HK1196275B - Antibacterial phage, phage peptides and methods of use thereof - Google Patents

Description

Antibacterial bacteriophages, bacteriophage peptides and methods of use thereof

1. Sequence listing

This application contains a sequence listing submitted in ASCII form via EFS-Web and incorporated herein by reference in its entirety. The ASCII copy was created on day 14/9/2011, named 16395us1.txt and sized 3,295,858 bytes.

2. Related application

This application claims priority to U.S. provisional application No. 61/384,015, filed on 9/17/2010, the contents of which are hereby incorporated by reference in their entirety.

3. Field of the invention

The present invention relates to the field of phage therapy for the treatment and control of bacterial infections. In particular, the present invention relates to novel bacteriophages F387/08, F391/08, F394/08, F488/08, F510/08, F44/10, F125/10, isolated polypeptides thereof, compositions comprising one or more novel bacteriophages and/or isolated polypeptides; and methods for treating and preventing bacterial infections caused by: such as Staphylococcus aureus (Staphylococcus aureus), Klebsiella pneumoniae (Klebsiella pneumoniae), Acinetobacter baumannii (Acinetobacter baumannii), Escherichia coli (Escherichia coli) and/or Pseudomonas aeruginosa (Pseudomonas aeruginosa).

4. Background of the invention

Bacteriophage (phage) is a virus that specifically infects and lyses bacteria. Phage therapy, a method for treating bacterial infectious diseases using whole phage viruses, was introduced in the 20 th century by Felix d' Herelle. Initially, phage therapy was intensively investigated and numerous studies were conducted to evaluate the potential of phage therapy for treating bacterial infections in humans and animals. Early success has prompted the development of a variety of commercial phage preparations. For example, in 1940, Eli lilly company produced 7 phage products for human use, including phage preparations for treating different diseases caused by Staphylococcus species (Staphylococcus sp.), escherichia coli and other pathogenic bacteria. These formulations are useful, for example, in the treatment of infections causing abscesses, suppurative wounds, vaginitis, acute and chronic upper respiratory infections and mastoiditis infections.

However, with the development of antibiotics in the 40's of the 20 th century, interest in phage-based therapies has declined in the western world. One of the most important factors contributing to this decline is the lack of standardized test protocols and production methods. The documented results of the failed interference studies, which developed industry-wide standards for testing phage therapy, led to perceived lack of efficacy and reliability issues with regard to the value of phage therapy. Further, problems associated with phage sample/specimen production complicate initial research and investigation. Various stabilizers and preservatives were initially used in an attempt to increase the activity of phage therapy. However, because the biology of bacteriophages and various stabilizers is poorly understood, many of the ingredients added in an attempt to prolong the viability of phage preparations prove to be toxic to humans or negatively impact long-term storage. Another problem in phage production relates to the purity level of commercial preparations of phage. At the time, phage therapy preparations generally consisted of the original lysate of the host bacteria that had been treated with the phage of interest. Thus, many preparations contain what are currently recognized as undesirable bacterial components such as endotoxins. Accordingly, adverse events are often associated with the formulation, particularly in patients receiving it intravenously. However, in eastern europe and former soviet union, where access to antibiotics is limited, the development and use of phage therapy continues with or in place of antibiotics.

However, with the emergence of antibiotic resistant strains of many bacteria, interest in phage-based therapy has returned in the western world. Although new classes of antibiotics have been developed, the prospect that bacteria will eventually develop resistance to new drugs has exacerbated the search for non-chemotherapeutic approaches for controlling, preventing and treating bacterial infections. There are three major phage-based strategies for using phage therapy in a clinical setting: 1) administering a toxic bacteriophage; 2) using endolysins or purified lysins encoded by bacteriophage, 3) using structural proteins of bacteriophages as metabolic inhibitors of key bacterial enzymes such as enzymes that synthesize peptidoglycans.

Thus, there is a need to develop novel bacteriophage and bacteriophage products as potential therapeutic and/or prophylactic agents for use against pathogenic bacteria in vivo. In particular, there is a need for bacteriophage capable of lysing nosocomial bacteria, including staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, and/or pseudomonas aeruginosa. Because most bacteriophages and bacteriophage peptides studied to date exhibit activity specific to the bacterial species (or subspecies) from which they are isolated, novel phage-based therapies can be particularly useful in a hospital setting, selectively targeting nosocomial pathogens without affecting normal surrounding flora.

5. Summary of the invention

The present invention relates to isolated bacteriophage and to isolated antibacterial polypeptides of bacteriophage origin for use in the treatment, prevention or management of a condition associated with infection by gram-positive or gram-negative bacteria. In particular, the isolated bacteriophage or polypeptide of the present invention may be used in a pharmaceutical composition for the treatment, prevention or management of infection by nosocomial pathogens, such as gram-negative bacteria including, but not limited to, klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, and pseudomonas aeruginosa; and gram positive bacteria include, but are not limited to, staphylococcus aureus. In particular embodiments, the pharmaceutical compositions of the invention are used to treat conditions associated with infection by antibiotic resistant strains of bacteria, such as methicillin resistant strains of staphylococcus aureus (MRSA). In particular embodiments, the isolated bacteriophage or polypeptide of the present invention is used to topically treat an infection by a nosocomial pathogen in a subject in need thereof. In other embodiments, the isolated bacteriophage or polypeptide of the present invention is used to diagnose an infectious agent in a sample derived from a patient (e.g., a tissue, blood, urine, saliva sample). In other embodiments, the isolated bacteriophage or polypeptide of the present invention is used as a prophylactic disinfectant or anti-infective agent for the preparation of solid surfaces, including skin or other epidermal surfaces.

In a particular embodiment, the invention provides an isolated bacteriophage, F391/08, having an amino acid sequence comprising seq id NO:1 (fig. 15A-15III) and exhibits antibacterial activity against one or more strains of klebsiella pneumoniae. In other embodiments, the invention provides an isolated bacteriophage, F394/08, having an amino acid sequence comprising SEQ ID NO:2 (fig. 16A-16Q) and exhibits antibacterial activity against one or more strains of acinetobacter baumannii. In still other embodiments, the invention provides an isolated bacteriophage, F488/08, having an amino acid sequence comprising SEQ ID NO:3 (fig. 17A-17 kkkkkkkk) and exhibits antibacterial activity against one or more strains of e. In still other embodiments, the invention provides an isolated bacteriophage, F510/08, having an amino acid sequence comprising SEQ ID NO:4 (fig. 18A-18X) and exhibits antibacterial activity against one or more strains of pseudomonas aeruginosa. In yet a further embodiment, the present invention provides an isolated bacteriophage, F44/10, having an amino acid sequence comprising SEQ ID NO:560 (figures 19A-19UUU) and exhibits antibacterial activity against one or more strains of staphylococcus aureus. In yet a further embodiment, the present invention provides an isolated bacteriophage, F387/08, having an amino acid sequence comprising SEQ ID NO:781 (fig. 20A-20 kkkkkk) and exhibits antibacterial activity against one or more strains of klebsiella pneumoniae. In yet a further embodiment, the present invention provides an isolated bacteriophage, F125/10, having a nucleotide sequence comprising SEQ id no:1074 (FIGS. 21A-21ZZZ) and exhibits antibacterial activity against one or more strains of Staphylococcus aureus.

The invention also encompasses isolated bacteria infected with one or more bacteriophage of the invention. In a particular embodiment, the invention provides an isolated klebsiella pneumoniae infected by a bacteriophage having an amino acid sequence comprising SEQ ID NO:1 or a nucleic acid sequence consisting of SEQ ID NO:1 in a genome. In other embodiments, the invention provides an isolated acinetobacter baumannii infected with a bacteriophage having an amino acid sequence comprising seq id NO:2 or a nucleic acid sequence consisting of SEQ ID NO:2 in a genome. In yet other embodiments, the invention provides an isolated escherichia coli infected with a bacteriophage having an amino acid sequence comprising SEQ ID NO:3 or a nucleic acid sequence consisting of SEQ ID NO:3 in a genome. In still other embodiments, the invention provides an isolated pseudomonas aeruginosa infected with one or more bacteriophage having an amino acid sequence comprising SEQ ID NO:4 or a nucleic acid sequence consisting of SEQ ID NO:4, or a nucleic acid sequence of seq id no. In still other embodiments, the invention provides an isolated staphylococcus aureus infected with one or more bacteriophage having an amino acid sequence comprising SEQ ID NO:560 or by the nucleic acid sequence of SEQ ID NO:560, or a nucleic acid sequence of the genome. In yet a further embodiment, the present invention provides an isolated klebsiella pneumoniae infected with one or more bacteriophage having a sequence comprising SEQ ID NO:781 or a nucleic acid sequence consisting of SEQ ID NO:781 to a genome. In yet a further embodiment, the present invention provides an isolated staphylococcus aureus infected with one or more bacteriophage having an amino acid sequence comprising SEQ ID NO:1074 or a nucleic acid sequence consisting of SEQ ID NO: 1074.

The invention encompasses polypeptides isolated from bacteriophage F387/08, F391/08, F394/08, F488/08, F510/08, F44/10, and/or F125/10 that exhibit antibacterial activity against one or more species or strains of gram-positive or gram-negative bacteria, such as Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, and/or Staphylococcus aureus. In particular embodiments, the polypeptides of the invention isolated or derived from F387/08 and F3910/08 exhibit antibacterial or antimicrobial activity, e.g., lytic killing activity, against at least klebsiella pneumoniae; those isolated or derived from F394/08 exhibit antibacterial or antimicrobial activity against at least acinetobacter baumannii; those isolated or derived from F488/08 exhibit antibacterial or antimicrobial activity against at least E.coli; those isolated or derived from F510/08 exhibit antibacterial or antimicrobial activity against at least Pseudomonas aeruginosa; those isolated or derived from F44/10 and F125/10 exhibit antibacterial or antimicrobial activity against at least Staphylococcus aureus.

In particular embodiments, the polypeptides of the invention comprise or consist of an isolated lysin or fragment thereof (e.g., a CHAP domain) that exhibits antibacterial activity against one or more species or strains of bacteria, such as gram-positive bacteria, e.g., staphylococcus aureus, and/or gram-negative bacteria, e.g., klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, and/or pseudomonas aeruginosa. In a particular embodiment, the polypeptide of the invention is an isolated lysin protein, such as endolysin or urolysin, comprising or consisting of the amino acid sequence: SEQ ID NO: 20. SEQ ID NO: 80. SEQ ID NO: 192. SEQ ID NO: 282. SEQ ID NO: 547. SEQ ID NO: 556. SEQ ID NO: 557. SEQ ID NO: 598. SEQ ID NO: 1216. or SEQ ID NO: 1261. the predicted functions of the lysin proteins include, for example, Ig-like virion protein (SEQ ID NO: 20), cell wall hydrolase (SEQ ID NO: 80), N-acetylmuramyl-L-alanine amidase (SEQ ID NO: 192), soluble lysozyme (SEQ ID NO: 282), T4-like lysozyme (SEQ ID NO: 547), endolysin (SEQ ID NO: 556), lambda Rz 1-like protein (SEQ ID NO: 557), endolysin (SEQ ID NO: 598), endolysin (SEQ ID NO: 1216), and urolysin (SEQ ID NO: 1261).

In other embodiments, the polypeptide of the invention comprises SEQ ID NO: 20. SEQ ID NO: 80. SEQ ID NO: 192. SEQ ID NO: 282. SEQ ID NO: 547. SEQ ID NO: 556. SEQ ID NO: 557. SEQ ID NO: 598. SEQ ID NO: 1216. or SEQ ID NO: 1261, wherein said fragment, variant or derivative has antibacterial or antimicrobial activity, e.g. lytic killing activity, against one or more strains of klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa and/or staphylococcus aureus. In a specific example according to this embodiment, SEQ ID NO: 20 and/or SEQ ID NO: 80, is shown to be directed against one or more strains of klebsiella pneumoniae, for example against a strain having an amino acid sequence comprising SEQ ID NO:1 or a nucleic acid sequence consisting of seq id NO:1 (e.g., lytic killing activity). In other examples according to this embodiment, seq id NO: 192, is shown to be directed against one or more strains of acinetobacter baumannii, e.g. against a strain having an amino acid sequence comprising SEQ ID NO:2 or a nucleic acid sequence consisting of SEQ ID NO:2 (e.g., lytic killing activity). In other examples according to this embodiment, SEQ ID NO: 282, e.g., against one or more strains of e.coli, e.g., against a strain having an amino acid sequence comprising SEQ ID NO:3 or a nucleic acid sequence consisting of SEQ ID NO:3 (b) an antibacterial or antimicrobial activity (e.g., lytic killing activity) of a bacteriophage of the genome consisting of the nucleic acid sequence of 3. In other examples according to this embodiment, SEQ ID NO: 547. SEQ ID NO: 556 and/or SEQ ID NO: 557 to the amino acid sequence of SEQ ID NO:4 or a nucleic acid sequence consisting of SEQ ID NO:4 (e.g., lytic killing activity). In other examples according to this embodiment, SEQ ID NO: 598. SEQ ID NO: 1216 and/or SEQ ID NO: 1261 is shown against one or more strains of staphylococcus aureus, e.g. against a strain having an amino acid sequence comprising seq id NO:560 or SEQ ID NO:1074 or a nucleic acid sequence consisting of SEQ ID NO:560 or SEQ ID NO:1074, or a nucleic acid sequence of a bacteriophage of the genome (e.g., lytic killing activity).

In a particular embodiment, the isolated polypeptide of the invention comprises or consists of the CHAP domain as follows: SEQ ID NO: 20. SEQ ID NO: 80. SEQ ID NO: 192. SEQ ID NO: 282. SEQ ID NO: 547. SEQ ID NO: 556. SEQ ID NO: 557. or SEQ ID NO: 598. in yet other embodiments, the polypeptide of the invention comprises SEQ ID NO: 20. SEQ ID NO: 80. SEQ ID NO: 192. SEQ ID NO: 282. SEQ ID NO: 547. SEQ ID NO: 556. SEQ ID NO: 557. or SEQ ID NO: 598, wherein the fragment, variant or derivative has antibacterial or antimicrobial activity, e.g., lytic killing activity, against at least one or more strains of klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa, and/or staphylococcus aureus.

In other embodiments, the polypeptide of the invention comprises or consists of: an isolated tail protein (e.g., tail component, fiber protein, tail length tape protein, adsorption-related tail protein, major tail sheath protein, substrate wedge subunit) or fragment thereof, having a biological function associated with a bacteriophage from which it is derived, e.g., antimicrobial or antibacterial activity (e.g., lytic killing activity), against at least one or more species or strains of klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa, and/or staphylococcus aureus.

In a particular embodiment, the polypeptide of the invention is an isolated tail protein comprising or consisting of the amino acid sequence: SEQ ID NO: 15. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 30. SEQ ID NOs: 32-35, SEQ ID NO: 180. SEQ ID NO: 183. SEQ ID NO: 185. SEQ ID NO: 190. SEQ ID NO: 231. SEQ ID NO: 232. SEQ ID NO: 235. SEQ ID NOs: 239-245, SEQ ID NO: 248. SEQ ID NO: 249. SEQ ID NO: 252. SEQ ID NO: 254. SEQ ID NOs: 433-437, SEQ ID NOs: 489-496, SEQ ID NO: 544. SEQ ID NO: 545. SEQ ID NO: 549. SEQ ID NO: 551. SEQ ID NO: 629. SEQ ID NO: 686. SEQ ID NO: 789. SEQ ID NOs: 796-800, SEQ ID NO: 806. SEQ ID NO: 854. SEQ ID NOs: 999-1004, SEQ ID NOs: 1053-1060, SEQ ID NO: 1077. SEQ ID NO: 1217. SEQ ID NO: 1250. or SEQ ID NO: 1266. in other embodiments, the polypeptide of the invention comprises SEQ ID NO: 15. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 30. SEQ ID NOs: 32-35, SEQ ID NO: 180. SEQ ID NO: 183. SEQ ID NO: 185. SEQ ID NO: 190. SEQ ID NO: 231. SEQ ID NO: 232. SEQ ID NO: 235. SEQ ID NOs: 239-245, SEQ ID NO: 248. SEQ ID NO: 249. SEQ ID NO: 252. SEQ ID NO: 254. SEQ ID NOs: 433-437, SEQ ID NOs: 489-496, SEQ ID NO: 544. SEQ ID NO: 545. SEQ ID NO: 549. SEQ ID NO: 551. SEQ ID NO: 629. SEQ ID NO: 686. SEQ ID NO: 789. SEQ ID NOs: 796-800, SEQ ID NO: 806. SEQ ID NO: 854. SEQ ID NOs: 999-1004, SEQ ID NOs: 1053-1060, SEQ ID NO: 1077. SEQ ID NO: 1217. SEQ ID NO: 1250. or SEQ ID NO: 1266, wherein said fragment, variant or derivative exhibits a biological function, such as antimicrobial or antibacterial activity (e.g. lytic killing activity), associated with a bacteriophage from which it is derived, said function being directed against one or more strains of klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa and/or staphylococcus aureus.

The predicted functions of the tail proteins include, for example, receptor-binding tail protein (SEQ ID NO: 15), major tail protein (SEQ ID NO: 26 and SEQ ID NO: 1077), minor tail protein (SEQ ID NO: 27), pore-forming tail tip protein (SEQ ID NO: 30), tail protein (SEQ ID NOs: 32-33), minor tail protein (SEQ ID NO: 34), phage tail protein (SEQ ID NO: 35), tail sheath protein (SEQ ID NO: 180), tail coil ruler protein (SEQ ID NO: 183), tail protein (SEQ ID NO: 185), fiber protein (SEQ ID NO: 190), tail tube protein (SEQ ID NO: 231), tail sheath monomer (SEQ ID NO: 232), tail sheath stabilizer and complete protein (SEQ ID NO: 235), short fiber (SEQ ID NO: 239), Substrate wedge complete tail (SEQ ID NOs: 240-241), substrate wedge complete tail socket (tail fiber socket) (SEQ ID NO: 242), substrate wedge subunit (SEQ ID NO: 243), substrate wedge initiator (SEQ ID NO: 244), substrate wedge (SEQ ID NO: 245), substrate receptacle subunit and tail lysozyme, cell penetrating device (SEQ ID NO: 248), substrate wedge complete (SEQ ID NO: 249), tail complete and sheath stabilizing protein (SEQ ID NO: 252), chaperonin long and short tail assembly (SEQ ID NO: 254), tail protein (SEQ ID NO: 433), tail protein (SEQ ID NO: 434), hinge connector long tail (SEQ ID NO: 435), tail hinge (SEQ ID NO: 436), proximal tail subunit (SEQ ID NO: 437), substrate wedge complete tail (SEQ ID NO: 489), Substrate (SEQ ID NO: 490), substrate receptacle subunit, tail length determinant (SEQ ID NO: 491), substrate distal receptacle subunit (SEQ ID NO: 492), substrate receptacle subunit (SEQ ID NO: 493), substrate receptacle assembly catalyst (SEQ ID NO: 494), substrate receptacle subunit (SEQ ID NO: 495), substrate wedge subunit (SEQ ID NO: 496), tailpiece protein (SEQ ID NO: 544-545), tail fiber protein (SEQ ID NO: 549 and SEQ ID NO: 551), major tail sheath protein (SEQ ID NO: 629 and SEQ ID NO: 1250), major tail protein (SEQ ID NO: 686), tailpiece protein (SEQ ID NO: 789), fibritin (SEQ ID NO: 796), short tail fiber (SEQ ID NO: 797), substrate wedge complete tail pin (SEQ ID NO: 798), Substrate wedge subunit and tail pin (SEQ ID NO: 799), substrate wedge tail filament linker (SEQ ID NO: 800), substrate socket subunit and lysozyme (SEQ ID NO: 806), lysozyme (SEQ ID NO: 854), phage perforin (holin) (SEQ ID NO: 999 and SEQ ID NO: 1217), distal long tail filament assembly catalyst (SEQ ID NO: 1000), L-shaped tail filament protein (SEQ ID NO: 1001), hinge linker of long tail filament distal linker (SEQ ID NO: 1002), hinge linker of long tail filament proximal linker (SEQ ID NO: 1003), long tail filament proximal subunit (SEQ ID NO: 1004), substrate initiator (SEQ ID NO: 1053), substrate tail tube cap (tail tube cap) (SEQ ID NO: 1054), substrate socket subunit, tail long tail filament (SEQ ID NO: 1055), Substrate distal receptacle subunit (SEQ ID NO: 1056), substrate receptacle subunits (SEQ ID NOs: 1057 and 1059), substrate receptacle assembly catalyst (SEQ ID NO: 1058), substrate wedge subunit (SEQ ID NO: 1060), and substrate protein (SEQ ID NO: 1266).

In particular embodiments, the invention comprises SEQ ID NO: 15. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 30. or SEQ ID NOs: 32-35, which is shown to be homologous to a polypeptide having an amino acid sequence comprising the nucleic acid sequence SEQ ID NO:1 or by the nucleic acid sequence SEQ ID NO:1, such as antimicrobial or antibacterial activity (e.g., lytic killing activity), against one or more strains of klebsiella pneumoniae. In other embodiments, the invention comprises SEQ ID NO: 180. SEQ ID NO: 183. SEQ ID NO: 185. or SEQ ID NO: 190, which is shown to be homologous to a polypeptide having an amino acid sequence comprising the nucleic acid sequence of SEQ ID NO:2 or by the nucleic acid sequence SEQ ID NO:2, e.g., antimicrobial or antibacterial activity (e.g., lytic killing activity), said function being directed against one or more strains of acinetobacter baumannii.

In particular embodiments, the invention comprises SEQ ID NO: 231. SEQ ID NO: 232. SEQ ID NO: 235. SEQ ID NOs: 239-245, SEQ ID NO: 248. SEQ ID NO: 249. SEQ ID NO: 252. SEQ ID NO: 254. SEQ ID NOs: 433-437, SEQ ID NOs: 489-495, or SEQ ID NO: 496 which is shown to hybridize to a polypeptide having an amino acid sequence comprising the nucleic acid sequence of SEQ ID NO:3 or a nucleic acid sequence consisting of SEQ ID NO:3, e.g., antimicrobial or antibacterial activity (e.g., lytic killing activity), said function being directed against one or more strains of e. In particular embodiments, the invention comprises SEQ ID NO: 544. SEQ ID NO: 545. SEQ ID NO: 549. or SEQ ID NO: 551 which shows a homology reaction with a polypeptide having an amino acid sequence comprising the nucleic acid sequence SEQ ID NO:4 or by the nucleic acid sequence SEQ ID NO:4, e.g., antimicrobial or antibacterial activity (e.g., lytic killing activity), against one or more strains of pseudomonas aeruginosa. In still other embodiments, the invention comprises SEQ ID NO: 629 or SEQ ID NO: 686, which is shown to be identical to a variant, fragment or derivative having an amino acid sequence comprising the nucleic acid sequence of SEQ ID NO:560 or by the nucleic acid sequence SEQ ID NO:560, for example antimicrobial or antibacterial activity (e.g., lytic killing activity), against one or more strains of staphylococcus aureus. In still other embodiments, the invention comprises SEQ ID NO: 789. SEQ ID NOs: 796-800, SEQ ID NO: 806. SEQ ID NO: 854. SEQ ID NOs: 999-1004, or SEQ ID NOs: 1053-1060, which is shown to hybridize to a polypeptide having an amino acid sequence comprising the nucleic acid sequence of SEQ ID NO:781 or consists of the nucleic acid sequence SEQ ID NO:781, which function is directed against one or more strains of klebsiella pneumoniae, such as antimicrobial or antibacterial activity (e.g., lytic killing activity). In still other embodiments, the invention comprises seq id NO: 1077. SEQ ID NO: 1217. SEQ ID NO: 1250. or SEQ ID NO: 1266, which is shown to be homologous to a polypeptide having an amino acid sequence comprising the nucleic acid sequence of SEQ ID NO:1074 or a nucleic acid sequence consisting of SEQ ID NO:1074, such as antimicrobial or antibacterial activity (e.g., lytic killing activity), against one or more strains of staphylococcus aureus.

In particular embodiments, the invention provides isolated polypeptides that exhibit antimicrobial or antibacterial activity (e.g., lytic killing activity) against one or more strains of bacteria, such as gram-positive bacteria (e.g., Staphylococcus aureus), gram-negative bacteria (e.g., Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, and Pseudomonas aeruginosa) or bacteria not classified as gram-positive or gram-negative, wherein the isolated polypeptide has an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a second amino acid sequence of the same length (i.e., consisting of the same number of residues) that is SEQ ID NO: 15. SEQ ID NO: 20. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 30. SEQ ID NOs: 32-35, SEQ ID NO: 80. SEQ ID NO: 180. SEQ ID NO: 183. SEQ ID NO: 185. SEQ ID NO: 190. SEQ ID NO: 192. SEQ ID NO: 231. SEQ ID NO: 232. SEQ ID NO: 235. SEQ ID NOs: 239-245, SEQ ID NO: 248. SEQ ID NO: 249. SEQ ID NO: 252. SEQ ID NO: 254. SEQ ID NO: 282. SEQ ID NOs: 433-437, SEQ ID NOs: 489-496, SEQ ID NO: 544. SEQ ID NO: 545. SEQ ID NO: 547. SEQ ID NO: 549. SEQ ID NO: 551. SEQ ID NO: 556. SEQ ID NO: 557. SEQ ID NO: 598. SEQ ID NO: 629. SEQ ID NO: 686. SEQ ID NO: 789. SEQ ID NOs: 796-800, SEQ ID NO: 806. SEQ ID NO: 854. SEQ ID NOs: 999-1004, SEQ ID NOs: 1053-1060, SEQ ID NO: 1077. SEQ ID NO: 1216. SEQ ID NO: 1217. SEQ ID NO: 1250. SEQ ID NO: 1261. SEQ ID NO: 1266 and/or fragments thereof.

The invention further provides an isolated polypeptide comprising or consisting of any of the amino acid sequences set forth in seq id no: SEQ ID NOs: 5-176, SEQ ID NOs: 177-223, SEQ ID NOs: 224-506, SEQ ID NOs: 507-559, SEQ ID NOs: 561-780, SEQ ID NOs:782-1073, and SEQ ID NOs: 1075-1300. In other embodiments, isolated polypeptides of the invention are provided recombinantly fused or chemically conjugated (e.g., covalently or non-covalently conjugated) to a therapeutic agent (e.g., a heterologous polypeptide or small molecule).

The invention also encompasses polynucleotides encoding the polypeptides of the invention. In particular embodiments, the invention provides a polypeptide comprising a nucleotide sequence encoding SEQ ID NOs: 5-176, SEQ ID NOs: 177-223, SEQ ID NOs: 224-506, SEQ ID NOs: 507-559, SEQ ID NOs: 561-780, SEQ ID NOs:782-1073, and SEQ ID NOs: 1075-1300. In other embodiments, the invention provides an isolated nucleic acid comprising a nucleic acid sequence encoding any of the following polypeptides, or active fragments, variants or derivatives thereof: SEQ ID NO: 15. SEQ ID NO: 20. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 30. SEQ ID NOs: 32-35, SEQ ID NO: 80. SEQ ID NO: 180. SEQ ID NO: 183. SEQ ID NO: 185. SEQ ID NO: 190. SEQ ID NO: 192. SEQ ID NO: 231. SEQ ID NO: 232. SEQ ID NO: 235. SEQ ID NOs: 239-245, SEQ ID NO: 248. SEQ ID NO: 249. SEQ ID NO: 252. SEQ ID NO: 254. SEQ ID NO: 282. SEQ ID NOs: 433-437, SEQ ID NOs: 489-496, SEQ ID NO: 544. SEQ ID NO: 545. SEQ ID NO: 547. SEQ ID NO: 549. SEQ ID NO: 551. SEQ ID NO: 556. SEQ ID NO: 557. SEQ ID NO: 598. SEQ ID NO: 629. SEQ ID NO: 686. SEQ ID NO: 789. SEQ ID NOs: 796-800, SEQ ID NO: 806. SEQ ID NO: 854. SEQ ID NOs: 999-1004, SEQ ID NOs: 1053-1060, SEQ ID NO: 1077. SEQ ID NO: 1216. SEQ ID NO: 1217. SEQ ID NO: 1250. SEQ ID NO: 1261. or SEQ ID NO: 1266, which polypeptide or active fragment, variant or derivative exhibits a biological function associated with a bacteriophage from which it is isolated and/or derived, such as an antimicrobial or antibacterial activity (e.g. lytic killing activity). The invention also relates to vectors comprising one or more of said nucleic acids. In a particular embodiment, the vector is an expression vector. The invention further provides host cells containing a vector comprising one or more polynucleotides encoding a polypeptide of the invention.

The invention comprises a method for producing a polypeptide of the invention or an active fragment thereof, in particular for use in a pharmaceutical composition, i.e. an antimicrobial composition. For example, a polypeptide of the invention can be isolated directly from a cell culture (e.g., a bacterial cell culture) infected with bacteriophage F387/08, F391/08, F394/08, F488/08, F510/08, F44/10, and/or F125/10. Alternatively, the polypeptides of the invention may be derived by recombinant methods using expression vectors comprising a nucleic acid sequence encoding a polypeptide of the invention, such as SEQ ID NO: 15. SEQ ID NO: 20. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 30. SEQ ID NOs: 32-35, SEQ ID NO: 80. SEQ ID NO: 180. SEQ ID NO: 183. SEQ ID NO: 185. SEQ ID NO: 190. SEQ ID NO: 192. SEQ ID NO: 231. SEQ ID NO: 232. SEQ ID NO: 235. SEQ ID NOs: 239-245, SEQ ID NO: 248. SEQ ID NO: 249. SEQ ID NO: 252. SEQ ID NO: 254. SEQ ID NO: 282. SEQ ID NOs: 433-437, SEQ ID NOs: 489-496, SEQ ID NO: 544. SEQ ID NO: 545. SEQ ID NO: 547. SEQ ID NO: 549. SEQ ID NO: 551. SEQ ID NO: 556. SEQ ID NO: 557. SEQ ID NO: 598. SEQ ID NO: 629. SEQ ID NO: 686. SEQ ID NO: 789. SEQ ID NOs: 796-800, SEQ ID NO: 806. SEQ ID NO: 854. SEQ ID NOs: 999-1004, SEQ ID NOs: 1053-1060, SEQ ID NO: 1077. SEQ ID NO: 1216. SEQ ID NO: 1217. SEQ ID NO: 1250. SEQ ID NO: 1261. or SEQ ID NO: 1266, or an active fragment, derivative or variant thereof. The polypeptides of the invention or fragments thereof may be produced by any method known in the art for the production of polypeptides, in particular by chemical synthesis or by recombinant expression techniques.

In a particular embodiment, the present invention relates to a method for the recombinant production of a bacteriophage protein, such as a lysin protein, tail protein or an active fragment, variant or derivative thereof, said method comprising: (i) culturing a host cell containing a vector comprising a nucleic acid sequence encoding the following amino acid sequence under conditions suitable for expression of the protein in the culture medium: SEQ ID NO: 15. SEQ ID NO: 20. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 30. SEQ ID NOs: 32-35, SEQ ID NO: 80. SEQ ID NO: 180. SEQ ID NO: 183. SEQ ID NO: 185. SEQ ID NO: 190. SEQ ID NO: 192. SEQ ID NO: 231. SEQ ID NO: 232. SEQ ID NO: 235. SEQ ID NOs: 239-245, SEQ ID NO: 248. SEQ ID NO: 249. SEQ ID NO: 252. SEQ ID NO: 254. SEQ ID NO: 282. SEQ ID NOs: 433-437, SEQ ID NOs: 489-496, SEQ ID NO: 544. SEQ ID NO: 545. SEQ ID NO: 547. SEQ ID NO: 549. SEQ ID NO: 551. SEQ ID NO: 556. SEQ ID NO: 557. SEQ ID NO: 598. SEQ ID NO: 629. SEQ ID NO: 686. SEQ ID NO: 789. SEQ ID NOs: 796-800, SEQ ID NO: 806. SEQ ID NO: 854. SEQ ID NOs: 999-1004, or SEQ ID NOs: 1053-1060, SEQ ID NO: 1077. SEQ ID NO: 1216. SEQ ID NO: 1217. SEQ ID NO: 1250. SEQ ID NO: 1261. or SEQ ID NO: 1266, or a fragment thereof; and (ii) recovering the protein from the culture medium. In particular embodiments, the nucleic acid sequence encoding the polypeptide of the invention is operably linked to a heterologous promoter.

The invention also encompasses methods for diagnosing causative agents in the clinical manifestations of bacterial infection. The isolated bacteriophage or polypeptide of the present invention may be used to assist in the determination of bacterial species in a patient sample by determining the sensitivity of bacteria in the sample to the bacteriophage and/or polypeptide of the present invention. Such methods further include methods of estimating the antibacterial activity of the isolated bacteriophage and/or polypeptide of the present invention. The antibacterial activity of a bacteriophage or polypeptide of the present invention, or the sensitivity of an unknown sample to such activity, may be assessed by any method known in the art and/or described herein. In particular embodiments, antimicrobial activity and/or sensitivity is assessed by: known bacteria and/or patient tissue, blood, fluid or swab samples are cultured according to standard techniques (e.g., in liquid culture or on agar plates), the culture is contacted with a bacteriophage and/or polypeptide of the invention and cell growth following the contact is monitored. For example, in liquid culture, bacteria (e.g., klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa, and/or staphylococcus aureus) can grow to an optical density ("OD") representing the midpoint of exponential growth in the culture; the culture is exposed to one or more concentrations of one or more bacteriophage and/or polypeptides of the invention, and the OD is monitored relative to a control culture. The reduced OD relative to the control culture represents a bacteriophage and/or polypeptide that exhibits antibacterial activity (e.g., exhibits lytic killing activity) against the bacterial species and/or strain in the test sample or culture. Similarly, bacterial colonies may be allowed to form on agar plates that are exposed to a bacteriophage or polypeptide of the invention, and subsequent growth of the colonies is estimated relative to control plates. A decrease in the size of the colonies or a decrease in the total number of colonies indicates that the bacteriophage and/or polypeptide has antibacterial activity against the test sample and/or the cultured species or strain.

The invention also relates to a pharmaceutical composition comprising or consisting of a bacteriophage having a nucleotide sequence comprising the nucleic acid sequence SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 560. SEQ ID NO: 781. or SEQ ID NO:1074 or a nucleic acid sequence consisting of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 560. SEQ ID NO: 781. or SEQ ID NO: 1074. In a particular embodiment, the pharmaceutical composition of the invention comprises, in addition to one or more other bacteriophage, a bacteriophage having a genome comprising the nucleic acid sequence of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 560. SEQ ID NO: 781. or SEQ ID NO:1074 or a nucleic acid sequence consisting of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 560. SEQ ID NO: 781. or SEQ ID NO: 1074. The one or more other bacteriophage may be one or more bacterial strains of the present invention (e.g., having a nucleic acid sequence comprising or consisting of the nucleic acid sequence according to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:560, SEQ ID NO:781, or SEQ ID NO: 1074), or a genome comprising or consisting of the nucleic acid sequence according to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:560, SEQ ID NO:781, or SEQ ID NO: 1074), or may be one or more strains thereof, or may be other than having a nucleic acid sequence according to SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 560. SEQ ID NO: 781. or SEQ ID NO:1074 of the genome of a bacteriophage known in the art. Further, one or more bacteriophage in the pharmaceutical composition of the present invention may target the same or different species or strains of bacteria. In particular embodiments, a pharmaceutical composition comprising one or more bacteriophage of the present invention further comprises one or more polypeptides of the present invention and/or other bacteriophage products as described herein or known in the art.

In a particular embodiment, the present invention provides pharmaceutical compositions comprising polypeptides or active fragments thereof isolated from bacteriophage having a nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 560. SEQ ID NO:781 and/or SEQ ID NO:1074 or a nucleic acid sequence consisting of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 560. SEQ ID NO:781 and/or SEQ ID NO: 1074. In a particular embodiment, the pharmaceutical composition of the invention comprises one or more polypeptides having the following amino acid sequences: SEQ ID NO: 15. SEQ ID NO: 20. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 30. SEQ ID NOs: 32-35, SEQ ID NO: 80. SEQ ID NO: 180. SEQ ID NO: 183. SEQ ID NO: 185. SEQ ID NO: 190. SEQ ID NO: 192. SEQ ID NO: 231. SEQ ID NO: 232. SEQ ID NO: 235. SEQ ID NOs: 239-245, SEQ ID NO: 248. SEQ ID NO: 249. SEQ ID NO: 252. SEQ ID NO: 254. SEQ ID NO: 282. SEQ ID NOs: 433-437, SEQ ID NOs: 489-496, SEQ ID NO: 544. SEQ ID NO: 545. SEQ ID NO: 547. SEQ ID NO: 549. SEQ ID NO: 551. SEQ ID NO: 556. SEQ ID NO: 557. SEQ ID NO: 598. SEQ ID NO: 629. SEQ ID NO: 686. SEQ ID NO: 789. SEQ ID NOs: 796-800, SEQ ID NO: 806. SEQ ID NO: 854. SEQ ID NOs: 999-1004, SEQ ID NOs: 1053-1060, SEQ ID NO: 1077. SEQ ID NO: 1216. SEQ ID NO: 1217. SEQ ID NO: 1250. SEQ ID NO: 1261. or SEQ ID NO: 1266. in other embodiments, the pharmaceutical compositions of the invention comprise a polypeptide that is SEQ ID NO: 15. SEQ ID NO: 20. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 30. SEQ ID NOs: 32-35, SEQ ID NO: 80. SEQ ID NO: 180. SEQ ID NO: 183. SEQ ID NO: 185. SEQ ID NO: 190. SEQ ID NO: 192. SEQ ID NO: 231. SEQ ID NO: 232. SEQ ID NO: 235. SEQ ID NOs: 239-245, SEQ ID NO: 248. SEQ ID NO: 249. SEQ ID NO: 252. SEQ ID NO: 254. SEQ ID NO: 282. SEQ ID NOs: 433-437, SEQ ID NOs: 489-496, SEQ ID NO: 544. SEQ ID NO: 545. SEQ ID NO: 547. SEQ ID NO: 549. SEQ ID NO: 551. SEQ ID NO: 556. SEQ ID NO: 557. SEQ ID NO: 598. SEQ ID NO: 629. SEQ ID NO: 686. SEQ ID NO: 789. SEQ ID NOs: 796-800, SEQ ID NO: 806. SEQ ID NO: 854. SEQ ID NOs: 999-1004, SEQ ID NOs: 1053-1060, SEQ ID NO: 1077. SEQ ID NO: 1216. SEQ ID NO: 1217. SEQ ID NO: 1250. SEQ ID NO: 1261. or SEQ ID NO: 1266, wherein said variant, derivative or fragment retains a biological function of the polypeptide from which it is derived, such as antimicrobial or antibacterial activity (e.g., lytic killing activity), preferably against one or more strains of klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa, and/or staphylococcus aureus.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient or stabilizer. In a particular embodiment, the pharmaceutical compositions of the invention are antibacterial compositions (as they exhibit antibacterial activity) or therapeutic compositions for treating, preventing and/or ameliorating the symptoms of a disease or disorder associated with infection by bacteria in a subject in need thereof. In a particular embodiment, the pharmaceutical composition of the invention is an antibacterial composition or a therapeutic composition for the treatment, prevention and/or amelioration of the symptoms of a disease or disorder associated with infection by klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa and/or staphylococcus aureus. In particular embodiments, the subject receiving the pharmaceutical composition of the invention is a mammal (e.g., a cow, sheep, equine, primate (e.g., human), rodent, lagomorph, or avian (e.g., chicken, duck, goose)).

The present invention provides methods for treating or preventing a bacterial infection comprising administering to a subject in need thereof a pharmaceutical composition comprising one or more bacteriophage or bacteriophage products (e.g., an isolated bacteriophage polypeptide or active fragment, variant, or derivative thereof), optionally in addition to one or more other bacteriophage or other bacteriophage products as described herein. In the context of the present invention, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to eliminate, lessen, reduce the severity of, slow the progression of, or delay or prevent the symptoms or underlying cause (e.g., bacterial infection) associated with the pathological state or condition. The pharmaceutical compositions of the present invention may be used to treat or manage infections associated with any bacterial infection, including, but not limited to, klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa, and/or staphylococcus aureus, and in particular embodiments, staphylococcus epidermidis (s.epidermis), staphylococcus aureus (s.auricularis), staphylococcus capitis (s.capitis), staphylococcus haemolyticus (s.haemolyticus), staphylococcus hominis (s.hominis), staphylococcus saprophyticus (s.saprophyticus), staphylococcus simulans (s.simulans), staphylococcus xylosus (s.xylosis), micrococcus luteus (m.luteus), bacillus subtilis (b.subtilis), bacillus pumilus (b.pustus), enterococcus faecalis (e.faecium), enterococcus flavus (e.hirae), enterococcus faecium (e.faecium), and combinations thereof. In particular embodiments, the pharmaceutical compositions may be used to treat conditions or disorders associated with bacterial infections, including, but not limited to, postoperative endophthalmitis (endophthalmitis), endocarditis, infections of the central nervous system, pneumonia, osteomyelitis (osteomyelitis), wound infections (e.g., diabetic foot ulcers), mastitis, septicemia, food poisoning, meningitis, skin infections, abscesses, toxic shock syndrome, bacteremia, and/or other conditions associated with nosocomial bacterial infections.

In particular embodiments, the invention provides the use of a bacteriophage or an isolated bacteriophage product (e.g., an isolated bacteriophage polypeptide or active fragment, variant, or derivative thereof) as a single agent therapy. In other embodiments, the invention provides the use of a bacteriophage or bacteriophage product (e.g., an isolated bacteriophage polypeptide or active fragment, variant, or derivative thereof) in combination with standard or experimental treatments for bacterial infection. Such combination therapies may enhance the efficacy of standard or experimental treatments. Examples of therapeutic agents that are particularly useful in combination with the bacteriophage and/or polypeptide of the invention are anti-inflammatory agents, standard chemotherapeutic antibiotic agents (e.g., penicillins, synthetic penicillins, bacitracins, methicillins, nafcillins, oxacillins (oxacillins), cloxacillins, vancomycin, teicoplanin, clindamycin, sulfamethoxazole, cefamandole, cefazolin, cefixime, cefmetazole, cefonicid (cefonicid), cefoperazone, ceforanide, cefotame, cefotaxime, cefotetan, cefoxitin, cefpodoxime acetate, ceftazidime, ceftizoxime, ceftriaxone (cefatrine) amoxef, cefuroxime, cephalexin, cephalosporin C sodium salt, cephalosporin, cephalothin sodium salt, cefapirin, cefepime C sodium salt, cephalosporin, cef, Cephradine, cefuroxime axetil, cephalosporin dihydrate, moxalactam, chlorocefamafate and a chelating agent), a local anesthetic and/or a corticosteroid. In still other embodiments, the compositions of the invention may be combined with one or more bacteriophage or bacteriophage products known in the art. The combination therapies encompassed by the present invention may be formulated as a single pharmaceutical composition, or may be administered in separate compositions but as part of an overall treatment regimen.

The pharmaceutical compositions of the present invention may be administered by any method known in the art to be suitable for the administration of antibacterial compounds, for example via oral or parenteral (e.g., inhalation, intramuscular, intravenous or epidermal) delivery. In a preferred embodiment, the pharmaceutical composition of the invention is administered topically, e.g. in a topical formulation. The compositions of the invention may be used topically for the treatment and/or prevention of common nosocomial infections, such as infections at the site of surgical incisions or associated with catheters or drainage. In other embodiments, the compositions of the present invention are used to treat bacterial infections of the skin or upper dermal layers (e.g., infections of diabetic ulcers or carbuncles on the feet).

The pharmaceutical compositions of the invention may also be used in traditional non-therapeutic applications, such as antibacterial agents in cosmetics, or in sprays or solutions for use on solid surfaces to prevent colonization by bacteria (i.e. as disinfectants).

The invention also relates to methods for screening peptides for antimicrobial activity. In one embodiment, the method comprises screening for antimicrobial activity a contiguous amino acid sequence of at least 6, 10, 15, 20, or 25 residues in length consisting of the nucleic acid sequence of SEQ id no: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 560. SEQ ID NO: 781. or SEQ ID NO:1074, as measured by the ability of the peptide to inhibit bacterial growth, for example in agar or liquid culture.

5.1 definition

The term "fragment" as used herein refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues of the amino acid sequence of a protein. In particular embodiments, the fragment is a functional fragment in that it retains at least one function (e.g., antimicrobial or antibacterial activity (e.g., lytic cell killing)) of the protein from which it is isolated.

As used herein, the terms "active bacteriophage product" and "bacteriophage product" refer to a polypeptide, or fragment, variant, or derivative thereof, isolated from a bacteriophage of the present invention that exhibits a biological function or activity (e.g., antimicrobial or antibacterial activity (e.g., lytic cell killing)) associated with the bacteriophage from which it is isolated or derived.

As used herein, the term "isolated" in the context of a peptide, polypeptide, or fusion protein, or refers to a peptide, polypeptide, or fusion protein that is substantially free of cellular material or contaminating proteins from the cell or tissue source from which it is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of a peptide, polypeptide or fusion protein in which the peptide, polypeptide or fusion protein is separated from cellular components of the cell from which it is isolated or recombinantly produced. Thus, a peptide, polypeptide, or fusion protein that is substantially free of cellular material includes preparations of the peptide, polypeptide, or fusion protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein"). When the peptide, polypeptide or fusion protein is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% by volume of the protein preparation. When the peptide, polypeptide or fusion protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals involved in the synthesis of the peptide, polypeptide or fusion protein. Accordingly, such preparations of the peptide, polypeptide, fusion protein or antibody have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the peptide, polypeptide or fusion protein of interest.

As used herein, the term "isolated" in the context of nucleic acid molecules refers to a first nucleic acid molecule that is separated from other nucleic acid molecules present in the natural source of the first nucleic acid molecule. Furthermore, an "isolated" nucleic acid molecule, e.g., a cDNA molecule, is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized, and may be free of other cdnas or other genomic DNA molecules, e.g., when it has been isolated from other clones in a nucleic acid library.

The term "purified" means a peptide, polypeptide, fusion protein, or nucleic acid molecule that has been measurably increased in concentration by any purification process, including but not limited to column chromatography, HPLC, precipitation, electrophoresis, and the like, to partially, substantially, nearly completely, or completely remove impurities, e.g., involving the preparation of a precursor or other chemical of the peptide, polypeptide, or fusion protein or nucleic acid molecule. One skilled in the art will appreciate the amount of purification necessary for a given use. For example, an isolated protein intended for use in a therapeutic composition intended for administration to a human typically must have a high purity in accordance with regulatory standards and good manufacturing procedures.

As used herein, the term "derivative" in the context of a polypeptide refers to a polypeptide comprising an amino acid sequence that has been altered by the introduction of amino acid residue substitutions, deletions or additions. The term "derivative" as used herein also refers to a polypeptide that has been modified, i.e., by covalent attachment of any type of molecule to the polypeptide. For example, but not limited to, the polypeptide may be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, and the like. Derivative polypeptides can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. Further, the derivative polypeptide may contain one or more atypical amino acids. The polypeptide derivative has a function similar or equivalent to that of the polypeptide from which it is derived. The term "derived" as used in reference to a polypeptide "derived" from an organism may also refer to the isolation of the polypeptide directly from the organism (e.g., a bacterial cell or phage).

As used herein, the term "host cell" refers to the particular subject cell transfected with a nucleic acid molecule and to the progeny or potential progeny of such a cell containing the nucleic acid molecule or a chromosomally integrated form thereof. Progeny of such cells may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental effects that may occur in subsequent generations or within the genome of the host cell into which the nucleic acid molecule is integrated. For expression of bacteriophage proteins and polypeptides, the host cell is preferably not the same species or strain from which the bacteriophage is isolated or cultured.

As used herein, the term "in combination with … …" refers to the use of more than one prophylactic and/or therapeutic agent. The use of the term "in combination with … …" does not limit the order in which prophylactic and/or therapeutic agents are administered to a subject having a disease or disorder. The first prophylactic or therapeutic agent can be administered prior to (e.g., the first 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks), concomitantly with, or subsequent to (e.g., the last 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks) administration of the second prophylactic or therapeutic agent (different from the first prophylactic or therapeutic agent) to a subject in need thereof, e.g., a subject having a disease or disorder.

As used herein, the terms "nucleic acid" and "nucleotide sequence" include single-and double-stranded DNA and/or RNA molecules, or combinations thereof. As used herein, the term "encoded by a nucleic acid" refers to an amino acid sequence resulting from the translation of a forward, reverse, complementary, or reverse complement sequence of a reference nucleic acid sequence using a standard genetic code (i.e., a standard codon triplet) as is well known in the art.

As used herein, the term "prophylactic agent" refers to a bacteriophage and/or polypeptide of the present invention, which may be used to prevent, treat, manage or ameliorate one or more symptoms of a disease or disorder, particularly a disease or disorder associated with a bacterial infection.

As used herein, the term "therapeutic agent" refers to a bacteriophage and/or polypeptide of the present invention that may be used to prevent, treat, manage, or ameliorate one or more symptoms of a disease or disorder, particularly a disease or disorder associated with a bacterial infection.

As used herein, the term "therapeutically effective amount" refers to an amount of a therapeutic agent sufficient to result in the amelioration of one or more symptoms of a disease or disorder, particularly a disease or disorder associated with a bacterial infection.

As used herein, the terms "treatment" and "treating" refer to an improvement in one or more symptoms of a disease or disorder, particularly a disease or disorder associated with a bacterial infection, resulting from the administration of one or more bacteriophage and/or polypeptides of the present invention. As noted above, "treatment" and related terms refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to eliminate, lessen, reduce the severity of, slow the progression of, or delay or prevent symptoms or underlying causes (e.g., bacterial infection) associated with the pathological state or condition.

As used herein, the terms "antibacterial activity" and "antimicrobial activity" with respect to a bacteriophage, an isolated bacteriophage protein (or variant, derivative or fragment thereof), or a bacteriophage product are used interchangeably to refer to the ability to kill and/or inhibit the growth or reproduction of a microorganism, particularly a bacterial species or strain infected with a bacteriophage. In particular embodiments, the antibacterial or antimicrobial activity is assessed by: bacteria such as gram positive bacteria (e.g., staphylococcus aureus), gram negative bacteria (e.g., klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, and pseudomonas aeruginosa) or bacteria not classified as gram positive or gram negative are cultured according to standard techniques (e.g., in liquid culture or on agar plates), the culture is contacted with a bacteriophage or polypeptide of the invention and cell growth is monitored following such contact. For example, in liquid culture, bacteria can grow to an optical density ("OD") representing the midpoint of exponential growth in the culture; the culture is exposed to one or more concentrations of one or more bacteriophage or polypeptides of the invention and the OD is monitored relative to a control culture. Reduced OD relative to control cultures represents a bacteriophage or polypeptide that exhibits antibacterial activity (e.g., exhibits lytic killing activity). Similarly, bacterial colonies may be allowed to form on agar plates that are exposed to a bacteriophage or polypeptide of the invention, and subsequent growth of the colonies is assessed with respect to control plates. A decrease in the size of the colonies or a decrease in the total number of colonies indicates that the bacteriophage or polypeptide has antibacterial activity.

As used herein, "CHAP domain" refers to a conserved amidase domain found in several phage-encoded peptidoglycan hydrolases and represents a "cysteine, histidine-dependent amidohydrolase/peptidase". See, e.g., rigden d, et al, Trends Biochem sci.2003 May 28 (5): 230-4. It is found in the amidase superfamily, which includes GSP amidases and peptidoglycan hydrolases. This family includes at least two different types of peptidoglycan cleavage activity: L-muramyl-L-alanine amidase and D-alanyl-glycyl endopeptidase activity. The CHAP domain typically contains conserved cysteine and histidine residues and hydrolyzes gamma-glutamyl containing substrates. These cysteine residues are thought to be essential for the activity of several of these amidases, and their sulfhydryl groups appear to act as nucleophiles in the catalytic mechanism of all enzymes containing this domain. The CHAP domain is typically found in combination with other domains that cleave peptidoglycans, e.g., act in a synergistic manner to cleave specialized substrates. See also, Bateman a, et al, Trends Biochem sci.2003 May 28 (5): 234-7.

6. Brief description of the drawings

FIGS. 1A-1B: comprises the amino acid sequence of SEQ ID NO:1, in the genome of F391/08. The predicted open reading frame ("ORF") in the approximately 113kb genome is represented by an arrow and numbered in black. The direction of the arrow indicates the direction of transcription. Color coding: black-ORFs for which functional assignment can be made to their products based on the known function of the homologous protein; grey-ORFs encoding products similar to proteins of unknown function; blank-ORFs encoding proteins that do not share significant homology with proteins in the available databases. Functionally assigned ORFs are also listed in this figure. The information in this figure is also included in tabular form in figure 2.

FIGS. 2A-2 II: characteristics of the bacteriophage F391/08 genome, including the designation of gene products and putative functions. The figure includes a list of ORFs of the genome and provides for each ORF (i) its position within the genome, (ii) the encoded amino acid sequence, (iii) a list of homologous proteins and conserved domains within their encoded polypeptides and (iv) a specification of putative functions. ORFs 1-172 listed in figure 2 encode SEQ ID NOs: 5-176.

FIG. 3: comprises the amino acid sequence of SEQ ID NO:2, in the genome of F394/08. The predicted open reading frame ("ORF") in the approximately 31kb genome is represented by an arrow and numbered in black. The direction of the arrow indicates the direction of transcription. Color coding: black-ORFs for which functional assignment can be made to their products based on the known function of the homologous protein; grey-ORFs encoding products similar to proteins of unknown function; blank-ORFs encoding proteins that do not share significant homology with proteins in the available databases. Functionally assigned ORFs are also listed in this figure. The information in this figure is also included in tabular form in figure 4.

FIGS. 4A-4K: characteristics of the bacteriophage F394/08 genome, including the designation of gene products and putative functions. The figure includes a list of ORFs of the genome and provides for each ORF (i) its position within the genome, (ii) the encoded amino acid sequence, (iii) a list of homologous proteins and conserved domains within their encoded polypeptides and (iv) a specification of putative functions. ORFs 1-47 listed in figure 4 encode SEQ ID NOs: 177-223.

FIGS. 5A-5B: comprises the amino acid sequence of SEQ ID NO:3, in the genome of F488/08. The predicted open reading frame ("ORF") in the genome of about 167kb is represented by an arrow and numbered in black. The direction of the arrow indicates the direction of transcription. Color coding: black-ORFs whose function can be assigned to their products based on the known function of the homologous protein; grey-ORFs encoding products similar to proteins of unknown function; blank-ORFs encoding proteins that do not share significant homology with proteins in the available databases. Functionally assigned ORFs are also listed in this figure. The information in this figure is also included in tabular form in figure 6.

FIGS. 6A-6 DDD: characteristics of the bacteriophage F488/08 genome, including the designation of gene products and putative functions. The figure includes a list of ORFs of the genome and provides for each ORF (i) its position within the genome, (ii) the encoded amino acid sequence, (iii) a list of homologous proteins and conserved domains within their encoded polypeptides and (iv) a specification of putative functions. ORFs 1-283 listed in FIG. 6 encode SEQ ID NOS: 224-506.

FIG. 7: comprises the amino acid sequence shown in SEQ ID NO:4, in the genome of F510/08. The predicted open reading frame ("ORF") in the approximately 43kb genome is represented by an arrow and numbered in black. The direction of the arrow indicates the direction of transcription. Color coding: black-ORFs for which functional assignment can be made to their products based on the known function of the homologous protein; grey-ORFs encoding products similar to proteins of unknown function; blank-ORFs encoding proteins that do not share significant homology with proteins in the available databases. Functionally assigned ORFs are also listed in this figure. The information in this figure is also included in tabular form in figure 8.

FIGS. 8A-8S: characteristics of the bacteriophage F510/08 genome, including the designation of gene products and putative functions. The figure includes a list of ORFs of the genome and provides for each ORF (i) its position within the genome, (ii) the encoded amino acid sequence, (iii) a list of homologous proteins and conserved domains within their encoded polypeptides and (iv) a specification of putative functions. ORFs 1-53 listed in figure 8 encode SEQ ID NOs: 507-559.

FIG. 9: comprises the amino acid sequence of SEQ ID NO:560, for the F44/10 genome organization. The predicted open reading frame ("ORF") in the genome of approximately 137kb is represented by an arrow and numbered in black. The direction of the arrow indicates the direction of transcription. Color coding: black-ORFs for which functional assignment can be made to their products based on the known function of the homologous protein; grey-ORFs encoding products similar to proteins of unknown function; blank-ORFs encoding proteins that do not share significant homology with proteins in the available databases. Functionally assigned ORFs are also listed in this figure. The information in this figure is also included in tabular form in figure 10.

FIGS. 10A-10 QQ: characteristics of the bacteriophage F44/10 genome, including the designation of gene products and putative functions. The figure includes a list of ORFs of the genome and provides for each ORF (i) its position within the genome, (ii) the encoded amino acid sequence, (iii) a list of homologous proteins and conserved domains within their encoded polypeptides and (iv) a specification of putative functions. ORFs 1-216 listed in FIG. 10 include the amino acid sequences of ORFs 1a, 1b, 82a, 82b, 82c, 114a and 114b, which encode SEQ ID NOS 561-780, respectively.

FIG. 11: schematic representation of the organization of the F387/08 genome comprising the nucleic acid sequence of SEQ ID NO 781. The predicted open reading frame ("ORF") in the genome of about 167kb is represented by an arrow and numbered in black. The direction of the arrow indicates the direction of transcription. Color coding: black-ORFs for which functional assignment can be made to their products based on the known function of the homologous protein; grey-ORFs encoding products similar to proteins of unknown function; blank-ORFs encoding proteins that do not share significant homology with proteins in the available databases. Functionally assigned ORFs are also listed in this figure. The information in this figure is also included in tabular form in figure 12.

FIGS. 12A-12 UUuu: characteristics of the bacteriophage F387/08 genome, including the designation of gene products and putative functions. The figure includes a list of ORFs of the genome and provides for each ORF (i) its position within the genome, (ii) the encoded amino acid sequence, (iii) a list of homologous proteins and conserved domains within their encoded polypeptides and (iv) a specification of putative functions. ORFs 1-292 listed in FIG. 12 encode the amino acid sequences of SEQ ID NOs:782-1073, respectively.

FIGS. 13A-13B: schematic representation of the organization of the F125/10 genome comprising the nucleic acid sequence of SEQ ID NO 1074. The predicted open reading frame ("ORF") in the approximately 145kb genome is represented by an arrow and numbered in black. The direction of the arrow indicates the direction of transcription. Color coding: black-ORFs for which functional assignment can be made to their products based on the known function of the homologous protein; grey-ORFs encoding products similar to proteins of unknown function; blank-ORFs encoding proteins that do not share significant homology with proteins in the available databases. Functionally assigned ORFs are also listed in this figure. The information in this figure is also included in tabular form in figures 14A-14 ZZZ.

FIGS. 14A-14 ZZZ: characteristics of the bacteriophage F125/10 genome, including the designation of gene products and putative functions. The figure includes a list of ORFs of the genome and provides for each ORF (i) its position within the genome, (ii) the encoded amino acid sequence, (iii) a list of homologous proteins and conserved domains within their encoded polypeptides and (iv) a specification of putative functions. The ORFs 1b-221, 1a, including 36a and 36b, 68a and 68b, and 153a and 153b listed in this figure encode SEQ ID NOs: 1075-1300.

FIGS. 15A-15 III: the nucleotide sequence of the genome of bacteriophage F391/08 (SEQ ID NO: 1).

FIGS. 16A-16Q: the nucleotide sequence of the genome of bacteriophage F394/08 (SEQ ID NO: 2).

FIGS. 17A-17 KKKKKK: the nucleotide sequence of the genome of bacteriophage F488/08 (SEQ ID NO: 3).

FIGS. 18A-18X: the nucleotide sequence of the genome of bacteriophage F510/08 (SEQ ID NO: 4).

FIGS. 19A-19 UUuu: the nucleotide sequence of the genome of bacteriophage F44/10 (SEQ ID NO: 560).

FIGS. 20A-20 KKKK: the nucleotide sequence of the genome of bacteriophage F387/08 (SEQ ID NO: 781).

FIGS. 21A-21 ZZZ: the nucleotide sequence of the genome of bacteriophage F125/10 (SEQ ID NO: 1074).

6.1 detailed description

The present invention relates to isolated bacteriophage and isolated polypeptide products thereof, having antibacterial activity against one or more species or strains of the nosocomial pathogens Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. In one embodiment, an isolated bacteriophage or polypeptide is provided that exhibits antimicrobial and/or antibacterial activity against a Methicillin Resistant Strain (MRSA) of staphylococcus aureus. In addition, the bacteriophage and polypeptides of the present invention may exhibit antibacterial or antimicrobial activity against one or more species or strains of pathogenic bacteria, including, but not limited to, staphylococcus epidermidis, staphylococcus aureus, staphylococcus capitis, staphylococcus haemolyticus, staphylococcus hominis, staphylococcus saprophyticus, staphylococcus simulans, staphylococcus xylosus, micrococcus luteus, bacillus subtilis, bacillus pumilus, enterococcus hilgardii, and enterococcus avium.

In certain embodiments, the present invention provides a polypeptide having a sequence comprising SEQ ID NO:1 or a nucleic acid sequence consisting of SEQ id no:1, or a bacteriophage of a genome consisting of the nucleic acid sequence of seq id no. A specific example according to this embodiment is the isolated bacteriophage F391/08, which targets a number of strains of the klebsiella species, including klebsiella pneumoniae and klebsiella oxytoca (k.oxytoca). Comprises the amino acid sequence of SEQ ID NO:1 is provided in figure 1. The Open Reading Frame (ORF) in the F391/08 genome is provided in FIG. 2. Also provided are the location of the ORF within the genome, the amino acid sequence encoded by the ORF, homologous or similar proteins and conserved domains within the encoded polypeptide, and the assignment of putative functions. ORFs 1-172 listed in figure 2 encode SEQ ID NOs: 5-176.

In certain embodiments, the present invention provides a polypeptide having a sequence comprising SEQ ID NO:781 or a nucleic acid sequence consisting of seq id NO:781 to a genome consisting of the nucleic acid sequence of seq id no. A specific example according to this embodiment is isolated bacteriophage F387/08, which targets a number of strains of the species Klebsiella, including Klebsiella pneumoniae and Klebsiella oxytoca. Comprises the amino acid sequence of SEQ ID NO:781 is provided in fig. 11. The Open Reading Frame (ORF) in the F387/08 genome is provided in FIG. 12. Also provided are the location of the ORF within the genome, the amino acid sequence encoded by the ORF, homologous or similar proteins and conserved domains within the encoded polypeptide, and the assignment of putative functions. ORFs 1-292 listed in FIG. 12 encode SEQ ID NOs: 782-1073.

Klebsiella pneumoniae is a gram-negative, non-motile, rod-shaped bacterium found in the normal flora of the mouth, skin and small intestine. As a podded, facultative anaerobe, the bacterium also occurs naturally in soil, and about 30% of the strains can fix nitrogen under anaerobic conditions. Clinically, it is the most important member of the genus Klebsiella of the Enterobacteriaceae (Enterobacteriaceae) family and is also closely related to Klebsiella oxytoca. Klebsiella infections tend to occur in people with a weakened immune system due to improper diet, for example in alcoholics and diabetics. Klebsiella is also an opportunistic pathogen in patients with chronic lung disease, enteropathogenicity, nasal mucosa atrophy and rhinoscleroma. New antibiotic resistant strains of klebsiella pneumoniae emerge and are increasingly found as nosocomial infections, for example due to contact with contaminated instruments.

In certain embodiments, the present invention provides a polypeptide having a sequence comprising SEQ ID NO:2 or a nucleic acid sequence consisting of SEQ id no:2, or a bacteriophage of a genome consisting of the nucleic acid sequence of seq id no. A specific example according to this embodiment is the isolated bacteriophage F394/08, which targets a number of strains of acinetobacter species, including acinetobacter baumannii, acinetobacter calcoaceticus (a.calco aceticus), and acinetobacter iwoffi. Comprises the amino acid sequence of SEQ ID NO:2 is provided in figure 3. The Open Reading Frame (ORF) in the F394/08 genome is provided in FIG. 4. Also provided are the location of the ORF within the genome, the amino acid sequence encoded by the ORF, homologous or similar proteins and conserved domains within the encoded polypeptide, and the assignment of putative functions. ORFs 1-47 listed in FIG. 4 encode SEQ ID NOs: 177-223.

Acinetobacter baumannii is a bacterial species that causes many serious clinical infections, particularly in individuals with compromised immune systems. Acinetobacter baumannii is a pleomorphic aerobic gram-negative rod-shaped bacterium that is commonly isolated from hospital settings and hospitalized patients. The bacteria typically enter the body in open wounds, ducts or breathing tubes. Acinetobacter baumannii typically colonizes aquatic environments and is usually cultured from saliva or respiratory secretions, wounds, and urine of hospitalized patients. In a hospital setting, acinetobacter baumannii typically colonizes lavage fluids and intravenous solutions. It is also known to be resistant to various antibiotics, and the number of nosocomial infections caused by acinetobacter baumannii has increased in recent years.

In certain embodiments, the present invention provides a polypeptide having a sequence comprising SEQ ID NO:3 or a nucleic acid sequence consisting of SEQ id no:3, or a nucleic acid sequence of the genome. A specific example according to this embodiment is the isolated bacteriophage F488/08, which targets a number of strains of the genus Escherichia species, including E.coli. Comprises the amino acid sequence of SEQ ID NO:3 is provided in figure 5. The Open Reading Frame (ORF) in the F488/08 genome is provided in FIG. 6. Also provided are the location of the ORF within the genome, the amino acid sequence encoded by the ORF, homologous or similar proteins and conserved domains within the encoded polypeptide, and the assignment of putative functions. ORF 1-283 listed in FIG. 6 encodes SEQ ID NOs: 224-506.

Coli is a gram-negative rod-shaped bacterium commonly found in the lower intestine of mammals, including the primarau facultative anaerobes of the human gastrointestinal tract. Most strains of escherichia coli are harmless and may form part of the normal flora of the intestine, where they may contribute to their host, for example by producing vitamin K2 and/or by preventing colonization of the intestine by pathogenic bacteria. However, certain virulent strains of escherichia coli can cause food poisoning, typically manifested as episodes of diarrhea. More toxic strains such as O157: H7 can cause severe illness and even death in the elderly, very young or immunocompromised. Strains such as O157: H7 and O121 and O104: H21 potentially produce lethal toxins. Virulent strains of escherichia coli can also cause gastroenteritis, urinary tract infections and neonatal meningitis, and in rare cases, hemolytic-uremic syndrome (HUS), peritonitis, mastitis, septicaemia and gram-negative pneumonia. Further, if e.coli bacteria escape the intestine through perforations (e.g. due to ruptured ceca and ulcers or surgical errors) and enter the abdomen, they often cause peritonitis which can be fatal if not immediately treated. Coli associated with the intestinal mucosa has also been observed in an increasing number of inflammatory bowel diseases, crohn's disease, and ulcerative colitis.

however, as gram-negative organisms, E.coli is resistant to many antibiotics that are effective against gram-positive organisms, and antibiotic resistance is an ongoing problem.

In certain embodiments, the present invention provides a polypeptide having a sequence comprising SEQ ID NO:4 or the nucleic acid sequence consisting of SEQ id no:4, or a nucleic acid sequence of seq id No. 4. A specific example according to this embodiment is the isolated bacteriophage F510/08, which targets a number of strains of the Pseudomonas species, including Pseudomonas aeruginosa. Comprises the amino acid sequence of SEQ ID NO:4 is provided in figure 7. The Open Reading Frame (ORF) in the F510/08 genome is provided in FIG. 8. Also provided are the location of the ORF within the genome, the amino acid sequence encoded by the ORF, homologous or similar proteins and conserved domains within the encoded polypeptide, and the assignment of putative functions. ORFs 1-53 listed in FIG. 8 encode SEQ ID NOs: 507-559.

Pseudomonas aeruginosa is a common gram-negative rod-shaped bacterium found in soil, water, skin flora, and most artificial environments. It not only thrives at standard atmospheric pressure, but also as a facultative anaerobe with little oxygen, and can infect damaged tissues or immunocompromised individuals. When such colonization occurs in key body organs such as the lung, urinary tract and kidney, the result can be fatal. Because it thrives on the surface, this bacteria is also found on and in medical devices, including catheters, causing cross-infection in hospitals and clinics. Pseudomonas aeruginosa is one of the most relevant opportunistic nosocomial pathogens, and it has been estimated that one of the ten nosocomial infections is from the pseudomonas genus. Pseudomonas aeruginosa is also the most common cause of infection by burn injuries, and the most frequent colonizers of medical devices such as catheters.

In certain embodiments, the present invention provides a polypeptide having a sequence comprising SEQ ID NO:560 or by seq id NO:560, or a bacteriophage of a genome consisting of the nucleic acid sequence of seq id no. A specific example according to this embodiment is the isolated bacteriophage F44/10, which targets many strains of Staphylococcus species, including Staphylococcus aureus. Comprises SEQ ID NO:560 is provided in FIG. 9. The Open Reading Frame (ORF) in the F44/10 genome is provided in FIG. 10. Also provided are the location of the ORF within the genome, the amino acid sequence encoded by the ORF, homologous or similar proteins and conserved domains within the encoded polypeptide, and the assignment of putative functions. As shown in this figure, ORFs 1-216 listed in figure 10 include 1a, 1b, 82a, 82b, 82c, 114a and 114b encoding SEQ ID NOs: 561-780.

In certain embodiments, the present invention provides a polypeptide having a sequence comprising SEQ ID NO:1074 or a nucleic acid sequence consisting of seq id NO:1074, and a genome consisting of the nucleic acid sequence of seq id no. A specific example according to this embodiment is isolated bacteriophage F125/10, which targets a number of strains of Staphylococcus species, including Staphylococcus aureus. Comprises SEQ ID NO:1074 is provided in figure 13 by F125/10 genome diagram organization. The Open Reading Frame (ORF) in the F125/10 genome is provided in FIG. 14. Also provided are the location of the ORF within the genome, the amino acid sequence encoded by the ORF, homologous or similar proteins and conserved domains within the encoded polypeptide, and the assignment of putative functions. As shown in this figure, ORFs 1-221 listed in figure 14 include 1a, 1b, 36a, 36b, 68a, 68b, 153a and 153b encode SEQ id nos: 1075-1300.

Staphylococcus aureus is a gram-positive coccoid facultative anaerobe that grows as a grape-like cluster with a characteristic gold color and is the most common cause of staphylococcus infections (staph infections). It is usually part of the human skin flora and is responsible for a range of infections including papules, carbuncles, scalded skin syndrome, pneumonia, gastroenteritis, meningitis, myelitis, endocarditis, toxic shock syndrome, bacteremia and septicaemia. It is also one of the five most common causes of nosocomial infections, often causing post-operative wound infections. It has been estimated that about 50,000 patients in the U.S. hospital are infected with staphylococcal infection. Of particular interest are methicillin-resistant staphylococcus aureus strains (MRSA). MRSA was still a rare occurrence in the hospital setting until the 90's of the 20 th century, when there was an outbreak of MRSA epidemic in hospitals where it is currently considered endemic, especially in the uk. Johnson a.p., et al, j.antimicrobial chemitherapy, 48 (1): 143-144(2001). Staphylococcus aureus has proven to be a very bitter bacterium and in one study it has been shown to survive almost three months on polyester, which is the main material used in hospital privacy curtains. Neely, a.n., et al, j.clin.microbiol, 38 (2): 724-726(2000).

The following organisms were deposited under the budapest treaty ("budapest treaty") on the collection of microorganisms for patent programs, internationally acknowledged, at 16.9.20011, with NCIMB Limited at Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB 219 YA, Scotland UK, and NCIMB has been assigned the corresponding NCIMB accession numbers as follows: host strains Pseudomonas aeruginosa 433/07B2, NCIMB 41861; host strains staphylococcus aureus 743/06B 1, NCIMB 41862; host strains Acinetobacter baumannii 1305/05B3, NCIMB 41863; pseudomonas aeruginosa phage F770/05, NCIMB 41864; acinetobacter baumannii phage F1245/05, NCIMB 41865; staphylococcus aureus phage F125/10, NCIMB 41866; staphylococcus aureus phage F44/10, NCIMB 41867; and Pseudomonas aeruginosa phage F510/08, NCIMB41868, all of which are incorporated herein by reference.

In a particular embodiment, the bacteriophage of the present invention comprises or consists of a genome that is substantially identical to the genome of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 560. SEQ ID NO: 781. or SEQ ID NO:1074, wherein the bacteriophage exhibits at least one biological activity, e.g., antimicrobial or antibacterial activity (e.g., lytic killing activity), of one or more of bacteriophage F391/08, F394/08, F488/08, F510/08, F387/08, FF44/10, and F125/10. Alternatively or additionally, the bacteriophage of the present invention may have a nucleotide sequence comprising SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 560. SEQ ID NO: 781. or SEQ ID NO:1074, including the sequence of any of the open reading frames described in figures 2, 4, 6, 8, 10, 12 and/or 14.

The invention also provides isolated bacteria infected with one or more bacteriophage of the invention. In a particular embodiment, the invention provides an isolated klebsiella pneumoniae infected by a bacteriophage having an amino acid sequence comprising SEQ ID NO:1 and/or SEQ ID NO:781 or a nucleic acid sequence consisting of SEQ ID NO:1 and/or SEQ ID NO:781 to a genome. In a particular embodiment, the invention provides an isolated acinetobacter baumannii infected with a bacteriophage having an amino acid sequence comprising SEQ ID NO:2 or a nucleic acid sequence consisting of SEQ ID NO:2 in a genome. In a particular embodiment, the invention provides an isolated escherichia coli infected with a bacteriophage having an amino acid sequence comprising SEQ ID NO:3 or a nucleic acid sequence consisting of SEQ ID NO:3 in a genome. In a particular embodiment, the present invention provides an isolated pseudomonas aeruginosa infected with a bacteriophage having an amino acid sequence comprising SEQ ID NO:4 or a nucleic acid sequence consisting of SEQ ID NO:4, or a nucleic acid sequence of seq id no. In a particular embodiment, the invention provides an isolated staphylococcus aureus infected with a bacteriophage having an amino acid sequence comprising SEQ ID NO:560 and/or SEQ ID NO:1074 or a nucleic acid sequence consisting of SEQ ID NO:560 and/or SEQ ID NO: 1074.

The present invention provides methods of producing and isolating bacteriophage having a nucleotide sequence comprising SEQ id no: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 560. SEQ ID NO: 781. or SEQ ID NO:1074 or a nucleic acid sequence consisting of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 560. SEQ ID NO: 781. or SEQ ID NO: 1074. In particular embodiments, the present invention provides methods of producing and/or isolating a bacteriophage having an amino acid sequence comprising SEQ ID NO:1 and/or SEQ ID NO:781 or a nucleic acid sequence consisting of SEQ ID NO:1 and/or SEQ ID NO:781, the method comprising (i) obtaining a culture of klebsiella pneumoniae, (ii) contacting the cell with a nucleic acid sequence comprising SEQ ID NO:1 and/or SEQ ID NO:781 or a nucleic acid sequence consisting of SEQ ID NO:1 and/or SEQ ID NO:781 by a bacteriophage of a genome consisting of the nucleic acid sequence; (iii) culturing until significant lysis of the culture is observed; and (iv) isolating the bacteriophage from the culture. In other embodiments, the invention provides methods of producing and/or isolating a bacteriophage having a nucleotide sequence comprising SEQ ID NO:2 or a nucleic acid sequence consisting of SEQ ID NO:2, the method comprising (i) obtaining a culture of acinetobacter baumannii, (ii) culturing the acinetobacter baumannii in a culture medium having a nucleic acid sequence comprising SEQ ID NO:2 or a nucleic acid sequence consisting of SEQ ID NO:2, or a nucleic acid sequence of seq id No. 2; (iii) culturing until significant lysis of the culture is observed; and (iv) isolating the bacteriophage from the culture. In yet other embodiments, the present invention provides methods of producing and/or isolating a bacteriophage having an amino acid sequence comprising SEQ ID NO:3 or a nucleic acid sequence consisting of SEQ ID NO:3, the method comprising (i) obtaining a culture of e.coli, (ii) isolating the cell from the genome with a nucleic acid sequence comprising SEQ ID NO:3 or a nucleic acid sequence consisting of SEQ ID NO:3, or a genome consisting of the nucleic acid sequence of seq id no; (iii) culturing until significant lysis of the culture is observed; and (iv) isolating the bacteriophage from the culture. In yet other embodiments, the present invention provides methods of producing and/or isolating a bacteriophage having an amino acid sequence comprising SEQ ID NO:4 or a nucleic acid sequence consisting of SEQ ID NO:4, the method comprising (i) obtaining a culture of pseudomonas aeruginosa, (ii) culturing the pseudomonas aeruginosa cell in a culture medium having a nucleic acid sequence comprising SEQ ID NO:4 or the nucleic acid sequence consisting of SEQ id no:4, or a genome consisting of the nucleic acid sequence of seq id no; (iii) culturing until significant lysis of the culture is observed; and (iv) isolating the bacteriophage from the culture.

In yet a further embodiment, the present invention provides a method of producing and/or isolating a bacteriophage having an amino acid sequence comprising SEQ ID NO:560 and/or SEQ ID NO:1074 or the nucleic acid sequence consisting of SEQ id no:560 and/or SEQ ID NO:1074, the method comprising (i) obtaining a culture of staphylococcus aureus, (ii) contacting the culture with a nucleic acid sequence comprising SEQ ID NO:560 and/or SEQ ID NO:1074 or a nucleic acid sequence consisting of seq id NO:560 and/or SEQ ID NO:1074 by infecting it with a bacteriophage of the genome consisting of the nucleic acid sequence of seq id no; (iii) culturing until significant lysis of the culture is observed; and (iv) isolating the bacteriophage from the culture.

Bacteriophage can be isolated from a bacterial sample using any of the methods described herein or known In the art (see, e.g., Carlson, "work with bacteriophages: common technologies and methods," In, Kutter and Sulakveldze (eds.) bacteriophages: Biology and applications, 5 th edition CRC Press (2005); incorporated by reference In its entirety).

The invention also provides polypeptides isolated from the bacteriophage of the invention. The isolated polypeptide may be a full-length bacteriophage protein or may be a fragment, variant, or derivative of a bacteriophage protein, provided that the fragment, variant, or derivative exhibits at least one biological activity associated with the bacteriophage or polypeptide from which it is derived. In particular embodiments, the polypeptide of the invention is isolated from bacteriophage F387/08or F391/08 (which are typically infected with Klebsiella pneumoniae), F394/08 (which are typically infected with Acinetobacter baumannii), bacteriophage F488/08 (which are typically infected with E.coli), bacteriophage F510/08 (which are typically infected with Pseudomonas aeruginosa), or bacteriophage F44/10or F125/40 (which are typically infected with Staphylococcus aureus).

In a particular embodiment, the polypeptide of the invention is a lysin isolated from a bacteriophage having a genome comprising or consisting of the amino acid sequence: SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 560. SEQ ID NO: 781. or SEQ ID NO:1074 (e.g., bacteriophage F391/08, F394/08, F488/08, F510/08, F44/10, F387/08, or F125/10, respectively). In a particular embodiment, the polypeptide of the invention is a lysin, such as an endolysin or a urolysin, having or consisting of an amino acid sequence comprising: SEQ ID NO: 20. SEQ ID NO: 80. SEQ ID NO: 192. SEQ ID NO: 282. SEQ ID NO: 547. SEQ ID NO: 556. SEQ ID NO: 557. SEQ ID NO: 598. SEQ ID NO: 1216 or SEQ ID NO: 1261. the predicted functions of lysins include, for example, Ig-like virion protein (SEQ ID NO: 20), cell wall hydrolase (SEQ ID NO: 80), endolysins; N-acetylmuramyl-L-alanine amidase (SEQ ID NO: 192), soluble lysozyme (SEQ ID NO: 282), T4-like lysozyme (SEQ ID NO: 547), endolysin (SEQ ID NO: 556), lambda Rz 1-like protein (SEQ ID NO: 557), endolysin (SEQ ID NO: 598), endolysin (SEQ ID NO: 1216), and urolysin (SEQ ID NO: 1261).

In other embodiments, an isolated polypeptide of the invention is an endolysin or a fragment, variant or derivative of a lysin isolated from a bacteriophage of the invention, which fragment, variant or derivative exhibits at least one biological activity, preferably an antibacterial activity (e.g., lytic killing activity) of the endolysin, lysin or bacteriophage from which it is isolated or derived. Accordingly, in a particular embodiment, the invention provides an isolated polypeptide which is an endolysin or a fragment, variant or derivative of a lysin isolated from a bacteriophage of the invention, which fragment, variant or derivative exhibits antibacterial or antimicrobial activity (e.g. lytic killing activity) against one or more of klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa or staphylococcus aureus. In other embodiments, the isolated polypeptide is an endolysin or a fragment, variant or derivative of a lysin isolated from a bacteriophage of the present invention that exhibits antibacterial or antimicrobial activity (e.g., lytic killing activity) against one or more bacterial species other than klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa, or staphylococcus aureus. In particular embodiments, the polypeptide of the invention comprises the amino acid sequence SEQ id no: 20 and/or SEQ ID NO: 80 or a fragment, variant or derivative thereof, or a polypeptide consisting of the amino acid sequence SEQ ID NO: 20 and/or SEQ ID NO: 80 or a fragment, variant or derivative thereof, said polypeptide being shown to be directed against one or more strains of klebsiella pneumoniae, for example against a polypeptide having a sequence comprising the nucleic acid sequence SEQ ID NO:1 or by the nucleic acid sequence SEQ ID NO:1 of a genome consisting of a bacteriophage. In other embodiments, the polypeptide of the invention comprises the amino acid sequence SEQ ID NO: 192 or a fragment, variant or derivative thereof, or a polypeptide consisting of the amino acid sequence SEQ ID NO: 192 or a fragment, variant or derivative thereof, said polypeptide being shown to be directed against one or more strains of acinetobacter baumannii, for example against a polypeptide having a sequence comprising the nucleic acid sequence SEQ ID NO:2 or by the nucleic acid sequence SEQ ID NO:2, or an antimicrobial or antimicrobial activity of a bacteriophage of the genome consisting of seq id no. In yet other embodiments, the polypeptide of the invention comprises the amino acid sequence SEQ ID NO: 282 or a fragment, variant or derivative thereof, or a polypeptide consisting of the amino acid sequence SEQ ID NO: 282 or a fragment, variant or derivative thereof, said polypeptide being shown to be directed against one or more strains of escherichia coli, e.g. against a polypeptide having a sequence comprising the nucleic acid sequence SEQ ID NO:3 or a nucleic acid sequence consisting of SEQ ID NO:3 of a genome consisting of a bacteriophage. In yet other embodiments, the polypeptide of the invention comprises the amino acid sequence SEQ ID NO: 547. SEQ ID NO: 556. SEQ ID NO: 557 or a fragment, variant or derivative thereof, or a polypeptide consisting of the amino acid sequence SEQ ID NO: 547. SEQ ID NO: 556. SEQ ID NO: 557 or a fragment, variant or derivative thereof, said polypeptide exhibiting activity against one or more strains of pseudomonas aeruginosa, for example against a polypeptide having a sequence comprising the nucleic acid sequence SEQ ID NO:4 or by the nucleic acid sequence SEQ ID NO:4 of a genome consisting of a bacteriophage. In yet a further embodiment, the polypeptide of the invention comprises the amino acid sequence of seq id NO: 598 or a fragment, variant or derivative thereof, or a polypeptide consisting of the amino acid sequence SEQ ID NO: 598 or a fragment, variant or derivative thereof, said polypeptide being shown to be directed against one or more strains of staphylococcus aureus, for example against a polypeptide having a sequence comprising the nucleic acid sequence SEQ ID NO:560 or by the nucleic acid sequence SEQ ID NO:560, or a bacteriophage. In yet a further embodiment, the polypeptide of the invention comprises the amino acid sequence SEQ ID NO: 1216 and/or SEQ ID NO: 1261 or a fragment, variant or derivative thereof, or a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1216 and/or SEQ ID NO: 1261 or a fragment, variant or derivative thereof, said polypeptide being shown to be directed against one or more strains of staphylococcus aureus, for example against a polypeptide having a sequence comprising the nucleic acid sequence SEQ ID NO:1074 or a nucleic acid sequence consisting of SEQ ID NO:1074 the antibacterial or antimicrobial activity of a bacteriophage of the genome consisting of said bacteriophage.

In a particular embodiment, the polypeptide of the invention comprises or consists of a CHAP domain isolated from an endolysin or lysin of a bacteriophage F387/08, F391/08, F394/08, F488/08, F510/08, F44/10or F125/10. Isolated CHAP domains have been shown to retain the antibacterial activity, e.g., lytic killing activity, of the endolysins or lysins from which they are derived; CHAP domains can be identified and isolated by methods conventional in the art (see, e.g., Rigden et al, 2003, Trends biochem. Sci.28: 230-234; Bateman et al, 2003, Trends biochem. Sci.28: 234-237, each of which is incorporated herein by reference in its entirety). In certain embodiments, the polypeptide of the invention comprises or consists of a CHAP domain isolated from a polypeptide having the amino acid sequence of SEQ ID NO: 20. SEQ ID NO: 80. SEQ ID NO: 192. SEQ ID NO: 282. SEQ ID NO: 547. SEQ ID NO: 556. SEQ ID NO: 557. SEQ ID NO: 598. SEQ ID NO: 1216. or SEQ ID NO: 1261. In other embodiments, the invention provides a fragment, variant or derivative of the CHAP domain isolated from the endolysin or lysin of bacteriophage F387/08, F391/08, F394/08, F488/08, F510/08, F44/10, F125/10, said fragment, variant or derivative exhibiting at least one biological activity of the CHAP domain from which it is derived, e.g., lytic cell killing.

In a particular embodiment, the polypeptide of the invention comprises or consists of: a tail protein (e.g., tail component, fiber protein, adsorption related tail protein, tail length tape protein, substrate wedge subunit) or a fragment, variant or derivative thereof isolated from a bacteriophage having a genome comprising or consisting of: SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 560. SEQ ID NO: 781. or SEQ ID NO:1074 (e.g., bacteriophage F391/08, F394/08, F488/08, F510/08, F44/10, F387/08, or F125/10, respectively), wherein the tail protein, or fragment, variant, or derivative thereof, has a biological function, e.g., antimicrobial or antibacterial activity (e.g., lytic killing activity), associated with the bacteriophage from which it is derived. In particular embodiments, the antimicrobial or antibacterial activity of the tail protein is against at least one or more species or strains of klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa, and staphylococcus aureus. In a particular embodiment, the polypeptide of the invention is a tail protein having an amino acid sequence comprising or consisting of: SEQ ID NO: 15. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 30. SEQ ID NOs: 32-35, SEQ ID NO: 180. SEQ ID NO: 183. SEQ ID NO: 185. SEQ ID NO: 190. SEQ ID NO: 231. SEQ ID NO: 232. SEQ ID NO: 235. SEQ ID NOs: 239-245, SEQ ID NO: 248. SEQ ID NO: 249. SEQ ID NO: 252. SEQ ID NO: 254. SEQ ID NOs: 433-437, SEQ ID NOs: 489-496, SEQ ID NO: 544. SEQ ID NO: 545. SEQ ID NO: 549. SEQ ID NO: 551. SEQ ID NO: 629; SEQ ID NO: 686. SEQ ID NO: 789. SEQ ID NOs: 796-800, SEQ ID NO: 806. SEQ ID NO: 854. SEQ ID NOs: 999-1004, SEQ ID NOs: 1053-1060, SEQ ID NO: 1077. SEQ ID NO: 1217. SEQ ID NO: 1250. or SEQ ID NO: 1266. in other embodiments, the isolated polypeptide of the invention is a fragment, variant or derivative of the following amino acid sequence: SEQ ID NO: 15. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 30. SEQ ID NOs: 32-35, SEQ ID NO: 180. SEQ ID NO: 183. SEQ ID NO: 185. SEQ ID NO: 190. SEQ ID NO: 231. SEQ ID NO: 232. SEQ ID NO: 235. SEQ ID NOs: 239-245, SEQ id no: 248. SEQ ID NO: 249. SEQ ID NO: 252. SEQ ID NO: 254. SEQ ID NOs: 433-437, SEQ ID NOs: 489-496, SEQ ID NO: 544. SEQ ID NO: 545. SEQ ID NO: 549. SEQ ID NO: 551. SEQ ID NO: 629; SEQ ID NO: 686. SEQ ID NO: 789. SEQ ID NOs: 796-800, SEQ ID NO: 806. SEQ ID NO: 854. SEQ ID NOs: 999-1004, SEQ ID NOs: 1053-1060, SEQ ID NO: 1077. SEQ ID NO: 1217. SEQ ID NO: 1250. or SEQ ID NO: 1266 which exhibits at least one biological activity or function, e.g. antimicrobial or antibacterial activity (e.g. lytic killing activity), of a bacteriophage from which it is isolated or derived. In a preferred embodiment, at least one biological activity or function of the fragment, variant or derivative is directed against one or more strains of klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa and staphylococcus aureus.

The predicted functions of the tail proteins include, for example, receptor-binding tail protein (SEQ ID NO: 15), major tail protein (SEQ ID NO: 26), minor tail protein (SEQ ID NO: 27), pore-forming tail tip protein (SEQ ID NO: 30), tail protein (SEQ ID NOs: 32-33), minor tail protein (SEQ ID NO: 34), phage tail protein (SEQ ID NO: 35), tail sheath protein (SEQ ID NO: 180), tail coil ruler protein (SEQ ID NO: 183), tail protein (SEQ ID NO: 185), tail fiber protein (SEQ ID NO: 190), tail tube protein (SEQ ID NO: 231), tail sheath monomer (SEQ ID NO: 232), tail sheath stabilizer and holoprotein (SEQ ID NO: 235), short tail fiber (SEQ ID NO: 239), substrate wedge full tail nail (SEQ ID NOs: 240-241), Substrate wedge complete fiber socket (SEQ ID NO: 242), substrate wedge subunit (SEQ ID NO: 243), substrate wedge initiator (SEQ ID NO: 244), substrate wedge (SEQ ID NO: 245), substrate receptacle subunit and tail lysozyme, cell penetrating device (SEQ ID NO: 248), substrate wedge complete (SEQ ID NO: 249), tail complete and sheath stabilizing protein (SEQ ID NO: 252), chaperone protein long and short tail assembly (SEQ ID NO: 254), tail protein (SEQ ID NO: 433), tail protein (SEQ ID NO: 434), hinge connector long tail (SEQ ID NO: 435), tail hinge (SEQ ID NO: 436), proximal tail subunit (SEQ ID NO: 437), tail tube substrate initiator (SEQ ID NO: 489), substrate (SEQ ID NO: 490), Substrate receptacle subunit, tail length determinant (SEQ ID NO: 491), substrate distal receptacle subunit (SEQ ID NO: 492), substrate receptacle subunit (SEQ ID NO: 493), substrate receptacle assembly catalyst (SEQ ID NO: 494), substrate receptacle subunit (SEQ ID NO: 495), substrate wedge subunit (SEQ ID NO: 496), tailpiece protein (SEQ ID NOs: 544-545), tail fiber protein (SEQ ID NO: 549 and SEQ ID NO: 551), primary tail sheath protein (SEQ ID NO: 629); major tail protein (SEQ ID NO: 686); the tailpipe protein (SEQ ID NO: 789); fibritin (SEQ ID NO: 796); short fiber (SEQ ID NO: 797); a substrate wedge complete tail (SEQ ID NO: 798); a substrate wedge subunit and a tail pin (SEQ ID NO: 799); substrate wedge-bulge fiber linker (SEQ ID NO: 800); substrate receptacle subunit and lysozyme (SEQ ID NO: 806); lysozyme (SEQ ID NO: 854); bacteriophage perforin (SEQ ID NO: 999); a distal long tail assembly catalyst (SEQ ID NO: 1000); l-shaped fiber protein (SEQ ID NO: 1001); a hinged linker of a long-tail distal linker (SEQ ID NO: 1002); a hinge link to a long-tail proximal link (SEQ ID NO: 1003); a long fiber proximal subunit (SEQ ID NO: 1004); substrate Tail pipe initiator (SEQ ID NO: 1053); a substrate tail cap (SEQ ID NO: 1054); a substrate receptacle subunit; the tail length determinant (SEQ ID NO: 1055); a substrate distal receptacle subunit (SEQ ID NO: 1056); substrate receptacle subunits (SEQ ID NOs: 1057 and 1059); substrate jack mount catalyst (SEQ ID NO: 1058); a substrate wedge subunit (SEQ ID NO: 1060); major tail protein (SEQ ID NO: 1077); bacteriophage perforin (SEQ ID NO: 1217); major tail sheath protein (SEQ ID NO: 1250); and substrate protein (SEQ ID NO: 1266).

In particular embodiments, the invention comprises SEQ ID NO: 15. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 30. or SEQ ID NOs: 32-35, which exhibits a biological function, such as antimicrobial or antibacterial activity (e.g., lytic killing activity), associated with a bacteriophage having a sequence comprising the nucleic acid sequence of SEQ ID NO:1 or by the nucleic acid sequence SEQ ID NO:1, said function being directed against one or more strains of klebsiella pneumoniae. In particular embodiments, the invention comprises SEQ ID NO: 789. SEQ ID NOs: 796-800, SEQ ID NO: 806. SEQ ID NO: 854. SEQ ID NOs: 999-1004, or SEQ ID NOs: 1053-1060 that exhibits a biological function, such as antimicrobial or antibacterial activity (e.g., lytic killing activity), associated with a bacteriophage having an amino acid sequence comprising the nucleic acid sequence of SEQ ID NO:781 or consists of the nucleic acid sequence SEQ ID NO:781, said function being directed against one or more strains of klebsiella pneumoniae. In other embodiments, the invention comprises SEQ ID NO: 180. SEQ ID NO: 183. SEQ ID NO: 185. or SEQ ID NO: 190, which exhibits a biological function, such as antimicrobial or antibacterial activity (e.g., lytic killing activity), associated with a bacteriophage having an amino acid sequence comprising the nucleic acid sequence of SEQ ID NO:2 or by the nucleic acid sequence SEQ ID NO:2, said function being directed against one or more strains of acinetobacter baumannii.

In particular embodiments, the invention comprises SEQ ID NO: 231. SEQ ID NO: 232. SEQ ID NO: 235. SEQ ID NOs: 239-245, SEQ ID NO: 248. SEQ ID NO: 249. SEQ ID NO: 252. SEQ ID NO: 254. SEQ ID NOs: 433-437, SEQ ID NOs: 489-495, or SEQ ID NO: 496 which exhibits a biological function, such as antimicrobial or antibacterial activity (e.g., lytic killing activity), associated with a bacteriophage having an amino acid sequence comprising the nucleic acid sequence of SEQ ID NO:3 or a nucleic acid sequence consisting of SEQ ID NO:3, said function being directed against one or more strains of escherichia coli. In certain embodiments, the invention comprises seq id NO: 544. SEQ ID NO: 545. SEQ ID NO: 549. or SEQ ID NO: 551 which exhibits a biological function associated with a bacteriophage, such as antimicrobial or antibacterial activity (e.g., lytic killing activity), having an amino acid sequence comprising the nucleic acid sequence of SEQ ID NO:4 or by the nucleic acid sequence SEQ ID NO:4, said function being directed against one or more strains of pseudomonas aeruginosa. In particular embodiments, the invention comprises SEQ ID NO: 629 or SEQ ID NO: 686 that exhibits a biological function, such as antimicrobial or antibacterial activity (e.g., lytic killing activity), associated with a bacteriophage having an amino acid sequence comprising the nucleic acid sequence of SEQ ID NO:560 or by the nucleic acid sequence SEQ ID NO:560, said function being directed against one or more strains of staphylococcus aureus. In particular embodiments, the invention comprises SEQ ID NO: 1077. SEQ ID NO: 1216. SEQ ID NO: 1217. SEQ ID NO: 1250. SEQ ID NO: 1261. or SEQ ID NO: 1266, which exhibits a biological function, such as an antimicrobial or antibacterial activity (e.g. lytic killing activity), associated with a bacteriophage having a nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NO:1074 or a nucleic acid sequence consisting of SEQ ID NO:1074, said function being directed against one or more strains of staphylococcus aureus.

In a particular embodiment, the isolated polypeptide of the invention is a variant of a bacteriophage polypeptide comprising or consisting of: an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a second amino acid sequence of the same length (i.e., consisting of the same number of residues) that is SEQ ID NO: 15. SEQ ID NO: 20. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 30. SEQ ID NOs: 32-35, SEQ ID NO: 80. SEQ ID NO: 180. SEQ ID NO: 183. SEQ ID NO: 185. SEQ ID NO: 190. SEQ ID NO: 192. SEQ ID NO: 231. SEQ ID NO: 232. SEQ ID NO: 235. SEQ ID NOs: 239-245, SEQ ID NO: 248. SEQ ID NO: 249. SEQ ID NO: 252. SEQ ID NO: 254. SEQ ID NO: 282. SEQ ID NOs: 433-437, SEQ ID NOs: 489-496, SEQ ID NO: 544. SEQ ID NO: 545. SEQ ID NO: 547. SEQ ID NO: 549. SEQ ID NO: 551. SEQ ID NO: 556. SEQ ID NO: 557. SEQ ID NO: 598. SEQ ID NO: 629. SEQ ID NO: 686. SEQ ID NO: 789. SEQ ID NOs: 796-800, SEQ ID NO: 806. SEQ ID NO: 854. SEQ ID NOs: 999-1004, SEQ ID NOs: 1053-1060, SEQ ID NO: 1077. SEQ ID NO: 1216. SEQ ID NO: 1217. SEQ ID NO: 1250. SEQ ID NO: 1261. SEQ ID NO: 1266 and/or a fragment thereof, and wherein said variant exhibits at least one biological function or activity (e.g. antimicrobial or antibacterial activity (e.g. lytic killing activity)) against a bacteriophage from which it is derived of one or more strains of a bacterium, such as a gram-positive bacterium (e.g. staphylococcus aureus), a gram-negative bacterium (e.g. klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa) or a bacterium not classified as gram-positive or gram-negative.

In particular embodiments, the invention provides a polypeptide having the sequence of SEQ ID NOs: 5-176, SEQ ID NOs: 177-223, SEQ ID NOs: 224-506, SEQ ID NOs: 507-559, SEQ ID NOs: 561-780, SEQ ID NOs:782-1073, and SEQ ID NOs: 1075-1300, and active biological fragments thereof. In a preferred embodiment, a variant polypeptide of the invention exhibits at least one biological activity associated with the polypeptide or bacteriophage from which it is isolated or derived, e.g., lytic activity against at least one or more strains of klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa, and/or staphylococcus aureus.

In other embodiments, the invention provides nucleic acids encoding SEQ ID NOs: 5-176, SEQ ID NOs: 177-223, SEQ ID NOs: 224-506, SEQ ID NOs: 507-559, SEQ ID NOs: 561-780, SEQ ID NOs:782-1073, and SEQ ID NOs: 1075-1300 and active fragments thereof. In other embodiments, the invention provides a nucleic acid sequence encoding any of the open reading frames identified in figures 2, 4, 6, 8, 10, 12 and/or 14.

In particular embodiments, the polypeptides of the invention are recombinantly fused or chemically conjugated (including covalent and non-covalent conjugation) to a therapeutic agent, e.g., a heterologous polypeptide or small molecule, to produce a fusion protein or chimeric polypeptide. The fusion need not be direct, but may occur through a linker sequence or through chemical conjugation. Non-limiting examples of therapeutic agents to which the polypeptides of the invention may be conjugated are peptide or non-peptide cytotoxins (including antimicrobial agents and/or antibiotics), tracers/marker molecules (e.g., radionuclides and fluorophores), and other antibiotics or antibacterial compounds known in the art.

6.2 antibiotic compositions

The isolated bacteriophage or polypeptide of the present invention may be administered alone or incorporated within a pharmaceutical composition for use in the treatment or prevention of a bacterial infection, such as an infection caused by a bacterium, including but not limited to klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa, and staphylococcus aureus. The polypeptide may be combined with a pharmaceutically acceptable carrier, excipient, or stabilizer. Examples of pharmaceutically acceptable carriers, excipients, and stabilizers include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants include ascorbic acid; a low molecular weight polypeptide; proteins such as serum albumin and gelatin; parent (R)Water polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN^TMPolyethylene glycol (PEG) and PLURONICS^TM. For example, in addition to the above ingredients, the pharmaceutical composition of the present invention (e.g., antibacterial composition) may further include lubricating agents, wetting agents, sweetening agents, flavoring agents, emulsifying agents, suspending agents and preserving agents.

The bacteriophage and/or polypeptide of the present invention may also be combined with one or more therapeutic and/or prophylactic agents (e.g., one or more lysins) for use in treating bacterial infections described herein and/or known in the art. The pharmaceutical composition of the invention may therefore comprise two or more isolated bacteriophages of the invention (having antibacterial activity against the same or different bacterial species or strains), a combination of a bacteriophage of the invention and a polypeptide, or a combination of a bacteriophage of the invention and/or a polypeptide and a bacteriophage and/or a polypeptide known in the art. In particular embodiments, the combined therapeutic components target two or more species or strains of bacteria, or exhibit different enzymatic activities. For example, lysins generally exhibit one of amidase, endopeptidase, muramidase or glucosaminidase activity. Accordingly, combinations of lysins exhibiting different activities may provide a synergistic enhancement of the therapeutic activity of the pharmaceutical compositions of the present invention.

In certain embodiments, a number of different bacteriophage are combined to provide a "phage mixture. In certain embodiments, the phage mixture comprises at least 2 phage, at least 3 phage, at least 4 phage, at least 5 phage, at least 6 phage, at least 7 phage, at least 8 phage, at least 9 phage, at least 10 phage, or more. In certain embodiments, the phage mixture comprises 2-20 phage, 2-15 phage, 2-10 phage, 3-8 phage, or 4-6 phage.

In certain embodiments, at least one bacteriophage of the mixture is a bacteriophage having a biological activity against at least one gram-negative bacterium, including but not limited to klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, and pseudomonas aeruginosa; and/or bacteriophage active against at least one gram positive bacterium including, but not limited to, staphylococcus aureus. In a particular embodiment, at least one bacteriophage of the mixture is F391/08, having a heavy chain comprising SEQ ID NO:1 and exhibits antibacterial activity against one or more strains of klebsiella pneumoniae. In a particular embodiment, at least one bacteriophage of the mixture is F394/08 having an amino acid sequence comprising SEQ ID NO:2 and exhibits antibacterial activity against one or more strains of acinetobacter baumannii. In a particular embodiment, at least one bacteriophage of the mixture is F488/08 having an amino acid sequence comprising SEQ ID NO:3 and exhibits antibacterial activity against one or more strains of escherichia coli. In a particular embodiment, at least one bacteriophage of the mixture is F510/08 having an amino acid sequence comprising SEQ ID NO:4 and exhibits antibacterial activity against one or more strains of pseudomonas aeruginosa. In a particular embodiment, at least one bacteriophage of the mixture is F44/10 having an amino acid sequence comprising SEQ ID NO:560 and exhibits antibacterial activity against one or more strains of staphylococcus aureus. In a particular embodiment, at least one bacteriophage of the mixture is F387/08 having an amino acid sequence comprising SEQ ID NO:781 and exhibits antibacterial activity against one or more strains of klebsiella pneumoniae. In a particular embodiment, at least one bacteriophage of the mixture is F125/10 having an amino acid sequence comprising SEQ ID NO:1074 and exhibits antibacterial activity against one or more strains of staphylococcus aureus.

In a particular embodiment, at least one bacteriophage of the mixture is F170/08, having a genome as disclosed in WO2010/090542, and exhibiting antibacterial activity against one or more strains of fecal or enterococcus faecium. In a particular embodiment, the at least one bacteriophage of the mixture is F168/08, having a genome as disclosed in WO2010/090542, and exhibiting antibacterial activity against one or more strains of fecal or enterococcus faecium. In a particular embodiment, at least one bacteriophage of the mixture is F770/05, having a genome as disclosed in WO2010/090542, and exhibiting antibacterial activity against one or more strains of pseudomonas aeruginosa. In a particular embodiment, at least one bacteriophage of the mixture is F1245/05, which has a genome as disclosed in WO2010/090542 and shows antibacterial activity against one or more strains of acinetobacter baumannii.

In certain preferred embodiments, the mixture comprises a bacteriophage that has biological activity against acinetobacter. For example, the mixture may comprise F394/08 and/or F1245/05 showing antibacterial activity against one or more strains of acinetobacter baumannii. In particular embodiments, the mixture of bacteriophages comprises at least one bacteriophage that exhibits antibacterial activity against one or more strains of acinetobacter baumannii, and at least one bacteriophage that exhibits antibacterial activity against a different bacterium. For example, in certain embodiments, the phage mixture comprises F394/08 and/or F1245/05 in combination with at least one other phage selected from the group consisting of F391/08, F488/08, F510/08, F44/10, F387/08, F170/08, F168/08, F770/05, and F125/10. In particular embodiments, the mixture of bacteriophages comprises at least one bacteriophage that exhibits antibacterial activity against one or more strains of klebsiella pneumoniae, and at least one bacteriophage that exhibits antibacterial activity against a different bacterium. For example, in certain embodiments, the phage mixture comprises F391/08 and/or F387/08 in combination with at least one other phage selected from F394/08, F488/08, F510/08, F44/10, F1245/05, F170/08, F168/08, F770/05, and F125/10. In particular embodiments, the phage mixture comprises at least one phage that exhibits antibacterial activity against one or more strains of E.coli, and at least one phage that exhibits antibacterial activity against a different bacterium. For example, in certain embodiments, the phage mixture comprises F488/08 in combination with at least one other phage selected from the group consisting of F391/08, F510/08, F44/10, F394/08, F387/08, F170/08, F168/08, F1245/05, F770/05, and F125/10.

In particular embodiments, the mixture of bacteriophages comprises at least one bacteriophage that exhibits antibacterial activity against one or more strains of pseudomonas aeruginosa, and at least one bacteriophage that exhibits antibacterial activity against a different bacterium. For example, in certain embodiments, the phage mixture comprises F510/08 and/or F770/05 in combination with at least one other phage selected from the group consisting of F391/08, F394/08, F488/08, F44/10, F387/08, F170/08, F168/08, F1245/05, and F125/10. In particular embodiments, the phage mixture comprises at least one phage that exhibits antibacterial activity against one or more strains of staphylococcus aureus, and at least one phage that exhibits antibacterial activity against a different bacterium. For example, in certain embodiments, the phage mixture comprises F44/10 and/or F125/10 in combination with at least one other phage selected from the group consisting of F391/08, F394/08, F488/08, F510/08, F387/08, F170/08, F168/08, F770/05, and F1245/05. In particular embodiments, the phage mixture comprises at least one phage that exhibits antibacterial activity against one or more strains of fecal or enterococcus faecium, and at least one phage that exhibits antibacterial activity against a different bacterium. For example, in certain embodiments, the phage mixture comprises F170/08 and/or F168/08 in combination with at least one other phage selected from the group consisting of F391/08, F394/08, F488/08, F510/08, F44/10, F387/08, F770/05, F1245/05, and F125/10.

In a particular embodiment, the phage mixture comprises at least four (4) phages selected from the group consisting of F391/08, F394/08, F488/08, F510/08, F44/10, F387/08, F170/08, F168/08, F770/05, F1245/05, and F125/10. In particular embodiments, the phage mixture comprises F391/08, F394/08, F488/08, and F510/08. In particular embodiments, the phage mixture comprises F44/10, F387/08, F170/08, and F168/08. In particular embodiments, the phage mixture comprises F391/08, F394/08, F770/05, and F1245/05. In particular embodiments, the phage mixture comprises F391/08, F394/08, F510/08, and/or F44/10. In particular embodiments, the phage mixture comprises F391/08, F394/08, F44/10, and/or F387/08. In particular embodiments, the phage mixture comprises F391/08, F394/08, F387/08, and/or F170/08. In particular embodiments, the phage mixture comprises F391/08, F394/08, F170/08, and F168/08. In particular embodiments, the phage mixture comprises F391/08, F394/08, F168/08, and/or F770/05. In particular embodiments, the phage mixture comprises F391/08, F394/08, F770/05, and F1245/05.

In particular embodiments, the phage mixture comprises F125/10, F391/08, F394/08, and F488/08. In particular embodiments, the phage mixture comprises F125/10, F394/08, F488/08, and F510/08. In particular embodiments, the phage mixture comprises F125/10, F488/08, F510/08, and F44/10. In particular embodiments, the phage mixture comprises F125/10, F44/10, F387/08, and F170/08. In particular embodiments, the phage mixture comprises F125/10, F170/08, F168/08, and F770/05. In particular embodiments, the phage mixture comprises F125/10, F770/05, F1245/05 and F391/08. In particular embodiments, the phage mixture comprises F125/10, F510/08, F44/10, F387/08. In particular embodiments, the phage mixture comprises F125/10, F387/08, F170/08, F168/08. In particular embodiments, the phage mixture comprises F125/10, F168/08, F770/05, and F1245/05. In particular embodiments, the phage mixture comprises F125/10, F1245/05, F391/08, and F394/08.

In particular embodiments, the phage mixture comprises F394/08, F488/088, F510/08, and/or F44/10. In particular embodiments, the phage mixture comprises F394/08, F488/088, F44/10, and/or F387/08. In particular embodiments, the phage mixture comprises F394/08, F488/088, F387/08, and/or F170/08. In particular embodiments, the phage mixture comprises F394/08, F488/088, F170/08, and/or F168/08. In particular embodiments, the phage mixture comprises F394/08, F488/088, F168/08, and/or F770/05. In particular embodiments, the phage mixture comprises F394/08, F488/088, FF770/05, and/or F1245/05. In particular embodiments, the phage mixture comprises F394/08, F488/088, F1245/05, and/or F391/08. In particular embodiments, the phage mixture comprises F488/08, F510/08, F44/10, and/or F387/08. In particular embodiments, the phage mixture comprises F488/08, F510/08, F387/08, and/or F170/08. In particular embodiments, the phage mixture comprises F488/08, F510/08, F170/08, and/or F168/08. In particular embodiments, the phage mixture comprises F488/08, F510/08, F168/08, and/or F770/05. In particular embodiments, the phage mixture comprises F488/08, F510/08, F770/05, and/or F1245/05. In particular embodiments, the phage mixture comprises F488/08, F510/08, F1245/05, and/or F391/08. In particular embodiments, the phage mixture comprises F488/08, F510/08, F391/08, and/or F394/08.

In particular embodiments, the phage mixture comprises F387/08, F170/08, F168/08, and/or F770/05. In particular embodiments, the phage mixture comprises F387/08, F170/08, F770/05, and/or F1245/05. In particular embodiments, the phage mixture comprises F387/08, F170/08, F1245/05, and/or F391/08. In particular embodiments, the phage mixture comprises F387/08, F170/08, F391/08, and/or F394/08. In particular embodiments, the phage mixture comprises F387/08, F170/08, F394/08, and/or F488/08. In particular embodiments, the phage mixture comprises F387/08, F170/08, F488/08, and/or F510/08. In particular embodiments, the phage mixture comprises F387/08, F170/08, F510/08, and/or F44/10. In particular embodiments, the phage mixture comprises F387/08, F170/08, F44/10, and/or F387/08. In particular embodiments, the phage mixture comprises F387/08, F170/08, F387/08, and/or F170/08. In particular embodiments, the phage mixture comprises F387/08, F170/08, and/or F168/08. In particular embodiments, the phage mixture comprises F387/08, F170/08, F168/08, and/or F770/05. In particular embodiments, the phage mixture comprises F387/08, F170/08, F770/05, and/or F1245/05. In particular embodiments, the phage mixture comprises F387/08, F170/08, F1245/05, and/or F391/08. In particular embodiments, the phage mixture comprises F387/08, F170/08, F391/08, and/or F394/08. In particular embodiments, the phage mixture comprises F387/08, F170/08, F394/08, and/or F488/08. In particular embodiments, the phage mixture comprises F387/08, F170/08, F488/08, and/or F510/08. In particular embodiments, the phage mixture comprises F387/08, F170/08, F510/08, and/or F44/10.

In certain embodiments, the phage mixture comprises F510/08, F44/10, F387/08, and/or F170/08. In certain embodiments, the phage mixture comprises F510/08, F44/10, F170/08, and/or F168/08. In certain embodiments, the phage mixture comprises F510/08, F44/10, F168/08, and/or F770/05. In certain embodiments, the phage mixture comprises F510/08, F44/10, F770/05, and/or F1245/05. In certain embodiments, the phage mixture comprises F510/08, F44/10, F1245/05, and/or F391/08. In certain embodiments, the phage mixture comprises F510/08, F44/10, F391/08, and/or F394/08. In certain embodiments, the phage mixture comprises F510/08, F44/10, F394/08, and/or F488/08.

In certain embodiments, the bacteriophage comprises F44/10, F387/08, F170/08, and/or F168/08. In certain embodiments, the bacteriophage comprises F44/10, F387/08, F168/08, and/or F770/05. In certain embodiments, the bacteriophage comprises F44/10, F387/08, F770/05, and/or F1245/05. In certain embodiments, the bacteriophage comprises F44/10, F387/08, F1245/05, and/or F391/08. In certain embodiments, the bacteriophage comprises F44/10, F387/08, F391/08, and/or F394/08. In certain embodiments, the bacteriophage comprises F44/10, F387/08, F394/08, and/or F488/08. In certain embodiments, the bacteriophage comprises F44/10, F387/08, F488/08, and/or F510/08.

In certain embodiments, the phage mixture composition may or may not involve phages selected for increased in vivo half-life, for example as disclosed in US 5,688,501, the contents of which are incorporated herein by reference.

In certain embodiments, the mixture comprises one or more polypeptides and/or fragments, variants or derivatives thereof isolated from one or more bacteriophage, particularly polypeptides, fragments, variants or derivatives thereof having antibacterial or antimicrobial activity. In certain embodiments, the polypeptide, or fragment, variant or derivative thereof comprises or consists of lysin (or fragment thereof, e.g., CHAP domain) and/or tail protein. In a more specific embodiment, the polypeptide corresponds to an isolated polypeptide, fragment, variant or derivative thereof as described herein and/or in WO2010/090542, the contents of which are incorporated herein by reference. In certain embodiments, the mixture is administered in the absence of an isolated polypeptide, e.g., in the absence of a lytic enzyme.

Other examples of other therapeutic agents that may be used in combination with the polypeptides of the invention include, but are not limited to, standard antibiotic agents, anti-inflammatory agents, antiviral agents, local anesthetics, and corticosteroids. In certain embodiments, the mixture is administered in the absence of an antibiotic.

Standard antibiotics that can be used with pharmaceutical compositions comprising the bacteriophage and/or polypeptide of the present invention include, but are not limited to, amikacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, rhodotreptomycin, streptomycin, tobramycin, apramycin, rifamycin, naproxycin, mupirocin, geldanamycin, ansamicin, carbacephem, imipenem, meropenem, ertapenem, faropenem, doripenem, panipenem/betamipron, biapenem, PZ-601, cephalosporin, cefotaxime, cefadroxil, cephalexin, cefalexin, cefaloxin, ceftiofur, cephaloridine, cefapirin, ceftriazine, cefazezine, cefazedone, cefazolin, cefradine, cefixadine, ceftazidime, cefaclor, cefixol, cefixime, Cefprozil, cefuroxime, ceftizoxime, cefmetazole, cefotetan, cefoxitin, cefcapene, cefixime, cefdinir, cefditoren, cefetamet, cefixime, cefditoren, ceftibuten, ceftiofur, cefoselin, ceftizoxime, ceftriaxone, cefoperazone, cefradine moxef, cefixime, cefradine, cefotaxime, cefotiam, cefozopran, cefquinome, flomoxef, ceftipid, azithromycin, clindamycin, dirithromycin, erythromycin, roxithromycin, aztreonam, penicillin and penicillin derivatives, actinomycin, bacitracin, colistin, polymyxin B, cinoxacin, flumequine, nalidixic acid, oxolinic acid, pipemidic acid, roxacin, enoxacin, roxacinofloxacin, roxacin, ceftiofur-A, flonicamid, cefprozil, cefditin, cefprozil, ceftiofur-A, ceft, Lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin, gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin, pazufloxacin, sparfloxacin, temafloxacin, tosufloxacin, clinafloxacin, gatifloxacin, gemifloxacin, sitafloxacin, trovafloxacin, prulifloxacin, acetazolamide, benzzolamide, bumetanide, celecoxib, chlorthalidone, clopamide, dichlofenamide, dorzolamide, phenetolazolide, furosemide, hydrochlorothiazide, indapamide, mafenfide, mefficist, metolazone, probenecroscovitine, sulfacetamide, sulfadoxine, sulfamethazine, sulfamethoxazole, sulfasalazine, sumatriptan, xipamide, tetracycline, aureomycin, doxycycline, meclocycline, and cloxacin, Methacycline, minocycline, rocycline, methicillin, nafcillin, oxacillin, cloxacillin, vancomycin, teicoplanin, clindamycin, sulfamethoxazole, and any combination thereof in an amount effective to, or additive to, or synergistically enhance the efficacy of a bacteriophage and/or polypeptide of the invention for a given infection.

Local anesthetics that can be used in the pharmaceutical compositions of the present invention include tetracaine, tetracaine hydrochloride, lidocaine hydrochloride, dimethylisoquinoline hydrochloride, dibucaine hydrochloride, chlorobenzyl clofibrate picrate, and pramoxine hydrochloride. Exemplary concentrations of local anesthetics are from about 0.025% to about 5% by weight of the total composition.

Corticosteroids that may be used in combination with the polypeptides, bacteriophage and/or pharmaceutical compositions of the present invention include betamethasone, dipropionate, fluocinolone acetonide, actinides, betamethasone valerate, triamcinolone actinides, clobetasol propionate, desoximetasone, diflorasone diacetate, amcinonide, flurandrenolide acetonide, hydrocortisone valerate, hydrocortisone butyrate and desonide. Exemplary concentrations of corticosteroids are from about 0.01% to about 1% by weight of the total composition.

In particular embodiments, a formulation comprising a bacteriophage and/or polypeptide of the invention further comprises SM buffer (0.05M Tris-HCl (pH 7.4-7.5); 0.1M NaCl; 10mM MgSO₄). In other embodiments, the formulation further comprises SM buffer and 10mM MgCl₂. In still other embodiments, the formulation further comprises SM buffer and about 20% or about 30% ethanol.

Pharmaceutical compositions comprising a bacteriophage and/or polypeptide of the present invention may be formulated in unit-dose or multi-dose formulations. Suitable formulations may be selected from ointments, solutions, suspensions or emulsions, extracts, powders, granules, sprays, lozenges, tablets or capsules; and may additionally include a dispersant or stabilizer.

The pharmaceutical compositions of the invention may be administered by inhalation, in the form of suppositories or pessaries, topically (e.g., in the form of lotions, solutions, creams, ointments or dusting powders), transdermally or transdermally (e.g., by using a skin patch), orally (e.g., as a tablet, which may contain excipients such as starch or lactose), as capsules, ovules, elixirs, solutions or suspensions, each optionally containing flavouring, colouring and/or excipients, or they may be injected parenterally (e.g., intravenously, intramuscularly or subcutaneously). For parenteral administration, the compositions may be used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration, the compositions may be administered in the form of tablets or lozenges formulated in a conventional manner. In preferred embodiments, the bacteriophage and/or polypeptide of the present invention is administered topically as a single agent or in combination with other antibiotic treatments, as described herein or known in the art.

The bacteriophage and/or polypeptide of the present invention may also be applied to the skin or transdermally. For topical application to the skin, the bacteriophage and/or polypeptide of the present invention may be combined with one or a combination of carriers, which may include, but are not limited to, aqueous liquids, alcohol-based liquids, water-soluble gels, lotions, ointments, non-aqueous liquid bases, mineral oil bases, blends of mineral oil and petrolatum, lanolin, liposomes, protein carriers such as serum albumin or gelatin, powdered cellulose carmel, and combinations thereof. Topical delivery means may include smears, sprays, bandages, time release patches, liquid absorbing wipes, and combinations thereof. The bacteriophage and/or polypeptide of the present invention may be applied directly or in a carrier to a patch, wipe, bandage, or the like. The patch, wipe, bandage, etc. may be moist or dry, with the phage and/or polypeptide (e.g., lysin) on the patch in lyophilized form. The carrier of the topical composition may comprise semi-solid and gel-like vehicles including polymeric thickeners, water, preservatives, active surfactants or emulsifiers, antioxidants, sunscreen lotions, and solvents or mixed solvent systems. U.S. patent No. 5,863,560 discloses many different carrier combinations that can assist in exposing the skin to an agent, and the contents of which are incorporated herein. The carrier may or may not be involved in a controlled release formulation, for example as disclosed in US 2008/0260697, the contents of which are incorporated herein by reference. The vector may or may not involve phage adsorbed on a substrate, for example as described in any of US 2008/0038322, US 2008/0138311, US 2009/0130196, EP 1812025, EP 1817043 and EP 1833497, the contents of which are incorporated herein by reference. In certain embodiments, the carrier may or may not be related to a viscous formulation, e.g. a gel, e.g. as disclosed in US 2009/0191254, the content of which is incorporated herein by reference.

For intranasal or administration by inhalation, the bacteriophage and/or polypeptide of the present invention is conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurized container, pump, nebulizer or atomizer, using a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as 1, 1, 1, 2-tetrafluoroethane (HFA134a. tm.) or 1, 1, 1, 2, 3, 3, 3-heptafluoropropane (HFA 227ea. tm.), carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be measured by providing a valve delivery measurement. The pressurised container, pump, spray or atomiser may contain a solution or suspension of the active compound, for example using a mixture of ethanol and propellant as the solvent, which may additionally contain a lubricant such as sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of a bacteriophage and/or polypeptide of the invention and a suitable powder base such as lactose or starch.

For administration in the form of suppositories or pessaries, the therapeutic compositions may be applied topically in the form of gels, hydrogels, lotions, solutions, creams, ointments or dusting powders. The compositions of the present invention may also be administered by the ocular route. For ophthalmic use, the compositions of the present invention may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or preferably as solutions in isotonic, pH adjusted sterile saline, optionally in combination with a preservative such as benzalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.

The dosage and desired drug concentration of the pharmaceutical composition of the present invention may vary depending on the particular use. Determination of the appropriate dosage or route of administration is within the skill of the ordinary practitioner. Animal experiments can provide reliable guidance for determining effective doses in human therapy. Interspecies scaling of effective doses can be performed by one of ordinary skill in The art according to The principles described by Mordenti, J. and Chappell, W. "The use of The identities scaling in The automation and New Drug Development, Yacobi et al, eds., Pergamon Press, New York 1989, pages 42-96.

6.3 therapeutic uses

The bacteriophage and polypeptides of the present invention have activity against a variety of strains of klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa, and/or staphylococcus aureus, for example as described in tables 1-7 in the examples below. Thus, the compositions of the present invention may be useful in methods of preventing and/or treating infections associated with Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa and/or Staphylococcus aureus in humans and animals. In other embodiments, the compositions of the invention may be used to treat infections associated with related species or strains of these bacteria, including but not limited to staphylococcus epidermidis, staphylococcus aureus, staphylococcus cephalus, staphylococcus haemolyticus, staphylococcus hominis, staphylococcus saprophyticus, staphylococcus simulans, staphylococcus xylosus, micrococcus luteus, bacillus subtilis, bacillus pumilus, enterococcus hirae.

In particular embodiments, the subject receiving the pharmaceutical composition of the invention is a mammal (e.g., a cow, sheep, equine, primate (e.g., human), rodent, lagomorph, or avian (e.g., chicken, duck, goose)). In the context of the present invention, "treatment" refers to a therapeutic treatment in which the objective is to eliminate, reduce the severity of, ameliorate, slow the progression of, or prevent symptoms or underlying causes (e.g., bacterial infection) associated with a pathological state or condition. "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to eliminate, lessen, reduce the severity of, slow the progression of, or delay or prevent the symptoms or underlying cause (e.g., bacterial infection) associated with the pathological state or condition. It is also contemplated that the bacteriophage and/or polypeptide of the present invention act as a prophylactic or preventative measure, preventing the onset of infection by one or more bacteria.

Klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa and staphylococcus aureus are responsible for many serious opportunistic infections, particularly in individuals with compromised immune systems. The pharmaceutical compositions of the present invention are contemplated for use in the treatment of any infection associated with klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa, and/or staphylococcus aureus, or any infection associated with other species or strains of bacteria, including but not limited to infections of the skin (including but not limited to skin ulcers, carbuncles, bedsores, and diabetic foot ulcers), infections in and around wounds, post-operative infections, infections associated with catheters and surgical drainage, and blood infections.

Diabetic foot ulcers are one of the major complications of diabetes, occurring in about 15% of all diabetic patients and resulting in about 85% of all lower leg amputations. (Brem, et al, J.clinical invest, 2007, 117 (5): 1219-1222). Diabetes hinders the normal steps of the wound healing process. Unhealed chronic diabetic ulcers are typically treated with extracellular matrix replacement therapy, advanced moist wound therapy, bio-engineered tissue or skin substitutes, growth factors, debridement, arterial revascularization, and/or negative pressure wound therapy. (Blume et al, Diabetes Care, 2008, 31: 631-636). Ulcers can become infected with opportunistic bacteria, which further exacerbates the condition. Accordingly, foot ulcers in diabetes also typically require antibiotics, e.g., against staphylococcus, as well as other anaerobic bacteria, e.g., klebsiella pneumoniae, escherichia coli, and/or pseudomonas aeruginosa.

One or more of the compositions of the present invention are useful in the treatment of diabetic foot ulcers. For example, the isolated phage or polypeptide of the invention can be used to treat an infection associated with a diabetic foot ulcer in a subject in need thereof. In particular embodiments, the composition for treating diabetic foot ulcers is a topical composition formulated for topical application, e.g., a composition for direct application to ulcers, wounds, lesions, and/or sores associated with diabetic foot ulcers.

In a particular embodiment, the composition for use with respect to diabetic foot ulcers comprises isolated F44/10 having a sequence comprising SEQ ID NO:560 and exhibits antibacterial activity against one or more strains of staphylococcus aureus. In certain embodiments, a composition comprising a polypeptide isolated from bacteriophage F44/10, or a fragment, variant, or derivative thereof, that exhibits antibacterial activity against one or more species or strains of staphylococcus aureus is used. In certain such embodiments, the polypeptide, or fragment, variant or derivative thereof, is a lysin, CHAP domain or tail protein that exhibits antibacterial activity against one or more species or strains of staphylococcus aureus. In particular embodiments, the composition for use with respect to diabetic foot ulcers comprises isolated F125/10 having a sequence comprising SEQ ID NO:1074 and exhibits antibacterial activity against one or more strains of staphylococcus aureus. In certain embodiments, a composition comprising a polypeptide, or a fragment, variant, or derivative thereof, isolated from bacteriophage F125/10 that exhibits antibacterial activity against one or more species or strains of staphylococcus aureus is used. In certain such embodiments, the polypeptide, or fragment, variant or derivative thereof, is a lysin, CHAP domain or tail protein that exhibits antibacterial activity against one or more species or strains of staphylococcus aureus.

In particular embodiments, a composition comprising a mixture of bacteriophages is used, for example wherein the mixture of bacteriophages comprises at least one bacteriophage that exhibits antibacterial activity against one or more strains of staphylococcus aureus, and at least one bacteriophage that exhibits antibacterial activity against a different bacterium. In particular embodiments, the phage mixture comprises F44/10 and/or F125/10 in combination with at least one other phage selected from the group consisting of F391/08, F394/08, F488/08, F510/08, F387/08, F170/08, F168/08, F770/05, and F1245/05. In a particularly preferred embodiment, the phage mixture comprises F44/10 and/or F125/10 in combination with one, two, three or more other phage selected from F391/08, F387/08, F488/08, F510/08 and/or F770/05.

In particular embodiments, the compositions for use with respect to diabetic foot ulcers comprise isolated F391/08 and/or F387/08 having the amino acid sequence comprising SEQ ID NO:1 or SEQ ID NO:781 and exhibits antibacterial activity against one or more strains of klebsiella pneumoniae. In certain embodiments, a composition comprising a polypeptide, or a fragment, variant, or derivative thereof isolated from bacteriophage F391/08 and/or F387/08 that exhibits antibacterial activity against one or more species or strains of klebsiella pneumoniae is used. In certain such embodiments, the polypeptide, or fragment, variant, or derivative thereof, is a lysin, CHAP domain or tail protein that exhibits antibacterial activity against one or more species or strains of klebsiella pneumoniae. In particular embodiments, a composition comprising a mixture of bacteriophages is used, for example wherein the mixture of bacteriophages comprises at least one bacteriophage showing antibacterial activity against one or more strains of klebsiella pneumoniae, and at least one bacteriophage showing antibacterial activity against a different bacterium. In particular embodiments, the phage mixture comprises F391/08 and/or F387/08 in combination with at least one other phage selected from F394/08, F488/08, F510/08, F44/10, F170/08, F168/08, F770/05, F1245/05, and F125/10. In a particularly preferred embodiment, the phage mixture comprises F391/08 and/or F387/08 in combination with one, two, three or more other phage selected from F44/10, F488/08, F510/08 and/or F770/05.

In particular embodiments, the composition for use with respect to diabetic foot ulcers comprises isolated F488/08 having an amino acid sequence comprising SEQ ID NO:3, and exhibits antibacterial activity against one or more strains of escherichia coli. In certain embodiments, a composition comprising a polypeptide isolated from bacteriophage F488/08, or a fragment, variant, or derivative thereof, that exhibits antibacterial activity against one or more species or strains of escherichia coli is used. In certain such embodiments, the polypeptide, or fragment, variant, or derivative thereof, is a lysin, CHAP domain, or tail protein that exhibits antibacterial activity against one or more species or strains of e. In particular embodiments, a composition comprising a mixture of bacteriophages is used, for example wherein the mixture of bacteriophages comprises at least one bacteriophage that exhibits antibacterial activity against one or more strains of E.coli, and at least one bacteriophage that exhibits antibacterial activity against a different bacterium. In particular embodiments, the phage mixture comprises F488/08 in combination with at least one other phage selected from the group consisting of F394/08, F510/08, F44/10, F170/08, F168/08, F770/05, F1245/05, F391/08F387/08, and F125/10. In a particularly preferred embodiment, the phage mixture comprises F488/08 in combination with one, two, three or more other phage selected from F391/08, F387/08, F44/10, F125/10, F510/08, and/or F770/05.

In particular embodiments, the compositions for use with respect to diabetic foot ulcers comprise isolated F510/08 and/or F770/05 having the amino acid sequence comprising SEQ ID NO:4 or a nucleic acid sequence as disclosed in WO2010/090542, and exhibits antibacterial activity against one or more strains of pseudomonas aeruginosa. In certain embodiments, a composition comprising a polypeptide, or fragment, variant, or derivative thereof, isolated from bacteriophage F510/08 and/or F770/05 that exhibits antibacterial activity against one or more species or strains of pseudomonas aeruginosa is used. In certain such embodiments, the polypeptide, or fragment, variant, or derivative thereof, is a lysin, CHAP domain, or tail protein that exhibits antibacterial activity against one or more species or strains of pseudomonas aeruginosa. In particular embodiments, compositions comprising a mixture of bacteriophages are used, for example wherein the mixture of bacteriophages comprises at least one bacteriophage that exhibits antibacterial activity against one or more strains of pseudomonas aeruginosa, and at least one bacteriophage that exhibits antibacterial activity against a different bacterium. In particular embodiments, the phage mixture comprises F510/08 and/or F770/05 in combination with at least one other phage selected from the group consisting of F394/08, F488/08, F44/10, F170/08, F168/08, F1245/05, F391/08, F387/08, and F125/10. In a particularly preferred embodiment, the phage mixture comprises F510/08 and/or F770/05 in combination with one, two, three or more other phage selected from F44/10, F488/08, F391/08 and/or F387/08.

Klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa and staphylococcus aureus are also associated with infections involving organ systems with high fluid content, and it is contemplated that the bacteriophage and/or polypeptides of the invention have therapeutic use in the prevention and treatment of these infections. For example, the pharmaceutical composition of the present invention may be used for the prevention or treatment of infections of the respiratory tract, cerebrospinal fluid, peritoneal fluid and urinary tract. The compositions of the invention may also be used for the prevention and/or treatment of nosocomial pneumonia, infections associated with Continuous Ambulatory Peritoneal Dialysis (CAPD), catheter-related bacteremia and nosocomial meningitis.

In a preferred embodiment, the bacteriophage and/or polypeptide of the present invention is used prophylactically in a hospital setting, in particular to prevent infections associated with wounds, ulcers and openings in the skin, for example due to catheterization and any other medical procedure or device.

In particular embodiments, the bacteriophage and/or polypeptide of the present invention is used as a single agent for the treatment or prevention of an infection associated with klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa, and/or staphylococcus aureus, and/or other bacterial species. In other embodiments of the invention, the bacteriophage and/or polypeptide of the invention is used in combination with other agents, including other bacteriophage (e.g., targeting a different species or strain of bacteria), or antibiotics targeting the same or a different species of bacteria, including bacteria selected from any gram-positive bacteria, any gram-negative bacteria, and any other group of bacteria not classified as gram-positive or gram-negative. The compositions of the present invention may also be used in combination with any other method of treating bacterial infections known to those skilled in the art.

Also contemplated by the present invention are methods of preventing and methods of treating infections caused by bacteria, including but not limited to klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa, and/or staphylococcus aureus, comprising administering to a mammal in need thereof a composition comprising or consisting of an amino acid sequence comprising or consisting of: SEQ ID NO: 20. SEQ ID NO: 80. SEQ ID NO: 192. SEQ ID NO: 282. SEQ ID NO: 547. SEQ ID NO: 556. SEQ ID NO: 557. SEQ ID NO: 598. SEQ ID NO: 1216. SEQ ID NO: 1261, wherein said fragment, variant or derivative exhibits antibacterial or antimicrobial activity against a bacterial species from which the parent bacteriophage is isolated. In a specific example according to this embodiment, the present invention provides a method of preventing or treating an infection caused by a bacterium, including but not limited to klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa, and/or staphylococcus aureus, comprising administering to a mammal in need thereof a composition comprising an isolated CHAP domain of lysin, or a fragment, variant, or derivative thereof, wherein the fragment, variant, or derivative exhibits at least one biological activity (e.g., lytic cell killing) of the CHAP domain from which it is isolated.

In particular embodiments, the present invention provides methods of preventing and/or treating an infection caused by a bacterium, including but not limited to klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa, and/or staphylococcus aureus, comprising administering to a mammal in need thereof a composition comprising a tail protein comprising or consisting of an amino acid sequence, or fragment, variant, or derivative thereof: SEQ ID NO: 15. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 30. SEQ ID NOs: 32-35, SEQ ID NO: 180. SEQ ID NO: 183. SEQ ID NO: 185. SEQ ID NO: 190. SEQ ID NO: 231. SEQ ID NO: 232. SEQ ID NO: 235. SEQ ID NOs: 239-245, SEQ ID NO: 248. SEQ ID NO: 249. SEQ ID NO: 252. SEQ ID NO: 254. SEQ ID NOs: 433-437, SEQ ID NOs: 489-496, SEQ ID NO: 544. SEQ ID NO: 545. SEQ ID NO: 549. SEQ ID NO: 551. SEQ ID NO: 629. or SEQ ID NO: 686. SEQ ID NO: 789. SEQ ID NOs: 796-800, SEQ ID NO: 806. SEQ ID NO: 854. SEQ ID NOs: 999-1004, SEQ ID NOs: 1053-1060, SEQ ID NO: 1077. SEQ ID NO: 1217. SEQ ID NO: 1250. SEQ ID NO: 1266, wherein said fragment, variant or derivative exhibits a biological activity associated with a bacteriophage from which it is derived.

In yet other embodiments, the present invention provides methods of preventing and/or treating an infection caused by a bacterium, including but not limited to klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa, and/or staphylococcus aureus, comprising administering to a mammal in need thereof a composition comprising a bacteriophage having a genome comprising or consisting of a nucleic acid sequence: SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 560. SEQ ID NO:781 and/or SEQ ID NO: 1074. also contemplated are combinations of lysins (or fragments, variants or derivatives thereof as described above) optionally with one or more bacteriophage of the invention or other treatments, e.g., antibiotics, and methods of treating and/or preventing bacterial infections using one or more of the combinations described herein.

As used herein, the term "in combination with … …" refers to the use of more than one prophylactic and/or therapeutic agent. The use of the term "in combination with … …" does not limit the order in which prophylactic and/or therapeutic agents are administered to a subject having a disease or disorder. The first prophylactic or therapeutic agent can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concurrently with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) administration of a second prophylactic or therapeutic agent (different from the first prophylactic or therapeutic agent) to a subject having a disease or disorder.

6.4 disinfectant and anti-infective uses

Bacterial pathogens are most commonly infected at mucosal sites (e.g., upper and lower respiratory, intestinal, urogenital, ocular, etc.). The mucosa itself is usually the reservoir, and sometimes the only reservoir, for many pathogenic bacteria found in the environment (e.g., pneumococci, staphylococci, and streptococci). There are few anti-infective drugs designed to control the carrier status of pathogens. However, studies have shown that by reducing or eliminating this reservoir in environments such as hospitals and nursing homes, the incidence of infection by these bacteria will be significantly reduced. Prevention of nosocomial infections involves routine and repeated cleaning of the affected surfaces.

The bacteriophage and/or polypeptide of the present invention may be used in an anti-infective composition for controlling the growth of bacteria, such as gram positive bacteria (e.g., staphylococcus aureus), gram negative bacteria (e.g., klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, and pseudomonas aeruginosa) or bacteria not classified as gram positive or gram negative, in order to prevent or reduce the occurrence of nosocomial infections. In addition to use in compositions for application to mucous membranes, the bacteriophage and/or polypeptide of the invention may also be incorporated into formulations such as gels, creams, ointments or sprays for the control or prevention of bacterial colonization on bodily surfaces (e.g. skin and mucous membranes) (e.g. for sterilization of the exposed skin of the surgical area or hands and healthcare workers and/or patients) and other solid surfaces (e.g. appliances, countertops and in particular hospital equipment).

6.5 use in nanotechnology

The bacteriophage and/or polypeptide of the present invention may also be used in nanotechnology, such as in the development of nanoscale devices. The combination of nanotechnology and molecular biology has led to a new generation of nanoscale-based devices, such as nanoscale conductors. Biological systems function based on the structure of macromolecules, primarily proteins and nucleic acids, organized on nanoscale structures. Accordingly, biological macromolecules may be useful in nanoscale applications. In particular, proteins with highly organized structures can be used in the development of nanoscale devices.

In certain embodiments, the polypeptides of the invention comprising or consisting of: a tail protein (e.g., tail component, fiber protein, adsorption related tail protein, tail length tape protein, substrate wedge subunit) or a fragment, variant or derivative thereof isolated from a bacteriophage having a genome comprising or consisting of: SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 560. SEQ ID NO: 781. or SEQ ID NO:1074 (e.g., bacteriophage F391/08, F394/08, F488/08, F510/08, F44/10, F387/08, or F125/10, respectively). For example, tail proteins from bacteriophage fibers have a highly organized structure and may be useful in nanoscale conductors. Such conductors may be used, for example, to place gold and/or other ions.

In a particular embodiment, the polypeptide of the invention used in nanotechnology is an isolated tail protein comprising or consisting of the amino acid sequence: SEQ ID NO: 15. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 30. SEQ ID NOs: 32-35, SEQ ID NO: 180. SEQ ID NO: 183. SEQ ID NO: 185. SEQ ID NO: 190. SEQ ID NO: 231. SEQ ID NO: 232. SEQ ID NO: 235. SEQ ID NOs: 239-245, SEQ id no: 248. SEQ ID NO: 249. SEQ ID NO: 252. SEQ ID NO: 254. SEQ ID NOs: 433-437, SEQ ID NOs: 489-496, SEQ ID NO: 544. SEQ ID NO: 545. SEQ ID NO: 549. SEQ ID NO: 551. SEQ ID NO: 629. SEQ ID NO: 686. SEQ ID NO: 789. SEQ ID NOs: 796-800, SEQ ID NO: 806. SEQ ID NO: 854. SEQ ID NOs: 999-1004, SEQ ID NOs: 1053-1060, SEQ ID NO: 1077. SEQ ID NO: 1217. SEQ ID NO: 1250. or SEQ ID NO: 1266. in other embodiments, the polypeptide of the invention comprises a fragment, variant or derivative of: SEQ ID NO: 15. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 30. SEQ ID NOs: 32-35, SEQ ID NO: 180. SEQ ID NO: 183. SEQ ID NO: 185. SEQ ID NO: 190. SEQ ID NO: 231. SEQ ID NO: 232. SEQ ID NO: 235. SEQ ID NOs: 239-245, SEQ ID NO: 248. SEQ ID NO: 249. SEQ ID NO: 252. SEQ ID NO: 254. SEQ ID NOs: 433-437, SEQ ID NOs: 489-496, SEQ ID NO: 544. SEQ ID NO: 545. SEQ ID NO: 549. SEQ ID NO: 551. SEQ ID NO: 629. SEQ ID NO: 686. SEQ ID NO: 789. SEQ ID NOs: 796-800, SEQ ID NO: 806. SEQ ID NO: 854. SEQ ID NOs: 999-1004, SEQ ID NOs: 1053-1060, SEQ ID NO: 1077. SEQ ID NO: 1217. SEQ ID NO: 1250. or SEQ ID NO: 1266, wherein said fragment, variant or derivative has a highly organised structure. Such polypeptides are useful, for example, in nanoscale conductors, as described above.

6.6 diagnostic methods

The invention also encompasses diagnostic methods for determining causative agents in bacterial infections. In a particular embodiment, the diagnosis of causative agents in the presentation of bacterial infection is performed by: (i) culturing a tissue, blood or fluid sample of the patient according to standard techniques; (ii) contacting the culture with one or more bacteriophage and/or polypeptides of the invention; and (iii) monitoring evidence of cell growth and/or lysis following said contacting. Because the activity of a bacteriophage and/or isolated product thereof (e.g., a polypeptide, or biologically active fragment, variant, or derivative thereof) tends to be species or strain specific, a susceptibility or lack of susceptibility to one or more bacteriophage and/or polypeptides of the present invention may be indicative of a species or strain of infectious bacteria. For example, in a nucleic acid sequence corresponding to a polypeptide having a sequence comprising the nucleic acid sequence of SEQ ID NO:1 or 781 or a nucleic acid sequence consisting of SEQ ID NO:1 or 781, or an isolated polypeptide or polypeptide derived therefrom, a reduced growth of the test culture may indicate that the test sample comprises klebsiella pneumoniae. Similarly, a polypeptide having a sequence comprising the nucleic acid sequence of SEQ ID NO:2 or by the nucleic acid sequence SEQ ID NO:2, or an isolated polypeptide product thereof or a polypeptide product derived therefrom, can be used to identify infection by acinetobacter baumannii; having a sequence comprising the nucleic acid sequence SEQ ID NO:3 or a nucleic acid sequence consisting of SEQ ID NO:3, or an isolated polypeptide product thereof or a polypeptide product derived therefrom, can be used to identify infection by e.coli; and having a nucleotide sequence comprising the nucleic acid sequence of SEQ ID NO:4 or by the nucleic acid sequence of seq id NO:4, or an isolated polypeptide product thereof or a polypeptide product derived therefrom, can be used to identify infection by pseudomonas aeruginosa; and has a sequence comprising the nucleic acid sequence SEQ ID NO:560 or 1074 or a nucleic acid sequence consisting of seq id NO:560 or 1074, or an isolated polypeptide product thereof or a polypeptide product derived therefrom, can be used to identify infection by staphylococcus aureus.

In addition, in certain embodiments, the bacteriophage and/or polypeptide of the present invention may be used in biosensors in the diagnostic context. As used herein, "biosensor" refers to an analytical device for detecting an analyte that combines a biological component with a physicochemical detector component. In particular, proteins involved in bacterial receptor recognition may be used in the development of diagnostic biosensors.

In certain embodiments, the polypeptides of the invention comprising or consisting of: a tail protein (e.g., tail component, fiber protein, adsorption related tail protein, tail length tape protein, substrate wedge subunit) or a fragment, variant or derivative thereof isolated from a bacteriophage having a genome comprising or consisting of: SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 560. SEQ ID NO: 781. SEQ ID NO:1074 (e.g., bacteriophage F391/08, F394/08, F488/08, F510/08, F44/10, F387/08, or F125/10, respectively). For example, phage tail proteins can specifically recognize one or more bacterial species and/or strains, and thus can be useful in biosensor diagnostics. Detection of a particular bacterial species and/or strain by one or more bacteriophage and/or polypeptides of the present invention may indicate a species or strain of infectious bacteria.

In a particular embodiment, the polypeptide of the invention used in the biosensor application is an isolated tail protein comprising or consisting of the amino acid sequence: SEQ ID NO: 15. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 30. SEQ ID NOs: 32-35, SEQ ID NO: 180. SEQ ID NO: 183. SEQ ID NO: 185. SEQ ID NO: 190. SEQ ID NO: 231. SEQ ID NO: 232. SEQ ID NO: 235. SEQ ID NOs: 239-245, SEQ ID NO: 248. SEQ ID NO: 249. SEQ ID NO: 252. SEQ ID NO: 254. SEQ ID NOs: 433-437, SEQ ID NOs: 489-496, SEQ ID NO: 544. SEQ ID NO: 545. SEQ ID NO: 549. SEQ ID NO: 551. SEQ ID NO: 629. SEQ ID NO: 686. SEQ ID NO: 789. SEQ ID NOs: 796-800, SEQ ID NO: 806. SEQ ID NO: 854. SEQ ID NOs: 999-1004, SEQ ID NOs: 1053-1060, SEQ ID NO: 1077. SEQ ID NO: 1217. SEQ ID NO: 1250. Or SEQ ID NO: 1266. in other embodiments, the polypeptide of the invention comprises a fragment, variant or derivative of: SEQ ID NO: 15. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 30. SEQ ID NOs: 32-35, SEQ ID NO: 180. SEQ ID NO: 183. SEQ ID NO: 185. SEQ ID NO: 190. SEQ ID NO: 231. SEQ ID NO: 232. SEQ ID NO: 235. SEQ ID NOs: 239-245, SEQ ID NO: 248. SEQ ID NO: 249. SEQ ID NO: 252. SEQ ID NO: 254. SEQ ID NOs: 433-437, SEQ ID NOs: 489-496, SEQ ID NO: 544. SEQ ID NO: 545. SEQ ID NO: 549. SEQ ID NO: 551. SEQ ID NO: 629. SEQ ID NO: 686. SEQ ID NO: 789. SEQ ID NOs: 796-800, SEQ ID NO: 806. SEQ ID NO: 854. SEQ ID NOs: 999-1004, SEQ ID NOs: 1053-1060, SEQ ID NO: 1077. SEQ ID NO: 1217. SEQ ID NO: 1250. or SEQ ID NO: 1266, wherein said fragment, variant or derivative is capable of specifically recognizing a bacterium, e.g. a specific species of bacterium and/or one or more specific strains. Such polypeptides are useful, for example, in biosensors for detecting specific bacteria and/or diagnosing specific infections, as described above.

Typically, a bacteriophage tail protein for use in a biosensor will detect its host bacteria, or one or more specific species and/or specific strains of host bacteria. Accordingly, in particular embodiments, the invention comprises a polypeptide corresponding to SEQ ID NO: 15. SEQ ID NO: 26. SEQ ID NO: 27. SEQ ID NO: 30. or SEQ ID NOs: 32-35, or a fragment, variant or derivative thereof, which recognizes and can detect one or more strains of klebsiella pneumoniae. Such detection may be indicative of a klebsiella pneumoniae infection. In particular embodiments, the invention comprises a polypeptide corresponding to SEQ ID NO: 180. SEQ ID NO: 183. SEQ ID NO: 185. or SEQ ID NO: 190, or a fragment, variant or derivative thereof, which recognizes and can detect one or more strains of acinetobacter baumannii. Such detection may be indicative of acinetobacter baumannii infection. In particular embodiments, the invention comprises a polypeptide corresponding to SEQ ID NO: 231. SEQ ID NO: 232. SEQ ID NO: 235. SEQ ID NOs: 239-245, SEQ ID NO: 248. SEQ ID NO: 249. SEQ ID NO: 252. SEQ ID NO: 254. SEQ ID NOs: 433-437, SEQ ID NOs: 489-495, or SEQ ID NO: 496, or a fragment, variant or derivative thereof, which recognizes and can detect one or more strains of escherichia coli. Such detection may be indicative of an e.

In particular embodiments, the invention comprises a polypeptide corresponding to SEQ ID NO: 544. SEQ ID NO: 545. SEQ ID NO: 549. or SEQ ID NO: 551, or a fragment, variant or derivative thereof, which recognizes and can detect one or more strains of pseudomonas aeruginosa. Such detection may be indicative of a pseudomonas aeruginosa infection. In particular embodiments, the invention comprises a polypeptide corresponding to SEQ ID NO: 629. SEQ ID NO: 686. SEQ ID NO: 1077. SEQ ID NO: 1217. SEQ ID NO: 1250. or SEQ ID NO: 1266, or a fragment, variant or derivative thereof, which recognizes and can detect one or more strains of staphylococcus aureus. Such detection may be indicative of a staphylococcus aureus infection. In particular embodiments, the invention comprises a polypeptide corresponding to SEQ ID NO: 789. SEQ ID NOs: 796-800, SEQ ID NO: 806. SEQ ID NO: 854. SEQ ID NOs: 999-1004, or SEQ ID NOs: 1053-1060, or a fragment, variant or derivative thereof, which recognizes and can detect one or more strains of klebsiella pneumoniae. Such detection may be indicative of a klebsiella pneumoniae infection.

In certain embodiments, the invention comprises the use of more than one tail protein, e.g., a combination of two or more tail proteins provided above, in a biosensor for the detection of more than one bacterial species and/or strain. The biosensor may also comprise additional proteins and/or other reagents for detecting the same or different bacteria.

6.7 amino acid variants

Amino acid sequence variants of the polypeptides of the invention may be prepared. In certain embodiments, they may be substitution, insertion and/or deletion variants. Deletion variants lack one or more residues of the native protein, which are generally not required for functional (e.g., antimicrobial or antibacterial activity). Insertional mutants generally involve the addition of material at a non-terminal point in the polypeptide. Substitution variants generally involve the exchange of one amino acid for another at one or more sites within a polypeptide, and may be designed to modulate one or more properties of the polypeptide, such as stability against proteolytic cleavage, preferably without loss (or substantial loss) of other function or property. Substitutions of this kind are preferably conservative, i.e. one amino acid is replaced by one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the following changes: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.

Once a general region of a gene is identified as encoding a particular antibacterial activity, for example as a lysin as described herein, point mutagenesis can be used to identify with greater specificity which amino acid residues are important in antibacterial activity. Thus, one skilled in the art is able to generate single base changes in, for example, a DNA strand to result in altered codons and/or missense mutations that preserve the desired function.

Preferably, mutation of amino acids of the protein results in an equivalent or even improved second generation molecule. For example, a particular amino acid may be substituted for other amino acids in the protein structure without a detectable or substantial loss of function (e.g., antibacterial or antimicrobial activity). In making such changes, the hydropathic index (hydropathic index) of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art. It is recognized that the relatively hydrophilic-hydrophobic character of amino acids contributes to the secondary structure of the produced protein, which in turn defines the interaction of the protein with other molecules, such as peptidoglycans within the outer shell of gram-positive bacteria. Each amino acid has been assigned a hydropathic index based on its hydrophobicity and charge characteristics; for example: isoleucine (+ 4.5); valine (+ 4.2); leucine (+ 3.8); phenylalanine (+ 2.8); cysteine/cystine (+ 2.5); methionine (+ 1.9); alanine (+ 1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan 0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). It is also understood in the art that substitutions of like amino acids can be made efficiently based on hydrophilicity. Like hydrophobicity, each amino acid has been assigned a hydrophilicity value: arginine (+ 3.0); lysine (+ 3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+ 0.3); asparagine (+ 0.2); glutamine (+ 0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4). Equivalent molecules can be obtained by substituting one amino acid for another, wherein their hydropathic and hydrophobic properties and/or their hydropathic indices are within ± 2, preferably ± 1 or most preferably ± 5 of each other.

In particular embodiments, the invention encompasses isolated polypeptides comprising 1, 2, 3, 4, 5,6, 7, 8, 9, or 10or more amino acid modifications (e.g., insertions, substitutions, deletions, etc.) relative to the amino acid sequences disclosed herein. In a preferred embodiment, the mutations are made such that the biological activity of a particular polypeptide is retained or substantially retained. For example, the invention comprises a polypeptide isolated from bacteriophage F387/08, F391/08, F394/08, F488/08, F510/068, F44/10, and/or F125/10, mutated to comprise 1, 2, 3, 4, 5,6, 7, 8, 9, or 10or more amino acid modifications relative to the amino acid sequences listed herein, and exhibiting antibacterial activity against gram-positive or gram-negative bacteria, such as against one or more species or strains of klebsiella pneumoniae, acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa, and/or staphylococcus aureus. In particular embodiments, the polypeptides of the invention derived from F387/08or F391/08 exhibit antibacterial or antimicrobial activity, e.g., lytic killing activity, against at least Klebsiella pneumoniae; those derived from F394/08 exhibit antibacterial or antimicrobial activity against at least acinetobacter baumannii; those derived from F488/08 exhibit antibacterial or antimicrobial activity against at least E.coli; those derived from F510/08 exhibit antibacterial or antimicrobial activity against at least Pseudomonas aeruginosa, and those derived from F44/10or F125/10 exhibit antibacterial or antimicrobial activity against at least Staphylococcus aureus.

6.8 polynucleotides encoding polypeptides of the invention

The present invention provides polynucleotides comprising a nucleotide sequence encoding a polypeptide of the present invention. The invention also encompasses polynucleotides that hybridize under high, medium or lower stringency hybridization conditions to polynucleotides encoding polypeptides of the invention and encoding modified polypeptides having antibiotic and/or other biological activity, e.g., as defined above.

Polynucleotides can be obtained and the nucleotide sequence of the polynucleotide determined by any method known in the art. For example, a polynucleotide encoding a polypeptide of the invention can be produced from a nucleic acid from a suitable source (e.g., bacteriophage F387/08, F391/08, F394/08, F488/08, F510/08, F44/10, and/or F125/10). Nucleotide sequences can be isolated from the phage genome by conventional methods known In the art (see, e.g., Carlson, "Working with bacteriophages: common techniques and methods apporaches," In, Kutter, and Sulakvelde (eds.) bacteriophages: Biology and Applications, 5 th edition CRC Press (2005); incorporated by reference In its entirety). If the source of the nucleic acid comprising the encoded polypeptide is not available, but the amino acid sequence of the polypeptide of the invention is known, the nucleic acid encoding the polypeptide can be chemically synthesized using any method known in the art and cloned into a replicable cloning vector.

Once the nucleotide sequence of a polypeptide of the present invention is determined, the nucleotide sequence of the polypeptide can be manipulated using methods well known in the art for manipulating nucleotide sequences, such as recombinant DNA techniques, site-directed mutagenesis, PCR, and the like (see, for example, the techniques described in Sambrook et al, 1990, Molecular Cloning, A Laboratory Manual, 2 nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, and Ausubel et al, eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, incorporated by reference in their entirety) to generate polypeptides having different amino acid sequences, e.g., to make amino acid substitutions, deletions, and/or insertions.

In certain embodiments, nucleotide sequences encoding one or more of the ORFs of figures 2, 4, 6, 8, 10, 12, and 14 are provided. In certain embodiments, there is provided a nucleotide sequence encoding a variant, fragment or derivative of one or more of the ORFs of figures 2, 4, 6, 8, 10, 12 and 14, wherein the variant, fragment or derivative is shown to be directed against one or more strains of klebsiella pneumoniae, for example against a strain having a sequence comprising the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:781 or consists of the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:781 antibacterial or antimicrobial activity (e.g., lytic killing activity) of bacteriophage of the genome of composition; and/or against one or more strains of acinetobacter baumannii, e.g. against a strain having a sequence comprising the nucleic acid sequence SEQ id no:2 or by the nucleic acid sequence SEQ ID NO:2 of a genome; and/or against one or more strains of escherichia coli, e.g. against a strain having a nucleic acid sequence comprising SEQ ID NO:3 or a nucleic acid sequence consisting of SEQ ID NO:3 of a genome; against one or more strains of pseudomonas aeruginosa, for example against a strain having a nucleic acid sequence comprising seq id NO:4 or by the nucleic acid sequence SEQ ID NO:4 of a genome; and/or against one or more strains of staphylococcus aureus, e.g. against a strain having a nucleic acid sequence comprising SEQ ID NO:560 or SEQ ID NO:1074 or a nucleic acid sequence consisting of SEQ ID NO:560 or SEQ ID NO:1074 of a genome.

6.9 recombinant expression of the molecules of the invention

Once a nucleic acid sequence encoding a molecule (e.g., a polypeptide) of the invention has been obtained, vectors for producing the molecule can be generated by recombinant DNA techniques using techniques well known in the art. Methods well known to those skilled in the art can be used to construct expression vectors containing the coding sequences of the molecules of the invention, as well as appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. (see, e.g., Sam brook et al, 1990, Molecular Cloning, A Laboratory Manual, 2 nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al, eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY).

The present invention provides expression vectors encoding the polypeptides of the invention. Expression vectors comprising the nucleotide sequences of the molecules identified by the methods of the invention can be transferred to host cells by conventional techniques (e.g., electroporation, lipofection, and calcium phosphate precipitation), and the transfected cells are subsequently cultured by conventional techniques to produce the molecules of the invention. In a preferred embodiment, the host cell is of a different species than the parent bacterium from which the bacteriophage comprising the sequence is derived. In particular embodiments, expression of the molecules of the invention is regulated by constitutive, inducible or tissue-specific promoters. In particular embodiments, the expression vector is pQE-30(Qiagen) or pET-29(a) (Novagen).

The host cell used to express the molecule identified by the method of the invention may be any bacterial cell (preferably not sensitive to a bacteriophage protein of the invention or a variant, derivative or fragment thereof). A variety of host expression vector systems can be used to express the molecules identified by the methods of the invention. Such host expression systems represent the vehicles by which the coding sequences of the molecules of the invention can be produced and subsequently purified, and also represent cells that, when transformed or transfected with the appropriate nucleotide coding sequences, express the molecules of the invention in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., bacillus subtilis) that are not susceptible to a bacteriophage protein of the present invention or a variant, derivative or fragment thereof, transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the coding sequence of the molecule identified by the methods of the present invention; yeast (e.g., Pichia pastoris) transformed with a recombinant yeast expression vector containing sequences encoding molecules identified by the methods of the invention; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing sequences encoding molecules identified by the methods of the invention; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus (CaMV) and Tobacco Mosaic Virus (TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmids) containing sequences encoding molecules identified by the methods of the invention), or mammalian cell systems (e.g., COS, CHO, BHK, 293T, 3T3 cells, lymphocytes (lymphotic cells) (see u.s.5,807,715), Per c.6 cells (human retinal cells developed by crucel) having a promoter derived from the genome of mammalian cells (e.g., metallothionein promoter) or derived from mammalian viruses (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter) containing sequences encoding molecules identified by the methods of the invention.

In bacterial systems that are not susceptible to the bacteriophage proteins of the present invention, or variants, derivatives or fragments thereof, a number of expression vectors may be advantageously selected depending on the intended use for the molecule to be expressed. For example, vectors that direct high level expression of an easily purified fusion protein product may be desirable when large quantities of such proteins are to be produced for the production of pharmaceutical compositions of polypeptides. Such vectors include, but are not limited to, the E.coli expression vector pUR278(Ruther et al, 1983, EMBO J.2: 1791) in which the protein sequence can be ligated into the vector separately in frame with the lacZ coding region, thereby allowing the production of a fusion protein; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res.13: 3101-3109; Van Heeke & Schuster, 1989, J.biol.chem.24: 5503-5509); and the like. pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione agarose beads, followed by elution in the presence of free glutathione. The pGEX vector is designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In the insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus is preferably grown in Spodoptera frugiperda (Spodoptera frugiperda) cells. The polypeptide coding sequence may be cloned individually into a non-essential region of the virus (e.g., the polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., the polyhedrin promoter).

Once a molecule (i.e., polypeptide) of the invention has been recombinantly expressed, it can be purified by any method known in the art for purifying polypeptides, such as by chromatography (e.g., ion exchange, affinity, and size exclusion column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying polypeptides or antibodies.

Certain modifications and improvements will occur to those skilled in the art upon a reading of the foregoing specification. It is to be understood that all such modifications and improvements have been deleted herein for the sake of brevity and readability but are properly within the scope of the following claims.

7. Examples of the embodiments

It is understood that the following examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Unless otherwise indicated, the bacteriophage of the present invention are isolated, processed and analyzed according to the following method.

7.1.1 purification of phages

According to Carlson, "Working with bacteriophage: common techniques and statistical approaches, "In, Kutter, and Sulakveldze (eds.) Bacteriophage: stock preparations of bacteriophage isolated from clinical samples were prepared according to the protocol described in Biologyand Applications, 5 th edition CRC Press (2005) (incorporated herein by reference in its entirety as "Carlson").

According to Carlson and Yamamoto et al, 2004, PNAS 101: 6415-6420, the bacteriophage stock preparation is concentrated by precipitation with PEG. Briefly, the stock formulations were incubated in 1M NaCl at 4 ℃ for one hour with agitation. Next, PEG 8000(AppliChem, Cheshire, MA) was gradually added to a final concentration of 10% (w/v). The composition was then incubated overnight at 4 ℃. After the incubation period, the composition was centrifuged at 10000Xg for 30 minutes at 4 ℃. The pellet was then resuspended at 1% w/v in SM buffer with gelatin (0.05M Tris-HCl at pH 7.4, 0.1M NaCl, 10mM MgSO)₄) And centrifuged again at 1000rpm for 10 minutes at 4 ℃. The supernatant containing the suspended phage was saved for further purification. The supernatant was purified using a CsCl gradient according to the method in Carlson.

CsCl was removed from the purified and concentrated phage bulk by dialysis. A dialysis Membrane, Cellu. Sep H1 High Grade Regenerated Cellulose Tubular Membrane (Cellu. Sep, River Street, USA), was prepared according to the manufacturer's instructions. Dialysis included a first incubation at 4 ℃ for 30 minutes in 100mM Tris-HCl and 3M NaCl (pH 7.4). This was followed by a second incubation at 4 ℃ for 30 minutes in 100mM Tris-HCl and 0.3M NaCl (pH 7.4). After dialysis, the suspended phages were removed from the inside of the dialysis bag and stored at 4 ℃.

7.1.2 extraction of phage DNA

To 5ml of the purified and concentrated bacteriophage sample, 20mM EDTA at pH 8.0, SDS at 0.5% (p/v) and proteinase K at a final concentration of 40. mu.g/ml were added. The mixture was incubated at 56 ℃ for one hour. Continuous extraction in phenol chloroform alcohol at a ratio of 25: 24: 1 was performed until the interface between the water and organic phases was clear. The aqueous phase was then treated with an equal volume of chloroform and centrifuged at 13,0000 13,0000x g for 10 minutes at 4 ℃. The aqueous phase was removed again and the DNA was precipitated by adding two volumes of absolute ethanol and incubating at 20 ℃ for thirty minutes. The samples were then centrifuged at 11,000x g for 30 minutes at 4 ℃. The pellet was washed with 70% ethanol at room temperature and resuspended in 50. mu.l of ultrapure water (Gibco, California). The DNA concentration was determined by measuring the absorbance at 260nm in an ND-1000 spectrophotometer. The integrity of the isolated phage DNA was analyzed by electrophoresis on a 1% agarose gel.

7.1.3 analysis of the phage genome

Sequencing of the bacteriophage genome allows identification of potential Open Reading Frames (ORFs) within the genome. The putative ORF of the bacteriophage was used to search the NCBI nucleotide pool database for homologous DNA sequences using the BLASTN program (see, e.g., Zhang et al, 2000, J.Compout.biol.7: 203-214).

7.2 example 1: bacteriophage F391/08

Comparison of the putative ORF of bacteriophage F391/08 genome with sequences in the NCBI nucleotide database revealed that only a small portion of the genome (. ltoreq.11% genome coverage) showed homology to known sequences. A schematic organization of the F391/08 genome is provided in FIG. 1. The F391/08ORF, its encoded amino acid sequence and known homologous proteins are provided in FIG. 2. The prediction of orf was performed by integrating the results obtained with the GeneMark. hmm and MetaGeneAntator programs (Besemer, J. and Borodovsky, M.1999.nucleic Acids Res., 27: 3911-3920; Noguchi, H. et al, 2008.DNA Res., 15: 387-396). Protein homology searches were performed using the NCBI non-redundant protein sequence database using the BLASTP program (Alschul, S.F. et al, 1997.Nucleic Acids Res., 25: 3389-33402). The protein conserved domain was predicted using NCBI specific BLAST (Marchler-Bauer, A. et al, 2007.Nucleic Acids Res.35: 237-240). The orf whose product exhibits homology to the same protein is indicated in fig. 2 with the same number, with the addition of lower case letters. Identification of putative transfer RNA genes (tRNA) was performed using the tRNAscan-SE program (Lowe, T.M. et al, 1997.Nucleic acids sRs., 25: 955-964).

Table 1 below provides the results of a spot test that evaluates the host range and activity of bacteriophage F391/08 against 100 strains of Klebsiella species (86 strains of Klebsiella pneumoniae; 12 strains of Klebsiella acidogenic; and 2 strains of Klebsiella species) isolated from clinical samples. Each spot contained 5. mu.l of bacteriophage suspension prepared from CsCl-purified lysate with indicated titer. The sensitivity of each strain to phages was estimated based on the relative scale ranging from turbid (+) to clear (++++) -lytic halos. Spots derived from isolated plaques are indicated as (pfu) and resistance to phage infection is indicated as (-). The percentage of strains showing a particular susceptibility phenotype is also indicated.

TABLE 1

7.3 example 2: bacteriophage F394/08

Comparison of the putative ORF of bacteriophage F394/08 genome with sequences in the NCBI nucleotide database revealed no significant homology to known sequences, except that observed for a small portion of the genome (. ltoreq.1% genome coverage). A schematic organization of the F394/08 genome is provided in FIG. 3. The F394/08 ORF, its encoded amino acid sequence and known homologous proteins are provided in FIG. 4. The prediction of orf was performed by integrating the results obtained with the GeneMark. hmm and MetaGeneAntator programs (Besemer, J. and Borodovsky, M.1999.nucleic Acids Res., 27: 3911-3920; Noguchi, H. et al, 2008. DNARRes., 15: 387-396). Protein homology searches were performed using the NCBI non-redundant protein sequence database using the BLASTP program (Alschul, S.F. et al, 1997.Nucleic Acids Res., 25: 3389-33402). The protein conserved domain was predicted using NCBI specific BLAST (Marchler-Bauer, A. et al, 2007.Nucleic Acids Res.35: 237-240). Rof whose product exhibits homology to the same protein is indicated by the same number in FIG. 4, with the addition of lower case letters. Identification of putative transfer RNA genes (tRNA) was performed using the tRNAscan-SE program (Lowe, T.M. et al, 1997.nucleic acids Res., 25: 955-964).

Table 2 below provides the results of a spot test that evaluates the host range and activity of bacteriophage F394/08 against 100 strains of Acinetobacter species (93 Acinetobacter baumannii strains; 6 Acinetobacter calcoaceticus strains; and 1 Acinetobacter lwoffii strain) isolated from clinical samples. Each spot contained 5. mu.l of bacteriophage suspension prepared from CsCl-purified lysate with indicated titer. The sensitivity of each strain to phages was estimated based on the relative scale ranging from turbid (+) to clear (++++) -lytic halos. Spots derived from isolated plaques are indicated as (pfu) and resistance to phage infection is indicated as (-). The percentage of strains showing a particular susceptibility phenotype is also indicated.

TABLE 2

7.4 example 3: bacteriophage F488/08

Comparison of the putative ORF of bacteriophage F488/08 genome with sequences in the NCBI nucleotide database revealed that about 94% of the bacteriophage F488/08DNA is highly similar to that of the enterobacteriophage RB14, with individual ORF identities ranging from 70-100%. A schematic organization of the F488/08 genome is provided in FIG. 5. The F488/08ORF, its encoded amino acid sequence, and known homologous proteins are provided in FIG. 6. The prediction of orf was performed by integrating the results obtained with the GeneMark. hmm and MetaGeneAntator programs (Besemer, J. and Borodovsky, M.1999.nucleic Acids Res., 27: 3911-3920; Noguchi, H. et al, 2008. DNARRes., 15: 387-396). Protein homology searches were performed using the NCBI non-redundant protein sequence database using the BLASTP program (Alschul, S.F. et al, 1997.Nucleic Acids Res., 25: 3389-33402). The protein conserved domain was predicted using NCBI specific BLAST (Marchler-Bauer, A. et al, 2007.Nucleic acids sRs.35: 237-240). The orf whose product exhibits homology to the same protein is indicated in fig. 4 with the same number, with the addition of lower case letters. Identification of putative transfer RNA genes (tRNA) was performed using the tRNAscan-SE program (Lowe, T.M. et al, 1997.Nucleic Acids Res., 25: 955-964).

Table 3 below provides the results of the spot test, which evaluates the host range and activity of bacteriophage F488/08 against 100 strains of E.coli (ECO) isolated from clinical samples. Each spot contained 5. mu.l of bacteriophage suspension with indicated titer prepared from lysate purified by ion exchange chromatography. The sensitivity of each strain to phages was estimated based on the relative scale ranging from turbid (+) to clear (++++) -lytic halos. Spots derived from isolated plaques are indicated as (pfu) and resistance to phage infection is indicated as (-). The percentage of strains showing a particular susceptibility phenotype is also indicated.

TABLE 3

7.5 example 4: bacteriophage F510/08

Comparison of the putative ORF of bacteriophage F510/08 genome with sequences in the NCBI nucleotide database revealed that about 95% of the bacteriophage F510/08 DNA is highly similar to that of Pseudomonas bacteriophage LUZ19, with individual ORF identities ranging from 89-97%. A schematic organization of the F510/08 genome is provided in FIG. 7. The F510/08 ORF, its encoded amino acid sequence and known homologous proteins are provided in FIG. 8. The prediction of orf was performed by integrating the results obtained with the GeneMark. hmm and MetaGeneAntator programs (Besemer, J. and Borodovsky, M.1999.nucleic Acids Res., 27: 3911-3920; Noguchi, H. et al, 2008.DNA Res., 15: 387-396). Protein homology searches were performed using the NCBI non-redundant protein sequence database using the BLASTP program (Alschul, S.F. et al, 1997.Nucleic Acids Res., 25: 3389-33402). The protein conserved domain was predicted using NCBI specific BLAST (Marchler-Bauer, A. et al, 2007.Nucleic acids sRs.35: 237-240). The orf whose product exhibits homology to the same protein is indicated in fig. 8 with the same number, with the addition of lower case letters. Identification of putative transfer RNA genes (tRNA) was performed using the tRNAscan-SE program (Lowe, T.M. et al, 1997.Nucleic Acids Res., 25: 955-964).

Table 4 below provides the results of the spot test, which evaluates the host range and activity of bacteriophage F510/08 against 100 Pseudomonas aeruginosa (PSA) strains isolated from clinical samples. Each spot contained 5. mu.l of bacteriophage suspension prepared from CsCl-purified lysate with indicated titer. The sensitivity of each strain to phages was estimated based on the relative scale ranging from turbid (+) to clear (++++) -lytic halos. Spots derived from isolated plaques are indicated as (pfu) and resistance to phage infection is indicated as (-). The percentage of strains showing a particular susceptibility phenotype is also indicated.

TABLE 4

7.6 example 5: bacteriophage F44/10

Comparison of the putative ORF of bacteriophage F44/10 genome with sequences in the NCBI nucleotide database revealed that about 81% of the bacteriophage F44/10DNA is highly similar to that of Staphylococcus phage K, with individual ORFs identical in the range of 80-99%. A schematic organization of the F44/10 genome is provided in FIG. 9. The F44/10ORF, its encoded amino acid sequence, and known homologous proteins are provided in FIG. 10. The prediction of orf was performed by integrating the results obtained with the GeneMark. hmm and MetaGeneAntator programs (Besemer, J. and Borodovsky, M.1999.nucleic Acids Res., 27: 3911-3920; Noguchi, H. et al, 2008. DNARRes., 15: 387-396). Protein homology searches were performed using the NCBI non-redundant protein sequence database using the BLASTP program (Alschul, S.F. et al, 1997.Nucleic Acids Res., 25: 3389-33402). The protein conserved domain was predicted using NCBI specific BLAST (Marchler-Bauer, A. et al, 2007.Nucleic acids sRs.35: 237-240). The orf whose product exhibits homology to the same protein is indicated with the same number in fig. 10, with the addition of lower case letters. As previously reported for Staphylococcus phage K, the putative polymerase genes (orf114a, orf114b) may contain intron-like sequences (O' Flaherty et al, 2004). Identification of putative transfer RNA genes (tRNA) was performed using the tRNAscan-SE program (Lowe, T.M. et al, 1997.Nucleic Acids Res., 25: 955-964).

Table 5 below provides the results of the spot test, which evaluated the host range and activity of bacteriophage F44/10 against 100 staphylococcus aureus (STA) strains isolated from clinical samples. Each spot contained 5. mu.l of bacteriophage suspension prepared from CsCl-purified lysate with indicated titer. The sensitivity of each strain to phages was estimated based on the relative scale ranging from turbid (+) to clear (++++) -lytic halos. Spots derived from isolated plaques are indicated as (pfu) and resistance to phage infection is indicated as (-). The percentage of strains showing a particular susceptibility phenotype is also indicated.

TABLE 5

7.7 example 6: bacteriophage F387/08

Comparison of the putative ORF of bacteriophage F387/08 genome with sequences in the NCBI nucleotide database revealed no significant homology to known sequences, except that observed for a small portion of the genome (< 12% genome coverage). A schematic organization of the F387/08 genome is provided in FIG. 11. The function-specific orf is indicated on the right and in fig. 11B-C. A DNA homology search was performed using the NCBI nucleotide collection database with the BLASTN program (Zhang, z. et al, 2000.j. comput.biol., 7: 203-214).

The F387/08ORF, the amino acid sequence encoded thereby and the known homologous proteins are provided in FIG. 12. The prediction of orf was performed by integrating the results obtained with the GeneMark. hmm and MetaGeneAntator programs (Besemer, J. and Borodovsky, M.1999.nucleic acids Res., 27: 3911-3920; Noguchi, H. et al, 2008.DNA Res., 15: 387-396). Protein homology searches were performed using the NCBI non-redundant protein sequence database using the BLASTP program (Alschul, S.F. et al, 1997.Nucleic Acids Res., 25: 3389-33402). The protein conserved domain was predicted using NCBI specific BLAST (Marchler-Bauer, A. et al, 2007.Nucleic Acids Res.35: 237-240).

Table 6 below provides the results of a spot test that evaluates the host range and activity of bacteriophage F387/08 against 100 strains of Klebsiella species (86 strains of Klebsiella pneumoniae; 12 strains of Klebsiella acidovorans; and 2 strains of Klebsiella species) isolated from clinical samples. Each spot contained 5. mu.l of bacteriophage suspension prepared from CsCl-purified lysate with indicated titer. The sensitivity of each strain to phages was estimated based on the relative scale ranging from turbid (+) to clear (++++) -lytic halos. Spots derived from isolated plaques are indicated as (pfu) and resistance to phage infection is indicated as (-). The percentage of strains showing a particular susceptibility phenotype is also indicated.

TABLE 6

7.8 example 7: bacteriophage F125/10

Comparison of the putative ORF of the bacteriophage F125/10 genome with sequences in the NCBI nucleotide database revealed that about 87% of the bacteriophage F125/10DNA is highly similar to that of Staphylococcus phage A5W, with individual ORF identities ranging from 77-99%. F125/1A schematic organization of the 0 genome is provided in fig. 13. The F125/10ORF, its encoded amino acid sequence and known homologous proteins are provided in FIG. 14. The prediction of orf was performed by integrating the results obtained with the GeneMark. hmm and MetaGeneAntator programs (Besemer, J. and Borodovsky, M.1999.nucleic Acids Res., 27: 3911-3920; Noguchi, H. et al, 2008.DNA Res., 15: 387-396). Protein homology searches were performed using the NCBI non-redundant protein sequence database using the BLASTP program (Alschul, S.F. et al, 1997.Nucleic Acids Res., 25: 3389-33402). The protein conserved domain was predicted using NCBI specific BLAST (Marchler-Bauer, A. et al, 2007. nucleic acids Res.35: 237-240). The orf whose product exhibits homology to the same protein is indicated with the same number in fig. 14, with the addition of lower case letters. As previously reported for Staphylococcus phage K (O' Flaherty et al, 2004, J.of Bacteriology 186 (9): 2862-2871) and phage Tport (Landthaler et al, 2002, Nucleic Acids Research 30 (9): 1935-1943), it was found that introns of genes involved in DNA metabolism were interrupted; and the putative telomerase large subunit genes (orf153a, orf153b) may contain an intron-like sequence (orf154^*)。

Table 7 below provides the results of the spot test, which evaluated the host range and activity of bacteriophage F125/10 against 98 staphylococcus aureus (STA) strains isolated from clinical samples. Each spot contained 5. mu.l of bacteriophage suspension prepared from CsCl-purified lysate with indicated titer. The sensitivity of each strain to phages was estimated based on the relative scale ranging from turbid (+) to clear (++++) -lytic halos. The dilution of phage derived from the isolated plaques is indicated as (pfu) and the resistance to phage infection is indicated as (-). The percentage of strains showing a particular susceptibility phenotype is also indicated.

TABLE 7

Claims (25)

1. A purified bacteriophage having a genome comprising at least 99% sequence identity to the nucleotide sequence of SEQ ID NO. 4 and having antimicrobial activity against Pseudomonas aeruginosa.

2. The bacteriophage of claim 1, wherein the genome comprises the nucleotide sequence of SEQ ID No. 4.

3. A pharmaceutical composition comprising the bacteriophage of claim 1 or 2 and a pharmaceutically acceptable carrier.

4. The pharmaceutical composition of claim 3, further comprising one or more additional strains of bacteriophage known to have antimicrobial activity against P.

5. A pharmaceutical composition comprising bacteriophage F510/08 and at least three additional purified bacteriophages selected from bacteriophage F391/08, bacteriophage F394/08, bacteriophage F488/08, bacteriophage F44/10, bacteriophage F387/08, bacteriophage F170/08, bacteriophage F168/08, bacteriophage F770/05, bacteriophage F125/10, and bacteriophage F1245/05;

wherein the bacteriophage F510/08 has a genome comprising at least 99% sequence identity to the nucleotide sequence of SEQ ID NO. 4;

wherein the bacteriophage F391/08 has a genome comprising at least 99% sequence identity to the nucleotide sequence of SEQ ID NO. 1;

wherein said bacteriophage F394/08 has a genome comprising at least 99% sequence identity to the nucleotide sequence of SEQ ID NO. 2;

wherein the bacteriophage F488/08 has a genome comprising at least 99% sequence identity to the nucleotide sequence of SEQ ID NO. 3;

wherein the bacteriophage F44/10 has a genome comprising at least 99% sequence identity to the nucleotide sequence of SEQ ID NO 560;

wherein the bacteriophage F387/08 has a genome comprising at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 781;

wherein the bacteriophage F125/10 has a genome comprising at least 99% sequence identity to the nucleotide sequence of SEQ ID NO 1074.

6. The pharmaceutical composition of claim 5, wherein the composition comprises bacteriophage F510/08 and at least three additional purified bacteriophages selected from the group consisting of bacteriophage F44/10, bacteriophage F391/08, bacteriophage F387/08, bacteriophage F488/08, bacteriophage F125/10, and bacteriophage F770/05.

7. The pharmaceutical composition of claim 6, wherein the composition comprises bacteriophage F44/10or bacteriophage F125/10 in addition to bacteriophage F510/08.

8. The pharmaceutical composition of claim 7, wherein the composition comprises bacteriophage F44/10 and bacteriophage F125/10 in addition to bacteriophage F510/08.

9. The pharmaceutical composition of any one of claims 5-8, wherein the composition is formulated for topical application.

10. Use of an effective amount of the pharmaceutical composition of any one of claims 3-4 or 5-9 in the manufacture of a medicament for treating or reducing the occurrence of a bacterial infection in a subject in need thereof by administering the medicament.

11. The use of claim 10, wherein the bacterial infection is by pseudomonas aeruginosa.

12. The use of claim 10or 11, wherein the infection is a nosocomial infection.

13. The use of claim 10or 11, wherein the infection is an infection of the skin.

14. The use of claim 13, wherein the skin infection is associated with burn injury, damaged skin, or diabetic foot ulcer.

15. The use of claim 13 or 14, wherein the pharmaceutical composition is administered topically.

16. The use of claim 10or 11, wherein the subject is a mammal.

17. The use of claim 16, wherein the mammal is a human.

18. Use of a bacteriophage according to claim 1 or 2 in the preparation of a medicament for use in a method of diagnosing a causative agent of a bacterial infection, comprising

(i) Culturing a tissue sample from a patient;

(ii) (ii) contacting the culture of step (i) with a medicament comprising a bacteriophage of claim 1 or 2;

(iii) evidence for monitoring growth and/or lysis of said culture

Wherein evidence of lysis of the culture indicates that the culture comprises Pseudomonas aeruginosa.

19. The use of claim 18, wherein the tissue sample is a sample of blood, urine, saliva, damaged skin, tissue biopsy, or swab collected from the patient.

20. A non-therapeutic method for reducing or inhibiting the colonization or growth of pseudomonas aeruginosa on a solid surface comprising contacting the surface with a bacteriophage of claim 1 or 2.

21. The method of claim 20, wherein the solid surface is a surface of a hospital instrument or a hospital equipment part.

22. The method of claim 21, wherein the instrument or device is part of a surgical instrument or surgical device.

23. Use of a bacteriophage of claim 1 or 2 in the preparation of a medicament for reducing or inhibiting colonization or growth of pseudomonas aeruginosa on a biological surface in contact therewith.

24. The use of claim 23, wherein the biological surface is mammalian skin, damaged skin, or mucous membranes.

25. The use of claim 24, wherein the mammal is a human.