, the bacteriophages disclosed herein are administered to a patient intraarterially, intravenously, intraurethrally, intramuscularly, orally, subcutaneously, by inhalation, or any combination thereof. In some embodiments, the bacteriophages disclosed herein are administered to a patient orally. In some embodiments, the bacteriophages disclosed herein are administered to a patient by topical, cutaneous, transdermal, transmucosal, implant, sublingual, buccal, rectal, vaginal, ocular, otic, or nasal administration. In some embodiments, the bacteriophages disclosed herein are administered to a patient by any combination of the aforementioned routes of administration.

[0140] In some embodiments, the compositions (bacteriophages) disclosed herein are administered before, during, or after the onset of a disease or condition. In some embodiments, the timing of administration of a bacteriophage-containing composition can vary. In some embodiments, the pharmaceutical compositions are used as prophylactics and are administered continuously to a subject with a disease or condition prone to prevent the onset of the disease or condition. In some embodiments, the pharmaceutical compositions are administered to a subject during or as soon as possible after the onset of symptoms. In some embodiments, administration of the composition begins within the first 48 hours after the onset of symptoms, within the first 24 hours after the onset of symptoms, within the first 6 hours after the onset of symptoms, or within 3 hours after the onset of symptoms. In some embodiments, the initial administration of the composition is by any practical route, including any of the routes described herein, using any of the formulations described herein. In some embodiments, the compositions are administered as soon as practicable after the onset of a disease or condition is detected or suspected, for the period necessary to treat the condition, e.g., from about 1 month to about 3 months. In some embodiments, the length of treatment will vary from subject to subject.

[0141] bacterial infection In certain embodiments, methods of treating bacterial infections are disclosed herein. In some embodiments, the bacteriophages disclosed herein treat or prevent in a human or animal subject a disease or illness mediated or caused by a bacterium disclosed herein. In some embodiments, the bacteriophages disclosed herein treat or prevent in a human or animal subject a disease or illness caused or exacerbated by a bacterium disclosed herein. Such bacteria are typically in contact with tissues of the subject, including tissues of the intestine, oral cavity, lungs, axilla, eye, vagina, anus, ear, nose, or throat. In some embodiments, the bacterial infection is treated by modulating the activity of the bacteria and / or by directly killing the bacteria.

[0142] In some embodiments, the bacterium is a Staphylococcus species. In some embodiments, the bacterium is Staphylococcus aureus.

[0143] In some embodiments, one or more Staphylococcus species present in the bacterial population are pathogenic.

[0144] In some embodiments, the bacteriophages disclosed herein are used to treat an infection, disease, or illness in the gastrointestinal tract of a subject. In some embodiments, the bacteriophages are used to modulate and / or kill target bacteria in the microbiome or gut flora of a subject. In some embodiments, the bacteriophages are used to selectively modulate and / or kill one or more target bacteria among a plurality of bacteria in the microbiome or gut flora of a subject. In some embodiments, the bacteriophages are used to selectively modulate and / or kill one or more target enteropathogenic bacteria among a plurality of bacteria in the microbiome or gut flora of a subject.

[0145] In some embodiments, the bacteriophages disclosed herein are used to treat infections, diseases, or conditions in the urinary tract of a subject. In some embodiments, the bacteriophages are used to modulate and / or kill target bacteria within the subject's urinary tract flora, including, but not limited to, Staphylococcus epidermidis, Enterococcus faecalis, and some alpha-hemolytic Streptococci. In some embodiments, the bacteriophages are used to selectively modulate and / or kill one or more target uropathogenic bacteria among multiple bacteria within the subject's urinary tract flora.

[0146] In some embodiments, the bacteriophages disclosed herein are used to treat an infection, disease, or illness in the skin of a subject, hi some embodiments, the bacteriophages are used to regulate and / or kill target bacteria on the skin of a subject.

[0147] In some embodiments, the bacteriophages disclosed herein are used to treat an infection, disease, or illness in a mucosa of a subject, hi some embodiments, the bacteriophages are used to regulate and / or kill target bacteria on the mucosa of a subject.

[0148] In some embodiments, the pathogenic bacteria is antibiotic resistant. In some embodiments, the pathogenic bacteria is methicillin resistant. In some embodiments, the pathogenic bacteria is methicillin-resistant Staphylococcus aureus.

[0149] In some embodiments, one or more target bacteria present in a bacterial population form a biofilm. In some embodiments, the biofilm comprises pathogenic bacteria. In some embodiments, the bacteriophages disclosed herein are used to treat biofilms.

[0150] In some embodiments, the bacterium comprises a Staphylococcus species, hi some embodiments, the bacterium is Staphylococcus aureus.

[0151] In some embodiments, the bacteriophage treats acne and other related skin infections.

[0152] In some embodiments, the Staphylococcus species is a multidrug-resistant (MDR) bacterial strain. An MDR strain is a bacterial strain that is resistant to at least one antibiotic. In some embodiments, the bacterial strain is resistant to antibiotic classes such as cephalosporins, fluoroquinolones, carbapenems, colistin, aminoglycosides, vancomycin, streptomycin, and methicillin. In some embodiments, the bacterial strain is Staphylococcus aureus. In some embodiments, the pathogenic bacterium is methicillin-resistant Staphylococcus aureus.

[0153] In some embodiments, the bacterium is Staphylococcus aureus. In some embodiments, the methods and compositions disclosed herein are for use in veterinary and medical applications, as well as research applications.

[0154] Microbiome "Microbiome," "microbiota," and "microbial habitat" are used interchangeably hereinafter and refer to the ecological community of microorganisms that live on or in a subject's body surfaces, cavities, and fluids. Non-limiting examples of microbiome habitats include the intestine, colon, skin, skin surface, skin orifices, vaginal cavity, umbilical region, conjunctival region, intestinal region, stomach, nasal cavities and passages, gastrointestinal tract, urogenital tract, saliva, mucus, and feces. In some embodiments, the microbiome comprises microbial material, including, but not limited to, bacteria, archaea, protists, fungi, and viruses. In some embodiments, the microbial material comprises Gram-negative bacteria. In some embodiments, the microbial material comprises Gram-positive bacteria. In some embodiments, the microbial material comprises Proteobacteria, Actinobacteria, Bacteroidetes, or Firmicutes.

[0155] In some embodiments, the bacteriophages disclosed herein are used to regulate or kill target bacteria in a subject's microbiome. In some embodiments, the bacteriophages are used to regulate and / or kill target bacteria in a microbiome by a CRISPR-Cas system, lytic activity, or a combination thereof. In some embodiments, the bacteriophages are used to regulate and / or kill target bacteria in a subject's microbiome. In some embodiments, the bacteriophages are used to selectively regulate and / or kill one or more target bacteria among a plurality of bacteria in a subject's microbiome.

[0156] In some embodiments, bacteriophages are used to modulate or kill one or more bacteria within the gastrointestinal microbiome or gut flora of a subject. Alterations to the microbiome or gut flora (e.g., dysbiosis) increase the risk of health conditions such as diabetes, psychiatric disorders, ulcerative colitis, colorectal cancer, autoimmune disorders, obesity, diabetes, central nervous system disorders, and inflammatory bowel disease. Exemplary bacteria associated with gastrointestinal disorders and diseases that can be modulated or killed by bacteriophages include strains, substrains, and enterotypes of Staphylococcus aureus.

[0157] In some embodiments, bacteriophages are used to modulate or kill one or more bacteria within the gastrointestinal microbiome or gut flora of a subject. Alterations to the microbiome or gut flora (e.g., dysbiosis) increase the risk of health conditions such as diabetes, psychiatric disorders, ulcerative colitis, colorectal cancer, autoimmune disorders, obesity, diabetes, central nervous system disorders, and inflammatory bowel disease. An exemplary list of bacteria associated with gastrointestinal diseases and disorders and that may be modulated or killed by bacteriophages includes Enterobacteriaceae, Pasteurellaceae, Fusobacteriaceae, Neisseriaceae, Veillonellaceae, Gemellaceae, Bacteroidales, Clostridiales, Erysipelothrix, Bifidobacteriales, Bacteroides, Faecalibacterium, Roseburia, Blautia, Ruminococcus, Coprococcus, Streptococcus, Dorea, Blautia, Ruminococcus, Lactobacillus, Enterococcus, Streptococcus, Actinomyces, Lactococcus, Roseburia, Blautia, Diarrister, Desulfovibrio, Escherichia , Lactobacillus, Coprococcus, Clostridium, Bifidobacteriales, Klebsiella, Granulicatella, Eubacterium, Anaerostipes, Parabacteroides, Coprobacillus, Gordonibacter, Collinsella, Bacteroides, Faecalibacterium, Anaerotruncus, Alistipes, Haemophilus, Anaerococcus, Veillonella, Arebotella, Akkermansia, Bilophila, Stellara, Egertella, Holdemannia, Gemella, Peptoniphilus, Rothia, Pediococcus, Citrobacter, Odoribacter, Enterobacteriaceae, Fusobacterium, Proteus, Escherichia coli, Fusobacterium nucleatum, Haemophilus parainfluenzae (Pasteurellaceae), Veillonella parvula, Eikenella corodenscorrodens (Neisseriaceae), Gemella moribillum, Bacteroides vulgatus, Bacteroides caccae, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium dentum, Blautia hansenii, Ruminococcus gnavus, Clostridium These include strains, substrains, and enterotypes of Bacillus nexile, Faecalibacterium prausnitzii, Ruminoccus torques, Clostridium bolteae, Eubacterium rectale, Roseburia intestinalis, and Coprococcus iomes.

[0158] In some embodiments, the bacteriophage is used to modulate or kill one or more bacteria within the epidermal microbiome or flora of a subject. Alterations to the microbiome or skin flora (e.g., dysbiosis) increase the risk of health conditions such as eczema or atopic dermatitis.

[0159] In some embodiments, the bacteriophage disclosed herein is administered to a subject to promote a healthy microbiome. In some embodiments, the bacteriophage disclosed herein is administered to a subject to restore the subject's microbiome to a health-promoting microbiome composition. In some embodiments, the composition comprising the bacteriophage disclosed herein includes a prebiotic or a third agent. In some embodiments, a microbiome-related disease or disorder is treated with the bacteriophage disclosed herein.

[0160] environmental therapy In some embodiments, the bacteriophages disclosed herein are further used in food and agricultural sanitation (including meat, fruit, and vegetable sanitation), hospital sanitation, household sanitation, vehicle and facility sanitation, industrial sanitation, etc. In some embodiments, the bacteriophages disclosed herein are used to eliminate antibiotic-resistant or other undesirable pathogens from medical environments, veterinary environments, animal husbandry environments, or any additional environments where bacteria are passed to humans or animals.

[0161] Environmental applications of phage in healthcare facilities include equipment such as endoscopes and environments such as ICUs, which are potential sources of hospital-acquired infections caused by pathogens that are difficult or impossible to disinfect. In some embodiments, the phages disclosed herein are used to treat equipment or environments inhabited by bacterial genera that have become resistant to commonly used disinfectants. In some embodiments, the phage compositions disclosed herein are used to disinfect inanimate objects. In some embodiments, the environments disclosed herein are sprayed, painted, or poured with an aqueous solution containing a phage titer. In some embodiments, the bacteriophages disclosed herein are applied by an aerosolizing agent that includes a dry dispersant to facilitate distribution of the bacteriophage to the environment. In some embodiments, the objects are immersed in a solution containing the bacteriophages disclosed herein.

[0162] hygiene In some embodiments, the bacteriophages disclosed herein are used as sanitizing agents in various fields. It should be noted that while the terms "phage" or "bacteriophage" may be used, where appropriate, this term should be interpreted broadly to include a single bacteriophage, multiple bacteriophages, such as a mixture of bacteriophages, and mixtures of bacteriophages with agents such as disinfectants, cleaning agents, surfactants, water, etc.

[0163] In some embodiments, bacteriophages are used to disinfect hospital facilities, including operating rooms, patient rooms, waiting rooms, laboratories, or other miscellaneous hospital equipment. In some embodiments, this equipment includes electrocardiographs, ventilators, cardiovascular assist devices, intra-aortic balloon pumps, infusion devices, other patient care devices, televisions, monitors, remote controls, telephones, beds, etc. In some situations, the bacteriophages are applied via aerosol canisters. In some embodiments, the bacteriophages are applied by wiping the phage on the object with a transfer vehicle.

[0164] In some embodiments, the bacteriophages described herein are used in conjunction with patient care devices. In some embodiments, the bacteriophages are used in conjunction with conventional ventilators or respiratory therapy devices to clean interior and exterior surfaces between patients. Examples of ventilators include devices that assist with artificial ventilation during surgery, devices that assist with artificial ventilation in incapacitated patients, and similar equipment. In some embodiments, conventional therapy includes automatic or powered devices, or manual bag-type devices such as those commonly found in emergency rooms and ambulances. In some embodiments, respiratory therapy includes inhalers for administering medications such as bronchodilators, such as those commonly used in chronic obstructive pulmonary disease or asthma, or devices for maintaining airway patency, such as continuous positive airway pressure devices.

[0165] In some embodiments, the bacteriophages described herein are used to clean surfaces in areas where highly contagious bacterial diseases, such as meningitis or enteric infections, are present and to treat people colonized in the area.

[0166] In some embodiments, water supplies are treated with the compositions disclosed herein. In some embodiments, the bacteriophages disclosed herein are used to treat contaminated water, water found in basins, wells, reservoirs, holding tanks, cisterns, conduits, and similar water distribution devices. In some embodiments, the bacteriophages are applied to industrial holding tanks where water, oil, cooling fluids, and other liquids accumulate in collection pools. In some embodiments, the bacteriophages disclosed herein are periodically dosed into industrial holding tanks to reduce bacterial growth.

[0167] In some embodiments, the bacteriophages disclosed herein are used to disinfect living areas such as homes, apartments, condominiums, dormitories, or any living area. In some embodiments, the bacteriophages are used to disinfect theaters, concert halls, museums, train stations, airports, pet areas such as pet beds, or public areas such as restrooms. In this capacity, the bacteriophages are dispensed from conventional devices, including pump sprayers, aerosol containers, skirt bottles, pre-moistened towels, etc., and applied (e.g., sprayed) directly to the area to be disinfected or transferred to the area via a transfer medium such as a towel or sponge. In some embodiments, the phages disclosed herein are applied to various rooms of the home, including the kitchen, bedrooms, bathrooms, garage, basement, etc. In some embodiments, the phages disclosed herein are in the same format as conventional cleaners. In some embodiments, the phages are applied in conjunction with (before, after, or simultaneously with) conventional cleaners, provided that the conventional cleaners are formulated to preserve appropriate bacteriophage biological activity.

[0168] In some embodiments, the bacteriophages disclosed herein are added to components of a paper product during or after processing of the paper product is complete, including, but not limited to, paper towels, toilet paper, and moist paper wipes.

[0169] food safety In some embodiments, the bacteriophages described herein are used in any food or dietary supplement to prevent contamination. Examples of food or pharmaceutical products are milk, yogurt, curd, cheese, fermented milk, fermented milk products, ice cream, fermented grain products, milk-based powders, infant formula or tablets, liquid suspensions, dry oral supplements, wet oral supplements, or dry tube-feeding foods.

[0170] The broad concept of bacteriophage hygiene can be applied to other agricultural applications and organisms, including produce, including fruits and vegetables, dairy products, and other agricultural products. For example, freshly cut produce often arrives at processing plants contaminated with pathogenic bacteria, resulting in outbreaks of foodborne illness that can be traced back to production. In some embodiments, application of bacteriophage preparations to produce significantly reduces or eliminates the likelihood of foodborne illness by applying a single phage or a mixture of phage specific to bacterial species associated with foodborne illness. In some embodiments, bacteriophages are applied at various stages of production and processing to reduce bacterial contamination at that time or to protect against contamination at a later time point.

[0171] In some embodiments, certain bacteriophages are applied to produce in restaurants, grocery stores, and produce distribution centers. In some embodiments, the bacteriophages disclosed herein are applied periodically or continuously to fruit and vegetable ingredients in salad bars. In some embodiments, the application of bacteriophages to salad bars or to sanitize the exterior of food products is a misting or spraying process, or a washing process.

[0172] In some embodiments, the bacteriophages described herein are used in matrices or support media, including packaging containing meat, produce, cut fruits and vegetables, and other food products. In some embodiments, a polymer suitable for packaging is impregnated with the bacteriophage preparation.

[0173] In some embodiments, the bacteriophages described herein are used in livestock feed and livestock rations. In some embodiments, on farms where livestock are raised, the livestock are provided with the bacteriophages in their drinking water, food, or both. In some embodiments, the bacteriophages described herein are sprayed onto carcasses and used to disinfect the carcass area.

[0174] The use of specific bacteriophages as biocontrol agents in produce offers many advantages. For example, bacteriophages are natural, non-toxic products that specifically lyse targeted foodborne pathogens without disrupting the ecological balance of natural microbiota in the way that typical chemical disinfectants do. Unlike chemical disinfectants, bacteriophages are natural products that co-evolve with their host bacteria, meaning that new phages active against recently emerged resistant bacteria can be quickly identified when needed, but identifying new effective disinfectants is a lengthy process that can last for several years.

[0175] Pharmaceutical Composition Certain embodiments disclosed herein include a pharmaceutical composition comprising (a) a nucleic acid sequence disclosed herein and (b) a pharmaceutically acceptable excipient. Certain embodiments disclosed herein include a pharmaceutical composition comprising (a) a bacteriophage disclosed herein and (b) a pharmaceutically acceptable excipient. Certain embodiments disclosed herein further include a pharmaceutical composition comprising (a) a composition disclosed herein and (b) a pharmaceutically acceptable excipient.

[0176] In some embodiments, the present disclosure provides pharmaceutical compositions and methods for administering the same for treating bacterial infections, archaeal infections, or disinfecting an area. In some embodiments, the pharmaceutical compositions include any of the reagents discussed above in a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions or methods disclosed herein treat bloodstream infections (BSIs) and / or inflammatory diseases (e.g., atopic dermatitis (AD)). In some embodiments, the pharmaceutical compositions or methods disclosed herein treat eczema. In some embodiments, the pharmaceutical compositions or methods disclosed herein treat atopic dermatitis.

[0177] In some embodiments, the compositions disclosed herein comprise a drug, pharmaceutical agent, carrier, adjuvant, dispersant, diluent, or the like.

[0178] In some embodiments, the bacteriophages disclosed herein are formulated for administration in a pharmaceutical carrier according to a suitable method. In some embodiments, in the manufacture of pharmaceutical compositions according to the present disclosure, the bacteriophages are mixed with, among other things, an acceptable carrier. In some embodiments, the carrier is solid (including powder) or liquid, or both, and is preferably formulated as a unit-dose composition. In some embodiments, one or more bacteriophages are incorporated into the compositions disclosed herein and prepared by any suitable method of pharmacy.

[0179] In some embodiments, a method of treating a subject in vivo comprises administering to the subject a pharmaceutical composition comprising a bacteriophage disclosed herein in a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered in a therapeutically effective amount. In some embodiments, administration of the bacteriophage to a human subject or animal in need thereof is by any means known in the art.

[0180] In some embodiments, the bacteriophages disclosed herein are for oral administration. In some embodiments, the bacteriophages are administered in solid dosage forms such as capsules, tablets, and powders, or in liquid dosage forms such as elixirs, syrups, and suspensions. In some embodiments, compositions and methods suitable for buccal (sublingual) administration include lozenges containing the bacteriophage in a flavored base, usually sucrose and acacia or tragacanth, and troches containing the bacteriophage in an inert base such as gelatin and glycerin or sucrose and acacia.

[0181] In some embodiments, the methods and compositions of the present disclosure are suitable for parenteral administration, including sterile aqueous and non-aqueous injection solutions of bacteriophages. In some embodiments, these preparations are isotonic with the blood of the intended recipient. In some embodiments, these preparations include antioxidants, buffers, bacteriostals, and solutes that render the composition isotonic with the blood of the intended recipient. In some embodiments, aqueous and non-aqueous sterile suspensions include suspending agents and thickening agents. In some embodiments, the compositions disclosed herein are provided in unit-dose or multi-dose containers, such as sealed ampoules and vials, and are stored in a freeze-dried (lyophilized) state requiring only the addition of a sterile liquid carrier, such as saline or water for injection, immediately prior to use.

[0182] In some embodiments, methods and compositions suitable for rectal administration are provided as unit-dose suppositories.In some embodiments, they are prepared by mixing bacteriophage with one or more conventional solid carriers, such as cocoa butter, and then shaping the resulting mixture.In some embodiments, methods and compositions suitable for topical application to the skin are in the form of ointments, creams, lotions, pastes, gels, sprays, aerosols, or oils.In some embodiments, the carriers used include petrolatum, lanolin, polyethylene glycol, alcohol, transdermal enhancers, and combinations of two or more thereof.

[0183] In some embodiments, methods and compositions suitable for transdermal administration are presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.

[0184] In some embodiments, methods and compositions suitable for administration to a subject's lungs, by nasal or other means, include any suitable means, for example, by an aerosol suspension of respirable particles comprising the bacteriophage composition, which is inhaled by the subject. In some embodiments, the respirable particles are liquid or solid. As used herein, "aerosol" includes any gas-borne suspended phase capable of being inhaled into the bronchioles or nasal passages. In some embodiments, liquid particle aerosols are generated by any suitable means, such as pressure-driven aerosol nebulizers or ultrasonic nebulizers. In some embodiments, solid particle aerosols comprising the composition are generated using any solid particulate drug aerosol generator, by techniques known in the pharmaceutical arts.

[0185] In some embodiments, methods and compositions suitable for administering bacteriophages disclosed herein to the surface of an object or subject include an aqueous solution. In some embodiments, such an aqueous solution is sprayed onto the surface of the object or subject. In some embodiments, the aqueous solution is used to cleanse or cleanse a bodily wound of a subject from foreign matter, including bacteria.

[0186] In some embodiments, the bacteriophage disclosed herein is administered to a subject in a therapeutically effective amount. In some embodiments, at least one bacteriophage composition disclosed herein is formulated as a pharmaceutical preparation. In some embodiments, the pharmaceutical preparation comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more bacteriophages disclosed herein. In some examples, the pharmaceutical preparation comprises a bacteriophage described herein and at least one excipient, diluent, or carrier.

[0187] In some embodiments, the pharmaceutical formulation comprises an excipient. Excipients are described in Handbook of Pharmaceutical Excipients, American Pharmaceutical Association (1986), and include, but are not limited to, solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, and lubricants.

[0188] Non-limiting examples of suitable excipients include, but are not limited to, buffering agents, preservatives, stabilizers, binders, compression agents, lubricants, chelating agents, dispersion enhancers, disintegrants, flavoring agents, sweetening agents, and coloring agents.

[0189] In some embodiments, the excipient is a buffering agent.Non-limiting examples of suitable buffering agents include, but are not limited to, sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate.In some embodiments, the pharmaceutical formulation comprises any one or more of the following buffering agents: sodium bicarbonate, potassium bicarbonate, magnesium hydroxide, magnesium lactate, magnesium gluconate, aluminum hydroxide, sodium citrate, sodium tartrate, sodium acetate, sodium carbonate, sodium polyphosphate, potassium polyphosphate, sodium pyrophosphate, potassium pyrophosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, trisodium phosphate, tripotassium phosphate, potassium metaphosphate, magnesium oxide, magnesium hydroxide, magnesium carbonate, magnesium silicate, calcium acetate, calcium glycerophosphate, calcium chloride, calcium hydroxide, and other calcium salts.

[0190] In some embodiments, the excipient is a preservative.Non-limiting examples of suitable preservatives include, but are not limited to, antioxidants such as α-tocopherol and ascorbate, and antibacterial agents such as parabens, chlorobutanol, and phenol.In some embodiments, antioxidants include ethylenediaminetetraacetic acid (EDTA), citric acid, ascorbic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), sodium sulfite, p-aminobenzoic acid, glutathione, propyl gallate, cysteine, methionine, ethanol, and N-acetylcysteine. In some embodiments, preservatives include validamycin A, TL-3, sodium orthovanadate, sodium fluoride, Na-tosyl-Phe-chloromethylketone, Na-tosyl-Lys-chloromethylketone, aprotinin, phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, protease inhibitors, reducing agents, alkylating agents, antibacterial agents, oxidase inhibitors, or other inhibitors.

[0191] In some embodiments, the pharmaceutical formulation comprises a binder as an excipient. Non-limiting examples of suitable binders include starch, pregelatinized starch, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamide, polyvinyloxoazolidone, polyvinyl alcohol, C 12 -C 18 Included are fatty acid alcohols, polyethylene glycols, polyols, sugars, oligosaccharides, and combinations thereof.

[0192] In some embodiments, the binder used in the pharmaceutical formulation is selected from starches such as potato starch, corn starch, wheat starch, sugars such as sucrose, glucose, dextrose, lactose, maltodextrin, natural and synthetic gums, gelatin, cellulose derivatives such as microcrystalline cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropylmethyl cellulose, carboxymethyl cellulose, methyl cellulose, ethyl cellulose, polyvinylpyrrolidone (povidone), polyethylene glycol (PEG), waxes, calcium carbonate, calcium phosphate, alcohols such as sorbitol, xylitol, mannitol, and water, or combinations thereof.

[0193] In some embodiments, the pharmaceutical formulation comprises a lubricant as an excipient. Non-limiting examples of suitable lubricants include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oil, Sterotex, polyoxyethylene monostearate, talc, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, and light mineral oil. In some embodiments, the lubricant present in the pharmaceutical formulation is selected from metal stearates (such as magnesium stearate, calcium stearate, and aluminum stearate), fatty acid esters (such as sodium stearyl fumarate), fatty acids (such as stearic acid), fatty alcohols, glyceryl behenate, mineral oil, paraffin, hydrogenated vegetable oil, leucine, polyethylene glycol (PEG), metal lauryl sulfates (such as sodium lauryl sulfate and magnesium lauryl sulfate), sodium chloride, sodium benzoate, sodium acetate, and talc, or combinations thereof.

[0194] In some embodiments, the excipient comprises a flavoring agent, which may include natural oils, extracts from plants, leaves, flowers, and fruits, and combinations thereof.

[0195] In some embodiments, the excipient comprises a sweetener. Non-limiting examples of suitable sweeteners include glucose (corn syrup), dextrose, invert sugar, fructose, and mixtures thereof (when not used as a carrier), saccharin and its various salts, such as the sodium salt, dipeptide sweeteners, such as aspartame, dihydrochalcone compounds, glycyrrhizin, Stevia Rebaudiana (stevioside), chloro derivatives of sucrose, such as sucralose, and sugar alcohols, such as sorbitol, mannitol, xylitol, and the like.

[0196] In some instances, the pharmaceutical formulation comprises a colorant. Non-limiting examples of suitable colorants include food, drug, and cosmetic colors (FD&C), drug and cosmetic colors (D&C), and external drug and cosmetic colors (Ext. D&C).

[0197] In some embodiments, the pharmaceutical formulations disclosed herein comprise a chelating agent, such as ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA), disodium, trisodium, tetrasodium, dipotassium, tripotassium, dilithium, and diammonium salts of EDTA, or barium, calcium, cobalt, copper, dysprosium, europium, iron, indium, lanthanum, magnesium, manganese, nickel, samarium, strontium, or zinc chelates of EDTA.

[0198] In some examples, the pharmaceutical formulation includes a diluent. Non-limiting examples of diluents include water, glycerol, methanol, ethanol, and other similar biocompatible diluents. In some embodiments, the diluent is an aqueous acid such as acetic acid, citric acid, maleic acid, hydrochloric acid, phosphoric acid, nitric acid, or sulfuric acid.

[0199] In some embodiments, the pharmaceutical formulation comprises a surfactant, which in some embodiments is selected from, but is not limited to, polyoxyethylene sorbitan fatty acid esters (polysorbates), sodium lauryl sulfate, sodium stearyl fumarate, polyoxyethylene alkyl ethers, sorbitan fatty acid esters, polyethylene glycol (PEG), polyoxyethylene castor oil derivatives, docusate sodium, quaternary ammonium compounds, amino acids such as L-leucine, sugar esters of fatty acids, glycerides of fatty acids, or combinations thereof.

[0200] In some examples, the pharmaceutical formulation includes an additional pharmaceutical agent. In some embodiments, the additional pharmaceutical agent is an antibiotic. In some embodiments, the antibiotic is from the group consisting of an aminoglycoside, ansamycin, carbacephem, carbapenem, cephalosporin (including first-, second-, third-, fourth-, and fifth-generation cephalosporins), lincosamide, macrolide, monobactam, nitrofuran, quinolone, penicillin, sulfonamide, polypeptide, or tetracycline.

[0201] In some embodiments, the antibiotic described herein is an aminoglycoside, such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, or paromomycin. In some embodiments, the antibiotic described herein is an ansamycin, such as geldanamycin or herbimycin.

[0202] In some embodiments, the antibiotic described herein is a carbacephem, such as loracarbef. In some embodiments, the antibiotic described herein is a carbapenem, such as ertapenem, doripenem, imipenem / cilastatin, or meropenem.

[0203] In some embodiments, the antibiotic described herein is a cephalosporin (first generation) such as cefadroxil, cefazolin, cephalexin, cephalothin, or cephalothin, or a cephalosporin (second generation) such as cefaclor, cefamandole, cefoxitin, cefprozil, or cefuroxime. In some embodiments, the antibiotic is a cephalosporin (third generation) such as cefixime, cefdinir, cefiditoren, cefoperazone, cefotaxime, cefpodoxime, ceftibuten, ceftizoxime, and ceftriaxone, or a cephalosporin (fourth generation) such as cefepime or ceftobiprole.

[0204] In some embodiments, the antibiotics described herein are lincosamides, such as clindamycin and azithromycin, or macrolides, such as azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, and spectinomycin.

[0205] In some embodiments, the antibiotic described herein is a monobactam, such as aztreonam, or a nitrofuran, such as furazolidone or nitrofurantoin.

[0206] In some embodiments, the antibiotic described herein is a penicillin, such as amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, oxacillin, penicillin G or V, piperacillin, temocillin, and ticarcillin.

[0207] In some embodiments, the antibiotic described herein is a sulfonamide such as mafenide, sulfonamide chrysoidine, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfamethizole, sulfamethoxazole, sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim, or trimethoprim-sulfamethoxazole (Co-trimoxazole) (TMP-SMX).

[0208] In some embodiments, the antibiotic described herein is a quinolone, such as ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, and temafloxacin.

[0209] In some embodiments, the antibiotic described herein is a polypeptide such as bacitracin, colistin, or polymyxin B.

[0210] In some embodiments, the antibiotic described herein is a tetracycline, such as demeclocycline, doxycycline, minocycline, or oxytetracycline.

[0211] Embodiment 1. A bacteriophage derived from a temperate bacteriophage that has been made lytic by removal, replacement, or inactivation of a lysogenic gene. 2. A bacteriophage according to embodiment 1, which has been made lytic by removal of any one of SEQ ID NOs: 30-32, 47-58. 3. The bacteriophage of embodiment 1, wherein the bacteriophage has been rendered lytic by removal of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of any one of SEQ ID NOs: 30-32, 47-58. 4. The bacteriophage of embodiment 1, wherein the bacteriophage has been made lytic by removal of regulatory elements of the lysogeny genes. 5. The bacteriophage of embodiment 1, wherein the bacteriophage has been made lytic by removing, modifying, or replacing the promoter of a lysogeny gene. 6. The bacteriophage of embodiment 1, wherein the bacteriophage has been made lytic by removal of a functional element of a lysogeny gene. 7. A composition comprising a Phytavirus bacteriophage and a Rosenblum virus bacteriophage, a Phytavirus bacteriophage and a Kaivirus bacteriophage, a Rosenblum virus bacteriophage and a Kaivirus bacteriophage, or a Phytavirus bacteriophage, a Rosenblum virus bacteriophage and a Kaivirus bacteriophage. 8. The composition of embodiment 7, comprising a Phytavirus bacteriophage that has been engineered to remove, replace, or inactivate a lysogeny gene or a promoter of a lysogeny gene. 9. The composition of embodiment 8, wherein the lysogeny gene encodes a suppressor. 10. The composition of any one of embodiments 7 to 9, comprising a Phytavirus bacteriophage and a Rosenblum virus bacteriophage. 11. The composition of any one of embodiments 7 to 9, comprising a Phytavirus bacteriophage and a Kaivirus bacteriophage. 12. The composition of any one of embodiments 7-9, comprising a Rosenblum virus bacteriophage and a Kai virus bacteriophage. 13. The composition of any one of embodiments 7-9, comprising a Phytavirus bacteriophage, a Rosenblumvirus bacteriophage, and a Kaivirus bacteriophage. 14. The composition of any one of embodiments 7-13, comprising a kaivirus bacteriophage, wherein the kaivirus bacteriophage comprises two or more kaivirus bacteriophages. 15. The composition of any one of embodiments 7 to 14, wherein the bacteriophage of the composition infects at least about 90% of a population of at least about 30 Staphylococcus bacteria. 16. A composition comprising a plurality of bacteriophages, including a first bacteriophage and a second bacteriophage, wherein the plurality of bacteriophages infects at least about 90% of a population of at least about 30 Staphylococcus bacteria. 17. Staphylococcus bacteria include b004604, b004605, b004606, b004607, b004608, b004609, b004610, b004611, b004612, b004613, b004614, b004615, b004616, b004617, b004618, b004619, b004620, b004621, b004622, b004623, b004624, b00462 5, b004626, b004627, b004628, b004629, b004630, b004631, b004632, b0046 33, b004634, b004635, b004636, b004637, b004638, b004639, b004640, b00 4641, b004642, b004643, b004644, b004645, b004646, b004647, b004648, b 004649, b004650, b004651, b004652, b004653, b004654, b004655, b004656 , b004657, b004658, b004659, b004660, b004661, b004662, b004663, b00466 4, b004665, b004666, b004667, b004668, b004669, b004670, b004671, b004 672, b004673, b004674, b004675, b004676, b004677, b004678, b004679, b0 04680, b004681, b004682, b004683, b004684, b004685, b004686, b004687, b004688, b004689, b004690, b004691, b004692, b004693, b004694, b004695 , b004696, b004697, b004698, b004699, b004700, b004701, b004702, b0047 03, b004704, b004705, b004706, b004707, b004708, b004709, b004710, b00 4711, b004712, b004713, b004714, b004715, b004716, b004717, b004718, b 004719, b004720, b004721, b004722, b004723, b004724, b004725, b004726,b004727, b004728, b004729, b004730, b004731, b004732, b004733, b004734, b004735, b004736, b004737, b004738, b004739, b004740, b004741, b0047 42、b004743、b004744、b004745、b004746、b004747、b004748、b004749、b004750、b004751、b004752、b004753、b004754、b004755、b004756、b004757、b0 04758、b004759、b004760、b004761、b004762、b004763、b004764、b004765、b004766、b004767、b004768、b004769、b004770、b004771、b004772、b004773 b004774, b004775, b004776, b004777, b004778, b004779, b004780, b004781, b004782, b004783, b004784, b004785, b004786, b004787, b004788, b004 789、b004790、b004791、b004792、b004793、b004794、b004795、b004796、b0 04797、b004798、b004799、b004800、b004801、b004802、b004803、b004804、b 004805、b004806、b004807、b004808、b004809、b004810、b004811、b004812 、b004813、b004814、b004815、b004816、b004817、b004818、b004819、b00482 0、b004821、b004822、b004823、b004824、b004825、b004826、b004827、b004 828、b004829、b004830、b004831、b004832、b004833、b004834、b004835、b00 4836、b004837、b004838、b004839、b004840、b004841、b004842、b004843、b 004844、b004845、b004846、b004847、b004848、b004849、b004850、b004851、b004852, b004853, b004854, b004855, b004856, b004857, b004858, b004859 , b004860, b004861, b004862, b004863, b004864, b004865, b004866, b00486 7, b004868, b004869, b004870, b004871, b004872, b004873, b004874, b0048 75, b004876, b004877, b004878, b004879, b004880, b004881, b004882, b004 883, b004884, b004885, b004886, b004887, b004888, b004889, b004890, b004891, b004892, b004893, b004894, b004895, b004896, b004897, b004898, b004899, b004900, b004901, b004902, b004903, b004904, b004905, b004906, b004907, b004908, b004909, b004910, and b004911. 18. The staphylococcus bacteria consortium is: 8, 5, 22, 15, 1, 30, 398, 105, 45, 672, 2250, 582, 72, 97, 239, 34, 87, 101, 109, 1159, 1165, 1181, 12, 121, 152, 1750, 20, 225, 25, 291, 3628, 59, 7, 779, 88, 10, 1011, 1049, 1156, 1351, 18. The composition of embodiment 16 or 17, comprising a Staphylococcus bacterium with an MLST of 149, 1637, 1649, 1757, 1842, 188, 1970, 2066, 256, 2867, 2945, 3149, 3182, 3510, 39, 395, 4317, 47, 4730, 50, 508, 573, 6, 630, 737, 828, 848, 923, or 93. 19. The composition of any one of embodiments 16-18, wherein the population of Staphylococcus bacteria comprises bacteria isolated from a bloodstream infection. 20. The composition of any one of embodiments 16-19, wherein at least about 40% of the population of Staphylococcus bacteria are multidrug resistant. 21. The composition of any one of embodiments 16-20, wherein the Staphylococcus bacteria comprises Staphylococcus aureus. 22. The composition of any one of embodiments 16-21, wherein infection is determined by plaque assay or growth inhibition assay. 23. The composition of any one of embodiments 16-22, wherein at least about 90% is at least about 95%. 24. The composition of any one of embodiments 16-22, wherein at least about 90% is at least about 98%. 25. The composition of any one of embodiments 16-22, wherein at least about 90% is at least about 99%. 26. At least about 30 Staphylococcus bacteria are present, including b004604, b004605, b004606, b004607, b004608, b004609, b004610, b004611, b004612, b004613, b004614, b004615, b004616, b004617, b004618, b004619, b004620, b004621, b004622, b004623, b004624, b004625, b004626, b004627, b004628, b004629, b004630, b004631, b00 4632, b004633, b004634, b004635, b004636, b004637, b004638, b004639, b 004640, b004641, b004642, b004643, b004644, b004645, b004646, b004647, b004648, b004649, b004650, b004651, b004652, b004653, b004654, b00465 5, b004656, b004657, b004658, b004659, b004660, b004661, b004662, b0046 63, b004664, b004665, b004666, b004667, b004668, b004669, b004670, b00 4671, b004672, b004673, b004674, b004675, b004676, b004677, b004678, b 004679, b004680, b004681, b004682, b004683, b004684, b004685, b004686 , b004687, b004688, b004689, b004690, b004691, b004692, b004693, b00469 4, b004695, b004696, b004697, b004698, b004699, b004700, b004701, b004 702, b004703, b004704, b004705, b004706, b004707, b004708, b004709, b00 4710, b004711, b004712, b004713, b004714, b004715, b004716, b004717, b 004718, b004719, b004720, b004721, b004722, b004723, b004724, b004725,b004726, b004727, b004728, b004729, b004730, b004731, b004732, b004733, b004734, b004735, b004736, b004737, b004738, b004739, b004740, b0047 41、b004742、b004743、b004744、b004745、b004746、b004747、b004748、b004749、b004750、b004751、b004752、b004753、b004754、b004755、b004756、b0 04757、b004758、b004759、b004760、b004761、b004762、b004763、b004764、b004765、b004766、b004767、b004768、b004769、b004770、b004771、b004772 b004773, b004774, b004775, b004776, b004777, b004778, b004779, b004780, b004781, b004782, b004783, b004784, b004785, b004786, b004787, b004 788、b004789、b004790、b004791、b004792、b004793、b004794、b004795、b0 04796、b004797、b004798、b004799、b004800、b004801、b004802、b004803、b 004804、b004805、b004806、b004807、b004808、b004809、b004810、b004811 、b004812、b004813、b004814、b004815、b004816、b004817、b004818、b00481 9、b004820、b004821、b004822、b004823、b004824、b004825、b004826、b004 827、b004828、b004829、b004830、b004831、b004832、b004833、b004834、b00 4835、b004836、b004837、b004838、b004839、b004840、b004841、b004842、b 004843、b004844、b004845、b004846、b004847、b004848、b004849、b004850、b004851, b004852, b004853, b004854, b004855, b004856, b004857, b004858, b004859, b004860, b004861, b004862, b004863, b004864, b004865, b004866, b 004867, b004868, b004869, b004870, b004871, b004872, b004873, b004874, b0 04875, b004876, b004877, b004878, b004879, b004880, b004881, b004882, b00 4883, b004884, b004885, b004886, b004887, b004888, b004889, b004890, b004891, b004892, b004893, b004894, b004895, b004896, b004897, b004898, b004899, b004900, b004901, b004902, b004903, b004904, b004905, b004906, b004907, b004908, b004909, b004910, and b004911. , 27. At least about 30 Staphylococcus bacteria are present in the following populations: 8, 5, 22, 15, 1, 30, 398, 105, 45, 672, 2250, 582, 72, 97, 239, 34, 87, 101, 109, 1159, 1165, 1181, 12, 121, 152, 1750, 20, 225, 25, 291, 3628, 59, 7, 779, 88, 10, 1011, 1049, 1156, 1351 , 149, 1637, 1649, 1757, 1842, 188, 1970, 2066, 256, 2867, 2945, 3149, 3182, 3510, 39, 395, 4317, 47, 4730, 50, 508, 573, 6, 630, 737, 828, 848, 923, or 93. 28. The composition of any one of embodiments 16-27, wherein the at least about 30 Staphylococcus bacteria comprise bacteria isolated from a bloodstream infection. 29. The composition of any one of embodiments 16-28, wherein at least about 40% of the at least about 30 Staphylococcus bacteria are multidrug-resistant. 30. The composition of any one of embodiments 16-25, wherein the first bacteriophage and the second bacteriophage are of different genera. 31. The composition of any one of embodiments 16-31, wherein the plurality of bacteriophages comprises a Phytavirus bacteriophage and a Rosenblumvirus bacteriophage, a Phytavirus bacteriophage and a Kaivirus bacteriophage, a Rosenblumvirus bacteriophage and a Kaivirus bacteriophage, or a Phytavirus bacteriophage, a Rosenblumvirus bacteriophage and a Kaivirus bacteriophage. 32. The composition of any one of embodiments 16-31, wherein the plurality of bacteriophages comprises a phytovirus. 33. The composition of embodiment 32, wherein the Phytavirus is engineered to remove, replace, or inactivate a lysogeny gene or a promoter of a lysogeny gene. 34. The composition of embodiment 33, wherein the lysogeny gene encodes a suppressor. 35. A composition comprising a plurality of bacteriophages, wherein the plurality of bacteriophages comprises a first bacteriophage that binds to a first receptor on Staphylococcus bacteria and a second bacteriophage that binds to a second receptor on Staphylococcus bacteria, and wherein the plurality of bacteriophages is more resistant by Staphylococcus bacteria than either the first or second bacteriophage alone. 36. The composition of embodiment 35, wherein the first receptor and the second receptor are different. 37. The composition of embodiment 35 or 36, wherein the first bacteriophage comprises a phytovirus. 38. The composition of embodiment 35 or 36, wherein the first bacteriophage comprises a Phytavirus and a Rosenblum virus. 39. The composition of any one of embodiments 35-38, wherein the second bacteriophage comprises a Kaivirus. 40. The composition of embodiment 35 or 36, wherein the plurality of bacteriophages comprises a Phytavirus bacteriophage and a Rosenblumvirus bacteriophage, a Phytavirus bacteriophage and a Kaivirus bacteriophage, a Rosenblumvirus bacteriophage and a Kaivirus bacteriophage, or a Phytavirus bacteriophage, a Rosenblumvirus bacteriophage and a Kaivirus bacteriophage. 41. The composition of any one of embodiments 35-40, wherein the multiple bacteriophages are more resistant to Staphylococcus than the first bacteriophage or the second bacteriophage alone. 42. The composition of any one of embodiments 35-41, wherein the plurality of bacteriophages of the composition infects at least about 90% of a population of at least about 30 Staphylococcus bacteria. 43. The composition of any one of embodiments 35-42, wherein the first bacteriophage and the second bacteriophage are of different genera. 44. The composition of embodiment 43, wherein the first bacteriophage and the second bacteriophage are capable of independently infecting at least 90% of a population of Staphylococcus bacteria. 45. The composition of any one of embodiments 35-44, wherein the first bacteriophage comprises a Phytavirus, a Rosenblumvirus, or a Kaivirus. 46. ​​The composition of any one of embodiments 35-45, wherein the second bacteriophage comprises a Phytavirus, a Rosenblumvirus, or a Kaivirus. 47. The composition of any one of embodiments 35-46, wherein the plurality of bacteriophages comprises a Phytavirus bacteriophage and a Rosenblumvirus bacteriophage, a Phytavirus bacteriophage and a Kaivirus bacteriophage, a Rosenblumvirus bacteriophage and a Kaivirus bacteriophage, or a Phytavirus bacteriophage, a Rosenblumvirus bacteriophage and a Kaivirus bacteriophage. 48. The composition of any one of embodiments 35-47, wherein the plurality of bacteriophages infects at least about 90% of a population of Staphylococcus bacteria. 49. The composition of embodiment 48, wherein the staphylococcus bacteria comprises Staphylococcus aureus. 50. The composition of any one of embodiments 48-49, wherein at least about 90% infection is determined by plaque assay or growth inhibition assay. 51. The composition of embodiment 50, wherein at least about 90% is at least about 95%. 52. The composition of embodiment 50, wherein at least about 90% is at least about 98%. 53. The composition of embodiment 50, wherein at least about 90% is at least about 99%. 54. The staphylococcus bacteria consortium is: 8, 5, 22, 15, 1, 30, 398, 105, 45, 672, 2250, 582, 72, 97, 239, 34, 87, 101, 109, 1159, 1165, 1181, 12, 121, 152, 1750, 20, 225, 25, 291, 3628, 59, 7, 779, 88, 10, 1011, 1049, 1156, 1351, 149, 1637, 16 54. The composition of any one of embodiments 35-53, comprising a Staphylococcus bacterium having an MLST of 49, 1757, 1842, 188, 1970, 2066, 256, 2867, 2945, 3149, 3182, 3510, 39, 395, 4317, 47, 4730, 50, 508, 573, 6, 630, 737, 828, 848, 923, or 93, or a combination of two or more thereof. 55. The composition of any one of embodiments 35-54, wherein the population of staphylococcus bacteria comprises bacteria isolated from a bloodstream infection. 56. The composition of any one of embodiments 35-55, wherein at least about 40% of the population of Staphylococcus bacteria are multidrug-resistant. 57. A phytavirus bacteriophage engineered to remove, replace, or inactivate a lysogeny gene or the promoter of a lysogeny gene. 58. The bacteriophage of embodiment 57, wherein the lysogeny gene encodes a suppressor. 59. The bacteriophage of embodiment 58, wherein the suppressor comprises an amino acid sequence that is at least about 80% identical to SEQ ID NO: 47 or 48. 60. A bacteriophage according to any one of embodiments 57 to 59, wherein removing the lysogenic genes comprises removing between about 1% and 100% of the lysogenic genes. 61. The bacteriophage of embodiment 60, wherein about 10 to about 1,200 base pairs of the lysogeny gene are deleted. 62. The bacteriophage of any one of embodiments 57-61, wherein the lysogeny gene is deleted, replaced, or inactivated. 63. The bacteriophage of embodiment 62, in which the lysogeny genes are removed. 64. A bacteriophage according to any one of embodiments 57 to 63, wherein the promoter of the lysogeny gene is removed, replaced or inactivated. 65. The bacteriophage of any one of embodiments 57-62, further comprising a Rosenblum virus. 66. The bacteriophage of any one of embodiments 57-65, further comprising a Kaivirus. 67. The bacteriophage of any one of embodiments 57-66, comprising a plurality of bacteriophages, including a Phytavirus bacteriophage, and wherein the plurality of bacteriophages infects at least about 90% of a population of at least about 30 Staphylococcus bacteria. 68. TreA, Ipi, DNAsel, RIP, FS3, PLNC8α, PLNC8β, LytM, LnqO, dispesin D, aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, α-galactosidase, β-galactosidase, glucoamylase, α-glucosidase 69. The composition of any one of embodiments 7 to 44, or the bacteriophage of any one of embodiments 57 to 68, comprising a nucleic acid encoding an exogenous peptide selected from an enzyme, a beta-glucosidase, a haloperoxidase, an invertase, a laccase, a lipase, a mannosidase, an oxidase, a pectinolytic enzyme, a peptidoglutaminase, a peroxidase, a phytase, a polyphenol oxidase, a proteolytic enzyme, a ribonuclease, a transglutaminase, a xylanase, a lyase, a glycosyl hydroxylase, a polyglucosamine (PGA) depolymerase, a colonic acid depolymerase, a 1,4-L-fucodise hydrolase, a colanic acid, or a depolymerizing arginase. 69. A composition according to any one of embodiments 7 to 44, or a bacteriophage according to any one of embodiments 57 to 68, comprising one or more components of a CRISPR-Cas system. 70. The composition or bacteriophage of embodiment 69, wherein the CRISPR-Cas system is a type IB CRISPR-Cas system derived from Listeria monocytogenes (LMIB). 71. A composition described in any one of embodiments 7 to 44, or a bacteriophage described in any one of embodiments 57 to 68, comprising a spacer sequence or a crRNA transcribed therefrom, wherein the spacer sequence is complementary to a target nucleic acid sequence derived from a target gene in a target bacterium. 72. The composition or bacteriophage of embodiment 71, wherein the target bacteria comprises Staphylococcus bacteria. 73. A method for killing bacteria in a subject infected with bacteria, the method comprising administering to the subject a plurality of bacteriophages comprising Rosenblum virus. 74. The method of embodiment 73, wherein the plurality of bacteriophages comprises a phytovirus. 75. The method of embodiment 73 or 74, wherein the plurality of bacteriophages comprises Kaivirus. 76. A method for killing bacteria in a subject infected with bacteria, the method comprising administering to the subject a plurality of bacteriophages, including a phytovirus. 77. The method of embodiment 76, wherein the plurality of bacteriophages comprises Rosenblum virus. 78. The method of embodiment 76 or 77, wherein the plurality of bacteriophages comprises Kaivirus. 79. A method for killing bacteria in a subject infected with bacteria, the method comprising administering to the subject a plurality of bacteriophages, including Kaivirus. 80. The method of embodiment 78, wherein the plurality of bacteriophages comprises Rosenblum virus. 81. The method of embodiment 78 or 79, wherein the plurality of bacteriophages comprises a phytovirus. 82. A bacteriophage containing at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1378 (PTA-127329). 83. A bacteriophage containing at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1381 (PTA-127330). 84. A bacteriophage containing at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p4815 (PTA-127331). 85. A bacteriophage containing at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1378e062 (PTA-127333). 86. A bacteriophage containing at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1378e074 (PTA-127334). 87. A bacteriophage containing at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p4815e037 (PTA-127335). 88. A bacteriophage containing at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1378e075 (PTA-127338). 89. A bacteriophage containing at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1381e017 (PTA-127339). 90. A bacteriophage containing at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p4815e053 (PTA-127340). 91. A bacteriophage containing at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1498e001 (PTA-127336). 92. A bacteriophage containing at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p3693e001 (PTA-127337). 93. A bacteriophage containing at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p3224e002 (PTA-127341). 94. A bacteriophage containing at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p5593e001 (PTA-127342). 95. A bacteriophage containing at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p3224e002 (PTA-127341). 96. A bacteriophage containing at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1468e003 (PTA-127343). 97. A bacteriophage containing at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1478e003 (TA-127344). 98. A bacteriophage containing at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1494e002 (PTA-127345). 99. A bacteriophage containing at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p2808 (PTA-127332). 100. A bacteriophage containing at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1473. 101. A composition according to any one of embodiments 7 to 43 or 55 to 59, or a bacteriophage according to any one of embodiments 1 to 6, 44 to 54, or 82 to 100, for use in treating a staphylococcal infection. 102. A composition for use in treating a staphylococcal infection comprising at least two bacteriophages, wherein a first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1378, and a second bacteriophage comprises at least one of p1381, p4815, p1378e062, p1378e074, p4815e 037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, p2808, or p1473. 103. A composition for use in treating a staphylococcal infection comprising at least two bacteriophages, wherein a first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p4815, and a second bacteriophage comprises at least one of p1381, p1378, p1378e062, p1378e074, p4815e 037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, p2808, or p1473. 104. A composition for use in treating a staphylococcal infection comprising at least two bacteriophages, wherein a first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1494e002, and a second bacteriophage comprises at least one of p1381, p1378, p4815, p1378e062, p1378e 074, p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p2808, or p1473. 105. A composition for use in treating a staphylococcal infection comprising at least two bacteriophages, wherein a first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p2808, and a second bacteriophage comprises at least one of p1381, p1378, p4815, p1378e062, p1378e074, A composition comprising at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, or p1473. 106. A composition according to any one of embodiments 7 to 43, 55 to 59, or 101 to 105, or a bacteriophage according to any one of embodiments 1 to 6, 44 to 54, or 82 to 100, for use in the manufacture of a medicament for treating a staphylococcal infection. 107. A composition for use in the manufacture of a medicament for treating a staphylococcal infection, comprising at least two bacteriophages, wherein a first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1378, and a second bacteriophage comprises at least one of p1381, p4815, p1378e062, p1378e074, A composition comprising at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, p2808, or p1473. 108. A composition for use in the manufacture of a medicament for treating a staphylococcal infection, comprising at least two bacteriophages, wherein a first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p4815, and a second bacteriophage comprises at least one of p1381, p1378, p1378e062, p1378e074, A composition comprising at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, p2808, or p1473. 109. A composition for use in the manufacture of a medicament for treating a staphylococcal infection, comprising at least two bacteriophages, wherein a first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1378, and a second bacteriophage comprises at least one of p1381, p4815, p1378e062, p1378e074, A composition comprising at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, p2808, or p1473. 110. A composition for use in the manufacture of a medicament for treating a staphylococcal infection, comprising at least two bacteriophages, wherein a first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p2808, and a second bacteriophage comprises at least one of p1381, p1378, p4815, p1378e062, p137 p4815e037, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, or p1473. 111. A composition comprising a plurality of bacteriophages, including a first bacteriophage and a second bacteriophage, wherein bacteria treated with the plurality of bacteriophages exhibit a reduced amount of regrowth compared to treatment with either the first bacteriophage or the second bacteriophage alone. 112. The composition of embodiment 111, wherein the first bacteriophage comprises a Phytavirus and the second bacteriophage comprises a Rosenblumvirus or a Kaivirus, or the first bacteriophage comprises a Rosenblumvirus and the second bacteriophage comprises a Phytavirus or a Kaivirus, or the first bacteriophage comprises a Kaivirus and the second bacteriophage comprises a Phytavirus or a Rosenblumvirus.

[0212] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description of the present disclosure herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.

[0213] Unless the context clearly indicates otherwise, it is specifically intended that the various features of the present disclosure described herein can be used in any combination. Furthermore, the present disclosure also contemplates that in some embodiments, any feature or combination thereof specified herein may be excluded or omitted. To illustrate, if a composition is described herein as including components A, B, and C, it is specifically intended that any of A, B, or C, or any combination thereof, alone or in any combination, be omitted and discarded.

[0214] Those skilled in the art will understand the interchangeability of the terms used to designate various CRISPR-Cas systems and their components due to the lack of consistency in the literature and ongoing efforts in the art to standardize such terms.Similarly, those skilled in the art will understand the interchangeability of the terms used to designate various anti-CRISPR proteins due to the lack of consistency in the literature and ongoing efforts in the art to standardize such terms.

[0215] As used in this specification and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. Also, as used herein, "and / or" refers to and includes all possible combinations of one or more of the associated listed items, as well as the lack of combination when interpreted in the alternative ("or").

[0216] As used herein, the term "about," when referring to a measurable value such as dosage or duration, refers to a variation of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount. As used herein, phrases such as "between X and Y" and "between about X and Y" should be interpreted to include X and Y. As used herein, phrases such as "between about X and Y" mean "between about X and about Y," and phrases such as "about X and Y" mean "about X to about Y."

[0217] The terms "comprise," "comprises," and "comprising," "includes," "including," "have," and "having," as used herein, specify the presence of stated features, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0218] As used herein, the transitional phrase "consisting essentially of" means that the claims should be construed to include the specific materials or steps recited in the claims and those that do not materially affect the basic and novel characteristics of the claimed disclosure. Thus, the term "consisting essentially of" when used in the claims of the present disclosure is not intended to be construed as the equivalent of "comprising."

[0219] The terms "consists of" and "consisting of," when used herein, exclude all features, steps, operations, elements, and / or components not otherwise directly recited. The use of "consisting of" limits only the features, steps, operations, elements, and / or components that are recited in that clause, but does not exclude other features, steps, operations, elements, and / or components from the claim as a whole.

[0220] As used herein, "chimera" refers to a nucleic acid molecule or polypeptide in which at least two components are derived from different sources (eg, different organisms, different coding regions).

[0221] As used herein, "complement" refers to 100% complementarity or identity with the reference nucleotide sequence, or to less than 100% complementarity (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc.). Complement or complementable can also be used to refer to the "complement" of a mutation or "complementing" it.

[0222] The term "complementary" or "complementarity" as used herein refers to the natural binding of polynucleotides by base pairing under permissive salt and temperature conditions. For example, the sequence "AGT" binds to the complementary sequence "TCA". The complementarity between two single-stranded molecules can be "partial," in that only a portion of the nucleotides bind, or it can be complete, in that all complementarity exists between the single-stranded molecules. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.

[0223] As used herein, the term "gene" refers to a nucleic acid molecule that can be used to produce mRNA, tRNA, rRNA, miRNA, anti-microRNA, regulatory RNA, etc. A gene may or may not be usable to produce a functional protein or gene product. A gene includes both coding and non-coding regions (e.g., introns, regulatory elements, functional elements, promoters, enhancers, termination sequences, and / or 5' and 3' untranslated regions). A gene is "isolated," meaning a nucleic acid that is substantially or essentially free from components normally found associated with the nucleic acid in its natural state. Such components include other cellular material, culture medium from recombinant production, and / or various chemicals used in the chemical synthesis of the nucleic acid.

[0224] As used herein, "target nucleotide sequence" refers to the portion of a target gene that is complementary to the spacer sequence of a recombinant CRISPR array.

[0225] As used herein, a "target nucleotide sequence" refers to a portion of a target gene that is fully complementary or substantially complementary (e.g., at least 70% complementary (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)) to a spacer sequence in a CRISPR array (i.e., a "protospacer sequence" adjacent to a target region in the genome or a protospacer adjacent motif (PAM) sequence).

[0226] As used herein, the term "protospacer adjacent motif" or "PAM" refers to a DNA sequence present on a target DNA molecule adjacent to a nucleotide sequence matching the spacer sequence. This motif is found in the target gene adjacent to the region to which the spacer sequence binds as a result of its complementarity to the region, and identifies the point at which base pairing with the spacer nucleotide sequence begins. The exact PAM sequence required varies in different CRISPR-Cas systems. Non-limiting examples of PAMs include CCA, CCT, CCG, TTC, AAG, AGG, ATG, GAG, and / or CC. In some cases, in type I systems, the PAM is located immediately 5' of the sequence matching the spacer, and therefore 3' of the sequence that base pairs with the spacer nucleotide sequence, allowing it to be directly recognized by the cascade. In some cases, in the B. halodurans type IC system, the PAM is YYC, where Y can be either T or C. In some cases, such as the Listeria monocytogenes type IB system, the PAM is CCW, where W can be A or T. Upon recognition of the cognate protospacer and PAM, Cas is recruited, which then cleaves and degrades the target DNA. In type II systems, the PAM is required for the Cas9 / sgRNA to form an R-loop to interrogate specific DNA sequences through Watson-Crick pairing of its guide RNA with the genome. PAM specificity is a function of the DNA binding specificity of the Cas9 protein (e.g., the protospacer-adjacent motif recognition domain at the C-terminus of Cas9).

[0227] As used herein, type I clustered regularly interspaced short palindromic repeats (CRISPR)-associated complex (cascade) for antiviral defense refers to a complex of polypeptides involved in processing pre-crRNA and subsequent binding to target DNA in a type I CRISPR-Cas system. These polypeptides include, but are not limited to, type I subtype IA, IB, IC, ID, IE, IF, and IU cascade polypeptides. Non-limiting examples of type IA polypeptides include Cas7 (Csa2), Cas8a1 (Csx13), Cas8a2 (Csx9), Cas5, Csa5, Cas6a, Cas3', and / or Cas3''. Non-limiting examples of type IB polypeptides include Cas6b, Cas8b (Csh1), Cas7 (Csh2), and / or Cas5. Non-limiting examples of IC-type polypeptides include Cas5d, Cas8c (Csd1), and / or Cas7 (Csd2). Non-limiting examples of ID-type polypeptides include Cas10d (Csc3), Csc2, Csc1, and / or Cas6d. Non-limiting examples of IE-type polypeptides include Cse1 (CasA), Cse2 (CasB), Cas7 (CasC), Cas5 (CasD), and / or Cas6e (CasE). Non-limiting examples of IF-type polypeptides include Cys1, Cys2, Cas7 (Cys3), and / or Cas6f (Csy4). Non-limiting examples of IF-type polypeptides include Cas8u2, Cas7, and / or fusion Cas5-Cas6 polypeptides. Non-limiting examples of IU-type polypeptides include Cas8a1 (Cst1), Cas7 (Cst2), Cas5 (Cst5t), and Cas3.In some embodiments, the recombinant nucleic acids described herein comprise, consist essentially of, or consist of a nucleotide sequence encoding a subset of Type I cascade polypeptides that function to process the CRISPR array and subsequently bind to target DNA using the spacer of the processed CRISPR RNA as a guide.

[0228] As used herein, " CRISPR array " refers to a nucleic acid molecule that comprises at least two repeat sequences, or a portion of each of the repeat sequences, and at least one spacer sequence.One of the two repeat sequences or a portion thereof is linked to the 5' end of the spacer sequence, and the other of the two repeat sequences or a portion thereof is linked to the 3' end of the spacer sequence.In recombinant CRISPR array, the combination of repeat sequence and spacer sequence is synthetic, and is made by humans but is not found in nature. In some embodiments, a "CRISPR array" refers to a nucleic acid construct that includes at least one repeat-spacer sequence from 5' to 3' (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more repeat-spacer sequences, and any range or value therein), wherein the 3' end of the 3' most repeat-spacer sequence of the array is linked to a repeat sequence, such that all spacers in the array are adjacent to a repeat sequence on both the 5' and 3'' ends.

[0229] As used herein, "spacer sequence" or "spacer" refers to a nucleotide sequence complementary to a target DNA (i.e., a target region in a genome, or a "protospacer sequence" adjacent to a protospacer adjacent motif (PAM) sequence). The spacer sequence is fully complementary or substantially complementary to the target DNA (e.g., at least about 70% complementary (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)).

[0230] As used herein, "repeat sequence" refers to any repeat sequence of a wild-type CRISPR locus or a repeat sequence of a synthetic CRISPR array (e.g., a repeat-spacer-repeat sequence), for example, separated by a "spacer sequence." Repeat sequences useful in the present disclosure are any known or later identified repeat sequence of a CRISPR locus, or synthetic repeats designed to function in a CRISPR system, e.g., a CRISPR type I system.

[0231] As used herein, the terms "CRISPR phage," "CRISPR-enhanced phage," and "crPhage" refer to a bacteriophage particle comprising bacteriophage DNA that includes at least one heterologous polynucleotide encoding at least one component of a CRISPR-Cas system (e.g., a CRISPR array, a crRNA, e.g., a P1 bacteriophage containing a targeting crRNA insertion). In some embodiments, the polynucleotide encodes at least one transcriptional activator of the CRISPR-Cas system. In some embodiments, the polynucleotide encodes at least one component of an anti-CRISPR polypeptide of the CRISPR-Cas system.

[0232] As used herein, the phrase "substantially identical" or "substantial identity" in the context of two nucleic acid molecules, nucleotide sequences, or protein sequences means a sequence that is at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152 Substantial identity refers to two or more sequences or subsequences that have 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and / or 100% nucleotide or amino acid residue identity. In some embodiments, substantial identity refers to two or more sequences or subsequences that have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, 96%, 97%, 98%, or 99% identity. When comparing sequences, typically one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the designated program parameters.

[0233] The optimal alignment of sequences for aligning a comparison window is performed by tools such as Smith and Waterman's local homology algorithm, Needleman and Wunsch's homology alignment algorithm, Pearson and Lipman's similarity search method, and optionally by computerized implementations of these algorithms, such as GAP, BESTFIT, FASTA, and TFASTA, available as part of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA). The "identity fraction" of the aligned segments of a test sequence and a reference sequence is the number of identical components shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined portion of the reference sequence. The sequence identity percentage is expressed as the identity fraction multiplied by 100. Comparison of one or more polynucleotide sequences is performed on the full-length polynucleotide sequence or a portion thereof, or on a longer polynucleotide sequence. In some instances, "percent identity" is determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.

[0234] In some embodiments, the recombinant nucleic acid molecules, nucleotide sequences, and polypeptides disclosed herein are "isolated." An "isolated" nucleic acid molecule, "isolated" nucleotide sequence, or "isolated" polypeptide is a nucleic acid molecule, nucleotide sequence, or polypeptide that exists away from its natural environment. In some examples, the isolated nucleic acid molecule, nucleotide sequence, or polypeptide exists in a purified form, at least partially separated from at least some of the other components of the naturally occurring organism or virus, e.g., cellular or viral structural components, or other polypeptides or nucleic acids normally found in association with the polynucleotide. In representative embodiments, the isolated nucleic acid molecule, isolated nucleotide sequence, and / or isolated polypeptide is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more pure.

[0235] The terms "treat," "treating," or "treatment" intend that the severity of the disease in a subject is reduced or at least partially ameliorated or altered, and that some relief, alleviation, or reduction is achieved in at least one clinical symptom, and / or there is a delay in the progression of the disease or condition and / or a delay in the onset of the disease or condition. With respect to an infection, disease, or condition, the term refers to a reduction in symptoms or other signs of the infection, disease, or condition. In some embodiments, treatment reduces symptoms or other signs of the infection, disease, or condition by at least about 5%, e.g., about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more.

[0236] As used herein, the terms "infection," "disease," or "illness" refer to any adverse, negative, or harmful physiological condition in a subject. In some embodiments, the origin of the "infection," "disease," or "illness" is the presence of a target bacterial population in and / or on a subject. In some embodiments, the bacterial population comprises one or more target bacterial species. In some embodiments, the one or more bacterial species in the bacterial population comprise one or more strains of one or more bacteria. In some embodiments, the target bacterial population causes an acute or chronic "infection," "disease," or "illness." In some embodiments, the target bacterial population causes a localized or systemic "infection," "disease," or "illness." In some embodiments, the target bacterial population causes an episodic "infection," "disease," or "illness." In some embodiments, the target bacterial population causes an "infection," "disease," or "illness" that is acquired through means including, but not limited to, respiratory inhalation, ingestion, skin and wound infections, bloodstream infections, middle ear infections, gastrointestinal infections, peritoneal infections, urinary tract infections, genitourinary tract infections, oral soft tissue infections, intraperitoneal infections, epidermal or mucosal absorption, eye infections (including contact lens contamination), endocarditis, infections in cystic fibrosis and non-cystic fibrosis bronchiectasis (NCFB), infections of indwelling medical devices such as joint prostheses, dental implants, catheters, and cardiac implants, sexual contact, and / or hospital-acquired and ventilator-associated bacterial pneumonia.

[0237] As used herein, the term "biofilm" refers to an accumulation of microorganisms embedded in a polysaccharide matrix. Biofilms form on solid biological or non-biological surfaces as well as at liquid-air interfaces and are medically important, accounting for more than 80 percent of microbial infections in the body.

[0238] The terms "prevent," "preventing," and "prevention" (and grammatical variations thereof) refer to preventing and / or delaying the onset of, and / or reducing the severity of, an infection, disease, illness, and / or clinical symptom in a subject compared to what would occur if the methods disclosed herein were not performed prior to the onset of the disease, disorder, and / or clinical symptom. Thus, in some embodiments, foods, surfaces, medical implements, and devices are treated with the compositions and methods disclosed herein to prevent infection.

[0239] As used herein, the term "individual" or "subject" includes any animal having or susceptible to an infection, disease, or illness involving bacteria. Thus, in some embodiments, the subject is a mammal, bird, reptile, amphibian, fish, crustacean, or mollusk. Mammalian subjects include, but are not limited to, humans, non-human primates (e.g., gorillas, monkeys, baboons, chimpanzees, etc.), dogs, cats, goats, horses, pigs, cows, sheep, etc., and laboratory animals (e.g., rats, guinea pigs, mice, gerbils, hamsters, etc.). Avian subjects include, but are not limited to, chickens, ducks, turkeys, geese, quail, pheasants, and pet birds (e.g., parakeets, parrots, macaws, cockatoos, canaries, etc.). Piscean subjects include, but are not limited to, species used in aquaculture (e.g., tuna, salmon, tilapia, catfish, carp, trout, cod, sea bass, perch, snapper, etc.). Crustacean subjects include, but are not limited to, species used in aquaculture (e.g., shrimp, prawns, lobsters, crayfish, crabs). Mollusc subjects include, but are not limited to, species used in aquaculture (e.g., abalone, mussels, oysters, clams, scallops). In some embodiments, suitable subjects include both males and females, and subjects of any age, including embryos (e.g., in utero or in ovo), infants, young children, adolescents, adults, and geriatric subjects. In some embodiments, the subject is a human.

[0240] As used herein, the term "isolated" in the context of a nucleic acid sequence is a nucleic acid sequence that exists apart from its natural environment.

[0241] As used herein, "pharmaceutically acceptable" means a material that is not biologically or otherwise unwanted, i.e., such a material may be administered to a subject without producing any unwanted biological effects, such as toxicity.

[0242] As used herein, the term "in vivo" is used to describe events that take place inside the body of a subject.

[0243] As used herein, the term "in vitro" is used to describe events that occur within a container for holding experimental reagents such that the material is separated from the biological source from which it is obtained. In vitro assays can include cell-based assays in which live or dead cells are utilized. In vitro assays can also include cell-free assays that do not utilize intact cells.

[0244] The terms "bacteriophage" and "phage" are used interchangeably herein. [Example]

[0245] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0246] Example 1: Engineered phages used in this application Recombinant bacteriophages were engineered with gene deletions in the predicted lysogenic module region. Figure 1 shows the predicted lysogenic region of phage p1473. Additionally, Figure 1 shows open reading frames encoding putative suppressor and anti-repressor proteins, as well as an open reading frame encoding a putative integrase. The first recombinant phage had a gene deletion introduced into the predicted lysogenic module region of the bacteriophage genome in the region containing the putative anti-repressor; two clones were isolated and designated Var009 and Var010. The second recombinant phage had a gene deletion introduced into the predicted lysogenic module region of the bacteriophage genome in the region containing the putative repressor; one clone was isolated and designated Var012. A final recombinant phage (Var042) was isolated with a deletion from Var012 and a second deletion that removed the predicted integrase gene. Two additional control mutants were generated with different deletions outside the lysogenic region as controls (not shown), which were isolated and designated Var002 and Var006, respectively. Figure 6 shows the sequence of the Var010 deletion. Figure 7 shows the sequence of the Var012 deletion, which is also the first deletion of Var042. Figure 8 shows the second deletion of Var042. Additional phages used in this application are listed in Table 1.

[0247] [Table 1-1]

[0248] [Table 1-2]

[0249] Example 2: Engineered phage mutants that exhibit a lytic phenotype in Staphylococcus aureus Figure 2 shows a dilution series of wild-type (WT) p1473 and several mutants plated onto a lawn of S. aureus using the double agar overlay method. As shown, the WT phage and mutants Var002 and Var006 produced small, cloudy plaques, while mutants Var009, Var010, and Var012 produced larger, clear plaques. Mutants Var002 and Var006 contain mutations outside the lysogenic region. Mutants with mutations outside the lysogenic region did not show changes in plaque morphology, indicating that the phage mutants remained temperate. Var009, Var010, and Var012, which contain mutations in the lysogenic region, showed clearer plaques and higher plaque formation efficiency for the indicated strains. These data confirm that Var009, Var010, and Var012 were successfully converted from a lysogenic to a lytic phenotype, since clear plaque morphology is a hallmark of lytic bacteriophages in S. aureus (Mitarai et al., J. Bacteriol. 2016. doi:10.1128 / JB.00965-15; Garcia et al., Appl. Environ. Microbiol. 2009. doi:10.1128 / AEM.01864-09), a known phenomenon in other examples of temperate phages that have been genetically converted to obligately lytic. The ability of VarO10 and VarO12 to form plaques more efficiently (i.e., when the phage is further diluted further down the plate) may indicate that the modified phage are not subject to interference from endogenous prophage present in the bacterial strain, as previously observed with other phages after conversion from temperate to obligately lytic (see Zhang et al., Microbiol. 2013. doi:10.1099 / mic.0.067116-0). Figure 3 shows a magnified image of Figure 2, showing larger plaque morphology in wild-type p1473, VarO10, and VarO12. In Figure 3, arrows point to individual phage plaques. As shown, VarO10 and VarO12 plaques exhibit a more distinct morphology compared to the wild-type.Furthermore, distinct regions where plaques are too numerous to count are more apparent for VarO10 and VarO12 than for WT phage.

[0250] Example 3: Confirmation of the conversion from a lysogenic to a lytic phenotype by bacteriophage killing assay Figure 4 shows a bacteriophage killing assay using strain b4063 (USA300 strain FPR3757) in LB challenged with wild-type p1473, VarO12, or VarO42, as measured by bacterial counts as a function of time. For bacteriophage killing assays using strain b4063 (USA300 strain FPR3757), bacteria were inoculated into LB in triplicate. For cells alone, nothing else was added. For other phages, wild-type p1473, VarO12, or VarO42 was added at an MOI of 1. Bacterial CFU were counted 0, 2, 4, 6, and 24 hours after inoculation. As shown in Figure 4, at 24 hours, bacterial counts recovered in p1473 WT-treated bacteria, while the two mutants showed sustained killing down to the limit of detection (LOD). Temperate phages often exhibit rapid rebound due to the proliferation of lysogens that are resistant to phage infection, and the lack of rebound in VarO12 and VarO42 is consistent with the inability of these mutants to form lysogens.

[0251] Example 4: Comparison of rebound against clinical isolates of Staphylococcus aureus Figure 5 shows growth curves of three S. aureus clinical isolates (b2991, b3022, and b3202) in LB challenged with p1473WT or p1473 VarO12. Growth of the clinical isolates in LB was monitored by periodically taking the OD at 600 nm over a 24-hour period. Control growth curves are uninfected culture controls for each strain, and test growth curves are for cultures challenged with WT or VarO12 at an MOI of 1. Arrows indicate regrowth of the clinical isolates in the WT-challenged cultures, while the VarO12-challenged cultures remain inhibited for up to 24 hours. Overall, experimental comparisons of three S. aureus clinical isolates (b2991, b3022, and b3202) in LB loaded with p1473 WT or p1473 Var012 reveal more regrowth in strains loaded with WT phage, with b2991 showing the best recovery over 24 h.

[0252] Example 5. Phage lytic activity when engineered with complete CRISPR-Cas constructs A top agar overlay was prepared by mixing 100 μL of an overnight saturated culture of b4063 with 6 mL of 0.375% agar in LB containing 10 mM MgCl2 and 10 mM CaCl2. After the top agar solidified, 2 μL drops of a 10-fold dilution series of p1473wt (wild type), p1473-Cas (Cas system only), and p1473-crArray (targeted CRISPR array + Cas system) were spotted onto the surface of the top agar. The plate was incubated at 37°C for approximately 18 hours and then imaged at 4x and 10x magnification using a Keyence BZ-X800 microscope.

[0253] Example 6: Engineering lytic bacteriophages Bacteriophage were engineered using a type IB CRISPR-Cas system (LMIB) and a CRISPR array (SEQ ID NO: 24). Engineered phage lysates were spotted onto bacterial overlays to obtain isolated (clonal) plaques. Seven plaques were picked and screened by PCR for the presence of the desired insert. Each plaque was screened using two pairs of PCR primers: one pair covering the upstream engineered junction (i.e., the site where the wild-type phage genome intersects with the engineered insert) and the significant insert region, and the other pair covering the downstream engineered junction and the significant insert region. Because one primer from each primer pair binds within the insert, unengineered phage do not produce a PCR band. In Figure 9, L indicates a DNA size ladder, numbers 1 through 7 indicate individual clonal plaques screened, and a and b indicate two primer pairs. Both primer pairs produced bands of the expected size for all plaques, confirming that all plaques were successfully engineered with the LMIB insert (SEQ ID NO: 23).

[0254] InqQ (SEQ ID NO: 7), P cat -lnqQ (SEQ ID NO: 16, 7), P SarA -lnqQ (SEQ ID NO: 17, 7), or P TmpG After engineering the three promoter mutants of -lnqQ (SEQ ID NO: 18, 7) into Kaivirus phage p4815, polymerase chain reaction (PCR) was used to confirm the insertion of the promoter + InqQ into the phage. The SarA promoter used the SodB RBS (SEQ ID NO: 21). The PCR reaction used one primer upstream of the insertion site and one primer in the insertion sequence. The results are shown in Figure 10. Sequencing of the phage DNA confirmed the insertion of the mutants in the phage.

[0255] Clinical isolate b2655 was treated with wild-type (WT) phage (square), P cat -lnqQ, P SarA -lnqQ, or P TmpGIn each sample, three bacterial replicates were inoculated with phage at a multiplicity of infection of 1. Cultures were grown at 37°C in a shaking plate reader, and the optical density at 600 nm was measured every 10 minutes for 18 hours. The results are shown in Figure 11. Phage carrying InqQ reduced bacterial growth compared to WT phage, starting at approximately 80 minutes and persisting throughout the experiment.

[0256] Example 7: Engineered bacteriophage containing DNAse I The p1378 Kaivirus bacteriophage was engineered to contain DNAse I (SEQ ID NO: 1) and the Pcat promoter (SEQ ID NO: 16). The DNA sequence encoding mature human DNase was modified to optimize codon usage and remove sequences not tolerated by the phage while maintaining amino acid identity. This sequence was placed downstream of the Pcat promoter. The Pcat-DNase I sequence was engineered into the phage, and the sequence was confirmed by Illumina next-generation sequencing of the engineered phage genome. The results are shown in Figure 12.

[0257] Example 8: Engineering of p1494 phage The phytophage p1494 was isolated. Region e002 (var0022) was deleted as shown in Figure 13A. Figure 13B shows a bacteriophage killing assay using strain b4063 (USA300 strain FPR3757) in LB loaded with wild-type and engineered phage, as measured by bacterial counts over time. Growth curves of USA300 strain FPR3757 with either no phage / cells only (circles), wild-type p1494 (squares), or lytic p1494e002 (triangles) at a multiplicity of 1. At 0, 3, 6, and 24 hours postinfection, the cultures were diluted and plated on LB agar to count surviving bacteria. Data show the mean and standard error of the mean for three replicates. Open shapes represent time points where bacteria were not recovered and plotted at the limit of detection (LOD) of the assay. Closed shapes represent time points where bacterial colonies were counted. No bacteria were recovered at any time point with the lytic p1494e002 phage.

[0258] Figure 13C shows growth curves for Staphylococcus aureus isolate b2655 with either no phage / cells only (circles), wild-type p1498 (squares), or lytic p1498e001 (triangles) at a multiplicity of infection of 1. At 0, 2, 4, 8, and 24 hours postinfection, cultures were diluted and plated on LB agar to enumerate surviving bacteria. Data show the mean and standard error of the mean for three replicates. Open shapes represent time points where bacteria were not recovered and plotted at the limit of detection (LOD) of the assay. Closed shapes represent time points where bacterial colonies were enumerated.

[0259] Example 9: Bacteriophage killing assay for cocktails Growth curves from treatment of bacterial cultures with individual phages or a four-phage cocktail containing CK811, p1378, p1494e002, p2808, and p4815. Cultures were grown at 37°C with aeration, and bacterial growth was monitored throughout the 20-hour experiment by measuring the optical density at 600 nm every hour. Results are shown in Figures 14A-C. In strain b4735, an isolate from a bloodstream infection, three individual phages, p1494e002 (inverted triangle), p2808 (diamond), and p4815 (star), show a reduction in bacterial growth, while a sustained reduction is only observed with CK811 (square). In strain b2655, an isolate from a respiratory infection, three individual phages, p1378 (triangle), p1494e002 (green inverted triangle), and p4815 (star), show reduced bacterial growth. Sustained inhibition of bacterial growth is observed only in samples treated with the four-phage cocktail, CK811 (square). In strain b4681, an isolate from a bloodstream infection, three individual phages, p1378 (red triangle), p2808 (purple diamond), and p4815 (orange star), show reduced bacterial growth, but sustained inhibition is observed only with CK811 (black square).

[0260] Example 10: Host range of bacteriophage cocktails The host range of the bacteriophage cocktail was tested using a panel of 308 strains of S. aureus clinical isolates from bloodstream infections. 136 (44%) of the isolates were multidrug-resistant. The strains contained at least 69 different multilocus sequencing types (MLSTs), as shown in Table 2. MLSTs were based on sequence analysis of fragments derived from seven S. aureus housekeeping genes: arcC, aroE, glpF, gmk, pta, tpi, and yqiL. S. aureus strains were typed using the method described in Deurenberg RH, Vink C., Kalenic S., Friedrich AW, Bruggeman CA, Stobberingh EE. The molecular evolution of methicillin-resistant Staphylococcus aureus. Clin. Microbiol. Infect. 2007;13:222-235. doi:10.1111 / j.1469-0691.2006.01573.x. The S. aureus strains used were b004604, b004605, b004606, b004607, b004608, b004609, b004610, b004611, b004612, b004613, b004614, b004615, b004616, b004617, b004618, b004619, b004620, b004621, b004622, b004623, b004624, b004625, b004626, b004627, b004628, b004629, b004630, b004631, and b004632. 632, b004633, b004634, b004635, b004636, b004637, b004638, b004639, b 004640, b004641, b004642, b004643, b004644, b004645, b004646, b00464 7, b004648, b004649, b004650, b004651, b004652, b004653, b004654, b00 4655, b004656, b004657, b004658, b004659, b004660, b004661, b004662,b004663、b004664、b004665、b004666、b004667、b004668、b004669、b00467 0、b004671、b004672、b004673、b004674、b004675、b004676、b004677、b0046 78、b004679、b004680、b004681、b004682、b004683、b004684、b004685、b00 4686、b004687、b004688、b004689、b004690、b004691、b004692、b004693、b0 04694、b004695、b004696、b004697、b004698、b004699、b004700、b004701、 b004702、b004703、b004704、b004705、b004706、b004707、b004708、b004709 、b004710、b004711、b004712、b004713、b004714、b004715、b004716、b0047 17、b004718、b004719、b004720、b004721、b004722、b004723、b004724、b004 725、b004726、b004727、b004728、b004729、b004730、b004731、b004732、b0 04733、b004734、b004735、b004736、b004737、b004738、b004739、b004740、b 004741、b004742、b004743、b004744、b004745、b004746、b004747、b004748、b004749、b004750、b004751、b004752、b004753、b004754、b004755、b00475 6、b004757、b004758、b004759、b004760、b004761、b004762、b004763、b004764、b004765、b004766、b004767、b004768、b004769、b004770、b004771、b00 4772、b004773、b004774、b004775、b004776、b004777、b004778、b004779、b004780、b004781、b004782、b004783、b004784、b004785、b004786、b004787、b004788, b004789, b004790, b004791, b004792, b004793, b004794, b00479 5, b004796, b004797, b004798, b004799, b004800, b004801, b004802, b004 803, b004804, b004805, b004806, b004807, b004808, b004809, b004810, b0 04811, b004812, b004813, b004814, b004815, b004816, b004817, b004818, b 004819, b004820, b004821, b004822, b004823, b004824, b004825, b004826 , b004827, b004828, b004829, b004830, b004831, b004832, b004833, b0048 34, b004835, b004836, b004837, b004838, b004839, b004840, b004841, b00 4842, b004843, b004844, b004845, b004846, b004847, b004848, b004849, b0 04850, b004851, b004852, b004853, b004854, b004855, b004856, b004857, b004858, b004859, b004860, b004861, b004862, b004863, b004864, b00486 5, b004866, b004867, b004868, b004869, b004870, b004871, b004872, b004 873, b004874, b004875, b004876, b004877, b004878, b004879, b004880, b00 4881, b004882, b004883, b004884, b004885, b004886, b004887, b004888, b 004889, b004890, b004891, b004892, b004893, b004894, b004895, b004896 , b004897, b004898, b004899, b004900, b004901, b004902, b004903, b004904, b004905, b004906, b004907, b004908, b004909, b004910, b004911. ,

[0261] [Table 2]

[0262] The results are presented in Table 3. The ability of Kaivirus, Fietavirus, and Rosenblumvirus to target the 308 strains in the panel was tested. As can be seen in Figure 15, for the majority of strains tested, at least two phages infected a particular target bacterium, reducing the possibility that the target bacterium could develop resistance to a particular phage and avoid infection by another bacteriophage.

[0263] [Table 3]

[0264] Example 11: Comparison of rebound against clinical isolates of Staphylococcus aureus Growth of clinical isolate b4681 in LB was monitored by periodically measuring the OD at 600 nm over a 24-hour period. Control growth curves are uninfected culture controls for each strain, and test growth curves are for cultures challenged with phage at an MOI of 1. Figures 16A-16E show growth curves for S. aureus clinical isolate b4681 in LB challenged with p1378, p4815, p2808, p1494e002, or the tri-genera cocktail. B4681 showed rebound or no hits with all single-phage treatments but not with the tri-genera phage cocktail.

[0265] Example 12: Comparison of rebound against clinical isolates of Staphylococcus aureus Growth of clinical isolates b4681, b4787, b4655, and b4689 in LB was monitored by periodically taking the OD at 600 nm over a 24-hour period. Control growth curves are uninfected culture controls for each strain, and test growth curves are for cultures challenged with phage at an MOI between 0.1 and 1.

[0266] Figures 17A-17F show the growth curves for b4681. Figures 18A-18F show the growth curves for b4787. Treatment with p1378 or p2808 resulted in growth of b4681 or b4787 over time (Figures 17A-17B, 18A-18B). Treatment with a two-generic cocktail (p1378, p2808) resulted in suppression of bacterial growth (Figures 17C, 18C). Treatment with p4815 or p498e001 further allowed growth of b4681 or b4787, whereas treatment with a three-generic cocktail (p1378, p2808, p4815, p498e001) resulted in reduced resistant growth (Figures 17D-17F, 18D-18F).

[0267] Growth of clinical isolates b4655 and b4689 demonstrates the additive effect of the cocktails. The growth curve for b4655 is shown in Figures 19A-19F, and the growth curve for b4689 is shown in Figures 20A-20F. Treatment with p1378 or 02808 reduced the growth of the clinical isolates (Figures 19A-19B, 20A-20B), but treatment with the two-genera cocktail was able to more rapidly suppress growth (Figures 19C, 20C). Treatment of b4655 with p4815 or p1498e001, or treatment of b4655 with p4815, also resulted in growth, but treatment with p1498e001 was able to inhibit the growth of b4689 (Figures 19D-19E, 20D-20E), and treatment with the three-genera cocktail rapidly suppressed growth (Figures 19F, 20F).

[0268] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0269]

Table 4

[0270]

Table 5-1

[0271]

Table 5-2

[0272]

Table 6-1

[0273]

Table 6-2

[0274]

Table 6-3

[0275]

Table 6-4

[0276]

Table 6-5

[0277]

Table 6-6

[0278]

Table 6-7

[0279]

Table 6-8

[0280]

Table 6-9

[0281]

Table 6-10

[0282]

Table 6-11

[0283]

Table 6-12

[0284]

Table 7

[0285]

Table 8-1

[0286]

Table 8-2

[0287]

Table 8-3

[0288]

Table 8-4

[0289]

Table 8-5

[0290]

Table 8-6

[0291]

Table 8-7

[0292]

Table 8-8

Claims

1. A composition comprising at least two bacteriophages, wherein a first bacteriophage is at least 80% identical to p1378 or p4815 and a second bacteriophage is at least 80% identical to p1494e002 or p2808.

2. 2. The composition of claim 1, wherein the first bacteriophage is at least 80% identical to p1378 and the second bacteriophage is at least 80% identical to p1494e002.

3. 2. The composition of claim 1, wherein the first bacteriophage is at least 80% identical to p1378 and the second bacteriophage is at least 80% identical to p2808.

4. 2. The composition of claim 1, wherein the first bacteriophage is at least 80% identical to p4815 and the second bacteriophage is at least 80% identical to p1494e002.

5. 2. The composition of claim 1, wherein the first bacteriophage is at least 80% identical to p4815 and the second bacteriophage is at least 80% identical to p2808.

6. 3. The composition of claim 2, comprising a third bacteriophage that is at least 80% identical to said p4815.

7. 7. The composition of claim 6, comprising a fourth bacteriophage that is at least 80% identical to said p2808.

8. 4. The composition of claim 3, comprising a third bacteriophage that is at least 80% identical to said p4815.

9. 5. The composition of claim 4, comprising a third bacteriophage that is at least 80% identical to said p2808.

10. 1. A composition comprising a plurality of bacteriophages, including a first bacteriophage and a second bacteriophage, wherein bacteria treated with the plurality of bacteriophages exhibit reduced amounts of regrowth compared to treatment with the first bacteriophage or the second bacteriophage alone, and wherein the first bacteriophage comprises a Rosenblumvirus and the second bacteriophage comprises a Phietavirus or a Kayvirus.

11. 10. The composition of claim 1, wherein the amount of regrowth is measured by optical density (OD) at a wavelength of 600 nm.

12. 12. The composition of claim 10 or 11, wherein the amount of regrowth of bacteria treated with the plurality of bacteriophages 12 hours after treatment is less than or equal to about 50%, 40%, 30%, 20%, or 10% of the regrowth of bacteria treated with the first bacteriophage.

13. 12. The composition of claim 10 or 11, wherein the amount of regrowth of bacteria treated with the plurality of bacteriophages 12 hours after treatment is less than or equal to about 50%, 40%, 30%, 20%, or 10% of the regrowth of bacteria treated with the second bacteriophage.

14. The composition of any one of claims 10 to 13, wherein the bacterium is a Staphylococcus bacterium.

15. 15. The composition of claim 14, wherein the bacteria comprises Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus salivarius, Staphylococcus argenteus, Staphylococcus hemolyticus, or Staphylococcus schweitzeri, or any combination of two or more thereof.

16. The composition of claim 15 , wherein the bacteria comprises Staphylococcus aureus.

17. A bacteriophage that has been engineered to be made lytic by removal, replacement, or inactivation of a lysogenic sequence, wherein the lysogenic sequence comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of a sequence selected from SEQ ID NOs: 49-58.

18. 18. The bacteriophage of claim 17, which is an engineered phytovirus.

19. 19. A composition comprising the bacteriophage of claim 17 or 18, and further comprising a Rosenblum virus and / or a Kai virus.

20. A method for treating a disease or condition associated with staphylococci, comprising administering a bacteriophage described in any one of claims 1 to 9, 17 to 18, or a composition described in any one of claims 10 to 13 or 19 to a subject in need of treatment for said disease or condition.

21. 21. The method of claim 20, wherein the staphylococcus bacteria causes and / or contributes to the disease or illness.

22. A bacteriophage comprising at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1378 (PTA-127329).

23. A bacteriophage comprising at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p4815 (PTA-127331).

24. A bacteriophage comprising at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1494e002 (PTA-127345).

25. A bacteriophage comprising at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p2808 (PTA-127332).

26. 1. A composition comprising at least two bacteriophages, wherein a first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1378, and a second bacteriophage comprises at least one of p1381, p4815, p1378e062, p1378e074, p4815e037, p1378e0 75, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, p2808, or p1473.

27. 1. A composition comprising at least two bacteriophages, wherein a first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p4815, and a second bacteriophage comprises at least one of p1381, p1378, p1378e062, p1378e074, p4815e037, p1378e0 75, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, p2808, or p1473.

28. 28. The composition of claim 26 or 27, wherein the second bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p4815, 01494e002, or p2808.

29. 30. The composition of claim 28, further comprising a third bacteriophage comprising at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p4815, 01494e002, or p2808.

30. 30. The composition of claim 29, further comprising a fourth bacteriophage comprising at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p4815, 01494e002, or p2808.

31. 1. A composition comprising at least two bacteriophages, wherein a first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p1494e002 and a second bacteriophage comprises at least one of p1381, p1378, p4815, p1378e062, p1378e074, p4815e0 37, p1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p2808, or p1473.

32. A composition comprising at least two bacteriophages, wherein a first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with p2808, and a second bacteriophage comprises at least one of p1381, p1378, p4815, p1378e062, p1378e074, p4815e037, p 1378e075, p1381e017, p4815e053, p1498e001, p3693e001, p3224e002, p5593e001, p1468e003, p1478e003, p1494e002, or p1473.

33. 33. The composition of any one of claims 26-32, wherein the at least two bacteriophages of the composition infect at least about 90% of a population of at least about 30 Staphylococcus bacteria.

34. 34. The composition of claim 33, wherein the at least about 90% infection is determined by a plaque assay or a growth inhibition assay.

35. The composition of any one of claims 33-34, wherein said at least about 90% is at least about 95%.

36. The composition of any one of claims 33-34, wherein said at least about 90% is at least about 98%.

37. The composition of any one of claims 33-34, wherein said at least about 90% is at least about 99%.

38. The staphylococcus bacteria consortium is 8, 5, 22, 15, 1, 30, 398, 105, 45, 672, 2250, 582, 72, 97, 239, 34, 87, 101, 109, 1159, 1165, 1181, 12, 121, 152, 1750, 20, 225, 25, 291, 3628, 59, 7, 779, 88, 10, 1011, 1049, 1156, 1351, 149, 1637, 16 38. The composition of any one of claims 33 to 37, comprising a Staphylococcus bacterium having an MLST of 49, 1757, 1842, 188, 1970, 2066, 256, 2867, 2945, 3149, 3182, 3510, 39, 395, 4317, 47, 4730, 50, 508, 573, 6, 630, 737, 828, 848, 923, or 93, or a combination of two or more thereof.

39. 39. The composition of any one of claims 33 to 38, wherein the population of Staphylococcus bacteria comprises bacteria isolated from a bloodstream infection.

40. 40. The composition of any one of claims 33-39, wherein at least about 40% of the population of Staphylococcus bacteria are multidrug resistant.

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