Phage therapy for escherichia coli infection

Abstract

The present invention relates to phage therapy. More specifically, the present invention relates to novel phages with high specificity against Escherichia coli strains, their manufacture, components thereof, compositions comprising them and their use in phage therapy.

Description

[0001] This invention relates to novel bacteriophages, compositions comprising said bacteriophages, their manufacture, and their uses. The invention is particularly applicable to the treatment of infections in mammals and to improving the condition of subjects by modifying their gut microbiota. Background Technology

[0002] Bacteriophages are small viruses that exhibit the ability to infect and kill bacteria without affecting cells from other organisms. First described by William Twort almost a century ago and independently discovered shortly thereafter by Félix d'Herelle, over 6,000 different bacteriophages have been discovered and morphologically described to date, including viruses of bacteria and archaea. The vast majority of these viruses are tailed, while a smaller number are polyhedral, filamentous, or pleomorphic. They can be classified according to their morphology, genetic contents (DNA or RNA), specific host, habitat (marine viruses or other habitats), and life cycle. As intracellular parasites of bacterial cells, bacteriophages exhibit different life cycles within their bacterial hosts: lytic, lysogenic, pseudolysogenic, and chronic infection (Weinbauer, 2004; Drulis-Kawa, 2012). Lysogenic bacteriophages, as a normal part of their life cycle, cause lysis of the host bacterial cell. Lysogenic phages (also known as temperate phages) can replicate using a lytic life cycle and cause host bacterial lysis, or they can incorporate their DNA into the host bacterial DNA and become non-infectious prephages. Regardless of the phage's life cycle type, the first step is attachment to receptors on the bacterial cell wall before the phage can enter the bacteria. This specific process affects the range of possible phage-bacteria interactions.

[0003] Bacteriophages are often used as research tools to modify bacteria in laboratory experiments.

[0004] Due to their target host cell specificity, bacteriophages have been considered for use as a therapy to treat acute and chronic infections, particularly in dermatology, ophthalmology, urology, dentistry, pediatrics, otolaryngology, or surgery. However, the concept of this therapeutic use of bacteriophages to treat bacterial infections has been highly controversial from the outset and has not been widely accepted by the public or the medical community. Early studies were widely criticized for the lack of suitable controls and inconsistent results. The lack of reproducibility and numerous conflicting results obtained in various published studies led the Council on Pharmacy and Chemistry of the American Medical Association to conclude that the therapeutic value of lysate filtrate is largely contradictory and unconvincing, and recommended further research to confirm its claimed benefits.

[0005] Since the introduction of antibiotics in the 1940s, this field of therapeutics has received little attention, especially in the Western world. However, the indiscriminate use of antibiotics has led to the widespread emergence and spread of antibiotic-resistant bacteria worldwide, causing increasingly serious problems. Therefore, overcoming the limited remaining treatment options for major multidrug-resistant microorganisms has become a major therapeutic challenge.

[0006] Furthermore, many pathogenic microorganisms reside within biofilms, which pose additional problems when designing novel antimicrobial agents. In this regard, bacteria that grow as biofilms rather than in single-celled (“plankton”) form tend to be particularly resistant to antimicrobial agents, and the host immune system is especially unable to provide a suitable response.

[0007] *Escherichia coli*, belonging to the genus *Escherichia* and family *Enterobacteriaceae*, is a Gram-negative, short rod-shaped bacterium that exhibits high diversity and frequency of occurrence in the human and animal microbiota. It has been revealed that although most *E. coli* strains are non-pathogenic, they can cause opportunistic infections. Furthermore, certain *E. coli* strains are highly pathogenic and can cause a wide variety of diseases and sepsis in mammals, including humans. Several reports have associated *Enterobacter* *Escherichia coli* with one of the most common infections in patients of all age groups: skin and soft tissue infections (SSTIs). In some moderate to severe cases, these infections require hospitalization and parenteral therapy. In particular, *Escherichia coli* has been found to be a pathogen of neonatal omphalitis (Fraser et al., 2006), cellulitis of the lower or upper extremities (Brzozowski et al., 1997; Corredoira et al., 1994), necrotizing fasciitis (Afifi et al., 2008; Krebs et al., 2001), surgical site infections (Tourmousoglou et al., 2008), and post-burn infections (Rodgers et al., 2000). A seven-year surveillance study of SSTIs covering three continents (Europe, Latin America, and North America) showed that *Escherichia coli* is an important pathogen, as it is the third most prevalent species isolated. Therefore, *Escherichia coli* should be treated with specific and targeted therapies, especially considering the significant decrease in antibiotic susceptibility of pathogenic *Escherichia coli* strains in recent years, their diversity, and their prominent presence in the gut microbiota.

[0008] Furthermore, *Escherichia coli* bacteria are capable of forming biofilms, which contribute to their increased resistance to antibiotics. These biofilms may contain more than one type of bacteria supported and surrounded by a secreted extracellular matrix, and help the bacteria colonize a wide variety of surfaces. Biofilms allow bacteria to attach to surfaces and reach population densities that would otherwise be unsustainable, providing increased resistance not only to antibiotics but also to many environmental stresses, including toxins such as heavy metals, bleach, and other cleaning agents. Bacteria in biofilms are known to be 100 to 1000 times more resistant to antibiotics than the same bacterial strains growing in planktonic form. This increased resistance means that bacteria that appear sensitive to antibiotics in laboratory tests may be resistant to therapy in a clinical context. Even if some bacteria are eliminated, biofilms can provide a reservoir of resistance, allowing for rapid colonization once antibiotics are no longer present. Therefore, biofilms are clearly an important factor in many human diseases. Chemotherapy is unsuitable for combating biofilms because this is precisely the situation they have evolved to resist. Physical abrasion does offer a means of disrupting biofilms. Unfortunately, many surfaces that support bacterial pathogenesis, such as bones, joints, and implanted medical devices, are not well-suited for harsh abrasion. For example, surfaces from wounds or burns are extremely sensitive and delicate. Even where abrasion is both suitable and routinely used, biofilm removal is limited. Dental plaque on tooth surfaces is a biofilm and is partially removed through daily brushing. However, bacteria remain on unbrushed surfaces (such as in the crevices between teeth) and can rapidly and effectively recombine on cleaned surfaces. It is clear that existing methods for removing biofilm have limited effectiveness.

[0009] Rapid adaptability and the ability to form biofilms are the main reasons why *Escherichia coli* is identified as an opportunistic pathogen. They have acquired hospital pathogen status and can be isolated from clinical samples obtained from wounds, sputum, bladder, urethra, vagina, ear, eye, and respiratory tract. The emergence of resistance to the most potent new antibiotics in these clinical *Escherichia coli* strains, even during treatment, makes the fight against hospital pathogens of *E. coli* extremely challenging.

[0010] Furthermore, it has been reported that the pathological or physiological condition of subjects is affected by the microbial balance in their gut microbiota. Therefore, improving or restoring the gut microbiota by eliminating Escherichia coli populations is also a valuable method for improving the condition of subjects.

[0011] Therefore, there is a great need for new antibacterial agents or compositions that can be used to eliminate Escherichia coli strains, even in tissues within bacterial biofilms, suitable for use in human or animal therapies and for the sterilization of materials. Invention Overview

[0013] The inventors have isolated and characterized novel bacteriophages exhibiting specific lytic activity against *Escherichia coli* (E. coli), which can be used as active agents in pharmaceutical or veterinary formulations, particularly for treating *E. coli* bacterial infections or for improving microbial balance in subjects. The novel bacteriophages of this invention exhibit strong lytic activity and high selectivity, and can be combined to induce controlled destruction of a very wide range of *E. coli* cells.

[0014] One object of the present invention is to provide an antibacterial composition comprising at least one, preferably at least two, bacteriophages having lytic activity against strains of Escherichia coli, said bacteriophages being selected from bacteriophages whose genome contains a nucleotide sequence of any one of SEQ ID NO: 1 to 15 or a sequence having at least 90% identity with it.

[0015] Another object of the present invention relates to a bacteriophage with lytic activity against Escherichia coli strains, said bacteriophage having a genome comprising a nucleotide sequence selected from any one of SEQ ID NO: 1 to 15 or a sequence having at least 90% identity with, preferably at least 97% identity with, such nucleotides. In certain embodiments, the bacteriophage of the present invention exhibits lytic activity against multidrug-resistant strains, particularly against antibiotic-resistant pathogenic Escherichia coli, such as preferably broad-spectrum β-lactamase (ESBL)-producing strains or vemurotoxin-producing Escherichia coli (VTEC) strains.

[0016] Another object of the present invention relates to bacteriophages and variants thereof selected from BP539, BP700, BP753, BP814, BP953, BP954, BP970, BP1002, BP1151, BP1155, BP1168, BP1176, BP1197, BP1226, or BP1229, whose genomes respectively contain the nucleotide sequences of SEQ ID NO: 1 to 15, wherein the variants retain the phenotypic characteristics of the bacteriophages, and wherein the bacteriophages and variants thereof have lytic activity against strains of Escherichia coli.

[0017] Another object of the present invention is a composition comprising at least one phage as defined above. In a particular embodiment, the composition of the present invention comprises at least two different phages as defined above, preferably at least three, and even more preferably at least four different phages as defined above. A particular composition of the present invention comprises a combination of all phages BP539, BP700, BP753, BP814, BP953, BP954, BP970, BP1002, BP1151, BP1155, BP1168, BP1176, BP1197, BP1226, and BP1229.

[0018] On the other hand, the present invention relates to a bacteriophage with lytic activity against pathogenic Escherichia coli strains, wherein the bacteriophage is specifically active against Escherichia coli, against antibiotic-resistant Escherichia coli strains, and has a productive lytic effect of less than 15.

[0019] The present invention also relates to isolated nucleic acid sequences contained in the bacteriophages of the present invention, and isolated polypeptides encoded by said isolated nucleic acids.

[0020] Another object of the present invention is a composition comprising a polypeptide as defined above.

[0021] Another object of the present invention is a composition comprising nucleic acids as defined above.

[0022] The compositions of the present invention typically also contain pharmaceutically or veterinarily applicable excipients or carriers. These may be liquid, semi-liquid, solid, or freeze-dried.

[0023] Another object of the present invention relates to bacteriophages, nucleic acids, polypeptides or compositions as defined above, for use in treating infections in mammals, improving the microbial flora in mammals, sterilizing materials, and / or killing Escherichia coli bacteria or impairing the integrity of bacterial biofilms.

[0024] The present invention also relates to the use of one or more lytic bacteriophages for improving the condition of a subject by modifying the microbial community in the subject. The microbial community can be modified by correcting, altering, or restoring an appropriate balance of microorganisms within the community.

[0025] The present invention also relates to a method for treating an infection in a mammal, the method comprising administering to the mammal at least one phage, nucleic acid, polypeptide or composition as defined above.

[0026] The present invention also relates to a method for treating a surface or material suspected of being contaminated with Escherichia coli, the method comprising applying to the surface or material at least one bacteriophage, nucleic acid, polypeptide, or composition as defined above. The surface or material may be, for example, the surface of any device, container, laboratory material, clothing, etc.

[0027] Another object of the present invention relates to a method for predicting or determining the efficacy of phage therapy in a subject, wherein the method includes the step of determining in vitro the lytic activity of one or more phages of the present invention against *Escherichia coli* strains from a sample of the subject, wherein the lytic activity of one or more phages of the present invention against at least one *E. coli* strain from the sample indicates effective treatment. The method may also optionally include the step of treating the subject with at least one phage having lytic activity against *E. coli* strains from a sample of the subject.

[0028] On the other hand, the present invention provides a method for selecting subjects or determining whether subjects are likely to benefit from phage therapy, wherein the method includes the step of determining in vitro the lytic activity of one or more phages of the present invention against Escherichia coli strains derived from samples of the subject, wherein the lytic activity of one or more phages of the present invention against at least one Escherichia coli strain indicates the responsive subject.

[0029] This invention can be used in any mammal, preferably in humans, or for processing any material, including laboratory materials or medical devices. Brief description of the attached diagram

[0031] Figure 1 The in vitro efficiency of the bacteriophage of the present invention against combinations of Escherichia coli strains under various MOIs.

[0032] Figure 2 The in vivo efficacy of the bacteriophage of the present invention against combinations of Escherichia coli strains at various dosages.

[0033] Figure 3 The in vivo efficacy of the bacteriophage of this invention against infection mediated by SH113 Escherichia coli strain. ΦIV: intravenous treatment; ΦIP: intraperitoneal treatment; ΦSC: subcutaneous treatment; Temp Inf.: untreated control; TemGenta.: gentamicin-treated control.

[0034] Figure 4 The in vivo efficacy of the bacteriophage of this invention against infection mediated by SH113 Escherichia coli strain: dose-effect. Φconc.: complete concentration (10 8 pfu / ml); Φ1 / 10: concentration diluted 10 times; Φ1 / 100: concentration diluted 100 times; Φ1 / 1000: concentration diluted 1000 times; Tem infect.: control for antibiotic treatment of infection. Invention Details

[0036] This invention relates to novel bacteriophages, their components, compositions comprising said bacteriophages, their manufacture, and their use as antibacterial agents, particularly for treating infections in mammals and improving the condition of said subjects by modifying the microbial flora in said subjects.

[0037] definition

[0038] To facilitate understanding of this invention, many terms are defined below.

[0039] When used herein, the term "phage" refers to a functional phage particle containing a nucleic acid genome packaged in a protein envelope or capsid. The term also refers to portions of a phage including, for example, a head, or assemblies of phage components that provide substantially the same functional activity.

[0040] The term "phenotypic characteristics" more preferably refers to the morphology and / or host range of a bacteriophage. Methods for determining bacteriophage phenotypes are known in the past and include, for example, determining bacterial host range and / or activity against biofilms produced by certain bacterial strains.

[0041] When used in this invention, the term "lytic activity" refers to the ability of a bacteriophage to cause lysis of bacterial cells. The lytic activity of a bacteriophage can be tested on strains of Escherichia coli using techniques known per se in the art (see also the Experimental Section).

[0042] The term "variant" of a reference phage refers to a phage that has variations in its genomic sequence and / or the polypeptide encoded by it compared to the reference phage, while retaining the same phenotypic characteristics as the reference phage. Variants typically contain, for example, silent mutations, conserved mutations, minor deletions, and / or minor duplications of genetic material, and retain the phenotypic characteristics of the reference phage. In a preferred embodiment, the variants of the present invention retain any observable features or characteristics dependent on the genome of the phage of the present invention, i.e., the phenotypic characteristics of the phage, and / or lytic activity against *Escherichia coli* strains. Preferred variants have less than 5% nucleic acid variation compared to the genome of the reference phage, even more preferably less than 4%, and even more preferably less than 2%. Alternatively or in combination, the variant preferably has less than 5% amino acid variation in the encoded polypeptide sequence compared to the polypeptide of the reference phage.

[0043] For nucleic acid sequences, the term "% identity" refers to the level of identity or homology between the sequences and can be determined by techniques known per se in the art. Typically, the % identity between two nucleic acid sequences is determined using computer programs, such as GAP (Program Manual for the Wisconsin Package, Version 8, August 1996, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711) provided in the GCG package (Needleman, SB and Wunsch, CD, (1970), Journal of Molecular Biology, 48, 443-453). Using settings adjusted to, for example, DNA sequences (specifically: a GAP generation penalty of 5.0 and a GAP expansion penalty of 0.3), nucleic acid molecules can be aligned with each other using Pileup alignment software available as part of the GCG package.

[0044] The term "fragment" of nucleic acid generally refers to a fragment having at least 10 consecutive nucleotides of said nucleic acid, more preferably a fragment having at least 15, 20, 25, 30, 35, 40, 50 or more consecutive nucleotides of said nucleic acid.

[0045] The term "fragment" of a polypeptide generally refers to a fragment having at least 5 consecutive amino acids of the polypeptide, more preferably having at least 10, 15, 20, 30, 40, 50 or more consecutive amino acids of the polypeptide.

[0046] The term "ESBL-resistant Escherichia coli strain" refers to antibiotic-resistant Escherichia coli, and more specifically, to Escherichia coli strains that produce broad-spectrum β-lactamases.

[0047] The term "VTEC" refers to another type of antibiotic-resistant Escherichia coli strain, more specifically, VTEC strains that produce vemurotoxin.

[0048] For bacteriophages, the term "specific" or "specific" refers to the type of host that the phage can infect. Specificity is typically mediated by the tail filaments of the phage, which are involved in the interaction with receptors expressed on the cell. More preferably, an "Escherichia coli-specific" phage refers to a phage that can infect one or more strains of Escherichia coli and cannot infect non-Escherichia coli bacteria under physiological conditions.

[0049] When used herein, the term “peptide” refers to a polypeptide of any size, including, for example, small peptides of 5 to 20 amino acids, longer polypeptides, proteins, or fragments thereof.

[0050] The term "PLE" or "productive lysis effect" refers to the ratio between the amount of a given phage released and the productive lysis time. Release amount and productive lysis time are parameters that define phage-host interactions and correspond, respectively, to the average yield of phage particles produced by a bacterium infected by a phage and the time taken for free phage to lyse the bacterial cell.

[0051] In the context of this specification, the term "isolated phage" should be understood to mean material taken from its native environment. For phages, the term specifically refers to, for example, phages that have been cultured, purified, and / or cultured separately from their native environment. For nucleic acids or peptides, the term "isolated" refers to, for example, nucleic acid molecules or peptides separated from at least some components of their native environment, such as proteins, lipids, and / or nucleic acids.

[0052] When used herein, the term "pharmaceutical or veterinary-grade" means any material (e.g., carrier, excipient, or medium) compatible with use in mammalian subjects. These materials include physiologically acceptable solutions or media that are harmless to the organism or do not elicit any significant specific or nonspecific immune response or disintegrate the biological activity of the active compound. To formulate the composition into a liquid dosage form, saline, sterile water, Ringer's solution, buffered physiological saline, albumin infusion solution, dextran solution, maltodextrin solution, glycerol, ethanol, and mixtures thereof can be used as pharmaceutical or veterinary-grade excipients or carriers. Other conventional additives such as thickeners, diluents, buffers, preservatives, surfactants, antioxidants, and antibacterial agents may be added if necessary. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to the composition to prepare injectable formulations such as aqueous solutions, suspensions, and emulsions; oral formulations such as pills, capsules, granules, or tablets; or powder formulations.

[0053] When used herein, “PFU” refers to plaque-forming unit, as clearly defined in the art. A lysing phage lyses the host cell, creating a clear zone (or plaque) on a culture plate. Theoretically, each plaque is formed by one phage, and the number of plaques multiplied by the dilution factor equals the total number of phages in the experimental preparation.

[0054] The term "treatment" or "therapy" refers to both curative and preventative treatment of a disease. Curative treatment is defined as treatment that causes a cure of the disease, or treatment that alleviates, improves, and / or eliminates, reduces, and / or stabilizes the symptoms of the disease or the suffering it causes directly or indirectly. Preventative treatment includes both treatment that causes prevention of the disease and treatment that reduces and / or delays the incidence of the disease or the risk of its occurrence.

[0055] The term "mammal" includes human subjects as well as non-human mammals such as pets (e.g., dogs, cats), horses, ruminants, sheep, goats, pigs, etc.

[0056] When used herein, the term "biofilm" refers to heterogeneous bacterial formations grown on various surfaces; preferably, bacterial communities embedded in an extracellular polysaccharide matrix attached to a solid biological or non-biological surface.

[0057] When used herein, the term “damage” refers to any alteration of integrity. Damage to a bacterial biofilm is understood as the invasion of the biofilm by a bacteriophage, infection or lysis of biofilm-associated bacteria, and / or partial or complete removal of the biofilm (i.e., by stopping colonization and / or disrupting the biofilm).

[0058] When used herein, the term "sample" refers to any sample containing cells. Examples of such samples include fluids such as blood, plasma, saliva, or urine, as well as biopsy, organ, tissue, or cell samples. Samples may be processed before use.

[0059] When used herein, the terms “subject” or “patient” refer to an animal, preferably a mammal, and even more preferably a human, including adults and children. However, the term “subject” also encompasses non-human animals, particularly mammals such as dogs, cats, horses, cows, pigs, sheep, and non-human primates.

[0060] When used herein, the term “efficacy” of treatment or “response” to phage therapy refers to treatment that results in a reduction in the number of Escherichia coli strains in a subject after phage therapy compared to the number of E. coli strains prior to treatment. A “good response” subject is a subject who has shown or will show clinically significant improvement when treated with phage therapy.

[0061] The term "mixture" or "composition" of bacteriophages refers to a combination of two or more different bacteriophages. The bacteriophages in the mixture / composition are preferably formulated together, i.e., in the same container or package, although they can also be used as a set of components, in which the bacteriophages (or some bacteriophages) are formulated or packaged separately and combined when used or administered.

[0062] Description of Implementation

[0063] This invention relates to novel phage therapy. More specifically, this invention relates to novel phages with high specificity against strains of Escherichia coli, their manufacture, their components, compositions comprising them, and their use in phage therapy.

[0064] bacteriophage:

[0065] In a first aspect, the present invention discloses the isolation and characterization of novel phages that exhibit lytic activity against Escherichia coli strains specifically and individually or in combination, displaying a remarkable host-wide spectrum. These phages are selected, isolated, and characterized from environmental samples. As indicated, the phages, individually and in combination, possess activity against Escherichia coli strains. They are remarkably effective against pathogenic Escherichia coli strains, such as antibiotic-resistant Escherichia coli strains. Furthermore, the phages of the present invention exhibit a significant productive lytic effect (“PLE”) of less than 15, more preferably less than 10, and even more preferably between 0.1 and 10. Moreover, the phages of the present invention are specific to Escherichia coli strains, i.e., they do not induce lysis in non-Escherichia coli bacteria. As will be further explained, the present invention shows that these phages can be combined and formulated into a state suitable for use as pharmaceutical or veterinary agents to exhibit a targeted and very strong antibacterial effect against a controlled range of Escherichia coli strains.

[0066] More specifically, the following bacteriophages have been selected and characterized. Their corresponding nucleic acid sequences have also been annotated.

[0067] Table 1

[0068] SEQ ID number bacteriophage SEQ ID NO: 1 BP539 SEQ ID NO: 2 BP700 SEQ ID NO: 3 BP753 SEQ ID NO: 4 BP814 SEQ ID NO: 5 BP953 SEQ ID NO: 6 BP954 SEQ ID NO: 7 BP970 SEQ ID NO: 8 BP1002 SEQ ID NO: 9 BP1151 SEQ ID NO: 10 BP1155 SEQ ID NO: 11 BP1168 SEQ ID NO: 12 BP1176 SEQ ID NO: 13 BP1197 SEQ ID NO: 14 BP1226 SEQ ID NO: 15 BP1229

[0069] The lysis profiles of these phages have been determined on a wide range of Escherichia coli strains. As disclosed in the table below, these phages were selected based on their potency and synergistic potential. The table presents the lysis effects of the phages on reference and pathogen-resistant strains to demonstrate their high lysis potential.

[0070] Table 2

[0071]

[0072] The shaded boxes are examples of strains of Escherichia coli that have been produced.

[0073] As can be seen from Table 2, the individual bacteriophages have a very strong lytic ability and can produce combinations (or mixtures) of these bacteriophages capable of killing all tested Escherichia coli strains, thereby producing a broad-spectrum antibacterial composition.

[0074] As an example, a mixture of all 15 bacteriophages of the present invention was able to effectively kill all the bacteria listed in Table 2.

[0075] Furthermore, the specificity of the phage has been tested on a variety of non-Escherichia coli strains. More specifically, the experimental portion confirmed that the phage of the present invention has no lytic effect on bacteria selected from Pseudomonas aeruginosa, Acinetobacter baumanii, Enterobacter aerogenes, Enterobacter asburiae, Enterobacter cloacae, Klebsiella pneumoniae, Porteus mirabilis, Staphylococcus aureus, Stenotrophomonas maltophila, and / or Serratia marcescens.

[0076] Therefore, a specific object of the present invention is a bacteriophage having lytic activity against Escherichia coli strains, wherein the genome of the bacteriophage comprises a nucleotide sequence selected from any one of SEQ ID NO: 1 to 15 or a sequence having at least 97% identity with it, preferably at least 98% or 99% identity with it.

[0077] Therefore, a specific object of the present invention is a bacteriophage with lytic activity against Escherichia coli strains, wherein the genome of the bacteriophage has a nucleotide sequence selected from any one of SEQ ID NO: 1 to 15 or is composed of said nucleotide sequence.

[0078] The phages of the present invention can be prepared using standard culturing, isolation, and purification methods. For example, *Escherichia coli* producing bacteria are cultured, infected with a phage sample, and then treated to remove bacterial cells and debris. The enriched phage solution can be plated in a medium, such as agar, containing an embedded susceptible *E. coli* host strain to obtain plaques. Individual plaques can then be picked for subsequent phage purification and amplification. One or more selective amplification cycles of the phages of the present invention can be performed, for example, by mixing the phage with competent *E. coli*, adding growth medium, and incubating under selected experimental growth conditions. After centrifugation, the clarified amplification supernatant is filtered and another selective amplification cycle is performed or the presence of lytic activity is tested. The phage titer in the suspension and the visualization of the plaque morphology of the phages of the present invention can be estimated using known methods, such as plaque counting. Furthermore, as is well known in the art, the bacteriophages of the present invention can be processed into various different forms (liquid, freeze-dried, etc.) by any suitable method for short-term, long-term, frozen or any other type of storage (see Clark, 1962).

[0079] The activity of the phages of the present invention can be evaluated using methods known in the art, such as plaque assays, also known as double agar assays, on the basis of growing the phages using potential host cells and then evaluating their ability to kill the host bacterial cells. In the plaque assay, after incubation in soft agar medium for a period of time, the phages induce lysis of the target *Escherichia coli* strain, producing a clear zone on the plate called a plaque. Preferably, the phages of the present invention, alone or in combination, exhibit lytic activity against pathogenic *Escherichia coli* strains, including antibiotic-resistant *Escherichia coli* strains such as ESBL *Escherichia coli* strains. Furthermore, these phages retain variants of the phage phenotypes (e.g., specificity and lytic activity) and can be produced and / or isolated using techniques known per se in the art.

[0080] In certain embodiments, the present invention relates to BP539 phage or any variant thereof. BP539 phage or any variant thereof can be produced or amplified, for example, in Escherichia coli strain ECOR54. BP539 or any variant thereof is specific to and has lytic activity against strains K12 / DH5, ECOR15, ECOR35, ECOR38, ECOR40, ECOR54, ECOR62, ECOR64 and / or ECOR71. The genome of BP539 comprises the sequence presented in SEQ ID NO: 1 or a sequence having at least 80% identity, more preferably at least 85% identity, more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 1. Isolated nucleic acid sequences from BP539 phage or variants thereof are also provided. The present invention also covers isolated polypeptides encoded by BP539 phage or variants thereof or by isolated nucleic acid sequences derived from BP539 phage of the present invention.

[0081] Another feature of the BP539 phage of the present invention is that the PLE is less than 15, more preferably less than 10, and even more preferably around 0.1.

[0082] In another specific embodiment, the present invention relates to BP700 phage or any variant thereof. BP700 phage or any variant thereof can be produced or amplified, for example, in Escherichia coli strain ECOR55. BP700 or any variant thereof is specific to and has lytic activity against strains ECOR46, ECOR55, ECOR60, and / or ECOR64. The genome of BP700 comprises the sequence proposed in SEQ ID NO:2 or a sequence having at least 80% identity, more preferably at least 85% identity, more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:2. Isolated nucleic acid sequences from BP700 phage or variants thereof are also provided. The present invention also covers isolated polypeptides encoded by BP700 phage or variants thereof or encoded by isolated nucleic acid sequences from BP700 phage of the present invention.

[0083] Another feature of the BP700 phage of the present invention is that the PLE is less than 15, more preferably less than 10, and even more preferably around 2.

[0084] On the other hand, the present invention relates to BP753 phage or any variant thereof. BP753 phage or any variant thereof can be produced or amplified, for example, in Escherichia coli strain ECOR56. BP753 or any variant thereof is specific to and has lytic activity against strains ECOR10, ECOR48, and / or ECOR56. The genome of BP753 comprises the sequence proposed in SEQ ID NO:3 or a sequence having at least 80% identity, more preferably at least 85% identity, more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:3. Isolated nucleic acid sequences from BP753 phage or variant thereof are also provided. The present invention also covers isolated polypeptides encoded by BP753 phage or variant thereof or encoded by isolated nucleic acid sequences from BP753 phage of the present invention.

[0085] Another feature of the BP753 phage of the present invention is that the PLE is less than 15, more preferably less than 10, and even more preferably around 2.

[0086] On the other hand, the present invention relates to BP814 phage or any variant thereof. BP814 phage or any variant thereof can be produced or amplified, for example, in Escherichia coli strain ECOR55. BP814 or any variant thereof is specific to and has lytic activity against strains ECOR46, ECOR50, ECOR55 and / or ECOR64. The genome of BP814 comprises the sequence proposed in SEQ ID NO:4 or a sequence having at least 80% identity, more preferably at least 85% identity, more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:4. Isolated nucleic acid sequences from BP814 phage or variants thereof are also provided. The present invention also covers isolated polypeptides encoded by BP814 phage or variants thereof or encoded by isolated nucleic acid sequences from BP814 phage of the present invention.

[0087] Another feature of the BP814 phage of the present invention is that the PLE is less than 15, more preferably less than 10, and even more preferably around 0.3.

[0088] In another specific embodiment, the present invention relates to BP953 phage or any variant thereof. BP953 phage or any variant thereof can be produced or amplified, for example, in Escherichia coli strain ECOR55. BP953 or any variant thereof is specific to and has lytic activity against strains K12 / DH5, ECOR1, ECOR46, ECOR50, ECOR54, ECOR55, ECOR59 and / or ECOR60. The genome of BP953 comprises the sequence presented in SEQ ID NO: 5 or a sequence having at least 80% identity, more preferably at least 85% identity, more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 5. Isolated nucleic acid sequences from BP953 phage or variant thereof are also provided. The present invention also covers isolated polypeptides encoded by BP953 phage or variant thereof or encoded by isolated nucleic acid sequences from BP953 phage of the present invention.

[0089] Another feature of the BP953 phage of the present invention is that the PLE is less than 15, more preferably less than 10, and even more preferably around 3.

[0090] In another specific embodiment, the present invention relates to BP954 phage or any variant thereof. BP954 phage or any variant thereof can be produced or amplified, for example, in Escherichia coli strain ECOR35. BP954 or any variant thereof is specific to and has lytic activity against strains ECOR24, ECOR35, ECOR38, ECOR40, ECOR46, and / or ECOR62. The genome of BP954 comprises the sequence presented in SEQ ID NO: 6 or a sequence having at least 80% identity, more preferably at least 85% identity, more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 6. Isolated nucleic acid sequences from BP954 phage or variants thereof are also provided. The present invention also covers isolated polypeptides encoded by BP954 phage or variants thereof or encoded by isolated nucleic acid sequences from BP954 phage of the present invention.

[0091] Another feature of the BP954 phage of the present invention is that the PLE is less than 15, more preferably less than 10, and even more preferably around 3.

[0092] On the other hand, the present invention relates to BP970 phage or any variant thereof. BP970 phage or any variant thereof can be produced or amplified, for example, in Escherichia coli strain K12 / DH5. BP970 or any variant thereof is specific to and has lytic activity against strains K12 / DH5, ECOR1, ECOR2, ECOR5, ECOR10, ECOR13, ECOR15, ECOR28 and / or ECOR72. The genome of BP970 comprises the sequence proposed in SEQ ID NO: 7 or a sequence having at least 80% identity, more preferably at least 85% identity, more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 7. Isolated nucleic acid sequences from BP970 phage or variant thereof are also provided. The present invention also covers isolated polypeptides encoded by BP970 phage or variant thereof or encoded by isolated nucleic acid sequences from BP970 phage of the present invention.

[0093] Another feature of the BP970 phage of the present invention is that the PLE is less than 15, more preferably less than 10, and even more preferably around 5.

[0094] In another specific embodiment, the present invention relates to BP1002 phage or any variant thereof. BP1002 phage or any variant thereof can be produced or amplified, for example, in Escherichia coli strain ECOR56. BP1002 or any variant thereof is specific to and has lytic activity against strains ECOR15, ECOR24, ECOR35, ECOR38, ECOR40, ECOR48, ECOR54, ECOR55, ECOR56, ECOR59, ECOR60 and / or ECOR64. The genome of BP1002 comprises the sequence proposed in SEQ ID NO:8 or a sequence having at least 80% identity, more preferably at least 85% identity, more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:8. Isolated nucleic acid sequences from BP1002 phage or variants thereof are also provided. The present invention also covers isolated polypeptides encoded by BP1002 phage or variants thereof or by isolated nucleic acid sequences derived from BP1002 phage of the present invention.

[0095] Another feature of the BP1002 bacteriophage of the present invention is that the PLE is less than 15, more preferably less than 10, and even more preferably around 5.

[0096] In another specific embodiment, the present invention relates to BP1151 phage or any variant thereof. BP1151 phage or any variant thereof can be produced or amplified, for example, in Escherichia coli strain ECOR56. BP1151 or any variant thereof is specific to and has lytic activity against strains K12 / DH5, ECOR2, ECOR13, ECOR15, ECOR24, ECOR35, ECOR38, ECOR40, ECOR46, ECOR48, ECOR50, ECOR54, ECOR56, ECOR59, ECOR60, ECOR62, ECOR64 and / or ECOR71. The genome of BP1151 comprises the sequence presented in SEQ ID NO: 9 or a sequence having at least 80% identity, more preferably at least 85% identity, more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 9. Also provided are isolated nucleic acid sequences from BP1151 phage or variants thereof. The invention also covers isolated polypeptides encoded by BP1151 phage or variants thereof, or encoded by isolated nucleic acid sequences from BP1151 phage of the present invention.

[0097] Another feature of the BP1151 bacteriophage of the present invention is that the PLE is less than 15, more preferably less than 10, and even more preferably around 10.

[0098] On the other hand, the present invention relates to BP1155 phage or any variant thereof. BP1155 phage or any variant thereof can be produced or amplified, for example, in Escherichia coli strain ECOR59. BP1155 or any variant thereof is specific to and has lytic activity against strains K12 / DH5, ECOR2, ECOR13, ECOR15, ECOR28, ECOR46, ECOR50, ECOR54, ECOR55, ECOR59 and / or ECOR71. The genome of BP1155 comprises the sequence proposed in SEQ ID NO: 10 or a sequence having at least 80% identity, more preferably at least 85% identity, more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 10. Isolated nucleic acid sequences from BP1155 phage or variants thereof are also provided. The present invention also covers isolated polypeptides encoded by BP1155 phage or variants thereof or by isolated nucleic acid sequences derived from BP1155 phage of the present invention.

[0099] Another feature of the BP1155 phage of the present invention is that the PLE is less than 15, more preferably less than 10, and even more preferably around 0.5.

[0100] On the other hand, the present invention relates to BP1168 phage or any variant thereof. BP1168 phage or any variant thereof can be produced or amplified, for example, in Escherichia coli strain ECOR59. BP1168 or any variant thereof is specific to and has lytic activity against strains K12 / DH5, ECOR13, ECOR15, ECOR28, ECOR35, ECOR46, ECOR50, ECOR54, ECOR55, ECOR59, ECOR71 and / or ECOR72. The genome of BP1168 comprises the sequence proposed in SEQ ID NO: 11 or a sequence having at least 80% identity, more preferably at least 85% identity, more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 11. Isolated nucleic acid sequences from BP1168 phage or variants thereof are also provided. The present invention also covers isolated polypeptides encoded by BP1168 phage or variants thereof or by isolated nucleic acid sequences derived from BP1168 phage of the present invention.

[0101] Another feature of the BP1168 phage of the present invention is that the PLE is less than 15, more preferably less than 10, and even more preferably around 0.8.

[0102] On the other hand, the present invention relates to BP1176 phage or any variant thereof. BP1176 phage or any variant thereof can be produced or amplified, for example, in Escherichia coli strain ECOR60. BP1176 or any variant thereof is specific to and has lytic activity against strains K12 / DH5, ECOR2, ECOR4, ECOR13, ECOR15, ECOR24, ECOR28, ECOR35, ECOR55, ECOR56, ECOR59 and / or ECOR60. The genome of BP1176 comprises the sequence proposed in SEQ ID NO: 12 or a sequence having at least 80% identity, more preferably at least 85% identity, more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 12. Isolated nucleic acid sequences from BP1176 phage or variants thereof are also provided. The present invention also covers isolated polypeptides encoded by BP1176 phage or variants thereof or by isolated nucleic acid sequences derived from BP1176 phage of the present invention.

[0103] Another feature of the BP1176 phage of the present invention is that the PLE is less than 15, more preferably less than 10, and even more preferably around 4.

[0104] On the other hand, the present invention relates to BP1197 phage or any variant thereof. BP1197 phage or any variant thereof can be produced or amplified, for example, in Escherichia coli strain K12 / DH5. BP1197 or any variant thereof is specific to and has lytic activity against strains K12 / DH5, ECOR2, ECOR13, ECOR15, ECOR28, ECOR46, ECOR48, ECOR50, ECOR54, ECOR59, ECOR60, ECOR71 and / or ECOR72. The genome of BP1197 comprises the sequence proposed in SEQ ID NO: 13 or a sequence having at least 80% identity, more preferably at least 85% identity, more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 13. Isolated nucleic acid sequences from BP1197 phage or variants thereof are also provided. The present invention also covers isolated polypeptides encoded by BP1197 phage or variants thereof or by isolated nucleic acid sequences derived from BP1197 phage of the present invention.

[0105] Another feature of the BP1197 phage of the present invention is that the PLE is less than 15, more preferably less than 10, and even more preferably around 1.

[0106] On one hand, the present invention relates to BP1226 phage or any variant thereof. BP1226 phage or any variant thereof can be produced or amplified, for example, in Escherichia coli strain ECOR59. BP1226 or any variant thereof is specific to and has lytic activity against strains K12 / DH5, ECOR2, ECOR13, ECOR28, ECOR48, ECOR59, ECOR60 and / or ECOR72. The genome of BP1226 comprises the sequence proposed in SEQ ID NO: 14 or a sequence having at least 80% identity, more preferably at least 85% identity, more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 14. Isolated nucleic acid sequences from BP1226 phage or variants thereof are also provided. The present invention also covers isolated polypeptides encoded by BP1226 phage or variants thereof or by isolated nucleic acid sequences derived from BP1226 phage of the present invention.

[0107] Another feature of the BP1226 phage of the present invention is that the PLE is less than 15, more preferably less than 10, and even more preferably around 5.

[0108] In another specific embodiment, the present invention relates to BP1229 phage or any variant thereof. BP1229 phage or any variant thereof can be produced or amplified, for example, in Escherichia coli strain K12 / DH5. BP1229 or any variant thereof is specific to and has lytic activity against strains K12 / DH5, ECOR2, ECOR13, ECOR15, ECOR28, ECOR46, ECOR48, ECOR50, ECOR54, ECOR55, ECOR59, and / or ECOR72. The genome of BP1229 comprises the sequence proposed in SEQ ID NO: 15 or a sequence having at least 80% identity, more preferably at least 85% identity, more preferably 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 15. Isolated nucleic acid sequences from BP1229 phage or variants thereof are also provided. The present invention also covers isolated polypeptides encoded by BP1229 phage or variants thereof or by isolated nucleic acid sequences derived from BP1229 phage of the present invention.

[0109] Another feature of the BP1229 phage of the present invention is that the PLE is less than 15, more preferably less than 10, and even more preferably around 6.

[0110] Nucleic acids and polypeptides

[0111] This invention also relates to nucleic acids or any fragments of such nucleic acids contained in the bacteriophages of this invention. The term "fragment" more preferably refers to a fragment containing (or constituting) an open reading frame. The nucleic acid may be DNA or RNA, and may be single-stranded or double-stranded.

[0112] The nucleic acid may be isolated from preserved bacteriophages or produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning), enzymatic or chemical synthesis, or a combination thereof, according to techniques generally known in the art. It also includes its homologous sequences and fragments, including but not limited to natural allelic variants and nucleic acid sequences in which nucleotides have been inserted, deleted, substituted, and / or inverted.

[0113] In a particular embodiment, the present invention relates to nucleic acids comprising a sequence selected from any of SEQ ID NO: 1-15 or a sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with any of SEQ ID NO: 1-15.

[0114] In another particular embodiment, the present invention relates to nucleic acids comprising a fragment of a sequence selected from any of SEQ ID NO: 1-15 or a fragment of a sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with any of SEQ ID NO: 1-15, said fragment comprising an open reading frame or a regulatory element such as a promoter.

[0115] In a particular embodiment, the present invention relates to nucleic acids having a sequence selected from any of SEQ ID NO: 1-15 or a sequence having at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with any of SEQ ID NO: 1-15, or nucleic acids composed of said sequences.

[0116] The nucleic acids of this invention can be in free form or cloned in a vector.

[0117] On the other hand, the present invention also relates to isolated polypeptides encoded by nucleic acid sequences selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15. The polypeptides can be produced by techniques known per se in the art, such as synthesis, recombinant techniques, or combinations thereof. The polypeptides can be isolated or purified and used as antibacterial agents or as reagents for in vitro analysis.

[0118] The composition of the present invention

[0119] One aspect of the invention relates to compositions comprising at least one, more preferably at least two or more phages as described above, and optionally pharmaceutically or veterinarily applicable excipients. As described, the phages of the present invention exhibit very strong lytic activity against Escherichia coli strains. Combinations of these phages can be generated to expand the host range and produce highly effective antibacterial compositions.

[0120] More specifically, the present invention relates to an antibacterial composition comprising at least two bacteriophages having lytic activity against strains of Escherichia coli, wherein the at least two bacteriophages are selected from bacteriophages whose genome contains a nucleotide sequence of any one of SEQ ID NO: 1 to 15 or has at least 90% identity with it, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it.

[0121] In a preferred embodiment, the composition of the present invention comprises at least three, or more preferably at least four, different phages selected from phages whose genomes contain nucleotide sequences of any one of SEQ ID NO: 1 to 15, or have at least 90% identity with them, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them. The composition of the present invention may comprise at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all 15 of the different phages disclosed above.

[0122] One aspect of the present invention relates to a composition comprising at least one bacteriophage selected from BP539, BP700, BP753, BP814, BP953, BP954, BP970, BP1002, BP1151, BP1155, BP1168, BP1176, BP1197, BP1226 and / or BP1229 and variants thereof.

[0123] The present invention also contemplates a composition comprising at least two different phages selected from BP539, BP700, BP753, BP814, BP953, BP954, BP970, BP1002, BP1151, BP1155, BP1168, BP1176, BP1197, BP1226 and / or BP1229 and variants thereof.

[0124] Preferably, the composition of the present invention comprises at least three different phages, more preferably at least four different phages, more preferably at least five different phages, more preferably at least six different phages, more preferably at least seven different phages, more preferably at least eight different phages, more preferably at least nine different phages, and more preferably at least ten different phages, wherein the phages are selected from BP539, BP700, BP753, BP814, BP953, BP954, BP970, BP1002, BP1151, BP1155, BP1168, BP1176, BP1197, BP1226 and / or BP1229 and variants thereof.

[0125] In a particular embodiment, the composition of the present invention comprises BP539 in combination with at least one other phage selected from BP700, BP753, BP814, BP1151, BP1176 and BP1168.

[0126] In another specific embodiment, the composition of the present invention comprises a combination of BP1002 with at least one other phage selected from BP1151, BP1155, BP1168, BP1176 and BP1197.

[0127] In another specific embodiment, the composition comprises a combination of BP1155 with at least one other phage selected from BP1168, BP1197, BP1226, BP1229 and BP1176.

[0128] In another preferred embodiment, the composition comprises BP1151 in combination with at least one other phage selected from BP1176, BP953, BP970, BP700 and BP1002.

[0129] In another preferred embodiment, the composition comprises BP953 and / or BP1168 and / or BP1176, optionally in further combination with at least one other phage of the present invention.

[0130] Specifically, the present invention relates to a composition comprising a combination of bacteriophages BP953 and BP1168. Such a composition can kill 100% of the tested Escherichia coli strains and nearly 70% of the 25 Escherichia coli species listed in Table 2 (see Example 3.1).

[0131] The present invention also relates to a composition comprising a combination of bacteriophages BP953+BP1168+BP1229. Such a composition can kill Escherichia coli of types O157, O144, and O104, including hemorrhagic strains (see Example 3.2).

[0132] The present invention also relates to a composition comprising a combination of bacteriophages BP953+BP1151+BP1155+BP1176. Such a composition can kill 80% of all tested Escherichia coli bacteria isolated from a hospital (see Example 3.3).

[0133] The present invention also relates to a composition comprising a combination of bacteriophages BP700+BP953+BP970+BP1002+BP1176. Such a composition can kill 100% of the tested ST131 type Escherichia coli bacteria, at least 80% of the tested BLSE type Escherichia coli bacteria, and at least 93% of the tested BMR type Escherichia coli bacteria (see Example 3.4).

[0134] The present invention also relates to a composition comprising a combination of bacteriophages BP1002+BP1151+BP1155+BP1168+BP1176. Such a composition can kill 100% of the tested Escherichia coli bacteria that cause meningitis (see Example 3.5).

[0135] The present invention also relates to a composition comprising a combination of bacteriophages BP539+BP700+BP753+BP814+BP1151+BP1176+BP1168. Such a composition can kill approximately 96% of the 24 Escherichia coli species in the ECOR repository in Table 2 (see Example 3.6).

[0136] This invention also relates to a composition comprising all bacteriophages BP539, BP700, BP753, BP814, BP953, BP954, BP970, BP1002, BP1151, BP1155, BP1168, BP1176, BP1197, BP1226 and / or BP1229 or a combination thereof. Such a composition can kill 100% of the 25 Escherichia coli species listed in Table 2 (see Example 3.7).

[0137] Specific examples of the compositions of the present invention include:

[0138] - Phages whose genome contains the nucleotide sequence of SEQ ID NO: 5 or a sequence having at least 90% identity with it, and phages whose genome contains the nucleotide sequence of SEQ ID NO: 11 or a sequence having at least 90% identity with it;

[0139] - Phages whose genome contains the nucleotide sequence of SEQ ID NO: 5 or a sequence having at least 90% identity with it, phages whose genome contains the nucleotide sequence of SEQ ID NO: 11 or a sequence having at least 90% identity with it, and phages whose genome contains the nucleotide sequence of SEQ ID NO: 15 or a sequence having at least 90% identity with it;

[0140] - Phages whose genome contains the nucleotide sequence of SEQ ID NO: 5 or a sequence having at least 90% identity with it, phages whose genome contains the nucleotide sequence of SEQ ID NO: 9 or a sequence having at least 90% identity with it, phages whose genome contains the nucleotide sequence of SEQ ID NO: 10 or a sequence having at least 90% identity with it, and phages whose genome contains the nucleotide sequence of SEQ ID NO: 12 or a sequence having at least 90% identity with it;

[0141] - Phages whose genome contains the nucleotide sequence of SEQ ID NO: 2 or a sequence having at least 90% identity with it, phages whose genome contains the nucleotide sequence of SEQ ID NO: 5 or a sequence having at least 90% identity with it, phages whose genome contains the nucleotide sequence of SEQ ID NO: 7 or a sequence having at least 90% identity with it, phages whose genome contains the nucleotide sequence of SEQ ID NO: 8 or a sequence having at least 90% identity with it, and phages whose genome contains the nucleotide sequence of SEQ ID NO: 12 or a sequence having at least 90% identity with it;

[0142] - Phages whose genome contains the nucleotide sequence of SEQ ID NO: 8 or a sequence having at least 90% identity with it; phages whose genome contains the nucleotide sequence of SEQ ID NO: 9 or a sequence having at least 90% identity with it; phages whose genome contains the nucleotide sequence of SEQ ID NO: 10 or a sequence having at least 90% identity with it; phages whose genome contains the nucleotide sequence of SEQ ID NO: 11 or a sequence having at least 90% identity with it; and phages whose genome contains the nucleotide sequence of SEQ ID NO: 12 or a sequence having at least 90% identity with it; or

[0143] - Phages whose genome contains the nucleotide sequence of SEQ ID NO: 1 or a sequence having at least 90% identity with it, phages whose genome contains the nucleotide sequence of SEQ ID NO: 2 or a sequence having at least 90% identity with it, phages whose genome contains the nucleotide sequence of SEQ ID NO: 3 or a sequence having at least 90% identity with it, phages whose genome contains the nucleotide sequence of SEQ ID NO: 4 or a sequence having at least 90% identity with it, phages whose genome contains the nucleotide sequence of SEQ ID NO: 9 or a sequence having at least 90% identity with it, phages whose genome contains the nucleotide sequence of SEQ ID NO: 12 or a sequence having at least 90% identity with it, and phages whose genome contains the nucleotide sequence of SEQ ID NO: 11 or a sequence having at least 90% identity with it.

[0144] Specific embodiments of the present invention relate to a composition comprising:

[0145] - The genome contains the nucleotide sequence of SEQ ID NO: 1 or a phage having at least 90% identity with it;

[0146] - A bacteriophage whose genome contains the nucleotide sequence of SEQ ID NO: 2 or a sequence having at least 90% identity with it;

[0147] - A bacteriophage whose genome contains the nucleotide sequence of SEQ ID NO: 3 or a sequence having at least 90% identity with it;

[0148] - The genome contains the nucleotide sequence of SEQ ID NO: 4 or a phage having at least 90% identity with it;

[0149] - A bacteriophage whose genome contains the nucleotide sequence of SEQ ID NO: 5 or a sequence having at least 90% identity with it;

[0150] - The genome contains the nucleotide sequence of SEQ ID NO: 6 or a phage having at least 90% identity with it;

[0151] - The genome contains the nucleotide sequence of SEQ ID NO: 7 or a phage having at least 90% identity with it;

[0152] - The genome contains the nucleotide sequence of SEQ ID NO: 8 or a phage having at least 90% identity with it;

[0153] - The genome contains the nucleotide sequence of SEQ ID NO: 9 or a phage having at least 90% identity with it;

[0154] - A bacteriophage whose genome contains the nucleotide sequence of SEQ ID NO: 10 or a sequence having at least 90% identity with it;

[0155] - A bacteriophage whose genome contains the nucleotide sequence of SEQ ID NO: 11 or a sequence having at least 90% identity with it;

[0156] - A bacteriophage whose genome contains the nucleotide sequence of SEQ ID NO: 12 or a sequence having at least 90% identity with it;

[0157] - A bacteriophage whose genome contains the nucleotide sequence of SEQ ID NO: 13 or a sequence having at least 90% identity with it;

[0158] - A bacteriophage whose genome contains the nucleotide sequence of SEQ ID NO: 14 or a sequence having at least 90% identity with it; and

[0159] - A bacteriophage whose genome contains the nucleotide sequence of SEQ ID NO: 15 or a sequence having at least 90% identity with it.

[0160] The compositions of the present invention exhibit lytic activity against the bacterial pathogen *Escherichia coli*. The compositions of the present invention may also contain other antibacterial agents, particularly other bacteriophages with different host specificities.

[0161] The preferred composition of the present invention has lytic activity against antibiotic-resistant Escherichia coli strains.

[0162] Other preferred compositions of the present invention exhibit lytic activity against more than 90% of the bacterial strains in the EcoR repository, which is a reference repository of Escherichia coli strains found in nature.

[0163] The antibacterial compositions of the present invention can take various forms, such as liquid, semi-liquid, solid or freeze-dried formulations.

[0164] In one embodiment of the invention, the composition comprises 10 e2 Up to 10 e12 Between PFUs, preferably 10 e5 Up to 10 e10 At least one of the phages of the present invention among PFUs would be ideal. When the antibacterial composition comprises several phages as defined above, the composition preferably comprises 10. e2 Up to 10 e12 Each of the present invention's phages present between PFUs.

[0165] The compositions of the present invention may contain any effective amount of the selected phage. Preferably, they contain 10 e2 Up to 10 e12 Between PFUs, preferably 10 e5 Up to 10 e10 Each of the aforementioned phages between PFUs. The relative amount of each type of phage in the composition of the present invention can be adjusted by a person skilled in the art. Generally, when the antibacterial composition contains several (n) different phages as defined above, the total relative amount %A of each phage in the composition is more preferably %A = (100 / n) i )xV, where n iV represents the number of different phage types, and is a variability factor between 0.2 and 5. Most preferably, V is between 0.3 and 3, even more preferably between 0.5 and 2, and generally between 0.8 and 1.5. In a typical embodiment, when the antibacterial composition contains several phages as defined above, the composition preferably contains 10... e2 Up to 10 e12 Each of the PFUs present in the present invention contains a phage of the present invention. Preferably, each type of phage is present in the composition of the present invention in approximately equal relative amounts.

[0166] The compositions of the present invention preferably contain a suitable diluent or carrier, such as a pharmaceutically or veterinarily applicable excipient or carrier. In addition to the selected bacteriophage, the compositions of the present invention may include any excipient or carrier, such as thickeners, diluents, buffers, preservatives, surfactants, etc. These include physiologically acceptable solutions or media that are harmless to the organism or do not induce any significant specific or nonspecific immune response in the organism or do not negate the biological activity of the bacteriophage. For liquid formulations, saline, sterile water, Ringer's solution, buffered physiological saline, albumin infusion, dextran solution, maltodextrin solution, glycerol, ethanol, and mixtures thereof can be used as pharmaceutically or veterinarily applicable excipients or carriers. Other conventional additives such as thickeners, diluents, buffers, preservatives, surfactants, antioxidants, and antibacterial agents may be added if necessary. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to the composition to prepare injectable formulations such as aqueous solutions, suspensions, and emulsions, oral formulations such as pills, capsules, granules, or tablets, or powder formulations. Preparations for topical drug delivery may include adhesive bandages, dressings, patches, films, ointments, lotions, creams, gels, drops, suppositories, sprays, aerosols, tampons, sanitary napkins, liquids, and powders. Preparations for disinfection or for medical use may also include aerosols or sprays.

[0167] The compositions of the present invention can be used in the medical field, including human or veterinary medicine, for example, to treat infections in mammals or to improve the condition of subjects.

[0168] The composition can be used to kill Escherichia coli bacteria in organisms for the treatment of infections. The composition can also be used to improve the condition of mammals by modifying their microbial flora. Specifically, the composition of the present invention can specifically remove Escherichia coli strains from the skin or mucous membranes of mammals, thereby modifying their microbial flora and restoring a suitable balance.

[0169] In certain embodiments, the invention also relates to a method for treating an infection in a mammal, the method comprising administering to the mammal a composition, bacteriophage, nucleic acid, or polypeptide as defined above.

[0170] In a particular embodiment, the method includes administering a phage selected from at least one, preferably at least two, or even more preferably at least three phages selected from BP539, BP700, BP753, BP814, BP953, BP954, BP970, BP1002, BP1151, BP1155, BP1168, BP1176, BP1197, BP1226 and / or BP1229 or variants thereof.

[0171] The present invention also relates to the use of the described compositions, bacteriophages, nucleic acids or peptides for the manufacture of pharmaceuticals for the treatment of infections in mammals or the restoration of microbial flora in said mammals.

[0172] The compositions or agents of the present invention can be administered via any convenient route, including intravenous, oral, transdermal, subcutaneous, mucosal, intramuscular, intrapulmonary, intranasal, parenteral, rectal, vaginal, and topical administration. In a preferred embodiment, the phage or composition is administered via a topical route, for example, by application to the skin of a subject. The composition can be administered directly or indirectly, for example, through a support. In this regard, the composition can be applied or sprayed onto the affected area, for example. The compositions of the present invention can also be administered orally or parenterally. The dosage suitable for application, spraying, or administration of the compositions of the present invention can be adjusted by those skilled in the art based on various factors including the formulation, route of administration, age, weight, sex, condition, diet, route of administration, and responsiveness of the mammal to be treated. Physicians with ordinary skills in the art can readily determine and prescribe the desired effective amount of the composition.

[0173] Dosage can also be adjusted by a professional technician to achieve lytic activity against antibiotic-resistant Escherichia coli strains. Depending on the route of administration, effective agents that achieve lytic activity in vivo typically include at least 10 e2 PFU / ml, preferably about 10 e2 Up to 10 e12 The concentration of PFU / ml. Dosing can be administered only once, or repeated if necessary.

[0174] The compositions of the present invention can be administered to treat Escherichia coli infections, typically respiratory, urinary, burn, traumatic, ear, skin and soft tissue, gastrointestinal, or postoperative infections.

[0175] As shown in the experimental section, the phages and compositions of the present invention are capable of selectively killing *Escherichia coli* bacteria in vitro or in vivo. The compositions can even disrupt mixtures of different *Escherichia coli* bacteria in vivo, even at low doses. Furthermore, the compositions of the present invention can effectively kill bacteria embedded in biofilms, which is particularly important for pathogenic bacteria. Additionally, the compositions and phages of the present invention strictly do not affect mammalian cells, and are therefore specific in vivo and without side effects.

[0176] This invention also relates to the use of the compositions, bacteriophages, nucleic acids, or peptides of this invention for the disinfection of materials. Due to their potent antibacterial effects and their ability to even disrupt the integrity of bacterial biofilms, the compositions of this invention can be used as disinfectants to eliminate bacteria on materials or at least reduce their number. Such methods can be applied to the treatment of a wide variety of biological or non-biological surfaces in both medical and non-medical contexts, including solid materials or devices such as contact lenses, surfaces of devices to be implanted in the body, tubes, catheters, laboratory containers, fabrics, etc.

[0177] The diagnostic / predictive test of this invention:

[0178] The present invention also relates to a method for predicting or determining the efficacy of phage therapy in a subject, wherein the method includes determining the lytic activity of one or more phages selected from BP539, BP700, BP753, BP814, BP953, BP954, BP970, BP1002, BP1151, BP1155, BP1168, BP1176, BP1197, BP1226 and / or BP1229 against a strain of *Escherichia coli* from a sample of the subject, such lytic activity indicating effective treatment. Preferably, the method optionally further includes treating the subject with one or more phages having lytic activity against a strain of *Escherichia coli* from a sample of the subject.

[0179] On the other hand, the present invention provides a method for selecting subjects or determining whether subjects are likely to benefit from phage therapy, wherein the method includes the step of determining the lytic activity of one or more phages selected from BP539, BP700, BP753, BP814, BP953, BP954, BP970, BP1002, BP1151, BP1155, BP1168, BP1176, BP1197, BP1226 and / or BP1229 against Escherichia coli strains from samples of said subject, wherein the lytic activity of one or more phages of the present invention against at least one Escherichia coli strain indicates the responsive subject.

[0180] Another object of the present invention relates to a method for predicting a subject's response to phage therapy, wherein the method includes the step of determining the lytic activity of one or more phages selected from BP539, BP700, BP753, BP814, BP953, BP954, BP970, BP1002, BP1151, BP1155, BP1168, BP1176, BP1197, BP1226 and / or BP1229 against a strain of *Escherichia coli* derived from a sample of the subject, wherein the lytic activity of one or more phages of the present invention against at least one strain of *Escherichia coli* indicates a good response to the therapy.

[0181] Other aspects and advantages of the invention will be disclosed in the experimental section below, which is merely illustrative. Example

[0182] Materials and methods

[0183] bacteriophage isolation and preparation

[0184] MDR Escherichia coli bacteria were used to isolate and enrich each virulent phage from environmental water. Environmental samples were mixed with an overnight culture of the bacteria in Luria Bertani (LB) and incubated with shaking at 37°C for 24 h to enrich specific phages. At the end of the incubation, a few drops of chloroform were added to the culture. The culture was centrifuged at 11,000 g for 5 min to remove bacterial cells and debris. The supernatant was passed through a 0.2 μm filter to remove residual bacterial cells. The enriched phage solution was plated on LB agar plates containing Escherichia coli. Plasma formation occurred on the plates after incubation at 37°C for 24 h. Individual plasma were picked for subsequent phage purification and amplification. The phages were then stored in suspension in LB broth or physiological saline at 4°C.

[0185] Plaque counting is used to estimate the titer of bacteriophages in a suspension (Postic, 1961). A 10-fold dilution of the suspension is applied to dried bacterial colonies of the propagating strain. The plates are read after an overnight incubation. Plaque counting also allows for visualization of plaque morphology.

[0186] Host range determined

[0187] The host range of bacteriophages was determined in a group of 26 *Escherichia coli* strains from the ECOR repository. 10 9 Bacterial cells were mixed with melted agar, and the mixture was poured onto solid agar to create a double-layer agar plate. After solidification, isolated phage stock solutions were spotted onto each plate containing a different bacterial strain. After allowing the spotted samples to be absorbed for 20 minutes, the plates were inverted and incubated at 37°C for 24 hours, and then the degree of lysis was recorded.

[0188] Electron microscopy

[0189] Each phage was photographed using a transmission electron microscope.

[0190] Sequencing, analysis and annotation of bacteriophage genomes

[0191] To isolate phage DNA, the phage was propagated as described above. The phage DNA was isolated by extraction with phenol:chloroform:isoamyl alcohol (25:24:1, V / V), ethanol precipitation, and redissolution in water. Whole-genome sequencing was performed, and the BLAST algorithm was used to determine similarity to genes described in the National Center for Biotechnology Information (NCBI) database. The genome was scanned to obtain potential open reading frames (ORFs).

[0192] Example 1: Bacteriophage-host characteristics and dynamics

[0193] A one-step growth experiment was performed as previously described to first determine the productive lysis time, adsorption rate, and then the phage release amount. To determine the adsorption rate, samples were taken at different time intervals to analyze free phage particles in the solution. For determining the productive time and phage release amount, *Escherichia coli* bacteria were mixed with the phage solution, and phage adsorption was allowed for 15 min. The mixture was immediately centrifuged at 5000 rpm for 10 min to remove free phage particles. The precipitate was resuspended in 5 aliquots of fresh LB medium, and the culture was incubated at 37°C. Samples were taken at 3-min intervals to determine the phage titer. These results allowed for the calculation of the number of phages produced per bacterium (release amount), productive time, and productive lysis effect (PLE), as shown in Table 3 below.

[0194] Table 3

[0195]

[0196] These results show that all phages possess strong viral production capacity and adsorption rates. Most phages have a PLE below 5, demonstrating remarkable distribution. Phage 539 is particularly effective in this regard. Furthermore, different PLEs and adsorption times allow for the production of mixtures with selected variability.

[0197] Example 2: Preparation of the Mixture

[0198] The following mixed compositions were constructed, each containing 10 -9 Up to 10 -11Each phage between PFUs:

[0199] Table 4

[0200] mixture bacteriophage I BP953+BP1168 II BP953+BP1168+BP1229 III BP953+BP1151+BP1155+BP1176 IV BP700+BP953+BP970+BP1002+BP1176 V BP1002+BP1151+BP1155+BP1168+BP1176 VI BP539+BP700+BP753+BP814+BP1151+BP1176+BP1168

[0201] The following two additional mixed compositions containing all the various phages were constructed, covering the most important diversity of Escherichia coli species.

[0202] Table 5A: Mixture Composition A :

[0203] bacteriophage BP539 BP700 BP 753 BP 814 BP953 BP954 BP970 BP1002 titer <![CDATA[2.72 E+09 ]]> <![CDATA[8.00 E+09 ]]> <![CDATA[2.27 E+08 ]]> <![CDATA[5.89 E+07 ]]> <![CDATA[4.53 E+10 ]]> <![CDATA[3.00 E+08 ]]> <![CDATA[4.02 E+08 ]]> <![CDATA[9.73 E+08 ]]>

[0204] bacteriophage BP1151 BP1155 BP1168 BP1176 BP1197 BP1226 B1229 titer <![CDATA[1.56 E+09 ]]> <![CDATA[3.00 E+10 ]]> <![CDATA[7.77 E+09 ]]> <![CDATA[1.00 E+10 ]]> <![CDATA[3.91 E+09 ]]> <![CDATA[4.44 E+06 ]]> <![CDATA[9.11 E+09 ]]>

[0205] Table 5B: Mixed Composition B :

[0206] bacteriophage BP539 BP700 BP 753 BP 814 BP953 BP954 BP970 BP1002 titer <![CDATA[8.85 E+08 ]]> <![CDATA[1.89 E+08 ]]> <![CDATA[7.26 E+07 ]]> <![CDATA[3.04 E+08 ]]> <![CDATA[9.47 E+08 ]]> <![CDATA[3.89 E+08 ]]> <![CDATA[9.56 E+07 ]]> <![CDATA[2.09 E+09 ]]>

[0207] bacteriophage BP1151 BP1155 BP1168 BP1176 BP1197 BP1226 B1229 titer <![CDATA[7.35 E+08 ]]> <![CDATA[2.57 E+09 ]]> <![CDATA[3.01 E+09 ]]> <![CDATA[1.77 E+09 ]]> <![CDATA[1.03 E+10 ]]> <![CDATA[2.00 E+09 ]]> <![CDATA[1.56 E+09 ]]>

[0208] Example 3: Bacterial sensitivity to the bacteriophage mixture of the present invention

[0209] Using the phage mixture of the present invention, at 2.10 9 The concentration of bacteriophages / ml was used to test various bacterial strains. Different bacterial concentrations were plated on a 2.10 mL solution. 9 The mixture was prepared by adding 1 phage / ml of phage mixture and incubating at 37°C for 24 hours.

[0210] The mixture was tested on different *Escherichia coli* bacteria listed in Table 2, as well as additional *Escherichia coli* bacteria, including meningitis-causing bacteria (37 strains), BLSE (5 strains) and ST131 (9 strains) types of *Escherichia coli* from the R. Debré collection, *Escherichia coli* bacteria derived from hospitalized patients (35 strains), and hemorrhagic *Escherichia coli* bacteria of types O157, O144, and O104 (3 strains). The percentage of bacterial species susceptible to the mixture is listed in Table 6 below:

[0211] Example 3.1: Efficacy of Mixture I

[0212] As shown in Table 6 below, Mixture I was able to destroy 100% of the tested Escherichia coli bacteria.

[0213] Table 6

[0214]

[0215] In addition, mixture I can also destroy nearly 70% of the 25 Escherichia coli bacteria listed in Table 2.

[0216] Example 3.2: Efficacy of Mixture II

[0217] As shown in Table 7 below, Mixture II was able to destroy 100% of the tested Escherichia coli bacteria.

[0218] Table 7

[0219]

[0220] In addition, Mixture II can also destroy 76% of the 25 Escherichia coli bacteria listed in Table 2.

[0221] Example 3.3: Efficacy of Mixture III

[0222] As shown in Table 8 below, Mixture III was able to destroy at least 80% of the tested Escherichia coli strains isolated from hospitalized patients.

[0223] Table 8

[0224]

[0225]

[0226] Example 3.4: Efficacy of Mixture IV

[0227] As shown in Table 9 below, Mixture IV was able to destroy ST131 and BLSE type Escherichia coli strains.

[0228] Table 9

[0229]

[0230] Example 3.5: The efficacy of mixture V

[0231] As shown in Table 10 below, mixture V is able to destroy 100% of the meningitis-causing Escherichia coli bacteria from the R Debré repository.

[0232] Table 10

[0233]

[0234]

[0235] Example 3.6: The efficacy of mixture VI

[0236] Mixture VI was able to destroy nearly 96% of the 24 Escherichia coli species listed in Table 2 in the ECOR repository.

[0237] Example 3.7: Efficacy of mixtures A and B

[0238] Both mixtures A and B can destroy 100% of the 25 Escherichia coli bacteria listed in Table 2.

[0239] The bacteria were further counted and used to calculate the resistance rate (number of bacteria after incubation / number of bacteria on the plate). Table 11 below shows the resistance rates using mixture A containing 15 different types of bacteriophages:

[0240] Table 11

[0241] bacteria Resistance rate (bacteria / ml) ECOR1 >1.00E-02 ECOR24 2.00E-05 ECOR60 4.00E-06 S22 1.18E-04 S106 <1.00E-06 S182 2.00E-06 SH5 1.00E-06 SH113 1.00E-06 Astrid 9 <1.00E-06 BSE 3 8.50E-05 BSE 7 5.00E-06 0157:133 2.63E-04 XXT 1.74E-04

[0242] All the bacteria tested were sensitive to the compositions of the present invention.

[0243] Example 4: Mixture Specificity

[0244] The specificity of the mixture was verified by testing on 10 bacterial species, including Pseudomonas aeruginosa, Acinebacter baumanii, Enterobacter aerogenes C, Enterobacter asburiae, Enterobacter cloacae, Klebsiella pneumoniae, Porteus mirabilis, Staphylococcus aureus, Stenotrophomonas maltophila, and Serratia marcescens.

[0245] Table 12 summarizes the lytic activity observed for each phage when used alone or as a mixture of 15 phages.

[0246] Table 12

[0247]

[0248] The table above clearly shows that there is no lytic activity against bacteria other than Escherichia coli strains. Therefore, the bacteriophages and mixtures of the present invention are highly specific to Escherichia coli strains.

[0249] Example 5: Efficiency of bacteriophages against Escherichia coli strains in vitro

[0250] Several strains from the EcoR repository were selected to represent the genetic diversity of *Escherichia coli* and various forms of antibiotic resistance. The strains were sensitive to or resistant to one or more antibiotics. They were grown individually or in combinations of 2 to 8 strains. Phage mixtures were grown at concentrations of 1 to 10. e-6 The MOI is added at a dilution rate of 1 to 1 million (bacteria / phage).

[0251] The results show Figure 1 And in Table 13 below.

[0252] Table 13: Efficacy of the obtained phage mixture against Escherichia coli mixture in vitro:

[0253] With 2.10 e7 CFU / ml density and various dilutions:

[0254] mixture MOI 1 MOI 0.1 MOI 0.01 MOI 0.001 MOI 0.0001 MOI 0.000001 1 type of bacteria ++ ++ ++ ++ + + 2 types of bacteria ++ ++ + + / - + / - + / - 3 types of bacteria ++ ++ + + + / - + / - 4 types of bacteria ++ ++ ++ + + / - + / - 5 types of bacteria ++ ++ ++ + + + / - 6 types of bacteria ++ ++ + + / - + / - + / - 7 types of bacteria ++ ++ ++ + + / - + / - 8 types of bacteria ++ ++ ++ + + / -

[0255] The composition of the present invention is capable of killing a mixture of eight different strains of Escherichia coli. At a dilution of 1 / 1000, the mixed composition remains effective against all eight strains.

[0256] Example 6: Efficiency of bacteriophages against Escherichia coli strains in vivo

[0257] The SH113 strain isolated from burn patients in 2011 was used in the following experiments.

[0258] The SH113 strain is resistant to ampicillin, ticarcillin, cefotaxime, cefotaxime, nalidixic acid, norfloxacin, ofloxacin, and ciprofloxacin.

[0259] SKH1 mice (or hairless mice) were used as a mouse model of Escherichia coli infection.

[0260] Instructions: (See Table 14 below)

[0261] - Immunosuppression was achieved in mice by administering 1.5 mg cyclophosphamide (Cy) IP injections every 2 days for a total of 3 times, starting from day 3 before infection.

[0262] - Burns were inflicted on the skin of mice using 2 μl of liquid mustard gas at a concentration of 30 mg / kg.

[0263] - Two days after the burn, infection was prevented by subcutaneous injection of bacterial suspension at the burn site.

[0264] Table 14

[0265]

[0266] The mixed composition was prepared according to Example 2 and applied with 10 [units of something] before day 0. e7 The phage mixture of 1 phage / ml is used to wet the pressure cloth.

[0267] Various concentrations of Escherichia coli strains were tested using 100 μl phage mixtures. For example... Figure 2 As shown above, all Escherichia coli strains were killed 6 hours after treatment.

[0268] Following subcutaneous injection of the SH113 strain of *Escherichia coli* into SKH1 mice, all mice died without further treatment. Remarkable survival was observed in mice treated with the phage mixture shown in Table 9 above (see Table 9). Figure 3 Survival rates were 100% for SKH1 mice treated subcutaneously or intravenously, and 65% for those treated intraperitoneally. In comparison, 80% survival was observed in SKH1 mice treated with double doses of the antibiotic gentamicin at day 0+6h and for seven consecutive days, including two injections on day 1.

[0269] At 1 / 10, 1 / 100 and 1 / 1000 of the mixture of the present invention (i.e., 10 per mouse) 5 A remarkable 100% survival rate was also achieved after subcutaneous treatment with PFU dilution (see [link]). Figure 4 ).

[0270] Therefore, the compositions of the present invention can treat infections in vivo and can induce 100% survival in infected mice.

[0271] References

[0272] Afifi, RY, and AAEl-Hindawi. 2008. Acute necrotizing fasciitis in Egyptian patients: a case series. Int. J. Surg. 66-14.

[0273] Brzozowski D. and DC Ross. 1997. Upper limb Escherichia coli cellulitis in the immunocompromised, J. Hand Surg. 22678-680

[0274] Clark WA, 1962, Appl Microbiol. Comparison of several methods for preserving bacteriophages, 1962 Sep; 10:466-71.

[0275] Corredoira, JM, J. Ariza, R. Pallares, J. Carratala, P.P. Viladrich, G. Rufi, R. Verdaguer, and F. Gudiol. 1994. Gram-negative bacillary cellulitis in patients with hepatic cirrhosis. Eur. J. Clin. Microbiol. Infect. Dis. 1319-24

[0276] Drulis-Kawa Z, Majkowska-Skrobek G, Maciejewska B, Delattre AS, Lavigne R, 2012, Learning from bacteriophages—advantages and limitations of phage and phage-encoded protein applications; 13(8):699-722.

[0277] Fraser, N., BWDavies and J. Cusack. 2006. Neonatal omphalitis: a review of its serious complications. Acta Paediatr. 95519-522.

[0278] Krebs, VL, KMKoga, EMDiniz, MECeccon, and FAVaz. 2001. Necrotizing fasciitis in a newborn infant: a case report, Rev. Hosp. Clin. Fac. Med. Sao Paulo 5659-62.

[0279] Needleman SB, Wunsch CD, A general method applicable to the search for similarities in the amino acid sequence of two proteins, 1970 Mar; 48(3):443-53.

[0280] Rodgers, GL, J. Mortensen, M. C. Fisher, A. Lo, A. Cresswell, and SSLong. 2000. Predictors of infectious complications after burn injuries in children. Pediatr. Infect. Dis.; 19(10): 990-5.

[0281] Stone R. 2002. Bacteriophage therapy. Stalin's forgotten cure, Science 298, 728–731 (DOI:10.1126 / science.298.5594.728)

[0282] Tourmousoglou, CE, ECYiannakopoulou, V. Kalapothaki, J. Bramis and J. St. Papadopoulos. 2008. Surgical-site infection surveillance in general surgery: a critical issue. J. Chemother. 20(3): 312-318.

[0283] Weinbauer MG. Ecology of prokaryotic viruses, FEMS Microbiol Rev 2004; 28:127-81.

Claims

1. An antibacterial composition comprising at least (a) an antibacterial agent against Escherichia coli (Escherichia coli). E. coli (a) a phage with lytic activity against strains of Escherichia coli, the genome of which consists of the nucleotide sequence of SEQ ID NO: 9; and (b) a phage with lytic activity against strains of Escherichia coli, the genome of which consists of the nucleotide sequence selected from any one of SEQ ID NO: 1 to 8 and 10 to 15.

2. The composition of claim 1, comprising at least (a) a phage having lytic activity against Escherichia coli strains, the genome of said phage consisting of the nucleotide sequence of SEQ ID NO: 9; and (b) two phages having lytic activity against Escherichia coli strains, the genome of said phage consisting of the nucleotide sequence selected from any one of SEQ ID NO: 1 to 8 and 10 to 15.

3. The composition of claim 1, comprising at least (a) a phage having lytic activity against Escherichia coli strains, the genome of said phage consisting of the nucleotide sequence of SEQ ID NO: 9; and (b) three phages having lytic activity against Escherichia coli strains, the genome of said phage consisting of the nucleotide sequence selected from any one of SEQ ID NO: 1 to 8 and 10 to 15.

4. The composition of claim 1, comprising any one of the following combinations of bacteriophages: - Phages whose genomes consist of the nucleotide sequence of SEQ ID NO: 5, phages whose genomes consist of the nucleotide sequence of SEQ ID NO: 9, phages whose genomes consist of the nucleotide sequence of SEQ ID NO: 10, and phages whose genomes consist of the nucleotide sequence of SEQ ID NO: 12; or - Phages whose genomes consist of the nucleotide sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12; or - Phages whose genomes consist of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 9, SEQ ID NO: 12, and SEQ ID NO:

11.

5. The composition of claim 1, comprising a combination of all phages BP539, BP700, BP753, BP814, BP953, BP954, BP970, BP1002, BP1151, BP1155, BP1168, BP1176, BP1197, BP1226 and BP1229, each of which consists of the nucleotide sequence of SEQ ID NO: 1 to 15.

6. The composition of any one of claims 1 to 5, which has lytic activity against antibiotic-resistant Escherichia coli strains.

7. The composition of any one of claims 1 to 5, which has lytic activity against more than 90% of the bacterial strains in the EcoR repository.

8. The composition of any one of claims 1 to 5, further comprising a pharmaceutically acceptable excipient or carrier.

9. The composition of any one of claims 1 to 5, wherein it is a liquid, semi-liquid, solid or lyophilized formulation.

10. The composition of any one of claims 1 to 5, comprising 10 e2 Up to 10 e12 Each phage between PFUs.

11. Use of the composition of any one of claims 1 to 10 in the preparation of a medicament for treating Escherichia coli infection in mammals.

12. Use of the composition of any one of claims 1 to 10 in the preparation of a medicament for improving the condition of a mammal by modifying the microbial flora of a mammal infected with Escherichia coli.

13. Use of the composition of any one of claims 1 to 10 for disinfecting materials infected with Escherichia coli.

14. A method for preparing a composition according to any one of claims 1 to 10, the method comprising separately producing the one or more phages and combining the phages with a suitable carrier or excipient.

15. A bacteriophage having lytic activity against strains of Escherichia coli, wherein the genome of said bacteriophage consists of the nucleotide sequence of SEQ ID NO:

9.

16. An isolated nucleic acid consisting of the nucleotide sequence of SEQ ID NO:

9.

17. Use of the bacteriophage of claim 15 or the nucleic acid of claim 16 in the preparation of a medicament for treating Escherichia coli infection in mammals.

18. Use of the bacteriophage of claim 15 or the nucleic acid of claim 16 in the preparation of a medicament for restoring the microbial flora of mammals infected with Escherichia coli.

19. Use of the bacteriophage of claim 15 or the nucleic acid of claim 16 for disinfection of materials infected with Escherichia coli.

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