Bacteriophages with improved antimicrobial activity

Abstract

Provided herein are bacteriophages engineered to express an exopolysaccharide (EPS) depolymerase for treating bacterial infections. In some embodiments, the EPS depolymerase comprises an alginate lyase. Also contemplated within the scope of the invention are compositions comprising one or more of the bacteriophages, methods for treating bacterial infections, and kits comprising the compositions described herein.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Patent Application No. 63 / 228,504, filed Aug. 2, 2021, which is incorporated by reference in its entirety for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on July 27, 2022, has a file name of "054249-520001WO_SL_ST26" and is 3.51 bytes in size. [Background technology]

[0003] With an increasing number of bacterial strains resistant to traditional small molecule antibiotic treatment regimes, there is a growing demand for alternative antibiotics. Bacteriophage therapy uses bacterial viruses or phages to target and destroy bacteria at various infection sites. Recent advances in biotechnology have allowed for the rapid expansion of existing phage libraries to create potent and specific phages that can target and destroy bacteria of interest. Pseudomonas aeruginosa (PA) is an opportunistic pathogen that can cause severe chronic and acute infections, especially in immunocompromised patients, and has the ability to form biofilms. Furthermore, strains of PA with antibiotic resistance exist, increasing the difficulty of treating these chronic infections. Occasionally, PA infections can occur in the presence of cystic fibrosis (CF) or noncystic fibrotic bronchiectasis (NCFB). Bacteriophage therapy approaches that circumvent traditional mechanisms of antibiotic resistance, prevent the toxic side effects of traditional small molecule therapies, and can be effective against biofilms have become particularly attractive. Summary of the Invention

[0004] Alginate, the main component of Pseudomonas aeruginosa biofilms, is a polysaccharide with two units, β-D-mannuronic acid (M) and α-L-guluronic acid (G). These units can be linked as homopolymers (polyG, polyM) or heteropolymers (polyM / G). Overproduction of alginate (also called "mucoid type") is particularly common in NCFB. Alginate lyases, which degrade alginate, have been classified into seven subfamilies of polysaccharide lyases (PL).

[0005] The activity of Sphingomonas A1-III, a member of the PL5 family, has been previously investigated. A1-III is known to degrade poly-M alginate into di- and trisaccharides. When expressed in two phage families, A1-III was shown to have activity against preformed biofilms of mucoid P. aeruginosa. The Alg2A version of alginate lyase, which belongs to the PL7 family and is expressed by Flavobacterium, was also expressed. Alg2A has previously been shown to degrade both poly-G and poly-M and showed higher activity against P. aeruginosa flora than A1-III. Furthermore, Alg2A improved antibiotic treatment of P. aeruginosa biofilms. Therefore, Alg2A was cloned and expressed from phage to compare its activity with that of A1-III. The data presented herein indicate that Alg2A has a stronger degradative effect on P. aeruginosa biofilms than A1-III.

[0006] Described herein are bacteriophages, compositions of bacteriophages, phage combinations, and their uses in medical and non-medical applications, including the treatment of bacterial infections and diseases.

[0007] In one aspect, the disclosure provides a bacteriophage engineered to express an exopolysaccharide (EPS) depolymerase.

[0008] In some embodiments, the EPS depolymerase is expressed from a nucleotide sequence selected from SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:36, or SEQ ID NO:59, or a sequence having at least 90% identity to the sequence of SEQ ID NO:20, at least 90% identity to the sequence of SEQ ID NO:21, at least 90% identity to the sequence of SEQ ID NO:22, at least 90% identity to the sequence of SEQ ID NO:23, at least 90% identity to the sequence of SEQ ID NO:24, at least 90% identity to the sequence of SEQ ID NO:25, at least 90% identity to the sequence of SEQ ID NO:36, or at least 90% identity to the sequence of SEQ ID NO:59.

[0009] In some embodiments, the EPS depolymerase is an alginate lyase. In some embodiments, the alginate lyase comprises Alg2A or A1-III. In some embodiments, the alginate lyase comprises Alg2A. In some embodiments, the alginate lyase comprises A1-III.

[0010] In some embodiments, the bacteriophage exhibits improved host range.

[0011] In some embodiments, the bacteriophage belongs to the PhiKMV virus genus. In some embodiments, the bacteriophage belongs to the Pakpunavirus genus. In some embodiments, the bacteriophage belongs to the Bruynoghevirus genus. In some embodiments, the bacteriophage belongs to the Pubnavirus genus.

[0012] In some embodiments, the bacteriophage targets Pseudomonas aeruginosa. In some embodiments, the bacteriophage targets one or more of Pseudomonas aeruginosa, antibiotic-resistant Pseudomonas aeruginosa, and multi-antibiotic-resistant Pseudomonas aeruginosa. In some embodiments, the bacteriophage infects and kills one or more of Pseudomonas aeruginosa, antibiotic-resistant Pseudomonas aeruginosa, and multi-antibiotic-resistant Pseudomonas aeruginosa.

[0013] In some embodiments, the bacteriophage reduces biofilm mass.

[0014] In another aspect, the disclosure provides a bacteriophage composition comprising one or more bacteriophages expressing an exopolysaccharide (EPS) depolymerase, wherein the one or more bacteriophages are selected from the group consisting of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, 9, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:73, a polynucleotide sequence having at least 90% identity to SEQ ID NO:26, a polynucleotide sequence having at least 90% identity to SEQ ID NO:27, a polynucleotide sequence having at least 90% identity to SEQ ID NO:28 a polynucleotide sequence having at least 90% identity to SEQ ID NO:29, a polynucleotide sequence having at least 90% identity to SEQ ID NO:30, a polynucleotide sequence having at least 90% identity to SEQ ID NO:31, a polynucleotide sequence having at least 90% identity to SEQ ID NO:32, a polynucleotide sequence having at least 90% identity to SEQ ID NO:33, a polynucleotide sequence having at least 90% identity to SEQ ID NO:34, a polynucleotide sequence having at least 90% identity to SEQ ID NO:35, a polynucleotide sequence having at least 90% identity to SEQ ID NO:37, a polynucleotide sequence having at least 90% identity to SEQ ID NO:38, a polynucleotide sequence having at least 90% identity to SEQ ID NO:39, a polynucleotide sequence having at least 90% identity to SEQ ID NO:40, a polynucleotide sequence having at least 90% identity to SEQ ID NO:41, a polynucleotide sequence having at least 90% identity to SEQ ID NO:42, a polynucleotide sequence having at least 90% identity to SEQ ID NO:43,A polynucleotide sequence having at least 90% identity to SEQ ID NO:44, a polynucleotide sequence having at least 90% identity to SEQ ID NO:45, a polynucleotide sequence having at least 90% identity to SEQ ID NO:46, a polynucleotide sequence having at least 90% identity to SEQ ID NO:47, a polynucleotide sequence having at least 90% identity to SEQ ID NO:48, a polynucleotide sequence having at least 90% identity to SEQ ID NO:49, a polynucleotide sequence having at least 90% identity to SEQ ID NO:50, a polynucleotide sequence having at least 90% identity to SEQ ID NO:51, a polynucleotide sequence having at least 90% identity to SEQ ID NO:52, a polynucleotide sequence having at least 90% identity to SEQ ID NO:53, a polynucleotide sequence having at least 90% identity to SEQ ID NO:54, a polynucleotide sequence having at least 90% identity to SEQ ID NO:55, a polynucleotide sequence having at least 90% identity to SEQ ID NO:56, a polynucleotide sequence having at least 90% identity to SEQ ID NO:57, a polynucleotide sequence having at least 90% identity to SEQ ID NO:58 a polynucleotide sequence having at least 90% identity to SEQ ID NO:60, a polynucleotide sequence having at least 90% identity to SEQ ID NO:61, a polynucleotide sequence having at least 90% identity to SEQ ID NO:62, a polynucleotide sequence having at least 90% identity to SEQ ID NO:63, a polynucleotide sequence having at least 90% identity to SEQ ID NO:64, a polynucleotide sequence having at least 90% identity to SEQ ID NO:65, a polynucleotide sequence having at least 90% identity to SEQ ID NO:66, a polynucleotide sequence having at least 90% identity to SEQ ID NO:67, a polynucleotide sequence having at least 90% identity to SEQ ID NO:68, a polynucleotide sequence having at least 90% identity to SEQ ID NO:69, a polynucleotide sequence having at least 90% identity to SEQ ID NO:70, a polynucleotide sequence having at least 90% identity to SEQ ID NO:71, or a polynucleotide sequence having at least 90% identity to SEQ ID NO:73.

[0015] In some embodiments, the EPS depolymerase is an alginate lyase.

[0016] In some embodiments, one or more of the bacteriophages are engineered, hi some embodiments, two or more of the bacteriophages are engineered.

[0017] In some embodiments, the second bacteriophage of the one or more bacteriophages comprises a naturally occurring phage, hi some embodiments, two or more bacteriophages of the one or more bacteriophages are naturally occurring phages.

[0018] In some embodiments, at least one of the bacteriophages targets Pseudomonas aeruginosa.

[0019] In some embodiments, the one or more bacteriophages of the composition target one or more of P. aeruginosa, antibiotic-resistant P. aeruginosa, and multi-antibiotic-resistant P. aeruginosa.

[0020] In some embodiments, one or more bacteriophages of the composition infect and kill one or more of P. aeruginosa, antibiotic-resistant P. aeruginosa, and multi-antibiotic-resistant P. aeruginosa.

[0021] In some embodiments, the composition further comprises a storage medium for storage at room temperature or at a temperature below 8° C. In some embodiments, the composition is stored at a temperature ranging from −20° C. to 25° C. In some embodiments, the composition is stored at 2° C. to 8° C. In some embodiments, the composition is stored at room temperature. In some embodiments, the storage medium is for storage at 4° C., 0° C., −20° C., or −80° C.

[0022] In some embodiments, the preservation medium comprises a cryoprotectant. In some embodiments, the cryoprotectant comprises glycerol. In some embodiments, the composition comprises about 5% to about 50% glycerol. In some embodiments, the preservation medium comprises about 20% glycerol. In some embodiments, the cryoprotectant comprises sucrose. In some embodiments, the composition comprises about 5% to about 30% sucrose. In some embodiments, the composition comprises about 10% sucrose. In some embodiments, the cryoprotectant comprises dimethyl sulfoxide (DMSO). In some embodiments, the DMSO is at a concentration of 2% to 10%.

[0023] In some embodiments, the composition further comprises a pharma- ceutically acceptable carrier, diluent, excipient, or a combination thereof.

[0024] In some embodiments, the composition is a liquid, semi-liquid, solid, frozen, or lyophilized formulation.

[0025] In some embodiments, the composition is at 1×10 per milliliter. 8 ~1×10 12 Contains PFU of each bacteriophage.

[0026] In some embodiments, one or more bacteriophages of the composition reduce biofilm mass.

[0027] In yet another aspect described herein, there is provided herein a method for treating a Pseudomonas aeruginosa infection comprising administering any of the compositions described herein to a subject in need of treatment for a Pseudomonas aeruginosa infection.

[0028] In some embodiments, the composition contains at least 3×10 per dose. 8 The total bacteriophage dose is administered in PFU.

[0029] In some embodiments, the method further comprises administration of an antibiotic, hi some embodiments, the antibiotic is selected from the group consisting of fluoroquinolones, carbapenems, aminoglycosides, ansamycins, cephalosporins, penicillins, beta-lactams, beta-lactamase inhibitors, folate pathway inhibitors, fucidanes, glycopeptides, glycylcyclines, lincosamides, lipopeptides, macrolides, quinolones, oxazolidinones, phenicol phosphonic acid, streptogramins, tetracyclines, sulfonamides, imipenem, meropenem, amikacin, ciprofloxacin, levofloxacin, tobramycin, azithromycin, aztreonam, colistin, inhaled tobramycin, inhaled aztreonam, and inhaled colistin.

[0030] In some embodiments, the methods further include administration of one or more CFTR modulators. In embodiments, the CFTR modulator can be selected from, but is not limited to, ivacaftor, lumacaftor / ivacaftor, tezacaftor / ivacaftor, elexacaftor / tezacaftor / ivacaftor, or any combination thereof.

[0031] In some embodiments, the bacterial infection is resistant to one or more antibiotics selected from fluoroquinolones, carbapenems, aminoglycosides, ansamycins, cephalosporins, penicillins, beta-lactams, beta-lactamase inhibitors, folate pathway inhibitors, fucidanes, glycopeptides, glycylcyclines, lincosamides, lipopeptides, macrolides, quinolones, oxazolidinones, phenicol phosphonic acids, streptogramins, tetracyclines, sulfonamides, imipenem, meropenem, amikacin, ciprofloxacin, levofloxacin, tobramycin, azithromycin, aztreonam, colistin, inhaled tobramycin, inhaled aztreonam, and inhaled colistin.

[0032] In some embodiments, the bacteriophage compositions are administered by inhalation. In some embodiments, the bacteriophage compositions are administered by nebulization. In some embodiments, the bacteriophage compositions are administered intravenously.

[0033] In some embodiments, the bacteriophage composition is administered at least once a day, hi some embodiments, the bacteriophage composition is administered for at least one day.

[0034] In some embodiments, the subject is a human.

[0035] In some embodiments, the subject has cystic fibrosis (CF). In some embodiments, the subject has non-cystic fibrosis bronchiectasis (NCFB).

[0036] In yet another aspect, the disclosure also provides an assay for determining alginate lyase activity of an engineered bacteriophage, comprising administering an effective amount of any of the engineered bacteriophages described herein to a Pseudomonas aeruginosa biofilm and determining a reduction in biofilm mass.

[0037] In yet another aspect, the disclosure provides a method for treating a bacterial infection, comprising: (a) selecting a subject having a bacterial infection; and (b) administering to the subject an effective amount of any of the bacteriophages described herein, or any of the bacteriophage compositions described herein, thereby treating the bacterial infection. In some embodiments, the subject is selected based on having undergone a previous treatment for the bacterial infection. For example, the subject is selected based on having already undergone at least one round of antibiotic treatment for the infection that did not completely clear the infection, or based on having an infection with a bacteria that is resistant to one or more antibiotics.

[0038] In some embodiments, the bacterial infection is a Pseudomonas infection. In some embodiments, the bacterial infection is a Pseudomonas aeruginosa infection.

[0039] In some embodiments, the bacterial infection is characterized by a biofilm.

[0040] In some embodiments, the subject has cystic fibrosis (CF). In some embodiments, the subject has non-cystic fibrosis bronchiectasis (NCFB).

[0041] In some embodiments, the composition contains at least 3×10 per dose. 8 The total bacteriophage dose is administered in PFU.

[0042] In some embodiments, the method further comprises administration of an antibiotic, hi some embodiments, the antibiotic is selected from the group consisting of fluoroquinolones, carbapenems, aminoglycosides, ansamycins, cephalosporins, penicillins, beta-lactams, beta-lactamase inhibitors, folate pathway inhibitors, fucidanes, glycopeptides, glycylcyclines, lincosamides, lipopeptides, macrolides, quinolones, oxazolidinones, phenicol phosphonic acid, streptogramins, tetracyclines, sulfonamides, imipenem, meropenem, amikacin, ciprofloxacin, levofloxacin, tobramycin, azithromycin, aztreonam, colistin, inhaled tobramycin, inhaled aztreonam, and inhaled colistin.

[0043] In some embodiments, the methods further include administration of one or more CFTR modulators. In embodiments, the CFTR modulator can be selected from, but is not limited to, ivacaftor, lumacaftor / ivacaftor, tezacaftor / ivacaftor, elexacaftor / tezacaftor / ivacaftor, or any combination thereof.

[0044] In some embodiments, the bacterial infection is resistant to one or more antibiotics selected from fluoroquinolones, carbapenems, aminoglycosides, ansamycins, cephalosporins, penicillins, beta-lactams, beta-lactamase inhibitors, folate pathway inhibitors, fucidanes, glycopeptides, glycylcyclines, lincosamides, lipopeptides, macrolides, quinolones, oxazolidinones, phenicol phosphonic acids, streptogramins, tetracyclines, sulfonamides, imipenem, meropenem, amikacin, ciprofloxacin, levofloxacin, tobramycin, azithromycin, aztreonam, colistin, inhaled tobramycin, inhaled aztreonam, and inhaled colistin.

[0045] In some embodiments, the bacteriophage composition is administered by inhalation, hi some embodiments, the bacteriophage composition is administered by nebulization.

[0046] In some embodiments, the bacteriophage composition is administered at least once a day, hi some embodiments, the bacteriophage composition is administered for at least one day.

[0047] In some embodiments, the subject is a human.

[0048] In yet another aspect, the disclosure provides a method for producing an engineered bacteriophage, the method comprising providing a bacteriophage and incorporating an exopolysaccharide (EPS) depolymerase into the bacteriophage.

[0049] In some embodiments, the EPS depolymerase is an alginate lyase. In some embodiments, the alginate lyase comprises Alg2A or A1-III. In some embodiments, the alginate lyase comprises Alg2A. In some embodiments, the alginate lyase comprises A1-III.

[0050] In some embodiments, the bacteriophage belongs to the PhiKMV virus genus. In some embodiments, the bacteriophage belongs to the Pakpunavirus genus. In some embodiments, the bacteriophage belongs to the Bruynoghevirus genus. In some embodiments, the bacteriophage belongs to the Pubnavirus genus.

[0051] In yet another aspect, the disclosure provides a kit comprising any of the bacteriophages described herein, or any of the bacteriophage compositions described herein, and instructions for their use.

[0052] In some embodiments, the kit further comprises an antibiotic.

[0053] In some embodiments, the kit further comprises a means for administering the bacteriophage or bacteriophage composition. In some embodiments, the means comprises a syringe, a transdermal patch, a sustained release device, a spray, a nebulizer, an inhaler, or a respirator. In some embodiments, the sustained release device comprises a mini-osmotic pump.

[0054] In some embodiments, the kit further comprises a second bacteriophage or bacteriophage composition.

[0055] In yet another aspect, the disclosure provides a bacteriophage composition comprising one or more bacteriophages engineered to express an exopolysaccharide (EPS) depolymerase, wherein the one or more bacteriophages belong to the PhiKMV virus genus, the Pakpunavirus genus, the Bruynoghevirus genus, and / or the Pubunavirus genus.

[0056] Embodiments will now be described, by way of example only, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0057] [Figure 1] FIG. 1 shows the different extent of bacterial clearance by different variants of engineered bacteriophage of SEQ ID NO:2 against pre-formed biofilms compared to saline control (PBS). [Diagram 2] Figure 4 shows the alginate lyase activity of different transgenic phages. (A) Top view of phages spotted onto preformed lawns of a mucoid alginate-expressing strain of P. aeruginosa (strain 15844) and activity is shown as a crater or depression in the lawn at the application site. (B) Bottom view of the same plate as in 4A and phages spotted onto preformed lawns of a mucoid alginate-expressing strain of P. aeruginosa (strain 15844) and activity is shown as a circular depression at the application site. [Diagram 3] 1 shows detection of alginate lyase (AlgL) A1-III-His6 in lysates of APBP1-1, APBP1-2, and APBP3-2 by Western blotting. [Figure 4] The positions of the N-terminal fragment A1-III of alginate lyase Aly from Sphingomonas sp. and several variations of this fragment are shown. [Diagram 5]The difference in expression and activity between A1-III fragments 70-399 and 54-412 is shown. (A) shows a Western blot of the 70-399 fragment, showing that the expression of this fragment is low in E. coli. (B) shows a Western blot of the 54-412 fragment, showing that this fragment is well expressed in E. coli. (C) shows the activity of different clones on 2% seaweed alginate plates. A1-C7 is a clone with the correct sequence of A1-III with a C-terminal His tag. A1-C4 is a clone with a frameshift mutation that does not result in expression of A1-III. Expression of A1-III was induced in mid-log phase after incubation with 0.5 mM IPTG for 30 min at room temperature (without shaking). A1-III-His6 was purified from cell lysates using Ni-NTA affinity columns and spotted onto plates containing 2% alginate along with alginate lyase commercially available from Sigma-Aldrich as a control. (D) shows the activity of A1-III protein expressed and purified from clone A1-C7 against a preformed clone of a mucoid alginate-expressing strain of P. aeruginosa (strain 15840). AL10 is 10 ng of alginate lyase purchased from Sigma-Aldrich as a control, A1-III 10 is 10 ng of purified A1-III, and 50, 1, 0.5, and 0.1 refer to the amount of A1-III spotted in ng. [Figure 6]We show that alginate lyase A1-III is expressed as a fusion protein with gp13 when integrated downstream of APBP4 gp13.1. (A) shows a Western blot (left) of lysates of engineered phage carrying the A1-III alginate lyase payload and a table (right) identifying the contents of each lane. Boxes indicate bands corresponding to the fusion protein. (B) shows the DNA sequence around the C-terminus of A1-III and explains how trans-reading of the TGA stop codon of A1-III-His6 gave rise to an in-frame fusion protein with gp13 via a 19 amino acid long "anti-terminator linker". (C) shows the predicted sequence of the A1-III-His6-linker-gp13 fusion protein generated in a lysate of APBP4-4. Residues in bold correspond to the A1-III-His6 sequence, those in underline correspond to gp13, those in grey indicate the putative linker, and the tryptophan residue highlighted in black is likely where cells misread the TGA stop codon in A1-III-His6. (D) APBP4-4 shows no activity against the mucoid flora of alginate-producing P. aeruginosa 15844 strain, indicating that the fusion protein is not functional. [Figure 7] Western blot of lysates of engineered phage carrying the A1-III alginate lyase payload (left) and a table identifying the contents of each lane (right). [Figure 8] The results of cloning two codon usage matrices of alginate lyase A1-III into phages are shown. (A) Sequence alignment between the two different codon usage matrices. (B) Alginate lyase activity of phages expressing each payload against a mucoid lawn of Pseudomonas aeruginosa 15844 strain. [Figure 9]Examples of alginate lyase activity profiles from lysates of engineered phages expressing different forms of A1-III are shown. (A) shows the activity of A1-III70-399 incorporated into APBP4-1. (B) shows the activity of A1-III70-399 incorporated into APBP17-1. (C) shows the activity of A1-III54-408 incorporated into APBP1-4. (D) shows the activity of A1-III54-408 incorporated into APBP4-5. (E) shows the activity of A1-III54-412-His6 incorporated into APBP18-1. (F) shows the activity of A1-III54-412-His6 incorporated into APBP1-2. (G) shows the activity of A1-III54-412-His6 incorporated into APBP3-6. (H) shows the activity of A1-III54-412-His6 incorporated into APBP1-1. (I) shows the activity of A1-III54-412-His6 incorporated into APBP3-2. [Figure 10] We show that flavobacterial Alg2A (accession no. AEB69783) expressed from multiple phages has alginate lyase activity whether it retains its N-terminal signal sequence, is tagged with a C-terminal His6-tag, or is encoded in four different genes. (A) shows an alignment of different versions of the Alg2A protein cloned into various phages. (B) shows alginate lyase activity by a phage expressing full-length Alg2A with a C-terminal His-tag on a mucoid lawn of the alginate-producing Pseudomonas aeruginosa 15844 strain. (C) shows alginate lyase activity of a phage expressing a shorter signal sequence deleted version of Alg2A23-288 that does not contain the His6-tag. (D) shows the percent identity between the different genes encoding Alg2A23-288 proteins and cloned into APBP3. FIG. 1(E) shows the alginate lyase activity of APBP3-derived phages expressing Alg2A23-288 of SEQ ID NO: 37, SEQ ID NO: 38, or SEQ ID NO: 39 against the mucoid flora of the alginic acid-producing Pseudomonas aeruginosa 15844 strain. [Figure 11]Figure 2 shows alginate lyase activity against pre-established mucoid lawns by a cocktail of phages incorporating either A1-III or Alg2A. [Figure 12] 1 shows that the growth of phages APBP1-1, APBP1-2, APBP3-1 and APBP3-2 is not altered by expression of alginate lyase protein A1-III. [Figure 13] The results of a host range improvement assay using different bacteriophages with or without alginate lyase activity are shown. Phage dilutions are increased 10-fold for each spot from top to bottom. (A) Each phage shows the same titer on their host, P. aeruginosa strain 7299. (B) On P. aeruginosa PS30, APBP3-1 shows clearing up to a dilution of 10-2, whereas its parent APBP3 does not form plaques on this strain. Similarly, APBP1-1 and APBP1-2 produce plaques on the host (P. aeruginosa PS30), whereas their parent APBP1 does not. (C) APBP1-1 and APBP1-2 show improved clearing on P. aeruginosa strain 7176 compared to their parent APBP1. (D) The apparent titers of APBP1-1 and APBP1-2 against P. aeruginosa 15843 strain are about 28 PFU / ml, whereas the apparent titer of APBP1 (their parent) is only about 10-fold lower at about 27 PFU / ml. (E) In P. aeruginosa 15839 strain, APBP3-1 shows clearing whereas its parent APBP3 does not form any plaques in this strain. APBP1-1 and APBP1-2 also produce plaques on the host, whereas their parent APBP1 does not. (F) APBP3-1 shows enhanced clearing compared to its parent APBP3 on this P. aeruginosa 15840 strain. APBP1-1 and APBP1-2 also show improved clearing compared to APBP1. [Figure 14] FIG. 13 is a graph showing the ability of different phage strains expressing no lyase (WT), A1-III (Eng-A1-III), or Alg2A (Eng-Alg2A) to disrupt biofilms. [Figure 15]The suitability of different loci in the APBP6 phage genome for integrating A1-III is shown. (A) Recombination between pLIX36 and APBP6 to integrate A1-III between gp038 and gp039. (B) Recombination between pLIX46 and APBP6 to integrate A1-III between gp005 and gp006. (C) Agarose gel showing insertion of A1-III into APBP6 when grown in a strain containing pLIX36 (left) and then passaged without pLIX36 (right). (D) Agarose gel showing insertion of A1-III into APBP6 when grown in a strain containing pLIX46 (left) and then passaged without pLIX46 (right). [Figure 16] Western blot showing expression of the alginate lyase protein Alg2A23-288 by engineered phages ABP4-6, ABP18-2, and ABP6-3 (left), and a table identifying the contents of each lane (right). [Figure 17] Western blot showing expression of alginate lyase proteins Alg2A1-288 and Alg2A23-288 by engineered phages APBP3-5 and APBP1-5, respectively (left), and a table identifying the contents of each lane (right). [Figure 18] Western blots of lysates of engineered phages carrying the A1-III54~412 or A1-III54~408 alginate lyase gene (left) and a table identifying the contents of each lane (right). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] As mentioned above, the world is facing an antibiotic crisis. Bacterial diseases are an ever-present concern, yet the rise of antibiotic resistance means that the number of effective antibiotics available is ever-shrinking. The embodiments and aspects of the present application provide innovative solutions to replace the use of standard antibiotics. These embodiments and inventions are the result of considerable and challenging inventive efforts and overcoming of technical challenges and obstacles.

[0059] As a result, the embodiments and aspects described herein generally relate to novel and inventive bacteriophages that are effective, for example, alone or in combination, to treat Pseudomonas infections. Methods of treating Pseudomonas bacterial infections are generally described, but methods of treating specific types of infections, such as respiratory infections, fibrosis, infections associated with pneumonia, etc. Storage and manufacturing of compositions and methods are also described. Various embodiments and aspects provide a novel and urgent solution to the worldwide antibiotic crisis.

[0060] It will be understood that the disclosure is not limited to the particular embodiments described, which may therefore vary widely. The scope of the disclosure will be limited only by the appended claims, and it will also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0061] The detailed description of the present disclosure is divided into different sections for the convenience of the reader only, and the disclosure content found in any section can be combined with the disclosure content of another section. Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0062] definition It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "bacteriophage composition" includes a plurality of such candidate agents, and reference to a "bacteriophage" is a reference to one or more bacteriophages and equivalents thereof known to those skilled in the art, and so forth.

[0063] In this disclosure, "comprises," "comprising," "containing," and "having," and similar terms, may have the meanings ascribed to them in U.S. Patent Law and may refer to "includes," "including," and similar terms. "Consisting essentially of" or "consists essentially of" have the meanings ascribed to them in U.S. Patent Law, but the term is open-ended, permitting the presence of more than what is recited, but excluding prior art embodiments, so long as the basic or novel characteristics of what is recited are not altered by the presence of more than what is recited. "Consisting of" means excluding more than trace elements and substantial method steps of other ingredients. Aspects defined by each of these transitional phrases are included within the scope of this disclosure.

[0064] As used herein, the term "consisting essentially of" means that only the explicitly indicated bacteriophage(s) is present in the bacteriophage composition, but that said composition may contain additional components other than the bacteriophage, such as, for example, a pharma- ceutical suitable carrier, diluent, excipient, antibiotic (e.g., chemical antibiotic), or combinations thereof.

[0065] As used herein, the term "about," when used before numerical designations, e.g., of temperature, time, amount, concentration, and the like (including ranges), indicates an approximation that may vary by (+) or (-) 10%, 5%, or 1%. All values ​​in this disclosure are preceded by the term "about" even if not explicitly stated.

[0066] When ranges (e.g., dosage ranges) are described herein, it is to be understood that the values ​​can include every individual value or range within the described range(s), including the endpoints.

[0067] As used herein, the terms "mutant" and "variant" are used interchangeably and refer to a bacteriophage that is genetically different from a reference bacteriophage, but still retains the ability to infect and kill a target bacterium, e.g., Pseudomonas aeruginosa. For example, a "mutant" may refer to a bacteriophage that is genetically mutated compared to one or more of SEQ ID NO:1-SEQ ID NO:11 and / or any of the other bacteriophages referenced or described herein, but still retains the ability to infect and kill the target bacterium, Pseudomonas aeruginosa. A mutant may include, for example, silent mutations, conservative mutations, minor deletions, and / or minor duplications of genetic material, and may retain phenotypic characteristics of the reference bacteriophage. In embodiments, a "mutant" may be a bacteriophage progeny. A bacteriophage progeny may be a bacteriophage obtained after lysing a Pseudomonas (e.g., Pseudomonas aeruginosa) target bacterium using a bacteriophage described herein (i.e., a "parent bacteriophage"). In other words, the bacteriophage progeny can be a second generation (or higher) bacteriophage. In one embodiment, the variant retains any observable characteristic or property dependent on the genome of the bacteriophage described herein, i.e., the phenotypic characteristics of said bacteriophage and / or the lytic activity against Pseudomonas species or strains. Preferred variants retain the ability to infect and kill P. aeruginosa target bacteria and have less than 10%, even more preferably less than 7%, more preferably less than 1% nucleic acid changes compared to the genome of the reference bacteriophage. Alternatively or in combination with the above, the variant preferably has less than 7% amino acid changes in the encoded polypeptide sequence compared to the polypeptide of the reference bacteriophage.

[0068] As used herein, the terms "percent identity", "percent sequence identity" and "percent identity" in reference to nucleic acid or amino acid sequences refer to the level of identity or homology between said sequences and can be determined by techniques well known in the art. Any of a wide range of sequence alignment methods can be used to determine percent identity, including but not limited to global, local and hybrid methods, such as segmental approaches. Protocols for determining percent identity are routine and within the skill of the artisan. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up the scores of each nucleotide pair and applying gap penalties. Non-limiting methods include, for example, CLUSTAL W (see Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680(1994)) and iterative refinement. Non-limiting methods include, for example, BLAST, Match-box (see, for example, Align-M, see, for example, Ivo Van Walle et al., Align-MA New Algorithm for Multiple Alignment of Highly Divergent Sequences, Bioinformatics 20(9):1428-1435 (2004)). This definition also refers to or can be applied to the complement of a test sequence. This definition also includes sequences that have deletions and / or additions, as well as sequences that have substitutions. As explained below, the preferred algorithm can take into account gaps and the like.Preferably, the identity exists over a region that is at least about 100 nucleotides in length, or more preferably over a region that is 100 to 1000 or more nucleotides in length.

[0069] As used herein (and as is well understood in the art), the term "treat" or "treatment" is used according to its plain and ordinary meaning and broadly includes any approach to obtain beneficial or desired results in a subject's condition, including clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, reduction in the extent of a disease, stabilization (i.e., not worsening) of disease pathology, prevention of disease transmission or spread, delay or slowing of disease progression, improvement or mitigation of disease pathology, reduction in disease recurrence, and remission, whether partial or complete, and whether detectable or undetectable. In other words, as used herein, "treatment" includes any cure, amelioration, or prevention of a disease. Treatment can prevent the onset of a disease, inhibit the spread of a disease, alleviate the symptoms of a disease, completely or partially eliminate the underlying cause of a disease, shorten the duration of a disease, or a combination thereof. As used herein, the terms "treat" or "therapeutic" are intended to encompass preventative treatment as well as corrective treatment (treatment of a subject already suffering from a disease).

[0070] As used herein, the term "administering" refers to, for example, oral, intravenous, parenteral, intraperitoneal, intramuscular, intrathecal, intranasal, intrapulmonary, or subcutaneous administration, or implantation of a sustained release device, such as, for example, a mini-osmotic pump, into a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, but is not limited to, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like. In embodiments, administration does not include administration of any active agent other than the active agent described. In embodiments, administration of the compositions described herein is by intravenous administration. In embodiments, administration of the compositions described herein is by intranasal administration, such as inhalation or spraying. In embodiments, administration may be pulmonary delivery by nasal or oral administration (eg, by aerosolization or nebulization).

[0071] "Co-administered" means that the compositions described herein are administered simultaneously, immediately before, or immediately after the administration of one or more additional therapies. The compounds provided herein may be administered to a patient alone or simultaneously. Co-administration is intended to include simultaneous or sequential administration of individual or combined compounds (compounds). Thus, the formulations may also be combined with other active substances (e.g., antibiotics) as needed.

[0072] As used herein, the term "lytic" or "lytic activity" refers to the property of a bacteriophage to cause the lysis of a bacterial cell. The lytic activity of a bacteriophage can be tested on bacteria (e.g., Pseudomonas aeruginosa strains) according to techniques well known in the art. The lytic cycle is named for the process that occurs when a phage infects a cell, replicates new phage particles, and ruptures the host's cell membrane. Some phages exhibit a lysogenic cycle in which the bacteriophage DNA is kept essentially dormant due to active inhibition of bacteriophage processes. Each time a bacterium divides, the phage's DNA is also copied. In this way, the virus can continue to replicate within the host without lysing it. At some point, when conditions change, the phage enters the lytic cycle. "Obligately lytic" refers to a phage that cannot enter the lysogenic cycle.

[0073] As used herein, the term "bacteriophage target" refers to any bacterial species that a particular bacteriophage can infect. The bacteriophage recognizes and binds to the cell surface of the target bacterium and injects its genetic material into the bacterial host. The genetic material from the infecting phage can be integrated into the bacterial genome. The bacteriophage can be lysogenic, in which case the viral genome is kept dormant within the bacterial host genome until a triggering event. The bacteriophage can also be lytic, in which case multiple copies of the infecting phage are produced by the machinery of the infecting bacterium, which are then released by lysis, extrusion, or budding. In an embodiment, the bacterial target is Pseudomonas aeruginosa.

[0074] As used herein, the term "bacterial host production strain" or "production strain" refers to a bacterium used to grow a bacteriophage. A method of bacteriophage production may require a production process that includes at least two functional units: host bacterium growth and bacteriophage propagation (or infection). Consideration of basic parameters of bacterial growth and phage infection, such as the substrate selected for the bacterium and the optimum temperature, is important for both bacterial growth and phage infection, as these factors may affect phage infectivity.

[0075] The use or method generally includes administering a bacteriophage or bacteriophage composition described herein to a subject. As used herein, the term "subject" or "patient" refers to a human or non-human animal. Preferably, the subject is a human. Preferably, the subject or patient is in need of treatment with the composition described herein, e.g., has a bacterial infection susceptible to treatment with the composition.

[0076] As used herein, the term "isolated" indicates that the bacteriophage is removed from its original environment in which it naturally occurs. In particular, an isolated bacteriophage is, for example, cultured and grown away from the environment in which it naturally occurs.

[0077] As used herein, the term "purified" indicates that the bacteriophage is removed from the natural and / or production host bacteria. In particular, purified bacteriophage has been substantially freed of production impurities, such as bacterial components, from its production or production environment. Bacterial components include, but are not limited to, bacterial host proteins, lipids, and / or bacterial endotoxins. The term "purified" may also refer to genetic purification, where a strain of bacteriophage is genetically homogenous. In some embodiments, purified bacteriophage includes bacteriophage that is at least 99% pure or includes at least 99% of a population of desired bacteriophage.

[0078] As used herein, the term "substantially purified" refers to a composition that contains less than 1%, less than 0.1%, less than 0.001%, or no detectable amounts of contaminants such as host bacterial proteins or endotoxins. Also, as used herein, the term "substantially pure" when used to describe a bacteriophage strain refers to the genetic purity of the composition such that the strain is greater than 99%, greater than 99.9%, greater than 99.999%, or greater than 100% of a particular genomic sequence.

[0079] Generally, a composition is substantially pure when at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% (or any subvalue or subrange therebetween) of the total material in a sample (by volume, wet or dry weight, or by mole percent or mole fraction) is free of impurities or genetic variants.

[0080] As used herein, a "synergistic amount" refers to the sum of a first amount (e.g., a bacteriophage) and a second amount (e.g., a different bacteriophage) that results in a synergistic effect (i.e., an effect that is greater than additive). Thus, the terms "synergy," "synergistic," "combined synergistic amount," and "synergistic therapeutic effect," as used interchangeably herein, refer to a measured effect of compounds administered in combination that is greater than the sum of the individual effects of each of the compounds provided herein administered alone as a single agent.

[0081] As used herein, the term "substantially free" refers to being free of less than 10% of the substance to be described. For example, the substance is 0.01% to 10% (including any subvalues ​​and subranges therein) (including endpoints), such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (or any subvalues ​​or subranges therebetween, including endpoints).

[0082] As used herein, the term "obtainable" also encompasses the term "obtained" as used herein. In one embodiment, the term "obtainable" means obtained.

[0083] As used herein, "pharmaceutical acceptable excipients", "pharmaceutical acceptable diluents" and "pharmaceutical acceptable carriers" refer to substances that aid in the administration and absorption of active agents to and by a subject and can be included in the compositions of the present disclosure without causing significant adverse toxic effects to the patient. Non-limiting examples of pharmaceutical acceptable excipients include water, NaCl, normal saline, lactated Ringer's solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, salt solutions (such as Ringer's solution), alcohols, oils, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and dyes. Such preparations can be sterilized and, if necessary, mixed with auxiliary substances such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, and / or aromatic substances that do not adversely react with the compounds of the present invention and the active agents. Those of skill in the art will recognize that other pharmaceutical excipients are useful in the present disclosure.

[0084] As used herein, the term "pharmaceutical acceptable salts" is intended to include salts of active compounds prepared using relatively non-toxic acids or bases, depending on the specific substituents found on the compounds described herein. When a compound of the present disclosure has a relatively acidic functional group, a base addition salt can be obtained by contacting a neutral form of such a compound with a sufficient amount of a desired base, either neat or in a suitable inert solvent. Examples of pharmaceutical acceptable base addition salts include sodium salts, potassium salts, calcium salts, ammonium salts, organic amino salts, or magnesium salts, or similar salts. When a compound of the present disclosure has a relatively basic functional group, an acid addition salt can be obtained by contacting a neutral form of such a compound with a sufficient amount of a desired acid, either neat or in a suitable inert solvent. Examples of pharma- ceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphate, dihydrogenphosphate, sulfuric, monohydrogensulfuric, hydroiodic, or phosphorous, as well as those derived from relatively non-toxic organic acids such as acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, etc. Also included are salts of amino acids such as arginates, and salts of organic acids such as glucuronic or galactunolonic acid.

[0085] As used herein, the term "persist" refers to the ability to exist or continue to exist beyond the usual, expected, or normal amount of time.

[0086] As used herein, "improved," "broadened," or "broader" with respect to the target range of a bacteriophage refers to an increase in host range. Host range is the number of cell types, strains, or host species that a virus / bacteriophage (or combination of viruses) can infect. An increase in host range or target bacterial range is an expansion of the absolute number of distinct cell types, cell lines, or cell species that a virus (or combination of viruses) can infect, compared to a reference and / or unmodified virus. In some examples, an increase in host range or an increase in target bacterial range is an increase in the number of bacterial strains or variants within a bacterial species that a virus (or combination of viruses) can infect. An increase in host range may be an increase in at least one or more strains, cell types, or species. Host range can be assayed, for example, by standard plaque assays well known in the art.

[0087] As used herein, "multiplicity of infection (MOI)" is the ratio of the number of virus particles to the number of host cells in a particular infection medium. A value of MOI=1 means that, on average, there is one host cell for every one phage particle.

[0088] As used herein, a "partially synthetic" phage refers to a phage in which a limited or percentage or substantial portion of the genome has been designed or engineered. As used herein, a "fully synthetic" phage refers to a phage in which the entire genome has been designed or engineered.

[0089] Further terms and phrases are defined below.

[0090] Engineered bacteriophages In some embodiments, the disclosure relates to one or more bacteriophages engineered to express heterologous genes. In some embodiments, the heterologous genes may include exopolysaccharide (EPS) depolymerase, an enzyme that breaks down polysaccharides into smaller fragments. Bacteriophages of the Podoviridae family often express so-called depolymerases as structural components of the virion. These enzymes are expressed as tail spike proteins. After specific binding to the capsular polysaccharide, exopolysaccharide (EPS) or lipopolysaccharide (LPS) of the host bacterium, the repeating units of the polysaccharide are specifically cleaved. Finally, the phage reaches the outer membrane and infects the cell using machinery that allows it to inject its DNA through both the membrane and the cell wall.

[0091] Depolymerases are structural components of the adsorption apparatus and facilitate the binding and digestion of the capsule. Their name indicates that the repeating units of the polysaccharide are cleaved and disintegrated. Biochemically, depolymerases are divided into two groups: lyases and hydrolases. Lyases, in contrast to hydrolases, cleave their substrate non-hydrolytically, meaning that no water molecules are released after substrate cleavage. Many of the well-characterized phage-encoded depolymerases that target EPS or LPS O-polysaccharides are lyases (Tomlinson and Taylor, 1985; Linnerborg et al., 2001; Olszak et al., 2017). These depolymerases generally have a large diversity of substrate specificities. However, specific cleavage sites exist in different polysaccharides, allowing the enzyme to act on two different substrates. The term depolymerase can refer generally to any protein that can degrade a polymer. In this respect, phage-encoded endolysins are also depolymerases, since they cleave the bacterial polysaccharide peptidoglycan in general in a hydrolase-like manner ( Schmelcher and Loessner, 2016 ).

[0092] In some embodiments, the EPS depolymerase comprises alginate lyase. Alginate is a linear polysaccharide that has been isolated from a wide range of organisms, from plants and bacteria to fungi. Alginate is also the main component of the cell wall of brown algae and the main source of fixed carbon for other organisms. Alginate lyase belongs to the family of lyases, specifically carbon-oxygen lyases that act on polysaccharides, and catalyzes the decomposition of alginate into various monosaccharide and polysaccharide products. The systematic name for this enzyme class is poly(β-D-1,4-mannuronide) lyase. Other commonly used names include, but are not limited to, alginate lyase I, alginate lyase, arginase I, arginase II, arginase, and alginate lyase III. This enzyme is involved in fructose and mannose metabolism.

[0093] In some embodiments, the disclosure provides a bacteriophage engineered to express Alg2A. In some embodiments, the disclosure provides a bacteriophage engineered to express A1-III. In some embodiments, the EPS depolymerase sequence described herein may comprise a portion or functional fragment of an EPS depolymerase gene. In some embodiments, the EPS depolymerase sequence may comprise the entire EPS depolymerase gene.

[0094] As used herein, the term "functional fragment" or "functional variant" refers to a molecule, including, for example, a nucleic acid or protein, that contains a nucleotide and / or amino acid sequence that has one or more nucleotide and / or amino acid sequence changes compared to the nucleotide and / or amino acid sequence of a parent or reference molecule. For proteins, functional variants are still capable of functioning in a manner similar to the parent molecule. In other words, modifications in the amino acid and / or nucleotide sequence of a parent molecule do not significantly affect or change the functional characteristics of the molecule encoded by the nucleotide sequence or containing the amino acid sequence. Functional variants may have conservative sequence modifications, including nucleotide and amino acid substitutions, additions, and deletions. These modifications can be introduced by standard techniques well known in the art, such as direct DNA synthesis, site-directed mutagenesis, and random mutagenesis by PCR. Functional variants also include, but are not limited to, derivatives that have significant similarity in primary structural sequence but that contain, for example, chemical and / or biochemical in vitro or in vivo modifications not found in the parent molecule. Such modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, formation of a GPI anchor, hydroxylation, iodination, methylation, myristoylation, oxidation, PEGylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination, among others.

[0095] The EPS depolymerase may be from any organism that expresses an EPS depolymerase, in some embodiments, the EPS depolymerase may be from a bacterial genus selected from, but not limited to, a member of the genus Sphingomonas, Flavobacterium, Pseudomonas, Klebsiella, Corynebacterium, Alteromonas, Zobelia, Aplysia, Vibrio, Saccharophagus, Stenotrophomonas, Streptomyces, Shewanella, Agrobacterium, and / or Azotobacter. Specifically, the EPS depolymerase may be derived from, but is not limited to, Sphingomonas (accession number BAB03312), Flavobacterium (accession number AEB69783), Pseudomonas (accession number 1VAV), Klebsiella (accession number 4OZX), Corynebacterium (accession number 1UAI), Alteromonas (accession number 1J1T), Zobelia (accession numbers 3ZPY, 4BE3), Pseudoalteromonas (accession number 4Q8K); The serotype may be derived from Aplysia (accession number 5GMT), Vibrio (accession number WP_017072010.1), Saccharofagus (accession number WP_011469755.1), Stenotrophomonas (accession number WP_049467230.1), Streptomyces (accession number NED67686.1), Shewanella (accession number WP_188926150.1), Agrobacterium (accession number WP_046801053.1), or Azotobacter (accession number Q9ZFG9).

[0096] In some embodiments, the EPS depolymerase is encoded by the nucleotide sequence of SEQ ID NO:20. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 90% identity to the sequence of SEQ ID NO:20. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 91% identity to the sequence of SEQ ID NO:20. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 92% identity to the sequence of SEQ ID NO:20. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 93% identity to the sequence of SEQ ID NO:20. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 94% identity to the sequence of SEQ ID NO:20. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 95% identity to the sequence of SEQ ID NO:20. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 96% identity to the sequence of SEQ ID NO:20. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 97% identity to the sequence of SEQ ID NO: 20. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 98% identity to the sequence of SEQ ID NO: 20. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 99% identity to the sequence of SEQ ID NO: 20.

[0097] In some embodiments, the EPS depolymerase is encoded by the nucleotide sequence of SEQ ID NO:21. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 90% identity to the sequence of SEQ ID NO:21. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 91% identity to the sequence of SEQ ID NO:21. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 92% identity to the sequence of SEQ ID NO:21. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 93% identity to the sequence of SEQ ID NO:21. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 94% identity to the sequence of SEQ ID NO:21. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 95% identity to the sequence of SEQ ID NO:21. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 96% identity to the sequence of SEQ ID NO:21. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 97% identity to the sequence of SEQ ID NO: 21. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 98% identity to the sequence of SEQ ID NO: 21. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 99% identity to the sequence of SEQ ID NO: 21.

[0098] In some embodiments, the EPS depolymerase is encoded by the nucleotide sequence of SEQ ID NO:22. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 90% identity to the sequence of SEQ ID NO:22. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 91% identity to the sequence of SEQ ID NO:22. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 92% identity to the sequence of SEQ ID NO:22. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 93% identity to the sequence of SEQ ID NO:22. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 94% identity to the sequence of SEQ ID NO:22. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 95% identity to the sequence of SEQ ID NO:22. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 96% identity to the sequence of SEQ ID NO:22. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 97% identity to the sequence of SEQ ID NO: 22. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 98% identity to the sequence of SEQ ID NO: 22. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 99% identity to the sequence of SEQ ID NO: 22.

[0099] In some embodiments, the EPS depolymerase is encoded by the nucleotide sequence of SEQ ID NO:23. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 90% identity to the sequence of SEQ ID NO:23. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 91% identity to the sequence of SEQ ID NO:23. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 92% identity to the sequence of SEQ ID NO:23. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 93% identity to the sequence of SEQ ID NO:23. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 94% identity to the sequence of SEQ ID NO:23. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 95% identity to the sequence of SEQ ID NO:23. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 96% identity to the sequence of SEQ ID NO:23. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 97% identity to the sequence of SEQ ID NO: 23. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 98% identity to the sequence of SEQ ID NO: 23. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 99% identity to the sequence of SEQ ID NO: 23.

[0100] In some embodiments, the EPS depolymerase is encoded by the nucleotide sequence of SEQ ID NO:24. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 90% identity to the sequence of SEQ ID NO:24. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 91% identity to the sequence of SEQ ID NO:24. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 92% identity to the sequence of SEQ ID NO:24. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 93% identity to the sequence of SEQ ID NO:24. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 94% identity to the sequence of SEQ ID NO:24. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 95% identity to the sequence of SEQ ID NO:24. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 96% identity to the sequence of SEQ ID NO:24. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 97% identity to the sequence of SEQ ID NO: 24. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 98% identity to the sequence of SEQ ID NO: 24. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 99% identity to the sequence of SEQ ID NO: 24.

[0101] In some embodiments, the EPS depolymerase is encoded by the nucleotide sequence of SEQ ID NO:25. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 90% identity to the sequence of SEQ ID NO:25. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 91% identity to the sequence of SEQ ID NO:25. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 92% identity to the sequence of SEQ ID NO:25. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 93% identity to the sequence of SEQ ID NO:25. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 94% identity to the sequence of SEQ ID NO:25. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 95% identity to the sequence of SEQ ID NO:25. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 96% identity to the sequence of SEQ ID NO:25. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 97% identity to the sequence of SEQ ID NO: 25. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 98% identity to the sequence of SEQ ID NO: 25. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 99% identity to the sequence of SEQ ID NO: 25.

[0102] In some embodiments, the EPS depolymerase is encoded by the nucleotide sequence of SEQ ID NO:36. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 90% identity to the sequence of SEQ ID NO:36. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 91% identity to the sequence of SEQ ID NO:36. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 92% identity to the sequence of SEQ ID NO:36. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 93% identity to the sequence of SEQ ID NO:36. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 94% identity to the sequence of SEQ ID NO:36. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 95% identity to the sequence of SEQ ID NO:36. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 96% identity to the sequence of SEQ ID NO:36. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 97% identity to the sequence of SEQ ID NO: 36. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 98% identity to the sequence of SEQ ID NO: 36. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 99% identity to the sequence of SEQ ID NO: 36.

[0103] In some embodiments, the EPS depolymerase is encoded by the nucleotide sequence of SEQ ID NO:59. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 90% identity to the sequence of SEQ ID NO:59. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 91% identity to the sequence of SEQ ID NO:59. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 92% identity to the sequence of SEQ ID NO:59. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 93% identity to the sequence of SEQ ID NO:59. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 94% identity to the sequence of SEQ ID NO:59. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 95% identity to the sequence of SEQ ID NO:59. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 96% identity to the sequence of SEQ ID NO:59. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 97% identity to the sequence of SEQ ID NO: 59. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 98% identity to the sequence of SEQ ID NO: 59. In some embodiments, the EPS depolymerase is encoded by a sequence having at least 99% identity to the sequence of SEQ ID NO: 59.

[0104] In some embodiments, the bacteriophage exhibits improved host range.

[0105] In some embodiments, the bacteriophage belongs to the PhiKMV virus genus. In some embodiments, the bacteriophage belongs to the Pakpunavirus genus. In some embodiments, the bacteriophage belongs to the Bruynoghevirus genus. In some embodiments, the bacteriophage belongs to the Pubunavirus genus. In some embodiments, the bacteriophage belongs to the Luzseptimavirus genus. In some embodiments, the bacteriophage belongs to the Litunavirus genus. In some embodiments, the bacteriophage belongs to the Nankokuvirus genus.

[0106] In some embodiments, the bacteriophage targets Pseudomonas. In some embodiments, the bacteriophage targets Pseudomonas aeruginosa. In some embodiments, the bacteriophage targets one or more Pseudomonas aeruginosa strains.

[0107] In some embodiments, the bacteriophage targets one or more of P. aeruginosa, drug-resistant P. aeruginosa, antibiotic-resistant P. aeruginosa, and multi-antibiotic-resistant P. aeruginosa, hi some embodiments, the bacteriophage infects and kills one or more of P. aeruginosa, antibiotic-resistant P. aeruginosa, and multi-antibiotic-resistant P. aeruginosa.

[0108] In some embodiments, the bacteriophage reduces biofilm mass. As used herein, the terms "reduce", "reduce", "reduction", "minimal", "low" or "lower" refer to a decrease to a value below basal levels, e.g., compared to a control. "Increase", "high", "higher", "maximum", "elevate" or "elevation" refer to an increase to a value above basal levels, e.g., compared to a control. An increase, elevation, decrease, or reduction compared to a control or baseline level 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%, or 100%. Each of the values ​​or ranges set forth herein can include any value or subrange therebetween, including the endpoints.

[0109] Bacteriophage Composition In some embodiments, the present disclosure relates to one or more bacteriophage compositions comprising one or more bacteriophages engineered to express a heterologous gene (e.g., an exopolysaccharide depolymerase) as described herein. In some embodiments, a target locus for insertion of a heterologous gene in a bacteriophage can be selected based on the deletion of a regulatory signal, a gene, an open reading frame (ORF), a terminator, a promoter, an origin of replication, or any other known element essential for the phage replication cycle. Thus, in some embodiments, a heterologous gene is not inserted at any such location. In some embodiments, a target locus for insertion of a heterologous gene in a bacteriophage can be selected based on the expected expression level from said locus during the phage replication cycle. In some embodiments, the target locus comprises a highly transcribed genomic region. In some embodiments, the heterologous gene can comprise an EPS depolymerase.

[0110] The heterologous gene may be positively inserted at any suitable location, e.g., a location that does not unnecessarily affect essential genes of the virus, as described above, and / or, e.g., a highly transcribed location, as described above. In some embodiments, the heterologous gene may be inserted at a specific locus and may replace an entire segment from the genomic sequence of the corresponding unmodified (wild-type) phage. Additionally or alternatively, in some embodiments, the heterologous gene may be inserted at a specific locus, while otherwise maintaining the entire genomic sequence. For example, the following table shows some non-limiting examples of such insertions into various bacteriophage genera. [Table 1-1] [Table 1-2]

[0111] The above are non-limiting examples of insertions that have been made. Although specific base pair positions or ranges are listed for some of the specific strains in Table 1, it should be understood that insertions and / or deletions can be made to any base pair within the listed range and / or remove any range of bases therein. Insertions can also be made within 10 base pairs before or after the listed insertion point or range.

[0112] The one or more bacteriophages may comprise a polynucleotide of SEQ ID NO:26, a polynucleotide of SEQ ID NO:27, a polynucleotide of SEQ ID NO:28, a polynucleotide of SEQ ID NO:29, a polynucleotide of SEQ ID NO:30, a polynucleotide of SEQ ID NO:31, a polynucleotide of SEQ ID NO:32, a polynucleotide of SEQ ID NO:33, a polynucleotide of SEQ ID NO:34, a polynucleotide of SEQ ID NO:35, a polynucleotide of SEQ ID NO:37, a polynucleotide of SEQ ID NO:38, a polynucleotide of SEQ ID NO:39, a polynucleotide of SEQ ID NO:40, a polynucleotide of SEQ ID NO:41, a polynucleotide of SEQ ID NO:42, a polynucleotide of SEQ ID NO:43, a polynucleotide of SEQ ID NO:44, a polynucleotide of SEQ ID NO:45, a polynucleotide of SEQ ID NO:46, a polynucleotide of SEQ ID NO:47, a polynucleotide of SEQ ID NO:48, a polynucleotide of SEQ ID NO:49, a polynucleotide of SEQ ID NO:50, a polynucleotide of SEQ ID NO:51, a polynucleotide of SEQ ID NO:52, a polynucleotide of SEQ ID NO:53, a polynucleotide of SEQ ID NO:54, a polynucleotide of SEQ ID NO:55, a polynucleotide of SEQ ID NO:56, a polynucleotide having at least 90% identity to SEQ ID NO:26, a polynucleotide having at least 90% identity to SEQ ID NO:27, a polynucleotide having at least 90% identity to SEQ ID NO:28, a polynucleotide having at least 90% identity to SEQ ID NO:29, a polynucleotide having at least 90% identity to SEQ ID NO:30, a polynucleotide having at least 90% identity to SEQ ID NO:31, a polynucleotide having at least 90% identity to SEQ ID NO:32,A polynucleotide sequence having at least 90% identity to SEQ ID NO:33, a polynucleotide sequence having at least 90% identity to SEQ ID NO:34, a polynucleotide sequence having at least 90% identity to SEQ ID NO:35, a polynucleotide sequence having at least 90% identity to SEQ ID NO:37, a polynucleotide sequence having at least 90% identity to SEQ ID NO:38, a polynucleotide sequence having at least 90% identity to SEQ ID NO:39, a polynucleotide sequence having at least 90% identity to SEQ ID NO:40, a polynucleotide sequence having at least 90% identity to SEQ ID NO:41, a polynucleotide sequence having at least 90% identity to SEQ ID NO:42, a polynucleotide sequence having at least 90% identity to SEQ ID NO:43, a polynucleotide sequence having at least 90% identity to SEQ ID NO:44, a polynucleotide sequence having at least 90% identity to SEQ ID NO:45, a polynucleotide sequence having at least 90% identity to SEQ ID NO:46, a polynucleotide sequence having at least 90% identity to SEQ ID NO:47, a polynucleotide sequence having at least 90% identity to SEQ ID NO:48 a polynucleotide sequence having at least 90% identity to SEQ ID NO:49, a polynucleotide sequence having at least 90% identity to SEQ ID NO:50, a polynucleotide sequence having at least 90% identity to SEQ ID NO:51, a polynucleotide sequence having at least 90% identity to SEQ ID NO:52, a polynucleotide sequence having at least 90% identity to SEQ ID NO:53, a polynucleotide sequence having at least 90% identity to SEQ ID NO:54, a polynucleotide sequence having at least 90% identity to SEQ ID NO:55, a polynucleotide sequence having at least 90% identity to SEQ ID NO:56, a polynucleotide sequence having at least 90% identity to SEQ ID NO:57, a polynucleotide sequence having at least 90% identity to SEQ ID NO:58, a polynucleotide sequence having at least 90% identity to SEQ ID NO:60, a polynucleotide sequence having at least 90% identity to SEQ ID NO:61, a polynucleotide sequence having at least 90% identity to SEQ ID NO:62, a polynucleotide sequence having at least 90% identity to SEQ ID NO:63,The polynucleotide sequence may include a polynucleotide sequence selected from a polynucleotide sequence having at least 90% identity to SEQ ID NO:64, a polynucleotide sequence having at least 90% identity to SEQ ID NO:65, a polynucleotide sequence having at least 90% identity to SEQ ID NO:66, a polynucleotide sequence having at least 90% identity to SEQ ID NO:67, a polynucleotide sequence having at least 90% identity to SEQ ID NO:68, a polynucleotide sequence having at least 90% identity to SEQ ID NO:69, a polynucleotide sequence having at least 90% identity to SEQ ID NO:70, a polynucleotide sequence having at least 90% identity to SEQ ID NO:71, and a polynucleotide sequence having at least 90% identity to SEQ ID NO:73.

[0113] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:26. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:26. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:26. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:26. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:26. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:26. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:26. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:26. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:26.

[0114] In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:27. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:27. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:27. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:27. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:27. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:27. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:27. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:27. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:27.

[0115] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:28. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:28. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:28. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:28. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:28. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:28. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:28. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:28. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:28.

[0116] In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:29. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:29. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:29. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:29. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:29. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:29. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:29. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:29. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:29.

[0117] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:30. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:30. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:30. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:30. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:30. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:30. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:30. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:30. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:30.

[0118] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:31. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:31. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:31. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:31. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:31. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:31. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:31. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:31. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:31.

[0119] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:32. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:32. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:32. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:32. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:32. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:32. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:32. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:32. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:32.

[0120] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:33. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:33. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:33. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:33. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:33. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:33. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:33. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:33. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:33.

[0121] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:34. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:34. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:34. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:34. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:34. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:34. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:34. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:34. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:34.

[0122] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:35. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:35. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:35. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:35. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:35. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:35. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:35. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:35. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:35.

[0123] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:37. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:37. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:37. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:37. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:37. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:37. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:37. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:37. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:37.

[0124] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:38. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:38. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:38. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:38. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:38. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:38. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:38. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:38. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:38.

[0125] In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:39. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:39. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:39. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:39. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:39. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:39. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:39. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:39. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:39.

[0126] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:40. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:40. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:40. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:40. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:40. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:40. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:40. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:40. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:40.

[0127] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:41. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:41. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:41. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:41. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:41. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:41. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:41. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:41. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:41.

[0128] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:42. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:42. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:42. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:42. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:42. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:42. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:42. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:42. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:42.

[0129] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:43. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:43. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:43. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:43. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:43. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:43. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:43. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:43. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:43.

[0130] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:44. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:44. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:44. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:44. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:44. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:44. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:44. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:44. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:44.

[0131] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:45. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:45. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:45. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:45. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:45. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:45. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:45. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:45. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:45.

[0132] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:46. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:46. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:46. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:46. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:46. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:46. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:46. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:46. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:46.

[0133] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:47. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:47. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:47. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:47. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:47. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:47. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:47. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:47. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:47.

[0134] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:48. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:48. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:48. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:48. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:48. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:48. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:48. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:48. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:48.

[0135] In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:49. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:49. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:49. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:49. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:49. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:49. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:49. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:49. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:49.

[0136] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:50. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:50. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:50. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:50. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:50. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:50. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:50. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:50. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:50.

[0137] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:51. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:51. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:51. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:51. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:51. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:51. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:51. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:51. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:51.

[0138] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:52. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:52. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:52. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:52. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:52. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:52. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:52. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:52. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:52.

[0139] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:53. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:53. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:53. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:53. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:53. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:53. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:53. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:53. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:53.

[0140] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:54. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:54. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:54. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:54. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:54. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:54. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:54. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:54. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:54.

[0141] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:55. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:55. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:55. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:55. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:55. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:55. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:55. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:55. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:55.

[0142] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:56. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:56. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:56. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:56. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:56. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:56. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:56. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:56. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:56.

[0143] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:57. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:57. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:57. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:57. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:57. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:57. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:57. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:57. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:57.

[0144] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:58. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:58. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:58. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:58. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:58. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:58. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:58. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:58. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:58.

[0145] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:60. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:60. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:60. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:60. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:60. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:60. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:60. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:60. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:60.

[0146] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:61. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:61. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:61. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:61. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:61. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:61. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:61. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:61. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:61.

[0147] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:62. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:62. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:62. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:62. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:62. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:62. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:62. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:62. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:62.

[0148] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:63. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:63. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:63. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:63. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:63. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:63. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:63. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:63. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:63.

[0149] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:64. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:64. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:64. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:64. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:64. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:64. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:64. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:64. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:64.

[0150] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:65. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:65. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:65. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:65. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:65. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:65. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:65. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:65. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:65.

[0151] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:66. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:66. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:66. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:66. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:66. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:66. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:66. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:66. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:66.

[0152] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:67. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:67. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:67. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:67. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:67. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:67. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:67. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:67. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:67.

[0153] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:68. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:68. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:68. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:68. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:68. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:68. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:68. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:68. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:68.

[0154] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:69. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:69. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:69. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:69. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:69. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:69. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:69. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:69. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:69.

[0155] In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:70. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:70. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:70. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:70. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:70. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:70. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:70. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:70. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:70.

[0156] In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:71. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:71. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:71. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:71. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:71. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:71. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:71. In some embodiments, the one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:71. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:71.

[0157] In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 91% identity to SEQ ID NO:73. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 92% identity to SEQ ID NO:73. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 93% identity to SEQ ID NO:73. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 94% identity to SEQ ID NO:73. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 95% identity to SEQ ID NO:73. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 96% identity to SEQ ID NO:73. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 97% identity to SEQ ID NO:73. In some embodiments, one or more bacteriophages can comprise a polynucleotide sequence having at least 98% identity to SEQ ID NO:73. In some embodiments, the one or more bacteriophages may comprise a polynucleotide sequence having at least 99% identity to SEQ ID NO:73.

[0158] In some embodiments, the EPS depolymerase is an alginate lyase, as described in detail elsewhere herein.

[0159] In some embodiments, one or more of the bacteriophages are engineered. In some embodiments, one or more of the bacteriophages are genetically engineered. As used herein, a "genetically engineered" or "genetically modified" bacteriophage can be a bacteriophage whose polynucleotide sequence has been modified by genetic engineering techniques. The genetic engineering of the polynucleotide sequence can be performed by any of the modern molecular biology techniques known in the art, including but not limited to homologous recombination, bacteriophage engineering, CRISPR-based engineering (e.g., CRISPR-Cas), reconstruction of the full-length phage genome in yeast, or by transformation of the full-length naked phage DNA into a host bacterium after in vitro DNA splicing, and any combination of these techniques.

[0160] In some embodiments, two or more of the bacteriophages are engineered. In some embodiments, three or more of the bacteriophages are engineered. In some embodiments, four or more of the bacteriophages are engineered. In some embodiments, five or more of the bacteriophages are engineered. In some embodiments, six or more of the bacteriophages are engineered. In some embodiments, seven or more of the bacteriophages are engineered. In some embodiments, eight or more of the bacteriophages are engineered. In some embodiments, nine or more of the bacteriophages are engineered. In some embodiments, ten or more of the bacteriophages are engineered.

[0161] In some embodiments, the second bacteriophage of the one or more bacteriophages comprises a natural phage. The terms "natural" and "wild type" refer to a form found in nature. For example, a natural or wild type nucleic acid molecule, nucleotide sequence or protein is present in a natural source, can be isolated therefrom, and has not been intentionally modified by human manipulation. Thus, a natural phage is a naturally occurring, unengineered phage. In some embodiments, the second bacteriophage may comprise a mutated natural phage and / or a partially or fully synthetic phage, particularly when the further bacteriophage has the ability to infect, kill or reduce bacterial infection, as described in detail in, for example, WO2016 / 100389, the entire contents of which are incorporated herein by reference.

[0162] In some embodiments, two or more of the bacteriophages are naturally occurring phages. In some embodiments, three or more of the bacteriophages are naturally occurring phages. In some embodiments, four or more of the bacteriophages are naturally occurring phages. In some embodiments, five or more of the bacteriophages are naturally occurring phages. In some embodiments, six or more of the bacteriophages are naturally occurring phages. In some embodiments, seven or more of the bacteriophages are naturally occurring phages. In some embodiments, eight or more of the bacteriophages are naturally occurring phages. In some embodiments, nine or more of the bacteriophages are naturally occurring phages. In some embodiments, ten or more of the bacteriophages are naturally occurring phages.

[0163] In some embodiments, one or more of the bacteriophages in the composition are modified and one or more are naturally occurring. In some embodiments, one or more of the bacteriophages in the composition are modified and two or more are naturally occurring. In some embodiments, one or more of the bacteriophages in the composition are modified and three or more are naturally occurring. In some embodiments, one or more of the bacteriophages in the composition are modified and four or more are naturally occurring. In some embodiments, one or more of the bacteriophages in the composition are modified and five or more are naturally occurring. In some embodiments, two or more of the bacteriophages in the composition are modified and one or more are naturally occurring. In some embodiments, three or more of the bacteriophages in the composition are modified and one or more are naturally occurring. In some embodiments, four or more of the bacteriophages in the composition are modified and one or more are naturally occurring. In some embodiments, five or more of the bacteriophages in the composition are modified and one or more of the bacteriophages are natural, in some embodiments any other combination of engineered and natural phages is contemplated.

[0164] In some embodiments, the bacteriophage targets Pseudomonas. In some embodiments, at least one of the bacteriophages targets Pseudomonas aeruginosa. In some embodiments, the bacteriophage targets one or more Pseudomonas aeruginosa strains.

[0165] In some embodiments, the one or more bacteriophages of the composition target one or more of P. aeruginosa, antibiotic-resistant P. aeruginosa, and multi-antibiotic-resistant P. aeruginosa, hi some embodiments, the one or more bacteriophages of the composition infect and kill one or more of P. aeruginosa, antibiotic-resistant P. aeruginosa, and multi-antibiotic-resistant P. aeruginosa, as described above.

[0166] In some embodiments, the composition further comprises a storage medium for storage at room temperature or a temperature of 8° C. or less. In embodiments, the bacteriophage composition comprises a storage medium for storage at a temperature of 7° C. or less. In embodiments, the bacteriophage composition comprises a storage medium for storage at a temperature of 6° C. or less. In embodiments, the bacteriophage composition comprises a storage medium for storage at a temperature of 5° C. or less. In embodiments, the bacteriophage composition comprises a storage medium for storage at a temperature of 4° C. or less. In embodiments, the bacteriophage composition comprises a storage medium for storage at a temperature of 3° C. or less. In embodiments, the bacteriophage composition comprises a storage medium for storage at a temperature of 2° C. or less. In embodiments, the bacteriophage composition comprises a storage medium for storage at a temperature of 1° C. or less. In embodiments, the bacteriophage composition comprises a storage medium for storage at a temperature of 0° C. or less. In embodiments, the bacteriophage composition comprises a storage medium for storage at a temperature of −20° C. or less. In embodiments, the bacteriophage composition comprises a storage medium for storage at a temperature of −80° C. or less.

[0167] In specific embodiments, the composition is stored at a temperature ranging from -20°C to 25°C, e.g., 20°C, 21°C, 22°C, 23°C, 24°C, or 25°C, or any value or subrange therebetween (including the endpoints).

[0168] In specific embodiments, the composition is stored at a temperature ranging from 2°C to 8°C, e.g., 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, or 8°C, or any value or subrange therebetween (including endpoints).

[0169] In a specific embodiment, the composition is stored at room temperature.

[0170] In some embodiments, the preservation medium comprises a cryoprotectant. In some embodiments, the cryoprotectant is glycerol, e.g., about 5% to about 50% glycerol, more preferably about 10% to about 30% glycerol, and most preferably about 20% glycerol. In other embodiments, the cryoprotectant is sucrose, e.g., about 5% to about 30% sucrose, and most preferably about 10% sucrose. In some embodiments, the cryoprotectant is dimethyl sulfoxide (DMSO), e.g., about 2% to about 10% DMSO. Suitable concentrations can be any value or subvalue within the recited ranges, including the endpoints.

[0171] In some embodiments, the bacteriophage compositions can be used directly, refrigerated, cryogenically dried, lyophilized, or stored frozen in an aqueous or other solution containing an appropriate cryoprotectant, as described above.

[0172] In some embodiments, the composition further comprises a pharma- ceutically acceptable carrier, diluent, excipient, or combination thereof, as described in detail elsewhere herein.

[0173] In some embodiments, the composition is a liquid, semi-liquid, solid, frozen, or lyophilized formulation. In some embodiments, the bacteriophage composition is in a liquid formulation. In some embodiments, the bacteriophage composition is in a semi-liquid formulation. In some embodiments, the bacteriophage composition is in a solid formulation. In some embodiments, the bacteriophage composition is in a frozen formulation. In some embodiments, the bacteriophage composition is in a lyophilized formulation.

[0174] In some embodiments, the composition is at 1×10 per milliliter. 8 ~1×10 12In some embodiments, the bacteriophage comprises 1 x 10 PFU of each bacteriophage per ml of the composition. 8 ~1×10 9 PFU, 1 × 10 8 ~1×10 10 PFU, 1 × 10 8 ~1×10 11 PFU, or 1 × 10 8 ~1×10 12 In some embodiments, the bacteriophage comprises 3×10 PFU of each phage per ml of composition. 8 ~1×10 9 PFU, 3 × 10 8 ~1×10 10 PFU, 3 × 10 8 ~1×10 11 PFU, or 3 × 10 8 ~1×10 12 In some embodiments, the bacteriophage comprises 3×10 PFU of each phage per ml of composition. 8 ~3×10 9 PFU, 3 × 10 8 ~3×10 10 PFU, or 3 × 10 8 ~3×10 12 In some embodiments, the bacteriophage comprises 1 x 10 PFU of each phage per ml of the composition. 9 ~1×10 10 PFU, or 1 × 10 9 ~1×10 11 PFU, or 1 × 10 9 ~1×10 12 In some embodiments, the bacteriophage concentration is 1×10 per ml of composition. 10 ~1×10 12 PFU of each phage. In some embodiments, the bacteriophage concentration is 1 x 10 per ml of composition. 12 ~1×10 12 In some embodiments, the bacteriophage is administered to a subject at least about 1 x 10 per ml of the composition. 8 PFU of each phage, at least about 3 x 10 8PFU of each phage, at least about 1 x 10 9 PFU of each phage, at least about 1 x 10 10 PFU of each phage, at least about 1 x 10 11 PFU, or at least about 1 × 10 12 In an embodiment, one or more bacteriophage(s) are combined to provide a dose of about 1×10 per ml of composition. 8 , about 3×10 8 , about 1×10 9 , about 1×10 10 , 1×10 11 , or 1 × 10 12 It can be the total concentration of each phage in PFU. The concentration includes any value, subvalue, range, or subrange within the stated range, including the endpoints.

[0175] In some embodiments, one or more bacteriophages of the composition reduce biofilm mass, as described in more detail in the Examples. Methods for Treating Infections

[0176] In some embodiments, the disclosure relates to a method for treating a Pseudomonas aeruginosa infection, comprising administering any of the compositions described herein to a subject in need of treatment for a Pseudomonas aeruginosa infection. As used herein, the term "in need of treatment" refers to a judgment made by a caregiver (e.g., in the case of humans, a physician, nurse, nurse practitioner, or individual; in the case of animals, including non-human mammals, a veterinarian) that a subject requires or will benefit from treatment. This judgment may be made based on a variety of factors that are within the expertise of the caregiver, including knowledge that the subject is ill or will become ill as a result of a condition treatable by a composition of the invention.

[0177] In some embodiments, provided herein is a method for treatment comprising administering a therapeutically effective amount of a bacteriophage or a therapeutically effective amount of a bacteriophage composition. In some embodiments, provided herein is a method for treatment comprising administering a bacteriophage to a subject, wherein the bacteriophage is at about 1×10 per ml of each bacteriophage. 8 ~Approx. 1×10 12 Methods are provided that include a range of bacteriophage concentrations in PFU / ml. In some embodiments, the bacteriophage concentration is 1×10 per ml of composition or greater. 8 ~1×10 9 PFU, 1 × 10 8 ~1×10 10 PFU, 1 × 10 8 ~1×10 11 PFU, or 1 × 10 12 PFU of each phage. In some embodiments, the bacteriophage concentration is 3×10 per ml of composition. 8 ~1×10 9 PFU, 3 × 10 8 ~1×10 10 PFU, 3 × 10 8 ~1×10 11 PFU, or 3 × 10 8 ~1×10 1 PFU of each phage. In some embodiments, the bacteriophage concentration is 3×10 per ml of composition. 8 ~3×10 9 PFU, or 3 × 10 8 ~3×10 10 PFU of each phage. In some embodiments, the bacteriophage concentration is 1 x 10 per ml of composition. 9 ~1×10 10 PFU, 1 × 10 9 ~1×10 11 PFU, or 1 × 10 9 ~1×10 12 PFU of each phage. In some embodiments, the bacteriophage concentration is 1 x 10 per ml of composition. 10 ~1×10 12PFU of each phage. In embodiments, one or more bacteriophage(s) may be combined and administered. In some embodiments, the one or more combined bacteriophages for administration may include two different engineered phages, such as any of those described herein. The two or more engineered phages may, for example, be engineered to express different EPS depolymerases or the same EPS depolymerase. The engineered phages may be from the same genus, for example as described herein, or from different genuses. It should be understood that in some embodiments, one or more phages engineered to express genes described herein may be used in a medical treatment along with one or more phages that have not been so engineered (e.g., phages that are not modified at all or that do not contain a depolymerase gene). In some embodiments, one or more bacteriophage(s) may be combined to provide a concentration of about 1×10 per ml of composition. 8 ~Approx. 5×10 12 The total concentration of each phage may be about 1×10 PFU per ml of composition. In some embodiments, one or more bacteriophage(s) may be combined to provide a total concentration of about 1×10 per ml of composition. 8 ~Approx. 3×10 12 The total concentration of each phage may be about 1×10 per ml of composition. In an embodiment, one or more bacteriophage(s) may be combined to provide a total of about 1×10 per ml of composition. 8 , 3×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , or 1 × 10 12 The total concentration of each phage may be in the form of PFU. In an embodiment, one or more bacteriophage(s) may be combined to provide a total of about 9×10 per ml of composition. 8 , 3×10 9 , 3×10 10 , 3×10 11 , or 3 × 10 12 It can be the total concentration of each phage in PFU. The concentration includes any value or range within the stated range, including the endpoints.

[0178] In some embodiments, the bacteriophage is administered to a subject at least about 1 x 10 per dose. 8 PFU of each phage, at least about 3 x 10 8 PFU of each phage, at least about 1 x 10 9 PFU of each phage, at least about 1 x 10 10 PFU of each phage, or at least about 1 x 10 11 In some embodiments, the composition is administered at a dose of at least 3×10 PFU of each phage per dose. 8 In some embodiments, the composition is administered in a dose of at least 3×10 PFU of total bacteriophage per dose. 9 In some embodiments, the composition is administered at a dose of about 1×10 total bacteriophage per dose. 8 PFU~5×10 12 The dosage is administered in PFU of total bacteriophage. The dosage includes any value or range within the stated range, including the endpoints.

[0179] In some embodiments, the method further comprises administration of an antibiotic, hi embodiments, the antibiotic is selected from fluoroquinolones, carbapenems, aminoglycosides, ansamycins, cephalosporins, penicillins, beta-lactams, beta-lactamase inhibitors, folate pathway inhibitors, fucidanes, glycopeptides, glycylcyclines, lincosamides, lipopeptides, macrolides, quinolones, oxazolidinones, phenicol phosphonic acid, streptogramins, tetracyclines, sulfonamides, imipenem, meropenem, amikacin, ciprofloxacin, levofloxacin, tobramycin, azithromycin, aztreonam, colistin, inhaled tobramycin, inhaled aztreonam, and inhaled colistin.

[0180] In some embodiments, the methods further include administration of one or more CFTR modulators. In embodiments, the CFTR modulator can be selected from, but is not limited to, ivacaftor, lumacaftor / ivacaftor, tezacaftor / ivacaftor, elexacaftor / tezacaftor / ivacaftor, or any combination thereof.

[0181] In some embodiments, the bacterial infection is resistant to one or more antibiotics or other treatments, hi some embodiments, the bacterial infection is resistant to one or more of fluoroquinolones, carbapenems, aminoglycosides, ansamycins, cephalosporins, penicillins, beta-lactams, beta-lactamase inhibitors, folate pathway inhibitors, fucidanes, glycopeptides, glycylcyclines, lincosamides, lipopeptides, macrolides, quinolones, oxazolidinones, phenicol phosphonic acid, streptogramins, tetracyclines, sulfonamides, imipenem, meropenem, amikacin, ciprofloxacin, levofloxacin, tobramycin, azithromycin, aztreonam, colistin, inhaled tobramycin, inhaled aztreonam, and inhaled colistin.

[0182] In some embodiments, the method may include treating a subject who has not been previously treated with one or more antibiotics, or who has been previously treated with one or more antibiotics, and optionally selecting the subject for treatment. For example, the phage therapy described according to any of the embodiments described herein may be the first treatment administered (alone or in combination with another type of treatment, such as antibiotic treatment) for a new infection, or may be the treatment administered following some other first treatment approach, such as antibiotic treatment. As described above, the method may include selecting a subject or patient based on the subject having already undergone a first treatment. The first treatment may be effective or only partially effective. The infection may, for example, be resistant or have developed resistance to another treatment.

[0183] In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and a fluoroquinolone. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and a carbapenem. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and an aminoglycoside. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and an ansamycin. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and a cephalosporin. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and a penicillin. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and a beta-lactam. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and a beta-lactamase inhibitor. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and a folate pathway inhibitor. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and a fucidane. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and a glycopeptide.In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and a glycylcycline. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and a lincosamide. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and a lipopeptide. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and a macrolide. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and a quinolone. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and an oxazolidinone. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and a phenicol phosphonic acid. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and a streptogramin. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and a tetracycline. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and a sulfonamide. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and imipenem.In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and meropenem. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and amikacin. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and ciprofloxacin. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and levofloxacin. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and tobramycin. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and azithromycin. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and aztreonam. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and colistin. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and inhaled tobramycin. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and inhaled aztreonam. In embodiments, provided herein is a method for treating a bacterial infection by administering in combination any of the bacteriophage compositions described herein and inhaled colistin.Any one or more of the treatments described herein may be explicitly excluded.

[0184] In some embodiments, the bacteriophage composition is administered by inhalation, hi some embodiments, the bacteriophage composition is administered by nebulization.

[0185] In embodiments, provided herein are methods of administering any of the bacteriophages or compositions described herein to a subject, wherein the administration occurs over a period ranging from about 3 to about 24 hours. In embodiments, the bacteriophage is administered to the subject every 3 hours. In embodiments, the bacteriophage is administered to the subject every 4 hours. In embodiments, the bacteriophage is administered to the subject every 5 hours. In embodiments, the bacteriophage is administered to the subject every 6 hours. In embodiments, the bacteriophage is administered to the subject every 7 hours. In embodiments, the bacteriophage is administered to the subject every 8 hours. In embodiments, the bacteriophage is administered to the subject every 9 hours. In embodiments, the bacteriophage is administered to the subject every 10 hours. In embodiments, the bacteriophage is administered to the subject every 11 hours. In some embodiments, the bacteriophage is administered to the subject every 12 hours. In embodiments, the bacteriophage is administered to the subject every 18 hours. In embodiments, the bacteriophage is administered to the subject every 24 hours. In some embodiments, the bacteriophage is administered to the subject at least once a day. Timing includes any value or range within the described ranges, including the endpoints.

[0186] In certain embodiments, the bacteriophage composition is administered at least every 6 hours.

[0187] In some embodiments, the bacteriophage composition is administered to the subject for at least one day. In embodiments, the bacteriophage composition is administered to the subject for a total of two days. In embodiments, the bacteriophage composition is administered to the subject for a total of three days. In embodiments, the bacteriophage composition is administered to the subject for a total of four days. In embodiments, the bacteriophage composition is administered to the subject for a total of five days. In embodiments, the bacteriophage composition is administered to the subject for a total of six days. In embodiments, the bacteriophage composition is administered to the subject for a total of seven days. In embodiments, the bacteriophage composition is administered to the subject for a total of ten days. In embodiments, the bacteriophage composition is administered to the subject for a total of fourteen days. In embodiments, the bacteriophage composition is administered to the subject for a total of twenty-one days. In embodiments, the bacteriophage composition is administered to the subject for a total of twenty-eight days. In embodiments, the bacteriophage composition is administered to the subject for a total of one day to about four weeks. The duration of administration can be any value or subrange within the recited ranges, including the endpoints.

[0188] In some embodiments, the bacteriophages and / or compositions described herein may be administered in combination with one or more purified alginate lyases.

[0189] In some embodiments, the subject is a human.

[0190] In some embodiments, the subject has cystic fibrosis (CF). In some embodiments, the subject has non-cystic fibrosis bronchiectasis (NFCB).

[0191] In other embodiments, the present disclosure also provides a method for treating a bacterial infection, comprising selecting a subject having a bacterial infection and administering to the subject an effective amount of any of the bacteriophages described herein.

[0192] The subject may be selected based on any of the following criteria: In some embodiments, the subject may have a bacterial infection that is a Pseudomonas infection. In some embodiments, the bacterial infection is a Pseudomonas aeruginosa infection. In some embodiments, the infection may include or be associated with, but is not limited to, cystic fibrosis and / or pneumonia. In some embodiments, the infection includes a bacterial infection in the presence of cystic fibrosis and / or pneumonia. In some embodiments, the infection may include or be associated with, but is not limited to, non-cystic fibrosis bronchiectasis (NCFB). In some embodiments, the infection includes a bacterial infection in the presence of NCFB and / or pneumonia. In some embodiments, the infection includes a mucoid type, or an overproduction of alginate. In some embodiments, the bacterial infection is characterized by a biofilm. In some embodiments, the subject may have an antibiotic-resistant Pseudomonas infection. In some embodiments, the subject may have a bacterial infection that is resistant to multiple antibiotics. In some embodiments, the subject may be selected based on having previously been treated for a bacterial infection with one or more antibiotics or another anti-bacterial treatment. In some embodiments, subjects can be selected on the basis of having an infection with a bacteria that has developed or is resistant to an initial antibiotic treatment or to one or more antibiotics.

[0193] Methods for producing engineered bacteriophages In some embodiments, the disclosure provides a method for producing an engineered bacteriophage, the method comprising providing a bacteriophage and incorporating an exopolysaccharide (EPS) depolymerase into the bacteriophage.

[0194] In some embodiments, the EPS depolymerase may be, but is not limited to, an alginate lyase. In some embodiments, the alginate lyase may include, but is not limited to, Alg2A or A1-III, as described in detail elsewhere herein. In some embodiments, the alginate lyase sequences described herein may include portions or functional fragments of an alginate lyase gene. In some embodiments, the sequences may include the entire alginate lyase gene.

[0195] In some embodiments, the bacteriophage belongs to the PhiKMV virus genus. In some embodiments, the bacteriophage belongs to the Pakpunavirus genus. In some embodiments, the bacteriophage belongs to the Bruynoghevirus genus. In some embodiments, the bacteriophage belongs to the Pubunavirus genus. In some embodiments, the bacteriophage belongs to the Luzseptimavirus genus. In some embodiments, the bacteriophage belongs to the Litunavirus genus. In some embodiments, the bacteriophage belongs to the Nankokuvirus genus.

[0196] In some embodiments, the bacteriophages and compositions described herein encode regulatory elements, which may include, but are not limited to, promoters, cis elements, enhancers, terminators, or introns. In some aspects of the invention, gene expression from an inserted gene or nucleic acid encoding an EPS depolymerase and / or phage is regulated by a promoter to which the nucleic acid is operably linked. The terms "promoter," "promoter region," or "promoter sequence" refer to a nucleic acid sequence to which an RNA polymerase can bind that initiates transcription of the gene in the 5' to 3' ("downstream") direction. A gene is "under the control" or "controlled" by a promoter when the RNA polymerase that binds to the promoter is the proximal origin of transcription of that gene. A promoter or promoter region typically provides recognition sites for RNA polymerase and other factors required for proper transcription initiation. Promoters can be isolated from the 5' untranslated region (5'UTR) of a genomic copy of a gene. Alternatively, promoters can be synthetically generated or designed by modifying known DNA elements. Chimeric promoters that combine the sequence of one promoter with the sequence of another promoter are also contemplated. Promoters can be defined by their expression patterns, for example, based on metabolic, environmental, or developmental conditions. Promoters can be used as regulatory elements to regulate the expression of operably linked transcribable polynucleotide molecules (e.g., coding sequences). In addition to the sequence recognized by RNA polymerase (and preferably other transcription factors), promoters can also contain regulatory elements (e.g., cis elements or enhancer domains) that affect the transcription of operably linked genes. A "viral promoter" is a natural or non-natural promoter that initiates the transcription of one or more genes located in a viral genome.

[0197] The promoter(s) may be constitutive promoter(s) and / or conditional promoter(s) and / or inducible promoter(s) and / or tissue-specific promoter(s). A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. A constitutive promoter is active regardless of the external environment, such as light and medium composition. In some examples, a constitutive promoter is active in the presence and absence of nutrients. For example, a constitutive promoter may be a promoter that is active (mediates transcription of a functionally linked gene) under nitrogen-depleted conditions and under conditions where nitrogen is not limiting (nitrogen-rich conditions). Conversely, an "inducible" promoter is a promoter that responds to a particular environmental condition (e.g., the presence or absence of a nutrient or regulator, the presence of light, etc.).

[0198] The promoters are those recognized by RNA polymerases pol I, pol II, pol III, T7, M13, and bacterial sigma factors (σ 70、 σ 54 , σ S , σ 32 , σ 19 , σ 28 , or σ 38 ), e.g. P rrn , P lac , P tac , P tet , P bla , P cat , P bad , P L or any natural or synthetic constitutive or inducible promoter known to those skilled in the art.

[0199] In some embodiments, the promoter is a promoter that naturally occurs in the bacteriophage.Therefore, a promoter that is operably linked to a gene that is not operably linked in its natural state (e.g., in the genome of a non-genetically engineered organism or virus) is referred to herein as a heterologous promoter, even if the promoter may originate from the same species (or possibly the same organism or virus) as the gene to which it is linked.Similarly, when referring to a protein localization sequence or protein domain of an engineered protein, "heterologous" means that the localization sequence or protein domain originates from a protein different from the protein that is incorporated by genetic engineering.

[0200] As used herein, the term "operably linked" refers to a configuration in which a control sequence is appropriately positioned relative to the coding sequence of a polynucleotide sequence such that the control sequence induces or regulates the expression of the coding sequence of a polypeptide and / or functional RNA. Thus, a promoter is operably linked to a nucleic acid sequence if it is capable of mediating transcription of the nucleic acid sequence. An expression cassette, when introduced into a host cell, can result in transcription and / or translation of the encoded RNA or polypeptide under appropriate conditions. Antisense or sense constructs that are not or cannot be translated are not excluded by this definition. In the case of transgene expression and suppression of endogenous genes, the skilled artisan will recognize that the inserted polynucleotide sequence need not be identical, but only substantially identical to the sequence of the gene from which it is derived. As described herein, these substantially identical variants are specifically encompassed by reference to a particular nucleic acid sequence.

[0201] In some aspects of the invention, gene expression from the inserted gene or nucleic acid encoding the EPS depolymerase and / or the phage is regulated by a terminator to which the nucleic acid is operably linked. As used herein, "terminator" or "terminator sequence" or "transcription terminator" refers to a regulatory region of a gene sequence that causes an RNA polymerase to terminate transcription. In some embodiments, the terminator is a terminator that naturally occurs in a bacteriophage. In some embodiments, the terminator is not a terminator that naturally occurs in a bacteriophage.

[0202] In some embodiments, the engineered viruses described herein can include codon-optimized sequences. As used herein, the term "codon-optimized" means that a polynucleotide, nucleic acid sequence, or coding sequence has been redesigned compared to a wild-type or reference polynucleotide, nucleic acid sequence, or coding sequence by selecting different codons without changing the amino acid sequence of the encoded protein. Thus, codon optimization generally refers to replacing codons with synonymous codons to optimize protein expression while keeping the amino acid sequence of the translated protein the same. Codon optimization of a sequence can, for example, increase the protein expression level of the encoded protein (Gustafsson et al., Codon bias and heterologous protein expression. 2004, Trends Biotechnol 22:346-53) and provide other advantages. Variables such as codon usage preference as measured by the codon adaptation index (CAI), e.g., the presence or frequency of U and other nucleotides, mRNA secondary structure, cis-regulatory sequences, GC content, and other variables can be correlated with protein expression levels (Villalobos et al., Gene Designer: a synthetic biology tool for constructing artificial DNA segments. 2006, BMC Bioinformatics 7:285).

[0203] Any method of codon optimization can be used to codon-optimize the polynucleotides and nucleic acid molecules provided herein, and any variable can be changed by codon optimization. Thus, any combination of codon optimization methods can be used. Exemplary methods include the high codon adaptation index (CAI) method, the Low U method, and the like. The CAI method selects the most frequently used synonymous codon for the entire protein-coding sequence. As an example, the most frequently used codon for each amino acid can be estimated from 74,218 protein-coding genes in the human genome. The Low U method generally targets U-containing codons that can be replaced with synonymous codons with fewer U moieties without changing other codons. If there are multiple replacement options, the more frequently used codon can be selected. Any polynucleotide, nucleic acid sequence, or codon sequence provided herein can be codon-optimized.

[0204] In some embodiments, any of the EPS depolymerases or alginate lyases described herein can be codon-optimized, if desired, for any of the sequences described herein. Any method known in the art for optimizing codons can be used.

[0205] The engineered phages described herein can be made using any method known in the art for genetically engineering viruses, including bacteriophages. Non-limiting examples are provided in U.S. Patent Nos. 5,811,093, 8,865,158, and 10,837,004, which are hereby incorporated by reference in their entireties for all methods, compositions, reagents, and all other information set forth therein.

[0206] In some embodiments, the disclosure also provides an assay for determining alginate lyase activity of an engineered bacteriophage, comprising administering an effective amount of any of the engineered bacteriophages described herein to a P. aeruginosa biofilm and determining a reduction in biofilm mass.

[0207] kit In some embodiments, the disclosure provides one or more kits comprising any of the bacteriophages or bacteriophage compositions described herein and instructions for use thereof, hi some embodiments, the kits can include one or more purified alginate lyases for co-administration with the bacteriophage compositions.

[0208] In some embodiments, the kit may further comprise an antibiotic. Suitable antibiotics include, but are not limited to, fluoroquinolones, carbapenems, aminoglycosides, ansamycins, cephalosporins, penicillins, beta-lactams, beta-lactamase inhibitors, folate pathway inhibitors, fucidanes, glycopeptides, glycylcyclines, lincosamides, lipopeptides, macrolides, quinolones, oxazolidinones, phenicol phosphonic acid, streptogramins, tetracyclines, sulfonamides, imipenem, meropenem, amikacin, ciprofloxacin, levofloxacin, tobramycin, azithromycin, aztreonam, colistin, inhaled tobramycin, inhaled aztreonam, and inhaled colistin, as described elsewhere herein.

[0209] In some embodiments, the kit further comprises one or more CFTR modulators. In embodiments, the CFTR modulator can be selected from, but is not limited to, ivacaftor, lumacaftor / ivacaftor, tezacaftor / ivacaftor, elexacaftor / tezacaftor / ivacaftor, or any combination thereof.

[0210] In some embodiments, the kit may further comprise a means for administering the bacteriophage or bacteriophage composition, which may include, but is not necessarily limited to, one or more of a syringe, a transdermal patch, a sustained release device, a spray, a nebulizer, an inhaler, or a respirator.

[0211] In some embodiments, the sustained release device may include a mini-osmotic pump.

[0212] In some embodiments, the kit may further comprise a second bacteriophage or bacteriophage composition. EXAMPLES

[0213] Example 1: Alginate lyase activity in various bacteriophages The expression level of the fragment 70-399 of alginate lyase A1-III was low and it had no activity, but the fragment 54-412 had activity.

[0214] The Sphingomonas alginate lyase gene, aly, produces a preproprotein that is proteolytically cleaved to produce AlgL A1-II and A1-III, the latter of which possesses the bacterial alginate-degrading activity of Aly.

[0215] Fragments 70–399 or 54–412 of the aly gene (alginate lyase) of Sphingomonas were cloned into an E. coli expression plasmid with a C-terminal his tag and purified using a Ni affinity column. Fragment 70–399 was nearly insoluble and required denaturation and refolding of aggregates for purification (Figure 5A). Fragment 54–412 of the aly gene (alginate lyase) of Sphingomonas was cloned into an E. coli expression plasmid with a C-terminal his tag and purified using a Ni affinity column. Fragment 54–412 was soluble (Figure 5B). The resulting purified proteins were >99% pure in denaturing gel tests and were tested for their ability to degrade alginate from seaweed or bacteria. Activity was compared to a commercially available alginate lyase preparation from Sigma-Aldrich (Reference A1603) prepared at 10 mg / ml. Activity is evaluated on agar plates containing 2% seaweed alginate. A 5 μl sample of the preparation or control was applied to the surface of the plate, dried, and then incubated at 30 °C for 4 h to overnight. The plate was then stained with 10% cetylpyridinium chloride (CPC), where activity was shown as a halo without a white precipitate (Figure 5C). Activity was also evaluated on preformed lawns of a mucoid alginate-expressing strain of P. aeruginosa, where activity is shown as a halo of a more rugged and more transparent lawn at the application site. Figure 5D shows the results for the lawn of strain 15840.

[0216] Phage expressing AlgL A1-III fragment 70–399 does not have alginate lyase activity Phage APBP4-4 is a mutant of APBP4 with the gene for AlgL A1-III fragment 70-399 inserted downstream of gp99, but does not exhibit alginate lyase activity. APBP17-1 is a mutant of APBP17 with the gene for AlgL A1-III fragment 70-399, but also does not exhibit alginate lyase activity.

[0217] Phages APBP1-1, APBP1-2, APBP3-1, and APBP3-2 exhibit stable alginate lyase activity APBP1-1 and APBP1-2 are mutants of Pubnavirus APBP1 in which the fragment 54-412 of Aly has been cloned immediately downstream of gene gp82. APBP1-1 has a version of algL A1-III (Pae) ​​that is codon-optimized for E. coli, whereas the version of the gene cloned in APBP1-2 is codon-optimized for Pseudomonas aeruginosa (Pae). Both have a 6-His-tag at their C-terminus. APBP3-1 and APBP3-2 are mutants of PhiKMV virus APBP3. APBP3-1 has the same algL A1-III allele as APBP1-2. APBP3-2 has the same algL A1-III allele as APBP1-1. algL A1-III has been inserted downstream of gp037. Each phage exhibits alginate lyase activity in a preformed lawn assay of Pae strain 15844, whereas the parent phage does not. Figure 2A shows a top view of phage spotted onto a preformed lawn of a mucoid alginate-expressing strain of P. aeruginosa (strain 15844), with activity shown as a crater or depressed lawn at the application site. Figure 2B shows a bottom view of the same plate as seen in Figure 13A, with phage spotted onto a preformed lawn of a mucoid alginate-expressing strain of P. aeruginosa (strain 15844), with activity shown as a circular depressed area at the application site.

[0218] Western blot detection of AlgL A1-III-His6 in lysates of APBP1-1, APBP1-2, APBP3-1 or APBP3-2 Total lysates of each phage were prepared for denaturing SDS-PAGE, electrophoresed, and transferred to a PVDF membrane, and His-tagged proteins were detected using a primary antibody against the His tag and an appropriate HRP-conjugated secondary antibody. The alginate lyase (AlgL) A1-III fragment 54–412 is shown as a band at approximately 37 kDa (Figure 3).

[0219] Growth of phages APBP1-1, APBP1-2, APBP3-1, and APBP3-2 is not altered by expression of AlgL A1-III APBP3-1 and APBP3-2 lysates, initiated from single plaques picked from lawns of their host P. aeruginosa strain 7299 and resuspended in exponentially growing cultures of P. aeruginosa strain 7299, reached titers similar to those of their parent phage APBP3. APBP1-1 and APBP1-2 grew to titers approximately 10-fold lower than that of the parent phage APBP1 (Figure 12).

[0220] The host range of phages APBP1-1, APBP1-2, APBP3-1, and APBP3-2 was expanded by expression of AlgL A1-III (see Figures 13A to 13F).

[0221] Example 2: Methods for producing engineered bacteriophages One method to generate engineered bacteriophages that express alginate lyase from their genome involves the construction of a plasmid carrying the alginate lyase gene with appropriate expression signals (promoter and / or ribosome binding site) cloned between two fragments of the target phage genome that direct the alginate lyase gene to the appropriate locus by homologous recombination with the corresponding locus of the wild-type phage. These so-called "homology arms" can be as short as 50 bp or as long as 2 kb, but are usually 500 bp. In addition, the plasmid carries a single guide RNA (sgRNA) that is under constitutive expression. The sgRNA is a synthetic guide RNA that works in concert with the Cas9 nuclease, which can be programmed to generate a double-stranded break a few base pairs downstream of the sequence complementary to the sgRNA. The sgRNA / Cas9 complex thus represents a sequence-directed nuclease that can be used as a counterselection system for specific sequences. Here, sgRNA / Cas9 was used to counterselect wild-type target phage and promote the growth of recombinants that had acquired the alginate lyase gene. This was done by choosing a gRNA sequence that was disrupted or deleted during the recombination process so that the sgRNA / Cas9 complex could not target the genome of the recombinant. The design of sgRNA is well known to those skilled in the art. Cas9 was obtained in a specially constructed P. aeruginosa strain that expresses Cas9 from an IPTG-inducible promoter that is sensitive to the target phage. A plasmid carrying alginate lyase and sgRNA surrounded by homology arms was transformed into an appropriate Cas9-expressing strain. The resulting transformants were grown in the presence of IPTG and infected with wild-type phage. The resulting lysate was then plated on a lawn of the host of the target phage and individual plaques were analyzed for the presence of the alginate lyase gene at the target locus. Apparent recombination efficiencies ranged from 1 to 100%, and were most typically around 10% (1 / 10 phage plaques were correct recombinants).Analysis for proper insertion of the alginate lyase gene can be functional PCR-based and / or can be performed by complete genome sequencing, for example using Illumina NGS.

[0222] Another method for generating engineered bacteriophages relies on the work of Ando et al. (US2015 / 0064770A1). Briefly, the entire genome of the target phage is amplified by PCR in overlapping segments of 20-500 bp (most ideally 50 bp). At each end of the phage genome, the oligonucleotides used for amplification bear homology to a yeast artificial chromosome (YAC). Co-transformation of all overlapping phage genome fragments and the appropriate YAC in yeast (Saccharomyces cerevisiae) results in the assembly of a circular plasmid capable of replicating in yeast, which can be selected using a selection marker present on the YAC fragment (most commonly leucine auxotrophy), within which the entire phage genome is reassembled. Since yeast and bacteria have completely different gene expression machinery, the phage genome is kept inactive. The reconstructed phage-YAC is then extracted from the yeast cells and transformed into a highly competent bacterial strain (most commonly E. coli DH10B or its derivatives) where it reactivates to produce progeny that can be plated, grown on a suitable host (in this case, P. aeruginosa) and analyzed for correctness. Because PCR oligonucleotides can be selected at any location on the phage genome to generate overlapping genomic fragments, it is possible to make insertions, deletions, or mutations at any location in the phage, and to make multiple modifications in one reconstruction experiment (e.g., deleting a sequence and replacing it with another, or deleting a sequence in one locus and inserting another at another location in the genome). With appropriate primer design, it is possible to insert the alginate lyase gene at any desired locus in the target phage genome, as long as the following requirements are met: 1) at least 20 bp of overlap is maintained between each successive PCR fragment, and 2) no essential phage functions in the reconstructed genome are inactivated:

[0223] Example 3: Specially engineered bacteriophages A1-III is an N-terminal fragment of Sphingomonas sp. alginate lyase Aly (accession number BAB03312) corresponds to amino acids 54-412 of Aly. To preserve the coding sequence of the engineered phage, cloning of fragment 70-399 (Figure 4) was performed but did not show alginate activity. However, both fragment 54-412 and fragment 54-408 yielded alginate lyase activity. In some constructs, the C-terminal His was removed for detection or purification using anti-His tag antibodies. 6 The inset in Figure 4 shows the differences between some of the sequences around the C-terminus of the different A1-III fragments examined at the amino acid level.

[0224] The Sphingomonas alginate lyase gene, aly, produces a preproprotein. The full-sized protein is proteolytically cleaved to produce AlgL A1-II and A1-III, the latter of which possesses the bacterial alginate hydrolysis activity of Aly. Fragments 70–399 or 54–412 of the Sphingomonas aly gene (alginate lyase) were cloned using a C-terminal His 6The fragments were cloned into an E. coli expression plasmid with the tag and purified using a Ni affinity column. Fragment 70–399 was mostly insoluble and required denaturation and refolding of aggregates for purification (Figure 5A). A1-III fragment 54–412 showed a band of the correct size on a Western blot, indicating that the fragment was expressed (Figure 5B). The resulting purified protein was >99% pure in denaturing gel tests and was tested for its ability to degrade alginate from seaweed or bacteria. Activity was compared to a commercial alginate lyase preparation from Sigma-Aldrich (reference A1603) prepared at 10 mg / ml. Activity was evaluated on agar plates containing 2% seaweed alginate. 5 μl samples of the preparations or controls were applied to the surface of the plates, allowed to dry and then incubated at 30 °C for 4 h to overnight. Finally, the plates were stained with calcium phosphate cement (CPC), and activity was shown as a halo without a white precipitate (Figure 5C). Activity was also evaluated on preformed lawns of a mucoid alginate-expressing strain of P. aeruginosa, where activity is shown as a halo of rugged, more transparent lawn at the application site. Results are shown on the mucoid lawn of the alginate-producing P. aeruginosa strain 15844 (Figure 5D).

[0225] Alginate lyase A1-III was expressed as a fusion protein with gp13 when integrated downstream of APBP4 gp13.1. Figure 6A shows a Western blot of lysates of engineered phages carrying the A1-III alginate lyase payload. Engineered phages were used to infect exponentially growing cultures of P. aeruginosa clinical isolate DCF47 at an MOI of 1. Phage and host bacterial cells were grown together, shaken at 37°C, and then harvested 250 min post-infection. Lysates were centrifuged to enrich for cell debris. Cell pellets and supernatants were separated and frozen at -80°C. Each sample was then prepared for denaturing SDS-PAGE, electrophoresed, transferred to a PVDF membrane, and A1-III protein was detected using a primary antibody against A1-III and an appropriate alkaline phosphatase secondary antibody. Alginate lyase A1-III fragment 54-412 was shown as a band of approximately 37 kDa. The band of APBP4-4 (APBP4 (gp13.1 A1-III gp13)) in lanes 11 and 12 appeared at 55 kDa instead of approximately 43 kDa, presumably due to read-through of the gene into which the A1-III payload was inserted. This indicates that a fusion protein was produced, rather than the correct size A1-III protein alone. Figure 6B shows the DNA sequence around the C-terminus of A1-III, and A1-III-His 6 Figure 6C illustrates how trans-reading of the TGA stop codon of A1-III-His produced an in-frame fusion protein with gp13 via a 19 amino acid long "anti-terminator linker." 6 The deduced sequence of the A1-III-His-linker-gp13 fusion protein is shown. Residues in bold are A1-III-His 6 The underlined residues correspond to gp13, the grey residues indicate the putative linker, and the black highlighted tryptophan residues indicate that cells 6This is likely due to misreading of the TGA stop codon in APBP4-4. APBP4-4 did not show activity against the mucoid flora of the alginate-producing P. aeruginosa strain 15844, indicating that the fusion protein was not functional (Figure 6D).

[0226] Engineered phages carrying the A1-III alginate lyase payload were generated. The engineered phages were used to infect exponentially growing cultures of P. aeruginosa strain 7193 or clinical isolate DCF47 at an MOI of 1. Phage and host bacterial cells were grown together, shaken at 37°C, and then harvested 250 min post-infection. The lysates were centrifuged to enrich for cell debris. Cell pellets and supernatants were separated and frozen at -80°C. Each sample was then prepared for denaturing SDS-PAGE, electrophoresed, transferred to a PVDF membrane, and A1-III protein was detected using a primary antibody against A1-III and an appropriate alkaline phosphatase secondary antibody. These engineered versions of APBP4 showed the correct size band (Figure 7), indicating expression of A1-III and the absence of fusion with surrounding proteins.

[0227] Two codon usage matrices of alginate lyase A1-III were cloned into phage. Both were cloned into the same locus in APBP1 to generate APBP1-1 and APBP1-2. SEQ ID NO:34 has a higher GC content than SEQ ID NO:35 and is considered to be more optimized for expression in P. aeruginosa, whereas SEQ ID NO:35 is more optimized for expression in E. coli K-12 strain. Overall, the two genes code for the same protein, but only share 80.1% identity at the DNA level (Figure 8A). Both codon-optimized A1-IIIs incorporated into phage APBP1 showed alginate lyase activity against mucoid flora of P. aeruginosa 15844 (Figure 8B).

[0228] Examples of alginate lyase activity profiles from lysates of engineered phages expressing various forms of A1-III are shown in Figures 9A-9I. Activity was assessed in preformed lawns of a mucoid alginate-expressing strain of P. aeruginosa, where activity is shown as a halo of rugged, more transparent lawn at the application site. Results shown in Figures 9A-9I were observed in lawns of strain 15844. Fragment 70-399 shows no activity when integrated into the genome of phage APBP4 (Figure 9A) or APBP17 (Figure 9B). The 54-408 and 54-412 fragments of A1-III show activity in five different phages: the 54-408 fragment of SEQ ID NO:35 cloned into APBP1 (FIG. 9C); the 54-412 fragment of SEQ ID NO:35 cloned into APBP4 (FIG. 9D); the C-terminal His fragment cloned into APBP18 (FIG. 9E) and APBP1 (FIG. 9F). 6 - the 54-412 fragment of SEQ ID NO: 35 carrying a tag; the C-terminal His cloned into APBP3 (Figures 9G and 9I) and APBP1 (Figure 9H) 6 - Fragment 54-412 of SEQ ID NO: 34 carrying a tag.

[0229] The next set of experiments demonstrated that flavobacterial Alg2A (accession no. AEB69783), expressed from multiple phages, was expressed either by retaining its N-terminal signal sequence or by integrating a C-terminal His 6 We showed that all phages expressing Alg2A, whether tagged with a His-tag or encoded by four different genes, have alginate lyase activity. All phages expressing full-length Alg2A with a C-terminal His-tag produce lysates that exhibit alginate lyase activity against a mucoid lawn of the alginate-producing P. aeruginosa strain 15844 (Figure 10B). 6 -Alg2A without tags 23~288Phages expressing truncated signal sequence deleted versions of alg2A all produce lysates that exhibit alginate lyase activity against a mucoid strain of alginate producing P. aeruginosa 15844 (Figure 10C). We synthesized alg2A genes using four different codon usage matrices and cloned them into phage APBP3. SEQ ID NO:36 is believed to be optimized for expression in E. coli K-12, SEQ ID NO:37 for E. coli B, SEQ ID NO:38 for S. pneumoniae, and SEQ ID NO:39 for S. enterica serovar Typhimurium. These genes are 75.9-77.9% identical to each other (Figure 10D). Alg2A in SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39 are believed to be optimized for expression in E. coli K-12, E. coli B, SEQ ID NO:39 for S. enterica serovar Typhimurium, and SEQ ID NO:40 for S. pneumoniae. 23~288 APBP3-derived phages expressing α-glucose lyase produced lysates that exhibited alginate lyase activity against mucoid flora of the alginate-producing P. aeruginosa strain 15844 (Figure 10E).

[0230] To determine whether the cocktail of phages incorporating either A1-III or Alg2A alginate lyase had enzymatic activity, 10 microliters of the phage mixture was spotted onto a preformed mucoid lawn of P. aeruginosa strain FRD1 and then incubated at 30° C. before being photographed. The spot on the left in FIG. 11 is a mixture of wild-type parent phages APBP4, APBP1, APBP2, APBP6, and APBP3. No clearing of the mucoid lawn is visible. The center spot is a mixture of the same phages engineered to express A1-III: APBP2-1, APBP3-6, APBP6-1, APBP1-4, APBP4-5. Clearing is visible on the shiny surface of the mucoid lawn, suggesting that the enzymes have digested the available alginate. The spot on the right is a mixture of phages engineered to express Alg2A:APBP2-2, APBP3-5, APBP6-3, APBP1-5, and APBP4-6. Crater-like clearings are visible on the shiny surface of the mucoid flora, suggesting increased alginate lyase activity.

[0231] Each of the tested phages reached the same titer as the parental phages APBP1 and APBP3, which did not express alginate lyase, suggesting that the growth of phages APBP1-1, APBP1-2, APBP3-1, and APBP3-2 was not altered by the expression of the alginate lyase protein A1-III (Figure 12). Titers were calculated from the count of plaque-forming units (PFU) and the dilution rate of each lysate.

[0232] To determine the effect of expression of alginate lyase A1-III on the host range of the phages, lysates of the parent phages and their engineered derivatives were first titered against their host, P. aeruginosa strain 7299. The dilution series was in 10-fold steps for all phages and all plates. As can be seen in FIG. 13A, phages APBP3-1, wild-type APBP3, APBP1-1, APBP1-2, and wild-type APBP1 all yielded the same titer. In P. aeruginosa PS30, APBP3-1 was titered at a dilution of 10. -2 APBP1-1 and APBP1-2 produce plaques on P. aeruginosa PS30 strain, whereas their parent APBP1 does not produce plaques on this strain (Figure S13B). Similarly, APBP1-1 and APBP1-2 produce plaques on P. aeruginosa PS30 strain, whereas their parent APBP1 does not produce plaques (Figure S13B). APBP1-1 and APBP1-2 show improved clearing on P. aeruginosa 7176 strain compared to their parent APBP1 (Figure S13C). APBP1-1 and APBP1-2 produce plaques on P. aeruginosa 15843 strain, whereas their parent APBP3 does not form plaques on this strain (Figure S13C). -7 (about 2E8 PFU / ml), whereas their parent APBP1 formed plaques at a dilution rate of about 10 -6APBP3-1 formed plaques up to 100% (approximately 2E7 PFU / ml) on P. aeruginosa 15839 strain, whereas its parent APBP3 did not form any plaques on this strain (Fig. 13E). APBP1-1 and APBP1-2 also formed plaques on P. aeruginosa 15839 strain, whereas their parent APBP1 did not form plaques on this strain (Fig. 13E). APBP3-1 showed enhanced clearing on P. aeruginosa 15840 strain compared to its parent APBP3 (Fig. 13F). APBP1-1 and APBP1-2 also showed improved clearing on P. aeruginosa 15840 strain compared to APBP1 (Fig. 13F). These results suggest that the host range of phages APBP1-1, APBP1-2, APBP3-1 and APBP3-2 is expanded by expression of alginate lyase A1-III.

[0233] Four different strains of P. aeruginosa were grown as biofilms on PEG using an MBEC (minimum biofilm eradicating concentration) assay kit (Innovotech MBEC assay kit or any other similar assay known in the art may be used). Three versions of the 5-phage cocktail (AP-PA02) were applied to the biofilms, respectively. After treatment, the biofilms were washed and stained with crystal violet. The dye was extracted and then analyzed for OD595 density. Two cocktails of engineered phages, including phages expressing either A1-III (Eng-A1-III) or Alg2A (Eng-Alg2A), showed increased biofilm destruction when compared to the wild-type phage cocktail (wild type, FIG. 14), with strain 1 showing an increase of up to 30%. These results suggest that biofilms treated with engineered phages are more disrupted than those treated with the corresponding wild-type phages.

[0234] Recombination between expression plasmid pLIX36 and APBP6 carrying A1-III to integrate A1-III between gp038 and gp039 is shown in Figure 15A. A strain carrying plasmid pLIX36 carrying 520 bp upstream of APBP6 base pair 20585 (upstream homology arm, end of gp038), A1-III gene, and 500 bp downstream of base pair 20586 (downstream homology arm, start of gp039) was infected with APBP6 to allow recombination between the plasmid and the phage to generate an engineered APBP6 derivative in which base pair T20585 was replaced with A1-III. Recombination between pLIX46 and APBP6 to integrate A1-III between gp005 and gp006 is shown in Figure 15B. A strain carrying the plasmid pLIX46, which contains 437 bp upstream of base pair 2501 of APBP6 (upstream homology arm, gp003-005), the A1-III gene, and 501 bp downstream of base pair 2540 (downstream homology arm, start of gp039), was infected with APBP6 to allow recombination between the plasmid and the phage to generate an engineered APBP6 derivative in which base pairs 2502-2539 were replaced by A1-III.

[0235] A PCR assay was performed to verify the presence of the inserted A1-III in the lysate of APBP6 grown on a pLIX36-containing strain and then passaged in a plasmid-free host. A band at approximately 1.1 kb indicates the presence of the engineered phage (Figure 15C). The lysate of APBP6 grown in the presence of pLIX36 was serially diluted and assayed by PCR for the presence of the engineered phage. The recombinant lysate was then passaged in a host of APBP6 without pLIX36 and assayed again by PCR (Figure 15C). Recombinants were no longer detected, indicating that the engineered phage was not viable when cultured in the absence of pLIX36. A PCR assay was also performed to verify the presence of the inserted A1-III in the lysate of APBP6 grown on a pLIX46-containing strain and then passaged in a plasmid-free host. A band at approximately 1.1 kb indicates the presence of the engineered phage (Figure 15D). APBP6 lysates grown in the presence of pLIX46 were serially diluted and assayed by PCR for the presence of the engineered phage. Recombinant lysates were then passaged in the APBP6 host lacking pLIX46 and assayed again by PCR. Recombinants were still detected, indicating that the engineered phage could survive when cultured in the absence of pLIX36 (Figure 15D). These results suggested that integration of A1-III into APBP6 is possible at both the gp038 / gp039 and gp005 / gp006 loci, but expression of active enzyme occurs only at the gp005 / gp006 loci.

[0236] Figure 16 shows the alg2A 23~288Western blots of lysates of engineered phages carrying the alginate lyase gene are shown. Engineered phages were used to infect exponentially growing cultures of their hosts, P. aeruginosa clinical isolates 7193 or DCF47, at an MOI of 1. Phages and host bacterial cells were grown together, shaken at 37°C, and then harvested 250 min post-infection. The lysates consisted of lysed cells and phages in the medium. To separate the lysates into pellets and supernatants, the cell debris was concentrated by centrifugation. The cell pellets and supernatants were separated and frozen at -80°C. Each sample was then prepared for denaturing SDS-PAGE, electrophoresed, transferred to a PVDF membrane, and Alg2A protein was detected using a primary antibody against A1-III and an appropriate alkaline phosphatase secondary antibody. The alginate lyase Alg2A fragment was shown as a band at approximately 32 kDa. The ELISA protein quantification data are shown in the far right column of the chart in Figure 16 and demonstrate that the protein levels are pharmacologically relevant. These results demonstrate that engineered phages ABP4-6, ABP18-2, and ABP6-3 inhibit the alginate lyase protein Alg2A. 23~288 This suggests that the expression of

[0237] Figure 17 shows the alg2A 23~288 Western blots of lysates of engineered phages carrying different versions of the alginate lyase gene are shown. Engineered phages were used to infect exponentially growing cultures of their hosts, P. aeruginosa clinical isolates 7193 or DCF47, at an MOI of 1. Phages and host bacterial cells were grown together, shaken at 37°C, and then harvested 250 min post-infection. The lysates consisted of lysed cells and phages in the medium. The cell debris was concentrated by centrifugation to separate the lysates into pellets and supernatants. The cell pellets and supernatants were separated and frozen at -80°C. Each sample was then prepared for denaturing SDS-PAGE, electrophoresed, transferred to a PVDF membrane, and Alg2A protein was detected using a primary antibody against A1-III and the appropriate alkaline phosphatase secondary antibody. The full-length Alg2A protein was shown as a 35 kDa band, whereas Alg2A23~288 The fragment migrates as a band of approximately 32 kDa. ELISA protein quantification data is shown in the rightmost column of the chart in Figure 17 and indicates that the protein levels are pharmacologic relevant. These results demonstrate that engineered phages APBP3-5 and APBP1-5 are capable of targeting the alginate lyase protein Alg2A. 1~288 and Alg2A 23~288 This suggests that they express

[0238] Figure 18 shows A1-III 54~412 or A1-III 54~408 Western blots of lysates of engineered phages carrying the alginate lyase gene are shown. Engineered phages were used to infect exponentially growing cultures of their hosts, P. aeruginosa clinical isolates 7193 or DCF47, at an MOI of 1. Phages and host bacterial cells were grown together, shaken at 37°C, and then harvested 250 min post-infection. The lysates consisted of lysed cells and phages in the medium. To separate the lysates into pellets and supernatants, the cell debris was concentrated by centrifugation. The cell pellets and supernatants were separated and frozen at -80°C. Each sample was then prepared for denaturing SDS-PAGE, electrophoresed, transferred to a PVDF membrane, and Alg2A protein was detected using a primary antibody against A1-III and an appropriate alkaline phosphatase secondary antibody. The alginate lyase A1-III fragment was shown as a band at approximately 40 kDa. These results suggest that the engineered phages APBP18-1, APBP4-7, and APBP6-1 bind to the alginate lyase protein A1-III. 54~412 and APBP3-6 express alginate lyase protein A1-III 54~412 -His 6 and APBP1-4 express alginate lyase protein A1-III 54~408 This suggests that the expression of

[0239] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the methods and systems of the present invention will become apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unnecessarily limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the claims.

Claims

1. a sequence having at least 90% identity to the sequence of SEQ ID NO: 20; a sequence having at least 90% identity to the sequence of SEQ ID NO: 21; a sequence having at least 90% identity to the sequence of SEQ ID NO: 22; a sequence having at least 90% identity to the sequence of SEQ ID NO: 23; a sequence having at least 90% identity to the sequence of SEQ ID NO: 24; a sequence having at least 90% identity to the sequence of SEQ ID NO: 25; a sequence having at least 90% identity to the sequence of SEQ ID NO: 36, or a sequence having at least 90% identity to the sequence of SEQ ID NO: 59; A bacteriophage engineered to express an alginate lyase encoded by one or more sequences selected from:

2. 2. The bacteriophage of claim 1, wherein the alginate lyase comprises Alg2A or A1-III.

3. 10. The bacteriophage of claim 1, wherein the bacteriophage exhibits an improved host range.

4. 2. The bacteriophage according to claim 1, which belongs to the genus PhiKMV virus, Pakpunavirus, Bruynoghevirus, or Pubunavirus.

5. 5. The bacteriophage of claim 4, which targets one or more of Pseudomonas aeruginosa, antibiotic-resistant Pseudomonas aeruginosa, and multi-antibiotic-resistant Pseudomonas aeruginosa.

6. The bacteriophage of claim 1 , which reduces biofilm mass.

7. a polynucleotide sequence having at least 90% identity to SEQ ID NO: 26; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 27; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 28; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 29; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 30; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 31; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 32; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 33; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 34; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 35; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 37; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 38; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 39; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 40; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 41; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 42; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 43; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 44; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 45; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 46; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 47; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 48; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 49; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 50; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 51; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 52; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 53; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 54; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 55; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 56; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 57; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 58; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 60; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 61; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 62; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 63; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 64; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 65; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 66; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 67; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 68; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 69; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 70; a polynucleotide sequence having at least 90% identity to SEQ ID NO: 71, or a polynucleotide sequence having at least 90% identity to SEQ ID NO: 73; A bacteriophage composition comprising one or more bacteriophages comprising one or more polynucleotide sequences selected from:

8. 8. The bacteriophage composition of claim 7, wherein the one or more polynucleotide sequences encode an alginate lyase gene selected from Alg2A and A1-III.

9. 8. The bacteriophage composition of claim 7, wherein one or more of the bacteriophages are engineered.

10. 8. The bacteriophage composition of claim 7, wherein a second bacteriophage of said one or more bacteriophages comprises a natural phage.

11. 8. The bacteriophage composition of claim 7, wherein two or more bacteriophages of said one or more bacteriophages are naturally occurring phages.

12. 12. The bacteriophage composition of claim 11, wherein one or more bacteriophages of the composition target one or more of Pseudomonas aeruginosa, antibiotic-resistant Pseudomonas aeruginosa, and multi-antibiotic-resistant Pseudomonas aeruginosa.

13. 8. The bacteriophage composition of claim 7, further comprising a storage medium for storage at room temperature or at a temperature below 8°C, said storage medium comprising a cryoprotectant.

14. 8. The bacteriophage composition of claim 7, which is stored at a temperature ranging from -20°C to 25°C.

15. 15. The bacteriophage composition of claim 14, stored at 2°C to 8°C.

16. 14. The bacteriophage composition of claim 13, wherein the cryoprotectant comprises about 5% to about 50% glycerol.

17. 8. The bacteriophage composition of claim 7, further comprising a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof.

18. 14. The bacteriophage composition of claim 13, wherein the cryoprotectant comprises about 5% to about 30% sucrose.

19. 14. The bacteriophage composition of claim 13, wherein the cryoprotectant comprises dimethyl sulfoxide (DMSO) at a concentration of 2% to 10%.

20. 8. The bacteriophage composition of claim 7, which is a liquid, semi-liquid, solid, frozen, or lyophilized formulation.

21. 1 x 10 per milliliter ８ ~1 x 10 １２ 8. The bacteriophage composition of claim 7, comprising each bacteriophage of a PFU.

22. 8. The bacteriophage composition of claim 7, wherein the one or more bacteriophages of the composition reduce biofilm mass.

23. The composition of claim 7 for treating a Pseudomonas aeruginosa infection.

24. The composition has at least 3 x 10 per dose ８ 24. The composition of claim 23, administered in a dosage of PFU total bacteriophage.

25. 24. The composition of claim 23, further comprising an antibiotic selected from the group consisting of a fluoroquinolone, a carbapenem, an aminoglycoside, an ansamycin, a cephalosporin, a penicillin, a beta-lactam, a beta-lactamase inhibitor, a folate pathway inhibitor, a fucidane, a glycopeptide, a glycylcycline, a lincosamide, a lipopeptide, a macrolide, a quinolone, an oxazolidinone, a phenicol phosphonate, a streptogramin, a tetracycline, a sulfonamide, an imipenem, a meropenem, an amikacin, a ciprofloxacin, a levofloxacin, a tobramycin, an azithromycin, an aztreonam, a colistin, an inhaled tobramycin, an inhaled aztreonam, and an inhaled colistin.

26. 24. The composition of claim 23, further comprising one or more CFTR modulators selected from ivacaftor, lumacaftor and ivacaftor, tezacaftor and ivacaftor, elexacaftor, tezacaftor, and ivacaftor, or any combination thereof.

27. 24. The composition of claim 23, wherein the bacterial infection has become resistant to one or more antibiotics selected from fluoroquinolones, carbapenems, aminoglycosides, ansamycins, cephalosporins, penicillins, beta-lactams, beta-lactamase inhibitors, folate pathway inhibitors, fucidanes, glycopeptides, glycylcyclines, lincosamides, lipopeptides, macrolides, quinolones, oxazolidinones, phenicol phosphonates, streptogramins, tetracyclines, sulfonamides, imipenem, meropenem, amikacin, ciprofloxacin, levofloxacin, tobramycin, azithromycin, aztreonam, colistin, inhaled tobramycin, inhaled aztreonam, and inhaled colistin.

28. 24. The composition of claim 23, wherein the bacteriophage composition is administered by inhalation, nebulization, and / or intravenous administration.

29. 24. The composition of claim 23, wherein the bacteriophage composition is administered at least once daily and / or for at least one day.

30. 24. The composition of claim 23, wherein the subject is a human.

31. 24. The composition of claim 23, wherein the subject has cystic fibrosis (CF) or non-cystic fibrosis bronchiectasis (NCFB).

32. The composition of any of claims 23 to 31, wherein the subject has been previously treated with one or more antibiotics.

33. 10. An assay for determining the alginate lyase activity of an engineered bacteriophage, comprising administering an effective amount of the engineered bacteriophage of claim 1 to a Pseudomonas aeruginosa biofilm and determining a reduction in biofilm mass.

34. 8. The composition of claim 7 for treating a bacterial infection, comprising: (a) selecting a subject with a bacterial infection; (b) the subject with a bacterial infection is treated with an effective amount of the composition.

35. 1. A method for producing an engineered bacteriophage, the method comprising: providing a bacteriophage; and incorporating into the bacteriophage a nucleic acid sequence encoding an alginate lyase.

36. 36. The method of claim 35, wherein the alginate lyase is selected from Alg2A and A1-III.

37. 10. A kit comprising the bacteriophage compositions of claim 7 and instructions for their use.

38. 38. The kit of claim 37, further comprising a means for administering the bacteriophage composition selected from a syringe, a transdermal patch, a sustained release device, a spray, a nebulizer, an inhaler, or a respirator.

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