Compositions comprising antibacterial bacteriophages and methods of use thereof

Abstract

The present invention is directed to phage therapy for the treatment and control of bacterial infections, in particular urinary tract infections caused by Escherichia coli. More specifically, the present invention is directed to novel bacteriophage strains, cocktails thereof and variants thereof; and to methods using the same in the treatment, reduction or prevention of bacterial infections. The cocktail is used as a first line treatment against urinary tract infections, in particular in cancer patients, diabetic patients, or patients with a weakened immune system. Disclosed herein is a pharmaceutical composition comprising one or more purified strains of bacteriophage, each purified strain having a genome with at least 98% sequence identity to a nucleotide sequence selected from SEQ. ID No. 1, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 and / or variants thereof. The pharmaceutical composition may also be used in combination with other therapies for urinary tract infections such as antibiotics. The pharmaceutical composition may also be used in a method of diagnosis of the causative agent of a bacterial infection. A bacteriophage comprising at least 95% sequence identity with SEQ. ID No. 1, SEQ ID NO. 5 or SEQ ID NO. 6 is also disclosed.

Description

[0001] COMPOSITIONS COMPRISING ANTIBACTERIAL BACTERIOPHAGES AND METHODS OF USE THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention is directed to the field of phage therapy for the treatment and control of bacterial infections, in particular urinary tract bacterial infections.

[0004] STATE OF THE ART

[0005] Bacteriophages (phages) are viruses that specifically infect and lyse bacteria. Phage therapy, a method of using whole phage viruses for the treatment of bacterial infectious diseases, was introduced in the 1920s by Felix d'Herelle. With the development of antibiotics in the 1940s, however, interest in phage-based therapeutics declined in the Western world. One of the most important factors that contributed to this decline was the lack of standardized testing and methods of production. The failure to develop industry wide standards for the testing of phage therapies interfered with the documentation of study results, leading to a perceived lack of efficacy, as well as problems of credibility, regarding the value of phage therapy. Another problem in phage production was related to the purity grade of commercial preparations of phage, with preparations containing undesired bacterial components, e.g., endotoxins. Accordingly, adverse events were often associated with the preparations, particularly in patients receiving them intravenously. Nevertheless, in Eastern Europe and the former Soviet Union, where access to antibiotics was limited, the development and use of phage therapy continued jointly with, or in place of, antibiotics. Further, with the rise of antibiotic resistant strains of many bacteria, interest in phage-based therapeutics has returned in the Western world. That is, even though novel 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 for controlling, preventing, and treating bacterial infections.

[0006] Phage therapy, and phage cocktails in particular, present an alternative to antibiotics for the treatment of bacterial infections, and in particular, for the treatment of urinary tract infections (UTIs), including complicated and non-complicated infections. Phage cocktails are well suited to treating infections caused by antibiotic-resistant bacteria.

[0007] UTI is a severe public health problem of the urinary tract, that can range from simple cases such as cystitis (lower UTI - bladder), pyelonephritis (upper UTI - kidneys), to severe cases such as uroseptic shock. This infection is mostly caused by bacteria although other pathogens may also be accountable. UTI affects 150 million people each year worldwide. It is responsible for about 30%~40% of nosocomial infections and 8% to 21% occur in Intensive-Care Units (ICU).

[0008] UTIs are more common in women and girls because their urethras are shorter and closer to the rectum, which makes it easier for bacteria to enter the urinary tract. Other factors that can increase the risk of UTIs include: (i) a previous UTI; (ii) sexual activity, and especially a new sexual partner; (iii) changes in the bacteria that live inside the vagina (vaginal flora), for example caused by menopause or use of spermicides; (iv) pregnancy; (v) age (older adults and young children are more likely to get UTIs); (vi) structural problems in the urinary tract, such as prostate enlargement; (vii) poor hygiene, particularly in children who are pottytraining; and (viii) immunosuppression, as in the case of cancer and transplant patients.

[0009] UTIs may affect individuals who are otherwise healthy and have no structural or neurological urinary tract abnormalities. These infections are differentiated into lower UTIs (cystitis) and upper UTIs (pyelonephritis) and have several risk factors associated including a prior UTI, sexual activity, vaginal infection, diabetes, obesity, and genetic susceptibility.

[0010] Complicated UTIs are associated with factors increasing colonization and decreasing efficacy of therapy and usually involve one or all of the following complicating factors: anatomic or functional abnormality of urinary tract (enlarged prostate, stone disease, diverticulum, neurogenic bladder, etc.); immunocompromised host; multi-drug resistant bacteria; additional factors that compromise the urinary tract or host defense, including urinary obstruction, urinary retention caused by neurological disease, renal failure, renal transplantation, pregnancy and the presence of foreign bodies such as calculi, indwelling catheters or other drainage devices.

[0011] UTIs in patients who have urinary tract catheters or those who were catheterized in the 48 hours before infection are known as catheter-related UTIs (CR-UTIs). CR-UTIs are the most frequent healthcare-associated UTIs (HAUTIs). CR-UTIs are the most frequent hospital infections. More than 1 million patients in acute-care hospitals and nursing homes develop such infections in the USA annually. The bacteriuria incidence that may be attributed to indwelling catheterization is 3-8% per day. 26% of hospitalized patients who have indwelling catheters for 2 to 10 days are expected to develop bacteriuria, 24% of patients who have bacteriuria will develop UTIs, and 3.6% will have bacteriemia from a urinary tract source. CR- UTIs are related to increased mortality and morbidity and are the most frequent causes of secondary bloodstream infections. A study conducted in 29 intensive care units (ICUs) from ten countries showed that CR-UTIs increased the length of ICU stay by 1.59 days on average, and the mortality risk by 15%. Catheterization duration is thought to be the most important risk factor for CR-UTIs. Over 4 million patients acquire healthcare-associated infections in the European Union every year, 20-30% of which are considered preventable. HAUTIs represent the largest subtype among all healthcare-associated infections. The prevalence of HAUTIs assessed in regional studies ranges from 12.9% in the US and 19.6% in Europe to up to 24% in developing countries. The dominant pathogen in all conditions was uropathogenic Escherichia coli (UPEC) (39.7%), although it comprises a lower proportion in UTIs Enterococcus spp. (11.5%), Klebsiella spp. (11.1%), and Pseudomonas aeruginosa (10.8%) encompassed approximately one-third of infections.

[0012] Antibiotherapy is routinely used to address UTI bacterial infections but the therapeutic options for the multidrug-resistant (MDR) bacteria, especially Gram-negative bacteria, are scarce. Drugs commonly recommended for simple UTIs include trimethoprim / sulfamethoxazole, fosfomycin, nitrofurantoin, cephalexin and ceftriaxone. Cefiderocol is a new cephalosporin designed to treat multidrug-resistant gram-negative bacteria that is used to treat complicated UTIs (including pyelonephritis) in patients with limited or no other treatment options. However, it is expected, as for other chemotherapeutics, that sooner or later bacteria will develop mechanisms of overcoming its activity.

[0013] Khawaldeh et al. reported on the success of adjunctive bacteriophage therapy after repeated failure of antibiotics alone. However, this article is silent relating to the use of the referred bacteriophages in infections caused by Escherichia coli (E. coli), does not disclose the bacteriophages per se and refers to a combinatory therapy of bacteriophages and antibiotics Leitner et al, 2017 hypothesize that intravesical bacteriophage treatment in patients with UTIs due to E. coli and other uropathogens, shows a 40% increase in success rate (normalization of urine culture defined as no evidence of bacteria, i.e. <104colony forming units / mL) as compared to placebo treatment within 7 days. This difference is considered to be clinically relevant. The author also hypothesizes that bacteriophage treatment is non-inferior to antibiotic treatment in terms of treatment success rates, with a non-inferiority margin of 35%. However, this article is directed to intravesical bacteriophages for treating UTIs in patients undergoing transurethral resection of the prostate specifically, which is not the purpose and / or use proposed for the present invention.

[0014] International patent application number PCT / PT2011 / 000031 (published as WO 2012036580) filed by the applicant discloses bacteriophage SEQ. ID NO.4, therein identified as F488 / 08.

[0015] EP 3352773 discloses compositions and methods used to treat, prevent, or ameliorate an infection, for example, an infection in the gastrointestinal tract, or bowel. The bacteriophages disclosed in EP 3352773 are genetically modified and are silent relating to its use in the treatment of UTIs in patients with cancer. EP 3091992 discloses new bacteriophages presenting the specific lytic activity to E. coli. EP 3091992 is silent regarding use of the bacteriophages of the present invention in the treatment of UTIs in patients with cancer, diabetes or weakened immune systems.

[0016] EP 3749342 discloses compositions and methods for treating or preventing a bacterial- associated cancer in a patient by administration of a bacteriophage composition. Preferably, the composition comprises at least one bacteriophage that targets the bacteria. The bacteriophage(s) may be specific for the bacteria or a strain. This document refers specifically to bacteriophages intended to lyse carcinogenic bacteria, i.e. oncogenic bacteria.

[0017] TECHNICAL PROBLEMS SOLVED BY THE INVENTION

[0018] The aim of the present invention is to provide novel and inventive E. coli bacteriophages that may be used in the form of a phage cocktail to treat bacterial infections. Optionally, the present invention may be used as a first-line treatment for cancer patients, diabetic patients or other patients that have compromised immune responses and hence are prone to UTIs caused by E. coli strains.

[0019] As is well known, cancer patients, are much more prone to bacterial infections than healthy, not immune compromised individuals. Many factors increase the susceptibility of immunosuppressed cancer patients to infection, such as neutropenia resulting from aggressive therapy, alteration of the normal flora due to frequent administration of antibiotics, and damage to epithelial surfaces caused by cytotoxic agents. The use of E. coli bacteriophages to combat infections caused by this microorganism substitutes the use of antibiotics reducing the administration of antibiotics in this population, thereby preventing the imbalance of the normal flora since the bacteriophage cocktail is highly specific. Another potential benefit of the proposed alternative treatment with the bacteriophages cocktails disclosed herein is the improved life expectancy of these patients as it has been shown that the use of antibiotics during oncological treatment reduces the average life expectancy of patients.

[0020] The present invention proposes a potential solution to treat UTIs in patients that have their immunity compromised. The bacteriophages are harmless to human cells contrary to cytotoxic compounds used to treat oncologic diseases. The cocktails of the present invention may be used to target one of the most prevalent microorganisms in UTIs, thereby reducing antibiotic use in these patients. In fact, the present cocktails may be used as a first line treatment against UTIs in cancer patients.

[0021] For all the above reasons the present invention may contribute to an increased life expectancy of cancer patients. Since the phages comprising the cocktails of the present invention are capable of infecting multidrug-resistant strains, their use may help reduce the transmission of multiresistant clones both within the target population and the broader community, where other immunocompromised individuals may also benefit.

[0022] SUMMARY OF THE INVENTION

[0023] Compositions comprising one or more purified strains of bacteriophage are disclosed herein. Each of said purified strains has a genome which comprises at least 98% sequence identity to the nucleotide sequence selected from the group consisting of SEQ. ID No. 1, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 and / or variants thereof which maintain the lytic activity of the bacteriophage, and has antibacterial activity against E.coli. The composition may be a pharmaceutical composition. Use of a therapeutically or prophylactically effective amount of the composition in the treatment, management or prevention of a bacterial infection in a subject in need thereof is also disclosed herein. The subject may be a mammal, optionally a human. Use of the composition in a method of diagnosis of the causative agent of a bacterial infection and / or a method of assessing whether a bacterial strain is susceptible to the lytic activity of a bacteriophage is also disclosed herein. Use of the composition in reducing or inhibiting colonization or growth of bacteria on a surface by contacting the surface with the composition is also disclosed herein. The surface may be a mucus membrane of a mammal, optionally a mucus membrane of the urinary tract of a human.

[0024] The bacteriophages used in the compositions disclosed herein have been deposited: SEQ ID NO. 1 has deposit accession number NCIMB 43922, SEQ ID NO. 4 has deposit accession number NCIMB 43928, SEQ ID NO. 5 has deposit accession number NCIMB 44322, and SEQ ID NO. 6 has deposit accession number NCIMB 44323. These bacteriophages can lyse E. coli strains 86 / 13-ECO1 (NCIMB accession number 43923), 63 / 12-ECO3 (NCIMB accession number 43927) and 18 / 20-ECO4 (NCIMB accession number 44321).

[0025] A therapeutically or prophylactically effective amount of a composition disclosed herein may be particularly useful in treating, reducing or preventing the occurrence of a bacterial infection caused by an E. coli bacterial strain, including bacterial strains showing resistance to one or more known antibiotics and / or that are highly pathogenic and / or that are capable of forming a biofilm.

[0026] A therapeutically or prophylactically effective amount of a composition disclosed herein may be particularly useful in treating, preventing or reducing the occurrence of UTIs, optionally complicated UTIs. In addition, a therapeutically or prophylactically effective amount of a composition disclosed herein may be particularly useful in treating, preventing or reducing the occurrence of UTIs (including complicated UTIs) in cancer patients, in diabetic patients, or in patients with weakened immune systems. Patients with weakened immune systems will include, for example, immunosuppressed individuals, such as those with autoimmune diseases or transplant recipients requiring immunosuppressive therapy.

[0027] Use of a therapeutically or prophylactically effective amount of the composition disclosed herein may comprise administering the composition as a suspension or as an emulsion to the bladder.

[0028] Use of a therapeutically or prophylactically effective amount of the composition disclosed herein may comprise administration of the composition as a first line treatment and readministration approximately 4-8 hours later.

[0029] Use of a therapeutically or prophylactically effective amount of the composition disclosed herein may comprise use in combination with an antibiotic agent having antibacterial activity against E.coli.

[0030] Use of the composition disclosed herein to diagnose the causative agent of a bacterial infection and / or assess whether a bacterial strain is susceptible to the lytic activity of a bacteriophage may comprise:

[0031] (i) culturing a samplefrom a patient;

[0032] (ii) contacting the culture of step (i) the composition; and

[0033] (iii) monitoring for evidence of cell growth or lysis of the culture, wherein evidence of lysis of the culture indicates that the culture comprises a bacterial strain known to be susceptible to the one or more purified strains of bacteriophage in the composition.

[0034] DEFINITIONS

[0035] As used herein, the term "phage cocktail" or "cocktail" refers to a composition comprising different bacteriophages. The cocktail may comprise at least two different purified bacteriophages, for example, two, three or four different purified bacteriophages, or variants thereof.

[0036] As used herein, the term "bacterial or bacteriophage isolate" should be considered to mean a bacteriophage that is removed from a biological sample. The term designates, particularly, a phage that is e.g., cultivated in vitro, purified, and / or formulated with any suitable product for formulation, such as diluent(s) or excipient(s).

[0037] The term "purified" with respect to a bacteriophage means that the phage has been measurably increased in concentration by any purification process, including but not limited to, isolation from the environment or culture, e.g., isolation from culture following propagation and / or amplification, centrifugation, etc., thereby partially, substantially, nearly completely, or completely removing impurities, such as host cells and host cell components. A person skilled in the art will appreciate the amount of purification necessary for a given use. For example, a purified phage meant for use in therapeutic compositions intended for administration to humans ordinarily must be of high purity in accordance with regulatory standards and good manufacturing processes.

[0038] As used herein, the term "variant" in the context of nucleotide sequences refers to a genomic nucleotide sequence that comprises or consists of a nucleotide sequence having a sequence identity of at least 98% or at least 99% with a reference nucleic acid sequence. A variant may be selected that maintains one or more functions of the reference nucleic acid sequence. For example, a variant bacteriophage may exhibit at least one biological activity, e.g., antibacterial activity, such as lytic killing activity, of the bacteriophage from which it is derived. A person skilled in the art will appreciate that nucleic acid replication in phages is less than 100% accurate, such that a given phage will show at least 1% variation as it replicates, including during its production as an antibacterial agent. The expected genome variation during the manufacture and use of phages may result in progeny that are variants having at least about 98%, or 99% sequence identity to the nucleotide sequence of the parent genome. It follows that, the bacteriophage of the invention may comprise or consist of a genome having at least about 98%, or at least 99% sequence identity to the nucleotide sequence of the parent phage, while retaining antibacterial activity against the target (host) bacteria of the parent phage. A "variant" in the context of a bacteriophage, is a bacteriophage, the genome of which has at least about 98%, or 99% sequence identity to the nucleotide sequence of the parent genome and maintains at least part of the lytic activity (i.e., lyses the same strain or strains of bacteria) as the parent bacteriophage. In certain aspects, the "variant" is identified solely by its lytic activity (for example, as described in the Examples herein) against 1, 2 or three strains of bacteria.

[0039] For example, the bacteriophage may be a variant of SEQ ID NO. 1, SEQ ID NO. 5 and SEQ ID NO. 6 can retain antibacterial and / or lytic activity against at least one of E. coli strains 86 / 13- ECO1, 63 / 12-ECO3 and 18 / 20-ECO4.

[0040] As used herein, the term "host cell" refers to the particular bacterial strain used to propagate bacteriophages. "Host cell" also refers to a bacterial cell infected with a bacteriophage, e.g., where the bacteriophage replicates. For the amplification of bacteriophage, the host cell may or may not be the same strain from which the bacteriophage was isolated or cultured. As used herein, the term "in combination" refers to the use of an additional prophylactic and / or therapeutic agent 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.

[0041] As used herein, the term "booster" refers to subsequent, repeat use of the same or substantially the same prophylactic and / or therapeutic agent, such as repeat doses of a bacteriophage, phage product, or phage cocktail of the invention.

[0042] 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, which can be used in the prevention, management, control, or reduction in the incidence of, one or more symptoms of a disease or disorder, in particular, a disease or disorder associated with a bacterial infection, more particularly, a disease or disorder associated with a urinary tract bacterial infection, most particularly a urinary tract infection in cancer patients.

[0043] As used herein, the terms "therapeutic agent" and "therapeutic agents" refer to an agent, such as a bacteriophage, phage product, or phage cocktail of the invention, which can be used in the treatment, management, or control of one or more symptoms of a disease or disorder, in particular, a disease or disorder associated with a bacterial infection, more particularly, a disease or disorder associated with a urinary tract bacterial infection, most particularly a urinary tract infection in cancer patients.

[0044] As used herein, the terms "treat", "treatment" and "treating" refer to obtaining a therapeutic benefit in a subject receiving a composition as disclosed herein. With respect to achieving a therapeutic benefit, the object is to eliminate, lessen, decrease the severity of, ameliorate, or slow the progression of the symptoms or underlying cause (e.g., bacterial infection) associated with the pathological condition or disorder. A "therapeutically effective amount" refers to the amount of a therapeutic agent sufficient to achieve at least one therapeutic benefit in a subject receiving the composition.

[0045] As used herein, the terms "prevent", "prevention" and "preventing" refer to obtaining a prophylactic benefit in a subject receiving a composition as disclosed herein. With respect to achieving a prophylactic benefit, the object is to delay, reduce the incidence of, or prevent the symptoms or underlying cause (e.g., bacterial infection) associated with the pathological condition or disorder. A "prophylactically effective amount" refers to that amount of a prophylactic agent sufficient to achieve at least one prophylactic benefit in a subject receiving the composition.

[0046] As used herein, the terms "antibacterial activity" and "antimicrobial activity", with reference to a bacteriophage or bacteriophage product (e.g., a phage cocktail), or a variant thereof, are used interchangeably to refer to the ability to kill and / or inhibit the growth of a microorganism, in particular, the bacteria of the species or strain that the bacteriophage infects. Antibacterial activity may be assessed by culturing bacteria, e.g., Gram-negative bacteria {e.g., E. coH) according to standard techniques (e.g., in liquid culture or on agar plates), contacting the culture with a bacteriophage, and / or variant thereof of the invention, or with a cocktail of bacteriophages, phage proteins, or variants thereof, and monitoring cell growth after said contacting. For example, in a liquid culture, the bacteria may be grown to an optical density ("OD") representative of a mid-point in exponential growth of the culture; the culture is exposed to one or more concentrations of one or more bacteriophages of the invention, bacteriophage products, or variants thereof, and the OD is monitored relative to a control culture. Decreased OD relative to a control culture is representative of phage(s) or phage product(s) exhibiting antibacterial activity (e.g., lytic killing activity). Similarly, bacterial suspensions can be allowed to grow on an agar plate, the plate exposed to one or more bacteriophages or phage products of the invention, or variants thereof, and subsequent growth evaluated related to control plates, without bacteriophage. Bacterial killing, shown by the presence of lysis halos, indicate phage(s) or phage product(s) with antibacterial activity. As used herein, the term "cancer patient" means an individual who has received a positive diagnosis of cancer and / or oncologic disease, including a patient who has not started therapy for cancer and / or oncologic disease, and a patient who has started a therapeutic regimen against cancer or oncologic disease, including but not limited to chemotherapy, immunotherapy and / or radiotherapy.

[0047] The term "sequence identity" as used herein is the percentage of identity obtained by BLAST (basic local alignment search tool from the National Center for Biotechnology Information (NCBI)) that finds regions of similarity between biological sequences. The program compares the nucleotide sequences to sequence databases and calculates the statistical significance (Agarwala et al. 2016).

[0048] BRIEF DESCRIPTION OF THE FIGURES

[0049] Embodiments will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0050] FIG. 1 depicts the growth kinetics assays of E. coli strain ECO4 in the presence of single phages for 24 hours. Three of the selected phages are represented by SEQ ID NO. 1, SEQ ID NO. 5 and SEQ ID NO. 6.

[0051] FIG. 2 shows the growth kinetics assays of f. coli strain ECO4 in the presence of two phages simultaneously for 24 hours. Three of the selected phages are represented in different combinations by SEQ ID NO. 1 + SEQ ID NO. 5, SEQ ID NO. 1 + SEQ ID NO. 6 and SEQ ID NO. 5 + SEQ ID NO. 6 against strain ECO4.

[0052] FIG. 3 represents the growth kinetics assays of f. coli strain ECO4 in the presence of a triple combination of phages for 24 hours. Three of the selected phages are represented in one combination by SEQ ID NO. 1 + SEQ ID NO. 5 + SEQ ID NO. 6 against strain ECO4.

[0053] FIG. 4 represents the bacterial susceptibility tests done to 30 surviving colonies at 24 hours from the growth promotion kinetics single and triple cultures. The percentages of phage resistance (R) or susceptibility (S) is presented.

[0054] FIG. 5 represents the individual host range of the three selected phages performed by double agar overlay plaque assay.

[0055] FIG. 6 is a tabular representation of bacterial strain 18 / 20 -ECO4 growth inhibition in the presence of three phage combination (SEQ ID NO. 1 + SEQ ID NO. 5 + SEQ ID NO. 6) and clinically representative antibiotics.

[0056] DETAILED DESCRIPTION

[0057] The present invention is directed to phage therapy for the treatment and control of bacterial infections, in particular urinary tract bacterial infection, more particularly UTIs in cancer patients. One aspect invention relates to novel and inventive bacteriophage strains, including E.coli phages comprising SEQ ID NO. 1, SEQ ID NO. 5 and SEQ ID NO. 6 as well as variants thereof.

[0058] Another aspect of the invention relates to compositions comprising one or more strains of purified bacteriophage of the invention, as well as combinations with other phages, preferably E. coli phages comprising SEQ ID NO. 4.

[0059] Another aspect relates to the compositions as used in a method of using the same in the treating, reducing or preventing the occurrence of bacterial infections, in particular, UTIs caused by E. coli in cancer patients.

[0060] Still other aspects of the invention relate to use of the compositions as diagnostic tools, and use in reducing or inhibiting colonization or growth of bacteria on a surface.

[0061] BACTERIOPHAGES AND VARIANTS THEREOF

[0062] Another aspect of the invention relates to novel E. coli bacteriophages that target a number of strains of E. coli. E. coli 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, the bacterium also naturally occurs in the soil. In one embodiment, it is provided the purified bacteriophage SEQ ID NO. 1, which targets several strains of E. coli, as well as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.

[0063] Bacteriophage and certain bacterial strains disclosed herein were deposited with the NCIMB and these deposits are:

[0064] PHAGE COCKTAILS

[0065] Phage cocktails provide advantages over the use of phages individually, e.g., to increase the lytic activity against a particular species or strain of bacteria and / or to decrease the possibility of the emergence of bacteria resistant to an individual bacteriophage. Different bacteriophages also can be mixed as cocktails to broaden their properties, preferably resulting in a collectively greater antibacterial spectrum of activity.

[0066] The present invention disclosed herein may provide cocktails comprising at least two different purified bacteriophages, with antibacterial activity against the same or different bacterial species or strains. Optionally, the present invention provides a cocktail comprising or consisting of the following purified bacteriophages SEQ ID NO. 1, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and / or variants thereof, including variants that have lytic activity against one or all of f. coli 86 / 13-ECO1, 63 / 12-ECO3 and / or 18 / 20-ECO4.

[0067] Preferably, the combination of different bacteriophages does not impair or reduce (or does not substantially or significantly impair or reduce) infection ability or host range and / or lytic activity of an individual bacteriophage in the presence of a different bacteriophage. More preferably, the efficacy of at least one bacteriophage in the cocktail is enhanced or improved due to the presence of at least one other bacteriophage in the cocktail, thereby producing an increased effect.

[0068] The bacteriophages used in the cocktails of the invention, can be obtained by any methods known in the art and / or disclosed herein. The present invention may provide for methods of production, amplification and purification of a bacteriophage SEQ ID NO. 1, for example, from the deposited strain disclosed herein. The present invention may further provide for methods of production, amplification and purification of a bacteriophage SEQ ID NO. 1, and SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, for example, from the deposited strain disclosed herein.

[0069] Further, bacteriophages may be isolated from a biological sample 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), incorporated herein by reference in its entirety). Specific bacterial strains that may be used include, e.g. E. coli 86 / 13-ECO1, 63 / 12-ECO3 and 18 / 20-ECO 4 (e.g., for isolating phage SEQ ID NO. 1, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6).

[0070] A person skilled in the art may use one or more methods to propagate or amplify a bacteriophage, particularly purified bacteriophage SEQ ID NO. 1, and SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6, as well of variants thereof, to obtain greater amounts of a given phage.

[0071] A method of producing and / or isolating additional phage may comprise (i) obtaining a culture of E. coli, (ii) infecting it with the bacteriophage SEQ ID NO. 1, SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO.6, or a variant thereof; (iii) culturing until significant lysis of the culture is observed; and (iv) isolating from the culture the bacteriophage progeny. The host cell used may be any bacterial strain, for example, any E. coli strain susceptible to infection by the phage that can be used to replicate it. In some embodiments, the host cell used may be, for example, E. coli 86 / 13-ECO1, 63 / 12-ECO3, and 18 / 20-ECO4 strain.

[0072] COMPOSITIONS

[0073] The purified bacteriophages disclosed herein, including variants thereof, and phage cocktails, may be administered alone or incorporated into a composition for use in the treatment or prophylaxis of bacterial infections, e.g., infections caused by bacteria including, but not limited to, E. coli. The bacteriophage(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; hydrophilic polymers such as polysaccharides (e.g., dextran, alginate, and chitosan); amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN™ and polyethylene glycol. The compositions of the present invention (e.g., antibacterial compositions) can also include a lubricant, an emulsifier, a suspending agent, and a preservative, e.g., in addition to one or more of the above ingredients. In specific aspects, the purified bacteriophage is formulated in a NaCI solution, for example 0.9% NaCI. The compositions disclosed herein may be formulated for oral, intravenous, intraurethral, or intravesical administration. Formulations for intravesical delivery may be in the form of suspension, emulsions, lyophilized preparations, liposomal preparations, and the like.

[0074] The bacteriophages and phage cocktails disclosed herein may be used in combination with one or more other therapeutic and / or prophylactic agents useful for the treatment of bacterial infection as described herein and / or known in the art, e.g. one or more other bacteriophages or an antibiotic. For example, a composition may comprise two or more purified bacteriophages disclosed herein (with antibacterial activity against the same or different bacterial species or strains) in combination with a bacteriophage known in the art. The therapeutic components of a combination may target two or more species or strains of bacteria.

[0075] The compositions disclosed herein may also be used in combination with one or more nonphage therapeutic and / or prophylactic agents useful for the treatment and / or prevention of bacterial infections as described herein and / or as known in the art (e.g. one or more traditional antimicrobial agents). Other therapeutic and / or prophylactic agents that may be used in combination with the phage(s) or phage product(s) disclosed herein include, but are not limited to, antibiotic agents, anti-inflammatory agents, antifungal agents, urinary anti- infectives, urinary antispasmodics, miscellaneous genitourinary tract agents or anesthetic agents. The compositions disclosed herein may comprise a phage cocktail , which is administered in the absence of a standard or traditional antibiotic agent. The phage cocktail may be administered with a broad-spectrum antibiotic. Standard or traditional antibiotic agents include, but are not limited to, amikacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin, streptomycin, tobramycin, apramycin, rifamycin, naphthomycin, mupirocin, geldanamycin, ansamitocin, carbacephems, imipenem, meropenem, ertapenem, faropenem, doripenem, panipenem / betamipron, biapenem, PZ-601, cephalosporins, cefacetrile, cefadroxil, cefalexin, cefaloglycin, cefalonium, cefaloridine, cefalotin, cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cefradine, 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, pencillin and penicillin derivatives, actinomycin, bacitracin, colistin, polymyxin B, cinoxacin, flumequine, nalidixic acid, oxolinic acid, piromidic acid, pipemidic acid, rosoxacin, ciprofloxacin, enoxacin, fleroxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin, gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin, pazufloxacin, sparfloxacin, temafloxacin, tosufloxacin, clinafloxacin, garenoxacin, gemifloxacin, stifloxacin, trovalfloxacin, prulifloxacin, acetazolamide, benzolamide, bumetanide, celecoxib, chlorthalidone, clopamide, dichlorphenamide, dorzolamide, ethoxyzolamide, furosemide, hydrochlorothiazide, indapamide, mafendide, mefruside, metolazone, probenecid, sulfacetamide, sulfadimethoxine, sulfadoxine, sulfanilamides, sulfamethoxazole, sulfasalazine, sultiame, sumatriptan, xipamide, tetracycline, chlortetracycline, oxytetracycline, doxycycline, lymecycline, meclocycline, methacycline, minocycline, rolitetracycline, methicillin, nafcillin, oxacilin, cioxacillin, vancomycin, teicoplanin, clindamycin, co-trimoxazole, flucioxacillin, dicloxacillin, ampicillin, amoxicillin, fosfomycin, nitrofurantoin and any combination thereof. The compositions disclosed herein may comprise an antibiotic agent having antibacterial activity against E. coli. The antibiotic agent can be present in an amount effective to additively or synergistically enhance the therapeutic and / or prophylactic effect of a phage, or phage cocktail for a given infection.

[0076] Each phage used in the compositions disclosed herein may be administered at a dosage of 109pfu / mL to 1011pfu / mL and, for example, may be present at 109pfu / mL in the composition. In addition or alternatively, the compositions disclosed herein can be administered in the form of a suppository orally (e.g., as a tablet, which may contain excipients such as starch or lactose, as a capsule, ovule, suspension, each optionally containing excipients), or they can be injected parenterally (e.g., intravenously). For parenteral administration, the compositions may be used in the form of a sterile aqueous suspension 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 can be formulated in a conventional manner. Topical formulations according to the invention may include a sterile buffer, such as a sterile PBS, water, or saline buffer, or a sterile SM buffer.

[0077] The compositions disclosed herein can be administered according to a dosage regimen. The dosage regime may involve administration of the composition every 6 or 8 hours (for example, a multiple dosing regimen for a topical phage cocktail on diabetic cutaneous wounds (Mendes JJ, et al., 2013, Wound Repair Regen 21 :595-603)). Preferably, initial administration is followed by a second or "booster" dose, involving re-administration of the pharmaceutical composition. For example, the booster may follow an initial dose after about 12 hours (example Leitner et al, 2017), 1 day, or 2 days.

[0078] THERAPEUTIC USE

[0079] Another aspect relates to the use of the compositions for treating, reducing and / or preventing bacterial infections. Phages present 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 efficiency against bacteria in biofilms (Phee A et al., 2013, J Endod 39(3):364-369); their lack of pathogenicity towards human and animal cells (Abedon ST et al., 2011, Bacteriophage l(2):66-85), and their activity in microaerophilic environments even with high bacterial load (Azeredo J, et al. 2008. Curr Pharm Biotechnol 9:261-266). Phage cocktails, in particular, can provide additional advantages over the use of individual phages, e.g., to increase lytic activity against a particular bacterial strain, to increase host range, and / or to decrease the possibility of bacterial resistance emerging to an individual bacteriophage. Indeed, different bacteriophages are mixed as cocktails to broaden their properties, preferably resulting in a collectively greater antibacterial spectrum, such as an expanded host range, which makes development of resistance less likely in the subject receiving the agent.

[0080] The compositions disclosed herein may have activity against a plurality of bacterial strains, such as a plurality of strains of E. coli. Such compositions provide methods of treating, reducing and / or preventing infections associated with E. coli in humans using a phage or phage cocktail. The compositions disclosed herein may also provide methods of treating, reducing and / or preventing infections associated with related strains of these bacteria.

[0081] E. coli is responsible for many severe opportunistic infections, particularly in individuals with compromised immune systems. The compositions disclosed herein are contemplated for treating, reducing and / or preventing infections associated with E. coli, or associated with other strains of bacteria, including, but not limited to, infections of the bladder and urinary tract system, including hospital-acquired bacterial infections such as post-operative infections, infections associated with catheters and surgical drains, and infections of the blood. The compositions disclosed herein may find use in treating, reducing and / or preventing bacterial infections associated with the bladder and urinary tract system in cancer patients comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a composition. Preferably, administration results in an improvement in urinary function.

[0082] A phage cocktail may provide methods of treating a surprising range of E. coli strains. For example, a phage cocktail comprising the Escherichia coli phages SEQ ID NO. 1, SEQ ID NO.

[0083] 5, and SEQ ID NO. 6 shows great efficacy against Escherichia coli clinically relevant strains such as ESBL with 42 % infection, in host range (Example 6 - 6.5 Host range).

[0084] The compositions disclosed herein may comprise a therapeutically and / or prophylactically effective amount of one of more phages. The amount required to bring about a therapeutic and / or prophylactic benefit will depend on the particular formulation, route of administration, condition being treated, whether other agents or therapies are used in combination with methods of the invention, and other factors.

[0085] Preferably, a phage cocktail comprising bacteriophages selected from SEQ ID NO. 1, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 is delivered to provide a significant decrease in E. coli.

[0086] Optionally, the bacteriophage, or phage cocktail disclosed herein is administered as a suspension or emulsion, while an additional agent is administered systemically. For example, a phage cocktail may be administered intravesically while an antibiotic agent is administered systemically, such as an antibiotic agent having activity against E. coli.

[0087] DIAGNOSTIC METHODS

[0088] Another aspect also relates to diagnostic methods for determining the causative agent in a bacterial infection using one or more phages or a phage cocktail as disclosed herein. Because the activity of phages and / or their isolated products tends to be species or strain specific, the susceptibility, or lack of susceptibility, to one or more phages, and phage cocktails can indicate the species or strain of bacteria causing the infection.

[0089] In some embodiments, a bacterial isolate is obtained from a patient and contacted with one or more of SEQ ID NO. 1, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, or a variant thereof. Decreased growth and / or lysis of the culture can indicate that the test sample comprises E. coli, in particular, a strain of E. coli susceptible to infection by the bacteriophage or bacteriophage cocktail used, as disclosed herein, thereby identifying the infective agent and allowing appropriate diagnosis and / or treatment.

[0090] The sample may be a tissue biopsy collected from the patient, or a fluid sample, such as blood, or urine. In preferred embodiments, the tissue sample is obtained from the urinary tract of the patient, e.g., a bladder biopsy sample. EXAMPLES

[0091] It is understood that the following examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.

[0092] Unless otherwise indicated, specific bacteriophages disclosed herein were isolated, processed and analyzed according to the following methods.

[0093] Example 1 - Bacterial Strains

[0094] Three phages represented by SEQ ID NO. 1, SEQ ID NO. 5 and SEQ ID NO. 6 were selected to be tested in a growth kinetics assay individually and in combination against E. coli planktonic cells. This will prove insightful in determining the efficacy of phage activity in a period of 24 hours. The selection of these phages at this point was in accordance with previously obtained results regarding, (1) host range; (2) RFLP profile, (3) genome sequencing and (4) endurance tests on salinity, pH and temperature. Given that E.coli strain ECO4 was the (common) host for the selected phages, the strain's growth was addressed in order to establish a parallel between the bacterial cell viability in the presence and absence of phage.

[0095] Example 2 - Single curves

[0096] On single phage lysis curves, three phages were tested individually against strain ECO4, during a 24-hour period. Bacterial viable cell counts were reduced more than 3 log units by phage SEQ ID NO. 6 after just 1 hour of culture. Four hours post phage inoculation, a slight increase in the bacteria CFU count started being recorded. Phage SEQ ID NO. 1 and SEQ ID NO. 6 displayed distinct behaviors in culture with different extents in CFU reduction and in the recovery of bacterial growth. Phage SEQ ID NO. 6 was the only phage exhibiting bacterial load below the input at 8 hours culture demonstrating a delay in cell growth recovery of the bacterial host (FIG. 1). After 24 hours post phage inoculation, there were no differences between the CFU counts of the bacteria control culture, host strain ECO4, and the bacteria culture challenged with individual phage SEQ ID NO. 1 and phage SEQ ID NO. 6. SEQ ID NO. 5 was the exception, surpassing by approximately 1 log unit regarding the final quantified bacterial counts.

[0097] Example 3 - Combined curves

[0098] The combined assays started with double phage combination. Three possible associations were tested phage SEQ ID NO. 1 + phage SEQ ID NO. 5, phage SEQ ID NO. 1 + phage SEQ ID NO. 6 and phage SEQ ID NO. 5 + phage SEQ ID NO. 6 (FIG. 2). A reduction of the host cell counts in almost 5 log units was accomplished for the combination phage SEQ ID NO. 5 + phage SEQ ID NO. 6 2-hours post phage inoculation. This culture maintained the low cell counts for 2 more hours before the recovery of the bacterial growth. The other cultures with phage combinations phage SEQ ID NO. 1 + phage SEQ ID NO. 5 and phage SEQ ID NO. 1 + phage SEQ ID NO. 6 although not so effective were able to reduce the viable cell counts by nearly 4 logs. In these double combinations the CFU increase was detected after 5 hours post phage inoculation. After 8 hours of phage inoculation, all cultures presented CFU counts below the input with a significant difference in the viable cell count when compared with the bacteria control ECO4 culture, ranging between a ~1 log unit difference (SEQ ID NO. 5 + SEQ ID NO. 6 and SEQ ID NO. 1 + SEQ ID NO. 6) to a 2 log unit difference (SEQ ID NO. 1 + SEQ ID NO. 5). After 24 hours SEQ ID NO. 5 + SEQ ID NO. 6 combination showed a significant difference (~2 log units) in the CFUs count when compared to the bacteria control culture. At this point it was noticed that combining phages two by two had a positive effect on the delay of the ECO4 regrowth in culture throughout 8 hours, being this even extended moderately for the 24-hour window depending on the phage combination

[0099] Triple combination lysis curve proceeded. One possible combination was tested: SEQ ID NO. 1 + SEQ ID NO. 5 + SEQ ID NO. 6 (FIG. 3).

[0100] It was observed a reduction in bacterial load of more than 5 log units during the first couple of hours post-phage inoculation, representing an increase of 0.5 logs lytic activity from the most effective double combination The host strain started exhibiting a refractory behavior after 4 hours, however at 8 hours post phage inoculation the cell counts were still below the input bacterial load by ~2 log units. After 24 hours culture the difference between the bacterial control and the phage combination culture was narrower, by only ~1 log unit. At this point the results indicated that the triple phage combination was more efficient in killing the bacterial host than any of the double phage combinations.

[0101] The results collected from the growth kinetic assays show that, although the double combination of SEQ ID NO. 5 + SEQ ID NO. 6 was highly effective, the addition of SEQ ID NO. 1 made the phage cocktail more efficient in reducing the CFU of ECO4 and limiting its growth up until 24 hours post phage inoculation (FIG. 3). The comparison between the lytic activity of double combinations versus the lytic activity of triple combinations was evaluated under the same conditions. Example 4 - Bacterial phage resistance

[0102] Bacterial phage resistance can appear in vitro quite rapidly (Mangalea et al, 2020) however, its study can orientate the selection of the phages that compose the therapeutic cocktails to act as an additional tool and prevent the rising of such resistance in vivo during treatments. In FIG. 4, the results from the phage susceptibility test were done on 30 surviving colonies of 18 / 20-ECO4 growth kinetic cultures at 24 hours post phage infection. As can be seen in graph A (FIG. 4) of the test results on the single curve colonies, most of the phages induced resistance to themselves, with percentages of 40, 60, and 80, SEQ ID NO. 6, SEQ ID NO. 5 and SEQ ID NO. 1, respectively. Interestingly none of the phages produced a culture entirely resistant to phage infection. Surprisingly, phage SEQ ID NO. 6 induced resistance to phage SEQ ID NO. 5. The colonies surviving phage SEQ ID NO. 6 culture presented 40% resistance not only to phage SEQ ID NO. 6 but also to phage SEQ ID NO. 5. This cross-resistance is not reciprocal as SEQ ID NO. 5 does not seem to generate resistance to SEQ ID NO. 6, as shown by the results of phage susceptibility. Surviving colonies of phage SEQ ID NO. 5 culture were 60% to that phage but only 10% resistant to phage SEQ ID NO. 6 (FIG. 4A).

[0103] The bacterial susceptibility to phage infection after the selective pressure of the three phages combined against planktonic cells of ECO4, SEQ ID NO. 1 + SEQ ID NO. 5 + SEQ ID NO. 6 (FIG. 3), revealed a surprising result as 80% of the surviving colonies were susceptible to phage SEQ ID NO. 1 (FIG. 4B). The same 30 colonies showed a profile with a considerable reduction of resistance to phage SEQ ID NO. 5 and phage SEQ ID NO. 6 when compared with the profiles of the respective single cultures. The results from these assays reiterate the advantage of having phage SEQ ID NO. 1 in the combination as it can maintain a susceptible population to phage infection for as long as 24 hours in vitro.

[0104] Example 5 - Antibiotic-Phaae Interaction Assay

[0105] To clarify potential effects, such as synergism or antagonism, when combining the selected phages and standard of care antibiotic therapy used to address UTIs, an adapted version of minimum inhibitory concentration (MIC) assay (broth microdilution) was used. The selection of antibiotics to be used in this assay relied on available standard-of-care information for noncomplicated, recurrent and nosocomial associated UTI infections: Ciprofloxacin, Amikacin, Amoxicillin-Clavulanic acid, Ceftazidime and Meropenem. Antibiotic quality control (QC) was performed using E. coli strains ATCC 25922 as a control. Additionally, the antimicrobial susceptibility profile of strain ECO4 that had been previously determined through disk diffusion and was further corroborated through MIC for the same antibiotics. All antibiotics were prepared according to EUCAST guidelines (available at https: / / www.eucast.org). QC results for antibiotic quality and MIC results were determined under EUCAST Routine and extended internal quality control for MIC determination and disk diffusion vl l. (available at https: / / www.eucast.org). All antibiotics were tested within a range of concentrations between 32 mg / L and 0.016 mg / L. Applied tested MOIs ranged from 1 until 0,001. Phage titers for phages SEQ ID NO. 1 + SEQ ID NO. 5 + SEQ ID NO. 6 were reassessed against ECO 18 / 20 to ensure adequate MOI determination. As shown in Table 18, the combined effect of the tested antibiotics was only evident equal to or under a MOI of 0,001. Antibiotic combination with the combined phage cocktail resulted in a lowering of MIC profile. The observations led to the conclusion that there might potentially be a synergistic effect between the tested antibiotics, except Ciprofloxacin and the selected combined phages at MOI 0.001, since the MIC values of the antibiotics are lower than the MIC of 18 / 20-ECO4. Overall, the bacterial (18 / 20-ECO4) growth inhibition in the presence of the three-phage combination and antibiotics suggests that the presence of antibiotics does not inhibit the activity of the phage.

[0106] Example 6 - Host range

[0107] Host range of E. coli strains previously characterized in-house was evaluated through an internal assay in order to evaluate phage susceptibility. The first set of experiments with 34 E. coli strains tested the group of phages from which phage SEQ ID NO. 1, phage SEQ ID NO. 5, and phage SEQ ID NO. 6 were selected. Double agar overlay plaque assay, the standard method to determine bacterial susceptibility to phage infection was used and FIG. 5 shows the overall results obtained for the three selected phages. The combined coverage reached 50% infection of clinically relevant E. coli strains.

[0108] The diverse panel of the clinically relevant tested strains included E. coli belonging to ST131, typically highly virulent and / or MDR strains (Pitout and Finn, 2020). Additionally, SEQ ID NO. 1 + SEQ ID NO. 5 + SEQ ID NO. 6 were able to suppress E. coli ESBL strains. Considering the high prevalence of ESBL-producing Enterobacteriaceae (5. coli being the most predominant with 82.5%) UTIs in over 40% of cancer patients (Wang et al, 2023), these results demonstrate the potential of the present invention to treat this particular population.

[0109] References:

[0110] 1. Wang C, Yang D, Wang Y, Ni W. Cefiderocol for the Treatment of Multidrug-Resistant Gram-Negative Bacteria: A Systematic Review of Currently Available Evidence. Front Pharmacol. 2022 Apr 12; 13:896971. doi: 10.3389 / fphar.2022.896971. Erratum in: Front Pharmacol. 2022 Aug 18; 13:976792. doi: 10.3389 / fphar.2022.976792.

[0111] 2. Khawaldeh, A., Morales, S., Dillon, B., Alavidze, Z., Ginn, A. N., Thomas, L., ... Iredell, J. R. (2011). Bacteriophage therapy for refractory Pseudomonas aeruginosa urinary tract infection. Journal of Medical Microbiology, 60(11), 1697-1700.

[0112] 3. Leitner L, Ujmajuridze A, Chanishvili N, Goderdzishvili M, Chkonia I, Rigvava S, Chkhotua A, Changashvili G, McCallin S, Schneider MP, Liechti MD, Mehnert U, Bachmann LM, Sybesma W, Kessler TM. Intravesical bacteriophages for treating urinary tract infections in patients undergoing transurethral resection of the prostate: a randomised, placebo- controlled, double-blind clinical trial. Lancet Infect Dis. 2021 Mar;21(3):427-436. doi: 10.1016 / 51473-3099(20)30330-3.

[0113] 4. Lossouarn J, Beurrier E, Bouteau A, Moncaut E, Sir

[0114] Silmane M, Portalier H, Zouari A, Cattoir V, Serror P, Petit M. 2024. The virtue of training : extending phage host spectra against vancomycin-resistant Enterococcus faecium strains using the Appelmans method. Antimicrob Agents Chemother 68:e01439-23. https: / / doi.org / 10.1128 / aac.01439-23.

[0115] 5. Gamal M. El-Sherbiny, Eman E. Farghal, Mohamed K. Lila, Yousseria M. Shetaia, S.S. Mohamed, Marwa MF. Elswify. 2024. Antibiotic susceptibility and virulence factors of bacterial species among cancer patients, Biotechnology Notes, 5:27-32 https: / / doi.org / 10.1016 / j.biotno.2024.02.002.

[0116] 6. Martins Lopes MS, Machado LM, Ismael Amaral Silva PA, Tome Uchiyama AA, Yen CT, Ricardo ED, Mutao TS, Pimenta JR, Shimba DS, Hanriot RM, Peixoto RD. Antibiotics, cancer risk and oncologic treatment efficacy: a practical review of the literature. Ecancermedicalscience. 2020 Sep 21; 14: 1106. doi: 10.3332 / ecancer.2020.1106.

[0117] 7. Mangalea MR, Duerkop BA. Fitness Trade-Offs Resulting from Bacteriophage Resistance Potentiate Synergistic Antibacterial Strategies. Infect Immun. 2020 Jun 22;88(7):e00926-19. doi: 10.1128 / IAI.00926-19.

[0118] 8. Johann D.D. Pitout, Thomas J. Finn, The evolutionary puzzle of Escherichia coli ST131, Infection, Genetics and Evolution, Volume 81, 2020, 104265, https: / / doi.0rg / lO.lOl6 / j.meegid. 2020.104265.

[0119] 9. Wang G, Zhu Y, Feng S, Wei B, Zhang Y, Wang J, Huang S, Qin S, Liu X, Chen B, Cui W. Extended-spectrum beta-lactamase-producing Enterobacteriaceae related urinary tract infection in adult cancer patients: a multicenter retrospective study, 2015-2019. BMC Infect Dis. 2023 Mar 6;23(1): 129. doi: 10.1186 / S12879-023-08023-3.

Claims

CLAIMS1. A composition comprising one or more purified strains of bacteriophage, each of said purified strains having a genome which comprises at least 98% sequence identity to the nucleotide sequence selected from the group consisting of SEQ. ID No. 1, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6 and / or variants thereof, and having antibacterial activity against Escherichia coli (E.coli).

2. A composition according to claim 1, wherein said composition further comprises a pharmaceutically acceptable carrier, excipient or stabilizer.

3. A composition according to claim 1 or 2, wherein said composition is formulated for oral, intravenous, intraurethral or intravesical administration.

4. The composition according to any one of claims 1-3, wherein each said bacteriophage is present in a concentration of from 109to IO10pfu / mL.

5. A therapeutically or prophylactically effective amount of a composition according to any one of claims 1-4 for use in treating, reducing and / or preventing the occurrence of a bacterial infection in a subject in need thereof.

6. A composition for use according to claim 5, wherein said bacterial infection is caused by a E.coli bacterial strain.

7. A composition for use according to claim 6, wherein said bacterial strain (i) shows resistance to one or more known antibiotics and / or (ii) is highly pathogenic and / or (iii) forms a biofilm.

8. A composition for use according to claim 6 or 7, wherein said subject is a mammal, optionally a human.

9. A composition for use according to any one of claims 6-8, wherein said bacterial infection is a urinary tract infection (UTI), optionally a complicated UTI.

10. A composition for use according to any one of claims 6-9, wherein said subject is a cancer patient, a diabetic patient, or a patient with a weakened immune system.

11. A composition for use according to any one of claims 6-10, wherein said composition is administered as a first line treatment and re-administered approximately 4-8 hours later.

12. A composition for use according to any one of claims 6-11, wherein an antibiotic agent having antibacterial activity against E.coli is used in combination with said composition to treat, reduce and / or prevent the occurrence of the bacterial infection in a subject in need thereof.

13. A composition according to any one of claims 1-4 for use in a method of diagnosis of the causative agent of a bacterial infection and / or a method of assessing whether an infectious agent is susceptible to the lytic activity of a bacteriophage.

14. A composition for use according to claim 13, wherein said method of diagnosis comprises:(i) culturing a sample from a patient;(ii) contacting the culture of step (i) with the composition; and(iii) monitoring for evidence of cell growth or lysis of the culture, wherein evidence of lysis of the culture indicates that the culture comprises a bacterial strain known to be susceptible to the one or more purified strains of bacteriophage in the composition.

15. A composition for use according to claim 14, wherein the sample is a urine sample or a tissue sample, optionally the tissue sample is a tissue biopsy collected from the urinary tract of said patient.

16. A composition for use according to claim 14 or 15, wherein the tissue sample is a tissue biopsy collected from a mucus membrane of the urinary tract of a human.

17. A composition according to any one of claims 1-4 for use in reducing or inhibiting colonization or growth of bacteria on a surface, wherein the use comprises contacting the surface with the composition.

18. A composition for use according to claim 17, wherein the surface is a mucus membrane of a mammal, optionally a mucus membrane of the urinary tract of a human.

19. A bacteriophage having a genome comprising at least 95% sequence identity with the nucleotide sequence of SEQ ID NO. 1, optionally at least 98% or at least 99% sequence identity, and having antibacterial activity against one or more strains of E.coli.

20. A bacteriophage having a genome comprising at least 95% sequence identity with the nucleotide sequence of SEQ ID NO. 5, optionally at least 98% or at least 99% sequence identity, and having antibacterial activity against one or more strains of E.coli.

21. A bacteriophage having a genome comprising at least 95% sequence identity with the nucleotide sequence of SEQ ID NO. 6, optionally at least 98% or at least 99% sequence identity, and having antibacterial activity against one or more strains of E.coli.

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