Patent Application
20220162270
The invention relates to the production of transduction particles, such as phage, as well as compositions comprising the particles and use of these. The particles are particularly useful for delivering toxic payloads into target bacteria for antibacterial action. The invention is also useful for the production of wild-type (“native”) phage and phage cocktails comprising different types of phage. Embodiments enable production of compositions of such particles for use in medical, environmental or food production settings.
The use of helper phage to package phagemid DNA into phage virus particles is known. An example is the M13KO7 helper phage, a derivative of M13, used in E coli host cells. Other examples are R408 and CM13.
Bacteriophages (phages) are a phylum of viruses that infect bacteria and are distinct from the animal and plant viruses. Phages can have either a “lytic” life cycle, a “lysogenic” life cycle that can potentially become lytic, or a “non-lytic” life cycle. Phages replicating through the lytic cycle cause lysis of the host target bacterial cell as a normal part of their life cycles. Phages replicating through the lysogenic cycles are called temperate phages. These can either replicate by means of the lytic life cycle and cause lysis of the host bacterium, or they can incorporate their DNA into the host bacterial DNA and become noninfectious prophages. Bacteriophages are bacterial viruses that only infect and multiply within their specific bacterial hosts. Host specificity is generally found at strain level, species level, or, more rarely, at genus level. This specificity allows for directed targeting of dangerous bacteria using phages. The adsorption of bacteriophages onto host cells is, in all but a few rare cases, a sine qua non condition for the onset of the infection process.
The natural capability of phages to infect and kill bacteria, together with the specificity of the phage-bacterial interactions, is the basic phenomena on which the concept of phage therapy is built. Therefore, phages that possess lytic life cycle are suitable candidates for phage therapy. The use of phage in food production has recently become an option for the food industry as a novel method for biocontrol of unwanted pathogens, enhancing the safety of especially fresh and ready-to-eat food products.
PCT/EP2018/082053 and U.S. Ser. No. 15/985,658 disclose the production of non-replicative transduction particles. PCT/EP2018/071454 discloses methods of transduction particle propagation.
International Patent Application No. WO 00/69269 discloses the use of certain phage strain for treating infections caused by Vancomycin-sensitive as well as resistant strains of Enterococcus faecium, and International Patent Application No. WO 01/93904 discloses the use of bacteriophage, alone or in combination with other anti-microbial means, for preventing or treating gastrointestinal diseases associated with the species of the genus Clostridium.
US Patent Application No. 2001/0026795 describes methods for producing bacteriophage modified to delay inactivation by the host defense system, and thus increasing the time period in which the phage is active in killing the bacteria.
US Patent Application No. 2002/0001590 discloses the use of phage therapy against multi-drug resistant bacteria, specifically methicillin-resistant Staphylococcus aureus, and International Patent Application No. WO 02/07742 discloses the development of bacteriophage having multiple host range.
The use of phage therapy for the treatment of specific bacterial-infectious disease is disclosed, for example, in US Patent Application Nos. 2002/0044922; 2002/0058027 and International Patent Application No. WO 01/93904.
US20160333348 describes the use of CRISPR/Cas systems delivered to host target bacterial cells using phage as vectors.
The commercial scale production of bacteriophage compositions for therapeutic use is still limited.
In current techniques, the titer of the phage composition is low, usually in the range of 109-1011 pfu/ml on a laboratory scale, and 107-109 on a commercial scale, whereas the titer typically required for phage therapy is 1012 pfu/ml. Additionally, to reach the desirable titer, very large volumes of liquid are required.
As transduction particle (eg, bacteriophage) use in industrial and medical settings grows there is a need for commercial quantities of such particles. Therefore, there is a need for a method for production of transduction particles that provides good yield titer and/or reduces manufacturing volume.
The invention provides:—
In a First Configuration
A method of producing transduction particles wherein the particles are capable of recognising a receptor on bacterial target cells for transduction of the cells, the method comprising producing the particles in bacterial producer cells, wherein the producer cells do not express the receptor on their surface.
In a Second Configuration
Use of the producer cells, for enhancing the production yield of transduction particles.
In a Third Configuration
A composition (optionally a pharmaceutical composition) comprising transduction particles obtained or obtainable by the method or use of the invention.
In a Fourth Configuration
A method of killing bacterial target cells, the method comprising contacting the cells with a composition according to the invention, wherein transduction particles infect the cells and introduce therein a nucleic acid or nucleotide sequence of interest (NSI), wherein the NSI comprises or encodes an antibacterial agent that kills the target cells, or wherein the NSI comprises or encodes a component of such an agent.
In a Fifth Configuration
A composition according to the invention for use in a method of treating or preventing a disease or condition in a human or animal subject, wherein the disease or condition is mediated by bacterial target cells, the method comprising administering the composition to the subject and contacting the target cells with a composition, whereby target cells are killed or the growth or proliferation of target cells is inhibited, thereby treating or preventing the disease or condition.
In a Sixth Configuration
A plurality of transduction particles obtainable by the method.
“Transduction particles” may be phage or smaller than phage and are particles that are capable of transducing nucleic acid (eg, encoding an antibiotic or component thereof) into target cells. Examples of transduction particles are phage or particles comprising a phage capsid. In an example, each particle is a replication-defective phage particle. In an example, each particle is a particle produced from a genomic island (eg, a pathogenicity island such as a SaPI) or a modified version thereof.
The invention relates to the production of transduction particles, such as phage, as well as compositions comprising the particles and use of these. The particles are particularly useful for delivering toxic payloads into target bacteria for antibacterial action. The invention is also useful for the production of wild-type (“native”) phage and phage cocktails comprising different types of phage. Embodiments enable production of compositions of such particles for use in medical, environmental or food production settings. Transduction particles, eg, phage, can be used in compositions, such as medicaments, herbicides and other agents where such particles may usefully be used. Thus, the invention provides the following Clauses and embodiments.
The particles can be capable of attaching to the receptor when surface expressed on target cells. In an example, the particles attach to the receptor on target cells and transduce DNA into the target cells. In an embodiment, the DNA comprises a NSI. In an example, the particles are phage-like or are phage. For example, the particles comprise a phage capsid in which the DNA is packaged.
In an example, the producer and target cells are bacterial cells. In another example, they are archaeal cells.
The producer cells are different from the target cells. At least, they differ in that the former do not (or substantially do not) surface express the receptor and the latter do.
Optionally, the producer and target cells are cell of the same strain, with the exception that the target cells surface express the receptor.
In an example the cells are E coli Nissle or K-12 (eg, K-12 MG1655) cells.
In an example, each producer cell comprises a nucleic acid comprising a nucleic acid sequence of interest (NSI), a phage packaging sequence (eg, pac or cos or a homologue thereof) and an origin of replication. In an embodiment, nucleic acid is a phagemid of plasmid. The nucleic acid is packaged in the producer cell to produce the particles of the invention. In an embodiment, the producer cell comprises the genome of a helper virus or phage which is used to provide essential functions required for packaging and/or replicating the nucleic acid in the producer cell. The skilled person will be familiar with helper viruses and helper phage.
In an example, each producer cell comprises (i) a helper phage genome; and (ii) a nucleic acid comprising a nucleic acid sequence of interest (NSI), a phage packaging sequence (eg, pac or cos), an origin of replication and optionally one or more nucleotide sequences each encoding a helper phage transactivation factor for activating the helper phage. In an embodiment, component (i) is comprised by the nucleic acid (ii). In an embodiment, nucleic acid (ii) is a phagemid of plasmid.
In an embodiment, the NSI encodes a protein of interest (POI), eg, an enzyme, a nuclease, an antibiotic, an antigen binding site, a marker (eg, an antibiotic marker or fluorescence marker) or a medicament or component thereof.
In an example, the NSI is comprised by a phagemid. In an example, the NSI is comprised by a plasmid.
Optionally, the guide comprises a spacer sequence that is capable of hybridising to a protospacer sequence comprised by target cells. In one embodiment, the target cells comprise endogenously active Cas (eg, Cas 3 or Cas9) that is operable with the guide RNA in the target cells to guide the Cas to the cognate protospacer sequence for modification (eg, cutting) of the sequence, and optionally killing of the target cell. In another embodiment, the Cas is instead or additionally encoded by DNA transduced into the target cell by the particle of the invention.
In an example, each producer cell is a rfa mutant, eg, wherein the producer cell is an E coli cell. In an embodiment, the rfa is rfaC. In an embodiment, the rfa is rfaD. In an embodiment, the rfa is rfaF. In an embodiment, the rfa is rfaG. In an embodiment, the rfa is rfaI. In an embodiment, the rfa is rfa J. See
Optionally there is a disruption of a rfa gene in the genome (eg, chromosome) of the producer cell, eg, rfaC, rfaD, rfaE, rfaF, rfaG, rfaP, rfaQ, rfaY, rfaB, rfaI or rfaJ. Optionally, the disruption is a disruption (eg, deletion) of rfaD and/or rfaE. Optionally, the producer cell here is an E coli cell. The disruption may be a knock-out of the gene, insertion of a heterologous nucleotide sequence in the gene, one or mutations (eg, deletions, substitutions or additions, or any combination in the gene) that renders the gene non-functional for production of the receptor, a component of the receptor, or a gene product that is essential for receptor production in the producer cell. Standard molecular biology techniques for effecting this will be apparent to the skilled person.
In an example, the receptor is any receptor mentioned in Tables 1-3 or otherwise mentioned herein. In an example, the particles comprise P2 phage capsids and the receptor is a P2 receptor.
Optionally the composition comprises an antibiotic that kills or is toxic to the target cells.
Optionally, any method or use of the composition, population or particles of the invention is a method or use in vitro or ex vivo. Optionally, the method or use is not performed in or on a human or animal. Optionally, the method or use is performed in or on a human or animal tissue, cell or serum sample in vitro.
Examples of receptors for use in the present invention are discussed below.
Proteinaceous receptors are mainly outer membrane proteins; sugar moieties include those that compose the cell wall, pellicles, teichoic and LTA. The receptor of the invention is, for, example selected from any of these.
Bacteriophage adsorption initiates the infection process. Through a series of interactions between binding proteins of the bacteriophage (phage) and receptors on the bacterial cell surface, the virus recognizes a potentially sensitive host and then positions itself for DNA ejection. Phage adsorption is thus not only a crucial step in the infection process, but also represents the initial point of contact between virus and host and dictates host range specificity.
Bacteriophage adsorption generally consists of three steps: initial contact, reversible binding and irreversible attachment (Duckworth 1987). The first step involves random collisions between phage and host caused by Brownian motion, dispersion, diffusion or flow (Kokjohn and Miller 1992). In the reversible step, binding to bacterial surface components is not definitive and the phage can desorb from the host. This process, firstly identified by Garen and Puck (1951) through experimental observations of phage detachment after elution, may serve to keep the phage close to the cell surface as it searches for a specific receptor (Kokjohn and Miller 1992). The specific connection between bacterial receptor and phage-binding domains is sometimes mediated by an enzymatic cleavage. This step triggers conformational rearrangements in other phage molecules that allow the insertion of the genetic material into the host (for further details on the mechanism of phage genome ejection, see the review by Molineux and Panja (2013)).
Numerous review studies have highlighted the extensive range of host-associated receptors (proteins, sugars and cell surface structures) that bacteriophages target during adsorption (Lindberg 1977; Schwartz 1980; Wright, McConnell and Kanegasaki 1980; Heller 1992; Frost 1993; Henning and Hashemolhosseini 1994; Vinga et al. 2006; Rakhuba et al. 2010; Chaturongakul and Ounjai 2014). The nature and location of the host cell receptors recognised by bacteriophages varies greatly depending on the phage and host. They range from peptide sequences to polysaccharide moieties. In fact, bacteriophages have been shown to bind to receptors located in the walls of both Gram-positive (Xia et al. 2011) and Gram-negative bacteria (Marti et al. 2013), in bacterial capsules or slime layers (Fehmel et al. 1975), and in appendages [e.g. pili (Guerrero-Ferreira et al. 2011) and flagella (Shin et al. 2012)]. This diversity in receptors and structures involved is a testament to the multiplicity of mechanisms developed by phages and hosts to overcome the evolutionary strategies adopted by their counterparts. It is not unexpected to encounter so many possibilities considering the diversity and staggering amount of phages estimated to populate the different environments of the planet (Clokie et al. 2011). Nevertheless, in all cases, adsorption has so far been shown to involve either constituents of the bacterial cell wall or protruding structures. In an embodiment, therefore, a receptor in the present invention can be any such receptor mentioned in this paragraph or elsewhere in this disclosure.
Optionally, the receptor comprises lipopolysaccharide (LPS), a heptose moiety, the host's glucosylated cell wall teichoic acid (WTA), YueB, or a receptor recognized by a tail fiber protein of the phage or gp21 of the phage.
Peptidoglycan, or murein, is an important component of the bacterial cell wall and is often involved in bacteriophage adsorption. It is a polymer composed of multiple units of amino acids and sugar derivatives—N-acetylglucosamine and N-acetylmuramic acid. These sugar constituents are connected through glycosidic bonds, forming glycan tetrapeptide sheets that are joined together through the cross-linking of amino acids. The cross-linking occurs through peptide bonds between diaminopimelic acid (an amino acid analog) and D-alanine, or through short peptide interbridges. These interbridges are more numerous in Gram-positive bacteria, leading to their characteristically thicker cell walls.
Another main component of the cell wall of Gram-positive bacteria that can be involved in phage adsorption is teichoic acid-polysaccharides composed of glycerol phosphate or ribitol phosphate and amino acids. They are bonded to the muramic acid of peptidoglycans. When teichoic acids are bonded to the lipids of the plasma membrane, they are called lipoteichoic acids (LTA). Further details of the composition of cell walls of bacteria can be found in Tortora, Funke and Case (2007), Willey, Sherwood and Woolverton (2008), Pommerville (2010) and Madigan et al. (2012).
The majority of the receptors so far identified are associated either with peptidoglycan or teichoic acid structures (Table 1). Out of 30 phages targeting Gram-positive bacteria reported in Table 1, only 10 utilize other structures for adsorption. Among these 10 phages, 9 display interactions with residues of either teichoic acid (phage SPP1) or peptidoglycan (phages 5, 13, c2, h, m13, kh, L and p2) for reversible binding. This highlights the important role these structures may play in the adsorption of phage to Gram-positive bacteria.
Optionally, the receptor of the invention is peptidoglycan, murein, teichoic acid or lipoteichoic acid (LTA). Optionally, each transduction particle of the invention is a first phage or comprises a capsid of a first phage, wherein the first phage is a phage of a family listed in Table 1 (and optionally the producer and/or target cell is the host for the phage as listed in Table 1 and/or the receptor is the receptor for the phage as listed in Table 1). In an embodiment, the producer and target cells are gram-positive cells. Optionally the producer and/or target cells are of a species or strain listed in Table 1 (where the producer and target cell species are different or the same). Preferably when the producer cell is a gram-positive bacteria, the receptor is a peptidoglycan. Alternatively, when the producer cell is a gram-positive bacteria, the receptor is a teichoic acid.
In Gram-negative bacteria, the peptidoglycan layer is relatively thin and is located inward of the outer membrane, the major component of the cell wall. These two layers are connected by Braun's lipoproteins. The outer membrane is a sophisticated structure composed of a lipid bilayer ornamented with proteins, polysaccharides and lipids; the latter two molecules form the LPS layer. LPSs are complexes that consist of three parts: lipid A, the core polysaccharide and the O-polysaccharide. Lipid A is, in general, composed of fatty acids attached to glucosamine phosphate disaccharides. The core polysaccharide is connected to the lipid A through a ketodeoxyoctonate linker. The core polysaccharide and the O-polysaccharide (O-chain or O-antigen) contain several units of sugar residues extending outward to the outer membrane. Cells that contain all three components of the LPS are denominated as smooth (S) type and those that lack the O-polysaccharide portion are distinguished as rough (R) type. In general, the saccharides composing the O-antigen are highly variable and those of the core polysaccharide are more conserved among species. Because of this, phages specific to only S-type strains tend to target the O-polysaccharide and, thus, have generally a narrower host range when compared to those able to adsorb to R-type cells (Rakhuba et al. 2010).
Table 2(a) compiles Gram-negative bacterial receptors located in the cell wall that interact with phage receptor-binding proteins (RBPs). Interestingly, in coliphages there is no preference for proteinaceous or polysaccharide receptors: some phages adsorb on cell wall proteins, some on sugar moieties and others require both structures for adsorption. In the case of Salmonella phages, the picture is not so different: some use proteins, some sugar moieties and some both types of receptors. On the other hand, Pseudomonas phages commonly adsorb onto polysaccharide receptors. Although definitive conclusions cannot be drawn from such a small sample size, it should be noted that Pseudomonas can have two LPS moieties, a short chain LPS named A band and a longer B-band LPS (Beveridge and Graham 1991).
Optionally, the receptor of the invention is a host cell wall protein. Optionally, the receptor is a saccharide. Optionally, the receptor comprises O-antigen, LPS lipid A or LPS core polysaccharide. In an example, the receptor is smooth LPS or rough LPS. Optionally, the host cells are S-type bacteria and the receptor comprises O-antigen of the host. Optionally, the host cells are R-type bacteria and the receptor comprises LPS lipid A of the host.
Optionally, the receptor is a host cell wall protein. Optionally, the receptor is a saccharide. Optionally, the receptor comprises O-antigen, LPS lipid A or LPS core polysaccharide. In an example, the receptor is smooth LPS or rough LPS. Optionally, the host cells are S-type bacteria and the receptor comprises O-antigen of the host. Optionally, the host cells are R-type bacteria and the receptor comprises LPS lipid A of the host.
In an example, the host is E coli and the transduction particles are coliphage (or comprise coliphage capsids), wherein the receptor is a polysaccharide receptor and/or a host cell wall protein receptor. In an example, the producer cells are engineered not to express E coli polysaccharide receptor and/or an E coli cell wall protein receptor.
In an example, the host is Salmonella, wherein the receptor is a polysaccharide receptor and/or a host cell wall protein receptor. In an example, the producer cells are engineered not to express Salmonella polysaccharide receptor and/or a Salmonella cell wall protein receptor.
In an example, the host is Klebsiella, wherein the receptor is a polysaccharide receptor and/or a host cell wall protein receptor. In an example, the producer cells are engineered not to express Klebsiella polysaccharide receptor and/or a Klebsiella cell wall protein receptor.
In an example, the host is Pseudomonas, wherein the receptor is a polysaccharide receptor. In an example, the second cells are engineered not to express Pseudomonas polysaccharide receptor.
Optionally, each transduction particle of the invention is a first phage or comprises a capsid of a first phage, wherein the first phage is a phage of a family listed in Table 2 (and optionally the producer cell is the host for the phage as listed in Table 2 and/or the receptor is the receptor for the phage as listed in Table 2).
In an embodiment, the producer and target cells are gram-negative cells. Preferably, the producer cells are E coli cells. Optionally the producer and/or target cells are of a species or strain listed in Table 2 (eg, where the cell species are different or the same).
Table 2(b) reports cases where phages not only adsorb onto bacterial surfaces but also enzymatically degrade the sugar moieties in the O-chain structure. It should be noted that all these phages belong to the Podoviridae family.
In this section, bacterial structures, other than cell wall moieties, that also serve as receptors for particles or phages are discussed. These include structures such as flagella, pili and capsules. They can be found in species from both Gram stains. See Table 3 for examples.
Optionally, the receptor of the invention is a flagellum, pilus or capsule component (eg, a component listed in Table 3 in the listed species or as found in a host that is of a different species to that listed). Optionally, the phage is a phage of a family listed in Table 3 (and optionally the host is the host for the phage as listed in Table 3 and/or the receptor is the receptor for the phage as listed in Table 3). Optionally, the phage is a phage listed in Table 3 (and optionally the host is the host for the phage as listed in Table 3 and/or the receptor is the receptor for the phage as listed in Table 3).
Flagella are long thin helical structures that confer motility to cells. They are composed of a basal body, a flagellar hook and a flagellar filament composed of subunits of flagellin proteins (Willey, Sherwood and Woolverton 2008). Table 3(a) reports phages attaching to flagellal proteins. The adhesion of phages to the filament structure is generally reversible and the flagellum's helical movement causes the phage to move along its surface until they reach the bacterial wall. Irreversible adsorption occurs, then, on receptors located on the surface of the bacterium, near the base of the flagellum (Schade, Adler and Ris 1967; Lindberg 1973; Guerrero-Ferreira et al. 2011). Interestingly, some phages (ϕCbK and ϕCb13) were observed to contain filaments protruding from their capsids that are responsible for reversible binding onto the host's flagellum; irreversible adsorption occurs only when the phage's tails interact with pili portals on the cell pole (Guerrero-Ferreira et al. 2011). Because for these phages irreversible adsorption occurs on the pilus, even if they interact with the flagellum, they were reported in Table 3(b), which focuses on phages interacting with receptors in pili and mating pair formation structures.
Pili are rod-shaped filamentous appendages used for bacterial conjugation (Lindberg 1973). They extend from the donor cell and attach to receptors on the wall of the recipient cell. A depolymerization of the pilus causes its retraction, bringing both cells closer to each other. Further adhesion of the cells is achieved through binding proteins on their surfaces; genetic material is transferred through this conjugating junction (Madigan et al. 2012). Adsorption to the pilus structure has been so far associated with phages that belong to orders different from Caudovirales (Table 3b). In fact, according to Frost (1993), the families Cystoviridae and Inoviridae compose the majority of phages that adsorb onto pili structures. Interestingly, phages can be selective towards certain parts of the pili. That is the case for F-type phages, whose adsorption occur only on the tip of the pilus (Click and Webster 1998). In other phages, such as 46, the attachment happens at the sides (shaft) of the structure (Daugelavicius et al. 2005).
Capsules are flexible cementing substances that extend radially from the cell wall. They act as binding agents between bacteria and/or between cells and substrates (Beveridge and Graham 1991). Slime layers are similar to capsules, but are more easily deformed. Both are made of sticky substances released by bacteria, and their common components are polysaccharides or proteins (Madigan et al. 2012). Adsorption of phages to capsules or slime layers is mediated by enzymatic cleavage of the exopolysaccharides that compose the layers. The hydrolysis of the layer is a reversible step, whereas irreversible binding is achieved through bonding of the phage with receptors on the cell wall (Rakhuba et al. 2010). As can be seen in Table 3(c), the few phages identified to have RBP recognizing exopolysaccharides are mostly of Podoviridae morphology.
In an example, the producer cell is a Salmonella (eg, S enterica Serovar Typhimurium) cell and the receptor is selected from flagella, vitamin B12 uptake outer membrane protein, BtuB and lipopolysaccharide-related O-antigen. In an example the receptor is a flagellum or BtuB and the first phage are Siphoviridae phage. In an example the receptor is O-antigen of LPS and the first phage are Podoviridae phage. Optionally, the receptor is FliC host receptor or FUjB receptor.
Optionally, the producer cell is a S enterica or P aeruginosa cell. Optionally, the receptor is the receptor of the host as listed in Table 4.
The O-antigen structure of Salmonella O66 has been established, which reportedly differs from the known O-antigen structure of Escherichia coli O166 only in one linkage (most likely the linkage between the O-units) and O-acetylation. The O-antigen gene clusters of Salmonella O66 and E. coli O166 were found to have similar organizations, the only exception being that in Salmonella O66, the wzy gene is replaced by a non-coding region. The function of the wzy gene in E. coli O166 was confirmed by the construction and analysis of deletion and trans-complementation mutants. It is proposed that a functional wzy gene located outside the O-antigen gene cluster is involved in Salmonella O66 O-antigen biosynthesis, as has been reported previously in Salmonella sero groups A, B and D1. The sequence identity for the corresponding genes between the O-antigen gene clusters of Salmonella O66 and E. coli O166 ranges from 64 to 70%, indicating that they may originate from a common ancestor. It is likely that after the species divergence, Salmonella O66 got its specific O-antigen form by inactivation of the wzy gene located in the O-antigen gene cluster and acquisition of two new genes (a wzy gene and a prophage gene for O-acetyl modification) both residing outside the O-antigen gene cluster.
In an example, the producer cells are E coli cells and do not comprise an expressible E coli (eg, Escherichia coli O166) wzy gene.
Optionally, the receptor is selected from lipopolysaccharides, teichoic acids (optionally a ManNAc(β1→4)GlcNAc disaccharide with one to three glycerol phosphates attached to the C4 hydroxyl of the ManNAc residue followed by a long chain of glycerol- or ribitol phosphate repeats), proteins and flagella.
Optionally, the receptor comprises an O-antigen of the producer cell species.
Optionally, the phage or particles of the invention are operable to express an endolysin or holin in the producer cells, optionally when phage or particles replicate in producer cells. This is useful for releasing the particles for subsequent purification away from cellular material to produce a composition of the invention.
In an embodiment, each particle is capable of infecting a target bacterium, the particle comprising a nucleotide sequence of interest (NSI) that is capable of expressing a protein or RNA in the target bacterium, or wherein the NSI comprises a regulatory element that is operable in the target bacterium. In an example, the NSI is capable of recombination with the target cell chromosome or an episome comprised by the target cell to modify the chromosome or episome. Optionally, this is carried out in a method wherein the chromosome or episome is cut (eg, at a predetermined site using a guided nuclease, such as a Cas, TALEN, zinc finger or meganuclease; or a restriction endonuclease) and simultaneously or sequentially the cell is infected by a particle that comprises first DNA comprising the NSI, wherein the DNA is introduced into the cell and the NSI or a sequence thereof is introduced into the chromosome or episome at or adjacent the cut site. In an example the first DNA comprises one or more components of a CRISPR/Cas system operable to perform the cutting (eg, comprising at least a nucleotide sequence encoding a guide RNA or crRNA for targeting the site to be cut) and further comprising the NSI.
In an embodiment, the presence in the target bacterium of the NSI or its encoded protein or RNA mediates target cell killing, or downregulation of growth or propagation of target cells, or mediates switching off of expression of one or more RNA or proteins encoded by the target cell genome, or downregulation thereof.
In an embodiment, the presence in the target bacterium of the NSI or its encoded protein or RNA mediates upregulation of growth or propagation of the target cell, or mediates switching on of expression of one or more RNA or proteins encoded by the target cell genome, or upregulation thereof.
In an embodiment, the NSI encodes a component of a CRISPR/Cas system that is toxic to the target bacterium.
In an embodiment, the first NSI is comprised by a vector (eg, a plasmid or shuttle vector).
An embodiment provides a method of treating an environment ex vivo, the method comprising exposing the environment to a population of transduction particles obtainable by the production method of the invention, wherein the environment comprises target bacteria and the particles (eg, phage or particles comprising a phage capsid) infect and kill the target bacteria. In an example an agent is further administered to the environment simultaneously or sequentially with the phage administration. In an example, the agent is an herbicide, pesticide, insecticide, plant fertilizer or cleaning agent.
A method of treating an infection of target bacteria in a human or animal subject is provided, the method comprising exposing the bacteria to a population of transduction particles obtainable by the production method, wherein the particles infect and kill the target bacteria.
Optionally, target bacteria herein are comprised by a microbiome of the subject, eg, a gut microbiome. Alternatively, the microbiome is a skin, scalp, hair, eye, ear, oral, throat, lung, blood, rectal, anal, vaginal, scrotal, penile, nasal or tongue microbiome.
In an example the subject is further administered a medicament simultaneously or sequentially with the transduction particle administration. In an example, the medicament is an antibiotic, antibody, immune checkpoint inhibitor (eg, an anti-PD-1, anti-PD-LI or anti-CTLA4 antibody), adoptive cell therapy (eg, CAR-T therapy) or a vaccine.
In an example, the invention employs helper phage for packaging the NSI. In an example, the helper phage are capable of packaging DNA comprising the NSI to produce first transduction particles, wherein the particles are different from the helper phage and the helper phage are incapable themselves of producing helper phage particles.
A composition is provided comprising a population of transduction particles of the invention, wherein the particles require helper phage according to the immediately preceding paragraph for replication of the transduction particles; and optionally wherein less than 20, 15, 10, 5, 4, 3, 2, 1, 0.5, 0.4, 0.2 or 0.1% of total transduction particles comprised by the composition are particles of such helper phage. In an example the composition comprises helper phage and less than 1% of total transduction particles comprised by the composition are particles of such helper phage. In an example the composition comprises helper phage and less than 0.5% of total transduction particles comprised by the composition are particles of such helper phage. In an example the composition comprises helper phage and less than 0.1% of total transduction particles comprised by the composition are particles of such helper phage. In an example the composition comprises helper phage and less than 0.01% of total transduction particles comprised by the composition are particles of such helper phage. In an example the composition comprises helper phage and less than 0.001% of total transduction particles comprised by the composition are particles of such helper phage. In an example the composition comprises helper phage and less than 0.0001% of total transduction particles comprised by the composition are particles of such helper phage. In an example the composition comprises helper phage and less than 0.00001% of total transduction particles comprised by the composition are particles of such helper phage. In an example the composition comprises helper phage and less than 0.000001% of total transduction particles comprised by the composition are particles of such helper phage.
In an example the composition comprises helper phage and less than 0.0000001% of total transduction particles comprised by the composition are particles of such helper phage. In an example the composition comprises helper phage and less than 0.00000001% of total transduction particles comprised by the composition are particles of such helper phage.
In an example, the composition or population comprises at least 103, 104, 105 or 106 transduction particles, as indicated a transduction assay, for example. In an example, the composition or population comprises at least 103 transduction particles and eg, no more than 1011 particles. In an example, the composition or population comprises at least 104 transduction particles and eg, no more than 1014 particles. In an example, the composition or population comprises at least 105 transduction particles and eg, no more than 1014 particles. In an example, the population comprises at least 106 transduction particles and eg, no more than 1014 particles. To have a measure of the particle concentration, for example, one can perform a standard transduction assay when the genome of the particles of the invention contains an antibiotic marker. Thus, in this case the particles of the invention are capable of infecting target bacteria and in a sample of 1 ml the composition of population comprises at least 103, 104, 105 or 106 transduction particles, which can be determined by infecting susceptible bacteria at a multiplicity of infection <0.1 and determining the number of infected cells by plating on a selective agar plate corresponding to the antibiotic marker in vitro at 20 to 37 degrees centigrade, eg, at 20 or 37 degrees centrigrade.
Optionally at least 99.9, 99.8, 99.7, 99.6, 99.5, 99.4, 99.3, 99.2, 99.1, 90, 85, 80, 70, 60, 50 or 40% of total transduction particles comprised by the composition are particles of particles of the invention.
In an example, genome of the particles of the invention comprises an f1 origin of replication.
In an example, the helper phage are E coli phage. In an example, the particles of the invention are E coli, C dificile, Streptococcus, Klebsiella, Pseudomonas, Acitenobacter, Enterobacteracea, Firmicutes or Bacteroidetes phage or comprise a capsid of such a genus, wherein the capsid packages a NSI. In an example, the helper phage are engineered M13 phage.
In an example, the genome of the particles of the invention comprises a phagemid, wherein the phagemid comprises a packaging signal for packaging the particles in the presence of the helper phage.
The particles of the invention may contain DNA comprising a nucleotide sequence of interest (NSI), eg, as defined herein, such as a NSI that encodes a component of a CRISPR/Cas system operable in target bacteria that can be infected by the particles. Once inside the target bacteria, optionally the particle DNA is incapable of being packaged to form transduction particles in the absence of the helper phage. This usefully contains the activity of the genome of the particles of the invention and its encoded products (proteins and/or nucleic acid), as well as limits or controls dosing of the NSI and its encoded products in an environment comprising the target bacteria that have been exposed to the particles of the invention. This is useful, for example to control the medical treatment of an environment comprised by a human or animal subject, plant or other environment (eg, soil or a foodstiff or food ingredient).
In an embodiment, each particle of the invention comprises one or more phage structural proteins and/or comprises a phage capsid. Examples of phage structural proteins are phage coat proteins, collar proteins and phage tail fibre proteins. In an example, the particle comprises a capsid and tail fibre proteins of first type of phage. For example, the phage type is an E coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Clostridium dificile, Helicobacter pylori, Staphylococcus aureus, Salmonella (eg, typhimurium) or Campylobacter phage.
Optionally, at least 95% (eg, 100%) of transduction particles comprised by the composition are particles of the invention.
In another embodiment, the composition comprises second transduction (eg, phage) particles, wherein the second particles are different from the first particles of the invention (ie, the particles recited in claim 1).
Optionally, the composition population comprises at least 103, 104, 105 or 106 phage particles, as indicated in a transduction assay.
Optionally, each particle of the invention comprises a vector for the NSA, wherein the vectors are plasmids or phagemids. For example, the vectors are shuttle vectors (eg, pUC vectors) that can be replicated in host bacteria.
Optionally, the genome of each particle of the invention comprises a packaging signal, such as a pac or cos sequence or homologue thereof.
Optionally, the transduction particles of the invention are temperate phage. Optionally, the transduction particles of the invention are lytic phage.
Optionally, the particles of the invention are capable of infecting target bacteria, the particles comprising a nucleotide sequence of interest (NSI) that is capable of expressing a protein or RNA (eg, gRNA or crRNA) in target bacteria, or wherein the NSI comprises a regulatory element that is operable in target bacteria.
Optionally, the presence in target bacteria of the NSI or its encoded protein or RNA mediates target cell killing, or downregulation of growth or propagation of target cells, or mediates switching off of expression of one or more RNA or proteins encoded by the target cell genomes, or downregulation thereof.
Optionally, the presence in target bacteria of the NSI or its encoded protein or RNA mediates upregulation of growth or propagation of target cells, or mediates switching on of expression of one or more RNA or proteins encoded by the target cell genomes, or upregulation thereof.
Optionally, the particles of the invention are capable of infecting target bacteria and each particle comprises engineered antibacterial means for killing target bacteria. By use of the term “engineered” it will be readily apparent to the skilled addressee that the relevant means has been introduced and is not naturally-occurring in the phage or particle. For example, the means is recombinant, artificial or synthetic.
In an example, each particle of the invention comprises a genomic island DNA or pathogenicity island (eg, saPI) DNA, wherein optionally the DNA comprises the NSI or engineered antibacterial means for killing target bacteria (eg, the DNA encodes a nuclease, such as Cas nuclease (eg, Cas9 or Cas3), and/or a guide RNA for expression in a target bacterium).
Optionally, the antibacterial means comprises one or more components of a CRISPR/Cas system.
Optionally, the component(s) comprise (i) a DNA sequence encoding a guide RNA (eg, a single guide RNA) or comprising a CRISPR array for producing guide RNA, wherein the guide RNA is capable of targeting the genome of target bacteria; (ii) a Cas nuclease-encoding DNA sequence; and/or (iii) a DNA sequence encoding one or more components of Cascade. In an example, a Cas herein is a Cas9. In an example, a Cas herein is a Cas3. The Cas may be identical to a Cas encoded by the target bacteria.
Optionally, the antibacterial means comprises a nucleic acid encoding a guided nuclease, such as a Cas nuclease, TALEN, zinc finger nuclease or meganuclease.
Optionally, the composition, population or transduction particles is each for use in medicine practised on a human or animal subject, or the composition is a pharmaceutical composition for use in medicine practised on a human or animal subject. In an example, the animal is a livestock or companion pet animal (eg, a cow, pig, goat, sheep, horse, dog, cat or rabbit). In an example, the animal is an insect (an insect at any stage of its lifecycle, eg, egg, larva or pupa). In an example, the animal is a protozoan. In an example, the animal is a cephalopod.
Optionally, the composition is a herbicide, pesticide, food or beverage processing agent, food or beverage additive, petrochemical or fuel processing agent, water purifying agent, cosmetic additive, detergent additive or environmental (eg, soil) additive or cleaning agent.
The inability in some embodiments of the particles of the invention to self-replicate and to require helper phage to do this usefully provides containment in the location (eg, gut) of action of the composition and/or in the environment of the subject, eg, when exposed to secretions such as urine and faeces of the subject that otherwise may contain replicated first phage. Inability of the helper phage in some embodiments to self-package limits availability of factors required by the particles to form packaged particles, hence providing containment by limiting propagation of the particles of the invention. This may be useful, for example, to contain an antibacterial activity provided by the particles, such as a CRISPR/Cas killing principle.
In an example, when the subject is a human, the subject is not an embryo.
Optionally, the environment is a microbiome of soil; a plant, part of a part (e.g., a leaf, fruit, vegetable or flower) or plant product (e.g., pulp); water; a waterway; a fluid; a foodstuff or ingredient thereof; a beverage or ingredient thereof; a medical device; a cosmetic; a detergent; blood; a bodily fluid; a medical apparatus; an industrial apparatus; an oil rig; a petrochemical processing, storage or transport apparatus; a vehicle or a container. In an example, the environment is an ex vivo bodily fluid (e.g., urine, blood, blood product, sweat, tears, sputum or spit), bodily solid (e.g., faeces) or tissue of a human or animal subject that has been administered the composition.
Optionally, the antibacterial means comprises one or more components of a CRISPR/Cas system.
For example, the component(s) comprise (i) a DNA sequence encoding a guide RNA (eg, a single guide RNA) or comprising a CRISPR array for producing guide RNA, wherein the guide RNA is capable of targeting the genome of target bacteria; (ii) a Cas (eg, Cas9, Cas3, Cpf1, CasX or CasY) nuclease-encoding DNA sequence; and/or (iii) a DNA sequence encoding one or more components of Cascade (eg, CasA).
Optionally, the antibacterial means comprises a nucleic acid encoding a guided nuclease, such as a Cas nuclease, TALEN, zinc finger nuclease or meganuclease.
In an example, the particles, population or composition of the invention is comprised by a medical container, eg, a syringe, vial, IV bag, inhaler, eye dropper or nebulizer. In an example, the particles, population or composition of the invention is comprised by a sterile container. In an example, the particles, population or composition of the invention is comprised by a medically-compatible container. In an example, the particles, population or composition of the invention is comprised by a fermentation vessel, eg, a metal, glass or plastic vessel.
In an example, the particles, population or composition of the invention is comprised by a medicament, e,g in combination with instructions or a packaging label with directions to administer the medicament by oral, IV, subcutaneous, intranasal, intraocular, vaginal, topical, rectal or inhaled administration to a human or animal subject. In an example, the particles, population or composition of the invention is comprised by an oral medicament formulation. In an example, the particles, population or composition of the invention is comprised by an intranasal or ocular medicament formulation. In an example, the particles, population or composition of the invention is comprised by a personal hygiene composition (eg, shampoo, soap or deodorant) or cosmetic formulation. In an example, the particles, population or composition of the invention is comprised by a detergent formulation. In an example, the particles, population or composition of the invention is comprised by a cleaning formulation, eg, for cleaning a medical or industrial device or apparatus. In an example, the particles, population or composition of the invention is comprised by foodstuff, foodstuff ingredient or foodstuff processing agent. In an example, the particles, population or composition of the invention is comprised by beverage, beverage ingredient or beverage processing agent. In an example, the particles, population or composition of the invention is comprised by a medical bandage, fabric, plaster or swab. In an example, the particles, population or composition of the invention is comprised by an herbicide or pesticide. In an example, the particles, population or composition of the invention is comprised by an insecticide.
In an example, each particle is a first phage particle or comprises a capsid of a first phage (and optionally also tail fibres of the first phage), wherein the first phage is a is a Corticoviridae, Cystoviridae, Inoviridae, Leviviridae, Microviridae, Myoviridae, Podoviridae, Siphoviridae, or Tectiviridae virus. In an example, the helper phage is a is a Corticoviridae, Cystoviridae, Inoviridae, Leviviridae, Microviridae, Myoviridae, Podoviridae, Siphoviridae, or Tectiviridae virus. In an example, the helper phage is a filamentous M13, a Noviridae, a tailed phage (eg, a Myoviridae, Siphoviridae or Podoviridae), or a non-tailed phage (eg, a Tectiviridae).
In an example, both the first phage are Corticoviridae. In an example, the first phage are Cystoviridae. In an example, the first phage are Inoviridae. In an example, the first phage are Leviviridae.
In an example, the first phage are Microviridae. In an example, the first phage are Podoviridae. In an example, the first phage are Siphoviridae. In an example, the first phage are Tectiviridae.
In an example, the CRISPR/Cas component(s) are component(s) of a Type I CRISPR/Cas system. In an example, the CRISPR/Cas component(s) are component(s) of a Type II CRISPR/Cas system. In an example, the CRISPR/Cas component(s) are component(s) of a Type III CRISPR/Cas system. In an example, the CRISPR/Cas component(s) are component(s) of a Type IV CRISPR/Cas system. In an example, the CRISPR/Cas component(s) are component(s) of a Type V CRISPR/Cas system. In an example, the CRISPR/Cas component(s) comprise a Cas9-encoding nucleotide sequence (eg, Spyogenes Cas9, S aureus Cas9 or S thermophilus Cas9). In an example, the CRISPR/Cas component(s) comprise a Cas3-encoding nucleotide sequence (eg, E coli Cas3, C dificile Cas3 or Salmonella Cas3). In an example, the CRISPR/Cas component(s) comprise a Cpf-encoding nucleotide sequence. In an example, the CRISPR/Cas component(s) comprise a CasX-encoding nucleotide sequence. In an example, the CRISPR/Cas component(s) comprise a CasY-encoding nucleotide sequence.
In an example, the genomes of the particles encode a CRISPR/Cas component or protein of interest from a nucleotide sequence comprising a promoter that is operable in the target bacteria.
In an example, the host bacteria and/or target bacteria are E coli. In an example, the host bacteria and/or target bacteria are C difficile (eg, the vector is a shuttle vector operable in E coli and the host bacteria are C difficile). In an example, the host bacteria and/or target bacteria are Streptococcus, such as S thermophilus (eg, the vector is a shuttle vector operable in E coli and the host bacteria are Streptococcus). In an example, the host bacteria and/or target bacteria are Pseudomonas, such as P aeruginosa (eg, the vector is a shuttle vector operable in E coli and the host bacteria are P aeruginosa). In an example, the host bacteria and/or target bacteria are Klebsiella, such as K pneumoniae (eg, the vector is a shuttle vector operable in E coli and the host bacteria are Klebsiella). In an example, the host bacteria and/or target bacteria are Salmonella, eg, S typhimurium (eg, the vector is a shuttle vector operable in E coli and the host bacteria are Salmonella).
Optionally, each producer and/or target bacterium is a gram negative bacterium (eg, a spirilla or vibrio). Optionally, each producer and/or target bacterium is a gram positive bacterium. Optionally, each producer and/or target bacterium is a mycoplasma, chlamydiae, spirochete or mycobacterium. Optionally, each producer and/or target bacterium is a Streptococcus (eg, pyogenes or thermophilus). Optionally, each producer and/or target bacterium is a Staphylococcus (eg, aureus, eg, MRSA). Optionally, each producer and/or target bacterium is an E. coli (eg, O157: H7) host, eg, wherein the Cas is encoded by the vecor or an endogenous host Cas nuclease activity is de-repressed. Optionally, each producer and/or target bacterium is a Pseudomonas (eg, aeruginosa). Optionally, each producer and/or target bacterium is a Vibro (eg, cholerae (eg, O139) or vulnificus). Optionally, each producer and/or target bacterium is a Neisseria (eg, gonnorrhoeae or meningitidis). Optionally, each producer and/or target bacterium is a Bordetella (eg, pertussis). Optionally, each producer and/or target bacterium is a Haemophilus (eg, influenzae). Optionally, each producer and/or target bacterium is a Shigella (eg, dysenteriae). Optionally, each producer and/or target bacterium is a Brucella (eg, abortus). Optionally, each producer and/or target bacterium is a Francisella host. Optionally, each producer and/or target bacterium is a Xanthomonas host. Optionally, each producer and/or target bacterium is an Agrobacterium host. Optionally, each producer and/or target bacterium is an Erwinia host. Optionally, each producer and/or target bacterium is a Legionella (eg, pneumophila). Optionally, each producer and/or target bacterium is a Listeria (eg, monocytogenes). Optionally, each producer and/or target bacterium is a Campylobacter (eg, jejuni). Optionally, each producer and/or target bacterium is a Yersinia (eg, pestis). Optionally, each producer and/or target bacterium is a Borelia (eg, burgdorferi). Optionally, each producer and/or target bacterium is a Helicobacter (eg, pylon). Optionally, each producer and/or target bacterium is a Clostridium (eg, drjicile or botulinum). Optionally, each producer and/or target bacterium is a Erlichia (eg, chafeensis). Optionally, each producer and/or target bacterium is a Salmonella (eg, typhi or enterica, eg, serotype typhimurium, eg, DT 104). Optionally, each producer and/or target bacterium is a Chlamydia (eg, pneumoniae). Optionally, each producer and/or target bacterium is a Parachlamydia host. Optionally, each producer and/or target bacterium is a Corynebacterium (eg, amycolatum). Optionally, each producer and/or target bacterium is a Klebsiella (eg, pneumoniae). Optionally, each producer and/or target bacterium is an Enterococcus (eg, faecalis or faecim, eg, linezolid-resistant). Optionally, each producer and/or target bacterium is an Acinetobacter (eg, baumannii, eg, multiple drug resistant).
Further examples of target cells and targeting of antibiotic resistance in such cells using the present invention are as follows:—
Genetic variation of bacteria and archaea can be achieved through mutations, rearrangements and horizontal gene transfers and recombinations. Increasing genome sequence data have demonstrated that, besides the core genes encoding house-keeping functions such as essential metabolic activities, information processing, and bacterial structural and regulatory components, a vast number of accessory genes encoding antimicrobial resistance, toxins, and enzymes that contribute to adaptation and survival under certain environmental conditions are acquired by horizontal gene transfer of mobile genetic elements (MGEs). Mobile genetic elements are a heterogeneous group of molecules that include plasmids, bacteriophages, genomic islands, chromosomal cassettes, pathogenicity islands, and integrative and conjugative elements. Genomic islands are relatively large segments of DNA ranging from 10 to 200 kb often integrated into tRNA gene clusters flanked by 16-20 bp direct repeats. They are recognized as discrete DNA segments acquired by horizontal gene transfer since they can differ from the rest of the chromosome in terms of GC content (% G+C) and codon usage.
Pathogenicity islands (PTIs) are a subset of horizontally transferred genetic elements known as genomic islands. There exists a particular family of highly mobile PTIs in Staphylococcus aureus that are induced to excise and replicate by certain resident prophages. These PTIs are packaged into small headed phage-like particles and are transferred at frequencies commensurate with the plaque-forming titer of the phage. This process is referred to as the SaPI excision replication-packaging (ERP) cycle, and the high-frequency SaPI transfer is referred to as SaPI-specific transfer (SPST) to distinguish it from classical generalized transduction (CGT). The SaPIs have a highly conserved genetic organization that parallels that of bacteriophages and clearly distinguishes them from all other horizontally acquired genomic islands. The SaPI1-encoded and SaPIbov2-encoded integrases are used for both excision and integration of the corresponding elements, and it is assumed that the same is true for the other SaPIs. Phage 80α can induce several different SaPIs, including SaPI11, SaPI2, and SaPlbov1, whereas φ11 can induce SaPlbov1 but neither of the other two SaPIs.
Reference is made to “Staphylococcal pathogenicity island DNA packaging system involving cos-site packaging and phage-encoded HNH endonucleases”, Quiles-Puchalt et al, PNAS Apr. 22, 2014. 111 (16) 6016-6021. Staphylococcal pathogenicity islands (SaPIs) are highly mobile and carry and disseminate superantigen and other virulence genes. It was reported that SaPIs hijack the packaging machinery of the phages they victimise, using two unrelated and complementary mechanisms. Phage packaging starts with the recognition in the phage DNA of a specific sequence, termed “pac” or “cos” depending on the phage type. The SaPI strategies involve carriage of the helper phage pac- or cos-like sequences in the SaPI genome, which ensures SaPI packaging in full-sized phage particles, depending on the helper phage machinery. These strategies interfere with phage reproduction, which ultimately is a critical advantage for the bacterial population by reducing the number of phage particles.
Staphylococcal pathogenicity islands (SaPIs) are the prototypical members of a widespread family of chromosomally located mobile genetic elements that contribute substantially to intra- and interspecies gene transfer, host adaptation, and virulence. The key feature of their mobility is the induction of SaPI excision and replication by certain helper phages and their efficient encapsidation into phage-like infectious particles. Most SaPIs use the headful packaging mechanism and encode small terminase subunit (TerS) homologs that recognize the SaPI-specific pac site and determine SaPI packaging specificity. Several of the known SaPIs do not encode a recognizable TerS homolog but are nevertheless packaged efficiently by helper phages and transferred at high frequencies. Quiles-Puchalt et al report that one of the non-terS-coding SaPIs, SaPIbov5, and found that it uses two different, undescribed packaging strategies. SaPIbov5 is packaged in full-sized phage-like particles either by typical pac-type helper phages, or by cos-type phages—i.e., it has bothpac and cossites and uses the two different phage-coded TerSs. This is an example of SaPI packaging by a cos phage, and in this, it resembles the P4 plasmid of Escherichia coli. Cos-site packaging in Staphylococcus aureus is additionally unique in that it requires the HNH nuclease, carried only by cos phages, in addition to the large terminase subunit, for cos-site cleavage and melting.
Characterization of several of the phage-inducible SaPIs and their helper phages has established that the pac (or headful) mechanism is used for encapsidation. In keeping with this concept, some SaPIs encode a homolog of TerS, which complexes with the phage-coded large terminase subunit TerL to enable packaging of the SaPI DNA in infectious particles composed of phage proteins. These also contain a morphogenesis (cpm) module that causes the formation of small capsids commensurate with the small SaPI genomes. Among the SaPI sequences first characterized, there were several that did not include either a TerS homolog or a cpm homolog, and the same is true of several subsequently identified SaPIs from bovine sources and for many phage-inducible chromosomal islands from other species. It was assumed, for these several islands, either that they were defective derivatives of elements that originally possessed these genes, or that terS and cpm genes were present but not recognized by homology.
Quiles-Puchalt et al observed that an important feature of ϕSLT/SaPlbov5 packaging is the requirement for an HNH nuclease, which is encoded next to the ϕSLT terminase module. Proteins carrying HNH domains are widespread in nature, being present in organisms of all kingdoms. The HNH motif is a degenerate small nucleic acid-binding and cleavage module of about 30-40 as residues and is bound by a single divalent metal ion. The HNH motif has been found in a variety of enzymes playing important roles in many different cellular processes, including bacterial killing; DNA repair, replication, and recombination; and processes related to RNA. HNH endonucleases are present in a number of cos-site bacteriophages of Gram-positive and -negative bacteria, always adjacent to the genes encoding the terminases and other morphogenetic proteins. Quiles-Puchalt et al have demonstrated that the HNH nucleases encoded by #12 and the closely related ϕSLT have nonspecific nuclease activity and are required for the packaging of these phages and of SaPIbov5. Quiles-Puchalt et al have shown that HNH and TerL are jointly required for cos-site cleavage. Quiles-Puchalt et al have also observed that only cos phages of Gram-negative as well as of Gram-positive bacteria encode HNH nucleases, consistent with a special requirement for cos-site cleavage as opposed to pac-site cleavage, which generates flush-ended products. The demonstration that HNH nuclease activity is required for some but not other cos phages suggests that there is a difference between the TerL proteins of the two types of phages-one able to cut both strands and the other needing a second protein to enable the generation of a double-stranded cut.
In the alternative, instead of a bacterium, each producer and/or target cell is an archaeal cell and instead of a phage there is a virus that is capable of infecting the archaeal cell (or each particle comprises a capsid (and optionally tail fibres) of such a virus).
Optionally, the transduction particles are non-self replicative particles. A “non-self replicative transduction particle” refers to a particle, (eg, a phage or phage-like particle; or a particle produced from a genomic island (eg, a SaPI) or a modified version thereof) capable of delivering a nucleic acid molecule encoding an antibacterial agent or component into a bacterial cell, but does not package its own replicated genome into the transduction particle. In an alternative herein, instead of a phage, there is used or packaged a virus that infects an animal, human, plant or yeast target cell. For example, an adenovirus when the target cell is a human cell.
Optionally, the genome of each particle is devoid of genes encoding phage structural proteins. These can be supplied instead by a helper phage during production. Optionally, the genome of each particle is devoid of one or more phage genes rinA, terS and terL.
Optionally, the genomic island is an island that is naturally found in target and/or producer bacterial cells (and optionally in particles of the invention, the genomic island DNA comprises the NSI). In an example, the genomic island is selected from the group consisting of a SaPI, a SaPI1, a SaPI2, a SaPIbov1 and a SaPibov2 genomic island. For example, the island is a modified pathogenicity island. Optionally, the pathogenicity island is an island that is naturally found in target and/or producer bacterial cells, eg, a Staphylococcus SaPI or a Vibro PLE or a P. aeruginosa pathogenicity island (eg, a PAPI or a PAGI, eg, PAPI-1, PAGI-5, PAGI-6, PAGI-7, PAGI-8, PAGI-9, PAGI-10, or PAGI-11. Optionally, the pathogenicity island is a SaPI (S aureus pathogenicity island); optionally, a helper phage is used during production in this case, wherein the helper phage is ϕ11, 80α, ϕ12 or ϕSLT. Staphylococcus phage 80α appears to mobilise all known SaPIs. Thus, in an example, the genome of each particle comprises modified SaPI and the helper phage is a 80α. Optionally, the pathogenicity island is a V. cholerae PLE (phage-inducible chromosomal island-like element) and optionally the first phage is ICP1. Optionally, the pathogenicity island is a E coli PLE.
Optionally, each particle genome comprises P4 DNA, eg, at least P4 packaging signal sequence. The particle may comprise DNA comprising a P4 packaging signal and the NSI or antibacterial means. In an embodiment, a helper phage is used to produce the particle, wherein the helper phage is a P2 phage or a modified P2 phage that is self-replicative defective; optionally present as a prophage in the producer cell genome.
Optionally, the transcription of particle nucleic acid is under the control of a constitutive promoter, for transcription of copies of the antibacterial agent or component or NSI in a target cell. Optionally, Constitutive transcription and production in target cells may be used where the target cells should be killed, eg, in medical settings.
Optionally, the transcription of particle nucleic acid is under the control of an inducible promoter, for transcription of copies of the antibacterial agent or component or NSI in a target cell. This may be useful, for example, to control switching on of the antibacterial activity against target bacterial cells, such as in an environment (eg, soil or water) or in an industrial culture or fermentation container containing the target cells. For example, the target cells may be useful in an industrial process (eg, for fermentation, eg, in the brewing or dairy industry) and the induction enables the process to be controlled (eg, stopped or reduced) by using the antibacterial agent against the target bacteria.
When the agent comprises a plurality of components, eg, wherein the agent is a CRISPR/Cas system, or is a CRISPR array encoding crRNA or a nucleic acid encoding a guide RNA (eg, single guide RNA) operable with a Cas in target cells, wherein the crRNA or gRNA guides the Cas to a target sequence in the cell to modify the target (eg, cut it or repress transcription from it). Optionally, the genes are comprised by the target cell chromosome and/or one or more cell episome(s).
Optionally, the agent is a guided nuclease system or a component thereof, wherein the agent is capable of recognising and cutting target cell DNA (eg, chromosomal DNA).
In examples, such cutting causes one or more of the following:—
Optionally, the guided nuclease system is selected from a CRISPR/Cas system, TALEN system, meganuclease system or zinc finger system. Optionally, the system is a CRISPR/Cas system and each particle genome encodes a (a) CRISPR array encoding crRNA or (b) a nucleic acid encoding a guide RNA (gRNA, eg, single guide RNA), wherein the crRNA or gRNA is operable with a Cas in target cells, wherein the crRNA or gRNA guides the Cas to a target nucleic acid sequence in the target cell to modify the target sequence (eg, cut it or repress transcription from it). Optionally, the Cas is a Cas encoded by a functional endogenous nucleic acid of a target cell. For example, the target is comprised by a DNA or RNA of the target cell. Optionally, the system is a CRISPR/Cas system and each particle genome encodes a Cas (eg, a Cas nuclease) that is operable in a target bacterial cell to modify a target nucleic acid sequence comprised by the target cell.
Any Cas herein may be a Cas3, Cas9, Cas13, CasX, CasY or Cpf1.
Optionally, the system is a CRISPR/Cas system and each particle genome encodes one or more Cascade Cas (eg, Cas, A, B, C, D and E).
Optionally, each particle genome further encodes a Cas3 that is operable in a target bacterial cell with the Cascade Cas.
Optionally, the producer and/or target cell is a cell of a first species or strain, wherein the first species or strain is a gram positive species or strain.
Optionally, the producer and/or target cell is a cell of a first species or strain, wherein the first species or strain is a gram negative species or strain.
Optionally, the first species or strain is selected from Table 6 For example, the first species or strain is selected from Shigella, E coli, Salmonella, Serratia, Klebsiella, Yersinia, Pseudomonas and Enterobacter. These are species that P2 phage can infect. Thus, in an embodiment, the particle genome comprises one or more P4 sequences (eg, a P4 packaging sequence) and the genome is packaged by a P2 phage capsid. Thus, the genome is packaged by P2 structural proteins and the resultant transduction particles can usefully infect a broad spectrum of species, ie, two or more of Shigella, E coli, Salmonella, Serratia, Klebsiella, Yersinia, Pseudomonas and Enterobacter. This provides a broadly-applicable delivery platform, where target cell antibacterial specificity can be achieved by encoding on the particle genome guide RNA(s) that specifically target one or more predetermined species within the group of Shigella, E coli, Salmonella, Serratia, Klebsiella, Yersinia, Pseudomonas and Enterobacter.
By “non-replicative” or “replicative-defective” it is meant that the particle is not capable by itself of self-replicating. For example, the particle genome is devoid of one or more nucleotide sequences encoding a protein (eg, a structural protein) that is necessary to produce a transduction particle.
In an example, the reduction in growth or proliferation of target cells is at least 50, 60, 70, 80, 90 or 95%.
In an example each producer cell and/or target cell is selected from a Staphylococcal, Vibrio, Pseudomonas, Clostridium, E coli, Helicobacter, Klebsiella and Salmonella cell.
Optionally, each particle comprise a plasmid comprising
Optionally, the antibacterial agent is a CRISPR/Cas system and the plasmid encodes a crRNA or guide RNA (eg, single gRNA) that is operable with a Cas in the target cells to guide the Cas to a target nucleotide sequence to modify (eg, cut) the sequence, whereby
Optionally, the antibacterial agent is a CRISPR/Cas system and the plasmid encodes a Cas that is operable with a crRNA or guide RNA (eg, single gRNA) in the target cells to guide the Cas to a target nucleotide sequence to modify (eg, cut) the sequence, whereby
Optionally, the plasmid further encodes said crRNA or gRNA.
A plurality of transduction particles obtainable by the method of the invention is provided for use in medicine, eg, for treating or preventing an infection of a human or animal subject by target bacterial cells, wherein transducing particles are administered to the subject for infecting target cells and killing the cells using the antibacterial agent.
Optionally, the particles are for administration to a human or animal for medical use.
Further Concepts of the invention are as follows:—
The present invention is optionally for an industrial or domestic use, or is used in a method for such use. For example, it is for or used in agriculture, oil or petroleum industry, food or drink industry, clothing industry, packaging industry, electronics industry, computer industry, environmental industry, chemical industry, aeorspace industry, automotive industry, biotechnology industry, medical industry, healthcare industry, dentistry industry, energy industry, consumer products industry, pharmaceutical industry, mining industry, cleaning industry, forestry industry, fishing industry, leisure industry, recycling industry, cosmetics industry, plastics industry, pulp or paper industry, textile industry, clothing industry, leather or suede or animal hide industry, tobacco industry or steel industry.
The present invention is optionally for use in an industry or the environment is an industrial environment, wherein the industry is an industry of a field selected from the group consisting of the medical and healthcare; pharmaceutical; human food; animal food; plant fertilizers; beverage; dairy; meat processing; agriculture; livestock farming; poultry fanning; fish and shellfish fanning; veterinary; oil; gas; petrochemical; water treatment; sewage treatment; packaging; electronics and computer; personal healthcare and toiletries; cosmetics; dental; non-medical dental; ophthalmic; non-medical ophthalmic; mineral mining and processing; metals mining and processing; quarrying; aviation; automotive; rail; shipping; space; environmental; soil treatment; pulp and paper; clothing manufacture; dyes; printing; adhesives; air treatment; solvents; biodefence; vitamin supplements; cold storage; fibre retting and production; biotechnology; chemical; industrial cleaning products; domestic cleaning products; soaps and detergents; consumer products; forestry; fishing; leisure; recycling; plastics; hide, leather and suede; waste management; funeral and undertaking; fuel; building; energy; steel; and tobacco industry fields.
In an example, each particle genome comprises a CRISPR array that encodes a respective guide RNA that targets target bacteria, wherein the array comprises one, or two or more spacers (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or more spacers) for targeting the genome of target bacteria.
In an example, the target bacteria are comprised by an environment as follows. In an example, the environment is a microbiome of a human, eg, the oral cavity microbiome or gut microbiome or the bloodstream. In an example, the environment is not an environment in or on a human. In an example, the environment is not an environment in or on a non-human animal. In an embodiment, the environment is an air environment. In an embodiment, the environment is an agricultural environment. In an embodiment, the environment is an oil or petroleum recovery environment, eg, an oil or petroleum field or well. In an example, the environment is an environment in or on a foodstuff or beverage for human or non-human animal consumption.
In an example, the environment is a human or animal microbiome (eg, gut, vaginal, scalp, armpit, skin or oral cavity microbiome). In an example, the target bacteria are comprised by a human or animal microbiome (eg, gut, vaginal, scalp, armpit, skin or oral cavity microbiome).
In an example, the particles, population or composition of the invention are administered intranasally, topically or orally to a human or non-human animal, or is for such administration. The skilled person aiming to treat a microbiome of the human or animal will be able to determine the best route of administration, depending upon the microbiome of interest. For example, when the microbiome is a gut microbiome, administration can be intranasally or orally. When the microbiome is a scalp or armpit microbiome, administration can be topically. When the microbiome is in the mouth or throat, the administration can be orally.
In any use or method herein, in an embodiment particles of the invention are contacted with the target bacteria at a multiplicity of infection (MOI) of at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600 or 700. For example, the MOI is from 20 to 200, from 20 to 100, fro 50 to 200, from 50 to 100, from 75 to 150, 100 or about 100, or 200 or about 200. In an example, this may be determined by obtaining a sample of the microbiome containing the target bacteria (eg, a sample of a waterway or gut microbiome of a subject) and determining the number of CFU/ml or mg in the sample and using this to titrate the phage dose at the desired MOI to be exposed to the microbiome or administered to the environment or subject to be treated.
In an example, the environment is harboured by a beverage or water (eg, a waterway or drinking water for human consumption) or soil. The water is optionally in a heating, cooling or industrial system, or in a drinking water storage container.
In an example, the producer and/or target bacteria are Firmicutes selected from Anaerotruncus, Acetanaerobacterium, Acetitomaculum, Acetivibrio, Anaerococcus, Anaerofilum, Anaerosinus, Anaerostipes, Anaerovorax, Butyrivibrio, Clostridium, Capracoccus, Dehalobacter, Dialister, Dorea, Enterococcus, Ethanoligenens, Faecalibacterium, Fusobacterium, Gracilibacter, Guggenheimella, Hespellia, Lachnobacterium, Lachnospira, Lactobacillus, Leuconostoc, Megamonas, Moryella, Mitsuokella, Oribacterium, Oxobacter, Papillibacter, Proprionispira, Pseudobutyrivibrio, Pseudoramibacter, Roseburia, Ruminococcus, Sarcina, Seinonella, Shuttleworthia, Sporobacter, Sporobacterium, Streptococcus, Subdoligranulum, Syntrophococcus, Thermobacillus, Turibacter and Weisella.
In an example, the particles, population, composition, use or method is for reducing pathogenic infections or for re-balancing gut or oral microbiota eg, for treating or preventing obesity or disease in a human or animal. For example, the particles, population, composition, use or method is for knocking-down Clostridium dificile or E col bacteria in a gut microbiota of a human or animal.
In an example, the disease or condition is a cancer, inflammatory or autoimmune disease or condition, eg, obesity, diabetes, IBD (eg, wherein the target cell is an E coli or Klebsiella cell), a GI tract condition or an oral cavity condition.
Optionally, the environment is comprised by, or the target bacteria are comprised by, a gut microbiota, skin microbiota, oral cavity microbiota, throat microbiota, hair microbiota, armpit microbiota, vaginal microbiota, rectal microbiota, anal microbiota, ocular microbiota, nasal microbiota, tongue microbiota, lung microbiota, liver microbiota, kidney microbiota, genital microbiota, penile microbiota, scrotal microbiota, mammary gland microbiota, ear microbiota, urethra microbiota, labial microbiota, organ microbiota or dental microbiota. Optionally, the environment is comprised by, or the target bacteria are comprised by, a plant (eg, a tobacco, crop plant, fruit plant, vegetable plant or tobacco, eg on the surface of a plant or contained in a plant) or by an environment (eg, soil or water or a waterway or aqueous liquid).
Optionally, the disease or condition of a human or animal subject is selected from
Neurodegenerative or CNS Diseases or Conditions for Treatment or Prevention by the Invention
In an example, the neurodegenerative or CNS disease or condition is selected from the group consisting of Alzheimer disease, geriopsychosis, Down syndrome, Parkinson's disease, Creutzfeldt-jakob disease, diabetic neuropathy, Parkinson syndrome, Huntington's disease, Machado-Joseph disease, amyotrophic lateral sclerosis, diabetic neuropathy, and Creutzfeldt Creutzfeldt-Jakob disease. For example, the disease is Alzheimer disease. For example, the disease is Parkinson syndrome.
In an example, wherein the method of the invention is practised on a human or animal subject for treating a CNS or neurodegenerative disease or condition, the method causes downregulation of Treg cells in the subject, thereby promoting entry of systemic monocyte-derived macrophages and/or Treg cells across the choroid plexus into the brain of the subject, whereby the disease or condition (eg, Alzheimer's disease) is treated, prevented or progression thereof is reduced. In an embodiment the method causes an increase of IFN-gamma in the CNS system (eg, in the brain and/or CSF) of the subject. In an example, the method restores nerve fibre and/or reduces the progression of nerve fibre damage. In an example, the method restores nerve myelin and/or reduces the progression of nerve myelin damage. In an example, the method of the invention treats or prevents a disease or condition disclosed in WO2015136541 and/or the method can be used with any method disclosed in WO2015136541 (the disclosure of this document is incorporated by reference herein in its entirety, eg, for providing disclosure of such methods, diseases, conditions and potential therapeutic agents that can be administered to the subject for effecting treatment and/or prevention of CNS and neurodegenerative diseases and conditions, eg, agents such as immune checkpoint inhibitors, eg, anti-PD-1, anti-PD-LI, anti-TIM3 or other antibodies disclosed therein).
Cancers for Treatment or Prevention by the Method
Cancers that may be treated include tumours that are not vascularized, or not substantially vascularized, as well as vascularized tumours. The cancers may comprise non-solid tumours (such as haematological tumours, for example, leukaemias and lymphomas) or may comprise solid tumours. Types of cancers to be treated with the invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukaemia or lymphoid malignancies, benign and malignant tumours, and malignancies e.g., sarcomas, carcinomas, and melanomas. Adult tumours/cancers and paediatric tumours/cancers are also included.
Haematologic cancers are cancers of the blood or bone marrow. Examples of haematological (or haematogenous) cancers include leukaemias, including acute leukaemias (such as acute lymphocytic leukaemia, acute myelocytic leukaemia, acute myelogenous leukaemia and myeloblasts, promyeiocytic, myelomonocytic, monocytic and erythroleukaemia), chronic leukaemias (such as chronic myelocytic (granulocytic) leukaemia, chronic myelogenous leukaemia, and chronic lymphocytic leukaemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myeiodysplastic syndrome, hairy cell leukaemia and myelodysplasia.
Solid tumours are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumours can be benign or malignant. Different types of solid tumours are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumours, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumour, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous eel! carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumour, cervical cancer, testicular tumour, seminoma, bladder carcinoma, melanoma, and CNS tumours (such as a glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medu!loblastoma, Schwannoma craniopharyogioma, ependymoma, pineaioma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases).
Autoimmune Diseases for Treatment or Prevention by the Method
Inflammatory Diseases for Treatment or Prevention by the Method
In an example, any composition of the invention comprises at least 1×103 transduction particles of the invention per ml or mg, such as when the composition is comprised by a fluid (eg, a liquid) or solid. In an example, any composition of the invention comprises at least 1×104 transduction particles of the invention per ml or mg. In an example, any composition of the invention comprises at least 1×105 transduction particles of the invention per ml or mg. In an example, any composition of the invention comprises at least 1×106 transduction particles of the invention per ml or mg. In an example, any composition of the invention comprises at least 1×107 transduction particles of the invention per ml or mg. In an example, any composition of the invention comprises at least 1×108 transduction particles of the invention per ml or mg. In an example, any composition of the invention comprises at least 1×109 transduction particles of the invention per ml or mg. In an example, any composition of the invention comprises at least 1×1010 transduction particles of the invention per ml or mg. In an example, any composition of the invention comprises at least 1×1011 transduction particles of the invention per ml or mg. In an example, any composition of the invention comprises at least 1×1012 transduction particles of the invention per ml or mg. In an example, any composition of the invention comprises at least 1×1013 transduction particles of the invention per ml or mg. In an example, any composition of the invention comprises at least 1×1014 transduction particles of the invention per ml or mg.
In an example, any composition of the invention comprises up to 1×1014 transduction particles of the invention per ml or mg, such as when the composition is comprised by a fluid (eg, a liquid) or solid. In an example, any composition of the invention comprises up to 1×1013 transduction particles of the invention per ml or mg. In an example, any composition of the invention comprises up to 1×1012 transduction particles of the invention per ml or mg. In an example, any composition of the invention comprises up to 1×1011 transduction particles of the invention per ml or mg. In an example, any composition of the invention comprises up to 1×1010 transduction particles of the invention per ml or mg. In an example, any composition of the invention comprises up to 1×109 transduction particles of the invention per ml or mg.
In an example, any composition of the invention comprises at least 1×103 to 1×1010, 1×1011, 1×1012, 1×1013 or 1×1014 transduction particles of the invention per ml or mg, such as when the composition is comprised by a fluid (eg, a liquid) or solid. In an example, any composition of the invention comprises at least 1×104 to 1×1010, 1×1011, 1×1012, 1×1013 or 1×1014 transduction particles of the invention per ml or mg. In an example, any composition of the invention comprises at least 1×105 to 1×1010, 1×1011, 1×1012, 1×1013 or 1×1014 transduction particles of the invention per ml or mg. In an example, any composition of the invention comprises at least 1×106 to 1×1010, 1×1011, 1×1012, 1×1013 or 1×1014 transduction particles of the invention per ml or mg. In an example, any composition of the invention comprises at least 1×107 to 1×1010, 1×1011, 1×1012, 1×1013 or 1×1014 transduction particles of the invention per ml or mg. In an example, any composition of the invention comprises at least 1×108 to 1×1010, 1×1011, 1×1012, 1×1013 or 1×1014 transduction particles of the invention per ml or mg. In an example, any composition of the invention comprises at least 1×109 to 1×1010, 1×1011, 1×1012, 1×1013 or 1×1014 transduction particles of the invention per ml or mg. In an example, any composition of the invention comprises at least 1×1010 to 1×1010, 1×1011, 1×1012, 1×1013 or 1×1014 transduction particles of the invention per ml or mg. In an example, any composition of the invention comprises at least 1×1011 to 1×1010, 1×1011, 1×1012, 1×1013 or 1×1014 transduction particles of the invention per ml or mg. In an example, any composition of the invention comprises at least 1×1012 to 1×1010, 1×1011, 1×1012, 1×1013 or 1×1014 transduction particles of the invention per ml or mg. In an example, any composition of the invention comprises at least 1×1013 to 1×1010, 1×1011, 1×1012, 1×1013 or 1×1014 transduction particles of the invention per ml or mg. In an example, any composition of the invention comprises at least 1×1014 to 1×1010, 1×1011, 1×1012, 1×1013 or 1×1014 transduction particles of the invention per ml or mg.
In an example, the composition comprises one or more doses of the transduction particles of the invention for administration to a subject for medical use, eg, to treat or prevent a disease or condition in the subject. In an example, the composition comprises a single dose. In an example, the composition comprises (or comprises at least) 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 doses. In an example, each dose is (or is at least) a 0.5, 1, 2, 3, 4, 5, 10, 20, 25, 30, 40, 50, 75, 100, 125, 200 or 250 mg or ml dose comprising said phage (ie, the dose is said amount and comprises phage and an excipient, diluent or carrier for example).
In an example, the composition comprises one or more doses of the transduction particles of the invention for administration to a subject for non-medical use, eg, for agricultural use. In an example, the composition comprises a single dose. In an example, the composition comprises (or comprises at least) 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 doses. In an example, each dose is (or is at least) a 0.5, 1, 2, 3, 4, 5, 10, 20, 25, 30, 40, 50, 75, 100, 125, 200, 250, 500, 750, 1000, 2000, 3000, 4000, 5000, 10000, 50000, 100000 mg or ml dose comprising said phage (ie, the dose is said amount and comprises phage and an excipient, diluent or carrier for example). The dose may be dissolved or diluted in a solvent (eg, an aqueous solvent or water) before use for contacting with target bacteria. In an example 1 imperial gallon comprises one dose of the transduction particles of the invention, eg, for agricultural use, such as crop spraying, or for animal or livestock use, such as use as a beverage.
Optionally, the NSI is comprised by a high copy number plasmid. Optionally, the NSI is comprised by a medium copy number plasmid. The meaning of low, medium and high copy number or and plasmids is known to the skilled addressee and these are terms of art. As is known by the skilled person, copy number denotes the average number of plasmid copies per cell. For example, a low copy number plasmid is a plasmid that exists in from 1 to 10 copies per bacterial cell in which the plasmid is harboured; a medium copy number plasmid exists in from 11 to 50 (eg, 11 to 40 or 20 to 30 or 40) copies per cell; and a high copy number is >50 (eg, up to 100, 200, 250, 300, 400, 500, 600 or 700) copies per cell. In an example, the plasmid or vector comprising first DNA is a medium copy number plasmid or vector. In an example, the plasmid or vector comprising first DNA is a high copy number plasmid or vector. An example of common on and plasmids is shown in Table 8.
This study relates to the production of transduction particles which contain a DNA sequence of interest, where the particles can usefully be used to infect target bacteria in to introduce the DNA for expression in the target bacteria. These particles can inject their DNA into the same set of bacterial strains as the original phage on which the particle design is based. Binding of the phage particles to the host cell requires the presence of one or more specific molecules on the surface of the cell, providing a phage receptor. We surprisingly see a very large increase in yield of particle production. Whilst not wishing to be bound by any theory, elimination of the phage receptor from the surface of the producer cells may prevent re-absorption of the produced particles thus increasing production yield significantly.
We used the well-studied P2 phage/Escherichia coli system as a model. To identify P2 receptor mutants, we tested a set of single knockout mutants from the KEIO collection (“Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection”, Tomoya Baba et al, DOI 10.1038/msb4100050, Molecular Systems Biology (2006) 2, 2006.0008) for P2 plaque formation in a standard soft agar overlay spot test. LB agar was prepared in petri dishes and covered by 3 ml soft agar overlay (LB+0.6% agar) containing 100 ul overnight cell culture. P2 vir phage lysate was spotted on the plates and after overnight incubation at 37° C. the plates were checked for plaque formation. We found that P2 does not plaque on a set of rfa mutants involved in LPS core biosynthesis (
To construct a receptor deletion mutant for further studies, we replaced the rfaD gene with a zeocin resistance marker in the E. coli C1a P2 lysogen using the Lambda Red system (“One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products”, Kirill A. Datsenko and Barry L. Wanner, PNAS Jun. 6, 2000 97 (12) 6640-6645; https://doi.org/10.1073/pnas.120163297). The zeocin marker of plasmid pEM7/zeo (Invitrogen) was PCR amplified using the primers rfaDupR (SEQ ID NO: 1) and rfaDdnR (SEQ ID NO: 2). E. coli C1a P2 lysogen cells were transformed with the plasmid pKD46 (GenBank: MF287367.1), carrying the Lambda Red system. The transformants were grown at 30° C. to mid log phase in LB containing 100 ug/ml ampicillin and induced with 0.4% arabinose for 2 h. Cells were washed with 20% glycerol and electroporated with the PCR fragment containing the zeo marker.
Recombinants were selected on LB zeo plates at 37° C., also eliminating the pKD46 plasmid, which has a temperature sensitive replication. Proper replacement of the rfaD gene with a zeo marker was sequence verified.
Both the parental strain (C1a P2 lysogen) and the receptor mutant (C1a P2 lysogen ΔrfaD) were transformed with a plasmid, containing (i) the arabinose inducible P4 phage transactivation region (to induce the P2 helper functions), (ii) the P4 packaging site, (iii) a spectinomycin resistance marker, and (iv) the CloDF13 replication origin.
To compare the yield of the transduction particles obtained in the two strains, overnight cell cultures were diluted 1:25 in LB medium containing 50 μg/ml Spectinomycin, 10 mM MgSO4, and 5 mM CaCl2. After 90 minutes shaking at 37° C., 0.8% arabinose was added to the cultures. Due to the induction of the chromosomal P2 phage, cells lysed after 3 hours. Cell debris was removed by centrifugation and the lysate was extracted with chloroform to remove any remaining cells.
The yield of production was quantified by measuring transduction of the spectinomycin marker. The lysates were serially diluted in LB medium containing 10 mM MgSO4, and 5 mM CaCl2 (10-fold steps) in 100 μl volume and the dilutions were mixed with 100 μl overnight E. coli C1a cell cultures. After 30 minutes at 37° C., 10 μl of each sample was spotted on LB spectinomycin plates. Colonies were counted after overnight incubation. Results are shown in
Removal of the phage receptor surprisingly increased the yield of transduction particles by more than 100 times. Therefore, this invention can greatly reduce the production cost of transduction particles or phages.
Bacillus anthracis
Bacillus subtilis
Bacillus subtilis
Bacillus thuringiensis
Lactobacillus delbrueckii
Lactobacillus plantarum
Lactobacillus plantarum
Lactococcus lactis
Lactococcus lactis
Lactococcus lactis
Listeria monocytogenes
Listeria monocytogenes
Listeria monocytogenes
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
aMonteville, Ardestani and Geller (1994) noted that since phages can also bind to glucose and galactose moieties in the cell wall, these might, to a lesser extent, be involved in the adsorption mechanism;
bPellicle is a protective polysaccharide layer that covers the cell surface of Lactococcus lactis(Chapot-Chartier et al. 2010).
Caulobacter crescentus
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Erwinia
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
K-12
Escherichia coli B
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Escherichia coli
Pseudoalteromonas
Pseudomonas aeruginosa
Pseudomonas aeruginosa
Pseudomonas aeruginosa
Pseudomonas aeruginosa
Pseudomonas syringae
Salmonella
Salmonella
Salmonella
Salmonella
Salmonella
Salmonella
Yersinia pestis
Yersinia pestis
Yersinia pestis
Yersinia pestis
Yersinia pestis
Yersinia pseudotuberculosis
Escherichia coli
Salmonella
Salmonella
Salmonella
Shigella
aSukupolvi (1984) suggested that LPS is also required for adsorption of phage Ox2 on E. coli and S. typhimurium, although the study verified that isolated OmpA is enough to inactivate the phage and that the binding is not increased with the addition of LPS to the protein.
bAccording to Rakhuba et al. (2010), TonB is not a receptor itself, but acts as a mediator of electrochemical potential transmission; Vinga et al. (2006) stated that TonB is a membrane protein required for genome entry; Letellier et al. (2004) explained that TonB is part of a protein complex involved in the energy transduction from the electron transfer chain in the cytoplasmic membrane to the outer membrane receptors and speculated that it possibly might be critical for the genome injection through its interaction with FhuA.
cRhakuba et al. (2010) mentioned proteins FhuA and TonB as the receptors for T7; Molineux (2001) reported that ‘Bayer patches’, described as adhesion sites between the cytoplasmic membrane and the outer envelope of Gram-negative bacteria, are the proposed receptors for T7.
dIn 2010 the same group suggested that the adsorption of the phage on the sugar moieties of the host is an initial interaction, and that the true receptor is a protein molecule or protein complex (Cvirkaite-Krupovic 2010).
eKdo, 2-keto-3-deoxy-octulosonic acid; Ko, D-glycero-D-talo-oct-2-ulosonic acid; Hep, heptulose (ketoheptose); Glc, glucose; Gal, galactose; GlcNAc, N-acetylglucosamine (from Filippov et al. 2011).
Salmonella
Salmonella
Salmonella
Caulobacter
crescentus
Escherichia
coli
Escherichia
coli
Pseudomonas
Pseudomonas
aeruginosa
Pseudomonas
aeruginosa
Pseudomonas
aeruginosa
Escherichia
coli
Klebsiella
Salmonella
Salmonella
Salmonella
S. enterica
P. aeruginosa
Firmicutes, Bacteroidetes, Salmonella, Klebsiella, Pseudomonas, Acintenobacter or Streptococcus cells.
Abiotrophia
Acidocella
Actinomyces
Alkalilimnicola
Aquaspirillum
Abiotrophia defectiva
Acidocella aminolytica
Actinomyces bovis
Alkalilimnicola ehrlichii
Aquaspirillum polymorphum
Acaricomes
Acidocella facilis
Actinomyces denticolens
Alkaliphilus
Aquaspirillum
Acaricomes phytoseiuli
Acidomonas
Actinomyces europaeus
Alkaliphilus oremlandii
putridiconchylium
Acetitomaculum
Acidomonas methanolica
Actinomyces georgiae
Alkaliphilus transvaalensis
Aquaspirillum serpens
Acetitomaculum ruminis
Acidothermus
Actinomyces gerencseriae
Allochromatium
Aquimarina
Acetivibrio
Acidothermus cellulolyticus
Actinomyces
Allochromatium vinosum
Aquimarina latercula
Acetivibrio cellulolyticus
Acidovorax
hordeovulneris
Alloiococcus
Arcanobacterium
Acetivibrio ethanolgignens
Acidovorax anthurii
Actinomyces howellii
Alloiococcus otitis
Arcanobacterium
Acetivibrio multivorans
Acidovorax caeni
Actinomyces hyovaginalis
Allokutzneria
haemolyticum
Acetoanaerobium
Acidovorax cattleyae
Actinomyces israelii
Allokutzneria albata
Arcanobacterium pyogenes
Acetoanaerobium noterae
Acidovorax citrulli
Actinomyces johnsonii
Altererythrobacter
Archangium
Acetobacter
Acidovorax defluvii
Actinomyces meyeri
Altererythrobacter ishigakiensis
Archangium gephyra
Acetobacter aceti
Acidovorax delafieldii
Actinomyces naeslundii
Altermonas
Arcobacter
Acetobacter cerevisiae
Acidovorax facilis
Actinomyces neuii
Altermonas haloplanktis
Arcobacter butzleri
Acetobacter cibinongensis
Acidovorax konjaci
Actinomyces odontolyticus
Altermonas macleodii
Arcobacter cryaerophilus
Acetobacter estunensis
Acidovorax temperans
Actinomyces oris
Alysiella
Arcobacter halophilus
Acetobacter fabarum
Acidovorax valerianellae
Actinomyces radingae
Alysiella crassa
Arcobacter nitrofigilis
Acetobacter ghanensis
Acinetobacter
Actinomyces slackii
Alysiella filiformis
Arcobacter skirrowii
Acetobacter indonesiensis
Acinetobacter baumannii
Actinomyces turicensis
Aminobacter
Arhodomonas
Acetobacter lovaniensis
Acinetobacter baylyi
Actinomyces viscosus
Aminobacter aganoensis
Arhodomonas aquaeolei
Acetobacter malorum
Acinetobacter bouvetii
Actinoplanes
Aminobacter aminovorans
Arsenophonus
Acetobacter nitrogenifigens
Acinetobacter calcoaceticus
Actinoplanes auranticolor
Aminobacter niigataensis
Arsenophonus
Acetobacter oeni
Acinetobacter gerneri
Actinoplanes brasiliensis
Aminobacterium
nasoniae
Acetobacter orientalis
Acinetobacter haemolyticus
Actinoplanes consettensis
Aminobacterium mobile
Arthrobacter
Acetobacter orleanensis
Acinetobacter johnsonii
Actinoplanes deccanensis
Aminomonas
Arthrobacter agilis
Acetobacter pasteurianus
Acinetobacter junii
Actinoplanes derwentensis
Aminomonas paucivorans
Arthrobacter albus
Acetobacter pornorurn
Acinetobacter lwoffi
Actinoplanes digitatis
Ammoniphilus
Arthrobacter aurescens
Acetobacter senegalensis
Acinetobacter parvus
Actinoplanes durhamensis
Ammoniphilus oxalaticus
Arthrobacter chlorophenolicus
Acetobacter xylinus
Acinetobacter radioresistens
Actinoplanes ferrugineus
Ammoniphilus oxalivorans
Arthrobacter citreus
Acetobacterium
Acinetobacter schindleri
Actinoplanes globisporus
Amphibacillus
Arthrobacter crystallopoietes
Acetobacterium bakii
Acinetobacter soli
Actinoplanes humidus
Amphibacillus xylanus
Arthrobacter cumminsii
Acetobacterium carbinolicum
Acinetobacter tandoii
Actinoplanes italicus
Amphritea
Arthrobacter globiformis
Acetobacterium dehalogenans
Acinetobacter tjernbergiae
Actinoplanes liguriensis
Amphritea balenae
Arthrobacter
Acetobacterium fimetarium
Acinetobacter towneri
Actinoplanes lobatus
Amphritea japonica
histidinolovorans
Acetobacterium malicum
Acinetobacter ursingii
Actinoplanes missouriensis
Amycolatopsis
Arthrobacter ilicis
Acetobacterium paludosum
Acinetobacter venetianus
Actinoplanes palleronii
Amycolatopsis alba
Arthrobacter luteus
Acetobacterium tundrae
Acrocarpospora
Actinoplanes philippinensis
Amycolatopsis albidoflavus
Arthrobacter methylotrophus
Acetobacterium wieringae
Acrocarpospora corrugata
Actinoplanes rectilineatus
Amycolatopsis azurea
Arthrobacter mysorens
Acetobacterium woodii
Acrocarpospora
Actinoplanes regularis
Amycolatopsis coloradensis
Arthrobacter nicotianae
Acetofilamentum
macrocephala
Actinoplanes
Amycolatopsis lurida
Arthrobacter nicotinovorans
Acetofilamentum rigidum
Acrocarpospora pleiomorpha
teichomyceticus
Amycolatopsis mediterranei
Arthrobacter oxydans
Acetohalobium
Actibacter
Actinoplanes utahensis
Amycolatopsis rifamycinica
Arthrobacter pascens
Acetohalobium arabaticum
Actibacter sediminis
Actinopolyspora
Amycolatopsis rubida
Arthrobacter
Acetomicrobium
Actinoalloteichus
Actinopolyspora halophila
Amycolatopsis sulphurea
phenanthrenivorans
Acetomicrobium faecale
Actinoalloteichus
Actinopolyspora mortivallis
Amycolatopsis tolypomycina
Arthrobacter
Acetomicrobium flavidum
cyanogriseus
Actinosynnema
Anabaena
polychromogenes
Acetonema
Actinoalloteichus
Actinosynnema mirum
Anabaena cylindrica
Atrhrobacter protophormiae
Acetonema longum
hymeniacidonis
Actinotalea
Anabaena flos-aquae
Arthrobacter
Acetothermus
Actinoalloteichus spitiensis
Actinotalea fermentans
Anabaena variabilis
psychrolactophilus
Acetothermus paucivorans
Actinobaccillus
Aerococcus
Anaeroarcus
Arthrobacter ramosus
Acholeplasma
Actinobacillus capsulatus
Aerococcus sanguinicola
Anaeroarcus burkinensis
Arthrobacter sulfonivorans
Acholeplasma axanthum
Actinobacillus delphinicola
Aerococcus urinae
Anaerobaculum
Arthrobacter sulfureus
Acholeplasma brassicae
Actinobacillus hominis
Aerococcus urinaeequi
Anaerobaculum mobile
Arthrobacter uratoxydans
Acholeplasma cavigenitalium
Actinobacillus indolicus
Aerococcus urinaehominis
Anaerobiospirillum
Arthrobacter ureafaciens
Acholeplasma equifetale
Actinobacillus lignieresii
Aerococcus viridans
Anaerobiospirillum
Arthrobacter viscosus
Acholeplasma granularum
Actinobacillus minor
Aeromicrobium
succiniciproducens
Arthrobacter woluwensis
Acholeplasma hippikon
Actinobacillus muris
Aeromicrobium erythreum
Anaerobiospirillum thomasii
Asaia
Acholeplasma laidlawii
Actinobacillus
Aeromonas
Anaerococcus
Asaia bogorensis
Acholeplasma modicum
pleuropneumoniae
Aeromonas
Anaerococcus hydrogenalis
Asanoa
Acholeplasma morum
Actinobacillus porcinus
allosaccharophila
Anaerococcus lactolyticus
Asanoa ferruginea
Acholeplasma multilocale
Actinobacillus rossii
Aeromonas bestiarum
Anaerococcus prevotii
Asticcacaulis
Acholeplasma oculi
Actinobacillus scotiae
Aeromonas caviae
Anaerococcus tetradius
Asticcacaulis biprosthecium
Acholeplasma palmae
Actinobacillus seminis
Aeromonas encheleia
Anaerococcus vaginalis
Asticcacaulis excentricus
Acholeplasma parvum
Actinobacillus succinogenes
Aeromonas
Anaerofustis
Atopobacter
Acholeplasma pleciae
Actinobaccillus suis
enteropelogenes
Anaerofustis stercorihominis
Atopobacter phocae
Acholeplasma vituli
Actinobacillus ureae
Aeromonas eucrenophila
Anaeromusa
Atopobium
Achromobacter
Actinobaculum
Aeromonas ichthiosmia
Anaeromusa acidaminophila
Atopobium fossor
Achromobacter denitrificans
Actinobaculum massiliense
Aeromonas jandaei
Anaeromyxobacter
Atopobium minutum
Achromobacter insolitus
Actinobaculum schaalii
Aeromonas media
Anaeromyxobacter
Atopobium parvulum
Achromobacter piechaudii
Actinobaculum suis
Aeromonas popoffii
dehalogenans
Atopobium rimae
Achromobacter ruhlandii
Actinomyces urinale
Aeromonas sobria
Anaerorhabdus
Atopobium vaginae
Achromobacter spanius
Actinocatenispora
Aeromonas veronii
Anaerorhabdus furcosa
Aureobacterium
Acidaminobacter
Actinocatenispora rupis
Agrobacterium
Anaerosinus
Aureobacterium barkeri
Acidaminobacter
Actinocatenispora
Agrobacterium
Anaerosinus glycerini
Aurobacterium
hydrogenoformans
thailandica
gelatinovorum
Anaerovirgula
Aurobacterium liquefaciens
Acidaminococcus
Actinocatenispora sera
Agrococcus
Anaerovirgula multivorans
Avibacterium
Acidaminococcus fermentans
Actinocorallia
Agrococcus citreus
Ancalomicrobium
Avibacterium avium
Acidaminococcus intestini
Actinocorallia aurantiaca
Agrococcus jenensis
Ancalomicrobium adetum
Avibacterium gallinarum
Acidicaldus
Actinocorallia aurea
Agromonas
Ancylobacter
Avibacterium paragallinarum
Acidicaldus organivorans
Actinocorallia cavernae
Agromonas oligotrophica
Ancylobacter aquaticus
Avibacterium volantium
Acidimicrobium
Actinocorallia glomerata
Agromyces
Aneurinibacillus
Azoarcus
Acidimicrobium ferrooxidans
Actinocorallia herbida
Agromyces fucosus
Aneurinibacillus aneurinilyticus
Azoarcus indigens
Acidiphilium
Actinocorallia libanotica
Agromyces hippuratus
Aneurinibacillus migulanus
Azoarcus tolulyticus
Acidiphilium acidophilum
Actinocorallia longicatena
Agromyces luteolus
Aneurinibacillus
Azoarcus toluvorans
Acidiphilium angustum
Actinomadura
Agromyces mediolanus
thermoaerophilus
Azohydromonas
Acidiphilium cryptum
Actinomadura alba
Agromyces ramosus
Angiococcus
Azohydromonas australica
Acidiphilium multivorum
Actinomadura atramentaria
Agromyces rhizospherae
Angiococcus disciformis
Azohydromonas lata
Acidiphilium organovorum
Actinomadura
Akkermansia
Angulomicrobium
Azomonas
Acidiphilium rubrum
bangladeshensis
Akkermansia muciniphila
Angulomicrobium tetraedrale
Azomonas agilis
Acidisoma
Actinomadura catellatispora
Albidiferax
Anoxybacillus
Azomonas insignis
Acidisoma sibiricum
Actinomadura chibensis
Albidiferax ferrireducens
Anoxybacillus pushchinoensis
Azomonas macrocytogenes
Acidisoma tundrae
Actinomadura chokoriensis
Albidovulum
Aquabacterium
Azorhizobium
Acidisphaera
Actinomadura citrea
Albidovulum inexpectatum
Aquabacterium commune
Azorhizobium caulinodans
Acidisphaera rubrifaciens
Actinomadura coerulea
Alcaligenes
Aquabacterium parvum
Azorhizophilus
Acidithiobacillus
Actinomadura echinospora
Alcaligenes denitrificans
Azorhizophilus paspali
Acidithiobacillus albertensis
Actinomadura fibrosa
Alcaligenes faecalis
Azospirillum
Acidithiobacillus caldus
Actinomadura formosensis
Alcanivorax
Azospirillum brasilense
Acidithiobacillus ferrooxidans
Actinomadura hibisca
Alcanivorax borkumensis
Azospirillum halopraeferens
Acidithiobacillus thiooxidans
Actinomadura kijaniata
Alcanivorax jadensis
Azospirillum irakense
Acidobacterium
Actinomadura latina
Algicola
Azotobacter
Acidobacterium capsulatum
Actinomadura livida
Algicola bacteriolytica
Azotobacter beijerinckii
Actinomadura
Alicyclobacillus
Azotobacter chroococcum
luteofluorescens
Alicyclobacillus
Azotobacter nigricans
Actinomadura macra
disulfidooxidans
Azotobacter salinestris
Actinomadura madurae
Alicyclobacillus
Azotobacter vinelandii
Actinomadura oligospora
sendaiensis
Actinomadura pelletieri
Alicyclobacillus vulcanalis
Actinomadura rubrobrunea
Alishewanella
Actinomadura rugatobispora
Alishewanella fetalis
Actinomadura umbrina
Alkalibacillus
Actinomadura
Alkalibacillus
verrucosospora
haloalkaliphilus
Actinomadura vinacea
Actinomadura viridilutea
Actinomadura viridis
Actinomadura yumaensis
Bacillus
Bacteroides
Bibersteinia
Borrelia
Brevinema
Bacteroides caccae
Bibersteinia trehalosi
Borrelia afzelii
Brevinema andersonii
Bacteriovorax
Bacteroides coagulans
Bifidobacterium
Borrelia americana
Brevundimonas
Bacteriovorax stolpii
Bacteroides eggerthii
Bifidobacterium adolescentis
Borrelia burgdorferi
Brevundimonas alba
Bacteroides fragilis
Bifidobacterium angulatum
Borrelia carolinensis
Brevundimonas aurantiaca
Bacteroides galacturonicus
Bifidobacterium animalis
Borrelia coriaceae
Brevundimonas diminuta
Bacteroides helcogenes
Bifidobacterium asteroides
Borrelia garinii
Brevundimonas intermedia
Bacteroides ovatus
Bifidobacterium bifidum
Borrelia japonica
Brevundimonas subvibrioides
Bacteroides pectinophilus
Bifidobacterium boum
Bosea
Brevundimonas vancanneytii
Bacteroides pyogenes
Bifidobacterium breve
Bosea minatitlanensis
Brevundimonas variabilis
Bacteroides salyersiae
Bifidobacterium catenulatum
Bosea thiooxidans
Brevundimonas vesicularis
Bacteroides stercoris
Bifidobacterium choerinum
Brachybacterium
Brochothrix
Bacteroides suis
Bifidobacterium coryneforme
Brachybacterium
Brochothrix campestris
Bacteroides tectus
Bifidobacterium cuniculi
alimentarium
Brochothrix thermosphacta
Bacteroides thetaiotaomicron
Bifidobacterium dentium
Brachybacterium faecium
Brucella
Bacteroides uniformis
Bifidobacterium gallicum
Brachybacterium
Brucella canis
Bacteroides ureolyticus
Bifidobacterium gallinarum
paraconglomeratum
Brucella neotomae
Bacteroides vulgatus
Bifidobacterium indicum
Brachybacterium rhamnosum
Bryobacter
Balnearium
Bifidobacterium longum
Brachybacterium
Bryobacter aggregatus
Balnearium lithotrophicum
Bifidobacterium
tyrofermentans
Burkholderia
Balneatrix
magnumBifidobacterium
Brachyspira
Burkholderia ambifaria
Balneatrix alpica
merycicum
Brachyspira alvinipulli
Burkholderia andropogonis
Balneola
Bifidobacterium minimum
Brachyspira hyodysenteriae
Burkholderia anthina
Balneola vulgaris
Bifidobacterium
Brachyspira innocens
Burkholderia caledonica
Barnesiella
pseudocatenulatum
Brachyspira murdochii
Burkholderia caryophylli
Barnesiella viscericola
Bifidobacterium
Brachyspira
Burkholderia cenocepacia
Bartonella
pseudolongum
pilosicoli
Burkholderia cepacia
Bartonella alsatica
Bifidobacterium pullorum
Bradyrhizobium
Burkholderia cocovenenans
Bartonella bacilliformis
Bifidobacterium ruminantium
Bradyrhizobium canariense
Burkholderia dolosa
Bartonella clarridgeiae
Bifidobacterium saeculare
Bradyrhizobium elkanii
Burkholderia fungorum
Bartonella doshiae
Bifidobacterium subtile
Bradyrhizobium japonicum
Burkholderia glathei
Bartonella elizabethae
Bifidobacterium
Bradyrhizobium liaoningense
Burkholderia glumae
Bartonella grahamii
thermophilum
Brenneria
Burkholderia graminis
Bartonella henselae
Bilophila
Brenneria alni
Burkholderia kururiensis
Bartonella rochalimae
Bilophila wadsworthia
Brenneria nigrifluens
Burkholderia multivorans
Bartonella vinsonii
Biostraticola
Brenneria quercina
Burkholderia phenazinium
Bavariicoccus
Biostraticola tofi
Brenneria quercina
Burkholderia plantarii
Bavariicoccus seileri
Bizionia
Brenneria salicis
Burkholderia pyrrocinia
Bdellovibrio
Bizionia argentinensis
Brevibacillus
Burkholderia silvatlantica
Bdellovibrio bacteriovorus
Blastobacter
Brevibacillus agri
Burkholderia stabilis
Bdellovibrio exovorus
Blastobacter capsulatus
Brevibacillus borstelensis
Burkholderia thailandensis
Beggiatoa
Blastobacter denitrificans
Brevibacillus brevis
Burkholderia tropica
Beggiatoa alba
Blastococcus
Brevibacillus centrosporus
Burkholderia unamae
Beijerinckia
Blastococcus aggregatus
Brevibacillus choshinensis
Burkholderia vietnamiensis
Beijerinckia derxii
Blastococcus saxobsidens
Brevibacillus invocatus
Buttiauxella
Beijerinckia fluminensis
Blastochloris
Brevibacillus laterosporus
Buttiauxella agrestis
Beijerinckia indica
Blastochloris viridis
Brevibacillus parabrevis
Buttiauxella brennerae
Beijerinckia mobilis
Blastomonas
Brevibacillus reuszeri
Buttiauxella ferragutiae
Belliella
Blastomonas natatoria
Brevibacterium
Buttiauxella gaviniae
Belliella baltica
Blastopirellula
Brevibacterium abidum
Buttiauxella izardii
Bellilinea
Blastopirellula marina
Brevibacterium album
Buttiauxella noackiae
Bellilinea caldifistulae
Blautia
Brevibacterium aurantiacum
Buttiauxella warmboldiae
Belnapia
Blautia coccoides
Brevibacterium celere
Butyrivibrio
Belnapia moabensis
Blautia hansenii
Brevibacterium epidermidis
Butyrivibrio fibrisolvens
Bergeriella
Blautia producta
Brevibacterium
Butyrivibrio hungatei
Bergeriella denitrificans
Blautia wexlerae
frigoritolerans
Butyrivibrio proteoclasticus
Beutenbergia
Bogoriella
Brevibacterium halotolerans
Beutenbergia cavernae
Bogoriella caseilytica
Brevibacterium iodinum
Bordetella
Brevibacterium linens
Bordetella avium
Brevibacterium lyticum
Bordetella bronchiseptica
Brevibacterium mcbrellneri
Bordetella hinzii
Brevibacterium otitidis
Bordetella holmesii
Brevibacterium oxydans
Bordetella parapertussis
Brevibacterium paucivorans
Bordetella pertussis
Brevibacterium stationis
Bordetella petrii
Bordetella trematum
Bacillus
B. acidiceler
B. aminovorans
B. glucanolyticus
B. taeanensis
B. lautus
B. acidicola
B. amylolyticus
B. gordonae
B. tequilensis
B. lehensis
B. acidiproducens
B. andreesenii
B. gottheilii
B. thermantarcticus
B. lentimorbus
B. acidocaldarius
B. aneurinilyticus
B. graminis
B. thermoaerophilus
B. lentus
B. acidoterrestris
B. anthracis
B. halmapalus
B. thermoamylovorans
B. licheniformis
B. aeolius
B. aquimaris
B. haloalkaliphilus
B. thermocatenulatus
B. ligniniphilus
B. aerius
B. arenosi
B. halochares
B. thermocloacae
B. litoralis
B. aerophilus
B. arseniciselenatis
B. halodenitrificans
B. thermocopriae
B. locisalis
B. agaradhaerens
B. arsenicus
B. halodurans
B. thermodenitrificans
B. luciferensis
B. agri
B. aurantiacus
B. halophilus
B. thermoglucosidasius
B. luteolus
B. aidingensis
B. arvi
B. halosaccharovorans
B. thermolactis
B. luteus
B. akibai
B. aryabhattai
B. hemicellulosilyticus
B. thermoleovorans
B. macauensis
B. alcalophilus
B. asahii
B. hemicentroti
B. thermophilus
B. macerans
B. algicola
B. atrophaeus
B. herbersteinensis
B. thermoruber
B. macquariensis
B. alginolyticus
B. axarquiensis
B. horikoshii
B. thermosphaericus
B. macyae
B. alkalidiazotrophicus
B. azotofixans
B. horneckiae
B. thiaminolyticus
B. malacitensis
B. alkalinitrilicus
B. azotoformans
B. horti
B. thioparans
B. mannanilyticus
B. alkalisediminis
B. badius
B. huizhouensis
B. thuringiensis
B. marisflavi
B. alkalitelluris
B. barbaricus
B. humi
B. tianshenii
B. marismortui
B. altitudinis
B. bataviensis
B. hwajinpoensis
B. trypoxylicola
B. marmarensis
B. alveayuensis
B. beijingensis
B. idriensis
B. tusciae
B. massiliensis
B. alvei
B. benzoevorans
B. indicus
B. validus
B. megaterium
B. amyloliquefaciens
B. beringensis
B. infantis
B. vallismortis
B. mesonae
B. berkeleyi
B. infernus
B. vedderi
B. methanolicus
B. beveridgei
B. insolitus
B. velezensis
B. methylotrophicus
B. bogoriensis
B. invictae
B. vietnamensis
B. migulanus
B. boroniphilus
B. iranensis
B. vireti
B. mojavensis
B. dipsosauri
B. borstelensis
B. isabeliae
B. vulcani
B. mucilaginosus
B. drentensis
B. brevis Migula
B. isronensis
B. wakoensis
B. muralis
B. edaphicus
B. butanolivorans
B. jeotgali
B. weihenstephanensis
B. murimartini
B. ehimensis
B. canaveralius
B. kaustophilus
B. xiamenensis
B. mycoides
B. eiseniae
B. carboniphilus
B. kobensis
B. xiaoxiensis
B. naganoensis
B. enclensis
B. cecembensis
B. kochii
B. zhanjiangensis
B. nanhaiensis
B. endophyticus
B. cellulosilyticus
B. kokeshiiformis
B. peoriae
B. nanhaiisediminis
B. endoradicis
B. centrosporus
B. koreensis
B. persepolensis
B. nealsonii
B. farraginis
B. cereus
B. korlensis
B. persicus
B. neidei
B. fastidiosus
B. chagannorensis
B. kribbensis
B. pervagus
B. neizhouensis
B. fengqiuensis
B. chitinolyticus
B. krulwichiae
B. plakortidis
B. niabensis
B. firmus
B. chondroitinus
B. laevolacticus
B. pocheonensis
B. niacini
B. flexus
B. choshinensis
B. larvae
B. polygoni
B. novalis
B. foraminis
B. chungangensis
B. laterosporus
B. polymyxa
B. oceanisediminis
B. fordii
B. cibi
B. salexigens
B. popilliae
B. odysseyi
B. formosus
B. circulans
B. saliphilus
B. pseudalcalophilus
B. okhensis
B. fortis
B. clarkii
B. schlegelii
B. pseudofirmus
B. okuhidensis
B. fumarioli
B. clausii
B. sediminis
B. pseudomycoides
B. oleronius
B. funiculus
B. coagulans
B. selenatarsenatis
B. psychrodurans
B. oryzaecorticis
B. fusiformis
B. coahuilensis
B. selenitireducens
B. psychrophilus
B. oshimensis
B. galactophilus
B. cohnii
B. seohaeanensis
B. psychrosaccharolyticus
B. pabuli
B. galactosidilyticus
B. composti
B. shacheensis
B. psychrotolerans
B. pakistanensis
B. galliciensis
B. curdlanolyticus
B. shackletonii
B. pulvifaciens
B. pallidus
B. gelatini
B. cycloheptanicus
B. siamensis
B. pumilus
B. pallidus
B. gibsonii
B. cytotoxicus
B. silvestris
B. purgationiresistens
B. panacisoli
B. ginsengi
B. daliensis
B. simplex
B. pycnus
B. panaciterrae
B. ginsengihumi
B. decisifrondis
B. siralis
B. qingdaonensis
B. pantothenticus
B. ginsengisoli
B. decolorationis
B. smithii
B. qingshengii
B. parabrevis
B. globisporus (eg, B.
B. deserti
B. soli
B. reuszeri
B. paraflexus
B. solimangrovi
B. rhizosphaerae
B. pasteurii
B. solisalsi
B. rigui
B. patagoniensis
B. songklensis
B. ruris
B. sonorensis
B. safensis
B. sphaericus
B. salarius
B. sporothermodurans
B. stearothermophilus
B. stratosphericus
B. subterraneus
B. subtilis (eg, B.
Caenimonas
Campylobacter
Cardiobacterium
Catenuloplanes
Curtobacterium
Caenimonas koreensis
Campylobacter coli
Cardiobacterium hominis
Catenuloplanes atrovinosus
Curtobacterium albidum
Caldalkalibacillus
Campylobacter concisus
Carnimonas
Catenuloplanes castaneus
Curtobacterium citreus
Caldalkalibacillus uzonensis
Campylobacter curvus
Carnimonas nigrificans
Catenuloplanes crispus
Caldanaerobacter
Campylobacter fetus
Carnobacterium
Catenuloplanes indicus
Caldanaerobacter subterraneus
Campylobacter gracilis
Carnobacterium alterfunditum
Catenuloplanes japonicus
Caldanaerobius
Campylobacter helveticus
Carnobacterium divergens
Catenuloplanes nepalensis
Caldanaerobius fijiensis
Campylobacter hominis
Carnobacterium funditum
Catenuloplanes niger
Caldanaerobius
Campylobacter hyointestinalis
Carnobacterium gallinarum
Chryseobacterium
polysaccharolyticus
Campylobacter jejuni
Carnobacterium
Chryseobacterium
Caldanaerobius zeae
Campylobacter lari
maltaromaticum
balustinum
Caldanaerovirga
Campylobacter mucosalis
Carnobacterium mobile
Citrobacter
Caldanaerovirga acetigignens
Campylobacter rectus
Carnobacterium viridans
C. amalonaticus
Caldicellulosiruptor
Campylobacter showae
Caryophanon
C. braakii
Caldicellulosiruptor bescii
Campylobacter sputorum
Caryophanon latum
C. diversus
Caldicellulosiruptor kristjanssonii
Campylobacter upsaliensis
Caryophanon tenue
C. farmeri
Caldicellulosiruptor owensensis
Capnocytophaga
Catellatospora
C. freundii
Capnocytophaga canimorsus
Catellatospora citrea
C. gillenii
Capnocytophaga cynodegmi
Catellatospora
C. koseri
Capnocytophaga gingivalis
methionotrophica
C. murliniae
Capnocytophaga granulosa
Catenococcus
C. pasteurii
[1]
Capnocytophaga haemolytica
Catenococcus thiocycli
C. rodentium
Capnocytophaga ochracea
C. sedlakii
Capnocytophaga sputigena
C. werkmanii
C. youngae
Clostridium
Coccochloris
Coccochloris elabens
Corynebacterium
Corynebacterium flavescens
Corynebacterium variabile
Clostridium
Clostridium absonum, Clostridium aceticum, Clostridium acetireducens, Clostridium acetobutylicum, Clostridium acidisoli, Clostridium aciditolerans, Clostridium acidurici, Clostridium aerotolerans, Clostridium
aestuarii, Clostridium akagii, Clostridium aldenense, Clostridium aldrichii, Clostridium algidicarni, Clostridium algidixylanolyticum, Clostridium algifaecis, Clostridium algoriphilum, Clostridium alkalicellulosi,
Clostridium aminophilum, Clostridium aminovalericum, Clostridium amygdalinum, Clostridium amylolyticum, Clostridium arbusti, Clostridium arcticum, Clostridium argentinense, Clostridium asparagiforme,
Clostridium aurantibutyricum, Clostridium autoethanogenum, Clostridium baratii, Clostridium barkeri, Clostridium bartlettii, Clostridium beijerinckii, Clostridium bifermentans, Clostridium bolteae, Clostridium
bornimense, Clostridium botulinum, Clostridium bowmanii, Clostridium bryantii, Clostridium butyricum, Clostridium cadaveris, Clostridium caenicola, Clostridium caminithermale, Clostridium carboxidivorans,
Clostridium carnis, Clostridium cavendishii, Clostridium celatum, Clostridium celerecrescens, Clostridium cellobioparum, Clostridium cellulofermentans, Clostridium cellulolyticum, Clostridium cellulosi,
Clostridium cellulovorans, Clostridium chartatabidum, Clostridium chauvoei, Clostridium chromiireducens, Clostridium citroniae, Clostridium clariflavum, Clostridium clostridioforme, Clostridium coccoides,
Clostridium cochlearium, Clostridium colletant, Clostridium colicanis, Clostridium colinum, Clostridium collagenovorans, Clostridium cylindrosporum, Clostridium difficile, Clostridium diolis, Clostridium
disporicum, Clostridium drakei, Clostridium durum, Clostridium estertheticum, Clostridium estertheticum estertheticum, Clostridium estertheticum laramiense, Clostridium fallax, Clostridium felsineum, Clostridium
fervidum, Clostridium fimetarium, Clostridium formicaceticum, Clostridium frigidicarnis, Clostridium frigoris, Clostridium ganghwense, Clostridium gasigenes, Clostridium ghonii, Clostridium glycolicum,
Clostridium glycyrrhizinilyticum, Clostridium grantii, Clostridium haemolyticum, Clostridium halophilum, Clostridium hastiforme, Clostridium hathewayi, Clostridium herbivorans, Clostridium hiranonis,
Clostridium histolyticum, Clostridium homopropionicum, Clostridium huakuii, Clostridium hungatei, Clostridium hydrogeniformans, Clostridium hydroxybenzoicum, Clostridium hylemonae, Clostridium jejuense,
Clostridium indolis, Clostridium innocuum, Clostridium intestinale, Clostridium irregulare, Clostridium isatidis, Clostridium josui, Clostridium kluyveri, Clostridium lactatifermentans, Clostridium lacusfryxellense,
Clostridium laramiense, Clostridium lavalense, Clostridium lentocellum, Clostridium lentoputrescens, Clostridium leptum, Clostridium limosum, Clostridium litorale, Clostridium lituseburense, Clostridium ljungdahlii,
Clostridium lortetii, Clostridium lundense, Clostridium magnum, Clostridium malenominatum, Clostridium mangenotii, Clostridium mayombei, Clostridium methoxybenzovorans, Clostridium methylpentosum,
Clostridium neopropionicum, Clostridium nexile, Clostridium nitrophenolicum, Clostridium novyi, Clostridium oceanicum, Clostridium orbiscindens, Clostridium oroticum, Clostridium oxalicum, Clostridium
papyrosolvens, Clostridium paradoxum, Clostridium paraperfringens (Alias: C. welchii), Clostridium paraputrificum, Clostridium pascui, Clostridium pasteurianum, Clostridium peptidivorans, Clostridium perenne,
Clostridium perfringens, Clostridium pfennigii, Clostridium phytofermentans, Clostridium piliforme, Clostridium polysaccharolyticum, Clostridium populeti, Clostridium propionicum, Clostridium proteoclasticum,
Clostridium proteolyticum, Clostridium psychrophilum, Clostridium puniceum, Clostridium purinilyticum, Clostridium putrefaciens, Clostridium putrificum, Clostridium quercicolum, Clostridium quinii,
Clostridium ramosum, Clostridium rectum, Clostridium roseum, Clostridium saccharobutylicum, Clostridium saccharogumia, Clostridium saccharolyticum, Clostridium saccharoperbutylacetonicum, Clostridium
sardiniense, Clostridium sartagoforme, Clostridium scatologenes, Clostridium schirmacherense, Clostridium scindens, Clostridium septicum, Clostridium sordellii, Clostridium sphenoides, Clostridium spiroforme,
Clostridium sporogenes, Clostridium sporosphaeroides, Clostridium stercorarium, Clostridium stercorarium leptospartum, Clostridium stercorarium stercorarium, Clostridium stercorarium thermolacticum,
Clostridium sticklandii, Clostridium straminisolvens, Clostridium subterminale, Clostridium sufflavum, Clostridium sulfidigenes, Clostridium symbiosum, Clostridium tagluense, Clostridium
tepidiprofundi, Clostridium termitidis, Clostridium tertium, Clostridium tetani, Clostridium tetanomorphum, Clostridium thermaceticum, Clostridium thermautotrophicum, Clostridium thermoalcaliphilum,
Clostridium thermobutyricum, Clostridium thermocellum, Clostridium thermocopriae, Clostridium thermohydrosulfuricum, Clostridium thermolacticum, Clostridium thermopalmarium,
Clostridium thermopapyrolyticum, Clostridium thermosaccharolyticum, Clostridium thermosuccinogenes, Clostridium thermosulfurigenes, Clostridium thiosulfatireducens, Clostridium tyrobutyricum,
Clostridium uliginosum, Clostridium ultunense, Clostridium villosum, Clostridium vincentii, Clostridium viride, Clostridium xylanolyticum, Clostridium xylanovorans
Dactylosporangium
Deinococcus
Delftia
Echinicola
Dactylosporangium aurantiacum
Deinococcus aerius
Delftia acidovorans
Echinicola pacifica
Dactylosporangium fulvum
Deinococcus apachensis
Desulfovibrio
Echinicola vietnamensis
Dactylosporangium matsuzakiense
Deinococcus aquaticus
Desulfovibrio desulfuricans
Dactylosporangium roseum
Deinococcus aquatilis
Diplococcus
Dactylosporangium thailandense
Deinococcus caeni
Diplococcus pneumoniae
Dactylosporangium vinaceum
Deinococcus radiodurans
Deinococcus radiophilus
Enterobacter
Enterobacter kobei
Faecalibacterium
Flavobacterium
E. aerogenes
E. ludwigii
Faecalibacterium prausnitzii
Flavobacterium antarcticum
E. amnigemis
E. mori
Fangia
Flavobacterium aquatile
E. agglomerans
E. nimipressuralis
Fangia hongkongensis
Flavobacterium aquidurense
E. arachidis
E. oryzae
Fastidiosipila
Flavobacterium balustinum
E. asburiae
E. pulveris
Fastidiosipila sanguinis
Flavobacterium croceum
E. cancerogenous
E. pyrinus
Fusobacterium
Flavobacterium cucumis
E. cloacae
E. radicincitans
Fusobacterium nucleatum
Flavobacterium daejeonense
E. cowanii
E. taylorae
Flavobacterium defluvii
E. dissolvens
E. turicensis
Flavobacterium degerlachei
E. gergoviae
E. sakazakii Enterobacter soli
Flavobacterium
E. helveticus
Enterococcus
denitrificans
E. hormaechei
Enterococcus durans
Flavobacterium filum
E. intermedins
Enterococcus faecalis
Flavobacterium flevense
Enterococcus faecium
Flavobacterium frigidarium
Erwinia
Flavobacterium mizutaii
Erwinia hapontici
Flavobacterium
Escherichia
okeanokoites
Escherichia coli
Gaetbulibacter
Haemophilus
Ideonella
Janibacter
Gaetbulibacter saemankumensis
Haemophilus aegyptius
Ideonella azotifigens
Janibacter anophelis
Gallibacterium
Haemophilus aphrophilus
Idiomarina
Janibacter corallicola
Gallibacterium anatis
Haemophilus felis
Idiomarina abyssalis
Janibacter limosus
Gallicola
Haemophilus gallinarum
Idiomarina baltica
Janibacter melonis
Gallicola barnesae
Haemophilus haemolyticus
Idiomarina fontislapidosi
Janibacter terrae
Garciella
Haemophilus influenzae
Idiomarina loihiensis
Jannaschia
Garciella nitratireducens
Haemophilus paracuniculus
Idiomarina ramblicola
Jannaschia cystaugens
Geobacillus
Haemophilus parahaemolyticus
Idiomarina seosinensis
Jannaschia helgolandensis
Geobacillus thermoglucosidasius
Haemophilus parainfluenzae
Idiomarina zobellii
Jannaschia
Geobacillus stearothermophilus
Haemophilus
Ignatzschineria
pohangensis
Geobacter
paraphrohaemolyticus
Ignatzschineria
Jannaschia rubra
Geobacter bemidjiensis
Haemophilus parasuis
larvae
Janthinobacterium
Geobacter bremensis
Haemophilus pittmaniae
Ignavigranum
Janthinobacterium
Geobacter chapellei
Hafnia
Ignavigranum ruoffiae
agaricidamnosum
Geobacter grbiciae
Hafnia alvei
Ilumatobacter
Janthinobacterium lividum
Geobacter hydrogenophilus
Hahella
Ilumatobacter fluminis
Jejuia
Geobacter lovleyi
Hahella ganghwensis
Ilyobacter
Jejuia pallidilutea
Geobacter metallireducens
Halalkalibacillus
Ilyobacter delafieldii
Jeotgalibacillus
Geobacter pelophilus
Halalkalibacillus halophilus
Ilyobacter insuetus
Jeotgalibacillus
Geobacter pickeringii
Helicobacter
Ilyobacter polytropus
alimentarius
Geobacter sulfurreducens
Helicobacter pylori
Ilyobacter tartaricus
Jeotgalicoccus
Geodermatophilus
Jeotgalicoccus halotolerans
Geodermatophilus obscurus
Gluconacetobacter
Gluconacetobacter xylinus
Gordonia
Gordonia rubripertincta
Kaistia
Labedella
Listeria ivanovii
Micrococcus
Nesterenkonia
Kaistia adipata
Labedella gwakjiensis
L. marthii
Micrococcus luteus
Nesterenkonia holobia
Kaistia soli
Labrenzia
L. monocytogenes
Micrococcus lylae
Nocardia
Kangiella
Labrenzia aggregata
L. newyorkensis
Moraxella
Nocardia argentinensis
Kangiella aquimarina
Labrenzia alba
L. riparia
Moraxella bovis
Nocardia corallina
Kangiella
Labrenzia alexandrii
L. rocourtiae
Moraxella nonliquefaciens
Nocardia
koreensis
Labrenzia marina
L. seeligeri
Moraxella osloensis
otitidiscaviarum
Kerstersia
Labrys
L. weihenstephanensis
Nakamurella
Kerstersia gyiorum
Labrys methylaminiphilus
L. welshimeri
Nakamurella multipartita
Kiloniella
Labrys miyagiensis
Listonella
Nannocystis
Kiloniella laminariae
Labrys monachus
Listonella anguillarum
Nannocystis pusilia
Klebsiella
Labrys okinawensis
Macrococcus
Natranaerobius
K. gramilomatis
Labrys
Macrococcus bovicus
Natranaerobius
K. oxytoca
portucalensis
Marinobacter
thermophilus
K. pneumoniae
Lactobacillus
Marinobacter algicola
Natranaerobius trueperi
K. terrigena
Marinobacter bryozoorum
Naxibacter
K. variicola
Laceyella
Marinobacter flavimaris
Naxibacter alkalitolerans
Kluyvera
Laceyella putida
Meiothermus
Neisseria
Kluyvera ascorbata
Lechevalieria
Meiothermus ruber
Neisseria cinerea
Kocuria
Lechevalieria aerocolonigenes
Methylophilus
Neisseria denitrificans
Kocuria roasea
Legionella
Methylophilus methylotrophus
Neisseria gonorrhoeae
Kocuria varians
Microbacterium
Neisseria lactamica
Kurthia
Listeria
Microbacterium
Neisseria mucosa
Kurthia zopfii
L. aquatica
ammoniaphilum
Neisseria sicca
L. booriae
Microbacterium arborescens
Neisseria subflava
L. cornellensis
Microbacterium liquefaciens
Neptunomonas
L. fleischmannii
Microbacterium oxydans
Neptunomonas japonica
L. floridensis
L. grandensis
L. grayi
L. innocua
Lactobacillus
L. acetotolerans
L. catenaformis
L. mali
L. parakefiri
L. sakei
L. acidifarinae
L. ceti
L. manihotivorans
L. paralimentarius
L. salivarius
L. acidipiscis
L. coleohominis
L. mindensis
L. paraplantarum
L. sanfranciscensis
L. acidophilus
L. collinoides
L. mucosae
L. pentosus
L. satsumensis
Lactobacillus agilis
L. composti
L. murinus
L. perolens
L. secaliphilus
L. algidus
L. concavus
L. nagelii
L. plantarum
L. sharpeae
L. alimentarius
L. coryniformis
L. namurensis
L. pontis
L. siliginis
L. amylolyticus
L. crispatus
L. nantensis
L. protectus
L. spicheri
L. amylophilus
L. crustorum
L. oligofermentans
L. psittaci
L. suebicus
L. amylotrophicus
L. curvatus
L. oris
L. rennini
L. thailandensis
L. amylovorus
L. delbrueckii subsp. bulgaricus
L. panis
L. reuteri
L. ultunensis
L. animalis
L. delbrueckii subsp.
L. pantheris
L. rhamnosus
L. vaccinostercus
L. antri
delbrueckii
L. parabrevis
L. rimae
L. vaginalis
L. apodemi
L. delbrueckii subsp. lactis
L. parabuchneri
L. rogosae
L. versmoldensis
L. aviarius
L. dextrinicus
L. paracasei
L. rossiae
L. vini
L. bifermentans
L. diolivorans
L. paracollinoides
L. ruminis
L. vitulinus
L. brevis
L. equi
L. parafarraginis
L. saerimneri
L. zeae
L. buchneri
L. equigenerosi
L. homohiochii
L. jensenii
L. zymae
L. camelliae
L. farraginis
L. iners
L. johnsonii
L. gastricus
L. casei
L. farciminis
L. ingluviei
L. kalixensis
L. ghanensis
L. kitasatonis
L. fermentum
L. intestinalis
L. kefiranofaciens
L. graminis
L. kunkeei
L. fornicalis
L. fuchuensis
L. kefiri
L. hammesii
L. leichmannii
L. fructivorans
L. gallinarum
L. kimchii
L. hamsteri
L. lindneri
L. frumenti
L. gasseri
L. helveticus
L. harbinensis
L. malefermentans
L. hilgardii
L. hayakitensis
Legionella
Legionella adelaidensis
Legionella drancourtii
Candidatus Legionella jeonii
Legionella quinlivanii
Legionella anisa
Legionella dresdenensis
Legionella jordanis
Legionella rowbothamii
Legionella beliardensis
Legionella drozanskii
Legionella lansingensis
Legionella rubrilucens
Legionella birminghamensis
Legionella dumoffii
Legionella londiniensis
Legionella sainthelensi
Legionella bozemanae
Legionella erythra
Legionella longbeachae
Legionella santicrucis
Legionella brunensis
Legionella fairfieldensis
Legionella lytica
Legionella shakespearei
Legionella busanensis
Legionella fallonii
Legionella maceachernii
Legionella spiritensis
Legionella cardiaca
Legionella feeleii
Legionella massiliensis
Legionella steelei
Legionella cherrii
Legionella geestiana
Legionella micdadei
Legionella steigerwaltii
Legionella cincinnatiensis
Legionella genomospecies
Legionella monrovica
Legionella taurinensis
Legionella clemsonensis
Legionella gormanii
Legionella moravica
Legionella tucsonensis
Legionella donaldsonii
Legionella gratiana
Legionella nagasakiensis
Legionella tunisiensis
Legionella gresilensis
Legionella nautarum
Legionella wadsworthii
Legionella hackeliae
Legionella norrlandica
Legionella waltersii
Legionella impletisoli
Legionella oakridgensis
Legionella worsleiensis
Legionella israelensis
Legionella parisiensis
Legionella yabuuchiae
Legionella jamestowniensis
Legionella pittsburghensis
Legionella pneumophila
Legionella quateirensis
Oceanibulbus
Paenibacillus
Prevotella
Quadrisphaera
Oceanibulbus indolifex
Paenibacillus thiaminolyticus
Prevotella albensis
Quadrisphaera
Oceanicaulis
Pantoea
Prevotella amnii
granulorum
Oceanicaulis alexandrii
Pantoea
Prevotella bergensis
Quatrionicoccus
Oceanicola
agglomerans
Prevotella bivia
Quatrionicoccus
Oceanicola batsensis
Paracoccus
Prevotella brevis
australiensis
Oceanicola granulosus
Paracoccus alcaliphilus
Prevotella bryantii
Quinella
Oceanicola nanhaiensis
Paucimonas
Prevotella buccae
Quinella
Oceanimonas
Paucimonas lemoignei
Prevotella buccalis
ovalis
Oceanimonas baumannii
Pectobacterium
Prevotella copri
Ralstonia
Oceaniserpentilla
Pectobacterium aroidearum
Prevotella dentalis
Ralstonia eutropha
Oceaniserpentilla haliotis
Pectobacterium atrosepticum
Prevotella denticola
Ralstonia insidiosa
Oceanisphaera
Pectobacterium betavasculorum
Prevotella disiens
Ralstonia mannitolilytica
Oceanisphaera donghaensis
Pectobacterium cacticida
Prevotella histicola
Ralstonia pickettii
Oceanisphaera litoralis
Pectobacterium carnegieana
Prevotella intermedia
Ralstonia
Oceanithermus
Pectobacterium carotovorum
Prevotella maculosa
pseudosolanacearum
Oceanithermus desulfurans
Pectobacterium chrysanthemi
Prevotella marshii
Ralstonia syzygii
Oceanithermus profundus
Pectobacterium cypripedii
Prevotella melaninogenica
Ralstonia solanacearum
Oceanobacillus
Pectobacterium rhapontici
Prevotella micans
Ramlibacter
Oceanobacillus caeni
Pectobacterium wasabiae
Prevotella multiformis
Ramlibacter henchirensis
Oceanospirillum
Planococcus
Prevotella nigrescens
Ramlibacter
Oceanospirillum linum
Planococcus citreus
Prevotella oralis
tataouinensis
Planomicrobium
Prevotella oris
Raoultella
Planomicrobium okeanokoites
Prevotella oulorum
Raoultella ornithinolytica
Plesiomonas
Prevotella pallens
Raoultella planticola
Plesiomonas shigelloides
Prevotella salivae
Raoultella terrigena
Proteus
Prevotella stercorea
Rathayibacter
Proteus vulgaris
Prevotella tannerae
Rathayibacter caricis
Prevotella timonensis
Rathayibacter festucae
Prevotella veroralis
Rathayibacter iranicus
Providencia
Rathayibacter rathayi
Providencia stuartii
Rathayibacter toxicus
Pseudomonas
Rathayibacter tritici
Pseudomonas aeruginosa
Rhodobacter
Pseudomonas alcaligenes
Rhodobacter sphaeroides
Pseudomonas anguillispetica
Ruegeria
Pseudomonas fluorescens
Ruegeria gelatinovorans
Pseudoalteromonas
haloplanktis
Pseudomonas mendocina
Pseudomonas
pseudoalcaligenes
Pseudomonas putida
Pseudomonas tutzeri
Pseudomonas syringae
Psychrobacter
Psychrobacter faecalis
Psychrobacter
phenylpyruvicus
Saccharococcus
Sagittula
Sanguibacter
Stenotrophomonas
Tatlockia
Saccharococcus thermophilus
Sagittula stellata
Sanguibacter keddieii
Stenotrophomonas
Tatlockia maceachernii
Saccharomonospora
Salegentibacter
Sanguibacter suarezii
maltophilia
Tatlockia micdadei
Saccharomonospora azurea
Salegentibacter salegens
Saprospira
Streptococcus
Tenacibaculum
Saccharomonospora cyanea
Salimicrobium
Saprospira grandis
Tenacibaculum
Saccharomonospora viridis
Salimicrobium album
Sarcina
Streptomyces
amylolyticum
Saccharophagus
Salinibacter
Sarcina maxima
Streptomyces
Tenacibaculum discolor
Saccharophagus degradans
Salinibacter ruber
Sarcina ventriculi
achromogenes
Tenacibaculum
Saccharopolyspora
Salinicoccus
Sebaldella
Streptomyces
gallaicum
Saccharopolyspora erythraea
Salinicoccus alkaliphilus
Sebaldella
cesalbus
Tenacibaculum
Saccharopolyspora gregorii
Salinicoccus hispanicus
termitidis
Streptomyces cescaepitosus
lutimaris
Saccharopolyspora hirsuta
Salinicoccus roseus
Serratia
Streptomyces cesdiastaticus
Tenacibaculum
Saccharopolyspora hordei
Salinispora
Serratia fonticola
Streptomyces cesexfoliatus
mesophilum
Saccharopolyspora rectivirgula
Salinispora arenicola
Serratia marcescens
Streptomyces fimbriatus
Tenacibaculum
Saccharopolyspora spinosa
Salinispora tropica
Sphaerotilus
Streptomyces fradiae
skagerrakense
Saccharopolyspora taberi
Salinivibrio
Sphaerotilus natans
Streptomyces fulvissimus
Tepidanaerobacter
Saccharothrix
Salinivibrio costicola
Sphingobacterium
Streptomyces griseoruber
Tepidanaerobacter
Saccharothrix australiensis
Salmonella
Sphingobacterium multivorum
Streptomyces griseus
syntrophicus
Saccharothrix coeruleofusca
Salmonella bongori
Staphylococcus
Streptomyces lavendulae
Tepidibacter
Saccharothrix espanaensis
Salmonella enterica
Streptomyces
Tepidibacter
Saccharothrix longispora
Salmonella subterranea
phaeochromogenes
formicigenes
Saccharothrix mutabilis
Salmonella typhi
Streptomyces
Tepidibacter thalassicus
Saccharothrix syringae
thermodiastaticus
Thermus
Saccharothrix tangerinus
Streptomyces tubercidicus
Thermus aquaticus
Saccharothrix texasensis
Thermus filiformis
Thermus thermophilus
Staphylococcus
S. arlettae
S. equorum
S. microti
S. schleiferi
S. agnetis
S. felis
S. muscae
S. sciuri
S. aureus
S. fleurettii
S. nepalensis
S. simiae
S. auricularis
S. gallinarum
S. pasteuri
S. simulans
S. capitis
S. haemolyticus
S. petrasii
S. stepanovicii
S. caprae
S. hominis
S. pettenkoferi
S. succinus
S. carnosus
S. hyicus
S. piscifermentans
S. vitulinus
S. caseolyticus
S. intermedius
S. pseudintermedius
S. warneri
S. chromogenes
S. kloosii
S. pseudolugdunensis
S. xylosus
S. cohnii
S. leei
S. pulvereri
S. condimenti
S. lentus
S. rostri
S. delphini
S. lugdunensis
S. saccharolyticus
S. devriesei
S. lutrae
S. saprophyticus
S. epidermidis
S. lyticans
S. massiliensis
Streptococcus
Streptococcus agalactiae
Streptococcus infantarius
Streptococcus orisratti
Streptococcus thermophilus
Streptococcus anginosus
Streptococcus iniae
Streptococcus parasanguinis
Streptococcus sanguinis
Streptococcus bovis
Streptococcus intermedius
Streptococcus peroris
Streptococcus sobrinus
Streptococcus canis
Streptococcus lactarius
Streptococcus pneumoniae
Streptococcus suis
Streptococcus constellatus
Streptococcus milleri
Streptococcus
Streptococcus uberis
Streptococcus downei
Streptococcus mitis
pseudopneumoniae
Streptococcus vestibularis
Streptococcus dysgalactiae
Streptococcus mutans
Streptococcus pyogenes
Streptococcus viridans
Streptococcus equines
Streptococcus oralis
Streptococcus ratti
Streptococcus
Streptococcus faecalis
Streptococcus tigurinus
Streptococcus salivariu
zooepidemicus
Streptococcus ferus
Uliginosibacterium
Vagococcus
Vibrio
Virgibacillus
Xanthobacter
Uliginosibacterium
Vagococcus carniphilus
Vibrio aerogenes
Virgibacillus
Xanthobacter agilis
gangwonense
Vagococcus elongatus
Vibrio aestuarianus
halodenitrificans
Xanthobacter
Ulvibacter
Vagococcus fessus
Vibrio albensis
Virgibacillus
aminoxidans
Ulvibacter litoralis
Vagococcus fluvialis
Vibrio alginolyticus
pantothenticus
Xanthobacter
Umezawaea
Vagococcus lutrae
Vibrio campbellii
Weissella
autotrophicus
Umezawaea tangerina
Vagococcus salmoninarum
Vibrio cholerae
Weissella cibaria
Xanthobacter flavus
Undibacterium
Variovorax
Vibrio cincinnatiensis
Weissella confusa
Xanthobacter tagetidis
Undibacterium pigrum
Variovorax boronicumulans
Vibrio coralliilyticus
Weissella halotolerans
Xanthobacter viscosus
Ureaplasma
Variovorax dokdonensis
Vibrio cyclitrophicus
Weissella hellenica
Xanthomonas
Ureaplasma
Variovorax paradoxus
Vibrio diazotrophicus
Weissella kandleri
Xanthomonas
urealyticum
Variovorax soli
Vibrio fluvialis
Weissella koreensis
albilineans
Ureibacillus
Veillonella
Vibrio furnissii
Weissella minor
Xanthomonas alfalfae
Ureibacillus composti
Veillonella atypica
Vibrio gazogenes
Weissella
Xanthomonas
Ureibacillus suwonensis
Veillonella caviae
Vibrio halioticoli
paramesenteroides
arboricola
Ureibacillus terrenus
Veillonella criceti
Vibrio harveyi
Weissella soli
Xanthomonas
Ureibacillus thermophilus
Veillonella dispar
Vibrio ichthyoenteri
Weissella thailandensis
axonopodis
Ureibacillus thermosphaericus
Veillonella montpellierensis
Vibrio mediterranei
Weissella viridescens
Xanthomonas
Veillonella parvula
Vibrio metschnikovii
Williamsia
campestris
Veillonella ratti
Vibrio mytili
Williamsia marianensis
Xanthomonas citri
Veillonella rodentium
Vibrio natriegens
Williamsia maris
Xanthomonas codiaei
Venenivibrio
Vibrio navarrensis
Williamsia serinedens
Xanthomonas
Venenivibrio stagnispumantis
Vibrio nereis
Winogradskyella
cucurbitae
Verminephrobacter
Vibrio nigripulchritudo
Winogradskyella
Xanthomonas
Verminephrobacter eiseniae
Vibrio ordalii
thalassocola
euvesicatoria
Verrucomicrobium
Vibrio orientalis
Wolbachia
Xanthomonas fragariae
Verrucomicrobium spinosum
Vibrio parahaemolyticus
Wolbachia persica
Xanthomonas fuscans
Vibrio pectenicida
Wolinella
Xanthomonas gardneri
Vibrio penaeicida
Wolinella succinogenes
Xanthomonas hortorum
Vibrio proteolyticus
Zobellia
Xanthomonas hyacinthi
Vibrio shilonii
Zobellia galactanivorans
Xanthomonas perforans
Vibrio splendidus
Zobellia uliginosa
Xanthomonas phaseoli
Vibrio tubiashii
Zoogloea
Xanthomonas pisi
Vibrio vulnificus
Zoogloea ramigera
Xanthomonas populi
Zoogloea resiniphila
Xanthomonas theicola
Xanthomonas
translucens
Xanthomonas
vesicatoria
Xylella
Xylella fastidiosa
Xylophilus
Xylophilus ampelinus
Xenophilus
Yangia
Yersinia mollaretii
Zooshikella
Zobellella
Xenophilus azovorans
Yangia pacifica
Yersinia philomiragia
Zooshikella ganghwensis
Zobellella denitrificans
Xenorhabdus
Yaniella
Yersinia pestis
Zunongwangia
Zobellella taiwanensis
Xenorhabdus beddingii
Yaniella flava
Yersinia pseudotuberculosis
Zunongwangia profunda
Zeaxanthinibacter
Xenorhabdus bovienii
Yaniella halotolerans
Yersinia rohdei
Zymobacter
Zeaxanthinibacter
Xenorhabdus cabanillasii
Yeosuana
Yersinia ruckeri
Zymobacter palmae
enoshimensis
Xenorhabdus doucetiae
Yeosuana aromativorans
Yokenella
Zymomonas
Zhihengliuella
Xenorhabdus griffiniae
Yersinia
Yokenella regensburgei
Zymomonas mobilis
Zhihengliuella
Xenorhabdus hominickii
Yersinia aldovae
Yonghaparkia
Zymophilus
halotolerans
Xenorhabdus koppenhoeferi
Yersinia bercovieri
Yonghaparkia alkaliphila
Zymophilus paucivorans
Xylanibacterium
Xenorhabdus nematophila
Yersinia enterocolitica
Zavarzinia
Zymophilus raffinosivorans
Xylanibacterium ulmi
Xenorhabdus poinarii
Yersinia entomophaga
Zavarzinia compransoris
Xylanibacter
Yersinia frederiksenii
Xylanibacter oryzae
Yersinia intermedia
Yersinia kristensenii
S. aureus str. MRSA252
S. aureus str. NY940
S. aureus str. RF122
S. aureus str. V329
S. aureus str. mu50
S. aureus str. mw2
S. aureus str. FRI909
. III)
S. aureus str. RN4282
S. aureus str. COL
S. aureus str. USA300
S. aureus str. n315
S. aureus str. mu50,
S. aureus str. RN3984
S. aureus European
S. aureus str. RF122
S. aureus strains
S. saprophyticus str.
S. saprophyticus, Staphylococcus saprophyticus.
.
Nomenclature used by Lindsay and Holden
.
This strain has not been
ShPI2 is located 180° away from the other SaPIs
S. haemolyticus genome .
GenBank accession NC 007622.
indicates data missing or illegible when filed
| Number | Date | Country | Kind |
|---|---|---|---|
| 1901099.0 | Jan 2019 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/051937 | 1/27/2020 | WO | 00 |
