WO2024115430A1 - Phages acinetobacter baumannii - Google Patents

Phages acinetobacter baumannii Download PDF

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Publication number
WO2024115430A1
WO2024115430A1 PCT/EP2023/083257 EP2023083257W WO2024115430A1 WO 2024115430 A1 WO2024115430 A1 WO 2024115430A1 EP 2023083257 W EP2023083257 W EP 2023083257W WO 2024115430 A1 WO2024115430 A1 WO 2024115430A1
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nucleic acid
acid sequence
phages
seq
depicted
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PCT/EP2023/083257
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English (en)
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Li Deng
Mohammadali KHAN MIRZAEI
Jinling XUE
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Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH)
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Publication of WO2024115430A1 publication Critical patent/WO2024115430A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/00021—Viruses as such, e.g. new isolates, mutants or their genomic sequences

Definitions

  • the present invention relates to a composition comprising isolated phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 , and further comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 11 , wherein the phages comprise a nucleic acid being part of its genome, wherein the nucleic acid comprises a nucleic acid sequence, which is at least 60% identical to the nucleic acid sequence encoding the amino acid sequence as depicted in SEQ ID NO: 1 and 11 and wherein the isolated phages are capable of infecting Acinetobacter baumannii.
  • the present invention also relates to a pharmaceutical composition comprising said composition, and to said composition or said pharmaceutical composition for use in therapy as well as for use in a method of treating or preventing an infection of a subject with Acinetobacter baumannii. Additionally, the present invention relates to kits and to a method of preparing said composition comprising isolated phages and a composition comprising isolated phages obtainable or obtained by said method.
  • Acinetobacter is often susceptible or resistant to many antibiotics, so that the threat level was escalated to urgent because of the emergence of easily spread resistance in Acinetobacter and the lack of current antibiotics, and antibiotics in development, to treat these infections.
  • Antimicrobial resistance is a major threat to global health and global economies. Worldwide, infections by multiple-drug resistant bacteria are estimated to kill more than 700,000 people, including 25,000 cases in Europe and 23,000 cases in the US every year (Laximinarayan et al., The Lancet Infect Diseases (2013), 13: 1057-1098). Antimicrobial resistance also places a tremendous burden on healthcare systems and society, with an annual cost due to healthcare expenditures and productivity losses estimated by the European Commission at approximately €1.5 billion in the EU.
  • bacteriophages also called phages
  • viruses that infect bacteria can be employed to fight AMR as a complement to antibiotic therapy
  • AMR antibiotic therapy
  • phages are the most abundant biological entities on earth, with ⁇ 10 31 phages in total and about 10 23 phage infections per second. Phages are highly specific in their choice of bacterial hosts, mediated by specific receptor proteins, thus will cause no collateral damage to the microbial companions that form our microbiome, which is increasingly appreciated as essential to our health (Shreiner et al., Curr Op Gastroenterol (2015), 31: 69-75).
  • phages After over a billion years of co-evolutionary struggle with their bacterial hosts, phages have evolved highly diverse proteins that inhibit or ‘hijack’ bacterial metabolic processes to their own benefit.
  • phageinsensitive mutants such as Acinetobacter baumannii.
  • the inventors successfully identified and characterized a composition comprising phages which have been isolated from samples inter alia by the proprietary high-throughput “viral tagging” as it is disclosed by WO2021/048257 incorporated by references herein and by classical phage isolation methods, and which phages comprised by the composition of the invention are against a multidrug resistant Acinetobacter baumannii (short. A. baumannii) bacteria so that such phages are capable of infecting A. baumannii. This results in replication of phage genome within said host cell and lysis of such bacteria.
  • the major problem associated with phage therapy namely the emergence of phage-insensitive mutants (BIMs) such as A.
  • baumannii which are bacterial mutants of phage-sensitive bacteria, due to mutations affecting bacterial-surface molecules, thereby interfering with the phage attachment, and thus being phage resistant, could be solved by preventing the development of Acinetobacter resistance with the particular composition of the present invention comprising said isolated phages.
  • the inventors were also able to mitigate biofilm formation in the A. baumannii strain, thus resulting in an efficient bacterial removal of the multiple-drug resistant A. baumannii and therefore minimizing the abovementioned problems with phage resistance and also AMR.
  • the isolated A. baumannii phages comprised within the composition of the invention preferably all hybridize with phage nucleic acid to two particular hooks as defined by the amino acid sequences of SEQ ID NOs: 1 and 11 (see Figure 3D).
  • the present invention comprises a composition comprising isolated phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1, and wherein the composition further comprises isolated phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 11 , wherein the phages comprise a nucleic acid being part of its genome, wherein the nucleic acid comprises a nucleic acid sequence, which is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least
  • Said signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in any one selected from the group consisting of SEQ ID NOs: 1-15, the isolated phages hybridize under stringent conditions to, are considered as the so called “hook(s)”.
  • the phages comprised in a sample as defined elsewhere herein were labeled by the viral tagging procedure as defined by WO2021/048257, before target bacteria (A. baumannii) were added into the samples comprising the labelled phages for phage isolation. After isolation of said A. baumannii phages by flow cytometry and sorting, A.
  • baumannii bacteria were lysed to extract viral DNA as explained further in the Example section and the phage genome was then analyzed performing VT metagenomics sequencing and bioinformatics analysis (including protein clustering) as also described in the Examples.
  • the genome of the isolated Acinetobacter phages, viral contigs generated by said viral tagging as defined by the Examples, and the Acinetobacter phage genomes from the NCBI RefSeq database were then used to create a gene catalogue, where the proteins of these genes were clustered into protein families and annotated.
  • the hook as defined by said signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 refers to a protein family of a metallo-phosphoesterase which is one of the most abundant proteins within the A. baumannii phages which have been analysed by metagenomics sequencing. Meaning a targeted A. baumannii phage isolation using such particular hook allows to isolate the most divergent phages which are then comprised in said composition of the invention.
  • the present invention further encompasses pharmaceutical composition comprising said composition as defined elsewhere herein.
  • the invention additionally comprises in a third aspect said composition as defined elsewhere herein or said pharmaceutical composition for use in therapy.
  • the invention envisages said composition or said pharmaceutical composition as defined elsewhere herein for use in a method of treating or preventing an infection of a subject with A baumannii.
  • the present invention also comprises a kit comprising the composition as defined elsewhere herein or the pharmaceutical composition as defined elsewhere herein.
  • the present invention also comprises a kit comprising a first nucleic acid comprising a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1, or a complementary sequence thereof, and further comprising a second nucleic acid comprising a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 11 , or a complementary sequence thereof.
  • the present invention comprises a kit comprising a first nucleic acid comprising a nucleic acid sequence, which is at least 90% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 or a complementary sequence thereof, and further comprising a second nucleic acid comprising a nucleic acid sequence, which is at least 90% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 11, or a complementary sequence thereof.
  • the invention also encompasses a method of preparing a composition comprising isolated phages, the method comprising: targeted isolating of phages comprised in a provided sample, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and further comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 11 , and wherein the phages comprise a nucleic acid being part of its genome, wherein the nucleic acid comprises a nucleic acid sequence, which is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%
  • FIG. 1 A heatmap of the presence-absence matrix shows protein families (x-axis) in different viral clusters (y-axis).
  • Fig. 2 (A) Genome of vB_AbM_HMGU, vB_AbS_HMGU, and vB_AbA_HMGU; arrows indicate the predicted CDSs and the directions of arrows indicate the direction of transcription. (B) Transmission electron microscopy of vB_AbS_HMGU. (C) Infection kinetics of vB_AbS_HMGU such as adsorption rate, latency period, and burst size of vB_AbS_HMGU.
  • FIG. 3 The fluorescent staining of the bacterial biofilm under different treatment scenarios. Live cell (green, depicted as arrow) and dead cells (red, depicted with circles); (A) negative control; (B) phage vB_AbM_HMGU; (C) vB_AbA_HMGU; (D) combination of phages vB_AbM_HMGU and vB_AbA_HMGU.
  • the term "at least" preceding a series of elements is to be understood to refer to every element in the series.
  • the term “at least one” refers to one, two, three or more such as four, five, six, seven, eight, nine, ten and more.
  • the term “about” means plus or minus 20%, preferably plus or minus 10%, more preferably plus or minus 5%, most preferably plus or minus 1%.
  • a “composition” comprising the phages which have been isolated as defined elsewhere herein, refers to any kind of composition which comprises these isolated phages, e.g. naturally- occurring I environmental samples, a nutritional composition, a cosmetic composition or the like.
  • Said composition can be a liquid (preferably aqueous), a solid, a gel, a powder, a paste, an ointment, a capsule, a food product etc. Further comprised herein is a dried or frozen form of the composition as defined herein.
  • compositions described herein may be stored directly in liquid form for later use, stored in a frozen state and thawed prior to use, or prepared in dried form, such as a lyophilized, air-dried, or spray-dried form, for later reconstitution into a liquid form or other form prior to use.
  • a composition described herein may be stored by any method known to one of skill in the art. Non-limiting examples include cooling, freezing, lyophilizing, and spray drying the formulation, wherein storage by cooling is preferred.
  • composition of the invention comprising said isolated phages may further comprise one or more ingredients, by the way of illustration and not limitation, such as an excipient, a preservative, an ingestible support, a flavour, a solubilizer, a wetting agent, a sweetener, a colorant, a coating agent, or an antioxidant.
  • the excipients of the composition may refer to diluents such as, e.g. water, saline, glycerol, ethanol, bacteriostatic water for injection (BWFI), Ringer's solution, dextrose solution, or aqueous solutions of salts and/or buffers etc.
  • composition of the present invention may further comprise at least one excipient as defined herein, preferably a buffer (pH buffering agent) as excipient.
  • excipients as defined herein, preferably a buffer (pH buffering agent) as excipient.
  • substances necessary for formulation purposes may be comprised in said composition as acceptable excipients such as emulsifying agents, stabilizing agent, and/or surfactants known to a person skilled in the art.
  • buffer or “pH buffering agent” as used herein, includes those agents that maintain the pH in a desired range.
  • a buffer is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugated acid. It has the property that the pH of the solution changes very little when a small amount of a strong acid or base is added. Buffer solutions are used as a means of keeping pH at a nearly constant value in a wide variety of chemical applications.
  • a buffer when applied in the composition of the invention preferably stabilizes the isolated phages.
  • PBS or sodium bicarbonate buffer is used as a buffer being further comprised by the composition of the present invention.
  • the term “isolated” refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) designed, produced, prepared, and/or manufactured by the hand of man.
  • the phages which are isolated may refer to the substance and/or entity separated from samples such as sewage water samples by applying at least any one of the defined hooks which have been explicitly designed / prepared by the inventors as explained above and in the Example section in more detail.
  • Such “targeted phage isolation” (“hook isolation”) refers to applying viral tagging as defined in the Example section and as it is disclosed by WO2021/048257, followed by phage isolation methods as defined elsewhere herein and qPCR, where the already designed hook(s) are used as primers.
  • a baumannii bacterial cells as target bacteria are applied, whereby such labeled phages get into contact with said target cells by binding to receptors on the surface of A. baumannii.
  • nucleic acid molecule can be used interchangeably with the term “nucleic acid” and means a polymer comprising one, two, or more nucleotides, e.g., single- or double stranded.
  • nucleic acids may comprise inter alia DNA molecules, RNA molecules, oligonucleotide thiophosphates, substituted ribooligonucleotides or PNA molecules.
  • such term may refer to DNA or RNA or hybrids thereof or any modification thereof that is known in the art (see, e.g., US 5525711, US 471 1955, US 5792608 or EP 302175 for examples of modifications).
  • the nucleic acid may be single- or double- stranded, linear or circular, natural or synthetic, and without any size limitation.
  • the nucleic acid may be genomic DNA, cDNA, mitochondrial DNA, mRNA, antisense RNA, ribozymal RNA or a DNA encoding such RNAs or chimeroplasts (Gamper, Nucleic Acids Research, 2000, 28, 4332 - 4339).
  • a nucleic acid described herein may also be a fragment of the nucleic acid. Particularly, such a fragment is a functional fragment. Examples for such functional fragments are nucleic acids which can serve as primers.
  • nucleic acid also comprising a nucleic acid sequence as known to a skilled person is part of the phage genome, and hybridizes under stringent conditions to a signature sequence or its complementary sequence as defined elsewhere herein.
  • the term “being part of its genome” means that the nucleic acid (nucleic acid comprising a nucleic acid sequence) of the isolated phages belongs to the phage genome I is (integrated into) phage genome.
  • phage genome DNA or RNA
  • A. baumannii in the invention is comprised in the head of the phage and is then injected into the target bacteria (A. baumannii in the invention) for replication of its genome within said host cell.
  • Such nucleic acid can also be considered as “phage nucleic acid”.
  • Such phage nucleic acid comprising a nucleic acid sequence may refer to a phage nucleic acid comprising a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence encoding the amino acid sequence as depicted in any one selected from the group consisting of SEQ ID NOs: 1-15, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence encoding the amino acid sequence as depicted in SEQ ID NO: 1 ; at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence encoding the amino acid sequence as depicted in SEQ ID NO: 2
  • nucleic acid sequence as known to a person skilled in the art can also be used interchangeably with the term “nucleic acid sequence”.
  • sequence identity is meant a property of sequences that measures their similarity or relationship.
  • sequence identity or “identity” as used in the present invention means the percentage of pair-wise identical residues - following (homology) alignment of a sequence - in the present invention the phage nucleic acid sequence - with a sequence in question (the nucleic acid sequence encoding the amino acid sequence as depicted in any one selected from the group consisting of SEQ ID NOs: 1-15) with respect to the number of residues in the longer of these two sequences. Identity is measured by dividing the number of identical residues by the total number of residues and multiplying the product by 100.
  • the percentage of sequence identity can, for example, be determined herein using the program BLASTP, version blastp 2.2.5 (November 16, 2002; cf. Altschul, S. F. et al. (1997) Nucl. Acids Res.25, 3389- 3402).
  • sequences e.g., nucleic acid sequences or amino acid sequences
  • identity may refer to the shorter sequence and that part of the longer sequence that matches said shorter sequence.
  • the degree of identity may preferably either refer to the percentage of nucleotide residues in the shorter sequence which are identical to nucleotide residues in the longer sequence or to the percentage of nucleotides in the longer sequence which are identical to nucleotide sequence in the shorter sequence.
  • identity levels of nucleic acid sequences or amino acid sequences may refer to the entire length of the respective sequence and is preferably assessed pair-wise, wherein each gap is to be counted as one mismatch.
  • nucleic acid/amino acid sequences having the given identity levels to the herein-described particular nucleic acid/amino acid sequences may represent derivatives/variants of these sequences which, preferably, have the same biological function. They may be either naturally occurring variations, for instance sequences from other varieties, species, etc., or mutations, and said mutations may have formed naturally or may have been produced by deliberate mutagenesis. Furthermore, the variations may be synthetically produced sequences. The variants may be naturally occurring variants or synthetically produced variants, or variants produced by recombinant DNA techniques.
  • Deviations from the above-described nucleic acid sequences may have been produced, e.g., by deletion, substitution, addition, insertion and/or recombination.
  • the term “addition” refers to adding a nucleic acid residue/amino acid to the beginning or end of the given sequence, whereas “insertion” refers to inserting a nucleic acid residue/amino acid within a given sequence.
  • the term “deletion” refers to deleting or removal of a nucleic acid residue or amino acid residue in a given sequence.
  • substitution refers to the replacement of a nucleic acid residue/amino acid residue in a given sequence.
  • hybridize refers to “bind” or “binding”, or “being complementary to”, e.g. with regard to the phages to be isolated from an A. baumannii phage colony by applying such hook(s) as defined herein where the phages hybridize to.
  • nucleic acid comprising a nucleic acid sequence
  • the nucleic acid within the phage genome binds to the defined hook, namely a signature sequence or its complementary sequence comprising a nucleic acid sequence which encodes an amino acid sequence as depicted in any one of SEQ ID NOs: 1- 15.
  • Such term may also comprise hybridizing to at least a portion of the signature sequence or its complementary sequence as defined herein, which means that said phage nucleic acid hybridizes or is complementary to at least about 3, 4, 5, 6, 7, 8, 9, 20, 11, 12, 13, 14 or 15 (preferably consecutive) nucleotides of the nucleotide sequence comprised by the signature sequence or its complementary sequence encoding any one of the amino acid sequence as shown in SEQ ID NOs: 1-15.
  • the nucleic acid being part of the phage genome hybridizes under stringent conditions to at least about 3, 4, 5, 6, 7, 8, 9, 20, 11 , 12, 13, 14 or 15 (preferably consecutive) nucleotides of the nucleotide sequence comprised by the signature sequence or its complementary sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 ;
  • the phage nucleic acid being part of the phage genome hybridizes under stringent conditions to at least about 3, 4, 5, 6, 7, 8, 9, 20, 11 , 12, 13, 14 or 15 (preferably consecutive) nucleotides of the nucleotide sequence comprised by the signature sequence or its complementary sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2;
  • the phage nucleic acid being part of the phage genome hybridizes under stringent conditions to at least about 3, 4, 5, 6, 7, 8, 9, 20, 11 , 12, 13, 14 or 15 (preferably consecutive) nucleotides of the nucleotide sequence comprised
  • hybridization means for hybridizations under stringent or nonstringent conditions. If not further specified, the conditions are preferably stringent. Said hybridization conditions may be established according to conventional protocols described, for example, in Sambrook, Russell “Molecular Cloning, A Laboratory Manual”, Cold Spring Harbor Laboratory, N. Y. (2001); Current Protocols in Molecular Biology, Update May 9, 2012, Print ISSN: 1934-3639, Online ISSN: 1934-3647; Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, N. Y.
  • Variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments.
  • Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations.
  • the inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
  • low stringent hybridization conditions for the detection of homologous or not exactly complementary sequences may, for example, be set at 6 x SSC, 1% SDS at 65 °C.
  • the length of the probe and the composition of the nucleic acid to be determined constitute further parameters of the hybridization conditions.
  • Hybridizing nucleic acid molecules also comprise fragments of the above described molecules. Such fragments may represent nucleic acid molecules which encode for a functional hook as described herein or a functional fragment thereof. Furthermore, nucleic acid molecules which hybridize with any of the aforementioned nucleic acid molecules also include complementary fragments, derivatives and variants of these molecules. Additionally, a hybridization complex refers to a complex between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration.
  • a hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., membranes, filters, chips, pins or glass slides to which, e.g., cells have been fixed).
  • a solid support e.g., membranes, filters, chips, pins or glass slides to which, e.g., cells have been fixed.
  • complementary or complementarity refer to the natural binding of polynucleotides under permissive salt and temperature conditions by basepairing.
  • the sequence "A-G-T” binds to the complementary sequence "T-C-A”.
  • Complementarity between two single-stranded molecules may be "partial", in which only some of the nucleic acids bind, or it may be complete when total complementarity exists between single-stranded molecules.
  • hybridizing nucleic acids I sequences preferably refers to sequences which display a sequence identity of at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98% more preferably at least 99%, more preferably at least 99.5%, and most preferably 100% identity with a nucleic acid I nucleic acid sequence as described herein, e.g.
  • hybridize or “hybridizing” as used herein preferably refers to a nucleic acid being part of its genome of the isolated phages as defined herein, wherein the nucleic acid comprises a nucleic acid sequence, which is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to the nucleic acid sequence encoding the amino acid sequence as depicted in any one of SEQ ID NOs: 1-15, preferably as depicted in SEQ ID NO: 1 and 11.
  • the term “signature sequence or its complementary sequence hereto” may also refer to a nucleic acid as defined elsewhere herein and is called signature sequence due to the fact that it refers to the particular hook the A. baumannii phages within the composition of the invention hybridize to and which can thus be used for targeted A. baumannii phage isolation. In such isolation such hook may act as a primer within qPCR.
  • Such hook is further defined as a nucleic acid comprising a nucleic acid sequence as defined herein encoding an amino acid sequence as depicted in any one selected from the group consisting of SEQ ID NOs: 1-15.
  • “Complementary” means in this context that the complementary sequence to the signature sequence has the base on each position in the complementary (i.e. A to T, C to G) and in the reverse order as the signature sequence as defined above.
  • Each signature sequence of its complementary sequence hereto as defined herein encoding an amino acid sequence as depicted in any one selected from the group consisting of SEQ ID NOs: 1-15 refers to a different “hook” which was designed for different phages after metagenomic sequencing.
  • the hook refers to the signature sequence encoding SEQ ID NO: 1 (which refers to a metallo-phosphoesterase)
  • the composition of the invention may comprise isolated phages (the same phages or different phages) which hybridize to such particular hook. The same applies to the other hooks as defined herein (see any one of SEQ ID NOs: 2-15).
  • the composition of the invention may comprise isolated phages (again same phages or different phages) which hybridize with the hook as defined by SEQ ID NO: 1 and with the other hook as defined by SEQ ID NO: 2 (which refers to a HNH endonuclease) or even when different phages are comprised in the composition, some bind to SEQ ID NO: 1 , others to SEQ ID NO: 2. Which different A. baumannii phages may thus be comprised by the composition of the invention (meaning which A.
  • baumannii phages may be isolated and then comprised by the composition of the invention), is defined by which hook is applied to which the phages hybridize to - however each phage comprised by the composition is capable of infecting bacterial cells (bacteria) A. baumannii.
  • This enables a novel composition comprising divergent phages capable of infecting A. baumannii which can be isolated by any one of the defined hooks.
  • infecting bacteria such as A. baumannii by phages means that such phages either break open (lyses) the bacterial cells, such as A. baumannii, immediately by using e.g. polysaccharide-degrading enzymes which enzymatically degrade the capsular outer layer of such bacteria and thus destroys such bacteria after immediate replication within said bacteria (if it is a lytic phage) or do not lyse the host cells, such as bacteria cells A. baumannii, immediately, but its viral genome integrates with host DNA and replicates along with it before then initiating the reproductive cycle, resulting in lysis of the host cell (if it is a lysogenic phage).
  • polysaccharide-degrading enzymes which enzymatically degrade the capsular outer layer of such bacteria and thus destroys such bacteria after immediate replication within said bacteria (if it is a lytic phage) or do not lyse the host cells, such as bacteria cells A. baumannii, immediately, but its viral
  • the phages infecting A. baumannii and which hybridize to the defined hook(s) may refer to lytic phages.
  • the phages bind to specific receptors on the surface of A. baumannii such as, but not limited to, outer membrane proteins, polysaccharides, lipopolysaccharides (LPS) and carbohydrate moieties.
  • the phages specifically infect only those bacteria, in the case of the invention A. baumannii, bearing certain receptors on their surface to which the phages bind to.
  • the present invention may thus comprise the composition as defined herein, wherein said composition has bactericidal activity against said A. baumannii. Meaning by applying such composition, e.g. comprised within a pharmaceutical composition, such composition comprising the particular phages is able to take action against the bacterial cell A. baumannii by infecting said bacteria as defined herein, thereby preventing the development of Acinetobacter resistance and thus resulting in an efficient bacterial removal of A. baumannii.
  • Such A. baumannii can refer to an antimicrobial resistance (AMR) bacterial cell.
  • AMR antimicrobial resistance
  • drug resistance comprises resistance to at least antibiotic agents known in the art, i.e. the ability of a bacterial cell to resist the effects of a drug or agent that could otherwise successfully treat said bacterial cell.
  • a baumannii may acquire AMR genes mediating resistance mostly to aminoglycosides, cephalosporins, carbapenems, sulfonamides, tetracyclines and macrolides (Wareth et al. 202, BMC Micobiol 10;21 (1):210).
  • the term “antimicrobial resistance” as used herein thus also comprises the term “antibiotic resistance” as known in the art.
  • the present invention may therefore also envisage the composition as defined herein, wherein the A. baumannii bacteria are AMR bacteria.
  • the invention relates to a composition comprising isolated phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 , wherein the phages are capable of infecting A. baumannii.
  • the invention also relates to a composition comprising isolated phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2, wherein the phages are capable of infecting A. baumannii.
  • the invention also relates to a composition comprising isolated phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 3, wherein the phages are capable of infecting A. baumannii.
  • the invention also relates to a composition comprising isolated phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 4, wherein the phages are capable of infecting A. baumannii.
  • the invention also relates to a composition comprising isolated phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 5, wherein the phages are capable of infecting A. baumannii.
  • the invention also relates to a composition comprising isolated phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 6, wherein the phages are capable of infecting A. baumannii.
  • the invention also relates to a composition comprising isolated phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 7, wherein the phages are capable of infecting A baumannii.
  • the invention also relates to a composition comprising isolated phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 8, wherein the phages are capable of infecting A. baumannii.
  • the invention also relates to a composition comprising isolated phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 9, wherein the phages are capable of infecting A. baumannii.
  • the invention also relates to a composition comprising isolated phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 10, wherein the phages are capable of infecting A. baumannii.
  • the invention also relates to a composition comprising isolated phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 11 , wherein the phages are capable of infecting A. baumannii.
  • the invention also relates to a composition comprising isolated phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 12, wherein the phages are capable of infecting A. baumannii.
  • the invention also relates to a composition comprising isolated phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 13, wherein the phages are capable of infecting A. baumannii.
  • the invention also relates to a composition comprising isolated phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 14, wherein the phages are capable of infecting A. baumannii.
  • the invention also relates to a composition comprising isolated phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 15, wherein the phages are capable of infecting A. baumannii.
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in any one selected from the group consisting of SEQ ID NOs: 2-15, wherein the phages are capable of infecting A.
  • Such phages within the composition may refer to the same phages, wherein each phage hybridizes to each hook (e.g. encoding SEQ ID NOs: 1 and 2) as well as to different phages, wherein some phages hybridize to the hook encoding SEQ ID NO: 1 and others hybridize to the hook encoding any one of SEQ ID NOs: 2- 5.
  • This explanation can be applied to all further embodiments disclosed below, also for embodiments concerning the hybridization to more than two hooks.
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2, wherein the phages are capable of infecting A.
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 3, wherein the phages are capable of infecting A.
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 4, wherein the phages are capable of infecting A baumannii.
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 5, wherein the phages are capable of infecting A.
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 6, wherein the phages are capable of infecting A.
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 7, wherein the phages are capable of infecting A.
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 8, wherein the phages are capable of infecting A baumannii.
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 9, wherein the phages are capable of infecting A.
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 10, wherein the phages are capable of infecting A.
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 11 , wherein the phages are capable of infecting A.
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 12, wherein the phages are capable of infecting A baumannii.
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 13, wherein the phages are capable of infecting A.
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 14, wherein the phages are capable of infecting A.
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 15, wherein the phages are capable of infecting A. baumannii.
  • compositions comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in any one selected from the group consisting of SEQ ID NOs: 1 and 3- 15, wherein the phages are capable of infecting A. baumannii.
  • compositions comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 3 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in any one selected from the group consisting of SEQ ID NOs: 1-2 and 4- 15, wherein the phages are capable of infecting A. baumannii.
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2 and further comprising (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2 and further
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2 and further comprising (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2 and further
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2 and further comprising (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2 and further
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2 and further comprising (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2 and further
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2 and further comprising (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2 and further
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2 and further comprising (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2 and further
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2 and further comprising (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2 and further
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2 and further comprising (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2 and further
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2 and further comprising (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2 and further
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2 and further comprising (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2 and further
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2 and further comprising (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2 and further
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2 and further comprising (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2 and further
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2 and further comprising (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2 and further
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2 and further comprising (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2 and further
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2, further comprising (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2, further comprising
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2, further comprising (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2, further comprising
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2, further comprising (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2, further comprising
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2, further comprising (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2, further comprising
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2, further comprising (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2, further comprising
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2, further comprising (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2, further comprising
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2, further comprising (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2, further comprising
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2, further comprising (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2, further comprising
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2, further comprising (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2, further comprising
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2, further comprising (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2, further comprising
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2, further comprising (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2, further comprising
  • the invention comprises a composition comprising isolated phages, wherein the (same or different) phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 and wherein the composition further comprises (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2, further comprising (the same or different) phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence hereto comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2, further comprising
  • the composition as defined elsewhere herein comprises one or more phages selected from the group consisting of vB_AbS_HMGU, vB_AbM_HMGU and vB_AbA_HMGU (see Figure 2), preferably the combination of vB_AbM_HMGU and vB_AbA_HMGU which showed a synergetic effect when used in combination against A. baumanii thus resulting in a higher efficiency compared to the corresponding single phage (see Figure 3).
  • Said particular phages have been named according to the ICTV guidelines for naming phages. The A.
  • baumannii phage labeled “AbA” binds to five signature sequences (or complementary sequences hereto) each comprising a nucleic acid sequence, wherein the first one encodes an amino acid sequence as depicted in SEQ ID NO: 1 (refers to a metallo-phosphoesterase), the second one encodes an amino acid sequence as depicted in SEQ ID NO: 2 (refers to a HNH endonuclease), the third one encodes an amino acid sequence as depicted in SEQ ID NO: 3 (refers to a terminase), the fourth one encodes an amino acid sequence as depicted in SEQ ID NO: 4 (refers to endolysin), the fifth one encodes an amino acid sequence as depicted in SEQ ID NO: 5 (refers to a head-tail adaptor), the signature sequence (or complementary sequence hereto) encoding an amino acid sequence as depicted in SEQ ID NO: 1 as the hook with regard to all 15 hooks depicted herein where the most phages hybridize to
  • the A. baumannii phage labeled “AbS” binds to other five signature sequences (or complementary sequences hereto) each comprising a nucleic acid sequence, wherein the first one encodes an amino acid sequence as depicted in SEQ ID NO: 6 (refers to endolysin), the second one encodes an amino acid sequence as depicted in SEQ ID NO: 7 (refers to a co-chaperonin GroES), the third one encodes an amino acid sequence as depicted in SEQ ID NO: 8 (refers to a SbcD-like subunit of palindrome specific endonuclease), the fourth one encodes an amino acid sequence as depicted in SEQ ID NO: 9 (refers to a thymidylate synthase), the fifth one encodes an amino acid sequence as depicted in SEQ ID NO: 10 (refers to a ribonucleoside-diphosphate reductase).
  • the A. baumannii phage labeled “AbM” binds to other five signature sequences (or complementary sequences hereto) each comprising a nucleic acid sequence, wherein the first one encodes an amino acid sequence as depicted in SEQ ID NO: 11 (refers to a thymidylate synthase), the second one encodes an amino acid sequence as depicted in SEQ ID NO: 12 (refers to a dCMP deaminase), the third one encodes an amino acid sequence as depicted in SEQ ID NO: 13 (refers to a phosphoesterase), the fourth one encodes an amino acid sequence as depicted in SEQ ID NO: 14 (refers to a phosphatase), the fifth one encodes an amino acid sequence as depicted in SEQ ID NO: 15 (refers to a endolysin), the signature sequence (or complementary sequence hereto) encoding an amino acid sequence as depicted in SEQ ID NO: 11 as the hook with regard to the hooks depicted in SEQ
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the novel composition including the isolated phages as defined herein.
  • Such novel composition of the invention can also be a pharmaceutical composition.
  • Such pharmaceutical composition may further comprise one or more of the ingredients (such as at least one excipient) as mentioned above for the composition of the invention. If at least one excipient is further comprised by the pharmaceutical composition, such excipient refers to at least one pharmaceutically acceptable excipient. Suitable pharmaceutical excipients are further described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. Said pharmaceutically acceptable excipient includes any excipient that does not itself elicit an adverse reaction harmful to the subject receiving the pharmaceutical composition.
  • composition of the invention additionally comprises at least one pharmaceutically acceptable excipient
  • said composition refers to a pharmaceutical composition.
  • Said pharmaceutical composition is thus used herein for therapeutic purposes.
  • present invention relates to the use of said composition as disclosed herein for the preparation of a pharmaceutical composition.
  • the term "pharmaceutical composition” relates to a composition for administration to a subject as defined herein, preferably a human.
  • Pharmaceutical compositions or formulations are usually in such a form as to allow the biological activity of the active ingredient to be effective and may therefore be administered to a subject for therapeutic use as described herein.
  • the pharmaceutical composition can be administered in a therapeutically effective amount as defined elsewhere by inhalation, injection, infusion, or orally.
  • the administration of said pharmaceutical composition as defined elsewhere herein may be performed intraperitoneally, intravenously, intraarterially, subcutaneously, intramuscularly, parenterally, transdermally, intraluminally, intrathecally, intranasally and/or directly into tissue due to the fact that infection with A baumannii can take place in different parts of the body I in multiple body sites of the subject.
  • the pharmaceutical compositions can be administered to the subject at a suitable dose.
  • the dosage regimen will be determined by the attending physician and by clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
  • Suitable excipients are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and lipid aggregates such as e.g. oil droplets or liposomes.
  • the excipient used in combination with the (pharmaceutical) composition of the present invention may be waterbased and forms an aqueous solution.
  • An oil-based excipient solution is an alternative to the aqueous excipient solution.
  • the pharmaceutical composition as defined herein may further comprise one or more adjuvants.
  • adjuvant is used according to its well-known meaning in connection with pharmaceutical compositions.
  • an adjuvant is an immunological agent that modifies, preferably enhances, the effect of such composition while having few, if any, desired immunogenic effects on the immune system when given per se.
  • Suitable adjuvants can be inorganic adjuvants such as, e.g., aluminium salts (e.g., aluminium phosphate, aluminium hydroxide), monophosphoryl lipid A, or organic adjuvants such as squalene or oil-based adjuvants, as well as virosomes.
  • composition of the invention also apply mutatis mutandis to the pharmaceutical composition.
  • the present invention further refers to said composition or said pharmaceutical composition comprising the novel composition of the invention as defined elsewhere herein for use as a medicament.
  • said composition or said pharmaceutical composition comprising the novel composition of the invention as defined elsewhere herein can also be used for therapy, i.e. treating or preventing an infection of a subject in need thereof with A. baumannii.
  • said composition or said pharmaceutical composition comprising the novel composition of the invention as defined elsewhere herein is particularly suitable for use in a method of preventing or treating an infection with A. baumannii of a subject in need thereof.
  • the term “treat”, “treating” or “treatment” as used herein means to reduce (slow down (lessen)), stabilize or inhibit or at least partially alleviate or abrogate the progression of the symptoms associated with the respective disease, such as an infection of a subject with A. baumannii.
  • it includes the administration of said (pharmaceutical) composition, preferably in the form of a medicament, to a subject, defined elsewhere herein.
  • Those in need of treatment include those already suffering from the disease, here an infection with A. baumannii as described elsewhere herein.
  • a treatment reduces (slows down (lessens)), stabilizes, or inhibits or at least partially alleviates or abrogates progression of a symptom that is associated with the presence and/or progression of such disease (such infection).
  • “Treat”, “treating”, or “treatment” refers to a therapeutic treatment.
  • treating or treatment refers to an improvement of the symptom(s) that is associated with said infection with A. baumannii as defined elsewhere herein in a subject in need thereof.
  • the term “treat”, “treating” or “treatment” refers to an anti-bacterial therapy that directly attacks said bacterial cells A.
  • baumannii the subject has been infected with, by the phages then infecting said bacterial cells A. baumannii (is then the host cell of the phages for replication) as defined herein.
  • the (pharmaceutical) composition as defined herein may also be used as anti-bacterial therapeutic.
  • prevent refers to prophylactic or preventative measures, wherein the subject is to prevent an abnormal, including pathologic, condition in the organism which would then lead to the defined disease, namely said infection with A. baumannii as defined herein.
  • said terms refer to a medical procedure whose purpose is to prevent such disease meaning inhibiting that a subject will likely suffer from any future infection with A. baumannii as defined herein. Meaning the compositions comprising said phages will be given preventive to the subject not yet being infected with A. baumannii. If the subject is then contacted and then infected with A.
  • the phages comprised in the already administered composition can then attack the bacteria right away thereby preventing a full infection with A. baumannii.
  • such terms also refer to the reduction in the risk of acquiring or developing such infection with A. baumannii in a patient.
  • Those in need of the prevention include those prone to having the disease, such as the infection with A. baumannii. In other words, those who are of a risk to develop such infection and will thus probably suffer from it in the near future. Subjects suffering from diabetes or COPD are more likely to get such infection.
  • the (pharmaceutical) composition as defined herein may also be used as a prophylaxis, not only as a therapeutic agent for a subject as defined herein that has already been infected with A. baumannii.
  • An infection with A. baumannii may refer to any infection which occurs in different parts of the body I in multiple body sites of the subject, including but not limited to the lung, the blood, the brain, the urinary tract, and also wounds.
  • an infection with A. baumannii may be any one of a nosocomial pneumonia, an inflammatory lung infection, a blood infection (due to the fact for example that the bacteria enter through a catheter placed in a subject’s vein), meningitis (after surgery on the brain or spine), urinary tract infection (infection of the kidneys, ureters or the bladder), or a skin or wound infection.
  • Such bacteria can easily spread by direct contact with a subject already infected with A.
  • microbiological cut-off values are set by studying the distribution of minimum inhibitory concentrations (MICs) of the chosen antimicrobials in bacterial populations belonging to a single taxonomical unit (species or genus). The part of the population that clearly deviates from the normal susceptible populations is categorised as resistant.
  • a bacterial cell is defined as susceptible when it is inhibited at a concentration of a specific antimicrobial equal or lower than the established cut-off value (S ⁇ x mg/L) defined by FEEDAP Panel.
  • a bacterial cell is defined as resistant when it is not inhibited at a concentration of a specific antimicrobial higher than the established cut-off value (R > x mg/L) defined by FEEDAP Panel.
  • the present invention may therefore also comprise the composition of the invention or the pharmaceutical composition for the use in a method of treating or preventing an infection with A. baumannii as defined herein, wherein the A. baumannii bacteria are AMR bacteria as defined elsewhere herein.
  • the term “subject” when used herein includes mammalian and non-mammalian subjects.
  • the subject of the present invention is a mammal, including human, domestic and farm animals, non-human primates, and any other animal that has mammary tissue.
  • the mammal is a mouse or a rat.
  • the mammal is a guinea pig or a rabbit.
  • the mammal is a cat.
  • the mammal is a dog.
  • the mammal is a monkey.
  • the mammal is a horse.
  • the mammal of the present invention is a human, most preferred an adult.
  • the subject is a living human who may receive treatment for such infection as described herein, it is also addressed as a “patient”.
  • the subject of the present invention suffers from said infection as described herein.
  • the term “suffering” as used herein means that the subject is not any more a healthy subject.
  • the term “healthy” means that the respective subject has no obvious or noticeable hallmarks or symptoms of the respective infection. This further means that the subject suffering from said infection is a subject “in need” of the respective treatment with the (pharmaceutical) composition as defined herein.
  • Such (pharmaceutical) composition is generally administered to the subject in a therapeutically effective amount.
  • Said therapeutically effective amount is sufficient to inhibit or alleviate the symptoms of said infection.
  • therapeutic effect or “therapeutically effective” is meant that the conjugate for use will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician.
  • therapeutically effective further refers to the inhibition of factors causing or contributing to the infection.
  • therapeutically effective amount includes that the amount of the composition when administered is sufficient to significantly improve the progression of the infection being treated or to prevent development of said infection. According to a preferred embodiment, the therapeutic effective amount is sufficient to alleviate or heal said infection as defined herein.
  • the therapeutically effective amount will vary depending on the (pharmaceutical) composition of the present invention, the infection with A. baumannii and its severity and on individual factors of the subject and/or also how the administration works. Therefore, the (pharmaceutical) composition of the present invention will not in all cases turn out to be therapeutically effective, because the method disclosed herein cannot provide a 100% safe prediction whether or not a subject may be responsive to the (pharmaceutical) composition, since individual factors are involved as well. It is to expect that age, body weight, general health, sex, diet, drug interaction and the like may have a general influence as to whether or not the (pharmaceutical) composition for use in the treatment of a subject suffering from said infection will be therapeutically effective.
  • administering means that the (pharmaceutical) composition as defined herein are given to the respective subject in an appropriate form and dose and using appropriate measures.
  • administration of the composition according to the present invention can be carried out by any method known in the art.
  • the (pharmaceutical) composition for the use in the treatment of said infection may also be administered in combination with an additional therapeutic agent (drug).
  • Drugs or therapeutic agents useful in this regard include without limitation drug-like molecules, proteins, peptides, and small molecules.
  • Protein therapeutic agents include, without limitation peptides, enzymes, antibodies, structural proteins, receptors and other cellular or circulating proteins as well as fragments and derivatives thereof, preferably an additional therapeutic agent I drug in the context of the present invention may be a drug for the use in an infection with A baumannii as described elsewhere herein, especially for combinatorial therapy in said infection.
  • Said combination according to the present invention can be administered as a combined formulation or separate from each other.
  • the present invention also provides for a method of treating or preventing an infection with A. baumannii in a subject in need thereof, the method comprising administering a therapeutically effective amount of said composition or said pharmaceutical composition to a subject in need thereof. Also comprised herein is the use of said (pharmaceutical) composition as defined herein for the manufacture of a medicament for therapeutic application in an infection with A. baumannii in a subject.
  • said (pharmaceutical) composition as defined herein for the manufacture of a medicament for therapeutic application in an infection with A. baumannii in a subject.
  • the definitions and embodiments made with regard to the first and second medical uses may also be applied, where necessary, in these embodiments as well.
  • nucleic acid which hybridizes under stringent conditions as defined herein to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in any one selected from the group consisting of SEQ ID NOs: 1-15 (the “hook(s)” of the invention, see below)
  • said nucleic acid may also comprise a nucleic acid sequence which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence encoding the amino acid sequence as depicted in any one selected from the group consisting of SEQ ID NOs: 1-15.
  • nucleic acid which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 , wherein said nucleic acid comprises a nucleic acid sequence which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence encoding the amino acid sequence as depicted in SEQ ID NO: 1 may also be comprised herein.
  • nucleic acid which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2, wherein said nucleic acid comprises a nucleic acid sequence which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence encoding the amino acid sequence as depicted in SEQ ID NO: 2.
  • nucleic acid which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 3, wherein said nucleic acid comprises a nucleic acid sequence which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence encoding the amino acid sequence as depicted in SEQ ID NO: 3.
  • nucleic acid which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 4, wherein said nucleic acid comprises a nucleic acid sequence which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence encoding the amino acid sequence as depicted in SEQ ID NO: 4.
  • nucleic acid which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 5, wherein said nucleic acid comprises a nucleic acid sequence which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence encoding the amino acid sequence as depicted in SEQ ID NO: 5.
  • nucleic acid which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 6, wherein said nucleic acid comprises a nucleic acid sequence which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence encoding the amino acid sequence as depicted in SEQ ID NO: 6.
  • nucleic acid which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 7, wherein said nucleic acid comprises a nucleic acid sequence which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence encoding the amino acid sequence as depicted in SEQ ID NO: 7.
  • nucleic acid which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 8, wherein said nucleic acid comprises a nucleic acid sequence which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence encoding the amino acid sequence as depicted in SEQ ID NO: 8.
  • nucleic acid which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 9, wherein said nucleic acid comprises a nucleic acid sequence which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence encoding the amino acid sequence as depicted in SEQ ID NO: 9.
  • nucleic acid which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 10, wherein said nucleic acid comprises a nucleic acid sequence which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence encoding the amino acid sequence as depicted in SEQ ID NO: 10.
  • nucleic acid which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 11, wherein said nucleic acid comprises a nucleic acid sequence which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence encoding the amino acid sequence as depicted in SEQ ID NO: 11.
  • nucleic acid which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 12, wherein said nucleic acid comprises a nucleic acid sequence which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence encoding the amino acid sequence as depicted in SEQ ID NO: 12.
  • nucleic acid which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 13, wherein said nucleic acid comprises a nucleic acid sequence which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence encoding the amino acid sequence as depicted in SEQ ID NO: 13.
  • nucleic acid which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 14, wherein said nucleic acid comprises a nucleic acid sequence which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence encoding the amino acid sequence as depicted in SEQ ID NO: 14.
  • nucleic acid which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 15, wherein said nucleic acid comprises a nucleic acid sequence which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleic acid sequence encoding the amino acid sequence as depicted in SEQ ID NO: 15.
  • such particular hook refers to a nucleic acid which comprises a nucleic acid sequence encoding an amino acid sequence as depicted in any one selected from the group consisting of SEQ ID NOs: 1-15, or a complementary sequence thereof.
  • the invention also comprises as a hook a nucleic acid comprising a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in any one selected from the group consisting of SEQ ID NOs: 1-15, or a complementary sequence thereof.
  • the invention also comprises as a hook a nucleic acid comprising a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1 , or a complementary sequence thereof.
  • the hook which encodes the metallo-phosphoesterase as defined by SEQ ID NO: 1 is the most abundant protein family within the isolated A. baumannii phages after having analysed the genome of the A. baumannii phages as defined elsewhere herein. Using such designed hook for targeted phage isolation, targets most A. baumannii phages, resulting in the most divergent A. baumannii phage composition.
  • the invention also comprises as a hook a nucleic acid comprising a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2, or a complementary sequence thereof.
  • the invention also comprises as a hook a nucleic acid comprising a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 3, or a complementary sequence thereof.
  • the invention also comprises as a hook a nucleic acid comprising a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 4, or a complementary sequence thereof.
  • the invention also comprises as a hook a nucleic acid comprising a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 5, or a complementary sequence thereof.
  • the invention also comprises as a hook a nucleic acid comprising a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 6, or a complementary sequence thereof.
  • the invention also comprises as a hook a nucleic acid comprising a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 7, or a complementary sequence thereof.
  • the invention also comprises as a hook a nucleic acid comprising a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 8, or a complementary sequence thereof.
  • the invention also comprises as a hook a nucleic acid comprising a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 9, or a complementary sequence thereof.
  • the invention also comprises as a hook a nucleic acid comprising a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 10, or a complementary sequence thereof.
  • the invention also comprises as a hook a nucleic acid comprising a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 11, or a complementary sequence thereof.
  • the invention also comprises as a hook a nucleic acid comprising a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 12, or a complementary sequence thereof.
  • the invention also comprises as a hook a nucleic acid comprising a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 13, or a complementary sequence thereof.
  • the invention also comprises as a hook a nucleic acid comprising a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 14, or a complementary sequence thereof.
  • the invention also comprises as a hook a nucleic acid comprising a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 15, or a complementary sequence thereof.
  • said nucleic acid which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in any one selected from the group consisting of SEQ ID NOs: 1-15 as defined herein or said nucleic acid of the hook as defined herein may also be conjugated to a detectable label. It is also comprised herein, that such nucleic acids may be attached to a detectable label or just labeled.
  • a “detectable label” as used herein may refer to a compound capable of targeting said nucleic acids and which can then be detected by any detection methods known to a person skilled in the art, which depends certainly on the label being used.
  • Said label may be selected from the group consisting of a biotin, a tag, a fluorophore, a radioactive phosphate (P 32 ) or radioactive sulfate (S 35 ), and any active site probe, i.e. a detectable moiety suitable for the labeling and detection of said nucleic acids.
  • the nucleic acids may also be conjugated to a combination of any one of the detectable labels mentioned above.
  • the nucleic acids may be conjugated to a detectable fluorophore and further labeled with a radioactive phosphate.
  • nucleotides of the nucleic acid sequences comprised by said nucleic acids may be labeled with biotin.
  • label is a tag
  • an oligohistidine-tag His 6 tag
  • the nucleic acid may be modified with for example nitrilotriacetate (NTA) which has high affinity to a His-tag.
  • the label when it is a fluorophore (also called fluorochrome or chromophore) in the present invention it may be any DNA fluorescent dye such as but not limited to Acridine Orange hydrochloride, 7-Aminoactinomycin D, DAPI, Hoechst 3342, Hoechst Janelia Fluor 526, Hoechst Janelia Fluor 646, Nucleic Acid Dye Green I, propidium iodide, or one of a fluorescent dye such as but not limited to Fluorescein (FITC), Alexa Fluor 350, 405, 488, 532, 546, 555, 568, 594, 647, 680, 700, 750, Pacific Blue, Coumarin, Pacific Green, Cy3, Texas Red, PE, PerCP-Cy5, PE-Cy7, Pacific Orange, or a fluorescent protein label such as R-PE or APC, or an expressed fluorescent protein such as CFP, EGFP, GFP or RFP
  • any DNA fluorescent dye such as but not limited to Acridine Orange hydro
  • the labeling with such fluorophore may be performed by labeling the nucleotides of the nucleic acids as it is known to a person skilled in the art.
  • the label is a radioactive phosphate (P 32 ) or sulfate (S 35 )
  • P 32 radioactive phosphate
  • S 35 sulfate
  • Said nucleic acids may be labeled at their 5' end (DNA 5’ end labeling), their 3' end (DNA 3’ end labeling), or throughout the nucleic acid depending on the application (for example labeling by PCR).
  • labeling by PCR
  • the conjugation (attachment) of a label preferably a fluorophore, even more preferably any DNA fluorescent dye
  • the nucleic acids may be conjugated via a linker I linked to a detectable label as defined herein. Said conjugation to the detectable label may also be covalently as defined elsewhere herein.
  • the nucleic acids are directly or indirectly conjugated to a detectable label, preferably covalently.
  • covalently refers to covalent bonds that are typically formed by the sharing of electron pairs between atoms. In accordance with the present invention and when the term “covalently” is used, a covalent bond is formed between the nucleic acids and the label as defined elsewhere herein by use of a linker of at least 1 amino acid in length as defined above.
  • the present invention also relates to a kit comprising the composition comprising the isolated phages of the present invention or said pharmaceutical composition comprising such composition.
  • a kit comprises said (pharmaceutical) composition
  • said compositions may be provided in a vial or a container, preferably also comprising in said vial or container at least one excipient as defined herein.
  • said kit may be associated with a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of the manufacture, use or sale of the product for human administration or diagnostics.
  • Said kit may comprise the (pharmaceutical) composition, preferably in a vial or container, in dried form, such as a lyophilized, air-dried, or spray-dried form (in form of a powder), for later reconstitution into a liquid form or other form prior to use. Further, said kit may also comprise the (pharmaceutical) composition, preferably in a vial or container, in a frozen state, being thawed prior to use. Further, said kit may also comprise the (pharmaceutical) composition, preferably in a vial or container, in liquid state.
  • the present invention also comprises a kit comprising the hook(s) as defined elsewhere herein.
  • a kit comprises said hook(s)
  • said hook(s) may be provided in a vial or a container, preferably also comprising in said vial or container at least one excipient as defined herein.
  • the kit may provide a vial or container comprising one of the defined hooks, meaning one kit comprises one of the defined hooks, or a kit may comprise at least one of the defined hooks, meaning in one kit there are more than one hooks (at least any one of SEQ ID NOs: 1-15) comprised.
  • such hooks can either be comprised separately in different vials or containers or comprised all together in one vial or container.
  • said kits may be associated with a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of the manufacture, use or sale of the product for human administration or diagnostics.
  • Said kits may comprise the hook(s), preferably in a vial or container, in dried form, such as a lyophilized, air-dried, or spray- dried form (in form of a powder), for later reconstitution into a liquid form or other form prior to use.
  • kits may also comprise the hook(s), preferably in a vial or container, in a frozen state, being thawed prior to use. Further, said kits may also comprise the hook(s), preferably in a vial or container, in liquid state.
  • the kit according to the present invention either comprising the (pharmaceutical) composition or the hook(s) may also comprise a detectable label as defined elsewhere herein.
  • a detectable label as defined elsewhere herein.
  • Such label may be selected from the ones as listed above.
  • said label may also be comprised in the one or more containers or vials of the kit as defined above comprising said (pharmaceutical) composition or in the one or more containers or vials of the kit as defined above comprising said hook(s); or in additional one or more vials or containers of said kits, preferably further comprising in said one or more vials or containers any excipient suitable for said label to be mixed with I contacted with.
  • said hook(s) may also be comprised in the same kit which comprises said (pharmaceutical) composition as defined above (with or without said label within the same kit).
  • the present invention also relates to a method of preparing a composition comprising isolated phages, the method comprising the following steps of first providing a sample which comprises the phages to be isolated from. Such step may also be optional. Such sample may be any one of a naturally environmental sample, a clinical sample, a wastewater treatment plant sample which includes but is not limited to a sewage water sample, or a hospital waste resources sample. In a next step, said phages are isolated from said sample as defined herein using any one of the defined hooks of the invention. “Targeted isolating” or “targeted isolation” (“hook isolation”) may be carried out by any method known in the art suitable to isolate phages based on selected gene or nucleic acid molecule sequences.
  • Such step may also include a viral tagging (also called phage labeling) step as defined by WO2021/048257 incorporated by references herein resulting in the phages being labeled, wherein the labelling may be selected from the group consisting of fluorescent labelling, antibody labelling and radioactive labelling (preferably fluorescent labelling).
  • target bacteria in the context of the invention A. baumannii
  • A. baumannii may be added to said sample comprising the already labeled phages, whereby said labeled phages come into contact with the target bacteria. This may be followed by single-cell sorting of such bacteria-phages into single pairs.
  • single phage-bacteria pairs may then be sorted onto solid bacterial lawns, or liquid growth media (e.g. in multi-well plate), and incubated under conditions allowing phage infection take place.
  • Agar from the infected area (plaque) in the former approach, or aliquots of phage suspensions in the latter approach may then be transferred to a vial (e.g. to 2 nd multi-well plates) containing premixed (e.g., barcoded) primer(s), which refer to at least any one of the defined hooks already designed before.
  • a vial e.g. to 2 nd multi-well plates
  • premixed primer(s) e.g., barcoded primer(s)
  • Such vial may also comprise besides the primer(s) a PCR Master mix for PCR or qPCR. This results in isolating the particular A. baumannii phages which hybridize to the particular hooks (primers in the qPCR) being used for said targeted phage isolation.
  • phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in any one selected from the group consisting of SEQ ID NOs: 1-15, preferably SEQ ID NO: 1 , which refers to the defined hook being used for targeted phage isolation.
  • such phages may comprise a nucleic acid being part of its genome, wherein the nucleic acid comprises a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 1.
  • such phages may comprise a nucleic acid being part of its genome, wherein the nucleic acid comprises a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 2.
  • such phages may comprise a nucleic acid being part of its genome, wherein the nucleic acid comprises a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 3.
  • such phages may comprise a nucleic acid being part of its genome, wherein the nucleic acid comprises a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 4.
  • such phages may comprise a nucleic acid being part of its genome, wherein the nucleic acid comprises a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 5.
  • such phages may comprise a nucleic acid being part of its genome, wherein the nucleic acid comprises a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 6.
  • such phages may comprise a nucleic acid being part of its genome, wherein the nucleic acid comprises a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 7.
  • such phages may comprise a nucleic acid being part of its genome, wherein the nucleic acid comprises a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 8.
  • such phages may comprise a nucleic acid being part of its genome, wherein the nucleic acid comprises a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 9.
  • such phages may comprise a nucleic acid being part of its genome, wherein the nucleic acid comprises a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 10.
  • such phages may comprise a nucleic acid being part of its genome, wherein the nucleic acid comprises a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 11.
  • such phages may comprise a nucleic acid being part of its genome, wherein the nucleic acid comprises a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 12.
  • such phages may comprise a nucleic acid being part of its genome, wherein the nucleic acid comprises a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 13.
  • such phages may comprise a nucleic acid being part of its genome, wherein the nucleic acid comprises a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 14.
  • such phages may comprise a nucleic acid being part of its genome, wherein the nucleic acid comprises a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in SEQ ID NO: 15.
  • the isolated phages are obtained and provided within a composition as defined herein, meaning such isolated phages are then added to a pre-prepared composition as defined herein.
  • the abovementioned can also be applied mutatis mutandis to a method of preparing a pharmaceutical composition as defined elsewhere herein.
  • Sewage water was sampled regularly and kept at 4 °C before processing. The samples were used for isolating phages using classical isolation and viral tagging (VT).
  • VT isolation and viral tagging
  • the phages were amplified by mixing 50 ml of wastewater with the same amount of double-strength lysis buffer (LB) and 10 ml of a single Escherichia coli Reference collection (ECOR) strain bacteria cultured overnight. After incubation overnight at 30°C, 10 ml of the mixture was centrifuged at 6000xg at +4°C for 15 minutes and sterile filtered through a 0.45 pm membrane filter. Then, the phage titer was measured in plaque assays. Sterile filtered phage lysates were diluted in SM buffer or LB to five different dilutions (10e5 to 10e9).
  • phages 100 pl of diluted phage and 200 pl of target bacteria (A baumannii) were mixed with 2 ml soft agar (SA), spread on pre-warmed LA plates, and incubated overnight at 30°C. The harvested phages were selected according to their plaque morphology.
  • Dyes used to stain viruses e.g. SYTO 9, SYBR Gold, SYBR Green II and SYBR Safe
  • TE buffer 10 mM Tris, 1 mM EDTA; pH 8.0
  • Viral samples were stained with dyes at 30 °C for 30 min for the environmental samples.
  • the ultracentrifugal devices (100 KDa cut-off; Amicon) were pretreated by incubating 1.5 ml of 0.2pm-filter-sterilized 1% BSA (Bioexpress, UT, cat# E531-1.5ML) in phosphate buffered saline (PBS) for 1 h at room temperature. Stained wastewater samples were washed six times by phage buffer in the pretreated ultracentrifugal devices. 50 pl phage buffer were added back for every 500 ml viral samples and sonicated (VWR Signature Ultrasonic cleaner B1500A-DTH, VWR) for 3 minutes using the settings of 50W at 42 kHz, resulted a 10-fold concentration of viruses from the original sample.
  • BSA Bioexpress, UT, cat# E531-1.5ML
  • PBS phosphate buffered saline
  • Stained and washed viruses were mixed with bacteria (A. baumannii) at concentrations and ratios desired for flow cytometer analysis, typically 10 5 cells per ml.
  • VT experiments were done with a negative control, which was prepared identically to the stained and washed virus samples except without viruses; this controlled for free dye creating the appearance of false positive ‘viral tagged cells’.
  • Bacterial cells for mixture 2 were stained with one microliter of dye, diluted 10X in sterile water, and incubated at room temperature in the dark for 30 min. Mixtures 1 to 3 were used as sorting controls and mixture 4 was the VT sample. All mixtures were incubated at 30 °C for 1 hour with mild rotary shaking to prevent sedimentation, and sample volumes were subsequently adjusted to 1 ml with SS before sorting using FACS. Non-specifically bound viruses were removed.
  • the negative sorting controls were processed first in the following order: unstained bacterial cells, bacterial cells mixed with unstained VLPs, bacterial cells mixed with unstained viruses, and bacterial cells mixed with stained VLPs.
  • the VT samples containing bacterial cells mixed with stained viruses were then processed, with 10,000 events usually recorded per sample to identify sorting gates which did not overlap with the negative controls.
  • the final sample processed prior to sorting was the stained bacterial cell control to confirm that there was no overlap with the identified VT sorting gates.
  • the flow cytometer was then cleaned prior to sorting of VT samples by repeatedly backflushing the fluidics and processing an ultra-pure water sample. For multi-cell sorts (50,000 or 100,000 cells), cells were collected in 1.5 ml low-bind tubes.
  • cells were sorted into 96 or 384 well plates with single-cell mode settings. The first, third and last column of the plate were left empty as negative controls, while 100 cells were collected into the second column as a positive amplification control. All sorted cells were stored at -80°C until further processing. Flow cytometry (.fsc) files were processed for visualisation using FlowVizand FlowCore R packages.
  • Either VT or purified community viral samples were combined with 10 pl of DNAse I buffer and 5 pl of DNAse I (Sigma-Aldrich, #AMPD1-1 KT) and incubated for 15 min at room temperature to degrade free bacterial DNA in the viral filtrate. DNAse was inactivated by adding 10 pl DNAse stop solution and incubation at 70°C for 10 min. Viral DNA was then extracted using a low biomass DNA extraction method, which could also be applied to single cells (see below) (see, e.g., Dzunkova et al., loc cit. for viral DNA extraction). All reagents used in the DNA extraction were autoclaved, sterilized by 0.1 pm filtration and UV-radiated for 60 min.
  • Each DNAsed virion sample (110 pl) was mixed with 77 pl of Lysis buffer prepared by combining 700 pl KOH stock (0.43g/10 ml), 430 pl DDT stock (Thermo Fisher, #R0861 , 0.8 g/10 ml) and 370 pl water, final pH 12. The sample was incubated for 10 min at room temperature, -80 °C for one hour and 55 °C for 5 min to complete virion lysis.
  • the alkaline reaction was then neutralized by adding 77 pl of Stop buffer (5 g Tris-HCI in 10 ml, pH 4.5) and 1 pl of Proteinase K (20mg/ml, Sigma-Aldrich, #3115887001) was added to the sample and incubated for 30 min at 55 °C.
  • Stop buffer 5 g Tris-HCI in 10 ml, pH 4.5
  • Proteinase K 20mg/ml, Sigma-Aldrich, #3115887001
  • the DNA was purified using 536 pl (1.8X sample volume) Agencourt AMPure beads (Beckman Coulter, #A63880) according to the manufacturer’s instructions. Each sample was finally resuspended in 30 pl of sterile water.
  • Illumina data quality-control consisted of trimming ends with a quality score lower than 25 as well as sequences containing ambiguous bases, only reads longer than 100 bp were kept. Additionally, because the Illumina sequencing was done from linker-amplified DNA, it was mixed 1 :1 with phiX174 DNA to minimize base-calling issues in Illumina software. Thus full- length reads matching (>98% identity) to the phiX174 genome were removed and the remaining reads were considered the target material, linkers removed and quality controlled (cf. Deng et al., Nature (2014), 513: 242-245).
  • ORFs Open reading frames
  • prodigal cf. Richards et al., Infect Genet Evol (2011), 11 : 1263-1275
  • ORFs were clustered using CD-HIT (cf. Devirgilliis et al., Genes Nutr (2011), 6: 275-284) with a cutoff of 75% identity.
  • Individual reads then were mapped to protein clusters using BLASTn, and evaluate a cutoff of 0.001, only non-redundant top hits were used.
  • Rarefaction curves were calculated using a custom perl script (Rarefaction.pl).
  • Chao-1 index was calculated from the protein cluster data as described in Chao and Lee (J A Stat Assoc (1992), 87: 210-217).
  • Shannon- Wiener (H’ - p In p), was calculated using PHACCS (see above) (cf. Angly et al., BMC Bioinformatics (2005), 6: 41).
  • Assembled contigs >1.5 Kb were annotated as follows: ORFs were predicted using Prodigal (above) and functionally annotated using manually curated data resulting from BLASTp analyses against the non-redundant protein database of Genbank, and custom databases of T4 phage gene clusters (T4-GCs) (cf. Sullivan, Environ Microbiol (2010), 12: 3035-3056) and Microbial Metabolic Genes (cf. Sharon et al., ISME J (2011), 5: 1178-1190).
  • BLASTx search was used against the phage genomes available in NCBI, and assigned taxonomy to metagenomic reads by the taxon lineage associated with their top hit (requiring e-value ⁇ 1e-3), read2family.pl available was used with the rest of the scripts.
  • the inventors used the genome of the isolated Acinetobacter phages, viral contigs generated by viral tagging, and the Acinetobacter phage genomes from the NCBI RefSeq database to create a gene catalogue.
  • the proteins of these genes were clustered into protein families (PFs) using MMseqs2, and PFs were annotated using the PHROG database.
  • phage genomes were clustered to genus level to create viral clusters (VCs) using vConTACT2. Then, a presence-absence matrix (PAM) of PFs in VCs was created, and the most prevalent PFs and their functions were manually checked.
  • the target PF was selected based on its prevalence in VCs and its function. For example, the inventors selected endolysin for designing the hooks for targeted isolation of phages as they were the most prevalent PF in VCs and had an important role in the bacteria lysing capacity of phages.
  • the inventors used in-depth comparative genomics and protein clustering to build a presence-absence matrix (see Figure 1) of protein families shared between the viral clusters from the viral-tagging (Deng et al., 2014; Dzunkova et al., 2019) data generated against A. baumannii, also Acinetobacter phages from the NCBI RefSeq database were included.
  • the inventors identified multiple proteins shared between these viral clusters (see Figure 1). Protein clusters from those genes that showed the highest similarities between viral clusters were selected (see Figure 1) as markers for developing a targeted isolation approach, which can be used to isolate novel phages from the environment and clinical samples with high antibacterial effects.
  • Example 2 Example 2.
  • the inventors Using the selected proteins/genes, the inventors targeted isolated multiple phages against A baumanii from which the inventors selected those with high efficiency against the target bacteria such as vB_AbM_HMGU (see Figure 2A Nr. 1), vB_AbS_HMGU (see Figure 2A Nr. 2), and vB_AbA_HMGU (see Figure 2A Nr. 3). The inventors then further characterized their genomic and morphological features (see Figure 2A and B). Phage vB_AbS_HMGU showed a short latency period and high adsorption rate, making it a good candidate for phage therapy (see Figure 2C).
  • phage vB_AbM_HMGU and vB_AbA_HMGU showed a synergetic effect when used in combination against a multi-resistant A. baumanii strain (see Figure 3D).
  • the inventors Using fluorescent staining, the inventors have shown that the combination of phages vB_AbM_HMGU and vB_AbA_HMGU leads to the destruction of biofilm structure and eradicating the bacterial cells; this was not achieved by applying each phage alone (see Figure 3).
  • vB_AbS_HMGU shows the highest similarity to our viral-tagging data (see Figure 1).
  • the endolysins genes are phage-encoded peptidoglycan hydrolases used by most phages to enzymatically disrupt the bacterial cell wall at the end of their replication cycle. They have the potential to be used as independent antibacterials and, therefore, can be of interest to the pharmaceuticals industry.
  • composition comprising isolated phages, wherein the phages comprise a nucleic acid being part of its genome, which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in any one selected from the group consisting of SEQ ID NOs: 1-15, wherein the phages are capable of infecting Acinetobacter baumannii.
  • the composition of item 1 wherein the composition has bactericidal activity against Acinetobacter baumannii.
  • the composition of item 1 or 2 wherein the Acinetobacter baumannii bacteria are antimicrobial resistance (AMR) bacteria.
  • AMR antimicrobial resistance
  • a pharmaceutical composition comprising the composition of any one of the preceding items.
  • composition of any one of items 1-5 or the pharmaceutical composition of item 6 for use in a method of treating or preventing an infection of a subject with Acinetobacter baumannii The composition or the pharmaceutical composition for the use of item 8, wherein the Acinetobacter baumannii bacteria are antimicrobial resistance (AMR) bacteria.
  • a nucleic acid which hybridizes under stringent conditions to a signature sequence or its complementary sequence comprising a nucleic acid sequence encoding an amino acid sequence as depicted in any one selected from the group consisting of SEQ ID NOs: 1- 15.
  • a nucleic acid comprising a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in any one selected from the group consisting of SEQ ID NOs: 1-15, or a complementary sequence thereof.
  • nucleic acid which comprises a nucleic acid being part of its genome, wherein the nucleic acid comprises a nucleic acid sequence, which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to a nucleic acid sequence encoding an amino acid sequence as depicted in any one selected from the group consisting of SEQ ID NOs: 1-15.
  • the method of item 13 wherein the sample is any one of a naturally environmental sample, a clinical sample, a wastewater treatment plant sample, or a hospital waste resources sample.
  • a composition comprising isolated phages obtainable or obtained by the method of any one of items 13-14.

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Abstract

La présente invention concerne une composition comprenant des phages isolés, les phages renfermant un acide nucléique faisant partie de leur génome, qui s'hybride dans des conditions rigoureuses à une séquence de signature ou à sa séquence complémentaire comprenant une séquence d'acide nucléique codant pour une séquence d'acide aminé telle que décrite dans SEQ ID No : 1, et renfermant en outre un acide nucléique faisant partie de leur génome, qui s'hybride dans des conditions rigoureuses à une séquence de signature ou à sa séquence complémentaire comprenant une séquence d'acide nucléique codant pour une séquence d'acide aminé telle que décrite dans SEQ ID No : 11, les phages renfermant un acide nucléique faisant partie de leur génome, l'acide nucléique comprenant une séquence d'acide nucléique identique à au moins 60 % à la séquence d'acide nucléique codant la séquence d'acide aminé telle que décrite dans SEQ ID No : 1 et 11 et les phages isolés pouvant infecter Acinetobacter baumannii. La présente invention concerne également une composition pharmaceutique comprenant ladite composition, et ladite composition ou ladite composition pharmaceutique destinée à être utilisée en thérapie ainsi que dans une méthode de traitement ou de prévention d'une infection d'un sujet par Acinetobacter baumannii. En outre, la présente invention concerne des trousses et un procédé de préparation de ladite composition comprenant des phages isolés, ainsi qu'une composition comprenant des phages isolés obtenue ou pouvant être obtenue par ledit procédé.
PCT/EP2023/083257 2022-11-28 2023-11-28 Phages acinetobacter baumannii WO2024115430A1 (fr)

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