WO2016066722A2

WO2016066722A2 - Bacteriophage combinations for human or animal therapy

Info

Publication number: WO2016066722A2
Application number: PCT/EP2015/075046
Authority: WO
Inventors: Ian Humphery-Smith; Alexander Yurievich ZURABOV
Original assignee: Ian Humphery-Smith; Zurabov Alexander Yurievich
Priority date: 2014-10-28
Filing date: 2015-10-28
Publication date: 2016-05-06
Also published as: WO2016066722A3

Abstract

The present invention provides a means of treating and preventing bacterial infection through numerous highly-specific bacteriophage strains that combine to fight a target infection. According to the invention, each composition or combined preparation contains classes of multiple bespoke building blocks/elements (specific bacteriophage strains) directed against: (a) the "target" pathogenic bacterial species in a given host tissue, organ or organ system; (b) bacteria which are likely to co- infect and / or colonise the patient in a site-specific manner, which are capable of producing disease (pathogenic) once that target species has been removed, particularly in a healthcare setting; (c) commonly-encountered bacteria likely to be encountered opportunistically throughout the body (in a non-site-specific manner), particularly in a healthcare setting; and (d) clonally-expanded multi-drug-resistant bacteria particularly of the same target species, as in (a) above, and especially those prevalent in a defined area.

Description

Bacteriophage Combinations for Human or

Animal Therapy

Field of the Invention

The present invention concerns bacteriophage combinations and their use in therapeutic strategies for, particularly, the treatment and prevention of nosocomial bacterial infections, or those in animal herds.

Background to the Invention

The gambit of bacterial species that constitute the human microbiome is consistently complex and comprised of endemic and transient species and populations that are in a continuous state of dynamic flux. Multiple sites across the surface and within the human body provide innumerable habitats for numerous bacterial species that are formed into a local communal assemblage inhabiting a particular biotope. The latter are never monogenic in nature and are comprised of communities made-up of multiple species of cultivable and non-cultivable commensals, symbionts and pathogens. Individual species may evolve from harmless commensals into life-threatening pathogens through a chain of colonisation as a consequence of the reduced or increased diversity of bacterial communities and host factors, such as, age, sex, immune-competence and general state of health of the host organism. Thus, bacteria can be occasionally pathogenic or often pathogenic depending upon their site of multiplication in both a facultative and obligatory manner. In turn, all manner of healthcare interventions can disrupt day-to-day status quo and the interspecies balance within bacterial communities to produce infectious states that are detrimental and/or life threatening to the human host, i.e. as a function of an infectious organism^'s relative virulence and infectivity.

Since the discovery of penicillin and streptomycin and their widespread use internationally since the end of the Second World War, the world has become accustomed to the use of a wide variety of broad-spectrum antibiotics as a means to treat a multitude of bacterial infections affecting humankind. Due to their over- and uncontrolled-use, the efficacy of these antibiotics against target microbial infections has become less and less effective over time and this is producing an alarming situation associated with huge economic cost to patients and healthcare providers alike^{1 ,2}. Today, multiple drug-resistant bacteria are commonplace, particularly within our healthcare facilities. A "nosocomial infection" or so called "hospital-acquired infection" can be defined as "An infection acquired in hospital by a patient who was admitted for a reason other than that infection. An infection occurring in a patient in a hospital or other health care facility in whom the infection was not present or incubating at the time of admission." This includes infections acquired in the hospital by appearing after discharge, and also occupational infections among staff of the healthcare facility³.

Patient care is provided in facilities which range from highly-equipped clinics and technologically-advanced university hospitals to front-line units with only basic facilities. Despite progress in public health and hospital care, infections continue to develop in hospitalised patients. Many factors promote infection among hospitalised patients: decreased immunity among patients; the increasing variety of medical procedures and invasive techniques creating potential routes of invention; and the transmission of the drug-resistant bacteria among crowded hospital populations, where poor infection control practices can facilitate transmission. Infections acquired in a healthcare setting may take the form of acute or chronic infection.

Nosocomial infections occur worldwide and affect both rich and poor countries alike. Infections acquired in healthcare settings are among the major causes of death and increased morbidity among hospitalized patients. They are a significant burden both for the patient and public health authorities, not to mention the increasingly-important economic burden of extended hospital stay³. A prevalence survey conducted under the auspices of WHO in 55 hospitals of 14 countries representing 4 WHO Regions (Europe, Eastern Mediterranean, South- East Asia and Western Pacific) showed an average of 8.7% of hospital patients had nosocomial infections³. At any moment, more than 1 .4 million people worldwide suffer from infectious complications acquired in hospital due to a dwindling arsenal of effective antibiotics to combat these superbugs.

New approaches to antibiotic therapy and prevention are much needed for both human and animal infections, because broad-spectrum antibiotics have become significantly less effective over the last 70 years due to the development of resistance by target bacterial species in our hospitals, in our communities and in agriculture. Each year some 2.2 and 3.0 million individuals in respectively the USA and Europe acquire infections from a healthcare facility. Approximately 1/20 to 1 /16 die as a result and the remainder suffer the consequences of increased morbidity and/or extended hospital stay of 3-19 days with an average of 7-9 days. The cost of extended hospital stay and litigation is a significant burden on already-stressed private and public healthcare systems. In developing countries, mortality rates are even higher, while Extreme Multiple Drug-Resistant Tuberculosis (X-MDR TB) could take-on epidemic proportions internationally, i.e. if not curtailed. Immunocompromised individuals as particularly susceptible and act as a community-based reservoir that further serves to expand the infection base. The most recent data available from the European Centre for Disease Prevention and Control (2013) estimated some 4, 100,000 healthcare-associated infections occur across the EU every year and of these approximately 37,000 die as a direct consequence.

The most frequent nosocomial infections are those of the urinary tract, all manner of post-operative wounds, pneumonia and bacteraemia. By far, the highest prevalence of nosocomial infections occurs in Intensive Care Units and in acute surgical, burns and orthopaedic wards. Infection rates are higher among patients with increased susceptibility due to old age, underlying disease and immunosuppression due to chemotherapy and/or disease.

Hospital-acquired infections add to functional disability and emotional stress of the patient and may, in some cases, lead to disabling conditions that reduce the quality of life. Nosocomial infections are also one of the leading causes of death, and the economic costs are considerable. The increased length of hospital stay for infected patients is one of the greatest contributors to cost. One study showed that the overall increase in the duration of hospitalisation for patients with surgical wound infections was 8.2 days, ranging from 3 days for gynaecology to 9.9 for general surgery and 19.8 for orthopaedic surgery^2,4,5. Prolonged stay not only increases direct costs to patients or payers, but also indirect costs due to lost work. The increased use of drugs, the need for isolation, and the use of additional laboratory and other diagnostic studies also contribute to costs: Hospital acquired infections add to the imbalance between resource allocation for primary and secondary health care by diverting scarce funds to the management of potentially preventable conditions. The advancing age of patients admitted to health care settings, the greater prevalence of chronic diseases among admitted patients, and the increased use of diagnostic and therapeutic procedures which affect the host defences will provide continuing pressure on nosocomial infections in the future.

It is also important to note that organisms causing nosocomial infections can be transmitted to the community through discharged patients, staff, and visitors. If organisms are multidrug resistant, they may also cause significant disease in the community.

Mastitis is a bovine disease caused principally by Staphylococcus aureus (the same species that causes significant loss of life in hospitals internationally), with secondary pathogens involved to varying degress, namely, Streptrococcus agalactiae and Streptococcus uberis. Once these causative infections have established themselves in one or more quarters of the cow udder, infection results in the destruction of milk-producing tissue. Tissue destruction (dead and decaying cells) then allows a plethora of other less invasive bacterial species to colonise the infected quarter and in turn produce symptoms that allow increased transmission of the disease within the milking herd. For example, 'pus^' dripping from the infected teat can be transmitted by insects, the milking parlour, bedding and more easily to other quarters of the same udder. The economic impact of mastitis is estimated to be approximately $2 billion USD per year. As in our hospitals and healthcare facilities, the over-use of antibiotics has been a dominant feature of the dairy industry since the end of the Second World War and involves repeated treatments as multiple times in the dairy cows life history. This includes multiple doses of antibiotics at weaning to prevent the onset of scouring, during the 'Drying-Off period prior to calving: and whenever mastitis events are detected during each lactation cycle. As a result, antibiotic resistance has become extremely prevalent among bacterial species infecting dairy cattle, namely, equally or more so than HAL Therefore, there remains a need in the art for an effective prophylactic or curative measure.

The history of the discovery of bacteriophage and their subsequent use in combating bacterial infections has been comprehensively reviewed by others in the art^6"18. The initial discovery of bacteriophages is attributed to Frederick Twort in 1915 and Felix Herelle in 1917. Herelle went on to demonstrate the efficacy of these novel biological elements in fighting all manner of infection in several settings that heightened international awareness of their potential to successfully eradicate disease. Most notable of Herelle's exploits was the eradication of an epidemic of Avian Typhoid fever in France. He also studied cholera patients in India. In 1927 over the course of a few months, Herelle was able to reduce the death toll due to cholera from 60% to 8% by inoculating wells with bacteriophage isolated from cholera patients. The origin of the curative properties of the far-from-clean waters of the Ganges River, as sought out by the Hindu faithful, can possibly be attributed to the gradual build-up and abundance in the waters of the Ganges of bacteriophage directed against cholera, and particularly their curative powers against this deadly infection during cholera epidemics. In 1934, Herelle moved to Tbilisi in Georgia to renew acquaintances with Georges Eliava. Together they were responsible for bacteriophage being employed routinely treat all manner of infections to the former Soviet Union. Outside the former Soviet Union, phages cocktails were to prove less reliable than broad spectrum antibiotics, i.e. once the latter became widely available after the Second World War; although during World War II, the Soviet Union used bacteriophages to treat many soldiers infected with various bacterial diseases e.g. dysentery and gangrene.

From early-on, however, it became evident to Herelle and others that the use of phage cocktails were often unreliable. This was likely due to many factors. Most notable is the fact that the exact constituents, dosages and batch-to-batch consistency of the preparations employed to fight infections with bacteriophage have rarely, if ever, been subjected to the same rigour of production under Good Manufacturing Procedures, i.e. as are applied to antibiotics or pharmaceutical products sold internationally. Thus, variability in patient outcomes can be attributed to the inherent variable of what was actually given to patients. In addition, the bacteriophage are inherently highly-specific to particular strains of bacteria. This limits their ability to target different strains of the same bacterial species beyond those for which they are specific. This specificity is a result of restricted receptor molecules expressed on the surface of the bacterial target. Thus, preparations that are efficient at killing one strain of a bacterial species may have zero effect on another strain of the same species that is producing exactly the same clinical symptoms in patients. Indeed, the nature of phage specificity is such that it can be exploited as a diagnostic test for bacterial strains^20"24. Thus, one bacteriophage may kill one strain of a particular target species and not another, while over time bacteria may evolve to express resistance to a given bacteriophage strain. Historically, many researchers have employed mixtures of bacteriophage to overcome to some extent the issue of limited efficacy due to individual strain specificity and thus non-target specific recognition by individual and/or mixtures of bacteriophages. Although conjectured earlier, it is only recently, however, that researchers have been able to clearly demonstrate the phenomenon whereby targeted bacterial species are less able to develop resistance to mixtures of bacteriophages, rather than against a single strain of bacteriophage, due to an onslaught of multiple gene-product targeting (phage-binding receptor variants) in parallel²⁵ .

The US pharmaceutical company Eli Lilly, attempted to commericalise bacteriophage during the 1940s, but this was short-lived and curtailed with the advent of antibiotics. Supporters of bacteriophage-based therapies have often attempted to garner support for their cause by stating that antibiotics are less desirable than bacteriophage because the former effectively kill both the deleterious and the beneficial bacterial flora simultaneously, while bacteriophage kill in a highly selective species and strain-specific manner.

It is noteworthy, however that humans and other mammalian species have over evolutionary time learnt to fight disease using both non-specific defenses and immune-modulated more specific means, while in all cases the latter phenomenon of 'specificity emergent'¹⁹, is delivered by a polyclonal (multifaceted approach, as opposed to a single 'magic bullet') at both the level of humoral and cellular immunity.

With regard to prior work towards standardising phage therapy with a view to Western regulatory approval, Merabashvili et a/.³¹ described, for the first time (to the best of the authors^' knowledge), the laboratory-based production of a defined bacteriophage cocktail (referred to as BFC-1 ), starting from the isolation and characterisation of the phage. The cocktail consisted of two bacteriophage strains specific for P. aeruginosa, and one specific for S. aureus, and was applied topically using a syringe spray to patients with infected burn wounds, in a small-scale safety trial. The phage were characterised molecularly in advance of use (by genomic and proteomic analysis), and the cocktail was subject a variety of quality control measures prior to administration.

Accordingly, building on such work, there is a need in the art for phage therapy to be subjected to greater stringency, an example being greater batch-to- batch reproducibility, as part of suggested modifications to the way in which such therapies currently undergo regulatory approval. Phage therapy has the potential to be a viable means of combatting the increasing morbidity and mortality resulting from Multiple Drug Resistant bacterial infections in healthcare facilities globally ^{17, 26"}

31 Summary of the Invention

The objective of the present invention is to reduce the overall level of, in particular, multiple drug-resistant nosocomial (hospital-acquired) infection in healthcare facilities so that use of antibiotic therapy can be reduced to an absolute minimum, i.e. the use of antibiotics can be restricted to the treatment of acute infections that resist the treatment / prophylactic protocol proposed by the present invention.

The present invention provides a means of treating and preventing bacterial infection through numerous highly-specific lytic bacteriophage strains that combine to fight a targeted infection.

Treatment or prophylaxis is achieved by the intravenous or intramuscular inoculation of a structured cocktail of different classes of bacteriophage strains, each defined with reference to their specific target bacterium, followed by repeated introductions of the same bacteriophage cocktail over time via the same and / or alternate routes of inoculation.

The approach is designed to emulate the activity of broad spectrum antibiotics capable of targeting both the target bacterium and other species and strains likely to co-infect site-specific infections and / or colonise a given site that would otherwise produce disease in particularly a healthcare setting.

Each composition (or combined preparation) contains classes of multiple bespoke building blocks/elements (specific bacteriophage strains) directed against: (a) the "target" pathogenic bacterial species in a given host tissue, organ or organ system; (b) bacteria which are likely to co-infect and / or colonise the patient in a site-specific manner, which are capable of producing disease (pathogenic) once that target species has been removed, particularly in a healthcare setting; (c) commonly-encountered bacteria likely to be encountered opportunistically throughout the body (in a non-site-specific manner), particularly in a healthcare setting; and (d) clonally-expanded multi-drug-resistant bacteria of the same target species, as in (a) above, and especially those prevalent in a defined area.

The invention is not patient specific in nature, but rather resembles vaccines, for example those produced annually for the protection of populations against an anticipated human Influenza virus strain, which may in the final analysis produce or not produce widespread disease in a given year.

Thus, the present invention enables disease-specific treatment and prophylaxis that would otherwise fail due to the highly strain-specific nature of bacteriophage killing and the short-lived nature of bacteriophage within the mammalian blood stream.

The approach employed for particularly nosocomial infections is equally applicable to intensive agricultural settings wherein animal species are housed or reared in association with large numbers of animals in a constrained space, thereby facilitating the rapid spread of bacterial disease. In particular, the disorders mastitis and metritis are especially relevant due to the typical overuse of antibiotics internationally. Therefore, the invention can be used to limit the use of particularly broad spectrum antibiotics to the barest minimum.

According to a first aspect of the invention, there is provided a composition comprising at least 10 different strains of lytic bacteriophage, for use in the treatment or prevention of a disorder in a subject, characterised by the infection of a tissue, organ or organ system by a pathogenic species of bacteria; wherein

(a) at least 3 strains of lytic bacteriophage have specificity for at least 3 different strains of said pathogenic species of bacteria;

(b) at least 5 strains of lytic bacteriophage have specificity for at least 5 different strains of one or more pathogenic species of bacteria, other than the species for which the strains of bacteriophage in (a) have specificity, which are capable of infecting said tissue, organ or organ system;

(c) a strain of lytic bacteriophage has specificity for a strain of bacteria which is a non-site-specific opportunistic pathogen, of a different species to the pathogenic species of bacteria for which the strains of bacteriophage in (a) and (b) have specificity; and

(d) a strain of lytic bacteriophage has specificity for a strain of bacteria which is resistant to at least three different antibiotics, and which is of the same species as said pathogenic species of bacteria for which the strains of bacteriophage in (a) have specificity;

wherein said treatment or prevention comprises:

(1 ) administering said composition to the subject intravenously, or intramuscularly, on at least one occasion; and

(2) administering said composition to the subject by any suitable means on at least two further separate occasions, the time between said two further occasions being no longer than 72 hours; wherein each strain of bacteriophage is present in the composition administered in each occasion in (1 ) and (2) at an infectious dose of at least 1 0⁴ plaque forming units/ml composition

Further aspects are defined in the Detailed Description of the Invention and Claims. Detailed Description of the Invention

Definitions

As used herein, the terms "bacteriophage" and "phage" (used interchangeably) have their conventional meaning as used in the art; that is, generally, a virus capable of infecting bacteria. Bacteriophage may have either lytic or lysogenic (or both) lifecycles. However, only the use of molecularly-defined lytic bacteriophage (as described below) is envisaged by the present invention, whereby their corresponding bacterial target strain has also been defined in advance of use.

As used herein, the term "strain", with reference to either bacteria or bacteriophage, has its conventional meaning as used in the art, that is, generally, a low taxonomic rank indicating a genetic variant or subtype of bacteria (within a defined species) or of bacteriophage. Most likely, but not necessarily, bacteriophage of a different strain will have varying specificity with regard to the infection of different strains of bacteria.

As used herein, the term "specificity", with reference to the activity of bacteriophage, refers to the preferential (but not necessarily exclusive) infection of a given strain of bacteria, most likely, but not necessarily, over multiple other strains of bacteria belonging to the same species.

As used herein, the term "therapy" encompasses both treatment and prevention of a disorder.

As used herein, the term "treatment" includes therapeutic measures that cure, slow down, and/or halt progression of a disorder.

As used herein, the term "prevention" includes therapeutic measures which are prophylactic or preventative in nature, performed to prevent and/or slow the development of a disorder.

As used herein, the term "subject" refers to any animal (particularly envisaged, a mammal) including, but not limited to, humans, non-human primates, livestock (whether bovine, caprine, ovine or otherwise), canines, felines, rodents and the like, which is to be the recipient of therapy according to the present invention. Human subjects are particularly envisaged according to the present invention and, as used herein, the term "patient" refers to such a subject, and more particularly one visiting or admitted to a healthcare facility.

As used herein, the term "pathogenic" has its conventional meaning as used in the art; that is, generally, the ability to cause disease.

As used herein, the term "site of infection" is to be used interchangeable with reference to the tissue(s), organ(s) or organ system(s) in which there is a bacterial infection. The terms "tissue", "organ" and "organ system" have their conventional meanings as used in the art.

As used herein, the term "infection [by a pathogenic species of bacteria]" , where the invention relates to treatment of a disorder (characterised by such infection), refers to the existence of a pathogenic species of bacteria preferentially at the site of infection, and not, or less so, in other tissue(s), organ(s) or organ system(s) which lack disease or pathology: wherein there exists a correlation between the severity of disease or pathology and the copy number of a nucleic acid sequence, at the site of infection, of sufficient uniqueness in sequence to said bacterial species so as to identify said bacterial species. Accordingly, said copy number will be decreased or become undetectable upon resolution of the disorder, and the opposite will occur upon relapse of the disorder. In practice, the relationship may be inferred by a number of techniques known in the art, including, but not limited to, real-time PGR, microarray, microbial culture and subsequent phenotypic analysis (for example gram staining), and symptomatic assessments performed by a skilled clinician. Accordingly, said techniques may comprise an assessment of variables other than copy number which enable the above relationship to be inferred, for example those which are indicative of presence, absence or number of a specific bacterial species at the site of infection. For certainty, tissue-sequence correlates may be sought at the cellular level, for example efforts may be made to demonstrate specific in situ hybridization of bacterial sequence to areas of tissue pathology and to visible bacteria, or to the site of infection (where bacteria are presumed to be located). Said one (or more than one) bacterial species is also referred to herein as the "target" bacterial species. Where the invention relates to the prevention of a disorder (characterised by such infection), said relationship may be identified in a subject other than that to be administered said prevention; wherein there is need to prevent an analogous infection (by the same pathogenic species of bacteria) in the subject that is to be administered said prevention, based on their circumstances (for example their geographic location). Alternatively, there may be a recognised need to prevent an infection in a subject, in which the skilled person would reasonably expect to the above relationship to exist, based on the circumstances of the subject. A non-target pathogenic bacterial species (for which strains of bacteriophage referred to in (b) have specificity) may be referred to as being "capable" of infecting a tissue, organ or organ system; meaning that the skilled person would understand said species to be able to elicit disease in the tissue, organ or organ system in the manner described above (it is especially envisaged that such bacterial species would be understood by the skilled person to be capable of causing such disease once the target bacterial species/strains thereof is/are removed).

As used herein, the term "opportunistic pathogen" refers to a non-target bacterial species and strains thereof, capable of co-infecting and / or colonising the same tissue, organ or organ system as the target bacterium in a non-site-specific manner and thereby produce disease (particularly in a healthcare setting where these opportunists may be encountered in many organ systems in a non-site- specific manner), when (i) the immune system of the subject is compromised, suppressed, down-regulated or otherwise inhibited by any degree; and / or (ii) as a result of an healthcare intervention (for example, harmless skin bacteria introduced into the blood of a subject at the site of catheter puncture or indeed production of an infected wound at the site of a catheter puncture). For the avoidance of doubt, opportunistic pathogens, as envisaged by the present invention, are not limited to bacteria found at or proximal to the site of infection; that is, they are non-site-specific and can be encountered anywhere in or on the body of the subject, particularly as a result of the subject being in a healthcare environment.

As used herein, the term "antibiotic" has its conventional meaning as used in the art. "Antibiotic resistance", as used herein, also has its conventional meaning as used in the art, generally indicating the ability of a bacterium to survive and replicate after exposure to what would otherwise be an effective quantity of an antibiotic; "susceptibility" indicates the absence of said ability.

As used herein, the term "healthcare facility" refers to premises in which medicine, or veterinary medicine, is practised by a skilled practitioner. With regard to premises for the practice of veterinary medicine, it is especially envisaged that these are associated with facilities for the rearing of animals in a high-density setting. "Hospital" herein refers to such premises for the practice of human medicine.

As used herein, the term "nosocomial infection" refers to an infection acquired in a hospital or other healthcare facility by a subject who was admitted, or visited the hospital or facility whether for therapy or otherwise, for reasons other than that infection, and in whom the infection was not present or incubating in prior to the admission or visit. The term encompasses infections which appear during a stay in a healthcare facility or after discharge, in addition to infections among staff of the hospital or present within the environment of the healthcare facility itself, e.g. ranging from a presence in or on food to surfaces, instruments and the air.

As used herein, the term "parenteral route" has its conventional meaning as used in the art; that is, generally a route of administration of a therapeutic agent other than enteral or topical administration, which enables the agent to act systemically.

As used herein, the terms "subcutaneous", "intradermal", "subdermal", "intraperitoneal", "intravenous", "intramuscular", "intramammary" and "intravesicular" have their conventional meanings as used in the art.

As used herein, the term "wound" has its conventional meaning as used in the art; encompassing both closed wounds such as contusions and haematomas, and open wounds, i.e. discontinuities in the skin or mucosa resulting from physical trauma and / or medical intervention, for example. It is especially envisaged that open wounds

As used herein, the terms "gastrointestinal tract", "respiratory system", "urino- genital tract", "multiple drug-resistant tuberculosis" (also herein referred to as "MDR- TB"), "bacterial meningitis", "mastitis" and "metritis" have their conventional meanings as used in the art.

As used, the term "composition comprising at least [a given number] of different strains of lytic bacteriophage" also encompasses any alternative dosage regimen which achieves the same therapeutic effect as a single composition; for example, the administration of a first composition concurrently with a second composition, whereby the combined constituents of the two compositions provide the given number of different strains of lytic bacteriophage. Aspects of the Invention

wherein said treatment or prevention comprises:

(2) administering said composition to the subject by any suitable means on at least two further separate occasions, the time between said two further occasions being no longer than 72 hours;

wherein each strain of bacteriophage is present in the composition administered in each occasion in (1 ) and (2) at an infectious dose of at least 10⁴ plaque forming units/ml composition

According to a second aspect of the invention, there is provided a composition comprising at least 10 different strains of lytic bacteriophage, for use in the treatment or prevention of a disorder in a subject, characterised by the infection of a tissue, organ or organ system by a pathogenic species of bacteria; wherein (a) at least 3 strains of lytic bacteriophage have specificity for at least 3 different strains of said pathogenic species of bacteria;

(d) a strain of lytic bacteriophage has specificity for a strain of bacteria which is resistant to at least three different antibiotics, and which is of the same species as said pathogenic species of bacteria for which the strains of bacteriophage in (a) have specificity; and wherein at least 20% of deaths attributable to said pathogenic species of bacteria in a given population, and within a given time period, are attributable to the strain of said pathogenic species of bacteria;

wherein said treatment or prevention comprises:

According to a third aspect, there is provided a process for the production of a combined preparation or composition comprising at least 10 different strains of lytic bacteriophage, for simultaneous, separate or sequential use in the prevention or treatment of a disorder characterised by the infection of a tissue, organ or organ system by pathogenic bacterial species; comprising the steps:

(i) species identification of a plurality of different bacterial strains which are present in one or more isolates; wherein said isolates have been obtained from said tissue, organ or organ system of one or more subjects having an infection by said pathogenic bacterial species; wherein steps (ii)-(vi) are performed if the species identification of (i) indicates the presence, in one or more of said isolates, of at least 10 different bacterial strains wherein:

(a) at least 3 of said at least 10 different bacterial strains are of said pathogenic bacterial species,

(b) at least 5 of said at least 10 bacterial strains are of one or more further pathogenic bacterial species, other than the species of (a),

(c) at least one of said at least 10 bacterial strains is of a bacterial species which is an opportunistic pathogen, other than the species of (a) and (b), and

(d) at least one of said at least 10 bacterial strains is of the same pathogenic bacterial species as of (a), and resistant to at least 3 different antibiotics;

(ii) determining the susceptibility of said at least 3 bacterial strains of (a) to a plurality of strains of lytic bacteriophage;

(iii) determining the susceptibility of said at least 5 bacterial strains of (b) to a plurality of strains of lytic bacteriophage;

(iv) determining the susceptibility of said at least one bacterial strain of (c) to a plurality of strains of lytic bacteriophage;

(v) determining the susceptibility of at said least one bacterial strain of (d) to a plurality of strains of lytic bacteriophage; and

(vi) selecting at least 10 strains of lytic bacteriophage to which the at least 10 bacterial strains are found to be susceptible in steps (ii)-(v), and preparing said combined preparation such that each of said strains of lytic bacteriophage are present at an infectious dose of at least 10⁴ plaque forming units (PFU)/ml per single dosage form.

According to a fourth aspect of the invention, there is provided a combined preparation or composition obtainable by the process of the fourth aspect of the invention.

According to a fifth aspect, there is provided a combined preparation comprising at least 12 different strains of lytic bacteriophage, for simultaneous, separate or sequential use in the treatment or prevention of mastitis or metritis in a subject; wherein

(a) at least one of strain of lytic bacteriophage has specificity for at least one strain of Staphylococcus aureus; (b) at least 5 strains of lytic bacteriophage each have specificity for at least one strain of Streptococcus agalacticae, Streptococcus uberis, Bacillus lichenformis. Bacillus cereus and Bacillus subtilis respectively;

(c) at least 4 strains of lytic bacteriophage each have specificity for at least one strain of Staphylococcus haemolyticus. Staphylococcus hyicus. Staphylococcus chromogenes. and Staphylococcus dysgalacticae respectively, and

(d) at least 2 strains of lytic bacteriophage each have specificity for at least one strain of Escherichia coli and Pseudomonas putida respectively;

and wherein each of said at least 12 strains of lytic bacteriophage is administered to the subject at an infectious dose of at least 10⁴ PFU/ml.

According to a sixth aspect of the invention, there is provided a composition comprising at least 10 different strains of lytic bacteriophage, for use in the therapy of a bacterial infection of a wound of the skin or mucosa in a subject; wherein

(a) at least 3 strains of bacteriophage have specificity for at least 3 different strains of Staphylococcus aureus and/or methicillin-resistant Staphylococcus aureus;

(b) at least 5 strains of bacteriophage have specificity for at least 5 different strains of one or more bacteria selected from the group consisting of Clostridium perfringen, Pseudomonas aeruginosa, Streptococcus pyogenes and vancomycin- resistant Enterococcus spp;

(c) a strain of bacteriophage has specificity for a strain of Salmonella enteriditis; and

(d) a strain of bacteriophage has specificity for a strain of Staphylococcus aureus that is resistant to at least three different antibiotics;

wherein said therapy comprises:

wherein each strain of bacteriophage is present in the composition administered in each occasion in (1 ) and (2) at an infectious dose of at least 10⁴ plaque forming units/ml composition According to a seventh aspect of the invention, there is provided a composition comprising at least 24 different strains of lytic bacteriophage, for use in the therapy of a bacterial infection associated with the gastrointestinal tract; wherein

(a) at least 12 strains of bacteriophage have specificity for at least 12 different strains of Clostridium difficile:

(b) at least 10 strains of bacteriophage have specificity for at least 10 different strains of one or more bacteria selected from the group consisting of Escherichia coli, Klebsiella spp, Enterococcus spp, Bacillus fragilis and Clostridium perfringens;

(d) a strain of bacteriophage has specificity for a strain of Clostridium difficile that is resistant to at least three different antibiotics in;

wherein said therapy comprises:

According to an eighth aspect of the invention, there is provided a composition comprising at least 19 different strains of lytic bacteriophage, for use in the therapy of a bacterial infection associated with the respiratory system in a subject; wherein

(a) at least 12 strains of bacteriophage have specificity for at least 12 different strains of Streptococcus pneumoniae;

(b) at least 5 strains of bacteriophage have specificity for at least 5 different strains of one or more bacteria selected from the group consisting of Haemophilus influenzae, Klebsiella pneumoniae and Staphylococcus aureus;

(c) a strain of bacteriophage has specificity for a strain of Pseudomonas aeruginosa] and

(d) a strain of bacteriophage has specificity for a strain of Streptococcus pneumoniae that is resistant to at least three different antibiotics; wherein said therapy comprises:

(1 ) administering said composition to the subject intravenously, or intramuscularly, on at least one occasion ; and

According to a ninth aspect of the invention, there is provided a composition comprising at least 22 different strains of lytic bacteriophage, for use in the therapy of a bacterial infection associated with the urino-genital tract in a subject; wherein

(a) at least 12 strains of bacteriophage have specificity for at least 12 different strains of Neisseria gonorrhoeae;

(b) at least 8 strains of bacteriophage have specificity for at least 8 different strains of one or more bacteria selected from the group consisting of Staphylococcus saprophyticus, Enterococcus faecalis, Enterococcus faecium, Proteus mirabilis, Klebsiella pneumoniae, Proteus vulgaris, Proteus penneri, Staphylococcus epidermidis, Pseudomonas aeruginosa, Mycobacterium tuberculosis and Group B Streptococcus agalactiae;

(c) a strain of bacteriophage has specificity for a strain of Escherichia coli; and

(d) a strain of bacteriophage has specificity for a strain of Neisseria gonorrhoeae that is resistant to at least three different antibiotics;

wherein said therapy comprises:

wherein each strain of bacteriophage is present in the composition administered in each occasion in (1 ) and (2) at an infectious dose of at least 10⁴ plaque forming units/ml composition According to a tenth aspect of the invention, there is provided a composition comprising at least 19 different strains of lytic bacteriophage, for use in the therapy of a multiple drug-resistant tuberculosis infection in a subject; wherein

(a) at least 12 strains of bacteriophage referred to in (a) have specificity for at least 12 different strains of Mycobacterium tuberculosis;

(b) at least 5 strains of bacteriophage have specificity for at least 5 different strains of one or more bacteria selected from the group consisting of Haemophilus influenzae, Klebsiella pneumonia and Staphylococcus aureus;

(c) a strain of bacteriophage has specificity for a strain of Streptococcus pneumoniae; and

(d) a strain of bacteriophage has specificity for a strain of Mycobacterium tuberculosis that is resistant to at least three different antibiotics;

wherein said therapy comprises:

According to an eleventh aspect of the invention, there is provided a composition comprising at least 22 different strains of lytic bacteriophage, for use in the therapy of a nosocomial Escherichia coli bacterial infection in a subject; wherein

(a) at least 12 strains of bacteriophage have specificity for at least 12 different strains of Escherichia coli;

(b) at least 8 strains of bacteriophage have specificity for at least 8 different strains of one or more bacteria selected from the group consisting of Acinetobacter baumannii, Staphylococcus aureus, Klebsiella pneumonia, Pseudomonas aeruginosa, Morganella morganii, Salmonella spp, Enterobacter cloacae, Staphylococcus hominis, Staphylococcus epidermidis and Staphylococcus saprophyticus;

(c) a strain of bacteriophage has specificity for a strain of Proteus mirabilis; and (d) a strain of bacteriophage has specificity for a strain of Escherichia coli that is resistant to at least three different antibiotics;

wherein said therapy comprises:

According to a twelfth aspect of the invention, there is provided a composition comprising at least 22 different strains of lytic bacteriophage, for use in the therapy of a bacterial meningitis infection in a subject; wherein

(a) at least 12 strains of bacteriophage have specificity for at least 12 different strains of Neisseria meningitidis;

(b) at least 8 strains of bacteriophage have specificity for at least 8 different strains of one or more bacteria selected from the group consisting of Listeria monocytogenes, Mycobacterium tuberculosis, Salmonella enterica and Serovar typhimurium;

(c) a strain of bacteriophage has specificity a strain of Listeria monocytogenes; and

(d) a strain of bacteriophage has specificity for a strain of Neisseria meningitidis that is resistant to at least three different antibiotics;

wherein said therapy comprises:

wherein each strain of bacteriophage is present in the composition administered in each occasion in (1 ) and (2) at an infectious dose of at least 10⁴ plaque forming units/ml composition The present invention also provides a method of treating or preventing a bacterial infection using the compositions or combined preparations according to the first, second and fourth to twelfth aspects of the invention; said method having the same optional and preferred features as are applicable to said aspects.

Preferred Embodiments of the Aspects of the Invention

All aspects of the invention require the selection of bacteriophage having specificity for defined strains of bacteria. This selection process is routine to the skilled person. For example, strains of bacteriophage may be purchased based on their bacterial target species (for example at http://www.lqcstandards~ atcc.orq/en Products/Cells and Microorganisms/Bacteria/ Bacteriophaqes.aspx ; as accessed in October 2014, the content of which is herein incorporated by reference). Alternatively or in addition, the selection of a suitable bacteriophage strain, when the skilled person is in possession of the bacterial strain to be targeted, may be achieved via an experiment which determines the ability of a bacteriophage strain to infect said bacterial strain (also referred to herein as the "susceptibility" of the bacterial strain to the bacteriophage strain). Any suitable method which exists in the art may be used, for example that described by Merabashvili et al. (2009)³¹ ; page 3; second column; "Selection of therapeutic bacteriophages" the content of which is herein incorporated by reference. Such methods generally, but not necessarily, rely on the observation of lysis zones in areas of bacterial growth following inoculation with different phage strains, which indicates successful infection by a strain of lytic bacteriophage. Typically, a panel of at least 10, for example, at least 20, at least 50 or at least 100 different lytic bacteriophage strains will be tested for specificity against a given bacterial strain.

Further according to all of said aspects, in advance of phage administration to a subject it is preferred that both the strain of bacteria and its respective strain of bacteriophage are molecularly-defined, in other words characterised at the molecular (preferably genetic) level. To achieve this, both phage and bacterial strain may undergo triple cloning, and subsequent sequencing of a nucleic acid (for example via PGR or RT-PCR), preferably multiple nucleic acids, of sufficient uniqueness to both the phage and bacterial strain so as to identify said strains, and so as to be able to resolve them from other variants at the same taxonomic level (for example, other strains of the same species in the case of bacteria). Furthermore, said nucleic acid (or a substantial portion thereof) may be subjected to a variety of restriction fragment length polymorphism (RFLP) and molecular probes so as to produce a reproducible and unique 'molecular fingerprint^' by methods known to one skilled in the art. Said nucleic acid may be the whole genome of the phage or bacterium, or a substantial portion thereof. Characterisation of both phage and bacterial strains in this manner is well known in the art, and may be performed using standard methods. The skilled person will be aware of specific genetic loci which may be sequenced to achieve this; for example through multi-locus sequence typing (MLST), for which target loci and sequencing protocols are publicly available (for example at www.pasteur.fr/mlst; as accessed in October 2014, the contents of which is herein incorporated by reference). As an example only, characterisation of Klebsiella pneumonia strains may be performed by sequencing of the gap A, infB, mdh, pgi, phoE, rpoB and tonB genes, as carried out by Wang et al. (2013)⁵²; page 2; second column; "Genotyping of K. pneumonia isolates by MLST analysis" the content of which is herein incorporated by reference. Proteomic characterisation of gene products for both phage and bacterial strains may also be performed.

Further according to all of said aspects, the composition or combined preparation comprising bacteriophage will preferably be subject to testing (both preclinical and clinical) inclusive of pharmacokinetics and toxicity and immunogenicity testing, ideally after elimination or reduction of endo- and exo-toxins levels (derived from lysed bacterial cells) from bacteriophage preparations. Quality control measures for the composition include: stability (shelf life); absence of pyrogenicity, sterility and cytotoxicity; confirmation of the absence of temperate bacteriophages; and transmission electron microscopy-based confirmation of the presence of the expected virion morphologic particles. Such assessments may be performed using standard methods which are available in the art, for example as described in Merabashvili et al. (2009)³¹.

Further according to said first, second and sixth to twelfth aspects, a combined preparation comprising the at least 10, the at least 24, the at least 19, or the at least 22 different strains of lytic bacteriophage (where applicable) for separate, sequential or simultaneous use in therapy, treatment or prevention (where applicable) is also envisaged, as an alternative to a single composition comprising said different strains.

Further according to said first to third and sixth to twelfth aspects, the at least 5 strains of bacteriophage referred to in (b) have specificity for at least 5 strains of one or more species of bacteria. For the avoidance of doubt, this is to be construed as meaning that, for example, if there are only 5 different strains of phage, they will have specificity for only 5 different bacterial strains, but said strains may be distributed across more than one species. For example, 2 targeted strains may belong to a first species, and 3 targeted strains may belong to a second species, and so forth. According to said fifth aspect, where it is stated that, for example, at least 5 strains of bacteriophage each have specificity for at least one strain of [a selection of 5 different bacterial species] respectively, it is meant that each phage strain is specific for a strain of one of the 5 species. In other words, strains of all 5 species are targeted.

Further according to said first to third and fifth to twelfth aspects, a strain of bacteriophage referred to in (d) has specificity for a strain of bacteria that is resistant to at least 3, preferably at least 5 different antibiotics (and is thus considered multidrug resistant). This may include any 3, preferably 5 conventional antibiotics known in the art. Preferably, the strain of bacteria is resistant to at least 3, more preferably at least 5 antibiotics selected from the group consisting of ampicillin, tazobactum, sulbactam, cefazolin, ceftriaxone, ceftazidime, cefeprime, cefotetan, ertapenam, imipenem, aztreonam, ciprofloxicin, levofloxacin, gentamycin, tobramycin, amikacin, nitrofurantoin, Penicillins, Cephalosporins, Fluoroquinolones, aminoglycosides, macrolides, tetracyclines, ketolides, lincosamides, Trimethoprim-Sulfamethoxazole, Laevomycetinums, glycopeptides, Sulfonamides, Spectinomycin, Trimethoprim, Chloramphenicol, Clindamycin, Ethambutol, Nitrofurantoin, Novobiocin, Tigecycline Oxazolidinone, inhibitors of cell wall synthesis, inhibitors of protein synthesis, inhibitors of membrane function, anti-metabolites, inhibitors of nucleic acid synthesis, Beta-Lactams and Aminoglycosides. Alternatively, the strain of bacteria is resistant to 3, or 5, antibiotics selected from a group consisting of 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24. 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43 or 44 of any of the above. Even more preferably, the strain of bacteria is resistant to at least 3, most preferably at least 5 antibiotics selected from the group consisting of ampicillin, tazobactum, sulbactam, cefazolin, ceftriaxone, ceftazidime, cefeprime, cefotetan, ertapenam, imipenem, aztreonam, ciprofloxicin, levofloxacin, gentamycin, tobramycin, amikacin, nitrofurantoin, Penicillins, Cephalosporins, Fluoroquinolones, aminoglycosides, macrolides, tetracyclines, ketolides, lincosamides, Trimethoprim-Sulfamethoxazole, Laevomycetinums, glycopeptides, Sulfonamides, Spectinomycin, Trimethoprim, Chloramphenicol, Clindamycin, Ethambutol, Nitrofurantoin, Novobiocin, Tigecycline and Oxazolidinone.

Further according to said first to third and fifth to twelfth aspects, said resistance (above) may be determined in vitro using standard microbial culture techniques (in the presence of the antibiotic agent), automated drug susceptibility testing, or through the sequencing and identification of known drug resistance- associated genes; for example as described by Wang et al. (2013)³¹ ; page 2: first column; "PCR amplification and sequencing for drug resistance genes". Resistance of a bacterial strain to a given antibiotic is unambiguous to the skilled person, and can be recognised by the skilled person after having performed the appropriate tests. However, for the avoidance of doubt, a strain of bacteria is to be considered "resistant" to a given antibiotic when its growth is inhibited in vitro by a concentration of antibiotic that is associated with a high likelihood of therapeutic failure; or preferably wherein the antibiotic has a minimum inhibitory concentration (MIC) in respect of said strain of at least 2mg/L, preferably at least 4mg/L, more preferably at least 6mg/L, even more preferably at least 10mg/L, as determined by standard methods known in the art (for example serial dilution). Where possible for an antibiotic-bacterium combination, alternatively "resistance" may be defined in accordance with predetermined clinical MIC breakpoints as determined by EUCAST (European Committee on Antimicrobial Susceptibility Testing) - available athttp://www.eucast.org/fileadmin/src/media/PDFs/EUCAST files/Breakpoint tables/ Breakpoint table v 4.0. pdf (accessed in October 2014, the contents of which is herein incorporated by reference); using analogous or preferably the same protocols described as suitable therein.

Further according to said first, second, fourth and sixth to twelfth aspects, the administration referred to in (2) is preferably by any suitable means other than intravenous and intramuscular administration; more preferably, those selected from the group consisting of enteral, rectal, intra-muscular, sub-dermal, aerosol, intranasal, topical, intramammary and peritoneal administration.

Further according to said first, second and fourth to twelfth aspects, repeat administration of the composition comprising bacteriophage is preferred. In this regard, the administration in both (1 ) and (2) is preferably to be performed on at least 2 separate occasions (or, equally preferred, one occasion of administration in (1 ), in combination with three occasions of administration in (2)); wherein the time between said occasions is no more than 72 hours, preferably no more than 48 hours. Preferably, the first occasion of the administration in (2) is within 48 or preferably 24 hours of the first, or only, occasion of the administration in (1 ). 3 sequential occasions is more preferred, and 5 sequential occasions is especially preferred for both (1 ) and (2); the time between any two occasions in sequence being no longer than 72 hours, preferably no longer than 48 hours (i.e. no two occasions in sequence of both (1 ) and (2) are longer than 72 or 48 hours apart). Furthermore, it is preferred that the occasions of the administration in (2) are within 24hours of their respective occasions in sequence of the administration in (1 ). It is further preferred that a further composition, characterised in that it comprises a bacteriophage as referred to in (d), is also administered intravenously, or intramuscularly, to the subject within 24 hours of the first occasion of the administration in (1 ).

According to said third aspect, in step (i) a plurality of bacterial strains are identified to at least the species level. The bacterial strains are those present in one or more isolates (for example, clinical samples) which have been obtained from one or more subjects having the infection to be treated or prevented. By way of example, isolates may have been obtained from the infected skin or mucosa, gastrointestinal tract, respiratory system, urino-genital tract, brain or spinal cord, or mammary tissue (more preferably ovine, bovine or caprine mammary tissue; most preferably bovine) depending on the location and nature of the infection to be treated or prevented. Any other sites of infection may be sampled as the relevant tissue, organ or organ system; however those listed above are preferred. The skilled person will be able to vary the number of isolates for representative sampling. Species identification may be performed by a variety of means available in the art including, but not limited to, the use of mass spectrometry, preferably matrix assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry; plating on selective growth media; DNA sequencing or DNA fingerprinting (although these are preferred means).

The process of the third aspect will proceed if the species identification of step (i) reveals the presence of at least 10 different bacterial strains in one or more of said isolates, comprising:

(a) at least 3 bacterial strains of the primary pathogenic bacterial species (also referred to as the "target" bacterial species - as defined above under "definitions"); (b) at least 5 bacterial strains of one of more further pathogenic bacterial species (also referred to as "non-target" pathogenic bacterial species - as defined under "definitions"); said further species being distinct from that of (a);

(c) at least one strain of a further pathogenic bacterial species which is an opportunistic pathogen (see "definitions"); also distinct from that of (a) and (b); and

(d) at least one bacterial strain which belongs to the same species as the pathogenic species of (a), but which is resistant to at least 3, preferably at least 5, different antibiotics;

For the avoidance of doubt, the bacterial strain of (d) is a distinct strain to that of (a), although they are of the same species.

Further according to said third aspect, based on the species identification of step (i), above, the skilled person will be able to classify the bacterial species as "pathogenic", or an "opportunistic pathogen" (where appropriate) based on knowledge in the art concerning bacterial characteristics. In order to determine antibiotic resistance of the bacterial strain according to (d), step (i) may further comprise, after species identification, in vitro antibiotic resistance testing, or sequencing of antibiotic resistance genes (in accordance with the methods outline above). Said in vitro antibiotic resistance testing is preferably performed using at least any 3, preferably at least any 5 antibiotics selected from the groups given above.

Further according to said third aspect, above, pathogenic bacterial species according to (a) which may be found to infect representative tissue, organ or organ systems may include Staphylococcus aureus in the case of mammary tissue (especially ovine, bovine or caprine, in particular bovine); Staphylococcus aureus or methicillin-resistant Staphylococcus aureus in the case of the skin or mucosa; Clostridium difficile in the case of the gastrointestinal tract; Streptococcus pneumoniae or Mycobacterium tuberculosis in the case of the respiratory system; Neisseria gonorrhhoeae in the case of the urino-genital tract; and Neisseria meningitidis in the case of the brain or spinal cord. Preferably, the process will proceed if at least 3 strains of a relevant species are identified in one or more of said isolates from the respective tissue, organ or organ system.

Further according to said third aspect, pathogenic bacterial species according to (b), which co-infect the tissue, organ or organ system, will be identifiable, if present, following step (i). By way of example, if the tissue, organ or organ system comprises mammary tissue (preferably bovine, ovine or caprine, more preferably bovine mammary tissue), Streptococcus uteris, Streptococcus agalactiae, Bacillus licheniformis, Bacillus cereus and Bacillus subtilis are representative bacterial species according to (b) which may be found. Furthermore, if the tissue, organ or organ system comprises the skin or mucosa, Clostridium perfringen, Pseudomonas aeruginosa, Streptococcus pyogenes and vancomycin-resistant Enterococcus spp are representative bacterial species according to (b) which may be found. Furthermore, if the tissue, organ or organ system comprises tissue of the gastrointestinal tract, Escherichia coli, Klebsiella spp, Enterococcus spp, Bacillus fragilis and Clostridium perfringen are representative bacterial species according to (b) which may be found. Furthermore, if the tissue, organ or organ system comprises tissue of the respiratory system, Haemophilus influenzae, Klebsiella pneumoniae and Staphylococcus aureus are representative bacterial species according to (b) which may be found. Furthermore, if the tissue, organ or organ system comprises tissue of the urino-genital tract Staphylococcus saprophyticus, Enterococcus faecalis, Enterococcus faecium, Proteus mirabilis, Klebsiella pneumoniae, Proteus vulgaris, Proteus penneri, Staphylococcus epidermidis, Pseudomonas aeruginosa, Mycobacterium tuberculosis and Group B Streptococcus agalacti are representative bacterial species according to (b) which may be found. Furthermore, if the tissue, organ or organ system comprises tissue of the brain or spinal cord Listeria monocytogenes, Mycobacterium tuberculosis, Salmonella enterica and Serovar typhimurium are representative bacterial species according to (b) which may be found. Preferably, the process will proceed if at least 5 strains of one or more of the above relevant species are identified in one or more of said isolates from the respective tissue, organ or organ system.

Further according to said third aspect, the bacterial species according to (c), which are opportunistic pathogens, will be identifiable, if present, following step (i). By way of example, opportunistic pathogens (which are not site-specific) include Salmonella enteritidis, Pseudomonas aeruginosa, Streptococcus pneumoniae, Proteus mirabilis. Listeria monocytogenes. Escherichia coli, Staphylococcus aureus. Staphylococcus haemolyticus, Staphylococcus hyicus. Staphylococcus chromogenes and Staphylococcus dysgalacticae. Preferably, the process will proceed if at least one strain of the above species of opportunistic pathogen are identified in the one or more isolates. Further according to said third aspect, once target bacterial species and strains have been molecularly defined, one is ideally seeking bacteriophage that possess lytic activity against more than a single molecularly-defined bacterial target strain, i.e. those strains that may be different at the level of their DNA code, but share a similar bacteriophage-binding receptor. As for all viral infections, the first step in the infection process is binding to target cells. If only one bacteriophage is capable of killing only one target bacterium, then this obliges a higher inoculum volume so as to ensure that all phage contained in a given cocktail are present above 10⁴ PFU/ ml and desirably at higher concentrations.

Further according to said third aspect, the identification of susceptibility of the relevant bacterial strains to a plurality of lytic bacteriophage strains in steps (ii) - (v) may be performed using standard methods known in the art. However, preferably the inoculation of bacteria lawns or areas of bacterial growths in either aerobic or anaerobic cultures is used, as is known in the art. Specifically, the observation of plaques or lysis zones in bacterial lawns or areas of bacterial growth is indicative of susceptibility. Based on the species identification in step (i), the skilled person is able to purposively select a panel of lytic bacteriophage for susceptibility testing (comprising at least 10, at least 20, at least 50 or at least 100 bacteriophage strains, for example). As detailed above, bacteriophage strains may be purchased based on their specificity for a given bacterial species or strain, to aid the selection of said panel.

Further according to said third aspect, the bacteriophage found to be effective during susceptibility testing (steps (ii)-(v)) are formulated as a combined preparation or composition suitable for simultaneous, or separate or sequential use in the treatment of the disorder in question. Their combination into a single composition (or bacteriophage cocktail) is preferred. The minimum infectious dose of each lytic bacteriophage strain per single dosage form of the combined preparation or composition is to be at least 10⁴ plaque forming units (PFU)/ml. For the avoidance of doubt, this is the infectious dose determined with respect to the specific bacterial strain, which is identified as susceptible to the lytic bacteriophage strain in question. The infectious dose may be determined during susceptibility testing (steps (ii)-(v), above). However, it may separately be determined by standard methods known in the art (as detailed below).

Further according to said third aspect, it is preferred that the bacterial strain of (d) is one "prevalent" in the community. The definition of prevalence (and of the community) is variable depending on whether the subject is human or animal. With a human subject present in a healthcare facility, the definitions are those given below in relation to the first and sixth to twelfth aspects of the invention. With an animal subject (preferably livestock, more preferably ovine, bovine or caprine livestock), the definitions are those given below in relation to the fifth aspect of the invention, but not limited to mastitis or metritis. In other words, the clonally- expanded strain is present in at least 20% and so forth of infections by said pathogenic species of bacteria in (a), within the herd.

According to said fourth aspect, a combined preparation or composition obtainable by, preferably obtained by, the process of the fourth aspect of the invention may be prepared using conventional excipients used in bacteriophage cocktails. Preferably, said combined preparation is for use in therapy; more preferably of a bacterial infection (even more preferably one characterised by infection by a bacterial species of (a) of a relevant tissue, organ or organ system as provided above).

According to said fifth aspect, preferably the treatment or prevention is of ovine, caprine or bovine mastitis or metritis; more preferably bovine. In the case of mastitis, the treatment or prevention of clinical mastitis (an established infection) and sub-clinical mastitis (prior to the showing of symptoms), are separate but equally preferred embodiments. Furthermore, the ovine, caprine or bovine subject may be present in various stages of rearing, for example when in the whole milking herd, during the pre-birth drying-off period, or during first pregnancy prior to joining the milking herd. The treatment or prevention of metritis is also a further separate but equally preferred embodiment of the fifth aspect of the invention.

Further according to said fifth aspect, the use of the invention is intended primarily to reduce the level of infection to a state which can be treated with conventional antibiotics. In this regard, the combined preparation may be administered in combination with one or more antibiotic agents. Preferably, the one or more antibiotic agents are administered after use of the combined preparation.

Further according to said fifth aspect, it is preferred that additional bacteriophage strains according to (a) and (b) are incorporated into the combined preparation. Preferably, the combined preparation comprises at least 3, preferably at least 6, preferably at least 9, and more preferably at least 12 further strains of lytic bacteriophage each respectively having specificity for at least one strain of Staphylococcus aureus. Preferably, the combined preparation further comprises at least 6, preferably at least 9, and more preferably at least 12 further strains of lytic bacteriophage each respectively having specificity for a different strain of one or more species selected from Streptococcus agalacticae, Streptococcus uberis. Bacillus lichenformis. Bacillus cereus or Bacillus subtilis. All of said additional strains are administered to the subject at an infectious dose of at least 10⁴ PFU/ml.

Further according to said fifth aspect, it is preferred that the composition of the invention is administered by intravenous or intramuscular administration on occasion (1 ), and intramammary administration on occasion (2).

Further according to said fifth aspect, it is preferred that the bacterial strains, for which the bacteriophage strains of (d) are specific, are each prevalent in the community of the subject. The bacterial strain may be considered as "prevalent" in the community (i.e. a clonally-expanded strain) if present in at least 20%, preferably at least 30%, more preferably at least 40%, even more preferably at least 50% of mastitis/metritis (where appropriate) infections within the herd in which the subject is present (in the case of a bovine, caprine, or ovine subject). This may be determined by representative sampling and analysis of bacterial isolates, using standard methods. More preferably, the bacterial strains for which the bacteriophage strains of (d) are specific are also resistant to at least 3, more preferably at least 5 antibiotics. This may be determined via in vitro antibiotic resistance testing, or sequencing of antibiotic resistance genes (in accordance with the methods outline above). Said in vitro antibiotic resistance testing is preferably performed using at least any 3, preferably at least any 5 antibiotics selected from the groups given above.

According to said first, second and sixth to twelfth aspects, wherein the infection is to be prevented, and wherein the subject is present in or admitted to a healthcare facility, the first occasions of the administrations in (1 ) and (2) are preferably within 48, more preferably within 24, even more preferably 12 hours following the entrance or admission of the subject into the healthcare facility. Furthermore, the composition of the invention may be further administered intravenously, or intramuscularly, to the subject within 48 hours prior to entrance or admission into the healthcare facility, and the composition may be even further administered intravenously, or intramuscularly, to the subject within 48 hours prior to leaving the healthcare facility.

Further according to all of said first, second and fourth to twelfth, it is preferred for the practice of the invention that each strain of bacteriophage is present in the administered composition at a consistent dosage (i.e. there is batch- to-batch consistency). Attention to such parameters has largely been overlooked by phage therapy historically. Thus, it is especially preferred that each strain of bacteriophage is present in the composition administered on every occasion in (1 ) at an infectious dose, as measured by the number of plaque forming units/ml composition (PFU/ml - as measured by standard techniques available in the art, for example a Plaque Forming Assay comprising serial phage dilution), that is within 30%, preferably within 20%, more preferably within 10%, for example within 5% of that of the same strain as present in the composition administered on the first occasion in (1 ); and wherein each strain of bacteriophage is present in the composition administered on every occasion in (2) at an infectious dose, as measured by the number of plaque forming units/ml composition, that is within 30%, preferably within 20%, more preferably within 10%, for example within 5% of that of the same strain as present in the composition administered on the first occasion in (2).

Further according to said first, second and fourth to twelfth aspects, it is essential that each strain of bacteriophage is present in the composition (or combined preparation) administered in every occasion in (1 ) and (2) (or per single dosage form, according to said third aspect, above) at an infectious dose of at least 10⁴ plaque forming units/ml composition. This can be determined using standard methods available in the art of virology (for example, the use of serial dilution and a plaque forming assay, as performed according to Merabashvili et al. (2009)³¹ ; page 2; second column; "Titration of bacteriophage suspensions using the agar overlay method").

Further according to said first, second and fourth to twelfth aspects, it is especially preferred that each strain of bacteriophage is present in the composition (or combined preparation) administered in every occasion in (1 ) and (2) (or per single dosage form, according to said third aspect, above) at a minimum of 10x the LD₅₀ for the strain of bacteriophage, in respect of its defined bacterial target. This can be determined using standard methods available in the art of virology.

Further according to said first, second and fourth to twelfth aspects, it is especially preferred that the copy number of the viral genome of each bacteriophage strain/ml composition (or total volume of combined preparation) (thus indicating the number of virus particles) is kept consistent between the respective occasions of administration in both (1 ) and (2). Accordingly, the copy number of the viral genome of each strain of bacteriophage present in the composition administered on every occasion in both (1 ) and (2) is within 30%, preferably within 20%, more preferably within 10%, for example within 5% or within 1 %, of that of the same strain as present in the composition administered on the first occasion in both (1 ) and (2), respectively. In this regard, real-time PGR targeting a strain-specific locus of the phage genome may be used to quantify copy number in a given volume of the composition

According to said first and second aspect of the invention, both treatment and prevention are encompassed. However, either treatment or prevention (and the features applicable to either form of therapy) may exist as separate embodiments of said first and second aspect.

Further according to said first and second aspects, the opportunistic pathogen(s) for which a strain of bacteriophage in (c) has specificity is non-site specific; meaning that it is not limited to those found in the relevant tissue, organ or organ system. Alternatively, the strain of bacteriophage in (c) has specificity for any opportunistic pathogen(s).

Further according to said first and sixth to twelfth aspects, wherein the use is for treatment that is to be administered in a healthcare facility, it is especially preferred that the strain of bacteria for which the strain of bacteriophage in (d) has specificity is prevalent in either the healthcare facility or the community. As described in Wang et al. (2013)⁵²; page 2; second column; "Definitions", infections acquired in a healthcare facility and the community may be differentiated from one another by considering the time of onset of the infection. Thus, infections presenting within 48 hours of admission of the subject (for example, through a first positive culture or a symptomatic indication) may be considered as community-acquired (thus a bacterial strain prevalent in the community will be targeted), and after 48 hours from admission may be considered healthcare facility-acquired (thus a bacterial strain prevalent in the healthcare facility will be targeted).

Further according to said first and sixth to twelfth aspects, wherein the use is for prevention that is to be administered in a healthcare facility, it is especially preferred that the strain of bacteria for which the strain of bacteriophage in (d) has specificity is also prevalent in either the healthcare facility, or the community.

Further according to said first and sixth to twelfth aspects, the strain of bacteriophage in (d) may be considered as having specificity for a strain of bacteria "prevalent" in the community (i.e. a clonally-expanded drug-resistant strain) if the strain of bacteria is characterised as being responsible for at least 20%, preferably at least 30%, more preferably at least 40%, even more preferably at least 50% of deaths caused by the species of bacteria for which the strains of bacteriophage selected in (a) have specificity, in healthcare facilities within a radius of 100 miles, or within a radius of 50 miles, or within a radius of 10 miles, from the healthcare facility in which the subject is administered said treatment or prevention; within 5 years, or within 3 years, or within 2 years, or within one year ending with the date on which said treatment or prevention is performed. Standard methods available in the art (such as those described above) may be used to identify bacterial strains (which are clonally-expanded to a level of prevalence as defined above) at the genetic level. These strains will invariably be known in advance due to their preponderance in a given area ^50-56 , due most probably to the clonal expansion of a particular strain or strains of antibiotic-resistant bacteria ⁵⁷ .

Further according to said first and sixth to twelfth aspects, the strain of bacteriophage in (d) may be considered as having specificity for a strain of bacteria "prevalent" in the healthcare facility (i.e. a clonally-expanded drug-resistant strain) if characterised as being responsible for at least 20%, preferably at least 30%, more preferably at least 40%, even more preferably at least 50% of deaths caused by the species of bacteria for which the strains of bacteriophage selected in (a) have specificity, in the healthcare facility within 5 years, or within 3 years, or within 2 years, or within one year ending with the date on which said treatment or prevention is performed. Standard methods available in the art (such as those described above) may be used to identify bacterial strains (which are clonally-expanded to a level of prevalence as defined above) at the genetic level. These strains will invariably be known in advance due to their preponderance in a given area ^50-56 , due most probably to the clonal expansion of a particular strain or strains of antibiotic- resistant bacteria ⁵⁷ .

Further according to said first and second aspects, the strains of bacteriophage in (b) may preferably be defined as follows: at least 5 strains of lytic bacteriophage having specificity for at least 5 different strains of one or more further pathogenic species of bacteria, other than the species for which the strains of bacteriophage in (a) have specificity; wherein if the tissue, organ or organ system comprises the skin or mucosa, said one or more further pathogenic species of bacteria are selected from the group consisting of Clostridium perfringen, Pseudomonas aeruginosa, Streptococcus pyogenes and vancomycin-resistant Enterococcus spp; wherein if the tissue, organ or organ system comprises tissue of the gastrointestinal tract, said one or more further pathogenic species of bacteria are selected from the group consisting of Escherichia coli, Klebsiella spp, Enterococcus spp, Bacillus fragilis and Clostridium perfringens; wherein if the tissue, organ or organ system comprises tissue of the respiratory system, said one or more further pathogenic species of bacteria are selected from the group consisting of Haemophilus influenzae, Klebsiella pneumoniae and Staphylococcus aureus: wherein if the tissue, organ or organ system comprises tissue of the urino-genital tract, said one or more said one or more further pathogenic species of bacteria are selected from the group consisting of Staphylococcus saprophyticus, Enterococcus faecalis, Enterococcus faecium, Proteus mirabilis, Klebsiella pneumoniae, Proteus vulgaris, Proteus penneri, Staphylococcus epidermidis, Pseudomonas aeruginosa, Mycobacterium tuberculosis and Group B Streptococcus agalacti; or wherein if the tissue, organ or organ system comprises tissue of the brain or spinal cord, said one or more further pathogenic species of bacteria are selected from the group consisting of Listeria monocytogenes, Mycobacterium tuberculosis, Salmonella enterica and Serovar typhimurium.

Further according to said first and second aspects, an opportunistic pathogen for which a strain of bacteriophage in (c) has specificity is preferably found in a healthcare environment. More preferably, the strain of bacteriophage in (c) has specificity for a strain of an opportunistic pathogen selected from the group consisting of Salmonella enteritidis, Pseudomonas aeruginosa. Streptococcus pneumoniae, Proteus mirabilis. Listeria monocytogenes, Escherichia coli and Staphylococcus aureus; or the group consisting of Salmonella enteritidis, Pseudomonas aeruginosa, Streptococcus pneumoniae, Proteus mirabilis, Listeria monocytogenes, Escherichia coli. Staphylococcus aureus. Staphylococcus haemolyticus. Staphylococcus hyicus. Staphylococcus chromogenes and Staphylococcus dysgalacticae In one embodiment, more than one, for example three different strains of bacteriophage may be employed which have specificity for more than one, for example three different strains of one or more species which is an opportunistic pathogen (preferably one or more species from the lists given above). Bacteriophage and Human and Animal Safety

The following summarises the state of the art concerning the suitability of bacteriophage for administration to humans and animals with regard to safety.

Bacteriophages are defined, as their name would suggest, as 'eaters of bacteria^'. They bind specific receptors found only on the surface of bacteria; and can therefore not directly infect mammalian or other eukaryotic cells. Bacteriophages are probably the most common 'self-replicating entity^' encountered on Earth, even more so than their bacterial hosts. Estimates run to some 10³¹ particles ³². Bacteriophages are found throughout our bodies and the surrounding environment and, as such, we eat them daily and play host to multitudes of different bacteriophage within our very own microbiomes within and on the human body.

A massive amount of scientific evidence has been gathered to demonstrate exactly how lytic bacteriophage reproduce and thereby kill bacteria. Several authors have detailed the history of the discovery of bacteriophage, their past use in fighting disease in humans and animals and the overall accumulation of our molecular knowledge that underwrites these processes ^6"18. Indeed, the ability of

bacteriophage to kill bacteria and resolve human infection has been known since many years. In 1917, Herelle reported the use of bacteriophage against bacterial dysentery (English translation after that detailed by Dublanchet ⁸ :

"The next morning, on opening the incubator, I experienced one of those rare moments of intense emotion.... I saw that the broth culture, which the night before had been very turbid (due to the presence of high numbers of bacteria growing in the broth solution), was perfectly clear (due to the killing action of the bactiophage lytic effect on and associated rupture of the bacterial cell wall): all the bacteria had vanished, they had dissolved away like sugar in water. As for the agar spread, it was devoid of all (bacterial) growth... in a flash, I had understood: What caused my clear spots was in fact an invisible microbe, a filterable virus, but a virus parasitic on bacteria... If this is true, the same thing has probably occurred during the night in the sick man... He should now be cured. In fact, during the night, his general condition had greatly improved and convalescence was beginning" In the early 1970s scientists in the USA and the US government realized that they had been inoculating humans with untold trillions of bacteriophages in live vaccines doses and this for many different vaccines administered to millions of the US population and individuals around the world for decades past. Without doubt, the many millions of doses of phage-containing vaccines administered internationally constitute collectively the very best large-scale ("Mega-Study") study conducted to date as concerns the overall safety of bacteriophages in humans.

The following attributes of bacteriophage have been known for many years and combine to demonstrate the anticipated safety of the therapeutic treatment regimen of the invention for human and animal inoculation.

Firstly, bacteriophage are potentially the most abundant life-form on the planet earth and by default are probably present and active in and against all microbial microbiomes in the human body. Where bacteria are present, one will also encounter bacteriophages. Thus, on a daily basis, humans either in our food and drink or on our skin or in our body orifices co-exist in the presence of billions and billions of bacteriophages. The latter recognize their bacterial host by surface receptors found uniquely on the surface of a specific strain of their bacterial host organism. They are not known to be capable of directly infecting mammalian cells. Bacteriophage have undergone co-evolution with their bacterial hosts so as to recognize and infect target bacterial cells with an extraordinary level of specificity and affinity of binding ³³. They also manifest remarkably low susceptibility to variations in temperature and pH, organic solvents and proteases, a capacity that is equally coupled with an ability to recognize the difference between living and dead cells ³³. Human beings as exposed daily to a deluge of bacteriophages. Many of these are destroyed by gastric juices when ingested orally, but many also make it across the intestinal wall and into the blood stream and even across the blood brain barrier. Elsewhere, bacteriophages have been demonstrated to harmlessly make their way to brain tissue of mice via the olfactory neuron route following intra-nasal inoculation and efficiently penetrating biological membranes in so doing ³⁴. This vindicates the earlier seminal work of Dubos ^{18, 35}, who was able to clearly demonstrate the passage of bacteriophage across the blood brain barrier to fight bacterial infection. To date, the presence of bacteriophage in our bodies is not known to cause detriment. Indeed, bacteriophages are actually beneficial and probably assist the body by naturally fighting-off infection agents and limiting the damage provoked within our bodies. Secondly, millions of bacteriophage have previously been given to millions of people as vaccination programs around the globe ^{36, 37}. The realization of this previously unknown contamination of numerous human vaccines with bacteriophage resulted in a high-profile exemption being issued by the US Food and Drug Admnistration (FDA) in 1975. This effectively meant that FDA was to formally approve the inclusion of bacteriophages of various and unknown kinds in human vaccines. Some years later, this resulted in FDA^'s tolerance of vaccine contaminants in a court case in 1987, that stated "each seed virus used in manufacture shall be demonstrated to be free of extraneous microbial agents except for unavoidable bacteriophage".

In February, 1975 Gina Bari Kolata ³⁸ , wrote an article entitled "Phage in Live Virus Vaccines: Are They Harmful to People? , stating that " in 1973, scientists at the Bureau of Biologies of the Food and Drug Administration (FDA) reported that all live virus vaccines are grossly contaminated with phage (viruses that infect bacteria). This finding presented a problem since federal regulations forbade extraneous material in vaccines, and no one knew whether phage are harmful to human beings or whether they could be removed from vaccines. The temporary solution was to amend the regulations so as to permit phage in vaccines" ³⁸.

As a direct consequence of these findings, namely, that bacteriophage were heavily contaminating human vaccines, the US FDA set about proving that bacteriophages as isolated from therapeutic vaccines were unlikely to cause harm to humans ³⁹.

Thirdly, multiple animal studies directed at treating bacteraemia with bacteriophage have been able to demonstrate the absence of an immune response in animals subjected to phage alone, i.e. as part of a placebo control studies in the absence of experimental infection ^40-46. The explanation of such results is probably linked to the extremely rapid clearance of bacteriophage from the blood by the body^'s reticulo-endothelial system ⁴⁷. Indeed, Inchley was able to demonstrate using radiolabeled T4 phage that the mouse liver phagocytized more than 99% of circulating phage within 30 minutes ⁴⁸. The conclusion must be that they do not persist long within the body. As reported by Merril and others this was seen as a major impediment to effect phage therapy.

Elsewhere, several authors have attempted to collect published accounts of the many thousands of human patients administered bacteriophage and effectively treated in the former Soviet Union ⁷' ⁹' ^{10, 17, 18}. In addition, one should note that of recent times the Russian state-owned corporation, MicroGen, sells millions of doses per annum of bacteriophage destined for human use. In 2013, MicroGen sold an estimate 26 million doses and this without widespread reports of detectable deleterious side- effects or a hecatomb in the Russian population.

Endotoxins and immunogens are sometimes cited as a potential cause for concern within preparations of bacteriophage destined for administration to humans. Today, there exist effective methods for the removal of these contaminants, for example, EndoTrap® Blue high endotoxin affinity ligand (Manufactured by Hyglos GmbH, Germany) which is a proteinaceous matrix derived from a bacteriophage that is covalently immobilized on agarose beads ³¹. It is equally important to note that during sepsis and associated bacteraemia, bacterial infections will be producing far more such contaminants within the body. By doing away with the cause of sepsis and unwanted endotoxin and immunogen production, the body can be allowed to rapidly return to health. Indeed several authors throughout history have remarked upon the dramatically rapid return to a healthy state following administration of bacteriophage. None better exemplifies this than original observations of d^'Herelle, as cited above ⁸, accounts of several other miraculously rapid recoveries ¹⁰ and that of Knouf et al. ⁴⁹ reporting on the treatment of typhoid patients in California prior to the availability of the antibiotic, chloramphenicol: "In 24-26 hours, patients that had been comatose and in the 'typhoid state^' amazed everyone by their cheerful, grateful attitude".

Example

Preliminary work underpinning the invention was performed in a milking herd, concerning mastitis induced by antibiotic resistant bacteria, specifically S. aureus. Specifically, samples taken from 21 cows over two farms (Farms A and B - Arshinovsk and Narochat respectively, Russian Federation) were analysed for susceptibility to antibiotics and a non-inventive bacteriophage cocktail, according to the following method.

Field sampling and subsequent growth of bacteria in aerobic and anaerobic culture was carried out on a variety of bacterial growth media. Bacterial colonies obtained from clinical milk samples were identified to the species level (Figure 1 ; positive identifications indicated with "+"). This was conducted using a Bruker LT MALDI-TOF Mass Spectrometer Biotyper for high-throughput screening, using standard protocols. Growth on additional media could alternatively be used to classify species, and DNA sequencing or fingerprinting to strain and/or species level can be performed (as detailed in the description).

Each bacterial sample was then subjected to susceptibility testing against a range of antibiotics (using antibiotic disks) under both aerobic and anaerobic conditions (Figures 2 and 3). The presence of susceptibility was determined based on a skilled assessment, as employed by those familiar with the art. However, the following approximations were used as a guide:

+ = Zone of bacterial growth inhibition < 0.5 cm in diameter.

++ = Zone of bacterial growth inhibition < 1 .0 cm in diameter.

+++ = Zone of bacterial growth inhibition > 1 .0 cm in diameter.

The assessment was performed, for example, following 48 hours co-culture with a titration of bacteriophage so as to permit plaque forming unit (PFU) detection somewhere within the serial titration (each on separate bacterial lawn culture plates); whereby the zone of inhibition is not too small or does not includes the entire bacterial culture plate.

Furthermore, each bacterial sample was subjected to susceptibility testing against a combined cocktail of two commercially-available bacteriophage cocktails (Phagoderm and Phagodent) with known activity against drug-resistant Staphylococcus aureus (the cause of mastitis in dairy cows and hospital-acquired infections in humans), in addition to 58 additional bacteriophage strains. The bacterial species for which the bacteriophage strains in the combined cocktail are specific are listed in Table 1 . The susceptibility of each bacterial sample to the combined bacteriophage cocktail is given in the final two columns of Figures 1 -4. Susceptibility scoring was performed in accordance with the approximations outlined above. Figure 4 summarises the bacterial species found to be highly prevalent. Three causative bacterial species were found to be present in all mastitis cases, whereas two species were found in 92.5% of cases.

Results and Conclusions

Across the two farms, the bacterial diversity detected was likely representative of well-established mastitis, before it was detectable by farm staff. Thus, more cows were affected in the herd than were detected, contributing to higher overall bacterial and somatic cell counts. Milk quality being produced by the herd would therefore likely be lowered.

As can be seen in Figures 1 -4, the approach of simply increasing the number of bacteriophage contained in a given cocktail demonstrated mixed, but general poor results. Only 9 out of 21 cows were likely to have been cured using the combined bacteriophage cocktail (i.e. the bacterial samples demonstrated sensitivity under both aerobic and anaerobic conditions: see Figures 1 -4). When compared with 22 different antibiotics tested under anaerobic growth conditions, 8 out of the 22 antibiotics performed better than the combined bacteriophage cocktail. Under aerobic growth conditions, 2 out of the 22 antibiotics performed better than the combined bacteriophage cocktail. As a result, the enlarged cocktail (containing a total of 161 different phage strains) was unable to demonstrate a sufficiently high efficacy in vitro to warrant field deployment. This was because the cocktail was not knowingly constructed based on a detailed knowledge of the target disease-causing agents, or with an appropriate virulent dose of specific phage strains (as per the invention).

Phage cocktails have traditionally met with varying degrees of success and failure. The choice of phage strain and the methods of phage cocktail preparation are critical to the final bacterial killing effect in vitro and in vivo. In vivo the final concentration of phage dosage is of paramount importance to the extent that adding an infinite number of phage does not necessarily increase bacterial killing effect (as shown in part in the Example). This is because phage that do not recognize relevant strains of the target infection serve to reduce the overall killing effect by increasing the dilution of "good killers" within the final cocktail. This issue is far more important in vivo in live humans or animals in an agricultural setting, where an insufficiency of virulent phage will not allow for adequate treatment due to inadequate dosage. The latter reduces the likelihood of bacteriophage encountering at least one target bacterial within the body volume of the infected host organism (humans or farm animal, for example) that will allow for exponential expansion of bacteriophage number at the site(s) of infection.

Table 1 Two commercially available bacteriophage cocktails each containing different bacteriophages strains directed against the same bacterial Species

The list of pathogens No. of phage strains lysing pathogens

Starting Point Preparations Additional

Phagodent Phagoderm phages tested against mastitis from farms

A & B

Acinetobacter towneri 2

Acinetobacter baumannii spp. 2

Actinomyces israelii 3

Actinomyces spp. 3

Aggregatibacter 3

actinomycetemcomitans

Alcaligenes faecalis 2

Bacillus amyloliquefaciens 2

Bacillus cereus 2

Bacillus licheniformis 2

Bacillus pumilus 2

Bacillus safensis 2

Bacillus subtilis 2

Bacteroides forsythus 3

Bacteroides fragilis 3

Bacteroides gracilis 3

Bacteroides spp. 3

Brevibacillus centrosporus 2

Campylobacter spp. 3 Citrobacter freundii 3

Corynebacterium xerosis 2

Corynebacterium spp. 3

Enterobacter spp. 3

Enterococcus faecalis 3

Enterococcus saccharolyticus 2

Escherichia coli 4 2

Fusobacterium spp. 3

Klebsiella spp. 3

Ochrobactrum intermedium 2

Paenibacillus amyloliticus 2

Porphyromonas gingivalis 3

Prevotella intermedia 3

Propionibacterium acnes 3

Proteus mirabilis spp. 3

Proteus vulgaris 3 3

Providencia rettgeri spp. 3

Pseudomonas putida 2

Pseudomonas aeruginosa 3 3

Rhodococcus rhodochrous 2

Serratia liquefaciens 2

Staphylococcus chromogenes 2

Staphylococcus epidermidis 2

Staphylococcus equorum 2

Staphylococcus haemolyticus 2

Staphylococcus hyicus 2

Staphylococcus sciuri 2

Staphylococcus aureus 3 3 2

Staphylococcus epidermidis 2

spp.

Streptococcus agalactiae 2

Streptococcus dysgalactiae 2

Streptococcus uberis 2

Streptococcus mitis 3 Streptococcus mutans 3

Streptococcus pyogenes 3

Streptococcus pyogenes spp. 3

Streptococcus salivarius 3

Treponema denticola 2

Trueperella pyogenes 2 (Arcanobacterium pyogenes)

Wohlfahrtiimonas chitiniclastica 2

Wolinella spp. 3

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Claims

1 . A composition comprising at least 10 different strains of lytic bacteriophage, for use in the treatment or prevention of a disorder in a subject, characterised by the infection of a tissue, organ or organ system by a pathogenic species of bacteria; wherein

(d) a strain of lytic bacteriophage has specificity for a strain of bacteria which is resistant to at least three different antibiotics, and which is of the same species as said pathogenic species of bacteria for which the strains of bacteriophage in (a) have specificity; wherein said treatment or prevention comprises:

(2) administering said composition to the subject by any suitable means on at least two further separate occasions, the time between said two further occasions being no longer than 72 hours; wherein each strain of bacteriophage is present in the composition administered in each occasion in (1 ) and (2) at an infectious dose of at least 10⁴ plaque forming units (PFU)/ml composition.

2. A composition comprising at least 10 different strains of lytic bacteriophage, for use in the treatment or prevention of a disorder in a subject, characterised by the infection of a tissue, organ or organ system by a pathogenic species of bacteria; wherein

(a) at least 3 strains of lytic bacteriophage have specificity for at least 3 different strains of said pathogenic species of bacteria:

(d) a strain of lytic bacteriophage has specificity for a strain of bacteria which is resistant to at least three different antibiotics, and which is of the same species as said pathogenic species of bacteria for which the strains of bacteriophage in (a) have specificity; and wherein at least 20% of deaths attributable to said pathogenic species of bacteria in a given population, and within a given time period, are attributable to the strain of said pathogenic species of bacteria; wherein said treatment or prevention comprises:

(1 ) administering said composition to the subject intravenously, or intramuscularly, on at least one occasion; and (2) administering said composition to the subject by any suitable means on at least two further separate occasions, the time between said two further occasions being no longer than 72 hours; wherein each strain of bacteriophage is present in the composition administered in each occasion in (1 ) and (2) at an infectious dose of at least 10⁴ PFU/ml composition.

3. A composition for use according to claim 1 or 2, wherein the intravenous or instramuscular administration in (1 ) is carried out on at least two separate occasions, the time between said two occasions being no longer than 72 hours.

4. A composition for use according to claim 1 or 2, wherein the administration in (2) is carried out on at least three sequential occasions, the time between any two occasions in sequence being no longer than 72 hours.

5. A composition for use according to any preceding claim, wherein the means of administration in (2) is any suitable means other than intravenous and intramuscular administration.

6. A composition for use according to claim 5, wherein the means of administration in (2) is selected from the group consisting of enteral, rectal, intra-muscular, sub- dermal, aerosol, intra-nasal, topical, intramammary and peritoneal administration.

7. A composition for use according to claim 1 , wherein said treatment or prevention is performed in a healthcare facility; wherein if the use is for treatment: if the infection presents within 48 hours of the admission of the subject to the healthcare facility, the strain of bacteria for which the strain of bacteriophage in (d) has specificity is one which is prevalent in the community; or if the infection presents after 48 hours of the admission of the subject to the healthcare facility, the strain of bacteria for which the strain of bacteriophage in (d) has specificity is one which is prevalent in the healthcare facility; or wherein if the use is for prevention: the strain of bacteria for which the strain of bacteriophage in (d) has specificity is one which is prevalent in the healthcare facility, or the community.

8. A composition for use according to claim 2, wherein in (d), at least 30%, preferably at least 40%, more preferably at least 50% of deaths attributable to said pathogenic species of bacteria in said population, and within said time period, are attributable to the strain of said pathogenic species of bacteria.

9. A composition for use according to claim 2 or 8, wherein the population in (d) is defined as the population of a city, in which the subject is present when receiving said treatment or prevention.

10. A composition for use according to claim 2 or 8, wherein the population in (d) is defined as the population of a country, in which the subject is present when receiving said treatment or prevention.

1 1 . A composition for use according to claim 2 or 8, wherein the population in (d) is defined as the population of a subcontinent, in which the subject is present when receiving said treatment or prevention.

12. A composition for use according to claim 2 or 8, wherein the population in (d) is defined as the population of a continent, in which the subject is present when receiving said treatment or prevention.

13. A composition for use according to claim 2 or 8, wherein the population in (d) is defined as the number of patients admitted to a healthcare facility, within said time period, in which the subject is present when receiving said treatment or prevention.

14. A composition for use according to claim 2 or 8, wherein said therapy is administered in a healthcare facility: and wherein the population in (d) is defined as all patients admitted to healthcare facilities, within said time period, within a radius of 100 miles of the healthcare facility where said treatment or prevention is administered.

15. A composition for use according to claim 2 or 8, wherein said therapy is administered in a healthcare facility; and wherein the population in (d) is defined as all patients admitted to healthcare facilities, within said time period, within a radius of 10 miles of the healthcare facility where said treatment or prevention is administered.

16. A composition for use according to any of claims 2 and 8-15 wherein said time period in (d) is one year ending with the date on which said treatment or prevention is administered.

17. A composition for use according to any of claims 2 and 8-15, wherein said time period in (d) is 2 years ending with the date on which said treatment or prevention is administered.

18. A composition for use according to any of claims 2 and 8-15, wherein said time period in (d) is 3 years ending with the date on which said treatment or prevention is administered.

19. A composition for use according to any of claims 2 and 8-15, wherein said time period in (d) is 5 years ending with the date on which said treatment or prevention is administered.

20. A composition for use according to any preceding claim, wherein the administration in both (1 ) and (2) is performed on 3, preferably 5 sequential occasions; the time between any two occasions in sequence being no longer than 72 hours, preferably no longer than 48 hours.

21 . A composition for use according to any of claims 3-20, wherein the occasions of the administration in (2) are within 24, preferably within 12 hours of their respective occasions in sequence of the administration in (1 ).

22. A composition for use according to any preceding claim, wherein a further composition, comprising a strain of bacteriophage as referred to in (d), is administered intravenously, or intramuscularly, to the subject within 24, preferably within 12 hours of the first occasion of the administration in (1 ).

23. A composition for use according to any preceding claim, wherein the use is for prevention, to be performed in a healthcare facility; wherein the first occasions of the administrations in (1 ) and (2) are within 48, preferably within 24, more preferably within 12 hours, even more preferably within 6 hours following the admission of the subject into the healthcare facility.

24. A composition for use according to any preceding claim, wherein the use is for prevention, to be performed in a healthcare facility; wherein said composition is further administered intravenously, or intramuscularly, to the subject within 48 hours prior to admission into the healthcare facility, and wherein said composition is even further administered intravenously, or intramuscularly, to the subject within 48 hours prior to leaving the healthcare facility.

25. A composition for use according to any preceding claim, wherein the strain of bacteria for which the strain of bacteriophage referred to in (d) has specificity is resistant to at least three antibiotics selected from the group consisting of ampicillin, tazobactum, sulbactam, cefazolin, ceftriaxone, ceftazidime, cefeprime, cefotetan, ertapenam, imipenem, aztreonam, ciprofloxicin, levofloxacin, gentamycin, tobramycin, amikacin, nitrofurantoin, Penicillins, Cephalosporins, Fluoroquinolones, aminoglycosides, macrolides, tetracyclines, ketolides, lincosamides, Trimethoprim- Sulfamethoxazole, Laevomycetinums, glycopeptides, Sulfonamides, Spectinomycin, Trimethoprim, Chloramphenicol, Clindamycin, Ethambutol, Nitrofurantoin, Novobiocin, Tigecycline Oxazolidinone, inhibitors of ceil wall synthesis, inhibitors of protein synthesis, inhibitors of membrane function, anti-metabolites, inhibitors of nucleic acid synthesis, Beta-Lactams and Aminoglycosides.

26. A composition for use according to any preceding claim, comprising at least 3 different strains of lytic bacteriophage as referred to in (c).

27. A composition for use according to any preceding claim, wherein the opportunistic pathogen, for which the strain of bacteriophage in (c) has specificity, is found in a healthcare environment.

28. A composition for use according to any preceding claim, comprising at least 3 different strains of lytic bacteriophage as referred to in (d).

29. A composition for use according to any preceding claim, wherein the strain of bacteria, for which the strain of bacteriophage in (d) has specificity, is found in a healthcare environment.

30. A composition for use according to any preceding claim, wherein the strain of bacteriophage in (c) has specificity for a strain of an opportunistic pathogen selected from the group consisting of Salmonella enteritidis, Pseudomonas aeruginosa. Streptococcus pneumoniae, Proteus mirabilis. Listeria monocytogenes, Escherichia coli and Staphylococcus aureus.

31 . A composition for use according to any of claims 3-30, wherein each strain of bacteriophage is present in the composition administered on each occasion in (1 ) at an infectious dose, as measured by the number of plaque forming units/ml composition, that is within 30%, preferably within 20%, more preferably within 10% of that of the same strain as present in the composition administered on the first occasion in sequence in (1 );

32. A composition for use according to any preceding claim, wherein each strain of bacteriophage is present in the composition administered on each occasion in (2) at an infectious dose, as measured by the number of plaque forming units/ml composition, that is within 30%, preferably within 20%, more preferably within 10% of that of the same strain as present in the composition administered on the first occasion in sequence in (2).

33. A composition for use according to any preceding claim, wherein each strain of bacteriophage is present in the composition administered in each occasion in (1 ) and (2) at a minimum of 10x the LD₅₀ for the strain of bacteriophage.

34. A composition for use according to any preceding claim, wherein the bacterial infection is associated with a wound of the skin or mucosa, the gastrointestinal tract, the respiratory system or the urino-genital tract; or wherein the bacterial infection is multiple drug-resistant tuberculosis, a nosocomial Escherichia coli infection or bacterial meningitis.

35. A composition for use according to claim 34, wherein the bacterial infection is associated with a wound of the skin or mucosa; and wherein at least 6, preferably at least 9, more preferably at least 12 strains of bacteriophage referred to in (a) have specificity for at least 6, preferably at least 9, more preferably at least 12 different strains of their specific bacterial species, which is preferably Staphylococcus aureus and/or methicillin-resistant Staphylococcus aureus; and at least 5 strains of bacteriophage referred to in (b) have specificity for at least 5 different strains of their one or more specific bacterial species, which is preferably one or more bacteria selected from the group consisting of Clostridium perfringen, Pseudomonas aeruginosa, Streptococcus pyogenes and vancomycin-resistant Enterococcus spp; preferably wherein a strain of bacteriophage referred to in (c) has specificity for a strain of Salmonella enteriditis; and a strain of bacteriophage referred to in (d) has specificity for a strain of Staphylococcus aureus.

36. A composition for use according to claim 34, wherein the bacterial infection is associated with the gastrointestinal tract; and wherein at least 12 strains of bacteriophage referred to in (a) have specificity for at least 12 different strains of their specific bacterial species, which is preferably Clostridium difficile; and at least 10, preferably at least 15, more preferably at least 20 strains of bacteriophage referred to in (b) have specificity for at least 10, preferably at least 15, more preferably at least 20 different strains of their one or more specific bacterial species, which is preferably one or more bacteria selected from the group consisting of Escherichia coli, Klebsiella spp, Enterococcus spp, Bacillus fragilis and Clostridium perfringens; preferably wherein a strain of bacteriophage referred to in (c) has specificity for a strain of Salmonella enteriditis; and a strain of bacteriophage referred to in (d) has specificity for a strain of Clostridium difficile.

37. A composition for use according to claim 34, wherein the bacterial infection is associated with the respiratory system: and wherein at least 12 strains of bacteriophage referred to in (a) have specificity for at least 12 different strains of their specific bacterial species, which is preferably Streptococcus pneumonia; and at least 5 strains of bacteriophage referred to in (b) have specificity for at least 5 different strains of their one or more specific bacterial species, which is preferably one or more bacteria selected from the group consisting of Haemophilus influenzae, Klebsiella pneumoniae and Staphylococcus aureus; preferably wherein a strain of bacteriophage referred to in (c) has specificity for a strain of Pseudomonas aeruginosa; and a strain of bacteriophage referred to in (d) has specificity for a strain of Streptococcus pneumoniae.

38. A composition for use according to claim 34, wherein the bacterial infection is associated with the urino-genital tract; and wherein at least 12 strains of bacteriophage referred to in (a) have specificity for at least 12 different strains of their specific bacterial species, which is preferably Neisseria gonorrhoeae; and at least 8 strains of bacteriophage referred to in (b) have specificity for at least 8 different strains of their one or more specific bacterial species, which is preferably one or more bacteria selected from the group consisting of Staphylococcus saprophyticus, Enterococcus faecalis, Enterococcus faecium, Proteus mirabilis, Klebsiella pneumoniae, Proteus vulgaris, Proteus penneri, Staphylococcus epidermidis, Pseudomonas aeruginosa, Mycobacterium tuberculosis and Group B Streptococcus agalactiae: preferably wherein a strain of bacteriophage referred to in (c) has specificity for a strain of Escherichia coli; and a strain of bacteriophage referred to in (d) has specificity for a strain of Neisseria gonorrhoeae.

39. A composition for use according to claim 34, wherein the bacterial infection is a multiple drug-resistant tuberculosis infection; and wherein at least 12 strains of bacteriophage referred to in (a) have specificity for at least 12 different strains of their specific bacterial species, which is preferably Mycobacterium tuberculosis; and at least 5 strains of bacteriophage referred to in (b) have specificity for at least 5 different strains of their one or more specific bacterial species, which is preferably one or more bacteria selected from the group consisting of Haemophilus influenzae, Klebsiella pneumonia and Staphylococcus aureus; preferably wherein a strain of bacteriophage referred to in (c) has specificity for a strain of Streptococcus pneumoniae; and a strain of bacteriophage referred to in (d) has specificity for a strain of Mycobacterium tuberculosis.

40. A composition for use according to claim 34, wherein the bacterial infection is a nosocomial Escherichia co// infection; and wherein at least 12 strains of bacteriophage referred to in (a) have specificity for at least 12 different strains of their specific bacterial species, which is preferably Escherichia coli; and at least 8 strains of bacteriophage referred to in (b) have specificity for at least 8 different strains of their one or more specific bacterial species, which is preferably one or more bacteria selected from the group consisting of Acinetobacter baumannii, Staphylococcus aureus, Klebsiella pneumonia, Pseudomonas aeruginosa, Morganella morganii, Salmonella spp, Enterobacter cloacae, Staphylococcus hominis, Staphylococcus epidermidis and Staphylococcus saprophyticus; preferably wherein a strain of bacteriophage referred to in (c) has specificity for a strain of Proteus mirabilis; and a strain of bacteriophage referred to in (d) has specificity for a strain of Escherichia coli.

41 . A composition for use according to claim 34, wherein the bacterial infection is a bacterial meningitis infection; and wherein at least 12 strains of bacteriophage referred to in (a) have specificity for at least 12 different strains of their specific bacterial species, which is preferably Neisseria meningitidis; and at least 8 strains of bacteriophage referred to in (b) have specificity for at least 8 different strains of their one or more specific bacterial species, which is preferably one or more bacteria selected from the group consisting of Listeria monocytogenes, Mycobacterium tuberculosis, Salmonella enterica and Serovar typhimurium; preferably wherein a strain of bacteriophage referred to in (c) has specificity for a strain of Listeria monocytogenes; and a strain of bacteriophage referred to in (d) has specificity for a strain of Neisseria meningitidis.

42. A process for the production of a combined preparation or composition comprising at least 10 different strains of lytic bacteriophage, for simultaneous, separate or sequential use in the prevention or treatment of a disorder characterised by the infection of a tissue, organ or organ system by pathogenic bacterial species: comprising the steps:

(c) at least one of said at least 10 bacterial strains is of a bacterial species which is an opportunistic pathogen, other than the species of (a) and (b), and (d) at least one of said at least 10 bacterial strains is of the same pathogenic bacterial species as of (a), and resistant to at least 3 different antibiotics:

43. A process according to claim 42, wherein the species identification of step (i) is performed via mass spectrometry, preferably matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry; plating on selective growth media; DNA sequencing or DNA fingerprinting.

44. A process according to claim 42 or 43, wherein said determining of susceptibility of steps (ii)-(iv) is performed via inoculation of bacterial lawns or areas of bacterial growth in aerobic or anaerobic culture with the strains of lytic bacteriophage, and observation of plaques or lysis zones in said bacterial lawns or areas of bacterial growth.

45. A process according to any of claims 42-44, wherein step (i) further comprises, following species identification, determining the resistance or susceptibility of said at least one bacterial strain of (d) to at least three antibiotics; or sequencing of antibiotic resistance genes.

46. A process according to any of claims 42-45, wherein the bacterial strain of (d) is prevalent in the community of said one or more subjects.

47. A combined preparation or composition which is obtainable by, preferably which has been obtained by, the process of claims 42-46.

48. A combined preparation or composition according to claim 47, for simultaneous, separate or sequential use in therapy, preferably in the treatment or prevention of a bacterial infection.

49. A combined preparation comprising at least 12 different strains of lytic bacteriophage, for simultaneous, separate or sequential use in the treatment or prevention of mastitis or metritis in a subject; wherein

(a) at least one strain of lytic bacteriophage has specificity for at least one strain of Staphylococcus aureus;

(b) at least 5 strains of lytic bacteriophage each have specificity for at least one strain of Streptococcus agalacticae. Streptococcus uberis. Bacillus lichenformis. Bacillus cereus and Bacillus subtilis respectively;

(c) at least 4 strains of lytic bacteriophage each have specificity for at least one strain of Staphylococcus haemolyticus. Staphylococcus hyicus. Staphylococcus chromogenes, and Staphylococcus dysgalacticae respectively; and

(d) at least 2 strains of lytic bacteriophage each have specificity for at least one strain of Escherichia coli and Pseudomonas putida respectively; and wherein each of said at least 12 strains of lytic bacteriophage is administered to the subject at an infectious dose of at least 10⁴ PFU/ml.

50. A combined preparation for use according to claim 49, in the treatment or prevention of ovine, caprine or bovine, preferably bovine, mastitis or metritis.

51 . A combined preparation for use according to claim 49 or 50, in the treatment or prevention of clinical or sub-clinical mastitis.

52. A combined preparation for use according to claim 51 where dependent on claim 50, wherein the subject is within the whole milking herd, during the pre-birth drying-off period, or during first pregnancy prior to joining the milking herd.

53. A combined preparation for use according to claim 51 or 52, in the treatment of clinical mastitis, in combination with one or more antibiotic agents.

54. A combined preparation for use according to claim 49 or 50, for use in the treatment or prevention of metritis.

55. A combined preparation for use according to any of claims 49-54, wherein the strains of bacteria for which the bacteriophage according to (d) have specificity are prevalent in the community of the subject; preferably wherein said strains of bacteria are furthermore resistant to at least 3, preferably at least 5 antibiotics.

56. A combined preparation according to any of claims 49-55, further comprising at least 3, preferably at least 6, preferably at least 9, more preferably at least 12 further strains of lytic bacteriophage according to (a); and wherein each of said further strains of bacteriophage is administered at an infectious dose of at least 10⁴ PFU/ml.

57. A combined preparation according to any of claims 50-56, further comprising at least 6, preferably at least 9, more preferably at least 12 further strains of lytic bacteriophage, each respectively having specificity for at least one strain of Streptococcus agalacticae, Streptococcus uberis. Bacillus lichenformis. Bacillus cereus or Bacillus subtil is; wherein each of said further strains of bacteriophage is administered at an infectious dose of at least 10⁴ PFU/ml

58. A combined preparation for use according to any of claims 50-57, which is administered via intravenous or intramuscular administration.

59. A combined preparation for use according to claim 58, wherein said treatment or prevention comprises (1 ) administering said combined preparation on at least one occasion via intravenous or intramuscular administration; and (2) administering said combined preparation on at least one further occasion by intramammary administration.

60. A combined preparation for use according to claim 59, wherein the administration in (2) is carried out on at least 2, preferably at least 3 sequential occasions; the time between any two occasions in sequence being no longer than 72 hours.

61 . A combined preparation for use according to claim 60, wherein the administration in both (1 ) and (2) is performed on 3, preferably 5 sequential occasions; the time between any two occasions in sequence being no longer than 72 hours, preferably no longer than 48 hours.

62. A combined preparation for use according to claim 61 , wherein the occasions of the administration in (2) are within 48, preferably within 24 hours of their respective occasions in sequence of the administration in (1 ).

63 A composition comprising at least 10 different strains of lytic bacteriophage, for use in the therapy of a bacterial infection of a wound of the skin or mucosa in a subject; wherein

(a) at least 3 strains of bacteriophage have specificity for at least 3 different strains of Staphylococcus aureus and/or methiciliin-resistant Staphylococcus aureus;

(b) at least 5 strains of bacteriophage have specificity for at least 5 different strains of one or more bacteria selected from the group consisting of Clostridium perihngen, Pseudomonas aeruginosa, Streptococcus pyogenes and vancomycin- resistant Enterococcus spp; (c) a strain of bacteriophage has specificity for a strain of Salmonella enteriditis; and

(d) a strain of bacteriophage has specificity for a strain of Staphylococcus aureus that is resistant to at least three different antibiotics; wherein said therapy comprises:

(2) administering said composition to the subject by any suitable means on at least two further separate occasions, the time between said two further occasions being no longer than 72 hours; wherein each strain of bacteriophage is present in the composition administered in each occasion in (1 ) and (2) at an infectious dose of at least 10⁴ plaque forming units/ml composition.

64. A composition for use according to claim 63, comprising least 13, preferably at least 16, more preferably at least 19 different strains of bacteriophage; the bacteriophage in (a) being characterised in that there are at least 6, preferably at least 9, more preferably at least 12 of said strains of bacteriophage, having specificity for at least 6, preferably at least 9, more preferably at least 12 different strains of Staphylococcus aureus and/or methicillin-resistant Staphylococcus aureus;

65. A composition comprising at least 24 different strains of lytic bacteriophage, for use in the therapy of a bacterial infection associated with the gastrointestinal tract; wherein

(a) at least 12 strains of bacteriophage have specificity for at least 12 different strains of Clostridium difficile; (b) at least 10 strains of bacteriophage have specificity for at least 1 0 different strains of one or more bacteria selected from the group consisting of Escherichia coli, Klebsiella spp, Enterococcus spp, Bacillus fragilis and Clostridium perfringens;

(d) a strain of bacteriophage has specificity for a strain of Clostridium difficile that is resistant to at least three different antibiotics in ; wherein said therapy comprises:

(1 ) administering said composition to the subject intravenously or intramuscularly on at least one occasion; and

66. A composition for use according to claim 65, comprising at least 29, preferably at least 34 different strains of bacteriophage; the bacteriophage in (b) being characterised in that there are at least 15, preferably at least 20 strains of bacteriophage, having specificity for at least 15, preferably at least 20 different strains of one or more bacteria selected from the group consisting of Escherichia coli, Klebsiella spp, Enterococcus spp, Bacillus fragilis and Clostridium perfringens.

67. A composition comprising at least 19 different strains of lytic bacteriophage, for use in the therapy of a bacterial infection associated with the respiratory system in a subject; wherein (a) at least 12 strains of bacteriophage have specificity for at least 12 different strains of Streptococcus pneumoniae;

(c) a strain of bacteriophage has specificity for a strain of Pseudomonas aeruginosa; and

68. A composition comprising at least 22 different strains of lytic bacteriophage, for use in the therapy of a bacterial infection associated with the urino-genital tract in a subject; wherein

(b) at least 8 strains of bacteriophage have specificity for at least 8 different strains of one or more bacteria selected from the group consisting of Staphylococcus saprophyticus, Enterococcus faecalis, Enterococcus faecium, Proteus mirabilis, Klebsiella pneumoniae, Proteus vulgaris, Proteus penneri. Staphylococcus epidermidis, Pseudomonas aeruginosa, Mycobacterium tuberculosis and Group B Streptococcus agalactiae;

(d) a strain of bacteriophage has specificity for a strain of Neisseria gonorrhoeae that is resistant to at least three different antibiotics; wherein said therapy comprises:

69. A composition comprising at least 19 different strains of lytic bacteriophage, for use in the therapy of a multiple drug-resistant tuberculosis infection in a subject; wherein

(b) at least 5 strains of bacteriophage have specificity for at least 5 different strains of one or more bacteria selected from the group consisting of Haemophilus influenzae, Klebsiella pneumonia and Staphylococcus aureus; (c) a strain of bacteriophage has specificity for a strain of Streptococcus pneumoniae; and

(d) a strain of bacteriophage has specificity for a strain of Mycobacterium tuberculosis that is resistant to at least three different antibiotics; wherein said therapy comprises:

70. A composition comprising at least 22 different strains of lytic bacteriophage, for use in the therapy of a nosocomial Escherichia coli bacterial infection in a subject; wherein

(b) at least 8 strains of bacteriophage have specificity for at least 8 different strains of one or more bacteria selected from the group consisting of Acinetobacter baumannii, Staphylococcus aureus, Klebsiella pneumonia, Pseudomonas aeruginosa, Morganella morganii, Salmonella spp, Enterobacter cloacae, Staphylococcus hominis, Staphylococcus epidermidis and Staphylococcus saprophyticus:

(c) a strain of bacteriophage has specificity for a strain of Proteus mirabilis; and (d) a strain of bacteriophage has specificity for a strain of Escherichia coli that is resistant to at least three different antibiotics; wherein said therapy comprises:

71 . A composition comprising at least 22 different strains of lytic bacteriophage, for use in the therapy of a bacterial meningitis infection in a subject; wherein

(d) a strain of bacteriophage has specificity for a strain of Neisseria meningitidis that is resistant to at least three different antibiotics; wherein said therapy comprises: (1 ) administering said composition to the subject intravenously, or intramuscularly, on at least one occasion; and

72. A composition for use according to any of claims 63-71 , wherein the strain of bacteria for which the strain of bacteriophage in (d) has specificity is resistant to at least three antibiotics selected from the group consisting of ampicillin, tazobactum, sulbactam, cefazolin, ceftriaxone, ceftazidime, cefeprime, cefotetan, ertapenam, imipenem, aztreonam, ciprofloxicin, levofloxacin, gentamycin, tobramycin, amikacin, nitrofurantoin, Penicillins, Cephalosporins, Fluoroquinolones, aminoglycosides, macrolides, tetracyclines, ketolides, lincosamides, Trimethoprim-Sulfamethoxazole, Laevomycetinums, glycopeptides, Sulfonamides, Spectinomycin, Trimethoprim, Chloramphenicol, Clindamycin, Ethambutol, Nitrofurantoin, Novobiocin, Tigecycline Oxazolidinone, inhibitors of cell wall synthesis, inhibitors of protein synthesis, inhibitors of membrane function, anti-metabolites, inhibitors of nucleic acid synthesis, Beta-Lactams and Aminoglycosides.

73. A composition for use according to any of claims 63-72, wherein the intravenous or intramuscular administration in (1 ) is carried out on at least two separate occasions, the time between said two occasions being no longer than 72 hours.

74. A composition for use according to any of claims 63-72, wherein the administration in (2) is carried out on at least three occasions in sequence by any suitable means, the time between any two occasions in sequence being no longer than 72 hours.

75. A composition for use according to any of claims 63-74, wherein the means of administration in (2) is selected from the group consisting of enteral, rectal, intramuscular, sub-dermal, aerosol, intra-nasal, topical, intramammary and peritoneal administration.

76. A composition for use according to any of claims 63-75, wherein the therapy is treatment of a bacterial infection.

77. A composition for use according to any of claims 63-75, wherein the therapy is prevention of a bacterial infection.