Bacterial producing cells and their use in production methods - Patents.com

Abstract

The present invention relates to a producer bacterial cell for producing phage particles or phage derived delivery vehicles, said producer bacterial cell stably comprising at least one phage structural gene and at least one phage DNA packaging gene, said phage structural gene and phage DNA packaging gene being derived from a first type of bacteriophage, and expression of at least one of said phage structural genes and / or at least one of said phage DNA packaging genes in said producer bacterial cell being controlled by at least one inducible mechanism, and said producer bacterial cell being from a bacterial species or strain different to the bacterial species or strain from which said first type of bacteriophage is derived and / or which is targeted by said first type of bacteriophage.

Description

[Technical Field]

[0001] The present invention relates to bacterial cells for producing phage particles and methods of using such bacterial cells. [Background technology]

[0002] Most current methods for producing phages or phage-derived delivery vehicles imply the use of bacterial species or strains that are the natural host of the phage as producer cells. Such methods can prove dangerous if such bacterial cells are pathogenic, e.g., if they produce toxins. Furthermore, many bacterial species cannot be easily manipulated, e.g., due to their growth conditions or the lack of efficient genetic tools for these bacteria. Since the same bacterial strain often contains several prophages that can result in the production of unwanted particles or unwanted recombination events, it can also be difficult to identify bacterial strains that contain only certain prophages and / or, for example, to derive and / or stably maintain prophages / phages in a given species or strain.

[0003] Therefore, there is a need for methods that allow for safe, easier and more efficient production of any phage or phage-derived particle.

[0004] The present invention fulfills this need.

[0005] The inventors considered that phages can be viewed as more or less large genetic circuits whose final output is the production of more phage particles. To do this, regardless of whether the phage is lytic, lysogenic or chronic (e.g., filamentous phage, e.g., M13), the inventors considered that the information encoded in their genomes can be broadly categorized depending on the function it performs: - Genes for insertion / excision purposes (for temperate phages). - Genes for purposes such as DNA replication, RNA transcription, etc. Indeed, for example, some lytic phages encode their own RNA or DNA polymerases. Some genes modify the host's RNA polymerase so that it can function beyond the terminator, and some other genes are involved in sequestration of the prophage sequence when it is present in a plasmid or linear plasmid form. - Genes for the purpose of packaging the newly synthesized phage genome into newly synthesized phage capsids: terminase and accessory proteins, ligase, etc. - structural genes for the purpose of building a protein capsid for the DNA: besides the strictly structural genes, e.g. capsid genes, tape measure, fiber, baseplate, etc., many other genes are needed to assemble proteins that can be packaged inside the capsid, be they as scaffolds or pilot proteins injected into the cell (e.g. RNA polymerase in phage N4 or some minor pilot proteins in other phages), as well as building blocks (chaperones, proteases). - Genes related to defense against host antiphage mechanisms, degradation / modification of host elements to complete the lytic cycle, super-exclusion mechanisms or genes advantageous for the host.

[0006] The DNA packaging and structural gene categories are deeply linked because the packaging machinery recognizes preassembled capsid heads and the DNA packaged into these heads, initiating and terminating DNA packaging.

[0007] The inventors hypothesized that by extracting and distinguishing all the modules defined above, a system could be constructed that contains all the excision / insertion, replication and regulatory elements from one phage and encodes the packaging / structural elements for another phage, because, as considered by the inventors, they can be viewed as independent genetic modules.

[0008] Treating them as independent genetic modules may also allow the construction of systems containing only the desired structural and / or regulatory elements of a phage, produced under the control of a master regulatory element (e.g., an inducible repressor) that may not be derived from the phage, in contrast to wild-type phage, where gene expression is tightly regulated by phage elements. For example, only the phage structural operon and DNA packaging machinery may be placed under the control of a repressor that responds to small molecules or physical / chemical signals (e.g., LacI, AraC, PhlF, lambda cI, etc.) that trigger the production of all elements necessary to generate a pure, mature phage delivery particle (phage or packaged phagemid). This "trimmed-down" version of the phage genome can be stably maintained in bacterial production strains. [Prior art documents] [Patent documents]

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[0011] Summary of the Invention The present invention arose from the unexpected discovery by the inventors that exchanging the structural operon of an Escherichia coli producer strain, encoding a system for generating pure lambda-packaged phagemids, with the structural operon of a prophage from a different species (e.g., Klebsiella pneumoniae) can drive the assembly and packaging of pure heterologous phagemid particles when supplemented with a plasmid containing the correct packaging signal (a cos site for Klebsiella pneumoniae phage). The inventors have therefore shown herein that phagemids can be produced that are structurally based on the K. pneumoniae prophage, but can be regulated and maintained in a lysogenic state by the lambda prophage machinery in the Escherichia coli producer strain.

[0012] We also showed that the structural operon of the P. freudenreichii prophage can be exchanged with that of a phage from a C. acnes strain. Using this approach, we demonstrated that exchanging the structural operon of the P. freudenreichii prophage with that of a phage from a C. acnes strain can drive the assembly and packaging of a pure C. acnes phagemid.

[0013] This approach represents a novel means for easier and / or safer production of phage particles and / or phage-derived delivery vehicles that target bacterial cells that are known to be pathogenic and / or difficult to manipulate and / or that for some reason are inefficient to use in the production of phage particles and / or phage-derived delivery vehicles.

[0014] The present invention therefore provides a producer bacterial cell for producing phage particles or phage-derived delivery vehicles, said producer bacterial cell stably comprising at least one phage structural gene and at least one phage DNA packaging gene, said phage structural gene and phage DNA packaging gene being derived from a first type of bacteriophage; expression of at least one of the phage structural genes and / or at least one of the phage DNA packaging genes in the producer bacterial cell is controlled by at least one inducible mechanism; The producer bacterial cell relates to a producer bacterial cell, wherein the producer bacterial cell is from a bacterial species or strain different from the bacterial species or strain from which the first type of bacteriophage is derived and / or which the first type of bacteriophage targets.

[0015] The present invention also provides a method for producing a phage particle or phage-derived delivery vehicle, comprising: (a) providing a bacterial production cell of the invention; and (b) inducing in the producer bacterial cell the expression of said at least one of said phage structural gene and said phage DNA packaging gene and the assembly of products expressed by said at least one phage structural gene and said at least one phage DNA packaging gene, thereby producing a phage particle or a phage-derived delivery vehicle. The present invention relates to a method, comprising:

[0016] Another object of the present invention is a hybrid helper phage system, comprising: (i) at least one phage DNA packaging gene derived from a first type of bacteriophage; (i') at least one phage structural gene derived from a bacteriophage of the first type; and (ii) at least one gene from a second type of bacteriophage involved in phage excision / insertion, phage DNA replication, and / or phage regulation; Including, the genes (i), (i') and (ii) are contained in a single nucleic acid molecule or in separate nucleic acid molecules; the first type of bacteriophage originates from and / or targets a bacterial species or strain different from the bacterial species or strain from which the second type of bacteriophage originates and / or targets, The hybrid helper phage system does not contain any expressed phage structural genes derived from the second type of bacteriophage. DETAILED DESCRIPTION OF THE INVENTION

[0017] Detailed Description of the Invention producing bacterial cells The present invention provides a producer bacterial cell for producing a phage particle or a phage-derived delivery vehicle, said producer bacterial cell stably comprising at least one phage structural gene and at least one phage DNA packaging gene derived from a first type of bacteriophage; expression of at least one of the phage structural genes and at least one of the phage DNA packaging genes in the producer bacterial cell is controlled by an inducible mechanism; The producer bacterial cell relates to a producer bacterial cell, wherein the producer bacterial cell is from a bacterial species or strain different from the bacterial species or strain from which the first type of bacteriophage is derived and / or which the first type of bacteriophage targets.

[0018] As used herein, the term "phage particle" refers to a functional or non-functional (e.g., non-replicative and / or replicative) virion.

[0019] As used herein, the term "phage-derived delivery vehicle" refers to any vehicle that allows for the transfer of a payload into bacteria and that is derived from a bacteriophage. In the context of the present invention, the term "phage-derived delivery vehicle" further encompasses bacteriophage-derived particles that do not contain any payload but are capable of targeting bacterial cells.

[0020] Phage-derived delivery vehicles can refer to bacteriophage-derived scaffolds, which can be obtained from natural, evolved, or engineered bacteriophages.

[0021] bacterial cells The producing bacterial cell of the present invention may be any bacterial species or strain, in particular those defined below under the section "Targeted Bacteria", provided that said bacterial species or strain is different from the bacterial species or strain from which said first type of bacteriophage is derived and / or targeted by said first type of bacteriophage.

[0022] However, the producing bacterial cells are preferably non-pathogenic bacterial cells. More preferably, the producing bacterial cells are bacterial cells that can be easily manipulated.

[0023] By "easily engineered" it is meant herein that the bacterial cells can be cultured and / or modified using well known techniques.

[0024] In certain preferred embodiments, the producer bacterial cells are E. coli bacterial cells. Alternatively, the producer bacterial cells may be Bacteroides bacterial cells, more particularly Bacteroides thetaiotaomicron bacterial cells, P. freudenreichii bacterial cells, Fusobacterium bacterial cells, or Streptococcus bacterial cells. In certain embodiments, the producer bacterial cells are P. freudenreichii bacterial cells.

[0025] The production bacterial cell of the present invention may be obtained by any technique known to those skilled in the art, in particular by introducing said phage structural genes and phage DNA packaging genes from a first type of bacteriophage into a bacterial cell by any technique known in the art.

[0026] The producing bacterial cells of the invention can typically be obtained by homologous recombination or recombineering, including, for example, MAGE, using, for example, CRISPR, TALEN, meganuclease and / or Zn finger technologies (Wannier et al., "Recombineering and MAGE." Nat Rev Methods Primers 1, 7 (2021)), or by using site-specific recombination with phage integrase, PASTE (Ioannidi et al., "Drag-and-drop genome insertion without DNA cleavage with CRISPR-directed integrases." Biorxiv 2021.11.01.466786 (2021) doi:10.1101 / 2021.11.01.466786) or transposon-associated CRISPR-Cas systems (Ma et al., Trends Microbiol 29, 565-568 (2021)).

[0027] Phage DNA packaging genes and phage structural genes The producer bacterial cells of the invention stably contain at least one phage structural gene and at least one phage DNA packaging gene derived from a first type of bacteriophage.

[0028] By "stably comprised" or "stably comprising," it is meant herein that the producer bacterial cell carries the phage structural and DNA packaging genes either integrated into its chromosome or on an episome that is maintained in the cell, typically through selection (e.g., using nutritional, auxotrophic, or drug resistance markers). Each gene stably comprised by the producer bacterial cell can be independently on a plasmid, on a helper phage, or integrated into the producer bacterial cell chromosome.

[0029] In certain embodiments, the producer bacterial cell stably contains at least two, three, four, or all of the phage structural genes from the first type of bacteriophage and at least one phage DNA packaging gene from the first type of bacteriophage.

[0030] In certain embodiments, the producer bacterial cell stably contains at least one phage structural gene from the first type of bacteriophage and at least two or all of the phage DNA packaging genes from the first type of bacteriophage.

[0031] In certain embodiments, the producer bacterial cell stably contains at least two, three, four, or all of the phage structural genes from the first type of bacteriophage and at least two or all of the phage DNA packaging genes from the first type of bacteriophage.

[0032] In certain embodiments, the producer bacterial cell stably contains all of the phage structural genes from the first type of bacteriophage and all of the phage DNA packaging genes from the first type of bacteriophage.

[0033] By "phage structural genes" is meant herein genes from a bacteriophage that are involved in the assembly of the bacteriophage protein capsid. Phage structural genes include genes that encode phage structural elements; genes that encode phage proteins that are involved in the assembly of phage structural elements; and genes that encode phage proteins that are packaged inside the capsid as scaffolds or as pilot proteins that are injected into targeted bacterial cells.

[0034] Phage structural elements are well known to those of skill in the art and depend on the type of bacteriophage from which they are derived. Phage structural elements can be proteins, but can also be RNA (e.g., some phages, such as phi29 from Bacillus subtilis, encode a structural scaffold made from RNA). Phage structural elements typically include capsid proteins, tape measure proteins, fibers, baseplate proteins, tail sheath proteins, whisker proteins, decoration proteins, etc.

[0035] Phage proteins involved in the assembly of structural elements are well known to those of skill in the art and depend on the type of bacteriophage from which they are derived and, optionally, on the structural elements encoded by other phage structural genes. Phage proteins involved in the assembly of structural elements typically include phage chaperone proteins and phage proteases.

[0036] The phage proteins packaged inside the capsid as scaffolds or as pilot proteins that are injected into target host cells are well known to those skilled in the art and depend on the type of bacteriophage from which they are derived. Examples of such phage proteins are the RNA polymerase or minor pilot protein from phage N4.

[0037] As will be appreciated by those skilled in the art, the presence of a particular phage structural gene in a producer bacterial cell of the present invention will depend on the bacteriophage from which said phage structural gene is derived.

[0038] By "phage DNA packaging genes" is meant herein genes from a bacteriophage that are involved in packaging the bacteriophage genome into a bacteriophage capsid. Phage DNA packaging genes are well known to those skilled in the art and include genes encoding phage terminase, genes encoding phage accessory proteins, genes encoding phage ligase, genes encoding phage exonucleases involved in DNA packaging, and genes encoding phage endonucleases involved in DNA packaging.

[0039] In certain embodiments, the producer bacterial cell further stably contains at least one gene involved in phage regulation from said first type of bacteriophage.

[0040] By "genes involved in phage regulation" is meant herein phage genes involved in phage interaction with the host. Examples of genes involved in phage regulation include phage genes encoding master repressors, phage genes encoding antitermination proteins, phage genes involved in superexclusion mechanisms, phage genes involved in defense against the host's anti-phage mechanisms, phage genes involved in the degradation and / or modification of host elements, e.g., to complete the lytic cycle, and phage genes that are beneficial to the host.

[0041] In certain embodiments, the producer bacterial cell stably contains phage genes involved in protection from the host's anti-phage mechanisms derived from the first type of bacteriophage.

[0042] In certain embodiments, the phage structural genes and phage DNA packaging genes from the first type of bacteriophage, and optionally the genes involved in phage regulation from the first type of bacteriophage, are comprised in at least one plasmid, chromosome and / or helper phage. In certain embodiments, the phage structural genes and phage DNA packaging genes from the first type of bacteriophage, and optionally the genes involved in phage regulation from the first type of bacteriophage, are comprised in at least two separate nucleic acid molecules, in particular at least two plasmids, chromosomes, helper phages or combinations thereof.

[0043] In certain embodiments, the phage structural genes and phage DNA packaging genes from the first type of bacteriophage, and optionally the genes involved in phage regulation from the first type of bacteriophage, are comprised in a hybrid helper phage system, as defined below.

[0044] In certain embodiments, the phage structural genes and phage DNA packaging genes from the first type of bacteriophage, and optionally the genes involved in phage regulation from the first type of bacteriophage, are contained in a helper phage.

[0045] Guidance mechanism In the context of the present invention, expression of at least one of the phage structural genes and / or at least one of the phage DNA packaging genes, as defined in the section above "Phage DNA packaging genes and phage structural genes," in the producer bacterial cell is controlled by at least one inducible mechanism.

[0046] In certain embodiments, expression of at least one, particularly at least two, at least three or all of the phage structural genes in the producer bacterial cell is controlled by at least one inducible mechanism, particularly by one inducible mechanism.

[0047] In certain embodiments, expression of at least one, particularly at least two, at least three, or all of the phage DNA packaging genes in the producer bacterial cell is controlled by at least one inducible mechanism, particularly by one inducible mechanism.

[0048] In certain embodiments, the same induction mechanism controls the expression of at least one of the phage structural genes and at least one of the phage DNA packaging genes.

[0049] In an alternative embodiment, expression of at least one of the phage structural genes and expression of at least one of the phage DNA packaging genes are controlled by different induction mechanisms.

[0050] By "inducible mechanism" is meant herein a mechanism encoded by a gene or group of genes contained, in particular stably contained, in said producing bacterial cell, which is capable of inducing expression of the genes they control in response to a given trigger.

[0051] In certain embodiments, the induction mechanism further controls the copy number of the at least one of the phage structural genes and / or the at least one of the phage DNA packaging genes, in other words, in certain embodiments, the induction mechanism further controls the replication of the at least one of the phage structural genes and / or the at least one of the phage DNA packaging genes, in particular the replication of nucleic acid molecules comprising the at least one of the phage structural genes and / or the at least one of the phage DNA packaging genes.

[0052] In certain embodiments, the induction mechanism further controls the assembly of products expressed by the at least one of the phage structural genes and the at least one of the phage DNA packaging genes.

[0053] Examples of such induction mechanisms include: - Protein repressor or activator-based inducible systems that respond to small molecules (e.g., sugars, quorum-sensing molecules, gases, synthetic molecules, peptides, amino acids, metabolites, etc.), physical signals (temperature, pressure, etc.), chemical signals (osmolarity, pH, etc.), biological signals (cell density, DNA damage, etc.); these systems may be activated by secondary proteins, e.g., orthogonal RNA polymerases or sigma factors. - A proteolytic system to activate or repress transcription from the promoter. - RNA-based inducible systems, such as aptamers that respond to the signals mentioned above, e.g. RNAi, CRISPRi, toehold systems, riboswitches, etc. - one or more nucleic acids comprising at least one gene from a second type of bacteriophage involved in phage excision / insertion, phage DNA replication, and / or phage regulation.

[0054] In certain embodiments, the induction mechanism comprises at least one gene from a second type of bacteriophage involved in phage excision / insertion, phage DNA replication, and / or phage regulation.

[0055] Thus, in certain embodiments, the producer bacterial cell further comprises at least one gene from a second type of bacteriophage involved in phage excision / insertion, phage DNA replication, and / or phage regulation.

[0056] Genes involved in phage excision / insertion, phage DNA replication, and / or phage regulation By "genes involved in phage excision / insertion" is meant herein genes from a temperate phage that are involved in the excision of a phage present as a prophage from the genome or episome of a bacterial cell and / or the insertion of a phage as a prophage into the genome or episome of a bacterial cell.

[0057] By "genes involved in phage DNA replication" is meant herein genes from lysogenic or lytic phages that are involved in the mechanism of replication of phage DNA. Examples of genes involved in phage DNA replication include genes encoding DNA polymerases and genes involved in segregation of prophage sequences when they are present in a plasmid or linear plasmid form.

[0058] By "genes involved in phage regulation" is meant herein phage genes involved in phage interaction with the host. Examples of genes involved in phage regulation include phage genes encoding master repressors, phage genes encoding antitermination proteins, phage genes involved in superexclusion mechanisms, phage genes involved in defense against the host's anti-phage mechanisms, phage genes involved in the degradation and / or modification of host elements, e.g., to complete the lytic cycle, and phage genes that are beneficial to the host.

[0059] In the context of the present invention, said genes involved in phage excision / insertion, phage DNA replication, and / or phage regulation are neither DNA packaging genes nor structural genes, as defined above.

[0060] In a preferred embodiment, the producer bacterial cell of the invention comprises at least one gene, preferably all genes, involved in phage excision / insertion from a bacteriophage of a second type; at least one gene, preferably all genes, involved in phage DNA replication from a bacteriophage of a second type; and / or at least one gene, preferably all genes, involved in phage regulation from a bacteriophage of a second type.

[0061] In the context of the present invention, said producer bacterial cells do not contain genes from the first type of bacteriophage involved in phage excision / insertion and / or phage DNA replication.

[0062] In certain embodiments, the genes involved in phage excision / insertion, phage DNA replication, and / or phage regulation from the second type of bacteriophage are comprised in at least one plasmid, chromosome, and / or helper phage. In certain embodiments, the genes involved in phage excision / insertion, phage DNA replication, and / or phage regulation from the second type of bacteriophage are comprised in at least two separate nucleic acid molecules, in particular at least two plasmids, chromosomes, helper phages, or combinations thereof.

[0063] In certain embodiments, the genes involved in phage excision / insertion, phage DNA replication, and / or phage regulation from the second type of bacteriophage are comprised in a hybrid helper phage system, as defined below.

[0064] In certain embodiments, the genes involved in phage excision / insertion, phage DNA replication, and / or phage regulation from the second type of bacteriophage are contained in a helper phage system, more particularly on the same helper phage system as the phage structural genes and phage DNA packaging genes from the first type of bacteriophage, and optionally the genes involved in phage regulation from the first type of bacteriophage.

[0065] In the context of the present invention, said second type of bacteriophage originates from and / or targets a bacterial species or strain different from the bacterial species or strain from which said first type of bacteriophage originates and / or which said first type of bacteriophage targets.

[0066] In certain embodiments, the producer bacterial cells are from the same bacterial species or strain from which the second type of bacteriophage is derived and / or which the second type of bacteriophage targets.

[0067] In more particular embodiments, the producing bacterial cells are E. coli bacterial cells. In another particular embodiment, the producing bacterial cells are P. freudenreichii bacterial cells.

[0068] Other Elements In certain embodiments, the production bacterial cells of the invention further comprise at least one gene involved in phage RNA transcription.

[0069] By "genes involved in phage RNA transcription" is meant genes from lysogenic or lytic phages that are involved in the mechanism of transcription of phage RNA. Examples of such genes include genes encoding phage RNA polymerases and phage genes that encode proteins that modify host RNA polymerases, typically so that they can function past terminators.

[0070] Bacteriophages and genes derived from them By "bacteriophage-derived gene" is meant herein that the sequence of a gene is obtained from a bacteriophage, and the sequence has optionally been modified, recoded, and / or optimized compared to the sequence originally present in the bacteriophage. For example, the sequence may have been recoded for codon exchange or optimization (e.g., some proteins of kappa prophage contain an amber TAG stop codon that is recognized by E. coli and is preferably changed to TAA or TGA) or to prevent recombination.

[0071] Bacteriophages are obligate intracellular parasites that multiply inside bacteria by utilizing some or all of the host's biosynthetic machinery. Inside bacteria, bacteriophages can be found either as prophage, whose genome is integrated into the bacterial chromosome, or as phage plasmids, whose genome is part of an extrachromosomal plasmid (such phage plasmids are disclosed, for example, in Ravin et al. (1999) Molecular Microbiology 34(5):980-994). Examples of bacteriophages that can be in the form of phage plasmids include phages P1, N15, SSU5, P7, D6, pMCR-1-P3, IEBH, phiGILI6c, Bam35c, pBClin15, VP882, KS-14, P88, pLP39, F116, D3, and phiSG1. Phage genomes come in a variety of sizes and shapes (e.g., linear or circular). Most phages range in size from 24 to 200 nm in diameter. Phages contain nucleic acid (i.e., genome) and proteins and may be enveloped by a lipid membrane. Depending on the phage, the nucleic acid genome can be either DNA or RNA and can exist in either circular or linear form. The size of the phage genome varies depending on the phage. The simplest phages have genomes only a few thousand nucleotides in size, while more complex phages can contain more than 100,000 nucleotides, and in rare cases, more than 1,000,000 nucleotides in their genomes. The number and amount of individual types of proteins in the phage particle vary depending on the phage.

[0072] In certain embodiments, the first type of bacteriophage is selected from the order Caudovirales, which, based on the taxonomy of Krupovic et al. (Krupovic et al., Arch Virol. 2016 Jan;161(1):233-47), consists of: - the Myoviridae family (for example, but not limited to, the genus Cp220virus, the genus Cp8virus, the genus Ea214virus, the genus Felixo1virus, the genus Mooglevirus, the genus Suspvirus, the genus Hp1virus, the genus P2virus, the genus Kayvirus, the genus P100virus, the genus Silviavirus, the genus Spo1virus, the genus Tsarbombavirus, the genus Twortvirus, the genus Cc31virus, the genus Jd18virus, the genus Js98virus, the genus Kp15virus, the genus Moonvirus, the genus Rb49virus, the genus Rb69virus, the genus S16virus, the genus Schizot4virus, the genus Sp18virus, the genus T4virus, the genus Cr3virus, the genus Se1virus, the genus V5virus, the genus Abouovirus, the genus Agatevirus, the genus Agrican357virus, the genus Ap22virus, the genus Arv1virus, the genus B4virus) , Bastillevirus genus, Bc431virus genus, Bcep78virus genus, Bcepmuvirus genus, Biquartavirus genus, Bxz1virus genus, Cd119virus genus, Cp51virus genus, Cvm10virus genus, Eah2virus genus, Elvirus genus, Hapunavirus genus, Jimmervirus genus, Kpp10virus genus, M12virus genus, Machinavirus genus, Marthavirus genus, Msw3virus genus, Muvirus genus, Myohalovirus genus, Nit1virus genus, P1virus genus, Pakpunavirus genus, Pbunavirus genus, Phikzvirus genus, Rheph4virus genus, Rsl2virus genus, Rslunavirus genus, Secunda5virus genus, Sep1virus genus, Spn3virus genus, Svunavirus genus, Tg1virus genus, Vhmlvirus genus and Wphvirus genus) - the family Podoviridae (for example, but not limited to, the genus Fri1virus, the genus Kp32virus, the genus Kp34virus, the genus Phikmvvirus, the genus Pradovirus, the genus Sp6virus, the genus T7virus, the genus Cp1virus, the genus P68virus, the genus Phi29virus, the genus Nona33virus, the genus Pocjvirus, the genus Tl2011virus, the genus Bcep22virus, the genus Bpp1virus, the genus Cba41virus, the genus Dfl12virus, Lus genus, Ea92virus genus, Epsilon15virus genus, F116virus genus, G7cvirus genus, Jwalphavirus genus, Kf1virus genus, Kpp25virus genus, Lit1virus genus, Luz24virus genus, Luz7virus genus, N4virus genus, Nonanavirus genus, P22virus genus, Pagevirus genus, Phieco32virus genus, Prtbvirus genus, Sp58virus genus, Una961virus genus, and Vp5virus genus - the family Siphoviridae (for example, but not limited to, the genera Camvirus, Likavirus, R4virus, Acadianvirus, Coopervirus, Pg1virus, Pipefishvirus, Rosebushvirus, Brujitavirus, Che9cvirus, Hawkeyevirus, Plotvirus, Jerseyvirus, K1gvirus, Sp31virus, Lmd1virus, Una4virus, Bongovirus, Reyvirus, Buttevirus, rs virus genus, Charlie virus genus, Redi virus genus, Baxter virus genus, Nymphadora virus genus, Bignus virus genus, Fishburne virus genus, Phayonce virus genus, Kp36 virus genus, Rogue1 virus genus, Rtp virus genus, T1 virus genus, Tls virus genus, Ab18 virus genus, Amigo virus genus, Anatole virus genus, Andromeda virus genus, Attis virus genus, Barnyard virus genus, Bernal13 virus genus, Biseptima virus Rus virus, Bron virus, C2 virus, C5 virus, Cba181 virus, Cbast virus, Ceci virus, Che8 virus, Chi virus, Cjw1 virus, Corndog virus, Cronus virus, D3112 virus, D3 virus, Decurro virus, Demosthenes virus, Doucette virus, E125 virus, Eiau virus, Ff47 virus, Gaia virus, Giles virus, Gordon virus, Gord tnk virus genus, Harrison virus genus, Hk578 virus genus, Hk97 virus genus, Jenst virus genus, Jwx virus genus, Kellezio virus genus, Korra virus genus, L5 virus genus, Lambda virus genus, Laroye virus genus, Liefie virus genus, Marvin virus genus, Mudcat virus genus, N15 virus genus, Nonag virus genus, Np1 virus genus, Omega virus genus, P12002 virus genus, P12024 virus genus, P23 virus genus, P70 virus genus, Pa6 virus genus,Pamx74 virus genus, Patience virus genus, Pbi1 virus genus, Pepy6 virus genus, Pfr1 virus genus, Phic31 virus genus, Phicbk virus genus, Phieta virus genus, Phifel virus genus, Phijl1 virus genus, Pis4a virus genus, Psa virus genus, Psimuna virus genus, Rdjl virus genus, Rer2 virus genus, Sap6 virus genus, Send513 virus genus, Septima3 virus genus, Seurat virus genus, Sextaec virus genus, Sfi11 virus genus, Sfi21dt1 virus genus, Sitara virus genera, Sk1virus, Slashvirus, Smoothievirus, Soupsvirus, Spbetavirus, Ssp2virus, T5virus, Tankvirus, Tin2virus, Titanvirus, Tm4virus, Tp21virus, Tp84virus, Triavirus, Trigintaduovirus, Vegasvirus, Vendettavirus, Wbetavirus, Wildcatvirus, Wizardvirus, Woesvirus, Xp10virus, Ydn12virus, and Yuavirus), - the Ackermannviridae family (including, but not limited to, the genera Ag3virus, Limestonevirus, Cba120virus, and Vi1virus);

[0073] In certain embodiments, the first type of bacteriophage is not part of the Caudovirales order but belongs to a family with an unclassified order, such as, but not limited to, the family Tectiviridae (e.g., Alphatectivirus, Betatectivirus), the family Corticoviridae (e.g., Corticovirus), the family Inoviridae (e.g., Fibrovirus, Habenivirus, Inovirus, Lineavirus, Plectrovirus, Saetivirus, Vespevirus, These are from the genus Rtiliovirus, the family Cystoviridae (e.g., the genus Cystovirus), the family Leviviridae (e.g., the genus Allolevivirus, the genus Levivirus), the family Microviridae (e.g., the genus Alpha3microvirus, the genus G4microvirus, the genus Phix174microvirus, the genus Bdellomicrovirus, the genus Chlamydiamicrovirus, the genus Spiromicrovirus) and the family Plasmaviridae (e.g., the genus Plasmavirus).

[0074] In certain embodiments, the first type of bacteriophage targets archaea and is not part of the Caudovirales but is from a family with an unclassified order, such as, but not limited to, Ampullaviridae, FuselloViridae, Globuloviridae, Guttaviridae, Lipothrixviridae, Pleolipoviridae, Rudiviridae, Salterprovirus, and Bicaudaviridae.

[0075] In certain embodiments, the second type of bacteriophage is selected from the bacteriophages defined above, with the proviso that said second type of bacteriophage is different from said first type of bacteriophage.

[0076] In certain embodiments, the first type of bacteriophage is derived from a first bacterial species or strain and the second type of bacteriophage is derived from a second bacterial species or strain, and the first and second bacterial species or strains are different.

[0077] By "bacteriophage derived from a particular bacterial species or strain" is meant herein a bacteriophage that specifically targets and / or hosts a particular bacterial species or strain.

[0078] A non-exhaustive list of bacterial genera and their known host-specific bacterial viruses is presented in the following paragraphs. Synonyms and orthographic variations are indicated in parentheses. Homophones are repeated the number of times they occur (e.g., D, D, d). Unnamed phages are indicated by "NN" and are given next to their genus and their number in parentheses.

[0079] Bacteria of the genus Actinomyces can be infected by the following phages: Av-I, Av-2, Av-3, BF307, CT1, CT2, CT3, CT4, CT6, CT7, CT8 and 1281.

[0080] Bacteria of the genus Aeromonas can be infected by the following phages: AA-I, Aeh2, N, PMl, TP446, 3, 4, 11, 13, 29, 31, 32, 37, 43, 43-10T, 51, 54, 55R.1, 56, 56RR2, 57, 58, 59.1, 60, 63, Aehl, F, PM2, 1, 25, 31, 40RR2.8t, (syn= 44R), (syn= 44RR2.8t), 65, PM3, PM4, PM5 and PM6.

[0081] バチルス (Bacillus) 、Al-KI、B、BCJAl、BCl、BC2、BLLl、BLl、BP142、BSLl、 BSL2、BSl、BS3、BS8、BS15、BS18、BS22、BS26、BS28、BS31、BS104、BS105、BS106、BTB、B1715V1、C、CK-I、Co ll, Corl, CP-53, CS-I, CSi, D, D, D, D5, entl, FP8, FP9, FSi, FS2, FS3, FS5, FS8, FS9, G, GH8, GT8, GV-I, GV-2, GT-4, g3, gl2, gl3, gl4, gl6, gl7, g21, g23, g24, g29, H2, kenl, KK-88, Kuml, Kyul, J7W-1, LP52, (syn= LP-52, L7, Mexl, MJ-I, mor2, MP-7, MPlO, MP12, MP14, MP15, Neol, N°2, N5, N6P, PBCl, PBLA, PBPl, P2, Sa, SF2, SF6, Shal, Sill, SP02, (syn=ΦSPP1), SPβ, STI, STi, SU-Il, t, TbI, Tb2, Tb5, TbIO, Tb26, Tb51, Tb53, Tb55, Tb77, Tb 97, Tb99, Tb560, Tb595, Td8, Td6, Tdl5, TgI, Tg4, Tg6, Tg7, Tg9, TgIO, TgIl, Tgl3, Tgl5, Tg21, Tinl, Tin7, Tin8, Tinl3, Tm3, Tocl, Togl, toll, TP-I, TP-10vir, TP-15c, TP-16c, TP-17c, TP-19, TP35, TP51, TP-84, Tt4, Tt6, type A、type B、type C、type D、type E、Tφ3、VA-9、W、wx23、wx26、Yunl、α、γ、pl l、φmed-2、φT、φμ-4、φ3T、φ75、φlO5、(syn=φlO5)、I A、IB、1-97A、1-97B、2、2、3、3、3、5、12、14、20、30、35 、36、37、38、41C、51、63、64、138D、I、II、IV、NN-バチルス(13)、alel、ARl、AR2、AR3、AR7、AR9、Bace-11、(syn=11) Bastille BLl BL2 BL3 BL4 BL5 BL6 BL8 BL9 BP1 BS28, BS80, Ch, CP-51, CP-54, D-5, darl, denl, DP-7, entl. FoSi, FoS2, FS4, FS6, FS7, G, gall, gamma, GEl, GF-2, GSi, GT -I、GT-2、GT-3、GT-4、GT-5、GT-6、GT-7、GV-6、gl5、19、110、I Si, K, MP9, MP13, MP21, MP23, MP24, MP28, MP29, MP30, MP32, M P34, MP36, MP37, MP39, MP40, MP41, MP43, MP44, MP45, MP47, M P50, NLP-I, No.l, N17, N19, PBSl, PKl, PMBl, PMB12, PMJl, S. SPOl, SP3, SP5, SP6, SP7, SP8, SP9, SPlO, SP-15, SP50 (syn= SP-50) SP82 SST subl SW Tg8 Tgl2 Tgl3 Tgl4 thul thuΛ thuS Tin4 Tin23 TP-13 TP33 TP50 TSP-I type V type VI, V, Vx, β22, φe, φNR2, φ25, φ63, 1, 1, 2, 2C, 3NT, 4, 5, 6 7, 8, 9, 10, 12, 12, 17, 18, 19, 21, 138, III, 4(B. megaterium))、4(B.Ch. sphaericus)) AR13 BPP-IO BS32 BS107 Bl B2 GA-I GP-IO GV-3 GV-5 g8 MP20 MP27 MP49N f PP5, PP6, SF5, Tgl8, TP-I, Versailles, φl5, φ29, 1-97, 837 / IV, mi-バルス(1), BatlO, BSLlO, BSLI l, BS6, BSI l, BS16, BS23, BSlOl, BS102, gl8, morl, PBLl, SN45, thu2, thu3, TmI, Tm2, ​​TP-20, TP21, TP52, type F, type G, typeIV, HN-BacMus(3), BLE, (syn=θc), BS2, BS4, BS5, BS7, BlO, B12, BS20, BS21, F, MJ-4, PBA12, AP50, AP50-04, AP50-11, AP50-23, AP50-26, AP50-27, and Bam35. The following Bacillus-specific phages were deleted: DLP10716, DLP-11946, DPB5, DPB12, DPB21, DPB22, DPB23, GA-2, M, No. IM, PBLB, PBSH, PBSV, PBSW, PBSX, PBSY, PBSZ, phi, SPa, type 1, and μ.

[0082] Bacteroides bacteria can be infected by the following phages: crAss-phage, ad I2, Baf-44, Baf-48B, Baf-64, Bf-I, Bf-52, B40-8, F1, β1, φAl, φBrOl, φBrO2, 11, 67.1, 67.3, 68.1, mt-Bacteroides (3), Bf42, Bf71, HN-Bdellovibrio (1), and BF-41.

[0083] Bacteria of the genus Bordetella can be infected by the following phages: 134 and NN-Bordetella (3).

[0084] Bacteria of the genus Borrelia can be infected by the following phages: NN-Borrelia (1) and NN-Borrelia (2).

[0085] Bacteria of the genus Brucella can be infected by the following phages: A422, Bk, (syn= Berkeley), BM29, FOi, (syn= FOl), (syn= FQl), D, FP2, (syn= FP2), (syn= FD2), Fz, (syn= Fz75 / 13), (syn= Firenze 75 / 13), (syn= Fi), Fi, (syn= Fl), Fim, (syn= FIm), (syn= Fim), FiU, (syn= FlU), (syn= FiU), F2, (syn= F2), F3, (syn= F3), F4, (syn= F4), F5, (syn= F5), F6, F7, (syn= F7), F25, (syn= F25), (syn=￡25), F25U, (syn= F25u), (syn= F25U), (syn= F25V), F44, (syn- F44), F45, (syn= F45), F48, (syn= F48), I, Im, M, MC / 75, M51, (syn= M85), P, (syn= D), S708, R, Tb, (syn= TB), (syn= Tbilisi), W, (syn= Wb), (syn= Weybridge), X, 3, 6, 7, 10 / 1, (syn= 10), (syn= F8), (syn= F8), 12m, 24 / 11, (syn= 24), (syn= F9), (syn= F9), 45 / 111, (syn= 45), 75, 84, 212 / XV, (syn= 212), (syn= Fi0), (syn= FlO), 371 / XXIX, (syn= 371), (syn= Fn), (syn= Fl l) and 513.

[0086] Bacteria of the genus Burkholderia can be infected by the following phages: CP75, NN-Burkholderia (1) and 42.

[0087] Bacteria of the genus Campylobacter can be infected by the following phages: C type, NTCC12669, NTCC12670, NTCC12671, NTCC12672, NTCC12673, NTCC12674, NTCC12675, NTCC12676, NTCC12677, NTCC12678, NTCC12679, NTCC12680, NTCC12681, NTCC12682, NTCC12683, NTCC12684, 32f, 111c, 191, NN-Campylobacter (2), Vfi-6, (syn= V19), VfV-3, V2, V3, V8, V16, (syn= Vfi-1), V19, V20 (V45), V45, (syn= Vfi-2), V5, V6, V7, V8, V9, V10, V11, V12, V13, V14, V15, V16, V17, V18, V19, V20 (V45), V19, V21, V22, V23, V24, V25, V26, V27, V28, V29, V30, V31, V32, V33, V34, V35, V36, V37, V38, V39, V39, V40, V41, V42, V43, V44, V45, V46, V47, V48, V49, V49, V50, V51, V52, V53, V54, V55, V56, V57, V58, V59, V59, V59, V60, V61, V62, V63, V64, V65, V-45) and NN-Campylobacter (1).

[0088] Bacteria of the genus Chlamydia can be infected by the following phages: Chpl.

[0089] Bacteria of the genus Clostridium can be infected by the following phages: CAKl, CA5, Ca7, CEβ, (syn= 1C), CEγ, Cldl, c-n71, c-203 Tox-, DEβ, (syn= ID), (syn= lDt0X+), HM3, KMl, KT, Ms, NAl, (syn= Naltox+), PA135Oe, Pfo, PL73, PL78, PL81, Pl, P50, P5771, P19402, lCt0X+, 2Ct0X\ 2D3 (syn= 2Dt0X+), 3C, (syn= 3Ctox+), 4C, (syn= 4Ct0X+), 56, III-l, NN-Clostridium (61), NBlt0X+, αl, CAl, HMT, HM2, PFl5 P-23, P-46, Q-05, Q-oe, Q-16, Q-21, Q-26, Q-40, Q-46, S111, SA02, WA01, WA03, Wm, W523, 80 , C, CA2, CA3, CPTl, CPT4, cl, c4, c5, HM7, H11 / A1, H18 / Ax, FWS23, Hi58ZA1, K2ZA1, K21ZS23, ML, NA2t0X; Pf2, Pf3, Pf4, S9ZS3, S41ZA1, S44ZS23, α2, 41, 112ZS23, 214 / S23, 233 / Ai, 234 / S23, 235 / S23, II-l, II-2, II-3, NN-Clostridium (12), CA1, Fl, K, S2, 1, 5, and NN-Clostridium (8).

[0090] Bacteria of the genus Corynebacterium can be infected by the following phages: CGKl (deleted), A, A2, A3, AlOl, A128, A133, A137, A139, A155, A182, B, BF, B17, B18, B51, B271, B275, B276, B277, B279, B282, C, capi, CCl, CGl, CG2, CG33, CL31, Cog, (syn= CG5), D, E, F, H, HI, hqi, hq2, 11ZH33, Ii / 31, J, K, K, (syn= Ktox"), L, L, (syn= Ktox"). Ltox+), M, MC-I, MC-2, MC-3, MC-4, MLMa, N, O, ovi, ov2, ov3, P, P, R, RP6, RS29, S, T, U, UB1, ub2, UH 1, UH3, uh3, uh5, uh6, β, (syn=βtox+), βhv64, βvir, γ, (syn=γtoχ-), γl9, δ, (syn=δ'ox+), p, (syn= ptoχ-), Φ9, φ984, ω, IA, 1 / 1180, 2, 2 / 1180, 5 / 1180, 5ad / 9717, 7 / 4465, 8 / 4465, 8ad / 10269, 10 / 9253, 13Z9253, 15 / 3148, 21 / 9253, 28, 29, 55, 2747, 2893, 4498 and 5848.

[0091] Bacteria of the genus Enterococcus are infected by the following phages: DF78, Fl, F2, 1, 2, 4, 14, 41, 867, Dl, SB24, 2BV, 182, 225, C2, C2F, E3, E62, DS96, H24, M35, P3, P9, SBlOl, S2, 2BII, 5, 182a, 705, 873, 881, 940, 1051, 1057, 21096C, NN-Enterococcus (1), PEl, Fl, F3, F4, VD13, 1, 200, 235, and 341.

[0092] Bacteria of the genus Erysipelothrix can be infected by the following phages: NN-erysipelothrix (1).

[0093] Bacteria of the genus Escherichia can be infected by the following phages: BW73, B278, D6, D108, E, El, E24, E41, FI-2, FI-4, FI-5, HI8A, Ffl8B, i, MM, Mu, (syn= mu), (syn= MuI), (syn= Mu-I), (syn= MU-I), (syn= MuI), (syn=μ), 025, PhI-5, Pk, PSP3, Pl, PlD, P2, P4 (deletion), Sl, Wφ, φK13, φR73 (deletion), φl, φ2, φ7, φ92, ψ (deletion), 7 A, 8φ, 9φ, 15 (deletion), 18, 28-1, 186, 299, HH-Escherichia (2), AB48, CM, C4, C16, DD-VI, (syn= Dd-Vi), (syn= DDVI), (syn= DDVi), E4, E7, E28, FIl, FI3, H, Hl, H3, H8, K3, M, N, ND-2, ND-3, ND4, ND-5, ND6, ND-7, Ox-I (syn= OXl), (syn= HF), Ox-2 (syn= 0x2), (syn= 0X2), Ox-3, Ox-4, Ox-5, (syn= 0X5), Ox-6, (syn= 66F), (syn=φ66t), (syn=φ66t-), 5 0111, PhI-I, RB42, RB43, RB49, RB69, S, SaI-I, Sal-2, Sal-3, Sal-4, Sal-5, Sal-6, TC23, TC45, TuII*-6, (syn= TuII*), TuIP-24, TuII*46, TuIP-60, T2, (syn= ganuTia), (syn=γ), (syn= PC), (syn= P.C.), (syn= T-2), (syn= T2), (syn= P4), T4, (syn= T-4), (syn= T4), T6, T35, αl, 1, IA, 3, (syn= Ac3), 3A, 3T+, (syn= 3), (syn= Ml), 5φ, (syn=φ5), 9266Q, CFO103, HK620, J, K, KlF, m59, no.A, no.E, no.3, no.9, N4, sd, (syn = Sd), (syn = SD), (syn = Sa)3(syn = sd), (syn = SD), (syn = CD), T3, (syn = T-3), (syn = T3), T7, (syn = T-7), (syn = T7), WPK, W31, ΔH, φC3888, φK3, φK7, φK12, φV-1, Φ04-CF, Φ05, Φ06, Φ07, φl, φl.2, φ20, φ95, φ263, φlO92, φl, φll, (syn = φW), Ω8, 1, 3, 7, 8, 26, 27, 28-2, 29, 30, 31, 32, 38, 39, 42, 933W, NN-Escherichia(1), Esc-7-11, AC30, CVX-5, Cl, DDUP, ECl, EC2, E21, E29, Fl, F26S, F27S, Hi, HK022, HK97, (syn = ΦHK97), HK139, HK253, HK256, K7, ND-I, no.D, PA-2, q, S2, Tl, (syn = α), (syn = P28), (syn = T-I), (syn = Tx), T3C, T5, (syn = T-5), (syn = T5), UC-I, w, β4, γ2, λ(syn = lambda), (syn = Φλ), ΦD326, φγ, Φ06, Φ7, Φ10, φ80, χ, (syn = χi), (syn = φχ), (syn = φχi), 2, 4, 4A, 6, 8A, 102, 150, 168, 174, 3000, AC6, AC7, AC28, AC43, AC50, AC57, AC81, AC95, HK243, KlO, ZG / 3A, 5, 5A, 21EL, H19-J, 933H, O157 typing phage 1-16, JES-2013, 121Q, 172-1, 1720a-02, ADB-2, AKFV33, av-05, bV_EcoS_AHP42, bV_EcoS_AHP24, bC_EcoS_AHS24, bV_EcoS_AKS96, CBA120..

[0094] Bacteria of the genus Fusobacterium are infected by the following phages: NN-Fusobacterium(2), fv83-554 / 3, fv88-531 / 2, 227, fv2377, fv2527 and fv8501.

[0095] Bacteria of the genus Haemophilus are infected by the following phages: HP1, S2 and N3.

[0096] Bacteria of the genus Helicobacter are infected by the following phages: HP1 and ^^-Helicobacter (1).

[0097] Bacteria of the genus Klebsiella are infected by the following phages: AIO-2, KI4B, Kl6B, Kl9, (syn= K19), Kl14, Kl15, Kl21, Kl28, Kl29, KI32, Kl33, Kl35, Kl106B, Kl171B, Kl181B, Kl832B, AIO-I, AO-I, AO-2, AO-3, FC3-10, K, Kl1, (syn= KI1), Kl2, (syn= K12), Kl3, (syn= K13), (syn= Kl 70 / 11), Kl4, (syn= K14), Kl5, (syn= K15), Kl6, (syn= K16), Kl7, (syn= K17), Kl8, (syn= K18), Kl19, (syn= K19), Kl27, (syn= K127), Kl31, (syn= K131), Kl35, Kl171B, II, VI, IX, CI-I, Kl4B, Kl8, Kl11, Kl12, Kl13, Kl16, Kl17, Kl18, Kl20, Kl22, Kl23, Kl24, Kl26, Kl30, Kl34, Kl106B, KIi65B, Kl328B, KLXI, K328, P5046, 11, 380, III, IV, VII, VIII, FC3-11, Kl2B, (syn= K12B), Kl25, (syn= K125), Kl42B, (syn= K142), (syn= K142B), Kl181B, (syn= KIl 81), (syn= K1181B), Kl765 / !, (syn= K1765 / 1), Kl842B, (syn= K1832B), Kl937B, (syn= K1937B), Ll, φ28, 7, 231, 483, 490, 632 and 864 / 100.

[0098] Bacteria of the genus Leptospira are infected by the following phages: LEl, LE3, LE4, and NN-leptospira (1).

[0099] Bacteria of the genus Listeria are infected by the following phages: A511, 01761, 4211, 4286, (syn=BO54), A005, A006, A020, A500, A502, A511, Al 18, A620, A640, B012, B021, B024, B025, B035, B051, B053, B054, B055, B056, BlOl, BI 1O, B545, B604, B653, C707, D441, HSO47, HIOG, H8 / 73, H19, H21, H43, H46, H107, H108, HI lO, H163 / 84, H312, H340, H387, H391 / 73, H684 / 74, H924A, PSA, U153, φMLUP5, (syn= P35), 00241, 00611, 02971A, 02971C, 5 / 476, 5 / 911, 5 / 939, 5 / 11302, 5 / 11605, 5 / 11704, 184, 575, 633, 699 / 694, 744, 9 00, 1090, 1317, 1444, 1652, 1806, 1807, 1921 / 959, 1921 / 11367, 1921 / 11500, 1921 / 11566, 1921 / 12460, 1921 / 12582, 1 967, 2389, 2425, 2671, 2685, 3274, 3550, 3551, 3552, 4276, 4277, 4292, 4477, 5337, 5348 / 11363, 5348 / 11646, 5348 / 12430, 5348 / 12434, 10072, 11355C, 11711A, 12029, 12981, 13441, 90666, 90816, 93253, 907515, 910716, and NN-Listeria (15).

[0100] Bacteria of the genus Morganella are infected by the following phages:47

[0101] Bacteria of the genus Mycobacterium are infected by the following phages: 13, AGl, ALi, ATCC 11759, A2, B.C3, BG2, BKl, BK5, butyricum, BI, B5, B7, B30, B35, Clark, Cl, C2, DNAIII, DSP1, D4, D29, GS4E, (syn= GS4E), GS7, (syn= GS-7), (syn= GS7), IPa, lacticola, Legendre, Leo, L5, (syn=ΦL-5), MC-I, MC-3, MC-4, minetti, MTPHI l, Mx4, MyF3P / 59a, phlei, (syn= phlei 1), phlei 4, and Polonus. II, rabinovitschi, smegmatis, TM4, TM9, TMlO, TM20, Y7, YlO, φ630, IB, IF, IH, 1 / 1, 67, 106, 1430, Bl, (syn= Bol), B24, D, D29, FK, FS, HP, Polonus I, Roy, Rl, (syn= Rl-Myb), (syn= Ri), 11, 31, 40, 50, 103a, 103b, 128, 3111-D, 3215-D and NN-Mycobacterium (1).

[0102] Bacteria of the genus Neisseria are infected by the following phages: Group I, group II and NP1.

[0103] Bacteria of the genus Nocardia are infected by the following phages: MNP8, NJ-L, NS-8, N5 and TtiN-Nocardia.

[0104] Bacteria of the genus Proteus are infected by the following phages: Pm5, 13vir, 2 / 44, 4 / 545, 6 / 1004, 13 / 807, 20 / 826, 57, 67b, 78, 107 / 69, 121, 9 / 0, 22 / 608, 30 / 680, PmI, Pm3, Pm4, Pm6, Pm7, Pm9, PmIO, PmI l, Pv2, πl, φm, 7 / 549, 9B / 2, 10A / 31, 12 / 55, 14, 15, 16 / 789, 17 / 971, 19A / 653, 23 / 532, 25 / 909, 26 / 219, 27 / 953, 32A / 909, 33 / 971, 34 / 13, 65, 5006M, 7480b, VI, 13 / 3a, Clichy 12, π2600, φχ7, 1 / 1004, 5 / 742, 9, 12, 14, 22, 24 / 860, 2600 / D52, Pm8 and 24 / 2514.

[0105] Bacteria of the genus Providencia are infected by the following phages: PL25, PL26, PL37, 9211 / 9295, 9213 / 921 Ib, 9248, 7 / R49, 7476 / 322, 7478 / 325, 7479, 7480, 9000 / 9402 and 9213 / 921 Ia.

[0106] Bacteria of the genus Pseudomonas are infected by the following phages: PfI, (syn = Pf-I), Pf2, Pf3, PP7, PRRl, 7s, im-Pseudomonas(1), AI-I, AI-2, B17, B89, CB3, Col 2, Col 11, Col 18, Col 21, C154, C163, C167, C2121, E79, F8, ga, gb, H22, K1, M4, N2, Nu, PB-I, (syn = PBl), pfl6, PMN17, PPl, PP8, Psal, PsPl, PsP2, PsP3, PsP4, PsP5, PS3, PS17, PTB80, PX4, PX7, PYO1, PYO2, PYO5, PYO6, PYO9, PYO10, PYO13, PYO14, PYO16, PYO18, PYO19, PYO20, PYO29, PYO32, PYO33, PYO35, PYO36, PYO37, PYO38, PYO39, PYO41, PYO42, PYO45, PYO47, PYO48, PYO64, PYO69, PYO103, PlK, SLPl, SL2, S2, UNL-I, wy, Yai, Ya4, Yan, φBE, φCTX, φC17, φKZ, (syn = ΦKZ), φ-LT, Φmu78, φNZ, φPLS-1, φST-1, φW-14, φ-2, 1 / 72, 2 / 79, 3, 3 / DO, 4 / 237, 5 / 406, 6C, 6 / 6660, 7, 7v, 7 / 184, 8 / 280, 9 / 95, 10 / 502, 11 / DE, 12 / 100, 12S, 16, 21, 24, 25F, 27, 31, 44, 68, 71, 95, 109, 188, 337, 352, 1214, HN-Pseudomonas(23), A856, B26, CI-I, CI-2, C5, D, gh-1, Fl16, HF, H90, K5, K6, K104, K109, K166, K267, N4, N5, O6N-25P, PE69, Pf, PPN25, PPN35, PPN89, PPN91, PP2, PP3, PP4, PP6, PP7, PP8, PP56, PP87, PPl14. PP206, PP207, PP306, PP651, Psp231a, Pssy401, Pssy 9220 psi PTB2 PTB20 PTB42 PXl PX3 PXlO PX12 PX14 PYO70, PYO71, R, SH6, SH133, tf, Ya5, Ya7, φBS, ΦKf77, φ- MC, ΦmnF82, φPLS27, φPLS743, φS-1, 1, 2, 2, 3, 4, 5, 6, 7, 7 8, 9, 10, 11, 12, 12B, 13, 14, 15, 14, 15, 16, 17, 18, 19, 20 20, 21, 21, 22, 23, 23, 24, 25, 31, 53, 73, 119x, 145, 0, 267, 284, 308, 525, NN-Ch BI-I, C22, D3, D37, D40, D62, D3112, F7, FlO, gd, ge, gξ Hwl2, Jb 19. KFl, L°, OXN-32P, O6N-52P, PCH-I, PC13-1, PC35-1, PH2, PH51 PH93, PH132, PMW, PM13, PM57, PM61, PM62, PM63, PM69, PM105, PMl 13 PM681, PM682, PO4, PPl, PP4, PP5, PP64, PP65, PP66, PP71, PP86, PP88, PP92, PP401, PP711, PP891 Pssy41, Pssy42, Pssy403, Pssy404, Pssy420, Pssy923, PS4, PS-IO, Pz, SDl, SLl, SL3, SL5, SM, φC5, φCl l, φCl l-1, φC13, φC15, φMO, φX, φO4, φl l φ240, 2, 2F, 5, 7m, 11, 13, 13 / 441, 14, 20, 24, 40, 45, 49, 61, 73, 148, 160, 198, 218, 222, 236, 242, 246, 249, 258, 269, 295, 297, 309, 318, 342, 350, 351, 357-1, 400-1, HN- GlOl, M6, M6a, Ll, PB2, Pssyl5, Pssy4210, Pssy4220, PYO12, PYO34, PYO4 9. PYO50, PYO51, PYO52, PYO53, PYO57, PYO59, PYO200, PX2, PX5, SL4, φO3, φO6, and 31214.

[0107] Bacteria of the genus Rickettsia are infected by the following phages: NN-Rickettsia.

[0108] Bacteria of the genus Salmonella are infected by the following phages: b, Beccles, CT, d, Dundee, f, FeIs 2, GI, GUI, GVI, GVIII, k, K, i, j, L, 01, (syn= 0-1), (syn= O1), (syn= OI), (syn= 7), 02, 03, P3, P9a, PlO, Sab3, Sab5, SanlS, Sanl7, SI, Taunton, ViI, (syn= ViI), 9, im Salmonella (1), NI, N-5, N-IO, N-17, N-22, 11, 12, 16-19, 20.2, 36, 449C / C17 8, 966A / C259, a, BAOR, e, G4, GUI, L, LP7, M, MG40, N-18, PSA68, P4, P9c, P22, (syn= P22), (syn= PLT22), (syn= PLT22), P22al, P22-4, P22-7, P22-11, SNT-I, SNT-2, SP6, Villi, ViIV, ViV, ViVI, ViVII, Worksop, Sj5, ε34, 1,37, 1(40), (syn=φl

[40] ), 1,422, 2, 2.5, 3b, 4, 5, 6,14(18), 8, 14(6,7), 10, 27, 28B, 30, 31, 32, 33, 34, 36, 37, 39, 1412, SNT-3, 7-11, 40.3, c, C236, C557, C625, C966N, g, GV, G5, Gl 73, h, IRA, Jersey, MB78, P22-1, P22-3, P22-12, Sabl, Sab2, Sab2, Sab4, Sanl, San2, San3, San4, San6, San7, San8, San9, Sanl3, Sanl4, Sanl6, Sanl8, Sanl9, San20, San21, San22, San23, San24, San25, San26, SasLl, SasL2, SasL3, SasL4, SasL5, SlBL, SII, ViII, φl, 1, 2, 3a, 3al, 1010, Ym-Salmonella (1), N-4, SasL6 and 27.

[0109] Bacteria of the genus Serratia are infected by the following phages: A2P, PS20, SMB3, SMP, SMP5, SM2, V40, V56, ic, ΦCP-3, ΦCP-6, 3M, 10 / la, 20A, 34CC, 34H, 38T, 345G, 345P, 501B, SMB2, SMP2, BC, BT, CW2, CW3, CW4, CW5, Lt232, L2232, L34, L.228, SLP, S MPA, V.43, σ, φCWl, ΦCP6-1, ΦCP6-2, ΦCP6-5, 3T, 5, 8, 9F, 10 / 1, 2OE, 32 / 6, 34B, 34CT, 34P, 37, 41, 56, 56D, 56P, 6O P, 61 / 6, 74 / 6, 76 / 4, 101 / 8900, 226, 227, 228, 229F, 286, 289, 290F, 512, 764a, 2847 / 10, 2847 / 1Oa, L.359 and SMBl.

[0110] Bacteria of the genus Shigella are infected by the following phages: Fsa, (syn = a), FSD2d, (syn = D2d), (syn = W2d), FSD2E, (syn = W2e), fv, F6, f7.8, H-Sh, PE5, P90, SfII, Sh, SHm, SHrv, (syn = HIV), SHvi, (syn = HVI), SHVvm, (syn = HVIII), SKγ66, (syn = gamma 66), (syn = yββ), (syn =γ66b), SKm, (syn = SIIIb)5(syn = UI), SKw, (syn = Siva), (syn = IV), SIC (trademark), (syn = SIVA.), (syn = IVA), SKvi, (syn = KVI), (syn = Svi), (syn = VI), SKvm, (syn = Svm), (syn = VIII), SKVΠIA, (syn = SvmA), (syn = VIIIA), STvi, STK, STx1, STxn, S66, W2, (syn = D2c), (syn = D20), φl, φIVb 3-SO-R, 8368-SO-R, F7, (syn = FS7), (syn = K29), FlO, (syn = FSlO), (syn = K31), I1, (syn = alfa), (syn = FSa), (syn = Kl 8), (syn = α), I2, (syn = a), (syn = K19), SG33, (syn = G35), (syn = SO-35 / G), SG35, (syn = SO-55 / G), SG3201, (syn = SO-3201 / G), SHn, (syn = HII), SHv, (syn = SHV), SHx, SHX, SKn, (syn = K2), (syn = KII), (syn = Sn), (syn = SsII), (syn = II), SKrv, (syn = Sm), (syn = SsIV), (syn = IV), SK1Va, (syn = Swab), (syn = SsIVa), (syn = IVa), SKV, (syn = K4), (syn = KV), (syn = SV), (syn = SsV), (syn = V), SKx, (syn = K9), (syn = KX), (syn = SX), (syn = SsX), (syn = X), STV, (syn = T35), (syn = 35-50-R), STvm, (syn =T8345)、(syn= 8345-SO-SR)、W1、(syn= D8)、(syn= FSD8)、W2a、(syn= D2A)、(syn= FS2a)、DD-2、Sf6、FSi=6、(syn=F F6). 1881-SO-R)、γ66、(syn= gamma 66a)、(syn= Ssγ66)、φ2、BIl、DDVII、(syn= DD7)、FSD2b、(syn= W2B)、=Fsyn2= F2)、FS4、(syn= F4)、(syn= F4)、FS5、(syn= F5)、(syn= F5)、FS9、(syn= F9)、(syn= F9)、FI l、P2-S0-S、(syn=G6)-SO、(syn=G36)、 G36). HXn)、SKI、KI、(syn= S1)、(syn= SsI)、(syn= Svπ)、(syn= SsVII)、(syn= KIX)、(syn= S1x) SsIX)、SKXII、(syn= KXII)、(syn= Sxn)、(syn= SsXII)、STi、STffl、STrv、STVi、STvπ、S70、S206、U2-S0-S、3 210-SO-S、3859-SO-S、4020-SO-S、φ3、φ5、φ7、φ8、ϕ9、φlO、φl l、ϕl3、ϕl4、ϕl8、SHm、(syn= Hπi)、SHχi、(syn= HXt) or SKxI、(syn= KXI)、(syn= Sχi)、=X= Ss(XπI)

[0111] Bacteria of the genus Staphylococcus are infected by the following phages: A, EW, K, Ph5, Ph9, PhIO, Phl3, Pl, P2, P3, P4, P8, P9, PlO, RG, SB-i, (syn = Sb-I), S3K, Twort, ΦSK311, φ812, 06, 40, 58, 119, 130, 131, 200, 1623, STCl, (syn = stcl), STC2, (syn = stc2), 44AHJD, 68, ACl, AC2, A6"C", A9"C", b581, CA-I, CA-2, CA-3, CA-4, CA-5, DI l, L39x35, L54a, M42, Nl, N2, N3, N4, N5, N7, N8, NlO, Nil, N12, N13, N14, N16, Ph6, Phl2, Phl4, UC-18, U4, U15, Sl, S2, S3, S4, S5, X2, Z1, φB5-2, φD, ω, 11, (syn = φl l), (syn = P11-M15), 15, 28, 28A, 29, 31, 31B, 37, 42D, (syn = P42D), 44A, 48, 51, 52, 52A, (syn = P52A), 52B, 53, 55, 69, 71, (syn = P71), 71A, 72, 75, 76, 77, 79, 80, 80α, 82, 82A, 83 A, 84, 85, 86, 88, 88A, 89, 90, 92, 95, 96, 102, 107, 108, 111, 129-26, 130, 130A, 155, 157, 157A, 165, 187, 275, 275A, 275B, 356, 456, 459, 471, 471A, 489, 581, 676, 898, 1139, 1154A, 1259, 1314, 1380, 1405, 1563, 2148, 2638A, 2638B, 2638C, 2731, 2792A, 2792B, 2818, 2835, 2848A, 3619, 5841, 12100, AC3, A8, AlO, A13, b594n, D, HK2, N9, N15, P52, P87, Sl, S6, Z4, φRE, 3A, 3B, 3C, 6, 7, 16, 21, 42B, 42C, 42E, 44, 47, 47A5 47C, 51, 54, 54x1, 70, 73, 75, 78, 81, 82, 88, 93, 94, 101, 105, 110, 115, 129 / 16, 174, 594n, 1363 / 14, 2460 and mS-Staphylococcus (1).

[0112] Bacteria of the genus Streptococcus are infected by the following phages: EJ-I, NN-Streptococcus(1), a, Cl, FL0Ths, H39, Cp-I, Cρ-5, Cp-7, Cp-9, Cp-IO, AT298, A5, alO / Jl, alO / J2, alO / J5, alO / J9, A25, BTI l, b6, CAl, c20-l, c20-2, DP-I, Dp-4, DTl, ET42, elO, FA101, FEThs, Fκ, FKKIOI, FKLIO, FKP74, FKH, FLOThs, FyIOl, fl, F10, F20140 / 76, g, GT-234, HB3, (syn= HB-3), HB-623, HB-746, M102, O1205, φO1205, PST, PO, Pl, P2, P3, P5, P6, P8, P9, P9, P12, P13, P14, P49, P50, P5 1, P52, P53, P54, P55, P56, P57, P58, P59, P64, P67, P69, P71, P73, P75, P76, P77, P82, P83, P88, sc, sch, sf, SfIl 1, (syn= SFiI l), (syn= φSFill), (syn= ΦSfil l), (syn= φSfil l), sfil9, (syn= SFil9), (syn= φSFil9), (syn= φSfil9), Sfi21, (syn= SFi21), (syn= φSFi21), (syn= φSfi21), ST0, STX, st2, ST2, ST4, S3, (syn=φS3), s265, φ17, φ42, φ57, φ80, φ81, φ82, φ83, φ84, φ85, φ86, φ87, φ88, φ89, φ90, φ91, φ92, φ93, φ94, φ95, φ96, φ97 , φ98, φ99, φlOO, φlOl, φlO2, φ227, φ7201, ωl, ω2, ω3, ω4, ω5, ω6, ω8, ωlO, 1, 6, 9, 1OF, 12 / 12, 14, 17SR, 19S, 24, 50 / 33, 50 / 34, 55 / 14, 55 / 15, 70 / 35, 70 / 36, 71 / ST15, 71 / 45, 71 / 46, 74F, 79 / 37, 79 / 38, 80 / J4, 80 / J9, 80 / ST16, 80 / 15, 80 / 47, 80 / 48, 101, 103 / 39, 103 / 40, 121 / 41, 121 / 42, 123 / 43, 123 / 44, 124 / 44, 337 / ST17, and mStreptococcus (34).

[0113] Bacteria of the genus Treponema are infected by the following phages: NN-Treponema (1).

[0114] The structure of the Vibrio strain is as follows: CTXΦ and fs (syn= si) fs2, Ivpf5, Vfl2, Vf33, VPIΦ, VSK, v6, 493, CP-Tl, ET25, kappa, K13 9. Labol) XN-69P, OXN-86, O6N-21P, PB-I, P147, rp-1, SE3, VA-I (syn= VcA-I), VcA-2, VPl, VP2, VP4, VP7, VP8, VP9, ​​VPlO, VP17, VP18, VP19, X29 (syn= 29). d'Herelle)、t、ΦHAWI-1、ΦHAWI-2、ΦHAWI-3、ΦHAWI-4、ΦHAWI-5、ΦHAWI-6、ΦHAWI-7、XHAWI- 8, ΦHAWI-9, ΦHAWI-10, ΦHCl-1, ΦHC1-2, ΦHC1-3, ΦHC1-4, ΦHC2-1,>HC2-2, ΦHC2-3, ΦHC2-4 ΦHC3-1, ΦHC3-2, ΦHC3-3, ΦHD1S-1, ΦHD1S-2, ΦHD2S-1, ΦHD2S-2, ΦHD2S-3, ΦHD2S-4, ΦHD2S- 5, ΦHDO-1, ΦHDO-2, ΦHDO-3, ΦHDO-4, ΦHDO-5, ΦHDO-6, ΦKL-33, ΦKL-34, ΦKL-35, ΦKL-36, ΦKWH -2, ΦKWH-3, ΦKWH-4, ΦMARQ-1, ΦMARQ-2, ΦMARQ-3, ΦMOAT-1, ΦO139, ΦPEL1A-1, ΦPEL1A-2, ΦP EL8A-1, ΦPEL8A-2, ΦPEL8A-3, ΦPEL8C-1, ΦPEL8C-2, ΦPEL13A-1, ΦPEL13B-1, ΦPEL13B-2, ΦP EL13B-3, ΦPEL13B-4, ΦPEL13B-5, ΦPEL13B-6, ΦPEL13B-7, ΦPEL13B-8, ΦPEL13B-9, ΦPEL13B -10, φVP143, φVP253, Φ16, φl38, 1-II, 5, 13, 14, 16, 24, 32, 493, 6214, 7050, 7227, II (syn= groupII), (syn==φ2), V, VIII, ~m-Vibrio (13), KVP20, KVP40, nt-1, O6N-22P, P68, el, e2, e3, e4, e5, FK, G, I, K, nt-6, Nl, N2, N3, N4, N5, O6N -34P, OXN-72P, OXN-85P, OXN-100P, P, Ph-I, PL163 / 10, Q, S, T, φ92, 1-9, 37, 51, 57, 70A-8, 72A-4, 72A-10, 110A-4, 333, 4996, I(syn= group I), III(syn= group III), VI, (syn= A-Saratov), ​​VII, IX, lOA-1, 110A-5, 110A-7, hv-1, OXN-52P, P13, P38, P53, P65, P108, Pill, TPl3 VP3, VP6, VP12, VP13, 70A-3, 70A-4, 70A-10, 72A-1, 108A-3, 109-B1, 110A-2, 149, (syn=φl49), IV, (syn= group IV), NN-Vibrio (22), VP5, VP11, VP15, VP16, α1, α2, α3a, α3b, 353B, and HN-Vibrio (7).

[0115] Bacteria of the genus Yersinia are infected by the following phages: H, HI, H-2, H-3, H-4, Lucas 110, Lucas 303, Lucas 404, YerA3, YerA7, YerA20, YerA41, 3 / M64-76, 5 / G394-76, 6 / C753-76, 8 / C239-76, 9 / F18167, 1701, 1710, PST, 1 / F2852-76, D'Herelle, EV, H, Kotljarova, PTB, R, Y, YerA41, φYerO3-12, 3, 4 / C1324-76, 7 / F783-76, 903, 1 / M6176, and Yer2AT.

[0116] In certain embodiments, the first type of bacteriophage is selected from the group consisting of the bacteriophages listed above, and the second type of bacteriophage is selected from the group consisting of the bacteriophages listed above, and said second type of bacteriophage is a different type of bacteriophage than the first type of bacteriophage.

[0117] In certain embodiments, the first and / or second type bacteriophage is selected from the group consisting of Salmonella virus SKML39, Shigella virus AG3, Dickeya virus Limestone, Dickeya virus RC2014, Escherichia virus CBA120, Escherichia virus PhaxI, Salmonella virus 38, Salmonella virus Det7, Salmonella virus GG32, Salmonella virus PM10, Salmonella virus SFP10, Salmonella virus SH19, Salmonella virus SJ3, Escherichia virus ECML4, Salmonella virus EGFR ... Irsu Marshall, Salmonella virus Maynard, Salmonella virus SJ2, Salmonella virus STML131, Salmonella virus ViI, Erwinia virus Ea2809, Klebsiella virus 0507KN21, Serratia virus IME250, Serratia virus MAM1, Campylobacter virus CP21, Campylobacter virus CP220, Campylobacter virus CPt10, Campylobacter virus IBB35, Campylobacter virus CP81, Campylobacter virus CP30A, Campylobacter Virus CPX, Campylobacter virus NCTC12673, Erwinia virus Ea214, Erwinia virus M7, Escherichia virus AYO145A, Escherichia virus EC6, Escherichia virus HY02, Escherichia virus JH2, Escherichia virus TP1, Escherichia virus VpaE1, Escherichia virus wV8, Salmonella virus FelixO1, Salmonella virus HB2014, Salmonella virus Mushroom, Salmonella virus UAB87, Citrobacter virus Moogle, Citrobacter virus Robactervirus Mordin, Escherichiavirus SUSP1, Escherichiavirus SUSP2, Aeromonasvirus phiO18P, Haemophilusvirus HP1, Haemophilusvirus HP2, Pasteurellavirus F108, Vibriovirus K139, Vibriovirus Kappa, Burkholderiavirus phi52237, Burkholderiavirus phiE122, Burkholderiavirus phiE202, Escherichiavirus 186, Escherichiavirus P4, Escherichiavirus P2, Escherichiavirus Wphi,Mannheimia virus PHL101, Pseudomonas virus phiCTX, Ralstonia virus RSA1, Salmonella virus Fels2, Salmonella virus PsP3, Salmonella virus SopEphi, Yersinia virus L413C, Staphylococcus virus G1, Staphylococcus virus G15, Staphylococcus virus JD7, Staphylococcus virus K, Staphylococcus virus MCE2014, Staphylococcus virus P108, Staphylococcus virus Rodi, Staphylococcus virus S2 53, Staphylococcal virus S25-4, Staphylococcal virus SA12, Listeria virus A511, Listeria virus P100, Staphylococcal virus Remus, Staphylococcal virus SA11, Staphylococcal virus Stau2, Bacillus virus Camphawk, Bacillus virus SPO1, Bacillus virus BCP78, Bacillus virus TsarBomba, Staphylococcal virus Twort, Enterococcal virus phiEC24C, Lactobacillus virus Lb338-1, Lactobacillus Bacillus virus LP65, Enterobacter virus PG7, Escherichia virus CC31, Klebsiella virus JD18, Klebsiella virus PKO111, Escherichia virus Bp7, Escherichia virus IME08, Escherichia virus JS10, Escherichia virus JS98, Escherichia virus QL01, Escherichia virus VR5, Enterobacter virus Eap3, Klebsiella virus KP15, Klebsiella virus KP27, Klebsiella virus Matisse, Klebsiella virus Miro, Cyto Citrobactervirus Merlin, Citrobactervirus Moon, Escherichiavirus JSE, Escherichiavirus phi1, Escherichiavirus RB49, Escherichiavirus HX01, Escherichiavirus JS09, Escherichiavirus RB69, Shigellavirus UTAM, Salmonellavirus S16, Salmonellavirus STML198, Vibriovirus KVP40, Vibriovirus nt1, Vibriovirus ValKK3, Escherichiavirus VR7, Escherichiavirus VR20, Escherichiavirus VR25,Escherichia virus VR26, Shigella virus SP18, Escherichia virus AR1, Escherichia virus C40, Escherichia virus E112, Escherichia virus ECML134, Escherichia virus HY01, Escherichia virus Ime09, Escherichia virus RB3, Escherichia virus RB14, Escherichia virus T4, Shigella virus Pss1, Shigella virus Shfl2, Yersinia virus D1, Yersinia virus PST, Acinetobacter virus 133, Aeromonas virus 65, Aeromonas virus Virus Aeh1, Escherichia virus RB16, Escherichia virus RB32, Escherichia virus RB43, Pseudomonas virus 42, Cronobacter virus CR3, Cronobacter virus CR8, Cronobacter virus CR9, Cronobacter virus PBES02, Pectobacterium virus phiTE, Cronobacter virus GAP31, Escherichia virus 4MG, Salmonella virus SE1, Salmonella virus SSE121, Escherichia virus FFH2, Escherichia virus FV3, Escherichia virus JES2013, Escherichia virus V5, Brevibacillus virus Abouo, Brevibacillus virus Davies, Bacillus virus Agate, Bacillus virus Bobb, Bacillus virus Bp8pC, Erwinia virus Deimos, Erwinia virus Ea35-70, Erwinia virus RAY, Erwinia virus Simmy50, Erwinia virus Special G, Acinetobacter virus AB1, Acinetobacter virus AB2, Acinetobacter virus AbC62, Acinetobacter virus A P22, Arthrobactervirus ArV1, Arthrobactervirus Trina, Bacillusvirus AvesoBmore, Bacillusvirus B4, Bacillusvirus Bigbertha, Bacillusvirus Riley, Bacillusvirus Spock, Bacillusvirus Troll, Bacillusvirus Bastille, Bacillusvirus CAM003, Bacillusvirus Bc431, Bacillusvirus Bcp1, Bacillusvirus BCP82, Bacillusvirus BM15, Bacillusvirus Deepblue, Bacillusvirus JBP901,Burkholderia virus Bcep1, Burkholderia virus Bcep43, Burkholderia virus Bcep781, Burkholderia virus BcepNY3, Xanthomonas virus OP2, Burkholderia virus BcepMu, Burkholderia virus phiE255, Aeromonas virus 44RR2, Mycobacterium virus Alice, Mycobacterium virus Bxz1, Mycobacterium virus Dandelion, Mycobacterium virus HyRo, Mycobacterium virus I3, Mycobacterium Muvirus Nappy, Mycobacterium virus Sebata, Clostridium virus phiC2, Clostridium virus phiCD27, Clostridium virus phiCD119, Bacillus virus CP51, Bacillus virus JL, Bacillus virus Shanette, Escherichia virus CVM10, Escherichia virus ep3, Erwinia virus Asesino, Erwinia virus EaH2, Pseudomonas virus EL, Halomonas virus HAP1, Vibrio virus VP882, Brevibacillus virus Jim mer, Brevibacillus virus Osiris, Pseudomonas virus Ab03, Pseudomonas virus KPP10, Pseudomonas virus PAKP3, Sinorhizobium virus M7, Sinorhizobium virus M12, Sinorhizobium virus N3, Erwinia virus Machina, Arthrobacter virus Brent, Arthrobacter virus Jawnski, Arthrobacter virus Martha, Arthrobacter virus Sonny, Edwardsiella virus MSW3, Edwardsiella virus PEi21, Escherichia virus S Mu, Shigella virus SfMu, Halobacterium virus phiH, Bacillus virus Grass, Bacillus virus NIT1, Bacillus virus SPG24, Aeromonas virus 43, Escherichia virus P1, Pseudomonas virus CAb1, Pseudomonas virus CAb02, Pseudomonas virus JG004, Pseudomonas virus PAKP1, Pseudomonas virus PAKP4, Pseudomonas virus PaP1, Burkholderia virus BcepF1, Pseudomonas virus 141, Pseudomonas virus Ab28,Pseudomonas virus DL60, Pseudomonas virus DL68, Pseudomonas virus F8, Pseudomonas virus JG024, Pseudomonas virus KPP12, Pseudomonas virus LBL3, Pseudomonas virus LMA2, Pseudomonas virus PB1, Pseudomonas virus SN, Pseudomonas virus PA7, Pseudomonas virus phiKZ, Rhizobium virus RHEph4, Ralstonia virus RSF1, Ralstonia virus RSL2, Ralstonia virus RSL1, Aeromonas virus 25, A Aeromonas virus 31, Aeromonas virus Aes12, Aeromonas virus Aes508, Aeromonas virus AS4, Stenotrophomonas virus IME13, Staphylococcus virus IPLAC1C, Staphylococcus virus SEP1, Salmonella virus SPN3US, Bacillus virus 1, Geobacillus virus GBSV1, Yersinia virus R1RT, Yersinia virus TG1, Bacillus virus G, Bacillus virus PBS1, Microcystis virus Ma-LMM01, Vibrio virus MAR, Vibriovirus Vibriovirus VHML, Vibriovirus VP585, Bacillusvirus BPS13, Bacillusvirus Hakuna, Bacillusvirus Megatron, Bacillusvirus WPh, Acinetobactervirus AB3, Acinetobactervirus Abp1, Acinetobactervirus Fri1, Acinetobactervirus IME200, Acinetobactervirus PD6A3, Acinetobactervirus PDAB9, Acinetobactervirus phiAB1, Escherichiavirus K30, Klebsiellavirus K5, Klebsiellavirus K11, Klebsiella Virus Kp1, Klebsiella virus KP32, Klebsiella virus KpV289, Klebsiella virus F19, Klebsiella virus K244, Klebsiella virus Kp2, Klebsiella virus KP34, Klebsiella virus KpV41, Klebsiella virus KpV71, Klebsiella virus KpV475, Klebsiella virus SU503, Klebsiella virus SU552A, Pantoea virus Limelight, Pantoea virus Limezero, Pseudomonas virus LKA1, Pseudomonas virus phiKMV,Xanthomonas virus f20, Xanthomonas virus f30, Xylella virus Prado, Erwinia virus Era103, Escherichia virus K5, Escherichia virus K1-5, Escherichia virus K1E, Salmonella virus SP6, Escherichia virus T7, Kluyvera virus Kvp1, Pseudomonas virus gh1, Prochlorococcus virus PSSP7, Synechococcus virus P60, Synechococcus virus Syn5, Streptococcus Cassvirus Cp1, Streptococcusvirus Cp7, Staphylococcusvirus 44AHJD, Streptococcusvirus C1, Bacillusvirus B103, Bacillusvirus GA1, Bacillusvirus phi29, Kurthiavirus 6, Actinomycesvirus Av1, Mycoplasmavirus P1, Escherichiavirus 24B, Escherichiavirus 933W, Escherichiavirus Min27, Escherichiavirus PA28, Escherichiavirus Stx2 II, Shigellavirus 7502Stx, Shigellavirus POCJ13, Escherichiavirus 191, Escherichiavirus PA2, Escherichiavirus TL2011, Shigellavirus VASD, Burkholderiavirus Bcep22, Burkholderiavirus Bcepil02, Burkholderiavirus Bcepmigl, Burkholderiavirus DC1, Bordetellavirus BPP1, Burkholderiavirus BcepC6B, Cernophagavirus Cba41, Cernophagavirus Cba172, Dinorceobactervirus DFL12, Erwiniavirus Ea9-2, Erwiniavirus Rus Frozen, Escherichia virus phiV10, Salmonella virus Epsilon15, Salmonella virus SPN1S, Pseudomonas virus F116, Pseudomonas virus H66, Escherichia virus APEC5, Escherichia virus APEC7, Escherichia virus Bp4, Escherichia virus EC1UPM, Escherichia virus ECBP1, Escherichia virus G7C, Escherichia virus IME11, Shigella virus Sb1, Achromobacter virus Axp3, Achromobacter virus JWAlpha, Edwardsiella virus KF1, Pseudomonas virus KPP25,Pseudomonas virus R18, Pseudomonas, Solanum virus Ab09, Pseudomonas virus LIT1, Pseudomonas virus PA26, Pseudomonas virus Ab22, Pseudomonas virus CHU, Pseudomonas virus LUZ24, Pseudomonas virus PAA2, Pseudomonas virus PaP3, Pseudomonas virus PaP4, Pseudomonas virus TL, Pseudomonas virus KPP21, Pseudomonas virus LUZ7, Escherichia virus N4, Salmonella virus 9NA, Salmonella virus SP069, Salmonella virus BTP1, Salmonella virus HK620, Salmonella virus Nellavirus P22, Salmonellavirus ST64T, Shigellavirus Sf6, Bacillusvirus Page, Bacillusvirus Palmer, Bacillusvirus Pascal, Bacillusvirus Pony, Bacillusvirus Pookie, Escherichiavirus 172-1, Escherichiavirus ECB2, Escherichiavirus NJ01, Escherichiavirus phiEco32, Escherichiavirus Septima11, Escherichiavirus SU10, Brucellavirus Pr, Brucellavirus Tb, Escherichiavirus Pollock, Salmonellavirus FSL SP-058, Salmonellavirus FSL SP-076, Helicobacter virus 1961P, Helicobacter virus KHP30, Helicobacter virus KHP40, Hamiltonella virus APSE1, Lactococcus virus KSY1, Phormidium virus WMP3, Phormidium virus WMP4, Pseudomonas virus 119X, Roseobacter virus SIO1, Vibrio virus VpV262, Vibrio virus VC8, Vibrio virus VP2, Vibrio virus VP5, Streptomyces virus Amela, Streptomyces virus phiCAM, Streptomyces virus Aaronocolus, Streptomyces virus Caliburn, Streptomyces virus Danzina, Streptomyces virus Hydra, Streptomyces virus Izzy, Streptomyces virus Lannister, Streptomyces virus Lika, Streptomyces virus Sujidade, Streptomyces virus Zemlya, Streptomyces virus ELB20,Streptomyces virus R4, Streptomyces virus phiHau3, Mycobacterium virus Acadian, Mycobacterium virus Baee, Mycobacterium virus Reprobate, Mycobacterium virus Adawi, Mycobacterium virus Bane1, Mycobacterium virus BrownCNA, Mycobacterium virus Chrisnmich, Mycobacterium virus Cooper, Mycobacterium virus JAMaL, Mycobacterium virus Nigel, Mycobacterium Mycobacterium virus Stinger, Mycobacterium virus Vincenzo, Mycobacterium virus Zemanar, Mycobacterium virus Apizium, Mycobacterium virus Manad, Mycobacterium virus Oline, Mycobacterium virus Osmaximus, Mycobacterium virus Pg1, Mycobacterium virus Soto, Mycobacterium virus Suffolk, Mycobacterium virus Athena, Mycobacterium virus Bernardo, Mycobacterium virus Rus Gadjet, Mycobacterium virus Pipefish, Mycobacterium virus Godines, Mycobacterium virus Rosebush, Mycobacterium virus Babsiella, Mycobacterium virus Brujita, Mycobacterium virus Che9c, Mycobacterium virus Sbash, Mycobacterium virus Hawkeye, Mycobacterium virus Plot, Salmonella virus AG11, Salmonella virus Ent1, Salmonella virus f18SE, Salmonella virus J ersey, Salmonella virus L13, Salmonella virus LSPA1, Salmonella virus SE2, Salmonella virus SETP3, Salmonella virus SETP7, Salmonella virus SETP13, Salmonella virus SP101, Salmonella virus SS3e, Salmonella virus wksl3, Escherichia virus K1G, Escherichia virus K1H, Escherichia virus K1ind1, Escherichia virus K1ind2, Salmonella virus SP31, Leuconostoc virus Lmd1, Leuconostoc virus LN03,Leuconostoc virus LN04, Leuconostoc virus LN12, Leuconostoc virus LN6B, Leuconostoc virus P793, Leuconostoc virus 1A4, Leuconostoc virus Ln8, Leuconostoc virus Ln9, Leuconostoc virus LN25, Leuconostoc virus LN34, Leuconostoc virus LNTR3, Mycobacterium virus Bongo, Mycobacterium virus Rey, Mycobacterium virus Butters, Mycobacterium virus Michelle, Mycobacterium virus Charlie, Mycobacterium virus Pipsqueaks, Mycobacterium virus Xeno, Mycobacterium virus Panchino, Mycobacterium virus Phrann, Mycobacterium virus Redi, Mycobacterium virus Skinnyp, Gordonia virus BaxterFox, Gordonia virus Yeezy, Gordonia virus Kita, Gordonia virus Zirinka, Gordonia virus Nymphadora, Mycobacterium Muvirus Bignuz, Mycobacterium virus Brusacoram, Mycobacterium virus Donovan, Mycobacterium virus Fishburne, Mycobacterium virus Jebeks, Mycobacterium virus Malithi, Mycobacterium virus Phayonce, Enterobacter virus F20, Klebsiella virus 1513, Klebsiella virus KLPN1, Klebsiella virus KP36, Klebsiella virus PKP126, Klebsiella virus Sushi, Escherichia virus Escherichia virus AHP42, Escherichia virus AHS24, Escherichia virus AKS96, Escherichia virus C119, Escherichia virus E41c, Escherichia virus Eb49, Escherichia virus Jk06, Escherichia virus KP26, Escherichia virus Rogue1, Escherichia virus ACGM12, Escherichia virus Rtp, Escherichia virus ADB2, Escherichia virus JMPW1, Escherichia virus JMPW2, Escherichia virus T1, Shigella virus PSf2, Shigella virus Shfl1,Citrobactervirus Stevie, Escherichiavirus TLS, Salmonellavirus SP126, Cronobactervirus Esp2949-1, Pseudomonasvirus Ab18, Pseudomonasvirus Ab19, Pseudomonasvirus PaMx11, Arthrobactervirus Amigo, Propionibacteriumvirus Anatole, Propionibacteriumvirus B3, Bacillusvirus Andromeda, Bacillusvirus Blastoid, Bacillusvirus Curly, Bacillusvirus Eoghan, Bacillusvirus Russ Finn, Bacillus virus Glittering, Bacillus virus Riggi, Bacillus virus Taylor, Gordonia virus Attis, Mycobacterium virus Barnyard, Mycobacterium virus Konstantine, Mycobacterium virus Predator, Mycobacterium virus Bernal13, Staphylococcus virus 13, Staphylococcus virus 77, Staphylococcus virus 108PVL, Mycobacterium virus Bron, Mycobacterium virus Fait h1, Mycobacterium virus Joedirt, Mycobacterium virus Rumpelstiltskin, Lactococcus virus bIL67, Lactococcus virus c2, Lactobacillus virus c5, Lactobacillus virus Ld3, Lactobacillus virus Ld17, Lactobacillus virus Ld25A, Lactobacillus virus LLKu, Lactobacillus virus phiLdb, Cernophaga virus Cba121, Cernophaga virus Cba171, Cernophaga virus Cba181, Cernophaga virus ST , Bacillus virus 250, Bacillus virus IEBH, Mycobacterium virus Ardmore, Mycobacterium virus Avani, Mycobacterium virus Boomer, Mycobacterium virus Che8, Mycobacterium virus Che9d, Mycobacterium virus Deadp, Mycobacterium virus Dlane, Mycobacterium virus Dorothy, Mycobacterium virus Dotproduct, Mycobacterium virus Drago, Mycobacterium virus Fruitloop,Mycobacterium virus Gumbie, Mycobacterium virus Ibhubesi, Mycobacterium virus Llij, Mycobacterium virus Mozy, Mycobacterium virus Mutaforma13, Mycobacterium virus Pacc40, Mycobacterium virus PMC, Mycobacterium virus Ramsey, Mycobacterium virus Rockyhorror, Mycobacterium virus SG4, Mycobacterium virus Shauna1, Mycobacterium virus Shilan, Mycobacterium Mycobacterium virus Spartacus, Mycobacterium virus Taj, Mycobacterium virus Tweety, Mycobacterium virus Wee, Mycobacterium virus Yoshi, Salmonella virus Chi, Salmonella virus FSLSP030, Salmonella virus FSLSP088, Salmonella virus iEPS5, Salmonella virus SPN19, Mycobacterium virus 244, Mycobacterium virus Bask21, Mycobacterium virus CJW1, Mycobacterium virus Eureka, Mycobacterium Mycobacterium virus Kostya, Mycobacterium virus Porky, Mycobacterium virus Pumpkin, Mycobacterium virus Sirduracell, Mycobacterium virus Toto, Mycobacterium virus Corndog, Mycobacterium virus Firecracker, Rhodobacter virus RcCronus, Pseudomonas virus D3112, Pseudomonas virus DMS3, Pseudomonas virus FHA0480, Pseudomonas virus LPB1, Pseudomonas virus MP22, Pseudomonas virus Pseudomonas virus MP29, Pseudomonas virus MP38, Pseudomonas virus PA1KOR, Pseudomonas virus D3, Pseudomonas virus PMG1, Arthrobacter virus Decurro, Gordonia virus Demosthenes, Gordonia virus Katyusha, Gordonia virus Kvothe, Propionibacterium virus B22, Propionibacterium virus Doucette, Propionibacterium virus E6, Propionibacterium virus G4, Burkholderia virus phi6442,Burkholderia virus phi1026b, Burkholderia virus phiE125, Edwardsiella virus eiAU, Mycobacterium virus Ff47, Mycobacterium virus Muddy, Mycobacterium virus Gaia, Mycobacterium virus Giles, Arthrobacter virus Captnmurica, Arthrobacter virus Gordon, Gordonia virus GordTnk2, Paenibacillus virus Harrison, Escherichia virus EK99P1, Escherichia virus HK578, Escherichia virus Escherichia virus JL1, Escherichia virus SSL2009a, Escherichia virus YD2008s, Shigella virus EP23, Sodalis virus SO1, Escherichia virus HK022, Escherichia virus HK75, Escherichia virus HK97, Escherichia virus HK106, Escherichia virus HK446, Escherichia virus HK542, Escherichia virus HK544, Escherichia virus HK633, Escherichia virus mEp234, Escherichia virus mEp235, Escherichia virus Rus mEpX1, Escherichia virus mEpX2, Escherichia virus mEp043, Escherichia virus mEp213, Escherichia virus mEp237, Escherichia virus mEp390, Escherichia virus mEp460, Escherichia virus mEp505, Escherichia virus mEp506, Brevibacillus virus Jenst, Achromobacter virus 83-24, Achromobacter virus JWX, Arthrobacter virus Kellezzio, Arthrobacter virus Kitkat, Arthrobacter virus B ennie, Arthrobactervirus DrRobert, Arthrobactervirus Glenn, Arthrobactervirus HunterDalle, Arthrobactervirus Joann, Arthrobactervirus Korra, Arthrobactervirus Preamble, Arthrobactervirus Pumancara, Arthrobactervirus Wayne, Mycobacteriumvirus Alma, Mycobacteriumvirus Arturo, Mycobacteriumvirus Astro, Mycobacteriumvirus Backyardigan,Mycobacterium virus BBPiebs31, Mycobacterium virus Benedict, Mycobacterium virus Bethlehem, Mycobacterium virus Billknuckles, Mycobacterium virus Bruns, Ma, Mycobacterium virus Bxb1, Mycobacterium virus Bxz2, Mycobacterium virus Che12, Mycobacterium virus Cuco, Mycobacterium virus D29, Mycobacterium virus Doom, Mycobacterium virus Ericb, Mycobacterium virus Euphoria, Mycobacterium virus George, Mycobacterium virus Gladiator, Mycobacterium virus Goose, Mycobacterium virus Hammer, Mycobacterium virus Heldan , Mycobacterium virus Jasper, Mycobacterium virus JC27, Mycobacterium virus Jeffabunny, Mycobacterium virus JHC117, Mycobacterium virus KBG, Mycobacterium virus Kssjeb, Mycobacterium virus Kugel, Mycobacterium virus L5, Mycobacterium virus Lesedi, Mycobacterium virus LHTSCC, Mycobacterium virus lockley, Mycobacterium virus Marcell, Mycobacterium virus Microwolf, Mycobacterium virus Mrgordo, Mycobacterium virus Museum, Mycobacterium virus Nepal, Mycobacterium virus Packman, Mycobacterium virus Peaches, Mycobacterium virus Perseus, Mycobacterium virus Pukovnik, Mycobacterium virus Rebeuca, Mycobacterium virus Redrock, Mycobacterium virus Ridgecb, Mycobacterium virus Rockstar, Mycobacterium Mycobacterium virus Saintus, Mycobacterium virus Skipole, Mycobacterium virus Solon, Mycobacterium virus Switzer, Mycobacterium virus SWU1, Mycobacterium virus Ta17a, Mycobacterium virus Tiger, Mycobacterium virus Timshel, Mycobacterium virus Trixie, Mycobacterium virus Turbido, Mycobacterium virus Twister, Mycobacterium virus U2, Mycobacterium virus Violet,Mycobacterium virus Wonder, Escherichia virus DE3, Escherichia virus HK629, Escherichia virus HK630, Escherichia virus Lambda, Arthrobacterium virus Laroye, Mycobacterium virus Halo, Mycobacterium virus Liefie, Mycobacterium virus Marvin, Mycobacterium virus Mosmoris, Arthrobacter virus Circum, Arthrobacter virus Mudcat, Escherichia virus N15, Escherichia virus 9g, Escherichia virus Escherichia virus JenK1, Escherichia virus JenP1, Escherichia virus JenP2, Pseudomonas virus NP1, Pseudomonas virus PaMx25, Mycobacterium virus Baka, Mycobacterium virus Courthouse, Mycobacterium virus Littlee, Mycobacterium virus Omega, Mycobacterium virus Optimus, Mycobacterium virus Thibault, Polaribacter virus P12002L, Polaribacter virus P12002S, Non-Ravens virus P12024L, Nonlavens virus P12024S, Thermus virus P23-45, Thermus virus P74-26, Listeria virus LP26, Listeria virus LP37, Listeria virus LP110, Listeria virus LP114, Listeria virus P70, Propionibacterium virus ATCC29399BC, Propionibacterium virus ATCC29399BT, Propionibacterium virus Attacne, Propionibacterium virus Keiki, Propionibacterium virus Kubed, Propionibacterium virus Propionibacterium virus Lauchelly, Propionibacterium virus MrAK, Propionibacterium virus Ouroboros, Propionibacterium virus P91, Propionibacterium virus P105, Propionibacterium virus P144, Propionibacterium virus P1001, Propionibacterium virus P1.1, Propionibacterium virus P100A, Propionibacterium virus P100D, Propionibacterium virus P101A, Propionibacterium virus P104A,Propionibacterium virus PA6, Propionibacterium virus Pacnes201215, Propionibacterium virus PAD20, Propionibacterium virus PAS50, Propionibacterium virus PHL009M11, Propionibacterium virus PHL025M00, Propionibacterium virus PHL037M02, Propionibacterium virus PHL041M10, Propionibacterium virus PHL060L00, Propionibacterium virus PHL067M01, Propionibacterium Propionibacterium virus PHL070N00, Propionibacterium virus PHL071N05, Propionibacterium virus PHL082M03, Propionibacterium virus PHL092M00, Propionibacterium virus PHL095N00, Propionibacterium virus PHL111M01, Propionibacterium virus PHL112N00, Propionibacterium virus PHL113M01, Propionibacterium virus PHL114L00, Propionibacterium virus PHL116M00, Propionibacterium virus PHL117M01, Propionibacterium virus PHL118M01, Propionibacterium virus PHL119M02, Propionibacterium virus PHL120M03, Propionibacterium virus PHL121M04, Propionibacterium virus PHL122M04, Propionibacterium virus PHL123M05, Propionibacterium virus PHL124L06, Propionibacterium virus PHL125M06, Propionibacterium virus PHL126M07, Propionibacterium virus PHL127M08, Propionibacterium virus PHL128M09, Propionibacterium virus PHL130M01, Propionibacterium virus PHL131M01, Propionibacterium virus PHL132M01, Propionibacterium virus PHL133M01, Propionibacterium virus PHL134L00, Propionibacterium virus PHL135M01, Propionibacterium virus PHL136M01, Propionibacterium virus PHL137M02, Pro Propionibacterium virus PHL117M00, Propionibacterium virus PHL117M01, Propionibacterium virus PHL132N00, Propionibacterium virus PHL141N00, Propionibacterium virus PHL151M00, Propionibacterium virus PHL151N00, Propionibacterium virus PHL152M00, Propionibacterium virus PHL163M00, Propionibacterium virus PHL171M01, Propionibacterium virus PHL179M00, Propionibacterium virus PHL163M00 Propionibacterium virus PHL194M00, Propionibacterium virus PHL199M00, Propionibacterium virus PHL301M00, Propionibacterium virus PHL308M00, Propionibacterium virus Pirate, Propionibacterium virus Procrass1, Propionibacterium virus SKKY, Propionibacterium virus Solid, Propionibacterium virus Stormborn, Propionibacterium virus Wizzo, Pseudomonas virus PaMx28,Pseudomonas virus PaMx74, Mycobacterium virus Patience, Mycobacterium virus PBI1, Rhodococcus virus Pepy6, Rhodococcus virus Poco6, Propionibacterium virus PFR1, Streptomyces virus phiBT1, Streptomyces virus phiC31, Streptomyces virus TG1, Caulobacter virus Karma, Caulobacter virus Magneto, Caulobacter virus phiCbK, Caulobacter virus Rogue, Caulobacter virus Swift, Staphylococcus virus 11, Staphylococcus virus 29, Staphylococcus virus 37, Staphylococcus virus 53, Staphylococcus virus 55, Staphylococcus virus 69, Staphylococcus virus 71, Staphylococcus virus 80, Staphylococcus virus 85, Staphylococcus virus 88, Staphylococcus virus 92, Staphylococcus virus 96, Staphylococcus virus 187, Staphylococcus virus 52a, Staphylococcus virus 80al pha, Staphylococcal virus CNPH82, Staphylococcal virus EW, Staphylococcal virus IPLA5, Staphylococcal virus IPLA7, Staphylococcal virus IPLA88, Staphylococcal virus PH15, Staphylococcal virus phiETA, Staphylococcal virus phiETA2, Staphylococcal virus phiETA3, Staphylococcal virus phiMR11, Staphylococcal virus phiMR25, Staphylococcal virus phiNM1, Staphylococcus virus phiNM2, Staphylococcus virus phiNM4, Staphylococcus virus SAP26, Staphylococcus virus X2, Enterococcus virus FL1, Enterococcus virus FL2, Enterococcus virus FL3, Lactobacillus virus ATCC8014, Lactobacillus virus phiJL1, Pediococcus virus cIP1, Aeromonas virus pIS4A, Listeria virus LP302, Listeria virus PSA, Methanobacterium virus psiM1, Roseobacter virus RDJL1,Roseobacter virus RDJL2, Rhodococcus virus RER2, Enterococcus virus BC611, Enterococcus virus IMEEF1, Enterococcus virus SAP6, Enterococcus virus VD13, Streptococcus virus SPQS1, Mycobacterium virus Papyrus, Mycobacterium virus Send513, Burkholderia virus KL1, Pseudomonas virus 73, Pseudomonas virus Ab26, Pseudomonas virus Kakheti25, Escherichia virus Cajan , Escherichia virus Seurat, Staphylococcus virus SEP9, Staphylococcus virus Sextaec, Streptococcus virus 858, Streptococcus virus 2972, Streptococcus virus ALQ132, Streptococcus virus O1205, Streptococcus virus Sfi11, Streptococcus virus 7201, Streptococcus virus DT1, Streptococcus virus phiAbc2, Streptococcus virus Sfi19, Streptococcus virus Sfi21, Pae Paenibacillus virus Diva, Paenibacillus virus Hb10c2, Paenibacillus virus Rani, Paenibacillus virus Shelly, Paenibacillus virus Sitara, Paenibacillus virus Willow, Lactococcus virus 712, Lactococcus virus ASCC191, Lactococcus virus ASCC273, Lactococcus virus ASCC281, Lactococcus virus ASCC465, Lactococcus virus ASCC532, Lactococcus virus Bibb29, Lactococcus virus bIL17 0, Lactococcus virus CB13, Lactococcus virus CB14, Lactococcus virus CB19, Lactococcus virus CB20, Lactococcus virus jj50, Lactococcus virus P2, Lactococcus virus P008, Lactococcus virus sk1, Lactococcus virus Sl4, Bacillus virus Slash, Bacillus virus Stahl, Bacillus virus Staley, Bacillus virus Stills, Gordonia virus Bachita, Gordonia virus ClubL, Gordonia virus OneUp,Gordoniavirus Smoothie, Gordoniavirus Soups, Bacillusvirus SPbeta, Vibriovirus MAR10, Vibriovirus SSP002, Escherichiavirus AKFV33, Escherichiavirus BF23, Escherichiavirus DT57C, Escherichiavirus EPS7, Escherichiavirus FFH1, Escherichiavirus H8, Escherichiavirus slur09, Escherichiavirus T5, Salmonellavirus 118970sal2, Salmonellavirus Shivani, Salmonellavirus SPC35 , Salmonella virus Stitch, Arthrobacter virus Tank, Tsukamurella virus TIN2, Tsukamurella virus TIN3, Tsukamurella virus TIN4, Rhodobacter virus RcSpartan, Rhodobacter virus RcTitan, Mycobacterium virus Anaya, Mycobacterium virus Angelica, Mycobacterium virus Crimd, Mycobacterium virus Fionnbarth, Mycobacterium virus Jaws, Mycobacterium virus Larva, Mycobacterium virus Ma cncheese, Mycobacterium virus Pixie, Mycobacterium virus TM4, Bacillus virus BMBtp2, Bacillus virus TP21, Geobacillus virus Tp84, Staphylococcus virus 47, Staphylococcus virus 3a, Staphylococcus virus 42e, Staphylococcus virus IPLA35, Staphylococcus virus phi12, Staphylococcus virus phiSLT, Mycobacterium virus 32HC, Rhodococcus virus RGL3, Paenibacillus virus Vegas, Gordonii Avirus Vendetta, Bacillusvirus Wbeta, Mycobacteriumvirus Wildcat, Gordoniavirus Twister6, Gordoniavirus Wizard, Gordoniavirus Hotorobo, Gordoniavirus Monty, Gordoniavirus Woes, Xanthomonasvirus CP1, Xanthomonasvirus OP1, Xanthomonasvirus phil7, Xanthomonasvirus Xop411, Xanthomonasvirus Xp10, Streptomycesvirus TP1604, Streptomycesvirus YDN12,Alphaproteobacterial virus phiJl001, Pseudomonas virus LKO4, Pseudomonas spp. Virus M6, Pseudomonas virus MP1412, Pseudomonas virus PAE1, Pseudomonas virus Yua, Pseudoalteromonas virus PM2, Pseudomonas virus phi6, Pseudomonas virus phi8, Pseudomonas virus phi12, Pseudomonas virus phi13, Pseudomonas virus phi2954, Pseudomonas virus phiNN, Pseudomonas virus phiYY, Vibrio virus fs1, Vibrio virus VGJ, Ralstonia virus RS603, Ralstonia virus RSM1, Ralstonia virus RSM3, Escherichia virus M13, Escherichia virus I22, Salmonella virus IKe, Acholeplasma virus L51, Vibrio virus fs2, Vibrio virus VFJ, Escherichia virus If1, Propionibacterium virus B5, Pseudomonas virus Pf1, Pseudomonas virus Pf3, Ralstonia virus PE226, Ralstonia virus RSS1, Spiroplasma virus SVTS2, Stenotrophomonas virus PSH1, Stenotrophomonas virus SMA6, Stenotrophomonas virus SMA7, Stenotrophomonas virus SMA9, Stenotrophomonas virus SMA10, Stenotrophomonas virus SMA11, Stenotrophomonas virus SMA12, Stenotrophomonas virus SMA13, Stenotrophomonas virus SMA14, Stenotrophomonas virus SMA15, Stenotrophomonas virus SMA16, Stenotrophomonas virus SMA17, Stenotrophomonas virus SMA18, Stenotrophomonas virus SMA19, Stenotrophomonas virus SMA20, Stenotrophomonas virus SMA21, Stenotrophomonas virus SMA22, Stenotrophomonas virus SMA31, Stenotrophomonas virus SMA41, Stenotrophomonas virus SMA52, Stenotrophomonas virus SMA63, Stenotrophomonas virus SMA15, Stenotrophomonas virus SMA16, Stenotrophomonas virus SMA17, Stenotrophomonas virus SMA18, Stenotrophomonas virus SMA19, Stenotrophomonas virus SMA19, Stenotrophomonas virus SMA20, Stenotrophomonas virus SMA21, Stenotrophomonas virus SMA31, Stenotrophomonas virus SMA41, Stenotrophomonas virus SMA52, Steno Vibriovirus SMA7, Stenotrophomonas virus SMA9, Vibriovirus CTXphi, Vibriovirus KSF1, Vibriovirus VCY, Vibriovirus Vf33, Vibriovirus VfO3K6, Xanthomonas virus Cf1c, Spiroplasma virus C74, Spiroplasma virus R8A2B, Spiroplasma virus SkV1CR23x, Escherichia virus FI, Escherichia virus Qbeta, Escherichia virus BZ13, Escherichia virus MS2, Escherichia virus alpha3, Escherichia virus Rus ID21, Escherichia virus ID32, Escherichia virus ID62, Escherichia virus NC28, Escherichia virus NC29, Escherichia virus NC35, Escherichia virus phiK, Escherichia virus St1, Escherichia virus WA45, Escherichia virus G4, Escherichia virus ID52, Escherichia virus Talmos, Escherichia virus phiX174, Bdellovibriovirus MAC1, Bdellovibriovirus MH2K, Chlamydia virus Chp1, Chlamydia virus Chp2,Chlamydia virus CPAR39, Chlamydia virus CPG1, Spiroplasma virus SpV4, Acholeplasma virus L2, Pseudomonas virus PR4, Pseudomonas virus PRD1, Bacillus virus AP50, Bacillus virus Bam35, Bacillus virus GIL16, Bacillus virus Wip1, Escherichia virus phi80, Escherichia virus RB42, Escherichia virus T2, Escherichia virus T3, Escherichia virus T6, Escherichia virus VT2-Sa, Escherichia virus VT1-Sakai, Escherichia virus VT2-Sakai, Escherichia virus CP-933V, Escherichia virus P27, Escherichia virus Stx2phi-I, Escherichia virus Stx1phi, Escherichia Virus Stx2phi-II, Escherichia virus CP-1639, Escherichia virus BP-4795, Escherichia virus 86, Escherichia virus Min27, Escherichia virus 2851, Escherichia virus 1717, Escherichia virus YYZ-2008, Escherichia virus EC026_P06, Escherichia virus ECO103_P15, Escherichia and the first bacteriophage is selected from the group consisting of Escherichiavirus ECO103_P12, Escherichiavirus ECO111_P16, Escherichiavirus ECO111_P11, Escherichiavirus VT2phi_272, Escherichiavirus TL-2011c, Escherichiavirus P13374, and Escherichiavirus Sp5; and the first bacteriophage is different from the second bacteriophage.

[0118] BW73, B278, 1 of 1 of 100 sq. ft. D6, D108, E, El, E24, E41, FI-2, FI-4, FI-5, H I8A, Ffl8B, i, MM, Mu, 025, PhI-5, Pk, PSP3, P l, PlD, P2, P4, Sl, Wφ, φK13, φl, φ2, φ7, φ92, 7 A, 8φ, 9φ, 18, 28-1, 186, 299, HH-Zarkol (2), AB48, CM, C4, C16, DD-VI, E4 E7, E28, FIl, FI3, H, Hl, H3, H8, K3, M, N, ND-2, ND-3, ND4, ND-5, ND6, ND-7, O x-I, Ox-2, Ox-3, Ox-4, Ox-5, Ox-6, PhI-I, RB42, RB43, RB49, RB69, S, SaI- I、Sal-2、Sal-3、Sal-4、Sal-5、Sal-6、TC23、TC45、TuII*-6、TuIP-24、TuII * 46 TuIP-60, T2, T4, T6, T35, αl, 1, IA, 3, 3A, 3T+, 5φ, 9266Q, CFO103, HK620, J, K, KlF, m59, no. A、no. Yes, no. 3、no. 9, N4, sd, T3, T7, WPK, W31, ΔH, φC3888, φK3, φK7, φK12, φV-1, Φ04-C F Φ05, Φ06, Φ07, φl, φl.2, φ20, φ95, φ263, φlO92, φl, φll, Ω8, 1, 3, 8, 26, 27, 28-2, 29, 30, 31, 32, 38, 39, 42, 933W -7-11 AC30 CVX-5 Cl DDUP ECl EC2 E21 E29 Fl F26S F27S Hi 、HK022、HK97、HK139、HK253、HK256、K7、ND-I、PA-2、q、S2、Tl、)、T3C T5, UC-I, w, β4, γ2, λ, ΦD326, φγ, Φ06, Φ7, Φ10, φ80, χ, 2, 4, 4A, 6, 8A 102, 150, 168, 174, 3000, AC6, AC7, AC28, AC43, AC50, AC57, AC81, A C95, HK243, KlO, ZG / 3A, 5, 5A, 21EL, H19-J, and 3933H are free-standing.

[0119] In certain embodiments, the first type of bacteriophage is a prophage.

[0120] In another specific embodiment, the first type of bacteriophage is a temperate bacteriophage, a filamentous phage, or a pseudotemperate phage.

[0121] By "temperate bacteriophage" or "lysogenic bacteriophage" is meant herein a bacteriophage that is able to infect bacteria or archaea, be stably maintained in the genome of the strain and / or as an episome, and in their lysogenic state replicate along with the cell without producing virions. It is well known to those skilled in the art which of the above listed bacteriophages are temperate phages.

[0122] By "filamentous phage" is meant herein a bacteriophage characterized by having a single-stranded DNA genome encased in a long protein capsid cylinder. Typically, bacteria infected by filamentous phage are not lysed during the phage life cycle and replication, but rather experience a reduced growth rate. Those skilled in the art will know which of the bacteriophages listed above are filamentous phage.

[0123] By "pseudo-lysogenic phage" is meant herein a bacteriophage that is in a stage of development arrested in a host cell, without replication synchronized with the cell cycle and stable maintenance in a cell line (as in lysogenization), that proceeds without doubling of the phage genome (as in lytic development) and without degradation of the viral genome, thereby allowing subsequent resumption of viral development.

[0124] In a preferred embodiment, the first type of bacteriophage is a K. pneumoniae prophage. In another preferred embodiment, the first type of bacteriophage is a C. acnes bacteriophage.

[0125] In a preferred embodiment, the second type of bacteriophage is a lambda bacteriophage. In another preferred embodiment, the second type of bacteriophage is a P. freudenreichii bacteriophage.

[0126] In a preferred embodiment, the first type of bacteriophage is a K. pneumoniae prophage and the second type of bacteriophage is a lambda bacteriophage. In another preferred embodiment, the first type of bacteriophage is a C. acnes bacteriophage and the second type of bacteriophage is a P. freudenreichii bacteriophage.

[0127] Additional bacterial genes As is well known to those skilled in the art, some phages use products produced by their bacterial host for the folding and / or assembly of their structural elements and / or for the proper packaging of their DNA.

[0128] Thus, in certain embodiments, the producing bacterial cell further comprises at least one bacterial gene from the bacterial species or strain from which the first type of bacteriophage is derived that is involved in folding and / or assembly of phage structural elements and / or that is involved in DNA packaging.

[0129] As will be appreciated by those skilled in the art, the bacterial genes involved in folding and / or assembly of phage structural elements will depend on the particular bacteriophage from which said phage structural elements are obtained, and will typically include bacterial genes encoding chaperones.

[0130] Similarly, the bacterial genes involved in phage DNA packaging depend on the particular bacteriophage from which they are obtained. Examples of such bacterial genes include genes encoding IHF proteins.

[0131] payload In certain embodiments, the producer bacterial cell further comprises a payload packaged in the phage particle or phage-derived delivery vehicle.

[0132] As used herein, the term "payload" refers to any nucleic acid sequence (DNA and / or RNA) or amino acid sequence, or a combination of both (e.g., but not limited to, peptide nucleic acids or peptide-oligonucleotide conjugates) that is transferred into bacteria using a delivery vehicle. In certain embodiments, the payload is a nucleic acid payload, more particularly a DNA and / or RNA payload, and even more particularly a DNA payload.

[0133] The term "payload" may also refer to a plasmid, vector, or cargo.

[0134] The payload can be a phagemid or phasmid derived from a natural, evolved, or engineered bacteriophage genome. The payload can also be composed only in part of a phagemid or phasmid derived from a natural, evolved, or engineered bacteriophage genome.

[0135] As used herein, the terms "phagemid" or "phasmid" are equivalent and refer to a recombinant DNA vector that contains at least one sequence of a bacteriophage genome and also allows packaging into a capsid, preferably incapable of producing progeny, more particularly a vector derived from both a plasmid and a bacteriophage genome. Phagemids of the present disclosure contain a phage packaging site and optionally an origin of replication (ori), particularly a bacterial and / or phage origin of replication. In one embodiment, the phagemid does not contain an origin of replication and therefore cannot replicate by itself when injected into bacteria. Alternatively, the phagemid contains a plasmid origin of replication, particularly a bacterial and / or phage origin of replication.

[0136] In certain embodiments, the payload is packaged in the form of a packaged phagemid.

[0137] As used herein, the term "packaged phagemid" refers to a phagemid that has been encapsulated in a bacteriophage scaffold, phage-derived delivery particle, or capsid. In particular, it refers to a bacteriophage scaffold, phage delivery particle, or capsid that lacks a bacteriophage genome. Packaged phagemids may also be produced using helper phage strategies, well known to those skilled in the art. The helper phage typically contains all the genes encoding the structural and functional proteins essential for the phagemid according to the invention to be encapsulated.

[0138] In certain embodiments, the payload is delivered into a targeted bacterial cell, as defined below.

[0139] In more particular embodiments, the payload is stably maintained in the targeted bacterial cell. In alternative embodiments, the payload does not replicate in the targeted bacterial cell.

[0140] A sequence of interest under the control of a promoter In certain embodiments, the payload comprises a sequence of interest, particularly under the control of a promoter.

[0141] As known to those skilled in the art, promoters can be classified as strong or weak according to their affinity for RNA polymerase. The strength of a promoter can depend on whether transcription initiation occurs at a high or low frequency at that promoter. Different promoters with different strengths can be used in the present invention, which can lead to different levels of gene / protein expression (e.g., the level of expression initiated from mRNA initiated from a weak promoter is lower than the level of expression initiated from a strong promoter).

[0142] It will be understood by those skilled in the art that promoter sequences may be selected from a large number of known bacterial genes expressed by various bacterial species. Methods for prokaryotic promoter prediction also exist, which may be based on DNA stability analysis as described in Kanhere and Bansal (BMC Bioinformatics 2005, 6:1). The selection of promoters for payloads used in the context of the present invention may therefore be based on the bacterium to be targeted.

[0143] In some embodiments, the nucleic acid of interest may be placed under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the nucleic acid of interest in its natural environment.

[0144] Examples of bacterial promoters for use according to the present invention include, but are not limited to, positively regulated E. coli promoters, such as the positively regulated σ70 promoter (e.g., inducible pBad / araC promoter, Lux cassette right promoter, modified lambda Prm promoter, plac Or2-62(positive), pBad / AraC with additional REN sites, pBad, P(Las)TetO, P(Las)CIO, P(Rhl), Pu, FecA, pRE, cadC, hns, pLas, pLux), the "s" promoter (e.g., Pdps), the σ32 promoter (e.g., heat shock), and the σ54 promoter (e.g., glnAp2); negatively regulated E. coli promoters, such as the negatively regulated σ70 promoter (e.g., promoter(PRM+), modified lambda Prm promoter, TetR - TetR-4C P(Las)TetO, P(Las)CIO, P(Lac)IQ, RecA_DlexO_DLac01, dapAp, FecA, Pspac-hy, pel, plux-cl, plux-lac, CinR, CinL, glucose-regulated, modified Pr, modified Prm+, FecA, Pcya, rec A(SOS), Rec A(SOS), EmrR-regulated, Betl-regulated, pLac_lux, pTet_Lac, pLac / Mnt, pTet / Mnt, LsrA / cI, pLux / cI, Lacl, LacIQ, pLacIQl, pLas / cI, pLas / Lux, pLux / Las, pRecA with LexA-binding site, reverse BBa_R0011, pLacI / ara-1, pLacIq, rrnB PI, cadC, hns, PfhuA, pBad / araC, nhaA, OmpF, RcnR), σ promoter (e.g., Lutz-Bujard LacO with alternative sigma factor σ 38), σ promoter (e.g., Lutz-Bujard LacO with alternative sigma factor σ 32), σ promoter (e.g., glnAp2); negatively regulated B. subtilis promoters, e.g., repressible B.Examples of suitable promoters include the B. subtilis σA promoter (e.g., Gram-positive IPTG-inducible, Xyl, hyper-spank), the σ promoter, and the BioFAB promoters disclosed in Mutalik VK et al. (Nature Methods, 2013, 10: 354-360; see especially the Supplementary Data) as well as those disclosed on the BioFAB website (http: / / biofab.synberc.org / data). Other inducible microbial and / or bacterial promoters may be used in accordance with the present invention. Inducible promoters for use in accordance with the present disclosure may be induced (or repressed) by one or more physiological conditions, such as pH, temperature, radiation, osmolality, saline gradients, cell surface binding, and the concentration of one or more exogenous or endogenous inducers. Exogenous inducers or inducers may include, but are not limited to, amino acids and amino acid analogs, sugars and polysaccharides, nucleic acids, protein transcriptional activators and repressors, cytokines, toxins, petroleum-based compounds, metal-containing compounds, salts, ions, enzyme substrate analogs, hormones, or combinations thereof. .

[0145] Particularly preferred bacterial promoters for use in accordance with the present invention are constitutive promoters regulated by σ70, such as those from the Anderson collection (http: / / parts.igem.org / Promoters / Catalog / Anderson) promoters: BBa_J23100, BBa_J23101, BBa_J23102, BBa_J23103, BBa_J23104, BBa_J23105, BBa_J23106, BBa_J23107, BBa_J23108, BBa_J23109, BBa_J23110, BBa_J23111, BBa_J23112, BBa_J23113, BBa_J23114, BBa_J23115, BBa_J23116, BBa_J23117, BBa_J23118, and BBa_J23119.

[0146] Other preferred bacterial promoters are those disclosed in Stanton et al. (2014) Nat. Chem. Biol. 10:99-105 (incorporated herein by reference), including, inter alia, TetR, IcaR(A), AmtR, BetI, SrpR, Orf2, BM3R1, ButR, PhlF, PsrA, HlyIIR, AmeR, LmrA, QacR, ScbR, McbR, LitR, HapR, SmcR, TarA, and variants thereof. In certain embodiments, the promoter is SrpR and / or PhlF, or a variant thereof.

[0147] In some embodiments of the invention, a promoter may or may not be used in combination with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence downstream of the promoter. The enhancer may be located in any functional position before or after the promoter.

[0148] In some embodiments, the payload may include a terminator sequence, or terminator. As used herein, a "terminator" is a nucleic acid sequence that causes transcription to stop. A terminator may be unidirectional or bidirectional. It consists of a DNA sequence involved in the specific termination of an RNA transcript by an RNA polymerase. The terminator sequence prevents transcriptional activation of a downstream nucleic acid sequence by an upstream promoter. Therefore, in certain embodiments, a terminator that terminates the production of an RNA transcript is envisioned. A terminator may be necessary to achieve a desired gene / protein expression level in vivo.

[0149] The most commonly used type of terminator is the forward terminator. When placed downstream of a nucleic acid of interest, which is typically transcribed, a forward transcription terminator causes transcription to cease. In some embodiments, a bidirectional transcription terminator is provided, which typically causes transcription termination in both the forward and reverse strands. In some embodiments, a reverse transcription terminator is provided, which typically terminates transcription only in the reverse strand. In prokaryotic systems, terminators typically fall into two categories: (1) rho-independent terminators and (2) rho-dependent terminators. Rho-independent terminators generally consist of a palindromic sequence that forms a stem-loop rich in GC base pairs, followed by a string of uracil bases.

[0150] Terminators for use according to the present invention include any transcription terminator described herein or known to those of skill in the art. Examples of terminators include, but are not limited to, gene termination sequences, such as the bovine growth hormone terminator, and viral termination sequences, such as the TO terminator, TE terminator, lambda T1, and T1T2 terminators found in bacterial systems. In some embodiments, the termination signal may be a sequence that cannot be transcribed or translated, such as the result of sequence truncation.

[0151] Terminators for use according to the present invention also include the terminators disclosed in Chen YJ et al. (2013, Nature Methods, 10: 659-664) and the BioFAB terminators disclosed in Cambray G et al. (Nucl Acids Res, 2013, 41(9): 5139-5148).

[0152] In one embodiment, the sequence of interest is a programmable nuclease circuit delivered to targeted bacteria. This programmable nuclease circuit may be capable of mediating in vivo sequence-specific elimination of bacteria containing a target gene of interest (e.g., a gene harmful to humans). Some embodiments of the present disclosure relate to engineered variants of the Type II CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated) system of Streptococcus pyogenes. Other programmable nucleases that can be used include other CRISPR-Cas systems, engineered TALEN (Transcription Activator-Like Effector Nuclease) variants, engineered zinc finger nuclease (ZFN) variants, naturally occurring, evolved, or engineered meganuclease or recombinase variants, and any combination or hybrid of programmable nucleases. As such, the engineered autonomously distributed circuits provided herein may be used to selectively cleave DNA encoding genes of interest, such as toxin genes, virulence factor genes, antibiotic resistance genes, remodeling genes, or regulatory genes (see WO2014124226 and U.S. Patent Application Publication No. 2015 / 0064138).

[0153] Other sequences of interest, preferably programmable, can be added to the payload for delivery to the targeted bacteria. Preferably, the sequences of interest added to the payload lead to cell death of the targeted bacteria. For example, the nucleic acid sequences of interest added to the payload may encode holins, endolysins, restriction enzymes, or toxins that affect the targeted bacteria.

[0154] Alternatively, the sequence of interest added to the payload does not lead to the death of the targeted bacteria. For example, the sequence of interest may encode a reporter gene that leads to a luminescent or fluorescent signal. Alternatively, the sequence of interest may comprise proteins and enzymes that perform a useful function, such as modifying the metabolism of the targeted bacteria, the composition of its environment, or affecting the host subject. More specifically, the sequence of interest may be an antigen that elicits an immune response in the host subject. The unique antigen may be released into the environment after inducing lysis of the target cell or may be secreted by the target cell. (Costa et al., Nat Rev Microbiol. 2015 Jun;13(6):343-59; Anne et al., Curr Top Microbiol Immunol. 2017;404:267-308)

[0155] In certain embodiments, the nucleic acid sequence of interest is selected from the group consisting of a Cas nuclease, a Cas9 nuclease, a guide RNA, a single guide RNA (sgRNA), a CRISPR locus, an enzyme such as a nuclease or kinase, a TALEN, a ZFN, a meganuclease, a recombinase, a transposase, a bacterial receptor, a membrane protein, a structural protein, a gene expressing a secreted protein, a gene expressing resistance to an antibiotic or drug in general, a gene expressing a toxic protein or virulence factor, and a gene expressing a virulence protein or factor, a bacterial secreted protein or transporter, a bacterial pore, or any combination thereof. These proteins can also be modified or engineered to include additional features, for example, but not limited to, adding or removing a function (e.g., dCas9), adding a secretion signal to a protein that is not normally secreted, or adding an exogenous peptide in a loop.

[0156] In certain embodiments, the nucleic acid sequence of interest encodes a guide RNA-assisted targeting (INTEGRATE) system, typically as disclosed in Vo et al., Nat Biotechnol. 2021 Apr;39(4):480-489, including, for example, a Type IF V. cholerae CRISPR-transposon or a Type VK S. hofmannii CRISPR-transposon. In certain embodiments, the nucleic acid sequence of interest includes a nucleic acid encoding a crRNA, a nucleic acid encoding a TniQ cascade, cas8, cas7, and cas6 proteins, a nucleic acid encoding a tnsA, tnsB, and tnsC protein, and further includes donor DNA, wherein the donor DNA encodes a protein of interest to be added to the genome of the targeted bacterium. In certain embodiments, the nucleic acids encoding the TniQ cascade, cas8, cas7, and cas6 proteins, and the nucleic acids encoding the tnsA, tnsB, and tnsC proteins are in the form of a single polycistronic nucleic acid. In another specific embodiment, the nucleic acid sequence of interest comprises a nucleic acid encoding a guide RNA, a cas12k protein, a tnsB and tnsC protein, and a TniQ cascade, and further comprises donor DNA, wherein the donor DNA encodes the protein of interest to be added to the genome of the targeted bacterium.

[0157] In certain embodiments, the payload used in the context of the present invention comprises a sequence of interest encoding a bacteriocin, which can be a proteinaceous toxin produced by bacteria to kill or inhibit the growth of other bacteria. Bacteriocins are categorized in several ways, including by the producing strain, common resistance mechanisms, and mode of killing. Such bacteriocins have been described from Gram-negative bacteria (e.g., microcins, colicin-like bacteriocins, and tailocins) and Gram-positive bacteria (e.g., Class I, Class II, Class III, or Class IV bacteriocins).

[0158] In one embodiment, the payload used in the context of the present invention further comprises a sequence of interest encoding a toxin selected from the group consisting of a microcin, a colicin-like bacteriocin, a teilosin, a class I, a class II, a class III, and a class IV bacteriocin. The circuit may also encode a transporter required for secretion of the toxin into the extracellular space.

[0159] In certain embodiments, corresponding immunity polypeptides (i.e., antitoxins) may be used to protect bacterial cells for the purposes of payload production and encapsidation (see review by Cotter et al., Nature Reviews Microbiology 11: 95, 2013), but are not present in the pharmaceutical compositions and targeted bacteria to which the payloads used in the context of the present invention are delivered.

[0160] In certain embodiments, the payload used in the context of the present invention comprises a sequence of interest encoding a CRISPR-Cas system.

[0161] CRISPR systems contain two distinct elements: i) an endonuclease, in this case a CRISPR-associated nuclease (Cas or "CRISPR-associated protein") and ii) a guide RNA. Depending on the type of CRISPR system, the guide RNA may be in the form of a chimeric RNA consisting of a combination of a CRISPR bacterial RNA (crRNA) and a trans-activating crRNA (tracrRNA) (Jinek et al., Science. 2012 Aug 17;337(6096):816-21). The guide RNA combines in a single transcript the targeting specificity of the crRNA, which corresponds to a "spacing sequence" that serves as a guide for the Cas protein, and the conformational properties of the tracrRNA. When the guide RNA and Cas protein are co-expressed in a cell, the target genomic sequence can be permanently disrupted (and, depending on the location, cause loss of the targeted sequence and surrounding sequences and / or cell death) or modified. The modification may be guided by a repair matrix.

[0162] CRISPR systems comprise two main classes depending on the mechanism of action of the nuclease: - Class 1 is composed of multisubunit effector complexes and includes types I, III and IV; - Class 2, like Cas9 nuclease, consists of single-unit effector modules and includes types II (II-A, II-B, II-C, II-C variant), V (VA, VB, VC, VD, VE, V-U1, V-U2, V-U3, V-U4, V-U5) and VI (VI-A, VI-B1, VI-B2, VI-C, VI-D).

[0163] A sequence of interest according to the present invention may comprise a nucleic acid sequence encoding a Cas protein. A variety of CRISPR enzymes are available for use as sequences of interest in payloads used in the context of the present invention. In some embodiments, the CRISPR enzyme is a Type II CRISPR enzyme, a Type II-A CRISPR enzyme, or a Type II-B CRISPR enzyme. In other embodiments, the CRISPR enzyme is a Type I CRISPR enzyme or a Type III CRISPR enzyme. In some embodiments, the CRISPR enzyme catalyzes DNA cleavage. In some other embodiments, the CRISPR enzyme catalyzes RNA cleavage. In one embodiment, the CRISPR enzyme may be linked to a guide RNA or single guide RNA (sgRNA). In certain embodiments, the guide RNA or sgRNA targets a gene selected from the group consisting of antibiotic resistance genes, virulence protein or factor genes, toxin protein or factor genes, bacterial receptor genes, membrane protein genes, structural protein genes, secreted protein genes, genes that generally express resistance to drugs, and genes that cause harmful effects in the host subject.

[0164] The sequence of interest may include a nucleic acid sequence encoding a guide RNA or sgRNA to guide a Cas protein endogenous to the targeted bacterium, either alone or in combination with a Cas protein and / or guide RNA encoded by the payload.

[0165] Non-limiting examples of Cas proteins as part of a multi-subunit effector or as single unit effectors include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cas11(SS), Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), C2c4, C2c8, C2c5, C2c10, C2c9, Cas13a (C2c2), Cas13b (C2c6), Cas13c (C2c7), Cas13d, Csa5, Csc1, Csc2, Csx1, Csx2, Csx3, Csx4, Csx5, Csx6, Csx7, Csx8, Csx9, Csx10, Csx11, Csx12, Csx13a, Csx12b, Csx13c, Csx13d, Csx11, Csx12, Csx13b, Csx13c, Csx13d, Csx11, Csx12, Csx13c, Csx13d, Csx11, Csx11, Csx12, Csx13a, Csx13b, Csx13c, Csx13d, Csx11, Csx11, Csx11, Csx12, Csx13c, Csx13d, Csx1 ... se1, Cse2, Csy1, Csy2, Csy3, Csf1, Csf2, Csf3, Csf4, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csn2, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, C In some embodiments, the CRISPR enzyme cleaves both strands of the target nucleic acid at a protospacer adjacent motif (PAM) site.

[0166] In certain embodiments, the CRISPR enzyme is any Cas9 protein, such as any naturally occurring bacterial Cas9, as well as any variant, homolog, or ortholog thereof.

[0167] By "Cas9" is meant the protein Cas9 (also called Csn1 or Csx12) or a functional protein, peptide, or polypeptide fragment thereof, i.e., one that has the ability to interact with a guide RNA and to exert an enzymatic activity (nuclease) that allows it to make a double-stranded break in the DNA of the target genome. "Cas9" can therefore refer to a modified protein, for example, one that has been truncated to remove domains of the protein that are not essential for the protein's predefined function, in particular domains that are not required for interaction with the gRNA.

[0168] Sequences encoding Cas9 (whole protein or fragments thereof) as used in the context of the present invention can be obtained from any known Cas9 protein (Fonfara et al., Nucleic Acids Res. 2014 Feb;42(4):2577-90; Koonin et al., Curr Opin Microbiol. 2017 Jun;37:67-78). Examples of Cas9 proteins useful in the present invention include, but are not limited to, the Cas9 proteins of Streptococcus pyogenes (SpCas9), Streptococcus thermophiles (St1Cas9, St3Cas9), Streptococcus mutans, Staphylococcus aureus (SaCas9), Campylobacter jejuni (CjCas9), Francisella novicida (FnCas9), and Neisseria meningitidis (NmCas9).

[0169] Sequences encoding Cpf1 (Cas12a) (whole protein or fragments thereof) as used in the context of the present invention can be obtained from any known Cpf1 (Cas12a) protein (Koonin et al., Curr Opin Microbiol. 2017 Jun;37:67-78). Examples of Cpf1 (Cas12a) proteins useful in the present invention include, but are not limited to, Cpf1 (Cas12a) proteins from Acidaminococcus sp., Lachnospiraceae bacterium, and Francisella novicida.

[0170] Sequences encoding Cas13a (whole protein or fragments thereof) as used in the context of the present invention can be obtained from any known Cas13a (C2c2) protein (Abudayyeh et al., Nature. 2017 Oct 12;550(7675):280-284). Examples of Cas13a (C2c2) proteins useful in the present invention include, but are not limited to, the Cas13a (C2c2) protein of Leptotrichia wadei (LwaCas13a).

[0171] Sequences encoding Cas13d (whole protein or fragments thereof) as used in the context of the present invention can be obtained from any known Cas13d protein (Yan et al., Mol Cell. 2018 Apr 19;70(2):327-339, e5). Examples of Cas13d proteins useful in the present invention include, but are not limited to, the Cas13d proteins of Eubacterium siraeum and Ruminococcus sp.

[0172] The sequence encoding Mad4 (whole protein or a fragment thereof) as used in the context of the present invention is disclosed in International Application WO 2018 / 236548.

[0173] The sequence encoding Mad7 (whole protein or a fragment thereof) as used in the context of the present invention is disclosed in International Application WO 2018 / 236548.

[0174] The sequence encoding Cms1 (whole protein or a fragment thereof) as used in the context of the present invention is disclosed in International Patent Application WO 2017 / 141173.

[0175] In certain embodiments, the nucleic acid sequence of interest is a CRISPR / Cas9 system for reducing or inactivating gene expression of a gene selected from the group consisting of antibiotic resistance genes, virulence factor or protein genes, toxin factor or protein genes, genes expressing bacterial receptors, membrane proteins, structural proteins, secreted proteins, genes expressing resistance to drugs in general, and genes causing deleterious effects to the host subject.

[0176] In one embodiment, the CRISPR system is used to target and inactivate virulence factors. Virulence factors can be any substance produced by a pathogen that alters host subject-pathogen interactions by increasing the degree of damage done to the host subject. Virulence factors are used by pathogens in many ways, including, for example, cell adhesion or establishment of a niche in a host subject, to evade the host subject's immune response, to facilitate invasion into and release from the host subject's cells, to obtain nutrients from the host subject, or to inhibit other physiological processes in the host subject. Virulence factors can include enzymes, endotoxins, adhesion factors, motility factors, factors involved in complement evasion, scavenging factors, and factors that promote biofilm formation. For example, such targeted virulence factor genes include, but are not limited to, E. coli virulence factor genes, such as EHEC-HlyA, Stx1 (VT1), Stx2 (VT2), Stx2a (VT2a), Stx2b (VT2b), Stx2c (VT2c), Stx2d (VT2d), Stx2e (VT2e), and Stx2f (VT2f), Stx2h (VT2h), stx2k, fimA, fimF, fimH, neuC, kpsE, sfa, foc, iroN, aer, iha, papC, papGI, papGII, papGIII, hlyC, cnf1, hra, sat, ireA, usp The targeted virulence factor gene can be ompT, ibeA, malX, fyuA, irp2, traT, afaD, ipaH, eltB, estA, bfpA, eaeA, espA, aaiC, aatA, TEM, CTX, SHV, csgA, csgB, csgC, ​​csgD, csgE, csgF, csgG, csgH, T1SS, T2SS, T3SS, T4SS, T5SS, or T6SS (secretion system). For example, such targeted virulence factor genes can be Shigella dysenteriae virulence factor genes, such as, but not limited to, stx1 and stx2. For example, such a targeted virulence factor gene can be a Yersinia pestis virulence factor gene, such as, but not limited to, yscF (plasmid-mediated (pCD1) T3SS outer needle subunit).For example, such a targeted virulence factor gene can be a Francisella tularensis virulence factor gene, such as, but not limited to, fslA. For example, such a targeted virulence factor gene can be a Bacillus anthracis virulence factor gene, such as, but not limited to, pag (anthrax toxin, cell-associated protective antigen). For example, such a targeted virulence factor gene can be a Vibrio cholera virulence factor gene, such as, but not limited to, ctxA and ctxB (cholera toxin), tcpA (toxin co-regulated pilus), and toxT (master virulence regulator). For example, such targeted virulence factor genes can be Pseudomonas aeruginosa virulence factor genes, such as, but not limited to, pyoverdin (e.g., sigma factor pvdS, biosynthetic genes pvdL, pvdl, pvdJ, pvdH, pvdA, pvdF, pvdQ, pvdN, pvdM, pvdO, pvdP, transporter genes pvdE, pvdR, pvdT, opmQ), siderophore pyochelin (e.g., pchD, pchC, pchB, pchA, pchE, pchF, and pchG), and toxins (e.g., exoU, exoS, and exoT). For example, such targeted virulence factor genes can be Klebsiella pneumoniae virulence factor genes, such as, but not limited to, fimA (adhesive, type I pilus major subunit) and cps (capsular polysaccharide). For example, such targeted virulence factor genes can be Acinetobacter baumannii virulence factor genes, such as, but not limited to, ptk (capsule polymerization) and epsA (assembly).For example, such targeted virulence factor genes can be Salmonella enterica Typhi virulence factor genes, such as, but not limited to, MIA (invasion, SPI-1 regulator), ssrB (SPI-2 regulator), and those associated with bile resistance, such as the efflux pump genes acrA, acrB, and tolC. For example, such targeted virulence factor genes can be Fusobacterium nucleatum virulence factor genes, such as, but not limited to, FadA and TIGIT. For example, such targeted virulence factor genes can be Bacteroides fragilis virulence factor genes, such as, but not limited to, bft. For example, such targeted virulence factor genes can be Cutibacterium acnes porphyrin genes, CAMP factors (CAMP1, CAMP2, CAMP3, CAMP4), hyaluronate lyases (HYL-IB / II, HYL-IA), lipases (GehA, GehB), hemolysins, sialidases, endoglycoceramidases, endo-β-N-acetylglucosaminidases, dermatan sulfate adhesins (DsA1, DsA2), proline-threonine repeats (PTRs), or acne-associated genomic loci 1, 2, 3 (plasmids), 4, such as those described in Tomida et al., mBio. 2013 Apr 30;4(3):e00003-13, e.g., tight adhesion These genes may be any virulence factor contained on the Tad locus, streptolysin S-related genes (sag), or nonribosomal peptide synthetases (NRPS).

[0177] In another embodiment, the CRISPR / Cas system encodes an antibiotic resistance gene, such as, but not limited to, GyrB, ParE, ParY, AAC(1), AAC(2'), AAC(3), AAC(6'), ANT(2"), ANT(3"), ANT(4'), ANT(6), ANT(9), APH(2"), APH(3"), APH(3'), APH(4), APH(6), APH(7"), APH(9), ArmA, RmtA, RmtB, RmtC, Sgm, AER, BLA1, CTX-M, KPC, SHV, TEM, BlaB, CcrA, IMP, NDM, VIM, ACT, AmpC, CMY, LAT, PDC, OXA β-lactamase, mecA, Omp36, OmpF, PIB, bla (blaI, blaR1) and mec (mecI, mecR1) operons, chloramphenicol acetyltransferase (CAT), chloramphenicol phosphotransferase, ethambutol-resistant arabinosyltransferase (EmbB), MupA, MupB, integral membrane protein MprF, Cfr 23S rRNA methyltransferase, rifampin ADP-ribosyltransferase (Arr), rifampin glycosyltransferase, rifampin monooxygenase, rifampin phosphotransferase, DnaA, RbpA, rifampin-resistant beta-subunit of RNA polymerase (RpoB), Erm 23SrRNA methyltransferase, Lsa, MsrA, Vga, VgaB, streptogramin Vgb lyase, Vat acetyltransferase, fluoroquinolone acetyltransferase, fluoroquinolone resistance DNA topoisomerase, fluoroquinolone resistance GyrA, GyrB, ParC, quinolone resistance protein (Qnr), FomA, FomB, FosC, FosA, FosB, FosX, VanA, VanB, VanD, VanR, VanS, lincosamide nucleotidyltransferase (Lin), EreA, EreB, GimA, Mgt, Ole, macrolide phosphotransferase (MPH) , MefA, MefE, Mel, streptothricin acetyltransferase (sat), Sul1, Sul2, Sul3, sulfonamide resistance FolP, tetracycline inactivation enzymes TetX, TetA, TetB, TetC, Tet30, Tet31, TetM, TetO, TetQ, Tet32, Tet36, MacAB-TolC, MsbA, MsrA, VgaB, EmrD, EmrAB-TolC, NorB, GepA, MepA, AdeABC, AcrD, MexAB-OprM, mtrCDE, EmrE, adeR, acrR, baeSR, mexR, phoPQ, mtrR, or any antibiotic resistance gene listed in the Comprehensive Antibiotic Resistance Database (CARD https: / / card.mcmaster.ca / ).

[0178] In another embodiment, the CRISPR / Cas system is used to target and inactivate bacterial toxin genes. Bacterial toxins can be classified as either exotoxins or endotoxins. Exotoxins are produced and actively secreted; endotoxins remain part of the bacterium. Responses to bacterial toxins can be accompanied by severe inflammation and can lead to sepsis. Such toxins can be, for example, botulinum neurotoxin, tetanus toxin, staphylococcal toxin, diphtheria toxin, anthrax toxin, alpha toxin, pertussis toxin, Shiga toxin, heat-stable enterotoxin (E. coli ST), colibactin, BFT (B. fragilis toxin), or any toxin described in Henkel et al. (Toxins from Bacteria in EXS. 2010; 100: 1-29).

[0179] In certain embodiments, the payload used in the context of the present invention comprises a sequence of interest encoding a base editing system.

[0180] Base editing (BE) refers to the ability to replace specific nucleotide base pairs on DNA or RNA molecules with alternative nucleotide base pairs. Until recently, the only way to make specific substitutions on DNA in vivo was to use template DNA recombination to create specific base pair changes at the target locus. Base editing technology relies on a completely different strategy: DNA is not exchanged; instead, an enzymatic reaction converts one nucleotide into another, leading to a mismatch at the dsDNA level, which is then corrected by the cellular machinery.

[0181] In some embodiments, the base editing system comprises one or more of the following enzymes and systems:

[0182] A) Cytosine base editors (CBEs) and adenosine base editors (ABEs) as described in Rees, HA & Liu, DR Nat Rev Genet 19, pp. 770-788 (2018).

[0183] To date, seven DNA base editors have been described: - Cytosine base editor (CBE) that converts C:G to T:A (Komor, A et al., Nature 533:420-4 (2016)) - Adenine base editor (ABE) converts A:T to G:C (Gaudelli, NM et al., Nature 551(7681) pp. 464-471 (2017)) - Cytosine guanine base editor (CGBE) that converts C:G to G:C (Chen, L et al., "Precise and programmable C:G to G:C base editing in genomic DNA." Biorxiv (2020); Kurt, I et al., "CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells." Nature Biotechnology (2020)) - Cytosine adenine base editor (CABE) converts C:G to A:T (Zhao, D. et al., "New base editors change C to A in bacteria and C to G in mammalian cells." Nature Biotechnology (2020)) - Adenine cytosine base editor (ACBE) that converts A:T to C:G (International Publication No. 2020181180) - Adenine thymine base editor (ATBE) that converts A:T to T:A (International Publication No. 2020181202) - Thymine adenine base editor (TABE) that converts T:A to A:T (WO 2020181193; WO 2020181178; WO 2020181195)

[0184] Base editors differ in the base-modifying enzymes they use: CBEs rely on ssDNA cytidine deaminases, including APOBEC1, rAPOBEC1, APOBEC1 mutants or evolved versions (evoAPOBEC1), and APOBEC homologs (APOBEC3A (eA3A), Anc689), cytidine deaminase 1 (CDA1), evoCDA1, FERNY, and evoFERNY.

[0185] ABE relies on the deoxyadenosine deaminase activity of the tandem fusion TadA-TadA*, which is an evolved version of the E. coli tRNA adenosine deaminase enzyme TadA, which can convert adenosine on ssDNA to inosine. TadA* includes TadA-8a–e and TadA-7.10.

[0186] Besides base-modifying enzymes, there are modifications we have made to base editors to increase the efficiency, precision, and modularity of editing: - Addition of one or two uracil DNA glycosylase inhibitor (UGI) domains to prevent base excision repair mechanisms from reverting base editing - Addition of mu-GAM reduces insertion-deletion rates by inhibiting non-homologous end joining (NHEJ) in cells - The use of nickase-active Cas9 (nCas9 D10A) to create a nick in the unedited strand, thereby facilitating its repair and the resulting fixation of the edited base. - For example, the use of different Cas proteins from different organisms, variants with different PAM motifs, or different fidelities or different families (e.g. Cas12a).

[0187] Non-limiting examples of DNA base editor proteins include BE1, BE2, BE3, BE4, BE4-GAM, HF-BE3, Sniper-BE3, Target-AID, Target-AID-NG, ABE, EE-BE3, YE1-BE3, YE2-BE3, YEE-BE3, BE-PLUS, SaBE3, SaBE4, SaBE4-GAM, Sa(KKH)-BE3, VQR-BE3, VRER-BE3, EQR-BE3, xBE3, Cas12a-BE, Ea3A-BE3, A3A-BE3, TAM, CRISPR-X, ABE 7.9, ABE7.10, ABE7.10*, xABE, ABESa, VQR-ABE, VRER-ABE, Sa(KKH)-ABE, ABE8e, SpRY-ABE, SpRY-CBE, SpG-CBE4, SpG-ABE, SpRY-CBE4, SpCas9-NG -Including ABE, SpCas9-NG-CBE4, enAsBE1.1, enAsBE1.2, enAsBE1.3, enAsBE1.4, AsBE1.1, AsBE1.4, CRISPR-Abest, CRISPR-Cbest, eA3A-BE3, AncBE4.

[0188] The cytosine guanine base editor (CGBE) consists of a nickase CRISPR fused to: - Cytosine deaminases (rAPOBEC) and base excision repair proteins (e.g., rXRCC1) (Chen, L et al., "Precise and programmable C:G to G:C base editing in genomic DNA." Biorxiv (2020); Chen et al., Nature Communications 12:1384 (2021)) - Rat APOBEC1 variant (R33A) protein and E. coli uracil DNA N-glycosylase (eUNG) (Kurt, I. et al., "CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells." Nature Biotechnology (2020))

[0189] Cytosine adenine base editors (CABEs) consist of Cas9 nickase, cytidine deaminase (e.g., AID), and uracil-DNA glycosylase (Ung) (Zhao, D. et al., "New base editors change C to A in bacteria and C to G in mammalian cells." Nature Biotechnology (2020)).

[0190] ACBEs include nucleic acid programmable DNA binding proteins and adenine oxidase (WO2020181180).

[0191] ATBEs consist of a Cas9 nickase and one or more adenosine deaminase or oxidase domains (WO2020181202).

[0192] TABEs consist of a Cas9 nickase and an adenosine methyltransferase, thymine alkyltransferase, or adenosine deaminase domain (WO20181193; WO2020181178; WO2020181195).

[0193] Base editor molecules can also consist of two or more of the above-listed editor enzymes fused to a Cas protein (e.g., a combination of ABE and CBE). These biomolecules are termed dual base editors and allow editing of two different bases (Grunewald, J et al., "A dual-deaminase CRISPR base editor enables concurrent adenine and cytosine editing," Nature Biotechnology (2020); Li, C et al., "Targeted, random mutagenesis of plant genes with dual cytosine and adenine base editors," Nature Biotechnology (2020)).

[0194] In certain embodiments, the base editing system comprises a cytosine base editor (CBE) and / or an adenosine base editor (ABE) as defined above.

[0195] B) The prime editor (PE), described in Anzalone, AV et al., Nature 576, pp. 149-157 (2019), consists of nCas9 fused to a reverse transcriptase used in combination with a prime editing RNA (pegRNA; a guide RNA containing a template region for reverse transcription).

[0196] Prime editing allows the introduction of insertions, deletions (indels), and 12-base transversions. Prime editing relies on the ability of a reverse transcriptase (RT) fused to a Cas nickase variant to convert an RNA sequence delivered by a prime-editing guide RNA (pegRNA) into DNA at a nick site generated by the Cas protein. The DNA flap generated from this process is then either included or excluded in the targeted DNA sequence.

[0197] The Prime Editing System includes: - Cas nickase variants, such as Cas9-H840A fused to a reverse transcriptase domain, e.g., M-MLV RT or mutant versions thereof (M-MLV RT(D200N), M-MLV RT(D200N / L603W), M-MLV RT(D200N / L603W / T330P / T306K / W313F). - Prime editing guide RNA (pegRNA)

[0198] To facilitate editing, a primed editing system can include expression of an additional sgRNA that targets Cas nickase activity to the unedited DNA strand, ideally only after degradation of the edited strand flap, by designing the sgRNA to anneal to the edited strand but not the original strand.

[0199] Non-limiting examples of prime editing systems include PE1, PE1-M1, PE1-M2, PE1-M3, PE1-M6, PE1-M15, PE1-M3inv, PE2, PE3, PE3b.

[0200] Cas9 Retron precISe Parallel Editing via homology Y ("CRISPEY"), a retron RNA fused to an sgRNA and co-expressed with Cas9 and a retron protein containing at least a reverse transcriptase (Sharon, E. et al., Cell 175, 544-557.e16 (2018)).

[0201] SCRIBE strategy: A retron system expressed in combination with a recombinase, also known as single-strand annealing protein (SSAP), promotes recombination of single-stranded DNA (Farzadfard, F. & Lu, TK Science 346, 1256272 (2014)). Such recombinases include, but are not limited to, phage recombinases such as lambda Red, recET, Sak, Sak4, and the newly described SSAP described in Wannier, TM et al., "Improved bacterial recombineering by parallelized protein discovery." Biorxiv 2020.01.14.906594 (2020) doi:10.1101 / 2020.01.14.906594.

[0202] A group II intron-based targetron system described in Karberg, M. et al., Nat Biotechnol 19, 1162-7 (2001) and adapted in many bacterial species.

[0203] Other retron-based gene targeting approaches are described in Simon, AJ, Ellington, AD & Finkelstein, IJ Nucleic Acids Res 47, 11007-11019 (2019).

[0204] C) CRISPR / Cas. In various embodiments, the sequence of interest encodes a fusion protein comprising a Cas9 (e.g., Cas9 nickase) domain and a deaminase domain. In some embodiments, the fusion protein comprises Cas9 and a cytosine deaminase enzyme, such as an APOBEC enzyme, or an adenosine deaminase enzyme, such as an ADAT enzyme, as disclosed, for example, in U.S. Patent Application Publication No. 2015 / 0166980. In one embodiment, the deaminase is an ACF1 / ASE deaminase.

[0205] In various embodiments, the APOBEC deaminase is selected from the group consisting of APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, and APOBEC3H deaminase. In various embodiments, the fusion protein comprises a Cas9 domain, a cytosine deaminase domain, and a uracil glycosylase inhibitor (UGI) domain.

[0206] In one embodiment, the deaminase is an adenosine deaminase that deaminates adenosine in DNA, e.g., as disclosed in U.S. Patent No. 10,113,163. In some embodiments, the fusion protein further comprises an inhibitor of base repair, e.g., a nuclease-dead inosine-specific nuclease (dISN), e.g., as disclosed in U.S. Patent No. 10,113,163. In various embodiments, the nucleic acid of interest encodes a fusion protein comprising a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase programmed with a primed editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit, e.g., as described in Anzalone et al.

[0207] In some embodiments, other programmable nucleases can be used. These include engineered TALENs (transcription activator-like effector nucleases) and variants, engineered zinc finger nuclease (ZFN) variants, naturally occurring, evolved, or engineered meganuclease or recombinase variants, and any combination or hybrid of programmable nucleases. Thus, the programmable nucleases provided herein can be used to selectively modify DNA sequences or genes of interest, such as DNA encoding toxin genes, virulence factor genes, antibiotic resistance genes, remodeling genes, or regulatory genes (see WO2014124226 and U.S. Patent Application Publication No. 2015 / 0064138).

[0208] In one embodiment, a base editing system or base editor is used to inactivate gene expression by editing one or several nucleotides involved in transcription or translation. More specifically, the base editing system or base editor targets one or several nucleotides in a promoter, RBS, or start codon.

[0209] In one embodiment, a base editing system or base editor is used to introduce a premature stop codon.

[0210] In one embodiment, a base editing system or base editor is used to introduce one or several rare codons.

[0211] In another embodiment, the base editing system or base editor is used to modulate gene expression by editing one or several nucleotides involved in transcription or translation. More specifically, the base editing system or base editor targets one or several nucleotides in the promoter, RBS, or start codon, leading to increased or decreased gene expression.

[0212] In another embodiment, base editing systems or base editors are used to revert mutations that lead to inactivation, a decrease, or an increase in the activity of a gene or pathway.

[0213] In another embodiment, a base editing system or base editor is used to revert a mutation that leads to increased pathogenicity.

[0214] In one embodiment, the base editing system or base editor is used to alter gene regulation by editing one or several nucleotides involved in gene regulation, such as operator sequences, transcription factor binding sites, riboswitches, RNAse recognition sites, protease cleavage sites, methylation sites, post-translational modification sites (phosphorylation, glycosylation, acetylation, pupylation, etc.).

[0215] In some embodiments, the target sequence encodes an RNA base editing system.RNA base editing is based on the same principle as DNA base editing, and the enzyme that catalyzes the conversion of one RNA base to another RNA base must be close to the target base to perform the conversion locally.In one embodiment, the enzyme used for RNA editing is an adenosine deaminase from the ADAR family, which converts adenosine to inosine in dsRNA structure.Some promising researches have used this specificity for dsRNA to program localized RNA base editing by using the ADAR deaminase domain (ADAR DD ) fused to an antisense oligo. More recently, the ability of some CRISPR-Cas systems to bind RNA molecules has been repurposed for RNA editing. The ADAR2 deaminase domain (REPAIRv1 contains the ADAR2 DD -E488Q, ADAR2 for REPAIRv2 DD Using a catalytically dead Cas13b enzyme (dPspCas13b) fused to a hyperactivating mutant of Cas13b (-E488Q-T375G), Cox et al. improved specificity and efficiency compared to previous RNA editing strategies (Cox, DBT et al., Science 358, 1019-1027 (2017)).

[0216] Non-limiting examples of RNA-based editor proteins include REPAIRv1 and REPAIRv2.

[0217] In one embodiment, an RNA base editor is used to inactivate expression of a gene by editing one or several nucleotides involved in translation. More specifically, the base editor targets one or several nucleotides in the 5'UTR, RBS, or start codon.

[0218] In one embodiment, an RNA base editor is used to introduce a premature stop codon.

[0219] In one embodiment, an RNA base editor is used to introduce one or several rare codons.

[0220] In another embodiment, RNA base editors are used to modulate gene expression by editing one or several nucleotides involved in translation. More specifically, the base editors target one or several nucleotides in the 5'UTR, RBS, or start codon, which leads to increased or decreased gene expression.

[0221] In another embodiment, RNA base editors are used to revert mutations that lead to inactivation or a decrease in the activity of a gene or pathway.

[0222] In another embodiment, base editors are used to revert mutations that lead to increased pathogenicity.

[0223] In a preferred embodiment, the sequence of interest exerts an effect only in the targeted bacterial cell, and more preferably, the sequence of interest is expressed only in the targeted bacterial cell.

[0224] origin of replication In certain embodiments, the copy number of the payload is controlled in the producer bacterial cell by the at least one inducible mechanism defined above. In alternative embodiments, another inducible mechanism controls the copy number of the payload in the producer bacterial cell.

[0225] Origins of replication known in the art have been identified from species-specific plasmid DNA (e.g., CoIE1, R1, pT181, pSC101, pMB1, R6K, RK2, and p15a), from bacterial viruses (e.g., φX174, M13, F1, and P4), and from bacterial chromosomal origins of replication (e.g., oriC).

[0226] In one embodiment, the payload used in the context of the present invention comprises a bacterial origin of replication that is functional in the targeted bacterium.

[0227] Alternatively, the payload used in the context of the present invention does not contain any functional bacterial origin of replication or contains an origin of replication that is inactive in the targeted bacterium, in such an embodiment, the payload used in the context of the present invention is not capable of replicating itself once introduced into the bacterium by a phage particle or phage-derived delivery particle.

[0228] In one embodiment, the origin of replication on the packaged payload is inactive in the targeted bacteria, i.e., the origin of replication is not functional in the bacteria targeted by the phage particle or phage-derived delivery vehicle, thereby preventing undesired plasmid replication.

[0229] In one embodiment, the payload comprises a bacterial origin of replication functional in the producing bacterial cell of the invention.

[0230] Bacteria-specific origin of replication Plasmid replication depends on host bacterial enzymes and plasmid-controlled cis- and trans-determinants. For example, some plasmids possess determinants that are recognized in nearly all Gram-negative bacteria and function correctly during replication initiation and regulation in each host bacterium. Other plasmids possess this ability only in some bacteria (Kues, U. and Stahl, U. 1989 Microbiol Rev. 53:491-516).

[0231] Plasmids replicate by three general mechanisms: theta-type, strand displacement, and rolling circle replication initiated at origins of replication (reviewed by Del Solar et al., 1998 Microbio and Molec Biol. Rev 62:434-464). These origins contain sites required for the interaction of plasmid- and / or host-encoded proteins.

[0232] The origin of replication used on the payload used in the context of the present invention may be of medium copy number, such as ColE1 from pBR322 (15-20 copies / cell) or R6K plasmid (15-20 copies / cell), or of high copy number, such as pUC oris (500-700 copies / cell), pGEM oris (300-400 copies / cell), pTZ oris (>1000 copies / cell) or pBluescript oris (300-500 copies / cell).

[0233] In one embodiment, the bacterial origin of replication is ColE1, pMB1 and variants (such as pBR322, pET, pUC), p15a, ColA, ColE2, pOSAK, pSC101, R6K, IncW (such as pSa), IncFII, pT181, P1, F IncP, IncC, IncJ, IncN, IncP1, IncP4, IncQ, IncH11, RSF1010, CloDF13, NTP16, R1, f5, pPS10, pC194, pE194, BBR1, pBC1, pEP2, pWVO1, pLF1311, pAP1, pWKS1, pLS1, pLS11, pUB6060, pJD4, pIJ101, pSN22, pAMbeta1, pIP5 01, pIP407, ZM6100(Sa), pCU1, RA3, pMOL98, RK2 / RP4 / RP1 / R68, pB10, R300B, pRO1614, pRO1600, pECB2, pCM1, pFA3, RepFIA, RepFIB, RepFIC, pYVE439-80, R387, phasyl, RA1, TF-FC2, pMV158 and pUB113.

[0234] More preferably, the bacterial origin of replication is an E. coli origin of replication selected from the group consisting of ColE1, pMB1 and variants (such as pBR322, pET, pUC), p15a, ColA, ColE2, pOSAK, pSC101, R6K, IncW (such as pSa), IncFII, pT181, P1, F IncP, IncC, IncJ, IncN, IncP1, IncP4, IncQ, IncH11, RSF1010, CloDF13, NTP16, R1, f5, pPS10.

[0235] More preferably, the bacterial origin of replication is selected in the group consisting of pC194, pE194, BBR1, pBC1, pEP2, pWVO1, pLF1311, pAP1, pWKS1, pLS1, pLS11, pUB6060, pJD4, pIJ101, pSN22, pAMbeta1, pIP501, pIP407, ZM6100(Sa), pCU1, RA3, pMOL98, RK2 / RP4 / RP1 / R68, pB10, R300B, pRO1614, pRO1600, pECB2, pCM1, pFA3, RepFIA, RepFIB, RepFIC, pYVE439-80, R387, phasyl, RA1, TF-FC2, pMV158 and pUB113.

[0236] Even more preferably, the bacterial origins of replication are ColE1 and p15a.

[0237] In one embodiment, the bacterial origin of replication is functional in Propionibacterium and Cutibacterium, more specifically Propionibacterium freudenreichii and Cutibacterium acnes, and is selected from the group consisting of pLME108, pLME106, p545, pRGO1, pZGX01, pPG01, pYS1, FRJS12-3, FRJS25-1, pIMPLE-HL096PA1, and A_15_1_R1. In certain embodiments, the bacterial origin of replication is selected from the bacterial origins of replication disclosed in U.S. Patent Application Publication No. 2022 / 135986 and U.S. Patent Application Publication No. 2022 / 135987.

[0238] Phage replication origin The payload used in the context of the present invention may comprise a complete phage genome complement as well as a phage origin of replication capable of initiating replication of the payload for subsequent encapsulation into a different capsid.

[0239] Phage origins of replication can also be engineered to act as bacterial origins of replication without the need for packaging any phage particles.

[0240] The phage origin of replication contained in the payload used in the context of the present invention can be any origin of replication found in a phage.

[0241] Preferably, the phage origin of replication can be the wild-type or non-wild-type sequence of M13, f1, phiX174, P4, Lambda, P2, 186, Lambda-like, HK022, mEP237, HK97, HK629, HK630, mEP043, mEP213, mEP234, mEP390, mEP460, mEPx1, mEPx2, phi80, mEP234, T2, T4, T5, T7, RB49, phiX174, R17, PRD1 P1-like, P2-like, P22, P22-like, N15, and N15-like bacteriophages.

[0242] More preferably, the phage origin of replication is selected in the group consisting of the M13, f1, φX174, P4, and Lambda phage origins of replication.

[0243] In a particular embodiment, the phage origin of replication is a P4 origin of replication.

[0244] In certain embodiments, the phage origin of replication is from a Propionibacterium phage: a BW-like phage, such as Doucette, B22, E6, or G4; a BV-like phage, such as Anatole, E1, or B3; a BX-like phage, such as PFR1 or PFR2; a filamentous B5 phage; or a BU-like phage (Cutibacterium acnes phage). In certain embodiments, the phage origin of replication is selected from the phage origins of replication disclosed in U.S. Patent Application Publication No. 2022 / 135986 and U.S. Patent Application Publication No. 2022 / 135987.

[0245] conditional origin of replication In certain embodiments, the payload comprises a conditional origin of replication that is inactive in the targeted bacteria but active in the producing bacterial cells.

[0246] In the context of the present invention, a "conditional origin of replication" refers to an origin of replication whose functionality can be controlled by the presence of a specific molecule.

[0247] In certain embodiments, a conditional origin of replication is an origin of replication whose replication is dependent on the presence of one or more given proteins, peptides, RNA, nucleic acids, molecules, or any combination thereof.

[0248] In certain embodiments, replication involving said origin of replication may further depend on a process that activates said replication, such as transcription.

[0249] In the context of the present invention, said conditional origin of replication is inactive in said targeted bacterium due to the absence of said given protein, peptide, RNA, nucleic acid, molecule or any combination thereof in said targeted bacterium.

[0250] In certain embodiments, the conditional origin of replication is active in the producer bacterial cell because the producer bacterial cell expresses the given protein, peptide, RNA, nucleic acid, molecule, or any combination thereof, hi certain embodiments, the protein, peptide, RNA nucleic acid, molecule, or any combination thereof is expressed in trans in the producer bacterial cell.

[0251] By "in trans" it is meant herein that the protein, peptide, RNA, nucleic acid, molecule or any combination thereof is not encoded on the same nucleic acid molecule as the nucleic acid molecule comprising the origin of replication. In certain embodiments, the protein, peptide, RNA, nucleic acid, molecule or any combination thereof is encoded on a chromosome or on a vector, particularly a plasmid. In certain embodiments, the vector comprises an antibiotic resistance marker. In alternative embodiments, the vector lacks an antibiotic resistance marker.

[0252] The conditional origin of replication may be selected depending on the particular bacterium to be targeted, as the conditional origin of replication is inactive in the targeted bacterium due to the absence of the given protein, peptide, RNA, nucleic acid, molecule, or any combination thereof, in the targeted bacterium.

[0253] The conditional origins of replication disclosed herein may originate from plasmids, bacteriophages or PICIs that preferably share the following characteristics: they contain repeated sequences, or iterons, in their origins of replication and encode at least one protein (i.e., Rep, protein O, protein P, pri) that interacts with said origin of replication for which they are specific.

[0254] By way of example, mention may be made of the conditional replication systems of the following plasmids and bacteriophages: RK2, R1, pSC101, F, Rts1, RSF1010, P1, P4, lambda, phi82, phi80.

[0255] In certain embodiments, the conditional origin of replication is selected from the group consisting of the R6Kλ DNA origin of replication and derivatives thereof, the IncPα oriV origin of replication and derivatives thereof, a ColE1 origin of replication modified to be under an inducible promoter, and an origin of replication from a phage-inducible chromosomal island (PICI) and derivatives thereof.

[0256] In certain embodiments, the conditional origin of replication is an origin of replication that is present in less than 50%, or less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of the bacteria in the microbiome of a host subject.

[0257] In another particular embodiment, said conditional origin of replication comprises or consists of a sequence that is less than 80% identical, in particular less than 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% identical to the sequence of an origin of replication of bacteria of the host subject's microbiome, in particular of bacteria in more than 50%, more particularly more than 60%, 70%, 80%, 90%, or more than 95% of the host subject's microbiome.

[0258] As used herein, the term "phage-inducible chromosomal island" or "PICI" refers to a mobile genetic element with a conserved genetic organization that encodes a pair of diverse regulatory genes, including a PICI master repressor. Typically, in Gram-positive bacteria, the PICI, located to the left of the rpr and transcribed in the same direction, encodes a small set of genes including the integrase (int) gene, while the PICI, located to the right of the rpr and transcribed in the opposite direction, encodes an excision function (xis) and, adjacent to these genes, a replication module consisting of a primase homolog (pri) and optionally a replication initiator (rep), followed by an origin of replication (ori). The PICI also encodes genes involved in phage interference and, optionally, a terminase small subunit homolog (terS), all of which are transcribed in the same direction.

[0259] In a particular embodiment, said conditional origin of replication is an origin of replication derived from a phage-induced chromosomal island (PICI).

[0260] Certain conditional replication origins are actually derived from PICI.

[0261] It has been shown that it is possible to derive novel conditionally replicating vectors, particularly based on replication origins from primase-helicase and PICI. These origins may be relatively rare in the target strain, and more advantageously, the primase-ori pair may be unique for each PICI, significantly reducing the likelihood of unwanted recombination or payload expansion events. They can be further modified to further limit the opportunity for recombination and remove restriction sites to bypass the target bacterial defense system.

[0262] In certain embodiments, the conditional origin of replication is derived from the origin of replication from PICI of Escherichia coli strain CFT073, as disclosed in Fillol-Salom et al. (2018) The ISME Journal 12:2114-2128.

[0263] In a particular embodiment, the conditional origin of replication is the primase ori from PICI of Escherichia coli strain CFT073, typically SEQ ID NO:1.

[0264] In another specific embodiment, the conditional origin of replication is a primase ori from PICI of Escherichia coli strain CFT073 that lacks at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 restriction sites selected from the group consisting of GAAABCC, GCCGGC, RCCGGY, GCNGC, TWCANNNNNNTGG (SEQ ID NO: 2), TGGCCA, ACCYAC, YGGCCR, AGACC, GCWGC, GGGANGC, GKAGATD, GCCGGYYD, GGCYAC, RGCCGGYYD, and VGCCGGYBD.

[0265] In a specific embodiment, the conditional origin of replication is the primase ori from PICI of Escherichia coli strain CFT073, lacking the restriction site GAAABCC. Preferably, the conditional origin of replication is that of SEQ ID NO: 3.

[0266] In another specific embodiment, the conditional origin of replication is a primase ori from PICI of Escherichia coli strain CFT073 lacking the restriction sites GAAABCC, GCCGGC, RCCGGY, GCNGC, TWCANNNNNNTGG (SEQ ID NO: 2), TGGCCA, ACCYAC, YGGCCR, AGACC, GCWGC, GGGANGC, GKAGATD, GCCGGYYD, GGCYAC, RGCCGGYYD, and VGCCGGYBD. Preferably, the conditional origin of replication is that of SEQ ID NO: 4.

[0267] In certain embodiments, wherein the origin of replication is derived from a phage-induced chromosomal island (PICI), the conditional origin of replication is active in the producer bacterial cell because the producer bacterial cell expresses a rep protein, particularly a primase-helicase, particularly the primase-helicase of SEQ ID NO: 5, typically encoded by a nucleic acid comprising or consisting of SEQ ID NO: 6.

[0268] These unique conditional replication origins are particularly compatible with lambda-based packaging and allow for sufficiently high titers (>10 10 / mL).

[0269] Preferably, the producing bacterial cells stably contain and are capable of replicating the payload.

[0270] In certain embodiments, when the conditional origin of replication of said payload is an origin of replication whose replication is dependent on the presence of a given protein, peptide, nucleic acid, RNA, molecule or any combination thereof, said donor bacterial cell expresses said protein, peptide, nucleic acid, RNA, molecule or any combination thereof. Preferably, said protein, peptide, nucleic acid, RNA, molecule or any combination thereof is expressed in trans, as defined above.

[0271] In certain embodiments, the producing bacterial cells stably contain nucleic acids encoding the proteins, peptides, nucleic acids, RNA, molecules, or any combination thereof.

[0272] In certain embodiments, when the origin of replication is derived from a phage-induced chromosomal island (PICI), the conditional origin of replication is active in the donor bacterial cell because the donor bacterial cell expresses a rep protein, in particular a primase-helicase, in particular the primase-helicase of SEQ ID NO: 5.

[0273] In a particular embodiment, the producing bacterial cell stably contains a nucleic acid encoding the rep protein, in particular the primase-helicase, which nucleic acid typically comprises or consists of SEQ ID NO:6.

[0274] Packaging part In certain embodiments, the payload is a nucleic acid payload that comprises a packaging site derived from a bacteriophage of the first type.

[0275] By "packaging site" herein is meant a DNA sequence on the phage genome required for packaging of the genome into virions. Host-specific bacteriophages (and their packaging sites) include, but are not limited to, SPP1 (SPP1 pac site), P1 (P1 pac site), T1 (T1 pac site), T7 (T7 concatemer junction), lambda (cos site), mu (mu pac site), P22 (P22 pac site), φ8 (φ8 pac site), Sf6 (Sf6 pac site), 149 (149 pac site), and A1122 (A1122-concatemer junction). For most bacteriophages, the packaging site is referred to as a pac site. In some cases, the packaging site is referred to as a concatemer junction (e.g., T7 concatemer junction). In all cases, the packaging site is substantially isolated from the naturally occurring sequences which flank it in the bacteriophage genome.

[0276] For some bacteriophages, the packaging site may be unknown. In these cases, the pac site can be determined by taking advantage of the property that plasmids containing a functional bacteriophage pac site are packaged. For example, the DNA sequence required for packaging of bacteriophage λ was determined by incorporating a small restriction fragment of λ phage genomic DNA into a plasmid (Hohn 1983 PNAS USA 80:7456-7460). After introduction into an in vivo packaging strain, the efficiency of packaging / transduction was quantitatively assessed. Similar strategies have been used to determine pac sites for a number of bacteriophages: λ (Miwa 1982 Gene 20:267-279); Mu (Croenen et al., 1985 Virology 144:520-522); filamentous bacteriophages including fl, fd, M13, and Ike (Russell et al., 1989 J Virol 1989 63:3284-3295); P22 (Petri et al., 1990 Gene 88:47-55; Wu et al., 2002 Molec Microbiol 45:1631-1646); T7 (Chung et al., 1990 J Mol Biol 216:927-938), and T3 (Hashimoto et al., 1992 Virology 187:788-795).

[0277] In certain embodiments, the packaging site is as disclosed in U.S. Patent Application Publication Nos. 2022 / 135986 and 2022 / 135987, which are incorporated herein by reference.

[0278] Other components of the payload The payloads used in the context of the present invention preferably lack antibiotic resistance markers.

[0279] Antibiotic resistance genes are well known in the art and include, but are not limited to, ampicillin resistance (Amp), chloramphenicol resistance (Cm), tetracycline resistance (Tet), kanamycin resistance (Kan), hygromycin resistance (Qiyg or hph genes), and zeocin resistance (Zeo).

[0280] In certain embodiments, the payload used in the context of the present invention comprises an auxotrophic marker. Auxotrophic markers in bacteria are described, for example, in U.S. Pat. Nos. 4,920,048, 5,691,185, 6,291,245, 6,413,768, and 6,752,994; U.S. Patent Application Publication No. 20050186666; Struhl et al. (1976) PNAS USA 73; 1471-1475; MacCormick et al. (1995) FEMS Microbiol. Lett. 127:105-109; Dickely et al. (1995) Mol. Microbiol. 15:839-847; Sorensen et al. (2000) Appl. Environ. Microbiol 66:1253-1258; and Fiedler & Skerra (2001) Gene 274: 111 118 and typically include DapA and ThyA. In a particular embodiment, the auxotrophic marker is ThyA.

[0281] In certain embodiments, the payload does not contain any restriction sites recognized by restriction enzymes frequently encoded by the targeted bacterial cells, hi other specific embodiments, the payload contains no more than 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 restriction sites recognized by restriction enzymes frequently encoded by the targeted bacterial cells or a population or group of targeted bacterial cells.

[0282] As used herein, the terms "restriction site" and "restriction enzyme site" are equivalent and refer to a location on a nucleic acid containing a unique nucleotide sequence recognized by a restriction enzyme. In particular, the nucleic acid contains a unique sequence that is bound and cleaved by a restriction enzyme. A restriction site is generally a palindromic sequence 4 to 8 base pairs in length. More precisely, a restriction site refers to a specific sequence and modification state that is bound and cleaved by a restriction enzyme. In particular, it refers to a specific unmodified sequence that is bound and cleaved by a restriction enzyme. In particular, the sequence is not methylated, hydroxymethylated, or glucosyl-hydroxymethylated. In this context, the restriction enzyme is of type I, II, or III. Alternatively, it can refer to a specific modified sequence that is bound and cleaved by a restriction enzyme, such as methylated, hydroxymethylated, and glucosyl-hydroxymethylated DNA. In this context, the restriction enzyme is of type IV.

[0283] As used herein, "recognized by" in reference to a restriction site and a restriction enzyme means that the restriction site is cleaved by the restriction enzyme.

[0284] In the restriction site sequences, N means that the nucleotide can be A, C, G, or T; B means that the nucleotide can be C, G, or T; Y means that the nucleotide can be C or T; W means that the nucleotide can be A or T; R means that the nucleotide can be A or G; D means A, G, or T.

[0285] As used herein, the terms "restriction enzyme" and "restriction endonuclease" are equivalent and refer to enzymes that cut nucleic acids at or near a restriction site. Restriction enzymes are commonly classified into four types (Type I to Type IV). The REBASE database allows listing the restriction sites that a given bacterium can recognize according to the restriction enzymes it expresses.

[0286] By "frequent" or "frequently" in a group of bacteria of interest is meant that at least 10, 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95 or 99% of the bacteria in the group encode a restriction enzyme.

[0287] A payload according to the present invention preferably contains 100 or fewer restriction sites. In a preferred embodiment, a payload according to the present invention contains 10 or fewer restriction sites. In a most preferred embodiment, a payload according to the present invention does not contain any restriction sites.

[0288] Targeted bacteria The bacteria targeted by the phage particles or phage-derived delivery particles of the present invention can be any bacteria present in a mammalian organism, a plant, or the environment, and can be any commensal, symbiotic, or pathogenic bacteria of the microbiota or microbiome.

[0289] A microbiome may include a variety of endogenous bacterial species, any of which may be targeted in accordance with the present disclosure. In some embodiments, the genus and / or species of endogenous bacterial cells targeted may depend on a first type of bacteriophage, as defined in the section above, "Bacteriophages and Genes Derived from Bacteriophages." For example, some bacteriophages exhibit tropism for or preferentially target specific host species of bacteria. Other bacteriophages do not exhibit such tropism and may be used to target numerous different genera and / or species of endogenous bacterial cells.

[0290] Examples of bacterial cells include, but are not limited to, bacteria from the following genera: Yersinia spp., Escherichia spp., Klebsiella spp., Acinetobacter spp., Bordetella spp., Neisseria spp., Aeromonas spp., Francisella spp., Corynebacterium spp., Citrobacter spp., Chlamydia spp., Haemophilus spp., Brucella spp.), Mycobacterium spp., Legionella spp., Rhodococcus spp., Pseudomonas spp., Helicobacter spp., Vibrio spp., Bacillus spp., Erysipelothrix spp., Salmonella spp., Streptomyces spp., Streptococcus spp., Staphylococcus spp., Bacteroides spp. spp.), Prevotella spp., Clostridium spp., Bifidobacterium spp., Clostridium spp., Brevibacterium spp., Lactococcus spp., Leuconostoc spp., Actinobacillus spp., Selenomonas spp.), Shigella spp., Zymomonas spp., Mycoplasma spp., Treponema spp., Leuconostoc spp., Corynebacterium spp., Enterococcus spp., Enterobacter spp., Pyrococcus spp., Serratia spp., Morganella spp., Parvimonas spp., Fusobacterium spp., Actinomyces spp.), Porphyromonas spp., Micrococcus spp., Bartonella spp., Borrelia spp., Brucelia spp., Campylobacter spp., Chlamydophilia spp., Cutibacterium spp., Propionibacterium spp., Gardnerella spp., Ehrlichia spp., Haemophilus spp., Leptospira spp. spp., Listeria spp., Mycoplasma spp., Nocardia spp., Rickettsia spp., Ureaplasma spp., Lactobacillus spp., Faecalibacterium spp., Ruminococcus spp., and mixtures thereof.

[0291] As such, the phage particles, phage delivery particles and / or phages may target (e.g., specifically target) bacterial cells from any one or more of the aforementioned bacterial genera, and specifically deliver a payload according to the present invention.

[0292] Preferably, the targeted bacteria may be selected from the group consisting of Yersinia, Escherichia, Klebsiella, Acinetobacter, Pseudomonas, Helicobacter, Vibrio, Salmonella, Streptococcus, Staphylococcus, Bacteroides, Clostridium, Shigella, Enterococcus, Enterobacter, Listeria, Cutibacterium, Propionibacterium, Fusobacterium, Porphyromonas, and Gardnerella.

[0293] In some embodiments, the targeted bacteria are anaerobic bacterial cells (e.g., cells that do not require oxygen for growth). Anaerobic bacterial cells include facultative anaerobic cells, such as, but not limited to, Escherichia coli, Shewanella oneidensi, Gardnerella vaginalis, and Listeria. Anaerobic bacterial cells also include obligate anaerobic cells, such as species of Bacteroides, Clostridium, Cutibacterium, Propionibacterium, Fusobacterium, and Porphyromonas. In humans, anaerobic bacteria are most commonly found in the gastrointestinal tract. Therefore, in certain embodiments, the targeted bacteria are bacteria most commonly found in the gastrointestinal tract. The bacteriophages, as well as the phage particles, phage delivery vehicles and / or phages used to prepare the hybrid helper phage, may target (e.g., specifically target) anaerobic bacterial cells according to their specific spectrum known to those skilled in the art for specific delivery of plasmids.

[0294] In some embodiments, the bacterial cells targeted include, but are not limited to, Bacteroides thetaiotaomicron, Bacteroides fragilis, Bacteroides distasonis, Bacteroides vulgatus, Clostridium leptum, Clostridium coccoides, Bacillus subtilis, Clostridium butyricum, Brevibacterium lactofermentum, Streptococcus agalactiae, Lactococcus lactis, Leuconostoc lactis, lactis, Actinobacillus actinobycetemcomitans, Cyanobacteria, Escherichia coli, Helicobacter pylori, Selnomonas ruminatium, Shigella sonnei, Zymomonas mobilis, Mycoplasma mycoides, Treponema denticola, Bacillus thuringiensis, Staphylococcus lugdunensis, Leuconostoc oenos oenos, Corynebacterium xerosis, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus caseicasei, Lactobacillus acidophilus, Enterococcus faecalis, Bacillus coagulans, Bacillus cereus, Bacillus popillae, Synechocystis strain PCC6803, Bacillus liquefaciens, Pyrococcus abyssi, Selenomonas nominantium, Lactobacillus hilgardii, Streptococcus ferus ferus, Lactobacillus pentosus, Bacteroides fragilis, Staphylococcus epidermidis, Streptomyces phaechromogenes, Streptomyces ghanaenis, Klebsiella pneumoniae, Enterobacter cloacae, Enterobacter aerogenes, Serratia marcescens, Morganella morganii, Citrobacter freundii freundii, Propionibacterium freudenreichii, Pseudomonas aeruginosa, Parvimonas micra, Prevotella intermedia, Fusobacterium nucleatum, Prevotella nigrescensnigrescens, Actinomyces israelii, Porphyromonas endodontalis, Porphyromonas gingivalis, Micrococcus luteus, Bacillus megaterium, Aeromonas hydrophila, Aeromonas caviae, Bacillus anthracis, Bartonella henselae, Bartonella quintana, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Campylobacter coli, Campylobacter fetus, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriaediphtheria, Cutibacterium acnes (formerly Propionibacterium acnes), Ehrlichia canis, Ehrlichia chaffeensis, Enterococcus faecium, Francisella tularensis, Haemophilus influenzae, Legionella pneumophila, Leptospira interrogans, Leptospira santarosai, Leptospira weilii, Leptospira noguchii, Listeria monocytogenes monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumonia, Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia asteroids, Rickettsia rickettsia, Salmonella enteritidis, Salmonella typhi, Salmonella paratyphi, Salmonella typhimurium typhimurium, Shigella flexnerii, Shigella dysenteriae, Staphylococcus saprophyticus, Streptococcus pneumoniaepneumoniae, Streptococcus pyogenes, Gardnerella vaginalis, Streptococcus viridans, Treponema pallidum, Ureaplasma urealyticum, Vibrio cholerae, Vibrio parahaemolyticus, Yersinia pestis, Yersinia enterocolitica, Yersinia pseudotuberculosis, Actinobacter baumannii baumannii), Pseudomonas aeruginosa, and mixtures thereof, and preferably, the target bacterium is selected from the group consisting of Escherichia coli, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter cloacae, and Enterobacter aerogenes, and mixtures thereof.

[0295] In some embodiments, the targeted bacterial cells include, but are not limited to, species of Anaerotruncus, Acetanaerobacterium, Acetitomaculum, Acetivibrio, Anaerococcus, Anaerofilum, Anaerosinus, Anaerostipes, Anaerovorax, Anaerovorax, Butyrivibrio, Clostridium, Capracoccus, Dehalobacter, Dialister, Dorea, Enterococcus, Ethanoligenens, Faecalibacterium, Fusobacterium, Gracilibacterium Gracilibacter, Guggenheimella, Hespellia, Lachnobacterium, Lachnospira, Lactobacillus, Leuconostoc, Megamonas, Moriella, Mitsuokella, Oribacterium, Oxobacter bacter, Papillibacter, Proprionispira, Pseudobutyrivibrio, Pseudoramibacter, Roseburia, Ruminococcus, Sarcina, Seinonella, Shuttleworthia, Sporobacter,The genus is selected from the group consisting of Sporobacterium, Streptococcus, Subdoligranulum, Syntrophococcus, Thermobacillus, Turibacter, Weisella, Clostridium, Bacteroides, Ruminococcus, Faecalibacterium, Treponema, Phascolarctobacterium, Megasphaera, Faecalibacterium, Bifidobacterium, Lactobacillus, Sutterella, and / or Prevotella.

[0296] In other embodiments, the targeted bacterial cells include, but are not limited to, Achromobacter xylosoxidans, Acidaminococcus fermentans, Acidaminococcus intestini, Acidaminococcus spp., Acinetobacter baumannii, Acinetobacter junii, Acinetobacter lwoffii, Actinobacillus capsulatus, Actinomyces naeslundii, Actinomyces neuii, Actinomyces odontolyticus, Actinomyces odontolyticus, Actinomyces radingae, Adlercreutzia equolifaciens, Aeromicrobium massiliense, Aggregatibacter actinomycetemcomitans, Akkermansia muciniphila, Aliagarivorans marinus, Alistipes finegoldii, Alistipes indistinctus, Alistipes inops, Alistipes onderdonchii onderdonkii, Alistipes putredinis, Alistipes senegalensis, Alistipes shahiishahii, Alistipes timonensis, Alloscardovia omnicolens, Anaerobacter polyendosporus, Anaerobaculum hydrogeniformans, Anaerococcus hydrogenalis, Anaerococcus prevotii, Anaerococcus senegalensis, Anaerofustis stercorihominis, Anaerostipes caccae, Anaerostipes hadras hadrus, Anaeroturunccus colihominis, Aneurinibacillus aneurinilyticus, Bacillus licheniformis, Bacillus massilioanorexius, Bacillus massiliosenegalensis, Bacillus simplex, Bacillus smithii, Bacillus subtilis, Bacillus thuringiensis, Bacillus timonensis, Bacteroides xylanisolvens xylanisolvens, Bacteroides acidifaciens, Bacteroides caccae, Bacteroides capillosus, Bacteroides cellulosiliticuscellulosilyticus, Bacteroides clarus, Bacteroides coprocola, Bacteroides coprophilus, Bacteroides dorei, Bacteroides eggerthii, Bacteroides faecis, Bacteroides finegoldii, Bacteroides fluxus, Bacteroides fragilis, Bacteroides gallinarum, Bacteroides intestinalis, Bacteroides nordii nordii, Bacteroides oleiciplenus, Bacteroides ovatus, Bacteroides pectinophilus, Bacteroides plebeius, Bacteroides salanitronis, Bacteroides salyersiae, Bacteroides, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus, Bacteroides xylanisorbens, Bacteroides pectinophilus ATCC ATCC), Barnesiella intestinihominis, Bavariicoccus seileri, Bifidobacterium adressentisadolescentis, Bifidobacterium angulatum, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium gallicum, Bifidobacterium longum, Bifidobacterium pseudocatenulatum Bacterium pseudocatenulatum, Bifidobacterium stercoris, Bilophila wadsworthia, Blautia faecis, Blautia hansenii, Blautia hydrogenotrophica, Blautia luti, Blautia obeum, Blautia producta, Blautia wexlerae, Brachymonas chironomi, Brevibacterium senegalense, Bryantella formatexigens formatexigens, butyrate-producing bacterium, Butyricicoccus pullicaecorum, Butyricimonas virosa, Butyrivibrio crossotuscrossotus, Butyrivibrio fibrisolvens, Caldicoprobacter faecalis, Campylobacter concisus, Campylobacter jejuni, Campylobacter upsaliensis, Catenibacterium mitsuokai, Cedecea davisae, Cellulomonas massiliensis, Cetobacterium somerae, Citrobacter braakii braakii, Citrobacter freundii, Citrobacter pasteurii, Citrobacter sp., Citrobacter youngae, Cloacibacillus evryensis, Clostridiales bacterium, Clostridioides difficile, Clostridium asparagiforme, Clostridium bartlettii, Clostridium boliviensis, Clostridium bolteae, Clostridium hasewai hathewayi, Clostridium hiranoni, Clostridium hylemonae, Clostridium leptum, Clostridium methylpentosum, Clostridium nexilenexile, Clostridium orbiscindens, Clostridium ramosum, Clostridium scindens, Clostridium sp., Clostridium genus, Clostridium spiroforme, Clostridium sporogenes, Clostridium symbiosum, Collinsella aerofaciens, Collinsella intestinalis, Collinsella stercoris, Collinsella tanakaei, Coprobacillus cateniformis cateniformis, Coprobacter fastidiosus, Coprococcus catus, Coprococcus comes, Coprococcus eutactus, Corynebacterium ammoniagenes, Corynebacterium amycolatum, Corynebacterium pseudodiphtheriticum, Cutibacterium acnes, Dermabacter hominis, Desulfitobacterium hafniens hafniense, Desulfovibrio fairfieldensis, Desulfovibrio piger, Dialister succinatiphilussuccinatiphilus, Dielma fastidiosa, Dorea formicigenerans, Dorea longicatena, Dysgonomonas capnocytophagoides, Dysgonomonas gadei, Dysgonomonas mossii, Edwardsiella tarda, Eggerthella lenta, Eisenbergiella tayi, Enorma massiliensis, Enterobacter aerogenes, Enterobacter asburiae asburiae, Enterobacter cancerogenus, Enterobacter cloacae, Enterobacter massiliensis, Enterococcus casseliflavus, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus flavescens, Enterococcus gallinarum, Enterococcus sp., Enterovibrio nigricans, Erysipelatoclostridium ramosum ramosum, Escherichia coli, Escherichia sp., Eubacterium biforme, Eubacterium dolichum, Eubacterium halliihallii), Eubacterium limosum, Eubacterium ramulus, Eubacterium rectale, Eubacterium silaeum, Eubacterium ventriosum, Exiguobacterium marinum, Exiguobacterium undae, Faecalibacterium cf, Faecalibacterium cf Faecalibacterium prausnitzii, Faecalitalea cylindroides, Ferrimonas balearica, Finegoldia magna, Flavobacterium daejeonense, Flavonifractor plautii, Fusicatenibacter saccharivorans, Fusobacterium gonidiaformans, Fusobacterium mortiferum, Fusobacterium necroforum necrophorum, Fusobacterium nucleatum, Fusobacterium periodonticum, Fusobacterium sp., Fusobacterium ulcerans, Fusobacterium varium, Gallibacterium anatis, Gemmiger formicilis, Gordonibacter pamelaeae, Hafnia alvei, Helicobacter bilis, Helicobacter canadensis, Helicobacter canis canis, Helicobacter cinaedi, Helicobacter macacae, Helicobacter pametensis, Helicobacter prolumpullorum, Helicobacter pylori, Helicobacter rodentium, Helicobacter winghamensis, Herbaspirillum massiliense, Holdemanella biformis, Holdemania fdiformis, Holdemania filiformis, Holdemania massiliensis, Holdemania filiformis, Hungatella hathewayi, Intestinibacter bartlettii, Intestinimonas butyriciproducens, Klebsiella oxytoca oxytoca, Klebsiella pneumoniae, Kurthia massiliensis, Lachnospira pectinoschiza, Lactobacillus acidophilus, Lactobacillus amylolyticus, Lactobacillus animalis, Lactobacillus antri, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus curvatus, Lactobacillus delbrueckii, Lactobacillus fermentum fermentum), Lactobacillus gasseri, Lactobacillus helveticushelveticus, Lactobacillus hilgardii, Lactobacillus iners, Lactobacillus intestinalis, Lactobacillus johnsonii, Lactobacillus murinus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus ruminis, Lactobacillus sakei, Lactobacillus salivarius, Lactobacillus altunensis ultunensis, Lactobacillus vaginalis, Lactobacillus plantarum subsp., Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Listeria grayi, Listeria innocua, Mannheimia granulomatis, Marvinbryantia formatexigens, Megamonas funiformis, Megamonas hypermegale, Methanobrevibacter smithii smithii), Methanobrevibacter smithiiFl, Micrococcus luteus, Microvirgula aerodenitrificansaerodenitrificans, Mitsuokella jalaludinii, Mitsuokella multacida, Mollicutes bacterium, Murimonas intestini, Neisseria macacae, Nitriliruptor alkaliphilus, Oceanobacillus massiliensis, Odoribacter laneus, Odoribacter splanchnicus, Ornithobacterium rhinotracheale, Oxalobacter formigenes, Paenibacillus barengoltzii, Paenibacillus chitinolyticus, Paenibacillus lautus, Paenibacillus motobuensis, Paenibacillus senegalensis, Paenisporosarcina quisquiliarum, Parabacteroides distasonis, Parabacteroides gordosteinii goldsteinii, Parabacteroides gordonii, Parabacteroides johnsonii, Parabacteroides merdae, Paraprevotella xylaniphilaxylaniphila, Parasutterella excrementihominis, Parvimonas micra, Pediococcus acidilactici, Peptoclostridium difficile, Peptoniphilus harei, Peptoniphilus obesi, Peptoniphilus senegalensis, Peptoniphilus timonensis, Phascolarctobacterium succinatutens, Porphyromonas asaccharolytica asaccharolytica, Porphyromonas uenonis, Prevotella baroniae, Prevotella bivia, Prevotella copri, Prevotella dentalis, Prevotella micans, Prevotella multisaccharivorax, Prevotella oralis, Prevotella salivae, Prevotella stercorea, Prevotella veroralis, Propionibacterium acnes acnes, Propionibacterium avidum, Propionibacterium freudenreichii, Propionimicrobium lymphophilum, Proteus mirabilismirabilis, Proteus penneri ATCC, Providencia alcalifaciens, Providencia rettgeri, Providencia rustigianii, Providencia stuartii, Pseudoflavonifractor capillosus, Pseudomonas aeruginosa, Pseudomonas luteola, Ralstonia pickettii, Rheinheimera perlucida, Rheinheimera texasensis, Riemerella columbina columbina, Romboutsia lituseburensis, Roseburia faecis, Roseburia intestinalis, Roseburia inulinivorans, Ruminococcus bicirculans, Ruminococcus bromii, Ruminococcus callidus, Ruminococcus champanellensis, Ruminococcus faecis, Ruminococcus gnavus, Ruminococcus lactalis lactaris, Ruminococcus obeum, Ruminococcus spp., Ruminococcus spp., Ruminococcus torques, Sarcina ventriciventriculi, Sellimonas intestinalis, Senegalimassilia anaerobia, Shigella sonnei, Slackia piriformis, Staphylococcus epidermidis, Staphylococcus lentus, Staphylococcus nepalensis, Staphylococcus pseudintermedius, Staphylococcus xylosus, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus anginosus anginosus, Streptococcus australis, Streptococcus caballi, Streptococcus castoreus, Streptococcus didelphis, Streptococcus equinus, Streptococcus gordonii, Streptococcus henryi, Streptococcus hyovaginalis, Streptococcus infantarius, Streptococcus infantis infantis, Streptococcus lutetiensis, Streptococcus merionis, Streptococcus mitismitis, Streptococcus mutans, Streptococcus oralis, Streptococcus ovis, Streptococcus parasanguinis, Streptococcus plurextorum, Streptococcus porci, Streptococcus pyogenes, Streptococcus salivarius ), Streptococcus sobrinus, Streptococcus thermophilus, Streptococcus thoraltensis, Streptomyces albus, Subdoligranulum variabile, Succinatimonas hippei, Sutterella parvirubra, Sutterella wadsworthensis, Terrisporobacter glycolicus, Terrisporobacter mayombei, Thalassobacillus devorans devorans, Timonella senegalensis, Turicibacter sanguinis, unknown genus, unknown genus, Varibaculum cambriense, Veillonella atypica, Veillonella dispar, Veillonella parvula, Vibrio cincinnatiensis, Virgibacillus salexigens, and / or Weissella paramesenteroides.

[0297] In other embodiments, the bacterial cells targeted are those commonly found in the skin microbiota, including, but not limited to, Acetobacter farinalis, Acetobacter malorum, Acetobacter orleanensis, Acetobacter sicerae, Achromobacter anxifer, Achromobacter denitrificans, Achromobacter marplatensis, Achromobacter spanius, Achromobacter xylosoxidans subsp. xylosoxidans, Acidovorax konjaci, Acidovorax radicis, Acinetobacter johnsonii, Actinomadura citrea, Actinomadura coerulea, Actinomadura fibrosa, Actinomadura fulvescens, Actinomadura jiaoheensis, Actinomadura luteofluorescens, Actinomadura mexicana mexicana), Actinomadura nitritigenes, Actinomadura verrucosospora, Actinomadura yumaensisyumaensis, Actinomyces odontolyticus, Actinomycetospora atypica, Actinomycetospora corticicola, Actinomycetospora rhizophila, Actinomycetospora rishiriensis, Aeromonas australiensis, Aeromonas bestiarum, Aeromonas bivalvium, Aeromonas encheleia, Aeromonas eucrenophila eucrenophila, Aeromonas hydrophila subsp. hydrophila, Aeromonas piscicola, Aeromonas popoffii, Aeromonas rivuli, Aeromonas salmonicida subsp. pectinolytica, Aeromonas salmonicida subsp. smithia, Amaryllidaceae ... ciceronei, Aminobacter lissarensis, Aminobacter niigataensis, Ancylobacter polymorphuspolymorphus, Anoxybacillus flavithermus subsp. yunnanensis, Aquamicrobium aerolatum, Archangium gephyra, Archangium gephyra, Archangium minus, Archangium violaceum, Arthrobacter viscosus, Bacillus anthracis, Bacillus australimaris, Bacillus drentensis, Bacillus mycoides, Bacillus pseudomycoides Bacillus pseudomycoides, Bacillus pumilus, Bacillus safensis, Bacillus vallismortis, Bosea thiooxidans, Bradyrhizobium huanghuaihaiense, Bradyrhizobium japonicum, Brevundimonas aurantiaca, Brevundimonas intermedia, Burkholderia aspalathi, Burkholderia choica, Burkholderia cordovensis cordobensis, Burkholderia diffusa, Burkholderia insulsa, Burkholderia rhynchosiaerhynchosiae, Burkholderia terrestris, Burkholderia udeis, Buttiauxella gaviniae, Caenimonas terrae, Capnocytophaga gingivalis, Chitinophaga dinghuensis, Chryseobacterium gleum, Chryseobacterium greenlandense, Chryseobacterium jejuense, Chryseobacterium piscium piscium, Chryseobacterium sediminis, Chryseobacterium tructae, Chryseobacterium ureilyticum, Chryseobacterium vietnamense, Corynebacterium accolens, Corynebacterium afermentans subsp. lipophilum, Corynebacterium minutissimum, Corynebacterium sundusvalens sundsvallense, Cupriavidus metallidurans, Cupriavidus nantongensis, Cupriavidus necator, Cupriavidus pampaepampae, Cupriavidus yeoncheonensis, Corynebacterium flaccumfaciens, Devosia epidermidihirudinis, Devosia riboflavina, Devosia riboflavina, Diaphorobacter oryzae, Dietzia psychralcaliphila, Ensifer adhaerens, Ensifer americanus, Enterococcus malodoratus, Enterococcus pseudoavium Pseudomonas pseudoavium, Enterococcus viikkiensis, Enterococcus xiangfangensis, Erwinia rhapontici, Falsirhodobacter halotolerans, Flavobacterium araucananum, Flavobacterium frigidimaris, Gluconobacter frateurii, Gluconobacter thailandicus, Gordonia alkanivorans, Halomonas aquamarina aquamarina, Halomonas axialensis, Halomonas meridiana, Halomonas olivarianaolivaria, Halomonas songnenensis, Halomonas variabilis, Herbaspirillum chlorophenolicum, Herbaspirillum frisingense, Herbaspirillum hiltneri, Herbaspirillum huttiense subsp. putei, Herbaspirillum lusitanum, Herminiimonas fonticola, Hydrogenophaga intermedia, Hydrogenophaga pseudoflava pseudoflava, Klebsiella oxytoca, Kosakonia sacchari, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus modestisalitolerans, Lactobacillus plantarum subsp. argentoratensis, Lactobacillus xiangfangensis, Lechevalieria roselyniae, Lentzea albida, Lentzea californiensis, Leuconostoc carnosum carnosum), Leuconostoc citreum, Leuconostoc gelidum subsp.gasicomitatum, Leuconostoc mesenteroides subsp. suionicum, Luteimonas aestuarii, Lysobacter antibioticus, Lysobacter koreensis, Lysobacter oryzae, Magnetospirillum moscoviense, Marinomonas alcarazii, Marinomonas primoryensis, Massilia aurea, Massilia jejuensis jejuensis, Massilia kyonggiensis, Massilia timonae, Mesorhizobium acaciae, Mesorhizobium qingshengii, Mesorhizobium shonense, Methylobacterium haplocladii, Methylobacterium platani, Methylobacterium pseudosasicola, Methylobacterium zatmanii, Microbacterium oxydans oxydans, Micromonospora chaiyaphumensis, Micromonospora chalcea, Micromonospora citrea, Micromonospora coccensiscoxensis, Micromonospora echinofusca, Micromonospora halophytica, Micromonospora kangleipakensis, Micromonospora maritima, Micromonospora nigra, Micromonospora purpureochromogenes, Micromonospora rhizosphaerae, Micromonospora saelicesensis, Microvirga subterranea, Microvirga zambiensis ... Mycobacterium alvei, Mycobacterium avium subsp. silvaticum, Mycobacterium colombiense, Mycobacterium conceptionense, Mycobacterium conceptionense, Mycobacterium farcinogenes, Mycobacterium fortuitum subsp. fortuitum, Mycobacterium goodii, Mycobacterium insubricum insubricum, Mycobacterium llatzerense, Mycobacterium neoaurum, Mycobacterium neworleansense, Mycobacterium obuense, Mycobacterium peregrinum, Mycobacterium saopaulense, Mycobacterium septicum, Mycobacterium setense, Mycobacterium smegmatis, Neisseria subflava subflava), Nocardia lijiangensis, Nocardia thailandica, Novosphingobium barchaimii, Novosphingobium lindaniclasticumlindaniclasticum, Novosphingobium lindaniclasticum, Novosphingobium mathurense, Ochrobactrum pseudogrignonense, Oxalicibacterium solurbis, Paraburkholderia glathei, Paraburkholderia humi, Paraburkholderia phenazinium, Paraburkholderia phytofirmans, Paraburkholderia sordidicola, Paraburkholderia terricola terricola, Paraburkholderia xenovorans, Paracoccus laeviglucosivorans, Patulibacter ginsengiterrae, Polymorphospora rubra, Porphyrobacter colymbi, Prevotella jejuni, Prevotella melaninogenica, Propionibacterium acnes subsp. elongatum, Proteus vulgaris vulgaris, Providencia rustigianii, Pseudoalteromonas agarivorans, Pseudoalteromonas atlanticaatlantica, Pseudoalteromonas paragorgicola, Pseudomonas asplenii, Pseudomonas asuensis, Pseudomonas benzenivorans, Pseudomonas cannabina, Pseudomonas cissicola, Pseudomonas congelans, Pseudomonas costantinii, Pseudomonas ficuserectae, Pseudomonas frederiksbergensis, Pseudomonas graminis graminis, Pseudomonas jessenii, Pseudomonas koreensis, Pseudomonas koreensis, Pseudomonas kunmingensis, Pseudomonas marginalis, Pseudomonas mucidolens, Pseudomonas panacis, Pseudomonas plecoglossicida, Pseudomonas poae, Pseudomonas pseudoalcaligenes, Pseudomonas putida, Pseudomonas reinekei reinekei), Pseudomonas rhizosphaerae, Pseudomonas seleniipraekipitansseleniipraecipitans, Pseudomonas umsongensis, Pseudomonas zhaodongensis, Pseudonocardia alaniniphila, Pseudonocardia ammonioxydans, Pseudonocardia autotrophica, Pseudonocardia kongjuensis, Pseudonocardia yunnanensis, Pseudorhodoferax soli, Pseudoxanthomonas dejonensis daejeonensis, Pseudoxanthomonas indica, Pseudoxanthomonas kaohsiungensis, Psychrobacter aquaticus, Psychrobacter arcticus, Psychrobacter celer, Psychrobacter marincola, Psychrobacter nivimaris, Psychrobacter okhotskensis, Psychrobacter okhotskensis, Psychrobacter piscatorii, Psychrobacter pulmonis pulmonis, Ramlibacter ginsenosidimutans, Rheinheimera japonicajaponica, Rheinheimera muenzenbergensis, Rheinheimera soli, Rheinheimera tangshanensis, Rheinheimera texasensis, Rheinheimera tilapiae, Rhizobium alamii, Rhizobium azibense, Rhizobium binae, Rhizobium daejeonense, Rhizobium endophyticum, Rhizobium etli, Rhizobium fabae fabae, Rhizobium freirei, Rhizobium gallicum, Rhizobium loessense, Rhizobium sophoriradicis, Rhizobium taibaishanense, Rhizobium vallis, Rhizobium vignae, Rhizobium yanglingense, Rhodococcus baikonurensis, Rhodococcus enclensis, Rhodoferax saidenbachensis saidenbachensis, Rickettsia canadensis, Rickettsia heilongjiangensis, Rickettsia honei, Rickettsia raoultii, Roseateles aquatilisaquatilis, Roseateles aquatilis, Salmonella enterica subsp. salamae, Serratia ficaria, Serratia myotis, Serratia vespertilionis, Shewanella aestuarii, Shewanella decolorationis, Sphingobium amiense, Sphingobium baderi, Sphingobium barthaii, Sphingobium chlorophenolicum, Sphingobium cupriresistsens cupriresistens, Sphingobium czechense, Sphingobium fuliginis, Sphingobium indicum, Sphingobium indicum, Sphingobium japonicum, Sphingobium lactosutens, Sphingomonas dokdonensis, Sphingomonas pseudosanguinis, Sphingopyxis chilensis, Sphingopyxis fribergensis, Sphingopyxis granulis granuli), Sphingopyxis indica, Sphingopyxis withfraliensiswitflariensis, Staphylococcus agnetis, Staphylococcus aureus subsp. aureus, Staphylococcus epidermidis, Staphylococcus hominis subsp. novobiosepticus, Staphylococcus nepalensis, Staphylococcus saprophyticus subsp. bovis, Staphylococcus sciuri subsp. carnaticus, Streptomyces caeruleatus, Streptomyces canarius canarius, Streptomyces capoamus, Streptomyces ciscaucasicus, Streptomyces griseorubiginosus, Streptomyces olivaceoviridis, Streptomyces panaciradicis, Streptomyces phaeopurpureus, Streptomyces pseudovenezuelae, Streptomyces resistomycificus, Tianweitania sediminis sediminis, Tsukamurella paurometabola, Variovorax guangxiensis, Vogesella alkaliphilaalkaliphila, Xanthomonas arboricola, Xanthomonas axonopodis, Xanthomonas cassavae, Xanthomonas cucurbitae, Xanthomonas cynarae, Xanthomonas euvesicatoria, Xanthomonas fragariae, Xanthomonas gardneri, Xanthomonas perforans, Xanthomonas pisi, Xanthomonas potpourri populi, Xanthomonas bacicolae vasicola, Xenophilus aerolatus, Yersinia nurmii, Abiotrophia defectiva, Acidocella aminolytica, Acinetobacter guangdongensis, Acinetobacter parvus, Acinetobacter radioresistens, Acinetobacter soli, Acinetobacter variabilis, Actinomyces cardiffensis, Actinomyces dentalis dentalis, Actinomyces europaeus, Actinomyces gerencseriae, Actinomyces graevenitzii, Actinomyces haliotis, Actinomyces johnsonii, Actinomyces massiliensis, Actinomyces meyeri, Actinomyces mayeri, Actinomyces naeslundii, Actinomyces neuii subsp. anitratus, Actinomyces odontolyticus, Actinomyces oris oris, Actinomyces turicensis, Actinomyces corticicola, Actinomyces schaalii, Aerococcus christenseniichristensenii, Aerococcus urinae, Aeromicrobium flavum, Aeromicrobium massiliense, Aeromicrobium tamlense, Aeromonas sharmana, Aggregatibacter aphrophilus, Aggregatibacter segnis, Aggregatibacter baldri, Albibacter methylovorans, Alcaligenes faecalis subsp. faecalis, Algoriphagus latkowskii ratkowskyi, Alkalibacterium olivapovliticus, Alkalibacterium pelagium, Alkalibacterium pelagium, Alloprevotella rava, Alsobacter metallidurans, Amaryllidoccus caprysensis, Amaryllidoccus veronensis, Anaerococcus hydrogenalis, Anaerococcus lactolyticus, Anaerococcus murdochii, Anaerococcus octavius, Anaerococcus prevotii, Anaerococcus vaginalis vaginalis, Aquabacterium citratiphilum, Aquabacterium olei, Aquabacterium olei, Aquabacterium parvumparvum, Aquincola tertiaricarbonis, Arcobacter venerupis, Arsenicicoccus bolidensis, Arthrobacter russicus, Asticcacaulis excentricus, Atopobium deltae, Atopobium parvulum, Atopobium rimae, Atopobium vaginae, Aureimonas altamirensis, Aureimonas rubiginis rubiginis, Azospira oryzae, Azospirillum oryzae, Bacillus circulans, Bacillus drentensis, Bacillus fastidiosus, Bacillus lehensis, Bacillus oceanisediminis, Bacillus rhizosphaerae, Bacteriovorax stolpii, Bacteroides coagulans coagulan, Bacteroides dorei, Bacteroides fragilis, Bacteroides ovatus, Bacteroides starchoris, Bacteroides uniformis, Bacteroides vulgatus, Bdellovibrio bacteriovorus, Bdellovibrio exovorus, Belnapia moabensis, Belnapia sorisoli, Blautia hansenii, Blautia obeum, Blautia wechslere, Bosea lathyri, Brachybacterium fresconis, Brachybacterium muris, Brevibacterium ammoniilyticum, Brevibacterium casei, Brevibacterium epidermidis, Brevibacterium iodinum, Brevibacterium luteolum, Brevibacterium paucivorans paucivorans, Brevibacterium pityocampae, Brevibacterium sanguinis, Brevundimonas albigilva, Brevundimonas diminuta, Brevundimonas vancanneytii, Caenimonas terrae, Calidifontibacter indicus, Campylobacter concisus, Campylobacter gracilis, Campylobacter hominis, Campylobacter rectus rectus, Campylobacter showae, Campylobacter ureolyticus, Capnocytophaga gingivalis, Capnocytophaga leadbetteri, Capnocytophaga ochraceaochracea, Capnocytophaga sputigena, Cardiobacterium hominis, Cardiobacterium valvarum, Carnobacterium divergens, Catonella morbi, Caulobacter henricii, Cavicella subterranea, Cellulomonas xylanilytica, Cellvibrio vulgaris, Chitinimonas taiwanensis, Chryseobacterium arachidis arachidis, Chryseobacterium daecheongense, Chryseobacterium formosense, Chryseobacterium formosense, Chryseobacterium greenlandens, Chryseobacterium indologenes, Chryseobacterium piscium, Chryseobacterium rigui, Chryseobacterium solani, Chryseobacterium taklimakanense, Chryseobacterium ureilyticum, Chryseobacterium ureilyticum, Chryseobacterium jiae zeae), Chryseomicrobium aureum, Cloacibacterium haliotis, Cloacibacterium normensisnormanense, Cloacibacterium normanens, Corynezola aerofaciens, Comamonas denitrificans, Comamonas trigena, Corynebacterium acupuncture, Corynebacterium afermentans subsp. lipophilum, Corynebacterium ammoniagenes, Corynebacterium amycolatum, Corynebacterium aurimucosum, Corynebacterium aurimucosum, Corynebacterium coyleae, Corynebacterium durum, Corynebacterium fleivrugense freiburgense, Corynebacterium glaucum, Corynebacterium glyciniphilum, Corynebacterium imitans, Corynebacterium jeikeium, Corynebacterium jeikeium, Corynebacterium kroppenstedtii, Corynebacterium lipophiloflavum, Corynebacterium massiliense, Corynebacterium mastitidis, Corynebacterium mattorcotii matruchotii, Corynebacterium minutisimum, Corynebacterium mucifaciens, Corynebacterium mustelae, Corynebacterium mycetoidesmycetoides, Corynebacterium pyruviciproducens, Corynebacterium simulans, Corynebacterium singulare, Corynebacterium sputi, Corynebacterium suicordis, Corynebacterium tuberculostearicum, Corynebacterium tuberculostearicum, Corynebacterium ureicelerivorans, Corynebacterium variabile variabile, Couchioplanes caeruleus subsp. caeruleus, Cupriavidus metallidurans, Curtobacterium herbarum, Dechloromonas agitata, Deinococcus actinosclerus, Deinococcus antarcticus, Deinococcus caeni, Deinococcus ficus, Deinococcus geothermalis, Deinococcus radiodurans, Deinococcus brumquiensis wulumuqiensis, Deinococcus xinjiangensis, Dermabacter hominis, Dermabacter vaginalis, Dermacoccus nishinomiyaensisnishinomiyaensis), Desemzia incerta, Desertibacter roseus, Dialister invisus, Dialister micraerophilus, Dialister Dialister propionicifaciens, Dietzia aurantiaca, Dietzia cercidiphylli, Dietzia timorensis, Dietzia timorensis, Dokdonella koreensis, Dolosigranulum pigrum, Eikenella corrodens, Elizabethkingia miricola, Elstera litoralis, Empedobacter brevis, Enhydrobacter aerosax aerosaccus, Enterobacter xiangfangensis, Enterococcus aquimarinus, Enterococcus faecalis, Enterococcus olivae, Erwinia rapontici, Eubacterium eligens, Eubacterium infirmum, Eubacterium rectare, Eubacterium saphenum, Eubacterium sulci, Exiguobacterium mexicanum, Facklamia tabacinasalis tabacinasalis), Falsilodobacter halotolerans, Finegoldia magna, Flavobacterium cutihirudinis, Flavobacterium linditorranslindanitolerans, Flavobacterium resistens, Friedmanniella capsulata, Fusobacterium nucleatum subsp. polymorphum, Gemella haemolysans, Gemella morbillorum, Gemella palaticanis, Gemella sanguinis, Gemmobacter aquaticus, Gemmobacter caeni, Gordonia jinhuaensis, Gordonia kloppenstettii kroppenstedtii, Gordonia polyisoprenivorans, Gordonia polyisoprenivorans, Granulicatella adiacens, Granulicatella elegans, Haemophilus parainfluenzae, Haemophilus sputorum, Halomonas sulfidaeris, Herpetosiphon aurantiacus, Hydrocarboniphaga effusa, Idiomarina maris, Janibacter anofelis anophelis, Janibacter hoylei, Janibacter indicus, Janibacter limosus, Janibacter mellonismelonis, Jeotgalicoccus halophilus, Jonquetella anthropi, Kaistia geumhonensis, Kingella denitrificans, Kingella oralis, Klebsiella oxytoca, Knoellia aerolata, Knoellia locipacati, Kocuria atrinae, Kocuria carniphila, Kocuria kristinae, Kocuria palustris, Kocuria tsurfanensis turfanensis, Lachnoanaerobaculum saburreum, Lachnoanaerobaculum sabreum, Lactobacillus crispatus, Lactobacillus iners, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. lactis, Lactococcus piscium, Lapillicoccus jejuensis, Lautropia mirabilis, Legionella beliardensis, Leptotrichia buccalis, Leptotrichia goodfellowii goodfellowii), Leptotrichia hofstadii, Leptotrichia hongkongensis, Leptotrichia sha'iishahii, Leptotrichia trevisanii, Leptotrichia weidii, Luteimonas terricola, Lysinibacillus fusiformis, Lysobacter spongiicola, Lysobacter xinjiangensis, Macrococcus caseolyticus, Marmoricola pocheonensis, Marmoricola scoriae, Massilia alkalitolerans, Massilia alkalitolerans, Massilia aurea, Massilia plicata plicata, Massilia timonae, Megamonas rupellensis, Meiothermus silvanus, Methylobacterium dankookense, Methylobacterium goesingense, Methylobacterium gesingens, Methylobacterium isbiliense, Methylobacterium jeotgali, Methylobacterium oxalidis, Methylobacterium platani, Methylobacterium pseudosacicola, Methyloversatilis universalis universalis), Microbacterium foliorum, Microbacterium hydrothermale, Microbacterium hydrothermale, Microbacterium lacticumlacticum, Microbacterium lacticum, Microbacterium laevaniformans, Microbacterium paludicola, Microbacterium petrolearium, Microbacterium phyllosphaerae, Microbacterium resistens, Micrococcus antarcticus, Micrococcus cohnii, Micrococcus flavus, Micrococcus lylae, Micrococcus terreus, Microlunatus aurantiacus aurantiacus, Micropruina glycogenica, Microvirga aerilata, Microvirga erilata, Microvirga subterranea, Microvirga vignae, Microvirga zambiensis, Microvirga aerodenitrificans, Mogibacterium timidum, Moraxella atlantae, Moraxella catarrhalis, Morganella morganii subsp. morganii, Morganella psychrotolerans, Murdochiella asaccharolytica, Mycobacterium asiaticum, Mycobacterium tuberculosischubuense, Mycobacterium crocinum, Mycobacterium gadium, Mycobacterium holsaticum, Mycobacterium iranicum, Mycobacterium longobardum, Mycobacterium neoaurum, Mycobacterium obense, Negativicoccus succinicivorans, Neisseria bacilliformis, Neisseria oralis, Neisseria sicca sicca, Neisseria subflava, Nesterenkonia lacusekhoensis, Nesterenkonia rhizosphaerae, Nevskia persephonica, Nevskia ramosa, Niabella yanshanensis, Niveibacterium umoris, Nocardia niwae, Nocardia thailandica, Nocardioides agariphilus, Nocardioides dilutus, Nocardioides camphaensis ganghwensis, Nocardioides hwasunensis, Nocardioides nanhaiensis, Nocardioides sediminis, Nosocomiicoccus ampulaeampullae, Noviherbaspirillum malthae, Novosphingobium lindaniclasticum, Novosphingobium rosa, Ochrobactrum rhizosphaerae, Olsenella uli, Ornithinimicrobium murale, Ornithinimicrobium tianjinense, Oryzobacter terrae, Ottowia beijingensis, Paenalcaligenes suwonensis, Paenibacillus agaridevorans agaridevorans, Paenibacillus phoenicis, Paenibacillus xylanexedens, Paludibacterium yongneupense, Pantoea cypripedii, Parabacteroides distasonis, Paraburkholderia andropogonis, Paracoccus alcaliphilus, Paracoccus angustae, Paracoccus kocurii, Paracoccus laeviglucosivorans, Paracoccus sediminis sediminis, Paracoccus sphaerophysae, Paracoccus yeei, Parvimonas micra, Parviterribacter multiflagellatusmultiflagellatus, Patulibacter ginsengitellae, Pedobacter aquatilis, Pedobacter ginsengisoli, Pedobacter xixiisoli, Peptococcus niger, Peptoniphilus coxii, Peptoniphilus gorbachii, Peptoniphilus hallei, Peptoniphilus Peptoniphilus koenoeneniae, Peptoniphilus lacrimalis, Peptostreptococcus anaerobius, Peptostreptococcus stomatis, Phascolarctobacterium faecium, Phenylobacterium haematophilum, Phenylobacterium kunshanense, Pluralibacter gergoviae, Polymorphobacter multimanifer multimanifer, Porphyromonas bennonis, Porphyromonas endodontalis, Porphyromonas gingivalis, Porphyromonas gingivicanis, Porphyromonas pasteri, Porphyromonas pogonae, Porphyromonas somerae, Povalibacter uvarum, Prevotella aurantiaca, Prevotella veroniae, Prevotella bivia, Prevotella buccae, Prevotella buccalis buccalis, Prevotella copri, Prevotella corporis, Prevotella denticola, Prevotella enoeca, Prevotella histicolahisticola, Prevotella intermedia, Prevotella jejuni, Prevotella jejuni, Prevotella maculosa, Prevotella melaninogenica, Prevotella melaninogenica, Prevotella micanthus, Prevotella multiformis, Prevotella nanceiensis, Prevotella nigrescens, Prevotella oris, Prevotella oulorum, Prevotella pallens, Prevotella pleuritidis, Prevotella saccharolytica, Prevotella saliviae, Prevotella shahii shahii, Prevotella timonensis, Prevotella veroralis, Propionibacterium acidifaciens, Propionibacterium acnes subsp. acnes, Propionibacterium acnes subsp. acnes, Propionibacterium acnes subsp. elongatum, Propionibacterium granulosum, Propionimicrobium lymphophilum, Propionispira arcuata, Pseudokineococcus lusitanus lusitanus, Pseudomonas aeruginosa, Pseudomonas chengduensis, Pseudonocardia benzenivorans, Pseudorhodoplanes sinuspersici, Psychrobacter sanguinissanguinis, Rhizobium ginsenosidemutans, Rheinheimera aquimaris, Rhizobium alvei, Rhizobium daejonens, Rhizobium larrymoorei, Rhizobium rhizoryzae, Rhizobium soli, Rhizobium taibaishanense, Rhizobium bigne, Rhodanobacter glycinis, Rhodobacter veldkampii, Rhodococcus enculensis, Rhodococcus fascians fascians, Rhodococcus fascians, Rhodovarius lipocyclicus, Rivicola pingtungensis, Roseburia inulinivorans, Rosenbergiella nectarea, Roseomonas aerilata, Roseomonas aquatica, Roseomonas mucosa, Roseomonas rosea, Roseomonas vinacea, Rothia aeria, Rothia amarae, Rothia dentocariosa dentocariosa, Rothia endophytica, Rothia mucilaginosa, Rothia nasimurium, Rubellimicrobium mesophilum, Rubellimicrobium roseum, Rubrobacter bracalensisbracarensis, Rudaea cellulosilytica, Ruminococcus gnavus, Runella zeae, Saccharopolyspora rectivirgula, Salinicoccus qingdaonensis, Scardovia wiggsiae, Sediminibacterium ginsengisoli, Selenomonas artemidis, Selenomonas infelix, Selenomonas noxia, Selenomonas sputigena sputigena, Shewanella astuari, Shuttleworthia satelles, Simonsiella muelleri, Skermanella aerolata, Skermanella stibiiresistens, Slackia exigua, Smaragdicoccus niigatensis, Sneathia sanguinegens, Solirubrobacter soli, Sphingobacterium caeni, Sphingobacterium daejonens daejeonense, Sphingobacterium hotanense, Sphingobacterium kyonggiense, Sphingobacterium multivorum, Sphingobacterium nematokidanematocida, Sphingobacterium spiritivorum, Sphingobium amiens, Sphingobium indicum, Sphingobium lactostensis, Sphingobium subterraneum, Sphingomonas abaci, Sphingomonas aestuarii, Sphingomonas canadensis, Sphingomonas daechungensis, Sphingomonas dokudonensis, Sphingomonas echinoides, Sphingomonas fonticola Sphingomonas fonticola, Sphingomonas formosensis, Sphingomonas gei, Sphingomonas hankookensis, Sphingomonas hankookensis, Sphingomonas koreensis, Sphingomonas kyeonggiensis, Sphingomonas laterariae, Sphingomonas mucosissima, Sphingomonas oligophenolica, Sphingomonas pseudosanguinis, Sphingomonas sediminicola sediminicola, Sphingomonas yantingensis, Sphingomonas yunnanensis, Sphingomonas indica, Spirosoma liguirigui, Sporacetigenium mesophilum, Sporocytophaga myxococcoides, Staphylococcus auricularis, Staphylococcus epidermidis, Staphylococcus epidermidis, Staphylococcus hominis subsp. novobiosepticus, Staphylococcus lugdunensis, Staphylococcus pettenkoferi, Stenotrophomonas koreensis, Stenotrophomonas rhizophylla rhizophila, Stenotrophomonas rhizophila, Streptococcus agalactiae, Streptococcus canis, Streptococcus cristatus, Streptococcus gordonii, Streptococcus infantis, Streptococcus intermedius, Streptococcus mutans, Streptococcus oligofermentans, Streptococcus oralis, Streptococcus sanguinis, Streptomyces iconiensis, Streptomyces youngensis yanglinensis, Tabrizicola aquatica, Tahibacter caeni, Tannerella forsythia, Tepidicella xavieri, Tepidimonas fonticaldi, Terracoccus luteusluteus, Tessaracoccus flavescens, Thermus thermophilus, Tianweitania sediminis, Tianweitania sediminis, Treponema amylovorum, Treponema denticola, Treponema lecithinolyticum, Treponema medium, Turicella otitidis, Turicibacter sanguinis, Undibacterium oligocarboniphilum, Undibacterium squillarum, Vagococcus salmoninarum salmoninarum, Varibaculum cambriens, Vibrio metschnikovii, Xanthobacter tagetidis, Xenophilus aerolatus, Xenophilus arseniciresistens, Yimella lutea, Zimmermannella alba, Zimmermannella bifida and / or Zoogloea caeni.

[0298] In other embodiments, the bacterial cells targeted are those commonly found in the vaginal microbiota, including, but not limited to, Acinetobacter antiviralis, Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter johnsonii, Actinobaculum massiliense, Actinobaculum sassaliii, Actinobaculum schaalii, Actinomyces europaeus, Actinomyces graebenitzii, Actinomyces israelii, Actinomyces mayerii, Actinomyces naeslundii, Actinomyces nouii, Actinomyces odontolyticus, Actinomyces turisensis, Actinomyces urogenitalis, Actinomyces viscosus, Aerococcus christensenii, Aerococcus urina, Aerococcus viridans, Aeromonas encheleia, Aeromonas salmonicida, Afipia masciliensis massiliensis, Agrobacterium tumefaciens, Algoriphagus aquatilis, Aliivibrio wodanis, Alistipes finegoldii, Alloiococcus otitis, Alloprevotella tannerae, Alloscardovia omnicolens, Altererythrobacter epoxidivorans, Ammoniphilus oxalaticusoxalaticus, Amnibacterium kyonggiense, Anaerococcus hydrogenalis, Anaerococcus lactolyticus, Anaerococcus muldchii, Anaerococcus obesiensis, Anaerococcus prevotii, Anaerococcus tetradius, Anaerococcus vaginalis, Anaeroglobus geminatus, Anoxybacillus pushchinoensis, Aquabacterium parvum, Arcanobacterium phocae, Arthrobacter aurescens aurescens, Asticacaulis excentricus, Atopobium minutum, Atopobium parvum, Atopobium limae, Atopobium vaginae, Avibacterium gallinarum, Bacillus acidicola, Bacillus atrophaeus, Bacillus cereus, Bacillus cibi, Bacillus coahuilensis, Bacillus gaemokensis, Bacillus methanolicus, Bacillus orellonius oleronius, Bacillus pumilus, Bacillus shackletonii, Bacillus sporothermodurans, Bacillus subtilis, Bacillus wakoensis, Bacillus weihenstephanensis, Bacteroides barnesiaebarnesiae, Bacteroides coagulans, Bacteroides dorei, Bacteroides faeces, Bacteroides forsythus, Bacteroides fragilis, Bacteroides nordii, Bacteroides obatas, Bacteroides seyriasiae, Bacteroides starcoris, Bacteroides uniformis, Bacteroides vulgatus, Bacteroides xylanisolvens, Bacteroides zoogleoformans, Barnesiella viscericola, Bhargavaea sessembensis cecembensis, Bifidobacterium alesentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium dentium, Bifidobacterium logum subsp. infantis, Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Bifidobacterium scardovii, Bilophila wordsworthia, Blautia hydrogenotrophica, Blautia obeum, Blautia producta, Brachybacterium faecium, Bradyrhizobium japonicum, Brevibacterium macbrerneri mcbrellneri, Brevibacterium otitidis, Brevibacterium paucivorans, Bulleidia extructa, Burkholderia fungorum, Burkholderia phenoliruptix, Caldicellulosiruptor saccharolyticus, Caldimonas taiwanensisCampylobacter taiwanensis, Campylobacter gracilis, Campylobacter hominis, Campylobacter sputorum, Campylobacter ureolyticus, Capnocytophaga ochracea, Cardiobacterium hominis, Chaetonella morbii, Chlamydia trachomatis, Chlamydophila abortus, Chondromyces robustus, Chryseobacterium aquaticum, Citrobacter yangae, Cloacibacterium normensis, Clostridium cavendishii, Clostridium colicanis, Clostridium jejuense jejuense, Clostridium perfringens, Clostridium ramosum, Clostridium sordellii, Clostridium viride, Comamonas trigena, Corynebacterium acupuncture, Corynebacterium appendicis, Corynebacterium koileae, Corynebacterium glucuronolyticum, Corynebacterium glutamicum glutamicum, Corynebacterium jeikeium, Corynebacterium kloppenstettii, Corynebacterium lipophylloflavum, Corynebacterium minutissimum, Corynebacterium mucifaciens, Corynebacterium nuruki, Corynebacterium pseudogenitalium, Corynebacterium pirubiciproducens, Corynebacterium singulare, Corynebacterium striatumstriatum, Corynebacterium tuberculostearicum, Corynebacterium xerosis, Cryobacterium psychrophilum, Corynebacterium fraccumfaciens, Cutibacterium acnes, Cutibacterium avidum, Cytophaga xylanolytica, Deinococcus radiophilus, Delftia tsuruhatensis, Desulfovibrio desulfuricans, Dialister invissus, Dialister micraerophilus, Dialister pneumosynthes pneumosintes, Diaryster propionicifaciens, Dickeya chrysanthemi, Dorea longicatena, Eggerthia lenta, Eggerthia catenaformis, Eikenella corrodens, Enhydrobacter aerosax, Enterobacter asbriae, Enterobacter cloacae, Enterococcus avium, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, Erwinia persicina, Erwinia rappontici, Erwinia toretana toletana, Escherichia fergusonii, Eubacterium brachy, Eubacterium eligens, Eubacterium nodatum, Eubacterium rectare, Eubacterium safenum, Eubacterium silaeum, Eubacterium salsi, Eubacterium yuriiyurii, Exiguobacterium acetylicum, Facklamia ignava, Faecalis bacterium prausnitzii, Filifactor alocis, Finegoldia magna, Fusobacterium gonidiaformans, Fusobacterium nucleatum, Fusobacterium periodonticum, Gardnerella vaginalis, Gemella asaccharolytica, Gemella bergeri, Gemella haemolysans, Gemella sanguinis, Geobacillus stearothermophilus, Geobacillus thermocatenulatus thermocatenulatus, Geobacillus thermoglucosidasius, Geobacter grbiciae, Granulocytella elegans, Haemophilus ducreyi, Haemophilus haemolyticus, Haemophilus parahaemolyticus, Haemophilus parainfluenzae, Hafnia alvei, Halomonas meridiana, Halomonas phoceae, Halomonas venusta, Herbaspirillum seropedicae, Janthinobacterium lividum lividum, Johnketella anthropi, Klebsiella granulomatis, Klebsiella oxytoca, Klebsiella pneumoniae, Lactobacillus acidophilus, Lactobacillus amylovorusamylovorus, Lactobacillus brevis, Lactobacillus coleohominis, Lactobacillus crispatus, Lactobacillus calvertus, Lactobacillus delbreckii, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus iners, Lactobacillus jensenii, Lactobacillus johnsonii, Lactobacillus kalixensis, Lactobacillus kefiranofaciens, Lactobacillus kimchicus, Lactobacillus kitasatonis kitasatonis, Lactobacillus mucosae, Lactobacillus panis, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus altunensis, Lactobacillus vaginalis, Lactococcus laevis cutis, Leptotrichia bucharis, Leuconostoc carnosum, Leuconostoc citreum, Leuconostoc garlicum, Leuconostoc lactis, Leuconostoc mesenteroides, Lysinimonas kribbensis, Mageeibacillus indolicus, Maribacter orientalis, Marinomonas protea, Marinospirillum insulare, Massilia timonae, Megasphaera elsdenii, Megasphaera micronuciformis, Mesorhizobium amorphae amorphae, Methylobacterium radiotolerans, Methylotenera versatilis, Microbacterium halophilum, Micrococcus luteus, Microterricola viridarii, Mobiluncus curtisii, Mobiluncus mulieris, Mogibacterium thymidum, Moorella glycerini, Moraxella osloensis, Morganella morganii, Moriella indoligenes, Murdsiella asacallolytica, Mycoplasma albi alvi), Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma muris, Mycoplasma salivariumsalivarium, Negatibicoccus succinicivorans, Neisseria flava, Neisseria gonorrhoeae, Neisseria mucosa, Neisseria subflava, Nevskia ramosa, Nevskia soli, Nitlily Group Tall Alkaliferus, Odoribacter plancnicus, Oligella urethralis, Olsenella uri, Paenibacillus amylolyticus, Paenibacillus humicus, Paenibacillus pabuli, Paenibacillus passadenensis pasadenensis, Paenibacillus pini, Paenibacillus validus, Pantoea agglomerans, Parabacteroides meldae, Paraburkholderia caryophylli, Paracoccus ii, Parastreptomyces abscessus, Parvimonas micra, Pectobacterium betavasculorum, Pectobacterium carotovorum, Pediococcus acidilactici, Pediococcus ethanolidurans ethanolidurans, Pedobacter alluvionis, Pedobacter wanjuense, Pelomonas aquatica, Peptococcus niger, Peptoniphilus asaccharolyticusasaccharolyticus, Peptoniphilus gorbatii, Peptoniphilus hallei, Peptoniphilus indolicus, Peptoniphilus lacrimalis, Peptoniphilus massiliensis, Peptostreptococcus anaerobius, Peptostreptococcus massiliae, Peptostreptococcus stomatis, Photobacterium angustum, Photobacterium frigidiphilum, Photobacterium phosphoreum, Porphyromonas asaccharolytica, Porphyromonas venonis, Porphyromonas catoniae catoniae, Porphyromonas endodontalis, Porphyromonas gingivalis, Porphyromonas somerae, Porphyromonas uenonis, Prevotella amnii, Prevotella berloniae, Prevotella bergensis, Prevotella bivia, Prevotella buccae, Prevotella buccalis, Prevotella colorans, Prevotella copri, Prevotella corporis, Prevotella dentalis, Prevotella denticola, Prevotella disiens, Prevotella intermedia, Prevotella loescheii, Prevotella marsii marshii), Prevotella melaninogenica, Prevotella micans, Prevotella nigrescens, Prevotella oris, Prevotella pleuritidis, Prevotella ruminicola (Prevotellaruminicola, Prevotella shahii, Prevotella starcorea, Prevotella timonensis, Prevotella veroralis, Propionimicrobium lymphophilum, Proteus mirabilis, Pseudomonas abietaniphila, Pseudomonas aeruginosa, Pseudomonas amygdali, Pseudomonas azotoformans, Pseudomonas chlororaphis, Pseudomonas cuatrocienegasensis, Pseudomonas fluorescens, Pseudomonas fulva fulva, Pseudomonas lutea, Pseudomonas musidorens, Pseudomonas oleovorans, Pseudomonas orientalis, Pseudomonas pseudoalcaligenes, Pseudomonas psychrophila, Pseudomonas putida, Pseudomonas synxantha, Pseudomonas syringae, Pseudomonas tolaasii, Pseudopropionibacterium propionicum, Rahnella aquatilis aquatilis, Ralstonia picketii, Ralstonia solanacearum, Raoultella planticola, Rhizobacter dauci, Rhizobium etori, Rhodococcus fascians, Rhodopseudomonas palustrispalustris, Roseburia intestinalis, Roseburia inulinivorans, Rottia mucilaginosa, Ruminococcus bloomii, Ruminococcus gnavus, Ruminococcus turchis, Sanguibacter keddieii, Sediminibacterium salmoneum, Selenomonas bovis, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Shewanella algae, Shewanella amazonensis, Shigella boydii boydii, Shigella sonnei, Slacchia exigua, Sneathia amnii, Sneathia sanguinegens, Solobacterium moorei, Sorangium cellulosum, Sphingobium amiens, Sphingobium japonicum, Sphingobium yanoikuyae, Sphingomonas wittichii, Sporosarcina aquimarina, Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus capitis capitis, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcus saprophyticus, Staphylococcus schleiferi, Staphylococcus simiae, Staphylococcus simulanssimulans, Staphylococcus warneri, Stenotrophomonas maltophilia, Stenoxybacter acetivorans, Streptococcus agalactiae, Streptococcus anginosus, Streptococcus australis, Streptococcus equinus, Streptococcus gallolyticus, Streptococcus infantis, Streptococcus intermedius, Streptococcus lutetiensis, Streptococcus marimalis, Streptococcus marimammalium, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus phocae, Streptococcus pseudopneumoniae, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus thermophilus, Strepella wadsworthensis, Tannerella forsythia, Terrahaemophilus aromaticivorans, Treponema denticola, Treponema maltophilum, Treponema parvum parvum, Treponema vincentii, Trueperella bernardiae, Turicella otaitidis, Ureaplasma parvum, Ureaplasma urealyticum, Varioborax cambriens, Variovorax paradoxus, Veillonella atypica, Veillonella dispar, Veillonella montpelierensismontpellierensis, Veillonella parvula, Virgibacillus proomii, Viridibacillus arenosi, Viridibacillus arvi, Weissella cibaria, Weissella soli, Xanthomonas campestris, Xanthomonas vesicatoria, Zobellia laminariae and / or Zoogloea ramigera.

[0299] In one embodiment, the targeted bacterium is Escherichia coli.

[0300] In one embodiment, the targeted bacterium is Cutibacterium acnes, more specifically, acne-associated Cutibacterium acnes from phylogenetic group IA1 or RT4, RT5, RT8, RT9, RT10 or clonal complex (CC) CC1, CC3, CC4, more specifically ST1, ST3, ST4.

[0301] As such, the first type of bacteriophage disclosed herein, and thus the phage particles or phage-derived delivery particles of the present invention, may target (e.g., specifically target) bacterial cells from any one or more of the above-mentioned genera and / or species of bacteria, in particular for the specific delivery of a payload.

[0302] In one embodiment, the targeted bacteria are pathogenic bacteria. The targeted bacteria can be virulent bacteria.

[0303] The targeted bacteria may be an antimicrobial-resistant bacterium selected from the group consisting of extended-spectrum beta-lactamase-producing (ESBL) Escherichia coli, ESBL Klebsiella pneumoniae, vancomycin-resistant Enterococcus (VRE), methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant (MDR) Acinetobacter baumannii, MDR Enterobacter spp., and combinations thereof. Preferably, the targeted bacteria may be selected from the group consisting of extended-spectrum beta-lactamase-producing (ESBL) Escherichia coli strains.

[0304] Alternatively, the targeted bacteria may be bacteria of the microbiome of a given species, preferably bacteria of the human microbiota.

[0305] In certain embodiments, the targeted bacterial cells are from a different species or strain than the producing bacterial cells.

[0306] Hybrid helper phage system and hybrid helper phage The present invention also provides a hybrid helper phage system, comprising: (i) at least one phage DNA packaging gene, as defined above in the section "Producer Bacterial Cells," derived from a first type of bacteriophage, as defined above in the section "Bacteriophages and Genes Derived From Bacteriophages"; (i') at least one phage structural gene, as defined above in the section "Producer Bacterial Cells," derived from said first type of bacteriophage; and (ii) at least one gene from a second type of bacteriophage, as defined above in the section "Bacteriophages and Genes Derived from Bacteriophages," involved in phage excision / insertion, phage DNA replication, and / or phage regulation, as defined above in the section "Producer Bacterial Cells." Including, the genes (i), (i') and (ii) are contained in a single nucleic acid molecule or in separate nucleic acid molecules; the first type of bacteriophage originates from and / or targets a bacterial species or strain different from the bacterial species or strain from which the second type of bacteriophage originates and / or targets, The hybrid helper phage system does not contain any expressed phage structural genes, as defined above in the section "Producer bacterial cells," derived from the second type of bacteriophage.

[0307] In the context of the present invention, the term "hybrid helper phage system" means a group of at least one nucleic acid molecule, preferably at least two separate nucleic acid molecules, comprising genes (i), (i') and (ii) as defined above, which allows the production of phage particles and / or phage-derived delivery vehicles by a producer bacterial cell comprising said system, and when the system comprises at least two separate nucleic acid molecules, said genes (i), (i') and (ii) are distributed on said at least two separate nucleic acid molecules.

[0308] As used herein, the term "nucleic acid" refers to a sequence of at least two nucleotides covalently linked together, which may be single-stranded or double-stranded, or contain portions of both single-stranded and double-stranded sequences. Nucleic acids of the present invention can be naturally occurring, recombinant, or synthetic. Nucleic acids can be in the form of circular or linear sequences, or a combination of both forms. Nucleic acids can be DNA, RNA, or a combination of both, both genomic and cDNA. Nucleic acids may contain any combination of deoxyribonucleotides and ribonucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, 5-hydroxymethylcytosine, and isoguanine. Other examples of modified bases that can be used in the present invention are detailed in Weigele et al., Chem Rev. 2016 Oct 26;116(20):12655-12687. The term "nucleic acid" also encompasses any nucleic acid analogs that may contain other backbones, including, but not limited to, phosphoramide, phosphorothioate, phosphorodithioate, O-methyl phosphoramidite linkages, and / or deoxyribonucleotide and ribonucleotide nucleic acids. Any combination of the above features of a nucleic acid is also encompassed by the present invention.

[0309] In particular embodiments, the genes (i), (i') and (ii) are contained in a bacterial chromosome, particularly a producer bacterial cell chromosome. In more particular embodiments, the genes (i), (i') and (ii) are contained in the same region of the bacterial chromosome. In alternative embodiments, the genes (i), (i') and (ii) are contained in separate regions of the bacterial chromosome.

[0310] In an alternative embodiment, the genes (i), (i') and (ii) are contained in separate plasmids. In another particular embodiment, the genes (i), (i') and (ii) are all contained in the same plasmid.

[0311] In another particular embodiment, the genes (i), (i') and (ii) are each independently contained in a bacterial chromosome or a plasmid.

[0312] In a more particular embodiment, said genes (i), (i') and (ii) are contained in a hybrid helper phage.

[0313] Thus, in certain embodiments, the hybrid helper phage system comprises: (i) at least one phage DNA packaging gene and at least one phage structural gene, as defined above in the section "Producer Bacterial Cells," derived from a first type of bacteriophage, as defined above in the section "Bacteriophages and Genes Derived from Bacteriophages," and (ii) at least one gene from a second type of bacteriophage, as defined above in the section "Bacteriophages and Genes Derived from Bacteriophages," involved in phage excision / insertion, phage DNA replication, and / or phage regulation, as defined above in the section "Producer Bacterial Cells." a hybrid helper phage comprising the first type of bacteriophage originates from and / or targets a bacterial species or strain different from the bacterial species or strain from which the second type of bacteriophage originates and / or targets; The hybrid helper phage does not contain any phage structural genes, as defined above in the section "Producer Bacterial Cells," derived from the second type of bacteriophage.

[0314] By "helper phage" is meant herein an engineered phage that provides all the necessary gene products for particle formation when a phagemid vector is used. Helper phage typically have a defective origin of replication or packaging signal and are therefore inefficient at self-packaging.

[0315] By "hybrid helper phage" is meant herein an engineered helper phage composed of elements from at least two different types of bacteriophage.

[0316] In certain embodiments, the hybrid helper phage of the invention is integrated as a prophage into the genome of the producer bacterial cell.

[0317] Production method The present invention further provides a method for producing a phage particle or phage-derived delivery vehicle, comprising the steps of: (a) providing a bacterial production cell of the invention; and (b) inducing in said producer bacterial cell the expression of at least one of said phage structural genes and at least one of said phage DNA packaging genes and the assembly of products expressed by said at least one phage structural gene and said at least one phage DNA packaging gene, thereby producing a phage particle or a phage-derived delivery vehicle. The present invention relates to a method, comprising:

[0318] The inducing step (b) can be carried out by any technique known to those skilled in the art, and in particular, as will be appreciated by those skilled in the art, the inducing step will depend on the particular induction mechanism controlling the expression of the at least one of the phage structural genes and phage DNA packaging genes in the producer bacterial cell.

[0319] More particularly, it will be understood by those skilled in the art that if the induction mechanism comprises at least one gene from a second type of bacteriophage involved in phage excision / insertion, phage DNA replication, and / or phage regulation, the inducing step will depend on the bacteriophage from which the sequence is derived. Typically, the inducing step can be thermal induction (for phages naturally induced by this signal or an engineered repressor, e.g., lambda cI), a small molecule inducer (depending on the phage), any signal that triggers an SOS response (e.g., addition of mitomycin), etc.

[0320] Production of teilosins and / or pyocins The approaches disclosed above are also applicable to the production of teilosins and / or pyocins, allowing for the safe and efficient production of such bacteriocins in engineerable production cells.

[0321] The present invention therefore also provides a bacterial producer cell for producing a teilosin and / or a pyocin, said bacterial producer cell stably comprising at least one teilosin and / or pyocin structural gene derived from a bacterial species or strain containing the teilosin and / or pyocin genes, expression of at least one of the teilosin and / or pyocin structural genes in the producing bacterial cell is controlled by at least one inducible mechanism; The present invention relates to a producing bacterial cell, wherein the producing bacterial cell is from a bacterial species or strain that is different from the bacterial species or strain containing the teilosin and / or pyocin genes from which the teilosin and / or pyocin structural genes are derived.

[0322] By "teilosin" is meant herein a multisubunit bacteriocin that resembles a bacteriophage tail. There are two classes of teilosin particles: flexible, non-contractile F-teilosins and rigid, contractile R-teilosins, which resemble and are evolutionarily related to Siphoviridae and Myoviridae phage tails, respectively. Examples of teilosins typically include F-type and R-type pyocins, carotovoricins, xenorhabdicins, and maltocins.

[0323] By "pyocin" herein is meant a bacteriocin produced by Pseudomonas aeruginosa. They can be produced spontaneously or can be induced by certain chemicals, such as mitomycin C. Three different types of pyocins have been identified: R-type, S-type, and F-type (Nakayama et al. (2000) Mol. Microbiol. 38:213-231). They differ by morphology and mode of killing. Their bactericidal activity is strain-specific. R-type pyocins resemble the inflexible, contractile tails of bacteriophages and belong to the teilosins disclosed above, and are further classified into five groups: R1, R2, R3, R4, and R5. F-type pyocins also resemble phage tails, being flexible but non-contractile rod-like structures with distal filaments, and also belong to the teilosins disclosed above. They are similar in structure and serological properties but differ in receptor specificity. Three subtypes of F-type pyocins have been reported: F1, F2, and F3. In certain embodiments, the pyocin is an R-type or F-type pyocin.

[0324] By "teilosin and / or pyocin structural genes" is meant herein genes from teilosin and / or pyocin-producing bacteria that are involved in the assembly of teilosins and / or pyocins. Teilosin and / or pyocin structural genes include the genes encoding the subunits and / or components of said teilosins and / or pyocins, as disclosed above, as well as genes encoding bacterial proteins that are involved in the assembly of teilosin and / or pyocin subunits and / or components.

[0325] In certain embodiments, the teilosin and / or pyocin structural genes are pyocin structural genes as defined above, and the bacterial species or strain containing the teilosin and / or pyocin genes from which the pyocin structural genes are derived is preferably a Pseudomonas aeruginosa bacterium.

[0326] In a particular embodiment, the teilosin and / or pyocin structural gene is a teilosin structural gene as defined above, in which embodiment the bacterial species or strain containing the teilosin and / or pyocin gene from which the teilosin structural gene is derived is preferably selected from bacteria as defined above in the section "Targeted Bacteria" that naturally contain the teilosin structural gene and preferably produce teilosin.

[0327] In certain embodiments, the teilosin and / or pyocin structural genes are contained in at least one plasmid, chromosome, and / or helper phage.

[0328] In the context of the present invention, said induction mechanism is as defined in the section "Producer bacterial cells" above.

[0329] More particularly, in one embodiment, at least one inducible mechanism controls the expression of all said teilosin and / or pyocin structural genes.

[0330] In certain embodiments, the at least one inducible mechanism further controls the copy number of the at least one of the teilosin and / or pyocin structural genes.

[0331] In certain embodiments, the at least one induction mechanism comprises at least one gene involved in teilosin and / or pyocin regulation, and the gene involved in teilosin and / or pyocin regulation is derived from a bacterial species or strain containing teilosin and / or pyocin genes that are different from those from which the teilosin and / or pyocin structural genes are derived.

[0332] Thus, in certain embodiments, the producing bacterial cell further comprises at least one gene involved in teilosin and / or pyocin regulation, wherein the gene involved in teilosin and / or pyocin regulation is derived from a bacterial species or strain containing teilosin and / or pyocin genes that are different from those from which the teilosin and / or pyocin structural genes are derived.

[0333] By "genes involved in teilosin and / or pyocin regulation" is meant herein genes that encode regulatory elements that control the induction and / or expression of teilosin and / or pyocin in naturally occurring teilosin and / or pyocin-producing bacterial cells.

[0334] Alternatively, the at least one induction mechanism comprises at least one gene derived from a bacteriophage, as defined above in the section "Bacteriophages and Genes Derived from Bacteriophages," involved in phage excision / insertion, phage DNA replication, and / or phage regulation, as defined above in the section "Producer Bacterial Cells," and the bacteriophage is derived from and / or targets a bacterial species or strain different from the bacterial species or strain from which the teilosin and / or pyocin structural genes are derived.

[0335] Thus, in certain embodiments, the producer bacterial cell further comprises at least one gene derived from a bacteriophage, as defined above in the section "Bacteriophages and Genes Derived from Bacteriophages," involved in phage excision / insertion, phage DNA replication, and / or phage regulation, as defined above in the section "Producer Bacterial Cells," and the bacteriophage is derived from and / or targets a bacterial species or strain different from the bacterial species or strain from which the teilosin and / or pyocin structural genes are derived.

[0336] In certain embodiments, the teilosins and / or pyocins are intended to lyse targeted bacterial cells, as defined above in the section "Targeted Bacterial Cells."

[0337] In certain embodiments, the targeted bacterial cells are from a different species or strain than the producing bacterial cells.

[0338] In certain embodiments, the producer bacterial cells are from the same bacterial species or strain as the bacterial species or strain from which the bacteriophage is derived and / or which the bacteriophage targets.

[0339] In a particular embodiment, the producing bacterial cell is an E. coli bacterial cell.

[0340] The present invention further provides a method for producing a teilosin and / or a pyocin, comprising the steps of: (a) providing a teilosin and / or pyocin producing bacterial cell as defined above; and (b) inducing in the producing bacterial cell the expression of said at least one of said teilosin and / or pyocin structural genes, as defined above, and the assembly of products expressed by said at least one teilosin and / or pyocin structural gene, as defined above in the section "Method of Production," thereby producing a teilosin and / or pyocin. The present invention relates to a method, comprising:

[0341] The present invention also provides a hybrid production system comprising: (i') at least one teilosin and / or pyocin structural gene, as defined above, derived from a bacterial species or strain containing the teilosin and / or pyocin gene, as defined above; and (ii) at least one gene derived from a bacteriophage, as defined above in the section "Bacteriophages and Genes Derived from Bacteriophages," that is involved in phage excision / insertion, phage DNA replication, and / or phage regulation, as defined above in the section "Producer Bacterial Cells"; or at least one gene involved in teilosin and / or pyocin regulation that is derived from a bacterial species or strain containing teilosin and / or pyocin genes that are different from those from which the teilosin and / or pyocin structural genes are derived. Including, the genes (i') and (ii) are contained in a single nucleic acid molecule or in separate nucleic acid molecules, as defined in the section "Hybrid helper phage systems and hybrid helper phages" above, the bacteriophage is derived from and / or targets a bacterial species or strain different from that from which the teilosin and / or pyocin structural genes are derived, The hybrid production system does not contain any expressed phage structural genes, as defined above in the section "Bacterial Producer Cells", derived from the bacteriophage.

[0342] By "hybrid production system" is meant herein a group of at least one nucleic acid molecule, preferably at least two separate nucleic acid molecules, comprising genes (i') and (ii) as defined above, which allows the production of teilosin and / or pyocin by a production bacterial cell comprising said system, and where the system comprises at least two separate nucleic acid molecules, said genes (i') and (ii) are distributed on said at least two separate nucleic acid molecules.

[0343] In a particular embodiment, said genes (i') and (ii) are comprised in a bacterial chromosome.

[0344] In an alternative embodiment, the genes (i') and (ii) are contained in separate plasmids.

[0345] In a further alternative embodiment, the hybrid production system comprises: (i') at least one teilosin and / or pyocin structural gene, as defined above, derived from a bacterial species or strain containing the teilosin and / or pyocin gene; and (ii) At least one gene derived from a bacteriophage, as defined above in the section "Bacteriophages and Genes Derived from Bacteriophages," involved in phage excision / insertion, phage DNA replication, and / or phage regulation, as defined above in the section "Producer Bacterial Cells." a hybrid helper phage comprising the bacteriophage is derived from and / or targets a bacterial species or strain different from that from which the teilosin and / or pyocin structural genes are derived, The hybrid helper phage does not contain any expressed phage structural genes, as defined above in the section "Producer Bacterial Cells," derived from the bacteriophage.

[0346] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0347] It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "cell" includes a plurality of such cells (e.g., a population of such cells). Similarly, a reference to a "nucleic acid" includes one or more such nucleic acids.

[0348] While the present invention has been described in conjunction with specific embodiments thereof, many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0349] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. Additionally, citation or identification of any reference in this application should not be construed as an admission that such reference is available as prior art to the present invention. Where section headings are used, they should not be construed as necessarily limiting.

[0350] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the invention in any way.

[0351] [Table 1-1]

[0352] [Table 1-2] [Brief explanation of the drawings]

[0353] [Figure 1] Organization of the lambda genome (lambda-packaged phagemid variant). Structural operons are marked in red, as well as the antitermination protein Q, which enables transcription of the late structural operon. Diagram adapted from Rajagopala et al., BMC Microbiol 11, 213 (2011). [Figure 2] Organization of the Klebsiella pneumoniae prophage genome (prophage variants). Structural and regulatory operons are marked in red, and some structural elements are labeled. [Figure 3] Lambda-K. pneumoniae prophage hybrid. The complete structural operon from the K. pneumoniae prophage (marked with a red line) occupies the place of the lambda late structural operon. The remainder of the lambda prophage, which regulates all other functions, is intact. [Figure 4] Titration of kappa-packaged phagemids with payload pTEST. From top to bottom and left to right: MG1656-OmpCO157, MG1655, MG-Kpne OmpC G1, Kpne F3, MG-Kpne OmpC 7, MG-Kpne OmpC G2, MG-Kpne OmpC G16, MG-Kpne OmpC G15, MG-Kpne OmpC G18. No hits were observed. [Figure 5] Titration of kappa-packaged phagemids on Klebsiella pneumoniae collections with payload pTEST. Top panel: Kpne collections treated with PBS only and plated on chloramphenicol to check for background Cm resistance (left, Kpne plate 1; right, Kpne plate 2). Bottom panel: Kpne collections treated with lysate from kappa + pTEST. No hits were observed. [Figure 6]Titration of kappa-packaged phagemids carrying payloads pTEST or pTEST-2 in the presence of HNH protein. Top panel: pTEST + HNH in trans. Bottom panel: pTEST-2 + HNH in trans. From top to bottom and left to right: Kpne F3, MG1656-OmpCO157, MG1655, MG-Kpne OmpC G7, MG-Kpne OmpC G2, MG-Kpne OmpC G1, MG-Kpne OmpC G18, MG-Kpne OmpC G16, MG-Kpne OmpC G15. [Figure 7] Titration of kappa-packaged phagemids carrying payloads pTEST or pTEST-2 in the presence of HNH protein on Klebsiella pneumoniae collections. Top panel: Left) Kpne collection 1 treated with PBS; Middle) pTEST + HNH in trans; Right) pTEST-2 + HNH in trans. Bottom panel, Left) Kpne collection 2 treated with PBS; Middle) pTEST + HNH in trans; Right) pTEST-2 + HNH in trans. [Figure 8] Titration of packaged phagemids on the Kpne collection. Top panel: left, PBS; right, Klebsiella pneumoniae collection 1 treated with pTEST-3 + HNH-operon in trans. Bottom panel, left, PBS; right, Klebsiella pneumoniae collection 2 treated with pTEST-3 + HNH-operon in trans. [Figure 9] Identification of P. freudenreichii phages using PCR. PCR for ORF3 and ORF5 was performed on all phage suspensions. BW4 from plaques 1-3 gave bands at the expected sizes for both orf3 and orf5. The ladder is GeneRuler 1 kb plus. [Figure 10] Immunity to superinfection with the lysogen Pf0s14253. Left panel: Top agar of Pf0s2841 with spots of four different BW-like phage suspensions. Right panel: Top agar of Pf0s14253 with spots of four different BW-like phage suspensions. [Figure 11] High induction of BW4 phage after mitomycin C treatment. Left panel: Top agar of Pf0s2841 with spots of culture supernatant from Pf0s14253 without mitomycin C (MMC) induction (ND: undiluted to 10-3 dilutions). Right panel: Top agar of Pf0s2841 with spots of culture supernatant from Pf0s14253 with 0.5 μg / ml mitomycin C induction (ND: undiluted to 10-7 dilutions). [Figure 12] Genome organization of BW4 and PAC7 bacteriophages. The BW4 and PAC7 genome organizations are similar, with both putative structural operons (represented by arrows) containing packaging, head, tail, and lysis modules. [Figure 13] Construction of a chimeric BW4-PAC7 prophage. Strain Pf1s22499 containing the BW4 prophage was transformed with the pAN514 suicide plasmid. Selection on chloramphenicol was used to select for a double crossover in the left homologous arm (LHA) and right homologous arm (RHA). The resulting prophage is a chimera containing the first BW4 gp1, followed by a structural operon with PAC7 gp1 to gp14, and the remainder of the BW4 structural genes (gp15 to gp25) after a chloramphenicol selection cassette (CmR). [Figure 14] Plasmid map of cosmid pAN594. [Figure 15] Titration of PAC7 phage-derived particles. Left panel: Titration from Pf1s22904 plated on erythromycin. Right panel: Titration from a control suspension of strain Pf1s22903 without any cosmid plated on erythromycin. [Figure 16]PCR confirmation of eight colonies streaked from a phage-derived particle titration of Pf1s22904 production. Top panel: SLTS PCR of eight colonies streaked from a phage-derived titration assay (Scholz 2014). The expected size is 612 bp. Bottom panel: pAN594-specific PCR of eight colonies. The expected size is 769 bp. The ladder is generic 1 kb plus. [Example]

[0354] Example 1 Exchange of the lambda structural operon with that of a phage from a different species The inventors have considered that phages can be viewed as more or less large genetic circuits whose final output is the production of more phage particles. To do this, regardless of whether the phages are lytic, lysogenic or chronic (e.g. filamentous phages, e.g. M13), the information encoded in their genomes can be broadly categorized depending on the function they perform: - Genes for insertion / excision purposes (for temperate phages). - Genes for purposes such as DNA replication, RNA transcription, etc. For example, some lytic phages encode their own RNA or DNA polymerase. Some genes modify the host's RNA polymerase so that it can function past the terminator, and some other genes are involved in sequestration of the prophage sequence when it is present in a plasmid or linear plasmid form. - Genes related to defense against host antiphage mechanisms, degradation / modification of host elements to complete the lytic cycle, super-exclusion mechanisms or genes advantageous for the host. - Genes for DNA packaging purposes: terminase and accessory proteins, ligase, etc. - structural genes for the purpose of building a protein capsid for the DNA: besides the strictly structural genes, e.g. capsid genes, tape measure, fiber, baseplate, etc., many other genes are needed to assemble proteins that can be packaged inside the capsid, be they as scaffolds or pilot proteins injected into the cell (e.g. RNA polymerase in phage N4 or some minor pilot proteins in other phages), as well as building blocks (chaperones, proteases).

[0355] The last two categories (DNA packaging and structural genes) are deeply connected because the packaging machinery recognizes the preassembled head and the DNA to be packaged, initiating and terminating DNA packaging.

[0356] We hypothesized that by extracting and distinguishing all the modules defined above, a system could in principle be constructed that contains all the excision / insertion, replication and regulatory elements from one phage and encodes the packaging / structural elements from another phage, since in principle they could be viewed as independent genetic modules.

[0357] In this example, "structural elements" are referred to for proteins required for DNA packaging and structural proteins required to assemble mature virions.

[0358] Such a system can be highly advantageous for a variety of approaches for the following reasons: - Structural modules from a phage that are not easily amplified or induced can be transferred to another phage (i.e., a prophage with an unknown inducer; a prophage found in a strain with a PICI / SaPI system; a phage with an unknown host, etc.); - particles whose structural genes originate from another species can be produced using species that are more suitable / safer for laboratory research / large-scale production; - Pure phagemid producer strains can be constructed using regulatory elements of well-characterized phages (eg lambda) etc. that drive the production of capsids of different phages.

[0359] This is the approach developed in this study. Using a production strain encoding a system for generating pure lambda phagemids, its structural operon was exchanged with that of a prophage derived from a different species (Klebsiella pneumoniae) (approximately 23 kb, from the small terminase to the STF gene). A diagram shows the changes made for the Klebsiella pneumoniae hybrid variant (Figures 1-3).

[0360] In this system, a thermolabile version of the prophage lambda contains all the regulatory elements required to excise the prophage, replicate the circularized excised genome, and drive expression of a long late operon, including the presence of antitermination protein Q. This, when supplemented with a plasmid containing the correct packaging signal (a cos site for Klebsiella pneumoniae phage), should drive the assembly and packaging of pure phagemid particles based entirely on other phages.

[0361] Analysis of Klebsiella pneumoniae prophage Kpne strain LMR_3612 (s17699) was analyzed using PHAST to extract prophage regions. One of them contains a predicted lambdoid prophage with some similarity to HK97. This prophage is called kappa. Of note, a similar Klebsiella oxytoca prophage has been described in the literature and is designated phiKO2 (Casjens et al., J Bacteriol. 2004 Mar;186(6):1818-32). The structural operon was found to span a continuous region encoding terminase, structural genes (capsid, tail, fiber, etc.), assembly protease, and chaperones. This region is approximately 30 kb long (SEQ ID NO: 7).

[0362] Building a Hybrid Starting with a production strain (s1965) containing a lambda prophage without a cos site, the lambda Red recombineering system was used to exchange the lambda prophage structural operon for a kappa one. Note that some codons were recoded to be more efficiently recognized by E. coli.

[0363] Production and Titration The resulting strain, containing the completely exchanged structural operon, was designated kappa. Next, a payload suitable for packaging by this phagemid was constructed. To do this, a 350-bp sequence (SEQ ID NO: 8) immediately upstream of the small terminase gene of the kappa prophage, a candidate for encoding the cos signal recognized by the kappa terminase, was inserted into the payload containing the chloramphenicol marker and sfGFP gene. This payload was designated pTEST (p1866, SEQ ID NO: 9). Note that some restriction sites found in the Klebsiella pneumoniae REBASE database were removed where possible. Strain kappa harboring the p1866 plasmid was grown overnight in LB + chloramphenicol, and production was performed the following day according to the following protocol.

[0364] The overnight culture was diluted 1:6 in a final volume of LB + 5 mM CaCl2 supplemented with chloramphenicol and grown at 30°C for 30 minutes with shaking. This was followed by a 45-minute heat shock at 42°C. Finally, the culture was grown at 37°C for 3 hours with shaking. After this period, the cells were harvested by centrifugation and lysed using 3 mL of B-PER Protein Extraction Reagent, followed by the addition of 600 mg of detergent removal biobeads and incubation at room temperature for 1 hour with gentle shaking. The lysate was then centrifuged at 10,000 g for 10 minutes, and the supernatant was filtered through a 0.2 micron pore-size membrane.

[0365] Two collections of Klebsiella pneumoniae strains (192 strains in total) belonging to different ST types were used to verify the production of phagemid particles. Overnight cultures of Klebsiella pneumoniae strains were diluted 1:100 in LB + CaCl2, grown for 2 hours at 37°C, and then diluted 1:20 before transduction. Ten microliters of phagemid lysate was added to 90 μL of each Klebsiella pneumoniae dilution and incubated at 37°C for 30 minutes. Finally, 10 μL of each transductant was plated on LB agar supplemented with chloramphenicol and incubated overnight at 37°C. Additionally, production titers were verified using Klebsiella pneumoniae strains F3 (s19091), MG1655 (s003), MG1656-OmpCO157 (s14269), and MG1656-dOmpC-dLamB, which harbor six different Klebsiella pneumoniae OmpC variants in trans.

[0366] In this first assay, no hits were observed in either the 192 Klebsiella pneumoniae strains or any of the other nine strains used for titration (Figures 4-5).

[0367] Following these results, we conducted a more detailed analysis of the kappa prophage to determine whether any structural or packaging elements were missing. Because this prophage appears to belong to the same family as HK97, we performed a literature search to identify any missing elements, identifying that HK97 (and generally many other non-lambda phages) require a small protein containing an HNH nuclease domain that aids in the processivity of cos site cleavage by the terminase complex (Moodley et al., Protein Sci. (2012) 21(6):809-818 and Kala et al., Proceedings of the National Academy of Sciences Apr 2014, 111(16) 6022-6027). Analysis of the kappa prophage region immediately upstream of the terminase gene (and putative cos site) revealed the presence of an operon containing the HNH protein, several other small ORFs of unknown function, and a Zn finger domain-containing protein immediately upstream of the putative cos site.

[0368] To test the hypothesis that the HNH protein is the deleted element in the production strain, this ORF (SEQ ID NO: 10 and SEQ ID NO: 11) was cloned into a plasmid (p1869, SEQ ID NO: 12) under the control of the inducible repressor PhlF and used to complement production. Additionally, in case the cos site present in pTEST was not complete, a second payload containing a larger region upstream of the terminase (SEQ ID NO: 13) was constructed. This second plasmid was designated pTEST-2 (p1867, SEQ ID NO: 14).

[0369] Phagemids were produced as in the initial experiments, but DAPG was added to the production when the culture was shifted to 42° C. to induce expression of the HNH protein. Screening of the collection was performed as described above.

[0370] The results showed that in the presence of the HNH protein, phagemids were readily produced (Figure 6). In this case, titration in a single MG1655 or Klebsiella pneumoniae F3 strain revealed that the phagemids were specific for Klebsiella and did not recognize E. coli at all. This demonstrates that the phagemids produced are structurally based on kappa but are regulated and maintained in a lysogenic state by the lambda prophage.

[0371] Similarly, when titrated in both Kpne collections, many hits were observed this time (Figure 7).

[0372] Two observations can be made from this experiment: - The titers are different when produced with pTEST or pTEST-2 payloads, both carrying the HNH protein in trans (estimated values ​​are 5 x 10 for pTEST). 5 TU / mL and 5 × 10 for pTEST-2 6 TU / mL). - Even at low titers, the spots in the Klebsiella pneumoniae collections were dense, indicating that delivery efficiency must be high.

[0373] Regarding the difference in titers for the tested payloads, the only change between both production runs is that the putative cos site present in pTEST-2 is longer than in pTEST; pTEST-2 also encodes a small ORF (part of the longer cos region) with two predicted Zn fingers (SEQ ID NO:15 and SEQ ID NO:16). If this protein is involved in packaging, this may explain why titers are higher in lysates containing pTEST-2 than in lysates containing pTEST, rather than due to length per se. For this reason, a third system was constructed, designated pTEST-3 (p1868, SEQ ID NO:18), in which a payload encoding a short cos site (to avoid encoding any protein in that region) (SEQ ID NO:17) was used. To complement the putative packaging proteins in trans, a so-called "HNH-operon" plasmid was constructed (p1872, SEQ ID NO:19), encoding the HNH protein (SEQ ID NO:10 and SEQ ID NO:11) and a putative Zn finger-containing protein (SEQ ID NO:15 and SEQ ID NO:16). The production was carried out as described above.

[0374] In this third case, a similar behavior as for pTEST-2 was observed, with many hits in the Kpne collection, successful production of phagemids and approximately 5×10 6 Titers in TU / mL were confirmed (Figure 8). Titers were obtained by analyzing dense spots on Klebsiella pneumoniae plates and titrating lysates using these strains. These results indicate that the minimal cos site is encoded in a shorter region than in pTEST and pTEST-2, and that the HNH-operon machinery is necessary and enhances the packaging reaction.

[0375] These results demonstrate that the E. coli production strain is capable of producing pure Klebsiella-specific phagemid particles. The specificity does not derive solely from the addition of a single structural element, such as the tail fiber. The complete protein composition of the produced phagemid is of K. pneumoniae origin, demonstrating that phagemid particles for other species can be assembled using E. coli as a production strain.

[0376] Example 2 Production of Cutibacterium acnes phage-derived particles Cutibacterium acnes is one of the most widespread and abundant species of skin (Kashaf et al., Nat Microbiol 7, pp. 169-179 (2022)), where it resides in the pilosebaceous system (PSU). Unlike on the stratum corneum, bacteria present in the PSU are surrounded by living cells, particularly keratinocytes, sebocytes, and different immune cells (Kabashima et al., Nat Rev Immunol 19, pp. 19-30 (2019)). Close contact between C. acnes and these cells can lead to beneficial or harmful interactions (Bruggemann et al., Front Microbiol 12, 673845 (2021)). Genetically modifying C. acnes was extremely challenging prior to the applicant's new tools disclosed in U.S. Patent Application Publication Nos. 2022 / 135986 and 2022 / 135987. In these patent applications, the inventors described for the first time the production of C. acnes phage-derived particles using C. acnes as the production strain.

[0377] In this example, we used a P. freudenreichii strain to produce C. acnes phage-derived particles by exchanging structural genes from the P. freudenreichii prophage with structural genes from the C. acnes phage.

[0378] result Isolation of BW4 phage P. freudenreichii and associated bacteriophages are known to be present in some dairy products (Gautier et al., (1995) Lait 75:427-434; Gautier et al., (1995) Appl. Environ. Microbiol. 61:2572-2576; Cheng et al., (2018) BMC Microbiology 18:19). We therefore screened for the presence of both Propionibacterium phage or P. freudenreichii lysogens in cheese samples.

[0379] Different types of cheese samples were crushed, resuspended in Reinforced Clostridial Medium, and incubated at 30°C for 2 days under anaerobic conditions. After incubation, a dilution of the culture was made in Lithium Glycerol Broth (WO 1994017201), a medium selective for Propionibacterium, and incubated at 30°C for 6 days. The final dilution in RCM + Mitomycin C was incubated at 30°C for 1 day to induce potential prophage. The induced cultures were filtered (0.2 μm) and spotted onto different indicator strains. One of the samples led to the formation of cloudy plaques on top agar of P. freudenreichii strain Pf0s2841. Three individual plaques were isolated by two consecutive pickings and streaking on Pf0s2841, and amplification was performed on top agar of Pf0s2841. For the three different plaques, amplification was approximately 10 10 This resulted in a phage suspension of 100 PFU / mL.

[0380] Two clusters of lysogenic dsDNA P. freudenreichii phages (BW and BV) have been previously identified (Cheng et al., (2018) BMC Microbiology 18:19). Two distinct fragments were extracted using PCR designed on the BW genome (KX620751) from Doucette phage: ORF3 containing AD1334 (SEQ ID NO: 20) / AD1335 (SEQ ID NO: 21) - ORF5 with AD1336 (SEQ ID NO: 22) / AD1337 (SEQ ID NO: 23). We were able to classify the isolated phages as BW-like (Figure 9). Sequencing of ORF5 revealed that all phages were almost certainly identical and therefore derived from the same BW-like phage, designated BW4.

[0381] Isolation of Pf0s2841 lysogens harboring BW4 phage We next isolated P. freudenreichii lysogens carrying the BW4 phage as a prophage. To do so, a BW4 phage suspension was spotted onto strain Pf0s2841 and incubated for three days. Turbid plaques were picked, resuspended, and streaked. After five days, single colonies were obtained, and several colonies were streaked. A second and third incubation was performed, and the presence of the phage gene was checked in each streak by PCR spanning the cohesive ends (AD1322 (SEQ ID NO: 24) / AD1323 (SEQ ID NO: 25)) after DNAse treatment to ensure the presence of the phage but not phage particles.

[0382] After the third streak, colonies were grown on top agar, and spots of undiluted BW-like phage suspension were spotted onto the putative lysogen strain (Pf0s14253) and the ancestral strain (Pf0s2841). After incubation, clearance was observed for both strains for the BW13 and BW14 spots, whereas clearance was observed only for Pf0s2841 for the BW4 spot (Figure 10). This indicates that strain Pf0s14253 is immune to BW4 phage superinfection and harbors the BW4 prophage. The absence of immunity for BW14 and BW13 indicates that these phages likely possess different immune repressors.

[0383] Induction of BW4 prophage To use the BW4 lysogenic strain as a producer strain for phage-derived particles, we first needed to test its ability to produce high concentrations of BW4 phage upon induction of the lytic cycle. To do so, Pf0s14253 was grown in the absence or presence of mitomycin C (MMC), an antibiotic known to induce prophage, and culture supernatants were titrated for the presence of BW4 phage particles in the indicator strain Pf0s2841. Higher amounts of BW4 phage particles were observed in conditions supplemented with mitomycin C (Figure 11), compared with 3.0 x 10 for conditions without mitomycin C. 3 7.4 × 10 for PFU / μL 7 PFU / μL, indicating a high dynamic range between the lytic and lysogenic cycles for the BW4 prophage under such conditions and confirming the potential of BW4 for the production of phage-derived particles.

[0384] Sequencing and annotation of BW4 phage To engineer the BW4 prophage for the production of C. acnes phage-derived particles, we sequenced the BW4 phage. DNA isolation (Promega Wizard DNA Clean-Up System) followed by Illumina sequencing was performed on the BW4 phage suspension. Raw reads were assembled into a single contig using Spades, and ends were corrected by Sanger sequencing (SEQ ID NO: 26). Annotation was performed using Phaster and manually curated based on homology to other BW-like phages (Cheng et al. (2018) BMC Microbiology 18:19).

[0385] As described in Cheng et al. (2018) BMC Microbiology 18:19, BW-like phages have a typical genome structure of other temperate phages, with a large putative structural operon (also called the lytic operon) organized into distinct functional modules, with, in transcriptional order, packaging, head, tail, and lysis modules. Surprisingly, the first gene of the putative operon (gp1) appears to be related to DNA replication based on HHpred, as it contains domains similar to bifunctional primase and polymerase proteins. Other parts of the BW4 phage genome contain genes required for prophage integration / excision, DNA replication, DNA recombination, regulation of the lytic / lysogenic cycle, and other accessory proteins. This modular structure supports the possibility of exchanging the genes required for BW4 phage capsid production and phage genome packaging with their equivalents from the C. acnes phage genome.

[0386] Isolation of C. acnes PAC7 phage C. acnes phages were isolated from the skin of healthy volunteers. Briefly, comedones were extracted using a patch (Biore) applied to the nose, resuspended in RCM, plated on MRS, and incubated at 37°C under anaerobic conditions. Plaques could be observed in some of the plates among the dense lawn of C. acnes. Potential phages were resuspended in DPBS (Dulbecco's Phosphate-Buffered Saline) and filtered to remove bacteria. The phage suspension was streaked onto the plate, and top agar containing strain Caos2345 was added. The plate was incubated for 2 days, and plaques were reisolated by three consecutive rounds of picking, streaking, and top agar plating. Finally, plaques were amplified on top agar containing strain Caos2345, and the resulting phage suspension was PEG-precipitated. High titers (>10) were observed when titrated on Caos2345. 6 A phage suspension of 1000 PFU / μL was obtained.

[0387] Sequencing and annotation of PAC7 phage DNA isolation (Promega Wizard DNA Clean-Up System) followed by Illumina sequencing was performed on the PAC7 phage suspension. Raw reads were assembled into a single contig using Spades, and ends were corrected by Sanger sequencing (SEQ ID NO: 27). Annotation was performed using Phaster and manually curated based on homology with other C. acnes phages (Marinelli et al., (2012) mBio 3:e00279-12). Similar to P. freudenreichii BW4 phage, a structural operon was identified containing modules for packaging, head and tail assembly, and cell lysis (Figure 12). The HNH endonuclease was identified as the last gene (gp45) of the phage. Such endonucleases have previously been shown to be essential for efficient packaging (Quiles-Puchalt et al., (2014) Proc Nat. Acad. Sci. 111:6016-6021).

[0388] Construction of a lysogenic strain carrying a chimeric BW4-PAC7 prophage The genes in the structural operon of the BW4 prophage, from the small terminase gp2 to the tape measure protein gp16, were replaced with the structural PAC7 genes gp1 to gp14 (Figure 13). This was achieved by homologous recombination using the P. freudenreichii suicide vector, plasmid pAN514 (SEQ ID NO: 28), cloned into E. coli DH10B. After transformation with the vector, a double crossover event was selected in P. freudenreichii (Pf1s22499) by selection on chloramphenicol. The integrity of the chimeric BW4-PAC7 structural operon was globally confirmed by PCR and Sanger sequencing of the entire chimeric structural operon.

[0389] Production and titration of PAC7-derived particles from a lysogenic strain harboring a chimeric BW4-PAC7 prophage To produce C. acnes phage-derived particles from P. freudenreichii BW4-PAC7 chimeric lysogens, Pf1s22903 was transformed with the pAN594 cosmid (FIG. 14) (disclosed in U.S. Patent Application Publication Nos. 2022 / 135986 and 2022 / 135987), which contains the packaging signal of the PAC7 phage (SEQ ID NO: 29), the five genes of the PAC7 tail module (gp15 to gp19) and the operon expressing the gp45 endonuclease (SEQ ID NO: 30), and origins of replication functional in P. freudenreichii and C. acnes. Transformants were streaked and grown in the presence of both chloramphenicol (1 μg / mL) to select for the presence of the prophage and erythromycin (2.5 μg / mL) to select for the presence of pAN594. An OD of approximately 0.4 was obtained. 600nm In the present study, cultures were supplemented with 0.5 μg / ml mitomycin C and grown overnight in anaerobic conditions at 30° C. After incubation, cells were harvested by centrifugation and lysed by bead-beating (2 × 20 min at 30 Hz with 0.1 mm glass beads), the supernatant was filtered, and the presence of phage-derived particles was titrated in C. acnes Ca0s2258.

[0390] Up to approximately 10 per μL 2 Potential transformants were obtained (Figure 15). Eight colonies were streaked onto Brain Heart Infusion (BHI) erythromycin (5 μg / mL), and PCR was used to confirm the presence of C. acnes and transformants harboring pAN594 (Figure 16).

[0391] We therefore demonstrated for the first time that C. acnes phage-derived particles capable of delivering DNA into Cutibacterium acnes can be produced by exchanging the structural genes of P. freudenreichii prophage with those of Cutibacterium acnes phage.

[0392] Materials and Methods: Strains used and generated

[0393] [Table 2]

[0394] Culture conditions All incubations of P. freudenreichii strains were carried out under anaerobic conditions at 30°C (Thermo Scientific™ Sachet Oxoid™ AnaeroGen).

[0395] All incubations of C. acnes strains were carried out in an anaerobic chamber at 37°C.

[0396] Construction of strain Pf1s22499 Deletion of the packaging signal from the BW4 prophage was achieved by homologous recombination and CRISPR-Cas selection of recombinants using the pAN241 P. freudenreichii vector, which was cloned into E. coli and then transformed into strain Pf0s14253. The pAN241 vector contains a template for homologous recombination (SEQ ID NO: 31) and an FnCpf1 transcription cassette with a crRNA targeting the cos of the BW4 prophage.

[0397] Transformation Protocol for P. freudenreichii Transformation of P. freudenreichii was adapted from Brede, DA et al., Appl Environ Microb 71, 8077-8084 (2005), substituting SLB (sodium lactate broth) medium for BHI.

[0398] Titration of phage-derived particles Strain Ca0s2258 was streaked onto BHI agar plates. Once dense growth was obtained on the plates, the liquid culture was placed in BHI. After overnight incubation, the turbid culture was concentrated 10-fold in BHI. 90 μl of cells were mixed with 10 μL of pure, 1 / 10-diluted, and 1 / 100-diluted solutions of phage-derived particles produced from Pf1s22904 or Pf1s22903 as a negative control. The samples were incubated at room temperature for 2 hours, followed by 1 / 10 serial dilutions in BHI. The samples were incubated at 37°C under anaerobic conditions for 2 hours before spotting 4 μL onto BHI + 5 μg / mL erythromycin. The plates were incubated at 37°C under anaerobic conditions for 7 days.

Claims

1. A producer bacterial cell for producing a phage particle or a phage derived delivery vehicle, said producer bacterial cell stably comprising at least one phage structural gene and at least one phage DNA packaging gene, said phage structural gene and phage DNA packaging gene being derived from a first type of bacteriophage; expression of at least one of the phage structural genes and / or at least one of the phage DNA packaging genes in the producer bacterial cell is controlled by at least one inducible mechanism; A production bacterial cell, wherein the production bacterial cell is from a bacterial species or strain different from the bacterial species or strain from which the first type of bacteriophage is derived and / or which the first type of bacteriophage is targeted.

2. 2. The production bacterial cell of claim 1, further comprising a payload packaged in the phage particle or phage-derived delivery vehicle.

3. 3. The production bacterial cell of claim 2, wherein the payload is a nucleic acid payload comprising a packaging site derived from the first type of bacteriophage.

4. The production bacterial cell of claim 2, wherein the payload is delivered into a targeted bacterial cell.

5. The production bacterial cell of claim 4, wherein the payload is stably maintained in the targeted bacterial cell.

6. The production bacterial cell of claim 4, wherein the payload does not replicate in the targeted bacterial cell.

7. 5. The bacterial production cell of claim 4, wherein the payload comprises a sequence of interest.

8. 8. The production bacterial cell of claim 7, wherein the sequence of interest produces an effect only in the targeted bacterial cell.

9. 9. The production bacterial cell of claim 8, wherein the targeted bacterial cell is from a different species or strain than the production bacterial cell.

10. 2. The producer bacterial cell of claim 1, wherein the same induction mechanism controls expression of at least one of the phage structural genes and at least one of the phage DNA packaging genes.

11. 2. The producer bacterial cell of claim 1, wherein expression of at least one of the phage structural genes and at least one of the phage DNA packaging genes are controlled by different induction mechanisms.

12. 2. The bacterial producer cell of claim 1, wherein at least one induction mechanism controls expression of all of the phage structural genes.

13. 2. The production bacterial cell of claim 1, wherein at least one inducible mechanism controls expression of all of the phage DNA packaging genes.

14. 2. The producer bacterial cell of claim 1, wherein the at least one inducible mechanism further controls the copy number of the at least one of the phage structural genes and / or the at least one of the phage DNA packaging genes.

15. The bacterial producer cell of claim 2, wherein the at least one inducible mechanism further controls the copy number of the payload in the bacterial producer cell.

16. The bacterial producer cell of claim 2, wherein another inducible mechanism controls the copy number of the payload in the bacterial producer cell.

17. 2. The producer bacterial cell of claim 1, wherein the phage structural genes and phage DNA packaging genes from the first type bacteriophage are contained in at least one plasmid, chromosome, and / or helper phage.

18. the production bacterial cell further comprises at least one gene from a second type of bacteriophage involved in phage excision / insertion, phage DNA replication, and / or phage regulation; 2. The production bacterial cell of claim 1, wherein the second type of bacteriophage originates from and / or targets a bacterial species or strain different from the bacterial species or strain from which the first type of bacteriophage originates and / or targets.

19. 19. The production bacterial cell of claim 18, which is from the same bacterial species or strain as the second type of bacteriophage is derived from and / or targets.

20. The production bacterial cell of claim 1 , wherein the production bacterial cell is an E. coli bacterial cell.

21. 2. The production bacterial cell of claim 1, wherein the production bacterial cell is a P. freudenreichii bacterial cell.

22. 22. The production bacterial cell of claim 21 , wherein the bacterial species or strain from which the first type of bacteriophage is derived and / or which the first type of bacteriophage targets is C. acnes.

23. 1. A method for producing a phage particle or a phage derived delivery vehicle, comprising: (a) providing a bacterial production cell according to any one of claims 1 to 22; and (b) inducing in said producer bacterial cell the expression of said at least one of said phage structural genes and said at least one of said phage DNA packaging genes and assembly of products expressed by said at least one phage structural gene and said at least one phage DNA packaging gene, thereby producing a phage particle or a phage derived delivery vehicle. A method comprising:

24. A hybrid helper phage system comprising: (i) at least one phage DNA packaging gene derived from a first type of bacteriophage; (i') at least one phage structural gene derived from a bacteriophage of the first type; and (ii) at least one gene from a second type of bacteriophage involved in phage excision / insertion, phage DNA replication, and / or phage regulation. Including, the genes (i), (i') and (ii) are contained in a single nucleic acid molecule or in separate nucleic acid molecules, the first type of bacteriophage originates from and / or targets a bacterial species or strain different from the bacterial species or strain from which the second type of bacteriophage originates and / or which the second type of bacteriophage targets; A hybrid helper phage system, wherein the hybrid helper phage system does not contain any expressed phage structural genes derived from the second type of bacteriophage.

25. 25. The hybrid helper phage system of claim 24, wherein the genes (i), (i') and (ii) are contained in a bacterial chromosome.

26. 25. The hybrid helper phage system of claim 24, wherein genes (i), (i') and (ii) are contained in separate plasmids.

27. The hybrid helper phage system comprises: (i) at least one phage DNA packaging gene and at least one phage structural gene derived from a first type of bacteriophage; and (ii) at least one gene from a second type of bacteriophage involved in phage excision / insertion, phage DNA replication, and / or phage regulation. a hybrid helper phage comprising the first type of bacteriophage originates from and / or targets a bacterial species or strain different from the bacterial species or strain from which the second type of bacteriophage originates and / or which the second type of bacteriophage targets; 25. The hybrid helper phage system of claim 24, wherein the hybrid helper phage does not contain any expressed phage structural genes derived from the second type of bacteriophage.

28. A production bacterial cell for producing a teilosin and / or a pyocin, said production bacterial cell stably containing at least one teilosin and / or pyocin structural gene from a bacterial species or strain containing the teilosin and / or pyocin genes, expression of at least one of the teilosin and / or pyocin structural genes in the producing bacterial cell is controlled by at least one inducible mechanism; A producing bacterial cell, wherein the producing bacterial cell is from a bacterial species or strain different from the bacterial species or strain containing the teilosin and / or pyocin genes from which the teilosin and / or pyocin structural genes are derived.

29. 29. The production bacterial cell of claim 28, wherein the teilosin and / or pyocin are intended to lyse a targeted bacterial cell.

30. 30. The production bacterial cell of claim 29, wherein the targeted bacterial cell is from a different species or strain than the production bacterial cell.

31. 30. The production bacterial cell of claim 28, wherein at least one induction mechanism controls expression of all of the teilosin and / or pyocin structural genes.

32. 30. The production bacterial cell of claim 28, wherein the at least one inducible mechanism further controls the copy number of the at least one of the teilosin and / or pyocin structural genes.

33. 29. The production bacterial cell of claim 28, wherein the teilosin and / or pyocin structural genes are contained in at least one plasmid, chromosome, and / or helper phage.

34. the production bacterial cell further comprises at least one gene derived from a bacteriophage involved in phage excision / insertion, phage DNA replication, and / or phage regulation; 30. The production bacterial cell of claim 28, wherein the bacteriophage is derived from and / or targets a bacterial species or strain different from the bacterial species or strain from which the teilosin and / or pyocin structural genes are derived.

35. 35. The production bacterial cell of claim 34, wherein the production bacterial cell is from the same bacterial species or strain from which the bacteriophage is derived and / or which the bacteriophage targets.

36. 30. The production bacterial cell of claim 28, wherein the production bacterial cell is an E. coli bacterial cell.

37. 1. A method for producing a teilosin and / or a pyocin, comprising: (a) providing a bacterial production cell according to any one of claims 28 to 36; and (b) inducing in the producing bacterial cell the expression of the at least one of the teilosin and / or pyocin structural genes and the assembly of a product expressed by the at least one teilosin and / or pyocin structural gene, thereby producing a teilosin and / or pyocin. A method comprising:

38. 1. A hybrid production system comprising: (i') at least one teilosin and / or pyocin structural gene from a bacterial species or strain containing a teilosin and / or pyocin gene; and (ii) at least one gene from a bacteriophage involved in phage excision / insertion, phage DNA replication, and / or phage regulation, or at least one gene involved in teilosin and / or pyocin regulation, said gene involved in teilosin and / or pyocin regulation being from a bacterial species or strain containing teilosin and / or pyocin genes different from those from which the teilosin and / or pyocin structural genes are derived. Including, the genes (i') and (ii) are contained in a single nucleic acid molecule or in separate nucleic acid molecules, the bacteriophage originates from and / or targets a bacterial species or strain different from that from which the teilosin and / or pyocin structural genes are derived, A hybrid helper production system, wherein the hybrid helper production system does not contain any expressed phage structural genes derived from the bacteriophage.

39. 39. The hybrid production system of claim 38, wherein genes (i') and (ii) are contained in a bacterial chromosome.

40. 39. The hybrid production system of claim 38, wherein genes (i') and (ii) are contained in separate plasmids.

41. The hybrid production system comprises: (i') at least one teilosin and / or pyocin structural gene from a bacterial species or strain containing a teilosin and / or pyocin gene; and (ii) at least one gene derived from a bacteriophage involved in phage excision / insertion, phage DNA replication, and / or phage regulation; a hybrid helper phage comprising the bacteriophage originates from and / or targets a bacterial species or strain different from that from which the teilosin and / or pyocin structural genes are derived, 39. The hybrid production system of claim 38, wherein the hybrid helper phage does not contain any expressed phage structural genes derived from the bacteriophage.

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