Cocktail compositions containing respiratory antibacterial phages and methods of using the same

Abstract

The present invention is directed to the field of phage therapy for the treatment and control of bacterial infections, particularly respiratory bacterial infections such as bacterial pneumonia. More specifically, the present invention is directed to novel bacteriophage strains and cocktails thereof, as well as variants thereof, and methods of using the same in the treatment and prevention of bacterial infections, including, by way of example, respiratory infections caused by Pseudomonas aeruginosa and / or Klebsiella pneumoniae. The cocktails are used as pharmaceutical compositions, alone or in further combination with other therapies, by way of example, antibiotics or other standard and non-standard therapies for respiratory infections.

Description

[Technical Field]

[0001] 1. Field of the Invention The present invention is directed to the field of phage therapy for the treatment and control of bacterial infections, particularly respiratory bacterial infections such as bacterial pneumonia. One aspect of the present invention is directed to a novel bacteriophage strain cocktail comprising Kle_F391 / 08 (SEQ ID NO: 1), Kle_F17 / 19 (SEQ ID NO: 2), and Kle_F58 / 19 (SEQ ID NO: 3), as well as variants thereof; and methods of using the same in the treatment and prevention of bacterial infections, including, for example, respiratory infections caused by Klebsiella pneumoniae. The cocktail may be administered in combination with other bacteriophage compositions, including cocktails directed against the same pathogen or different pathogens, such as Pseudomonas aeruginosa. The cocktail is used as a pharmaceutical composition, alone or in further combination with other therapies, such as antibiotics or other standard and non-standard therapies for respiratory infections. [Background technology]

[0002] 2. Background Bacteriophages (phages) are viruses that specifically infect and lyse bacteria. Phage therapy, a method using whole phage viruses to treat bacterial infectious diseases, was introduced by Felix d'Herelle in the 1920s. However, with the development of antibiotics in the 1940s, interest in phage-based therapeutics declined in Western countries. One of the most important factors contributing to this decline was the lack of standardized testing and production methods. The failure to develop industry-wide standards for phage therapy testing hindered the documentation of research results, leading to perceived lack of efficacy and credibility issues regarding the value of phage therapy. Another problem in phage production concerns the purity grade of commercially available phage preparations, which contain unwanted bacterial components, such as endotoxins. Consequently, the preparations are often associated with adverse events, especially in patients who receive the preparations intravenously.

[0003] Nevertheless, the development and use of phage therapy continued in tandem with or instead of antibiotics in Eastern Europe and the former Soviet Union, where access to antibiotics was limited. Furthermore, the emergence of antibiotic-resistant strains of many bacteria has revived interest in phage-based therapeutics in Western countries. Thus, even though new classes of antibiotics may be developed, the prospect that bacteria will eventually develop resistance to the new drugs has intensified the search for non-chemotherapeutic means to control, prevent, and treat bacterial infections.

[0004] Phage therapy, and in particular phage cocktails, offers an alternative to antibiotics for the treatment of bacterial infections, and in particular respiratory infections, including hospital-acquired pulmonary infections. Respiratory infections are responsible for over 4 million deaths annually. Hospital-acquired bacterial pneumonia (HABP) is an acute pulmonary infection and one of the most common types of infections acquired in intensive care unit settings, with an increased mortality rate (ranging from 33 to 41%) (Guzman-Herrador B, et al., 2014, J Hosp Infect 86(1):53-56). Nosocomial pulmonary infections are typically caused by Gram-negative Enterobacteriaceae, such as methicillin-resistant Staphylococcus aureus (MRSA), Klebsiella pneumoniae, or Gram-negative non-Enterobacteriaceae, such as Pseudomonas aeruginosa and Acinetobacter species (Quartin AA, et al., 2013, BMC Infect Dis 13:561-566; and Di Pasuale M, et al., 2014, Crit Care Med 42(2):303-312).

[0005] Antibiotic therapy is routinely used in HABP; however, there are few treatment options for multi-resistant (MDR) bacteria, especially Gram-negative bacteria. No new classes of drugs have been introduced recently, and the few currently available options include colistin, tigecycline, and fosfomycin. For severe hospital-acquired infections, there are few antibiotic options (Orsi GB, et al., 2011, Expert Rev Anti Infect Ther 9(8):653-679).

[0006] Aerosolization of antibiotics can result in higher antibiotic delivery to the lung parenchyma compared with intravenous administration of antibiotics (Luyt CE, et al., 2009, Crit Care 13(6):R200). However, to date, there has been no clear clinical benefit to using aerosolized antibiotics, such as colistin, in the treatment of pulmonary infections due to side effects from direct antibiotic toxicity to the airways and lung parenchyma. Side effects include, for example, side effects caused by systemic absorption of antibiotics, such as mucosal irritation and nephrotoxicity of aminoglycosides and polymyxins (Luyt CE, et al., 2013, Expert Rev Anti Infect Ther 11(5):511-521; and Quon BS, et al., 2014, Ann Am Thorac Soc 11(3):425-434).

[0007] Various studies have attempted to treat bacterial lung infections using bacteriophages administered via various routes (Hoe S, et al., 2013, J Aerosol Med Pulm Drug Deliv 26:317-335; Morello E. et al., 2011, PLoS One 6(2): el6963; and Debarbieux L, et al., 2010, J Infect Dis 201(7): 1096-1104). However, there is little published experimental evidence of aerosolized bacteriophages curing established infections (Ryan EM, et al., 2011, J Pharm Pharmacol 63:1253-1264). Previously published studies have not evaluated the effects of aerosolized bacteriophages in confirmed infections, and most have investigated outcomes only a few hours after infection (Wilson KR, et al., 200, Microbiology 153(Pt 4):968-979; and Alemayehu D. et al., 2012, MBio 3(2):e00029-12). Furthermore, few phage cocktails have antimicrobial activity against a variety of bacteria, likely due to the difficulty of combining phages of different specificities while maintaining storage stability. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Guzman-Herrador B, et al., 2014, J Hosp Infect 86(l):53-56 [Non-patent document 2] Quartin AA, et al., 2013, BMC Infect Dis 13:561-566 [Non-patent document 3] Di Pasuale M, et al., 2014, Crit Care Med 42(2):303-312 [Non-patent document 4] Orsi GB, et al., 2011, Expert Rev Anti Infect Ther 9(8):653-679 [Non-Patent Document 5] Luyt CE, et al., 2009, Crit Care 13(6):R200 [Non-Patent Document 6] Luyt CE, et al., 2013, Expert Rev Anti Infect Ther 11(5):511 -521 [Non-Patent Document 7] Quon BS, et al., 2014, Ann Am Thorac Soc ll(3):425-434 [Non-Patent Document 8] Hoe S, et al., 2013, J Aerosol Med Pulm Drug Deliv 26:317-335[[ID=]) [Non-Patent Document 9] <00000]96>Morello E. et al., 2011, PLoS One 6(2): el6963 [Non-Patent Document 10] Debarbieux L, et al., 2010, J Infect Dis 201(7): 1096-1104 [Non-Patent Document 11] Ryan EM, et al., 2011, J Pharm Pharmacol 63:1253-1264 [Non-Patent Document 12] Wilson KR, et al., 200, Microbiology 153(Pt 4):968-979 [Non-Patent Document 13] Alemayehu D. et al., 2012, MBio 3(2):e00029-12 [Summary of the Invention] [Problems to be Solved by the Invention]

[0009] Thus, there is a need to develop novel phage products as therapeutic and / or prophylactic agents for in vivo use against pathogenic bacteria, particularly pulmonary bacteria. There is also a need for better treatments, particularly aerosolized treatments, for respiratory infections. In particular, there is a need for bacteriophage cocktails that can lyse bacteria responsible for hospital-acquired respiratory infections, including Pseudomonas aeruginosa and / or Klebsiella pneumoniae bacteria. The present application addresses these and other needs. [Means for solving the problem]

[0010] 3. Summary of the Invention Novel Klebsiella pneumoniae bacteriophages and their uses in treating bacterial infections are provided. Pharmaceutical compositions comprising the bacteriophage cocktails disclosed herein, or combinations of three or more of the bacteriophages described herein, can be used in the treatment, management, or prevention of bacterial infections, particularly Pseudomonas aeruginosa and / or Klebsiella pneumoniae infections.

[0011] Also provided are novel Klebsiella pneumoniae bacteriophage cocktails and their use in treating bacterial infections. Pharmaceutical compositions comprising the bacteriophage cocktails disclosed herein, or combinations of three or more of the bacteriophages described herein, can be used in the treatment, management, or prevention of bacterial infections, particularly Pseudomonas aeruginosa and / or Klebsiella pneumoniae infections. Such pharmaceutical compositions can be particularly useful in the treatment, management, or prevention of respiratory infections, and the compositions can be formulated for pulmonary delivery.

[0012] One aspect of the present invention relates to novel Klebsiella pneumoniae bacteriophages. Purified Klebsiella pneumoniae phages Kle_F17 / 19 and Kle_F58 / 19, each having a genome comprising or consisting of the nucleic acid sequence SEQ ID NO: 2 and SEQ ID NO: 3, are provided, which have antibacterial activity against Klebsiella pneumoniae, as well as variants thereof that maintain the lytic activity of these bacteriophages. These bacteriophages have been deposited: Kle_F17 / 19 has accession number NCIMB 43534, and Kle_F58 / 19 has accession number NCIMB 43535. These bacteriophages are particularly capable of lysing Klebsiella pneumoniae strains 57 / 15 (NCIMB accession number 43536) and 237 / 14 (NCIMB accession number 43537).

[0013] In one embodiment, the present invention provides an isolated bacteriophage F17 / 19 having a genome comprising the nucleic acid sequence of SEQ ID NO:2 and exhibiting antibacterial activity against one or more strains of Klebsiella pneumoniae.

[0014] In one embodiment, the present invention provides an isolated bacteriophage F58 / 19 having a genome comprising the nucleic acid sequence of SEQ ID NO: 3 and exhibiting antibacterial activity against one or more strains of Klebsiella pneumoniae.

[0015] Another aspect of the invention relates to a composition comprising three or more different purified bacteriophages in a cocktail combination. In one aspect, the bacteriophage cocktail comprises three Kle_F391 / 08, Kle_F17 / 19, and Kle_F58 / 19 bacteriophages (including variants thereof that maintain the lytic activity of the bacteriophages) and has antibacterial activity against Kle_F391 / 08. This cocktail may, in other aspects of the invention, further comprise or be administered in combination with a cocktail of one, two, three, or four or more additional bacteriophages, e.g., against Pseudomonas aeruginosa. In one aspect, a cocktail of Kle_F391 / 08 (deposited as NCIMB 42918), Kle_F17 / 19, and Kle_F58 / 19 is co-formulated with and / or administered in combination with a cocktail of Pseudomonas aeruginosa bacteriophages Psa_F99 / 10 (deposited as NCIMB 42915), Psa_F27 / 12 (deposited as NCIMB 42916), and Psa_F95 / 13 (deposited as NCIMB 42917) (disclosed in U.S. Patent Application Publication No. 2019 / 0290709). In other embodiments, a bacteriophage cocktail disclosed herein is administered with one or more other purified bacteriophages that have antibacterial activity against Klebsiella pneumoniae, Pseudomonas aeruginosa, or bacteria that are not Klebsiella pneumoniae or Pseudomonas aeruginosa. In an even more preferred embodiment, the composition is formulated for administration as an aerosol and for pulmonary delivery.

[0016] Another aspect of the present invention relates to pharmaceutical compositions comprising a bacteriophage or phage product of the present invention and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises one or more additional bacteriophages or phage products having antibacterial activity against Klebsiella pneumoniae. In some embodiments, the composition is formulated in a dosage form in which the bacteriophage is present in an amount that provides a multiplicity of infection (MOI) of about 1 to about 10 upon administration of the composition to a subject in need thereof. In certain embodiments, each phage is present in an amount of about 1 to about 10. 9pfu~10 10 pfu, e.g., 10 9 pfu / ml In a preferred embodiment, the composition is formulated for administration as an aerosol.

[0017] Another aspect of the present invention relates to methods for treating, reducing the incidence of, or managing a bacterial infection in a subject in need thereof, comprising administering to the subject a therapeutically or prophylactically effective amount of a pharmaceutical composition of the present invention, as well as uses thereof. In some embodiments, the bacterial infection is caused by a Klebsiella pneumoniae bacterial strain, and / or a Pseudomonas aeruginosa bacterial strain in certain embodiments, including bacterial strains that exhibit resistance to one or more known antibiotics and / or exhibit the ability to form biofilms. In preferred embodiments, the bacterial infection to be treated or reduced in incidence is a respiratory infection, more preferably hospital-acquired bacterial pneumonia or a respiratory infection associated with cystic fibrosis. In particularly preferred embodiments, the composition is administered as an aerosol to the lungs. In some embodiments, the composition is re-administered about 4, 6, or 8 hours after the initial administration.

[0018] Another aspect of the present invention relates to a method for diagnosing the causative agent of a bacterial infection and / or assessing whether the infectious agent is susceptible to the lytic activity of a bacteriophage, comprising the steps of: (i) culturing a sample from a patient, such as a swab or sputum, or other sample suitable for culturing the bacteria causing the infection; (ii) contacting the culture of step (i) with a bacteriophage or phage product disclosed herein; and (iii) monitoring the culture for evidence of growth or lysis, where evidence of lysis of the culture indicates that the culture contains a bacterial strain known to be susceptible to the bacteriophage or phage product used in step (ii). In some embodiments, the sample is a tissue biopsy or swab taken from the patient's respiratory tract. For example, the sample may include bronchoalveolar lavage fluid or bronchial secretions.

[0019] Yet another aspect of the present invention provides methods for reducing or inhibiting bacterial colonization or growth on a surface, the method comprising contacting the surface with a bacteriophage or phage product of the present invention. In some embodiments, the surface is a mammalian mucosa, preferably the mucosa of the human respiratory tract. In some embodiments, the surface is a non-living surface, preferably a surface of a hospital instrument or equipment, more preferably a surface of a surgical instrument or equipment.

[0020] 3.1 Definition As used herein, the term "isolated" with respect to a nucleic acid molecule refers to a first nucleic acid molecule that is separated from other nucleic acid molecules that are present in the natural source of the first nucleic acid molecule. An "isolated" nucleic acid molecule, such as an "orf" or phage genome, is substantially free of other cellular material, or culture medium if produced by recombinant techniques, or substantially free of chemical precursors or other chemicals if chemically synthesized; for example, it may be free of other DNA or other genomic DNA molecules if purified and isolated from other clones in a nucleic acid library or from an isolated phage. Furthermore, "isolated" genomic DNA may be substantially free of other viruses or cellular material, or culture medium if produced by recombinant techniques or isolated from phage, or substantially free of chemical precursors or other chemicals if chemically synthesized, and may be free of other DNA or other genomic DNA molecules if it is purified and isolated from a preparation containing other bacteriophage or cellular material.

[0021] The term "purified" with respect to bacteriophage means that the concentration of the phage has been measurably increased by any purification process, including, but not limited to, isolation from an environment or culture, e.g., isolation from the culture after growth and / or amplification, centrifugation, or otherwise, whereby impurities such as host cells and host cell components have been partially, substantially, nearly completely, or completely removed. One of skill in the art will understand the amount of purification necessary for a given use. For example, purified phage intended for use in therapeutic compositions intended for administration to humans typically must be of high purity in accordance with regulatory standards and good manufacturing practices.

[0022] The term "purified" with respect to a peptide, polypeptide, fusion protein, or nucleic acid molecule means that the concentration of the peptide, polypeptide, fusion protein, or nucleic acid molecule has been measurably increased by any purification process, including, but not limited to, column chromatography, HPLC, precipitation, electrophoresis, etc., thereby partially, substantially, nearly completely, or completely removing impurities, such as precursors or other chemicals involved in preparing the peptide, polypeptide, fusion protein, or nucleic acid molecule. One of skill in the art will understand the amount of purification necessary for a given use. For example, isolated and purified genomic DNA or protein or polypeptide intended for use in a therapeutic composition intended for administration to humans must typically be of high purity in accordance with regulatory standards and good manufacturing practices.

[0023] As used herein, the terms "bacteriophage product" or "active bacteriophage product" refer to a protein or fragment or variant thereof, as well as the nucleic acid encoding same, that is isolated from or derived from a bacteriophage of the invention and that retains a biological function or activity (e.g., antimicrobial activity such as lytic cell killing) associated with the bacteriophage from which the product is isolated or derived.

[0024] As used herein, the term "variant" with respect to a nucleotide sequence refers to a nucleotide sequence comprising or consisting of a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to a reference nucleic acid sequence. Variants may be selected that maintain one or more functions of the reference nucleic acid sequence. For example, a variant bacteriophage may exhibit at least one biological activity of the bacteriophage from which it is derived, such as antimicrobial activity, such as lytic killing activity. Those skilled in the art will understand that because nucleic acid replication in phages is less than 100% accurate, a given phage will exhibit at least a 1% variability in its replication, including during its production as an antibiotic. Expected genomic variations during phage production and use may result in progeny that are variants having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of the parent genome. In certain embodiments, a bacteriophage of the invention will comprise or consist of a genome having at least about 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the nucleotide sequence of the parent phage, while retaining the antimicrobial activity of the parent phage against the target (host) bacteria. A "variant" with respect to a bacteriophage is a bacteriophage whose genome has at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of the parent genome and that retains lytic activity like the parent bacteriophage (i.e., lyses the same bacterial strain(s)). In certain embodiments, a "variant" is simply identified by its lytic activity against one, two, or three strains of bacteria (eg, as described in the Examples herein).

[0025] For example, in certain embodiments, the bacteriophage is a variant of Kle_F391 / 08, Kle_F17 / 19, or Kle_F58 / 19 and retains antibacterial and / or lytic activity against one, two, or three of Klebsiella pneumoniae strains 57 / 17, 237 / 14, and 397 / 07.

[0026] The term "progeny" with respect to any of the novel phages herein refers to a bacteriophage replica containing progeny produced following subculture of a bacteriophage of a specific nucleic acid identified herein or by methods known to those of skill in the art, or a bacteriophage having a restriction fragment length polymorphism (RFLP) DNA profile substantially equivalent to that of a bacteriophage of a specific nucleic acid identified herein. The term "having substantially equivalent or equal RFLP" is expressed to account for inter-organism variability according to the method suggested by Tenover et al. (Tenover, F.C. et al. Interpreting Chromosomal DNA Restriction Patterns Produced by Pulsed-Field Gel Electrophoresis: Criteria for Bacterial Strain Typing. J. Clin. Microbiol 33:2233-2239 (1995)). Tenover et al. suggest an acceptable level of variability, provided that the genomes of identically grown organisms are restricted with a restriction enzyme and then electrophoresed. Progeny having equivalent RFLP DNA profiles according to the criteria suggested by Tenover et al. can be considered equivalent bacteriophages that are substantially equivalent to the bacteriophages of the specific nucleic acids identified herein, i.e., equivalent bacteriophages that contain a nucleic acid sequence having substantially the nucleotide sequence of any of SEQ ID NOs: 1-3.

[0027] As used herein, the term "host cell" refers to a particular subject cell transfected with a nucleic acid molecule, and to progeny or potential progeny of such cells that contain the nucleic acid molecule or a version thereof integrated into a chromosome. The progeny of such cells may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental influences that may occur in subsequent generations, or due to integration of the nucleic acid molecule into the host cell genome. "Host cell" also refers to cells, such as bacterial cells, that are infected with bacteriophage, e.g., whole phages, and in which the bacteriophage survives and replicates. For the production of bacteriophage, the host cell may or may not be of the same species or strain from which the bacteriophage was isolated or cultured.

[0028] As used herein, the term "fragment" 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 the full-length protein. In specific embodiments, a fragment is a functional fragment in that it retains at least one function of the protein from which it is isolated, e.g., antimicrobial activity such as lytic cell killing.

[0029] As used herein, the terms "in combination" or "in further combination" or "further in combination" refer to the use of additional prophylactic and / or therapeutic agents with a bacteriophage or phage product of the invention, including a phage cocktail of different bacteriophages of the invention. The use of the term "in combination" does not restrict the order in which prophylactic and / or therapeutic agents are administered to a subject. A first prophylactic or therapeutic agent can be administered prior to (for example, 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), simultaneously with, or subsequent to (for example, 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) the administration of a second prophylactic or therapeutic agent (different from the first prophylactic or therapeutic agent) to a subject.

[0030] As used herein, the term "boost" or "booster" refers to subsequent repeated use of the same or substantially the same prophylactic and / or therapeutic agent, such as repeated doses of a bacteriophage, phage product, or phage cocktail of the invention. The prophylactic or therapeutic agent may be first 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) the second administration of the same or substantially the same prophylactic or therapeutic agent to the subject.

[0031] As used herein, the terms "prophylactic agent" and "prophylactic agents" refer to an agent, such as a bacteriophage, phage product, or phage cocktail of the invention, that can be used in preventing, managing, controlling, or reducing the incidence of one or more symptoms of a disease or disorder, particularly a disease or disorder associated with a bacterial infection, more particularly a disease or disorder associated with a respiratory bacterial infection, such as, but not limited to, hospital-acquired bacterial pneumonia or a respiratory bacterial infection associated with cystic fibrosis.

[0032] As used herein, the terms "therapeutic agent" and "therapeutics" refer to an agent, such as a bacteriophage, phage product, or phage cocktail of the invention, that can be used in the treatment, management, or control of one or more symptoms of a disease or disorder, particularly a disease or disorder associated with a bacterial infection, more particularly a disease or disorder associated with a respiratory bacterial infection, such as, but not limited to, hospital-acquired bacterial pneumonia, or a respiratory bacterial infection associated with cystic fibrosis.

[0033] As used herein, the terms "treat," "treatment," and "treating" refer to achieving a therapeutic effect in a subject to which a pharmaceutical composition is administered. In achieving a therapeutic effect, the goal is to eliminate, reduce, lessen the severity of, ameliorate, or slow the progression of the symptoms or underlying causes (e.g., bacterial infection) associated with a pathological condition or disorder. A "therapeutically effective amount" refers to an amount of a therapeutic agent, such as a bacteriophage or phage product, in a pharmaceutical composition of the invention sufficient to achieve at least one therapeutic effect in a subject to which the pharmaceutical composition is administered.

[0034] As used herein, the terms "prevent," "prevention," and "preventing" refer to achieving a prophylactic effect in a subject to which a pharmaceutical composition is administered. In achieving a prophylactic effect, the goal is to delay, reduce the incidence of, or prevent the symptoms or underlying causes (e.g., bacterial infection) associated with a pathological condition or disorder. A "prophylactically effective amount" refers to an amount of a prophylactic agent, such as a bacteriophage or phage product, in a pharmaceutical composition of the invention sufficient to achieve at least one prophylactic effect in a subject to which the pharmaceutical composition is administered.

[0035] As used herein, the terms "antibacterial activity" and "antimicrobial activity" of a bacteriophage or bacteriophage product (e.g., a phage protein) or variant or fragment thereof are used interchangeably and refer to the ability of the bacteriophage to kill and / or inhibit the growth or reproduction of microorganisms, particularly bacteria of the species or strain it infects. In certain embodiments, antibacterial activity is assessed by culturing bacteria, e.g., Gram-negative bacteria (e.g., Pseudomonas aeruginosa or Klebsiella pneumoniae), according to standard techniques (e.g., in liquid culture or on agar plates), contacting the culture with a bacteriophage, phage protein or variant thereof, or a cocktail of bacteriophages, phage proteins or variants thereof of the invention, and monitoring cell growth after said contact. For example, in liquid culture, bacteria may be grown to an optical density ("OD") representing the midpoint in the exponential growth of the culture; the culture is exposed to one or more concentrations of one or more bacteriophages, bacteriophage products, or variants thereof of the invention, and the OD is monitored relative to a control culture. A decrease in OD relative to the control culture is indicative of phage(s) or phage product(s) exhibiting antimicrobial activity (e.g., lytic killing activity). Similarly, bacterial colonies may be allowed to form on agar plates, the plates exposed to one or more bacteriophages or phage products, or variants thereof, of the invention, and subsequent colony growth assessed relative to the control plate. A decrease in colony size or total number of colonies is indicative of phage(s) or phage product(s) having antimicrobial activity. [Brief explanation of the drawings]

[0036] 4. Brief description of the drawings The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Figure 1]Figure 1 illustrates the morphological features of bacteriophages Kle_F391 / 08 (A), Kle_F17 / 19 (B), and Kle_F58 / 19 (C). These bacteriophages were characterized using a transmission electron microscope (Hitachi H-7650). [Figure 2-1] ~ [Figure 2-2]

[0033] Figure 1 shows a diagram of the F391 / 08 genome. Predicted orfs in the approximately 113 kb genome are represented by arrows and numbered in black. The direction of the arrow indicates the direction of transcription. Color code: black - open reading frames (orfs) for which a functional assignment can be made to the product based on homologous proteins; gray - orfs encoding products similar to proteins of unknown function; white arrows - orfs encoding proteins that do not share significant homology with proteins in the database. [Figure 3] Figure 1 shows a diagram of the Kle_F17 / 19 genome organization. Predicted orfs in the approximately 45 kb genome are represented by arrows and numbered in black. The direction of the arrow indicates the direction of transcription. Color code: black - orfs for which a functional assignment can be made to the product based on homologous proteins; grey - orfs encoding products similar to proteins of unknown function; white arrows - orfs encoding proteins that do not share significant homology with proteins in the database. [Figure 4-1] ~ [Figure 4-2] Figure 1 shows a diagram of the Kle_F58 / 19 genome organization. Predicted orfs in the approximately 170 kb genome are represented by arrows and numbered in black. The direction of the arrow indicates the direction of transcription. Color code: black - orfs for which a functional assignment can be made to the product based on homologous proteins; grey - orfs encoding products similar to proteins of unknown function; white arrows - orfs encoding proteins that do not share significant homology with proteins in the database. [Figure 5]Figure 1 shows single lysis curves for Kle_F58 / 19 bacteriophages (MOI 10) on Kle pneumoniae 57 / 17. Viable cell counts were quantified by 10-fold serial dilutions and monitored at 1-hour intervals over an 8-hour period and again at 24 hours ppi. [Figure 6] Figure 10 depicts a single lysis curve for Kle_F17 / 19 bacteriophage (MOI 10) on Kle pneumoniae 237 / 14. Viable cell counts were quantified by 10-fold serial dilutions and monitored at 1-hour intervals over an 8-hour period and again at 24 hours ppi. [Figure 7] Figure 1 shows the combined lysis curves of F391 / 08, Kle_F17 / 19, and Kle_F58 / 19 bacteriophages with an MOI of approximately 10. Viable cell counts were quantified by 10-fold serial dilutions and monitored at 1-hour intervals over an 8-hour period and again at 24 hours ppi. [Figure 8] Figure 1 illustrates bacterial burden 24 hours after infection with strain 2 and nebulized purified phage cocktail / antibiotic (co-) or NaCl (mock) treatment. Bacterial burden is given as cfu per precision-cut lung slice. Bars represent the mean ± SD for two technical replicates in an n=1 experiment. [Figure 9] Figure 1 shows tissue viability 24 hours after infection with strain 2 and aerosolized purified phage cocktail / antibiotic (co-) or NaCl (mock) treatment. Tissue viability was assessed by calcein staining and is given as relative light units. Bars represent the mean ± SD for two technical replicates (each measured in duplicate) in an n=1 experiment. [Figure 10]Figure 1 shows phage titers for rat PCLS efficacy of nebulized bacteriophage treatment. Phage titers in PCLS at 1 and 24 hours pi were measured using a plaque assay with the indicated detection strains. Phage titers are given as plaque-forming units (PFU) per PCLS. Bars represent the mean ± SD of 2-4 technical replicates (1-2 exposure plates with 2 PCLS each, shown as individual points) for n=1 experiment. [Figure 11] Figure 1 shows bacterial load on rat PCLS efficacy of nebulized bacteriophage treatment. Bacterial load 1 or 24 hours after infection and subsequent treatment with nebulized phage cocktail or NaCl (vehicle). Bacterial load is given as CFU per PCLS. Bars represent the mean ± SD of four technical replicates (two exposure plates with two PCLS each, mean per plate shown as dots) for n=1 experiment. [Figure 12] Figure 1 shows phage titers for rat PCLS efficacy of nebulized bacteriophage treatment and antibiotic co-treatment (phage lysate). Phage titers were measured using a plaque formation assay at 1 and 24 hours after infection with strain 1 and treatment with the phage cocktail. Phage titers are given as plaque-forming units. Bars represent the mean ± SD of two technical replicates (shown as individual points) for n=3 experiments. [Figure 13]Figure 1 shows bacterial burden on rat PCLS efficacy of nebulized bacteriophage and antibiotic co-treatment. Bacterial burden 24 hours pi after treatment with nebulized NaCl or phage cocktail, alone or in combination with in-liquid antibiotic co-treatment. Bacterial burden is given as CFU per PCLS. Bars represent the mean ± SD of two technical replicates (shown as individual points) for n=3 experiments. *** indicates significance of p<0.001 compared to the control column (infection + NaCl 24 hours pi) according to one-way ANOVA and Sidak's multiple comparison post-hoc test. ### indicates significance of p<0.001 according to unpaired two-tailed t-test for direct comparison of the indicated groups. [Figure 14] Figure 1 shows tissue viability for rat PCLS efficacy of nebulized bacteriophage treatment and antibiotic co-treatment. Tissue viability 24 hours after infection and treatment with NaCl (vehicle), nebulized phage cocktail alone, or antibiotic co-treatment. Tissue viability was assessed by calcein staining and is given as relative light units (RLU). Bars represent the mean ± SD of two technical replicates (shown as individual points), each measured in duplicate, for n = 3 experiments. **; *** indicate significance of p<0.01; 0.001 compared to the control column (infection + NaCl 24 hours p.i.) according to one-way ANOVA and Sidak's multiple comparison post-hoc test. DETAILED DESCRIPTION OF THE INVENTION

[0037] 5. Detailed Description The present invention is directed to phage therapy for the treatment and control of bacterial infections, particularly respiratory bacterial infections such as those associated with bacterial pneumonia and cystic fibrosis. One aspect of the invention relates to novel bacteriophage strains, including Kle_F17 / 19 and Kle_F58 / 19, and variants thereof. Another aspect of the invention relates to a cocktail composition of one or more bacteriophages and / or phage products of the invention, as well as combinations with other phages, including Kle_F391 / 08 (previously disclosed in International Publication No. PCT / PT2011 / 000031). Another aspect of the invention relates to a cocktail composition of Kle_F391 / 08, Kle_F17 / 19, and Kle_F58 / 19, and variants thereof. Yet another aspect relates to pharmaceutical compositions of the phage(s) as well as methods for their use in the treatment and prevention of bacterial infections, particularly respiratory infections caused by Klebsiella pneumoniae. Yet another aspect of the invention relates to the use of phages and combinations thereof as diagnostic tools and bactericidal agents.

[0038] 5.1. Bacteriophages and their variants Another aspect of the present invention relates to novel Klebsiella bacteriophages that target several strains of Klebsiella pneumoniae. Klebsiella pneumoniae is a Gram-negative, non-motile, rod-shaped bacterium found in the normal flora of the mouth, skin, and intestines. As an encapsulated facultative anaerobe, it also naturally occurs in soil. Clinically, it is the most important member of the genus Klebsiella in the family Enterobacteriaceae. Klebsiella infections tend to occur in people with weakened immune systems due to an improper diet, such as alcoholics and diabetics. Klebsiella is also an opportunistic pathogen for patients with chronic lung disease, nasal atrophy, cystic fibrosis, and nasal sclerosis. New antibiotic-resistant strains of Klebsiella pneumoniae are emerging and are increasingly found as hospital-acquired infections, for example, due to contact with contaminated equipment.

[0039] Klebsiella pneumoniae is one of the most important causative pathogens of respiratory tract infections in humans, accounting for 25–43% of nosocomial pneumonia cases caused by Gram-negative bacteria alone (Chibber S et al., 2008, J Med Microbiol 57(12): 1508–1513). The high prevalence of multidrug-resistant bacteria limits the efficacy of current antibiotics and increases the likelihood of colonization by resistant strains in patients. Capsular polysaccharides are an important virulence factor for Klebsiella species and a limiting factor for phage infection. 78 capsular types have been described in the literature (Hus CR, et al., 2013, PLoS One 8(8):e70092), and phages that infect these species overcome this barrier. Virulent strains of K. pneumoniae, such as those in pyogenic liver abscesses, are primarily associated with the K1 and K2 capsular serotypes (Cleg S et al., 2016, Microbiol Spectr 4(1); and Lin TZ et al., 2014, J Infect Dis 210:1734-1744), although the K1 capsular serotype is associated with community-acquired isolates rather than nosocomial isolates (Tsay RW et al., 2002, Arch Intern Med 162(9): 1021-1027). Nevertheless, depending on the type of infection, strains may display a diverse range of capsular serotypes, and the distribution of K. pneumoniae capsular serotypes varies worldwide (Hus CR et al., 2013, PLoS One 8(8):e70092).

[0040] In one embodiment, purified bacteriophage F17 / 19 (name used interchangeably with "Kle_F17 / 19") is provided that targets several strains of Klebsiella pneumoniae.

[0041] In another embodiment, purified bacteriophage F58 / 19 (name used interchangeably with "Kle_F58 / 19") is provided, which also targets several Kle pneumoniae strains.

[0042] In certain embodiments, a bacteriophage of the invention comprises or consists of a genome having at least 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO:2 or SEQ ID NO:3, 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 bacteriophages F17 / 19 and F58 / 19.

[0043] The bacteriophages and certain bacterial strains disclosed herein were deposited with NCIMB Ltd., Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, UK on December 13, 2019.

[0044] [Table 1]

[0045] 5.2. Cocktail Composition Certain aspects of the present invention relate to cocktail compositions of different bacteriophages. A "cocktail" can include at least two different purified bacteriophages, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or more different purified bacteriophages or variants thereof. The cocktail may be used alone or in further combination with other therapies, such as antibiotics and / or antifungals.

[0046] Phage cocktails offer advantages over the use of individual phages, such as increased lytic activity against specific species or strains of bacteria and / or reduced likelihood of emergence of bacteria resistant to individual bacteriophages. Also, different bacteriophages can be mixed in cocktails to broaden their properties, preferably resulting in a collectively greater spectrum of antimicrobial activity. However, few phage cocktails have antimicrobial activity against different bacteria, likely due to the difficulty of combining bacteriophage strains of different specificities while maintaining the infectivity and / or lytic activity of individual bacteriophages in the presence of distinct strains.

[0047] In some embodiments, the present invention provides a cocktail composition comprising at least three different purified bacteriophages having antibacterial activity against the same or different bacterial species or strains. In certain embodiments, the present invention provides a cocktail composition comprising or consisting of purified bacteriophages Kle_F391 / 08, Kle_F17 / 19, and Kle_F58 / 19, or variants thereof, including variants having lytic activity against all of Klebsiella pneumoniae 57 / 17, 237 / 14, and / or 397 / 07.

[0048] In certain embodiments, compositions are provided that comprise or consist of at least two different purified bacteriophages, including at least one of F17 / 19 or F58 / 19 or variants thereof.

[0049] In some preferred embodiments, the combination does not impair or reduce (or does not substantially or significantly impair or reduce) the infectivity or host range and / or lytic activity of the individual bacteriophages in the presence of the separate bacteriophage strains. In certain preferred embodiments, the efficacy of at least one phage in the cocktail combination is enhanced or improved due to the presence of at least one other phage in the cocktail combination, resulting in a synergistic effect.

[0050] In some embodiments, the cocktail composition comprises at least three phages that exhibit antibacterial activity against Klebsiella pneumoniae. In certain embodiments, the present invention provides a cocktail composition comprising at least three different purified bacteriophages, F391 / 08, F17 / 19, and F58 / 19, or consisting of the three purified bacteriophages, F391 / 08, F17 / 19, and F58 / 19, or variants thereof.

[0051] In another particularly preferred embodiment, the composition comprises or consists of purified bacteriophages F391 / 08, F17 / 19, and F58 / 19 or variants thereof, and three purified bacteriophages F99 / 10, F27 / 12, and F95 / 13 that have antibacterial activity against Pseudomonas aeruginosa. Alternatively, the composition comprising or consisting of purified bacteriophages F391 / 08, F17 / 19, and F58 / 19 is administered in combination with a composition comprising or consisting of purified bacteriophages F99 / 10, F27 / 12, and F95 / 13. F99 / 10, F27 / 12, and F95 / 13 are disclosed in U.S. Patent Application Publication No. 2019 / 0290709. These bacteriophages and certain P. aeruginosa strains were deposited with NCIMB Ltd. (Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, UK) on December 1, 2017.

[0052] [Table 2]

[0053] In some embodiments, a cocktail composition is provided that includes at least two different purified bacteriophages against one or more strains of Klebsiella species, more preferably strains including Klebsiella pneumoniae, one of which is either F17 / 19 or F58 / 19, and at least one selected from purified bacteriophages F92 / 15, F105 / 15, F134 / 15, and F141 / 15, all of which are disclosed in U.S. Patent Application Publication No. 2019 / 0290709.

[0054] In some embodiments, the present invention provides bacteriophage F168 / 08 (disclosed in WO 2010 / 090542) having antibiotic activity against one or more strains of E. faecalis and / or E. faecium, bacteriophage F170 / 08 (disclosed in WO 2010 / 090542) having antibiotic activity against one or more strains of E. faecalis and / or E. faecium, bacteriophage F770 / 05 (disclosed in WO 2010 / 090542) having antibacterial activity against one or more strains of Pseudomonas aeruginosa, bacteriophage F197 / 08 (disclosed in WO 2010 / 090542) having antibacterial activity against one or more strains of Staphylococcus aureus ... bacteriophage F86 / 06 (disclosed in WO 2010 / 090542) having antibacterial activity against one or more strains of Staphylococcus aureus; bacteriophage F87s / 06 (disclosed in WO 2010 / 090542) having antibacterial activity against one or more strains of Staphylococcus aureus; bacteriophage F91a / 06 (disclosed in WO 2010 / 090542) having antibacterial activity against one or more strains of Staphylococcus aureus; Acinetobacter baumannii bacteriophage F1245 / 05 (disclosed in WO 2010 / 090542) having antibacterial activity against one or more strains of Acinetobacter baumannii; bacteriophage strain F394 / 08 (disclosed in WO 2012 / 036580) having antibacterial activity against one or more strains of Acinetobacter baumannii;bacteriophage F488 / 08 (disclosed in WO 2012 / 036580) having antibacterial activity against one or more strains of Bacillus subtilis (Bacteroides aeruginosa), bacteriophage F510 / 08 (disclosed in WO 2012 / 036580) having antibacterial activity against one or more strains of Pseudomonas aeruginosa, bacteriophage F44 / 10 (disclosed in WO 2012 / 036580) having antibacterial activity against one or more strains of Staphylococcus aureus, Provided is a cocktail composition comprising at least F17 / 19 or F58 / 19 further combined with at least one additional phage selected from the group consisting of bacteriophage F387 / 08 (disclosed in WO 2012 / 036580) having antibacterial activity against one or more strains of Klebsiella pneumoniae, and bacteriophage F125 / 10 (disclosed in WO 2012 / 036580) having antibacterial activity against one or more strains of Staphylococcus aureus.

[0055] Bacteriophages of the invention and / or for use in the cocktail compositions of the invention can be obtained by any method known in the art and / or any method disclosed herein. In some embodiments, the invention provides methods for the production and purification of bacteriophages F391 / 08, F17 / 19, and / or F58 / 19, e.g., from the deposited strains disclosed herein.

[0056] Furthermore, bacteriophages can be isolated from bacterial samples using any method described herein or known in the art (see, e.g., Carlson, "Working with bacteriophages: common techniques and methodological approaches," in Kutter and Sulakvelidze (Eds) Bacteriophages: Biology and Applications, 5th ed. CRC Press (2005)). Particular bacterial strains that can be used include, for example, Klebsiella pneumoniae strains 57 / 17, 397 / 07 or 237 / 14 (for example, to isolate phages F391 / 08, F17 / 19 or F58 / 19), Pseudomonas aeruginosa strains 391 / 08, 92 / 15, 105 / 115 or 121 / 15 (for example, to isolate phages F99 / 10, F110 / 10, F27 / 12, F83 / 13 and / or F95 / 13); or bacteriophages can also be isolated from any other bacterial strain that is susceptible to infection by one or more of the bacteriophages and in which the bacteriophage replicates.

[0057] Also, one skilled in the art may use one or more methods for propagating or amplifying bacteriophages, particularly purified bacteriophages F391 / 08, F17 / 19, or F58 / 19, as well as variants thereof, to obtain larger quantities of a given phage. In some embodiments, a method for producing and / or isolating additional phages may include (i) obtaining a culture of K. pneumoniae, (ii) infecting it with bacteriophage F391 / 08, F17 / 19, or F58 / 19, or variants thereof, (iii) culturing until significant lysis of the culture is observed, and (iv) isolating the bacteriophage from the culture. The host cells used may be any bacterial strain, for example, any K. pneumoniae strain that is susceptible to infection by phages and that can be used to replicate phages. In some embodiments, the host cells used may be, for example, Klebsiella pneumoniae strain 57 / 17, Klebsiella pneumoniae strain 237 / 14, or Klebsiella pneumoniae strain 397 / 07.

[0058] 5.3. Pharmaceutical Compositions The purified bacteriophages and variants thereof and phage cocktail compositions disclosed herein can be administered alone or incorporated into pharmaceutical compositions for use in the treatment or prevention of bacterial infections, for example, infections caused by bacteria, including, but not limited to, Pseudomonas aeruginosa and Klebsiella pneumoniae. The bacteriophage(s) or phage product(s) 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 phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low-molecular-weight polypeptides; proteins such as serum albumin and gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; 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®, polyethylene glycol (PEG), and PLUONICS®. In addition to the above-mentioned components, pharmaceutical compositions (e.g., antibacterial compositions) of the present invention may also contain, for example, lubricants, wetting agents, emulsifiers, suspending agents, and preservatives. In a specific embodiment, purified bacteriophages are formulated in an NaCl solution, e.g., 0.9% NaCl.

[0059] In some embodiments, the pharmaceutical composition is formulated for administration as an aerosol. Formulations for aerosol delivery can be in the form of dry powders, microparticles, nanoparticles, solutions, lyophilized preparations, liposomal preparations, and the like. Liposomal formulations protect the bacteriophage from the harsh conditions of sputum, allowing for improved penetration into biofilms and / or more sustained drug release within the respiratory tract. Formulations for aerosol delivery typically contain sterile water and little or no preservatives to reduce side effects such as bronchial irritation and bronchospasm. Formulations for aerosol delivery preferably have an osmolarity that is the same as or substantially the same as the osmolarity of the airway surface liquid.

[0060] The phage and bacteriophage cocktails disclosed herein may be combined with one or more other therapeutic and / or prophylactic agents useful for treating bacterial infections described herein and / or known in the art, such as one or more other bacteriophages or antibiotics. For example, a pharmaceutical composition of the invention may include two or more purified bacteriophages disclosed herein (having antibacterial activity against the same or different bacterial species or strains) and a bacteriophage known in the art. In specific embodiments, the therapeutic components of the combination target two or more species or strains of bacteria.

[0061] The pharmaceutical compositions of the present invention may also be combined with one or more non-phage therapeutic and / or prophylactic agents useful for treating and / or preventing bacterial infections, as described herein, and / or one or more non-phage therapeutic and / or prophylactic agents known in the art (e.g., one or more traditional antibiotics) useful for treating and / or preventing bacterial infections. Other therapeutic and / or prophylactic agents that may be used in combination with the phage(s) or phage product(s) of the present invention include, but are not limited to, antibiotics, anti-inflammatory agents, antiviral agents, antifungal agents, or local anesthetics. In some preferred embodiments, the pharmaceutical compositions are formulated for the treatment and / or prevention of pulmonary infections and include one or more additional therapeutic and / or prophylactic agents selected from antibiotics, antifungal agents, and local anesthetics. In some embodiments, the pharmaceutical compositions comprise the phage cocktail combinations of the present invention administered in the absence of standard or traditional antibiotics.

[0062] In certain embodiments, the bacteriophage cocktail is administered in combination with a broad-spectrum antibiotic with activity against Gram-negative bacteria, such as an anti-pseudomonal β-lactam (e.g., piperacillin, tazobactam, ceftazidime, or meropenem) and / or an aminoglycoside (e.g., amikacin), and / or an antibiotic against Gram-positive bacteria, such as vancomycin or an oxazolidinone (e.g., linezolid) and / or colistin. Standard or traditional antibiotics include amikacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin, streptomycin, tobramycin, apramycin, rifamycin, naphthomycin, mupirocin, geldanamycin, ansamitocin, carbacephems, imipenem, meropenem, ertapenem, faropenem, doripenem, panipenem / betamipron, bipenem, bisphosphonate ... Apenem, PZ-601, cephalosporin, cephacetrile, cefadroxil, cephalexin, cephaloglycin, cephalonium, cephaloridine, cephalothin, cephapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cephradine, cefroxadine, ceftezole, cefaclor, cefonicid, cefprozil, cefuroxime, cefuzonam, cefmetazole, cefotetan, cefoxitin , cefcapene, cefdaloxime, cefdinir, cefditoren, cefetamet, cefixime, cefmenoxime, cefteram, ceftibuten, ceftiofur, ceftiolene, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime latamoxef, cefclidine, cefepime, cefluprenam, cefoselis, cefozopran, cefpirome, cefquinome, flomoxef, ceftobiprole, Azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, aztreonam, penicillin and penicillin derivatives, actinomycin, bacitracin, colistin, polymyxin B, cinoxacin, flumequine, nalidixic acid, oxolinic acid, piromidic acid, pipemidic acid, losoxacin, ciprofloxacin, enoxacin, fleroxacin, lomefloxacin,Nadifloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin, gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin, pazufloxacin, sparfloxacin, temafloxacin, tosufloxacin, clinafloxacin, garenoxacin, gemifloxacin, sitafloxacin, trovafloxacin, prulifloxacin, acetazolamide, benzolamide, bumetanide, celecoxib, chlorthalidone, clopamide, diclofenamide, dorzolamide, ethoxazolamide, furosemide, hydrochlorothiazide, indapamide, mafenide (mafendide), mefruside, metolazone, probenecid, sulfacetamide, sulfadimethoxine, sulfadoxine, sulfanilamide, sulfamethoxazole, sulfasalazine, sulthiame, sumatriptan, xipamide, tetracycline, chlortetracycline, oxytetracycline, doxycycline, lymecycline, meclocycline, methacycline, minocycline, rolitetracycline, methicillin, nafcillin, oxacillin, cloxacillin, vancomycin, teicoplanin, clindamycin, cotrimoxazole, flucloxacillin, dicloxacillin, ampicillin, amoxicillin, and any combination thereof.

[0063] In some embodiments, pharmaceutical compositions of the invention comprise an antibiotic agent having antibacterial activity against Pseudomonas aeruginosa and / or Klebsiella pneumoniae. In some other embodiments, pharmaceutical compositions of the invention comprise an antibiotic agent having antibacterial activity against bacteria other than Pseudomonas aeruginosa and / or Klebsiella pneumoniae. In preferred embodiments, the antibiotic agent is used in an amount effective to additively or synergistically enhance the therapeutic and / or prophylactic effect of the phage, phage product, or phage cocktail of the invention for a given infection.

[0064] Standard antifungal agents include amphotericin B, such as liposomal amphotericin B and nonliposomal amphotericin B.

[0065] In some preferred embodiments, the pharmaceutical compositions of the present invention are formulated for administration as an aerosol and further comprise one or more antibiotics for aerosol delivery, including, for example, inhaled aminoglycosides such as tobramycin, such as tobramycin solution or tobramycin dry powder, gentamicin, and amikacin; inhaled polymyxins such as colistin solution or colistin dry powder and colistimethate sodium; and inhaled monobactams such as aztreonam solution or nebulized aztreonam lysine; as well as aerosolized levofloxacin, ceftazidime, fosfomycin, gentamicin, vancomycin, amphotericin, capreomycin, fifampin, isoniazid, and ciprofloxacin (Quon BS et al., 2014, Annals ATS 11(3):425-434). In some embodiments, the aerosolized pharmaceutical compositions of the present invention also further comprise one or more antifungal agents, such as liposomal amphotericin B, for aerosol delivery.

[0066] In some embodiments, pharmaceutical compositions of the present invention are formulated for use in treating and / or preventing bacterial infections caused by Klebsiella species, such as Klebsiella pneumoniae. In some such embodiments, the pharmaceutical composition comprises a cocktail composition containing one or more purified bacteriophages of F17 / 19 or F58 / 19 and, optionally, F391 / 08 or variants thereof, and in certain embodiments, comprises or consists of F17 / 19, F58 / 19, and F391 / 08 or variants thereof. The composition may further comprise or consist of purified bacteriophages F99 / 10, F27 / 12, and F95 / 13 or variants thereof that have activity against Pseudomonas aeruginosa. In some embodiments, the pharmaceutical composition may further comprise an additional agent, for example, an antibiotic with antibacterial activity against Klebsiella pneumoniae and / or Pseudomonas aeruginosa and / or an antibiotic with antibacterial activity against bacteria other than Klebsiella pneumoniae or Pseudomonas aeruginosa. In some embodiments, the composition is formulated in a dosage form in which the bacteriophage is present in an amount that provides a multiplicity of infection (MOI) of about 1 to about 10 upon administration of the composition to a subject in need thereof. In certain embodiments, each phage is present in an amount that provides a multiplicity of infection (MOI) of about 1 to about 10. 9 pfu~10 10 pfu, for example, in a composition containing 10 9 pfu / ml In a preferred embodiment, the composition is formulated for administration as an aerosol.

[0067] Pharmaceutical compositions containing the purified bacteriophage cocktail of the present invention can be formulated in unit dose or multi-dose formulations. Preferred formulations are those that can be delivered as an aerosol, as discussed above. Other suitable formulations include suspensions, emulsions, lotions, solutions, creams, ointments or dusting powders, or in skin patches.

[0068] Additionally or alternatively, the pharmaceutical compositions provided herein may be administered orally in the form of a suppository or pessary (e.g., as tablets which may contain excipients such as starch or lactose, or as capsules, ovules, elixirs, solutions, or suspensions, each of which may contain flavorings, colorings, 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 which may be formulated in a traditional manner. Topical formulations generally contain a sterile buffer such as sterile PBS, water, or buffered saline, or sterile SM buffer.

[0069] Using the modes of administration described herein and / or known in the art, a desired dose of the phage, phage product, and / or phage cocktail of the present invention can be delivered according to an appropriate dosage regimen. Dosage and dosage regimens may vary depending on the specific formulation, route of administration, condition being treated, and other factors. Animal experiments, for example, may provide reliable guidance for determining effective doses in human treatment, within the skill of an ordinary physician. Scaling of effective doses between species can be performed by those skilled in the art, for example, following the principles described by Mordenti, J. et al., "The use of interspecies scaling in toxicokinetics," in Toxicokinetics and New Drug Development, Yacobi et al., Eds., Pergamon Press, New York, 1989, pp. 42-96. For example, the efficacy of the pharmaceutical compositions of the present invention can be assessed using a mouse model of acute pulmonary infection, as detailed in the Examples below.

[0070] The pharmaceutical compositions of the present invention can be administered according to a dosage regimen. In some embodiments, the dosage regimen includes administration of the cocktail composition of the present invention every 6 or 8 hours (e.g., a multi-dose regimen of topical phage cocktail for diabetic skin wounds (Mendes JJ, et al., 2013, Wound Repair Regen 21:595-603)). In a preferred embodiment, the initial administration is followed by a second or "booster" dose, including a re-administration of the pharmaceutical composition. For example, the booster can follow the initial dose about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 10 hours, 12 hours, 1 day, or 2 days later. In a preferred embodiment, in treating respiratory or pulmonary infections, including, but not limited to, hospital-acquired bacterial pneumonia (HABP), the booster dose is used about 4, about 5, about 6 hours, or about 8 hours after the initial dose.

[0071] 5.4. Therapeutic Use Another aspect relates to the use of purified bacteriophages and bacteriophage cocktails in pharmaceutical compositions for preventing and / or treating bacterial infections. Phages show great potential for treating bacterial infections due to their specificity and effectiveness in lysing pathogenic bacteria, including those associated with multidrug resistance (Larche J, et al., 2012, Antimicrob Agents Chemother 56(12):6175-6180), their potential effectiveness against bacteria in biofilms (Phee A et al., 2013, J Endod 39(3):364-369); their lack of pathogenicity to human and animal cells (Abedon ST et al., 2011, Bacteriophage l(2):66-85), and their activity in microaerobic environments even with high bacterial loads (Azeredo J, et al. 2008. Curr Pharm Biotechnol 9:261-266). In particular, phage cocktails may offer additional advantages over the use of individual phages, such as increased lytic activity against specific bacterial strains, increased host range, and / or reduced likelihood of bacterial resistance emerging for individual bacteriophages. Indeed, different bacteriophages are mixed as a cocktail to broaden their properties, preferably resulting in a collectively greater antibacterial spectrum, such as an extended host range, which makes the development of resistance less likely in subjects receiving the agent.

[0072] In specific embodiments, the subject to which the pharmaceutical composition of the invention is administered is a mammal (e.g., a cow, sheep, goat, horse, primate (e.g., a human), rodent, lagomorph, or bird (e.g., a chicken, duck, or goose)). In preferred embodiments, the subject to which the pharmaceutical composition of the invention is administered is a human, particularly a patient suffering from or at risk of suffering from a respiratory or pulmonary infection, including hospital-acquired bacterial pneumonia (HABP), ventilator-acquired pneumonia (VAP), and healthcare-associated pneumonia (HCAP), or a cystic fibrosis-associated infection. In certain embodiments, the infection is caused by an MDR, XDR, or PDR infectious agent.

[0073] In preferred embodiments, pharmaceutical compositions of the invention have activity against multiple bacterial strains. In some preferred embodiments, pharmaceutical compositions comprise phage cocktail combinations that have activity against multiple strains of Pseudomonas aeruginosa and / or Klebsiella pneumoniae. Accordingly, the invention provides methods for treating and / or preventing infections associated with Pseudomonas aeruginosa and / or Klebsiella pneumoniae in both humans and animals using the phages, phage products, or phage cocktail compositions of the invention. In other aspects, the invention provides methods for treating and / or preventing infections associated with related species or strains of these bacteria.

[0074] Pseudomonas aeruginosa and Klebsiella pneumoniae are responsible for many severe opportunistic infections, particularly in immunocompromised individuals. The pharmaceutical compositions of the present invention are intended for treating and / or preventing any infection associated with Pseudomonas aeruginosa and / or Klebsiella pneumoniae or associated with other bacterial species or strains, including, but not limited to, infections of the lungs and respiratory tract, post-operative infections, infections associated with catheters and surgical drains, and blood infections. In a preferred embodiment, the pharmaceutical compositions of the present invention find use in treating and / or preventing bacterial infections associated with the lungs and respiratory tract.

[0075] Respiratory and pulmonary infections include, but are not limited to, infections associated with cystic fibrosis, such as cystic fibrosis bronchiectasis; pneumonia, including hospital-acquired bacterial pneumonia, ventilator-associated pneumonia, and bronchopneumonia; non-cystic fibrosis bronchiectasis; bronchitis; chronic obstructive pulmonary disease; mycobacteriosis, post-lung transplant infection; infections associated with tuberculosis; empyema with chest wall fistula; pleuritis with fistula, lung abscess; rhinitis; suppurative cyst; and pulmonary sepsis. Symptoms of respiratory or pulmonary infection include, for example, coughing, wheezing, sputum production, dyspnea (difficulty breathing), dysphonia (difficulty speaking), and an overall decrease in quality of life. In a particularly preferred embodiment, the respiratory or pulmonary infection is hospital-acquired bacterial pneumonia (HABP).

[0076] For HABP, the time of onset during hospitalization is an indicator of risk for specific pathogens and outcomes. In early-onset cases, e.g., within the first 4 days of hospitalization, the most frequent pathogens are antibiotic-susceptible endogenous microflora, such as gram-negative and community-acquired Staphylococcus aureus, as well as Streptococcus pneumoniae and Haemophilus influenzae. In late-onset cases, e.g., cases occurring more than 5 days after hospitalization, gram-negative bacteria account for the majority of cases, many of which, including Pseudomonas aeruginosa, Klebsiella pneumoniae, certain strains of Enterobacter species, and certain strains of Acinetobacter species, as well as certain Staphylococcus aureus infections, are antibiotic-resistant, particularly in neurosurgical patients, diabetic patients, and patients with chronic renal impairment (2005, Am J Respir Crit Care Med Vol 171(4):388:416). Strains of Pseudomonas aeruginosa and Klebsiella pneumoniae, among others, are associated with late-onset HABP.

[0077] Klebsiella pneumoniae and Pseudomonas aeruginosa are also associated with infections involving other organ systems with high fluid content, and it is contemplated that the phage cocktails of the present invention have therapeutic and / or prophylactic use for such infections. For example, pharmaceutical compositions of the present invention may be used to prevent or treat infections of the cerebrospinal fluid, ascites, and urinary tract.

[0078] In some embodiments, the present invention provides a method for treating and / or preventing a respiratory or pulmonary infection, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a pharmaceutical composition of the invention. In preferred embodiments, administration results in improved breathing, e.g., returning labored or rapid breathing to normal.

[0079] In particularly preferred embodiments, the present invention provides methods for treating a surprising range of Klebsiella pneumoniae, and in certain embodiments, Pseudomonas aeruginosa, bacterial strains using phage cocktails of the invention. For example, a phage cocktail comprising Klebsiella pneumoniae phages F391 / 08, F17 / 19, and F58 / 19, for example, exhibits efficacy against a highly diverse range of clinical Klebsiella pneumoniae strains (53% infections) when compared to congener Klebsiella pneumoniae phages. In other embodiments, a phage cocktail further comprising Pseudomonas aeruginosa phages F99 / 10, F27 / 12, and F95 / 13, for example, exhibits efficacy against a highly diverse range of clinical Pseudomonas aeruginosa strains exhibiting heterogeneous capsular serotypes (57%) when compared to certain other Pseudomonas aeruginosa phages.

[0080] In a preferred embodiment, administration involves administering the pharmaceutical composition via aerosol to one or more respiratory tracts of a subject, e.g., via inhalation. Administration via inhalation can improve drug delivery to the target site of infection (i.e., the respiratory tract) and / or limit the potential for systemic side effects. Administration of the pharmaceutical composition as an aerosol includes, but is not limited to, administration via inhalation, intranasal instillation, tracheal catheterization, delivery to the pleural cavity of the lung, or bronchoscopy (Abedon ST, 2015, Bacteriophage, 5(1):el020260-1 to el020260-13). During administration of the pharmaceutical composition as an aerosol, the bacteriophage may remain viable and be contained within particles of an appropriate size to reach the lower respiratory tract. For example, in particularly preferred embodiments, the majority of the aerosolized particles are less than 5 μm in diameter, for example, at least 50%, 60%, 70%, or 80% of the particles are less than 5 μm in diameter, more preferably about 2 μm in diameter.

[0081] For intranasal or inhalation administration, the bacteriophages and / or phage products of the invention can be delivered in the form of dry powders, microparticles, nanoparticles, solutions, lyophilized preparations, liposomal preparations, and the like. Typically, formulations containing the phages, phage products, and / or phage cocktails of the invention are in the form of a dry powder inhalant or aerosol spray delivered from a pressurized container, pump, spray, or nebulizer with the use of a suitable propellant, e.g., a hydrofluoroalkane such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, 1,1,1,2-tetrafluoroethane (HFA 134A™) or 1,1,1,2,3,3,3-heptafluoropropane (FIFA 227EA™), carbon dioxide, or other suitable gas.

[0082] In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. The pressurized container, pump, spray, or nebulizer can contain a solution or suspension of the active compound using a mixture of ethanol as the solvent and a propellant, for example, which can additionally contain a lubricant, for example, sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator can be formulated to contain a powder mix of the phage, phage product, and / or phage cocktail of the invention and a suitable powder base such as lactose or starch.

[0083] Nebulization can be achieved using any means known in the art or described herein. Typically, nebulization is achieved by a jet nebulizer, which uses air or oxygen under high pressure to generate an aerosol. Other nebulizers include vibrating mesh nebulizers driven by piezoelectric actuators, which reduce size variability and reduce nebulization time (see, e.g., Aeroneb, Nebutec 4, Nebutec 6). Another approach involves mechanical ventilation, in which a nebulizer is connected to the inspiratory limb of the ventilator circuit. Yet another approach uses emulsion-based "spray-drying" to convert a solution or emulsion from a fluid state into microparticles with a uniform size distribution (approximately 1-5 μm). Two typical classes of nebulizers include AeroEclips (Trudell Medical International), a jet nebulizer in which atomization occurs only when the patient inhales; and Omron (Omron, MicroAir U22), a battery-powered mesh nebulizer that relies on the vibration of piezoelectric crystals to force the drug through a fine mesh and create an aerosol (Sahota et al., 2015, J. Aerosol Medicine and Pulmonary Drug Delivery 28(0): 1-8). In some preferred embodiments, the SYSTAM L290 (SYSTAM, Villeneuve-Sur-Lot, France) nebulizer is used. This nebulizer produces an ultrasonic aerosol in which approximately 70% of the particles have a diameter of less than 5 μm.

[0084] The pharmaceutical compositions of the invention will comprise a therapeutically and / or prophylactically effective amount of one or more of the phages or phage products described herein. A therapeutically and / or prophylactically effective amount refers to the amount necessary to provide a therapeutic and / or prophylactic effect, respectively, in the subject to which it is administered. The therapeutically and / or prophylactically effective amount will depend on the particular formulation, the route of administration, the condition being treated, whether other drugs or treatments are used in combination with the methods of the invention, and other factors.

[0085] In some embodiments, pharmaceutical compositions are delivered to a subject in need thereof to provide one or more bacteriophages in an amount corresponding to a multiplicity of infection (MOI) of about 1 to about 10. The MOI is determined by assessing the approximate bacterial burden in the lungs, or calculating the bacterial burden in the lungs of a particular patient, or using estimates for a given type of respiratory infection; then providing the phage in an amount calculated to provide the desired MOI (e.g., 2 x 10 pfu / g of lungs provides an MOI of 10). The MOI may be selected based on the "rule of 10," which indicates that bacterial density is significantly reduced when an average of 10 phages are absorbed per bacterium (Abedon ST, 2009, Foodborne Pathog Dis 6:807-815; and Kasman LM, et al., 2002, J Virol 76:5557-5564); whereas lower titer phage administration (e.g., using an MOI less than 10) is less likely to be successful (Goode D, et al., 2003, App Environ Microbiol 69:5032-5036; Kumari S, et al., 2010, J Infect Dev Ctries 4:367-377).

[0086] In some preferred embodiments, a phage cocktail comprising F391 / 08, F17 / 19, and F58 / 19, delivered to provide an MOI of 1-10 of each phage, results in about an 80%, 85%, 95%, 97%, 98%, or nearly about a 100% reduction (viable cell count reduction to 0) of Klebsiella pneumoniae in the lungs. In some preferred embodiments, a phage cocktail further comprising (or administered in combination with) purified bacteriophages F99 / 10, F27 / 12, and F95 / 13, delivered to provide an MOI of 1-10 of each phage, results in about an 80%, 85%, 95%, 97%, 98%, or nearly about a 100% reduction of Pseudomonas aeruginosa in the lungs. In some particularly preferred embodiments, the phage cocktail surprisingly exhibits synergistic bacteriolytic activity compared to each bacteriophage by itself.

[0087] In some embodiments, lower doses may surprisingly provide advantages over higher doses. For example, in some embodiments, an MOI of 1 or about 1 maintains lower levels of bacteria in the lungs for a longer period of time than an MOI of 10 or about 10. For example, lower MOIs of F391 / 08, F17 / 19, and F58 / 19 may achieve lower Klebsiella pneumoniae loads, and lower MOIs of F99 / 10, F110 / 10, and / or F27 / 12 may achieve lower Pseudomonas aeruginosa loads in the lungs of infected animals for longer periods of time after treatment, e.g., 12, 15, 18, 24, 30, 36, or more hours after treatment. Without being bound by theory, this may be due to a delayed emergence of bacterial resistance in response to lower doses of phage.

[0088] In some other embodiments, an MOI as low as about 0.2-0.4 can result in efficacy, e.g., a statistically significant reduction in the amount of Klebsiella pneumoniae and / or Pseudomonas aeruginosa in the lungs of infected animals. Without being bound by theory, efficacy may be due to active therapy. That is, a phage dose of 10 provides phage that sufficiently exceed the target bacterial population to reduce bacterial burden without the need for phage replication or life cycle completion. Lower phage doses may rely on active therapies involving phage infection / replication cycles to reduce target bacteria (Loc Carrillo C, et al., 2005, Appl Environ Microbiol 71:6554-6563; see also Cairns BJ, et al., 2009, PLoS Pathog 5:el000253; and Hooton SP, et al., 2011, Int J Food Microbiol 151:157-163).

[0089] In certain embodiments, the purified bacteriophage or bacteriophage cocktail compositions of the present invention are used as a single agent to treat or prevent infections caused by Pseudomonas aeruginosa and / or Klebsiella pneumoniae, such as respiratory or pulmonary infections. In other embodiments, the bacteriophages or bacteriophage cocktails disclosed herein are further used in combination with other agents, including standard antibiotics targeting the same or different types of bacteria, including 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 means of treating bacterial infections, particularly respiratory infections, known to those of skill in the art.

[0090] In some particularly preferred embodiments, the present invention provides methods of treating and / or preventing respiratory or pulmonary infections, comprising administering a phage cocktail of the present invention in combination with standard and / or non-standard treatments. Standard treatments for respiratory infections include inhaled and / or systemic antibiotics such as tobramycin, amikacin, colistin, aztreonam, as well as levofloxacin, ceftazidime, fosfomycin, gentamicin, vancomycin, amphotericin, capreomycin, fifampin, isoniazid, and ciprofloxacin; and inhaled and / or systemic antifungal agents such as amphotericin B.

[0091] In some embodiments, the phage, phage product, or phage cocktail composition of the invention is administered as an aerosol, while the additional agent is administered systemically. For example, in some preferred embodiments, the phage cocktail composition of the invention is administered by inhalation, while the antibiotic, such as an antibiotic with activity against Pseudomonas aeruginosa and / or Klebsiella pneumoniae, is administered systemically. In some embodiments, the phage cocktail composition of the invention is administered by inhalation along with the additional agent, which is also administered as an aerosol. For example, in some preferred embodiments, the phage cocktail pharmaceutical composition of the invention is administered as an aerosol to the lung along with another antibiotic or antifungal agent.

[0092] In some embodiments, the present invention provides methods of treating and / or preventing a respiratory or pulmonary infection, comprising administering a phage, phage product, or phage cocktail composition of the present invention in combination with a non-standard treatment for the respiratory infection, which is generally used when the respiratory infection is refractory to one or more standard treatments.

[0093] 5.5. Use of disinfectants and anti-infectives Bacterial pathogens most frequently infect mucous membranes (e.g., through the mucous membranes of the upper or lower respiratory tract, the urogenital system, the ocular structures, and the like). The mucous membranes themselves are often the reservoir, sometimes the only reservoir, for pathogenic bacteria found in the environment. Although there are very few anti-infective drugs designed to control this reservoir of pathogenic bacteria, studies have shown that reducing or eliminating this reservoir significantly reduces the incidence of infection, especially in settings such as hospitals and nursing homes.

[0094] The phages, phage products, and phage cocktails of the present invention can be used in anti-infective compositions to control the growth of bacteria, particularly Klebsiella pneumoniae and Pseudomonas aeruginosa, to prevent or reduce the incidence of hospital-acquired infections. The anti-infective compositions find use in reducing or inhibiting bacterial colonization or growth on surfaces that come into contact with the bacteria. The phages, phage products, and phage cocktails of the present invention can be incorporated into compositions formulated for application to biological surfaces, such as skin and mucous membranes, as well as to non-biological surfaces.

[0095] Anti-infective formulations for use on biological surfaces include, but are not limited to, gels, creams, ointments, sprays, and the like. In certain embodiments, the anti-infective formulations are used to sterilize surgical fields or the hands and / or exposed skin of healthcare workers and / or patients. In preferred embodiments, the biological surface is a mammalian mucosa, more preferably a human mucosa. In particularly preferred embodiments, the biological surface is a mucosa of the respiratory tract, such as the nasal mucosa, pharynx, larynx, trachea, bronchi, and / or the lining of the lungs.

[0096] Anti-infective formulations for use on non-living surfaces include sprays, solutions, suspensions, wipes impregnated with the solutions or suspensions, and the like. In certain embodiments, the anti-infective formulations are used on solid surfaces in hospitals, nursing homes, ambulances, and the like, including, for example, instruments, countertops, and medical devices, hospital equipment. In preferred embodiments, the non-living surface is the surface of a hospital instrument or hospital equipment. In particularly preferred embodiments, the non-living surface is a surgical instrument or surgical equipment.

[0097] 5.6. Diagnostics The present invention also encompasses diagnostic methods for determining the causative agent in a bacterial infection. In certain embodiments, diagnosis of the causative agent of a bacterial infection is performed by (i) culturing a sample from a patient, e.g., a swab, sputum, or other sample suitable for culturing the bacteria causing the infection; (ii) contacting the culture with one or more phages, phage products, and phage cocktails of the present invention; and (iii) monitoring the culture for evidence of cell growth and / or lysis. Because the activity of phages and / or their isolated products (e.g., polypeptides, biologically active fragments or variants thereof, or nucleic acids encoding same) tends to be species- or strain-specific, susceptibility or lack of susceptibility to one or more phages, phage products, and phage cocktails of the present invention can be indicative of the species or strain of bacteria causing the infection.

[0098] In some embodiments, a test culture is obtained from a patient and contacted with one or more of F391 / 08, F17 / 19, or F58 / 19, or variants thereof. Reduced growth and / or lysis of the culture indicates that the test sample contains K. pneumoniae, particularly a strain of K. pneumoniae that is susceptible to infection by the bacteriophage or bacteriophage cocktail used as disclosed herein, thereby allowing identification of the infectious agent and appropriate diagnosis and / or treatment.

[0099] The sample may be a tissue biopsy or swab taken from the patient, or a fluid sample such as blood, tears or urine, In a preferred embodiment, the tissue sample is obtained from the patient's respiratory tract, for example a mucus sample, sputum or a swab taken from the nostrils. [Example]

[0100] 6. Working Example 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 those skilled in the art and are to be included within the spirit and scope of this application and the appended claims.

[0101] Unless otherwise indicated, the specific bacteriophages disclosed herein were isolated, processed, and analyzed according to the following methods: Furthermore, the studies described below were approved locally by the Animal Ethics Committee of the Instituto de Medicina Molecular, Portugal, in accordance with Portuguese law, and nationally by the Portuguese General Directorate of Veterinary Services (Direccao Geral de Veterinaria).All animals in the studies were kept in accordance with European Directive 86 / 609 / EC (Council of the European Communities. Council Directive 86 / 609 / EEC of 24 November 1986 on the approximation of laws, regulations and administrative provisions of the Member States regarding the protection of animals used for experimental and other scientific purposes. Off J Eur Communities L358:l-28), Portuguese Law (Portaria 1005 / 92) (Portuguese Agricultural Ministry. Portaria no. 1005 / 92 of 23 October on the protection of animals used for experimental and other scientific purposes. Diario da Republica I - Serie B 245:4930-4942), and the Guide for the Care and Use of Laboratory Animals (NRC 2011) (Institute for Laboratory Animal Research. 2011. Guide for the care and use of laboratory animals. Washington (DC): National Academies Press.)

[0102] One goal of this study was to investigate the antimicrobial activity of nebulized bacteriophage cocktails against Pseudomonas aeruginosa and Klebsiella pneumoniae in a murine experimental model of acute lung infection. Nebulization of aerosolized bacteriophages allowed for direct delivery to affected lung areas, overcoming certain side effects of nebulized antibiotics. [Example]

[0103] 6.1. Bacterial strains A selected group of Klebsiella pneumoniae bacterial isolates (n = 36) was collected at at least seven different healthcare facilities between 2005 and 2019. Overall, these isolates were collected from hospital settings (n = 25), outpatients (n = 9), or of unknown origin (n = 2) and from a variety of biological sources: urine (n = 10), respiratory secretions (n ​​= 5), unknown (n = 8), ascites (n = 5), and other (n = 8). This panel was assessed for antibiotic susceptibility testing by disk diffusion against a selected panel of clinically important antibiotics: β-lactam antibiotics—ampicillin, ceftazidime, piperacillin with tazobactam, and meropenem; fluoroquinolone—ciprofloxacin; aminoglycoside—gentamicin; and sulfonamide—trimethoprim with sulfamethoxazole. Results were interpreted according to the cutoff values ​​recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST, http: / / mic.eucast.org / Eucast2 / ). Isolates were considered multidrug resistant (MDR) if they showed non-susceptibility to three or more structurally unrelated classes of antibiotics (Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, et al. 2011. Clin Microbiol Infect 2012; 18: 268-281).

[0104] This set of K. pneumoniae bacterial isolates (n = 36) was also characterized by MLST as previously reported to assess genetic diversity, clonal groups, and pandemic status. The resulting sequences were then analyzed by Finch TV and assigned to their respective MLST sequences using tools available on the MLST website (https: / / pubmlst.org). Each bacterial isolate was streaked onto tryptone soy agar plates (TSA, Biokar Diagnostics, Pantin Cedex, France) that were incubated at +37°C for 18 hours. All clinical strains were stored at -70°C in tryptone soy broth (TSB, Biokar Diagnostics, Pantin Cedex, France) containing 15% glycerol (w / v) until needed.

[0105] For in vitro experiments, -70°C frozen stocks were grown overnight on TSA for 18 hours at 37°C. Single colonies were then grown overnight in TSB at 37°C with agitation. Fresh bacterial suspensions (dilutions of the overnight cultures) were prepared and incubated at 37°C with agitation. Bacteria were harvested when they reached the exponential growth phase (optical density at 600 nm of 0.3-0.5). Approximately 2.0 × 10 6 cfu / mL of inoculum was used in the lysis curve. [Example]

[0106] 6.2. Bacteriophage Sources, Amplification, and Cocktails 6.2.1. Bacteriophage origin Kle_F17 / 19, Kle_F58 / 19 virulent bacteriophages were isolated from wastewater in the Lisbon area and amplified in Kle_F391 / 08, Kle_F17 / 19, and Kle_F58 / 19 clinical strains.

[0107] To isolate lytic bacteriophages against K. pneumoniae, several clinical strains were used. Wastewater samples from various sources in the Lisbon metropolitan area were tested to determine the presence of bacteriophages by their ability to infect clinical strains of K. pneumoniae using a double agar overlay plaque assay (Kropinski A, Mazzocco A, Waddell TE, Lingohr E, Johnson RP. 2009. Enumeration of bacteriophages by double agar overlay plaque assay. Methods Mol Biol 501:69-76).

[0108] Briefly, bacterial strains were grown overnight in TSB at +37°C with agitation. Fresh bacterial suspensions (dilutions of the overnight cultures) were prepared, incubated at +37°C with agitation, and harvested when the exponential growth phase was reached (optical density at 600 nm: 0.3-0.5). Each culture was added to a water sample, and the mixture was incubated at +37°C for 30 minutes, to which 3 ml of pre-equilibrated soft agar 0.7% was added. The agar-water-bacteria suspension was layered onto a 1.5% TSA plate, allowed to solidify at room temperature, and incubated at +37°C. After 18 hours of incubation, the plates were checked for the presence of phage plaques (clear zones) within the bacterial lawn, indicating the presence of bacteriophage. Bacteriophage plaques were picked using a sterile pipette tip, transferred to SM buffer, and stored at +4°C.

[0109] 6.2.2. Bacteriophage amplification Newly isolated bacteriophages were subjected to a process of propagation, amplification, and purification (three sequential elutions) on indicator strains before assessing their host range. The susceptibility of 36 Klebsiella pneumoniae bacterial isolates to specific bacteriophage infection was determined using a double-agar overlay plaque assay. The susceptibility of the 36 bacterial isolates to specific bacteriophages was determined by observing phage plaques within the bacterial lawn. Bacteriophages with the highest percentage of infection across the host range were selected and advanced to a novel process of amplification, concentration by high-speed centrifugation, purification on a cesium chloride (CsCl) gradient, extraction of bacteriophage genomic DNA, and restriction fragment length polymorphism analysis.

[0110] Phages with distinct restriction profiles and broader host ranges were selected for sequencing. The complete genome sequence was determined by pyrosequencing using an Illumina HiSeq2000 genome analyzer.

[0111] After bioinformatics analysis, the most promising bacteriophages were selected for the composition of a therapeutic cocktail. The morphology of Kle_F391 / 08, Kle_F17 / 19, and Kle_F58 / 19 bacteriophages was analyzed at the Histology and Comparative Pathology Laboratory of the Portuguese Institute of Molecular Medicine (Lisbon, Portugal). These data were combined with genomic analysis and the bacteriophages were classified according to the Ackermann classification (Ackermann HW. 2009. Phage classification and characterization. Methods Mol Biol 501:127-140.). [Example]

[0112] 6.3. Bacteriophage analysis 6.3.1. Phenotypic characterization Phenotypic characterization of the collected bacteria showed that among β-lactam antibiotics, 100% of isolates were nonsusceptible to ampicillin, 97.2% were nonsusceptible to piperacillin with tazobactam, 86.1% were nonsusceptible to ceftazidime, and 77.8% were nonsusceptible to meropenem. For non-β-lactam antibiotics, this group of bacterial isolates showed 91.7% nonsusceptibility to ciprofloxacin, 72.2% nonsusceptibility to gentamicin, and 88.9% nonsusceptibility to trimethoprim with sulfamethoxazole. Overall, 33 / 36 (91.7%) of the isolates were MDR (Table 3).

[0113] [Table 3] JPEG0007812496000004.jpg184170JPEG0007812496000005.jpg172170

[0114] In addition, antibiotic susceptibility test readouts suggested the production of extended-spectrum β-lactamases (ESBLs) or carbapenemases (Table 1). Overall, the majority of isolates showed non-susceptibility to last-resort antibiotics such as carbapenems (23 / 30, 76.6%), MDR (n=22 / 30, 73.3%), and production of clinically important carbapenemases or ESBLs such as VIM-2, KPC-2, or even NDM-1, which are widespread worldwide (16 / 30, 53.3%).

[0115] The identification of clonal lineages showed high genetic diversity represented through 19 different STs, with the detection of high-risk clones (ST11, ST13, ST14, ST15 or ST258) that show widespread dissemination worldwide and are actually responsible for outbreaks and high morbidity and mortality (Brink, A J., 2019. Epidemiology of carbapenem-resistant Gram-negative infections globally. Curr Opin Infect Dis 32, 609-616.; Karakonstantis, S., Kritsotakis, E. I, Gikas, A., 2019. Pandrug-resistant Gram-negative bacteria: a systematic review of current epidemiology, prognosis and treatment options. J Antimicrob Chemother.; Li, J., Li, Y., Song, N., Chen, Y., 2019. Risk factors for carbapenem-resistant Klebsiella pneumoniae J Glob Antimicrob Resist.;Navon-Venezia, S., Kondratyeva, K., Carattoli, A., 2017. Klebsiella pneumoniae: a major worldwide source and shuttle for antibiotic resistance. FEMS Microbiol Rev 41, 252-275.;Sarda, C., Fazal, F., Rello, J., 2019. Management of ventilator-associated pneumonia (VAP) caused by resistant gram-negative bacteria: which is the best strategy to treat? Expert Rev Respir Med 13, 787-798).

[0116] 6.3.2. Bacteriophage Morphology Using transmission electron microscopy, purified bacteriophages were classified based on their virion morphology. Kle_F17 / 19, Kle_F58 / 19, and Kle_F391 / 08 bacteriophages belong to the Caudovirales order. F391 / 08 exhibited an icosahedral head structure with a noncontractile, elongated tail that was often flexible. Based on these features, along with its genomic characteristics, we classified this bacteriophage as a member of the Siphoviridae family. Kle_F17 / 19 and Kle_F58 / 19 bacteriophages, which exhibited a contractile tail and an icosahedral head structure (capsid), with a baseplate structure and tail fibers, were classified as members of the Myoviridae family (Figure 1).

[0117] 6.3.3. Bacteriophage host range The K. pneumoniae-selected bacteriophages were tested on 36 clinical strains isolated from human clinical samples collected and identified in Lisbon-area hospitals. Of this panel of diverse, clinically important K. pneumoniae isolates, 53.8% were infected with at least one of the phages comprising the cocktail (Table 4).

[0118] [Table 4]

[0119] Indeed, Klebsiella pneumoniae is an important MDR pathogen affecting humans and a major source of nosocomial infections with high morbidity and mortality due to limited treatment options. These strains can be resistant to virtually all available classes of antibiotics, posing significant challenges to clinicians due to limited treatment options (Brink, AJ, 2019. Epidemiology of carbapenem-resistant Gram-negative infections globally. Curr Opin Infect Dis 32, 609-616; Navon-Venezia, S., Kondratyeva, K., Carattoli, A., 2017. Klebsiella pneumoniae: a major worldwide source and shuttle for antibiotic resistance. FEMS Microbiol Rev 41, 252-275).

[0120] Since the first emergence of carbapenemase-producing Klebsiella pneumoniae, nearly 100 international outbreaks caused by high-risk clones have been reported. The largest outbreak was caused by clonal group 258, which was responsible for 68% of all outbreaks and consisted of three STs: ST258, ST11, and ST512. The second most prevalent clonal group, responsible for approximately 20% of all outbreaks, was clonal group 15, consisting of ST14 and ST15. Other important STs contribute to outbreaks and clinically significant infections to a lesser extent (ST147, ST37, ST101, ST17) (Navon-Venezia, S., Kondratyeva, K., Carattoli, A., 2017. Klebsiella pneumoniae: a major worldwide source and shuttle for antibiotic resistance. FEMS Microbiol Rev 41, 252-275).

[0121] Overall, infections caused by carbapenem-resistant K. pneumoniae high-risk clones are a major health problem and frequently cause high mortality worldwide. K. pneumoniae phages were able to effectively eliminate isolates belonging to five of the nine high-risk clones listed above (ST11, ST14, ST15, ST258, and ST147) that not only exhibit carbapenem resistance caused by acquired carbapenemases but also show worldwide prevalence.

[0122] 6.3.4 Genomic analysis We performed whole-genome sequencing of the genomic DNA of Klebsiella pneumoniae F391 / 08, Kle_F17 / 19, and Kle_F58 / 19 bacteriophages. Upon annotation, circular maps were prepared for each genome, showing predicted ORFs encoding hypothetical proteins and their putative functions (Figures 2-4). An initial NCBI nucleotide blast analysis (BLASTN) of the complete genome sequences of selected bacteriophages was performed. Klebsiella pneumoniae bacteriophage F391 / 08 revealed high and significant homology to Klebsiella phage vB_Kpn_IME260 (NCBI reference sequence: KX845404.2). Kle_F17 / 19 bacteriophage showed the highest similarity to the bacteriophage Klebsiella phage KOX1 (NCBI reference sequence: KY780482.1). The bacteriophage Kle_F58 / 19 genome sequence showed high homology to other sequences in the NCBI database, with the highest similarity found with Klebsiella phage vB_KpnM_Potts1 (NCBI reference sequence: MN013081.1).

[0123] Bacteriophage F391 / 08 (Figure 2), with a genome size of 113,073 bp, shared up to 95% sequence identity with Klebsiella phage vB_Kpn_IME260 at 96% genome coverage. 173 ORFs were predicted, 28% of which were assigned putative functions, of which 25 were tRNA genes. None of the predicted ORFs shared significant homology with any sequence in the NCBI Nonredundant Protein Sequence Database. 90 of the predicted ORFs showed homology to proteins in the NCBI Nonredundant Protein Sequence Database, but no putative functions could be assigned. Genome analysis of the Kle_F17 / 19 bacteriophage revealed a 45,423-bp sequence (Figure 3) that exhibited up to 96% identity with the Myoviridae Klebsiella phage KOX1 over 94% of the genome (Brown TL, Petrovski S, Hoyle D, Chan HT, Lock P, Tucci J. 2017. Characterization and formulation into solid dosage forms of a novel bacteriophage lytic against Klebsiella oxytoca. PLoS ONE 12(8): e0183510). Of the 64 predicted ORFs, 50% had assigned putative functions. No putative functions could be assigned for 32 of the ORFs. The bacteriophage Kle_F58 / 19 (Figure 4), with a 169,725-bp genome, showed high similarity to the Myoviridae Klebsiella phage vB_KpnM_Potts1. Sequence identity was up to 97.5% in 98% of the genome sequence. 292 ORFs were predicted, 45.9% of which were assigned putative functions, of which 7 were tRNA genes. Approximately 45% of the predicted ORFs shared homology with sequences in the NCBI non-redundant protein sequence database, however, no function was assigned. Only one ORF shared no significant homology with any sequence in the NCBI non-redundant protein sequence database.

[0124] No significant similarity could be found in the sequences of these bacteriophages to known virulence or toxin proteins or to elements typically associated with lysogeny (integrase, repressor and antirepressor).

[0125] 6.3.5 Lytic activity of bacteriophage cocktails The lytic activity of the new K. pneumoniae F391 / 08, Kle_F17 / 19, and Kle_F58 / 19 bacteriophages was assessed against planktonic cultures to characterize the bacteriophage cocktail.

[0126] Conventional lysis curves were generated under controlled conditions using a previously determined bacterial inoculum. Approximately 2 × 10 6 Cultures were prepared with an inoculum of 1000 cfu / mL. Two different K. pneumoniae strains, 57 / 17 and 237 / 14, were used because it was not possible to find a highly virulent strain in our bacterial collection that could be simultaneously infected by three bacteriophages. Each bacteriophage was tested individually (Figures 5 and 6) and in combination (Figure 7) at an MOI approaching 10. Viable bacterial counts were monitored at 1-hour intervals over an 8-hour period and again at 24 hours post-inoculation (ppi) with the bacteriophages.

[0127] Bacteriophage F391 / 08 was tested individually at an MOI approaching 10 (Fig. 5), and at 1 hour post-inoculation (ppi), the cultures were significantly more resistant to bacterial control cultures (8.4 × 10 6 cfu / mL) compared to a reduction of approximately 3 logs (2.3 × 10 3 Within the first 4 hours, F391 / 08 showed 10 4 A viable bacterial count below cfu / mL could be maintained. The bacterial load began to increase during incubation, and at 8 hours ppi the culture exceeded the initial bacterial load (3.8 × 10 7 cfu / mL). However, at 24 hours ppi, the live count was 1.2 × 10 11 cfu / mL), an extremely significant reduction of 8.8 × 107 cfu / mL, demonstrating that F391 / 08 retains the ability to infect KLE57 / 17 even in stationary phase.

[0128] Bacteriophage Kle_F17 / 19 was tested at an MOI of approximately 10 against K. pneumoniae 237 / 14, and the live bacterial count at 1 hour was compared to the control bacterial culture (1.0 × 10 7 cfu / mL) was reduced by approximately 5 log units (5.0 × 10 2 By 4 hours ppi, the Kle_F17 / 19 bacteriophage was circulating at 10 cfu / mL in the culture, similar to that observed for F391 / 08 in its host. 4 The bacterial load was maintained below 1.1 x 10 cfu / mL. At 8 hours ppi, the viable bacterial count was 1.1 x 10 8 cfu / mL and continued until the end of culture incubation (24 h ppi), when the viable bacterial count reached 3.1 × 10, almost 1 log higher than that determined for the control culture. 10 cfu / mL.

[0129] The effect of Kle_F58 / 19 bacteriophage (Figure 6) on strain 57 / 17 was more rapid than that of phage F391 / 08. At 1 hour ppi, the viable bacterial count was 2.0 x 10 2 This was an approximately 4 log reduction compared to the control bacterial culture, which reached cfu / mL. By 8 hours ppi, viable bacteria had increased to 1.8 x 10 7 At 24 hours ppi, bacteriophage Kle_F58 / 19 was less effective against the host than F391 / 08, but was nevertheless able to achieve a 96% reduction in viable cells, a highly significant reduction compared to the control bacterial culture.

[0130] Individually, the three bacteriophages had high lytic capacity but showed slight differences in efficacy over time in each host, likely reflecting differences in their adsorption kinetics, latency period, and burst size (data not shown).

[0131] Figure 7 shows the combined lysis curves of bacteriophages F391 / 08 and Kle_F58 / 19 in Kle pneumoniae 57 / 17 at an MOI of approximately 10. A significant decrease in bacteriophage activity was observed at 1 hour ppi (1.0 x 10 1 cfu / mL). During the next 8 hours, the combined activity of the bacteriophages increased with some variability, but 4 Maintaining a viable cell count below cfu / mL extended the observed duration of action for each of the bacteriophages individually. Despite bacterial regrowth, cell counts at 24 hours ppi were significantly higher than the bacterial load of the control culture (4.2 × 10 10 cfu / mL), a significant reduction (approximately 3 log units) of 5.1 × 10 7 cfu / mL.

[0132] In addition to the lysis curves with F391 / 08 and Kle_F58 / 19, a second combination culture was prepared with the three bacteriophages F391 / 08, Kle_F58 / 19, and Kle_F17 / 19 (MOI approximately 10) with the goal of observing any effect of Kle_F17 / 19 on the efficacy of F391 / 08 and Kle_F58 / 19. Although K. pneumoniae 57 / 17 is not susceptible to infection by bacteriophage Kle_F17 / 19, the presence of this phage appeared to induce a stabilizing effect on the efficacy of bacteriophages F391 / 08 and Kle_F58 / 19 when combined during the first 6 hours of culture (Figure 9). At 1 hour ppi, the decrease in cell count was not as evident compared to the curves for F391 / 08 and Kle_F58 / 19; however, the behavior over the next few hours suggests an interaction with Kle_F17 / 19. At 8 hours ppi, the bacterial load was 5.3 x l0 9cfu / mL compared to the control culture, 5.3 × l0 3 At the end of the culture incubation, the determined cell count was 4.7 × 10 cfu / mL, a concentration close to the bacterial input at the start of the culture. 7 cfu / mL.

[0133] As previously reported by Loc-Carrillo and Abedon in 2011 (Loc-Carrillo C, Abedon ST. 2011. Pros and cons of phage therapy. Bacteriophage 1(2): 111-114), different bacteriophages can be mixed in cocktails to extend their effectiveness, typically resulting in a collectively greater spectrum of antibacterial activity. This study demonstrated exactly that.

[0134] 6.3.6. Conclusion The bacteriophage cocktail characterized and tested in this study consisted of three Klebsiella bacteriophages: F391 / 08, Kle_F17 / 19, and Kle_F58 / 19. Genome sequence analysis did not identify any known genes associated with integration, toxins, or antibiotic resistance, which are important features for safe bacteriophage use. Comparative genomic analysis revealed that Klebsiella bacteriophages F391 / 08, Kle_F17 / 19, and Kle_F58 / 19 share significant similarity with Klebsiella phages vB_Kpn_IME260, KOX1, and vB_KpnM_Potts1, respectively.

[0135] The Klebsiella pneumoniae bacteriophage vB_Kpn_IME260 is a T5-like virus (Xing S, Pan X, Sun Q, Pei G, An X, Mi Z, Huang Y, Zhao B, Tong Y. 2017. Complete genome sequence of a novel multidrug-resistant Klebsiella pneumonia phage, vB_Kpn_IME260. Genome Announc 5:e00055-170). The K. oxytoca bacteriophage KOX1 is most homologous to Kle_F17 / 19, and Brown et al. (Brown TL, Petrovski S, Hoyle D, Chan HT, Lock P, Tucci J. 2017. Characterization and formulation into solid dosage forms of a novel bacteriophage lytic against Klebsiella oxytoca. PLoS ONE 12(8): e0183510) described KOX1 as a myovirus. No information about the vB_KpnM_Potts1 bacteriophage, except for its morphology, is available in the literature. Transmission electron microscopy analysis confirmed that bacteriophages Kle_F58 / 19 and Kle_F17 / 19 belong to the Myoviridae family.

[0136] Another important selection criterion for bacteriophages is their host range, which should be as broad as possible, especially including bacterial species that are clinically prevalent (Gill JJ, Hyman P. 2010. Phage choice, isolation, and preparation for phage therapy. Curr Pharm Biotechnol 11(1):2-14). The extent of lytic activity of Klebsiella pneumoniae bacteriophages F391 / 08, Kle_F17 / 19, and Kle_F58 / 19 was tested against 36 clinical strains with high genomic diversity isolated in hospitals in the Lisbon metropolitan area during the past year.

[0137] Time-kill curves are often used to study the antibacterial effects and dosing regimens of single and combined drug compounds prior to in vivo efficacy studies (NCCLS. 1999. Methods for determining bactericidal activity of antimicrobial agents: approved guideline. 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087 USA: NCCLS). In a recent study, lysis curves were prepared to examine the lytic ability of individual and combined bacteriophages on bacterial cultures. A single bacterial inoculum was used. This value was carefully chosen based on previous evidence (Mendes JJ, Leandro C, Motolla C, Barbosa R, Silva F, Oliveira M, Vilela C, Melo-Cristino J, Gorski A, Pimentel M, Sao-Jose C, Cavaco-Silva P, Garcia M. 2014. In vitro design of a novel lytic bacteriophage cocktail with therapeutic potential against organisms causing diabetic foot infections. J Med Microbiol 63:1055-1065). All cultures had initial bacterial reduction during the first 1-2 hours after phage inoculation, with subsequent regrowth becoming noticeable after 6 hours and even more evident after 24 hours. Individually, all three bacteriophages exhibited high lytic efficacy against planktonic cells. With the exception of Kle_F17 / 19, the 95-99% bacterial reduction in 24-hour cultures indicated their increased individual ability to control Klebsiella pneumoniae burden. The combined activity of two of the three phages comprising the cocktail demonstrated synergistic bacteriolytic activity against the bacterial strain.The MOI chosen was based on the "rule of 10," which indicates that if the goal is a significant reduction in bacterial density, efforts should be made to ensure that 10 orders of magnitude of bacteriophage are adsorbed to the average bacterium (Abedon ST. 2009. Kinetics of phage-mediated biocontrol of bacteria. Foodborne Pathog Dis 6:807-815; Kasman LM, Kasman A, Westwater C, Dolan J, Schmidt MG, Norris JS. 2002. Overcoming the phage replication threshold: a mathematical model with implications for phage therapy. J Virol 76:5557-5564). Regrowth was observed in planktonic cells exposed to bacteriophage. [Example]

[0138] 6.3.7. In vitro interactions of Pseudomonas aeruginosa bacteriophage cocktail with antibiotics In vitro techniques were performed to evaluate potential interactions between the F99 / 10, F27 / 12, and F95 / 13 bacteriophage cocktail and a selection of antibiotics frequently used as empirical treatment for hospital-acquired pneumonia. While the cocktail targets Gram-negative bacteria, empirical antibiotic treatment often includes drugs for the treatment of pneumonia caused by Gram-positive pathogens; therefore, molecules targeted to the treatment of Staphylococcus aureus were also targeted.

[0139] Briefly, using a broth microdilution method, the minimum inhibitory concentrations (MICs) for β-lactams (piperacillin, ceftazidime, and meropenem with tazobactam), aminoglycosides (amikacin), glycopeptides (vancomycin), and oxazolidinones (linezolid) were determined at various MOIs (0.1, 1, and 10) with and without the presence of single and combined bacteriophages. The method was developed and optimized as proof of concept for P. aeruginosa using a single strain (PSA_1992 / 05; NCIMB accession 42914) infected by all three phages. The MIC range selected was 0.016–32 mg / L, allowing susceptibility and resistance breakpoints for all seven antibiotics to be included and detected. Various time points (4, 6, 12, and 18 hours) were used to optimize the technique, with the final reading determined at 18 hours. All experiments were performed in duplicate. Studies were optimized for individual bacteriophages, and the final assay containing data for the complete cocktail is presented here.

[0140] Briefly, the final tests performed with the cocktail (bacteriophages Psa_F99 / 10, Psa_F27 / 12, and Psa_F95 / 13) confirm the results obtained with each individual phage (data not shown). The potency of the cocktail was sufficient to produce a conversion of the MIC of PSA_1992 / 05 from significantly higher MIC values ​​to 0.016 mg / L or less for all antibiotics tested and for all three MOIs (0.1, 1, and 10) tested in this study (Table 5).

[0141] [Table 5]

[0142] This data demonstrates the increased efficacy of the combined F99 / 10, F27 / 12, and F95 / 13 cocktail in the presence of these antibiotics. No negative effects were observed produced by the addition of the phage cocktail.

[0143] Overall, vancomycin and linezolid are antibiotics that do not exhibit any activity against Gram-negative bacteria, but their presence did not inhibit the activity of the cocktail. Major classes of antibiotics used to treat hospital-acquired pneumonia were evaluated in combination with phages alone and as a cocktail for proof-of-concept and optimization purposes. Overall, no interactions were detected that could result in inhibition of phages or a significant decrease in antibiotic activity. [Example]

[0144] 6.3.8. Feasibility study of inhaled phage application Nebulization has been used continuously for inhalation therapy, i.e., for the use of bacteriophages in animal models by means of powder formulations (Chang, RYK, Chen, K., Wang, J., Wallin, M., Britton, W., Morales, S., Kutter, E., Li, J., Chan, HK, 2018. Proof-of-Principle Study in a Murine Lung Infection Model of Antipseudomonal Activity of Phage PEV20 in a Dry-Powder Formulation. Antimicrob Agents Chemother 62, 2). Here, for liquid formulations, a feasibility study for inhaled phage application was developed using various options of vibrating mesh nebulizers. These consisted of hospital-grade nebulizers used not only in hospital wards but also in intensive care units that could be appropriately attached to any standard ventilator circuit.

[0145] To compare three vibrating mesh nebulizer devices (Aeroneb, Nebutec 4, and Nebutec 6), 10 9 Cocktails containing pfu / ml of each phage, F99 / 10, F27 / 12, and F95 / 13, were prepared using phage suspension and DPBS (first experiment) or 0.9% NaCl solution (second experiment) as diluents. Three independent nebulization experiments were performed for each diluent using all three devices. For each nebulization, 1 ml of the phage cocktail was nebulized within a 1-minute time frame, followed by a 60-minute settling time. Phages were collected in Petri dishes containing 15 ml of the respective collection buffer. Phage titers were determined using specific detection (titration) strains for each phage. Droplet sizes for each device were experimentally determined by nebulization and laser diffraction and were consistent with manufacturer information. Furthermore, aerosol delivery rates were experimentally determined, and lung deposition potential was calculated based on the measured data. It was suggested that both Aeroneb and Nebutec 4 achieved comparable deposition and were superior to Nebutec 6 (data not shown). It is worth noting that due to droplet adhesion to the atomization cylinder, only 75% ± 8.6% of the atomized liquid was successfully deposited on the Petri dish. Overall, phage recovery was higher with NaCl as the diluent compared to DPBS. Furthermore, the Aeroneb device produced the most uniform recovery rates across all three phage species (data not shown).

[0146] Based on the results, the Aeroneb device and NaCl as the vehicle were selected. Overall, the results obtained selected the Aeroneb vibrating mesh nebulizer device, which was then applied to the remaining efficacy and toxicity studies conducted in the PCLS ex vivo model (see Example 6). Phage titer recovery and log reduction were consistent between the two formulations, among the different phages, and consistent with expected phage loss throughout the system. [Example]

[0147] 6.3.9. Efficacy and Toxicity Studies in a Precision Cut Lung Slice (PCLS) Ex Vivo Model To evaluate the efficacy and toxicity of bacteriophages F99 / 10, F27 / 12, and F95 / 13, a comprehensive ex vivo study was carefully designed in a PCLS Pseudomonas aeruginosa infection model. The system was designed and optimized in rat PCLS treated with a cocktail of purified F99 / 10, F27 / 12, and F95 / 13 by immersion and nebulization, with and without antibiotic co-treatment. The system was then transferred to a final setup in human PCLS. Experiments were performed with human PCLS to evaluate bacteriophage efficacy and toxicity after inhalation application to P. aeruginosa -infected human lung tissue, either alone or in combination with antibiotics. P. aeruginosa and the nebulized phage cocktail were applied directly onto the air-liquid interface culture tissue, while antibiotics (at an inhibitory concentration of less than 0.5 μg / ml) were applied by immersion (i.e., to the culture medium). In addition, to exclude the cytotoxic effect of phages on human lung tissue, uninfected controls were treated with nebulized phage cocktail. Similar to the results obtained with rat PCLS (data not shown), both nebulized phage treatment and antibiotics reduced the bacterial burden in infected human PCLS to a similar extent, while their simultaneous treatment resulted in a strong synergistic effect in bacterial reduction.

[0148] Phage treatment alone reduced bacterial load by more than 1 log, while antibiotic treatment alone resulted in an approximately 4-log reduction in cfu. In combination, a synergistic effect was observed, resulting in a greater than 5-log reduction in bacterial load (Figure 8). Upon analysis of tissue viability, no cytotoxic effect of purified phage on uninfected tissue could be observed. Treatment of infected PCLS with purified phage resulted in complete rescue of the loss of viability induced by P. aeruginosa infection. Antibiotic treatment alone or in combination with phage treatment restored tissue viability to a comparable extent (Figure 9). In uninfected human PCLS, phage could be detected at numbers equivalent to the inoculum 24 hours after treatment. In contrast, phage replication was observed in infected samples but not in infected samples co-treated with antibiotics, consistent with the observed difference in bacterial load (data not shown).

[0149] Overall, studies conducted in a P. aeruginosa PCLS ex vivo model demonstrated that treatment of infected rat / human slices with a cocktail of F99 / 10, F27 / 12, and F95 / 13 not only demonstrated efficacy through immersion and nebulization treatments, but also restored lung tissue viability while exhibiting no toxic effects, even in the presence of combination antibiotic therapy (meropenem, amikacin, and vancomycin). Analysis of uninfected slices treated with the cocktail in multiple conditions (immersion vs. nebulization; rat vs. human; phage lysate vs. purified phage) demonstrated no negative effect of the cocktail treatment on lung tissue viability, reinforcing the absence of predictable toxicity, specifically cytotoxicity, caused by phage treatment in the lung. This evidence is also consistent with previous studies that have shown no adverse effects of phage therapy in other infection models and compassionate use cases (McCallin, S., Sacher, JC, Zheng, J, Chan, BK, 2019. Current State of Compassionate Phage Therapy. Viruses 11, 343; Oliveira, A., Sereno, R., Nicolau, A., Azeredo, J, 2009. In vivo toxicity study of phage lysate in chickens. Br Poult Sci 50, 558-563).

[0150] For the different administration routes, nebulized phage treatment successfully reduced the bacterial load of infected PCLS, although the expected bacteriophage loss occurred in the nebulizer, thus demonstrating comparable efficacy to immersion treatment. An increase in phage titer was observed toward 24 hours. Determination of phage titer in the inoculum revealed that the number of phage in samples with and without the antibiotic mixture was comparable, thus demonstrating that the presence of antibiotics did not affect phage titer or activity. On the other hand, it was also possible to confirm that the cocktail did not interfere with the activity of the antibiotics. Indeed, phage / antibiotic cotreatment completely restored tissue viability. Overall, this combination was also supported by all safety endpoints in infected and uninfected rat and human lung slices. [Example]

[0151] 6.3.10. Precision-Cut Lung Slice (PCLS) Studies The efficacy of inhaled application of Klebsiella pneumoniae F391 / 08, Kle_F17 / 19, and Kle_F58 / 19 bacteriophages was investigated for respiratory bacterial infection in an ex vivo model of Klebsiella pneumoniae infection in precision-cut lung slices (PCLS). The study included confirmation of the biological activity of the bacteriophages after nebulization, establishment of a Klebsiella pneumoniae infection model, and assessment of phage activity after nebulization in rat PCLS.

[0152] Experiments were performed at the Department of Preclinical Pharmacology, Fraunhofer ITEM (Hannover, Germany). Animal sacrifice for organ removal for PCLS preparation was registered with the competent authority (Lower Saxony Federal State Office for Consumer Protection and Food Safety) and was carried out in accordance with the German Animal Protection Act (Tierschutzgesetz of 18 May 2006, BGB1.1 S. 1206, 1313; adopted 28 July 2014, BGB1.I S. 1308) and Directive 2010 / 63 / EU of the European Parliament and of the Council on the protection of animals used for scientific purposes.

[0153] Female rats (Wistar WU, 8–21 weeks old at the time of experimentation) were obtained from Charles River (Sulzfeld, Germany). The animals were kept under conventional housing conditions (22°C, 55% humidity, and a 12-hour day / night rhythm) until use for PCLS preparation. Rats were sacrificed by an overdose of sodium pentobarbital (Narcoren, Merial GmbH, Hallbergmoos, Germany) and exsanguination via the vena cava. The whole lungs were filled with a warm, low-melting agar / medium solution (1.5% (v / v) final concentration) at 37°C. The filled lobes were cooled in ice-cold PBS to allow the agar to polymerize. 10-mm-diameter cylinders were extracted from the lung tissue and cut into approximately 300-μm-thick slices using a Krumdieck microtome (Alabama Research and Development, Munford, AL, USA). Slices were harvested in cold EBSS at 4°C and then transferred to Petri dishes containing PCLS culture medium and incubated under cell culture conditions (37°C, 5% CO2) for approximately 30 minutes. Slices were then washed four times with PCLS culture medium for 30 minutes and finally incubated overnight under cell culture conditions before infection experiments. K. pneumoniae test infection and detection strains (57 / 17, 131 / 15, 130 / 14, and 25 / 14) were grown overnight in TSB medium at 37°C and 150 rpm. The overnight culture was diluted 1:30 and incubated for an additional hour at 37°C and 150 rpm to produce exponentially growing cultures. The detection strains were used for plaque formation assays at an OD600 of 0.3-0.5. A culture of infection strain 57 / 17 was diluted to 2 x 10 6 The cfu concentration was confirmed by adjusting the CFU / ml and inoculating the inoculum sample.

[0154] Bacteriophage nebulization was performed using a vibrating mesh nebulizer (AeroNeb). 9 Cocktails containing PFU / ml of each phage were prepared in 0.9% NaCl, and aerosols were generated for nebulization. 1 mL of the phage cocktail was nebulized within a 1-minute time frame, followed by settling time.

[0155] For the nebulization pre-test (without PCLS), a phage cocktail of phages F391 / 08, Kle_F17 / 19, and Kle_F58 / 19 was prepared, and the phages were nebulized and harvested into Petri dishes containing 15 ml of medium as harvest buffer (0.9% NaCl). The biological activity of the phages was determined by a double agar overlay plaque assay.

[0156] Efficacy of nebulized phage in rat PCLS was performed to assess whether phage nebulization in PCLS is effective in a K. pneumoniae infection model. Therefore, rat PCLS were cultured at the air-liquid interface (ALI) in 12-well Transwell plates (500 μm reservoir volume) and infected with K. pneumoniae strain 57 / 17 (1 × 10 5 CFU / PCLS were applied directly onto the tissue. One hour after infection (pi), the inoculum was removed, the PCLS was washed, and the Transwell was transferred onto a new companion plate well containing 500 μl of fresh medium in the lower chamber. 1 × 10 per phage 10 A phage cocktail containing PFU / mL was prepared (diluent: 0.9% NaCl). Using an exposure system with an Aeroneb device and customized adapters for four Transwells, 1 ml of the phage cocktail or vehicle (0.9% NaCl) was sprayed onto PCLS (nebulization time: 1 min, sedimentation time: 60 min). Separate exposure systems and adapters were used for phage or vehicle spraying to prevent contamination. PCLS were post-incubated overnight at 37°C and 5% CO2, and samples were taken and analyzed at 24 h pi. Additionally, in this initial experiment, separate samples were taken immediately after phage spraying to determine the actual deposited PFU per well.

[0157] After confirming the efficacy of bacteriophage nebulization in the K. pneumoniae PCLS model, the experiment was extended to test the efficacy of the bacteriophage cocktail alone and in combination with antibiotic co-treatment. Preparation, culture, and infection of rat PCLS, as well as phage treatment by nebulization, were performed as previously described. For antibiotic combination treatment, meropenem, vancomycin, and amikacin were added to the culture medium of each PCLS at final concentrations of 8, 8, or 1 μg / ml, respectively. Antibiotic treatment was performed simultaneously with phage treatment at 1 h p.i. Therefore, washed PCLS Transwells were transferred to new companion plates whose basolateral compartments contained medium or medium containing the antibiotic cocktail, and the apical side was exposed to bacteriophage or saline aerosols as described above.

[0158] PCLS were analyzed for cfu content, tissue viability and phage replication.

[0159] For bacterial load and phage titer analysis, PCLS were lysed with 1% Triton X-100 / PBS for 30 minutes at 4°C. Tissue residues were removed, and the lysate was centrifuged at 8000 x g for 10 minutes at 4°C to pellet the bacteria. The clarified lysate was used for phage titer determination. The pellet was resuspended in 250 μl of PBS / Tween and used for bacterial load determination.

[0160] For specific detection of phages F391 / 08, Kle_F17 / 19, and Kle_F58 / 19, phage titers were determined by plaque assay using Klebsiella pneumoniae strains 131 / 15, 130 / 14, and 25 / 14, respectively. Each sample treated with the phage cocktail was co-plated with each detection strain. As described above, phages were released from PCLS by tissue lysis and separated from bacteria by centrifugation. Duplicate 1:10 dilutions of phage samples in PBS / Tween were prepared in Eppendorf tubes and mixed by inversion to avoid mechanical disturbance. 100 μl of each phage dilution was added to 100 μl of each phage dilution.

[0161] The detector strain culture was added to 3 ml of soft agar adjusted to 48°C, and the mixture was spread onto one TSA plate. At the end of each PFU experiment, all detector strains were inoculated to eliminate phage contamination of the bacterial culture. After overnight incubation of the agar plates at 37°C, phage plaques were counted. To calculate PFU per PCLS, the plaque count was multiplied by the respective dilution factor and a factor of 2.5, which reflects the ratio of the total volume of the phage sample (250 μl) to the inoculated volume (100 μl).

[0162] To quantify bacterial load from PCLS lysates, several 1:10 dilutions of each sample were prepared in PBS / Tween, and 50 μl of each dilution was plated onto half of a TSA plate. After overnight incubation of the agar plates at 37°C, colonies were counted. To calculate CFU per PCLS, colony counts were multiplied by the respective dilution factor and a factor of 5, which reflects the ratio of the total volume of bacterial suspension (250 μl) to the plated volume (50 μl).

[0163] The viability of uninfected and infected tissues was assessed by calcein staining. Calcein AM penetrates intact cells and hydrolyzes intracellularly to produce calcein, a fluorescent compound that is retained in the cell cytoplasm. Increased numbers of dead or damaged cells within PCLS result in a decrease in calcein staining intensity. PCLS incubated in 70% ethanol for 15 minutes at room temperature were used as a reference ("dead control"). All PCLS were washed once with warm medium and incubated with calcein AM staining solution (4 μM in DMEM / F12) at 37°C and 150 rpm in the dark for 45 minutes. After three washes with PBS, PCLS were lysed with 1% Triton-X100 for 30 minutes at 4°C in the dark. Next, 50 μl of tissue lysate was transferred to a black-walled 96-well plate, and fluorescence (excitation wavelength: 485 nm, emission wavelength: 535 nm) was measured using a microplate reader. All samples were measured in duplicate. [Example]

[0164] 6.3.11. Nebulization Studies in a Klebsiella pneumoniae Infection Model of PCLS Prior to the first nebulization test, the bioactivity of the phages after nebulization was assayed.

[0165] 1×10 6 A phage cocktail of 1 x 10 PFU / ml was prepared in 0.9% NaCl. After nebulization, the recovered phage titer showed high losses during deposition, therefore a new cocktail was prepared at 1 x 10 9 PFU / ml of each phage was prepared. Additional nebulization tests were performed to estimate the expected phage titer after nebulization. Detailed results are shown in Table 3.

[0166] [Table 6]

[0167] In summary, with an initial phage cocktail of 1 x 10 PFU / ml, approximately 1.5 to 2.5 logs of residual activity can be expected after nebulization (phage 391 / 08 - 2 logs, phage Kle_F17 / 19 - 1.5 logs, phage Kle_F58 / 19 - 2.5 logs). To account for this reduction, a phage cocktail of 1 x 10 PFU / ml can be used. 10 It was decided to perform efficacy experiments with higher phage cocktail titers in PFU / ml.

[0168] Based on these results, it was concluded that bacteriophage activity after nebulization was confirmed and that despite partial loss of activity, the phage titer after nebulization should be sufficient to demonstrate efficacy in the PCLS infection model.

[0169] For treatment with nebulized bacteriophage, rat PCLS were cultured at the air-liquid interface in a Transwell, and the bacterial inoculum as well as the nebulized phage (or NaCl as a vehicle treatment) were deposited directly onto the tissue. Nebulization was performed as previously described.

[0170] 1×1010 Nebulization of the phage cocktail containing PFU / ml of each phage yielded approximately 1 × 10 PFU / ml per well (per PCLS) for phages 391 / 08 and Kle_F17 / 19 immediately after nebulization (1 hour pi). 7 PFU, and approximately 1 × 10 for phage Kle_F58 / 19 4 Phages 391 / 08 and Kle_F58 / 19 replicated strongly in K. pneumoniae 57 / 17-infected PCLS, resulting in deposited titers of approximately 3 x 10 PFU / PCLS, respectively. 8 PFU and reached titers of 2 × 10 pfu, while phage Kle_F17 / 19 even showed a slight decrease in titer (2 × 10 6 PFU) (Figure 10). This was expected, since phage Kle_F17 / 19 is unable to infect K. pneumoniae 57 / 17. Nevertheless, it was decided to test a cocktail of three phages on this strain to gather results from any toxic effects that may be induced by this phage treatment.

[0171] 1 × 10 PCLS 5 CFU of K. pneumoniae inoculum was allowed to infect for 1 hour, followed by removal, washing, and transfer to new wells to avoid overgrowth of non-tissue-associated planktonic bacteria. This procedure resulted in an actual bacterial load of approximately 10 immediately after exposure to the vehicle (NaCl). 4 After 1 hour of exposure to the nebulized phage cocktail, the CFU count was 10 2 CFU / PCLS were already reduced to approximately 4 × 10 CFU / PCLS, indicating rapid lysis of bacteria by the bacteriophage (Figure 8). Strong bacterial growth was observed at approximately 4 × 10 CFU / PCLS at 24 hours pi in vehicle-treated PCLS. 8 Treatment with the nebulized phage cocktail resulted in an approximately 2-log reduction in bacterial burden at 24 hours pi. Thus, nebulized phage treatment was successful in reducing the bacterial burden in K. pneumoniae-infected PCLS (FIG. 11).

[0172] To demonstrate proof of concept for bacteriophage efficacy after inhalation application, alone or in combination with antibiotics, rat PCLS cultured at the air-liquid interface were infected with Klebsiella pneumoniae strain 57 / 17 and the phage cocktail was sprayed directly onto the air-liquid interface-cultured tissue. For simultaneous treatment, antibiotics (subinhibitory concentrations) were applied by immersion (i.e., into the culture medium in the basolateral compartment of the Transwell) during phage treatment. One plate with four replicate wells was used per condition. Two wells were used for CFU / PFU determination, and the other two wells were used for tissue viability determination (calcein staining). Nebulization of the phage cocktail resulted in the deposition of approximately 10 PFU for phage 391 / 08 and Kle_F17 / 19 and approximately 10 PFU for phage Kle_F58 / 19 immediately after nebulization (1 h p.i.) (Figure 12).

[0173] Phages 391 / 08 and Kle_F58 / 19 then replicated in K. pneumoniae-infected PCLS, resulting in a 2-3 log increase in phage titer at 24 h p.i., while phage Kle_F17 / 19 titers remained stable. Samples with antibiotic co-treatment showed similar phage titers, as would be expected given the observed unaffected bacterial load.

[0174] Nebulized phage treatment successfully reduced CFU load in infected PCLS by approximately 1.5 logs (Figure 13), whereas treatment with subinhibitory antibiotics (immersion) did not reduce CFU load. Notably, combined antibiotic and nebulized bacteriophage cocktail treatment demonstrated a strong synergistic effect, reducing CFU load by approximately 4 logs (Figure 13).

[0175] Infection with K. pneumoniae induced a significant loss of tissue viability in PCLS at 24 hours pi (Figure 14). Treatment with nebulized phage cocktail significantly inhibited this infection-induced loss of tissue viability (Figure 14). Treatment with subinhibitory doses of antibiotics had no effect on tissue viability, as expected from unchanged CFU. Because phage treatment alone already significantly improved tissue viability, the synergistic effect of phage / antibiotic cotreatment on bacterial burden was not reflected in a further increase in tissue viability.

[0176] Precision-cut lung slices (PCLS) are fresh lung tissue sections that, as organotypic models, contain all cell types present in the lung, allowing detailed investigation of lung tissue responses. Their high level of complexity provides a physiologically relevant model. Data from PCLS studies demonstrated clear efficacy of nebulized bacteriophage treatment of Klebsiella pneumoniae-infected lung tissue slices. Phage cocktail administration reduced bacterial burden and restored tissue viability. Additional synergistic effects for CFU reduction of tested antibiotic-resistant Klebsiella pneumoniae strains were observed with combination treatment with subinhibitory doses of antibiotics.

[0177] Certain modifications and improvements will occur to those skilled in the art upon reading the above description, and all such modifications and improvements have been omitted from this specification for the sake of brevity and readability, but it is to be understood that they are properly included within the scope of the following claims.

Claims

1. or having a genome comprising a nucleotide sequence having at least 98% sequence identity to the nucleotide sequence of SEQ ID NO:2; having a genome comprising a nucleotide sequence having at least 99% sequence identity to the nucleotide sequence of SEQ ID NO:3; and a purified bacteriophage having antibacterial activity against one or more strains of Klebsiella pneumoniae.

2. A purified bacteriophage described in claim 1, having a genome comprising the nucleotide sequence of SEQ ID NO: 2 or 3.

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

4. 4. The pharmaceutical composition of claim 3, further comprising a purified bacteriophage having a genome comprising at least 85% sequence identity to the nucleotide sequence of SEQ ID NO:

1.

5. The pharmaceutical composition described in claim 4, wherein the purified bacteriophage has a genome comprising at least 85% sequence identity to the nucleotide sequence of SEQ ID NO: 1, and the genome comprises the nucleotide sequence of SEQ ID NO:

1.

6. 6. The pharmaceutical composition of any one of claims 3 to 5, further comprising one or more of the purified bacteriophages deposited under accession numbers NCIMB 42915 (F99 / 10), NCIMB 42916 (F27 / 12), and NCIMB 42917 (F95 / 13).

7. 7. The pharmaceutical composition of claim 6, further comprising purified bacteriophages deposited under accession numbers NCIMB 42915 (F99 / 10), NCIMB 42916 (F27 / 12), and NCIMB 42917 (F95 / 13).

8. 8. The pharmaceutical composition of any one of claims 3 to 7, further comprising one or more additional purified bacteriophages having antibacterial activity against Klebsiella pneumoniae.

9. The pharmaceutical composition according to any one of claims 3 to 8, further comprising one or more additional purified bacteriophages having antibacterial activity against Pseudomonas aeruginosa.

10. The pharmaceutical composition of any one of claims 3 to 9, which is formulated for administration as an aerosol.

11. 11. The pharmaceutical composition of any of claims 3 to 10, wherein each bacteriophage is present in an amount that provides a multiplicity of infection (MOI) of 1 to 10 upon administration of the pharmaceutical composition to a subject in need thereof.

12. Each bacteriophage is 10 ９ The pharmaceutical composition according to any one of claims 3 to 11, wherein the composition is present in pfu.

13. The pharmaceutical composition of any one of claims 3 to 12, which is formulated for administration as an aerosol.

14. A pharmaceutical composition according to any of claims 3 to 13 for use in treating or reducing the incidence of a bacterial infection in a subject in need thereof.

15. A pharmaceutical composition for use according to claim 14, administered in combination with a pharmaceutical composition comprising at least one purified bacteriophage deposited under accession numbers NCIMB 42915 (F99 / 10), NCIMB 42916 (F27 / 12), and NCIMB 42917 (F95 / 13).

16. The bacterial infection is caused by bacterial strains of Pseudomonas aeruginosa or Klebsiella pneumoniae and / or forms a biofilm; A pharmaceutical composition for use according to claim 14 or 15.

17. A pharmaceutical composition for use according to claim 16, wherein the bacterial strain exhibits resistance to one or more known antibiotics.

18. 18. The pharmaceutical composition for use according to claim 16 or 17, wherein the bacterial infection is a respiratory infection.

19. The pharmaceutical composition for use according to claim 18, wherein the respiratory infection is hospital-acquired bacterial pneumonia or an infection associated with cystic fibrosis.

20. The pharmaceutical composition for use according to claims 14 to 19, which is administered as an aerosol to the lungs.

21. A pharmaceutical composition for use according to claim 20, administered to the lungs of a subject in need thereof in an amount providing a multiplicity of infection (MOI) of 1 to 10.

22. 22. The pharmaceutical composition for use according to claim 21, wherein the pharmaceutical composition is re-administered 4 to 8 hours after the initial administration.

23. The pharmaceutical composition for use according to any one of claims 14 to 22, wherein the subject is a mammal.

24. A pharmaceutical composition for use according to claim 23, wherein the mammal is a human.

25. A method for generating data for diagnosing a causative pathogenic agent of a bacterial infection, comprising: (i) culturing a tissue sample from a patient; (ii) contacting the culture of step (i) with a purified bacteriophage of claim 1 or 2; and (iii) monitoring the culture for evidence of growth or lysis, wherein evidence of lysis of the culture indicates that the culture contains a bacterial strain known to be susceptible to the bacteriophage or protein used in step (ii); The method comprising:

26. 26. The method of claim 25, wherein the tissue sample is a tissue biopsy or swab taken from the patient's respiratory tract.

27. 11. An in vitro method for reducing or inhibiting bacterial colonization or growth on a surface, comprising contacting the surface with a purified bacteriophage of claim 1 or 2 or a pharmaceutical composition of any of claims 3 to 10.

28. 28. The method of claim 27, wherein the surface is a non-biological surface.

29. The method of claim 28, wherein the non-biological surface is a surface of a hospital instrument or facility.

30. The method described in claim 29, wherein the surface of an in-hospital instrument or in-hospital equipment is the surface of a surgical instrument or surgical equipment.

31. A pharmaceutical composition according to any one of claims 3 to 13 for use in reducing or inhibiting symptoms associated with bacterial colonisation or proliferation on mucous membranes in contact with the bacteria.

32. 32. The pharmaceutical composition for use according to claim 31, wherein the mucosa is mammalian skin or mucosa, or human skin or mucosa.

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