Patent Application
20190160120
This application claims priority benefit to United Kingdom Patent Application Nos. GB1719896.1 filed on Nov. 29, 2017 and GB1808063.0 filed on May 17, 2018, the contents of which are incorporated herein by reference in their entireties.
The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 786212000400SEQLIST.txt, date recorded: May 21, 2018, size: 71 KB).
The invention relates to the production of phage using DNAs (eg, plasmids and helper phage, or plasmids with chromosomally integrated helper phage genes), as well as the phage, helper phage, kits, compositions and methods involving these.
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.
The invention relates to the production of phage and provides:
In a First Configuration
In a Seond Configuration
A population of helper phage, wherein the helper phage are capable of packaging first phage, wherein the first phage are different from the helper phage and the helper phage are incapable of self-replication.
In a third Configuration
A composition comprising a population of first phage, wherein the first phage require helper phage according to the First Configuration for replication; and wherein less than [20%] of total phage comprised by the composition are such helper phage.
In a Fourth Configuration
A method of producing first phage, wherein the first phage require helper phage to replicate, the method comprising
In a Fifth Configuration
A phage production system, for producing phage (eg, the first phage of any preceding claim) comprising a nucleotide sequence of interest (NSI-phage), the system comprising components (i) to (iii):
A composition for use in antibacterial treatment of bacteria, the composition comprising an engineered mobile genetic element (MGE) that is capable of being mobilised in a first bacterial host cell of a first species or strain, the cell comprising a first phage genome, wherein in the cell the MGE is mobilised using proteins encoded by the phage and replication of first is inhibited, wherein the MGE encodes an antibacterial agent or encodes a component of such an agent.
A nucleic acid vector comprising the MGE integrated therein, wherein the vector is capable of transferring the MGE or a copy thereof into a host bacterial cell.
A non-self replicative transduction particle comprising said MGE or vector of the invention.
A composition comprising a plurality of transduction particles, wherein each particle comprises a MGE or vector according to the invention, wherein the transduction particles are capable of transferring the MGEs, or nucleic acid encoding the agent or component, or copies thereof into target bacterial cells, wherein
A composition comprising a plurality of non-self replicative transduction particles, wherein each particle comprises a MGE or plasmid according to the invention, wherein the transduction particles are capable of transferring the MGEs, or nucleic acid encoding the agent or component, or copies thereof into target bacterial cells, wherein the agent is a CRISPR/Cas system and the component comprises a nucleic acid encoding a crRNA or a guide RNA that is operable with a Cas in a target bacterial cell to guide the Cas to a target nucleic acid sequence of the cell to modify the sequence, whereby
A method of producing a plurality of transduction particles, the method comprising combining the composition of the invention with host bacterial cells of said first species, wherein the cells comprise the first phage, allowing a plurality of said MGEs to be introduced into host cells and culturing the host cells under conditions in which first phage-encoded proteins are expressed and MGE copies are packaged by first phage proteins to produce a plurality of transduction particles, and optionally separating the transduction particles from cells and obtaining a plurality of transduction particles separated from cells.
A bacterial host cell comprising a first phage and a MGE, vector or particle of the invention, wherein the agent is toxic to cells of the same species as the host cell, and wherein the host cell has been engineered so that the agent is not toxic to the host cell.
A bacterial host cell comprising a first phage, wherein the cell is comprised by a kit, the kit further comprising a composition of the invention, wherein the agent is toxic to cells of the same species as the host cell, and wherein the host cell has been engineered so that the agent is not toxic to the host cell.
A bacterial host cell comprising a first phage and a MGE, vector or particle of the invention, wherein the agent is not toxic to the host cell, but the agent is toxic to second cells of a species or strain that is different from the species or strain of the host cell, wherein the MGE is mobilizable in transduction particles producible by the host cell that are capable of transferring the MGE or a copy thereof into a said second cell, whereby the second cell is exposed to the antibacterial agent.
A bacterial host cell comprising a first phage, wherein the cell is comprised by a kit, the kit further comprising a composition of the invention, wherein the agent is not toxic to the host cell, but the agent is toxic to second cells of a species or strain that is different from the species or strain of the host cell, wherein the MGE is mobilizable in transduction particles producible by the host cell that are capable of transferring the MGE or a copy thereof into a said second cell, whereby the second cell is exposed to the antibacterial agent.
A bacterial host cell comprising a MGE, vector or particle of the invention and nucleic acid under the control of one or more inducible promoters, wherein the nucleic acid encodes all structural proteins necessary to produce a transduction particle that packages a copy of the MGE or plasmid, wherein the agent is not toxic to the host cell, but the agent is toxic to second cells of a species or strain that is different from the species or strain of the host cell, wherein the MGE is mobilizable in transduction particles producible by the host cell that are capable of transferring the MGE or a copy thereof into a said second cell, whereby the second cell is exposed to the antibacterial agent.
A plasmid comprising
A bacterial host cell comprising the genome of a helper phage that is incapable of self-replication, optionally wherein the genome is present as a prophage, and a plasmid according to the invention, wherein the helper phage is operable to package copies of the plasmid in transduction particles, wherein the particles are capable of infecting bacterial target cells to which the antibacterial agent is toxic.
A method of making a plurality of transduction particles, the method comprising culturing a plurality of host cells according to the invention, optionally inducing a lytic cycle of the helper phage, and incubating the cells under conditions wherein transducing particles comprising packaged copies of the plasmid are created, and optionally separating the particles from the cells to obtain a plurality of transduction particles.
A plurality of transduction particles obtainable by the method of the invention 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.
A method of making a plurality of transduction particles, the method comprising
A plurality of transduction particles obtainable by the method.
The invention relates to the production of phage using DNAs (eg, plasmids with helper phage), as well as the phage, helper phage, compositions and methods involving these. The invention finds utility, for example, for containing phage in environments ex vivo and in vivo, reducing the risk of acquisition of antibiotic resistance or other genes by phage, as well as controlling dosing of phage in an environment. The contamination of useful phage populations by helper phage may in examples also be restricted or eliminated, thereby controlling phage propagation and enhancing the proportion of desired phage in phage compositions, such as medicaments, herbicides and other agents where phage may usefully be used. Thus, the invention provides the following embodiments.
A kit comprising
For example the second DNA is devoid of a packaging signal for packaging second DNA. Additionally or alternatively, the second DNA is devoid of a nucleotide sequence required for replication of helper phage. Optionally, the nucleotide sequence enodes a sigma factor or comprises a sigma factor recognition site, a DNA polymerisation recognition site, or a promoter of a gene required for helper phage DNA replication when the second DNA is comprised by a helper prophage.
In an example, the second DNA is comprised by an M13 or M13-based helper phage. M13 encodes the following proteins required for phage packaging:
In this example, the second DNA is devoid of one or more of the genes coding for these proteins, eg, is devoid of a gene endoding pIII, a gene encoding pV, a gene endoding pVII, a gene endoding pVIII, a gene endoding pIX, a gene endoding pI, a gene endoding pIV and/or a gene endoding XI.
In an embodiment, the phage particle of (i) is capable of infecting a target bacterium, the phage 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 phage particle that comprises the first DNA, 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 DNA is a first DNA as defined in any preceding paragraph.
In an embodiment, the first DNA is comprised by a vector (eg, a plasmid or shuttle vector).
In an embodiment, the second DNA is comprised by a vector (eg, a plasmid or shuttle vector), helper phage (eg, a helper phagemid) or is integrated in the genome of a host bacterial cell.
An embodiment provides a bacterial cell comprising the first and second DNAs. Optionally, the cell is devoid of a functional CRISPR/Cas system before transfer therein of a first DNA, eg, a first DNA comprising a component of a CRISPR/Cas system that is toxic to the target bacterium. An embodiment provides an antibacterial composition comprising a plurality of cells, wherein each cell is optionally according to this paragraph, for administration to a human or animal subject for medical use.
A method of producing phage is provided, the method comprising expressing in a host bacterial cell the phage protein genes, wherein packaged phage are produced that comprise the first DNA, wherein the phage require the second DNA for replicaton thereof to produce further phage particles. Optionally, the method comprises isolating the phage particles.
A composition comprising a population of phage particles obtainable by the method is provided for administration to a human or animal subject for treating an infection of target bacterial cells, wherein the phage are capable of infecting and killing the target cells.
A method of treating an environment ex vivo, the method comprising exposing the environment to a population of phage particles obtainable by the method is provided, wherein the environment comprises target bacteria and the phage infect and kill the target bacteria. In an example thje subject is further administered an agent simultaneously or sequentially with the phage administration. In an example, the agent is a herbicide, pesticide, insecticide, plant fertilizer or cleaning agent.
Optionally, the method is for containing the treatment in the environment.
Optionally, the method is for controlling the dosing of the phage treatment in the environment.
Optionally, the method is for reducing the risk of acquisition of foreign gene sequence(s) by the phage in the environment.
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 phage particles obtainable by the production method, wherein the phage infect and kill the target bacteria.
Optionally, the method for treating is for containing the treatment in the subject.
Optionally, the method for treating is for containing the treatment in the environment in which the subject exists.
Optionally, the method for treating is for controlling the dosing of the phage treatment in the subject.
Optionally, the method for treating is for reducing the risk of acquisition of foreign gene sequence(s) by the phage in the subject.
Optionally, the method for treating is for reducing the risk of acquisition of foreign gene sequence(s) by the phage in the environment in which the subject exists.
Optionally, target bacteria herein are comprised by a microbiome of the subject, eg, a gut microbiome. Altertnatively, 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 thje subject is further administered a medicament simultaneously or sequentially with the phage administration. In an example, the medicament is an antibiotic, antibody, immune checkpoint inhibitor (eg, an anti-PD-1, anti-PD-L1 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 phage nucleic acid of interest. Thus, the invention provides the following illustrative Aspects:
In an example, the population comprises at least 103,104,105 or 106 phage particles, as indicated a transduction assay, for example. To have a measure of the first phage concentration, for example, one can perform a standard transduction assay when the first phage genome contains an antibiotic marker. Thus, in this case the first phage are capable of infecting target bacteria and in a sample of 1 ml the population comprises at least 103,104,105 or 106 transducing 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 centigrade.
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 phage particles comprised by the composition are particles of first phage.
In an example, the first phage genome comprises an f1 origin of replication.
In an example, the helper phage are E coli phage. In an example, the first phage are E coli, C Streptococcus, Klebsiella, Pseudomonas, Acitenobacter, Enterobacteracea, Firmicutes or Bacteroidetes phage. In an example, the helper phage are engineered M13 phage.
In an example, the first phage genome comprises a phagemid, wherein the phagemid comprises a packaging signal for packaging first phage particles in the presence of the helper phage.
The first phage particles may contain 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 first phage particles. Once inside the target bacteria, the first phage DNA is incapable of being packaged to form first phage particles in the absence of the helper phage. This usefully contains the activity of the first phage genome and its encoded products (protieins 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 first phage. 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).
Thus, the prophage is integrated in the chromosome of a host cell.
Examples of phage structural proteins are phage coat proteins, collar proteins and phage tail fibre proteins.
This can be determined, for example, using DNA probes (designed on the basis of the known heper phge genome sequence) with PCR, as is conventional. In an example, the composition may comprise residual helper prophage DNA, but essentially otherwise is devoid of helper DNA.
In another embodiment, the composition comprises second phage particles, wherein the second phage are different from the first phage and are not helper phage.
Thus, the genome is capable of nucleic acid replication but not packaging of helper phage.
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. For example, the means is recombinant, artificial or synthetic.
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.
In an example, the animak 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.
The inability of the first phage to self-replicate and to require helper phage or second DNA 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 or second DNA to self-package limits availability of factors required by the first phage to form packaged particles, hence providing containment by limiting first phage propagation. This may be useful, for example, to contain an antibacterial acitivity provided by the first phage, such as a CRISPR/Cas killing principle.
The cell may, for example, act as a carrier for the genome of the first phage, wherein the first phage DNA is capable of horizontal transfer from the carrier to the target bacteria once the carrier bacteria have been administered to an environment to be treated, eg, a soil or a human gut or other environment described herein. In an example, the environment is comprised by a human or animal subject and the carrier are commensal or probiotic in the subject. For example the carrier bacteria are Lactobacillus (eg, L reuteri or L lactis), E coli or Streptococcus (eg, S thermophilus) bacteria. The horizontal transfer can be transfer of a plasmid (such as a conjugative plasmid) to the target bacteria or first phage infection of the target bacteria, wherein the first phage have been prior packaged in the carrier. The use of a carrier is useful too for oral administration or other routes where the carrier can provide protection for the phage, helper or composition from the acid stomach or other harsh environments in the subject. Furthermore, the carrier can be formulated into a beverage, for example, a probiotic drink, eg, an adapted Yakult (trademark), Actimel (trademark), Kevita (trademark), Activia (trademark), Jarrow (trademark) or similar drink for human consumption.
In an example, when the subject is a human, the subject is not an embryo.
For example, this is useful for reducing the risk of antibiotic resistance genes by the phage, such as when the phage are in the presence of other phage or plasmids in the environment.
52. A method of controlling the dosing of first phage in an environment (e.g., ex vivo), the method comprising
In an embodiment, the DNA is comprised by a phagemid or cloning vector (eg, a shuttle vector, eg, a pUC vector).
There may be a modest amount of helper phage DNA replication to enable first phage protein production efficiently, or should replication of helper phage DNA may be eliminated totally eliminated.
This is useful as a safety measure to reduce or eliminate first phage activity outside the subject.
Usefully, propagation of the first phage is restricted or eliminated, so dosing in the subject can be controlled, or even pre-determined within a narrow expected range. This is useful, for example, for medicaments comprising the first phage or composition, and may be aid approval of such medicines before FDA and simiar authorities.
Alternatively, the dosing is dosing of an environment, such as soil etc disclosed herein, wherein limitation of the first phage or composition activity is also desirable to limit spread of activities in natural and other terrains.
Whereby the system is capable of producing a product comprising a population of NSI-phage, wherein each NSI-phage requires a said helper phage for propagation, optionally wherein the NSI-phage in the product are not mixed with helper phage or less than 20% of total phage comprised by the product are said helper phage.
The invention includes within its concept relatively low level of helper phage particle production if there is a residual capability of helper phage to replicate to produce particles, such as for example in the case that a helper phage packaging signal or other HPF nucleotide sequence in the helper phage genome is mutated (eg, by deletion, substitution or addition of nucleotides therein) to knock down the ability to form phage particles. Preferably, there is no production of helper phage particles, such as by deleting all or part of the sequence from the helper phage genome or inactivating the sequence.
In an example, the kit, DNA(s), first phage, helper phage or composition is comprised by a medical container, eg, a syringe, vial, IV bag, inhaler, eye dropper or nebulizer. In an example, the kit, DNA(s), first phage, helper phage or composition is comprised by a sterile container. In an example, the kit, DNA(s), first phage, helper phage or composition is comprised by a medically-compatible container. In an example, the kit, DNA(s), first phage, helper phage or composition is comprised by a fermentation vessel, eg, a metal, glass or plastic vessel.
In an example, the kit, DNA(s), first phage, helper phage or composition 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 kit, DNA(s), first phage, helper phage or composition is comprised by an oral medicament formulation. In an example, the kit, DNA(s), first phage, helper phage or composition is comprised by an intranasal or ocular medicament formulation. In an example, the kit, DNA(s), first phage, helper phage or composition is comprised by a personal hygiene composition (eg, shampoo, soap or deodorant) or cosmetic formulation. In an example, the kit, DNA(s), first phage, helper phage or composition is comprised by a detergent formulation. In an example, the kit, DNA(s), first phage, helper phage or composition is comprised by a cleaning formulation, eg, for cleaning a medical or industrial device or apparatatus. In an example, the kit, DNA(s), first phage, helper phage or composition is comprised by foodstuff, foodstuff ingredient or foodstuff processing agent. In an example, the kit, DNA(s), first phage, helper phage or composition is comprised by beverage, beverage ingredient or beverage processing agent. In an example, the kit, DNA(s), first phage, helper phage or composition is comprised by a medical bandage, fabric, plaster or swab. In an example, the kit, DNA(s), first phage, helper phage or composition is comprised by a herbicide or pesticide. In an example, the kit, DNA(s), first phage, helper phage or composition is comprised by an insecticide.
In an example, 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 and helper phage are Corticoviridae. In an example, both the first and helper phage are Cystoviridae. In an example, both the first and helper phage are Inoviridae. In an example, both the first and helper phage are Leviviridae. In an example, both the first and helper phage are Microviridae. In an example, both the first and helper phage are Podoviridae. In an example, both the first and helper phage are Siphoviridae. In an example, both the first and helper 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, S pyogenes 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 first DNA, first phage or vector 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 dificile (eg, the vector is a shuttle vector operable in E coli and the host bacteria are C dificile). 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 (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, host and/or target bacteria is a gram negative bacterium (eg, a spirilla or vibrio). Optionally, host and/or target bacteria is a gram positive bacterium. Optionally, host and/or target bacteria is a mycoplasma, chlamydiae, spirochete or mycobacterium. Optionally, host and/or target bacteria is a Streptococcus (eg, pyogenes or thermophilus). Optionally, host and/or target bacteria is a Staphylococcus (eg, aureus, eg, MRSA). Optionally, host and/or target bacteria 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, host and/or target bacteria is a Pseudomonas (eg, aeruginosa). Optionally, host and/or target bacteria is a Vibro (eg, cholerae (eg, O139) or vulnificus). Optionally, host and/or target bacteria is a Neisseria (eg, gonnorrhoeae or meningitidis). Optionally, host and/or target bacteria is a Bordetella (eg, pertussis). Optionally, host and/or target bacteria is a Haemophilus (eg, influenzae). Optionally, host and/or target bacteria is a Shigella (eg, dysenteriae). Optionally, host and/or target bacteria is a Brucella (eg, abortus). Optionally, host and/or target bacteria is a Francisella host. Optionally, host and/or target bacteria is a Xanthomonas host. Optionally, host and/or target bacteria is a Agrobacterium host. Optionally, host and/or target bacteria is a Erwinia host. Optionally, host and/or target bacteria is a Legionella (eg, pneumophila). Optionally, host and/or target bacteria is a Listeria (eg, monocytogenes). Optionally, host and/or target bacteria is a Campylobacter (eg, jejuni). Optionally, host and/or target bacteria is a Yersinia (eg, pestis). Optionally, host and/or target bacteria is a Borelia (eg, burgdorferi). Optionally, host and/or target bacteria is a Helicobacter (eg, pylori). Optionally, host and/or target bacteria is a Clostridium (eg, dificile or botulinum). Optionally, host and/or target bacteria is a Erlichia (eg, chaffeensis). Optionally, host and/or target bacteria is a Salmonella (eg, typhi or enterica, eg, serotype typhimurium, eg, DT 104). Optionally, host and/or target bacteria is a Chlamydia (eg, pneumoniae). Optionally, host and/or target bacteria is a Parachlamydia host. Optionally, host and/or target bacteria is a Corynebacterium (eg, amycolatum). Optionally, host and/or target bacteria is a Klebsiella (eg, pneumoniae). Optionally, host and/or target bacteria is an Enterococcus (eg, faecalis or faecim, eg, linezolid-resistant). Optionally, host and/or target bacteria 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 integrates 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 80a can induce several different SaPIs, including SaPI1, SaPI2, and SaPIbov1, whereas φ11 can induce SaPIbov1 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 both pac 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/SaPIbov5 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 aa 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 φ12 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.
The invention, also involves, in certain configurations the use of mobile genetic elements (MGEs). Thus, there are provided the following Clauses. Any of the other configurations, Aspects, Examples or description of the invention above or elsewhere herein are combinable mutatis mutandis with any of these Clauses:
In the alternative, instead of a bacteria, the host cell is a archaeal cell and instead of a phage there is a virus that is capable of infecting the archaeal cell.
In an example, the MGE is capable of integration into the genome of the host cell comprising the genome of a first phage, for example integration in the chromosome of the host cell and/or an episome thereof. Optionally, the MGE inhibits first phage replication.
In an example, first phage replication is totally inhibited. In an example, it is reduced by at least 50, 60, 70, 80 or 90% compared to replication in the absence of the MGE in host cells. This can be assessed by a standard in vitro plaque assay to determine the relative amount of first phage plaque formation.
Optionally, in the presence of the agent,
Viruses undergo lysogenic and lytic cycles in a host cell. If the lysogenic cycle is adopted, the phage chromosome can be integrated into the bacterial chromosome, or it can establish itself as a stable plasmid in the host, where it can remain dormant for long periods of time. If the lysogen is induced, the phage genome is excised from the bacterial chromosome and initiates the lytic cycle, which culminates in lysis of the cell and the release of phage particles. The lytic cycle leads to the production of new phage particles which are released by lysis of the host.
“Transduction particles” may be phage or smaller than phage and are particles that are capable of transducing nucleic acid encoding the antibiotic or component thereof into target bacterial cells.
Examples of structural proteins are phage proteins selected from one, more or all of the major head and tail proteins, the portal protein, tail fibre proteins, and minor tail proteins.
The MGE comprises a packaging signal sequence operable with proteins encoded by the first phage to package the MGE (or at least nucleic acid thereof encoding the agent or one or more components thereof) into transduction particles that are capable of infecting host cells of the same species or strain as the first host cell.
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 cell. For example, an adenovirus when the cell is a human cell.
Optionally, the MGE is devoid of one or more phage genes rinA, terS and terL. In an example, in a host cell a protein complex comprising the small terminase (encoded by terS) and large terminase (encoded by terL) proteins is able to recognise and cleave a double-stranded DNA molecule of the MGE at or near the pac site (cos site or other packaging signal sequence comprised by the MGE), and this allows the MGE or plasmid DNA molecule to be packaged into a phage capsid. When first phage as prophage in the host cell is induced, the lytic cycle of the phage produces the phage's structural proteins and the phage's large terminase protein. The MGE or plasmid is replicated, and the small terminase protein encoded by the MGE or plasmid is expressed. The replicated MGE or plasmid DNA containing the terS (and the nucleotide sequence encoding the antibacterial agent or component) are packaged into phage capsids, resulting in non-self replicative transduction particles carrying only MGE or plasmid DNA.
Optionally, the genomic island is an island that is naturally found in bacterial cells of the first species or strain. 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.
Optionally, the pathogenicity island is an island that is naturally found in bacterial cells of the first species or strain, 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-
This is useful where, not only does the presence of the MGE reduce first phage replication in the host cell, but also the MGE is taken up and may provide a survival, growth or other benefit to the host cell, promoting uptake and/or retention of MGEs by host cells. In an example, expression of the antibacterial agent in the host cell is under the control of an inducible promoter or weak promoter to allow for a period where uptake of MGEs into host cells may be favoured owing to the presence of the nucleotide sequence that is beneficial to cells of the first species or strain.
The terS homologues are sequences which, like terS, recognise the SaPI-specific pac site (or other packaging sequence) comprised by the MGE or plasmid and determine packaging specificity for packaging the MGE.
Examples of terminase genes are pacA, pacB, terA, terB and terL.
Optionally, the phage is P2. Optionally, the first phage is a T7 or T7-like phage that recognises direct repeat sequences comprised by the MGE for packaging.
The terS homologues are sequences which, like terS, recognise the SaPI-specific pac site (or other packaging sequence) comprised by the MGE or plasmid and determine packaging specificity for packaging the MGE.
In an example, the terS comprises the sequence of SEQ ID NO: 2:
Optionally the cpmA and B are from any SaPI disclosed herein. In an example any SaPI is a SaPI disclosed in
In an example, the MGE comprises a modified SaPI and comprises one, more or all genes cp1, cp2, and cp3.
Optionally, Constitutive transcription and production of the agent in target cells may be used where the target cells should be killed, eg, in medical settings.
Optionally, the transcription of MGE nucleic acid is under the control of an inducible promoter, for transcription of copies of the agent or component in a host 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 host cells, wherein the crRNA or gRNA guides the Cas to a target sequence in the host cell to modify the target (eg, cut it or repress transcription from it).
In examples, such cutting causes one or more of the following:
Optionally, the Cas is a Cas encoded by a functional endogenous nucleic acid of a host cell. For example, the target is comprised by a DNA or RNA of the host cell.
In an example, the first species of strain is a Staphylococcus (eg, S aureus) species or strain and optionally the MGE is a modified SaPI; and optionally the first phage is a φ80α or φ11. In an example, the first species of strain is a Vibrio (eg, V cholerae) species or strain and optionally the MGE is Vibrio (eg, V cholerae) PLE.
These are species that P2 phage can infect. Thus, in an embodiment, the MGE comprises one or more P4 sequences (eg, a P4 packaging sequence) and the first phage is P2. Thus, the MGE is packaged by P2 structural proteins and the resultant transduction particles can infect a broad spectrum of species, ie, two or more of Shigella, E coli, Salmonella, Serratia, Klebsiella, Yersinia, Pseudomonas and Enterobacter.
Suitable vectors are plasmids (eg, conjugative plasmids) or viruses (eg, phage or packaged phagemids).
A shuttle vector is a vector (usually a plasmid) constructed so that it can propagate in two different host species. Therefore, DNA inserted into a shuttle vector can be tested or manipulated in two different cell types.
By “non-replicative” it is meant that the MGE is not capable by itself of self-replicating. For example, the MGE is devoid of one or more nucleotide sequences encoding a protein (eg, a structural protein) that is necessary to produce a transduction particle comprising a copy of the MGE.
In an example, the reduction in growth or proliferation of host cells is at least 50, 60, 70, 80, 90 or 95%. The antibiotic can be any antibiotic disclosed herein.
In an example, the reduction in growth or proliferation of host cells is at least 50, 60, 70, 80, 90 or 95%. The antibiotic can be any antibiotic disclosed herein.
This is useful for packaging DNAs into smaller capsids.
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 farming; fish and shellfish farming; 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, the ifirst DNA, first phage or vector comprises a CRISPR array 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 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 DNAs, phage 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 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 host and/or target bacteraia 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 kit, DNA(s), first phage, helper phage, 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 first phage, helper phage, composition, use or method is for knocking-down Clostridium dificile or E coli bacteria in a gut microbiota of a human or animal.
In an example, the packaging signal, NPF and/or HPF consists or comprises SEQ ID NO: 1 or a structural or functional homologue thereof.
In an example, the packaging signal, NPF and/or HPF consists or comprises SEQ ID NO: 1 or a nucleotide sequence that is at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical thereto.
In an example, the disease or condition is a cancer, inflammatory or autoimmune disease or condition, eg, obesity, diabetes IBD, 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 acqueous 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 treatement and/or prevention of CNS and neurodegenerative diseases and conditions, eg, agents such as immune checkpoint inhibitors, eg, anti-PD-1, anti-PD-L1, 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
Background
We designed a strategy for efficient production of phage particles comprising components of a CRISPR/Cas system for killing target E coli Nissle strain bacteria. So our phage composition will consist of a lysate primarily containing CRISPR/Cas system components packaged in phage particles which will be devoid of phage protein-encoding sequences and which will have no or a very low proportion of helper phage. Also the strategy will work alternatively in less well characterised phage/bacterial strain combinations.
Outline of strategy for CRISPR/Cas component packaging in hitherto unknown phages
Example of the above specifically for E coli Nissle using phage P2:
Nissle is useful due to its GRAS (Generally Regarded as Safe) status and P2 has a relatively broad host range (most E coli, Shigella, Klebsiella, Salmonella in addtition to DNA delivery into e.g. Pseudomonas; Kahn et al 1991).
We will use pUC19 or other high copy number cloning vector. Temperate phage P2 can lysogenize Nissle. Most E coli K strains have an inactive CRISPR/Cas system and can be infected by P2 and thus all regular cloning hosts can be used (here exemplified by E coli TOP10).
P2 is introduced into TOP10 to produce a lysogen. P2 cannot be induced with mitomycin C or UV but we will use the epsilon anti-repressor from the parasite phage P4 that derepresses P2 and makes it go into lytic phase. We will express this gene from an inducible promoter in the production host strain.
The 325 bp packaging signal sequence as follows will be used
The packaging sequence will be deleted in the P2 prophage of the lysogenic production TOP10 strain.
A pUC19 shuttle vector encoding a guide RNA that targets the genome of the target Nissle strain (or alternatively comprising a CRISPR array for producing such a guide RNA) will be constructed and the packaging signal will be added. If the target Nissle harbours it own endogenous CRISPR/Cas system, we will use an activation strategy to activate the endogenous Cas3 by including Cas activating genes in the vector. If not, we will include an exogenous Cas3-encoding nucleotide sequence (and optionally one or more nucleotide sequences encoding one or more required Cascade components) in the vector for expression in the target Nissle. We will transform the vector into the TOP10 production strain, induce the P4 anti-repressor and harvest phage comprising the CRISPR/Cas component(s).
Since the induced (helper) phage DNA does not contain a packaging signal we will be able to isolate particles with only the vector DNA packaged. Thus, we will obtain a composition comprising such phage which can be used to infect target Nissle bacteria and introduce the CRISPR/Cas component(s) therein for killing the target bacteria.
Overview of possible different MGE packaging strategies follow.
Applicable to different types of phages:
Identify packaging signal and structural genes in the helper phage (delivery vehicle)
Delete packaging signal in helper phage and place on plasmid comprising MGE
Place both helper and plasmid in production strain
Induce structural gene transcription of helper to get production of helper-phage-packaged MGEs
For using parasitic mobile elements (P4 phage or SaPI etc) activation of helper phage structural genes is done by induction of a helper phage activator obtained from the parasitic element Delta in P4 or one, more or al of ptiA/B/M in SaPI.
If one wants smaller size particle one can choose to package in a parasite-size capsid (typically 10-20 kb) by including in the MGE or vector P4 Sid and psu or cpmA/B from a SaPI.
One can use defective helper phages where at least the packaging signal has been removed and structural genes are either on a plasmid or integrated as a cryptic prophage in the production host. If for some reason one cannot use this approach and need to use functional helper phages, one will include in the MGE or vector the genes on the parasite that hijack the phage packaging machinery to preferentially package parasite DNA (in our case CGV) over phage DNA.
List of the minimal genes one could Include on a plasmid vector from P4. P4 sequence: see world wide web.ncbi.nlm.nih.gov/nuccore/x51522
The homology between P2 and P4 pasted below; this may be used as a packaging signal in the MGE or vector:
For small capsid size (packages 11.4 kb instead of 33.5 kb) Sid and/or Psu can be included in the MGE or vector:
To activate helper phage P2, Delta can be included in a host cell genome (provided separately in a host cell, not on the MGE or vector to be packaged)
Minimum genes to include in the host chromosome/episome from P2. P2 sequence (acc.number: NC_001895)
Minimal genes to include from a SaPI on a vector or MGE. Several different SaPI systems exist.
If one uses a defective helper phage with deleted packaging signal one can use that signal from the helper phage. In this example from S. aureus phi11 (acc. number: AF424781), as follows:
For small capsid size (packages 15.8 kb instead of 43.6 kb), one can include cpmA and/or cpmB in the MGE or vector.
To activate helper phage phi11 one can include one, more or all of ptiA, B and M (provided separately in a host cell and not on the MGE or vector to be packaged)
Minimum genes to include in the host chromosome/episome from phi11.
A list of phage that work with SaPIs
Different SaPIs are linked to different helper phages (see
One can mutates the helper phage to only contain structural genes to direct the phage to package in smaller capsids. If only looking at the genes responsible for small capsid packaging (cpmA and cpmB) these are highly conserved among staphylococci indicating that they will function to redirect packaging in a variety of p hages broader than the list below (
Abiotrophia
Abiotrophia
defectiva
Acaricomes
Acaricomes
phytoseiuli
Acetitomaculum
Acetitomaculum
ruminis
Acetivibrio
Acetivibrio
cellulolyticus
Acetivibrio
ethanolgignens
Acetivibrio
multivorans
Acetoanaerobium
Acetoanaerobium
noterae
Acetobacter
Acetobacter
aceti
Acetobacter
cerevisiae
Acetobacter
cibinongensis
Acetobacter
estunensis
Acetobacter
fabarum
Acetobacter
ghanensis
Acetobacter
indonesiensis
Acetobacter
lovaniensis
Acetobacter
malorum
Acetobacter
nitrogenifigens
Acetobacter
oeni
Acetobacter
orientalis
Acetobacter
orleanensis
Acetobacter
pasteurianus
Acetobacter
pornorurn
Acetobacter
senegalensis
Acetobacter
xylinus
Acetobactcrium
Acetobacterium
bakii
Acetobacterium
carbinolicum
Acetobacterium
dehalogenans
Acetobacteriam
fimetarium
Acetobacterium
malicum
Acetobacterium
paludosum
Acetobacterium
tundrae
Acetobaclerium
wieringae
Acetobacterium
woodii
Acetofilamentum
Acetofilamentum
rigidum
Acetohalobium
Acetohalobium
arabaticum
Acetomicrobium
Acetomicrobium
faecale
Acetomicrobium
flavidum
Acetonema
Acetonema
longum
Acetothermus
Acetothermus
paucivorans
Acholeplasma
Acholeplasma
axanthum
Acholeplasma
brassicae
Acholeplasma
cavigenitalium
Acholeplasma
equifetale
Acholeplasma
granularum
Acholeplasma
hippikon
Acholeplasma
laidlawii
Acholeplasma
modicum
Acholeplasma
morum
Acholeplasma
multilocale
Acholeplasma
oculi
Acholeplasma
palmae
Acholeplasma
parvum
Acholeplasma
pleciae
Acholeplasma
vituli
Achromobacter
Achromobacter
denitrificans
Achromobacter
insolitus
Achromobacter
piechaudii
Achromobacter
ruhlandii
Achromobacter
spanius
Acidaminobacter
Acidaminobacter
hydrogenoformans
Acidaminococcus
Acidaminococcus
fermentans
Acidaminococcus
intestini
Acidicaldus
Acidicaldus
organivorans
Acidimicrobium
Acidimicrobium
ferrooxidans
Acidiphilium
Acidiphilium
acidophilum
Acidiphilium
angustum
Acidiphilium
cryptum
Acidiphilium
multivorum
Acidiphilium
organovorum
Acidiphilium
rubrum
Acidisoma
Acidisoma
sibiricum
Acidisoma
tundrae
Acidisphaera
Acidisphaera
rubrifaciens
Acidithiobacillus
Acidithiobacillus
albertensis
Acidithiobacillus
caldus
Acidithiobacillus
ferrooxidans
Acidithiobacillus
thiooxidans
Acidobacterium
Acidobacterium
capsulatum
Acidocella
Acidocella
aminolytica
Acidocella
facilis
Acidomonas
Acidomoms
methanolica
Acidothermus
Acidothermus
cellulolyticus
Acidovorax
Acidovorax
anthurii
Acidovorax
caeni
Acidovorax
cattleyae
Acidovorax
citrulli
Acidovorax
defluvii
Acidovorax
delafieldii
Acidovorax
facilis
Acidovorax
konjaci
Acidovorax
temperans
Acidovorax
valerianellae
Acinetobacter
Acinetobacter
baumannii
Acinetobacter
baylyi
Acinetobacter
bouvetii
Acinetobacter
calcoaceticus
Acinetobacter
gerneri
Acinetobacter
haemolyticus
Acinetobacter
johnsonii
Acinetobacter
junii
Acinetobacter
lwoffi
Acinetobacter
parvus
Acinetobacter
radioresistens
Acinetobacter
schindleri
Acinetobacter
soli
Acinetobacter
tandoii
Acinetobacter
tjernbergiae
Acinetobacter
towneri
Acinetobacter
ursingii
Acinetobacter
venetianus
Acrocarpospora
Acrocarpospora
corrugata
Acrocarpospora
macrocephala
Acrocarpospora
pleiomorpha
Actibacter
Actibacter
sediminis
Actinoalloteichus
Actinoalloteichus
cyanogriseus
Actinoalloteichus
hymeniacidonis
Actinoalloteichus
spitiensis
Actinobaccillus
Actinobacillus
capsulatus
Actinobacillus
delphinicola
Actinobacillus
hominis
Actinobacillus
indolicus
Actinobacillus
lignieresii
Actinobacillus
minor
Actinobacillus
muris
Actinobacillus
pleuropneumoniae
Actinobacillus
porcinus
Aclinobacillus
rossii
Actinobacillus
scotiae
Actinobacillus
seminis
Actinobacillus
succinogenes
Actinobaccillus
suis
Actinobacillus
ureae
Actinobaculum
Actinobaculum
massiliense
Actinobaculum
schaalii
Actinobaculum
suis
Actinomyces
urinale
Actinocatenispora
Actinocatenispora
rupis
Actinocatenispora
thailandica
Actinocatenispora
sera
Actinocorallia
Actinocorallia
aurantiaca
Actinocorallia
aurea
Actinocorallia
cavernae
Actinocorallia
glomerata
Actinocorallia
herbida
Actinocorallia
libanotica
Actinocorallia
longicatena
Actinomadura
Actinomadura
alba
Actinomadura
atramentaria
Actinomadura
bangladeshensis
Actinomadura
catellatispora
Actinomadura
chibensis
Actinomadura
chokoriensis
Actinomadura
citrea
Actinomadura
coerulea
Actinomadura
echinospora
Actinomadura
fibrosa
Actinomadura
formosensis
Actinomadura
hibisca
Actinomadura
kijaniata
Actinomadura
latina
Actinomadura
livida
Actinomadura
luteofluorescens
Actinomadura
macra
Actinomadura
madurae
Actinomadura
oligospora
Actinomadura
pelletieri
Actinomadura
rubrobrunea
Actinomadura
rugatobispora
Actinomadura
umbrina
Actinomadura
verrucosospora
Actinomadura
vinacea
Actinomadura
viridilutea
Actinomadura
viridis
Actinomadura
yumaensis
Actinomyces
Actinomyces
bovis
Actinomyces
denticolens
Actinomyces
europaeus
Actinomyces
georgiae
Actinomyces
gerencseriae
Actinomyces
hordeovulneris
Actinomyces
howellii
Actinomyces
hyovaginalis
Actinomyces
israelii
Actinomyces
johnsonii
Actinomyces
meyeri
Actinomyces
naeslundii
Actinomyces
neuii
Actinomyces
odontolyticus
Actinomyces
oris
Actinomyces
radingae
Actinomyces
slackii
Actinomyces
turicensis
Actinomyces
viscosus
Actinoplanes
Actinoplanes
auranticolor
Actinoplanes
brasiliensis
Actinoplanes
consettensis
Actinoplanes
deccanensis
Actinoplanes
derwentensis
Actinoplanes
digitatis
Actinoplanes
durhamensis
Actinoplanes
ferrugineus
Actinoplanes
globisporus
Actinoplanes
humidus
Actinoplanes
italicus
Actinoplanes
liguriensis
Actinoplanes
lobatus
Actinoplanes
missouriensis
Actinoplanes
palleronii
Actinoplanes
philippinensis
Actinoplanes
rectilineatus
Actinoplanes
regularis
Actinoplanes
teichomyceticus
Actinoplanes
utahensis
Actinopolyspora
Actinopolyspora
halophila
Actinopolyspora
mortivallis
Actinosynnema
Actinosynnema
mirum
Actinotalea
Actinotalea
fermentans
Aerococcus
Aerococcus
sanguinicola
Aerococcus
urinae
Aerococcus
urinaeequi
Aerococcus
urinaehominis
Aerococcus
viridans
Aeromicrobium
Aeromicrobium
erythreum
Aeromonas
Aeromonas
allosaccharophila
Aeromonas
bestiarnm
Aeromonas
caviae
Aeromonas
encheleia
Aeromonas
enteropelogenes
Aeromonas
eucrenophila
Aeromonas
ichthiosmia
Aeromonas
jandaei
Aeromonas
media
Aeromonas
popoffii
Aeromonas
sobria
Aeromonas
veronii
Agrobacterium
Agrobacterium
gelatinovorum
Agrococcus
Agrococcus
citreus
Agrococcus
jenensis
Agromonas
Agromonas
oligotrophica
Agromyces
Agromyces
fucosus
Agromyces
hippuratus
Agromyces
luteolus
Agromyces
mediolanus
Agromyces
ramosus
Agromyces
rhizospherae
Akkermansia
Akkermansia
muciniphila
Albidiferax
Albidiferax
ferrireducens
Albidovulum
Albidovulum
inexpectatum
Alcaligenes
Alcaligenes
denitrificans
Alcaligenes
faecalis
Alcanivorax
Alcanivorax
borkumensis
Alcanivorax
jadensis
Algicola
Algicola
bacteriolytica
Alicyclobacillus
Alicyclobacillus
disulfidooxidans
Alicyclobacillus
sendaiensis
Alicyclobacillus
vulcanalis
Alishewanella
Alishewanella
fetalis
Alkalibacillus
Alkalibacillus
haloalkaliphilus
Alkalilimnicola
Alkalilimnicola
ehrlichii
Alkaliphilus
Alkaliphilus
oremlandii
Alkaliphilus
transvaalensis
Allochromatium
Allochromatium
vinosum
Alloiococcus
Alloiococcus
otitis
Allokutzneria
Allokutzneria
albata
Altererythrohacter
Altererythrobacter
ishigakiensis
Altermonas
Altermonas
haloplanktis
Altermonas
macleodii
Alysiella
Alysiella
crassa
Alysiella
filiformis
Aminobacter
Aminobacter
aganoensis
Aminobacter
aminovorans
Aminobacter
niigataensis
Aminobacterium
Aminobacterium
mobile
Aminomonas
Aminomonas
paucivorans
Ammoniphilus
Ammoniphilus
oxalaticus
Ammoniphilus
oxalivorans
Amphibacillus
Amphibacillus
xylanus
Amphritea
Amphrilea
balenae
Amphritea
japonica
Amycolatopsis
Amycolatopsis
alba
Amycolatopsis
albidoflavus
Amycolatopsis
azurea
Amycolatopsis
coloradensis
Amycolatopsis
lurida
Amycolatopsis
mediterranei
Amycolatopsis
rifamycinica
Amycolatopsis
rubida
Amycolatopsis
sulphurea
Amycolatopsis
tolypomycina
Anabaena
Anabaena
cylindrica
Anabaena
flos-aquae
Anabaena
variabilis
Anaeroarcus
Anaeroarcus
burkinensis
Anacrobaculum
Anaerobaculum
mobile
Anaerobiospirillum
Anaerobiospirillum
succiniciproducens
Anaerobiospirillum
thomasii
Anaerococcus
Anaerococcus
hydrogenalis
Anaerococcus
lactolyticus
Anaerococcus
prevotii
Anaerococcus
tetradius
Anaerococcus
vaginalis
Anaerofustis
Anaerofustis
stercorihominis
Anaeromusa
Anaeromusa
acidaminophila
Anaeromyxobacter
Anaeromyxobacter
dehalogenans
Anaerorhabdus
Anaerorhabdus
furcosa
Anaerosinus
Anaerosinus
glycerini
Anaerovirgula
Anaerovirgula
multivorans
Ancalomicrobium
Ancalomicrobium
adetum
Ancylobacter
Ancylobacter
aquaticus
Aneurinibacillus
Aneurinibacillus
aneurinilyticus
Aneurinibacillus
migulanus
Aneurinibacillus
thermoaerophilus
Angiococcus
Angiococcus
disciformis
Angulomicrobium
Angulomicrobium
tetraedrale
Anoxybacillus
Anoxybacillus
pushchinoensis
Aquabacterium
Aquabacterium
commune
Aquabacterium
parvum
Aquaspirillum
Aquaspirillum
polymorphum
Aquaspirillum
putridiconchylium
Aquaspirillum
serpens
Aquimarina
Aquimarina
latercula
Arcanobacterium
Arcanobacterium
haemolyticum
Arcanobacterium
pyogenes
Archangium
Archangium
gephyra
Arcobacter
Arcobacter
butzleri
Arcobacter
cryaerophilus
Arcobacter
halophilus
Arcobacter
nitrofigilis
Arcobacter
skirrowii
Arhodomonas
Arhodomonas
aquaeolei
Arsenophonus
Arsenophonus
nasoniae
Arthrobacter
Arthrobacter
agilis
Arthrobacter
albus
Arthrobacter
aurescens
Arthrobacter
chlorophenolicus
Arthrobacter
citreus
Arthrobacter
crystallopoietes
Arthrobacter
cumminsii
Arthrobacter
globiformis
Arthrobacter
histidinolovorans
Arthrobacter
ilicis
Arthrobacter
luteus
Arthrobacter
methylotrophus
Arthrobacter
mysorens
Arthrobacter
nicotianae
Arthrobacter
nicotinovorans
Arthrobacter
oxydans
Arthrobacter
pascens
Arthrobacter
phenanthrenivorans
Arthrobacter
polychromogenes
Atrhrobacter
protophormiae
Arthrobacter
psychrolactophilus
Arthrobacter
ramosus
Arthrobacter
sulfonivorans
Arthrobacter
sulfureus
Arthrobacter
uratoxydans
Arthrobacter
ureafaciens
Arthrobacter
viscosus
Arthrobacter
woluwensis
Asaia
Ascua
bogorensis
Asanoa
Asanoa
ferruginea
Asticcacaulis
Asticcacaulis
biprosthecium
Asticcacaulis
excentricus
Atopobacter
Atopobacter
phocae
Atopobium
Atopobium
fossor
Atopobium
minutum
Atopobium
parvulum
Atopobium
rimae
Atopobium
vaginae
Aureobacterium
Aureobacterium
barkeri
Aurobacterium
Aurobacterium
liquefaciens
Avibacterium
Avibacterium
avium
Avibacterium
gallinarum
Avibacterium
paragallinarum
Avibacterium
volantium
Azoarcus
Azoarcus
indigens
Azoarcus
tolulyticus
Azoarcus
toluvorans
Azohydromonas
Azohydromonas
australica
Azohvdromonas
lata
Azomonas
Azomonas
agilis
Azomonas
insignis
Azomonas
macrocytogenes
Azorhizobium
Azorhizobium
caulinodans
Azorhizophilus
Azorhizophilus
paspali
Azospirillum
Azospirillum
brasilense
Azospirillum
halopraeferens
Azospirillum
irakense
Azotobacter
Azolobacter
beijerinckii
Azotobacter
chroococcum
Azotobacter
nigricans
Azotobacter
salinestris
Azotobacter
vinelandii
Bacillus
Bacteriovorax
Bacteriovorax
stolpii
Bacteroides
Bacteroides
caccae
Bacteroides
coagulans
Bacteroides
eggerthii
Bacteroides
fragilis
Bacteroides
galacturonicus
Bacteroides
helcogenes
Bacteroides
ovatus
Bacteroides
pectinophilus
Bacteroides
pyogenes
Bacteroides
salyersiae
Bacteroides
stercoris
Bacteroides
suis
Bacteroides
tectus
Bacteroides
thetaiotaomicron
Bacteroides
uniformis
Bacteroides
ureolyticus
Bacteroides
vulgatus
Balnearium
Balnearium
lithotrophicum
Balneatrix
Balneatrix
alpica
Balneola
Balneola
vulgaris
Barnesiella
Barnesiella
viscericola
Bartonella
Bartonella
alsatica
Bartonella
bacilliformis
Bartonella
clarridgeiae
Bartonella
doshiae
Bartonella
elizabethae
Bartonella
grahamii
Bartonella
henselae
Bartonella
rochalimae
Bartonella
vinsonii
Bavariicoccus
Bavariicoccus
seileri
Bdellovibrio
Bdellovibrio
bacteriovorus
Bdellovibrio
exovorus
Beggiatoa
Beggiatoa
alba
Beijerinckia
Beijerinckia
derxii
Beijerinckia
fluminensis
Beijerinckia
indica
Beijerinckia
mobilis
Belliella
Belliella
baltica
Bellilinea
Bellilinea
caldifistulae
Belnapia
Belnapia
moabensis
Bergeriella
Bergeriella
denitrificans
Beutenbergia
Beutenbergia
cavernae
Bibersteinia
Bibersteinia
trehalosi
Bifidobacterium
Bifidobacterium
adolescentis
Bifidobacterium
angulatum
Bifidobacterium
animalis
Bifidobacterium
asteroides
Bifidobacterium
bifidum
Bifidobacterium
boum
Bifidobacterium
breve
Bifidobacterium
catenulatum
Bifidobacterium
choerinum
Bifidobacterium
coryneforme
Bifidobacterium
cuniculi
Bifidobacterium
dentium
Bifidobacterium
gallicum
Bifidobacterium
gallinarum
Bifidobacterium
indicum
Bifidobacterium
longum
Bifidobacterium
magnum
Bifidobacterium
merycicum
Bifidobacterium
minimum
Bifidobacterium
pseudocatenulatum
Bifidobacterium
pseudolongum
Bifidobacterium
pullorum
Bifidobacterium
ruminantium
Bifidobacterium
saeculare
Bifidobacterium
subtile
Bifidobacterium
thermophilum
Bilophila
Bilophila
wadsworthia
Biostraticola
Biostraticola
tofi
Bizionia
Bizionia
argentinensis
Blastobacter
Blastobacter
capsulatus
Blastobacter
denitrificans
Blastococcus
Blastococcus
aggregatus
Blastococcus
saxobsidens
Blastochloris
Blastochloris
viridis
Blastomonas
Blastomonas
natatoria
Blastopirellula
Blastopirellula
marina
Blautia
Blautia
coccoides
Blautia
hansenii
Blautia
producta
Blautia
wexlerae
Bogoriella
Bogoriella
caseilytica
Bordetella
Bordetella
avium
Bordetella
bronchiseptica
Bordetella
hinzii
Bordetella
holmesii
Bordetella
parapertussis
Bordetella
pertussis
Bordetella
petrii
Bordetella
trematum
Borrelia
Borrelia
afzelii
Borrelia
americana
Borrelia
burgdorferi
Borrelia
carolinensis
Borrelia
coriaceae
Borrelia
garinii
Borrelia
japonica
Bosea
Bosea
minatitlanensis
Bosea
thiooxidans
Brachybacterium
Brachybacierium
alimentarium
Brachybacterium
faecium
Brachybacterium
paraconglomeratum
Brachybacterium
rhamnosum
Brachybacterium
tyrofermentans
Brachyspira
Brachyspira
alvinipulli
Brachyspira
hyodysenteriae
Brachyspira
innocens
Brachyspira
murdochii
Brachyspira
pilosicoli
Bradyrhizobium
Bradyrhizobium
canariense
Bradyrhizobium
elkanii
Bradyrhizobium
japonicum
Bradyrhizobium
liaoningense
Brenneria
Brenneria
alni
Brenneria
nigrifluens
Brenneria
quercina
Brenneria
quercina
Brenneria
salicis
Brevibacillus
Brevibacillus
agri
Brevibacillus
borstelensis
Brevibacillus
brevis
Brevibacillus
centrosporus
Brevibacillus
choshinensis
Brevibacillus
invocatus
Brevibacillus
laterosporus
Brevibacillus
parabrevis
Brevibacillus
reuszeri
Brevibacterium
Brevibacterium
abidum
Brevibacterium
album
Brevibacterium
aurantiacum
Brevibacterium
celere
Brevibacterium
epidermidis
Brevibacterium
frigoritolerans
Brevibacterium
halotolerans
Brevibacterium
iodinum
Brevibacterium
linens
Brevibacterium
lyticum
Brevibacterium
mcbrellneri
Brevibacterium
otitidis
Brevibacterium
oxydans
Brevibacterium
paucivorans
Brevibacterium
stationis
Brevinema
Brevinema
andersonii
Brevundimonas
Brevundimonas
alba
Brevundimonas
aurantiaca
Brevundimonas
diminuta
Brevundimonas
intermedia
Brevundimonas
subvibrioides
Brevundimonas
vancanneytii
Brevundimonas
variabilis
Brevundimonas
vesicularis
Brochothrix
Brochothrix
campestris
Brochothrix
thermosphacta
Brucella
Brucella
canis
Brucella
neotomae
Bryobacter
Bryobacter
aggregatus
Burkholderia
Burkholderia
ambifaria
Burkholderia
andropogonis
Burkholderia
anthina
Burkholderia
caledonica
Burkholderia
caryophylli
Burkholderia
cenocepacia
Burkholderia
cepacia
Burkholderia
cocovenenans
Burkholderia
dolosa
Burkholderia
fungorum
Burkholderia
glathei
Burkholderia
glumae
Burkholderia
graminis
Burkholderia
kururiensis
Burkholderia
multivorans
Burkholderia
phenazinium
Burkholderia
plantarii
Burkholderia
pyrrocinia
Burkholderia
silvatlanlica
Burkholderia
stabilis
Burkholderia
thailandensis
Burkholderia
tropica
Burkholderia
unamae
Burkholderia
vietnamiensis
Buttiauxella
Buttiauxella
agrestis
Buttiauxella
brennerae
Buttiauxella
ferragutiae
Buttiauxella
gaviniae
Buttiauxella
izardii
Buttiauxella
noackiae
Buttiauxella
warmboldiae
Butyrivibrio
Butyrivibrio
fibrisolvens
Butyrivibrio
hungatei
Butyrivibrio
proteoclasticus
Bacillus
B. acidiceler
B. acidicola
B. acidiproducens
B. acidocaldarius
B. acidoterrestris
B. aeolius
B. aerius
B. aerophilus
B. agaradhaerens
B. agri
B. aidingensis
B. akibai
B. alcalophilus
B. algicola
B. alginolyticus
B. alkalidiazotrophicus
B. alkalinitrilicus
B. alkalisediminis
B. alkalitelluris
B. altitudinis
B. alveayuensis
B. alvei
B. amyloliquefaciens
B. a. subsp. amyloliquefaciens
B. a. subsp. plantarum
B. dipsosauri
B. drentensis
B. edaphicus
B. ehimensis
B. eiseniae
B. enclensis
B. endophyticus
B. endoradicis
B. farraginis
B. fastidiosus
B. fengqiuensis
B. firmus
B. flexus
B. foraminis
B. fordii
B. formosus
B. fortis
B. fumarioli
B. funiculus
B. fusiformis
B. galactophilus
B. galactosidilyticus
B. galliciensis
B. gelatini
B. gibsonii
B. ginsengi
B. ginsengihumi
B. ginsengisoli
B. globisporus
B. g. subsp. Marinus)
B. aminovorans
B. amylolyticus
B. andreesenii
B. aneurinilyticus
B. anthracis
B. aquimaris
B. arenosi
B. arseniciselenatis
B. arsenicus
B. aurantiacus
B. arvi
B. aryabhattai
B. asahii
B. atrophaeus
B. axarquiensis
B. azotofixans
B. azotoformans
B. badius
B. barbaricus
B. bataviensis
B. beijingensis
B. benzoevorans
B. beringensis
B. berkeleyi
B. beveridgei
B. bogoriensis
B. boroniphilns
B. borstelensis
B. brevisMigula
B. butanolivorans
B. canaveralius
B. carboniphilus
B. cecembensis
B. cellulosilyticus
B. centrosporus
B. cereus
B. chagannorensis
B. chitinolyticus
B. chondroitinus
B. choshinensis
B. chungangensis
B. cibi
B. circulans
B. clarkii
B. clausii
B. coagulans
B. coahuilensis
B. cohnii
B. composti
B. curdlanolyticus
B. cycloheptanicus
B. cytotoxicus
B. daliensis
B. decisifrondis
B. decolorationis
B. deserti
B. glucanolyticus
B. gordonae
B. gottheilii
B. graminis
B. halmapalus
B. haloalkaliphilus
B. halochares
B. halodenitrificans
B. halodurans
B. halophilus
B. halosaccharovorans
B. hemicellulosilyticus
B. hemicentroti
B. herbersteinensis
B. horikoshii
B. horneckiae
B. horti
B. huizhouensis
B. humi
B. hwajinpoensis
B. idriensis
B. indicus
B. infantis
B. infernus
B. insolitus
B. invictae
B. iranensis
B. isabeliae
B. isronensis
B. jeotgali
B. kaustophilus
B. kobensis
B. kochii
B. kokeshiiformis
B. koreensis
B. korlensis
B. kribbensis
B. krulwichiae
B. laevolacticus
B. larvae
B. laterosporus
B. salexigens
B. saliphilus
B. schlegelii
B. sediminis
B. selenatarsenatis
B. selenitireducens
B. seohaeanensis
B. shacheensis
B. shackletonii
B. siamensis
B. silvestris
B. simplex
B. siralis
B. smithii
B. soli
B. solimangrovi
B. solisalsi
B. songklensis
B. sonorensis
B. sphaericus
B. sporothermodurans
B. stearothermophilus
B. stratosphericus
B. subterraneus
B. subtilis
B. s. subsp. Spizizeni; or
B. s. subsp. Subtilis)
B. taeanensis
B. tequilensis
B. thermantarcticus
B. thermoaerophilus
B. thermoamylovorans
B. thermocatenulatus
B. thermocloacae
B. thermocopriae
B. thermodenitrificans
B. thermoglucosidasius
B. thermolactis
B. thermoleovorans
B. thermophilus
B. thermoruber
B. thermosphaericus
B. thiaminolyticus
B. thioparans
B. thuringiensis
B. tianshenii
B. trypoxylicola
B. tusciae
B. validus
B. vallismortis
B. vedderi
B. velezensis
B. vietnamensis
B. vireti
B. vulcani
B. wakoensis
B. weihenstephanensis
B. xiamenensis
B. xiaoxiensis
B. zhanjiangensis
B. peoriae
B. persepolensis
B. persicus
B. pervagus
B. plakortidis
B. pocheonensis
B. polygoni
B. polymyxa
B. popilliae
B. pseudalcalophilus
B. pseudofirmus
B. pseudomycoides
B. psychrodurans
B. psychrophilns
B. psychrosaccharolyticus
B. psychrotolerans
B. pulvifaciens
B. pumilus
B. purgationiresistens
B. pycnus
B. qingdaonensis
B. qingshengii
B. reuszeri
B. rhizosphaerae
B. rigui
B. ruris
B. safensis
B. salarius
B. lautus
B. lehensis
B. lentimorbus
B. lentus
B. licheniformis
B. ligniniphilus
B. litoralis
B. locisalis
B. luciferensis
B. luteolus
B. luteus
B. macauensis
B. macerans
B. macquariensis
B. macyae
B. malacitensis
B. mannanilyticus
B. marisflavi
B. marismortui
B. marmarensis
B. massiliensis
B. megaterium
B. mesonae
B. methanolicus
B. methylotrophicus
B. migulanus
B. mojavensis
B. mucilaginosus
B. muralis
B. murimartini
B. mycoides
B. naganoensis
B. nanhaiensis
B. nanhaiisediminis
B. nealsonii
B. neidei
B. neizhouensis
B. niabensis
B. niacini
B. novalis
B. oceanisediminis
B. odysseyi
B. okhensis
B. okuhidensis
B. oleronius
B. oryzaecorticis
B. oshimensis
B. pabuli
B. pakistanensis
B. pallidus
B. pallidus
B. panacisoli
B. panaciterrae
B. pantothenticus
B. parabrevis
B. pciraflexus
B. pasteurii
B. patagoniensis
Caenimonas
Caertimonas
koreensis
Caldalkalibacillus
Caldalkalibacillus
uzonensis
Caldanaerobacter
Caldanaerobacter
subterraneus
Caldanaerobius
Caldanaerobius
fijiensis
Caldanaerobius
polysaccharolyticus
Caldanaerobius
zeae
Caldanaerovirga
Caldanaerovirga
acetigignens
Caldicellulosiruptor
Caldicellulosiruptor
bescii
Caldicellulosiruptor
kristjanssonii
Caldicellulosiruptor
owensensis
Campylobacter
Campylobacter
coli
Campylobacter
concisus
Campylobacter
curvus
Campylobacter
fetus
Campylobacter
gracilis
Campylobacter
helveticus
Campylobacter
hominis
Campylobacter
hyointestinalis
Campylobacter
jejuni
Campylobacter
lari
Campylobacter
mucosalis
Campylobacter
rectus
Campylobacter
showae
Campylobacter
sputorum
Campylobacter
upsaliensis
Capnocytophaga
Capnocytophaga
canimorsus
Capnocytophaga
cynodegmi
Capnocytophaga
gingivalis
Capnocytophaga
granulosa
Capnocytophaga
haemolytica
Capnocytophaga
ochracea
Capnocytophaga
sputigena
Cardiobacterium
Cardiobacterium
hominis
Carnimonas
Carnimoncis
nigrificans
Carnobacterium
Carnobacterium
alterfunditum
Carnobacterium
divergens
Carnobacterium
funditum
Carnobacterium
gallinarum
Carnobacterium
maltaromaticum
Carnobacterium
mobile
Carnobacterium
viridans
Caryophanon
Caryophanon
latum
Caryophanon
tenue
Catellatospora
Catellatospora
citrea
Catellatospora
methionotrophica
Catenococcus
Catenococcus
thiocycli
Catenuloplanes
Catenuloplanes
atrovinosus
Catenuloplanes
castaneus
Catenuloplanes
crispus
Catenuloplanes
indicus
Catenuloplanes
japonicus
Catenuloplanes
nepalensis
Catenuloplanes
niger
Chryseobacterium
Chryseobacterium
balustinum
Citrobacter
C. amalonaticus
C. braakii
C. diversus
C. farmeri
C. freundii
C. gillenii
C. koseri
C. murliniae
C. pasteurii[1]
C. rodentium
C. sedlakii
C. werkmanii
C. youngae
Clostridium
Coccochloris
Coccochloris
elabens
Corynebacterium
Corynebacterium
flavescens
Corynebacterium
variabile
Curtobacterium
Curtobacterium
albidum
Curtobacterium
citreus
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
chouvoei,
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
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
Dactylosporangium
aurantiacum
Dactylosporangium
fulvum
Dactylosporangium
matsuzakiense
Dactylosporangium
roseum
Dactylosporangium
thailandense
Dactylosporangium
vinaceum
Deinococcus
Deinococcus
aerius
Deinococcus
apachensis
Deinococcus
aquaticus
Deinococcus
aquatilis
Deinococcus
caeni
Deinococcus
radiodurans
Deinococcus
radiophilus
Delftia
Delflia
acidovorans
Desulfovibrio
Desulfovibrio
desulfuricans
Diplococcus
Diplococcus
pneumoniae
Echinicola
Echinicola
pacifica
Echinicola
vietnamensis
Enterobacter
E. aerogenes
E. amnigenus
E. agglomerans
E. arachidis
E. asburiae
E. cancerogenous
E. cloacae
E. cowanii
E. dissolvens
E. gergoviae
E. helveticus
E. hormaechei
E. intermedius
Enterobacter
kobei
E. ludwigii
E. mori
E. nimipressuralis
E. oryzae
E. pulveris
E. pyrinus
E. radicincitans
E. taylorae
E. turicensis
E. sakazakii
Enterobacter
soli
Enterococcus
Enterococcus
durans
Enterococcus
faecalis
Enterococcus
faecium
Erwinia
Erwinia
hapontici
Escherichia
Escherichia
coli
Faecalibacterium
Faecalibacterium
prausnitzii
Fangia
Fangia
hongkongensis
Fastidiosipila
Fastidiosipila
sanguinis
Fusobacterium
Fusobacterium
nucleatum
Flavobacterium
Flavobacterium
antarcticum
Flavobacterium
aquatile
Flavobacterium
aquidurense
Flavobacterium
balustinum
Flavobacterium
croceum
Flavobacterium
cucumis
Flavobacterium
daejeonense
Flavobacterium
defluvii
Flavobacterium
degerlachei
Flavobacterium
denitrificans
Flavobacterium
filum
Flavobacterium
flevense
Flavobacterium
frigidarium
Flavobacterium
mizutaii
Flavobacterium
okeanokoites
Gaetbulibacter
Gaetbulibacter
saemankumensis
Gallibacterium
Gallibacterium
anatis
Gallicola
Gallicola
barnesae
Garciella
Garciella
nitratireducens
Geobacillus
Geobacillus
thermoglucosidasius
Geobacillus
stearothermophilus
Geobacter
Geobacter
bemidjiensis
Geobacter
bremensis
Geobacter
chapellei
Geobacter
grbiciae
Geobacter
hydrogenophilus
Geobacter
lovleyi
Geobacter
metallireducens
Geobacter
pelophilus
Geobacter
pickeringii
Geobacter
sulfurreducens
Geodermatophilus
Geodermatophilus
obscurus
Gluconacetobacter
Gluconacetobacter
xylinus
Gordonia
Gordonia
rubripertincta
Haemophilus
Haemophilus
aegyptius
Haemophilus
aphrophilus
Haemophilus
felis
Haemophilus
gallinarum
Haemophilus
haemolyticus
Haemophilus
influenzae
Haemophilus
paracuniculus
Haemophilus
parahaemolyticus
Haemophilus
parainfluenzae
Haemophilus
paraphrohaemolyticus
Haemophilus
parasuis
Haemophilus
pittmaniae
Hafnia
Hafnia
alvei
Hahella
Hahella
ganghwensis
Halalkalibacillus
Halalkalibacillus
halophilus
Helicobacter
Helicobacter
pylori
Ideonella
Ideonella
azotifigens
Idiomarina
Idiomarina
abyssalis
Idiomarina
baltica
Idiomarina
fontislapidosi
Idiomarina
loihiensis
Idiomarina
ramblicola
Idiomarina
seosinensis
Idiomarina
zobellii
Ignatzschineria
Ignatzschineria
larvae
Ignavigranum
Ignavigranum
ruoffiae
Ilumatobacter
Ilumatobacter
fluminis
Ilyobacter
Ilyobacter
delafieldii
Ilyobacter
insuetus
Ilyobacter
polytropus
Ilyobacter
tartaricus
Janibacter
Janibacter
anophelis
Janibacter
corallicola
Janibacter
limosus
Janibacter
melonis
Janibacter
terrae
Jannaschia
Jannaschia
cystaugens
Jannaschia
helgolandensis
Jannaschia
pohangensis
Jannaschia
rubra
Janthinobacterium
Janthinobacterium
agaricidamnosum
Janthinobacterium
lividum
Jejuia
Jejuia
pallidilutea
Jeotgalibacillus
Jeotgalibacillus
alimentarius
Jeotgalicoccus
Jeotgalicoccus
halotolerans
Kaistia
Kaistia
adipata
Kaistia
soli
Kangiella
Kangiella
aquimarina
Kangiella
koreensis
Kerstersia
Kerstersia
gyiorum
Kiloniella
Kiloniella
laminariae
Klebsiella
K. granulomatis
K. oxytoca
K. pneumoniae
K. terrigena
K. variicola
Kluyvera
Kluyvera
ascorbata
Kocuria
Kocuria
roasea
Kocuria
varians
Kurthia
Kurthia
zopfii
Labedella
Labedella
gwakjiensis
Labrenzia
Labrenzia
aggregata
Labrenzia
alba
Labrenzia
alexandrii
Labrenzia
marina
Labrys
Labrys
methylaminiphilus
Labrys
miyagiensis
Labrys
monachus
Labrys
okinawensis
Labrys
portucalensis
Lactobacillus
Laceyella
Laceyella
putida
Lechevalieria
Lechevalieria
aerocolonigenes
Legionella
Listeria
L. aquatica
L. booriae
L. cornellensis
L. fleischmannii
L. floridensis
L. grandensis
L. grayi
L. innocua
Listeria ivanovii
L. marthii
L. monocytogenes
L. newyorkensis
L. riparia
L. rocourtiae
L. seeligeri
L. weihenstephanensis
L. welshimeri
Listonella
Listonella
anguillarum
Macrococcus
Macrococcus
bovicus
Marinobacter
Marinobacter
algicola
Marinobacter
bryozoorum
Marinobacter
flavimaris
Meiothermus
Meiothermus
ruber
Methylophilus
Methylophilus
methylotrophus
Microbacterium
Microbacterium
ammoniaphilum
Microbacterium
arborescens
Microbacterium
liquefaciens
Microbacterium
oxydans
Micrococcus
Micrococcus luteus
Micrococcus lylae
Moraxella
Moraxella
bovis
Moraxella
nonliquefaciens
Moraxella
osloensis
Nakamurella
Nakamurella
multipartita
Nannocystis
Nannocystis
pusilla
Natranaerobius
Natranaerobius
thermophilus
Natranaerobius
trueperi
Naxibacter
Naxibacter
alkalitolerans
Neisseria
Neisseria
cinerea
Neisseria
denitrificans
Neisseria
gonorrhoeae
Neisseria
lactamica
Neisseria
mucosa
Neisseria
sicca
Neisseria
subflava
Neptunomonas
Neptunomonas japonica
Nesterenkonia
Nesterenkonia
holobia
Nocardia
Nocardia
argentinensis
Nocardia
corallina
Nocardia
otitidiscaviarum
Lactobacillus
L. acetotolerans
L. acidifarinae
L. acidipiscis
L. acidophilus
Lactobacillus
agilis
L. algidus
L. alimentarius
L. amylolyticus
L. amylophilus
L. amylotrophicus
L. amylovorus
L. animalis
L. antri
L. apodemi
L. aviarius
L. bifermentans
L. brevis
L. buchneri
L. camelliae
L. casei
L. kitasatonis
L. kunkeei
L. leichmannii
L. lindneri
L. malefermentans
L. catenaformis
L. ceti
L. coleohominis
L. collinoides
L. composti
L. concavus
L. coryniformis
L. crispatus
L. crustorum
L. curvatus
L. delbrueckii subsp. bulgaricus
L. delbrueckii subsp. delbrueckii
L. delbrueckii subsp. lactis
L. dextrinicus
L. diolivorans
L. equi
L. equigenerosi
L. farraginis
L. farciminis
L. fermentum
L. fornicalis
L. fructivorans
L. frumenti
L. mali
L. manihotivorans
L. mindensis
L. mucosae
L. murinus
L. nagelii
L. namurensis
L. nantensis
L. oligofermentans
L. oris
L. panis
L. pantheris
L. parabrevis
L. parabuchneri
L. paracasei
L. paracollinoides
L. parafarraginis
L. homohiochii
L. iners
L. ingluviei
L. intestinalis
L. fuchuensis
L. gallinarum
L. gasseri
L. parakefiri
L. paralimentarius
L. paraplantarum
L. pentosus
L. perolens
L. plantarum
L. pontis
L. protectus
L. psittaci
L. rennini
L. reuteri
L. rhamnosus
L. rimae
L. rogosae
L. rossiae
L. ruminis
L. saerimneri
L. jensenii
L. johnsonii
L. kalixensis
L. kefiranofaciens
L. kefiri
L. kimchii
L. helveticus
L. hilgardii
L. sakei
L. salivarius
L. sanfranciscensis
L. satsumemis
L. secaliphilus
L. sharpeae
L. siliginis
L. spicheri
L. suebicus
L. thailandensis
L. ultunensis
L. vaccinostercus
L. vaginalis
L. versmoldensis
L. vini
L. vitulinus
L. zeae
L. zymae
L. gastricus
L. ghanensis
L. graminis
L. hammesii
L. hamsteri
L. harbinensis
L. hayakitensis
Legionella
Legionella
adelaidensis
Legionella
anisa
Legionella
beliardensis
Legionella
birminghamensis
Legionella
bozemanae
Legionella
brunensis
Legionella
busanensis
Legionella
cardiaca
Legionella
cherrii
Legionella
cincinnatiensis
Legionella
clemsonensis
Legionella
donaldsonii
Legionella
drancourtii
Legionella
dresdenensis
Legionella
drozanskii
Legionella
dumoffii
Legionella
erythra
Legionella
fairfieldensis
Legionella
fallonii
Legionella
feeleii
Legionella
geestiana
Legionella
genomospecies
Legionella
gormanii
Legionella
gratiana
Legionella
gresilensis
Legionella
hackeliae
Legionella
impletisoli
Legionella
israelensis
Legionella
jamestowniensis
Legionella
jordanis
Legionella
lansingensis
Legionella
londiniensis
Legionella
longbeachae
Legionella
lytica
Legionella
maceachernii
Legionella
massiliensis
Legionella
micdadei
Legionella
monrovica
Legionella
moravica
Legionella
nagasakiensis
Legionella
nautarum
Legionella
norrlandica
Legionella
oakridgensis
Legionella
parisiensis
Legionella
pittsburghensis
Legionella
pneumophila
Legionella
quateirensis
Legionella
quinlivanii
Legionella
rowbothamii
Legionella
rubrilucens
Legionella
sainthelensi
Legionella
santicrucis
Legionella
shakespearei
Legionella
spiritensis
Legionella
steelei
Legionella
steigerwaltii
Legionella
taurinensis
Legionella
tucsonensis
Legionella
tunisiensis
Legionella
wadsworthii
Legionella
waltersii
Legionella
worsleiensis
Legionella
yabuuchiae
Oceanibulbus
Oceanibulbus
indolifex
Oceanicaulis
Oceanicaulis
alexandrii
Oceanicola
Oceanicola
batsensis
Oceanicola
granulosus
Oceanicola
nanhaiensis
Oceanimonas
Oceanimonas
baumannii
Oceaniserpentilla
Oceaniserpentilla
haliotis
Oceanisphaera
Oceanisphaera
donghaensis
Oceanisphaera
litoralis
Oceanithermus
Oceanithermus
desulfurans
Oceanithermus
profundus
Oceanobacillus
Oceanobacillus
caeni
Oceanospirillum
Oceanospirillum
linum
Paenibacillus
Paenibacillus
thiaminolyticus
Pantoea
Pantoea
agglomerans
Paracoccus
Paracoccus
alcaliphilus
Paucimonas
Paucimonas
lemoignei
Pectobacterium
Pectobacterium
aroidearum
Pectobacterium
atrosepticum
Pectobacterium
betavasculorum
Pectobacterium
cacticida
Pectobacterium
carnegieana
Pectobacterium
carotovorum
Pectobacterium
chrysanthemi
Pectobacterium
cypripedii
Pectobacterium
rhapontici
Pectobacterium
wasabiae
Planococcus
Planococcus
citreus
Planomicrobium
Planomicrobium
okeanokoites
Plesiomonas
Plesiomonas
shigelloides
Proteus
Proteus
vulgaris
Prevotella
Prevotella
albensis
Prevotella
amnii
Prevotella
bergensis
Prevotella
bivia
Prevotella
brevis
Prevotella
bryantii
Prevotella
buccae
Prevotella
buccalis
Prevotella
copri
Prevotella
dentalis
Prevotella
denticola
Prevotella
disiens
Prevotella
histicola
Prevotella
intermedia
Prevotella
maculosa
Prevotella
marshii
Prevotella
melaninogenica
Prevotella
micans
Prevotella
multiformis
Prevotella
nigrescens
Prevotella
oralis
Prevotella
oris
Prevotella
oulorum
Prevotella
pallens
Prevotella
salivae
Prevotella
stercorea
Prevotella
tannerae
Prevotella
timonensis
Prevotella
veroralis
Providencia
Providencia
stuartii
Pseudomonas
Pseudomonas
aeruginosa
Pseudomonas
alcaligenes
Pseudomonas
anguillispetica
Pseudomonas
fluorescens
Pseudoalteromonas
haloplanktis
Pseudomonas
mendocina
Pseudomonas
pseudoalcaligenes
Pseudomonas
putida
Pseudomonas
tutzeri
Pseudomonas
syringae
Psychrobacter
Psychrobacter
faecalis
Psychrobacter
phenylpyruvicus
Quadrisphaera
Quadrisphaera
granulorum
Quatrionicoccus
Quatrionicoccus
australiensis
Quinella
Quinella
ovalis
Ralstonia
Ralstonia
eutropha
Ralstonia
insidiosa
Ralstonia
mannitolilytica
Ralstonia
pickettii
Ralstonia
pseudosolanacearum
Ralstonia
syzygii
Ralstonia
solanacearum
Ramlibacter
Ramlibacter
henchirensis
Ramlibacter
tataouinensis
Raoultella
Raoultella
ornithinolytica
Raoultella
planticola
Raoultella
terrigena
Rathayibacter
Rathayibacter
caricis
Rathayibacter
festucae
Rathayibacter
iranicus
Rathayibacter
rathayi
Rathayibacter
toxicus
Rathayibacter
tritici
Rhodobacter
Rhodobacter
sphaeroides
Ruegeria
Ruegeria
gelatinovorans
Saccharococcus
Saccharococcus
thermophilus
Saccharomonospora
Saccharomonospora
azurea
Saccharomonospora
cyanea
Saccharomonospora
viridis
Saccharophagus
Saccharophagus
degradans
Saccharopolyspora
Saccharopolyspora
erythraea
Saccharopolyspora
gregorii
Saccharopolyspora
hirsuta
Saccharopolyspora
hordei
Saccharopolyspora
rectivirgula
Saccharopolyspora
spinosa
Saccharopolyspora
taberi
Saccharothrix
Saccharothrix
australiensis
Saccharothrix
coeruleofusca
Saccharothrix
espanaensis
Saccharothrix
longispora
Saccharothrix
mutabilis
Saccharothrix
syringae
Saccharothrix
tangerinus
Saccharothrix
texasensis
Sagittula
Sagittula
stellata
Salegentibacter
Salegentibacter
salegens
Salimicrobium
Salimicrobium
album
Salinibacter
Salinibacter
ruber
Salinicoccus
Salinicoccus
alkaliphilus
Salinicoccus
hispanicus
Salinicoccus
roseus
Salinispora
Salinispora
arenicola
Salinispora
tropica
Salinivibrio
Salinivibrio
costicola
Salmonella
Salmonella
bongori
Salmonella
enterica
Salmonella
subterranea
Salmonella
typhi
Sanguibacter
Sanguibacter
keddieii
Sanguibacter
suarezii
Saprospira
Saprospira
grandis
Sarcina
Sarcina
maxima
Sarcina
ventriculi
Sebaldella
Sebaldella
termitidis
Serratia
Serratia
fonticola
Serratia
marcescens
Sphaerotilus
Sphaerotilus
natans
Sphingobacterium
Sphingobacterium
multivorum
Staphylococcus
Stenotrophomonas
Stenotrophomonas
maltophilia
Streptococcus
Streptomyces
Streptomyces
achromogenes
Streptomyces
cesalbus
Streptomyces
cescaepitosus
Streptomyces
cesdiastaticus
Streptomyces
cesexfoliatus
Streptomyces
fimbriatus
Streptomyces
fradiae
Streptomyces
fulvissimus
Streptomyces
griseoruber
Streptomyces
griseus
Streptomyces
lavendulae
Streptomyces
phaeochromogenes
Streptomyces
thermodiastaticus
Streptomyces
tubercidicus
Tatlockia
Tatlockia
maceachernii
Tatlockia
micdadei
Tenacibaculum
Tenacibaculum
amylolyticum
Tenacibaculum
discolor
Tenacibaculum
gallaicum
Tenacibaculum
lutimaris
Tenacibaculum
mesophilum
Tenacibaculum
skagerrakense
Tepidanacrobaeter
Tepidanaerobacter
syntrophicus
Tepidibacter
Tepidibacter
formicigenes
Tepidibacter
thalassicus
Thermus
Thermus
aquaticus
Thermus
filiformis
Thermus
thermophilus
Staphylococcus
S. arlettae
S. agnetis
S. aureus
S. auricularis
S. capitis
S. caprae
S. carnosus
S. caseolyticus
S. chromogenes
S. cohnii
S. condimenti
S. delphini
S. devriesei
S. epidermidis
S. equorum
S. felis
S. fleurettii
S. gallinarum
S. haemolyticus
S. hominis
S. hyicus
S. intermedius
S. kloosii
S. leei
S. lentus
S. lugdunensis
S. lutrae
S. lyticans
S. massiliensis
S. microti
S. muscae
S. nepalensis
S. pasteuri
S. petrasii
S. pettenkoferi
S. piscifermentans
S. pseudintermedius
S. pseudolugdunensis
S. pulvereri
S. rostri
S. saccharolyticus
S. saprophyticus
S. schleiferi
S. sciuri
S. simiae
S. simulans
S. stepanovicii
S. succinus
S. vitulinus
S. warneri
S. xylosus
Streptococcus
Streptococcus
agalactiae
Streptococcus
anginosus
Streptococcus
bovis
Streptococcus
canis
Streptococcus
constellatus
Streptococcus
downei
Streptococcus
dysgalactiae
Streptococcus
equines
Streptococcus
faecalis
Streptococcus
ferus
Streptococcus
infantarius
Streptococcus
iniae
Streptococcus
intermedius
Streptococcus
lactarius
Streptococcus
milleri
Streptococcus
mitis
Streptococcus
mutans
Streptococcus
oralis
Streptococcus
tigurinus
Streptococcus
orisratti
Streptococcus
parasanguinis
Streptococcus
peroris
Streptococcus
pneumoniae
Streptococcus
pseudopneumoniae
Streptococcus
pyogenes
Streptococcus
ratti
Streptococcus
salivariu
Streptococcus
thermophilus
Streptococcus
sanguinis
Streptococcus
sobrinus
Streptococcus
suis
Streptococcus
uberis
Streptococcus
vestibularis
Streptococcus
viridans
Streptococcus
zooepidemicus
Uliginosibacterium
Uliginosibacterium
gangwonense
Ulvibacter
Ulvibacter
litoralis
Umezawaea
Umezawaea
tangerina
Undibacterium
Undibacterium
pigrum
Ureaplasma
Ureaplasma
urealyticum
Ureibacillus
Ureibacillus
composti
Ureibacillus
suwonensis
Ureibacillus
terrenus
Ureibacillus
thermophilus
Ureibacillus
thermosphaericus
Vagococcus
Vagococcus
carniphilus
Vagococcus
elongatus
Vagococcus
fessus
Vagococcus
fluvialis
Vagococcus
lutrae
Vagococcus
salmoninarum
Variovorax
Variovorax
boronicumulans
Variovorax
dokdonensis
Variovorax
paradoxus
Variovorax
soli
Veillonella
Veillonella
atypica
Veillonella
caviae
Veillonella
criceti
Veillonella
dispar
Veillonella
montpellierensis
Veillonella
parvula
Veillonella
ratti
Veillonella
rodentium
Venenivibrio
Venenivibrio
stagnispumantis
Verminephrobacter
Verminephrobacter
eiseniae
Verrucomicrobium
Verrucomicrobium
spinosum
Vibrio
Vibrio
aerogenes
Vibrio
aestuarianus
Vibrio
albensis
Vibrio
alginolyticus
Vibrio
compbellii
Vibrio
cholerae
Vibrio
cincinnatiensis
Vibrio
coralliilyticus
Vibrio
cyclitrophicus
Vibrio
diazotrophicus
Vibrio
fluvialis
Vibrio
furnissii
Vibrio
gazogenes
Vibrio
halioticoli
Vibrio
harveyi
Vibrio
ichthyoenteri
Vibrio
mediterranei
Vibrio
metschnikovii
Vibrio
mytili
Vibrio
natriegens
Vibrio
navarrensis
Vibrio
nereis
Vibrio
nigripulchritudo
Vibrio
ordalii
Vibrio
orientalis
Vibrio
parahaemolyticus
Vibrio
pectenicida
Vibrio
penaeicida
Vibrio
proteolyticus
Vibrio
shilonii
Vibrio
splendidus
Vibrio
tubiashii
Vibrio
vulnificus
Virgibacillus
Virgibacillus
halodenitrificans
Virgibacillus
pantothenticus
Weissella
Weissella
cibaria
Weissella
confusa
Weissella
halotolerans
Weissella
hellenica
Weissella
kandleri
Weissella
koreensis
Weissella
minor
Weissella
paramesenteroides
Weissella
soli
Weissella
thailandensis
Weissella
viridescens
Williamsia
Williamsia
marianensis
Williamsia
maris
Williamsia
serinedens
Winogradskyella
Winogradskyella
thalassocola
Wolbachia
Wolbachia
persica
Wolinella
Wolinella
succinogenes
Zobellia
Zobellia
galactanivorans
Zobellia
uliginosa
Zoogloea
Zoogloea
ramigera
Zoogloea
resiniphila
Xanthobacter
Xanthobacter
agilis
Xanthobactcr
aminoxidans
Xanthobacter
autotrophicus
Xanthobacter
flavus
Xanthobacter
tagetidis
Xanthobacter
viscosus
Xanthomonas
Xanthomonas
albilineans
Xanthomonas
alfalfae
Xanthomonas
arboricola
Xanthomonas
axonopodis
Xanthomonas
campestris
Xanthomonas
citri
Xanthomonas
codiaei
Xanthomonas
cucurbitae
Xanthomonas
euvesicatoria
Xanthomonas
fragariae
Xanthomonas
fuscans
Xanthomonas
gardneri
Xanthomonas
hortorum
Xanthomonas
hyacinthi
Xanthomonas
perforans
Xanthomonas
phaseoli
Xanthomonas
pisi
Xanthomonas
populi
Xanthomonas
theicola
Xanthomonas
translucens
Xanthomonas
vesicatoria
Xylella
Xylella
fastidiosa
Xylophilus
Xylophilus
ampelinus
Xenophilus
Xenophilus
azovorans
Xenorhabdus
Xenorhabdus
beddingii
Xenorhabdus
bovienii
Xenorhabdus
cabanillasii
Xenorhabdus
doucetiae
Xenorhabdus
griffiniae
Xenorhabdus
hominickii
Xenorhabdus
koppenhoeferi
Xenorhabdus
nematophila
Xenorhabdus
poinarii
Xylanibacter
Xylanibacter
oryzae
Yangia
Yangia
pacifica
Yaniella
Yaniella
flava
Yaniella
halotolerans
Yeosuana
Yeosuana
aromativorans
Yersinia
Yersinia
aldovae
Yersinia
bercovieri
Yersinia
enterocolitica
Yersinia
entomophaga
Yersinia
frederiksenii
Yersinia
intermedia
Yersinia
kristensenii
Yersinia
mollaretii
Yersinia
philomiragia
Yersinia
pestis
Yersinia
pseudotuberculosis
Yersinia
rohdei
Yersinia
ruckeri
Yokenella
Yokenella
regensburgei
Yonghaparkia
Yonghaparkia
alkaliphila
Zavarzinia
Zavarzinia
compransoris
Zooshikella
Zooshikella
ganghwensis
Zunongwangia
Zunongwangia
profunda
Zymobacter
Zymobacter
palmae
Zymomonas
Zymomonas
mobilis
Zymophilus
Zymophilus
paucivorans
Zymophilus
raffinosivorans
Zobellella
Zobellella
denitrificans
Zobellella
taiwanensis
Zeaxanthinibacter
Zeaxanthinibacter
enoshimensis
Zhihengliuella
Zhihengliuella
halotolerans
Xylanibacterium
Xylanibacterium
ulmi
| Number | Date | Country | Kind |
|---|---|---|---|
| GB1719896.1 | Nov 2017 | GB | national |
| GB1808063.0 | May 2018 | GB | national |
