WO2022013314A1

WO2022013314A1 - Bacteriophage cocktails and uses thereof

Info

Publication number: WO2022013314A1
Application number: PCT/EP2021/069659
Authority: WO
Inventors: Lorenzo CORSINI; Zehra Claire VISRAM; Michele MUTTI; Marcela RESTREPO; David SAEZ MORENO; Susana Maria LAVADO DE OLIVEIRA GARDETE HARTMANN; Alexander BELCREDI
Original assignee: Biontech R&D (Austria) Gmbh
Priority date: 2020-07-14
Filing date: 2021-07-14
Publication date: 2022-01-20

Abstract

The present invention relates to novel cocktails of bacteriophages and methods of using the same, including medical and non-medical uses. More specifically, the invention relates to a composition comprising a first bacteriophage and a second bacteriophage, wherein the first bacteriophage is a mosaic bacteriophage, i.e. having a genome / nucleotide sequence comprising genome fragments / nucleotide sequence originating from multiple ancestor bacteriophages. The cocktails may be used as composition in non-medical methods of killing and/or inhibiting the growth of bacteria, such as, e.g., on a surface, or may be used as pharmaceutical composition in the treatment and/or prevention of bacterial infections, including implant-associated infections caused by, e.g., Staphylococcus aureus. Means and methods for generating a mosaic bacteriophage are also provided.

Description

BACTERIOPHAGE COCKAILS AND USES THEREOF

Infections of implantable medical devices are associated with bacterial biofilms that form on the implanted foreign materials and are impervious to antibiotic and immune cell penetration, leading to chronic and difficult-to-treat infections. Biofilms are adherent communities of microorganisms held together by a polymeric matrix composed of polysaccharides, proteins and/or nucleic acids. The distinct gene expression pattern, as well as the physical structure of biofilms increases bacterial resistance to many negative stimuli including chemical disinfectants, pH extremes, host immune defenses and antibiotics. In particular, the treatment of prosthetic joint infections (PJI) (i.e., infection of knee and hip joint prostheses) is exceedingly difficult because it typically involves reoperations to remove the infected prosthesis, prolonged courses of systemic antibiotics, and delayed reimplantation of a new prosthesis, all of which contribute to extended disability and rehabilitation and increased morbidity, mortality, and healthcare costs. Most PJI and other implant-related infections are thought to occur by invading bacteria during surgery or in the immediate postoperative period. However, hematogenous infections, which represent up to 20% of PJI, are especially problematic, because they can occur at any time after implantation by bacteria from a remote source of infection or exposure, seeding on a previously well-functioning prosthesis through the bloodstream. Staphylococcus aureus is a particularly clinically relevant pathogen because it is the most common cause of PJI or other implant associated infections in humans and is the responsible pathogen in 30-40% of patients with implant associated infections. Antibiotics such as Gentamycin (for MSSA) or Vancomycin/Rifampicin (for MRSA) are being used in the clinical routine, but it can be shown in vitro that they do not eradicate the biofilms on the implant. Therefore, surgical removal of the biofilms is always required, which is carried out by debridement and in many cases additionally by exchanging the implant. Re-infection rates range between 10% and 60% depending on the patient group. Furthermore, S. aureus clinical isolates are increasingly becoming resistant to antibiotics, underscoring the unmet clinical need for therapeutic alternatives to conventional antibiotics. In Europe alone, more than 30,000 people die every year from antibiotic resistances (Cassini et al., 2019).

Bacteriophage (phage) are viruses that specifically infect and lyse bacteria. Phage therapy, a method of using whole phage viruses for the treatment of bacterial infectious diseases, was introduced in the 1920s by Felix d’Herelle, and has been proposed as an alternative to antibiotics. Many natural phages, however, infect and lyse only a fraction of strains within a species. Phages are indeed highly specific to one strain or few strains of a bacterial species and this specificity makes them unique in their antibacterial action. Therefore, phages have been considered as "smart" antibacterial agents rather than "dummy" ones like antibiotics. The ability of phages to recognise precisely their hosts, renders them favourable antibacterial agents especially because broad-spectrum antibiotics kill both the target bacteria and all the beneficial bacteria present in the farm or in the organism body (Merril et al., 2003). The advantages of using phages against bacteria as lytic agents are numerous. However, the inability to cover all strains of certain bacterial species along with the easy development of evolutionary resistance by bacteria against their phages, have made phage therapy or phage biocontrol unsuccessful (Yieu, 1975) and eventually led to replacement of phage therapy, in most countries, with antibiotic treatment (Barrow and Soothill, 1997).

Phage cocktails may provide advantages to the use of phages individually, e.g., to increase the lytic activity against a particular bacterial strain, and to decrease the possibility of emergence of bacteria resistant to an individual bacteriophage. That is, different bacteriophage can be mixed as cocktails to broaden their properties, preferably resulting in a collectively greater antibacterial spectrum of activity e.g., an expanded host range, to which development of resistance is less likely. Nonetheless, to date, S. aureus strains of more than 25 different clonal complexes and lineages have been identified in the natural epidemiology of S. aureus in human infections, leading to phage cocktails that must comprise many bacteriophages to achieve wide host-range. However, a high number of different bacteriophages within the same composition may lead to instability and/or negative or adverse crossed effects between the bacteriophages. In addition, these phage cocktails often need to be combined with other agents, such as, e.g., antibiotics, to be fully efficient.

There are currently no two-bacteriophage compositions in the art which are able to eradicate suspensions of a of S. aureus strains from a majority (e.g. at least 6) of clinically relevant clonal complexes and lineages. While some disclosures describe broad host ranges of S. aureus bacteriophages and cocktails, the bacterial panels on which these were measured are not well characterized and do not systematically relate to the diversity of clonal complexes and lineages of S. aureus observed in human infections (e.g. EP2833899, US7745194, US2017/0065649). In that respect it is important to be noted that a panel of strains that are not characterized, even if said panel comprises a few hundred strains, if all these strains have been isolated from a single hospital, it is likely that these strains are very similar, and come from only 2 or 3 different clonal complexes. It follows that, even if the result is characterized as a “high host range”, it is not clinically relevant, as it is high in a non-diverse set / panel of strains. Therefore, the panels used in these disclosures could in reality have been composed of only few clonal complexes and the broad host ranges reported might not be representative for the diversity of S. aureus strains causing human infections. Furthermore, the host ranges in these previous disclosures have been defined as the percent of strains on which a phage is able to form plaques, or where the phage is capable of temporarily (i.e. for a few hours, such as 3-4h hours) reducing the bacterial titer. For the treatment of human patients, however, it is crucial that the outgrowth of bacterial resistance after phage treatment is suppressed in the long-term, or at least not possible after a longer time period, such as, e.g., a 24 hour-time period. Therefore, there is a particular need for such a two-bacteriophages cocktail which can be used to cure a S. aureus implant-associated infection in a reliable way, without the need for extensive surgery (Zimmerli and Sendi, 2017). This is also of high identified interest to the World Health Organization and this need is designated as a “high priority” pathogen issue by the WHO; see. e.g., https://www.who.int/news-room/detail/27-02-2017-who-publishes-list-of-bacteria-for-which- new-antibiotics-are-urgently-needed.

The technical problem is solved and the above-mentioned needs are addressed by the embodiments herein and as characterized in the claims.

The present invention relates to the provision of “mosaic bacteriophages” described herein with unexpected properties and structure which make them particularly suitable for various uses and methods. These mosaic bacteriophages are to be used in context of this invention in form of combination preparations also described herein as “cocktails” or “bacteriophage cocktails”. In accordance of the invention, the mosaic bacteriophages described herein are combined with other bacteriophages. This includes but is not limited to also the combination of said mosaic bacteriophages. A non-limiting example of the combination of mosaic bacteriophages comprises the combination of “PM4” plus “PM93”. An example of the combination of the mosaic bacteriophage described herein with another bacteriophage is “PM4” plus “phage Romulus”. Again, these are non-limiting examples of the present invention. In particular, the specific mosaic bacteriophages provided by the present invention are particularly useful in combination in bacteriophage compositions, i.e. bacteriophage “cocktails”, for treating, decontaminating or detecting bacterial infections and disorders, in particular in relation with Staphylococcus aureus.

In particular, the present invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage comprising at least one genome fragment of a first ancestor bacteriophage, at least one genome fragment of a second ancestor bacteriophage and at least one genome fragment of a third ancestor bacteriophage, wherein said first ancestor bacteriophage and said second ancestor bacteriophage are each an 812/K-like bacteriophage as defined herein and said third ancestor bacteriophage is an ISP-like bacteriophages as defined herein and (ii) a second bacteriophage. As is evident to a person skilled in the art, the terms “bacteriophage(s)” and “phage(s)” are used herein and in context of the invention interchangeably.

It was surprisingly found that by specifically combining at least two specific bacteriophages originating from different species, wherein at least one of the said bacteriophages is a mosaic bacteriophage as described herein, a bacteriophage composition / bacteriophage cocktail with unexpected properties, in terms of host-range and/or biofilms activities may be obtained. As shown herein, the bacteriophage compositions / bacteriophage cocktails provided by the present invention have the capacity to lyse up to 88% of the Staphylococcus aureus strains of a panel of 110 Staphylococcus aureus selected such that the frequency of the clonal complexes in the panel reflects the natural epidemiology of S. aureus in human infections as found in several literature cases (Arias et al., 2017; Kanjilal et al.; 2018; Luedicke et al.; 2010; Rasmussen et al.; 2013). As used herein, the term “mosaic bacteriophage” refers to a bacteriophage which comprises genetic information from at least two, preferably at least three (or more) different ancestor bacteriophages as defined herein. In a preferred embodiment, these “at least three ancestor bacteriophages” are two bacteriophages selected from the group consisting of the “812/K-like bacteriophages” as define herein and one bacteriophage selected from the group consisting of the “ISP-like bacteriophages” as defined herein. In a particular embodiment of this invention, the two bacteriophages selected from the group consisting of the “812/K-like bacteriophages” are “05” and “04” as defined herein and the one bacteriophage selected from the group consisting of the “ISP-like bacteriophages” is “03” as defined herein. Generally, and in accordance with the definition provided herein above, a “mosaic bacteriophage” describes a bacteriophage of which the polynucleotide sequence is the result of horizontal gene transfer (see, e.g., Dion et ah, 2020). In context of this invention and the current disclosure, a “mosaic bacteriophage” as described herein does not include full-length phage genomic sequences naturally found in nature, such as, for example, phage ISP (GenBank accession No. FR852584.1), phage 812 (GenBank accession No. NC_029080) and phage K (GenBank accession No. KF766114.1). Moreover, in context of this invention and the current disclosure, a “mosaic bacteriophage” as described herein does not include the full-length genomic sequence of the ancestor bacteriophages as described herein, namely 03 (SEQ ID NO: 3), 04 (SEQ ID NO: 2) and 05 (SEQ ID NO: 1).

Mosaic bacteriophages can be obtained by any suitable methods known to those skilled in the art. Preferably, the mosaic bacteriophage of the invention may be obtained / obtainable by the means and methods provided herein, such as, e.g., the herein provided PMAP, which will be described in detail in one aspect of the invention below. Accordingly, in the context of the present invention, a mosaic bacteriophage may also be defined as a progeny (or “offspring”) of multiple (e.g., two or more, three or more) ancestor bacteriophages which intercrossed their genomes upon superinfections. The term “superinfections” as used herein, means a coinfection of the targeted bacteria by two or more bacteriophages in the context of the herein provided breeding method / protocol. Thereby, superinfections enable horizontal gene transfer between two or more bacteriophages. That is, in one aspect of the invention, the mosaic bacteriophage of the invention is obtainable by coinfection of specific targeted bacteria by three specific ancestor bacteriophages. Said three specific ancestor bacteriophages are preferably selected from “812/K-like bacteriophages” and from “ISP-like bacteriophages”. In a preferred embodiment, said three ancestor bacteriophages are two “812/K-like bacteriophages” as defined herein and one “ISP-like bacteriophage” as defined herein. In a particular embodiment of this invention, said three specific ancestor bacteriophages are “05”, “04” and “03”, as respectively defined herein. As a result of such a breeding method (i.e. PMAP), which enables horizontal gene transfer between said three specific ancestors bacteriophages, a mosaic bacteriophage is obtained, having a mosaic genome comprising at least one genome fragment of the first ancestor bacteriophage (an 812/K-like bacteriophage, preferably “05”), at least one genome fragment of the second ancestor bacteriophage (an 8I2/K-like bacteriophage, preferably “04”) and at least one genome fragment of the third ancestor bacteriophage (an ISP- like bacteriophage, preferably “03”). Illustrative examples of mosaic bacteriophages according to the present invention are, inter alia, PM4, PM5, PM7, PM9, PM22, PM23, PM25, PM28, PM32, PM34, and PM36 (see Table 4). Surprisingly, the mosaic bacteriophage of the invention shows the ability to lyse bacteria, used and/or not used in the breeding method, that were previously insensitive to any one of said three ancestor bacteriophages, alone or in combination in a cocktail. As a particular example, none of the ancestor bacteriophages “03”, “04” and “05” shows the ability to lyse the bacterial strain CC22-MRSA-IY “Barmin” (A257), whereas all the mosaic bacteriophages according to the present invention shows the ability to lyse said bacterial strain (see Figure 2). Also surprisingly, such a mosaic bacteriophage further shows the ability to reduce by 90% or more up to 100% of preformed bio films of the 10 different bacterial strains tested, each from a different clonal complex (i.e. 10 different CCs tested), and among which many were previously insensitive to any one of said three ancestor bacteriophages. Thus, the mosaic bacteriophages of the invention surprisingly show exceedingly broad host-ranges, also on biofilms, which are overadditive over the host-ranges of the ancestor bacteriophages, individually or in combination. Furthermore, surprisingly and unexpectedly, it was further shown that the virulence of the mosaic bacteriophage according to the invention (illustrated with PM4) is strongly increased on bacterial strains in comparison to its ancestors (see Example 8). The increased virulence lead to more efficient killing of the bacteria, and therefore a higher potency of the mosaic bacteriophages according to the invention compared to their ancestors, when used to treat bacterial infections, for example in humans.

Additionally, and importantly, the bacteriophage composition / bacteriophage cocktail of the present invention comprises a mosaic bacteriophage as described herein (thereafter the “first bacteriophage”) and further comprises another bacteriophage (thereafter the “second bacteriophage”). Surprisingly, said second bacteriophage shows a host-range which is highly complementary to the host-range of the inventive mosaic bacteriophage, resulting in the broad host-range bacteriophage composition / bacteriophage cocktail of the present invention. In some instances, said first bacteriophage and said second bacteriophage may even act synergistically. As can be seen in Figure 2 of the present application, as an example, the bacteriophage cocktail PM4 plus PM93 is able to lyse, inter alia , MRSSA 2017-012 strains (CC772) and MSSA124308 strains (CC101), while none of the individual bacteriophages PM4 and PM93 have the ability to lyse these strains individually. Therefore, the combination of these two specific bacteriophages in the composition / cocktail of the invention may show antimicrobial effects that are overadditive over said specific bacteriophages individually, i.e. synergistic effects. As a result, the bacteriophage composition / bacteriophage cocktails of the present invention have the capacity to eradicate suspensions of a wide range of S. aureus strains, and advantageously of almost all the S. aureus clonal complexes involved in human infections, i.e. at least 10, preferably at least 15, more preferably at least 20 of the S. aureus clonal complexes involved in human infections (see Figure 2). As shown in the appended Examples, the second bacteriophage may also synergize with the first bacteriophage (i.e. the mosaic bacteriophage as provided herein) in killing bacteria in biofilms (i.e. reduction in biofilm CFU). This property is highly relevant for the application of phage therapy to implant-associated infections, which are very frequently caused by S. aureus. There is no phage cocktail described in the art which has a similarly strong and broad biofilm efficacy as the cocktails described in this disclosure. The present invention therefore provides a bacteriophage composition which can be used, inter alia, to cure a S. aureus implant-associated infection in a reliable way.

Accordingly, the technical problem identified herein above has successfully been solved by the means and methods provided herein, in particular by the provision of combined compositions, i.e. the herein described bacteriophage cocktails, comprising at least one mosaic bacteriophage as described herein. Surprising advantageous effects of the herein described bacteriophage cocktails comprise, inter alia, the use of these novel bacteriophage products as therapeutic, prophylactic and/or preventive agents in bacterial infection and/or in destruction of bacteria, for example pathogenic bacteria, in suspensions and/or in biofilms. The novel bacteriophage products are particularly useful in treatments, particularly topical treatments, for example in implant-associated infections. These inventive bacteriophage cocktails, preferably two-phage cocktails, are capable of lysing a wide range of bacteria responsible for implant-associated infection, including Staphylococcus aureus, and preferably eradicate or inhibit the growth of said bacteria over the course of 24h, i.e. not allowing resistance formation over this period. In accordance with the present invention, the herein described first bacteriophage is a mosaic bacteriophage, comprising at least one genome fragment of each of three specific ancestor bacteriophages.

The terms "genome fragment" and “stretch” as used herein interchangeably and refer to a nucleotide sequence which is identical to a part of the whole genome sequence / nucleotide sequence of a bacteriophage and comprises at least 50 consecutive nucleotides of said whole genome sequence, more preferably at least 75, 100, 150, 200, 250, 300, 350, 400, 500, and up to 1000, more preferably up to 1500, 2000, 5000, 10000, 12000, 13000, 14000, or 15000 consecutive nucleotides of said whole genome sequence.

The terms “ancestor bacteriophage”, “parental bacteriophage”, “original bacteriophage” and “wild-type bacteriophage” are used herein interchangeably and refer to bacteriophages in their wild form, i.e. it refers to material in the form as it can be found in its original environment in which it naturally occurs and which has not been genetically modified by hand of man. Preferably, the term “ancestor bacteriophage” (or parental/original/wild-type bacteriophage) as used herein refers to isolated material that has been removed from its original environment in which it naturally occurs.

It must be understood that the order of said three specific ancestor bacteriophages is of no importance in the context of the present invention. For clarity purposes only, they will be thereafter referred to as the “first ancestor bacteriophage”, the “second ancestor bacteriophage” and the “third ancestor bacteriophage”.

As shown in the appended examples the first (mosaic) bacteriophages can be generated by the breeding of the three specific bacteriophages as defined herein and are able to lyse up to 60% Staphylococcus aureus strains of a panel of 110 A aureus strains selected so that the frequency of the clonal complexes in said panel reflects the natural epidemiology of S. aureus in human infections. In comparison, the added lysing properties of the individual ancestor bacteriophages only lyses up to 25% of said panel of 110 S. aureus strains, as illustrated herein (see Table 6). Importantly, the first (mosaic) bacteriophage of the present invention have the advantage to lyse an over additive number of bacterial strains, meaning that said first (mosaic) bacteriophage eradicate bacterial strains which none of said ancestor bacteriophages can lyse. For example, appended Figure 2 shows that all mosaic bacteriophages eradicate the bacteria strain “A257” (available in the publicly accessible section of the Leibniz-Institute DSMZ (Braunschweig, Germany) under the strain name “Bamim” Isolate A257 ( Stapholococcus aureus subsp: aureus) and under the accession number DSM 111210; see also Table 4, Bacterial strain 56, CC22- MRSA-IY, “Bamim” (A257)) which in contrast none of said ancestor bacteriophages can eradicate. Accordingly, the invention also provides for a mosaic bacteriophage with unexpected host range properties, in particular, over additive lysing properties in comparison to the specific ancestor/parental bacteriophages as defined herein and over additive lysing properties when combined with a second bacteriophage as described herein. As demonstrated in the appended examples, the nucleotide sequences / genomes of these mosaic bacteriophages displaying the surprising broad host range have been analyzed and it has been surprisingly found that they all share structural features, i.e. nucleotide sequences / stretches / genome fragments. Accordingly, without being bound by any theory, it is thought that the advantageous activity displayed by the first (mosaic) bacteriophage of the invention is linked to the presence of specific stretches in the genome of said mosaic bacteriophage. The mosaic bacteriophages described herein are therefore in accordance with the present invention characterized by specific genomic information and combination of such genomic information which is inherited from the ancestor bacteriophages. For example, and in one particular embodiment of the invention, the first mosaic bacteriophage as described herein (that can be obtainable by the inventive means and methods provided herein) comprises at least one genome fragment / nucleotide sequence inherited from the first ancestor bacteriophage such as, e.g., the nucleotide sequence as provided in SEQ ID NO: 9 {i.e. “stretch 13” of Table 11 inherited from “05”), at least one genome fragment / nucleotide sequence inherited from the second ancestor bacteriophage such as, e.g., the nucleotide sequence as provided in SEQ ID NO: 17 {i.e. “stretch 6” of Table 11 inherited from “04”) and at least one genome fragment / nucleotide sequence inherited from the third ancestor bacteriophage such as, e.g., the nucleotide sequence as provided in SEQ ID NO: 31 {i.e. “stretch 10” of Table 11 inherited from “03”). In the appended examples “05”, “04” and “03” have been used as the three specific (ancestor) bacteriophages to be bred in combination in the means and methods for generating a first “mosaic” bacteriophage as provided herein. However, it is believed that any bacteriophages which are homologs to “05”, “04” and “03”, respectively, and comprise in their genome/nucleotide sequence the important “stretches” identified in Table 11 may be used in the breeding method of the invention.

Thus, in one aspect of the invention, the herein described first bacteriophage is a mosaic bacteriophage, comprising at least one genome fragment of a first ancestor bacteriophage, at least one genome fragment of a second ancestor bacteriophage and at least one genome fragment of a third ancestor bacteriophage, wherein said first ancestor bacteriophage and said second ancestor bacteriophage are each an 812/K-like bacteriophage and said third ancestor bacteriophage is an ISP-like bacteriophages. In one preferred embodiment, said first ancestor bacteriophage is “05” (SEQ ID NO: 1), also known as BT3 or SA3, said second ancestor bacteriophage is “04”, also known as phage 812 (SEQ ID NO: 2) and said third ancestor bacteriophage is “03” (SEQ ID NO: 3).

Phage 812 (herein also designated as “04”) is known and publicly available under NCBI accession number MH844528 (version MH844528.1 of December 02, 2018) or available at the Felix d’Herelle Reference Center for Bacterial Viruses, https://www.phage.ulaval.ca/en/home/, under accession number #HER: 475). Phage 812 is herein referred as “04” and has the nucleotide sequence as provided in SEQ ID NO: 2. As used herein, the term “812/K-like bacteriophage” refers to a bacteriophage comprising a nucleotide sequence homology of at least 97.5% identity over the full length to the nucleotide sequence representing the genome of said phage 812 as provided in SEQ ID NO: 2. Examples of 812/K-like bacteriophages include but are not limited to, phage 812 itself (SEQ ID NO: 2) and its variants (such as, e.g., as provided by NCBI accession number MH844528, loc. cit., or NCBI accession number NC_029080 version NC 029080.1 of June 04, 2019 or Felix d’Herelle Reference Center for Bacterial Viruses, https://www.phage.ulaval.ca/en/home/, under accession number #HER: 475), phage K (such as, e.g., as provided by NCBI accession number KF766114 version KF766114.1 of June 05, 2014) and J-Sa36 (such as, e.g., as provided by NCBI accession number MK417516 version MK4 17516.1 of January 30, 2019).

Alternatively or additionally, the “812/K-like bacteriophage” described in context of the present invention is also characterized by a nucleotide sequence homology of at least 80% identity over the full length to the nucleotide sequence of phage 812 as provided in NCBI (NCBI accession number MH844528, Version 1 (MH844528.1), December 02, 2018), or as provided in SEQ ID NO: 2 and by comprising in its genome

(a) the following nucleotide sequence (referred herein also as “nucleotide stretch” or

“stretch”):

(b) a nucleotide sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or completely (100%) to the full length nucleotide sequence as provided herein above (SEQ ID NO: 75) and which comprises at least 50 nucleotides, at least 55 nucleotides, at least 60 nucleotides, at least 65 nucleotides, or at least 70 nucleotides of the above 75 nucleotide sequence (SEQ ID NO: 75). In a preferred embodiment said at least 50 “nucleotide stretch” etc. are concrete fragments of SEQ ID NO: 75 and, accordingly, said nucleotides represent a consecutive nucleotide sequence/stretch of SEQ ID NO: 75.

In accordance with this invention, the nucleotide sequence/stretch as provided in SEQ ID NO: 75 is not found in “ISP-like bacteriophages”.

Accordingly, the “812/K-like bacteriophage” as described herein are defined either by a 97,5% sequence identity over the full length to SEQ ID NO: 2 or by a 80% sequence identity over the full length to SEQ ID NO: 2 and by comprising the nucleotide sequence as provided in SEQ ID NO: 75 as defined above.

As an example, “05” as defined herein has a nucleotide sequence identity of 89% over the full length to the nucleotide sequence as provided in SEQ ID NO: 2 and comprises a nucleotide sequence in its genome which is 100% identical to the nucleotide sequence as provided in SEQ ID NO: 75. Accordingly, the herein defined phage 05 is a preferred example of an “812/K-like bacteriophage”. Likewise, “04” as defined herein, comprising a nucleotide sequence identity of 100% over the full length to the nucleotide sequence as provided in SEQ ID NO: 2 and comprising a nucleotide sequence in its genome which is 100% identical to the nucleotide sequence as provided in SEQ ID NO: 75, is another preferred example of an “812/K-like bacteriophage”.

Phage ISP is known and publicly available under NCBI accession number FR852584 (version FR852584.1 of September 19, 2011). As used herein, the term “ISP-like bacteriophage” refers to a bacteriophage comprising a nucleotide sequence homology of at least 97.5% identity over the full length to the nucleotide sequence representing the genome of said phage ISP as provided by NCBI accession number FR852584 (version FR852584.1 of September 19, 2011). Examples of ISP-like bacteriophages include, but are not limited to, phage ISP itself (phage “ISP” NCBI FR852584.1, loc. cit.), Sa83 (such as, e.g., as provided by NCBI accession number MK417514 version MK417514.1 of January 30, 2019), Sa87 (such as, e.g., as provided by NCBI accession number MK417515 version MK417515.1 of January 30, 2019), pSa-3 (such as, e.g., as provided by NCBI accession number KY581279 version KY581279.1 of April 06, 2017), StaphlN (such as, e.g., as provided by NCBI accession number JX080300 version JX080300.2 of March 28, 2014), Sa30 (such as, e.g., as provided by NCBI accession number MK331931 version MK331931.1 of February 06, 2019), G1 (such as, e.g., as provided by NCBI accession number AY954969 version AY954969.1 of April 15, 2005), A5W (such as, e.g., as provided by NCBI accession number EU418428 version EU418428.2 of March 12, 2014), P4W (such as, e.g., as provided by NCBI accession number JX080305 version JX080305.2 of March 28, 2014) or Fi200W (such as, e.g., as provided by NCBI accession number JX080303 version JX080303.2 of March 28, 2014).

Alternatively or additionally, the “ISP-like bacteriophage” described in context of the present invention is characterized by a nucleotide sequence homology of at least 80% identity over the full length to the nucleotide sequence of bacteriophage ISP as provided in NCBI (NCBI accession code FR852584, Version 1 (FR852584.1), September 19, 2011), and by comprising in its genome

(a) the following nucleotide sequence (referred herein also as “nucleotide stretch” or “stretch”):

(b) a nucleotide sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical or completely (100%) to the full length of the nucleotide sequence as provided herein above (SEQ ID NO: 76) and it comprises at least 80 nucleotides, at least 85 nucleotides, at least 90 nucleotides, at least 91 nucleotides, at least 92 nucleotides, at least 93 nucleotides, at least 94 nucleotides, at least 95 nucleotides, at least 96 nucleotides, at least 97 nucleotides, at least 98 nucleotides or at least 99 nucleotides of the above 100 nucleotide sequence (SEQ ID NO: 76). In a preferred embodiment said at least 80 “nucleotide stretch” etc. are concrete fragments of SEQ ID NO: 76 and, accordingly, said nucleotides represent a consecutive nucleotide sequence/stretch of SEQ ID NO: 76. In accordance with this invention, the nucleotide sequence/stretch as provided in SEQ ID NO: 76 is not found in “812/K-like bacteriophages”.

Accordingly, the “ISP-like bacteriophage” as described herein are defined either by a 97,5% sequence identity over the full length to the nucleotide sequence of the bacteriophage “ISP” as provided in NCBI (NCBI accession code FR852584, Version 1 (FR852584.1), September 19, 2011) or by a 80% sequence identity over the full length to the nucleotide sequence of the bacteriophage “ISP” as provided in NCBI (NCBI accession code FR852584, loc. cit.) and by comprising the nucleotide sequence as provided in SEQ ID NO: 76 as defined above.

A preferred example of an ISP-like bacteriophage is the herein disclosed phage 03 having the nucleotide sequence as provided in SEQ ID NO: 3. The phage 03 thus comprises a nucleotide sequence in its genome which is 100% identical to the nucleotide sequence as provided in SEQ ID NO: 71. The nucleotide sequence of the phage 03 is described for the first time in the EP patent applications EP 20 185700.0 and EP 20 185697.8 and has a nucleotide sequence identity of 98.87% over the full length to the nucleotide sequence representing the genome of phage ISP (publicly available as phage “ISP” NCBI accession code FR852584, Version 1 (FR852584.1), September 19, 2011) when aligned using the blastn Suite of NCBI

(https://blast.ncbLnlm.nih.goy /Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LI NK_LOC=blasthome) using ISP as the query. According to the present disclosure, the full length of the nucleotide sequence representing the genome of phage ISP (publicly available as phage “ISP” NCBI accession code FR852584, Version 1 (FR852584.1), September 19, 2011) is 138339 pb. Thus, 03 and ISP are two distinct bacteriophages. This is further confirmed by the fact that the genome sequence of the bacteriophage 03 comprises the nucleotide sequence as provided in SEQ ID NO: 21, which is a nucleotide sequence specific to the phage 03 and which cannot be found in any other known bacteriophages. In other words, the nucleotide sequence as provided in SEQ ID NO: 21 is absent from the genome of the bacteriophage ISP (the sequence identity to the corresponding stretch on ISP is only 74%), confirming that 03 and ISP are two distinct bacteriophages.

In accordance with the present invention, the first ancestor bacteriophage as described herein is preferably a 812/K-like bacteriophage. Said first ancestor bacteriophage may have the genome selected from the group consisting of: (a) a genome comprising the genome fragment as provided in SEQ ID NO: 5 or a genome fragment with at least 90% identity with SEQ ID NO: 5;

(b) a genome comprising a genome fragment with at least 85% identity with SEQ ID NO: 6, a genome fragment with at least 60% identity with SEQ ID NO: 9 and/or a genome fragment with at least 99% identity with SEQ ID NO: 11 ;

(c) a genome with at least 80% identity with SEQ ID NO: 1 and comprising a combination of the genome fragments as provided in SEQ ID NOs: 5, 6, 9 and 11 ;

(d) a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 7, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 8, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 10, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 12, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 13, and/or a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 14; and

(e) a genome with at least 95% identity with SEQ ID NO: 1, wherein the % identity with a sequence is expressed over the full sequence length of said sequence.

In the context of the present invention, the term “genome” refers to a “nucleotide sequence”. Therefore, the term “genome” may be replaced by the term “nucleotide sequence”. Likewise, the term “genome fragment” also refers to a “nucleotide sequence” and the term “genome fragment” may also be replaced by the term “nucleotide sequence” in the present disclosure.

As used herein, the term "% sequence identity", has to be understood as follows: Two sequences to be compared are aligned to give a maximum correlation between the sequences. This may include inserting "gaps" in either one or both sequences, to enhance the degree of alignment. A % identity may then be determined over the whole length of each of the sequences being compared (so-called global alignment), that is particularly suitable for sequences of the same or similar length, or over shorter, defined lengths (so-called local alignment), that is more suitable for sequences of unequal length. In the above context, an amino acid sequence having a "sequence identity" of at least, for example, 95% to a query amino acid sequence, is intended to mean that the sequence of the subject amino acid sequence is identical to the query sequence except that the subject amino acid sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain an amino acid sequence having a sequence of at least 95% identity to a query amino acid sequence, up to 5% (5 of 100) of the amino acid residues in the subject sequence may be inserted or substituted with another amino acid or deleted. Methods for comparing the identity and homology of two or more sequences are well known in the art.

In context of this invention, “% of identity” or “homology” means said identity/homology over the full/whole corresponding reference sequence (i.e., over 100% of the corresponding reference sequence). Thus, in the context of this invention, a bacteriophage A having a nucleotide sequence identity of 95% over the full length to the nucleotide sequence representing the genome of a bacteriophage B means that the bacteriophage A has a genome sequence with 95% identity with the genome sequence of the bacteriophage B over the whole length of the genome sequence of the bacteriophage B (i.e., over 100% of the genome sequence of the bacteriophage B). In the context of the present invention, “whole length” and “full length” are used interchangeably.

Any of a variety of sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, local methods and hybrid methods, such as segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up scores of individual nucleotide pairs and by imposing gap penalties. Non-limiting methods include, e.g., CLUSTAL W (Thompson et al., 1994), and iterative refinement. Non-limiting methods include, e.g. Blast (Zhang et al., 2000). Some algorithms, including Blast, return two values, the “Query Cover “ and the “Percent Identity”. The Query Cover value indicates the share of the query sequence which the Percent Identity refers to. For example, a sequence which is 100% identical to half of the query sequence and completely different on the rest of the query sequence, would have Query Cover and Percent Identity values of 50% and 100%, respectively. In these cases, the two values were multiplied to obtain a “% sequence identity” valid across the whole query sequence (% sequence identity would be 50% for the example described above).

Non-limiting methods include, e.g., BLAST, Match-box, Align-M (see, e.g. Van Walle I et al., 2004). Furthermore, the percentage to which two sequences are identical can for example be determined by using a mathematical algorithm. A preferred, but not limiting, example of a mathematical algorithm which can be used is the algorithm of Karlin et al. (Karlin et al., 1993). Such an algorithm is integrated in the BLAST family of programs, e.g. BLAST or NBLAST program (Altschul et al., 1990 and 1997), accessible through the home page of the NCBI at world wide web site ncbi.nlm.nih.gov and FASTA (Pearson, 1990 and Pearson and Lipman, 1988). Sequences which are identical to other sequences to a certain extent can be identified by these programs. Furthermore, programs available in the Wisconsin Sequence Analysis Package, version 9.1 (Devereux et al., 1984), for example the programs BESTFIT and GAP, may be used to determine the % identity between two polypeptide sequences. BESTFIT uses the "local homology" algorithm of (Smith and Waterman, 1981) and finds the best single region of similarity between two sequences. If herein reference is made to an amino acid sequence sharing a particular extent of sequence identity to a reference sequence, then said difference in sequence is preferably due to conservative amino acid substitutions. Preferably, such sequence retains the activity of the reference sequence, e.g. albeit maybe at a slower rate. In addition, if reference is made herein to a sequence sharing "at least" at certain percentage of sequence identity, then 100% sequence identity is preferably also encompassed.

Accordingly, the first ancestor bacteriophage as described herein may have a genome comprising a genome fragment with at least 85%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, or 100% identity with SEQ ID NO: 5. Said first ancestor bacteriophage may also have a genome comprising a genome fragment with at least 85%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, or 100% identity with SEQ ID NO: 6. Said first ancestor bacteriophage may also have a genome comprising a genome fragment with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, or 100% identity with SEQ ID NO: 9. Said first ancestor bacteriophage may also have a genome comprising a genome fragment with at least 99% or 100% identity with SEQ ID NO: 11. Said first ancestor bacteriophage may have a genome comprising the genome fragment as provided in SEQ ID NO: 6, the genome fragment as provided in SEQ ID NO: 9 and/or the genome fragment as provided in SEQ ID NO: 11, and optionally the genome fragment as provided in SEQ ID NO: 5. Said first ancestor bacteriophage may also have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 7, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 8, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 10, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 12, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 13, and/or a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 14. Said first ancestor bacteriophage may further have a genome with at least 85%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, or 100% identity with SEQ ID NO: 1.

In some preferred embodiments, the first ancestor bacteriophage as described herein has a genome comprising the genome fragment as provided in SEQ ID NO: 5, the genome fragment as provided in SEQ ID NO: 6, the genome fragment as provided in SEQ ID NO: 9 and the genome fragment as provided in SEQ ID NO: 11.

In some even more preferred embodiments, the first ancestor bacteriophage as described herein has the genome as provided in SEQ ID NO: 1. In one embodiment of this invention, the first ancestor bacteriophage is “05”, as defined herein.

In one preferred embodiment, the first ancestor bacteriophage described herein is the herein disclosed phage “05”, an 812/K-like bacteriophage with the genome sequence/nucleotide sequence as provided in SEQ ID NO: 1. The genome sequence/nucleotide sequence of bacteriophage “05” comprises the nucleotide sequence as provided in SEQ ID NO: 5, which is a nucleotide sequence specific to the phage “05” and which cannot be found in any other known bacteriophages. Furthermore, the genome sequence/nucleotide sequence of bacteriophage “05” comprises the nucleotide sequence:

; see also SEQ ID NO: 75, which is a nucleotide sequence

specific to the group of the “812/K-like bacteriophages” as defined herein and meaning that the phage “05” belongs to said group. In addition, the genome sequence/nucleotide sequence of bacteriophage “05” comprises the nucleotide sequences as provided in SEQ ID NOs: 6, 9 and 11, which are the “stretches” found in the nucleotide sequence of the bred “mosaic” bacteriophages with improved properties. The genome fragment/nucleotide sequence as provided in SEQ ID NO: 6 encodes for the amino acid sequences as provided in SEQ ID NOs: 7 and/or 8, the genome fragment/nucleotide sequence as provided in SEQ ID NO: 9 encodes for the amino acid sequence as provided in SEQ ID NO: 10, and the genome fragment/nucleotide sequence as provided in SEQ ID NO: 11 encodes for the amino acid sequences as provided in SEQ ID NOs: 12, 13 and/or 14. Since the function of the proteins and/or peptides encoded by these “stretches” may be linked to the improved properties of the bred “mosaic” bacteriophages, without being bound by any theory, it is believed that variant sequences encoding the same amino acid sequences may be alternatives. Due to the redundancy of the genetic code it is evident for the skilled person that several nucleotide sequences can encode for the same amino acid sequence and therefore can be considered as the herein mentioned (suitable) alternatives.

In accordance with the present invention, the second ancestor bacteriophage as described herein is a 812/K-like bacteriophage. Said second ancestor bacteriophage may have the genome selected from the group consisting of:

(a) a genome comprising the genome fragment as provided in SEQ ID NO: 15;

(b) a genome comprising a genome fragment with at least 99% identity with SEQ ID NO: 17;

(c) a genome with at least 80% identity with SEQ ID NO: 2 and comprising a combination of the genome fragments as provided in SEQ ID NOs: 15 and 17;

(d) a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 16, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 18, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 19, and/or a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 20; and

(e) a genome with at least 95% identity with SEQ ID NO: 2, wherein the % identity with a sequence is expressed over the full sequence length of said sequence.

Accordingly, the second ancestor bacteriophage as described herein may have a genome comprising the genome fragment as provided in SEQ ID NO: 15. Said second ancestor bacteriophage may also have a genome comprising a genome fragment with at least 85%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, or 100% identity with SEQ ID NO: 17. Said second ancestor bacteriophage may have a genome comprising the genome fragment as provided in SEQ ID NO: 15, and/or the genome fragment as provided in SEQ ID NO: 17. Said second ancestor bacteriophage may also have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 16, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 18, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 19, and/or a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 20. Said second ancestor bacteriophage may further have a genome with at least 85%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, or 100% identity with SEQ ID NO: 2. In some preferred embodiments, the second ancestor bacteriophage as described herein has a genome comprising the genome fragment as provided in SEQ ID NO: 15 and the genome fragment as provided in SEQ ID NO: 17.

In some even more preferred embodiments, the second ancestor bacteriophage as described herein has the genome as provided in SEQ ID NO: 2. In one embodiment of this invention, the first ancestor bacteriophage is “04”, as defined herein.

The genome sequence/nucleotide sequence of bacteriophage “04” comprises the nucleotide sequence as provided in SEQ ID NO: 15, which is a nucleotide sequence specific to the phage “04” and which cannot be found in any other known bacteriophages. Furthermore, the genome sequence/nucleotide sequence of bacteriophage “04” comprises the nucleotide sequence:

”; see also SEQ ID NO: 75, which is a nucleotide sequence

specific to the group of the “812/K-like bacteriophages” as defined herein and meaning that the phage “04” belongs to said group. In addition, the genome sequence/nucleotide sequence of bacteriophage “04” comprises the nucleotide sequences as provided in SEQ ID NO: 17, which is the “stretch” found in the nucleotide sequence of the bred “mosaic” bacteriophages with improved properties. The genome fragment/nucleotide sequence as provided in SEQ ID NO: 15 encodes for the amino acid sequence as provided in SEQ ID NOs: 16 and the genome fragment/nucleotide sequence as provided in SEQ ID NO: 17 encodes for the amino acid sequences as provided in SEQ ID NOs: 18, 19 and/or 20. Since the function of the proteins encoded by these “stretches” may be linked to the improved properties of the bred “mosaic” bacteriophages without being bound by any theory it is believed that variant sequences encoding the same amino acid sequences may be suitable alternatives. Due to the redundancy of the genetic code it is evident for the skilled person that several nucleotide sequences can encode for the same amino acid sequence and therefore can be considered as the herein mentioned (suitable) alternatives.

In accordance with the present invention, the third ancestor bacteriophage as described herein is an ISP-like bacteriophage. Said third ancestor bacteriophage may have the genome selected from the group consisting of:

(a) a genome comprising the genome fragment as provided in SEQ ID NO: 21; (b) a genome comprising a genome fragment with at least 99% identity with SEQ ID NO: 22, a genome fragment with at least 99% identity with SEQ ID NO: 26, a genome fragment with at least 99% identity with SEQ ID NO: 31, a genome fragment with at least 99% identity with SEQ ID NO: 34, a genome fragment with at least 99% identity with SEQ ID NO: 38 and/or a genome fragment with at least 99% identity with SEQ ID NO: 41;

(c) a genome with at least 80% identity with SEQ ID NO: SEQ ID NO: 3 and comprising a combination of the genome fragments as provided in SEQ ID NOs: 21, 22, 26, 31, 34, 38, 41, 45, 49, 53, and 57;

(d) a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 23, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 24, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 25, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 27, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 28, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 29, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 30, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 32, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 33, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 35, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 36, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 37, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 39, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 40, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 42, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 43, and/or a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 44, and optionally a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 46, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 47, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 48, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 50, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 51, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 52, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 54, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 55, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 56, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 58, and/or a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 59; and

(e) a genome with at least 95% identity with SEQ ID NO: 3, wherein the % identity with a sequence is expressed over the full sequence length of said sequence.

Accordingly, the third ancestor bacteriophage as described herein may have a genome comprising the genome fragment as provided in SEQ ID NO: 21. Said third ancestor bacteriophage may also have a genome comprising a genome fragment with at least 85%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, or 100% identity with SEQ ID NO: 22. Said third ancestor bacteriophage may also have a genome comprising a genome fragment with at least 85%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, or 100% identity with SEQ ID NO: 26. Said third ancestor bacteriophage may also have a genome comprising a genome fragment with at least 85%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, or 100% identity with SEQ ID NO: 31. Said third ancestor bacteriophage may also have a genome comprising a genome fragment with at least 85%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, or 100% identity with SEQ ID NO: 34. Said third ancestor bacteriophage may also have a genome comprising a genome fragment with at least 85%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, or 100% identity with SEQ ID NO: 38. Said third ancestor bacteriophage may also have a genome comprising a genome fragment with at least 85%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, or 100% identity with SEQ ID NO: 41. Said third ancestor bacteriophage may have a genome comprising the genome fragment as provided in SEQ ID NO: 22, the genome fragment as provided in SEQ ID NO: 26, the genome fragment as provided in SEQ ID NO: 31, the genome fragment as provided in SEQ ID NO: 34, the genome fragment as provided in SEQ ID NO: 38 and/or the genome fragment as provided in SEQ ID NO: 41, and optionally the genome fragment as provided in SEQ ID NO: 21. Said third ancestor bacteriophage may also have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 23, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 24, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 25, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 27, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 28, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 29, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 30, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 32, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 33, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 35, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 36, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 37, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 39, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 40, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 42, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 43, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 44, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 46, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 47, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 48, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 50, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 51 , a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 52, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 54, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 55, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 56, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 58, and/or a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 59. Said third ancestor bacteriophage may further have a genome with at least 85%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, or 100% identity with SEQ ID NO: 3.

In some preferred embodiments, the third ancestor bacteriophage as described herein has a genome comprising the genome fragment as provided in SEQ ID NO: 21, the genome fragment as provided in SEQ ID NO: 22, the genome fragment as provided in SEQ ID NO: 26, the genome fragment as provided in SEQ ID NO: 31, the genome fragment as provided in SEQ ID NO: 34, the genome fragment as provided in SEQ ID NO: 38, and the genome fragment as provided in SEQ ID NO: 41. In some even more preferred embodiments, the third ancestor bacteriophage as described herein has the genome as provided in SEQ ID NO: 3. In one embodiment of this invention, the third ancestor bacteriophage is “03”, as defined herein.

The genome sequence/nucleotide sequence of bacteriophage “03” comprises the nucleotide sequence as provided in SEQ ID NO: 21, which is a nucleotide sequence specific to the phage “03” and which cannot be found in any other known bacteriophages. The genome sequence/nucleotide sequence of bacteriophage “03” comprises the nucleotide sequence:

ID NO: 76, which is a nucleotide sequence specific to the group of the “ISP-like bacteriophages” as defined herein and meaning that the phage “03” belongs to said group. In addition, the genome sequence/nucleotide sequence of bacteriophage “03” comprises the nucleotide sequences as provided in SEQ ID NOs: 22, 26, 31, 34, 38, 41, 45, 49, 53 and 57, which are the “stretches” found in the nucleotide sequence of the bred “mosaic” bacteriophages with improved properties. The genome fragment/nucleotide sequence as provided in SEQ ID NO: 22 encodes for the amino acid sequences as provided in SEQ ID NOs: 23, 24 and/or 25, the genome fragment/nucleotide sequence as provided in SEQ ID NO: 26 encodes for the amino acid sequences as provided in SEQ ID NOs: 27, 28, 29 and/or 30, the genome fragment/nucleotide sequence as provided in SEQ ID NO: 31 encodes for the amino acid sequences as provided in SEQ ID NOs: 32 and/or 33, the genome fragment/nucleotide sequence as provided in SEQ ID NO: 34 encodes for the amino acid sequences as provided in SEQ ID NOs: 35, 36 and/or 37, the genome fragment/nucleotide sequence as provided in SEQ ID NO: 38 encodes for the amino acid sequences as provided in SEQ ID NOs: 38, 39 and/or 40, the genome fragment/nucleotide sequence as provided in SEQ ID NO: 41 encodes for the amino acid sequences as provided in SEQ ID NOs: 42, 43 and/or 44, the genome fragment/nucleotide sequence as provided in SEQ ID NO: 45 encodes for the amino acid sequences as provided in SEQ ID NOs: 46, 47 and/or 48, the genome fragment/nucleotide sequence as provided in SEQ ID NO: 49 encodes for the amino acid sequences as provided in SEQ ID NOs: 50, 51 and/or 52, the genome fragment/nucleotide sequence as provided in SEQ ID NO: 53 encodes for the amino acid sequences as provided in SEQ ID NOs: 54, 55 and/or 56 and the genome fragment/nucleotide sequence as provided in SEQ ID NO: 57 encodes for the amino acid sequences as provided in SEQ ID NOs: 58 and/or 59. Since the function of the proteins encoded by these “stretches” may be linked to the improved properties of the bred “mosaic” bacteriophages without being bound by any theory it is believed that variant sequences encoding the same amino acid sequences may be suitable alternatives. Due to the redundancy of the genetic code it is evident for the skilled person that several nucleotide sequences can encode for the same amino acid sequence and therefore can be considered as the herein mentioned (suitable) alternatives.

Therefore, in one preferred aspect of the present invention, the first bacteriophage as described herein is a mosaic bacteriophage having a genome comprising at least one genome fragment of the ancestor bacteriophage having the genome as provided in SEQ ID NO: 1, at least one genome fragment of the ancestor bacteriophage having the genome as provided in SEQ ID NO: 2, and at least one genome fragment of the ancestor bacteriophage having the genome as provided in SEQ ID NO: 3.

In the appended examples, the ancestor bacteriophage having the genome as provided in SEQ ID NO: 1 is referred to as “05”, the ancestor bacteriophage having the genome as provided in SEQ ID NO: 2 is referred to as “04” and the ancestor bacteriophage having the genome as provided in SEQ ID NO: 1 is referred to as “03”.

In one particular aspect, the ancestor bacteriophages are,

(a) a first ancestor bacteriophage which is an 812/K-like bacteriophage from the virus family Herelleviridae having the genome/nucleotide sequence as provided in SEQ NO: 1, referred herein also to/as “05”,

(b) a second ancestor bacteriophage which also is an 812/K-like bacteriophage from the virus family Herelleviridae having the genome/nucleotide sequence as provided in SEQ NO: 2, referred herein also to/as “04”, and

(c) a third ancestor bacteriophage which is an ISP-like bacteriophage from the virus family Herelleviridae having the genome/nucleotide sequence as provided in SEQ NO: 3, referred herein also to/as “03”.

The mosaic bacteriophage as described herein may also have a genome comprising more than one genome fragments of one, two or each of the above described first, second, and third ancestor bacteriophages. Thus, in some embodiments, the mosaic bacteriophage described herein has a genome comprising at least one genome fragment of the first ancestor bacteriophage as described in any of the embodiments and aspect herein, at least one genome fragment of the second ancestor bacteriophage as described in any of the embodiments and aspect herein and at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten genome fragments of the third ancestor bacteriophage as described in any of the embodiments and aspect herein. In one particular example, the mosaic bacteriophage as described herein has a genome comprising at least one genome fragment of the ancestor bacteriophage having the genome as provided in SEQ ID NO: 1, at least one genome fragment of the ancestor bacteriophage having the genome as provided in SEQ ID NO: 2, and at least at least two, at least three, at least four, at least five, or at least six, preferably at least four, genome fragments of the ancestor bacteriophage having the genome as provided in SEQ ID NO: 3.

Accordingly, in some preferred embodiments, the mosaic bacteriophage as described herein has a backbone genome which originates from the third ancestor bacteriophage as described herein, preferably from 03 as defined herein, and comprises at least one genome fragment of the first ancestor bacteriophage and one genome fragment of the second bacteriophage. In some embodiments, the mosaic bacteriophage described herein may further comprises at least two genome fragments of the above described first ancestor bacteriophage.

As used herein, the term “backbone genome” refers to the genome of the ancestor bacteriophage which constitutes the majority, preferably more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75% or more than 80%, of the nucleotides of a bacteriophage which has been bred by intercrossing multiple ancestor bacteriophages. As an illustrative example, a mosaic bacteriophage having a backbone originating from the third ancestor as described herein, such as, e.g., a backbone originating from “03” refers to a mosaic bacteriophage having a genome sequence/nucleotide sequence comprising the nucleotide sequences as provided in SEQ ID NOs: 26, 31, 34, 38, and 41.

A mosaic bacteriophage in the context of the present disclosure is preferably a bred bacteriophage.

As used herein, the term “bred bacteriophage” or “bred phage” refers to a bacteriophage which has been obtained by breeding. As used herein, the term “breeding” means inducing the generation of bred bacteriophage variants and selecting those variants with desired properties. The generation of bacteriophage variants is achieved by promoting the generation of mutations, deletions or intercrossings by various techniques known to those skilled in the art, including, but not limited to, by induction of superinfections through infecting a single bacterial strain with one or more bacteriophages, preferably ancestor bacteriophages, or by propagation of the bacteriophage, preferably ancestor bacteriophage, in presence or absence of a substance which promotes mutations. The selection of bacteriophage variants with selected properties may be achieved by engineering any suitable evolutionary filter, for example propagation on multiple different strains (as an evolutionary filter to increase the host range), or by propagation on bacterial cells growing as biofilm (as an evolutionary filter to select for biofilm activity). An example of a breeding method that can be used to obtain bred bacteriophages is the herein provided breeding method (thereafter referred as “PhagoMed-Modified Appelmans Protocol” or “PMAP”), as described in Example 2. Importantly, when a unique ancestor bacteriophage is used in the input phage mixture, such as for PM56 or PM93, the obtained bacteriophage is a bred bacteriophage but not a mosaic bacteriophage. Alternatively, when two or more different ancestor bacteriophages are used in combination in the input phage mixture, such as for PM4, PM22 or PM32, the obtained bacteriophage is a bred bacteriophage and likely also a mosaic bacteriophage, i.e. a bred mosaic bacteriophage.

It was surprisingly found by the present invention that breeding one or more specific ancestor bacteriophage(s) according to the PhagoMed-Modified Appelmans Protocol provided herein results in a bred bacteriophage which has improved properties compared with the ancestor bacteriophage(s). The herein provided PhagoMed-Modified Appelmans Protocol allows bacteriophages to recombine their genomes to yield new, bred bacteriophages with enhanced properties. The resulting bred bacteriophage may be a mosaic bacteriophage, i.e. having a mosaic genome comprising genome fragments of two or more bacteriophage species, with vastly improved antimicrobial activities compared to the wild type bacteriophages, particularly in terms of host-range, biofilm activity and virulence. For example, appended Figure 2A/1 shows that all mosaic bacteriophages eradicate the MRSA strain “A257” (CC22) which in contrast none of the wild-type bacteriophages can eradicate. The MRSA strain “A257” (CC22) as described herein is available in the publicly accessible section of the Leibniz-Institute DSMZ (Braunschweig, Germany) under the strain name “Bamim” Isolate A257 {Staphylococcus aureus subsp: aureus) and under the accession number DSM 111210. Another example, appended Figure 5A-M shows that the bred mosaic bacteriophages described herein (exemplified by PM4) may inhibit or suppress the growth of a bacterial strain with an increased virulence as compared to at least one of the wild-type bacteriophage (i.e. ancestor bacteriophages). Surprisingly and unexpectedly such a bred mosaic bacteriophage shows highly broad host-range and/or synergism in killing cells in biofilms when combined with another bacteriophage (therein also referred to as the “second bacteriophage”), wherein said another bacteriophage is different from said mosaic bacteriophage and may advantageously also be a bred bacteriophage.

In the context of the invention a broad host range represents the capacity of a bacteriophage or a bacteriophage cocktail to lyse multiple bacterial strains (e.g. capable of lysing greater than 10, preferably greater than 20, more preferably greater than 30, even more preferably greater than 40, even more preferably greater than 50, most preferably greater than 60 different bacterial strains) and/or to lyse multiple clonal complexes (e.g. capable of lysing greater than 5, preferably greater than 10, more preferably greater than 15, even more preferably greater than 18, most preferably greater than 20 different Staphylococcus aureus clonal complexes). As shown in the appended examples the inventive bacteriophage cocktails of the invention are able to lyse up to 88% Staphylococcus aureus strains of a panel of 110 S. aureus strains selected so that the frequency of the clonal complexes in said panel reflects the natural epidemiology of S. aureus in human infections and are able to lyse up to 22 of the clonal complexes reflecting the natural epidemiology of S. aureus in human infections. The natural epidemiology of S. aureus in human infections is known to a skilled artisan and can be found in several literature cases (Arias et al., 2017; Kanjilal et al, 2018; Luedicke et al, 2010; Rasmussen et al, 2013). An example of such a panel is illustrated herein, see Table 1 in the appended examples. As is evident to a person skilled in the art, also other panels of bacterial strains may be used, provided that they reflect the natural epidemiology of S. aureus in human infections. A panel of bacterial strains with this property (i.e. of reflecting the natural epidemiology of S. aureus in human infections) is also referred to herein as reflecting a “controlled diversity”. Importantly, the bacteriophage cocktails of the present invention have the advantage to lyse an over additive number of bacterial strains, meaning that a combination of a first (mosaic) bacteriophage and a second bacteriophage eradicates bacterial strains which none of said first and said second bacteriophage can eradicate individually. As an example, the bacteriophage cocktail PM4 plus PM93 is able to lyse, inter alia, MRSSA 2017-012 strains (CC772) and MSSA124308 strains (CC101), while none of the individual bacteriophages PM4 and PM93 have the ability to lyse these strains (see Figure 2B/2). Accordingly, the invention provides for a bacteriophage cocktail comprising at least a first (mosaic) bacteriophage and a second bacteriophage and which is characterized by unexpected host range properties, in particular, over additive lysing properties in comparison to the first (mosaic) bacteriophage and the second bacteriophage, taken individually. Illustrative examples of the first (mosaic) bacteriophage according to the present invention are PM4, PM5, PM7, PM9, PM22, PM23, PM25, PM28, PM32, PM34, and PM36 and illustrative examples of the second bacteriophage according to the present invention are PM56, PM93, PM94, 01 and 02 (see Tables 7, 8, 10 and 11).

As demonstrated in the appended examples, the nucleotide sequences / genomes of the mosaic bacteriophages of the invention, displaying a surprising broad host range alone and/or in combination with a second bacteriophage, have been analyzed and it has been surprisingly found that they all share structural features, i.e. nucleotide sequences / stretches / genome fragments. Without being bound by any theory, it is believed that the bred mosaic bacteriophages recombine their genomes through the herein provided PhagoMed-Modified Appelmans Protocol by incorporating the genome fragments of each ancestor bacteriophages which are of importance for lysing a wide range of bacterial strains and/or bacterial biofilms, preferably S. aureus strains or bio films, leading to an optimized new bacteriophage surprisingly capable of lysing bacterial strains and/or biofilms that are phage insensitive to any one of the ancestor bacteriophages, individually or in combination in a bacteriophage cocktail. Without being bound by any theory, the genome fragments that, in combination in the genome of a bred mosaic bacteriophage confer such a surprising broad host range, have been identified by the present application and are disclosed herein (see Table 11).

Accordingly, in some preferred embodiments, the mosaic bacteriophage as described herein has a genome comprising a genome fragment with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, or 100% identity with SEQ ID NO: 9. Said mosaic bacteriophage may have a genome comprising a genome fragment with at least 85%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, or 100% identity with SEQ ID NO: 6. Said mosaic bacteriophage may have a genome comprising a genome fragment with at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identity with SEQ ID NO: 17. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 23. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 24. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 25. Said mosaic bacteriophage may have a genome comprising a genome fragment with at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identity with SEQ ID NO: 22. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 27. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 28. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 29. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 30. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 25. Said mosaic bacteriophage may have a genome comprising a genome fragment with at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identity with SEQ ID NO: 26. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 25. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 32. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 33. Said mosaic bacteriophage may have a genome comprising a genome fragment with at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identity with SEQ ID NO: 31. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 35. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 36. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 37. Said mosaic bacteriophage may have a genome comprising a genome fragment with at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identity with SEQ ID NO: 34. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 39. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 40. Said mosaic bacteriophage may have a genome comprising a genome fragment with at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identity with SEQ ID NO: 38. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 42. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 43. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 44. Said mosaic bacteriophage may have a genome comprising a genome fragment with at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identity with SEQ ID NO: 41. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 46. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 47. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 48. Said mosaic bacteriophage may have a genome comprising a genome fragment with at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identity with SEQ ID NO: 45. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 50. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 51. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 52. Said mosaic bacteriophage may have a genome comprising a genome fragment with at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identity with SEQ ID NO: 49. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 54. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 55. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 56. Said mosaic bacteriophage may have a genome comprising a genome fragment with at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identity with SEQ ID NO: 53. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 58. Said mosaic bacteriophage may have a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 59. Said mosaic bacteriophage may have a genome comprising a genome fragment with at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, or 100% identity with SEQ ID NO: 57.

In one preferred embodiment, said at least one genome fragment / nucleotide sequence of the first (ancestor) bacteriophage is the nucleotide sequence as provided in SEQ ID NO: 9 (stretch 13 of Table 7). Alternatively, said at least one genome fragment / nucleotide sequence of the first (ancestor) bacteriophage may be the nucleotide sequence as provided in SEQ ID NO: 6 (stretch 2 of Table 7). Also alternatively, said at least one genome fragment / nucleotide sequence of the first (ancestor) bacteriophage may be the nucleotide sequence as provided in SEQ ID NO: 11 (stretch 25 of Table 7). In some embodiments, the mosaic bacteriophage of the invention comprises any combinations of these genome fragments / nucleotide sequences of the first (ancestor) bacteriophage.

In one preferred embodiment, said at least one genome fragment / nucleotide sequence of the second (ancestor) bacteriophage is the nucleotide sequence as provided in SEQ ID NO: 17 (stretch 6 of Table 7).

In one preferred embodiment, said at least one genome fragment / nucleotide sequence of the third (ancestor) bacteriophage is the nucleotide sequence as provided in any one of SEQ ID NOs: 26, 31, 34, 38 and 41 (stretches 8, 10, 14, 16, and 20 of Table 7, respectively). Thus, in one embodiment, said at least one genome fragment / nucleotide sequence of the third (ancestor) bacteriophage is the nucleotide sequence as provided in SEQ ID NO: 26 (stretch 8 of Table 7). In one embodiment, said at least one genome fragment / nucleotide sequence of the third (ancestor) bacteriophage is the nucleotide sequence as provided in SEQ ID NO: 31 (stretch 10 of Table 7). In one embodiment, said at least one genome fragment / nucleotide sequence of the third (ancestor) bacteriophage is the nucleotide sequence as provided in SEQ ID NO: 34 (stretch

14 of Table 7). In one embodiment, said at least one genome fragment / nucleotide sequence of the third (ancestor) bacteriophage is the nucleotide sequence as provided in SEQ ID NO: 38 (stretch 16 of Table 7). In one embodiment, said at least one genome fragment / nucleotide sequence of the third (ancestor) bacteriophage is the nucleotide sequence as provided in SEQ ID NO: 41 (stretch 20 of Table 7).

Alternatively, said at least one genome fragment / nucleotide sequence of the third (ancestor) bacteriophage is the nucleotide sequence as provided in any one of SEQ ID NOs: 22, 45, 49, 53 and 57 (stretches 4, 12, 18, 21 and 23 of Table 7, respectively). Thus, in one embodiment, said at least one genome fragment / nucleotide sequence of the third (ancestor) bacteriophage is the nucleotide sequence as provided in SEQ ID NO: 22 (stretch 4 of Table 7). In one embodiment, said at least one genome fragment / nucleotide sequence of the third (ancestor) bacteriophage is the nucleotide sequence as provided in SEQ ID NO: 45 (stretch 12 of Table 7). In one embodiment, said at least one genome fragment / nucleotide sequence of the third (ancestor) bacteriophage is the nucleotide sequence as provided in SEQ ID NO: 49 (stretch 18 of Table 7). In one embodiment, said at least one genome fragment / nucleotide sequence of the third (ancestor) bacteriophage is the nucleotide sequence as provided in SEQ ID NO: 53 (stretch 21 of Table 7). In one embodiment, said at least one genome fragment / nucleotide sequence of the third (ancestor) bacteriophage is the nucleotide sequence as provided in SEQ ID NO: 57 (stretch 23 of Table 7). In some embodiments, the mosaic bacteriophage of the invention comprises any combinations of these genome fragments / nucleotide sequences of the third (ancestor) bacteriophage.

Accordingly, in one preferred embodiment, the mosaic bacteriophage of the invention has a genome sequence / nucleotide sequence which comprises the nucleotide sequence as provided in SEQ ID NO: 9, the nucleotide sequence as provided in SEQ ID NO: 17, and the nucleotide sequence as provided in any one of SEQ ID NOs: 22, 26, 31, 34, 38, 41, 45, 49, 53 and 57. In some even more preferred embodiments, the mosaic bacteriophage as described herein has a genome comprising the genome fragment as provided in SEQ ID NO: 9, the genome fragment as provided in SEQ ID NO: 17, and at least one genome fragment selected from the group consisting of SEQ ID NOs: 26, 31, 34, 38, and 41. In one even more preferred embodiment, the mosaic bacteriophage of the invention has a genome sequence / nucleotide sequence which comprises the nucleotide sequence as provided in SEQ ID NO: 9, the nucleotide sequence as provided in SEQ ID NO: 17, and the nucleotide sequence as provided in SEQ ID NO: 31.

Said mosaic bacteriophage may have a genome comprising the genome fragment as provided in SEQ ID NO: 9, the genome fragment as provided in SEQ ID NO: 17, and at least two, at least three or at least four genome fragments selected from the group consisting of SEQ ID NOs: 26, 31, 34, 38, and 41. Said mosaic bacteriophage preferably has a genome comprising all the genome fragments as provided in SEQ ID NOs: 9, 17, 26, 31, 34, 38, and 41. Said genome of said mosaic bacteriophage may further comprise the genome fragment as provided in SEQ ID NO: 6 and/or SEQ ID NO: 22. Alternatively or additionally, said genome of said mosaic bacteriophage may further comprise one or genome fragments selected from the group consisting of SEQ ID NOs: 45, 49, 53, 57.

Illustrative examples of the mosaic bacteriophages having a genome sequence / nucleotide sequence comprising these stretches / genome fragments / nucleotide sequences as described above are shown in the appended examples (see Table 7), and are referred to as PM4, PM32, PM22, PM28, PM34, PM36 and PM25.

A skilled person readily understands that any ancestor bacteriophages comprising such genome fragments in their genome can be used as suitable alternatives in the input phage mixture in order to obtain similar bred phages by the herein provided PMAP protocol, i.e. bred mosaic phages characterized by a similar genome structure and a similar host-range.

As explained in the appended examples, the different “stretches”, i.e. genome fragments of interest that can be found in the mosaic bred phages of the invention, and particularly those which originate from the third ancestor bacteriophage as described herein, may be found in other ISP-like bacteriophages such as , e.g., phage ISP, phage Sbl and phage Gl, and/or in other 812/K-like bacteriophage such as, e.g., phage K. Therefore, a skilled person readily understands that a possibility to obtain a bacteriophage having a genome comprising the genome fragment as provided in SEQ ID NO: 22 (i.e. Stretch 4 of Table 11), the genome fragment as provided in SEQ ID NO: 26 (i.e. Stretch 8 of Table 11), the genome fragment as provided in SEQ ID NO: 31 (i.e. Stretch 10 of Table 11), the genome fragment as provided in SEQ ID NO: 34 (i.e. Stretch 14 of Table 11), the genome fragment as provided in SEQ ID NO: 38 (i.e. Stretch 16 of Table 11), and the genome fragment as provided in SEQ ID NO: 41 (i.e. Stretch 20 of Table 11) is to use the bacteriophage 03 in the phage mixture in combination with the bacteriophages 04 and 05. Another possibility, however, would be to use the phage ISP instead of the phage 03, or to use the phage Sbl in combination with the phage Gl instead of the phage 03, in combination with the bacteriophages 04 and 05. Table 11 also demonstrates that, although some genome fragments may be found in phage K, or any other 812/K-like phages, ISP-like phages are more suitable to be used, alone or in combination, instead of the ISP-like phage 03 in the input phage mixture. Indeed, phage K, alone, will not be suitable to replace the ancestor phage 03 in the input phage mixture in order to obtain bred mosaic bacteriophages comprising stretches 4 (SEQ ID NO: 22) and 8 (SEQ ID NO: 26), for example.

As shown in the appended examples, the most active mosaic bacteriophage, i.e., the mosaic bacteriophage with the broadest host-range (KHR), is PM4 (bacteriophage deposited under the accession No. DSM33478 and/or having the genome as provided in SEQ ID NO: 4), followed by PM9 (bacteriophage having the genome as provided in SEQ ID NO: 61), PM5 (bacteriophage having the genome as provided in SEQ ID NO: 71), PM34 (bacteriophage having the genome as provided in SEQ ID NO: 67), PM22 (bacteriophage having the genome as provided in SEQ ID NO: 62), PM28 (bacteriophage having the genome as provided in SEQ ID NO: 68), PM23 (bacteriophage having the genome as provided in SEQ ID NO: 73), PM7 (bacteriophage having the genome as provided in SEQ ID NO: 72) and PM32 (bacteriophage deposited under the accession No. DSM33479 and/or having the genome as provided in SEQ ID NO: 60). These particularly useful mosaic bacteriophages also show a broad host range in lysis/eradication, i.e they are particularly useful in the lysis of (several) bacteria, in particular several S. aureus strains. All these mosaic bacteriophages are surprisingly more potent in this respect than their ancestor bacteriophages “05”, “04” and “03”. As also explained herein and shown for example in appended Table 11, these mosaic bacteriophages have also, in one embodiment, in common, that they comprise distinct genome sequences/”stretches” from their ancestors. Such stretches comprise “stretch 6” of Table 11 (derived from “04”; shown herein as SEQ ID NO: 17), “stretch 13” of Table 11 (derived from “05”; shown herein as SEQ ID NO: 9), “stretch 10” of Table 11 (derived from “03”; shown herein as SEQ ID NO: 31). As also explained herein and shown for example in appended Figure 2, these mosaic bacteriophages have also, in one embodiment, in common, that they display distinct lysing properties from their ancestors. An example of such lysing properties comprises the property of lysing/propagating on the bacterial strain CC22-MRSA “Bamim” (A257).

Thus, in a preferred aspect of the present invention, the first bacteriophage as described herein is a mosaic bacteriophage, wherein said mosaic bacteriophage is:

(a) a bacteriophage having a genome with at least 98% identity with the genome of the bacteriophage deposited under the accession No. DSM33478 [PM4],

(b) the bacteriophage deposited under the accession No. DSM33478 [PM4],

(c) a bacteriophage having a genome with at least 98% identity with SEQ ID NO: 4 [PM4],

(d) the bacteriophage having the genome as provided in SEQ ID NO: 4 [PM4],

(e) a bacteriophage having a genome with at least 98% identity with the genome of the bacteriophage deposited under the accession No. DSM33479 [PM32],

(f) the bacteriophage deposited under the accession No. DSM33479 [PM32],

(g) a bacteriophage having a genome with at least 98% identity with SEQ ID NO: 60 [PM32],

(h) the bacteriophage having the genome as provided in SEQ ID NO: 60 [PM32],

(i) a bacteriophage having a genome with at least 98% identity with SEQ ID NO: 61 [PM9],

(j) the bacteriophage having the genome as provided in SEQ ID NO: 61 [PM9],

(k) a bacteriophage having a genome with at least 98% identity with SEQ ID NO: 62 [PM22], (l) the bacteriophage having the genome as provided in SEQ ID NO: 62 [PM22],

(m) a bacteriophage having a genome with at least 98% identity with SEQ ID NO: 67 [PM34],

(n) the bacteriophage having the genome as provided in SEQ ID NO: 67 [PM34],

(o) a bacteriophage having a genome with at least 98% identity with SEQ ID NO: 68 [PM28],

(p) the bacteriophage having the genome as provided in SEQ ID NO: 68 [PM28],

(q) the bacteriophage having the genome as provided in SEQ ID NO: 71 [PM5],

(r) a bacteriophage having a genome with at least 98% identity with SEQ ID NO: 71 [PM5],

(s) the bacteriophage having the genome as provided in SEQ ID NO: 72 [PM7],

(t) a bacteriophage having a genome with at least 98% identity with SEQ ID NO: 72 [PM7],

(u) the bacteriophage having the genome as provided in SEQ ID NO: 73 [PM23], or

(v) a bacteriophage having a genome with at least 98% identity with SEQ ID NO: 73 [PM23]

The mosaic bacteriophage PM4 was deposited under the accession No. DSM33478, at the Leibniz-Institute DSMZ (Deutsche Sammhmg von Mikroorgansimen und Zellkulturen GmbH, in Braunschweig/Germany) on March 19, 2020, as an international deposit according to the provisions of the Budapest Treaty, see the appended deposit receipt.

The mosaic bacteriophage PM32 was deposited under the accession No. DSM33479, at the Leibniz-Institute DSMZ (Deutsche Sammhmg von Mikroorgansimen und Zellkulturen GmbH, in Braunschweig/Germany) on March 19, 2020, as an international deposit according to the provisions of the Budapest Treaty, see the appended deposit receipt.

Thus, in certain embodiments, the mosaic bacteriophage described herein is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome of the bacteriophage as provided in any one of SEQ ID NOs: 4, 60, 61, 62, 67, 68 and 71-73.

Importantly, it is to be noted that PM4 has a genome with 99.99% identity with the genome of PM5, PM7 and PM9 over the whole length of the genome of PM4, PM4 has a genome with 98.92% identity with the genome of PM22 over the whole length of the genome of PM4 (99.92% identity over 99% of the genome of PM4), PM4 has a genome with 98.91% identity with the genome of PM23 over the whole length of the genome of PM4, PM4 has a genome with 97.87% identity with the genome of PM25 over the whole length of the genome of PM4 (99.87% identity over 98% of the genome of PM4), PM4 has a genome with 98.96% identity with the genome of PM28 over the whole length of the genome of PM4 (99.96% identity over 99% of the genome of PM4), PM4 has a genome with 98.88% identity with the genome of PM32 over the whole length of the genome (99.88% identity over 99% of the genome of PM4), PM4 has a genome with 96.90% identity with the genome of PM34 over the whole length of the genome of PM4 (99.90% identity over 97% of the genome of PM4), and PM4 has a genome with 96.83% identity with the genome of PM36 over the whole length of the genome of PM4 (99.82% identity over 97% of the genome of PM4), when aligned with Blast2 sequences (Zheng Zhang, Scott Schwartz, Lukas Wagner, and Webb Miller (2000), "A greedy algorithm for aligning DNA sequences", J Comput Biol 2000; 7(l-2):203-14.). It can therefore be concluded that any bacteriophage having a genome with at least 98% identity with the genome of PM4 is likely to achieve a broad host range in a phage cocktail in combination with a Romulus/Remus- like bacteriophage and/or a bred phage thereof such as, e.g, PM56 and PM93.

Thus, in some preferred embodiments, the mosaic bacteriophage described herein is a bacteriophage having a genome with at least 98% with the genome of the bacteriophage as provided in SEQ ID NO: 4 and/or with the genome of the bacteriophage as deposited under the accession No. DSM33478 [PM4]

In certain embodiments, the mosaic bacteriophage described herein is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome of the bacteriophage deposited under the accession No. DSM33478 [PM4] In one particular and preferred embodiment, said mosaic bacteriophage may be the bacteriophage deposited under the accession No. DSM33478 [PM4]

In certain embodiments, the mosaic bacteriophage described herein is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome of the bacteriophage deposited under the accession No. DSM33479 [PM32] In one particular and preferred embodiment, said mosaic bacteriophage may be the bacteriophage deposited under the accession No. DSM33479 [PM32]

In certain embodiments, the mosaic bacteriophage described herein is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 4 [PM4] In one particular and preferred embodiment, said mosaic bacteriophage may be the bacteriophage having the genome as provided in SEQ ID NO: 4 [PM4]

In certain embodiments, the mosaic bacteriophage described herein is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 60 [PM32] In one particular and preferred embodiment, said mosaic bacteriophage may be the bacteriophage having the genome as provided in SEQ ID NO: 60 [PM32]

In certain embodiments, the mosaic bacteriophage described herein is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 61 [PM9] In one particular and preferred embodiment, said mosaic bacteriophage may be the bacteriophage having the genome as provided in SEQ ID NO: 61 [PM9]

In certain embodiments, the mosaic bacteriophage described herein is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 62 [PM22] In one particular and preferred embodiment, said mosaic bacteriophage may be the bacteriophage having the genome as provided in SEQ ID NO: 62 [PM22] In certain embodiments, the mosaic bacteriophage described herein is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 67 [PM34] In one particular and preferred embodiment, said mosaic bacteriophage may be the bacteriophage having the genome as provided in SEQ ID NO: 67 [PM34]

In certain embodiments, the mosaic bacteriophage described herein is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 68 [PM28] In one particular and preferred embodiment, said mosaic bacteriophage may be the bacteriophage having the genome as provided in SEQ ID NO: 68 [PM28]

In certain embodiments, the mosaic bacteriophage described herein is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 71 [PM5] In one particular and preferred embodiment, said mosaic bacteriophage may be the bacteriophage having the genome as provided in SEQ ID NO: 71 [PM5]

In certain embodiments, the mosaic bacteriophage described herein is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 72 [PM7] In one particular and preferred embodiment, said mosaic bacteriophage may be the bacteriophage having the genome as provided in SEQ ID NO: 72 [PM7] In certain embodiments, the mosaic bacteriophage described herein is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 72 [PM23] In one particular and preferred embodiment, said mosaic bacteriophage may be the bacteriophage having the genome as provided in SEQ ID NO: 72 [PM23]

As shown in the appended examples, the mosaic bacteriophages of the invention may also be characterized by their lysing properties. For example, the mosaic bacteriophages of the invention may be characterized in that it lyses at least one bacterial strain that none of the ancestor bacteriophages lyses or is capable to lyse. A particular example of said “at least one bacterial strain” that the mosaic bacteriophage of the invention may lyse (that none of the ancestor bacteriophages may lyse) is the herein provided bacterial strain CC22-MRSA-IV “Bamim” (A257). Thus, in one preferred embodiment, the mosaic bacteriophage of the invention is characterized in that it lyses the particular bacterial strain bacterial strain CC22- MRSA-IY “Bamim” (A257) available in the publicly accessible section of the Leibniz-Institute DSMZ (Braunschweig, Germany) under the strain name “Bamim” Isolate A257 ( Stapholococcus aureus subsp: aureus) and under the accession number DSM 111210.

Another example of the lysing properties by which the mosaic bacteriophage of the invention may be characterized is their kinetic host range. As demonstrated in the appended examples, the mosaic bacteriophages described herein have an antibacterial activity against a surprisingly broad kinetic host-range, such as, e.g., against at least 30%, at least 35%, preferably at least 40%, more preferably at least 45%, even more preferably at least 49% of the Staphylococcus aureus strains of a panel of 110 Staphylococcus aureus selected such that the frequency of the clonal complexes in the panel reflects the natural epidemiology of S. aureus in human infections as found in several literature cases. Also demonstrated in the appended examples, the mosaic bacteriophages described herein have an antibacterial activity against a surprisingly broad (kinetic) host range, such as, e.g., against at least 5, at least 10, at least 15, preferably at least 20 different Staphylococcus aureus clonal complexes reflecting the natural epidemiology of S. aureus in human infections. Illustrative examples of bacterial strains that can be comprised in the panels of bacterial strains as described herein are the S. aureus strains CC239-MRSA-III (2017-046), CC30-MRSA-IV (2011-278), CC25-MSSA (B91), CC12-MSSA (A161), CC22-MRSA-IY (A257, “Bamim”) and CC22-MRSA-IY (B311, “Bamim”) (each of these strains are available in the publicly accessible section of the Leibniz-Institute DSMZ, Braunschweig, Germany, under the accession numbers as indicated in Table 11) and the S. aureus strain CC25-MSSA (124605) (deposited under the stipulations of the Budapest treaty under the accession number DSM 33467).

Another example of the lysing properties by which the mosaic bacteriophage of the invention may be characterized is their biofilm host range. Also demonstrated in the appended Examples, the mosaic bacteriophages described herein have an antibacterial activity against a surprisingly broad biofilm host-range, such as, e.g., against at least 30%, at least 40%, preferably at least 50%, more preferably at least 60%, even more preferably at least 70% of the Staphylococcus aureus pre-formed biofilms of a panel of 10 Staphylococcus aureus strains selected such as to have a high diversity of the clonal complexes. Also demonstrated in the appended examples, the mosaic bacteriophages described herein have an antibacterial activity against a surprisingly broad (biofilm) host range, such as, e.g., against at least 3, at least 4, preferably at least 5, more preferably at least 6, even more preferably at least 7 different Staphylococcus aureus clonal complexes.

Another example of the lysing properties by which the mosaic bacteriophage of the invention may be characterized is their virulence.

In this disclosure, the term virulence means the phage property quantified by the virulence curve as defined in (Storms et al., 2020). In epidemiology, a common measurement of virulence is the reproduction number (R0) — defined as the average number of additional hosts that the virus will spread to after infecting a single host. An equivalent measurement is not applicable to the virulent phage life cycle. The number of phage progeny released per infected cell is given by the burst size; when replicating in a healthy, densely growing bacterial culture, the R0 of a phage is essentially equal to its burst size. However, burst size alone is not a proper indicator of phage virulence. For example, in a study of the efficacy of phage therapy, virulence was found to be a function of, among others, adsorption rate, burst size, the latent period, and host cell growth rate. In this context, virulence can be defined as the ability of a phage to kill or damage a host population. According to the definition of (Storms et al., 2020), a virulence curve is the local virulence (VMOI) plotted as a function of MOI. The individual values of VMOI are calculated according to the formula _VMOI = 1 - (Al-B)/ (A0-B) where:

• MOI is the multiplicity of infection at the start of the infection.

• Al is the area under the ODr.oo curve of the bacterial culture, infected with phages at a particular multiplicity of infection MOI, where OD600 is measured for 24h every 5 minutes at 37°C.

• B is the area under the ODeoo curve of the medium control, not infected with phages or bacteria, where OD600 is measured for 24h every 5 minutes at 37°C.

• AO is the area under the ODr.oo curve of the bacterial culture not infected with phages, where OD600 is measured for 24h every 5 minutes at 37°C.

The virulence curve allows for example to compare the different MOI values at which two phages have the same VMOI, thereby comparing the virulence of the two phages.

As demonstrated in the appended examples, the mosaic bacteriophages described herein may inhibit or suppress the growth of a bacterial strain at a low multiplicity of infection (MOI: number of phages per bacterial cell) (e.g. capable of inhibiting or suppressing the growth of a bacterial strain at a MOI lower than 10,000, preferably lower than 1,000, more preferably lower than 100, even more preferably lower than 50, even more preferably lower than 20, most preferably at a MOI as low as 10) for 24h. Thus, in one embodiment, the mosaic bacteriophage of the invention inhibits or suppresses the growth of a bacterial strain at a MOI lower than 10,000, preferably lower than 1,000, more preferably lower than 100, even more preferably lower than 50, even more preferably lower than 20, most preferably at a MOI as low as 10. In accordance with the present invention, the preferred bacterial strain to be used to assess the virulence of the mosaic bacteriophage of the present invention is any one of the S. aureus strains selected from the group consisting of : CC239-MRSA-III (2017-046), CC30-MRSA-IY (2011- 278), CC25-MSSA (B91), CC12-MSSA (A161), CC22-MRSA-IV (A257, “Bamim”) and CC22-MRSA-IY (B311, “Bamim”) (each of these strains are available in the publicly accessible section of the Leibniz-Institute DSMZ, Braunschweig, Germany, under the accession numbers as indicated in Table 13). As used herein, the terms “antibacterial activity” and “antimicrobial activity”, with reference to a bacteriophage (or variant or fragment thereof) or bacteriophage product, are used interchangeably to refer to the ability to kill and/or inhibit the growth or reproduction of a microorganism, in particular, the bacteria of the species or strain that the bacteriophage infects. In certain embodiments, antibacterial or antimicrobial activity is assessed by culturing bacteria, e.g. Gram-positive bacteria (e.g. S. aureus), Gram-negative bacteria (e.g. A. baumannii, E. coli, and/or P. aeruginosa) or bacteria not classified as either Gram-positive or Gram-negative, according to standard techniques (e.g. in liquid culture or on agar plates), contacting the culture with a bacteriophage or variant thereof of the invention and monitoring cell growth after said contacting. For example, in a liquid culture, the bacteria may be grown to an optical density (“OD”) representative of a midpoint in exponential growth of the culture. The culture is exposed to one or more concentrations of one or more bacteriophage(s) of the invention, or variants thereof, and the OD is monitored relative to control culture. Decreased OD relative to a control culture is representative of a bacteriophage exhibiting antibacterial activity (e.g. exhibits lytic killing activity). Similarly, bacterial colonies can be allowed to form on an agar plate, the plate exposed to one or more bacteriophage of the invention, or variants thereof, and subsequent growth of the colonies evaluated related to control plates. Decreased size of colonies, or decreased total numbers of colonies, indicate a bacteriophage with antibacterial activity.

As used herein, the term “lytic” or “lytic activity” designates the property of a bacteriophage to cause lysis of a bacterial cell. The lytic activity of a bacteriophage can be tested on bacteria (e.g. S. aureus strains) according to techniques known in the art, including but not limited to plaque assays or spot test. The lytic cycle is named for the process that occurs when a phage has infected a cell, replicated new phage particles, and bursts through the host cell membrane. Some phage exhibit a lysogenic cycle during which the bacteriophage DNA remains practically dormant due to active repression of bacteriophage processes. Whenever the bacteria divides, the DNA of the phage is copied as well. In this way, the virus can continue replicating within its host without lysing the host.

In accordance with the present invention, the bacteriophages as described herein has lytic activity against at least one or more bacterial strains. In the context of the invention, bacterial strains may be Staphylococcus strains, preferably Staphylococcus aureus strains. Examples of Staphylococcus aureus strains that may be lysed by the bacteriophages described herein are listed in Table 1 herein. Illustrative examples of S. aureus strains lysed by the inventive (mosaic) bacteriophages comprise CC22-MRSA-IY, “Bamim” (e.g. A257, A258, B311), CC12-MSSA (A161) and CC25-MSSA (124605). As shown in the appended examples and in illustrative Figure 2, neither “05”, “04” nor “03” are capable of lysing/eradicating the “Bamim” strain “A257”, whereas all the mosaic bacteriophages provided herein do lyse this strain. Accordingly, the strain CC22-MRSA-IY, “Bamim” (A257) may be particularly useful to select the mosaic bacteriophages of the invention and is used in that context as a preferred embodiment of the invention. In one specific embodiment the panel on which and with which the KHR may be assessed may comprise such “Bamim” strains. The person skilled in the art is aware that several isolates of such stains exist and in the present invention, inter alia, isolates “A257”, “A258” or “B311” have been employed. Again, in avoidance of any doubt, these are merely examples of bacterial strains that may be comprised in the panel to be employed for the establishment of KHR.

As shown in the appended examples and in illustrative Figure 2, all the mosaic bacteriophages can propagate on the strain CC25-MSSA (124605), Accordingly, the strain CC25-MSSA (124605) may be particularly useful for the propagation of the mosaic bacteriophages of the invention and is also part of this invention. Thus, in preferred embodiments, bacterial strains used to propagate the phage, including, but not limited to CC25-MSSA (124605) are specifically contemplated. The strain CC25-MSSA (124605) has been deposited under the stipulations of the Budapest treaty under the accession number DSM 33467 with the Leibniz- Institute DSMZ (Deutsche Sammlung von Mikroorgansimen und Zellkulturen GmbH, in Braunschweig/Germany) on March 19^th, 2020 (see appended deposit receipt). Thus, in some preferred embodiments, the bacterial strain used for propagating is strain CC25-MSSA (124605) as deposited under the accession No. DSM 33467.

In addition, in the context of the present invention mosaic and/or bred bacteriophages as described herein have improved properties compared with ancestor or wild-type bacteriophages, particularly in terms of host-ranges and biofilms reduction.

As used herein, the term “host-range” refers to the breadth of bacterial strains a bacteriophage is capable of infecting, with limits on host range stemming from the phage, the bacterial strains, environmental characteristics and the technique of measuring the host range (Hyman and Abedon, 2010). Techniques of measuring a host range include plaquing, spot testing, or broth- based/kinetic measures of phage and bacterial population growth. As the host range values may vary depending on the method by which they were measured, in this disclosure, the method will always be illustrated herein in the context of the host range values discussed, for example a “plaquing host-range” or a “kinetic host range”. The terms are further defined herein and also illustrated in the experimental part of the present invention. In one embodiment of the invention, the mosaic bacteriophages of the present invention are characterized by their improved “host range” as compared to their ancestors, in particular improved over “05”, “04” and/or “03”, and even combinations thereof. In the appended examples of the present application, the host- range have been measured as a Kinetic Host Range (KHR) or a Biofilm Host Range (BHR).

The term “Biofilm Host-Range” (BHR) as used herein is defined as a measure for the lytic activity of a given phage on bacteria growing as biofilm. The lytic activity on biofilm is measured as described in Example 4. It is defined as the % of strains of a panel of at least 10 strains, where the reduction in viable cells by phage treatment is more than 1 log CFU, 2 log CFU or 3 log CFU as further specified where BHR is described. The panel for measuring BHR comprises less bacterial strains than for the KHR measurement as the workload required to measure BHR is substantially higher than for KHR. The panel may comprise strains of at least 5, preferably 10 clonal complexes selected from the ones most frequently causing infections in humans, as described above.

The term “Kinetic Host Range” (KHR) as used herein is a measure for the lysis and/or eradication capacity of a given bacteriophage, mixture of bacteriophages or compositions comprising bacteriophages (“bacteriophage cocktail(s)”). This “host range”/”kinetic host range” may be measured/established by techniques known in the art and as described herein and as detailed herein below. In particular, said “KHR” may be defined as the ratio of bacterial strains eradicated/lysed either per se (for example “X % of bacteria lysed/eradicated of a 100% (given) panel of provided target/host bacterial strains/test bacterial strains). Therefore, the KHR reflects the percentage of lysed/eradicated target bacterial strains /host bacterial strains/test bacterial strains in comparison to a corresponding control panel of the same target bacterial strains /host bacterial strains/test bacterial strains not contacted with/exposed to the bacteriophage/the “bacteriophage mixture” to be assessed. Accordingly, a value of said KHR may be expressed by the percentage of a panel of bacteria target hosts lysed by the bacteriophage/the “bacteriophage mixture”. Said value may also be compared to a given control and/or a reference bacteriophage/“bacteriophage mixture”/”phage mixture” (“cocktail”). The test panel of target bacterial strains /host bacterial strains/test bacterial strains preferably reflects a distribution of clonal complexes of bacterial strains similar to what is found in infections, preferably in human infections. The target bacterial strains/host bacterial strains/test bacterial strains panel may comprise at least 10, preferably at least 20, preferably at least 30, preferably at least 40, preferably at least 50, preferably at least 60, preferably at least 70, preferably at least 80, preferably at least 90, preferably at least 100 different bacterial strains. As documented in the appended examples, the currently employed panel of the target bacterial strains/host bacterial strains/test bacterial strains comprise about 100 to 150 strains, in particular the 110 strains as shown in appended Table 4. Preferably, said panel(s) comprise Staphylococcus aureus strains. The person skilled in the art is readily in a position to obtain and provide for such “panels” of target/host strains for the assessment of a KHR. In one embodiment, the target/host panel comprises bacteria isolated form patient samples, i.e. form patients suffering from and/or being infected by corresponding bacterial strains. As shown in the appended examples, illustratively a panel of 110 S. aureus strains was compiled from patient isolates at the University Clinic Dresden, Germany, in 2010 and 2011, and was employed herein in the assessment of the “KHR”. The genomes of each strain of this “Dresden” panel was sequenced and genotyped to define their clonal complex. In this particular case, said 110 strains were selected such that the frequency of the clonal complexes in the panel reflects the natural epidemiology of S. aureus in human infections as found in several literature cases (Arias et al., 2017; Kanjilal et al., 2018; Luedicke et al. ; 2010; Rasmussen et al., 2013). Yet, it is evident for the skilled artisan that also other “panels” may be employed. Preferably, in context of this invention, said panel comprises S. aureus strains. Most preferably, said panel reflects a distribution of clonal complexes similar to what is found in infections, preferably in human infections. A person skilled in the art is readily in the position to obtain such corresponding and useful panel(s) and/or bacterial strains collection(s), for example from the national reference centers for antimicrobial resistance, or public collections of microorganisms.

In context of the present invention and as known in the art, a lysis/eradication of bacteria by a bacteriophage or a bacteriophage cocktail may be considered as achieved/successful, when said bacteriophage or said bacteriophage cocktail to be assessed reduces the number of (bacterial) cells (preferably grown in a suspension, i.e. in a broth allowing bacterial growth) by at least 90% or more (preferably at least 99,5% or even 100% of the bacterial “host” cells) compared to an untreated control (i.e. a parallel control sample of this bacterial culture that is not exposed to said bacteriophage/mixture of bacteriophages but is cultured under the same culturing conditions). The corresponding exposure conditions of said bacterial host cells (herein also defined as “reaction”) may be, inter alia, as follows: If not specified otherwise, the starting concentration of the (host) bacteria may be about 5xl0⁷ CFU/ml, with a starting ratio of bacteria to plaque forming units of 1:10 (multiplicity of infection/MOI = 10). The exposure of bacteriophage(s) with the host bacteria (the “reaction”) may preferably be carried out at 37 °C, over the course of preferably 24 hours. If desired, the person skilled in the art may lengthen or shorten this “reaction” time. A useful (bacterial) broth may be Brain-Heart-Infusion (BHI) broth. Further useful broth for the “reaction” comprise but are not limited to Luria-Bertani (LB) broth or Tryptic Soy Broth (TSB). As is evident for the person skilled in the art, in order to reduce the number of bacterial cells (i.e. “lysis”) or to even achieve complete eradication of the bacteria (in said suspension), the bacteriophage or bacteriophage cocktail must have parameters of the lytic cycle (such as absorption rate and burst size) that allow the bacteriophage particles to outcompete and quantitatively reduce the number of bacterial cells. Furthermore and in parallel to said lytic cycle, the rate of spontaneous resistance of the bacteria against the bacteriophage(s) in the phage mixture should be below one cell in the starting suspension, here for example said 5xl0⁷ (the number of CFU at the start of the experiment). The Kinetic Host Range (KHR) of the bacteriophage/the bacteriophage cocktail may be calculated as discussed herein above and/or as shown in Example 1, i.e. as the percent of strains for which after a given time (for example here 24h), lysis/eradication can be detected/measured. Such a detection/measurement may be achieved by the evaluation of the optical density of the bacterial suspension (for example at a given wavelength in nm, as shown in the appended examples at “OD600”). “OD600” as used herein is a measure of the scattering of monochromatic light with a wavelength of 600nm by a bacterial suspension, calculated as the base- 10 logarithm of the ratio of incident and transmitted light and measured with a Tecan Infinite F NANO + microplate reader. As described for example in Stevenson et al, 2016, the relationship between OD600 and the concentration of bacterial cells is linear within a specific concentration range. The measurements of OD600 described herein were conducted in 96-well plates filled with 200ul of liquid. OD600 values reported herein for bacterial suspensions or used to calculate derived quantities (e.g., “OD ratio”) were adjusted by subtracting the blank OD600 value, measured for the same volume of broth without bacteria. Calibration measurements were conducted and show that an OD600 value of 0.15 corresponds to a cell density of approximately 10⁸ CFU/ml for S. aureus cells, and that the linear range in this setting holds between OD600 values of 0.010 and 0.700. In context of this invention, a successful lysis/eradication of a given bacterial suspension is achieved when the optical density of the bacteriophage treated/bacteriophage exposed sample is preferably less than about 10% of the corresponding optical density of the untreated bacterial growth control under the same condition {i.e., the control sample comprising the same “test” bacteria, i.e. bacteria of the same strain and/or origin without the exposure to bacteriophages).

As used herein, “eradication” is defined as lack of bacterial re-growth after lysis of a bacterial suspension after 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, preferably 24 hours of lysis/reaction, and where the cell density was reduced and/or stayed at a level not detectable by OD600. The duration of the lysis/reaction must be chosen such that, if resistant cells emerge, they have sufficient time to replicate to detectable optical density (OD), which for S. aureus in rich medium (e.g. BHI, LB, TSB) and at 37°C is typically the case after 9 hours. Therefore, “eradication” as used herein does not by itself mean that sterility was demonstrated, but that a bacterial population collapsed to values below the detection level and was not able to re-grow within the time indicated above, which, considering the growth rates of the microorganism under investigation (e.g. S. aureus) means that sterility is likely. As mentioned herein above, the person skilled in the art may modify the time and may also decide to expose the (host) bacteria for a longer time with the bacteriophage, the bacteriophages and/or the mixtures/cocktails of bacteriophages without deferring from the gist of the present invention.

In context of this invention and as employed herein above, “sterility” means freedom from the presence of viable microorganisms.

In some preferred embodiments, the mosaic and/or bred bacteriophages herein provided have lytic activity against bacterial strains and/or bacterial biofilms which are phage insensitive to any one of the first, the second and/or the third ancestor bacteriophage(s) as defined herein.

The term “phage sensitive” means a bacterial strain and/or bacterial biofilm that is sensitive to infection and/or killing by bacteriophage and/or in growth inhibition, and on which plaques can form with a suitable assay. Likewise, the term “phage insensitive” or “phage resistant” or “phage resistance” or “resistant profile” is understood to mean a bacterial strain and/or bacterial bio film that is insensitive, and preferably highly insensitive to infection and/or killing by phage and/or growth inhibition, and on which plaques cannot form with a suitable assay.

Thus, in some preferred embodiments, the mosaic bacteriophage as described herein has lytic activity against at least one bacterial strain that none of the first ancestor bacteriophage as described herein, the second ancestor bacteriophage as described herein and the third bacteriophage as described herein lyses, meaning that said at least one strain is phage insensitive to said first ancestor bacteriophage, said second ancestor bacteriophage and said third bacteriophage individually. In some even more preferred embodiments, said at least one bacterial strain is further phage insensitive to a bacteriophage composition comprising said first ancestor bacteriophage, said second ancestor bacteriophage and said third bacteriophage in combination. An example of such a strain is the strain CC22-MRSA-IY, “Bamim” (A257) as explained in detail herein. That is, in one more preferred embodiment, the mosaic bacteriophage as described herein lyses the bacterial strain CC22-MRSA-IV “Bamim” (A257).

Thus, in one even more preferred embodiment, the mosaic bacteriophage as described herein has a genome sequence / nucleotide sequence that comprises the nucleotide sequence as provided in SEQ ID NO: 9, the nucleotide sequence as provided in SEQ ID NO: 17, the nucleotide sequence as provided in SEQ ID NO: 31 and which is able to lyse/propagate on the bacterial strain CC22-MRSA-IV “Bamim” (A257).

The invention further provides a composition comprising (i) a first bacteriophage, which is any one of the “mosaic bacteriophage” as described above, and (ii) a second bacteriophage, wherein said second bacteriophage is preferably a Romulus/Remus-like bacteriophage.

Thus, in one preferred aspect of the invention, the second bacteriophage as described herein is a Romulus/Remus-like bacteriophage.

As used herein interchangeably, the terms “Romulus/Remus-like bacteriophage” and “Remus/Romulus-like bacteriophage” refer to a bacteriophage comprising a nucleotide sequence homology of at least 95% over the full length to the nucleotide sequence representing the genome of phage Remus as provided by NCBI accession number JX846612 (version JX846612.1 of 15 August 2013) or as provided by SEQ ID NO: 63. The wild-type Remus bacteriophage is also referred to herein as “01” and has the nucleotide sequence as provided in SEQ ID NO: 63. The wild-type Romulus bacteriophage is also referred to herein as “02” and has the nucleotide sequence as provided in SEQ ID NO: 64. Examples of Romulus/Remus-like bacteriophages include but are not limited to, phage Remus itself (such as, e.g., provided by NCBI accession number JX846612 ( loc . cit.) or as provided in in SEQ ID NO: 63), and phage Romulus (such as, e.g. , as provided by NCBI accession number JX846613 version JX846613.1 of 12 March 2013 or as provided in in SEQ ID NO: 64). According to the present invention, the second bacteriophage may be an ancestor bacteriophage, or a bred and/or a mosaic bacteriophage.

Examples of the genome of an ancestor Romulus/Remus-like bacteriophage are provided herein in SEQ ID NOs: 63 (wild-type Remus bacteriophage, referred to as “01” in the appended examples) and 64 (wild-type Romulus bacteriophage, referred to as “02” in the appended examples). Examples of the genome of a bred Romulus/Remus-like bacteriophage are provided herein in SEQ ID NOs: 65 (bred progeny of Romulus bacteriophage, also referred to as “PM56” in the appended examples) and 66 (bred progeny of Remus bacteriophage, also referred to as “PM93” in the appended examples).

As shown in the appended Examples, the most active Romulus/Remus-like bacteriophage, i.e., the Romulus/Remus-like bacteriophage with the broadest host-range, is PM56 (bacteriophage having the genome as provided in SEQ ID NO: 65), followed by PM94 (bacteriophage having the genome as provided in SEQ ID NO: 74), PM93 (bacteriophage having the genome as provided in SEQ ID NO: 66), 02 (bacteriophage having the genome as provided in SEQ ID NO: 64), and 01 (bacteriophage having the genome as provided in SEQ ID NO: 63).

Thus, in a preferred aspect of the invention, the second bacteriophage as described herein is:

(a) a bacteriophage having a genome with at least 98% identity with SEQ ID NO: 63 [01],

(b) the bacteriophage having the genome as provided in SEQ ID NO: 63 [01],

(c) a bacteriophage having a genome with at least 98% identity with SEQ ID NO: 64 [02],

(h) the bacteriophage having the genome as provided in SEQ ID NO: 64 [02],

(i) a bacteriophage having a genome with at least 98% identity with SEQ ID NO: 65 [PM56],

(j) the bacteriophage having the genome as provided in SEQ ID NO: 65 [PM56],

(k) a bacteriophage having a genome with at least 98% identity with SEQ ID NO: 66 [PM93],

(l) the bacteriophage having the genome as provided in SEQ ID NO: 66 [PM93]

(m) a bacteriophage having a genome with at least 98% identity with SEQ ID NO: 74 [PM94],

(n) the bacteriophage having the genome as provided in SEQ ID NO: 74 [PM94]

Thus, in certain embodiments, the second bacteriophage as described herein is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome of the bacteriophage as provided in any one of SEQ ID NOs: 63 to 66 and 74 [PM94]

In certain embodiment, the second bacteriophage as described herein is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 63 [01] In one particular and preferred embodiment, said second bacteriophage may be the ancestor bacteriophage Remus having the genome as provided in SEQ ID NO: 63 [01]

In certain embodiment, the second bacteriophage as described herein is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 64 [02] In one particular and preferred embodiment, said second bacteriophage may be the ancestor bacteriophage Romulus having the genome as provided in SEQ ID NO: 64 [02]

In certain embodiment, the second bacteriophage as described herein is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 65 [PM56] In one particular and preferred embodiment, said second bacteriophage is the bred bacteriophage having the genome as provided in SEQ ID NO: 65 [PM56]

In certain embodiment, the second bacteriophage as describe herein is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 66 [PM93] In one particular and preferred embodiment, said second bacteriophage is the bred bacteriophage having the genome as provided in SEQ ID NO: 66 [PM93] In certain embodiment, the second bacteriophage as describe herein is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 74 [PM94] In one particular and preferred embodiment, said second bacteriophage is the bred bacteriophage having the genome as provided in SEQ ID NO: 74 [PM94]

As shown in the appended Examples, the bred bacteriophages have an antibacterial activity against broader host-ranges as compared with the ancestor bacteriophages. The best Romulus/Remus-like ancestor bacteriophage has indeed an antibacterial activity against 45% of the Staphylococcus aureus strains of a panel of 110 Staphylococcus aureus selected such that the frequency of the clonal complexes in the panel reflects the natural epidemiology of S. aureus in human infections, whereas the bred Romulus/Remus-like bacteriophages have an antibacterial activity against at least 56% of the same panel.

Thus, in a preferred aspect of the present invention, the second bacteriophage is a bred bacteriophage. Said second bacteriophage may preferably be a bred bacteriophage which has been obtained by the herein provided breeding method. In further preferred aspect of the present invention, the second bacteriophage is not a mosaic bacteriophage.

In accordance with the present invention, the bred bacteriophages as described herein have lytic activity against at least one bacterial strain that, when a unique ancestor phage was used in the input phage mixture, that said ancestor bacteriophage cannot lyse, or when multiple ancestor phages was used in combination in the input phage mixture, that none of said multiple ancestor bacteriophages lyses, meaning that said at least one strain is phage insensitive to said ancestor bacteriophage, or to any one of said multiple ancestor bacteriophages, individually. In some embodiments, said at least one bacterial strain is further phage insensitive to a bacteriophage composition comprising said multiple ancestor bacteriophages in combination. Bacterial strains may be Staphylococcus strains, preferably Staphylococcus aureus strains. Examples of Staphylococcus aureus strains that may be lysed by the mosaic and/or bred bacteriophages described herein are listed in Table 1 herein. Thus, in some preferred embodiments, the second bacteriophage as described herein is a bred bacteriophage, wherein said bred bacteriophage originates from a breeding method where a unique ancestor bacteriophage has been used in the input mixture, and wherein said bred bacteriophage lyses at least one strain that is phage insensitive to said unique ancestor bacteriophage. In one particular embodiment, said unique ancestor bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 63, and said bred bacteriophage may optionally be the bacteriophage having the genome as provided in SEQ ID NO: 66 or 74. In another particular embodiment, said unique ancestor bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 64, and said bred bacteriophage may optionally be the bacteriophage having the genome as provided in SEQ ID NO: 65.

As shown in the appended Examples, the most active bacteriophage composition / bacteriophage cocktail, i.e. the composition / cocktail with the broadest (kinetic) host-range, is PM4 plus PM93, followed by PM9 plus PM93, PM23 plus PM56, PM28 plus PM94, PM32 plus 02, PM7 plus PM56, PM32 plus PM56, PM22 plus 02, PM23 plus PM93, wherein all of those composition / cocktails shows lytic activity against more than 70% of the Staphylococcus aureus strains, and/or more than 15 different complex complexes, of a panel of 110 Staphylococcus aureus selected such that the frequency of the clonal complexes in the panel reflects the natural epidemiology of A aureus in human infections.

In line with the considerably high host-range of the above bacteriophage compositions / bacteriophage cocktails, these (and their functional variants) are preferred in the present invention.

Accordingly, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 4 [PM4] and wherein the second bacteriophage is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 66 [PM93]

Thus, in some embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 95% identity with the genome as provided in SEQ ID NO: 4 [PM4] and wherein the second bacteriophage is a bacteriophage having a genome with at least 95% identity with the genome as provided in SEQ ID NO: 66 [PM93]

In some preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 97% identity with the genome as provided in SEQ ID NO: 4 [PM4] and wherein the second bacteriophage is a bacteriophage having a genome with at least 97% identity with the genome as provided in SEQ ID NO: 66 [PM93]

In some more preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 98% identity with the genome as provided in SEQ ID NO: 4 [PM4] and wherein the second bacteriophage is a bacteriophage having a genome with at least 98% identity with the genome as provided in SEQ ID NO: 66 [PM93]

In some even more preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 99% identity with the genome as provided in SEQ ID NO: 4 [PM4] and wherein the second bacteriophage is a bacteriophage having a genome with at least 99% identity with the genome as provided in SEQ ID NO: 66 [PM93] In some most preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 4 [PM4] and wherein the second bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 66 [PM93]

The invention further provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 61 [PM9] and wherein the second bacteriophage is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 66 [PM93]

Thus, in some embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 95% identity with the genome as provided in SEQ ID NO: 61 [PM9] and wherein the second bacteriophage is a bacteriophage having a genome with at least 95% identity with the genome as provided in SEQ ID NO: 66 [PM93]

In some preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 97% identity with the genome as provided in SEQ ID NO: 61 [PM9] and wherein the second bacteriophage is a bacteriophage having a genome with at least 97% identity with the genome as provided in SEQ ID NO: 66 [PM93]

In some more preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 98% identity with the genome as provided in SEQ ID NO: 61 [PM9] and wherein the second bacteriophage is a bacteriophage having a genome with at least 98% identity with the genome as provided in SEQ ID NO: 66 [PM93]

In some even more preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 99% identity with the genome as provided in SEQ ID NO: 61 [PM9] and wherein the second bacteriophage is a bacteriophage having a genome with at least 99% identity with the genome as provided in SEQ ID NO: 66 [PM93]

In some most preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 61 [PM9] and wherein the second bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 66 [PM93]

The invention further provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 73 [PM23] and wherein the second bacteriophage is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 65 [PM56]

Thus, in some embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 95% identity with the genome as provided in SEQ ID NO: 73 [PM23] and wherein the second bacteriophage is a bacteriophage having a genome with at least 95% identity with the genome as provided in SEQ ID NO: 65 [PM56]

In some preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 97% identity with the genome as provided in SEQ ID NO: 73 [PM23] and wherein the second bacteriophage is a bacteriophage having a genome with at least 97% identity with the genome as provided in SEQ ID NO: 65 [PM56]

In some more preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 98% identity with the genome as provided in SEQ ID NO: 73 [PM23] and wherein the second bacteriophage is a bacteriophage having a genome with at least 98% identity with the genome as provided in SEQ ID NO: 65 [PM56]

In some even more preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 99% identity with the genome as provided in SEQ ID NO: 73 [PM23] and wherein the second bacteriophage is a bacteriophage having a genome with at least 99% identity with the genome as provided in SEQ ID NO: 65 [PM56]

In some most preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 73 [PM23] and wherein the second bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 65 [PM56]

The invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 68 [PM28] and wherein the second bacteriophage is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 74 [PM94]

Thus, in some embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 95% identity with the genome as provided in SEQ ID NO: 68 [PM28] and wherein the second bacteriophage is a bacteriophage having a genome with at least 95% identity with the genome as provided in SEQ ID NO: 74 [PM94]

In some preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 97% identity with the genome as provided in SEQ ID NO: 68 [PM28] and wherein the second bacteriophage is a bacteriophage having a genome with at least 97% identity with the genome as provided in SEQ ID NO: 74 [PM94]

In some more preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 98% identity with the genome as provided in SEQ ID NO: 68 [PM28] and wherein the second bacteriophage is a bacteriophage having a genome with at least 98% identity with the genome as provided in SEQ ID NO: 74 [PM94]

In some even more preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 99% identity with the genome as provided in SEQ ID NO: 68 [PM28] and wherein the second bacteriophage is a bacteriophage having a genome with at least 99% identity with the genome as provided in SEQ ID NO: 74 [PM94]

In some most preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 68 [PM28] and wherein the second bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 74 [PM94]

The invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is an ancestor bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 60 [PM32] and wherein the second bacteriophage is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 64 [02]

Thus, in some embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 95% identity with the genome as provided in SEQ ID NO: 60 [PM32] and wherein the second bacteriophage is a bacteriophage having a genome with at least 95% identity with the genome as provided in SEQ ID NO: 64 [02]

In some preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 97% identity with the genome as provided in SEQ ID NO: 60 [PM32] and wherein the second bacteriophage is a bacteriophage having a genome with at least 97% identity with the genome as provided in SEQ ID NO: 64 [02]

In some more preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 98% identity with the genome as provided in SEQ ID NO: 60 [PM32] and wherein the second bacteriophage is a bacteriophage having a genome with at least 98% identity with the genome as provided in SEQ ID NO: 64 [02]

In some even more preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 99% identity with the genome as provided in SEQ ID NO: 60 [PM32] and wherein the second bacteriophage is a bacteriophage having a genome with at least 99% identity with the genome as provided in SEQ ID NO: 64 [02]

In some most preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 60 [PM32] and wherein the second bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 64 [02]

The invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 72 [PM7] and wherein the second bacteriophage is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 65 [PM56]

Thus, in some embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 95% identity with the genome as provided in SEQ ID NO: 72 [PM7] and wherein the second bacteriophage is a bacteriophage having a genome with at least 95% identity with the genome as provided in SEQ ID NO: 65 [PM56] In some preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 97% identity with the genome as provided in SEQ ID NO: 72 [PM7] and wherein the second bacteriophage is a bacteriophage having a genome with at least 97% identity with the genome as provided in SEQ ID NO: 65 [PM56]

In some more preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 98% identity with the genome as provided in SEQ ID NO: 72 [PM7] and wherein the second bacteriophage is a bacteriophage having a genome with at least 98% identity with the genome as provided in SEQ ID NO: 65 [PM56]

In some even more preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 99% identity with the genome as provided in SEQ ID NO: 72 [PM7] and wherein the second bacteriophage is a bacteriophage having a genome with at least 99% identity with the genome as provided in SEQ ID NO: 65 [PM56]

In some most preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 72 [PM7] and wherein the second bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 65 [PM56]

The invention further provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 60 [PM32] and wherein the second bacteriophage is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 65 [PM56]

Thus, in some embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 95% identity with the genome as provided in SEQ ID NO: 60 [PM32] and wherein the second bacteriophage is a bacteriophage having a genome with at least 95% identity with the genome as provided in SEQ ID NO: 65 [PM56]

In some preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 97% identity with the genome as provided in SEQ ID NO: 60 [PM32] and wherein the second bacteriophage is a bacteriophage having a genome with at least 97% identity with the genome as provided in SEQ ID NO: 65 [PM56]

In some more preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 98% identity with the genome as provided in SEQ ID NO: 60 [PM32] and wherein the second bacteriophage is a bacteriophage having a genome with at least 98% identity with the genome as provided in SEQ ID NO: 65 [PM56] In some even more preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 99% identity with the genome as provided in SEQ ID NO: 60 [PM32] and wherein the second bacteriophage is a bacteriophage having a genome with at least 99% identity with the genome as provided in SEQ ID NO: 65 [PM56]

In some most preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 60 [PM32] and wherein the second bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 65 [PM56]

The invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 62 [PM22] and wherein the second bacteriophage is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 64 [02]

Thus, in some embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 95% identity with the genome as provided in SEQ ID NO: 62 [PM22] and wherein the second bacteriophage is a bacteriophage having a genome with at least 95% identity with the genome as provided in SEQ ID NO: 64 [02] In some preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 97% identity with the genome as provided in SEQ ID NO: 62 [PM22] and wherein the second bacteriophage is a bacteriophage having a genome with at least 97% identity with the genome as provided in SEQ ID NO: 64 [02]

In some more preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 98% identity with the genome as provided in SEQ ID NO: 62 [PM22] and wherein the second bacteriophage is a bacteriophage having a genome with at least 98% identity with the genome as provided in SEQ ID NO: 64 [02]

In some even more preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 99% identity with the genome as provided in SEQ ID NO: 62 [PM22] and wherein the second bacteriophage is a bacteriophage having a genome with at least 99% identity with the genome as provided in SEQ ID NO: 64 [02]

In some most preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 62 [PM22] and wherein the second bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 64 [02]

The invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 73 [PM23] and wherein the second bacteriophage is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 66 [PM93]

Thus, in some embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 95% identity with the genome as provided in SEQ ID NO: 73 [PM23] and wherein the second bacteriophage is a bacteriophage having a genome with at least 95% identity with the genome as provided in SEQ ID NO: 66 [PM93]

In some preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 97% identity with the genome as provided in SEQ ID NO: 73 [PM23] and wherein the second bacteriophage is a bacteriophage having a genome with at least 97% identity with the genome as provided in SEQ ID NO: 66 [PM93]

In some more preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 98% identity with the genome as provided in SEQ ID NO: 73 [PM23] and wherein the second bacteriophage is a bacteriophage having a genome with at least 98% identity with the genome as provided in SEQ ID NO: 66 [PM93] In some even more preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 99% identity with the genome as provided in SEQ ID NO: 73 [PM23] and wherein the second bacteriophage is a bacteriophage having a genome with at least 99% identity with the genome as provided in SEQ ID NO: 66 [PM93]

In some most preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 73 [PM23] and wherein the second bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 66 [PM93]

The invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 4 [PM4] and wherein the second bacteriophage is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 65 [PM56]

Thus, in some embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 95% identity with the genome as provided in SEQ ID NO: 4 [PM4] and wherein the second bacteriophage is a bacteriophage having a genome with at least 95% identity with the genome as provided in SEQ ID NO: 65 [PM56] In some preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 97% identity with the genome as provided in SEQ ID NO: 4 [PM4] and wherein the second bacteriophage is a bacteriophage having a genome with at least 97% identity with the genome as provided in SEQ ID NO: 65 [PM56]

In some more preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 98% identity with the genome as provided in SEQ ID NO: 4 [PM4] and wherein the second bacteriophage is a bacteriophage having a genome with at least 98% identity with the genome as provided in SEQ ID NO: 65 [PM56]

In some even more preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 99% identity with the genome as provided in SEQ ID NO: 4 [PM4] and wherein the second bacteriophage is a bacteriophage having a genome with at least 99% identity with the genome as provided in SEQ ID NO: 65 [PM56]

In some most preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 4 [PM4] and wherein the second bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 65 [PM56]

The invention further provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 72 [PM7] and wherein the second bacteriophage is a bacteriophage having a genome with at least 95% identity (preferably at least 96% identity, more preferably at least 97% identity, even more preferably at least 98%, even more preferably at least 99% identity, even more preferably at least 99.5% identity, and most preferably at least 99.7% identity) with the genome as provided in SEQ ID NO: 66 [PM93]

Thus, in some embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 95% identity with the genome as provided in SEQ ID NO: 72 [PM7] and wherein the second bacteriophage is a bacteriophage having a genome with at least 95% identity with the genome as provided in SEQ ID NO: 66 [PM93]

In some preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 97% identity with the genome as provided in SEQ ID NO: 72 [PM7] and wherein the second bacteriophage is a bacteriophage having a genome with at least 97% identity with the genome as provided in SEQ ID NO: 66 [PM93]

In some more preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 98% identity with the genome as provided in SEQ ID NO: 72 [PM7] and wherein the second bacteriophage is a bacteriophage having a genome with at least 98% identity with the genome as provided in SEQ ID NO: 66 [PM93] In some even more preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is a bacteriophage having a genome with at least 99% identity with the genome as provided in SEQ ID NO: 72 [PM7] and wherein the second bacteriophage is a bacteriophage having a genome with at least 99% identity with the genome as provided in SEQ ID NO: 66 [PM93]

In some most preferred embodiments, the invention provides a composition comprising (i) a first bacteriophage, which is a mosaic bacteriophage, and (ii) a second bacteriophage, which is a bred bacteriophage, wherein the first bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 72 [PM7] and wherein the second bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 66 [PM93]

A skilled person of the art readily understand that the present invention covers variants without altering the gist of the invention. As used herein, the term “variant” in the context of nucleotide sequences refers to a nucleotide sequence that comprises or consists of a nucleotide sequence having a sequence identity of at least 75%, 80%, 85%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% with a reference nucleic acid sequence over the entire length of said reference nucleic acid sequence. A variant may preferably be selected that maintains one or more functions of the reference nucleic acid sequence. For example, a “genome fragment variant” encoding a protein may differ by one or more nucleotides to the genome fragment from which it is derived but still retaining the function of the encoded protein. Where the encoded protein is only hypothetical, the genome fragment may differ by one or more nucleotides to the genome fragment from which it is derived but still retaining the identical amino acid sequence encoded by said the nucleotide sequence of said genome fragment (e.g. due to the redundancy of the genetic code). Likewise, the term “variant” in the context of bacteriophage refers to a bacteriophage having at least 75%, 80%, 85%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity across its entire genome when compared to the bacteriophage from which it is derived. A variant bacteriophage may be selected that maintains one or more function(s) of the reference nucleic acid sequence. For example, a “variant bacteriophage” may exhibit at least one biological activity, e.g. antimicrobial or antibacterial activity (e.g. lytic killing activity), of the bacteriophage from which it is derived. In a preferred embodiment, a variant bacteriophage may differ genetically by one or more nucleotides to the bacteriophage from which it is derived but still remaining the ability to infect and lyse, more preferably eradicate, the same target bacterial strains as the bacteriophage from which it is derived, thereby having the same host-range. Such a bacteriophage with similar structure and similar antibacterial activity, particularly similar host-range of one of the bacteriophages described herein may also be referred to as “functional variants”.

Accordingly, the invention further provides a composition comprising the first bacteriophage {i.e. a mosaic bacteriophage) as described herein or any functional variants thereof and the second bacteriophage as described herein or any functional variants thereof.

In some preferred embodiments, a composition comprising the first bacteriophage as described herein in combination with the second bacteriophage as described herein show synergistic effects on elimination of bacterial strains. This means that the combination of the first and the second bacteriophage has the surprising capability to lyse at least one bacterial strain that is phage insensitive to the first bacteriophage and the second bacteriophage, individually. Accordingly, the bacteriophage compositions / bacteriophage cocktails of the present invention may be characterized by an antibacterial activity which are not merely the sum of the antibacterial activities of each of the first and second bacteriophage individually, but which is over-additive, which can also be defined as synergism, and is therefore not predictable.

An essential feature of the composition / cocktail of the invention is the ability to lyse bacterial strains. Bacteria that may be lysed by the herein provided bacteriophage cocktails / compositions (i.e. targeted bacteria) include any bacterial pathogen that poses a health threat to a subject. Examples of such bacterial pathogen that poses a health threat to a subject are multidrug resistant bacterial strains. As understood herein, the terms, “multidrug resistant”, “multiple drug resistant”, “multiple drug resistance” (MDR) and like terms may be used interchangeably herein, and are familiar to one of skill in the art, i.e., a multiple drug resistant bacterium is an organism that demonstrates resistance to multiple antibacterial drugs, e.g., antibiotics. In a particularly preferred embodiment, bacterial strains to be lysed by the bacteriophage compositions / bacteriophage cocktails of the invention include Staphylococcus bacterial strains, more preferably Staphylococcus aureus bacterial strains. In the context of the present invention, Staphylococcus phages may belong to the viral families Herelleviridae. Staphylococcus phages of the Herelleviridae family include phage K, Gl, Twort, A5W, Sb-1, ISP, SA5, GH15, JD007, SA11, vB SauM Remus, vB SauM Romulus, S25-3, S25-4, philPLA-RODI, philPLA-CIC, phiSA012, Teaml, P108, MCE-2014, 812, SA1, StaphylN, MSA6, 676Z, P4W, and Fi200w (Cui et al., 2017 and Yandersteegen et al., 2013). The terms “ Staphylococcus aureus“ and “ S . aureus ” are used interchangeably herein for the bacterial species which belongs to the bacterial genus of Staphylococcus. The bacterial species S. aureus encompasses “Methicillin-susceptible Staphylococcus aureus ” (MSSA) strain, “Methicillin-resistant Staphylococcus aureus ” (MRSA) strain, i.e. Staphylococcus aureus that is resistant to certain antibiotics, in particular methicillin. MRSA may also be resistant to other antibiotics including but not limited to oxacillin, penicillin, and amoxicillin, and also encompasses “multidrug-resistant S. aureus ” (MDRSA) strain, i.e. Staphylococcus aureus that is resistant to at least oxacillin, lincosamides, erythromycin, and chloramphenicol. The targeted Staphylococcus aureus strain may be resistant to vancomycin, or rifampicin.

The composition of the present invention is highly advantageous for use as an antimicrobial agent in view of its very potent antimicrobial activity, particularly against Staphylococcus. As shown in the appended Examples, the composition of the present invention lyses up to 88% of the bacterial strains of a panel of bacterial strains, wherein said panel is composed such that the frequency of the clonal complexes in the panel reflects the natural epidemiology of S. aureus in human infections as found in several literature cases (Arias et al., 2017; Kanjilal et al.; 2018; Luedicke et al.; 2010; Rasmussen et al.; 2013). Said panel may preferably comprise at least 70, (preferably at least 80, more preferably at least 90, even more preferably at least 100, and most preferably 110) bacterial strains and may further comprise more than 10 (preferably more than 15, more preferably more than 20, most preferably 25) of the clonal complexes which most frequently cause infection in humans. An example of such a panel of controlled diversity that may be used in the context of the present invention is provided in Table 1. Alternatively, any other panel could have been used which consists of S. aureus strains of a distribution of clonal complexes similar to what is found in human infections. A person skilled in the art is readily in the position to obtain such useful panel and corresponding panel and/or bacterial strains collection, for example from the national reference centers for antimicrobial resistance, or public collections of microorganisms e.g. FDA / CDC (Food and Drug Administration / Centers for Disease Control and Prevention, part of the U.S. Department of Health & Human Services, https://www.cdc.gov/drugresistance/resistance-bank/index.html), DSMZ (Deutsche Sammlung von Mikroorgansimen und Zellkulturen GmbH, in Braunschweig/Germany).

As used herein, the term “controlled diversity” refers to a property of a panel of bacterial strains, by which the number of bacterial strains belonging to each sub-group of species used reflects the natural frequency of that sub-group in human infections. For S. aureus, this means that the number of strains of each clonal complex represented in the panel, or the number of strains with MRSA/MSSA status, should reflect the natural frequency of that clonal complex or the natural frequency of MRSA/MSSA found in epidemiological studies in humans (Arias et al., 2017, Kanjilal et al.; 2018, Luedicke et al.; 2010, Rasmussen et al.; 2013).

As used herein, the term “clonal complex” with reference to S. aureus means a group of bacterial strains with common ancestral relation as defined in public databases such as PubMLST (https://pubmlst.org/saureus/). Specifically, clonal complexes are defined as those sequence types (STs) that match the central genotype (ST) at four or more loci unless they more closely match another central genotype (Jolley et al., 2018).

As used herein, “clonal complexes which most frequently cause infection in humans” include, but are not limited to, CC45, CC30, CC8, CC22, CC5, CC398, CC25, CC15, CC12, CC101, CC1, CC239, CC6, CC80, CC9, CC88, CC121, CC49, CC59, CC60, CC7, CC772, CC96, CC97 CC395.

Thus, in one embodiment, the composition of the invention lyses one or more bacterial strains, preferably Staphylococcus strains, more preferably Staphylococcus aureus strains. Preferably, said composition lyses at least 50%, at least 70%, at least 75%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, or at least 87% of a panel of Staphylococcus aureus strains. A panel of bacterial strains may be easily composed by a skilled person of the art. One example of possible bacterial strains panel is the panel of 110 A aureus strains of Table 1. Thus, in one embodiment, the composition of the invention lyses at least 50% (preferably at least 70%, more preferably at least 75%, even more preferably at least 80%, even more preferably at least 82%, even more preferably at least 84%, even more preferably 86%, and most preferably at least 88%) of the Staphylococcus aureus strains of Table 1. In one further embodiment, the composition of the invention additionally or alternatively lyses at least 5 (preferably at least 10, more preferably at least 15, even more preferably at least 16, even more preferably at least 17, even more preferably at least 18, even more preferably 19, even more preferably 20, even more preferably 21, and most preferably at least 22) different Staphylococcus aureus strains clonal complexes.

In one embodiment, the composition of the invention reduces preformed biofrlms of one or more bacterial strains, preferably Staphylococcus strains, more preferably Staphylococcus aureus strains.

In one preferred embodiment, said composition reduces preformed biofilms by 90% or more on at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of a panel of Staphylococcus aureus strains. A panel of bacterial strains may be easily composed by a skilled person of the art. One example of possible bacterial strains panel is the panel of 10 A aureus strains of Table 8. Thus, in one embodiment, the composition of the invention reduces preformed biofilms by 90% or more on at least 50% (preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably 95%, and most preferably 100%) of the Staphylococcus aureus strains of Table 8. In one further embodiment, the composition of the invention additionally or alternatively reduces preformed biofilms by 90% or more at least 5 (preferably at least 6, more preferably at least 7, even more preferably at least 8, even more preferably at least 9, and most preferably at least 10) different Staphylococcus aureus strains clonal complexes.

In another preferred embodiment, said composition reduces preformed biofilms by 99% or more on at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% of a panel of Staphylococcus aureus strains. A panel of bacterial strains may be easily composed by a skilled person of the art. One example of possible bacterial strains panel is the panel of 10 S. aureus strains of Table 8. Thus, in one embodiment, the composition of the invention reduces preformed biofrlms by 99% or more on at least 10% (preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, and most preferably 60%) of the Staphylococcus aureus strains of Table 8. In one further embodiment, the composition of the invention additionally or alternatively reduces preformed biofilms by 99% or more at least 1 (preferably at least 2, more preferably at least 3, even more preferably at least 4, even more preferably at least 5, and most preferably at least 6) different Staphylococcus aureus strains clonal complexes. In another preferred embodiment, the composition of the invention synergistically reduces preformed biofilms of a particular bacterial strain, preferably a Staphylococcus strain, more preferably a Staphylococcus aureus strain.

Because of the high antibacterial activity of the composition of the invention, and in particular its particularly broad host-range and potent biofilm activity, said composition may be used in various medical and non-medical methods, in particular those aiming to inhibit growth of the targeted bacteria.

Accordingly, in another aspect, the invention provides the use of the composition of the present invention in a non-medical method of killing, eradicating and/or inhibiting the growth of bacteria and/or reducing biofilms formed by bacteria, preferably Staphylococcus , more preferably Staphylococcus aureus. Said use may be on a surface, on a crop or on a food product. Preferably said use is on a surface, such as, e.g., the skin of a mammal, equipment, medical equipment, prostheses, implant, bedding, furniture, walls, floors, or combinations thereof. Thus, in one embodiment, the composition of the invention may be used in a non-medical method of disinfecting or sterilizing an implant, before the implant is introduced into a patient in need thereof.

Further provided is a non-medical method of killing, eradicating and/or inhibiting the growth of bacteria and/or reducing biofilms formed by bacteria comprising applying the composition of the invention. Said bacteria may be Staphylococcus, preferably Staphylococcus aureus. Said composition may be to be applied to a food product, a crop or a surface. Preferably, said composition is to be applied on a surface. Said surface may be the skin of a mammal, equipment, medical equipment, prostheses, implant, bedding, furniture, walls, floors, or combinations thereof.

As used herein, the term “composition” encompasses “phage mixtures” as disclosed herein which include, but are not limited to, compositions a comprising, or alternatively consisting essentially of, or alternatively consisting of, a plurality of the same purified bacteriophage or a plurality of different purified bacteriophages. As used herein, the term “purified” refers to a preparation that is substantially free of unwanted substances in the composition, including, but not limited to biological materials e.g., other bacteriophages, whole bacteria, bacterial components, toxins such as for example, endotoxins, nucleic acids, proteins, carbohydrates, lipids, or subcellular organelles, and/or other impurities, e.g., metals or other trace elements, that might interfere with the effectiveness of the mixture. The term “purified” with respect to a bacteriophage means that the phage has been measurably increased in concentration by any purification process, including but not limited to, isolation from the environment or culture, e.g., isolation from culture following propagation and/or amplification, centrifugation, etc., thereby partially, substantially, nearly completely, or completely removing impurities, such as host cell components. One of skill in the art will appreciate the amount of purification necessary for a given use. For example, an isolated bacteriophage meant for use in therapeutic compositions intended for administration to humans ordinarily must be of high purity in accordance with regulatory standards and good manufacturing processes. The term “purified” as used herein may also indicate that the bacteriophage is removed from manufacturing host bacteria. In particular, a purified bacteriophage has production impurities, such as bacterial components, removed from its manufacturing or production environment. Bacterial components include but are not limited to bacterial host proteins, lipids, and/or bacterial endotoxin. The term “purified” may also refer to genetic purification in which the strain of bacteriophage is genetically homogenous.

The composition of the invention may also be a pharmaceutical composition. Thus, in one embodiment, the composition of the invention is pharmaceutical composition. The invention also provides a pharmaceutical composition comprising the composition of the invention and optionally a pharmaceutically acceptable carrier.

A “pharmaceutical composition” as used herein is familiar to one of skill in the art and typically comprises active pharmaceutical ingredients formulated in combination with inactive ingredients selected from a variety of conventional pharmaceutically acceptable excipients, carriers, buffers, and/or diluents. The term “pharmaceutically acceptable” is used to refer to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. Examples of pharmaceutically acceptable excipients, carriers, buffers, and/or diluents are familiar to one of skill in the art and can be found, e.g., in Remington ’s Pharmaceutical Sciences (latest edition), Mack Publishing Company, Easton, Pa. For example, pharmaceutically acceptable excipients include, but are not limited to, wetting or emulsifying agents, pH buffering substances, binders, stabilizers, preservatives, bulking agents, absorbents, disinfectants, detergents, sugar alcohols, gelling or viscosity enhancing additives, flavoring agents, and colors. Pharmaceutically acceptable carriers include macromolecules such as proteins, polysaccharides, polyactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, trehalose, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Pharmaceutically acceptable diluents include, but are not limited to, water, saline, and glycerol.

The pharmaceutical composition of the present invention may be for use in phage therapy.

As used herein, “phage therapy” refers to any therapy to treat a bacterial infection or bacterial caused disease, which may involve the administration to a subject requiring treatment (e.g. a patient) of one or more therapeutic composition that can be used to infect, kill or inhibit the growth of a bacterium, which comprises one or more viable phage as an antibacterial agent (e.g. a composition comprising one phage strain or a phage “cocktail”) and which may further comprise, or otherwise be administered in combination with a further therapeutic composition comprising, one or more antibiotics, one or more bactericides, and/or one or more other therapeutics molecules such as small molecules or biologies that have bactericidal activity. Where more than one therapeutic composition is involved in the phage therapy then the compositions may have a different host range (e.g. one may have a broad host range and one may have a narrow host range, and/or one or more of the compositions may act synergistically with one another). Further, as understood by one skill in the art, the therapeutic composition(s) used in a phage therapy will also typically comprise a range of inactive ingredients selected from a variety of conventional pharmaceutically acceptable excipients, carriers, buffers, and/or diluents.

Accordingly, the invention provides the pharmaceutical composition as described herein for use in the treatment and/or prevention of a bacterial infection, preferably a Staphylococcus bacterial infection, more preferably a Staphylococcus aureus bacterial infection.

As used herein, the terms “treat”, “treatment” and “treating” refer to obtaining a therapeutic benefit in a subject receiving a pharmaceutical composition. With respect to achieving a therapeutic benefit, the object is to eliminate, lessen, decrease the severity of, ameliorate, or slow the progression of the symptoms or underlying cause (e.g., bacterial infection) associated with the pathological condition or disorder. As used herein, the terms “prevent”, “prevention” and “preventing” refer to obtaining a prophylactic benefit in a subject receiving a pharmaceutical composition. With respect to achieving a prophylactic benefit, the object is to delay or prevent the symptoms or underlying cause (e.g. bacterial infection) associated with the pathological condition or disorder. A “prophylactic effective amount” refers to that amount of prophylactic agent, such as a bacteriophage composition of the invention, sufficient to achieve at least one prophylactic benefit in a subject receiving the pharmaceutical composition.

The pharmaceutical compositions of the invention may be administered topically (e.g., in the form of a lotion, solution, cream, ointment, or dusting powder), or epi- or transdermally (e.g., by use of a skin patch). Additionally or alternatively, the pharmaceutical compositions of the invention can be administered by inhalation, in the form of a suppository or pessary, orally (e.g., as a tablet, which may contain excipients such as starch or lactose, as a capsule, ovule, elixir, solution, or suspension, each optionally containing flavoring, coloring agents, and/or excipients), or they can be injected parenterally (e.g., intravenously, intramuscularly or subcutaneously). For parenteral administration, the compositions may be used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration, the compositions may be administered in the form of tablets or lozenges, which can be formulated in a conventional manner. In a preferred embodiment, the pharmaceutical composition of the present invention is formulated for topical administration, either as a single agent, or in combination with other therapeutic and/or prophylactic agents, as described herein or known in the art.

In some more preferred embodiments, the composition or pharmaceutical composition of the invention (comprising one or more mosaic bacteriophages of the invention) is to be administered subcutaneously or orally, in a form of a bolus, continuous administration, infusion or the like. Additionally, the composition or pharmaceutical composition of the invention (comprising one or more mosaic bacteriophages of the invention) can be administered via any suitable modes / means / methods known to the skilled person, such as, e.g., as described in WO2010/033546), W02013/141730), and US20170065649).

The pharmaceutical composition of the present invention may also be combined with one or more non-phage therapeutic and/or prophylactic agents, useful for the treatment and/or prevention of bacterial infections, as described herein and/or known in the art (e.g. one or more antibiotic agents, and/or a thrombolytic agent). Other therapeutic and/or prophylactic agents that may be used in combination with the composition of the invention include, but are not limited to, antibiotic agents, thrombolytic agents, anti-inflammatory agents, antiviral agents, local anesthetic agents, growth factors, and corticosteroids. Thus, in some embodiments, the pharmaceutical composition of the invention is to be co-administered with an antibiotic agent.

Standard antibiotics that may be used with pharmaceutical compositions comprising the phage cocktail of the invention include, but are not limited to, amikacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, rhodostreptomycin, streptomycin, tobramycin, apramycin, rifamycin, naphthomycin, mupirocin, geldanamycin, ansamitocin, carbacephems, imipenem, meropenem, ertapenem, faropenem, doripenem, panipenem/betamipron, biapenem, PZ-601, cephalosporins, cefacetrile, cefadroxil, cefalexin, cefaloglycin, cefalonium, cefaloridine, cefalotin, cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, ceffadine, cefroxadine, ceftezole, cefaclor, cefonicid, cefprozil, cefuroxime, cefuzonam, ceffnetazole, cefotetan, cefoxitin, cefcapene, cefdaloxime, cefdinir, cefditoren, cefetamet, cefixime, cefmenoxime, cefteram, ceftibuten, ceftiofur, ceftiolene, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime latamoxef, cefclidine, cefepime, cefluprenam, cefoselis, cefozopran, cefpirome, cefquinome, flomoxef. ceftobiprole, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, aztreonam, pencillin and penicillin derivatives, actinomycin, bacitracin, colistin, polymyxin B, cinoxacin, flumequine, nalidixic acid, oxolinic acid, piromidic acid, pipemidic acid, rosoxacin, ciprofloxacin, enoxacin, fleroxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin, gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin, pazufloxacin, Sparfloxacin, temafloxacin, tosufloxacin, clinafloxacin, garenoxacin, gemifloxacin, stifloxacin, trovalfloxacin, prulifloxacin, acetazolamide, benzolamide, bumetanide, celecoxib, chlorthalidone, clopamide, dichlorphenamide, dorzolamide, ethoxyzolamide, furosemide, hydrochlorothiazide, indapamide, mafendide, mefruside, metolazone, probenecid, sulfacetamide, sulfadimethoxine, sulfadoxine, sulfanilamides, sulfamethoxazole, sulfasalazine, sultiame, sumatriptan, xipamide, tetracycline, chlortetracycline, oxytetracycline, doxycycline, lymecycline, meclocycline, methacycline, minocycline, rolitetracycline, methicillin, nafcillin, oxacilin, cloxacillin, vancomycin, teicoplanin, clindamycin, co-trimoxazole, flucloxacillin, dicloxacillin, ampicillin, amoxicillin and any combination thereof in amounts that are effective to additively or synergistically enhance the therapeutic and/or prophylactic effect of the composition of the invention for a given infection.

In some more preferred embodiments, the pharmaceutical composition of the invention is to be co-administered with a thrombolytic agent. Standard thrombolytic agents that may be used with pharmaceutical compositions comprising one or mosaic bacteriophage(s) of the invention include, but are not limited to, tissue plasminogen activator (tPA), urokinase- type plasminogen activator (uPA), Streptokinase, Staphylokinase Anistreplase, Reteplase, Tenecteplase, Urokinase, Alteplase, variants and analogues thereof, functional fragments thereof or combinations thereof.

Further provided is the use of the composition of the invention or the pharmaceutical composition of the invention for the manufacture of a medicament for the treatment of a bacterial infection, preferably a Staphylococcus bacterial infection, more preferably a Staphylococcus aureus bacterial infection.

The present invention further provides a method of treating and/or preventing a bacterial infection, preferably a Staphylococcus bacterial infection, more preferably a Staphylococcus aureus bacterial infection, in a subject in need thereof comprising administering to said subject a therapeutically effective amount of the composition of the invention or the pharmaceutical composition of the invention.

As understood herein, an “effective amount” of a pharmaceutical composition of the instant invention refers to an amount of the composition suitable to elicit a therapeutically beneficial response in the subject, e.g., eradicating a bacterial pathogen in the subject. Such response may include e.g., preventing, ameliorating, treating, inhibiting, and/or reducing one or more pathological conditions associated with a bacterial infection.

The term “therapeutically effective amount” as used herein, pertains to that amount of an active compound, or a combination, material, antigen, composition or dosage form comprising an active compound, which is effective for producing some desired effect, commensurate with a reasonable benefit/risk ratio. As used herein, a “subject” is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. In other preferred embodiments, the “subject” is a rodent (e.g., a guinea pig, a hamster, a rat, a mouse), murine (e.g., a mouse), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), a primate, simian (e.g., a monkey or ape), a monkey (e.g., marmoset, baboon), or an ape (e.g., gorilla, chimpanzee, orangutan, gibbon). In other embodiments, non-human mammals, especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g., murine, primate, porcine, canine, or rabbit animals) may be employed. Preferably, a “subject” encompasses any organisms, e.g., any animal or human, that may be suffering from a bacterial infection, particularly an infection caused by a multiple drug resistant bacterium such as Staphyloccocus.

As understood herein, a “subject in need thereof’ includes any human or animal suffering from a bacterial infection, including but not limited to a multiple drug resistant bacterial infection. Indeed, while it is contemplated herein that the methods may be used to target a specific pathogenic species, the method can also be used against essentially all human and/or animal bacterial pathogens, including but not limited to multiple drug resistant bacterial pathogens. Thus, in a particular embodiment, by employing the methods of the present invention, one of skill in the art can design and create personalized phage mixtures against many different clinically relevant bacterial pathogens, including multiple drug resistant (MDR) bacterial pathogens.

Modes of administration described herein and/or known in the art may be used to deliver desired dosages of the phage cocktails of the invention and in accordance with suitable dosage regimens. Dosages and dosage regimens may vary depending on the particular formulation, route of administration, condition being treated, and other factors. Animal experiments can provide reliable guidance for the determination of effective doses in human therapy, e.g., as within the skill of the ordinary physician. Interspecies scaling of effective doses can be performed by one of ordinary skill in the art following the principles described, e.g., by Mordend, J. et al. “The use of interspecies scaling in toxicokinetics” in Toxicokinetics and New Drug Development, Yacogi, A et al., Eds., Pergamon Press, New York 1989, pp 42-96.

The topical mode of delivery may include a smear, a spray, a bandage, a wound dressing, a time-release patch, a liquid-absorbed wipe, and combinations thereof. Thus, further provided is a bandage or wound dressing comprising the composition or the pharmaceutical composition of the invention.

The invention also provides a kit comprising the composition of the invention or the pharmaceutical composition of the invention. Also provided is a kit comprising (a) a composition, a pharmaceutical composition or bandage herein provided and (b) instructions for use of same (e.g. in medicine).

The bred (mosaic) bacteriophages described herein may be obtained /generated and/or are obtainable by the inventive means and methods described herein, in particular, by the “PhagoMed-Modified Appelmans Protocol” or “PMAP” {i.e. a bacteriophage breeding method/protocol).

The invention further provides a breeding method for generating a bred (mosaic or non-mosaic) bacteriophage comprising the step of propagating a phage mixture on each bacterial strains of a panel of bacterial strains, wherein said phage mixture comprises one or more specific ancestor bacteriophage(s).

As used herein, the term “propagating” or “propagation” means infection of a bacterial strain with a bacteriophage or bacteriophage mixture in such a way that the number of plaque-forming viral particles increases compared to before the infection. In other words, the bacteriophages grow within the bacteria. As they grow, the bacteriophages may end up killing the bacterial host as the next generation of bacteriophages is released. In such cases, it means that the bacterial host cell is lysed, which is the last step in the propagation cycle of the phage. Propagating one or more bacteriophage(s) on a particular bacterial cell can be done in broth suspension, or by the double agar layer (DAL) method as is known to a person skilled in the art. Corresponding details are also provided herein and in the appended examples. For example, particular preferred bacterial strains for propagating are known to the skilled artisan. A particular preferred bacterial strain for propagating, without being limiting, may be the strain CC25-MSSA (124605). In accordance, with the present invention, all the bacteriophages described herein, namely the ancestor bacteriophages and/or bred (mosaic) bacteriophages have the ability to propagate on the strain CC25-MSSA (124605), even if the stage of “lysis” may not be achieved for all of them. In other words, even if some of the bacteriophages described herein cannot eradicate the strain CC25-MSSA (124605) (i.e. show no lysis in Fig. 2), in accordance with the present invention, these bacteriophages (as well as all the bacteriophages described herein) can propagate on this strain resulting in a high phage titer (data not shown). Accordingly, the present invention also relates to a method for generating a mosaic bacteriophage by breeding the three ancestor bacteriophages as described herein (in particular and in one embodiment the “05”, “04” and “03”) wherein the step of propagation is carried out and or implemented in or on the strain CC25-MSSA (124605). This particularly useful strain is also part of this invention and has been deposited under the stipulations of the Budapest treaty under the accession number DSM 33467 with the Leibniz-Institute DSMZ (Deutsche Sammlung von Mikroorgansimen und Zellkulturen GmbH, in Braunschweig/Germany) on March 19^th, 2020 (see appended deposit receipt). This strain was, inter alia , used in context of the present means and methods, in particular in the propagation of inventive (mosaic) bacteriophages PM4 and PM32.

Breeding as well as propagation of bacteriophages as performed in the inventive methods may be carried out with bacterial strains from the genus “Staphylococcus in particular the species “Staphylococcus aureus ” / “S. aureus ”. Corresponding strains are readily available for the skilled artisan and may comprise pathogenic bacteria. The person skilled in the art is readily aware of the fact that other strains may be employed in the context of “breeding” / “propagation” of desired bacteriophages. For example, a selection of bacterial strains from the University Hospital Dresden has been used in the appended examples for such purposes. Also useful in this context are for example, “Methicillin-susceptible Staphylococcus aureus ” (MSSA) bacterial strains as defined herein below such as, e.g., the S. aureus strains CC25- MSSA (124605) and CC12-MSSA (A161). Also useful in this context are for example, “Methicillin-resistant Staphylococcus aureus ” (MRSA) bacterial strains as defined herein, such as e.g., the S. aureus strains CC22-MRSA-IY, “Bamim” (A257) and CC22-MRSA-IV, “Barnim” (B311). Also useful in this context are for example, “multidrug-resistant S. aureus ” (MDRSA) bacterial strains as defined herein below. In context of this invention, particularly useful strains for these purposes and to be preferably employed in context of “breeding” and/or “propagating” according to the present invention have been identified, namely (i) the S. aureus strain CC25-MSSA (124605) deposited under the stipulations of the Budapest treaty under the accession number DSM 33467, (ii) the S. aureus strain CC12-MSSA (A161) available in the publicly accessible section of the Leibniz-Institute DSMZ (Braunschweig, Germany) under the strain name Isolate A161 {Stapholococcus aureus subsp: aureus) and under the accession number DSM 111212, (iii) the S. aureus strain CC22-MRSA-IV, “Bamim” (A257) available in the publicly accessible section of the Leibniz-Institute DSMZ (Braunschweig, Germany) under the strain name “Bamim” Isolate A257 ( Stapholococcus aureus subsp: aureus) and under the accession number DSM 111210 and (iv) the S. aureus strain CC22-MRSA-IV, “Barnim” (B311) available in the publicly accessible section of the Leibniz-Institute DSMZ (Braunschweig, Germany) under the strain name “Bamim” Isolate B311 ( Stapholococcus aureus subsp: aureus ) and under the accession number DSM 111211. Each of the above identified strains represent individually a preferred embodiment to be employed in the context of “breeding” or “propagating” according to the present invention.

A phage mixture in the context of the herein provided breeding method may comprise one or more bacteriophages. In order to obtain a bred mosaic bacteriophage, the phage mixture shall contain at least two different bacteriophages. Preferably, the phage mixture comprises one, two, or three ancestor bacteriophages. Alternatively, or additionally, the phage mixture may comprise one or more bred bacteriophage(s).

Thus, in one particular embodiment, the invention provides a breeding method for generating a bred mosaic bacteriophage, comprising the step of propagating a phage mixture on each bacterial strains of a panel of bacterial strains, wherein said phage mixture comprises three ancestor bacteriophages which are respectively the first ancestor bacteriophage as described in any one of the embodiments and aspects herein, the second ancestor bacteriophage as described in any one of the embodiments and aspects herein, and the third ancestor bacteriophage as described in any one of the embodiments and aspects herein. Accordingly, the present invention also provides for means and methods for the generation of inventive mosaic bacteriophages which comprise genome fragments / nucleotide sequences of three ancestor bacteriophages whereby, in a preferred embodiment, two of these ancestor bacteriophages are from the 812/K- like bacteriophages as defined herein and one is from the ISP-like bacteriophages as defined herein. Embodiments described herein above for the inventive composition / bacteriophage cocktails apply in this context mutatis mutandis. Therefore, preferably, said first, said second and said third ancestor bacteriophages are bacteriophages of the Herelleviridae family. Preferred embodiments of 812/K-like bacteriophages and/or ISP-like bacteriophages to be employed as ancestor bacteriophages in the herein provided breeding method are provided also here and above and characterizing structural features are also evident here above and herein below. Again, corresponding embodiments of the mosaic bacteriophage described herein also apply for the method for generating said mosaic bacteriophage. In one preferred aspect of the invention, said first ancestor bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 1 (i.e. “05”), said second ancestor bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 2 (i.e. “04”) and said third ancestor bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 3 (i.e. “03”).

In another particular embodiment, the invention provides a method of generating a bred non mosaic bacteriophage, comprising the step of propagating a phage mixture on each bacterial strains of a panel of bacterial strains, wherein said phage mixture comprises a unique ancestor bacteriophage, wherein said unique ancestor bacteriophage is preferably the bacteriophage having the genome as depicted in SEQ ID NO: 63 or 64.

The panel of bacterial strains used in the herein provided breeding method is composed by selecting strains of different clonal complexes and lineages, resulting in a high bacterial diversity or controlled diversity. In a certain embodiment, the panel of bacterial strains comprises more than 6 of the clonal complexes which most frequently cause infections in humans. Preferably, said panel comprises at least 10 bacterial strains. Said panel may consists of 10 to 50 bacterial strains, preferably 15 to 30, more preferably 17 to 24 bacterial strains. Preferably, the bacterial strains are Staphylococcus strains, more preferably Staphylococcus aureus strains. An example of panel that can be used in the herein provided breeding method is a subpanel of the panel of bacterial strains of Table 1. Examples of such subpanels of bacterial strains are shown in Table 3 herein. Alternatively, any other panel with a controlled diversity of S. aureus strains could have been used. It would be evident to the skilled person to obtain such useful and corresponding panel and/or bacterial strains collection, for example from the national reference centers for antimicrobial resistance, or public collections of microorganisms (e.g. FDA / CDC (Food and Drug Administration / Centers for Disease Control and Prevention, part of the U.S. Department of Health & Human Services, https://www.cdc.gov/drugresistance/resistance-bank/index.html), DSMZ (Deutsche Sammlung von Mikroorgansimen und Zellkulturen GmbH, in Braunschweig/Germany).

Preferably, the breeding method of the invention comprises at least two cycles of propagation, preferably at least 3, more preferably at least 5, even more preferably at least 10 cycles of propagation. Propagation may be continued until the phage mixture shows lytic activity against at least one previously unsusceptible bacterial strain. If no improvement, is visible after 4-6 cycles, the bacterial strains may be exchanged. If an improvement in the host range, i.e. the phage mixture shows lytic activity against one or more previously unsusceptible bacterial strains, the bred bacteriophages are isolated from the phage mixture.

In accordance with the method of breeding presented herein, one cycle of propagation may comprise one round of mixing a phage mixture comprising the combination of the at least three specific (ancestor) bacteriophages as defined herein with a bacterial strain (first round/cycle) or may comprise one round of mixing a phage mixture composed by pooling the lysates of each clear well and the two turbid below, as illustrated in Example 2 and Figure 1, with a bacterial strain (each round/cycle after the first round/cycle). Preferably, one cycle of propagation may comprise one round of mixing a phage mixture comprising the combination of the at least three specific (ancestor) bacteriophages with each bacterial strain of the panel of bacterial strain as described herein (first round/cycle) or may comprise one round of mixing a phage mixture composed by pooling the lysates of each clear well and the two turbid below, with each bacterial strain of the panel of bacterial strain as described herein (each round/cycle after the first round/cycle). Thus, in one embodiment of the methods of the invention, breeding comprises mixing the combination of ancestor bacteriophages with each bacterial strain of a first panel of bacterial strains. That is, breeding comprises a first cycle of propagation, wherein the first cycle of propagation comprises composing a phage mixture comprising the combination of ancestor bacteriophages and mixing said phage mixture with each bacterial strain of a first panel of bacterial strains. In a further embodiment, breeding comprises at least one cycle of propagation, preferably at least 2, more preferably at least 5, even more preferably at least 10 cycles of propagation. That is, breeding preferably further comprises at least a second/subsequent cycle of propagation (i.e. a first cycle of propagation and at least one subsequent cycle of propagation amounting to at least 2 cycles of propagation in total), more preferably at least 4 subsequent cycles of propagation (i.e. at least 5 cycles of propagation in total), even more preferably at least 9 subsequent cycles of propagation (i.e. at least 10 cycles of propagation in total). In particular, each of said second and subsequent cycles of propagation comprises composing a phage mixture by pooling the lysates of each clear well from the preceding cycle, where a clear well indicates that lysis has occurred in said well, and the two turbid wells with a bacteriophage titer 10X and 100X lower than the last clear well, and mixing said phage mixture with each bacterial strain of said first panel of bacterial strains (as, e.g., illustrated in Example 2 and Figure 1). In a preferred embodiment, said bacterial strain(s) is/are Staphylococcus bacterial strain(s), more preferably S. aureus bacterial strain(s). Said combination of the at least three specific (ancestor) bacteriophages may comprise a first and a second ancestor bacteriophage which are 812/K-like bacteriophages and a third ancestor bacteriophage which is an ISP-like bacteriophage. Preferably said first, second and third ancestor bacteriophages may have the SEQ ID NOs: 1, 2 and 3, respectively. As illustrated in the examples, said first, second and third ancestor bacteriophages are referred herein as “05”, “04” and “03”, respectively. The cycle of propagation further may comprise the step of pooling of wells, where lysis of a bacterial strain has been indicated, with at least the two turbid wells (see Example 2 and Figure 1). Subsequently, in the next propagation cycle (subsequent propagation cycle) the combination of the pooled wells may be mixed with again with said bacterial strain.

In one particular embodiment, the step of propagating as described herein is followed by a step of selecting a “bred bacteriophage” or a “bred phage”, for example the ones with the broadest “kinetic host range” on a panel of more than one bacterial strains (e.g., one of the panel of Table 3) or the ones with the ability to lyse a particular targeted bacterial strain (e.g. the strain Bamim A257), wherein the selected bred bacteriophage may be a mosaic bacteriophage (i.e. contains genomic information of at least two, preferably at least three, of the ancestor bacteriophages used as input in the step of breeding). Thus, in accordance with the present invention, a bred bacteriophage may be selected in accordance with the present invention based on its lysis activity (functional selection) and/or based on its genome mosaicity (structural selection). The selection of a bred mosaic bacteriophage may comprise first isolating said mosaic bacteriophage as described below to get monoclonal bacteriophages, then propagating the isolated bacteriophages to generate sufficient quantities for further processing. The selection of a bred mosaic bacteriophage may further comprise the measurement of the kinetic host range and additional analyses like genomic sequencing, based on which the most useful bred mosaic bacteriophages, for example the ones with the broadest host range and with successful mosaic recombination, are selected.

Thus, in one embodiment the step of selecting a bred (mosaic) bacteriophage as described herein comprises the step of “isolating” the bred (mosaic) bacteriophage.

As used herein, the term “isolating” comprises picking of phage plaques and serial re-streaking, to separate a phage from genetically different phages. Specifically, isolating a bacteriophage in the context of the breeding method of the invention can be done by known methods of the art including, but not limited to single plaque isolation. When isolating from environmental or patient samples, the phages are typically first enriched by incubating in a suspension of suitable bacteria in broth for multiple hours. When isolating from a breeding experiment, enrichment is not necessary because the phage titer is already sufficient for plaque isolation. Then, individual plaques are generated with the double layer agar method known to a person skilled in the art. To separate phages in a plaque from potentially contaminating phages, the phages are serially re-streaked at least 3 times, for example by touching the plaque with a sterile loop and streaking the loop on a fresh double agar layer plate with a suitable bacterial strain in the top layer, to generate new plaques.

In some embodiment, the breeding of the invention further comprises measuring the lytic activity of the isolated bred bacteriophage on a panel of bacterial strains. Said panel of bacterial strains may comprise the panel of bacterial strains used for breeding or consist of the panel of bacterial strains used for breeding and at least one additional bacterial strain different from the bacterial strains used for breeding or might be different of the panel of bacterial strains used for breeding, i.e. not comprising any of the bacterial strains used for breeding or comprising only partially the bacterial strains used for breeding.

The breeding method of the invention may further comprise selecting the bred bacteriophage which lyses at least one bacterial strain of said panel of bacterial strains that was phage resistant to the bacteriophages of the phage mixture before breeding, meaning that the host-range has been improved.

Examples of useful bacterial strains in the context of the selection of a bred (mosaic) bacteriophage as described herein comprise but are not limited to CC22-MRSA-IY “Bamim” (e.g. A257, A258 and B311), CC12-MSSA (A161), and CC8-MRSA-IY (2017-067). Such strains are used in this context in the appended examples, see Table 4 appended hereto.

As can be seen in Figure 2, none of the ancestor bacteriophages “03”, “04” and “05” shows the ability to lyse the bacterial strain bacterial strain CC22-MRSA-IV “Barmin” (A257), whereas all the mosaic bacteriophages according to the present invention shows the ability to lyse said bacterial strain. Therefore, this strain is particularly useful in selecting the inventive (mosaic) bacteriophage of the invention. Thus, in one embodiment, the step of selecting the bred (mosaic) bacteriophage as described herein comprises mixing the bred bacteriophage (which has been plaque isolated) with the strain the bacterial strain CC22-MRSA-IV “Barmin” (A257), and selecting the bred (mosaic) bacteriophage when lysis has occurred. Methods for measuring whether a bacteriophage lyses a specific bacterial strain are known to the skilled artisan and include, but are not limited to, the method used in the appended examples. This particularly useful strain to select bred (mosaic) bacteriophages according to the invention is available in the publicly accessible section of the Leibniz-Institute DSMZ (Braunschweig, Germany) under the strain name “Bamim” Isolate A257 ( Stapholococcus aureus subsp: aureus ) and under the accession number DSM 111210.

Thus, in one preferred embodiment, a bred mosaic bacteriophage is selected according to the selecting step described herein when said bred bacteriophage lyses at least one bacterial strain that none of the ancestor bacteriophage lyses. Preferably, said bacterial strain is a S. aureus “Bamim” strain. More preferably, said bacterial strain is the bacterial strain CC22-MRSA-IY “Bamim” (A257). That is, in one more preferred embodiment, the bred mosaic bacteriophage is selected according to the selecting step described herein when said bred bacteriophage lyses the bacterial strain CC22-MRSA-IV “Bamim” (A257).

Furthermore, the bred bacteriophage selected according to the selecting step described herein is a mosaic bacteriophage, meaning that it must have a nucleotide sequence (or genome sequence) comprising at least one nucleotide sequence (or genome fragment) derived from the first (ancestor) bacteriophage as described herein, at least one nucleotide sequence (or genome fragment) derived from the second (ancestor) bacteriophage as described herein, and at least one nucleotide sequence (or genome fragment) derived from the third (ancestor) bacteriophage as described herein. Thus, in one preferred embodiment, the bred bacteriophage selected according to the selecting step described herein is a mosaic bacteriophage having a nucleotide sequence (or genome sequence) comprising the nucleotide sequence as provided in SEQ ID NO: 9, the nucleotide sequence as provided in SEQ ID NO: 17, and at least one nucleotide sequence selected from the group consisting of the nucleotide sequences as provided in SEQ ID NOs: 26, 31, 34, 38, and 41. In one more preferred embodiment, the bred bacteriophage selected according to the selecting step described herein is a mosaic bacteriophage having a nucleotide sequence (or genome sequence) comprising the nucleotide sequence as provided in SEQ ID NO: 9, the nucleotide sequence as provided in SEQ ID NO: 17, and the nucleotide sequence as provided in SEQ ID NO: 31.

Thus, in one even more preferred embodiment, the step of selecting as described herein comprises selecting a bacteriophage having a genome sequence / nucleotide sequence that comprises the nucleotide sequence as provided in SEQ ID NO: 9, the nucleotide sequence as provided in SEQ ID NO: 17, the nucleotide sequence as provided in SEQ ID NO: 31 and which is able to lyse/propagate on the bacterial strain CC22-MRSA-IV “Bamim” (A257).

A further useful bacterial strain in context of this invention is the “propagation strain” CC25- MSSA (124605), disclosed herein and as deposited under DSM 33467 at the Leibniz-Institute DSMZ (Deutsche Sammlung von Mikroorgansimen und Zellkulturen GmbH, in Braunschweig/Germany) on March 19th, 2020, as an international deposit according to the provisions of the Budapest Treaty, see the appended deposit receipt.

Accordingly, in one embodiment, the step of selecting as described herein comprises selecting a bacteriophage having a genome sequence / nucleotide sequence that comprises the nucleotide sequence as provided in SEQ ID NO: 9, the nucleotide sequence as provided in SEQ ID NO: 17, the nucleotide sequence as provided in SEQ ID NO: 31 and which is able to lyse and/or propagate on the bacterial strain CC25-MSSA (124605).

In another aspect, the invention provides a mosaic bacteriophage obtained or obtainable by the above described means and methods for generating a mosaic bacteriophage. Accordingly, the inventive (mosaic) bacteriophage may be defined as a progeny or an offspring of the ancestor bacteriophages.

In a further aspect, the invention provides a bred bacteriophage having the nucleotide sequence as provided in any one of SEQ ID NOs: 4, 60, 61, 62, 65, 66, 67, 68, 69, 70, 71, 72, 73, and 74, or a bacteriophage which has been deposited under the accession No. DSM33478 or DSM33479. In a further aspect, the present invention provides any one of said bred bacteriophage for use in composition as active ingredient.

In a further aspect, the invention provides a mosaic bacteriophage having the nucleotide sequence as provided in any one of SEQ ID NOs: 4, 60, 61, 62, 67, 68, 69, 70, 71, 72 and 73. In some preferred embodiments, said mosaic bacteriophage is for use in combination with another bacteriophage, preferably a bred bacteriophage, more preferably the bred bacteriophage having the nucleotide sequence as provided in any one of SEQ ID NOs: 65, 66 and 74.

The present invention provides also for a progeny of the mosaic and/or bred bacteriophage as described herein which may have the same phenotypic characteristics and the same or higher lytic activity against Staphylococcus aureus strains as the parental mosaic and/or bred bacteriophage it has been bred from. ’’Lytic activity” or “eradiction” may be assessed by methods known in the art and as described and illustrated herein. This lytic activity may comprise an advantageous host range. Accordingly, one potential measurement of “lytic activity” relates to the assessment of the “Kinetic Host Range” as provided herein above and as illustrated in the appended examples. Therefore, any “progeny” of the mosaic and/or bred bacteriophages may maintain and should comprise the surprisingly advantageous effects of the mosaic bacteriophages of the present invention. Thus, said progeny may also be used as active ingredients in the composition or pharmaceutical composition of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art.

As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of nucleic acid molecules. “A bacterial strain” can mean at least one bacterial strain, as well as a plurality of bacterial strains, i.e., more than one bacterial strain. As understood by one of skill in the art, the term “bacteriophage” can be used to refer to a single bacteriophage or more than one bacteriophage.

As used herein the terms “obtainable” and “obtained by” are used interchangeably herein.

As used herein the terms “features” and “properties” are used interchangeably herein.

Where used herein, the term “and/or” when used in a list of two or more items means that any one of the listed characteristics can be present, or any combination of two or more of the listed characteristics can be present. For example, if a composition is described as containing characteristics A, B, and/or C, the composition can contain A feature alone; B alone; C alone; A and B in combination, A and C in combination; B and C in combination; or A, B and C in combination.

The present invention can “comprise” (open ended) or “consist essentially of’ the components of the present invention as well as other ingredients or elements described herein. As used herein, “comprising” means the elements recited, or their equivalent in structure or function, plus any other element or elements which are not recited. The terms “having” and “including” are also to be constructed as open ended unless the context suggests otherwise. As used herein, “consisting essentially of’ means that the invention may include ingredients in addition to those recited in the claim, but only if the additional ingredients do not materially alter the basic and novel characteristics of the claimed invention. A gist of the present invention relates to the following items:

1. A composition comprising

(i) a first bacteriophage, which is a mosaic bacteriophage having a genome which comprises a nucleotide sequence with at least 80% identity with the nucleotide sequence as provided in SEQ ID NO: 9, a nucleotide sequence with at least 80% identity with the nucleotide sequence as provided in SEQ ID NO: 17 and a nucleotide sequence with at least 80% with the nucleotide sequence as provided in SEQ ID NO: 31; and

(ii) a second bacteriophage.

2. The composition according to item 1, wherein said mosaic bacteriophage is functional, wherein the function comprises the ability to lyse at least one bacterial strain, preferably a Staphylococcus strain, more preferably a Staphylococcus aureus strain, even more preferably the Staphylococcus aureus CC22-MRSA-IY “Bamim” (A257) strain publicly available under the accession number DSM111210, and/or wherein the function comprises the ability to lyse at least 50% of a panel of bacterial strains, preferably wherein said panel of bacterial strains is a panel of Staphylococcus aureus strains, more preferably wherein said panel comprises at least 100 Staphylococcus aureus strains and comprises at least 20 of the clonal complexes which most frequently cause infections in humans, even more preferably wherein said panel of Staphylococcus aureus strains comprises at least 110 bacterial strains and comprises at least 25 of the clonal complexes which most frequently cause infections in humans, and/or wherein the function comprises the ability to lyse at least 6 of the Staphylococcus aureus clonal complexes which most frequently cause infections in humans, and/or wherein the function comprises the ability to propagate on at least one bacterial strain, preferably a Staphylococcus strain, more preferably a Staphylococcus aureus strain, even more preferably the Staphylococcus aureus CC25-MSSA (124605) strain deposited under the accession number DSM33467.

3. The composition according to item 1 or 2, wherein the first bacteriophage is the bacteriophage deposited under the accession No. DSM33478, a bacteriophage having at least 98% identity with the genome of the bacteriophage deposited under the accession No. DSM33478, the bacteriophage deposited under the accession No. DSM33479, a bacteriophage having at least 98% identity with the genome of the bacteriophage deposited under the accession No. DSM33479, the bacteriophage having the genome comprising the nucleotide sequence as provided in any one of SEQ ID NOs: 4, 60 to 62 and 67 to 73, or a bacteriophage having a genome comprising a nucleotide sequence with at least 98% identity with nucleotide sequence as provided in any one of SEQ ID NOs: 4, 60 to 62 and 67 to 73. The composition according to any one of items 1 to 3, wherein the second bacteriophage is the bacteriophage having the genome comprising the nucleotide sequence as provided in any one of SEQ ID NOs: 65, 66 and 74, or a bacteriophage having a genome comprising a nucleotide sequence with at least 98% identity with nucleotide sequence as provided in any one of SEQ ID NOs: 65, 66, and 74. The composition according to any one of items 1 to 4, wherein

(a) the first bacteriophage has the genome comprising a nucleotide sequence with at least 98% identity with the nucleotide sequence as provided in SEQ ID NO: 4 [PM4] and the second bacteriophage has the genome comprising a nucleotide sequence with at least 98% identity with the nucleotide sequence as provided in SEQ ID NO: 66 [PM93];

(b) the first bacteriophage has the genome comprising a nucleotide sequence with at least 98% identity with the nucleotide sequence as provided in SEQ ID NO: 61 [PM9] and the second bacteriophage has the genome comprising a nucleotide sequence with at least 98% identity with the nucleotide sequence as provided in SEQ ID NO: 66 [PM93];

(c) the first bacteriophage has the genome comprising a nucleotide sequence with at least 98% identity with the nucleotide sequence as provided in SEQ ID NO: 73 [PM23] and the second bacteriophage has the genome comprising a nucleotide sequence with at least 98% identity with the nucleotide sequence as provided in SEQ ID NO: 65 [PM56];

(d) the first bacteriophage has the genome comprising a nucleotide sequence with at least 98% identity with the nucleotide sequence as provided in SEQ ID NO: 68 [PM28] and the second bacteriophage has the genome comprising a nucleotide sequence with at least 98% identity with the nucleotide sequence as provided in SEQ ID NO: 74 [PM94];

(e) the first bacteriophage has the genome comprising a nucleotide sequence with at least 98% identity with the nucleotide sequence as provided in SEQ ID NO: 60 [PM32] and the second bacteriophage has the genome comprising a nucleotide sequence with at least 98% identity with the nucleotide sequence as provided in SEQ ID NO: 64 [02];

(f) the first bacteriophage has the genome comprising a nucleotide sequence with at least 98% identity with the nucleotide sequence as provided in SEQ ID NO: 72 [PM7] and the second bacteriophage has the genome comprising a nucleotide sequence with at least 98% identity with the nucleotide sequence as provided in SEQ ID NO: 65 [PM56];

(g) the first bacteriophage is has the genome comprising a nucleotide sequence with at least 98% identity with the nucleotide sequence as provided in SEQ ID NO: 60 [PM32] and the second bacteriophage has the genome comprising a nucleotide sequence with at least 98% identity with the nucleotide sequence as provided in SEQ ID NO: 65 [PM56];

(h) the first bacteriophage has the genome comprising a nucleotide sequence with at least 98% identity with the nucleotide sequence as provided in SEQ ID NO: 62 [PM22] and the second bacteriophage has the genome comprising a nucleotide sequence with at least 98% identity with the nucleotide sequence as provided in SEQ ID NO: 64 [02];

(i) the first bacteriophage has the genome comprising a nucleotide sequence with at least 98% identity with the nucleotide sequence as provided in SEQ ID NO: 73 [PM23] and the second bacteriophage has the genome comprising a nucleotide sequence with at least 98% identity with the nucleotide sequence as provided in SEQ ID NO: 66 [PM93];

(j) the first bacteriophage has the genome comprising a nucleotide sequence with at least 98% identity with the nucleotide sequence as provided in SEQ ID NO: 4 [PM4] and the second bacteriophage has the genome comprising a nucleotide sequence with at least 98% identity with the nucleotide sequence as provided in SEQ ID NO: 65 [PM56]; or

(k) the first bacteriophage has the genome comprising a nucleotide sequence with at least 98% identity with the nucleotide sequence as provided in SEQ ID NO: 72 [PM7] and the second bacteriophage has the genome comprising a nucleotide sequence with at least 98% identity with the nucleotide sequence as provided in SEQ ID NO: 66 [PM93] The composition according to any one of items 1 to 5, wherein said composition is functional, wherein the function comprises the ability to eradicate one or more bacterial strains, preferably Staphylococcus strains, more preferably Staphylococcus aureus strains, and/or wherein the function comprises the ability to lyse at least 50%, at least 70%, at least 75%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, or at least 87% of a panel of Staphylococcus aureus strains, preferably wherein said panel comprises at least 100 Staphylococcus aureus strains and comprises at least 20 of the clonal complexes which most frequently cause infections in humans, more preferably wherein said panel comprises at least 110 bacterial strains and comprises at least 25 of the clonal complexes which most frequently cause infections in humans and/or wherein the function comprises the ability to lyse at least 6 of the Staphylococcus aureus clonal complexes which most frequently cause infections in humans, and/or wherein the function comprises the ability to reduce by at least 90% or by at least 99% one or more pre-formed biofilms, preferably Staphylococcus pre-formed bio films, more preferably Staphylococcus aureus pre-formed biofilms, and/or wherein the function comprises the ability to reduce by at least 90%, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% pre-formed bio films of a panel of pre-formed biofilms, preferably wherein said panel is a panel of 10 pre-formed bio films, more preferably wherein said panel is a panel of 10 preformed Staphylococcus aureus biofilms and comprises 10 of the clonal complexes which most frequently cause infections in humans, and/or wherein the function comprises the ability to reduce by at least 99%, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, or 60% or more pre-formed biofilms of a panel of pre-formed biofilms, preferably wherein said panel is a panel of 10 pre formed biofilms, more preferably wherein said panel is a panel of 10 preformed Staphylococcus aureus biofilms and comprises 10 of the clonal complexes which most frequently cause infections in humans. A pharmaceutical composition comprising the composition according to any one of items 1 to 6 and optionally a pharmaceutically acceptable carrier. The pharmaceutical composition according to item 7 for use in therapy. The pharmaceutical composition according to item 7 for use in the treatment of a bacterial infection, preferably a Staphylococcus infection, more preferably a Staphylococcus aureus infection, even more preferably a Staphylococcus aureus infection involving bio film formation. Use of the composition according to any one of items 1 to 6 in a non-medical method of killing, eradicating and/or inhibiting the growth of bacteria, preferably wherein the bacteria is Staphylococcus, more preferably wherein the bacteria is Staphylococcus aureus. A non-medical method of reducing and/or preventing pre-formed bio films, comprising applying the composition according to any one of items 1 to 6, preferably wherein the biofilm is a Staphylococcus biofilm, more preferably wherein the biofilm is a Staphylococcus aureus biofilm. The use according to item 10 or the method according to item 11 wherein the composition according to any one of items 1 to 6 is to be applied to a food product, a crop or a surface, preferably wherein the surface is the skin of a mammal, equipment, medical equipment, prostheses, implant, bedding, furniture, walls, floors, or combinations thereof. A bandage or wound dressing comprising the composition according to any one of items 1 to 6, or the pharmaceutical composition according to item 7. A bacteriophage which has the nucleotide sequence as provided in any one of SEQ ID NOs: 4, 60 to 62 and 65 to 74, the bacteriophage which has been deposited under the accession No. DSM33478, or the bacteriophage which has been deposited under the accession No. DSM33479 or any progeny thereof. 15. A kit comprising : a. The composition according to any one of items 1 to 6, the pharmaceutical composition according to item 7, the bandage according to item 13 or the bacteriophage or progeny according to item 14; and b. Instructions for use of same (e.g. in medicine).

The present invention is further described by reference to the following non-limiting figures and examples.

The Figures show:

Figure 1. PhagoMed-Modified Appelmans Protocol (PMAP)

FIG. 1 shows a schematic representation of the PhagoMed-Modified Appelmans Protocol (PMAP) according to the invention. Briefly, one or more ancestor bacteriophages are pooled to create the input phage mixture, which is diluted or undiluted and mixed with each bacterial strain of the subpanel of 17-24 Staphyloccoccus aureus strains in a 96-well microtiter plate. As shown in the right-hand side of the Figure, the top half of the 96-well plate (4 rows x 12 columns) is used for 12 bacterial strains mixed with the input phage mixture in 4 different dilutions. The bottom half is filled the same way with 12 different strains, so that one plate may fit up to 24 bacterial strains, each mixed with 4 different dilutions of the same phage mixture. After incubation at 37 °C for 24h, OD600 is measured and each well showing lysis (clear wells) are pooled along with the two first non-lysed (turbid wells) past the point on lysis, i.e. all wells within the black lines as exemplary shown in the bottom of the Figure are pooled, pelleted by centrifugation and filtered. The resulting mixed lysate is used instead of the input phage mixture for the next round of the protocol. The process is typically repeated at least 10 rounds.

Figure 2. KHR measurements for wild-type and bred phages as well as two-phage cocktails

FIG. 2 shows the result of the full KHR measurement of 30 original (i.e. ancestor) phages, bred phages and two-phage cocktails as tested on the panel of 110 A aureus strains described in Table 1. The bacterial strains are depicted in the top row, grouped by clonal complex, with the MRSA/MSSA status shown in the second row. In Fig. 2A (parts 1 and 2), the phages tested are depicted on the left, grouped by originals or the experiment they were derived from (Table 4). In Fig. 2B (parts 1 and 2), the two-phage cocktails together with their constituent individual phages are depicted. Cells are colored dark gray or light gray when after 24h, the OD600 of the phage treated sample was less than 10% of the OD600 of the untreated bacterial growth control in 2/2 duplicates or 1/2 duplicates, respectively. Phage 05 was tested only on 11 strains, and all strains not tested are marked by black shading. White cells represent that the OD600 of the phage treated sample was more than 10% of the OD600 of the untreated bacterial growth control in 2/2 duplicates. The KHR is calculated as the percent of strains for which after 24h, the OD600 of the phage treated sample is less than 10% of the OD600 of the untreated bacterial growth control under the same condition (i.e. bacteria of the same strain without phages), in 2/2 duplicates.

Figure 3. Biofilm CFU reduction effects of the bacteriophage cocktails of the invention as compared to the individual phages. The effect of 3 bred bacteriophage and 2 cocktails are shown on 4 different S. aureus strains. Although the total phage concentration is identical in all treatment groups, the cocktails PM4+PM56 and PM4+PM93 have an over-additive or synergistic effect on each strain. Statistics were calculated by one-way Anova against the control group in Graphpad Prism 9. P-values are shown and further illustrated by asterisks, n.s. indicates not significant (p > 0.05).

Figure 4. Genomic alignment of PM4 with ancestor bacteriophages

FIG. 4 shows a genomic alignment of the terminal repeat region (nucleotides 129,270 to 148,627 ofPM4) of the progeny phage PM4 aligned to the corresponding region of its ancestors 03, 04 and 05. The thick dotted line depicts which ancestor is most homologous to PM4 in which region. Any difference of 03, 04, or 05 vs. PM4 is depicted in black, while regions identical to PM4 are gray. At the bottom, the origin of each region of PM4 is shown, along with the recombination sites.

Figure 5. Comparison of the virulence of PM4 and its ancestors 03, 04 and 05.

(A) illustrates the calculation of local virulence VMOI. Phage virulence at a defined MOI is termed local virulence Yio (the subscript indicates the MOI), calculated with the formula on top of the graph. A1 and AO are the areas under the OD600 curves of the treated and untreated samples, respectively. B is the blank area. (B), (D), (F), (H), (J), (L) depict the lytic activity of the bred bacteriophage PM4 and its ancestors at different starting MOIs and on six different strains: CC239-MRSA-III (2017-046), CC30-MRSA-IY (2011-278), CC25-MSSA (B91), CC12-MSSA (A161), CC22-MRSA-IV (A257, “Bamim”) and CC22-MRSA-IV (B311, “Bamim”), respectively (each of these strains are available in the publicly accessible section of the Leibniz-Institute DSMZ, Braunschweig, Germany, under the accession numbers as indicated in Table 13). The graphs show optical density measurements at 600 nm (OD600) of bacterial suspensions in presence or absence (GC: growth control) of the bacteriophages in a 96-well plate for 24 h at 37 °C. The bacterial strain name is depicted as title, the bacteriophages on the left and the different MOIs at the top of each column. The concentration of the phages was kept constant at 5 x 10⁸ PFU/mL and starting CFU/mL were modified to reach the different MOI concentrations at the start of the kinetic. (C), (E), (G), (I), (K), (M) depict virulence curves of the three ancestor phages and PM4. The virulence curves were calculated by plotting the local virulence as a function of the starting MOI used in the experiment.

EXAMPLES

The following Examples illustrate the invention.

Example 1 - Lytic activity of the individual wild-type bacteriophages and their Kinetic Host Range

Five “wild-type” (also referred therein as “original” or “ancestor” interchangeably) bacteriophages have been used, namely 01, 02, 03, 04 and 05.

Phage 01 and 02 are derivatives of Staphylococcus phages Remus and Romulus, respectively, as described in Vandersteegen et al., 2013 (J Virol. 2013 Mar;87(6):3237-47), or in NCBI (NCBI accession number JX846612 version JX846612.1 of August 15, 2013 for Remus and NCBI accession number JX846613 version JX846613.1 of March 12, 2013 for Romulus). Specifically, the sequences of the phages used for the present inventions are provided in SEQ ID NO: 63 and SEQ ID NO: 64 for 01 (Remus) and 02 (Romulus), respectively. 01 has a sequence length of 142 kbp, with terminal repeats of 7340 bp at either end. 02 has a sequence length of 137 kbp, with terminal repeats of 5322 bp.

Phage 03 is an ISP-like bacteriophage with a nucleotide sequence homology of 97,5% identity to Staphylococcus phage ISP, wherein Staphylococcus phage ISP is known under NCBI accession number FR852584 (version FR852584.1 of September 19, 2011). Phage 03 was isolated de novo at University Lausanne, Switzerland, by the laboratory of Prof. Gregory Resch, and is disclosed for the first time by the present disclosure. Specifically, 03 has the nucleotide sequence as provided in SEQ ID NO: 3, i.e. a sequence length of 145076 bp, with terminal repeats of 7441 bp at either end. The genome sequence/nucleotide sequence of bacteriophage “03” comprises the nucleotide sequence:

ID NO: 76, which is a nucleotide sequence specific to the group of the “ISP-like bacteriophages” as defined herein and meaning that the phage “03” belongs to said group.

Phage 04 has the nucleotide sequence as provided in SEQ ID NO: 2 and is the wild type Staphylococcus phage 812 obtained from the d’Herelle collection of Laval University in Montreal, Canada, also known under NCBI accession number MH844528 (version MH844521.1 of December 02, 2018). The genome sequence/nucleotide sequence of bacteriophage “04” comprises the nucleotide sequence:

, which is a nucleotide sequence

specific to the group of the “812/K-like bacteriophages” as defined herein and meaning that the phage “04” belongs to said group.

Phage 05 was isolated de novo at Leibniz Institut Deutsche Sammlung Zellkulturen und Mikroorganismen GmbH (DSMZ) in Braunschweig, Germany and is disclosed for the first time by the present disclosure. Specifically, 05 has the nucleotide sequence as provided in SEQ ID NO: 1, i.e. a sequence length of 146,878 and terminal repeats of 5438 bp at either end. The genome sequence/nucleotide sequence of bacteriophage “05” comprises the nucleotide

sequence:

”; see also SEQ ID NO: 75, which is a nucleotide sequence

specific to the group of the “812/K-like bacteriophages” as defined herein and meaning that the phage “05” belongs to said group.

A panel of 110 S. aureus strains was compiled from patient isolates of the University clinic Dresden in Germany in 2010 and 2011. The genomes of each strain were sequenced and genotyped to define their clonal complex. Of the strains available at the university clinic, 110 strains were selected such that the frequency of the clonal complexes in the panel reflects the natural epidemiology of S. aureus in human infections as found in several literature cases (Arias et al., 2017; Kanjilal et al.; 2018; Luedicke et al.; 2010; Rasmussen et al.; 2013). The strains are listed in Table 1 below. Alternatively, any other panel can be used which consists of S. aureus strains of a distribution of clonal complexes similar to what is found in human infections. A person skilled in the art is readily in the position to obtain such useful panel and corresponding panel and/or bacterial strains collection, for example from the national reference centers for antimicrobial resistance, or public collections of microorganisms.

[Table 1]

‘Bacterial strain CC22-MRSA-IV, “Barnim” (A257) is available in the publicly accessible section of the Leibniz- Institute DSMZ (Braunschweig, Germany) under the strain name “Barnim” Isolate A257 ( Stapholococcus aureus subsp: aureus) and under the accession number DSM 111210.

“Bacterial strain CC22-MRSA-IV, "Barnim" (B311) is available in the publicly accessible section of the Leibniz- Institute DSMZ (Braunschweig, Germany) under the strain name “Barnim” Isolate B311 ( Stapholococcus aureus subsp: aureus) and under the accession number DSM 111211.

“‘Bacterial strain CC12-MSSA (A 161) is available in the publicly accessible section of the Leibniz-lnstitute DSMZ (Braunschweig, Germany) under the strain name Isolate A161 ( Stapholococcus aureus subsp: aureus) and under the accession number DSM 111212.

““Bacterial strain CC25-MSSA (124605) has been deposited under the stipulations of the Budapest treaty under the accession number DSM 33467 with the Leibniz-lnstitute DSMZ (Deutsche Sammlung von Mikroorgansimen und Zellkulturen GmbH, in Braunschweig/Germany) on March 19th, 2020 (see appended deposit receipt). The lytic activity of the five wild-type (or original/ancestor) bacteriophages, namely 01, 02, 03, 04 and 05 on each of the 110 S. aureus strains of the panel as described above was measured in duplicate as follows. Using 96- or 384-well plates, 10 mΐ phage solution adjusted to 5xl0⁸ PFU/ml with 0.85% saline buffer, was added to 90 mΐ of a suspension of planktonic bacteria at OD600 = 0.01 (corresponds to 5xl0⁶ CFU/ml) in BHI medium (MOI=10 at the start of the reaction). Reaction plates were incubated at 37 °C for 24 hours in an incubator without shaking. OD600 was measured with a TECAN microplate reader.

The Kinetic Host Range (KHR) of each bacteriophage was calculated as the percent of strains for which after 24h, the OD600 of the phage treated sample was less than 10% of the OD600 of the untreated bacterial growth control for both duplicates.

The results of the KHR measurement are shown in Table 2 below.

[Table 2]

As can be seen from Table 2, no individual wild type phage exceeds 45% KHR.

Example 2 - Breeding to generate phages with improved host range

As a next step, it has been investigated whether it would be possible to achieve a KHR of more than 50% of the S. aureus strains panel of Table 1. For that purpose, the wild-type or original bacteriophages have been genetically modified by breeding to improve their properties. The following breeding method (therein also referred to as the “PhagoMed-Modified Appelmans Protocol” or “PMAP”) has been used.

Using a 96-well microtiter plate, 100 pL of bacterial culture of a single strain in BHI at OD600=0.2 was mixed with 100 mΐ of an input phage mixture comprising a single phage or a phage cocktail in serial 10-fold dilutions (undiluted to 10³, starting at lxlO⁹ pfu/ml). This yields final concentrations of 5xl0⁸ pfu/ml phage and 5xl0⁷ CFU/ml bacteria in 200 mΐ suspension for the wells with the highest phage concentration. In this way, the top half of the 96-well plate (4 rows x 12 columns) is used for 12 bacterial strains mixed with the input phage mixture in 4 different dilutions. The bottom half is filled the same way but with 12 different strains, so that one plate may fit 24 bacterial strains, each mixed with 4 different dilutions of the same phage mixture (as shown in the right-hand side of Fig. 1). Each phage mixture fills a separate 96-well plate. The filled plates are incubated at 37 °C for 24h, after which OD600 is measured. In each plate, and for each bacterial strain, the wells with OD600<0.1 are identified, indicating lysis of the strain and no massive outgrowth of bacterial resistance. For each bacterial strain, these clear wells and the two turbid wells below (i.e., with a starting phage titer lOx and lOOx lower than the last clear well, as illustrated in the bottom of Fig. 1) are pooled, pelleted by centrifugation at -5000 g for 20min and sterile filtered through a 0.22 pm syringe filter. The pooled lysates are used to infect the next round of breeding with the same system on 96-well plates, again with 4 different dilutions (undiluted to 10^-3). The process is typically repeated at least 10 times. If no improvement, i.e. no clear wells on previously unsusceptible bacterial strains, is visible after 4- 6 rounds, the bacterial strains are exchanged.

For breeding the bacteriophage according to the above method, subpanels of 17 to 24 Staphyloccoccus aureus strains were composed by selecting strains of different clonal complexes and lineages, resulting in a high genomic diversity. The specific subpanels of bacterial strains used for breeding in this Example are shown in Table 3 below.

[Table 3]

* Strains highlighted in bold were exchanged in some intermediate rounds of breeding.

Separate PMAP breeding experiments have been performed. The input phage mixture and the bred phages that were isolated from each experiment are summarized in Table 4 below.

[Table 4]

Isolation of the plaques was conducted only if during the PMAP breeding cycles (or rounds) clear improvements in the host range were visible, and was started from the pooled lysate of round 10 or later. To isolate phages from the pooled/mixed lysate, phage plaques are created using the Double Agar Layer (DAL) method as follows. 6 ml BHI top agar (0.7%) is melted and cooled to 55 °C, then mixed with 400 pL of a bacterial mid-log suspension at OD600=0.5 (corresponds to 2.5E+08 CFU/ml) and poured onto a solid BHI agar (1.5%) monolayer of 35 ml in a square 1-well plate. Plates are dried for 20 minutes in a sterile biosafety cabinet with open lids. A sterile loop was briefly immersed into the final lysate of any PMAP round, and a drop of liquid was streaked out on the solidified DAL plate. Plaques are visible after overnight incubation at 37 °C. To fully isolate monoclonal phages, selected plaques are touched with a fresh sterile loop and gently re-streaked on the top agar of a new DAL plate. The isolation process is repeated by re-streaking at least 3 times. From the final pooled lysates of each PMAP experiment (Table 4), phages are isolated by this method on different bacterial strains (e.g., see Table 5). These strains were selected from the group of Table 1, including strains which were resistant, intermediate and sensitive to the respective lysate. As depicted in Table 5 below, the bred phages were isolated on 3 specific strains.

[Table 5]

Example 3 - Lytic activity of bred phages and cocktails

The lytic activity of bred phages and several two-phages cocktails has been determined using the method of Example 1. For two-phage cocktails, the titer of the individual phages was adjusted such that the total phage concentration of the cocktail was equal to the total phage concentration for single phages (i.e. the concentration of the individual phages in the cocktail was half the concentration used for phages tested individually). As the ability of phages to eradicate/outcompete bacteria in suspension depends on the starting multiplicity of infection (MOI), the setup avoids skewing the KHR of cocktails by a higher initial MOI compared to single-phage experiments.

The results of the KHR measurement and lytic activity of the bred single phages and cocktails for each S. aureus strain are shown in Fig. 2A (part 1 and 2) and 2B (part 1 and 2), respectively.

As it can be seen in Figure 2, it has been surprisingly found that the bacterial strain CC25- MSSA (124605) is particularly useful to be used in the context of propagation. This particularly useful strain is also part of this invention and has been deposited under the stipulations of the Budapest treaty under the accession number DSM 33467 with the Leibniz-Institute DSMZ (Deutsche Sammlung von Mikroorgansimen und Zellkulturen GmbH, in Braunschweig/Germany) on March 19^th, 2020 (see appended deposit receipt). This strain was, inter alia, used in context of the present PMAP, in particular in the propagation of the inventive (mosaic) bacteriophages, such as, e.g., PM4 and PM32. As it can be seen in Figure 2, all mosaic bacteriophages can propagate on said strain and high phage titers may be obtained. Example 4 - Comparison of the KHR of two-phage cocktails composed of wild-type and bred phages

The KHRs of combinations of wild-type bacteriophages have been estimated from the KHRs of individual phages depicted in Table 6 below.

[Table 6]

As can be seen from the comparison of Tables 2 and 6, the host-ranges of ISP-like phages and 812/K-like phages are not complementary and since these two phage “families” tend to lyse the same strains. This is clearly seen with the combination of 03 plus 04 and 03 plus 05 which both show no improvement in comparison with 03 alone. Therefore, it can be concluded that 04 and 05 merely lyse a subset of strains already lysed by 03, and a combination of these bacteriophages in a cocktail does not lead to a broader host-range. On the contrary, combining a Romulus/Remus-like phage with an ISP-like phage or an 812/K-like phage increases the KHR of the cocktail compared to each of the individual phages. The highest KHR achievable with cocktails of the wild-type bacteriophages is 62% with a combination of the ISP-like phage 03 with the Romulus/Remus-like phage 02.

In contrast, bred phages can be combined with other bred phages or also with wild type phages to create cocktails with very broad host range. This is because the breeding process as described in Example 2 creates bred phages which lyse strains not lysed by any ancestor, or other bred phages, in a surprising and unpredictable way. Therefore, the bred phages are highly complementary to each other. As some bred phages surprisingly also lose lytic activity on some strains while still increasing the total number of strains lysed, it is even possible to combine bred phages and wild type phages in a complementary way. The measured and calculated KHRs of several two-phage cocktails are depicted in Table 7 below. [Table 7]

As can be clearly seen from Table 7, bacteriophage cocktails composed with bred bacteriophages show a vastly broader kinetic host range than the respective bacteriophage cocktails composed only of the wild-type bacteriophages, as shown in Table 6. Importantly, KHRs above 80% on the 110 S. aureus strains of the panel of Table 1 can be achieved. The highest measured KHR of 88% is achieved for the combination of PM4 and PM93. These two phages also display the highest level of complementarity, as the cocktail lyses 28 percentage points more strains than the best individual phage (PM4, 60% of strains). Such broad host ranges have never been achieved in the art before the present invention. Strikingly, host ranges above 70% can only be achieved by combining a Remus/Romulus-like phage with an ISP-like phage. Combinations within the groups would not achieve KHRs higher than 70% even if the individual bred phages included in the cocktails are vastly superior to the ancestors. For example, the combination cocktail of PM4 and PM32 has a KHR of 62%, only 2 percentage points better than PM4. However, both constituent phages have vastly superior KHRs to any of their ancestors (see Fig. 1).

Example 5 - Biofilm activities of ancestor phages, bred phages and cocktails.

As a next step, the biofilm activity of the original phages, bred phages, and several cocktails, was tested in vitro. Biofilms were prepared by incubating up to 15 porous sintered glass beads in 50ml tubes, filled with 1ml BHI per bead (max 15ml to allow for sufficient aeration), each tube inoculated with 100 pL of a lxlO⁸ CFU/ml suspension of one of the 10 S. aureus strains tested (see Table 8). The tubes were incubated at 37 °C for 24 hours to grow a stable biofilm. Then, the supernatants were removed and the beads in each of the tubes washed 3 times with 15ml 0.85% saline. Beads were then transferred into individual wells of a 96 deep well plate, each pre-filled with 900 pL BHI. Each treatment group (untreated growth control for each bacterial strain or the phage-treatment groups) consisted of a minimum of 4 beads for statistical purposes. Treatments were started by adding 100 pL saline (untreated control) or phage mixture adjusted to 10^s pfii/ml (total phage concentration, i.e. half the value for each phage in a two- phage cocktail) then the plates were covered with a lid and incubated at 37°C for 24h. After the treatment step, broth from each well was aspirated and each bead washed 3 times with 1 ml 0.85% saline, then 1 ml of 0.85% saline buffer was added to each bead. The plates were covered tightly with self-adhesive PCR film to avoid spills and cross-contamination in the next step. Plates were vortexed for 30 seconds, then sonicated with a Bandelin Sonorex Digiplus (2018) at maximum intensity for 1 minute. To remove drops from the PCR self-adhesive film, each plate was then spun for 1 min at 3800 rounds per min. After removing the foil, the supernatants were vigorously pipetted up and down to remove residual biofilm from the beads and the walls of the wells. Then supernatants were transferred to 96-U-well plates and serial 10-fold dilutions were prepared (adding 180 pL of 0.85% saline buffer to 20 pL of supernatant). 20 pi of each dilution were spotted onto solid 90mm TSA agar plates, the plates were dried and incubated for 18-24 hours at 37 °C. After incubation, colonies were counted and the number of surviving CFU after treatment was calculated.

The reduction in biofilm CFU upon phage treatment was measured for multiple individual phages or cocktails and for 10 bacterial strains, each combination at least with 4 biological replicates. Each original was tested, plus the bred phages with the most promising KHR as depicted in Fig. 2, along with combinations of bred phages or bred phages and ancestors. For each combination of individual phage (or phage cocktail) and bacterial strain, Table 8 below depicts the negative base- 10 logarithm of the reduction in biofilm CFU upon phage treatment, which in turn was calculated as the ratio of the median of surviving biofilm CFU in phage- treated samples and the median of bio film CFU in the untreated control. Phage/strain combinations where phages kill more than 90% of the bio film CFU (between 1 and 2 log 10 units) are highlighted in light gray, combinations where more than 99% (2 and more log 10 units) are killed are highlighted in darker gray. As depicted in Table 8, of the ancestor phages (01 -05), 02 achieves a >90% reduction on 60% of strains, more than any other ancestor phage. 03 acts in a more specific way, in that it achieves a >99% reduction on more strains than any other ancestor phage (20%), but with >90% killing on just 30% of strains it is not as broadly effective as 02. 01, 04 and 05 have weaker bio film activities. None of the ancestors has an average reduction in biofilm CFU of more than 1 log units (90% killing) across all 10 A aureus strains. In contrast, all of the bred phages except PM93 and PM56 have average log reductions above 1 log unit across the 10 strains. At the same time, seven of them (namely, PM5, PM7, PM9, PM22, PM23, PM28 and PM94) achieve >90% reduction on 60% or more of strains and at the same time >99% reduction on 10% or more strains. Therefore, considering also the higher KHR, these seven phages are clearly superior to the ancestors.

The two-phage cocktails were selected to include at least one Remus/Romulus-like phage or their ancestors (02, PM93, PM56) and at least one ISP-like phage or its ancestors (PM4, PM7, PM22, PM32), to test whether there is a synergistic interaction. The weakest two-phage cocktail by average CFU reduction, 1.45 log units, is achieved by PM22 plus 02 and PM32 plus PM56, which is higher than the log reduction of any single phage (max of 1.44 log units for PM5). The highest log reduction of a two-phage cocktail is 2.38 (PM4 plus PM56). The latter cocktail even achieves a >90% killing across 100% of the tested strains and a >99% killing over 60% of strains.

Importantly, all of the mosaic bacteriophages obtained by breeding a combination of the three ancestor bacteriophages 03, 04 and 05 according to the breeding method provided herein, namely PM4, PM5, PM7, PM9, PM22, PM23, PM28, and PM32, show >90% reduction on 50% or more of the bacterial strains tested whereas the ancestor bacteriophages 03, 04 and 05 show >90% reduction on 30%, 0% or 10% of the same bacterial strains, respectively. Thus, as can be clearly seen from Table 8 above, the mosaic bacteriophages described herein show a vastly broader biofilm host range (BHR) than any one of the ancestor bacteriophages 03, 04 and 05.

Example 6 - Synergistic biofilm killing by specific phage cocktails

As a next step, it was tested whether the high biofilm killing activity of certain cocktails described in Example 5 is based on a synergistic interaction of certain phages. The total phage concentration in experiments with cocktails was identical to the total phage concentration used for experiments with individual phages, because each phage in a two-phage cocktail was set to half the concentration of the phages tested individually. Therefore, if the phages had a purely additive effect, one would expect that the biofilm CFU reduction effect of the cocktail amounts to the average of the effects of the individual phages. However, as can be seen by the higher average CFU reduction values of cocktails in Table 8 and as depicted in Fig. 3, there are instances where the cocktail had a much stronger effect than the average of the constituent phages. A synergy is defined for cases where the biofilm CFU reduction of the cocktail is at least twice (0.3 log 10 units) the average effect of the individual phages (phages in the cocktail at half the concentration of the phages tested individually). Fig. 3 shows such synergies for the cocktails PM4 plus PM93 and PM4 plus PM56, for the four S. aureus strains “CC8-MRSA- IV/USA300 (A57)’ “CC12-MSSA (A161)”, “ST228-MRSA-I, "Suddeutscher" (057)”, and “CC5-MRSA-II, "Rhein-Hessen" (B94)”. All are common pathogenic S. aureus strains, and “USA300” has been reported to be the predominant strain involved in PJI in the United States (Kourbatova EY, Halvosa JS, King MD, Ray SM, White N, Blumberg HM. Emergence of community-associated methicillin-resistant Staphy- lococcus aureus USA 300 clone as a cause of health care-associated infections among patients with prosthetic joint infections. Am J Infect Control 2005; 33:385-91). For example, for strain “CC12-MSSA (A161)”, none of the 3 individual phages PM4, PM93 or PM56 induces a statistically significant biofilm CFU killing, while each of the cocktails induces highly significant multi-log killing between 3.2 and 4.3 loglO units. This indicates that the effect of each cocktails is based on surprising and unpredictable synergistic interactions between the constituent phages. Another example is strain “ST228-MRSA-I, "Suddeutscher" (057)”, on which PM4, PM56 and PM93 achieve a statistically weakly significant killing of less than 1 log 10 unit, while, while both cocktails PM4 plus PM56, PM4 plus PM93 achieve statistically significant biofilm reductions of 2.29, 1.79 loglO units, respectively, again indicating synergy. For strain “CC8-MRSA-IY/USA300 (A57)”, the effect of the cocktail PM4 plus PM56 is based on synergistic interaction of the constituent phages in that the killing by the cocktail is more than twice the average effect of the constituent phages. The effect of PM4 plus PM93 (killing of 1.33 loglO units, see Table 6) lies between the effects of the constituent phages and is therefore not synergistic. Also on strain “CC5-MRSA-II, "Rhein-Hessen" (B94)”, the cocktail PM4 plus PM56, shows a synergistic interaction of the phages. The same analysis was done for all of the 10 tested bacterial strains, and shows that the two cocktails PM4 plus PM93 and PM4 plus PM56 show a synergistic behavior on 70% and 90% of the strains, respectively. In conclusion, the biofilm efficacy of three cocktails displays a highly synergistic interaction between the constituent phages.

The synergy analysis was extended to all tested cocktails consisting of a combination of at least one Remus/Romulus-like phage or their ancestors (02, PM93, PM56) and one mosaic bacteriophage (PM4, PM9, PM22, PM32). As depicted in Table 9, several such cocktails were found with synergies on 70%-90% of the tested strains. It is therefore concluded that Remus/Romulus-like phages and their ancestors synergize with the mosaic bacteriophages as provided herein in killing biofilm cells. This property is highly relevant for the application of phage therapy to implant-associated infections, which are very frequently caused by S. aureus. There is no phage cocktail described in the prior art which has a similarly strong and broad biofilm efficacy as the cocktails described in this disclosure.

Example 7 - Mosaicity of the bred phages

Phage DNA was isolated according to the phage genomic DNA extraction protocol of Jason J. Gill at the Center for Phage Technology at Texas A&M University (rev.7/12/11). PEG/NaCl- sample mixtures were carefully mixed by inverting, incubated overnight at 4 °C. After PEG precipitation, the standard protocol of the DNeasy Blood and Tissue Kit (Qiagen, 69582) was followed, with an elution volume of 50m1. Genomic DNA was sequenced with MiSEQ Illumina platform and a 2x300 bp kit (Illumina). 2x300 bp paired-ends Illumina reads were assembled with SPAdes 3.13.0, with the cov-cutoff’ variable set at 500. The quality of the assembly was evaluated by visualizing the assembly graphs with Bandage 0.8.1.

Selected bacteriophages were additionally sequenced by the means of Nanopore MinlON (01, 02, 03, 04, 05, PM4, PM9, PM32, PM56, PM93). This was done because Illumina reads (~300bp) are too short to identify the length and position of the ~3000-7000bp terminal repeats, so that the correct assembly of the contig of a phage which has terminal repeats from only Illumina reads is not possible. Not all phages were sequenced with both methods due to resource constraints, and because, with the ancestor phages and selected bred phages assembled correctly, it is possible to draw conclusions also for the phage genomes assembled based on Illumina reads only. DNA was isolated as described by Dziuginta and Moodley (Dziuginta, J., and Moodley, A. (2018). A Rapid Bacteriophage DNA Extraction Method. Methods Protoc. 1, 3-7). Alternatively, the DNA was extracted with Norgen kit (NORGEN Biotech, #46800). In case of PM93, phages were first precipitated with PEG 8000, 10%, resuspended in saline and then processed with Norgen kit. DNA was eluted from the column with 50-75 uL of pure water. The sequencing library was prepared by using the Rapid Barcoding Kit (Oxford Nanopore Technologies, #SQK-RBK004). The sequencing was performed on a flow cell with pore technology 9.4 (FLO-MIN106), for 113 hours and 59 minutes. The reads were de-multiplexed and basecalled by using the MinlON Release 19.12.2. Basecalling was performed with configuration “FLO-MINI 06 / FLO-FLG001 DNA - High-Accuracy” in MinlON software, and FASTQ format was selected as output file. Only reads longer than 9 kb (ONT reads) were used, and imported in Geneious Prime® 2020.0.4 in FASTQ format for downstream analysis. On the Illumina contigs, the terminal repeat was located by identifying the region with above- the-average coverage of Illumina reads. The Illumina contig was manually edited by eliminating the terminal repeat and splitting in two parts (left and right fragment). The ONT reads were mapped to the fragments, extracted and assembled by using the Canu 1.9 algorithm. The contigs were corrected for sequencing errors by mapping the Illumina reads with high sensitivity. After the sequence correction, the contigs (bearing one LTR each) were assembled together. Genomes were annotated with PROKKA and a phage-specific database. Genomes were compared by using the MAUVE alignment tool in Geneious Prime® 2020.0.4.

As a result, PM4 has been determined to have a genome consisting of the nucleotide sequence as provided in SEQ ID NO: 4, PM5 has been determined to have a genome consisting of the nucleotide sequence as provided in SEQ ID NO: 71, PM7 has been determined to have a genome consisting of the nucleotide sequence as provided in SEQ ID NO: 72, PM9 has been determined to have a genome consisting of the nucleotide sequence as provided in SEQ ID NO: 61, PM22 has been determined to have a genome consisting of the nucleotide sequence as provided in SEQ ID NO: 62, PM23 has been determined to have a genome consisting of the nucleotide sequence as provided in SEQ ID NO: 73, PM25 has been determined to have a genome consisting of the nucleotide sequence as provided in SEQ ID NO: 69, PM28 has been determined to have a genome consisting of the nucleotide sequence as provided in SEQ ID NO: 68, PM32 has been determined to have a genome consisting of the nucleotide sequence as provided in SEQ ID NO: 60, PM34 has been determined to have a genome consisting of the nucleotide sequence as provided in SEQ ID NO: 60, PM36 has been determined to have a genome consisting of the nucleotide sequence as provided in SEQ ID NO: 67, PM56 has been determined to have a genome consisting of the nucleotide sequence as provided in SEQ ID NO: 65, PM93 has been determined to have a genome consisting of the nucleotide sequence as provided in SEQ ID NO: 66 and PM94 has been determined to have a genome consisting of the nucleotide sequence as provided in SEQ ID NO: 74, as summarized in Table 10 below.

[Table 10]

Among the bred bacteriophages disclosed herein, PM4 and PM32 have been deposited under the stipulations of the Budapest treaty under the accession numbers “DSM 33478” and “DSM 33479”, respectively, with the Leibniz-Institute DSMZ (Deutsche Sammlung von Mikroorgansimen und Zellkulturen GmbH, in Braunschweig/Germany) on March 19^th, 2020 (see appended deposit receipts).

Importantly, it is to be noted that PM4 has a genome with 99.99% identity with the genome of

PM5, PM7 and PM9 over the whole length of the genome, PM4 has a genome with 98.92% identity with the genome of PM22 over the whole length of the genome (99.92% identity over

99% of the genome), PM4 has a genome with 98.91% identity with the genome of PM23 over the whole length of the genome, PM4 has a genome with 97.87% identity with the genome of PM25 over the whole length of the genome (99.87% identity over 98% of the genome), PM4 has a genome with 98.96% identity with the genome of PM28 over the whole length of the genome (99.96% identity over 99% of the genome), PM4 has a genome with 98.88% identity with the genome of PM32 over the whole length of the genome (99.88% identity over 99% of the genome), PM4 has a genome with 96.90% identity with the genome of PM34 over the whole length of the genome (99.90% identity over 97% of the genome), and PM4 has a genome with 96.83% identity with the genome of PM36 over the whole length of the genome (99.82% identity over 97% of the genome), when aligned with Blast2 sequences (Zheng Zhang, Scott Schwartz, Lukas Wagner, and Webb Miller (2000), "A greedy algorithm for aligning DNA sequences", J Comput Biol 2000; 7(1 -2):203- 14.). It can therefore be concluded that any bacteriophage having a genome with at least 98% identity with the genome of PM4 is likely to achieve a broad host range in a phage cocktail in combination with a Romulus/Remus-like bacteriophage and/or a bred phage thereof such as, e.g, PM56 and PM93.

As mentioned above, PM4 has been deposited under the stipulations of the Budapest treaty under the accession number “DSM 33478” with the Leibniz-Institute DSMZ (Deutsche Sammlung von Mikroorgansimen und Zellkulturen GmbH, in Braunschweig/Germany) on March 19^th, 2020 (see appended deposit receipt) and its nucleotide sequence has further been determined as consisting of 148.625 bp as depicted in SEQ ID NO: 4. PM4 has 234 predicted protein coding sequences (CDS) and 4 predicted tRNAs. After annotation with Prokka and subsequent annotation of individual CDSs by BLAST, a function or homology to previously characterized genes can be assigned to 122 CDS (52%). It has direct terminal repeats (DTR) of 8343 bp at either end of the genome (bp 1-8343 and 140282 - 148625), on each of which there are 22 predicted CDSs.

Overall, the pairwise identity of PM4 to 03, 05 and 04 is 95.8%, 83.5% and 92%, respectively. The genome sequence of PM4 can be characterized by an ISP-like backbone genome, where, as depicted in Fig. 4 and Table 11 below, there are one or more integrated genome fragments of 04 and 05 especially in the terminal repeats. As an example of ISP-like bacteriophage, 03 is herein provided. However, a person skilled in the art readily understands that another ISP- like bacteriophage (or multiple other ISP-like bacteriophages in combination) may be used in the breeding method provided herein instead of 03, provided that said ISP-like bacteriophage (or said multiple other ISP-like bacteriophages in combination) contains in its genome the genome fragments identified below (see Table 12). The genome sequence of PM4 has been investigated in more details and might be divided into 25 stretches (or 25 genome fragments). The origin (i.e. 03, 04 or 05) of each stretch has been identified and compared to the corresponding stretches of other bred bacteriophage genomes obtained by the same method but in separate experiments (see Table 4). The results are shown in Table 11 below. The positions on the left-hand side of the Table correspond to the position on the genome of PM4 as depicted in SEQ ID NO: 4. The first row depicts the KHR measured for each bred phages. The content of the Table indicates the origin of each stretch, i.e. whether the stretch is a genome fragment from 03, 04 or 05. The genome sequence from 1 to 8343 is identical to the genome sequence from f 40285 to f 48627, therefore the same stretch numbering has been used. When appropriate, the SEQ ID NO providing the nucleotide sequence of the corresponding PM4 stretch has been indicated on the first left-hand side column.

[Table 11]

As depicted in Table 11 above, the nucleotide sequences / genomes of the mosaic bacteriophages displaying the surprising broad host-range have been analyzed and it has been surprisingly found that they all share structural features, i.e. nucleotide sequences / stretches / genome fragments. Accordingly, without being bound by any theory, it is thought that the advantageous activity displayed by the mosaic bacteriophages of the invention is linked to the presence of specific stretches in the genome of said mosaic bacteriophages. The mosaic bacteriophages are therefore characterized by specific genomic information and combination of such genomic information which is inherited from the ancestor bacteriophages. For example, the mosaic bacteriophages obtained by the PMAP, comprise at least one genome fragment / nucleotide sequence inherited from the first ancestor bacteriophage such as, e.g., the nucleotide sequence as provided in SEQ ID NO: 9 (i.e. “stretch 13” of Table 11 inherited from “05”), at least one genome fragment / nucleotide sequence inherited from the second ancestor bacteriophage such as, e.g., the nucleotide sequence as provided in SEQ ID NO: 17 (i.e. “stretch 6” of Table 11 inherited from “04”) and at least one genome fragment / nucleotide sequence inherited from the third ancestor bacteriophage such as, e.g., the nucleotide sequence as provided in SEQ ID NO: 31 (i.e. “stretch 10” of Table 11 inherited from “03”) or alternatively the nucleotide sequence as provided in any one of SEQ ID NOs: 26, 34, 38, and 41 (i.e. stretches 8, 14, 16, and 20 of Table 11, respectively, inherited from “03”). As depicted in Table 11 above, the bred bacteriophages with the broadest host range have a genome which mainly originates from the ISP-like ancestor bacteriophage (03). Therefore, without being bound by any theory, the bacteriophages of the invention might preferably comprise a backbone genome which originates from an ISP-like bacteriophage (such as, e.g., 03). From this ISP-like (e.g., 03) backbone genome, some of the stretches such as stretches 8, 10, 12, 14, 16, 20, 21 and 23 are shared by all the compared bred bacteriophages whereas oher stretches are independently coming from different ancestors. Therefore, without being bound by any theory, it is though that the ISP-like (e.g., 03) backbone genome might comprise stretches of importance for the broad host range activity of the bred bacteriophages of the invention, namely stretches 8, 10, 14, 16 and 20. Importantly, all the identified as important stretches which originate from the specific ISP-like ancestor bacteriophage used herein, namely 03, are 100% identical on a DNA and/or a protein level to corresponding stretches in the genome of phage ISP or other ISP-like or K-like bacteriophages such as phage K, Sbl and G1 as shown in Table 11 below.

[Table 12]

'The codes in brackets represent the NCBI accession numbers

Stretch 14 does not exhibit 100% pairwise identity with another ISP-like phage at the DNA level, however, for every single one of the 12 CDSs included in this stretch, a 100% match on an amino acid level with either phage K or G1 can be found. Likewise, each of the CDSs on stretch 16 is 100% identical on an amino acid level to corresponding CDSs in ISP or phage K. Therefore, a skilled person understands that such stretches might be obtained by an ISP-like ancestor bacteriophage other than 03 or a K-like ancestor bacteriophage in combination with a an ISP-like ancestor bacteriophage other than 03. Thus, 03 is merely an example of ISP-like ancestor bacetriophage that may be used as ancestor bacteriophage in the input phage mixture of the herein provided breeding method in order to obtain the inventive mosaic bacteriophages of the invention. Moreover, it can be observed that all bred mosaic bacteriophages which have been compared have integrated in their genomes one stretch which originate form 04 (stretch 6) and one stretch which originate from 05 (stretch 13). Since the bred bacteriophages compared in Table 11 have been otbained by separate experiments (see Table 4), and these two stretches have different boundaries on the different phages (data not shown), the incorporation of the two stretches in the ISP-like backbone genome should have been obtained by at least two independent recombination events. Without being bound by any theory, it is thought that these stretches are of high importance for the broad host range activity of the mosaic bacteriophages of the invention.

Furthermore, it appears from Table 11 that the bred bacteriophages PM4, PM22, PM28, PM32 and PM34, with broader host-range than the bacteriophages PM25 and PM36, can be distinguished from the two latter by stretch 2 which originates from 05, and stretch 4 which originates from 03. Without being bound by any theory, it is therefore thought that these two stretches might be of importance for the broad host range activity of the mosaic bacteriophages of the invention.

Preferably, said mosaic bacteriophage possessing a broad host range is bacteriophage having a genome with at least 98% identity with the genome of PM4 and comprising the stretch 6, 8, 10, 13, 14, 16 or 20 of PM4, respectively represented by SEQ ID NOs: 17, 26, 31, 9, 34, 38, and 41, or any combinations thereof.

Example 8 - Increased virulence of the bred phages compared to ancestors

The increased KHR of the bred phages over the ancestors indicates that, at a multiplicity of infection (MOI: number of phages per bacterial cell) of 10, these phages inhibited the growth of more S. aureus strains than the ancestor phages (Example 2). The strains that were not inhibited over 24 h were either not susceptible, or bacteriophage insensitive mutants (BIMs) were formed over the course of the infection. Therefore, these effects were dissected for a subset of six strains, by characterizing the virulence of PM4, 03, 04 and 05. For each of these strains, CC239-MRSA-III (2017-046), CC30-MRSA-IY (2011-278), CC25-MSSA (B91), CC12- MSSA (A161), CC22-MRSA-IV (A257, “Bamim”) and CC22-MRSA-IY (B311, “Bamim”), in Example 2 (see Figure 2), growth in suspension was suppressed by PM4 but not by any of the tested ancestors. The method of determining the virulence curves was adapted from (Storms et al., 2020). S. aureus strains CC239-MRSA-III (2017-046), CC30-MRSA-IV (2011-278), CC25-MSSA (B91), CC12-MSSA (A161), CC22-MRSA-IY (A257, “Bamim”) and CC22-MRSA-IV (B311, “Bamim”) were grown to log phase (each of these strains are available in the publicly accessible section of the Leibniz-Institute DSMZ, Braunschweig, Germany, under the accession numbers as indicated in Table 13). Then, using a 96-well plate, the bacteria were mixed with phages PM4, 03, 04 and 05, each at a final concentration of 5 x 10⁸ PFU/mL in the wells, or buffer as a control. The bacterial concentration was adjusted to final concentrations in the wells of 5 x 10⁸ CFU/mL (MOI=l), 5 x 10⁷ CFU/mL (MOI=10), 5 x 10⁶ CFU/mL (MOI=100), 5 x 10⁵ CFU/mL (MOI= 1,000), 5 x 10⁴ CFU/mL (MOI= 10,000), and 5 x 10³ CFU/mL (MOI= 100,000). The growth medium was Brain-Heart Infusion broth (BHI), Carl Roth. The reaction plates were incubated at 37 °C for 24 h in a Tecan microplate reader. ODr_>oo was measured every 5 min and further analyzed in Excel.

The S. aureus strains used in this example are available in the publicly accessible section of the Leibniz-Institute DSMZ (Braunschweig, Germany) under the strain name and accession numbers as indicated in Table 13 below.

[Table 13]

The area under the OD600 curves was calculated from time of infection until 24 h, for the phage and for the buffer-treated samples. The blank area (background OD) was subtracted in both cases. The local virulence of each phage at each MOI was calculated as one minus the ratio of the area under the OD600 curve of the phage-treated and untreated samples, as depicted in Figure 5 A. Each 24h time kinetic was measured in triplicate.

As shown in Figure 5B, phage PM4 fully suppresses the growth of strain CC239-MRSA-III (2017-046) at a MOI as low as 10 for 24 h, while the ancestors 03 and 04 each need a MOI of 10,000 to achieve the same effect. 05 cannot suppress the growth of this strain at any MOI. Figure 5C depicts the local virulence curve calculated from the data of experiments of Figure 5B and shows that the virulence of PM4 is higher than the virulence of any of its ancestors at any MOI for this strain. Analogous measurements were done for strains CC30-MRSA-IY (2011-278) and CC25-MSSA (B91) in Figure 5D,E and Figure 5F,G, respectively, with similar results: the virulence of PM4 is strongly increased on all these strains in comparison to its ancestors. Further measurements showed a similar increase of virulence on strains CC12- MSSA (A161), in Figure 5H, I CC22-MRSA-IV (A257, “Bamim”), in Figure 5J,K and CC22- MRSA-IY (B311, “Bamim”) in Figure 5L, M. It is to be noted that the inability of 04 to suppress growth at MOI=10 for strains CC12-MSSA (A161), CC22-MRSA-IV (A257, “Bamim”) and CC22-MRSA-IV (B311, “Bamim”) under the conditions described in Example 2 (see Figure 2) is not a contradiction to the data presented in Figure 5 H, J, and L. In fact, in this Example 8, both the starting phage concentration (5xl0⁸ PFU/ml) and the starting bacterial concentration at MOI=10 (5xl0⁷ CFU/ml) was ten times higher than under the conditions of Example 2. It can be reasonably expected by the person in the art that the phage infection is faster and more efficient when the concentration of phages and bacteria are increased by a factor 10. The increased KHR of PM4 vs. its ancestors (see Example 2), on the full panel of 110 strains at MOI = 10, indicates that the vimlence increased on more than the three strains analyzed in detail in Figure 5. Absence of bacterial growth over 24 h for 60% of the strains when treated with PM4 also indicates that no (fast-growing) BIMs could form on any of these strains.

As the KHR was increased on multiple bred phages, based on the detailed analysis for PM4 the person skilled in the art can reasonably conclude that also the vimlence of the other bred phages increased. The increased vimlence of the bred phages hints at a more efficient replication cycle, were either of the adsorption rate, the latency period and/or the burst size are improved. This would lead to more efficient killing of the bacteria, and therefore a higher potency of the bred phages compared to their ancestors, when used to treat bacterial infections, for example in humans.

Example 9 - Comparative alignments

The % pairwise identity of bacteriophages described herein, particularly PM4, PM9, PM32, PM56 and PM93 to known published phages has been calculated by using the blastn suite on NCBI (https://blast.ncbi.nlm.nih.gov/Blast. cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LI NK_LOC=blasthome) and selecting the option to align two or more sequences. The % query coverage and % identity have been multiplied to get the overall % overall identity shown in Table 14 below.

[Table 14]

When compared with blastn, PM4 has a genome sequence with 96.9% identity with the genome sequence of Sa83 over the whole length of the genome sequence of PM4 (99.9% identity over 97% of the genome sequence). According to the present disclosure, “over the whole length of the genome sequence of PM4” means over 100% of the genome sequence of PM4 as provided in SEQ ID NO: 4. While the identity percentage is a high number, there are striking differences between the two phages. Importantly, the nucleotide sequence as provided in SEQ ID NO: 9 does not exist in the genome sequence of Sa83. In other words, while the genome sequence of the bacteriophage PM4 comprises the nucleotide sequence as provided in SEQ ID NO: 9, which is one of the “stretches” found in the bred mosaic bacteriophage with improved properties (stretch 13 from Table 11), the nucleotide sequence as provided in SEQ ID NO: 9 is absent from the genome of the bacteriophage Sa83 (the sequence identity to the corresponding stretch on Sa83 is only 53.8%), confirming that PM4 and Sa83 are two distinct bacteriophages. Likewise, while the genome sequence of the bacteriophage PM4 comprises the nucleotide sequence as provided in SEQ ID NO: 17, which is another one of the “stretches” found in the bred mosaic bacteriophage with improved properties (stretch 6 from Table 11), the nucleotide sequence as provided in SEQ ID NO: 17 is absent from the genome of the bacteriophage Sa83 (the sequence identity to the corresponding stretch on Sa83 is 98.4%).

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Claims

1. A composition comprising

(i) a first bacteriophage, which is a mosaic bacteriophage comprising at least one genome fragment of a first ancestor bacteriophage, at least one genome fragment of a second ancestor bacteriophage and at least one genome fragment of a third ancestor bacteriophage, wherein said first ancestor bacteriophage and said second ancestor bacteriophage are each an 812/K-like bacteriophage and said third ancestor bacteriophage is an ISP-like bacteriophage; and

(ii) a second bacteriophage.

2. The composition according to claim 1, wherein the first ancestor bacteriophage has the genome selected from the group consisting of:

(a) a genome comprising the genome fragment as provided in SEQ ID NO: 5 or a genome fragment with at least 90% identity with SEQ ID NO: 5;

(b) a genome comprising a genome fragment with at least 85% identity with SEQ ID NO: 6, a genome fragment with at least 65% identity with SEQ ID NO: 9 and/or a genome fragment with at least 99% identity with SEQ ID NO: 11;

(c) a genome with at least 80% identity with SEQ ID NO: 1 and comprising a combination of the genome fragments as provided in SEQ ID NOs: 5, 6, 9 and 11;

3. The composition according to claim 1 or 2, wherein the first ancestor bacteriophage is a bacteriophage having a genome with at least 99% identity with SEQ ID NO: 1.

4. The composition according to any one of claims 1 to 3, wherein the first ancestor bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 1.

5. The composition according to any one of claims 1 to 4, wherein the second ancestor bacteriophage has the genome selected from the group consisting of:

(a) a genome comprising the genome fragment as provided in SEQ ID NO: 15;

6. The composition according to any one of claims 1 to 6, wherein the second ancestor bacteriophage is a bacteriophage having a genome with at least 99% identity with SEQ ID NO: 2.

7. The composition according to any one of claims 1 to 6, wherein the second ancestor bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 2.

8. The composition according to any one of claims 1 to 8, wherein the third ancestor bacteriophage has the genome selected from the group consisting of:

(a) a genome comprising the genome fragment as provided in SEQ ID NO: 21;

(b) a genome comprising a genome fragment with at least 99% identity with SEQ ID NO: 22, a genome fragment with at least 99% identity with SEQ ID NO: 26, a genome fragment with at least 99% identity with SEQ ID NO: 31, a genome fragment with at least 99% identity with SEQ ID NO: 34, a genome fragment with at least 99% identity with SEQ ID NO: 38and/or a genome fragment with at least

99% identity with SEQ ID NO: 41; (c) a genome with at least 80% identity with SEQ ID NO: 3 and comprising a combination of the genome fragments as provided in SEQ ID NOs: 21, 22, 26. 31, 34, 38, 41, 45, 49, 53, and 57;

(d) a genome comprising a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 23, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 24, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 25, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 27, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 28, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 29, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 30, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 32, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 33, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 35, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 36, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 37, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 39, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 40, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 42, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 43, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 44, and optionally a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 46, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 47, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 48, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 50, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 51, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 52, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 54, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 55, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 56, a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 58, and/or a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 59; and

9. The composition according to any one of claims 1 to 8, wherein the third ancestor bacteriophage is a bacteriophage having a genome with at least 99% identity with SEQ ID NO: 3.

10. The composition according to any one of claims 1 to 9, wherein the third ancestor bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 3.

11. The composition according to any one of claims 1 to 10, wherein the mosaic bacteriophage has a backbone genome which originates from the third ancestor bacteriophage and comprises at least one genome fragment of the first ancestor bacteriophage and one genome fragment of the second bacteriophage.

12. The composition according to any one of claims 1 to 11, wherein the mosaic bacteriophage has a genome which comprises the genome fragment as provided in SEQ ID NO: 9, or a genome fragment with at least 65% identity with SEQ ID NO: 9.

13. The composition according to any one of claims 1 to 12, wherein the mosaic bacteriophage has a genome which comprises the genome fragment as provided in SEQ ID NO: 6, or a genome fragment with at least 85% identity with SEQ ID NO: 6.

14. The composition according to any one of claims 1 to 13, wherein the mosaic bacteriophage has a genome which comprises the genome fragment as provided in SEQ ID NO: 17, or a genome fragment with at least 98% identity with SEQ ID NO: 17.

15. The composition according to any one of claims 1 to 14, wherein the mosaic bacteriophage has a genome which comprises a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 23.

16. The composition according to any one of claims 1 to 15, wherein the mosaic bacteriophage has a genome which comprises a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 24.

17. The composition according to any one of claims 1 to 16, wherein the mosaic bacteriophage has a genome which comprises a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 25.

18. The composition according to any one of claims 1 to 17, wherein the mosaic bacteriophage has a genome which comprises the genome fragment as provided in SEQ ID NO: 22, or a genome fragment with at least 98% identity with SEQ ID NO: 22.

19. The composition according to any one of claims 1 to 18, wherein the mosaic bacteriophage has a genome which comprises a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 27.

20. The composition according to any one of claims 1 to 19, wherein the mosaic bacteriophage has a genome which comprises a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 28.

21. The composition according to any one of claims 1 to 20, wherein the mosaic bacteriophage has a genome which comprises a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 29.

22. The composition according to any one of claims 1 to 21, wherein the mosaic bacteriophage has a genome which comprises a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 30.

23. The composition according to any one of claims 1 to 22, wherein the mosaic bacteriophage has a genome which comprises the genome fragment as provided in SEQ ID NO: 26, or a genome fragment with at least 98% identity with SEQ ID NO: 26.

24. The composition according to any one of claims 1 to 23, wherein the mosaic bacteriophage has a genome which comprises a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 32.

25. The composition according to any one of claims 1 to 24, wherein the mosaic bacteriophage has a genome which comprises a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 33.

26. The composition according to any one of claims 1 to 25, wherein the mosaic bacteriophage has a genome which comprises the genome fragment as provided in SEQ ID NO: 31, or a genome fragment with at least 98% identity with SEQ ID NO: 31.

27. The composition according to any one of claims 1 to 26, wherein the mosaic bacteriophage has a genome which comprises a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 35.

28. The composition according to any one of claims 1 to 27, wherein the mosaic bacteriophage has a genome which comprises a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 36.

29. The composition according to any one of claims 1 to 28, wherein the mosaic bacteriophage has a genome which comprises a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 37.

30. The composition according to any one of claims 1 to 29, wherein the mosaic bacteriophage has a genome which comprises the genome fragment as provided in SEQ ID NO: 34, or a genome fragment with at least 98% identity with SEQ ID NO: 34.

31. The composition according to any one of claims 1 to 30, wherein the mosaic bacteriophage has a genome which comprises a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 36.

32. The composition according to any one of claims 1 to 31, wherein the mosaic bacteriophage has a genome which comprises a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 37.

33. The composition according to any one of claims 1 to 32, wherein the mosaic bacteriophage has a genome which comprises the genome fragment as provided in SEQ ID NO: 38, or a genome fragment with at least 98% identity with SEQ ID NO: 38.

34. The composition according to any one of claims 1 to 33, wherein the mosaic bacteriophage has a genome which comprises a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 42.

35. The composition according to any one of claims 1 to 34, wherein the mosaic bacteriophage has a genome which comprises a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 43.

36. The composition according to any one of claims 1 to 35, wherein the mosaic bacteriophage has a genome which comprises a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 44.

37. The composition according to any one of claims 1 to 36, wherein the mosaic bacteriophage has a genome which comprises the genome fragment as provided in SEQ ID NO: 41, or a genome fragment with at least 98% identity with SEQ ID NO: 41.

38. The composition according to any one of claims 1 to 37, wherein the mosaic bacteriophage has a genome which comprises a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 46.

39. The composition according to any one of claims 1 to 38, wherein the mosaic bacteriophage has a genome which comprises a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 47.

40. The composition according to any one of claims 1 to 39, wherein the mosaic bacteriophage has a genome which comprises a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 48.

41. The composition according to any one of claims 1 to 40, wherein the mosaic bacteriophage has a genome which comprises the genome fragment as provided in SEQ ID NO: 45, or a genome fragment with at least 98% identity with SEQ ID NO: 45.

42. The composition according to any one of claims 1 to 41, wherein the mosaic bacteriophage has a genome which comprises a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 50.

43. The composition according to any one of claims 1 to 42, wherein the mosaic bacteriophage has a genome which comprises a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 51.

44. The composition according to any one of claims 1 to 43, wherein the mosaic bacteriophage has a genome which comprises a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 52.

45. The composition according to any one of claims 1 to 44, wherein the mosaic bacteriophage has a genome which comprises the genome fragment as provided in SEQ ID NO: 49, or a genome fragment with at least 98% identity with SEQ ID NO: 49.

46. The composition according to any one of claims 1 to 45, wherein the mosaic bacteriophage has a genome which comprises a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 54.

47. The composition according to any one of claims 1 to 46, wherein the mosaic bacteriophage has a genome which comprises a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 55.

48. The composition according to any one of claims 1 to 47, wherein the mosaic bacteriophage has a genome which comprises a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 56.

49. The composition according to any one of claims 1 to 48, wherein the mosaic bacteriophage has a genome which comprises the genome fragment as provided in SEQ ID NO: 53, or a genome fragment with at least 98% identity with SEQ ID NO: 53.

50. The composition according to any one of claims 1 to 49, wherein the mosaic bacteriophage has a genome which comprises a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 58.

51. The composition according to any one of claims 1 to 50, wherein the mosaic bacteriophage has a genome which comprises a genome fragment encoding the amino acid sequence as provided in SEQ ID NO: 59.

52. The composition according to any one of claims 1 to 51, wherein the mosaic bacteriophage has a genome which comprises the genome fragment as provided in SEQ ID NO: 57, or a genome fragment with at least 98% identity with SEQ ID NO: 57.

53. The composition according to any one of claims 1 to 52, wherein the mosaic bacteriophage lyses one or more of the Staphylococcus aureus strains listed in Table 1.

54. The composition according to any one of claims 1 to 53, wherein the mosaic bacteriophage lyses at least 50 % of the Staphylococcus aureus strains listed in Table 1.

55. The composition according to any one of claims 1 to 54, wherein the mosaic bacteriophage lyses at least one bacterial strain that none of the first ancestor bacteriophage, the second ancestor bacteriophage and the third bacteriophage lyses.

56. The composition according to claim 55, wherein said at least one bacterial strain is a Staphylococcus strain.

57. The composition according to claim 56, wherein said Staphylococcus strain is a Staphylococcus aureus strain.

58. The composition according to any one of claims 1 to 57, wherein the mosaic bacteriophage is a bacteriophage having at least 98% identity with the genome of the bacteriophage deposited under the accession No. DSM33478 [PM4]

59. The composition according to claim 58, wherein the mosaic bacteriophage is the bacteriophage deposited under the accession No. DSM33478 [PM4]

60. The composition according to claim 58, wherein the mosaic bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 4 [PM4]

61. The composition according to claim 58, wherein the mosaic bacteriophage is the bacteriophage deposited under the accession No. DSM33479 [PM32]

62. The composition according to claim 58, wherein the mosaic bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 60 [PM32]

63. The composition according to claim 58, wherein the mosaic bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 61 [PM9]

64. The composition according to claim 58, wherein the mosaic bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 62 [PM22]

65. The composition according to any one of claims 1 to 64, wherein the second bacteriophage is a Romulus/Remus-like bacteriophage.

66. The composition according to any one of claims 1 to 65, wherein the second bacteriophage is a bacteriophage having at least 98% identity with the genome as provided in SEQ ID NO: 63 [01] or SEQ ID NO: 64 [02]

67. The composition according to claim 66, wherein the second bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 63 [01/Remus].

68. The composition according to claim 66, wherein the second bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 64 [02/Romulus].

69. The composition according to claim 66, wherein the second bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 65 [PM56]

70. The composition according to claim 66, wherein the second bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 66 [PM93]

71. The composition according to any one of claims 1 to 70, wherein the first bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 4 [PM4] and wherein the second bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 66 [PM93]

72. The composition according to any one of claims 1 to 70, wherein the first bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 61 [PM9] and wherein the second bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 66 [PM93]

73. The composition according to any one of claims 1 to 70, wherein the first bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 73 [PM23] and wherein the second bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 65 [PM56]

74. The composition according to any one of claims 1 to 70, wherein the first bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 68 [PM28] and wherein the second bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 74 [PM94]

75. The composition according to any one of claims 1 to 70, wherein the first bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 60 [PM32] and wherein the second bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 64 [02]

76. The composition according to any one of claims 1 to 70, wherein the first bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 72 [PM7] and wherein the second bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 65 [PM56]

77. The composition according to any one of claims 1 to 70, wherein the first bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 60 [PM32] and wherein the second bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 65 [PM56]

78. The composition according to any one of claims 1 to 70, wherein the first bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 62 [PM22] and wherein the second bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 64 [02]

79. The composition according to any one of claims 1 to 70, wherein the first bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 73 [PM23] and wherein the second bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 66 [PM93]

80. The composition according to any one of claims 1 to 70, wherein the first bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 4 [PM4] and wherein the second bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 65 [PM56]

81. The composition according to any one of claims 1 to 70, wherein the first bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 72 [PM7] and wherein the second bacteriophage is the bacteriophage having the genome as provided in SEQ ID NO: 66 [PM93]

82. The composition according to any one of claims 1 to 81, wherein the composition lyses one or more of the Staphylococcus aureus strains.

83. The composition according to any one of claims 1 to 82, wherein the composition lyses at least 50%, at least 70%, at least 75%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, or at least 87% of a panel of Staphylococcus aureus strains.

84. The composition according to any one of claims 1 to 82, wherein the composition lyses at least 6 different Staphylococcus aureus clonal complexes of a panel of Staphylococcus aureus strains of different clonal complexes.

85. The composition according to claim 83 or 84, wherein the panel of Staphylococcus aureus strains is composed of the Staphylococcus aureus strains listed in Table 1.

86. The composition according to any one of claims 1 to 85, wherein the composition reduces by at least 90 % one or more pre-formed biofilms.

87. The composition according to any one of claims 1 to 86, wherein the composition reduces by at least 90%, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% pre-formed biofilms of a panel of 10 pre-formed biofilms.

88. The composition according to claim 87, wherein the panel of 10 pre-formed biofilms is a panel of 10 different biofilms from bacterial strains of 10 different clonal complexes.

89. The composition according to any one of claims 1 to 88, wherein the composition reduces by at least 99 % one or more pre-formed biofilms.

90. The composition according to any one of claims 1 to 89, wherein the composition reduces by at least 99%, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, or 60% or more pre-formed biofilms of a panel of 10 pre-formed biofilms.

91. The composition according to claim 90, wherein the panel of 10 pre-formed biofilms is a panel of 10 different biofilms from bacterial strains of 10 different clonal complexes.

92. The composition according to any one of claims 86 to 91, wherein the pre-formed biofilms are Staphylococcus pre-formed biofilms, preferably Staphylococcus aureus pre-formed biofilms.

93. The composition according to any one of claims 1 to 92, wherein the first bacteriophage and the second bacteriophage acts synergistically on reducing pre-formed biofilms.

94. The composition according to any one of claims 1 to 93 which is a pharmaceutical composition.

95. Use of the composition according to any one of claims 1 to 93 in a non-medical method of killing and/or inhibiting the growth of bacteria such as, e.g., on a surface.

96. The use according to claim 95, wherein the bacteria is Staphylococcus.

97. The use according to claim 96, wherein the bacteria is Staphylococcus aureus.

98. Use of the composition according to any one of claims 1 to 93 in a non-medical method of reducing and/or preventing pre-formed biofilms.

99. The use according to claim 98, wherein the biofilm is a Staphylococcus biofilm.

100. The use according to claim 99, wherein the biofilm is a Staphylococcus aureus biofilm.

101. A pharmaceutical composition comprising the composition according to any one of claims 1 to 93 and a pharmaceutically acceptable carrier.

102. The pharmaceutical composition according to claim 101 for use in the treatment of a bacterial infection.

103. The pharmaceutical composition for use according to claim 102, wherein said bacterial infection is a Staphylococcus bacterial infection.

104. The pharmaceutical composition for use according to claim 103, wherein said bacterial infection is a Staphylococcus aureus bacterial infection.

105. The pharmaceutical composition for use according to any one of claims 102 to 104, which is to be administered topically.

106. The pharmaceutical composition for use according to any one of claims 102 to 105, which is to be co-administered with an antibiotic agent.

107. Use of the composition according to any one of claims 1 to 93 or the pharmaceutical composition according to claim 94 or 101 for the manufacture of a medicament for the treatment of a bacterial infection.

108. The use according to claim 107, wherein the bacterial infection is a Staphylococcus infection.

109. The use according to claim 108, wherein the bacterial infection is a Staphylococcus aureus infection.

110. A method of treating or preventing a Staphylococcus aureus bacterial infection in a subject in need thereof comprising administering to said subject a therapeutically effective amount of the composition according to any one of claims 1 to 78 or the pharmaceutical composition according to claim 94 or 101.

111. A non-medical method of killing and/or inhibiting the growth of bacteria comprising applying the composition according to any one of claims 1 to 93.

112. The method according to claim 111, wherein the bacteria is Staphylococcus.

113. The method according to claim 112, wherein the bacteria is Staphylococcus aureus.

114. A non-medical method of reducing and/or preventing pre-formed biofilms comprising applying the composition according to any one of claims 1 to 93.

115. The method according to claim 114, wherein the biofilm is a Staphylococcus biofilm.

116. The method according to claim 115, wherein the biofilm is a Staphylococcus aureus biofilm.

117. The method according to any one of claims 111 to 116, wherein said composition is to be applied to a food product, a crop or a surface.

118. The method according to claim 117, wherein the surface is the skin of a mammal, equipment, medical equipment, prostheses, implant, bedding, furniture, walls, floors, or combinations thereof.

119. A bandage or wound dressing comprising the composition according to any one of claims 1 to 93, or the pharmaceutical composition according to claim 94 or 101.

120. A kit comprising the composition according to any one of claims 1 to 93, or the pharmaceutical composition according to claim 94 or 101.

121. A kit compri sing : a. A composition, a pharmaceutical composition or a bandage according to any one of the preceding claims; and b. Instructions for use of same (e.g. in medicine).

122. A bacteriophage which has the genomic sequence of any one of SEQ ID NOs: 4 60 to 62 and 65 to 74or a bacteriophage which has been deposited under the accession No. DSM33478 [PM4] or DSM33479 [PM32] or any progeny thereof.